Stereoelectronic Effects in Nucleosides and Nucleotides and Their Structural Implications

Stereoelectronic Effects in Nucleosides and Nucleotides and

their Structural Implications (including Appendix on literature up to 2005)

Christophe Thibaudeau, Parag Acharya and Jyoti Chattopadhyaya*

*To whom correspondence should be addressed.

E-mail: [email protected]

F +4618554495 T +46184714577

www.boc.uu.se

Department of Bioorganic Chemistry Biomedical Center, Uppsala University, Sweden

Christophe Thibaudeau, Parag Acharya and Jyoti Chattopadhyaya* Dept of Bioorganic Chemistry Uppsala University Biomedical Center, Box 581 S-751 23 Uppsala

Dr C. Thibaudeau has completed his Ph.D in Feb, 1999 at the Dept of Bioorganic Chemistry under the supervision of Prof J. Chattopadhyaya

Dr P. Acharya has completed his Ph.D in Dec, 2003 at the Dept of Bioorganic Chemistry under the supervision of Prof J. Chattopadhyaya

Dr J. Chattopadhyaya is professor of Bioorganic Chemistry at the Uppsala University

Use the following for citation in your reference:

3 The intrinsic flexibility of pentoses in natural PREFACE nucleosides and nucleotides, owing to their lower energy barrier for interconversions compared to the The three essential components of DNA and RNA are hexopyranoses, is dictated by the energetics of the aglycone, the pentofuranose sugar in β-D stereochemistry stereoelectronic effects, which simply can be tuned and the phosphodiester. The phosphates at the backbone and modulated by choice of substituents and their makes DNA/RNA to behave as polyelectrolyte, the pentose ionization state as well as by their complexation with sugar gives the intrinsic flexibility with relatively low energy potential ligands present in the medium. barrier for conformational interconversions (according to the Stereoelectronic effects operate by appropriate demand of the environment)compared to the hexopyranoses, orbital overlap between the donor and acceptor and the aglycones help in the self-assembly process throgh the through-bond and through-space interactions. The stacking and hydrogen bonding. Thus, the stability of the strength of the stereoelectronic effects induced folded structure of nucleic acids is usually attributed to the stabilization is proportional to the square of the interplay of various forces such as hydrogen bonding, overlap of the donor and acceptor orbitals, and is stacking, electrostatics, hydrophobics and hydration. inversely proportional to their energy difference. Are the physico-chemical roles of the aglycone, sugar Finally, a new section has been added and phosphate correlated and interdependent? If so, what are covering the latest in the field (Appendix: Chapter their thermodynamics? Energetically, how do they respond to 10), which makes this monograph current till the end the local change of the environment, and affect the of 2002. recognition process that culminate into function? We believe this monograph is of considerable It has long been qualitatively known that they are not value to those medicinal and pharmaceutical isolated structural elements. The electronic nature of the chemists in the academia or in the industry, who aglycone dictates certain preferred sugar torsions, which are wish to understand fundamental mechanism involved again correlated with certain phosphate torsions – they are in the design of structure of modified nucleos(t)ides, interdependent. X-ray crystallographic and NMR studies have and use this knowledge to rationally develop demonstrated how the sugar and phosphate moieties can antisense, RNAi or triplexing agents or specific adopt different conformations in DNA and RNA. It has been enzyme inhibitors. shown that the rotations about C-O and P-O ester bonds are We thank Swedish Natural Science Research restricted, and certain sugar-phosphate torsions are preferred Council (Vetenskapsrådet), Swedish Organization over the others. for Strategic Research (SSF) and Uppsala University Prior to the work summarized here from the Uppsala for generous financial support in our research group, very little was known about the dynamic projects interdependency of the conformational changes between the three essential components of DNA and RNA or the energetics involved in this process. Through our solution NMR studies, we have attempted to show that the electronic nature of the aglycone as well as those of all other substituents of the sugar moiety dictate the intrinsic character of the pentose-sugar conformation, which in turn dictate the phosphate backbone torsions. The important aspect of this dynamic interdependency of the aglycone-sugar-phosphate orientation is that it can be modulated by the change of the environment with a relatively much smaller energy penalty compared to the hexopyranose counterparts. This intrinsic flexibile character of the pentose ring is perhaps the evolutionary basis for their adoption in nucleic acids for storage of genetic information, almost error-free transcription, as well as for selective gene expression in the translation machinery. The present evidences suggest that the mechanism of this modulation of the pentose conformation in DNA and RNA is stereolectronic in character, and the concerted conformational change is unidirectional, originating from the aglycone to the sugar and further to the phosphate. In this monograph, we have explored the nature of the stereoelectronic forces arising from the gauche and anomeric interactions, that are partly responsible for the self- organization of nucleosides and nucleotides. Most importantly, we have experimentally measured the strength and the interplay of these interactions, for the first time, and shown the significance of their effects in dictating the overall dynamics and the structure of nucleos(t)ides and their analogs. This process has enabled engineering of specific conformations in a predictable manner in nucleos(t)ides by having appropriate substituent(s) in the sugar moiety.

Table of Contents

page

1. Introduction. Stereoelectronic effects in hexopyranoses 8

1.1 Equatorial monosubstituted cyclohexane 9 1.2 The Edward-Lemieux effect 9 1.3 ∆∆G° versus ∆∆H° estimates for the anomeric effect 10 1.4 The generalized anomeric effect 11 1.5 Influence of the nature and configuration of substituents on the anomeric effect 11 1.6 Effect of the polarity of the solvent on the anomeric effect 13 1.7 The reverse anomeric effect 13 1.8 Hyperconjugation as the origin of the anomeric effect 43 1.9 The nature of the electron lonepairs 16 1.10 Dipole-dipole (electrostatic) interactions as origin of the anomeric effect 17 1.11 Alternative explanations for the origin of the anomeric effect 18 1.12 The gauche effect 19 1.13 The energetics of the gauche effect 20 1.14 Possible origins of the gauche effect 20

2. Stereoelectronic effects in nucleosides and nucleotides 22

2.1 Structure of nucleic acids 22 2.2 The pseudorotation concept 22 2.3 The two-state N S equilibrium in β-nucleosides 25 2.4 The two-state N S equilibrium in α-nucleosides 27 2.5 The two-state N S equilibrium in carbocyclic nucleosides 27 2.6 Energy barriers of the pseudorotation cycle of β-D-nucleosides 34 2.7 Steric effect of the nucleobase on the sugar conformation 35 2.8 O4'-C1'-N1/9 stereoelectronic effect (the anomeric effect) 36 2.9 Effect of the aglycone on the conformation of nucleos(t)ides and oligos 38

2.9.1 Configuration-dependent sugar conformation in furanosides 39 2.9.2 Effect of electron-withdrawing nucleobases on pseudorotamer populations 39 2.9.3 Effect of the protonated nucleobase on pseudorotamer populations 40 2.9.4 Effect of base-modifications on the stability of nucleic acids 42

2.10 The gauche effects in α- and β-nucleosides 43

2.10.1 2'-Deoxynucleosides 43 2.10.2 Ribonucleosides and nucleotides 44

2.11 The gauche effects of sugar substituents and the self-organization of DNA/RNA 44 2.11.1 Studies on nucleosides 44 2.11.2 Studies on oligonucleotides 46

3. Methods to quantitate stereoelectronic effects in nucleos(t)ides 46

3.1 Thermodynamics of the two-state N S equilibria 46 3.2 1H-NMR spectra [temperature, pD, ligand-dependent spectra of nucleos(t)ides] 47 3 3.3 Pseudorotational analyses of JHH with PSEUROT and some practical hints 48

3.3.1 Incorporation of coupling constant errors in PSEUROT calculations 48 3.3.2 Parameters to be fixed or optimized during PSEUROT calculations 48 3.3.3 Electronegativity of the substituents on each HCCH fragment 49 3.3.4 Priority rule to number the substituents on the HAC1C2HB fragment 49 Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected]

5 3.3.5 Translation of HCCH torsion angles into endocyclic torsion angles 50 3.3.6 General operational conditions for PSEUROT 50

3.4 Generalised Karplus-type equation 53

3 3.4.1 EOS Karplus-Altona equation for JHH 53 3.4.2 Reparametrized EOS equation for carbocyclic nucleosides 54 3 3.4.3 Karplus equation for interpretation of JHF 55 3 3.4.4 Refined Karplus equation for JHH based on Fourier formalism 55

3 3 3.5 Translation of experimental JHH and JHF into pseudorotational parameters 56 3.6 Principle of iterations with PSEUROT 66 3.7 Estimation of ∆Hº, ∆Sº and ∆Gº of the N S equilibrium 61

3.7.1 Methodology 61 3.7.2 Accuracy of thermodynamics 62 3.7.3 Influence of λN1/9 on the thermodynamics 62 3.7.4 Influence of the nature of the aglycone dictates the thermodynamics 66

3 3.8 New Karplus equation to interpret JHF coupling constants 66

3 3.8.1 Dataset of ( JHF,ΦHF) pairs for monofluoronucleosides 67 3.8.2 Parametrization of the Karplus equation 68 3 3.8.3 Pseudorotational analyses of JHF in fluoronucleosides to validate the Karplus equation 69

4. The quantitation of the stereoelectronic effects in nucleos(t)ides 71

4.1 Quantitation of the anomeric and gauche effects by regression analyses 71

4.1.1 Stereoelectronic effects in neutral β-D-Ns 71 4.1.2 Effect of the 5'CH2OH versus 5'CH2OMe 72 4.1.3 Effect of the nucleobase 73 4.1.4 Gauche effects 73 4.1.5 Energetic equivalence of mirror-image β-D-dNs and β-L-dNs 74 4.1.6 3'-phosphate has stronger gauche effect than 3'-hydroxy 74 4.1.7 Stereoelectronic effects in neutral α-D-N 75 4.1.8 Weakening of the effects of 5'CH2OH and 5'CH2OMe in α-nucleosides 75 4.1.9 Weakening of the effect of the nucleobase in α-nucleosides 75 4.1.10 Weaker 3'-hydroxy gauche effect in α- compared with β-D-Ns 76 4.1.11 Limitations of regression analysis to quantitate stereoelectronic effects 76

4.2 Quantitation of the anomeric and gauche effects by pairwise comparisons 78 4.3 Strengths of ∆H° and -T∆S° to ∆G° of pseudorotation of neutral nucleosides 78 4.4 Nucleobase-dependent anomeric effects in neutral nucleosides 78

4.4.1 Effect of the 5'CH2OH versus 5'CH2OMe 82 4.4.2 The effect of the nucleobase is sugar-dependent 82

4.5 The gauche effect of the 3'-substituent in neutral dNs 85

4.5.1 The influence of the C1'-substituent 85 4.5.2 3'-substituent electronegativity dictates 3'-gauche effect 86 4.5.3 Stronger gauche effect in nucleosides than in 1,2-difluoroethane 90

4.6 The 2'-OH effect in ribonucleos(t)ides is nucleobase-dependent 90 4.7 3'-gauche effect modulation by 2'-OH in ribonucleos(t)ides 92 4.8 Drive of pseudorotation in β-nucleosides by the nature of the nucleobase 93 Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 6

4.8.1 The two-state N S equilibrium is evidenced by pKa values from ∆G° 93 4.8.2 Identical pKas of the nucleobases in 2',3'-dideoxy, 2'-deoxy and ribo series 93 4.8.3 Anomeric effect in β-D-ddNs is modulated by the nature of the nucleobase 95 4.8.4 The orbital mixing as the origin of the O4'-C1'-N1/9 anomeric effect 95 4.8.5 No reverse anomeric effect in pentofuranosyl nucleosides 97 4.8.6 Variable tunablity of anomeric effect in β-D-ddNs, β-D-dNs and β-D-rNs 97 4.8.7 Correlation of the electronic nature of aglycone with pseudorotational state 99

5. Comparison of stereoelectronic effects in α- and β-D-nucleosides 102

5.1 The relative magnitude of the anomeric and gauche effects in Neutral state 102

5.1.1 Anti orientation of the nucleobase in α/β-D-ddN and α/β-D-dN 103 5.1.2 The balance of ∆H° and -T∆S° in neutral α- and β-D-N 104 5.1.3 Weakening of 5'-substituent effect in α- compared with β-nucleosides 104 5.1.4 3'-gauche effect weakens anomeric effect in α-D-dN compared to α-D-ddN 105 5.1.5 Weakening of the 3'-gauche effect in α-D/L-dN compared with β-D/L-dN 105

5.2 The relative magnitude of the anomeric and gauche effects in the ionic states 105

5.2.1 Virtually identical pKa values of the nucleobase in α- and β-nucleosides 105 5.2.2 Predominant enthalpy over entropy in the ionic states of α-D-ddN and -dN 105 5.2.3 Weaker anomeric effect in α-D-ddN gives poorer flexibility 105 5.2.4 The interplay of pD-independent ∆H° and ∆S° in α-D-dN 106 5.2.5 Poor correlation of the nature of aglycone with pseudorotation in α-D-N 106

6. Quantitation of the anomeric effects in C- and N-nucleosides 107

6.1 The anomeric effect in C-nucleosides 107

6.1.1 Effect of the C1'-pyrimidine aglycone on the conformation of the sugar 108 6.1.2 Effect of the C1'-purine aglycone on the drive of the sugar conformation 110 6.1.3 pD-tunable anomeric effect in C-nucleosides 110 6.1.4 Transmission of the nature of the C-aglycone drives the N S equilibrium in C-nucleosides 111 6.1.5 Estimates for the thermodynamics of the N S equilibrium 111 6.1.6 Enhanced anomeric effect upon protonation of the aglycone 111 6.1.7 Weaker anomeric effect upon deprotonation of the aglycone 112 6.1.8 Quantitation of the anomeric effect in C-nucleosides 112 6.1.9 Comparison of pD-induced flexibility in C- and N-nucleosides 112 6.1.10 Correlation of the effect of the aglycone and its electronic nature 113

6.2 Quantitation of anomeric effect in N-nucleosides using C-nucleoside as reference 114

7. The interdependency of the sugar and phosphate conformation 118

7.1 Methods to assess the preferred conformation across the phosphate backbone 119 7.2 No correlation of sugar and phosphate conformation in 2'-dN-3'-ethylphosphates 120 7.3 Interaction of 2'-OH with vicinal 3'-phosphate in ribonucleotides 121

8. Application of stereoelectronic effects in oligonucleotides 124

8.1 Design of antisense oligonucleotides via the gauche engineering 124 8.2 Fused nucleosides to engineer preorganized DNA structures 127 8.3 Enzyme recognition by fused carbocyclic nucleosides of fixed conformation 127 8.4 The conformational transmission in the self-cleaving Lariat-RNA 127 Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected]

7 8.5 The conformational transmission by dihydrouridine in RNA 129 8.6 Preferential recognition of 3'-anthraniloyladenosine by Elongation Factor Tu 131 8.7 Conformational changes in nucleotides induced by interaction with metal ions 133 8.8 RNA as molecular wire 136 8.9 The importance of O4' in the self-organization of oligo-DNA 146

148 9. References

10. Appendix (New Additions of references during 2003-4) 160 10.1 The nature of Gauche Effect between 2'-OH and Glycosyl-Nitrogen. 10.2 The pseudorotational barrier of the pentose moiety of nucleosides and nucleotides.

10.3 The hydrogen bonding, hydration and pKa of 2'-OH in adenosine and adenosine 3'- ethylphosphate. 10.4 Latest articles (1998 – 2002) on the aspects of Anomeric (AE) and Gauche (GE) Effects and discussions/comments on these works 10.5 The sensitivity of the RNase H discriminates the local structure changes owing to conformational transmission induced by 3'-endo sugar constrained nucleotides in the antisense strand of the antisense-RNA hybrid duplex. 10.6 The influence of flouro substitution at the sugar moiety in modulating the furanose onformation of flourinated nucleosides.

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 8 1. Introduction. Stereoelectronic effects in hexopyranoses Stereoelectronic effects1-7 can be defined as phenomenological effects, which allow to explain "seemingly abnormal" conformational preferences. Their influence upon the structure of transition states and the course of chemical reactions is known. The extensive application by Deslongchamps2 and Kirby1 of the principles underlying anomeric hyperconjugation to rationalize and predict reaction pathways has given rise to the theories of stereoelectronic control, kinetic anomeric effect and to the antiperiplanar lone-pair hypothesis. The preponderant role of steroelectronic effects in the course of biochemical reactions has been pointed out for many enzymes, such as ribozymes8-10, serine proteases11, lysozyme12, or ribonuclease9,10,13. Most of the studies in the field of stereoelectronic effects have been devoted to six-membered rings, and the state-of-the-art for such systems has been the subject of many review articles1-7. On the other hand, to the best of our knowledge, no unifying model regarding the influence of stereoelectronic effects on the conformation of heterocyclic five- membered rings at the monomeric or oligomeric level has yet come out in the literature. This is possibly due to the fact that, in contrast with rather unflexible and consequently conformationally biased six-membered rings, saturated five-membered rings, such as the pentofuranose moiety in nucleosides and nucleotides, are generally involved in complex conformational equilibria. Some early experimental studies have shown in a qualitative manner how the anomeric and gauche effects control the bias of conformational equilibria of the pentofuranose moiety in nucleos(t)ides and of other saturated five-membered rings. A significant progress has however only been accomplished during the last decade with the development of new methodologies in our laboratory which give accurate estimates of the magnitude of energetics of stereoelectronic effects driving the two-state North South pseudorotational equilibrium in nucleos(t)ides14-45. At the oligomeric level, recent investigations46-54 on modified oligonucleotides have been conducted as a part of the pursuit for developing antisense compounds with increased nuclease- resistance compared with the parent DNA and RNA, and with improved hybridization with the target RNA. The analysis of the thermodynamic properties and of the three-dimensional structure55-61 of these oligonucleotides has shown that a particular modification of one of the three components of the constituent nucleotides, i.e. the nucleobase, the sugar moiety and the phosphate backbone alters both their stability and overall structure. The actual participation of stereoelectronic effects to the observed structural changes has been adressed only qualitatively46,49,62-64. The potential power of the application of the gauche / anomeric engineering strategy to design new oligonucleotides endowed with therapeutic properties is evident. A detailed appraisal of the qualitative and quantitative results from the early as well as from state-of-the-art studies on the manifestation, magnitude and origin of stereoelectronic effects in the pentofuranose ring in nucleosides and nucleotides is therefore required in order to understand its role in the self organization of DNA, RNA and their analogues. This is the purpose of the present monograph, which is organized as follows: (i) We first introduce the concept of stereoelectronic effects by summarizing the most important findings of conformational studies on pyranose derivatives (Section 1). (ii) The main results of qualitative conformational studies on nucleosides and nucleotides showing the influence of the nature of the substituents at C1'-C4' on the conformation of the constituent pentofuranose sugar are subsequently presented and analyzed in detail (Section 2). (iii) Sections 3 - 7 Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected]

9 are devoted to the quantitation of the thermodynamics of stereoelectronic effects driving pseudorotational equilibria in nucleos(t)ides using the methodology (described in Section 3) developed by us in Uppsala. In Sections 3 - 7, we show how specific conformational preferences of nucleos(t)ides can be engineered by altering either the nature or the configuration of one of their constituents, i.e. heterocyclic nucleobase, pentofuranose moiety or C2' and C3' substituents, or the pH of the medium. Evidences regarding the transmission of information between the basic components within the nucleotidyl unit are presented in Sections 4 - 7. (iv) We finally present recent works, both from our laboratory and others, on the impact of the anomeric and gauche effects on the threedimensional structure and biological function of oligonucleotides (Section 8). Stereoelectronic effects can be classified into a few categories: (i) The original Edward- Lemieux effect65-67 which designates the preference of the electronegative methoxy or acetoxy group at C2 in 2-substituted tetrahydropyran for the axial over the equatorial orientation. (ii) The generalized anomeric effect (GAE), which is an extension of the original Edward-Lemieux effect in the case of any acyclic or cyclic R-X-A-Y system. (iii) The reverse anomeric effect allows to rationalize a conformational preference opposite to that expected on the basis of the (generalized) anomeric effect, i.e. an increased tendency of the anomeric substituent in R-X-A-Y fragment to assume an equatorial orientation in comparison with its steric effect. (iv) The gauche effect refers to a stereoelectronic preference for conformations in which the best donor lonepair or bond is antiperiplanar to the best acceptor bond.

1.1 Equatorial monosubstituted cyclohexane Except in rare cases (such as bulky V-shaped alkyl groups68), the most stable conformation of monosubstituted cyclohexane (1) is the chair form E1 in which the substituent X takes up an equatorial orientation (Fig 1A). This is owing to the fact that in the axial chair conformation A1, X exerts energetically unfavourable steric repulsions with the axially oriented H3 and H5. The tendency of X in cyclohexane to adopt an equatorial orientation is dictated by its effective size and can be estimated from the Gibbs free-energy (i.e. ∆G°steric(cylcohex.)) of the A1 E1 equilibrium (Eq 1): ∆G°steric(cyclohex.) = -RT ln [E1]/[A1] ..... Eq 1. 69 13 In a study based on temperature-dependent C-NMR spectra of CFCl3-CDCl3 solutions, it -1 -1 has been shown that ∆G°steric(cyclohex.) at 300 K is ≈ -7.3 kJmol (X = Me), -7.5 kJmol (X = Et) and -9.3 kJmol-1 (X = iPr).

1.2 The Edward-Lemieux effect The acid hydrolysis of methyl β-pyranosides of glucose, mannose and galactose, in which the anomeric methoxy group is equatorial, is faster than that of the α-counterparts, in which it is axial, which has led Edward65 to propose that the axial orientation of the anomeric substituent in pyranosides is more stable than the equatorial one. This is opposite to what one would predict on the basis of simple steric effect rule (vide supra). Edward has attributed this unusual conformational behaviour to the destabilization of the equatorial conformer by electrostatic repulsions between the anomeric substituent and the ring dipole induced by the presence of the endocyclic oxygen atom. In his studies on various acetylated α- versus β-aldohexopyranoses, Lemieux66,67,70 also found that the acetoxy substituent at C2 adopts preferentially an axial orientation. He introduced the term "anomeric

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 10 effect" to designate this preference. An extension of the original65-67,70 Edward-Lemieux effect to the analysis of the conformational equilibrium of 2-methoxytetrahydropyran (2) allows to predict 71 correctly that the axial chair A2 is more stable than its equatorial counterpart E2 (Fig 1B). The Gibbs free-energy (∆G°heterocycle) of the A2 E2 conformational equilibrium in 2 is calculated according to Eq 2: ∆G°heterocycle = -RT ln [E2]/[A2] ..... Eq 2

Figure 1. Axial Equatorial anomeric conformational (A) H X equilibrium in six-membered rings. (A) The drive of the A H 1 H ΔGo = -A steric (cyclohex.) X E1 equibrium in monosubstituted cyclohexane (1) toward the H X equatorial conformer E1 minimizes steric repulsions of the 1: E chair 1: A1 chair 1 anomeric group with H3 and H5. (B) The O-C-Ome anomeric effect in 2-methoxytetrahydropyran (2) pushes its conformational (B) O o ΔGheterocycle equilibrium toward the A2 form in spite of unfavourable steric O OMe repulsions, which are reduced in the E2 chair. OMe 2: E chair 2: A2 chair 2 The original definition of the anomeric effect therefore implies that the stereoelectronic component of the O-C-O effet prevails over the -1 counteracting steric effect. The Gibbs free-energy of the anomeric effect (∆∆G°AE, kJmol ) in 2 is estimated using Eq 3: ∆∆G°AE = ∆G°heterocycle - F * ∆G°steric(cyclohex.) - 0.08 ..... Eq 3 where F (= 1.53) stands for Franck's correction factor72 and accounts for the fact that in 2 steric repulsions generated by the axial orientations of X, H3 and H5 are greater than in the parent monosubstituted cyclohexane (1), owing to the shorter C-O bond in 2 compared with C-C bond in (1).

1.3 ∆∆G° versus ∆∆H° estimates for the anomeric effect The question of which thermodynamic quantity [i.e. either enthalpy (∆∆H°), or Gibbs free- energy (∆∆G°), Table 1] is adequate to estimate the magnitude of the anomeric effect that drives the conformational equilibrium of 2-substituted tetrahydropyran has been adressed in many studies71,75,91, 92. An initial work by Booth et al 91 based on temperature-dependent 13C-NMR spectra showed that - T∆S° overrides the negligible ∆H° in the drive of the conformational equilibrium of 2- methoxytetrahydropyran (2) in CDCl3-CFCl3. It was therefore suggested that the stabilization of A2 71 over E2 form of 2 was the result of the entropy term only. Lemieux however pointed out that such a negligible ∆H° value is only obtained when 2-methoxytetrahydropyran is solvated by hydrogen- bonding or polar solvents, and he showed that in CCl4 the origin of the anomeric effect is mainly enthalpic. The relative preference69 for equatorial over axial chair forms of monosubstituted cyclohexane (1) carrying either Me, Et or iPr group is strongly temperature-dependent, as the result of entropy effects. It is therefore preferable to estimate the magnitude of the anomeric effect in terms of ∆∆H°ΑΕ (Eq 4) rather than ∆∆G°AE (Eq 3) in order to eliminate any misleading entropy-based 92 contribution :m∆∆H°AE = ∆H°heterocycle - ∆H°steric(cyclohex.) ..... Eq 4 where ∆H°steric(cyclohex.) and ∆H°heterocycle represent the ∆H° contributions to ∆G° of the conformational equilibria for substituted cyclohexane (Fig 1A) and tetrahydropyran (Fig 1B), respectively. Juaristi et al 86 have recently estimated the strength of the enthalpic S-C-P(O) anomeric effect in 2-(diphenylphosphinoyl)-1,3-dithiane by replacing ∆H°steric(cyclohex.) in Eq 4 by 72 F*∆H°steric(cyclohex.) in order to take into consideration Franck's argument regarding different Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected]

11 effective steric sizes of the anomeric substituents in substituted cyclohexane and tetrahydropyran (Table 1). Enthalpic anomeric stabilizations have been found experimentally for substituted heterocyclic six-membered rings, such as 2-substituted tetrahydropyrans, 2-carbomethoxy- and 2-cyano- piperidines87 and they have been extensively reviewed4,6 (Table 1).

1.4 The generalized anomeric effect The concept of generalized anomeric effect93-96 results from an extension of the original Edward-Lemieux effect to describe the preference for gauche over trans orientation of R-X with respect to A-Y in R-X-A-Y fragments. R-X-A-Y may be either acyclic or constitute a part of a ring other than the original substituted tetrahydropyran. X designates an element with at least one pair of non-bonding electrons, R and A have intermediate electronegativities and Y is more electronegative than A. Typically1-6, R stands for C or H, A is a tetrahedral (anomeric) center such as C or Si, X is either O, N or S whereas Y designates F, Cl, Br, O, N or P. Further manifestations and extensions of the generalized anomeric effect include the “benzylic anomeric effect” 97 which allows to explain the preferred perpendicular orientation of C-X bond with respect to the plane of the benzene ring (X = S(O)Me, SO2Me, Cl, etc) in ArCH2X compounds. The preference of chlorine and methoxy in α- chloro and α-methoxycyclohexanone oximes for an axial orientation has been attributed to the “vinylogous anomeric effect” 98, whereas the “syn anomeric effect” is responsible for the relatively stable synperiplanar orientation of the nitrogen lonepair with respect to the C-F bond in fluoromethylamine, as shown by ab initio calculations99. When R-X-A-Y is part of a heterocycle and both X and Y possess lonepairs of electrons, the conformation of the heterocycle is driven by the fine balance between the endo and exo anomeric effects3, which operate in opposite directions: The endo anomeric effect involves one of the lonepairs of the endocyclic atom X, which tends to be antiperiplanar with respect to the exocyclic A-Y bond, whereas for the exo anomeric effect it is one of the electron pairs of the exocyclic Y atom which adopts an anti orientation with respect to the endocyclic R-X bond. Depending on the relative magnitudes of endo and exo anomeric effects, the overall estimate (i.e. endo + exo ) for the magnitude of the anomeric effect may be either positive or negative3,4,6.

1.5 Influence of the nature and configuration of substituents on the anomeric effect The magnitude of the anomeric effect depends on the nature of the anomeric group, or other substituents and on their relative configurations. It is also modulated by the polarity and nature of the solvent. Conformational studies on acetylated and benzoylated β-D-ribo and xylopyranose derivatives have shown that the magnitude of the anomeric effect is regularly increased as the electron- withdrawing character of the anomeric substituent increases, H100,101 < OMe102 < OAc103 < OBz104 < Halogen101. Similarly, other experimental studies have shown that the preference of the anomeric substituent X for axial orientation in 2-substituted tetrahydropyran decreases steadily when X is 73,74 73,74 76 80 81 81 changed: Br , Cl > OMe > OH > (CH2)2N > MeHN . The relative

Table 1. The magnitude of the anomeric effect in heterocyclic 6-membered ringsa X

Y R' Y o ΔGheterocycle Z X Z R' R Axial R Equatorial b,c b,d e R R' Y Z X ∆∆G°AE1 (ref.) ∆∆G°AE2 ∆∆H°AE (ref.) H H O CH2 Br f ≈ 9.6 (73) / 13.4 (74) 10.8 l - H H O CH2 Cl f ≈ 9.6 (73) / 11.3 (74) 10.8 l 8.9 m (75) H H O CH2 MeO 8.0 (76) 10.3 3.1 m (75) H H O CH2 EtO 7.5 (76) 9.8 - H H O CH2 Me3CO 5.8 (76) 8.1 - H H O CH2 PhO 6.7 (77) 8.3 - H H O CH2 AcO 5.8 (78) 7.6 - H H O CH2 MeS 6.2 (79) 8.5 - H H O CH2 HO 3.9 (80) 6.2 2.5 m (75) H H O CH2 (CH2)2N 4.1 (81) 7.6 - H H O CH2 MeHN 1.5 (81) 4.4 ≈ 0 m (75) H H O CH2 MeO2C -0.5 (82) 2.4 -

Me H O CH2 HO 5.0 (78) 7.3 - Me H O CH2 MeO 7.7 (83) 10.0 - Me H O CH2 EtO 7.2 (83) 9.5 - Me H O CH2 AcO g 5.7 (84) 7.6 - Me H O CH h 0.1 (85) 3.0 - 2 MeO2C Me H O CH2 Cl f 11.3 (74) 12.3 - Me H O CH2 Br f 13.4 (74) 14.6 - Me H O CH2 I 13.0 (74) 14.0 -

H Me O CH2 MeO 7.2 (83) 9.5 - H Me O CH2 EtO 7.0 (83) 9.2 - H Me O CH2 AcO g 6.0 (84) 7.8 - H Me O CH2 MeS 5.6 (83) 7.9 - H Me O CH2 Me3CS 5.8 (83) 8.1 - H Me O CH h 0.1 (85) 3.0 - 2 MeO2C

H H S S i n POPh2 14.2 (86)

H H O CH2 COOMe j 0.5 n (6,87) H H NH CH2 COOMe j 4.5 n (6,87) H H O CH2 CN k 2.3 n (6,87) H H NH CH2 CN k 10.1 n (6,87) a -1 In kJ mol . Other data on the anomeric effect can also be found in ref.3. NMR spectra have been recorded in CCl4 ° b ° unless stated otherwise. ∆G steric(cyclohex.) values (Eq 1, Fig. 1A) are taken from refs. 88-90. ∆∆G AE1 and ° c ° ∆∆G AE2 values represent the free-energy of the Y-C-X anomeric effect in each compound. ∆∆G AE1 values have been ° ° ° ° estimated using the equation: ∆∆G AE1 = ∆G heterocycle - ∆G steric(cyclohex.) where ∆G heterocycle is the free-energy of ° the Axial Equatorial equilibrium of the heterocyclic six-membered ring (Eq 2, Fig 1B). ∆G steric(cyclohex.) is the ° estimate for the steric effect of X in the substituted cyclohexane counterpart. For (CH2)2N, ∆G steric(cyclohex.) has been Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected]

13 ° assumed equal to that of (CH3)2N. See ref. 3 and the refs. indicated in parentheses in col. 6 for the original ∆G heterocycle d ° values. ∆∆G AE2 has been calculated according to Eq 3. See ref. 3 and the refs. indicated in parentheses in col. 6 for ° e ° the original ∆G heterocycle values. ∆∆H AE values designate the enthalpy of the Y-C-X anomeric effect in each f g h i j k l compound. Neat. In acetic acid. In methanol. In CDCl3. In ether/toluene-d8. In CFCl3/CDCl3. ° m ° Using ∆G heterocycle values from ref. 73. In CFCl3/CDCl3. ∆∆H AE has been estimated from the simple relation: ° ° ° ° ∆∆H AE = ∆H heterocycle - ∆H steric(cyclohex.) (Eq 4) where ∆H heterocycle is the enthalpy of the Axial Equatorial ° equilbrium of the heterocyclic six-membered ring. ∆H steric(cyclohex.) is the enthalpy of the steric effect of X in the n ° ° ° substituted cyclohexane counterpart. ∆∆H AE has been derived from the equation: ∆∆H AE = ∆H heterocycle - F x ° ∆G steric(cyclohex.), by analogy with the method described in footnote d for free-energy estimates of the anomeric effect (see ref. 6 and 86 for the values of F). populations of chair conformations with axially or equatorially oriented anomeric substituent in pyranose derivatives are also controlled by the electronegativity of the substituents at C4 and C53.

1.6 Effect of the polarity of the solvent on the anomeric effect The influence of the polarity of the solvent upon the magnitude of the anomeric effect has been experimentally evidenced in numerous NMR studies on substituted heterocyclic 6-membered rings such as 2-hydroxy-80,84, 2-alkoxy-71,83,105 and 2-alkylthio-tetrahydropyran79,83, 2-alkoxy-1,3- dioxane83, 5-halogen-, 5-methoxy- and 5-ethoxy-2-isopropyl-1,3-dioxane95. Polar solvents stabilize the relatively more polar conformation, i.e. the equatorial chair, more efficiently than the apolar ones. Therefore the anomeric effect is weakened as the dielectric constant of the solvent increases. In the 81 case of other substituents at C2 in tetrahydropyran, such as N(CH2)2 , the modulation of the bias of the conformational equilibrium is much reduced. The influence of the solvent on the magnitude of the anomeric effect should however be considered with caution, especially when there are no accurate estimates for the actual change of the size (or bulkiness, i.e. A-value) of the anomeric group as a function of the polarity of the solvent.

1.7 The reverse anomeric effect An electronegative anomeric group in substituted tetrahydropyrans preferentially adopts an axial orientation, despite unfavorable steric interactions, owing to the anomeric effect. However, it has been claimed that if this electronegative substituent is positively charged, the bias of the conformational equilibrium is just opposite to that predicted in terms of anomeric effect, i.e. the anomeric group prefers the equatorial orientation more than expected on the basis of steric effect alone 7,106-110. This tendency has been attributed to the reverse anomeric effect (see Section 4.8 for a comparison with the pentofuranose system with the protonated aglycone30). The concept of reverse anomeric effect has been introduced by Lemieux106 to explain the preference of pyridinium in N-(tetra-O-acetyl-α-D- glucopyranosyl)-pyridinium or 4-methylpyridinium bromides and N-(tri-O-acetyl-α-D-2-deoxy-2- iodo-mannopyranosyl)-pyridinium perchlorate in aqueous solution for an equatorial orientation, as shown by his conformational analysis based on the interpretation of vicinal proton-proton coupling constants. A reverse anomeric effect has also been noted in the case of other pyranosides107,108 with a pyridinium group at C2. However, whether the preference of pyridinium for the equatorial orientation is the result of (i) a specific stereoelectronic interaction, or (ii) simply the necessity of the anomeric

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 14 substituent to avoid steric repulsions disfavouring an axial position as a result of a possible increase of steric bulk of the anomeric group in the protonated (P) compared to neutral (N) state, or (iii) a specific solvation of the cation, is still a matter of debate7, especially since no reverse anomeric effect has been observed for the corresponding glucopyranosylammonium ions, in which the bulkiness of the anomeric group is comparatively significantly reduced111. 3 The increase (experimentally evidenced by the change of vicinal JHH by 2-3 Hz) of the population of chair conformers with equatorial anomeric substituents in N-(tetra-O-acetyl-α-D- glucopyranosyl)imidazolium94, N-(tetra-O-acetyl-α-D-mannopyranosyl) imidazolium94, and N-(tri-O- acetyl-α-D-xylopyranosyl)imidazolium107 in comparison with their neutral imidazole counterparts in lipophilic media (i.e. in CDCl3 and Me2CO-d6) has also been attributed to the reverse anomeric effect. In this context, it is important to point out that the positive charge on the remote nitrogen in imidazolium does not increase dramatically the effective size of imidazolium in comparison with imidazole, as shown recently by Perrin et al. 112 with help of a 1H-NMR titration method at low temperature. However, the situation in the lipophilic medium changes completely when the coupling constants of the nonacetylated parent compounds are measured in aqueous solution 113: No significant 3 change (well within the experimental accuracy) in JHH is found between their P and N states. Another investigation by Perrin's laboratory114 has shown that the tendency of imidazolium in N-(D- glucopyranosyl)imidazolium and its tetraacetate to adopt an axial orientation in DMSO-d6, CD3OD and aqueous solution is slightly greater than that of imidazole in the neutral counterparts. This result, which is in agreement with what one would expect basing on the simple anomeric effect rule, violates the concept of the reverse anomeric effect. It also contradicts the initial results of Lemieux94. On the other hand, Thatcher et al110 have shown that in the case of N-(tri-O-acetyl-α-D- xylopyranosyl)imidazole in CDCl3 there is a clear enhancement (by ≈ 35 %) of the population of 1 equatorial chair C4 upon addition of trifluoroacetic acid (TFA) with respect to the neutral counterpart, and this enhancement is not the result of the change of the ionic strength of the solution induced by addition of TFA. This work confirms the initial findings of Paulsen et al107 and supports the existence of a reverse anomeric effect for the xylopyranose derivative.

1.8 Hyperconjugation as the origin of the anomeric effect As yet, no consensus has been reached regarding the origin of the anomeric effect in the hexose system, but recent evidences from our lab on the pentose systems in nucleosides and nucleotides point to the orbital mixing as the most probable reason for the anomeric effect (see Section 4.8). Two models have received outstanding recognition among all those proposed for the origin of the anomeric effect: (i) The anomeric effect has often been interpreted as the result of a stabilizing 115,116 hyperconjugative interaction between the filled orbital of one of the electron lonepairs (nX) of the endocyclic heteroatom X with the antibonding orbital (σ∗ ) of the A-Y bond. A-Y (ii) Alternatively, the anomeric effect can also be attributed to the attempt of the system to minimize electrostatic repulsions65,117,118 between the dipole formed by the A-Y bond and the dipole induced by the presence of the heteroatom X. (iii) A few other explanations have also been proposed, such as Eliel's119 "rabbit-ear effect", which also involves electrostatic interactions, however this time

15 directly between lonepairs of electrons. Finally, 4e- orbital mixing destabilizing effects120 have also been advocated. Lucken originally explained116 the unusually low nuclear quadrupole resonance frequencies of 35Cl in X-C-Cl fragments of α-halogenoethers by an overlap between a p-type orbital of the heteroatom X and the antibonding orbital (σ* ) of the C-Cl bond. His original interpretation has C-Cl given rise to the very popular molecular orbital overlap model which is currently used to explain the anomeric effect [Fig 2(A-C2)]. In α-halogenoethers, the preference of the halogen atom for an axial orientation is concomittant with a characteristic shortening of the C-O bond and a lengthening of the C-Cl bond115 compared with tetrahedral values (Fig 2E).

1 nsp3 (A) O 2 1n 3 nsp3 sp Figure 2. The rationalization of the anomeric effect in O 115 σ*C-Cl 6-membered rings with help of the delocalization and A2 E2 Cl 2n 3 Cl sp molecular orbital overlap116 models. (A) Overlap of an electron lonepair orbital of the endocyclic oxygen with (B) σ*C-Cl the σ*C-Cl antibonding orbital of C-Cl bond in 2- ΔE (σ*C-Cl - nO) nO 3 Hyperconjugative chlorotetrahydropyran (using sp hybridized oxygen stabilization lonepairs). (B) The magnitude of the anomeric effect is 2 2 proportional to S (S is the overlap between the oxygen AE α S /ΔE (σ* - nO) C-Cl lonepair orbital and the σ* orbital) and inversely 1 C-Cl 1 n 3 H1 nsp3 sp (C1) proportional to the energy difference between both H β 1 β 4,5 O O orbitals, i.e. ∆E(σ*C-Cl - nO) . The nO σ*C-Cl 2n 3 sp Cl 2 interaction results in the formation of two new orbitals. α nsp3 α (C1) and (C2) Orientation of the electron lonepairs of the A(C1) Cl E(C1) 3 1 1 1 endocyclic oxygen [either sp hybridized, i.e. nsp3 and n 2 nsp2 (p-type) sp (s-type) H (C2) 1 2 2 1 2 H1 nsp3 in (C1) or sp hybridized, i.e. nsp2 (p-type) and nsp2 β O β O 2n 2 (s-type) in (C2)] with respect to the exocyclic C-Cl bond. sp (s-type) Cl The nO σ*C-Cl overlap is maximal in the axial conformer A(C2) Cl 2 nsp2 E(C ) (s-type) 2 [A(C1) in (C1) or A(C2) in (C2)] in which the torsion angle β [defined as 1n 3-O1-C2-Cl in (C1) or 1n 2 (D) sp sp (p- O O type)-O1-C2-Cl in (C2)] is closest to 180°, whereas in the other conformer [E(C1) in (C1) or E(C2) in (C2)] the − Cl Cl oxygen lonepairs are either gauche (for both nsp3 orbitals 2 F in E(C1) and for the n 2 orbital in E(C2)) or cis Å sp (p-type) (E) 8 BzO 9 9 Å .3 1 2 1.33 1 O .4 (in the case of the n 2 orbital in E(C2)) to the O OAc 06 sp (s-type) Å F AcO 7 Å exocyclic C2-Cl bond. The anomeric effect therefore .36 AcO 1 favours the axial over the equatorial conformer. When the BzO OBz oxygen lonepairs orbitals are sp2 hybridized as in (C2), the magnitude of the anomeric effect is expected to be reduced in comparison with a situation in which lonepairs are sp3 hybridized as in (C1), owing to the fact that β is closer to 180° in A(C1) than in A(C2). (D) The anomeric effect has been alternatively explained by the hyperconjugation of one of the endocyclic oxygen lonepairs to the exocyclic C-Cl bond, which corresponds to the double bond no-bond resonance in terms of valence bond theory. (E) Shortening of the endocyclic bond C2-O and concomitant lengthening of the exocyclic bond C2-F in the axial conformer A3 of tri-O- benzoyl-2-fluorotetrahydropyran compared with the equatorial counterpart E4 of tri-O-acetyl-2-fluorotetrahydropyran, supporting the hyperconjugative origin of the anomeric effect101.

The hyperconjugation model (Fig 2D), initially proposed by Romers et al115 allows to explain the O- C-O anomeric effect in 2-substituted-tetrahydropyran (as well as the generalized anomeric effect in other systems) by the delocalization of one of the lonepairs of the endocyclic oxygen into the

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 16 antibonding orbital of the exocyclic C-O bond. This hyperconjugation, which corresponds to a double-bond no-bond resonance in terms of valence-bond theory (Fig 2D), is most efficient when the lonepair orbital and the antibonding orbital of the exocyclic C-O bond are in the same plane, i.e. when the lonepair adopts an antiperiplanar orientation with respect to the exocyclic C-O bond [Fig 2, (C1) & (C2)]. This is only achieved in the axial conformer A2, which is therefore more stable than the equatorial counterpart E2 (Fig 2A). The energy stabilization resulting from the n → σ* interaction is inversely proportional to the energy difference [∆E(σ* - n )] between both orbitals and proportional C-Cl O to square of their overlap (Fig 2B). The electron-donating abilities of orbitals decrease in the order: nC- > nN > nO > σC-S > σC-H > σC-C > σC-O > σC-F, whereas the electron accepting capability of 4,5 orbitals is reduced in the order: σ∗C-Cl > σ∗C-S > σ∗C-F > σ∗C-O > σ∗C-C > σ∗C-H . Delocalization is expected to result in the lengthening of the exocyclic C-O bond, whereas the concomittant shortening of the endocyclic C-O bond and opening of the O-C-O bond angle with respect to "standard" (tetrahedral) values are observed due to the reduced and enhanced p-character of the endocyclic and exocyclic C-O bonds, respectively. The hyperconjugation model is therefore strongly supported by the various examples of structural data found in the literature, such as in the case of pyranose derivatives, which show significant changes of the lengths of the exocyclic C-O (or C-Y in the general case) and endocyclic C- O bonds as well as of the value of O-C-O (or O-C-Y) bond angle (Fig 2E)1,121,122 in chair conformers with axially versus equatorially oriented anomeric groups.

1.9 The nature of the electron lonepairs The question of the adequate hybridization state (i.e. sp2 versus sp3) of the electron lonepairs orbitals has given rise to some controversy. The use of sp3 hybridized lonepairs allows to predict correctly conformational preferences induced by the generalized anomeric effect1. However, experimental and theoretical studies123-126 have led to the conclusion that the nonbonding oxygen lonepairs are nonequivalent: One of them occupies a higher energy orbital with high p-type character, whereas the other occupies an orbital of lower energy with predominant s-type character. In recent studies on the anomeric effect117,127,128, the sp2 hybridization model of the electron lonepairs orbitals has therefore gained widespread recognition. Basing on the results of their statistical analysis of X-ray crystal structures of COCOC molecular fragments, Cossé-Barbi and Dubois have shown129 that the five-membered furanose ring orients polar anomeric groups in axial or pseudoaxial position (95%) much more efficiently than the six-membered pyranose ring (56%). They argued that this stronger anomeric effect in furanose compared with pyranose is the result of the better ability of one of the lonepairs of the endocyclic oxygen to participate in stabilizing hyperconjugation, which can be easily understood with help of the sp2 (not sp3) hybridization model of lonepairs. Indeed, sp2 hybridization makes the n → σ* O C-X interaction more efficient in furanose than in pyranose130, since it results in a perfect antiperiplanar orientation of the p-type lonepair orbital of the endocyclic oxygen atom with respect to the exocyclic C-X bond in the former but not in the latter [compare panel (C2) in Fig 2 with panel (C) in Fig 9]. If the lonepairs of the endocyclic oxygen were sp3 hybridized, the situation would be just reverse, i.e.

17 the overlap between n and σ* orbital would be optimal in the pyranose system but not for the O C-X furanose counterpart [compare panel (C1) in Fig 2 with panel (B) in Fig 9].

1.10 Dipole-dipole (electrostatic) interactions as origin of the anomeric effect 65 Already in 1955, Edward proposed that the destabilization of the E2 chair of 2- methoxytetrahydropyran (2) (with equatorially oriented 2-OCH3 group) with respect to the axial counterpart A2 is the result of electrostatic repulsions between 2-OCH3 and the ring dipole. The ring dipole is itself the resultant of individual dipole moments from endocyclic C-O bonds and the lonepairs of the endocyclic oxygen. More recently, the electrostatic model has been refined and the anomeric effect has been attributed to electrostatic repulsions117,118 between the exocyclic C-X dipole and the ring dipole in the E2 chair (Fig 3A). The repulsion between two dipoles is function of their 74,131 magnitudes as well as the distance and the angle between them . In the equatorial conformer E2, both dipoles are nearly parallel and repel each other much more than in the axial form A2 where the angle between them is nearly 90°. As a result, the E2 conformer is destabilized by electrostatic repulsions compared to the A2 counterpart and the anomeric group 2-OCH3 preferentially adopts an axial orientation. The experimentally found (Section 1.6) dependence of the magnitude of the anomeric effect upon the polarity of the solvent has been considered as the best evidence for the electrostatic origin of the anomeric effect: Polar solvents stabilize the more polar conformation (i.e. the equatorial chair E2) more efficiently than solvents with lower dielectric constants, therefore the strength of the anomeric effect will be reduced in polar compared to apolar solvents. In a recent calorimetric study of the heats of hydrolysis of the anomeric forms of 4,6-dimethyl-2-methoxytetrahydropyran, Wiberg and Marquez118 have also shown that the nature of the solvent strongly influences ∆H° and ∆G° of the isomerization equilibrium, which led them to conclude that the anomeric effect is mainly induced by the electrostatic repulsion between the C-O dipoles, the magnitude of which decreases when the dielectric constant of the solvent is increased. However, there are also known examples of an increase in the strength of the anomeric effect induced by an increased polarity of the solvent, such as for trans-2,5-bis(trimethylsiloxy)-1,4- dioxane132. This particular result cannot be explained by the electrostatic model. Instead, it has been rationalized in terms of hyperconjugative interactions (Section 1.8). More importantly, in contrast with the hyperconjugation model, the electrostatic model does not allow to explain the changes of bond lengths and bond angles that are commonly associated with the existence of the anomeric effect in heterocyclic 6-membered rings (Section 1.8). The question of the relative importance of hyperconjugative interactions versus dipole-dipole repulsions as the origin of anomeric effect in non-polar solvents has been raised in a recent comparative conformational study of 2-methoxy-1,3-dimethylhexahydropyrimidine (3) and 2- 117 methoxy-1,3-dioxane (4) . It was argued that since the dipole moment of CH3-NH2 is smaller than that of CH3-OH, substitution of nitrogen in 3 for oxygen in 4 should lead to increased electrostatic repulsions in 4 compared to 3. Conversely, as evident from the lower ionization potential of CH3NH2 in comparison with that of CH3OH, replacing oxygen in 4 by nitrogen in 3 should raise the energy of the filled nN lonepair orbital in the later with respect to that of nO orbital in the former and therefore

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 18 strengthen the n→σ* hyperconjugative interactions in 3 compared to 4. As a result, if the anomeric effect operates mainly through dipole-dipole (electrostatic) interactions, then replacing oxygen by nitrogen should reduce the strength of the anomeric effect in 3 compared to 4. Conversely, if hyperconjugative interactions predominate, then the strength of the anomeric effect should be increased in 3 compared to 4. Perrin's conformational analysis based on temperature-dependent 1H and 13C NMR spectra showed that the proportion of axial conformer is nearly the same in 3 and 4. However, after correction of the results for the steric effects induced by the presence of methyl groups in 3 (supported by molecular mechanics and AM1 calculations), it turned out that the anomeric effect is weaker in 3, and it was therefore concluded that the anomeric effect is mainly the result of electrostatic interactions. Additionally, it was also suggested that the bond length changes that are often invoked as a key evidence for the existence of hyperconjugative interactions (Section 1.8) can also be explained in terms of dipole-dipole interactions. A subsequent reinvestigation of the origin of the anomeric effect by Salzner128 based on ab initio geometry optimizations (at HF/6-31G* level) of various conformers of hexahydropyrimidine (5), 2-hydroxypiperidine (6) and 2- hydroxyhexahydropyrimidine (7) and on the NBO analysis of the resulting wave functions showed that indeed the magnitude of the anomeric effect in 6 and 7 is much reduced in comparison with the oxygen analogs. However, it was also shown in that work that the total energies of the conformers do not correlate with total dipole moments, which discredited the predominance of electrostatic repulsions. Instead, the weaker anomeric effects in 6 and 7 in comparison with the oxygen counterparts were interpreted as the results of the competition between OH/NH bond repulsions and hyperconjugative stabilizations.

1.11 Alternative explanations for the origin of the anomeric effect 119 According to Eliel's original rabbit-ear effect model , the anomeric effect in R-X-A-Y fragment arises from repulsions between electron lonepairs of X and Y heteroatoms (Fig 3B). This model has been used in particular to explain the drive of the conformation of 2- alkoxytetrahydropyran76,95 and tripepiperideine133 by the O-C-O and N-C-N anomeric effects, respectively. Alternatively, it has also been suggested by Box120 that four electrons orbital mixing destabilizing interactions allow to account for X-C-Y anomeric effect controlling the conformation of glycosides: according to this model, the occupied (2+2) electron lonepair orbitals of X and Y heteroatoms interact to form two new orbitals, occupied by the four electrons (Fig 3C). However, the energy destabilization of the system induced by the newly formed high-energy orbital is greater than the stabilization gained by the formation of the new low-energy orbital, therefore such lonepair- lonepair interactions, on the overall, are destabilizing, and the system attempts to adopt a conformation in which there are fewer lonepair orbital interactions. As a result, the anomeric substituent adopts preferentially an axial orientation.

19 1.12 The gauche effect The "gauche effect"1,134-143 designates the tendency of R-X-Y-R' and X-C-C-Y fragments (where X and Y represent electronegative atoms or groups) to adopt preferentially gauche over antiperiplanar orientations in spite of unfavourable steric repulsions and electrostatic interactions (i.e. the relative energy difference between trans and gauche conformers, ∆Et-g, is negative, as depicted in Fig 4). For 1,2-disubstituted ethane in the gas phase, there is a linear relationship between ∆Et-g and the sum of Huggins electronegativities of both substituents135. 1,2-dimethoxyethane exists preferentially in a gauche conformation in solution, but the stabilization of the gauche conformer is much reduced in the gas phase144. On the other hand, ab initio calculations predict almost identical energies for the gauche and trans rotamers142. A study of the energy relationship of the isomers of 1,2-difluorethylene, 1,2-difluorodiazene and several other halogen- and oxygen-substrituted ethylenes145 show that the cis isomer has the lower energy, which is generally referred to as the cis effect.

O (A) Figure 3. The anomeric effect in 2-substituted O tetrahydropyran as the result of electrostatic interactions. X X Lonepairs on the endocyclic and exocyclic oxygen atoms are A2 chair E2 chair represented assuming sp3 hybridization. (A) Destabilization (B) of the equatorial chair E with respect to the axial counterpart B1 B2 B3 2 Me 65,117,118 A by dipole-dipole repulsions . (B) Prediction of the O O Me O 2 O Me OO relative stabilities of B1 - B6 conformers of 2- methoxytetrahydropyran (2) using Eliel's rabbit-ear model95

B4 B5 B6 of interaction between the electron lonepairs of the endocyclic and exocyclic oxygens. ("black" lonepair: in the O O O front, "empty": at the back, "grey": in the plane of the Me O O O drawing). The equatorial conformers B1 - B3 as well as the Me Me axial forms B4 - B5 are destabilized owing to one or two (for the B2 form only) repulsion(s) between lonepairs of the nO (endo) - nO (exo) endocyclic and exocyclic oxygens. The axial conformer B6, ΔE > ΔE (C) 2 1 in which the anomeric group OMe adopts an axial

ΔE2 orientation, is the most stable, since only in this situation there is no destabilizing lonepair-lonepair interaction. (C) n n O (endo) O (exo) 120 ΔE1 The four electrons interaction model . The interaction

nO (endo) + nO (exo) between two filled lonepairs orbitals of the endocyclic [nO (endo)] and exocyclic [nO (exo)] oxygens produces two new orbitals. This interaction is destabilizing, since the stabilization (∆E1) of the system gained from the formation of [nO(endo) + nO(exo)] orbital is weaker than the corresponding destabilization (∆E2) owing to the newly formed high-energy [nO(endo) - nO(exo)] orbital .

“Attractive” and “repulsive” gauche effects define a greater preference of a X-C-C-Y fragment for gauche and anti orientations, respectively, than that expected on the basis of steric effects and electrostatic interactions alone. It has been shown138-140 that the extent of stabilization of the 1,2- diaxial versus 1,2-diequatorial conformers of 1,2-trans-disubstituted cyclohexane is dictated by the fine balance between steric, electrostatic and gauche interactions between vicinal X and Y substituents: For strongly electronegative X and Y substituents (i.e. O/O, F/Cl, F/Br, F/I or F/O pairs), a strong attractive gauche effect operates, whereas when the electronegativity of X and Y is intermediate (such as O/Cl, O/I or Cl/I pairs), the observed conformational preferences can be explained by steric and electrostatic repulsions alone. In contrast, for S/S, S/Cl, S/Br or S/O Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 20 disubstitution, the population of gauche rotamers is less than expected (owing to the repulsive gauche effect). In 5-substituted-1,3-dioxan, conformers with axially oriented C5 substituent are preferred as the result of the attractive gauche effect146, whereas in 5-methoxy- and 5-methylthio-1,3-dithianes repulsive S/O and S/S gauche effects are operating147.

1.13 The energetics of the gauche effect As stated above, the gauche effect implies the electronic energy preference of a gauche rotamer over the corresponding anti rotamer1. The experimental energetic relationship of various rotamers for 1,2-difluoroethane (as derived from Raman148,149, infrared149, NMR spectroscopy150 and electron diffraction151-153 studies) or 1,2-dimethoxyethane (by NMR spectroscopy144) in the gas phase has been well worked out. These experimental works have shown that the gauche rotamer has a lower energy than the anti counterpart by 2.4 - 3.4 kJmol-1 (in the case of difluoro substitution), which compares well with results obtained from theoretical studies141,154 (i.e. ab initio calculations at the MP2/6-311++G** and TZ+2D+P levels).

4 cis Y cis X X Y eclipsed Figure 4. Plot of the relative energy of 1,2- 3 eclipsed y X rg X disubstituted ethane conformers as a function of the e n Y trans Y Φ torsion angle, showing the stabilization [∆E E 2 X XCCY (t- e iv gauche + gauche − + - t Y < 0] of gauche and gauche rotamers over the trans la X Y Y g) e 1 X R counterpart, as the result of the attractive X-C-C-Y ΔE (t-g) gauche effect. 0 0 60 120 180 240 300 360

ΦX-C-C-Y (° )

1.14 Possible origins of the gauche effect The origin of the gauche effect is still a matter of controversy: (i) It has been shown that the preferred gauche orientation in ethylene glycol142,155 results from hydrogen bonding. (ii) The repulsive gauche effects for some 1,2-disubstituted cyclohexane derivatives (e.g. for S/S disubstitution pattern) have been attributed to the through-space interaction (or "hockey sticks effect") between lonepair orbitals on the X and Y heteroatoms in the X-C-C-Y fragment138,156,157. Such an overlap between two doubly-occupied lonepairs is destabilizing: It results in the formation of a bonding orbital and its antibonding counterpart, however the destabilizing influence of the latter is greater than the stabilizing effect of the former (Fig 5, Panel A1). On the other hand, Hoffmann158 suggested that the lonepair interaction does not only take place through-space but it has also a significant through bond component (Fig 5, Panels A2 and A3). The actual repulsive or attractive159 character of the gauche effect is the result of the fine balance between through-space and through- bond contributions. Hoffmann's analysis is supported by the results of a photoelectron spectroscopic study of 3,7,9-trihetero derivatives of bicyclo[3.3.1]nonane, showing the preponderant influence of the through-bond component160. (iii) σ →σ∗ orbital interactions between the best donor σ orbital and the best acceptor σ* orbital have also been suggested136,161. The efficiency of these interactions is proportional to the

21 square of the overlap between the donor and acceptor orbitals and inversely proportional to the difference between their energies. An example of σC-H →σ∗C-F orbital overlap in the gauche conformer of 1,2-difluoroethane is shown in Fig 5B-C.

138,156-160 Figure 5. (A1)-(A3) Through-space (A1) and through-bond (A2 and A3) interactions between lonepair orbitals (Φ1 and Φ2) on the X and Y heteroatoms in the X-C-C-Y (A1) X Y fragment, as the origin of attractive or repulsive gauche effects. Through-space (A1) four electron interactions Φ - Φ 1 2 σ* result in the formation of a bonding orbital (Φ1 + Φ2), X Y X Y which is stabilizing (∆E1 < 0) whereas the antibonding Φ1 - Φ2 (A2) counterpart (Φ - Φ ) is destabilizing (∆E > 0). ∆E > ΔE2 1 2 2 2 ∆E1 and therefore the system is destabilized on the Y Φ overall. (Φ + Φ ) can in turn interact with σ [through- X 2 1 2 C-C ΔE1 ΔE Φ1 4 σ bond component of the origin of the gauche effect, (A3)]. This four-electron interaction is again destabilizing since X Y X Y ∆E > ∆E . On the other hand, the concomittant two- Φ +Φ ΔE3 4 3 1 2 Φ1 +Φ2 (A3) electron interaction between (Φ1 - Φ2) and σ*C-C is X Y stabilizing, as shown in Panel (A2). (B) and (C): σC-H

→σ*C-F orbital overlap to explain the gauche effect in 136,161 (B) F H H H H H H H 1,2-difluoroethane . The σ →σ* overlap is most efficient when it involves the best donor and best acceptor H H F H F F F H σ and σ* orbitals, respectively. In the case of the trans F- trans (1) gauche (2) (3) conformer, the σ orbital is σC-F, which is much poorer donor than σC-H in the corresponding gauche conformer F H [Section 1.8]. Therefore, the σ →σ* interaction H H H H C-H C-F stabilizes the gauche conformer with respect to the trans (C) 180ο counterpart, and by bond-no bond resonance the olefin (3) HH HF is envisioned as a canonical form. (D) The gauche effect F F ο 143,162 60 is shown in relation with bent bonds . In the trans F F F form, the C-C bond paths are bent in the opposite (D) direction (as shown by the dashed lines), which leads to a CC CC reduced overlap and a weaker C-C bond. On the contrary, in the gauche rotamer, bond paths are bent essentially in F trans (1) gauche (2) the same direction. Therefore the trans rotamer is destabilized with respect to the gauche counterpart.

Wolfe originally defined the gauche effect134 as "a tendency to adopt that structure which has the maximum number of gauche interactions between the adjacent electron pairs and/or polar bonds", which he rationalized in terms of a nuclear-electron attraction prevailing over nuclear-nuclear and electron-electron repulsions. This definition enables to predict correctly the preferred gauche orientation of two vicinal electron lonepairs R and R' in a R-X-Y-R' system or of X-C and C-Y polar bonds in X-C-C-Y fragment, but not the antiperiplanar orientation136 of a lonepair with respect to a vicinal polar bond, i.e. owing to the anomeric effect (Sections 1.2 - 1.6). It is therefore preferable to define1 gauche effect as "a stereoelectronic preference for conformations in which the best donor lonepair or bond is antiperiplanar to the best acceptor bond". (iii) The preferential gauche arrangement in 1,2-difluoroethane has also been explained by the fact that in this rotamer, the C-C bond paths (i.e. the regions with maximum electron density connecting both carbon atoms163) are bent in the same direction, whereas in the relatively less stable trans form bending occurs in opposite directions, which results into a reduced overlap and a weaker C-C bond143 (Fig 5D). This theory is supported by the

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 22 comparison of geometrical parameters for the gauche and anti rotamers of 1,2-difluoroethane, as obtained from an experimental study based upon high-resolution infrared spectroscopy162 or from ab initio calculations (using hybrid-DFT164 or RHF/6-311++G** level149) .

2. Stereoelectronic effects in nucleosides and nucleotides

2.1 Structure of nucleic acids Endowed with unique capabilities, such as the storage of the genetic information, induction of cellular differentiation, propagation of tumor viruses as well as splicing and autocatalysis, deoxyribo (DNA) and ribonucleic acids (RNA) are polymers built up of monomers, the nucleosides, which are covalently linked through 3'→5' phophodiester linkages165. Nucleosides consist of a pentofuranose sugar and a heterocyclic nucleobase (adenin-9-yl, guanin-9-yl, cytosin-1-yl, thymin-1-yl, uracil-1-yl or modified C-166 or N-heterocycle165) connected by the covalent C1'-N1/9 or C1'-C (glycosyl) bond. In natural DNA and RNA, the D-sugar enantiomer and β-glycosyl linkage are preferred over the L- and α-counterparts. The conformation along the phosphate backbone in nucleotides can be fully defined167,168 by the torsion angles α, β, γ, δ, ε and ζ (Section 7.1). The stability of the three-dimensional structure of nucleic acids is usually attributed to only a few types of forces: (i) Strong intermolecular H-bonds between complementary nucleobases; (ii) Intramolecular base-base stacking interactions; (iii) Electrostatic interactions and hydrophobic forces between adjacent base pairs and across the nucleotide chain165, and hydration. The purpose of the next sections is to present recent progress in our understanding of the interplay of stereoelectronic gauche and anomeric effects in the drive of the conformation of simple nucleosides and nucleotides and their analogs as well as the self-organization of oligonucleotides.

2.2 The pseudorotation concept Saturated heterocyclic six-membered rings are much less flexible than their five-membered counterparts169. The activation energy barrier for the conversion of one chair form of cyclohexane into the other is170,171 about 42 kJmol-1. 1H- and 13C NMR spectra of 2-methoxy-1,3- dimethylhexahydropyrimidine (3)117 measured in various solvents have shown that the ∆G* value for the ring inversion between two chair forms is ≈ 37 ± 2 kJmol-1 (the two interconverting chair conformers could be isolated at low temperature). On the other hand, the activation energy barrier for the interconversion between the two preferential puckering modes, i.e. N- and S-type pseudorotamers, of the pentofuranose moiety in purine nucleosides is much reduced in comparison, i.e. below 20 - 25 kJmol-1 (vide infra). The pseudorotation concept has been introduced by Kilpatick et al. 172 to describe the continuous interconversions between an infinite number of indefinite puckered forms of the cyclopentane ring. Pseudorotation173 allows cyclopentane to relieve strains which would be induced by 120° bond angles and eclipsed methylene groups if it would adopt a planar geometry. A barrier to planarity of cyclopentane of 22 kJmol-1 has been reported176. The concept of pseudorotation has been applied for the first time to furanose by Hall et al. 177 in their conformational analyses of pentofuranosyl fluorides. Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected]

23 Kilpatrick originally described172 the puckered states of cyclopentane by sequential out-of- plane displacements (of the carbon atoms) from the least square plane of the unpuckered ring. The displacement zi of the ith ring carbon in a particular puckered conformer was calculated using Eq 5: 1/2 zi = (2/5) q cos 2 (Φ + 2πi/5) ..... Eq 5 where, q and Φ represent the maximum puckering amplitude and the phase angle of pseudorotation of the pseudorotamer, respectively. Eq 5 holds exactly in the case of the infinitesimal diplacement in an equilateral pentagon. Cremer and Pople have later on devised a generalized set of puckering coordinates valid for equilateral and non-equilateral five-membered rings178. This formalism is however quite cumbersome since it requires the

North (N) H N HOH C 2 O Figure 6. The pseudorotation wheel (E = R H H H R' envelope; T = twist) for pentofuranosyl D- 3 T 3 2E 2 E 1 nucleosides. The hyperspace of geometries T 0º 3 2 342º 18º 4T 1 324º ο 36º accessible to N- and S-type pseudorotamers is E 40 4E 306º 54º West (W) 1 T 20ο 0 East (E) within the shaded areas for β-D-dNs, β-D-rNs 0 288º 72º 4T ο 0 HOH2C and β-D-arabinonucleosides (-1° < PN < 34°, H N CH2OH 0E 270º 0 90º E H O H H 4 ο 137° < P < 194°, 30° < Ψ (N), Ψ (S) < H T 252º 20 108º 0 N S m m 0 1T R R O 234º 126º H 174 R' 4 40ο R' H 46°) and within the empty oval shaped H E 1E 216º 144º 4 198º 162º 2 areas for α-D-dNs, α-D-rNs, α-D-xylo- and α- 3T 180º T 2 1 3E 2 E D-arabinofuranosyl nucleosides175 (-18° < P 3T N H < 19°, 168° < P < 224°, 28° < Ψ (N), Ψ (S) HOH2C S m m N O H H R' < 49°). H R South (S) knowledge of 15 cartesian coordinates. Altona et al. have solved this problem by proposing an alternative description179-181 of a puckered geometry of the pentofuranose ring in nucleic acid derivatives by two parameters P and Ψm, related in turn to the five endocyclic torsion angles νi (i = 0...4) (Eq 6a): νi = Ψm cos (P+4π(i-2)/5) ..... Eq 6a P, the phase angle of pseudorotation (0° < P < 360°), shows which part of the ring is mostly puckered, whereas Ψm, the maximum puckering amplitude, provides information about the largest deviation of the endocyclic torsions from zero. The ensemble of puckered forms that may be adopted by the pentofuranose moiety in nucleos(t)ides is represented in the form of the pseudorotation cycle (Fig 6). In nucleosides, the endocyclic torsions νi (i = 0...4) are defined as follows: ν0 [C4'-O4'-C1'- C2'], ν1 [O4'-C1'-C2'-C3'], ν2 [C1'-C2'-C3'-C4'], ν3 [C2'-C3'-C4'-O4'] and ν4 [C3'-C4'-O4'-C1']. P and Ψm are derived from Eqs 6b and 6c: tan P = [(ν4 + ν1) - (ν3 + ν0)] / [2ν2 (sin 36° + sin 72°)] .....Eq 6b Ψm = ν2 / cos P .....Eq 6c

D-series L-series (i) (ii)

OR' OR' HO HO

O O O O RO B B OH B B OR HO North (N) α-D-sugar South (S) α-D-sugar South (S) α-L-sugar North (N) α-L-sugar (C3'-endo-C2'-exo) (C2'-endo-C3'-exo) (C2'-endo-C3'-exo) (C2'-exo-C3'-endo)

(iii) (iv)

OR' OR' HO HO B B B B O O O O RO OH

OR HO North (N) β-D-sugar South (S) β-D-sugar South (S) β-L-sugar North (N) β-L-sugar (C3'-endo-C2'-exo) (C2'-endo-C3'-exo) (C2'-endo-C3'-exo) (C2'-exo-C3'-endo)

B = adenin-9-yl, guanin-9-yl, cytosin-1-yl, thymin-1-yl or uracil-1-yl (in RNA) Scheme 1: The D- and L- mirror image relationship for the two-state dynamic N S sugar equilibrium in α-D- dNs (i) , α-L-dNs (ii), β-D-dNs (iii) and β-L-dNs (iv). In L-nucleosides234, the N sugar is redefined as being the form with maximal negative value for the endocyclic torsion [C1'-C2'-C3'-C4']. Note that in α-D-dNs (i) and α-L- dNs (ii), the aglycone B becomes more pseudoaxial as the anomeric effect becomes stronger in the S-type conformation, whereas in β-D-dNs (iii) and β-L-dNs (iv), this is achieved in the N-type conformation. Hence, the sign for the energetic drive of the anomeric effect in α-nucleosides is opposite to that of β-counterparts (Sections 4 and 5).

For practical purposes, Eq 6a may be regarded as nearly exact for equilateral five-membered rings, since it reproduces the values of the endocyclic torsion angles of a cyclopentane pseudorotamer with an accuracy better than 0.1°182. In a study of 178 β-D-furanosides, the endocyclic torsion angles could be calculated using Eq 6a with an r.m.s. error of 0.4 - 0.9° 183,184. For non-equilateral rings, a more accurate expression (r.m.s. = 0.2 - 0.4°) for the dependence of the endocyclic torsions on P and 183-186 Ψm is given by Eq 7 : νi = ai * Ψm cos (P+εi+4π(i-2)/5) ..... Eq 7 where, ai and εi represent correction factors that are included to overcome differences in endocyclic bond lengths. Altona's model has not only been used to describe the conformation of the pentofuranose moiety in nucleosides and nucleotides187-226 but also the puckered states of cyclopentane179, the pyrrolidine ring in L-proline and in its derivatives182,227, the ring D in steroids180, and other five- membered rings115,228-231. Ab initio calculations232 have shown that pseudorotation of cyclopentane occurs without any substantial change in potential energy whereas in unsymmetrically substituted saturated five- membered rings potential energy thresholds induced by the presence of the exocyclic substituents limit the flexibility of the system and lead to prefered puckering modes233.

25 2.3 The two-state N S equilibrium in β-D-nucleosides In D-nucleosides, in the typical N (P = 0°) and S (P = 180°) conformations, the [C1'-C2'-C3'- C4'] torsion angle has a maximal positive and negative value, respectively, whereas in the L- enantiomers the signs, as defined by Hoffmann and Altona234, are opposite as a result of the mirror- image relationship of D- and L-enantiomers (Scheme 1).

A perusal174 of 178 crystal structures of β-D-ribo-, 2'-deoxyribo- and arabinonucleosides shows that the phase angle values of their puckered pentofuranose moieties are not evenly distributed over the whole 0 - 360° range but are instead clustered in the North (N, -1° < P < 34°, centered around C3'-endo) and South (S, 137° < P < 194°, centered around C2'-endo) regions, while Ψm is within the 30° - 46° range with an average value of 38.6 ± 3° (Fig 6). The N and S regions are nearly equally populated for ribonucleosides whereas for 2'-deoxyribonucleosides there is a clear preference for S- type geometries (≈ 3:1 for the ratio of S versus N pseudorotamer populations). Only a few E-type pseudorotamers and no W-type conformers were found among these crystal structures, which is consistent with an analysis based upon simple steric effects: W-type conformations are energetically highly disfavoured owing to the pseudoaxial orientation of both the nucleobase and the 5'CH2OH group and to the fact that the C2' and C3' substituents are eclipsed. Analogously, the energy destabilization of E-type conformations can be attributed to the eclipsed orientation of the C2' and C3' substituents. 1H-NMR studies in aqueous solution193-204,206-208,212,216,219,220,222,224-226,235-240, have shown that the puckered geometries of the furanose moiety in nucleos(t)ides interconvert rapidly (in the NMR timescale) since only time-average chemical shifts and coupling constants can be extracted from the spectra. This means that the activation energy barrier between the interconverting pseudorotamers is significantly reduced in comparison with heterocyclic six-membered rings, which often adopt a single chair conformation (vide infra).

However, two distinctly identifiable and dynamically interconverting N and S conformations have been observed in some B Z DNA241,242, A Z RNA243,244 or A-form B-form lariat RNA245,246 transitions. The NMR results studies as well as de Leeuw et al's statistical analysis of the distribution of P values of the crystal structures of nucleos(t)ides (vide supra) suggest that the conformation of the pentofuranose moiety of nucleos(t)ides in aqueous solution is adequately described by a two-state N S equilibrium model, since no third state is found yet. This is also supported by the following observations, based upon the results of our own conformational studies on nucleosides and

14-44 nucleotides : (i) PN, PS, Ψm(N) and Ψm(S), as obtained from pseudorotational analyses of

3 19 experimental vicinal proton-proton coupling constants ( JHH) for β-D-2',3'-dideoxynucleosides are 3 virtually the same in the analyses which have been performed using the JHH data at any single 3 temperature or in other analyses based upon the whole set of temperature-dependent JHH coupling constants (i.e. at seven temperatures within the 283 K - 353 K range). (ii) Van't Hoff plots of the

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 26 ratios of mole fractions of S- and N-type sugars (xS/xN) as a function of the inverse of temperature give straight lines for all nucleosides with correlation coefficients ≥ 0.95. (iii) We have recently shown that ∆H˚, ∆S° and ∆G° of the N S equilibrium in β-30,32,36, and α-nucleosides36,37 are pD- dependent (Sections 4 - 6). The shift of the N S equilibrium in β- (or α-) nucleosides toward more N- (or S-type) sugars at acidic pDs upon protonation of the nucleobase, and toward S-type (or N-type) conformers at alkaline pDs upon deprotonation, results from the transmission of pD-tunable electronic character of the nucleobase to steer the sugar conformation through modulable anomeric and gauche effects. The pDs at the inflection points of the plots of pD-dependent ∆G° values gave the pKas of the nucleobases, and these values were virtually the same as those published247 or independently determined through monitoring of pD-dependent chemical shifts of non-exchangeable aromatic and anomeric protons30,32,36,37. Raman spectroscopy study on A and T containing DNAs248 also support that the two-state N S equilibrium model also holds for the pentofuranose sugar moiety.

Throughout this whole book and in all papers cited from this laboratory, the signs of the thermodynamic quantities have arbitrarily chosen in such a way that the positive values indicate the drive of the N S equilibrium to N, whereas the negative values describe the drive to S. Interconversions between N- and S-type puckered furanose conformers most likely occur along the pseudorotational cycle (i.e. via puckered geometries) rather than through a planar intermediate, which is disfavoured as the result of high strain energies181,249,250. The fact that only a few E-type puckered geometries and no W-type pseudorotamer were identified among the crystal strutures of 178 β-D nucleos(t)ides suggests that N- to S- interconversion proceeds via an E-type puckered geometry intermediate, not through the W region. The intrinsic flexibility of the pentofuranose moiety is absent in cyclic nucleosides such as 2',3'-O-, 8,2'-O-, 8,3'-O-anhydronucleosides or in cyclic nucleotides165. This results in a reduced puckered amplitude, for instance in 2',3'-cyclic nucleotides251-253 or to unusual puckering modes of 3',5'-cyclic nucleotides, both in solution254 and in the solid state255-257. The effects of packing forces and hydrogen bonds upon the observed puckering modes of nucleosides in the solid state are well known165. Solution and solid state conformations of pentofuranose differ dramatically for some nucleosides: Adenosine258 crystallizes as the N-type conformer, whereas its hydrochloride salt crystallizes as the S-type form259 and our NMR studies30 show a clear preference (≈ 67% at room temperature) for S-type conformers at neutral pD, and the pseudorotational equilibrium is shifted to more N-type sugars at acidic pD (≈ 55% South conformer at 17 298 K). 3'-azidothymidine in D2O is involved in an equipopulated two-state N S equilibrium, despite the fact that it crystallizes in the S-type puckered conformers: P = 175° with Ψm = 32° and P = 260-265 266,267 215° with Ψm = 36° . 3'-fluorothymidine crystallizes in the form of S-type conformers, however the average phase angle value for these conformers (P ≈ 171°, Ψm ≈ 34°) deviates from that 1 17,26 found for the preferred S-type sugar geometry (≥ 90%, P ≈ 160°, Ψm ≈ 34°) by H NMR studies . For all β-D-2',3'-dideoxynucleosides (ddNs), the pentofuranose sugar is mostly puckered as the N-

27 19 type conformer in D2O solution (≥ 75% at 298K), whereas in the solid state, it adopts S-type 268 269 geometries (P = 194°, Ψm = 37° for ddA , P = 208° and Ψm = 34° for ddC ). An integrated conformational study on 4'-thiothymidine and (E)-5-(2-bromovinyl)-2'-deoxy-4'- 3 thiouridine in solution (based on the interpretation of JHH with help of the pseudorotation concept and on the analysis of nOe enhancements) and in the solid state (from X-ray crystallography) has been recently reported by our lab270. It was found that the crystal structures of both thymidine derivatives are grossly similar with a 4'-thiofuranose moiety in S-type conformation (P = 180° and Ψm = 48° for both compounds). Owing to the nonequilateral nature of 4'-thionucleosides, the pseudorotation equation (Eq 7) was reparametrized basing on ab initio calculations. The solution structure of the major conformer of 4'-thiothymidine and its derivative were similar to the crystal structure, showing that the presence of S4' vis-a-vis O4' does not result in any additional flexibility of the thiofuranose ring. The similarity of the bias of the N S equilibrium in thionucleosides and thymidine show that the magnitude of interplay of gauche and anomeric effects is comparable in both, thereby showing the insignificant effect of S vis-a-vis O. It is also noteworthy that the bond lengths of C1'-S and C4'-S are essentially the same as found in many other natural C-nucleosides271-282 and N-nucleosides174,283, suggesting that the identification of the anomeric effect on the basis of unequal bond lengths of C4'- X4' versus C1'-X4' is an oversimplification5.

2.4 The two-state N S equilibrium in α-nucleosides A recent perusal175 of the few crystal structures available in the literature for α-nucleosides has shown that their constituent pentofuranose moieties also tend to adopt predominantly conformations belonging to the C2'-endo and C3'-endo domains (-18° < PN < 19°, 168° < PS < 224°, 28° < Ψm(N), Ψm(S) < 49°) with a few exceptions found in the C4'-endo region. Therefore, the hypothesis of the two-state N S equilibrium has also been retained during our conformational studies on α-nucleosides (Section 5). In that work175, basing on the results of potential energy calculations on both α-purine and α-pyrimidine nucleosides, it was suggested that interconversion between N- and S-type α-sugars proceeds through the O4'-exo rather than through the higher O4'-endo activation energy barrier, in contrast with what has been claimed for β-counterparts (vide infra).

2.5 The two-state N S equilibrium in carbocyclic nucleosides The chemical and enzymatic vulnerability of the glycosyl bond of natural nucleosides has inspired the synthesis of a wide gamut of carbocyclic nucleosides284-291 in which a cyclopentane ring replaces the ribose moiety. However, these more stable carbocyclic nucleosides are generally less potent284 than their natural counterparts. This is attributed to the more flexible nature of the cyclopentane ring where both anomeric and gauche effects inherent to the natural nucleosides17,19- 30,32,33,35-38,40-44 are lacking. Efforts to reduce the flexibility of the cyclopentane ring have resulted in the design of conformationally constrained carbocyclic nucleosides292,293 (see Sections 8.2 and 8.3). The X-ray crystal structures of four conformationally unconstrained carbocyclic 294-297 294 nucleosides are so far known, i.e. for (-)-aristeromycin (P = 89.0° and Ψm = 40.8), (+)- 295 carbathymidine (P = 118.6° and Ψm = 41.2°), 1-(c-2-fluoro-t-4-hydroxy-c-3-hydroxymethyl-r-1- 296 cyclopentyl)uracil (P = 95.7° and Ψm = 43.1°) and the carbocyclic analogue of 2'-

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 28 297 59 deoxyguanosine (P = 293.3° and Ψm = 35.8°). The crystal structures of 6'-α-methylcarbocyclic thymidine in four different forms (P = 147.4° and Ψm = 47.6°; P = 141.9° and Ψm = 49.1°; P = 153.6° and Ψm = 44.5°; P = 111.2° and Ψm = 37.6°) and of 6'-α-hydroxycarbocyclic thymidine (P = 127.9° and Ψm = 44.2°; P = 141.1° and Ψm = 45.6°; P = 140.0° and Ψm = 48.2°; P = 123.4° and Ψm = 49.0°) as constituents in modified oligo-DNA duplexes have also been determined using X-ray crystallography. These data suggest that the cyclopentyl rings in carbocyclic nucleosides are prone to adopt a greater variety of conformations than the pentofuranosyl counterpart in the corresponding natural N- nucleosides. The greater flexibility of carbocyclic nucleosides compared with natural pentofuranosyl nucleosides may be attributed to the absence of O4' oxygen in the former, whereas in the latter O4' is involved in stereoelectronic interactions with the glycosyl nitrogen (i.e. the O4'-C1'-N1/9 anomeric effect) and with O2' and/or O3' (i.e. through gauche effects). However, any possible contribution of different solvation and electrostatic potential in the cyclopentyl compared with pentofuranosyl analogues cannot be excluded. The conformational variations of cyclopentane moieties in carbocyclic nucleosides in the solid state clearly suggested that a knowledge of their dependable solution conformations is highly desirable in order to compare the solution and the solid state structures. 3 Hence, a modified Karplus equation correlating the JHH values to the corresponding proton-proton torsion angle specifically in carbocylic nucleosides is now available (Eq 8b in Section 3.4.2). ∆G° of the protonation deprotonation equilibrium drives the two-state N S equilibrium of the furanose moiety in N-30,36,37 and C-nucleosides32 through the modulation of the interplay of gauche and anomeric effects (Sections 4 - 6). It has been found that the electronic nature of the aglycone is reflected, through the anomeric effect, in ∆G° of the N S equilibrium of the constituent pentofuranose moiety. In fact, the pKa value of the constituent nucleobase can be independently measured from a pD-dependent change of ∆G° of the N S equilibrium, which is closely similar to 41 3 the pKa value obtained from the pD-dependent chemical shift plots. We have examined JHH of aristeromycin and its 2' and 3'-deoxy derivatives at pD ≈2 and ≈7 in the 278 K - 358 K range to examine whether the protonation deprotonation equilibrium of the adenin-9-yl base also drives the pseudorotational equilibrium of their constituent cyclopentane moieties. As expected, temperature- 3 dependent JHH values are virtually unchanged from acidic to neutral pDs (± 0.15 Hz, Table 1 in ref. 41) suggesting that the change of the electronic nature of the aglycone in aristeromycin has no effect on the drive of the constituent cyclopentane conformation, thereby establishing the role of the anomeric effect in the drive of the sugar conformation in N- and C-nucleosides17,19-30,32,33,35-38,40-42.

The comparison of the Energy barrier of the pseudoroation in natural pentofuranosyl nucleoside versus the carbocyclic counterpart. Our ab initio calculations (Fig 7) at HF/3-21G* level with the GAUSSIAN 94 program298 on various pseudorotamers (with defined P values in the 0 - 360° range at a constant Ψm for all conformers of a particular compound) of aristeromycin (8), 2'-deoxyaristeromycin (9), 3'- deoxyaristeromycin (10), 2',3'-dideoxyaristeromycin (11) and their natural pentofuranosyl counterparts have allowed us to further shed light on the validity of the two-state N S (or W)

29 equilibrium model for the cyclopentane ring in carbocylic nucleosides (as suggested by the presence of two major energy wells, vide infra) and to assess the effect of the substitution of CH2 for O4' upon the energy barrier of pseudorotation in the modified versus natural nucleosides. This comparison is intended to improve our understanding of the energy penalty of stereoelectronic gauche and anomeric effects in natural nucleosides and nucleotides. (i) 2',3'-dideoxynucleosides constitute the system of choice to analyze and compare the relative flexibility of sugar conformation in carbocyclic versus natural pentofuranosyl nucleosides since 2'- and 3'-OH groups are absent, which prevents any H-bonding interaction between themselves or with the nucleobase [see subsections (iii) and (iv) below]. Both for 2',3'-dideoxyaristeromycin (11) and β- D-ddA (30), N-type pseudorotamers (P = 0°) are slightly preferred over the S-type counterparts (P = 180°) [by ≈ 0.7 kcalmol-1 for 11 and ≈ 1.2 kcalmol-1 for 30 (Compare Panels (A) and (B) in Fig 7]. However, whereas for β-D-ddA, the activation energy barrier (∆∆G°‡) in the E region for the N to S interconversion is ≈ 7.6 kcalmol-1 (see Section 2.6 for an upper limit of energy of activation of pseudorotation found experimentally) the same transition in 2',3'-dideoxyaristeromycin is more facile (∆∆G°‡ ≈ 3.7 kcalmol-1) which uniquevocally shows that the O4'-C1'-N9 anomeric effect efficiently hampers free pseudorotation in the former. If we now for the sake of convenience consider that 2',3'- dideoxyaristeromycin and β-D-ddA (30) are isosteric, then a simple subtraction of their above ∆∆G°‡ values gives us an idea of the influence of the anomeric effect on the energy barrier of the pseudorotation in β-D-ddA (30), which is 3.9 kcalmol-1. For nearly all optimized pseudorotamers of 11 and 30, the nucleobase is in anti orientation and β is found in the anti range while γ+ rotamers are preferred [Panels (I) and (J) in Fig 7]. (ii) For β-D-dA (37), among all possible pseudorotamers, N and S-type sugar conformations (both having the same energy) are energetically preferred as shown by the energy profile [Panel (D) in Fig 7]. The activation energy barrier from N to S through E is reduced (∆∆G°‡ ≈ 4.7 kcalmol-1) in comparison with β-D-ddA (7.6 kcalmol-1, see panel (B) in Fig 7), showing the effect of 3'-OH. The t t t constituent nucleobase remains in the anti orientation and γ , ε3 and β rotamers (see the legend of Fig 7 for definition of the ε3 torsion angle) are preferred throughout the whole pseudorotational cycle [Panel (L) in Fig 7]. For 2'-deoxyaristeromycin (9), among all pseudorotamers, the S- or W-type conformers (210° < P < 240°) are energetically preferred by ≈ 3 kcalmol-1 over the N-type counterparts (330° < P < 60°) [Panel (C) in Fig 7]. However, in contrast with the case of β-D-dA, the interconversion between the energetically preferred N- and S-/W-type conformations takes place through a very small barrier in the E-region (∆∆G°‡ ≈ 1.1 kcalmol-1) which is consistent with the absence of the O4'-C1'-N9 anomeric and [O3'-C3'-C4'-O4'] gauche effects, as discussed for 2',3'-

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 30 OH OR OH OR' OR H6' Base A O H O O O Base O H OH 6" H Base Base OH 30: β-D-ddA (R = H) RR' R' R" OR 17: α-D-ddA 21: α-D-dA (R = R' = H) 27: α-L-dA 31: β-D-ddG (R = H) 8: Aristeromycin (R = R' =OH) 12: R = R' = R" = H 18: α-D-ddG 22: 3'-OMe-α-D-dA (R = Me; R' = H) 28: α-L-dC 32: 5'-OMe-β-D-ddG (R = Me) 9: 2'-deoxy-aristeromycin 13: R = R" = H; R' = OH (R = OH; R' = H) 19 : α-D-ddC 23: 3',5'-diOMe-α-D-dA (R = R' = Me) 29: α-L-T 33: β-D-ddC (R = H) 14 : R = H; R' = R" = OH 10: 3'-deoxy-aristeromycin 20 : α-D-ddT 24: α-D-dG (R = R' = H) 34: β-D-ddT (R = H) (R = H; R' = OH) 15: R = R" = H; R' = OMe 25: α-D-dC (R = R' = H) 35: β-D-ddU (R = H) 11: 2',3'-dideoxy-aristeromycin 16 : R = Me; R' = OMe; R" = H (R = R' = H) 26: α-D-T (R = R' = H) 36: β-D-ddI (R = H) 37: β-D-dA (R = R' = H) OH H O H NH2 H NH2 O O Base N N N 38: 3'-OMe-β-D-dA 7 6 NH 7 6 N 6 N OH N 8 5 1 N 8 5 1 8 7 5 1 (R = Me; R' = H) OH 9 4 3 2 OH 9 4 3 2 OH 9 4 3 2 Base N N N OR' 39: 3',5'-diOMe-β-D-dA O O O Base OH OH OH O (R = R' = Me) 56: Formycin B 57: Formycin A 58: 9-deaza-A 40: β-D-dIm (R = R' = H) 46: β-L-dA 50: β-D-A 41: β-D-dG (R = R' = H) OH OH OH OH OH OH 47: β-L-dG 51: β-D-G OR NH2 O 42: β-D-dC (R = R' = H) 48: β-L-dC 52: β-D-C N 2 1 2 3NH RN1 3 NR' OH 6 A 43: β-D-T (R = R' = H) 49: β-L-T 53: β-D-rT 6 5 4 5 4 60: ψ-U: R = R' = H O OH O OH O 44: β-D-dU (R = R' = H) 54: β-D-U O O 61: 1-Me-ψ-U: 45: 5-F-β-D-dU (R = R' = H) 55: 5-F-β-D-U 59: ψ-isoC R = Me, R' = H 59b: ψ-isoC, HCl 62: 1,3-diMe-ψ-U: OH OH OH OH OH OH β OH OH OH R = R' = Me 63: β-D-3'-dA Base Base Base β Base O O O O γ γ O Base = NH2 N O δ δ + + N N Na + Na N NH NH Na ε Na+ ε N N −O − −O O − O O ζ O OH O O OH N N ζ N N N NH2 N P P imidazol- P H P β 2 β H2 adenyl-9-yl (A) guanin-9-yl (G) 1-yl (Im) hypoxanthin-9-yl (I) O OH O α O C O OH α O O C O O O CH3 NH2 64: β-D-dAMP 69: β-D-dAMPEt 74: β-D-AMP 79: β-D-rAMPEt H C F CH3 3 NH NH 65: β-D-dGMP 70: β-D-dGMPEt 75: β-D-GMP 80: β-D-rGMPEt N NH 66: β-D-dCMP 71: β-D-dCMPEt 76: β-D-CMP 81: β-D-rCMPEt N O N O N O N O 67: β-D-TMP 72: β-D-TMPEt 77: β-D-rTMP 82: β-D-rTMPEt 5-F-uracil-1-yl [5-F-dU cytosin-1-yl (C) thymin-1-yl (T) 68: β-D-dUMP 73: β-D-dUMPEt 78: β-D-rUMP 83: β-D-rUMPEt uracil-1-yl (U) (45) & 5-F-U ( 55)]

31 OH OR OH H H R H 1 OMMTr 5" 5" 5" B H5" H5" B H B H B B H5' O 5' O 5' O H5' O H5' O H3' F2' H3' H2' F3' H2' H3' HO H3' F2' H4' H1' H4' H1' H4' H1' H4' H1' H4' H1'

OH F2" F3" H2" H3" OR F3" H2" H3" H2"

84: B = A (diFA) 88: R = OH, B = T (FLT) 92: R = R1 = H, B = A (FXA) 95: B = T (F3AT) 96: B = U (F2'ddU) 85: B = G (diFG) 89: R = OTr, B = T (FLT5) 93: R = H; R1 = MMTr, 86: B = T (diFT) 90: R = NH , B = T (AFLT) B = A (FXA5) 2 94: R = CS-OPh; 87: B = C (diFC) 91: R = OH, B = U (F3"ddU) R1 = MMTr, B = A (FXA25) OH OR Cl H H HA Cl 5" 5" B COOCH O B F 3 H5' O H5' O Cl 101 : R1 = F A; R2 = H B; COOCH3 COOH Cl R1 R = H ; R = F ; H3' H2' F3' H2' 3 A 4 B H4' H1' H4' H1' COOH R3 102 : R1 = H A; R2 = H B; R3 = F A; R4 = F B; F H H HA Cl R2 H3" 2" H3" H2" B B Cl 103 : R1 = F A; R2 = F B; 97: B = U (F2"ddU) 98:R = H, B = U (F3'ddU) 99 F 100 R4 R3 = H A; R4 = F B;

F F H H COOH F N F N F N F F F H H H H eq H4eq H6eq H4eq H2eq H4eq OH F 2 F R COO H5" F H ax H ax T H4 F H2ax H4ax H6ax H4ax 2 4 H5' O F 104 105 : R = H 107 F2 108 106 : R = COOH OR OH Me Me H5" H5" B B X F HA HB H5' O H5' O HO Me Me F3' H2' H3' H2' 113 : X = NH 2 H H H H Me 4' 1' 4' 1' 114 : X = OMe OH 115 : X = NO 2 Hax FMe H3" H2" OH F2" 116 : X = OCF 3 109 110 111 :R = H, B = A (F3'ddA) 112 : B = C (F2"C)

A = adenin-9-ylG = guanin-9-yl C = cytosin-1-yl T = thymin-1-yl U = uracil-1-yl

) (I) 2',3'-dideoxyaristeromycin (11) ) (J) β-D-ddA (30) o o 60 ( 60 ( (A) 2',3'-dideoxyaristeromycin (11) (B) β-D-ddA (30) le γ le 0 γ 16 16 g 0 g n n -60 a -60 a χ n χ n-120 ) 12 ) 12 o-120 o -1 i i l -1 l rs -180 rs -180 o o o β o β m T-240 -240 l 8 l m 8 T a a c c -60 0 60 120 180 240 -60 0 60 120 180 240 (k k o o 4 ( P ( ) P ( ) E E 4 (K) 2'-deoxyaristeromycin (9) (L) β-D-dA (37) ) 200 0 0 o ( -60 ) s o ( γ -90 e190 -60 0 60 120 180 240 -60 0 60 120 180 240 le l g χ g ε3 o n -120 n P ( ) P (o) a a180 -150 n β ε3 n β io -180 io χ (C) 2'-deoxyaristeromycin (9) (D) β-D-dA (37) rs rs 170 o -210 γ o 16 16 T T -240 160 ) -60 0 60 120 180 240 -60 0 60 120 180 240 1 12 ) 12 o - l -1 l P ( ) o o o P ( ) m l 8 m 8 (M) 3'-deoxyaristeromycin (10) a l (N) β-D-3'-dA (63) c a 5 k c 6 ( (k ) ) 4 4 d E E (Å (Å 4 (5'-OH-O4') ) 5 ) d d n d n d o (2'-OH-N3) o 3 (2'-OH-N3) 0 0 -b 4 -b (H (H -60 0 60 120 180 240 -60 0 60 120 180 240 d 3 d 2 o P ( ) P (o) ) 60 ) 60 o ( o (E) 3'-deoxyaristeromycin (10) (F) β-D-3'-dA (63) ( s 0 ε s 0 16 16 le χ 2 le g -60 g ε n n -60 2 β ) a a 1 12 ) 12 n -120 - l -1 l o β n -120 o o si io χ m r -180 s -180 l 8 m 8 o γ r a l T o γ c a -240 T -240 k kc ( ( -60 0 60 120 180 240 -60 0 60 120 180 240 E 4 4 o E P ( ) P (o) 0 0 (O) aristeromycin (8) (P) β-D-A (50) 6 -60 0 60 120 180 240 -60 0 60 120 180 240 6 d ) (2'-OH-N3) ) d P (o) P (o) (Å (Å (2'-OH-N3) ) ) d d d (5'-OH-O4') n n 4 o 4 d o (G) aristeromycin (8) (H) β-D-A (50) -b (3'-OH-O2') -b d (H (H d (3'-OH-O2') 16 16 d d (2'-OH-O3') 2 2 d(2'-OH-O3') ) 12 ) 12 -1 l -1 l ) 60 ) o ε o ε o o ( 2 ( -60 3 m m s 0 s l 8 l 8 le le a a g χ g ε c c n -60 n -120 2 (k (k a a 4 4 ε E E n -120 3 n β io β io -180 rs -180 rs γ 0 0 o γ o χ T -240 T -240 -60 0 60 120 180 240 -60 0 60 120 180 240 -60 0 60 120 180 240 -60 0 60 120 180 240 P (o) P (o) P (o) P (o) Figure 7. The plots of the energy (E), of various torsion angles and of interatomic distances for ab initio optimized pseudorotamers of 2',3'-dideoxyaristeromycin (11) [Panels (A) and (I)], 2'-deoxyaristeromycin (9) [Panels (C) and (K)], 3'-deoxyaristeromycin (10) [Panels (E) and (M)], aristeromycin (8) [Panels (G) and (O)] and of their pentofuranosyl counterparts β-D-ddA (30) [Panels (B) and (J)], β-D-dA (37) [Panels (D) and (L)], β-D-3'-dA (63) [Panels (F) and (N)] and β-D-A (50) [Panels (H) and (P)] as a function of the phase angle of pseudorotation (P). The calculations have been performed by constraining only two endocyclic torsions (ν0 and ν4) to the appropriate values in order to sweep P from 0 to 360° in 30° (for 8 - 10, 37, 50 and 63 or 20° Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected]

33 292 (for 11 and 30) intervals at a common Ψm value [41° for 8 - 11 (as in the crystal structure of 8), 40° for 30 and 36° (as in the crystal structure of adenosine259) for 37, 50 and 63] whereas the rest of the molecule has been freely optimized. For 298 all conformers, the starting geometries used as input for the optimization with GAUSSIAN program were as follows (ε2 and ε3 designate the [2'-OH-O2'-C2'-C1'] and [3'-OH-O3'-C3'-C4'] torsion angles, respectively): χ = 120°, β = γ = 180° for

2',3'-dideoxyaristeromycin (11); χ = -170°, β = γ = 180° for β-D-ddA (30); χ = 120°, β = γ = ε3 = 180° for 2'- deoxyaristeromycin (9); χ = -170°, β = γ = ε3 = 180° for β-D-dA (37); χ = 120°, β = γ = ε2 = 180° for 3'- deoxyaristeromycin (10); χ = -170°, β = γ = ε2 = 180° for β-D-3'-dA (63); χ = 120°, β = γ = ε2 = 180° and ε3 = -60° for aristeromycin (8) and χ = -170°, β = γ = ε2 = 180° and ε3 = -60° for β-D-A (50). dideoxyaristeromycin under the subsection (i). It is also noteworthy that whereas the values of the γ, ε3 and β torsion angles (in the anti domain) do not change drastically from one pseudorotamer of 2'- deoxyaristeromycin to the other, the nucleobase adopts a syn orientation in the high energy W-type pseudorotamers while for all other sugar conformations it is anti [Panel (K) in Fig 7]. (iii) For 3'- deoxyaristeromycin (10), the W-type (P = 240°) pseudorotamers are preferred among all possible geometries and they are more stable by ≈ 6 kcalmol-1 than the N-type counterparts giving rise to a local energy minimum at P ≈ 330° [Panel (E) in Fig 7]. The interconversion between N- and S-type pseudorotamers is possible, provided that the energy barrier in the E-region (∆∆G°‡ ≈ 4.3 kcalmol-1) is overcome. It is noteworthy that the preference for W-type (P = 240°) geometries of 3'- + deoxyaristeromycin results from the gauche orientation of 2'-OH hydroxy group (ε2 ≈ 60°) which enables its hydrogen-bonding interaction [d(2'-OH-N3) ≈ 2.5 Å] with the nucleobase in antiperiplanar orientation [Panel (M) in Fig 7]. On the other hand, the relatively higher energy of other pseudorotamers in the W-region (P = 270° and P = 300°) is associated with a syn orientation of the nucleobase. The energy profile presented in Panel (F) for the natural counterpart β-D-3'-dA (63) is far more complex than that of 3'-deoxyaristeromycin and suggests a strong preference for E-type (P = 90°) or S-type (P = 180° or 210°) pseudorotamers (by ≈ 5 - 5.5 kcalmol-1) over the local energy minimum in + - + + the N-region (P = 0°). The preference for E-type (ε2 with β ) and S-type (ε2 with β ) pseudorotamers is owing to a stabilizing H-bonding interaction between 2'-OH proton and N3 [d(2'-OH-N3) ≈ 2.0 Å] and between 5'-OH and O4' [d(5'-OH-O4') ≈ 2.0 Å] (for E-type conformations) [Panel (N) in Fig 7]. (iv) For aristeromycin (8), the S/W type conformers (120° < P < 240°) are preferred over the N- type (330° < P < 30°) geometries by ≈ 10 kcalmol-1 [Panel (G) in Fig 7]. The N- to S/W-type geometries interconversions preferably take place through the activation energy barrier in the E (∆∆G°‡ ≈ 4.8 kcalmol-1 from N to S; ≈ 13.0 kcalmol-1 from S to N) rather than the W (∆∆G°‡ ≈ 5.8 kcalmol-1 from N to S; ≈ 14.1 kcalmol-1 from S to N) region of the pseudorotation cycle. The increased stability of S/W-type pseudorotamers with respect to other geometries is due to 2'-OH....N3 and 3'-OH....O2' + t hydrogen-bonding interactions which are permitted by ε2 and ε3 orientations [Panel (O) in Fig 7]. On the other hand for P values in the range 270° < P < 90°, a single H-bonding interaction is possible between 2'-OH and O3'. As for 2'-deoxy and 3'-deoxyaristeromycin (see (ii) and (iii)), in the relatively unstable conformers in the W-region (P = 270° and P = 300°), a syn orientation of the nucleobase is observed, whereas for the energy minima the nucleobase is anti. In fact, the energy profile [Panel (G) in Fig 7] and the dependence of the glycosyl torsion angle χ on the value of P appear to be very similar. The plot presented Panel (H) in Fig 7 shows that the energy of various pseudorotamers of β-D-A (50)

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 34 is much less sensitive to the phase angle value than for the aristeromycin counterpart [Panel (G) in Fig 7], possibly because of the fact that all pseudorotamers are stabilized by the H-bonding interaction between 2'-OH and O3' [Panel (P) in Fig 7]. The absolute energy minimum is found for N-type conformations of β-D-A (P = 0°) which are preferred over secondary minima in the E/S-regions at P = 120° (by 0.9 kcalmol-1) and P = 210° (by 2.3 kcalmol-1). The activation energy barriers between N- and E- (P = 120°) (through P = 60°: 5.5 kcalmol-1 and 4.6 kcalmol-1) and from E- to S- (P = 210°) (through P = 150°: 3.5 kcalmol-1 and 2.2 kcalmol-1) are reduced in comparison with what was found for aristeromycin. For all pseudorotamers, γt rotamers are preferred and the nucleobase is in anti t - orientation with ε2 for 270° < P < 90° and ε2 for 90° < P < 270°. The preference for P = 0° among all pseudorotamers in the N region correlates nicely with the fact that in this situation ε2 is in the maximal trans orientation. However, this trans orientation does not result into an H-bond between 2'-OH and N3 as shown by the fact that the d(2'-OH-N3) distance is greater than 4 Å at any P value. On the other hand, the presence of the secondary energy minimum at P = 120° may be attributed to a hydrogen-bond between 5'-OH and O4' permitted by the β- orientation. In summary, the energy plots in Fig 7 show the energy penalties induced by some specific conformational properties of the cyclopentane ring owing to the absence of O4' in carbocyclic nucleosides vis-a-vis natural pentofuranosyl nucleosides, in which O4' is the cause of both gauche and anomeric effects. Work is now in progress in our laboratory to perform the above calculations at a higher basis set in order to delineate the influence of hydration on the pseudorotational energy and conformational profile in the carbocylic as well as in pentofuranosyl nucleosides.

2.6 Energy barriers of the pseudorotation cycle of β-D-nucleosides Early quantum chemical, CNDO or classical potential energy calculations250,299-302 and more recent calculations using consistent force field method303 (permitting bond stretching and bond angle bending) on unsubstituted ribo and deoxyribofuranose have shown that the N-type and S-type sugars are preferred among all pseudorotamers, and that they have almost the same energy. Calculations on 1- amino-ribose, -2-deoxyribose or -3-deoxyribose304,305 and on nucleosides themselves306 have shown that the activation energy barrier for N- to S- interconversions is clearly greater in the W (≈ 24 kJ/mol for 2-deoxyribofuranose, ≈ 31 kJ/mol for ribofuranose) than in the E region (≈ 7.5 kJ/mol for 2- deoxyribofuranose, ≈ 16 kJ/mol for ribofuranose). The measurement307 of 13C longitudinal relaxation times of single tertiary carbons showed that the energy of activation of the N- to S-type sugar interconversion in purine nucleosides is ≈ 20 ± 2 kJ/mol, whereas for pyrimidines the internal motions are slow compared with the rotational diffusion of the whole molecule, as a result of a possible H-bonding between the 5'CH2OH group and the base, and the authors suggested that this might raise the apparent activation energy of pseudorotation for uridine and cytosine above 25 kJ/mol. A recent temperature-dependent 2H and 13C relaxation study from our laboratory308 on selectively deuterated thymidines and allofuranoses as well as their comparisons with the conformationally constrained analogues and abasic sugars did not allow us to determine the activation energy barrier of pseudorotation because the internal motions are heavily coupled with the overall molecular reorientations, which prevents dissection of the observed activation energy barrier of 20-23 Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected]

35 (±0.9) kJmol-1 into contribution from pseudorotational interconversions, rotation around the glycosyl and C4'-C5' torsions and overall tumbling. Nevertheless, this experimental estimate308 gives an upper limit for the pseudorotational barrier (see Appendix for detailed discussion on the estimates of the barrier).

2.7 Steric effect of the nucleobase on the sugar conformation The inspection of the three molecular fragments in nucleos(t)ides (i.e. the aglycone, the sugar and the phosphate) allows us to estimate the bias of the two-state N S pseudorotational equilibrium of the constituent pentofuranose moiety on the basis of various steric and stereoelectronic effects. The N-nucleobase influences the pentofuranose conformation through its inherent steric effect and counteracting stereoelectronic interactions within O4'-C1'-N1/9 fragment. In β-D-nucleosides, among all pseudorotamers, steric repulsions penalize O4'-exo (W-type) conformation304-306,309 most owing to the 1,3-diaxial orientation of the nucleobase and 5'CH2OH group and to the eclipsed arrangement of C2' and C3' substituents (Fig 6). In O4'-endo (E-type) pseudorotamers, the steric repulsions between the nucleobase and the 5'CH2OH group are minimized, however the substituents at C2' and C3', just as in the case of W-type conformers, are in the unfavorable eclipsed orientation (Fig. 6). Conformational analyses of nucleos(t)ides in aqueous solution are performed on the basis of a two- state N S equilibrium model (vide supra). In terms of steric effect alone, for β-D-nucleosides, S-type pseudorotamers are energetically favoured in comparison with N-type counterparts, since the pseudoequatorially oriented nucleobase in the former exerts less steric repulsions with the other substituents on the pentofuranose moiety than when it is pseudoaxial in the later (Figs 6 and 8A). In α-D-nucleosides, the nucleobase and the 5'CH2OH group are on opposite faces of the pentofuranose sugar, therefore their steric interactions will be minimal in comparison with the situation in β-nucleosides. In N-type conformers, the nucleobase and 3'-OH are pseudoequatorial while 2'-OH is pseudoaxial, whereas the opposite is true for S-type pseudorotamers. Both in W- and E-type conformers, 2'-OH and 3'-OH are pseudoaxial, whereas the nucleobase is pseudoequatorial in W-type geometry and pseudoaxial in the E-type counterpart. In α-D-2'-deoxynucleosides, the nucleobase exerts stronger steric repulsions with 3'-OH and H4' in N-type than in S-type pseudorotamers. Therefore, in solution, taking into consideration of a two-state N S equilibrium model, S-type pseudorotamers would be energetically favoured over the N-type counterparts if the steric effect alone were considered. Potential energy calculations on α- and β-2'-deoxy (or ribo) adenosine and thymidine have shown that syn conformations of the nucleobase in the α-anomers are energetically less favourable over the entire range of phase angle values, and that the energy barrier of anti syn interconversions is higher in the α-ribo than in the α-2'-deoxy series. Highly hypothetical syn orientations of a nucleobase in α-2'-deoxynucleosides may be stabilized by hydrogen-bonding interactions between N3 (or O2) and 3'-OH. From the previous observations, it can be derived that as the bulk of the nucleobase is increased, one expects the bias of the two-state N S pseudorotational equilibrium to be shifted toward more S-type and N-type conformations in β- and α-nucleosides, respectively.

2.8 O4'-C1'-N1/9 stereoelectronic effect (the anomeric effect) The anomeric effect in heterocyclic six-membered rings is commonly explained in terms of stabilizing hyperconjugative interactions (molecular orbital overlap model)115,116 or, alternatively, by destabilizing dipole-dipole repulsions65,117,118 (Section 1.3 and Table 1 for the energetics of the anomeric effect in 2-substituted hexopyranoses and derivatives). Similarly, the O4'-C1'-N1/9 anomeric effect in nucleosides and nucleotides may be explained either (i) by stabilizing n →σ∗ orbital interactions between the orbital of one of the O4' C1'-N1/N9 endocyclic O4' electron lonepairs (n ) and the antibonding orbital of the C1'-N1/9 glycosyl bond O4' (σ∗ ) (Fig 9), or (ii) by destabilizing electrostatic repulsions between two dipoles [i.e. the dipole C1'-N1/N9 of the furanose ring, on one hand (which is itself the resultant of C4'-O4', O4'-C1' individual dipole moments and of the dipole induced by O4' lonepairs) and the dipole oriented from C1' to N1/9, on the other (Fig 8B-C)]. In β-D-nucleosides, O4'-C1'-N1/9 stereoelectronic interactions are most efficient in W-type sugar geometries, where one of the O4' lonepairs is in optimal antiperiplanar orientation with respect to the C1'-N1/9 bond, and at the same time dipole-dipole repulsions are minimal since the angle between both dipoles is nearly 90°. However, for the steric reasons stated above, West-type conformers have neither been found in crystal structures of β-D-nucleos(t)ides nor in the conformational equilibria in solution. Conversely, in E-type pseudorotamers, both O4' lonepairs are gauche with respect to the C1'- N1/9 bond, which makes hyperconjugation (or molecular orbital overlap) least efficient; Simultaneously, dipole-dipole repulsions are maximal, owing to the fact that both dipoles are nearly parallel.

NH2 Figure 8: Steric effect of the base and (A) N N rationalization of the anomeric effect in H H H H H nucleos(t)ides in terms of electrostatic repulsions, H N9 C N N HO C N NH as examplified for β-D-dA (37). (A) The drive of HO O ' O 9 2 HO 4 H H H the two-state N S equilibrium toward S- over OH N N N-type pseudorotamers by the steric effect of the H NH2 nucleobase. (B) & (C) Stronger electrostatic N repulsions between the pentofuranose ring dipole N (black arrow) and the C1'-N9 dipole (white (B) arrow) in S-than in N-type sugars. Both dipoles N N N HOH2C are nearly parallel in the S-type pseudorotamers HOH2C N O O NH2 HO whereas they are nearly perpendicular in N-type sugars (β << α, Panel C). OH N N N β α N The anomeric effect is therefore often (C) C4' C ' H ' C ' invoked as one of the factors responsible 2 1 2 H1' C4' for the activation energy barrier encountered in the East region of the pseudorotational cycle for β-D-nucleosides, which, besides the stronger barrier in the West region, also hampers free pseudorotation of the constituent pentofuranose. The comparison of the geometries of N- and S-type pseudorotamers of β-D-nucleosides shows that dipole-dipole repulsions (Fig 8B-C) and O4'-C1'-N1/9 hyperconjugative interactions (Fig 9) are

37 reduced and enhanced, respectively, in the former compared to the latter. Therefore, O4'-C1'-N1/9 stereoelectronic interactions drive the two-state N S pseudorotational equilibrium in β-D- nucleosides, in aqueous solution, toward N-type conformations.

N-type sugar NH2 S-type sugar (A) N N

N Figure 9. Rationalization of the O4'-C1'- 9 N N HOH2C N1/9 anomeric effect in nucleos(t)ides in HOH2C σ*C1'-N9 N9 O O NH2 terms of molecular orbital overlap and HO 1 hyperconjugation, as examplified for β-D- 1n 2 nsp2 (p-type) N N sp (p-type) OH dA (37). The O4' lonepairs orbitals are N 2n 3 (B) 9 N9 sp 2 2 represented using either the sp [i.e. higher β = 132ο nsp3 ο 1 β1 = 96 energy 1n lonepair with sp2(p-type) O4' O4' C2' H1' predominant p-type character and lower 1 C ' 1n 3 C4' n 3 4 sp C ' sp energy 2n lonepair with 2 H1' sp2(s-type) (C) 2 N9 N9 3 nsp2 (s-type) predominant s-type character] or sp [i.e. 2 nsp2 (s-type) 1n and 2n lonepairs with the same β = 162ο 3 3 2 O ' O ' C ' sp sp 4 ο 4 2 H1' β2 = 126 energy] hybridization models123-126,129,130 C ' C4' 4 1n 2 1 C2' sp (p-type) (Section 1.9). (A) Molecular orbital overlap nsp2 (p-type) H ' 1 model116: The overlap (i.e. n σ* ) NH2 NH2 O4' C1'-N9 N N of a lonepair orbital of O4' [1n ] (D) N N sp2 (p-type) N N with the σ*C1'-N9 antibonding orbital of the 9 N δ− 9 N HOH C HOH C δ+ glycosyl bond results in the stabilization of 2 O 2 O HO HO N- over S-type pseudorotamers and shifts the pseudorotational equilibrium toward N. (B) & (C) Influence of sp3 (i.e. 1n and sp3 σ* (E) C1'-N9 2 2 1 n 3, Panel (B)) versus sp ( n 2 (p-type) nO4' ΔE(σ*C1'-N9 - nO4') sp sp and 2n , Panel (C)) hybridization of Stabilization due to sp2 (s-type) anomeric effect (AE) the O4' lonepairs orbitals on their relative orientation with respect to the glycosyl bond and on the efficiency of the O4'-C1'-N9 stereoelectronic interactions. For a typical N-type pseudorotamer (the angles in the projections are calculated for P = 0° and Ψm = 40°, assuming perfect trigonal symmetry), the nO4' σ*C1'-N9 interaction is possible due to the near antiperiplanar orientation of either 1n (β ≈ 132°) or 1n (β ≈ 162°) with respect to sp3 1 sp2 (p-type) 2 σ*C1'-N9 orbital, whereas in the S-type sugar counterpart (the angles are calculated for P = 160°, Ψm = 40°) the efficiency of the interaction is much reduced as the result of the more accute β1 and β2 angles compared to those in the N-type sugar. (D) Double-bond No-bond resonance resulting from hyperconjugation of one of the O4' lonepairs to the glycosyl C1'-N9 bond115. This model is consistent with the shortening of O4'-C1' bond compared to O4'-C4' bond observed in the crystal structures174,310,311 of β-nucleos(t)ides. (E) This overlap, which results in the formation of a stabilizing (occupied) and a destabilizing (unoccupied) orbitals, becomes more efficient as the square of the overlap (S) between both orbitals increases and as the difference (∆E) between their respective energies decreases4,5. A maximal interaction requires an antiperiplanar orientation of nO4' relative to the C1'-N9 bond.

The significant change (Fig 2E)1,121,122 of the endocyclic and exocyclic C-O bond lengths in combination with concommitant closing or opening of the corresponding bond angles in crystal structures of chair conformers of various 2-substituted-tetrahydropyrans with axially versus equatorially oriented anomeric substituents is considered as a strong evidence for the hyperconjugative origin of the anomeric effect in such systems. A perusal174,310,311 of the crystal structures of β-D-N- nucleosides also shows that the C1'-O4' bond is 0.03 - 0.04 Å shorter than the C4'-O4' bond, which

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 38 goes hand in hand with a possible hyperconjugative origin for the anomeric effect in nucleosides (Fig 9).

2.9 Effect of the aglycone on the conformation of nucleos(t)ides and oligos

History of the discovery of stereoelectronic effects in nucleos(t)ides: The first set of eight papers qualitatively indicating the influence of the electronic character of the C1'-aglycone on the conformation of the constituent pentofuranose sugar in nucleos(t)ides were the result of almost simultaneous independent observations: (i) That the UV spectrum of 1-methylcytosine and cytosine nucleosides varies with the nature of the carbohydrate component is the first milestone paper that established the cross-talk between the aglycone and the constituent sugar312. (ii) Guschlbauer et al showed313 that the confomation of the ribose moiety changes with the protonation of the guanin-9-yl base at N7 in 2'-GMP. (iii) As shown314 by Sarma et al., the sugar conformation is indeed different in oxidized and reduced β-nicotinamide mononucleotides. (iv) Remin and Shugar showed315 that the 3 protonation of cytidine or arabinocytidine changes the J1'2' value by about 0.2 Hz. (v) Altona and Sundaralingam did an important qualitative correlation on the nature of sugar pucker depending on the nature of the aglycone basing on their X-ray crystal structures data181. (vi) Subsequently, Altona and Sundaralingam extended their X-ray correlation data to the 3 coupling constant correlation, showing again the qualitative dependence of J1'2' with the nature of the aglycone316. (vii) The preference of S versus N conformer is pH-dependent, as demonstrated for the first time by Guschlbauer et al on guanosine phosphates317. (viii) A similar observation was also made by Hruska et al on 2'-O-methyladenosine318.

Further qualitative evidences: In 1987, we first showed14 basing on thermodynamic estimates on a set of four isomeric 2'/3'- deoxynucleosides that the net result of the gauche effect and the anomeric effect is of major importance in determining the overall furanose conformation. Beside the subsequent quantitative works from our labs (Sections 4 - 6), many qualitative studies have been conducted to understand how the electronic nature,309,319-323 protonation state198, bulkiness or substitution pattern207,239,324-327 and configuration of the nucleobase175,328-346 or of the C1 substituent234 modulate the sugar conformation in nucleosides and nucleotides as well as in furanosides347-351. The conformational analysis46 in solution of a dimer 352 3 containing a 4'-oxofuran derivative , based on pseudorotational analysis of vicinal JHH, has shown that the modified nucleoside adopts almost exclusively (89 %) S-type puckered geometries, as a result of the cooperative drive by the O5'-C4'-O4' anomeric effect and the [O3'-C3'-C4'-O4'] gauche effect (vide infra).

2.9.1 Configuration-dependent sugar conformation in furanosides 3 3 3 At the sugar level, spectroscopic studies based upon the interpretation of JHH, JCH and JCC coupling constants347,349,353 have shown that the α- and β-anomers of D-ribofuranose (and of their 1- methyl derivatives) adopt preferentially S- and N-type puckered geometries, respectively, owing to the O1-C1-O4 anomeric effect, which places the anomeric group in pseudoaxial or nearly pseudoaxial

39 orientation. The contribution of the exo-anomeric effect to the relative stabilities and preferred conformations of α- and β-D-erythrofuranose and -threofuranose, as obtained from ab initio calculations, has also been adressed348.

2.9.2 Effect of electron-withdrawing nucleobases on pseudorotamer populations 1H-NMR studies have shown that in 2-substituted tetrahydropyran, the magnitude of the O-C-O anomeric effect increases as the anomeric group becomes more electronegative (Section 1.5). In nucleosides and nucleosides, similar trends have also been found: (i) Egert et al., in their integrated approach321 consisting of an analysis of crystallographic data and 1H-NMR spectra as well as quantum-chemical calculations on 5-substituted uridines, have shown that electron-withdrawing substituents at C5 induce a lengthening and shortening of N1-C1' and C1'- O4' bonds, respectively, in comparison with the reference compound, uridine. For electron-donating substituents, the opposite situationwas observed. They invoked a possible charge transfer from one of the O4' lonepairs to the C5 substituent to rationalize these tendencies. We, on the other hand, argue that electron-withdrawing (or electron-donating) substituents at C5 are expected to strengthen (or weaken) the nO4' →σ∗C1'-N9 interactions (i.e. the anomeric effect), which would in turn also lead to the experimentally observed lengthening (shortening) and shortening (lengthening) of C1'-N1 and O4'-C1' bond lengths, respectively. In that work, the concomitant opening (closing) of the glycosyl torsion angle χ in 5-substituted uridines with electron-withdrawing (electron-donating) substituents at C5, respectively, with respect to uridine, was explained by the fact that small χ values favour nO4' →πC5-C6 and nO4' →π*C5-C6 interactions, which are destabilizing and stabilizing, respectively. For electron- withdrawing at C5, the stabilizing nO4' →π*C5-C6 interactions are strengthened while the destabilizing nO4' →πC5-C6 interactions is weakened (in comparison with the reference compound uridine), leading to the overall stabilization of the system, therefore such substituents favour small values of χ. In contrast, electron-donating groups at C5 will induce a strengthening of the destabilizing nO4' →πC5-C6 interactions and a weakening of the nO4' →π*C5-C6 interactions (i.e. an overall destabilization of the system) and in turn greater χ values will be found in comparison with uridine. A qualitative correlation between the preference of the sugar moiety in 5-substituted uridine derivatives for N-type versus S-type geometries and the value of χ was also proposed. (ii) In their conformational study on 5-substituted uridine derivatives in aqueous solution by 1H-NMR spectroscopy, Uhl et al.322 have shown that the population of N-type pseudorotamers (% N) increases from 44% to ≈ 90% going from NH2 to NO2 substituent at C5, and a plot of % N as a function of the Hammett constant, σp, of the substituent at C5, gave a straightline. This is also consistent with our proposition that electron-withdrawing (electron-donating) groups at C5 are expected to strengthen (weaken) nO4' →σ∗C1'-N9 interactions (i.e. the anomeric effect) and therefore stabilize (destabilize) the N- over S-type pseudorotamers. This is clearly evident from our quantitative analysis showing that the protonation of the nucleobase in adenosine, guanosine and cytidine nucleosides and in their deoxy derivativies strengthen the anomeric effect (i.e. increase of N-type conformation) , whereas it is weakened by the deprotonation (i.e. decrease of N-type conformation), compared to the neutral state (see Section 4 for detailed study).

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 40 (iii) We have quantitatively shown that the sugar conformation can be steered by altering the electronic character of the nucleobase as well as by the change of the electronegativity of the sugar substituent (see section 4). Subsequently, Rosemeyer and Seela have confirmed this by showing225 that the extent of the preference for N-type pseudorotamers of 7-substituted 7-deaza-2'-deoxyadenosines increases as the Hammett contant σm of the 7-substituent increases. This result is consistent with the expected increase in the strength of the O4'-C1'-N9 anomeric effect as the glycosyl nitrogen becomes a better electron-acceptor.

2.9.3 Effect of the protonated nucleobase on pseudorotamer populations The protonation of a nucleobase in a nucleotide directly controls its hydrogen-bonding capabilities, therefore the overall three-dimensional structure of oligonucleotides is dictated by the pH of the medium. For instance, the local DNA triple helix formed354,355 by the binding of a natural homopyrimidine oligonucleotide to a target DNA duplex is much more stable in the acidic solution than at neutral pH, owing to the fact that Hoogsteen base-pairing with the third pyrimidine strand requires that the cytosin-1-yl nucleobases be protonated. However, substitution of cytidine in the Hoogsteen strand for 5-methylcytosine or 5-bromouridine allows to increase its affinity for the DNA duplex under physiological pH356,357. pH-dependent conformational transitions and stabilities of C.A and G.A mismatches in DNA358-362 have been extensively studied. It has also been suggested363,364 + that although the secondary structure of the oligoDNA d(A -G)10 is presumably helical, it is stabilized not by stacking bases or hydrogen-bonding base pairs but instead by ionic bonds between positively charged 2'-deoxyadenosine residues and distal negatively charged phosphates. The formation of unusual parralel double-stranded DNA duplex365-367 or four-strand tetrads (the i-motif)368,369 with two parallel stranded base-paired duplexes at low pH has also been experimentally evidenced. The acid-base character of nucleobases in nucleosides and nucleotides varies widely165,247,370- 381 165 165 : Whereas adenosine (pKa ≈ 3.5) and cytidine (pKa ≈ 4.2) can be easily protonated in the 165 382-384 acidic solution at N1 and N3, respectively, uridine (pKa ≈ 9.4), 5-fluoro-2'-deoxyuridine (pKa 165 ≈ 7.8) and thymidine (pKa ≈ 9.9) are deprotonated at N3 in alkaline solution. Guanosine is either 379 165 protonated at N7 (pKa ≈ 1.9) in the acidic medium or deprotonated at N1 in the alkaline solution 385,386 (pKa ≈ 9.4). pKa values for C-nucleosides are known for formycin A (pKa = 4.4 (protonation at ,388 N3) and 9.6 (deprotonation at N7)), formycin B387 (pKa = 8.8 (deprotonation at N1) and 10.4 (deprotonation at N7)), pseudoisocytidine389 (pKa = 3.7 (protonation at N1) and 9.0 (deprotonation at N3)) and pseudouridine390 (pKa = 9.0 (mixed deprotonation at N1 and N3)). We have recently reported 34 for the first time the pKa values corresponding to protonation at N3 in 9-deazaadenosine (pKa = 6.0) and formycin B (pKa = 1.3). Most pKa values of nucleobases in nucleos(t)ides are rather different from the physiological pH, therefore one might conclude that pH-induced conformational transitions are not likely to occur in DNA and RNA near physiological pH (≈ 6.8 - 7.3), unless the pKa of a particular nucleobase changes drastically as a result of change of the microenvironment359,360,363,369,396-398. This can be examplified by the pKa values of adenin-9-yl and cytosin-1-yl moieties, which are significantly different at some oligonucleotides than those found in their monomers: For example, the pKa of adenin-9-yl in the A25 residue (located close to the cleavage site in a lead-dependent ribozyme) is 397,398 6.5 which is unusually high compared with the pKa of adenin-9-yl in adenosine (3.5). Clearly, Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected]

41 any slight deviation of the medium from the physiological pH may impose an ionic character on nucleobases with standard pKa values. Additionally, transition or soft metal ions, by binding to the nucleobase391-395 may also impose a change in the ionic character of the nucleobase, equivalent to the effect of its protonation or deprotonation (depending upon which of the sites in a nucleobase a metal ion prefers to bind!). The affinity of a pyrimidine oligonucleotide399 cytidine residues for a target DNA duplex increases by 10-fold when the pH is changed from 7.6 to 5.8, whereas the equilibrium binding constant for another pyrimidine oligonucleotide in which 5-methylcytosin-1-yl nucleotides have been incorporated increases by a factor 20 upon the same change in pH. An analysis of these data in terms of two-state model revealed that the above pyrimidine oligonucleotides with cytidine and its 5-methyl counterpart form triple helical structures with apparent pKas of 5.5 [(C+GC) triplets] and 5.7 5 [(m C+GC) triplets], respectively. These pKa values are respectively 1.2 and 1.3 unit higher than those known for the nucleoside counterparts, 2'-dC (pKa = 4.3) and 5-methyl-2'-dC (pKa = 4.4), respectively. From the above studies397-399, it is clear that some specific adenin-9-yl and cytosin-1-yl nucleotides, under certain folded structural states would form the protonated species at a pH close to the physiological pH. Since the magnitude of the O4'-C1'-N1/9 anomeric effect is enhanced in the P compared to the N state of the nucleobase in nucleosides (see the sections below), those residues are more prone to take a N-type conformation, which in turn may dictate the local phosphate backbone to adopt a specific conformation, which together may constitute a recognition element for a ligand binding. Owing to the key role played by protonation of the constituent nucleobases in the biological function of DNA and RNA, it appeared necessary to obtain reliable estimates of the actual magnitude of the anomeric effect in the N, P and D states of the nucleobases in nucleosides and nucleotides (Sections 4 - 6). As stated in Section 2.9, protonation of the nucleobase in various purine and pyrimidine nucleosides and nucleotides, such as adenin-9-yl at N1 in 2'-O-methyladenosine318 or arabinoadenosine derivatives198, guanin-9-yl at N7 in 2'-, 3'- and 5'-phosphates of guanosine313,317, cytosin-1-yl at N3 in arabinocytidine or its methyl derivatives315 results in the shift of the N S pseudorotational equilibrium of the constituent pentofuranose moiety toward more N-type pseudorotamers (as 3 experimentally evidenced by the change in JHH coupling constants), in which the nucleobase adopts a pseudoaxial orientation. In six-membered rings, on the other hand, it has been shown that the preference of imidazolium or pyridinium as anomeric groups in 2-substituted pyranose derivatives for equatorial positions is greater than that of their neutral counterparts94,106-108. This has been attributed to the reverse anomeric effect (Section 1.7). If the reverse anomeric effect were to play any role in the drive of the sugar conformation in β-D-nucleosides at acidic pH, one should observe an increase in the population of S- type pseudorotamers, in which the nucleobase adopts a pseudoequatorial orientation, in the acidic compared to the neutral pH. Since the above qualitative studies198,313,315,317,318,400 as well as our recent quantitative works30,32,36,37,44 (described in detail in Sections 4 - 6 and 8.8) show opposite trends, i.e. a greater preference for N-type conformations in the P compared with the N state, one can therefore conclude that no reverse anomeric effect operates in the protonated pentofuranosyl β-

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 42 nucleosides. Instead, the shift of the two-state N S pseudorotational equilibrium in nucleosides toward N-type conformers at acidic pH can be easily explained in terms of pD-dependent magnitude of the anomeric effect318: As the nucleobase becomes protonated, a partial positive charge is created at the glycosyl nitrogen. As a result, nO4' →σ∗C1'-N9 interactions become more efficient. Owing to the strengthening of the anomeric effect, the tendency of the nucleobase to adopt pseudoaxial orientations in N-type pseudorotamers is greater in protonated nucleosides than in their neutral counterparts. Protonation of guanosine at N7 promotes delocalization of the lonepair of the glycosyl nitrogen N9, making it slightly positively charged, which has a certain azine-type character, and therefore its 15N chemical shift resonates downfield by 6.7 ppm compared to N9 in neutral guanine moiety401. Similarly, upon protonation of N3 in 1-methylimidazole, the delocalization of the N1 lonepair results in its partial positive character, which promtes its 7.4 ppm downfield shift compared to the counterpart in the neutral form402. The delocalization of the N9 lonepair in N1-protonated adenosine to stabilize N1H+ species also results in partly positively charged N9, which resonates downfield by 6.6 ppm in comparison with N9 in neutral adenine403. On the other hand, in cytidine the chemical shift of N1 is hardly affected (the downfield shift is only 1.1 ppm404) upon protonation of cytosin-1-yl at N3. Upon its deprotonation at N3 in 3'-O-methylarabinouridine405, uracil-1-yl tends to prefer more pseudoequatorial orientations than in the N state, as reflected in the increase of the population of S-type pseudorotamers. This can be attributed to the fact that deprotonation of uracil-1-yl reduces the ability of the glycosyl nitrogen to be involved in nO4' →σ∗C1'-N9 interactions, i.e. a weakening of the anomeric effect, simply because of the electron-rich character of the conjugate base of uracil moiety.

2.9.4 Effect of base-modifications on the stability of nucleic acids The modification of the chemical nature of nucleobases affects the overall stability of DNA/RNA heteroduplexes has been reviewed47,49. However, the influence of the O4'-C1'-N1/9 anomeric effect (Section 4) as a result of base modification, or its modulation by the change of the aromatic character by the change of pH of the medium (i.e. protonation-deprotonation equilibrium) or by its binding to any specific ligand (i.e. association-dissociation equilibrium) may affect the stability of the duplexes, which has not been addressed hithertofore in the literature. The stabilization of DNA/RNA duplexes (with respect to parent unmodified duplexes), as a result of modification of some constituent nucleobase(s) in the oligodeoxyribonucleotide chain, may be attributed to either of the following events: (i) Increased stacking interactions in the case of 5-propynyl dU406, 5-(amino-ethyl-3-acrylimido) dU49,407, 7-halo-7-deaza408,409 and 7-propyne-7-deaza410 purines modifications, or (ii) to the shielding of the negative phosphate charges by 5-amino-hexyl-substituted pyrimidines411, or (iii) to the possibility to form an additional hydrogen-bond in the case of 2- aminoadenosine412,413. On the other hand, the destabilization of the heteroduplex DNA/RNA upon modification of the nucleobase has been explained either by (i) loss of hydrophobic interactions (upon substitution of thymin-1-yl by uracil-1-yl)414, or (ii) the inability of the thymin-1-yl nucleobase to adopt anti orientation around the glycosyl torsion when it is substituted at C6414, or (iii) to the loss of hydrogen-bonding sites in O2 or O4 substituted thymin-1-yl415.

2.10 The gauche effects in α- and β-nucleosides Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected]

43 In addition to O4'-C1'-N1/9 stereoelectronic interactions and the associated steric effect of the nucleobase, the gauche effect also plays an important role to explain the preferred conformation of the pentofuranose moiety in all nucleosides and nucleotides. The origin and the energetics of the gauche effect in simple 1,2-disubstituted ethanes have been discussed in Sections 1.12 - 1.14. In this section, we only present the relevant 1,4-gauche interactions which are involved in the drive of the sugar conformation in nucleosides and nucleotides.

2.10.1 2'-Deoxynucleosides

In α- and β-2'-deoxynucleosides, the gauche effect of [O4'-C4'-C3'-O3'] fragment stabilizes pseudorotamers in which the O4'-C4' and O3'-C3' bonds are in a gauche orientation (i.e. W- and S- type), over those where they are in a trans arrangement (i.e. E- and N-type)304,305. However, as discussed in Section 2.3-2.5, W-type pseudorotamers are neither observed in the solid state nor in solution, owing to the high energy penalty resulting from the eclipsed orientation of C2' and C3' substituents, therefore the [O4'-C4'-C3'-O3'] gauche effect alone drives the sugar conformation of nucleos(t)ides preferentially toward S-type pseudorotamers. In α-2'-deoxynucleosides, since O4'-C1'- N1/9 stereoelectronic interactions cooperate with the [O4'-C4'-C3'-O3'] gauche effect, one expects that the conformation of the pentofuranose sugar will be biased toward S-type conformations. The results of our recent study37 on the thermodynamics of the N S equilibrium in α-D/L-2'-deoxynucleosides are in agreement with this simple reasoning (Section 5). However, we have also shown that the magnitude of stereoelectronic interactions in α-series is in general considerably reduced than in the β- counterparts. In β-dNs, the anomeric effect drives the two-state N S pseudorotational equilibrium in solution toward N-type forms, whereas the [O4'-C4'-C3'-O3'] gauche effect favours S-type geometries (vide supra). The experimentally observed preference of β-dNs and β-2'-deoxy mono- and oligonucleotides for S-type conformations165 therefore suggests that the [O4'-C4'-C3'-O3'] gauche effect is the predominant factor controlling the conformation of the constituent sugar moieties, prevailing over the counteracting anomeric effect. This is consistent with our recent estimates of the magnitudes of anomeric and gauche effects in such systems20,30.

2.10.2 Ribonucleosides and nucleotides In ribonucleosides and nucleotides, three additional gauche effects determine the preferred orientations within [O2'-C2'-C1'-O4'], [O2'-C2'-C1'-N1/9] and [O2'-C2'-C3'-O3'] fragments. Both in α- and β-ribonucleos(t)ides, among all possible pseudorotamers, W-type geometries are the most favoured by the [O4'-C4'-C3'-O3'] and [O4'-C1'-C2'-O2'] gauche effects, since only in that situation both fragments are in a gauche orientation. Conversely, E-type pseudorotamers are the most disfavoured, owing to the trans orientation of O4'-C4' and O4'-C1' bonds with respect to C3'-O3' and C2'-O2', respectively. However, W-type pseudorotamers are energetically penalized (vide supra ), and in aqueous solution, the sugar moiety is involved in a two-state N S equilibrium. The [O4'-C4'-C3'- O3'] and [O4'-C1'-C2'-O2'] fragments are in a trans and a gauche orientation in N-type pseudorotamers, respectively, whereas in the S-type counterparts, this opposite is true, therefore the [O4'-C4'-C3'-O3'] and [O4'-C1'-C2'-O2'] gauche effects cancel each other. The [O2'-C2'-C1'-N1/9] gauche effect drives the pseudorotational equilibrium of the sugar moiety in β-ribonucleosides toward S-type Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 44 pseudorotamers, whereas in α-ribonucleosides C1'-N1/9 and C2'-O2' bonds are in gauche orientation both in N- and S-type conformations. The [O4'-C4'-C5'-O5'] gauche effect is one of the factors (besides the steric or hydrogen-bonding interactions between 4'-CH2OH group and the nucleobase) that controls the preferred conformation across the γ torsion angle. Several models have been proposed to account for the gauche effects within X-C-C-Y fragments, X and Y being electronegative elements or groups (Section 1.14). A simple explaination is based on σ → σ* interactions between best donor σ and best acceptor σ* orbitals as shown for 2'- deoxynucleosides in Fig 10 (A & B). Possible contributions from bond-bending, or a combination of through-space and through bonds interactions to the mechanism of the gauche effect can however not be excluded (Section 1.14). In all studies on the effect of 1,4-gauche interactions upon the sugar conformation in nucleosides and nucleotides, the estimates obtained for the magnitudes of these gauche effects actually correspond to overall strength, consisting of steric, stereoelectronic and electrostatic components.

2.11 The gauche effects of sugar substituents and the self-organization of DNA/RNA

2.11.1 Studies on nucleosides 1H- and 13C-NMR studies on N-nucleosides17,46,193,195-199,201-204,206,208, 213,216,219,222,226,235,416-423, and on C-nucleosides224,237 as well as theoretical works304,305 have qualitatively shown that the preferred conformation of the pentofuranose moiety in nucleosides, nucleotides and their derivatives is strongly affected by the electronic nature and relative configuration of the substituents at C2' and C3' positions. We, on the other hand, have shown in our recent quantitative studies, that as the nucleobase in β-D-N- and C-nucleosides becomes electron-deficient in the protonated state, the strength of O4'-C1'- N1/9 stereoelectronic interactions increases, whereas in the deprotonated state, the N9/1 becomes more electron-rich resulting in the weakening of O4'-C1'-N1/9 stereoelectronic interactions. This has been discussed in details in Sections 4-6. Systematic analyses of the conformational preferences of 2'-deoxy-2'-substituted uridine199,235 and adenosine206,417,418 derivatives have allowed us to derive linear relationships between the population of the N-type pseudorotamer and the electronegativity of the 2'-substituent: As the 2'- substituent becomes more electronegative, the population of N-type pseudorotamers linearly increases as a result of the increased strength of the 2'-substituent gauche effects. 2'-thionucleosides416 prefer more S-type geometries than in 2'-deoxynucleosides. Similarly, the two-state N S equilibrium in 2'- 421 methylthionucleosides is strongly (> 70 %) biased toward S-type conformations in CD3OD, and the effect of 2'-SMe has been attributed both to its reduced electronegativity (i.e. resulting in weaker [S2'- C2'-C1'-O4'] and [S2'-C2'-C1'-N1/9] gauche effects) and increased steric bulk (resulting in the destabilization of N-type pseudorotamers). The population of S-type pseudorotamers in 3'-deoxy-3'-substituted arabinofuranosyladenine201 is also dictacted by the tuning of the gauche effect of [X3'-C3'-C4'-O4'], which is in turn controlled by the electronegativity of the 3'-substituent (X). Similarly, substitution of 3'-OH in β-D-2'-

45 213 422 17,219,424 deoxynucleosides by a more electronegative 3'-OPO2 , 3'-NO2 or 3'-F results in the increased preference of the sugar moiety for S-type conformations. From a comparison of the conformational properties of natural and 3'-modified thymidine dimers it has been found423 that S-type pseudorotamers are more and more stabilized as the 3'-substituent is changed in the following order: 3'- NH2 < 3'-N3 < 3'-O-H < 3'-O-P < 3'-O-S. The two-state N S equilibrium in 2',3'-cis-fused furano- and pyrrolidino-β-D-nucleosides is driven to S- (in case of C3'-O or C3'-N substitution) or N-type sugars (for C2'-O or C2'-N).

(A) Figure 10. Rationalization of the [X3'-C3'- H3' C4'-O4'] gauche effect in a 2',3'-dideoxy-3'- σ C3'H3' Base HOH2C substituted-β-D-nucleoside in terms σ σ* HOH C 2 σ*C4'O4' Base interactions between best donor (σC3'-H3') and O O H σ*C4'O4' best acceptor (σ* ) orbitals. The σ σ* σ C4'-O4' X C4'O4' interactions stabilize the S- over N-type σC3'H3' X pseudorotamers [Panel (A)], owing to the fact North, trans [O4'-C4'-C3'-X] South, gauche [O4'-C4'-C3'-X] that in the former the C3'H3' and C4'O4' bonds are almost antiperiplanar, and therefore the torsion angle between σ and σ* (B) C3'-H3' C4'- σ H ' O4' orbitals is much reduced in the S-type C4'O4' σ 3 σ C ' C3'H3' ο C3'H3' 5 H3' β1 = -93 sugar geometries (β ≈ -37°) compared to the O ' C ' β = -37ο C1' 4 5 1 C2' N-type counterparts (β ≈ -93°) [Panel (B)] C1' C2' O4' σ*C4'O4' (Torsions angles have been calculated from the values of the endocyclic torsion angle ν σC4'O4' 3 σ*C4'O4' H4' H4' X X from ab initio optimized (at HF/6-31G*) HF/6-31G* geometry; X = F HF/6-31G* geometry; X = F geometries of typical N- (PN = 21°; Ψm(N) = 35°) and S-type (PS = 143°; Ψm(S) = 34°) (C) pseudorotamers of 3'-fluorothymidine, σ*C4'O4' assuming simple trigonal symmetry. (C) The

σC3'H3' extent of the energy stabilization of S-type ΔE (σ*C4'O4' - σC3'H3') Stabilization due to pseudorotamers through [X3'-C3'-C4'-O4'] gauche effect (GE) gauche effect is proportional to the square of the overlap between σC3'-H3' and σ*C4'-O4' 2 2 GE α S / ΔE (σ*C4'O4' - σC3'H3') where S = overlap between σ*C4'O4' and σC3'H3' orbitals (i.e. S) and inversely proportional between the difference between their energies. The sugar moiety prefers S- (or N) type conformers more in the furano than in the pyrrolidino analog, owing to the stronger gauche effect of [O4'-C4'-C3'-O3'] (or [O4'-C1'-C2'-O2'] and [O2'-C2'- C1'-N]) in the former compared with [O4'-C4'-C3'-N3'] (or [O4'-C1'-C2'-N2'] and [N2'-C2'-C1'-N]) in the latter419,420. The gauche effect of the fluorine substituent, due to its high electronegativity, has a profound stereoelectronic effect on the stereochemical orientation of the neighbouring groups, thereby fluorine substituent governs the overall conformation of the sugar ring199,206,208,222.The sugar moieties in 2'-α-fluoro-2',3'-β-D-dideoxyuridine and 3'-β-fluoro-2',3'-β-D-dideoxyuridine adopt exclusively N- type conformations, owing to the cooperative drive of the [F2"(α)-C2'-C1'-O4'] and [F3'(β)-C3'-C4'- O4'] gauche effects, respectively with the O4'-C1'-N1/9 anomeric effect. In contrast, as a result of configuration-dependent gauche effect, the two-state N S equilibrium in 2'-β-fluoro-2',3'-β-D- dideoxyuridine and 3'-α-fluoro-2',3'-β-D-dideoxyuridine are strongly biased to the S-type conformers because of the predominance of the [F2'(β)-C2'-C1'-O4'] and [F3"(α)-C3'-C4'-O4'] gauche effects, respectively, over the anomeric effect226. 3'-methyl-thymidine in a TT dimer adopts preferentially N-

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 46 type conformations425, owing to the lack of [O3'-C3'-C4'-O4'] gauche effect, whereas 3'-OH in the thymidine counterpart drives the sugar conformation toward S-type pseudorotamers.

2.11.2 Studies on oligonucleotides The application of the gauche effect concept in the design of oligonucleotides with specific conformational properties will be discussed in detail in Section 8.1.

3. Methods to quantitate stereoelectronic effects in nucleos(t)ides In our laboratory, a new strategy has been recently elaborated to obtain reliable accurate estimates for the magnitudes of the O4'-C1'-N1/9 anomeric effect and the gauche effects of [O4'-C4'- C3'-O3'], [O4'-C1'-C2'-O2'] and [O2'-C2'-C1'-N1/9] fragments controlling the sugar conformation in nucleosides and nucleotides14-45. Our method is based on two essential steps: (i) The initial calculation of the thermodynamics (i.e. ∆H°, ∆S° and ∆G°) of the two-state N S equilibrium in 12 - 83 (Section 3.1-3.7). (ii) We have subsequently correlated the experimental ∆H° values with the structural features of 12 - 83 either through semi-quantitative regression analysis or pairwise comparisons (Section 3.10 and following).

3.1 Thermodynamics of the two-state N S equilibrium ∆H°, ∆S° and ∆G° of the two-state N S equilibria of abasic sugars 12 - 1620,37, α-D-2',3'- dideoxynucleosides (α-D-ddNs) 17 - 2036, α-D-2'-deoxynucleosides (α-D-dNs) 21 - 2637, α-L-2'- deoxynucleosides (α-L-dNs) 27 - 2937, β-D-2',3'-dideoxynucleosides (β-D-ddNs) 30 - 3620,36, β-D-2'- deoxynucleosides (β-D-dNs) 37 - 4520,30, β-L-2'-deoxynucleosides (β-L-dNs) 46 - 4937, β-D- ribonucleosides (β-D-rNs) 50 - 5520,30, β-D-ribo-C-nucleosides (β-D-C-rNs) 56 - 6224,25,32, β-D-3'-dA 6327,426, 3'-monophosphates of β-D-2'-deoxynucleosides (β-D-dNMPs) 64 - 6823, 3'-ethylphosphates of β-D-2'-deoxynucleosides (β-D-dNMPEts) 69 - 7323, 3'-monophosphates of β-D-ribonucleosides (β- D-rNMPs) 74 - 7828 and 3'-ethylphosphates of β-D-ribonucleosides (β-D-rNMPEts) 79 - 8328 have 3 1 been derived in two steps: (i) Vicinal proton-proton coupling constants ( JHH) extracted from their H- NMR spectra have been initially translated into the parameters defining the geometry of the N- [PN and Ψm(N)] and S-type [PS and Ψm(S)] sugar pseudorotamers as well as their mole fraction at the equilibrium using the PSEUROT203,209,427 program. (ii) Van't Hoff analysis of the temperature- dependent mole fractions of the N- and S-type conformations has subsequently allowed to derive ∆H°, ∆S° and ∆G° values of their N S equilibria at each pD, and in the case of nucleosides exhibiting pD- dependent conformational preferences, we have subsequently estimated the values of ∆H°, ∆S° and ∆G° in each of their P, N and deprotonated (D) states from a nonlinear fitting procedure.

3.2 1H-NMR spectra [temperature, pD, ligand-dependent spectra of nucleos(t)ides] 1 One-dimensional H-NMR spectra of 5 - 20 mM D2O solutions of 12 - 83 have been recorded between 278 K and 358 K (in 5 K or 10 K steps) at one or several pDs within the 0.5 - 12.0 range at 600 MHz or 500 MHz using Bruker DRX 600, DRX 500 and AMX 500 spectrometers. For abasic sugars 12 - 16, β-D-ddU (35), β-D-ddI (36) and mononucleotides 64 - 73 and 75 - 83, the spectra have been recorded at one (neutral) pD only. At neutral pD, 50% of all molecules of 3'-

47 monophosphates β-D-dNMPs 64 - 68 and β-D-rNMPs 74 - 78 are expected to have a net charge of -1 165 (pKa ≈ 1.5 for the first dissociation of the monophosphate moiety ) whereas the remaining 50% will 165 carry a charge of -2 (pKa ≈ 6.7 for its second dissociation ). However, the corresponding 3'- ethylphosphates β-D-dNMPEts 69 - 73 and β-D-rNMPEts 79 - 83 carry a single net negative charge at the neutral pD. In order to assess any possible influence of the ionization state of the phosphate moiety upon the bias of the two-state N S equilibrium in β-D-rNMPs compared with their β-D-rNMPEts counterparts, we have also recorded 1H-NMR spectra of β-D-AMP (74) at several pDs in the range from 6.5 to 8.4 (0.5 pD unit resolution). Under this pD range, only the ionization state of the phosphate moiety is expected to change, not that of the constituent adenin-9-yl, since its pKa is ≈ 3.5 (section 3 3 2.8). We found that JHH and JHP remain constant throughout the whole 6.5 - 8.4 pD range, showing that the ionization state of the 3'-monophosphate moiety does not affect the preferred conformation of the pentofuranose moiety. For L-nucleosides 27 - 29 and 46 - 49 and for D-nucleosides 22, 23, 38 and 39, the 1H-NMR spectra have been recorded at two or three pDs (one in each of their P, N and/or D states). For all 1H-NMR experiments, 8 - 32 scans were typically recorded using a spectral width of ≈ 10 ppm. The FIDs were processed using a slight gaussian apodization in order to enhance resolution, and the final spectra consisted of 64 K datapoints. The pD values correspond actually to pH* values, since they have been obtained simply by reading the values displayed on a pH meter (equipped with a calomel electrode calibrated with pH 4 and 7 standard buffers in H2O) without any further correction for the deuterium isotope effect. The pD of each sample has been adjusted by the simple addition of microliter volumes of D2SO4 or NaOD solutions (typically 0.1 - 0.5 N). The small (< 0.1 ppm) and irregular change in the chemical shifts of all resonances from 278 K to 358 K suggested that aggregation was negligible. The 1H resonances were assigned through decoupling experiments and 1 1 2 one-dimensional nOe difference experiments. H- H coupling constants (i.e. geminal JHH and vicinal 3 428 JHH) have been verified with help of the DAISY simulation and iteration program package. Identical values have been found at 1mM and 20 mM concentrations. 3 The errors on experimental JHH coupling constants (± 0.1 Hz in most cases, except for some α-D-ddNs and β-D-ddNs at acidic pDs owing to the near isochronocity of some of H2', H2", H3' and H3" multiplets36) have been estimated from the fit of simulated (or iterated) spectra to the corresponding experimental 1H-NMR spectra. The influence of these errors upon the geometries and relative populations of N- and S-type pseudorotamers engaged in the two-state equilibrium in aqueous solution has been investigated during the pseudorotational analyses with a slightly modified version of PSEUROT program (vide infra).

3 3.3 Pseudorotational analyses of JHH with PSEUROT and some practical hints Some practical hints to perform dependable PSEUROT calculations on natural and unnatural systems for quantitative estimation of the thermodynamics of intramolecular stereoelectronic effects are perhaps important for new users. Before discussing in details the steps involved in performing dependable PSEUROT calculations, we would like to urge a potential user of PSEUROT to consider the following guidelines in addition to those described in the manual on PSEUROT 6.0 (Prof C. Altona, Gorlaeus Laboratories, University of Leiden, 2300 Leiden, The Netherlands).

3.3.1 Incorporation of coupling constant errors in PSEUROT calculations Measurements of experimental coupling constants are prone to errors of at least 0.1 Hz. In the original PSEUROT program this is not taken into consideration. We have therefore modified version 5.4 of PSEUROT to accomodate such error by generating datasets of coupling constants (subsequently used to perform individual calculations with PSEUROT) with a gaussian distribution around the experimental values (see our website: http://bioorgchem.boc.uu.se/WWW_secret/manual_pages/local/ps54ran.html). Readers are directed to Section 3.6 for details.

3.3.2 Parameters to be fixed or optimized during PSEUROT calculations PSEUROT allows to perform a multilinear fit of the experimental coupling constants to P and Ψm of the N and S conformers and their mole fractions in either of the following ways: (i) All parameters can be freely optimized during the calculation. This can be done only when the number of observables (i.e. temperature-dependent coupling constants) is greater than the number of unknowns (P and Ψm of the N and S conformers and mole fraction xN or xS of a pseudorotamer). This is a very important consideration! It is also noteworthy that this is also the reason why pseudorotational calculations on 2'- or 3'-dNs are more relibale than on the ribo counterparts. (ii) In the case of two-state N S equilibrium strongly biased (i.e. ≥ 70%) to either of the N- or S-type pseudorotamers, it is advised to obtain as much information as possible on the geometry of the major conformer, and also the evidence of why two-state equilibrium is a valid concept on the unnatural compound in question. This can be achieved by performing a set of PSEUROT runs in which P(minor) and Ψm(minor) of the minor pseudorotamer are constrained to different values in each calculation. P(minor) and Ψm(minor) are chosen in such a way that over the whole set of calculations the ensemble of P(minor) and Ψm(minor) values that one can reasonably expect to be accessible to the minor pseudorotamer is surveyed. The hyperspace of "reasonable" geometries that can be adopted by the minor pseudorotamer may be assessed basing on either a perusal of crystal structures of the compound of interest or some of its derivatives, or on the results of ab initio calculations at a higher basis set such HF/6-31G* (even semiemperical or molecular mechanics based geometry can show some guidelines when it is not possible to perform ab intio calculations). Further hints can also be found in the manual of PSEUROT version 6.0, p. 24, which can be obtained from Prof. Altona's group. (iii) In contrast, when there is no clear preference for either N- or S-type pseudorotamers, a PSEUROT calculation is performed in which Ψm(N) and Ψm(S) are fixed to an identical value during each calculation, and this value is incremented in the following runs with PSEUROT in such a way that all possible "reasonable" values are surveyed throughout the whole set of calculations. (iv) In the case of conformationally constrained compounds, it is required that the coupling constants show some variation as a function of temperature. If not, it is impossible to derive thermodynamics of stereoelectronic effects for these compounds.

3.3.3 Electronegativity of the substituents on each HCCH fragment

49 In the earlier versions of PSEUROT (e.g. 3B), the generalized equation (referred to as the EOS equation, Eq 8 in Section 3.4) was based on Huggins group electronegativities. In more recent versions, the Karplus equations incorporate the values of substituent parameters λ429,430 to take into account the effect of the electronegativity of the substituents on the HCCH fragment of the coupling protons, which present several advantages over the Huggins electronegativities (see Section 3.4 for details). In a situation where λ is not known for a particular substituent (which is very often the case in unnatural nucleosides used in the antisense work, for instance!), it is advised to use as a starting point the value for the chemical group (among the 50 presented in the article429) whose chemical nature is closest of that of the group of interest in the unnatural nucleoside. An alternative would consist in performing a series calculations in which hypothetical λ values (between the limiting values in the scale 0 to 1.4) are successively used in order to assess the effect of the unknown λ value on the thermodynamics of the N S equilibrium. For compounds with a known pKa value, one can determine the pH-dependent thermodynamics of the N S equilibrium using a series of λ values, and figure out which one gives the correct pKa value of the aglycone or of any other ionizeable substituent. In our recent studies on the pD-dependent modulation of the thermodynamics of the N S equilibrium in N- and C-nucleosides (Sections 4 - 6) through tunable gauche and anomeric effects, it was necessary to assess the influence of the change of the electron-density of the glycosyl nitrogen upon its λ value. Our unpublished results (see Section 3.7(c) for details) show that the effect of a particular λ(N1/9) value chosen within the 0.0 - 1.4 range on ∆H° and ∆S° of the N S pseudorotational equilibrium is not significant.

3.3.4 Priority rule to number the substituents on the HAC1C2HB fragment

The original convention431 proposed by Altona's group to differentiate the substituents on the HAC1C2HB fragment according to their relative orientations with respect to the coupling protons HA and HB is better shown under the form of Scheme 2 (see also p. 14 in the manual of PSEUROT version 6.0). The substituent S1 on C1 is said to be "positive" (ζ = +1 in the Karplus equation) since the projected valency angle between HA and S1, counting clockwise from HA, amounts to ≈ +120°. Conversely, S2 is a "negative" substituent, since the projected valency angle between HA and S2, counting anticlockwise from HA, amounts to ≈ -120° (ζ = +1 in the Karplus equation). Analogously, S3 and S4 are "positive" and "negative" substituents, respectively, owing to the fact that the projected valency angles between HB and S3, on one hand, and between HB and S4, on the other (counting clockwise and anticlockwise from HB, respectively) are ≈ +120° and ≈ -120°.

HA S3 HB C 2 3.3.5 Translation of HCCH C 1 torsion angles into endocyclic S S S 2 1 4 torsion angles In the second step of Φ H Φ A HB PSEUROT (Step 4a in Scheme 3 (+) S3 H (+) S H B 1 A and step 2 in Scheme 4), proton- α2 = -120° α = -120° α1 = +120° 4 α3 = +120° (-) S S (+) proton (HCCH) torsion angles are 2 1 (-) S4 S3 (+)

S4 (-) S2 (-) translated into the corresponding

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Scheme 2.Dept Definition of Bioorganic of "positive" Chemistry, and "negative" Box 581, substituents Uppsala on University, the H AC1C S-751232HB fragment Uppsala, Sweden, Ver 160205 [email protected] 50 endocyclic torsion angles (ν0 .... ν4) via linear relationships such as: Φ = Aνi + Bi (Eq 11 in Section 3.5). For natural pentofuranosyl nucleosides, A and B have been determined for the β-D-ribose, β-D- 2'-deoxyribose, β-D-arabinose, β-D-lyxose, β-D-xylose, α-D-lyxose and α-L-xylose configurations from linear regressions based on torsion angles extracted from crystal structures174,182,205 (see also the discussion in the manual of PSEUROT version 6.0). For other modified nucleosides, A and B pairs can be derived from correlation plots of HCCH versus νi torsion angles as extracted from ab initio optimized geometries (e.g. at HF/3-21G* or higher basis sets) of various pseudorotamers of the nucleoside of interest with selected P values (for instance, 12 structures with 0° < P < 330° at 30° resolution and a common puckering amplitude). Care is to be taken so as to ensure that each pseudorotamer optimized ab initio will have the desired phase angle value, i.e. by constraining the values of two endocyclic torsions (e.g. ν0 and ν4) during the calculation.

3.3.6 General operational conditions for PSEUROT 3 In our conformational studies on nucleosides and nucleotides, JHH coupling constants have been interpreted in terms of a two-state N S equilibrium with help of the program PSEUROT203,209,427. The validity of the two-state equilibrium has been experimentally evidenced by the analysis of the distribution of sugar conformations in the X-ray crystal structures of nucleosides, as well as by the results of NMR studies in solution both in our own lab and elsewhere32,36,37,174, 230,241- 244,245,248 (Section 2.3 and the following).

(2) (1) Ab Initio PSEUROT Program The two-state N S equilibrium is a valid model for 3 Calculations pseudorotational analyses of JHH of the nucleoside X on X 3 Experimental JHH Coupling Constants for a nucleoside X

(3a) Karplus-Altona 3 Equation Endocyclic Torsion (4b) Experimental JHH in Φ(HCCH) torsion angles constrained nucleosides Angle (ν0 - ν4)

Φ(HCCH) torsion angles A New 7 (10) (5b) Parameter (3b) (9) AH, BH parameters Karplus (4a) from ab initio type equation calculations or Phase Angle of Pseudorotation Corresponding crystal structure (P) and Maximum Puckering 3 Experimental JHH Φ(HCCH) torsions Amplitude (Ψm) and Coupling Constants angles from X-ray Endocyclic Torsion Angle (ν0 - ν4) Equilibrium Populations structures

(5a) Pseudorotational Concept (11) (12)

Phase Angle of Pseudorotation (P) and Phase Angle of Pseudorotation (P) and (7) Φ(HCCH) (6) Maximum Puckering Amplitude (Ψ ) X-ray Crystal m Maximum Puckering Amplitude (Ψ ) torsion angles Structure of X and Equilibrium Populations for X (8) m

203,209,427 3 Scheme 3: Iterative structure elucidation of nucleosides using NMR-PSEUROT analyses of JHH coupling constants, ab initio calculations and comparisons. Ab initio calculations on aristeromycin (8), 2'-deoxyaristeromycin (9) and 3'-deoxyaristeromycin (10)41 allowed us to validate the two-state N S equilibrium model (Steps 1 & 2) in such compounds and to derive AH and BH values required for step 4a (see below). Three translation steps are involved in 3 PSEUROT: (i) Experimental JHH are first used to calculate ΦHCCH torsion angles (Step 3a) using Eqs 8a-9a; (ii) ΦHCCH 174 are used to calculate the endocyclic torsion angles (νi) (Step 4a). AH and BH are published for β-D-dNs/rNs. For α-D- dNs, 120° was subtracted from AH and BH known for Φ1'2' (Φ1'2") in parent β-D-dNs. For α-/β-L-dNs, AH and BH are the

51 same (but with opposite signs) as those of α-/β-D counterparts234. For β-D-dNMPs/dNMPEts, we used the same values as for β-D-dNs. For β-D-rNMPs/rNMPEts and β-D-C-rNs we assumed AH and BH identical to those of β-D-rNs. For β-D- ddNs, AH and BH were derived from regression analysis of HCCH torsion angles versus the corresponding νi (taken from 268,269,432-434 crystal structures ). For α-D-ddNs, we have subtracted 120° from the values found for Φ1'2' (Φ1'2") for the parent β-D-ddNs. (iii) PN, PS, Ψm(N), Ψm(S) and xS are finally calculated (Step 5a) from all νi values using Eq 6. For aristeromycin, we found41 that the geometry of the cyclopentyl ring in the solid state (steps 6 and 7) is different (step 8) 431 from the solution state geometry derived from PSEUROT analyses (based on the original Karplus-Altona Eqs 8a-9a). To verify whether this was due to a poor parametrization of Eqs 8a-9a for H2CCH2 fragments, we specifically reparametrized them (Eq 8b-9b, steps 9 & 10) for carbocyclic nucleosides. PSEUROT analyses (steps 3b - 5b) based on Eqs 8b-9b produced the same geometries (step 11) for the cyclopentyl ring in aristeromycin, its 2' and 3'-deoxy derivatives as the initial analyses (steps 3a-5a), however the r.m.s. errors were reduced when Eqs 8b-9b were used.

Five parameters are necessary to define the position of the equilibrium at a certain temperature or pD: The phase angles of the N- (PN) and S-type (PS) pseudorotamers and their respective puckering amplitudes [Ψm(N) and Ψm(S)] as well as the mole fraction of one of the conformers (xN or xS). In the 3 3 case of 12 (10 experimental JHHs), of α-D-ddNs 17 - 20 and β-D-ddNs 30 - 36 (8 JHHs) and of 13, 3 15 and 16 (7 JHHs), the system is overdetermined and PN, PS, Ψm(S), Ψm(S) and xS can in principle be estimated simultaneously from a single set of coupling constants. For α-D-dNs 21 - 26, α-L-dNs 27 - 29, β-D-dNs 37 - 45, β-L-dNs 46 - 49, β-D-dNMPs 64 - 68, β-D-dNMPEts 69 - 73 and β-D-3'-dA 3 (63), five JHHs are available at each temperature. Since there are as many unknowns as knowns, if the calculation is performed without constraining one of the unknowns to some value estimated by another method (for instance from X-

Experimental 3J PSEUROT Program HF Coupling Constants

3 Experimental JHH 3 Coupling Constants JHF and Corresponding Proton-Fluorine Torsion Angles from Conformationally Fixed Compounds (1) Karplus-Altona Equation

(6) (8) Proton-Fluorine Proton-Proton Torsion Angle Torsion Angle

New Karplus-Type Equation AH, BH parameters 3 (2) from ab initio for JHF Coupling Constants calculations or (4) (7) crystal structure

3 Limiting JHF Proton-Fluorine Endocyclic Torsion Angle (ν - ν ) 0 4 Coupling Constants Torsion Angle

(5) (9) Pseudorotational (3) AF , BF parameters from Concept Extrapolation of experimental ab initio calculation or 3 crystal structure JHF to pure major conformer

Phase Angle of Pseudorotation (P) and Maximum Puckering Amplitude (Ψm) Endocyclic Torsion Angle (ν0 - ν4) and Equilibrium Populations (10)

39 Scheme 4: The "PSEUROT+JHF" program , as an extension of the original PSEUROT program for pseudorotational 3 3 3 analysis of JHF in combination with JHH coupling constants. We first performed pseudorotational analyses of JHH coupling constants of monofluoronucleosides to find out P, Ψm and xS (at various temperatures) of the N and S forms 3 (Steps 1 - 3). From the plot of temperature-dependent xS as a function of temperature-dependent experimental JHF, we 3 derived limiting JHF values for the major N or S pseudorotamer of each nucleoside (step 5). The corresponding HCCF 3 torsion angles for all JHF coupling constants were calculated from the corresponding endocyclic torsion angles (step 4), basing upon AF and BF values (Eq 11 applied to ΦHCCF torsion angles) derived from ab initio optimized geometries of our 3 monofluoronucleosides. A dataset of 57 pairs of the above limiting ( JHF , ΦHCCF) from our monofluoronucleosides and from conformationally constrained cyclic fluorinated organic compounds (step 6), which were added in order to better 3 define the values of JHF for ΦHCCF around ± 90° and 180°, was subsequently used to parametrize a new Karplus-type 3 equation specifically for JHF (Step 7), which includes a term accounting for HCC and FCC bond angle changes. The 3 validity of Eq 8c was proven by the fact that pseudorotational analyses based on JHF alone (steps 8 - 10) of a set of other monofluoro nucleosides yielded nearly the same values for P, Ψm of the N and S pseudorotamers as our initial analyses 3 which relied exclusively upon JHH data (Steps 1 - 3). Eq 8c was finally used in combination with the Karplus-Altona 430 equation to derive for the first time accurate estimates for P, Ψm of the N and S pseudorotamers and xS (at various temperatures) for difluoronucleosides 84 - 87 (steps 1 - 3 and 8 - 10). ray crystal structures or ab initio calculations), the accuracy of the results will strongly depend upon the 3 accuracy of the experimental JHH coupling constants. For all other compounds, only 3 (or 4 in the

53 3 case of abasic sugar 14) JHHs are available at each temperature and the iteration with PSEUROT is only possible if at least two (or one) parameter(s) are (is) fixed. Calculations with PSEUROT are based on three principal steps (Schemes 3 & 4): (i) 3 Translation of experimental JHH coupling constants into the corresponding proton-proton torsion angles (Φ) [Step 3a in Scheme 3, step 1 in Scheme 4] with help of the generalised Karplus-Altona equation. In some modified nucleosides it might be necessary to reparametrize this Karplus-Altona equation to take account of the influence of a specific substituent on the bond length, bond angle, through-space transmission effect (i.e. the Barfield effect) etc; (ii) ΦHCCH are themselves used to calculate the corresponding endocyclic torsion angles of the pentofuranose moiety (ν0 ... ν4) (Step 4a in Scheme 3, step 2 in Scheme 4); (iii) PN, PS, Ψm(N) and Ψm(S) of the N-type and S-type pseudorotamers as well as xS are ultimately derived from all endocyclic torsion angles obtained in step (ii) using the law of pseudorotation (Eq 6, Step 5a in Scheme 3, step 3 in Scheme 4).

3.4 Generalised Karplus-type equation Karplus-type equations (Step 3a in Scheme 3, Step 1 in Scheme 4) allow to translate vicinal coupling constants into the torsion angle between the coupling nuclei, and their use in conformational analysis is well known215,217,435-437.

3 3.4.1 EOS Karplus-Altona equation for JHH The generalised EOS equation (Eq 8a) developed by Altona's group431 describes the 3 dependence of a vicinal JHH coupling constant upon the corresponding proton-proton torsion angle, the Electronegativity and relative Orientations of the Substituents in the H-C-C-H fragment (EOS).

3 J = P cos2Φ+ P cosΦ + P + Δ χ [P + P cos2 (ζ Φ + P |∆χ |)] ... Eq 8a HH 1 2 3 ∑ i(g) 4 5 i 6 i(g) i=1−m 3 Φ designates the H-C-C-H torsion angle. The first three-terms in Eq 8a show the dependence of JHH upon the H-C-C-H torsion angle alone, using the same formalism as that originally proposed by Karplus et al438. The introduction of a phase-shifted cosine square function in the remaining terms allows to take into consideration the influence of the electronegativity of the non-hydrogen substituents on the H-C-C-H fragment and of their relative orientations with respect to the coupling protons. It is assumed that the effects of m substituents are additive. The value of ζi (± 1) reflects the orientation of the substituent i with respect to the coupling protons. Δχi(g) represents the difference between the group electronegativities of the substituent i and of hydrogen, which is used as a reference, in the 439 Huggins scale . Δχi(g) is calculated according to Eq 9a: Δχi (g) = Δχi(α) - P7 ∑Δ χj (β-substituent) j=1−n ..... Eq 9a. Δχi(g) takes into account both the electronegativity of the α-substituent [Δχi(α)] and χ the electron-withdrawing or donating effect of n β-substituents [Δ j (β−substituent)]. No term in Eq 8a takes into account a possible effect of H-C-C bond angles and C-C bond distances440,441, or the possible influence of through space orbital interactions 442-444. P1 - P7 have been optimized separately 3 for HCCH, H2CCH and H2CCH2 fragments using a set of 315 experimental JHH coupling constants and Φ values (as derived from molecular mechanics calculations), of conformationally constrained six-

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 54 membered rings, which implies that the torsion angles are clustered into the gauche and trans regions. 3 Eq 8a allows to predict the experimental JHH with an accuracy (r.m.s.) of 0.48 Hz. 3 Recently, Eq 8a has been reparametrized430 using a set of 299 pairs of ( JHH, Φ). In that work, Huggins Δχi(g) values (Eq 9a) were replaced by empirical substituent parameters (λ) and the overall 3 r.m.s. error between experimental and theoretical JHH values dropped down from 0.48 Hz to 0.36 Hz. Additionally, it was found that a separate parametrization for HCCH, H2CCH and H2CCH2 fragments was no longer necessary.

3.4.2 Reparametrized EOS equation for carbocyclic nucleosides We have recently determined41 the solution conformations of aristeromycin (8), 2'- 3 deoxyaristeromycin (9) and 3'-deoxyaristeromycin (10) from pseudorotational analyses of JHH coupling constants using PSEUROT version 3B203-203 program, which is based on the Karplus-Altona EOS equation (Eq 8a). We found serious discrepancies between the X-ray crystal structure (P = 89°, Ψm = 41°) of aristeromycin (8) and its structure calculated by NMR-PSEUROT conformational 3/4 0/4 analysis (35° < P [ T - T] < 65°, 35° < Ψm < 45°) (128° < P [1E] < 131°, 34° < Ψm < 36°), as well as relatively high errors in the NMR-PSEUROT analyses for aristeromycin and its 2'-deoxy and 3'-deoxy derivatives [∆Jmax ≤ 1.6 Hz (i.e. maximal difference between experimental and PSEUROT- 3 calculated JHH) and r.m.s. error ≤ 0.7 Hz]. These observations have prompted us to reparametrize (Eq 8b) the Karplus equation implemented in the PSEUROT program by using torsion angles derived from solid state geometries of conformationally constrained nucleosides and their corresponding 3 experimental JHH. 3J = 13.41 cos2Φ -0.98 cosΦ + 1.37 HH 2 + ∑Δ χi(g) [0.20 - 2.26 cos (ζiΦ + 0.39 |∆χi(g)|)] ...... Eq 8b i=1−m where, Δχi (g) = Δχi(α) + 0.071 ∑Δ χj (β-substituent) ..... Eq 9b j=1−n The χ2 value for the fitting of the experimental Φ vs 3J data using Eq. 8b is 7.2 Hz2, HH HH which corresponds to an r.m.s. error of 0.40 Hz. For comparison, the r.m.s. error of the original Haasnoot-Altona's equation (Eq 8a) was in the range 0.36 - 0.51 Hz, depending upon the substitution patterns of H-C-C-H fragments (0.48 Hz in the case where all H-C-C-H fragments are considered together for a common set of parameters). With the help of our new Karplus-type equation [Eq. 8b], 3 the difference between the experimental and calculated JHH couplings, ∆Jmax, was below 0.5 Hz for 40 data points, and in the range from 0.8 -1.3 Hz for five data points. It should also be noted that in our approach we have a common equation for all H-C-C-H fragments. Moreover, the small P7 value indicates the reduced influence of β-substituents. Therefore, for practical reasons, β-substituents may even be excluded when the determination of the electronegativities of the substituents is performed. The results of the PSEUROT analyses performed with the standard Haasnoot-Altona Karplus equation (EOS equation: Eq 8a) are also very comparable in terms of geometry with those based on our reparametrized equation (Eq. 8b). Both series of PSEUROT analyses suggest that the predominant conformation of the cyclopentane ring in carbocyclic nucleosides is defined by 128° < P < 140° for aristeromycin (8), 105° < P < 116° for 2'-deoxyaristeromycin (9) and 118° < P < 127° for 3'-

55 deoxyaristeromycin (10), with the puckering amplitude in the range from 34° to 40°. However, PSEUROT analyses based on our Karplus equation produced a smaller r.m.s. error by ≤ 0.14 Hz and ∆Jmax error by ≤ 0.5 Hz than those performed with the standard Haasnoot-Altona's equation.

3 3.4.3 Karplus equation for interpretation of JHF In the case of difluoronucleosides 84 - 87, the conformation of the constituent sugar moiety 3 3 cannot be determined on the basis of the unique experimental JHH available, i.e. J3'4' . However, four 3 additional vicinal coupling constants, i.e. proton-fluorine couplings JHF, can also be extracted from 1 3 their H-NMR spectra. It was clear to us that these JHF could also be used to find out the preferred conformation of the sugar moiety in 84 - 87 in solution through pseudorotational analyses, provided that we have at our disposal an accurate Karplus-type equation to translate them into the corresponding HCCF torsion angles. It is this observation that has prompted us to parametrize39 a new seven-term 3 Karplus equation specifically for JHF coupling constants using a dataset consisting of monofluoronucleosides and conformationally constrained fluorinated organic compounds (Eq 8c) [see Section 3.8 for details]. Eq 8c has been constructed basing upon Eq 8a, however two significant improvements have been made in comparison with the original formalism: (i) We have used λ substituent parameters to account for the electronegativity of the substituents on the H-C-C-F fragment of interest, in view of Altona's latest work430 and (ii) we have incorporated a cosine squared 3J term that allows to reproduce the experimentally observed variation in HF coupling constants as a function of the HCC (aHCC) and HCF (aFCC) bond angle values. 3 2 2 JHF = 40.61 cos Φ - 4.22 cosΦ + 5.88 + Σ λi [-1.27 - 6.20 cos (ξi Φ + 0.20 λi)] - 3.72 [(aFCC + aHCC)/2 - 110] ....Eq. (8c) Using Eq 8c, we have been able to elucidate the conformation of a series of mono and 3 3 difluorinated nucleosides using a combination JHH and JHF coupling constants (Section 3.8). Our study has shown that the geometries of the N- and S-type pseudorotamers derived from calcualtions with PSEUROT based on either type of coupling constants were nearly identical.

3 3.4.4 Refined Karplus equation for JHH based on Fourier formalism The formalism proposed in Eq 8a suffers itself from the following limitations. (i) It implies a strict additivity of the effects of the substituents, in spite of the fact that linear correlations between the 3 value of JHH and the sum of the substituent electronegativities for monosubstituted ethanes break down for 1,1-disubstituted ethanes carrying highly electronegative substituents. (ii) It poorly 3 reproduces the small experimental JHH values (< 1.2 Hz) for torsion angles in the ≈ ± 90° range. As a result, Eq 8a has only been incorporated in the earlier versions of the PSEUROT program (version 3B). In more recent versions (including version 5.4427, which has been used to perform the pseudorotational 3 analyses for 12 - 83), the dependency of JHH upon the HCCH torsion angle Φ and the nature of the substituents is formulated as a truncated Fourier series429,445,446 in Φ (up to 3Φ) with coefficients expanded as a Taylor series in the empirical substituent parameter λ, as shown in Eq 10a-b. 3J = C + C cosΦ + C cos2Φ + C cos3Φ + S sin2Φ .... Eq 10a HH 0 1 2 3 2 where C0 - C3 and S2 are calculated according to Eq 10b, as follows:

C0 = 7.01 - 0.58 Σi λi - 0.24 (λ1λ2 + λ3λ4); C1 = -1.08; C2 = 6.54 - 0.82 Σi λi + 0.20 (λ1λ4 + λ2λ3); 2 C3 = -0.49; S3 = 0.68 Σi (ζiλ i) ... Eq 10b The new formalism is clearly advantageous: Terms reflecting the individual influence of each substituent are still included, as in Eq 8, however new λ cross-terms are also introduced to take into account possible interdependent substituent effects. Additionally, the influence of β-substituents upon 3 JHH is incorporated in the value of empirically optimized λ parameters. The λ scale has been developed429 through a least-squares fitting procedure, basing upon the change in the value of vicinal 3 JHH as a function of the substitution pattern in mono- and 1,1-disubstituted ethanes, using two references, i.e. λ(H) = 0.0 and λ(OR) = 1.4. 3 During the pseudorotational analyses performed on experimental JHH for 12 - 83, the following λ values429,446 for the substituents at C1' - C4' have been used: λ(H) = 0.0; λ(C1') = 0.62 in nucleosides or 0.67 in abasic sugars; λ(C2' or C3' deoxy) = 0.67; λ(C2' or C3' ribo) = 0.62; λ(C4') = - - 0.62; λ(C5') = 0.68; λ(O4') = 1.27; λ(OH) = 1.26; λ(OMe) = 1.27; λ(OPO3H /OPO3Et ) = 1.27; λ(C- aglycone at C1') = 0.45. For the glycosyl nitrogen of the nucleobase in nucleosides, we have used λ = 0.58 at all pD values.

3 3 3.5 Translation of experimental JHH and JHF into pseudorotational parameters Proton-proton torsion angles (Φ) derived from step 3a in Scheme 3 and Step 1 in Scheme 4 can in turn be used to calculate endocyclic torsion angles (ν0 .... ν4) of the pentofuranose moiety in 12 - 83, using simple linear relationships (Eq 11, Step 4a in Scheme 3 and Step 2 in Scheme 4): Φ = Aνi + Bi .... Eq 11 In the case of β-D-dNs and β-D-rNs, the values of A and B parameters, taken from the literature174, have been determined from linear regressions, basing on torsion angles extracted from the crystal structures of nucleosides. For α-D-dNs, we have subtracted 120° from the values published for Φ1'2' (and Φ1'2") in the parent β-D-dNs, whereas for α- and β-L-dNs, A and B have been obtained by reversing the signs of both A and B values used for their α- and β-D counterparts, respectively, as suggested by Altona et al234. For β-D-dNMPs and β-D-dNMPEts, we have used the same A and B values as for β-D-dNs, whereas A and B for β-D-rNMPs, β-D-C-rNs and β-D-rNMPEts have been assumed identical to those known for β-D-rNs. For β-D-ddNs, A and B values have been determined from regression analysis of some proton-proton torsion angles Φ as a function of the corresponding νi values. Φ and νi values have been extracted from the crystal structures of β-D-ddA268, β-D-ddC269, β-D-ddT432, β-D-ddU433 and β-D-2',3'-dideoxyribavirin434. Finally, for α-D-ddNs, we have subtracted 120° from the values found for Φ1'2' (Φ1'2") for the parent β-D-ddNs. Step 3 in PSEUROT translate the values of the endocyclic torsion angles νi found in Step 2 into PN, Ψm(N) of the N-type pseudorotamer and PS, Ψm(S) of the S-type sugar via the law of pseudorotation (Eq 6a).

3.6 Principle of iterations with PSEUROT PSEUROT iterates the values of PN, Ψm(N), PS, Ψm(S), and of the mole fractions of the conformers (xS) using a Netwon-Raphson minimization procedure, in such a way that a best fit is 3 obtained between experimental JHH and those back-calculated using the best fit PN,S, Ψm(N,S) and xS

57 3 values. Initially, PSEUROT calculates theoretical JHH values using the values of P and Ψm of both N- and S-type pseudorotamers and their respective populations defined in the user's input. The 3 experimental and calculated JHH are compared. In the following iteration steps, random changes are made in P and Ψm values, and/or in the populations, depending upon the user's input, which specifies the parameters to be optimized or constrained during the calculation. The discrepancy between 3 experimental and calculated JHH is monitored and optimized during the iteration procedure. When the best fit is found, the optimal PN, Ψm(N), PS and Ψm(S) of the N- and S-type conformers and their relative populations are printed out, together with the error analysis, which shows both individual 3 differences between experimental and calculated JHH as well as an overall root mean square error (r.m.s.).

3 Incorporation of the error in experimental JHH during the PSEUROT calculation: We have modified the original version 5.4 of the PSEUROT program 203,209,427 to assess the propagation of 3 errors in experimental JHH throughout the calculations and during the subsequent treatment of the results. Our modified program32 retains all features of the original version 5.4, all changes are 3 additions. The estimated error, expressed as standard deviation (σ), of each JHH and the desired 3 number of sets of randomly varied JHH to be generated and subsequently analyzed by pseudorotational analyses, are included in the input file. Typically, for each set of contrained P and Ψm values (vide infra), 1000 data sets are generated and individually analyzed. Each generated dataset contains 3 3 randomly varied JHHs but over all data sets, each JHH is normally distributed around its experimental value with the given σ. The output from our modified program consists of statistical data [average, σ and skew of the calculated geometrical parameters, of the mole fractions and of the "randomized" 3 JHHs] and of the results from all the individual pseudorotational analyses (the calculated P and Ψm values and mole fractions). We have discarded results which fall outside given ranges for: (i) The individual errors between experimental and calculated coupling constants, i.e. Jcalc-Jexp, (ii) the root 3 mean square error (r.m.s.) in JHH, (iii) PN, PS, Ψm(N) and Ψm(S). PN, PS, Ψm(N) and Ψm(S) values are considered as "reasonable" when they are within the ranges found for the sugar moieties in the crystal structures of nucleosides and nucleotides, i.e. typically174: -40° < PN < 40°, 120° < PS < 200°, 30° (or 25° for α-nucleosides175) < Ψm(N) < 45° and 30° (or 25° for α-nucleosides175) < Ψm(S) < 45°. 3 Typically, Jcalc-Jexp and r.m.s. in JHH are considered as acceptable when they do not exceed Jcalc-Jexp and r.m.s., respectively, of the best fit analyses by more than 0.1 Hz.

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 58 Table 2. ∆H° and -T∆S° contributions (kJmol-1)a-c to ∆G298 of the two-state N S pseudorotational equilibrium in abasic sugars 12 - 16 and d,e nucleosides 17 - 63 with fully protonated, neutral and fully deprotonated nucleobases and the corresponding pKa values

Compound Fully protonated nucleobase pKa from: Neutral nucleobase pKafrom: Fully deprotonated nucleobase

ΔH -ΤΔS 298 ∆G° d δ1H e ΔH -ΤΔS 298 ∆G° d δ1H e ΔH -ΤΔS 298 P; P; ΔGP; N; ΔG N; D; D; ΔG D; 12 - - - - - 0.4 (0.3) -0.3 (0.3) 0.1 (0.4) - - - - - 13 ------4.1 (0.3) 1.5 (0.3) -2.6 (0.4) - - - - - 14 - - - - - 0.4 (0.1) 1.5 (1.0) 1.9 (1.0) - - - - - 15 ------4.6 (0.4) 1.2 (0.3) -3.4 (0.5) - - - - - 16 ------4.2 (0.4) 0.9 (0.3) -3.3 (0.5) - - - - - α-D-ddA (17) -1.7 (0.1) 0.2 (0.1) -1.5 (0.1) - 3.7 -1.7 (0.1) 0.2 (0.1) -1.5 (0.1) - - - - - α-D-ddG (18) -8.7 6.0 -2.7 2.7 2.7 -0.4 (0.2) -0.9 (0.2) -1.2 (0.1) - 9.7 -0.4 (0.2) -0.9 (0.2) -1.2 (0.1) α-D-ddC (19) -2.9 (0.2) 1.4 (0.2) -1.5 (0.1) - 4.1 -2.9 (0.2) 1.4 (0.2) -1.5 (0.1) - - - - - α-D-ddT (20) ------1.1 (0.1) 0.6 (0.1) -0.5 (0.1) 10.1 9.7 -0.5 (0.1) 0.3 (0.1) -0.2 (0.1) α-D-dA (21) -5.0 (0.4) 2.1 (0.3) -2.8 (0.1) - 3.6 -5.0 (0.4) 2.1 (0.3) -2.8 (0.1) - - - - - 3'-OMe-α-D-dA (22) -5.8 (1.5) 0.8 (1.5) -4.9 (0.3) - - -6.4 (0.8) 1.9 (0.7) -4.5 (0.3) - - - - - 3',5'-diOMe-α-D-dA (23) -5.3 (1.3) 0.8 (1.2) -4.7 (0.3) - 3.8 -5.8 (0.8) 1.6 (0.7) -4.1 (0.3) - - - - - α-D-dG (24) -10.7 (2.0) 6.4 (2.0) -4.4 (0.3) 2.6 2.5 -4.5 (0.5) 2.7 (0.5) -1.9 (0.1) - 9.5 -3.4 (0.5) 1.4 (0.5) -1.9 (0.1) α-D-dC (25) -7.1 (0.3) 2.5 (0.8) -4.3 (0.2) 4.1 4.2 -7.1 (0.3) 4.0 (0.5) -3.1 (0.2) - - - - - α-D-T (26) ------4.0 (0.2) 2.0 (0.5) -2.1 (0.2) 9.8 9.8 -4.0 (0.2) 2.7 (0.4) -1.1 (0.1) α-L-dA (27) -5.5 (0.9) 2.7 (0.9) -2.8 (0.3) - - -6.0 (0.5) 3.2 (0.6) -2.9 (0.4) - - - - - α-L-dC (28) -7.3 (0.6) 3.0 (0.6) -4.2 (0.2) - - -7.2 (0.5) 4.1 (0.6) -3.1 (0.2) - - - - - α-L-T (29) ------4.2 (0.4) 2.1 (0.5) -2.1 (0.2) - - -3.4 (0.2) 2.5 (0.4) -1.0 (0.1) β-D-ddA (30) 9.2 (0.1) -5.0 (0.1) 4.1 (0.1) 3.6 3.8 3.5 (0.1) -0.9 (0.1) 2.6 (0.1) - - - - -

Table 2 (Continued)

Compound Fully protonated nucleobase pKa from: Neutral nucleobase pKafrom: Fully deprotonated nucleobase

ΔH -ΤΔS 298 ∆G° d δ1H e ΔH -ΤΔS 298 ∆G° d δ1H e ΔH -ΤΔS 298 P; P; ΔGP; N; ΔG N; D; D; ΔG D; β-D-ddG (31) 23.6 -17.1 6.7 2.5 2.5 3.4 (0.4) -0.3 (0.2) 2.9 (0.1) 9.6 9.6 1.4 (0.1) 0.5 (0.1) 1.8 (0.1) 5'-OMe-β-D-ddG (32) 23.4 -16.8 6.8 2.5 2.5 4.3 (0.5) -0.6 (0.5) 3.6 (0.4) 9.7 9.4 2.3 (0.1) 1.0 (0.1) 3.3 (0.1) β-D-ddC (33) 9.6 (0.2) -4.9 (0.1) 4.6 (0.1) 4.2 4.3 6.6 (0.1) -3.0 (0.1) 3.5 (0.1) - - - - - β-D-ddT (34) - - - - - 5.4 (0.1) -2.2 (0.1) 3.2 (0.1) 9.8 9.9 3.5 (0.1) -1.1 (0.1) 2.4 (0.1) β-D-ddU (35) - - - - - 5.7 (0.3) -2.1 (0.4) 3.6 (0.5) - - - - - β-D-ddI (36) - - - - - 6.2 (0.2) -2.9 (0.3) 3.3 (0.4) - - - - - β-D-dA (37) -0.7 (0.1) -0.4 (0.1) -1.1 (0.1) 3.5 3.6 -3.9 (0.2) 1.8 (0.2) -2.1 (0.3) - - - - - 3'-OMe-β-D-dA (38) -1.4 (0.6) -0.7 (0.6) -2.2 (0.2) - - -4.6 (0.4) 1.4 (0.4) -3.2 (0.2) - - - - - 3',5'-diOMe-β-D-dA (39) -1.0 (0.6) -0.7 (0.6) -1.7 (0.2) - 3.5 -3.6 (0.4) 1.5 (0.4) -2.1 (0.2) - - - - - β-D-dImb (40) 0.1 (0.1) -0.2 (0.1) -0.1 (0.1) 6.0 6.0 -2.2 (0.1) 0.8 (0.1) -1.4 (0.1) - - - - - β-D-dG (41) 2.1 (0.1) -2.2 (0.2) -0.1 (0.2) 2.3 2.2 -2.8 (0.2) 1.1 (0.2) -1.7 (0.3) 9.5 9.5 -4.9 (0.2) 2.2 (0.2) -2.7 (0.3) β-D-dC (42) 0.0 (0.1) -0.8 (0.1) -0.8 (0.1) 4.2 4.3 -0.7 (0.1) -0.5 (0.1) -1.3 (0.1) - - - - - β-D-T (43) ------1.4 (0.2) 0.1 (0.1) -1.3 (0.2) 9.7 9.8 -1.9 (0.2) 0.3 (0.2) -1.6 (0.3) β-D-dU (44) ------0.6 (0.2) -0.4 (0.1) -1.1 (0.2) 9.5 9.4 -1.3 (0.2) -0.1 (0.3) -1.4 (0.4) 5-F-β-D-dU (45) ------0.8 (0.1) -0.4 (0.1) -1.2 (0.1) 7.8 7.7 -1.1 (0.1) -0.4 (0.1) -1.5 (0.1) β-L-dA (46) -1.2 (0.6) 0.1 (0.6) -1.1 (0.1) - - -3.9 (0.3) 1.8 (0.4) -2.2 (0.2) - - - - - β-L-dG (47) 1.3 (0.8) -1.6 (0.8) -0.3 (0.1) - - -2.7 (0.3) 0.9 (0.4) -1.8 (0.2) - - -4.3 (0.4) 1.7 (0.4) -2.6 (0.2) β-L-dC (48) -0.2 (0.3) -0.6 (0.3) -0.8 (0.1) - - -0.7 (0.3) -0.5 (0.3) -1.2 (0.1) - - - - - β-L-T (49) ------1.2 (1.1) 0.0 (1.0) -1.2 (1.5) - - -2.3 (0.7) 0.6 (0.6) -1.7 (0.9) β-D-A (50) -0.2 (0.1) -0.4 (0.2) -0.5 (0.2) 3.5 3.5 -4.4 (0.2) 2.6 (0.1) -1.8 (0.2) - - - - - β-D-G (51) 5.4 (0.2) -4.2 (0.5) 1.5 (0.5) 2.1 2.1 -3.3 (0.2) 1.8 (0.2) -1.5 (0.3) 9.5 9.6 -7.6 (0.5) 4.8 (0.2) -2.8 (0.5)

Compound Fully protonated nucleobase pKa from: Neutral nucleobase pKafrom: Fully deprotonated nucleobase

ΔH -ΤΔS 298 ∆G° d δ1H e ΔH -ΤΔS 298 ∆G° d δ1H e ΔH -ΤΔS 298 P; P; ΔGP; N; ΔG N; D; D; ΔG D; β-D-C (52) 5.2 (0.2) -3.3 (0.4) 1.9 (0.4) 4.0 4.1 2.3 (0.1) -0.8 (0.5) 1.5 (0.5) - - - - - β-D-rT (53) - - - - - 1.3 (0.1) -1.4 (0.3) -0.1 (0.3) 9.9 9.8 -0.2 (0.1) -0.2 (0.3) -0.4 (0.3) β-D-U (54) - - - - - 2.0 (0.2) -1.7 (0.3) 0.3 (0.4) 9.6 9.4 0.3 (0.1) -0.2 (0.1) 0.1 (0.1) 5-F−β-D-U (55) - - - - - 2.3 (0.1) -1.8 (0.2) 0.5 (0.3) 8.0 7.6 0.8 (0.1) -0.6 (0.3) 0.2 (0.3) Formycin B (56) -0.5 (0.1) -0.8 (0.1) -1.3 (0.1) 1.4 1.3 -8.1 (0.1) 4.8 (0.1) -3.3 (0.1) 8.9 8.8 -8.8 (0.1) 5.3 (0.1) -3.5 (0.1) Formycin A (57) -2.4 (0.1) 0.4 (0.1) -2.0 (0.1) 4.5 4.4 -8.1 (0.5) 4.7 (0.4) -3.4 (0.1) - 9.5 -8.1 (0.5) 4.7 (0.4) -3.4 (0.1) 9-deaza-A (58) -7.4 (0.2) 3.7 (0.3) -3.6 (0.1) 5.9 6.0 -14.2 (0.4) 9.1 (0.4) -5.0 (0.1) - - - - - Ψ-isoC (59) 4.2 (0.1) -3.8 (0.2) 0.5 (0.1) 3.6 3.6 -1.9 (0.1) 0.6 (0.1) -1.4 (0.1) 9.2 9.0 -7.9 (0.2) 4.9 (0.2) -3.0 (0.1) Ψ-U (60) - - - - - 0.7 (0.2) -1.4 (0.2) -0.6 (0.1) 9.4 9.1 -4.5 (0.1) 2.2 (0.1) -2.3 (0.1) 1-Me-Ψ-U (61) - - - - - 1.1 (0.1) -1.8 (0.1) -0.7 (0.1) 9.9 9.7 -3.0 (0.1) 1.5 (0.1) -1.5 (0.1) 1,3-diMe-Ψ-U (62) - - - - - 2.0 (0.1) -2.3 (0.1) -0.3 (0.1) - - - - - β-D-3'-dA (63) 6.9 (0.2) -3.2 (0.3) 3.8 (0.1) 3.4 3.5 1.3 (0.1) 0.5 (0.1) 1.8 (0.1) - - - - - a The errors are shown in parentheses. -TΔS° and ΔG°are at 298 K except for 17, 18, 30 -32 (288 K) owing to decomposition at acidic pD36,37. b ∆H°, -TΔS° and ΔG° are from ref.20 (for 12 - 14), ref. 37 (15, 16, 21 - 29, 37 - 39, 46 - 49), ref. 36 (17 - 20, 30 - 34), ref.447 (35), ref. 30 (40 - 45, 50 - 55), ref. 32 (56 - 62), ref. 27(and our unpublished result for 20 acidic pDs) for 63. Ealier estimates for 30 - 34 in the N state were published . The largest difference between them and the values in table 1 (from ref. 36) is 1.4 kJ/mol for ΔHN; of 33, which is nearly within the sum of the errors of the estimates. c The plateaus in the P, N and D states for ∆H°, -TΔS° and ΔG° of 18, 20, 24 - 26, 30 - 34, 37, 40 - 45, 50- 61 and 63 result from the fit of experimental pD-dependent values to Eq 12. For 22, 23, 27 - 29, 38, 39, 46 - 49, the errors are those of individual ΔH°, -TΔS° and ΔG° at a certain pD d (ref. 37). These pKas were calculated from plots of experimental ∆G° versus pD and from Hill plots of pD versus log(∆∆G°tot - ∆∆G° / ∆∆G°) (refs. 30, 32-37). For 18, 31 and 32 1 ° ° ° e at acidic pDs, the pKa calculated from pD-dependent H chemical shifts (col. 6) was used as a constraint in the determination of ∆H , -TΔS and ΔG in P, N and D states. These 1 (average) pKa values corespond to the pDs at the inflection point(s) of plots of H chemical shifts versus pD (298 K for 17 - 20, 288 K for 30 - 34) and from the corresponding Hill plots.

3.7 Estimation of ∆H°, ∆S° and ∆G° of the N S equilibrium

3.7.1 Methodology Each calculation performed with PSEUROT (using a single set of experimental temperature- 3 dependent JHH at one particular pD) produces one set of temperature-dependent mole fractions of the N- and S-type pseudorotamers.

Table 3. ∆H° and -T∆S° contributionsa to ∆G298 of the two-state NS pseudorotational equilibrium in mononucleotides 64 - 83

Compound ΔH° ∆S° -T∆S298 ∆G298 % S278 % S358 ∆ %S β-D-dAMP (64) -5.4 (0.3) -9.9 (0.7) 3.0 (0.2) -2.4 (0.4) 76 65 -11 β-D-dGMP (65) -4.1 (0.2) -6.5 (0.7) 1.9 (0.2) -2.2 (0.3) 73 64 -9 β-D-dCMP (66) -3.2 (0.1) -6.8 (0.5) 2.0 (0.1) -1.2 (0.2) 64 56 -8 β-D-TMP (67) -2.6 (0.1) -4.3 (0.4) 1.3 (0.1) -1.3 (0.2) 65 59 -6 β-D-dUMP (68) -2.7 (0.1) -3.8 (0.8) 1.1 (0.2) -1.6 (0.3) 67 61 -6 β-D-dAMPEt (69) -5.5 (0.2) -7.9 (0.7) 2.4 (0.2) -3.1 (0.3) 81 71 -10 β-D-dGMPEt (70) -4.8 (0.2) -7.0 (0.7) 2.1 (0.2) -2.7 (0.3) 77 68 -9 β-D-dCMPEt (71) -3.6 (0.2) -5.3 (0.9) 1.6 (0.3) -2.0 (0.3) 72 64 -8 β-D-TMPEt (72) -3.0 (0.1) -3.2 (1.1) 1.0 (0.3) -2.0 (0.3) 71 65 -6 β-D-dUMPEt (73) -2.7 (0.2) -2.3 (0.6) 0.7 (0.2) -2.0 (0.3) 71 65 -6 β-D-AMP (74) -4.9 (0.4) -9.1 (0.7) 2.7 (0.2) -2.2 (0.5) 74 63 -11 β-D-GMP (75) -4.5 (0.2) -10.8 (1.6) 3.2 (0.5) -1.3 (0.5) 66 55 -11 β-D-CMP (76) 0.8 (0.2) -0.6 (1.8) 0.2 (0.5) 1.0 (0.6) 40 42 2 β-D-rTMP (77) -0.9 (0.2) -1.6 (0.9) 0.5 (0.3) -0.4 (0.3) 55 53 -2 β-D-UMP (78) 1.2 (0.2) 2.1 (1.2) -0.6 (0.4) 0.6 (0.4) 43 46 3 β-D-AMPEt (79) -6.9 (0.8) -13.6 (1.1) 4.1 (0.3) -2.8 (0.9) 79 66 -13 β-D-GMPEt (80) -5.8 (0.4) -12.3 (1.1) 3.7 (0.3) -2.1 (0.5) 74 62 -12 β-D-CMPEt (81) -1.5 (0.2) -5.3 (1.0) 1.6 (0.3) 0.1 (0.4) 50 47 -3 β-D-rTMPEt (82) -2.5 (0.3) -5.3 (1.2) 1.6 (0.4) -0.9 (0.5) 61 55 -6 β-D-UMPEt (83) -1.6 (0.1) -3.1 (0.6) 0.9 (0.2) -0.7 (0.2) 58 54 -4 a ΔH° (kJmol-1), ΔS° (Jmol-1K-1), -TΔS° (298 K, kJmol-1) and ΔG° (298 K, kJmol-1) have been taken from ref. 23 for 64 - 67, from ref. 447 for 68 and 73 and from ref. 28 for 74 - 83. For 69 - 72, the values in Table 3 are refined (using their sodium salts) in comparison with the original data in ref. 23 (ammonium salts).

At each pD, the input file for the PSEUROT calculations has been prepared in such a way that (i) the hyperspace of geometries that is accessible to the constrained P and Ψm values of the minor pseudorotamer and/or to the constrained Ψm of both N- and S-type conformations was well 3 covered, and (ii) the errors in the experimental JHH were taken into account by generating ≈ 1000 sets of "randomized" temperature-dependent coupling constants for each geometrical constraint. Therefore, the total number of calculations at each pD was typically ≈ 5000 - 20000. The mole fractions from each calculation are used to construct a van't Hoff plot. The average slopes and Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 62 intercepts from ≈ 5000 - 20000 van't Hoff plots are used to calculate ∆H° and ∆S° (and their errors) of the N S equilibrium of 12 - 83 at the particular pD. The free-energy ∆G° (T), at the temperature T, of the two-state N S equilibrium has been calculated either by (i) adding ∆H° and -T∆S° contributions or (ii) directly from the average [lnaverage (xS / (1 - xS))] of the 5000 - 20000 2 2 individual ln (xS / (1 - xS)). The error on ∆G° (T) is given by: σ∆G° = [σ ∆H° + σ -T∆S°] (case (i)) or σ∆G° = -R.T.lnaverage (xS / (1 - xS)) (case (ii)).

∆H°, ∆S° and ∆G° of the two-state N S equilibrium of the sugar moiety in compounds 12 - 16, β- D-ddU (35), β-D-ddI (36) and mononucleotides 64 - 83 (at a single neutral pD), in L-nucleosides (27 - 29 and 46 - 49) and in D-nucleosides 22, 23, 38 and 39 (at two or three pDs, one in each of their P, N and D states) are presented in Tables 2 and 3. For all other nucleosides, the conformational analyses has been performed over part of or the entire 0.5 - 12.0 pD range, and we only report in Table 2 the limiting values of the thermodynamics of their two-state N S equilibria in each of the P, N and D states, which have been derived according to the procedure described in the present section.

3.7.2 Accuracy of thermodynamics The error on ∆H°, -TΔS° and ΔG° values corresponds to the standard deviation of the average of the corresponding individual ∆H°, -TΔS° and ΔG° values. For all compounds, the standard deviations on ∆H°, -TΔS° and ΔG° values are typically ≈ 0.5 - 1.0 kJ/mol, which have 3 been estimated during pseudorotational analyses in which the JHH error of ≈ 0.1 Hz for all compounds except for some36 ddNs and abasic sugars (0.2 Hz) was taken into consideration. The experimental accuracy of pD-dependent ΔH°, -TΔS° and ΔG° values were evident from the fact that they gave the pKa values of the nucleobase within an accuracy ±0.2 pD unit in average (in comparison with the literature values) with some exceptions where the ΔG° change as a function of pD was less than 1.0 kJ/mol (such as pyrimidine nucleosides).

3.7.3 Influence of λN1/9 on the thermodynamics In their recent investigation of the effect of solvent, pH, temperature and concentration upon 3 the values of the substituent parameters, λ, Altona et al have shown430 that the JHH coupling constant to methyl in ethylamine and propylamine in D2O increases by 0.16 Hz and 0.26 Hz, + respectively, in going from pD 12.5 (NH2) to pD 7.5 (NH3 ), therefore it was suggested that the λ value of NH2 decreases from 1.10 to 0.82 upon protonation. In view of this observation, we have examined the influence of the selected λ value for the glycosyl nitrogen N9 of the nucleobase in β-D-dG (41), in each of its P (pD = 1.0), N (pD = 7.5) and D (pD = 11.4) states, on ∆H°and ∆S° contributions to the free-energy ∆G° of its two-state N S equilibrium. At each pD, we have performed four calculations with PSEUROT. The input for each of these calculations only differs in the value of λ(N9) which has been taken successively as 0.3, 0.58, 0.88 and 1.2. The individual ∆H° and ∆S° values at each pD for the twelve calculations are collected in Table 4. A perusal of the data in Table 4 suggests the followings: Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected]

(D) β-D-dImb (40 ) 50

-0.5 β-D-dG (41 ) 55 5-F-β-D-dU (45 ) β-D-dC (42 ) β-D-dU (44 ) -1.0 60 β-D-dA (37 )

-1.5 65 β-D-T (43 ) 69 -2.0

73 -2.5

77 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 pD (E) 2.0 β-D-C (52 ) 31

1.0 40 5-F-β-D-U (55 ) 0.0 β-D-U (54 ) 50

β-D-rT (53 ) -1.0 β-D-A (50 ) 60

-2.0 69 β-D-G (51 ) -3.0 77 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 pD (F) Ψ-isoC (59 ) 0.0 1,3-diMe-Ψ-U (62 ) 50 1-Me-Ψ-U (61 ) Formycin B (56 )

-2.0 Formycin A (57 ) Ψ-U (60 ) 69

83 -4.0 9-deaza-A 58( )

92 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 pD

Figure 11 (Cont'd): Experimental ∆G° values (298 K) of N S equilibria in α-D-ddNs 17 - 20 [Panel (A)], α-D- dNs 21 and 24 - 26 [Panel (B)], β-D-ddNs 30 - 34 [Panel (C)], β-D-dNs 37, 40 - 45 [Panel (D)], β-D-rNs 50 - 55 [Panel (E)] and β-D-C-rNs 56 - 62 [Panel (F)] as a function of pD. The sigmoidal plots (except for α-D-ddA, α-D-ddC, α-D- dA and 1,3-diMe-Ψ-U, pD-independent values) were obtained by fitting the experimental data to Eq 12, giving the pKa(s) of the nucleobases at the inflection point(s) and limiting ∆G° values in the P, N and D states (Table 2).

(A) 0.5 45

0.0 50

-0.5 α-D-ddT (20 ) 55

-1.0 60

-1.5 α-D-ddG (18 ) 65

-2.0 α-D-ddA (17 ) & α-D-ddC (19 ) 69

-2.5 73

-3.0 77 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 pD (B) 0.0 50 -0.5 55 -1.0 α-D-T (26 ) 60

-1.5 65 -2.0 69 α-D-dG (24 ) -2.5 α-D-dC (25 ) 73 -3.0 77

-3.5 80 -4.0 83

-4.5 α-D-dA (21 ) 86 88 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 pD (C) 7.0 6 7 6.0 8 10 5.0 12 β-D-ddC (33 ) 14 4.0 17 β-D-ddA (30 ) 5'-OMe-β-D-ddG (32 ) 20 3.0 23 β-D-ddT (34 ) 27 2.0 31 β-D-ddG (31 ) 35 1.0 40 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 pD Figure 11 (See the legend p. 74)

(i) The largest influence of a change of λ(N9) upon ∆H° is found at alkaline pD [∆H°λ=1.2 - -1 -1 ∆H°λ=0.3 = -1.3 kJmol ], which is nearly within the sum (0.9 kJmol ) of the standard deviations of ∆H° values at both pDs. At pD 1.0 and 7.5 ∆H° is virtually pD-independent. (ii) By comparing the ∆H° values that have been calculated at pD 1.0 and 7.5 using 0.58 as the value for λ(N9), we find that the N-type pseudorotamers are stabilized by ∆∆H°(P-N, λ=0.58] = 4.6 kJmol-1 in going from the N to the P state of guanin-9-yl in β-D-dG, as the result of the strenghtening of the anomeric effect (see the discussion in section 4.8). (iii) If we now assume that λ(N9) is reduced from 0.58 at pD 7.5 to 0.3 at pD 1.0, as suggested by Altona et al430, and if we compare again the corresponding ∆H° values obtained with this new λ value, we find that the extent of the stabilization of N-type conformers at pD 1.0 with respect to pD -1 7.5 is the same as in (i), i.e. ∆∆H°(P-N, λ(pD 1.0)=0.3] = 4.6 kJmol . (iv) If we assume that λ(N9) is not affected by the deprotonation of guanin-9-yl, we find that S-type pseudorotamers are more stabilized at pD 11.4 than at pD 7.5 by ∆∆H°(D-N, λ=0.58] = -2.2 kJmol-1. (v) If we now assume that λ(N9) is increased from 0.58 to 0.88 in going from pD 7.5 to pD 11.4, the stabilization of S-type conformers is even greater, i.e. ∆∆H°(D-N, λ(pD 11.4)=0.88] = -2.7 kJmol-1. In conclusion, the extent of the stabilization or destabilization of N-type pseudorotamers upon protonation and deprotonation of guanin-9-yl in β-D-dG is independent of the value of λ(N9)! Note however that this conclusion has been only validated for our system, and should be carefully checked for any other unproven system.

Table 4. Influence of the value of the substituent parameter for the glycosyl nitrogen λ(N9) in β-D-dG (41) in each of the P, N and D states of the constituent guanin-9-yl upon the thermodynamicsa of the two-state N S equilibrium of the constituent sugar moiety

P state (pD = 1.0) N state (pD = 7.5) D state (pD = 11.4)

λ(N9) ΔH°P -TΔS°P ΔG°P ΔH°N -TΔS°N ΔG°N ΔH°D -TΔS°D ΔG°D

0.3 1.8 (0.9) -1.8 (0.9) 0.0 (0.1) -2.6 (0.3) 1.1 (0.3) -1.5 (0.2) -4.6 (0.4) 2.2 (0.4) -2.4 (0.2)

0.58 1.8 (0.9) -2.0 (1.0) -0.2 (0.1) -2.8 (0.3) 1.0 (0.4) -1.8 (0.2) -5.0 (0.4) 2.3 (0.4) -2.7 (0.2)

0.88 1.8 (1.0) -2.2 (1.0) -0.4 (0.1) -3.0 (0.4) 1.1 (0.4) -1.9 (0.2) -5.5 (0.4) 2.5 (0.4) -2.9 (0.2)

1.2 1.8 (1.0) -2.2 (1.0) -0.5 (0.1) -3.2 (0.4) 1.5 (0.3) -1.7 (0.1) -5.9 (0.5) 2.9 (0.5) -3.0 (0.2)

a In kJmol-1. -T∆S° and ∆G° in the P, N and D states are given at 298 K. All thermodynamic parameters have been calculated using our methodology for each particular value of λ(N9) (Section 3). Standard deviations of each thermodynamic quantity take into account the influence of the uncertainty (± 0.1 Hz) in experimental 3 JHH, as discussed in Section 3.6.

3.7.4 Influence of the nature of the aglycone dictates the thermodynamics In the case of α-D-ddA (17), α-D-ddC (19) and α-D-dA (21), ∆H°, ∆S° and ∆G° were found to be virtually pD-independent and the values reported in Table 2 for each of the P, N and D states have been calculated by averaging the experimental data at all pDs. On the other hand, the experimental ∆H°, ∆S° and/or ∆G° values of the N S equilibria in α-D-ddG (18), α-D-ddT (20), α-D-dG (24), α-D-dC (25), α-D-T (26), β-D-ddNs 30 - 34, β-D-dNs 37 and 40 - 45, β-D-rNs 50 - 55, β-D-ribo-C-nucleosides 56 - 61 and β-D-3'-dA (63) are dictated by the pD of the aqueous solution30,32,36,37,426, as shown by the sigmoidal plots in Fig 11. The curves through the experimental points in Fig 11 result from a nonlinear least-squares fitting of the experimental data to the Henderson-Hasselbach equation (Eq 12), which gives limiting values of the thermodynamics of two-state N S equilibria in each of the P and D states and the pKa(s) of the constituent nucleobase at the inflection point(s). [Note that the errors on the limiting ΔH°, -TΔS°, and ΔG° values reported in Table 4 actually correspond to standard deviations of averages of individual experimental ∆H°, ∆S° and ∆G° values at several pDs on the plateau in this particular state, whereas the standard deviation for ∆H°, ∆S° and ∆G° values at each pD are higher (typically ≈ 0.5 - 1.0 kJmol-1), as shown in the Tables of pD-dependent thermodynamics in the original publications.] [A] 1− α ..... pD = pKa + log = pKa + log Eq 12 [AH+ ] α In Eq 12, α represents the fraction of the protonated species, and it has been calculated from the change in experimental ∆H°, ∆S° and ∆G° value at a given pD relative to the reference neutral state divided by the total change in their respective values between the N state and the P or D state. The pKa(s) of the nucleobase in 18, 20, 24 - 26, 30 - 34, 37, 40 - 45, 50 - 61 and 63 has been determined either (i) directly from the nonlinear fit of the pD-dependent experimental ∆G° values to Eq 12, as discussed above, or (ii) from Hill plots of pD as a function of the logarithm of the ratio of the protonated to the unprotonated species. The Hill plots gave straight lines with Pearson's correlation coefficients typically above 0.9 and slopes close to 1, which is characteristic of a protonation deprotonation equilibrium involving a single protonation site (see Table 2, cols. 5 and 10 for the pKa(s) of nucleobases derived from pD-dependent ∆G° values). The pKa values determined from pD-dependent ∆G° values of the N S equilibrium in nucleosides fit very well to the literature values as well as those obtained from pD-dependent proton chemical shifts.

3 3.8 New Karplus equation to interpret JHF coupling constants Substitution of a hydroxyl group or hydrogen in nucleosides for a fluorine atom does not change significantly the steric effect of the functionality, however, the strongly electronegative fluorine atom involved in powerful gauche interactions governs the overall conformation of the sugar moiety199,206,210,222,448-449 Fluorine has been widely incorporated into nucleosides to design new therapeutic agents (e.g. FLT (88): anti-HIV activity450; diFC (87): cytotoxic against Chinese hamster ovary and tumor cells451; F2"C (112): inhibits the growth of human lymphoblastic cell lines452) with specific puckering modes453-457. Incorporation of 2'-deoxy-2'-fluoro nucleosides in

67 DNA458 stabilizes the antisense DNA:RNA duplex, which adopts an A-form conformation, owing to the stabilization of N-type sugars by the [F2'-C2'-C1'-O4'] gauche effect459. 2'-fluoro-2'- deoxyguanosine in a hexadeoxynucleotide induces a A-DNA Z-DNA conformational transition460. Incorporation of 2'-fluoro-2'-deoxyarabinothymidine (S-type conformation) in the Dickerson-Drew dodecamer stabilizes the duplex, but 2'-fluoro-2'-deoxyribothymidine destabilizes it by inducing the formation an A-B junction in the middle of the duplex64. When nucleosides are highly substituted (such as difluoronucleosides 84 - 87), it is not to assess the conformation of their 3 3 3 sugar moieties on the basis of a single or a few JHH (in 84 - 87, only J3'4' is available). JHF coupling constants have only been qualitatively used to assess the structure and conformation of pyranose derivatives461-463 and fluoronucleosides204,235 owing to the fact that no Karplus-type 3 equation allows to translate quantitatively experimental JHF data into HCCF torsion angles (ΦHF). 3 2 Several simplified equations such as JHF = A cos ΦHF + B cos ΦHF + C taking into account only the torsion angle dependence were parametrized464-468.

3 3.8.1 Dataset of ( JHF,ΦHF) pairs for monofluoronucleosides 3 We have parametrized a Karplus equation for the interpretation of JHF coupling constants using the strategy depicted in Scheme 4. We have initially performed pseudorotational analyses of 3 203,427 temperature-dependent JHH for monofluoronucleosides (88 - 98) using the PSEUROT program (steps 1 - 3 in Scheme 4). In the second step in PSEUROT (step 2 in Scheme 4), νi torsion angles are translated into the corresponding HCCH torsion angles using simple a relationships: ΦHH = AH νi + BH. AH and BH were obtained from ab initio calculations using the GAUSSIAN 94 program298 on 88, 90 - 92 and 95 - 98 (Table 3 and experimental Section in ref. 39). The plots of ΦHH versus νi for 88, 90 - 92 and 95 - 98 as extracted from their ab initio optimized geometries gave straight lines with correlation coefficients above 0.95. From the results of these initial pseudorotational analyses, the following conclusions could be drawn: (i) All compounds are strongly conformationally biased either to the N- or S-type sugar geometries. (ii) In all nucleosides, the presence of strongly electronegative fluorine atom at C2' or C3' on the α- or β-face makes the C-F bond adopt a gauche orientation with C1'-O4' or C4'-O4'. (iii) This gauche effect is stronger over any other stereoelectronic effect and is responsible for the bias of the sugar conformational equilibrium toward N- or S-type pseudorotamers, which is evidenced by the following observations: In F3"ddU (91) and F3'ddU (98), the same anomeric effect stabilizes N- type conformers. However, S-type pseudorotamers are preferred in 91 owing to predominant [F3"- C3'-C4'-O4'] gauche effect favouring S-type sugars, whereas 98 is locked in N-type conformation owing to cooperative anomeric effect and [F3'-C3'-C4'-O4'] gauche effect. As in F3"ddU (91), the pentofuranose sugar in FLT (88) and its derivatives 89 and 90 prefers S-type conformations owing to the stronger [F3"-C3'-C4'-O4'] gauche effect. In F2'ddU (96), the [F2'-C2'-C1'-N1] fragment is in gauche orientation both in N- and S-type pseudorotamers, and the stabilization of S- over N-type sugar is owing to the stronger [F2'-C2'-C1'-O4'] gauche effect over the anomeric effect. In F2"ddU (97) the combined influence of the [F2"-C2'-C1'-O4'] gauche effect and of the anomeric effect overrides the counteracting [F2"-C2'-C1'-N1] gauche effect and N-type conformers are preferred.

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 68 Basing on the results (PN, PS, Ψm(N), Ψm(S) and temperature-dependent xS) of these 3 pseudorotational analyses, we have built a dataset composed of 29 pairs of ( JHF,ΦHF) for the major pseudorotamers of 88 - 98 in two steps (steps 4 and 5 in Scheme 4): (i) From linear relationships ΦHF = AF νi + BF (based on torsion angles extracted from ab initio optimized geometries of 88, 90 - 92 and 95 - 98, vide supra) we calculated ΦHF torsion angles from the endocyclic torsions νi for each HCCF fragment in the major pseudorotamers of 88 - 98. (ii) We 3 found a linear correlation between temperature-dependent xS and JHF values, which allowed us to 3 calculate limiting JHF values for pure N- or S-type (major) pseudorotamers in 88 - 98 (Table 4 in ref. 39). 3 An analysis of the statistical distribution of the 29 pairs of ( JHF,ΦHF) for 88 - 98 revealed that the torsion angles are not distributed evenly over the whole 0 - 360° range, instead most of them are found in the cis and trans regions. Additionally, neither torsion angles around ≈ ± 90° nor 3 3 JHF above 45.3 Hz were present in this dataset. It is however known that the limiting JHF coupling constants can be ≈ 45 - 47 Hz for the trans substitution pattern of decalins469,470. Therefore a parametrization based on this dataset would not have allowed to define precisely the position of the minima (≈ ± 90°) and maxima (≈ 180°) of the Karplus curve in these regions. We have therefore extended (step 6 in scheme 4, Ref. 39) our dataset by incorporating 28 additional 3 466,467,469-471 pairs of ( JHF,ΦHF) from conformationally constrained (cyclic) compounds 99 - 109 . ΦHF were extracted from the structures of 99 - 109, whose geometry was optimized ab initio with GAUSSIAN 94298. Among these 28 pairs, 3 (entries #31, 33, 42 in Table 4 in ref. 39) allowed to 3 precise the ( JHF,ΦHF) values around ± 90° while 9 (entries #43, 44, 47, 48, 51 - 54, 57) were found 3 in the trans region (with JHaxF = 46 Hz in 109).

3.8.2 Parametrization of the Karplus equation 3 A perusal of the 57 pairs of ( JHF,ΦHF) in Table 4 (in ref. 39) shows variations of up to 16 3 Hz between the value of JHF either in the cis or trans region for the same torsion angle in different 3 3 3 compounds (compare JH2"F3" in F3AT (95) and JH3'F2' in F2'ddU (96), on one hand; JH2'F3" in 3 AFLT (90) and JH4'F3' in FXA (92)). This can be attributed to different substitution patterns on H2"-C2'-C3'-F3" in 95 and H3'-C3'-C2'-F2' in 96, in one hand, and H2'-C2'-C3'-F3" in 90 versus H4'-C4'-C3'-F3' in 92, in the other. This led us to conclude that a simple 3-term Karplus-type 3 2 equation (i.e. of the form JHF = A cos ΦHF + B cos ΦHF + C) will not reproduce our experimental 3 JHF accurately on the basis of the torsion angle value alone. It has been suggested that the 3 electronegativity of the substituents on the HCCF fragment affects the value of the JHF coupling constant461,472. In order to account for the influence of the nature and configuration of the 3 substituents on the HCCF fragments on the value of JHF, we have first parametrized (step 7 in scheme 4) a Karplus-type equation, whose form is that originally proposed by Altona et al (Eq 8a)431 (Section 3.4). In Eq 8a, we have however used λ substituent parameters429 to account for the effect of the electronegativity of the substituents on the H-C-C-F fragments instead of the original Huggins group electronegativities (Δχi(g) ). Through a MonteCarlo fitting procedure, we optimized the values of P1 - P6. Large 3 discrepancies up to 10 Hz between experimental JHF and those back-calculated with help of Eq 8a Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected]

69 3 were found, suggesting that its form is not adequate. By examining the variations in JHF values for a specific ΦHF value, we then realized that the coupling constants are also dictated by the values of HCC and FCC bond angles, as suggested in early qualitative studies466-468. After examining several forms of equations to reproduce the effects of bond angle, we found that it is best reflected in a cosine square function of the torsion angle (Eq 8c, Section 3.4). The r.m.s of the fit of our experimental data to the coupling constants calculated using Eq 8c is 1.38 Hz and the largest individual error is 2.9 Hz (Table 4 in ref. 39). Thus, the accuracy of our newly developed Karplus 3 equation (r.m.s. ≈ 1.4 Hz for JHF in the trans region ≈ 46 - 47 Hz, i.e. ≈ 3 % error) is comparable to 3 431 3 that used commonly to interpret JHH data (r.m.s. ≈ 0.4 - 0.5 Hz for JHH in the trans region ≈ 8 - 9 Hz, i.e. ≈ 4 % error). Other combinations of parameters in Eq 8c led to similar r.m.s error, 3 however, plots of JHF for particular substitution patterns (in particular highly substituted, with substituents of high electronegativity) a function of torsion angle showed negative (below -0.5 Hz) 3 JHF values for ΦHF ≈ ± 90° (Fig. 1 in ref. 39).

3 3.8.3 Pseudorotational analyses of JHF in fluoronucleosides to validate the Karplus equation. In order to demonstrate that Eq 8c can be used as a reliable tool in conformational analysis of fluoronucleosides, we have subsequently proceeded in four steps:

3 3 Plots of JHF versus JHH for qualitative conformational analysis of 88 - 98: We have first 3 3 constructed plots of JHF versus JHH data for different P values in the 0° to 360° range (at different Ψm) for mononucleosides 88 - 98 (Figs 3 and 4 in Ref. 39). The position of the experimental 3 3 temperature-dependent pairs of ( JHF, JHH) for 88 - 98 suggested a high preference of their pentofuranose moieties for N- and S-type pseudorotamers and the extent of this preference was in 3 good agreement with that obtained from our initial pseudorotational analyses based on JHH data only.

3 3 Pseudorotational analyses on 88 - 98 based on a combination of JHF and JHH data (Steps 8 - 10 in scheme 4): We have slightly modified (see the experimental Section on "PSEUROT+JHF" in Ref. 39 and our web site) the original PSEUROT version 3B program in order to make it possible to 3 perform a pseudorotational analysis using either (i) temperature-dependent JHH data or 3 3 3 temperature-dependent JHF data alone or (ii) a combination of JHH and JHF coupling constants. (i) The results (PN, PS, Ψm(N), Ψm(S) values and temperature-dependent xS as well as r.m.s. 3 3 error and individual errors in JHF or JHH) of the pseudorotational analyses performed on 88 - 98 3 are compiled in Table 5 in Ref. 39. The comparison of the results from analyses based on JHH or 3 JHF data only shows that the largest difference between P values is 35° (for AFLT), whereas for Ψm values it is at most 13° (for AFLT), and for xS values we found a largest error of 13 % unit (for F2'ddU). The r.m.s. error of both types of analysis is similar and ≤ 0.8 Hz. The largest individual 3 3 error between experimental and back-calculated JHH and JHF is 1.2 Hz and 2.0 Hz, respectively. 3 3 (ii) The analyses based on JHH and JHF data have been performed in two ways. During the 3 3 multilinear fitting process with PSEUROT+JHF, the errors in JHH and JHF data have been either 3 attributed the same "scale factor (1.0) or in a second series of analyses, the error in JHF was scaled Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 70 3 down to 20 % of that in JHH in order to account for the fact that the former are in average ≈ 5 times greater than the latter. The introduction of scale factors did not affect the geometry of the 3 pseudorotamers in the final output of the PSEUROT calculation. The analyses based on JHH and 3 JHF show that the P and Ψm values obtained from the analyses described in step (i) and (ii) are comparable r.m.s. errors below 1.2 Hz were found.

Qualitative and quantitative pseudorotational analyses on monofluoronucleosides 111, 112 and difluoronucleosides 84 - 87: In order to further verify the validity of Eq 8c, we have applied it to the conformational analysis of another set of monofluoro (111 and 112) and difluoronucleosides 3 3 (84 - 87) through qualitative plots of JHF as a function of JHH data (vide supra, Fig 5 in Ref. 39) for phase angles values from 0° to 360° or to the quantitative pseudorotational analysis using our modified PSEUROT+JHF program (Table 5 in Ref. 39). The position of the experimental pairs of 3 3 ( JHF, JHH) data suggest that the pentofuranose sugar adopts almost exclusively (or predominantly) N-type conformations in F3'ddA (or in F2"C). For 84 - 87, conformational analyses based on the 3 single JH3'H4' are at best misleading. Our qualitative plots for 84 - 87 show for the first time that the conformation of their sugar moieties is strongly biased toward N-E-type pseudorotamers (50° < P < 90°), as suggested by the clusering of all experimental coupling constants in this region. Our 3 3 more elaborate pseudorotational analyses on JHH and/or JHF data for 84 - 87, 111 and 112 precises the conclusions drawn on the basis of the above plots. Note that the results of the pseudorotational 3 analyses are again virtually independent of the type of vicinal coupling constants used ( JHH and/or 3 JHF) (i) The sugar moiety in F3'ddA (111) is indeed locked in N-type conformation (≥ 94 % at 298

K) with PN in the range from 11° to 20° while Ψm is between 34° and 37°. The r.m.s. of all analyses 3 3 3 is ≤ 0.7 Hz with -1.3 Hz as the largest error in JHH and JHF in analyses performed using JHH and 3 JHF data. (ii) Similarly, N-type pseudorotamers are strongly preferred in F2"C (112) (by > 73%) with PN ≈ 36° and Ψm(N) ≈ 33° - 37°. The r.m.s. error is ≤ 1.7 Hz for analyses based on all coupling 3 constants with 3.2 Hz as the largest individual error in JHF. (iii) In difluoronucleosides 84 - 87,

C4'-exo to O4'-endo-C4'-exo pseudorotamers (61° < P < 76°, Ψm ≈ 35° - 46°) are favoured by 78 - 94 %. The r.m.s. of all calculations is below 1.5 - 2.0 Hz, while individual errors are at most 3.4 Hz 3 for JHF data. In conclusion, this is the first report of an accurate Karplus-type equation for the 3 interpretation of JHF coupling constants in terms of torsion angles that can be successfully used to

71 define the solution conformational characteristics and relative populations of conformers engaged in an equilibrium either in nucleosides, nucleotides or organic compounds in general.

4. The quantitation of the stereoelectronic effects in nucleos(t)ides

A study of our first experimental thermodynamic estimates14 of pseudorotations, as early as 1987, on 9-(2'-deoxy-β-D-threo-ribofuranosyl)-adenine and 9-(3'-deoxy-β-D-threo-ribofuranosyl)- adenine and their comparison with the natural 2'-deoxyadenosine and 3'-deoxyadenosine revealed that the net result of the gauche effect and the anomeric effect is of major importance in determining the overall furanose conformation in nucleosides. This has subsequently led us to examine the interplay of gauche and anomeric effects determining overall sugar conformation in nucleosides, in general, more in details. The strength of the gauche and anomeric effects operating in 12 - 83 is reflected in the value of ∆H° contribution to ∆G° of their N S equilibrium (N E in 12 and 1420). Our ∆H° estimates correspond to the overall contribution (i.e. stereoelectronic and counteracting steric effect) of each molecular fragment. -T∆S° of the N S equilibrium in 12 - 83 shows the difference in the entropy between both pseudorotamers at the temperature T. The effect of a possible change in the solvation and/or bulk of the nucleobases or other substituents at C2'-C5' in 17 - 83 at different pDs (and/or owing to different configuration at C1') on the modulation of ∆H° and/or -T∆S° of their N S equilibria in either of their P or D states compared with the N state cannot be assessed in a straightforward quantitative manner.

4.1 Quantitation of the anomeric and gauche effects by regression analyses To quantitate the gauche and anomeric effects that drive the sugar conformation in 12 - 73, we have initially correlated their structural features with the experimental values of their two-state N S equilibria via the regression analyses (A) - (E) (using the program SYSTAT473 (Table 5)).

4.1.1 Stereoelectronic effects in neutral β-D-Ns

The 26 ΔH°N values of the N E equilibrium (in 12 and 14) or N S equilibrium in 13, 15 and 16, β-D-ddNs 30 - 35, β-D-dNs 37 - 39 and 41 - 45, β-D-rNs 50 - 55 and β-D-3'-dA (63) have been dissected into following components: (i) The (steric + gauche) effect of 5'CH2OH; (ii) The effect of 5'CH2OMe compared with 5'CH2OH; (iii) The [HO2'-C2'-C1'-O4'] gauche effect; (iv) and (v) The [O2'-C2'-C1'-N9(purine)] and [O2'-C2'-C1'-N1(pyrimidine)] gauche effects for purine and pyrimidine ribonucleosides, respectively; (vi) The [HO3'-C3'-C4'-O4'] gauche effect; (vii) The [MeO3'-C3'-C4'-O4'] gauche effect and (viii) - (xiii) the overall (steric + stereoelectronic) effects of adenin-9-yl, guanin-9-yl, cytosin-1-yl, thymin-1-yl, uracil-1-yl, 5-fluorouracil-1-yl. The ΔH°N values of β-D-ddI (36) and β-D-dImb (40) were not used, since no other nucleoside has hypoxanthin-9-yl or imidazol-1-yl as nucleobase. The Pearson's correlation coefficient of regression analysis (A), performed using these 26 ΔH°N values, is 0.993 and the standard error of estimates 0.6 kJmol-1. The largest individual errors between experimental and back-calculated [using estimates -1 -1 from regression (A)] ΔH°N values was found for 12 (-1.2 kJmol ) and β-D-ddA (30) (1.0 kJmol ).

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 72 Table 5. Estimationa of the strengths of the anomeric and gauche effects driving the sugar conformation in 12 - 73 from regression analysis with the SYSTAT program473

Regression model (A) (B) (C) (D) (E) SE 5'CH2OH 1.6 (0.4) 1.6 (0.3) 0.1 (0.3) 1.6 (0.3) 1.7 (0.3) SE 5'CH2OMe 0.7 (0.4) 0.7 (0.4) 0.5 (0.4) 0.9 (0.6) 0.6 (0.4) AE (A) 0.9 (0.4) 0.9 (0.3) -1.7 (0.3) 1.2 (0.3) 1.0 (0.3) AE (G) 2.0 (0.5) 2.0 (0.4) -0.6 (0.4) 1.5 (0.4) 2.0 (0.4) AE (C) 4.4 (0.5) 4.3 (0.4) -3.2 (0.3) 3.7 (0.4) 3.9 (0.4) AE (T) 3.5 (0.5) 3.5 (0.4) -0.6 (0.3) 2.1 (0.4) 3.6 (0.4) AE (U) 4.1 (0.5) 4.1 (0.4) - 3.5 (0.6) 4.0 (0.4) AE (5-FU) 4.1 (0.6) 4.1 (0.5) - 3.2 (0.8) 4.0 (0.5) GE[HO3'-C3'-C4'-O4'] -6.3 (0.3) -6.4 (0.2) -3.9 (0.2) -5.4 (0.4) -6.3 (0.2) GE[MeO3'-C3'-C4'-O4'] -6.7 (0.5) -6.7 (0.4) -4.7 (0.4) -6.9 (0.6) -6.7 (0.4) -1/-2 GE[ RO3PO3'-C3'-C4'-O4'] - - - - -8.3 (0.3) GE[O2'-C2'-C1'-O4']] 5.1 (0.7) 5.1 (0.6) - 4.2 (1.0) 5.1 (0.6) GE[O2'-C2'-C1'-N9(pur.)] -5.9 (0.8) -5.9 (0.7) - -5.6 (1.1) -6.0 (0.7) GE[O2'-C2'-C1'-N1(pyr.)] -2.5 (0.8) -2.4 (0.7) - -1.6 (1.1) -2.3 (0.7) Multiple R 0.993 0.993 0.992 0.974 0.992 σ estimates 0.6 0.5 0.4 1.0 0.5 a See the text for the assumptions used to build each regression model. All estimates are given in kJmol-1 (their standard deviations are indicated in parentheses). SE 5'CH2OH designates the overall (steric + gauche) effect of 5'CH2OH substituent, whereas SE 5'CH2OMe represents the additonal stabilization of N-type conformations by 5'CH2OMe in comparison with 5'CH2OH. AE (A), AE (G), AE (C), AE (T), AE (U) and AE (5-FU) give the overall strength of the (steric + stereoelectronic) effect of adenin-9-yl, guanin-9-yl, cytosin-1-yl, thymin-1-yl, uracil-1-yl and 5-fluoro-uracil-1- yl in 17 - 73. GE[HO3'-C3'-C4'-O4'], GE[MeO3'-C3'-C4'-O4'] and GE[O2'-C2'-C1'-O4'] are estimates of the strength of -1/-2 the [HO3'-C3'-C4'-O4'], [MeO3'-C3'-C4'-O4'] and [O2'-C2'-C1'-O4'] gauche effects. GE[ RO3PO3'-C3'C4'-O4'] in regression analysis (E) represents the magnitude of the [RO3PO3'-C3'C4'-O4'] gauche effect in β-D-dNMPs 64 - 68 (R = H, charge = -1 and -2 in 1:1 ratio, Section 7) and β-D-dNMPEt (R = Et, charge is -1) 69 - 73, which has been assumed to be the same for each nucleobase and independent of the nature of R. GE[O2'-C2'-C1'-N9(pur.)] and GE[O2'-C2'-C1'- N1(pyr.)] represent the strength of the [O2'-C2'-C1'-N9(pur.)] and [O2'-C2'-C1'-N1(pyr.)] gauche effects in purine and pyrimidine ribonucleos(t)ides, respectively. The multiple R is the Pearson's correlation coefficient. "σ estimates" corresponds to the average standard deviation of all the estimated stereoelectronic effects for each regression analysis.

4.1.2 Effect of the 5'CH2OH versus 5'CH2OMe According to regression analysis (A), the steric and [O5'-C5'-C4'-O4'] gauche effects of 5'CH OH drive the conformation of abasic sugars and nucleosides toward the N [∆H° = 2 (5'CH2OH) -1 1.6 kJmol ]. Substitution of 5'CH2OH in 15, 31 and 38 for 5'CH2OMe in 16, 32 and 39 slightly reinforces the drive of sugar conformation toward N-type forms [i.e. ∆H° > (5'CH2OMe) ∆H° ]. The stronger influence of 5'CH OMe than 5'CH OH might be attributed to the (5'CH2OH) 2 2 increased steric bulk of OMe compared with OH. ∆H° is stronger (by 1.2 kJmol-1) than (5'CH2OH) the experimental ΔH°N value of 12, where only 5'CH2OH controls the sugar conformation. This might suggest that the effect of 5'CH2OH is stronger in β-D-nucleosides than in 12, owing to the proximity of the nucleobase in the former. However, the contribution of 5'CH2OH to the drive of Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected]

73 the sugar conformation in β-D-nucleosides is much weaker than that of all other stereoelectronic effects.

4.1.3 Effect of the nucleobase The nucleobase promoted drive of the N S equilibrium in nucleos(t)ides is the result of two counteracting contributions: stereoelectronic interactions within the O4'-C1'-N1/9 fragment, i.e. ∗ nO4' → σ C1'-N1/N9 interactions and/or electrostatic repulsions between the dipole of the furanose ring and the C1'-N1/9 dipole, and the inherent steric effect of the nucleobase (Section 2.7). Stereoelectronic interactions are expected to drive the sugar conformation toward N-type pseudorotamers, in which the nucleobase is pseudoaxial, whereas the steric effect stabilizes S-type forms in which the nucleobase adopts a pseudoequatorial orientation (Section 2.7). The fact that ΔH° estimates resulting from regression analysis (A) for the overall effects of the nucleobases in β- D-ddNs, β-D-dNs, β-D-rNs and 63 have been found positive shows that the C4'-O4'-C1'-N1/9 fragments adopt preferentially a gauche over trans orientation owing to predominating stereoelectronic over steric interactions, therefore an anomeric effect is operating. The magnitude of the overall effect of the nucleobase is dictated by its electronic and steric nature, which are as follows: adenin-9-yl (0.9 kJmol-1) < guanin-9-yl (2.0 kJmol-1) < thymin-1-yl (3.5 kJmol-1) < uracil- 1-yl (4.1 kJmol-1) ≈ 5-fluoro-uracil-1-yl (4.1 kJmol-1) < cytosin-1-yl (4.4 kJmol-1). The effects of thymin-1-yl, uracil-1-yl, 5-fluorouracil-1-yl and cytosin-1-yl are stronger than those of adenin-9-yl and guanin-9-yl because in the former, N1, part of the electron-deficient pyrimidine ring, is more ∗ predisposed to accept electrons through nO4'→σ C1'-N orbital interactions than N9 in the latter which is part of the electron-rich imidazole ring. Similar correlations between the strength of the anomeric effect involving a substituent X at C2 in heterocyclic six-membered rings and the electronegativity of X have already been established (Section 1.5). However, it seems that 5- fluorouracil-1-yl behaves as an exception, since its overall effect is the same as that of uracil-1-yl, although one would expect fluorine to reinforce the electron-withdrawing character of its glycosyl ∗ nitrogen N1 and therefore its nO4' →σ C1'-N1 stereoelectronic interactions.

4.1.4 Gauche effects The conformation of the pentofuranose moiety in β-D-dNs is driven toward the S owing to the fact that the [HO3'-C3'-C4'-O4'] (or [MeO3'-C3'-C4'-O4'] in 38 and 39) gauche effect prevails over the counteracting overall effect of their nucleobases, as evident from the comparison of their respective ∆H° estimates. The magnitudes of the [HO3'-C3'-C4'-O4'] and [MeO3'-C3'-C4'-O4'] gauche effects is almost the same, which is consistent with the comparable electronegativities of 3'- OH and 3'-OMe26. In β-D-rNs compared with β-D-dNs, two additional gauche effects operate within [HO2'-C2'-C1'-O4'] and [HO2'-C2'-C1'-N1/9] fragments. Our regression analysis shows that ∆H° of the [HO3'-C3'-C4'-O4'] and [HO2'-C2'-C1'-O4'] gauche effects in β-D-rNs almost cancel each other, which is consistent with the fact that the experimental ΔH°N values of the two-state N S equilibria in abasic sugars 12 and 14 are identical. Therefore, the actual preference (as reflected in their respective experimental ΔH°N values) of β-D-rNs for N- or S-type conformations can be attributed to the fine balance of the overall effect of their nucleobases and of the [HO2'-C2'-C1'- N1/9] gauche effect. In purine nucleosides β-D-A and β-D-G, N9 is part of the π-electron-rich Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 74 imidazole ring, and its ability to participate to a gauche interaction with O2' is much greater than that of N1 in the π-electron deficient pyrimidine counterparts 52 - 55. This is reflected in the stronger [HO2'-C2'-C1'-N9(purine)] gauche effect compared to the [HO2'-C2'-C1'-N1(pyrimidine)] gauche effect. In sharp contrast, the ability of the purine nucleobase in β-D-A and β-D-G to shift the pseudorotational equilibrium of the constituent pentofuranose sugar toward N-type conformations is clearly reduced with respect to that of the pyrimidine ring in 52 - 55. Consequently, ΔH°N values of the N S equilibria in purine and pyrimidine β-D-rNs reflect the stabilization of S- and N-type pseudorotamers, respectively. In β-D-3'-dA, no [HO3'-C3'-C4'-O4'] gauche effect is operating and the pseudorotational equilibrium is driven toward N-type forms owing to the cooperativity of the effects of the nucleobase, the 5'CH2OH substituent and of the [HO2'-C2'-C1'-O4'] gauche effect, which override the counteracting [O2'-C2'-C1'-N9(purine)] gauche effect.

4.1.5 Energetic equivalence of mirror-image β-D-dNs and β-L-dNs The mirror image relationship of the natural DNA and of the unnatural β-L-counterpart 474- 476 has been proven by the identical X-ray crystal structures of D- and L-d(CGCGCG) hexamers474,477. The oligomerization of activated guanosine mononucleotides in the presence of poly(C) template is easily achieved if both the template and the monomers are of the same handedness, whereas it is much less efficient with the racemic D/L mixture of the monomer, since monomers of opposite handedness act as chain terminators due to their incorporation at the 2'(3') end of the oligomer478. The comparison of the thermodynamics of the two-state N S equilibria in β-D-dNs 37 and 41 - 43 and in their β-L-dNs counterparts 46 - 49 (either with neutral, fully protonated or deprotonated nucleobases) shows that D- and L-nucleosides cannot be energetically distinguished within the timeframe and accuracy of our method based on pseudorotational analyses 3 of JHH, since they exhibit virtually identical pD-dependent conformational preferences (Table 2). -1 The largest individual difference (0.8 kJmol ) is found between ΔH°N values of β-D-dG (41) and β-L-dG (47) with fully protonated guanin-9-yl and it is within the accuracy (σ = 0.8 kJmol-1) of the experimental ΔH°N value of β-L-dG. This suggests that the magnitudes of the stereoelectronic anomeric and gauche effects that drive the sugar conformation in D- and L-nucleosides are identical. In order to improve the statistical significance of the estimates derived from the regression analysis (A), we have performed a second multilinear regression analysis [regression (B)] basing on an extended dataset consisting of 30 experimental ΔH°N values (i.e. 26 from the dataset used for regression (A) as well as the 4 ΔH°N values for β-L-dNs 46 - 49). The estimates for the gauche and anomeric effects from regression (B) are virtually identical (within ± 0.1 kJmol-1) to those derived from regression (A) (Table 5, col. 2). The Pearson correlation coefficients of regressions (B) and (A) are nearly identical (0.993 versus 0.994). The standard error of estimates determined from regression (B) is 0.5 kJmol-1 [as compared with 0.6 kJmol-1 for regression (A)]. The largest error between experimental and theoretical ΔH°N values [i.e. calculated using the estimates from regression (B) or (A)] for nucleosides is found for β-D-ddA (1.0 kJmol-1). For all β-L-dNs [regression (B)], the error is within 0.3 kJmol-1.

4.1.6 3'-phosphate has stronger gauche effect than 3'-hydroxy Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected]

75 The magnitudes of the gauche and anomeric effects operating in all β-D/L-nucleosides in β-D- dNMPs 64 - 68 and β-D-dNMPEts 69 - 73 (Table 5) have been derived from the regression analysis

(E), which was performed using 40 experimental ΔH°N values assuming identical strengths for the -1/-2 -1 [ HO3PO3'-C3'-C4'-O4'] and [ EtO3PO3'-C3'-C4'-O4'] gauche effects in 64 - 68 and 69 - 73. The estimates resulting from regression analysis (E), its correlation coefficient and the standard error on the estimates are virtually the same as of regression (B) (Table 5). The average [3'- phosphate-C3'-C4'-O4'] gauche effect is about -8.3 kJmol-1, i.e. -2.0 kJmol-1 stronger than the [HO3'-C3'-C4'-O4'] counterpart.

4.1.7 Stereoelectronic effects in neutral α-D-N In order to quantitate the anomeric and gauche effects driving the conformation of α- nucleosides in the N state and compare them with those obtained for β-nucleosides [regression

(B)]), we have performed the regression analysis (C) using 17 ΔH°N values for abasic sugars 12, 13,

15 and 16, α-D-ddNs 17 - 20, α-D-dNs 21 - 26 and α-L-dNs 27 - 29. These ΔH°N values have been correlated with the magnitudes of: (i) the effect of 5'CH2OH; (ii) the additional influence of 5'CH2OMe compared with 5'CH2OH; (iii) the [HO3'-C3'-C4'-O4'] gauche effect, (iv) the [MeO3'- C3'-C4'-O4'] gauche effect and the overall effects of (v) adenin-9-yl, (vi) guanin-9-yl, (vii) cytosin- 1-yl and (viii) thymin-1-yl. Since, as in the β-anomers, the ∆H° values of the two-state N S equilibrium in each of the α-D-/-L- pairs are identical [within the accuracy of the individual -1 estimates, i.e. with 1.0 kJmol as largest deviation for ΔH°N of α-D-dA (21) versus α-L-dA (27)], we have considered that the anomeric and gauche effects operate with an identical strength in both α-D- and -L-series. The correlation coefficient of the regression analysis (C) is 0.992 [0.993 for regression (B)] and the standard error of the estimates is 0.4 kJmol-1 (0.5 kJmol-1 for regression (B)]. As a result, the residual errors between theoretical ∆H° values for 12, 13 and 15 - 29 [calculated using the estimates derived from the regression analysis (C)] and the corresponding experimental ΔH°N values are well within the accuracy of the later.

4.1.8 Weakening of the effects of 5'CH2OH and 5'CH2OMe in α-nucleosides The comparison of the data in cols. 3 and 4 for regressions (B) (β-nucleosides) and (C) (α- nucleosides) in Table 5 suggests that in α-nucleosides, the contribution of the effects of 5'CH2OH and 5'CH2OMe to the drive of the two-state N S equilibrium is reduced in comparison with the β- counterparts. This is possibly due to the fact that in the former the 5'-substituent and the nucleobase are on opposite faces of the pentofuranose ring whereas they are both on the β-face in the later.

4.1.9 Weakening of the effect of the nucleobase in α-nucleosides The data in Table 5 indicate that the magnitude of the overall effect of the nucleobase is weaker in α- compared with β-nucleosides by 1.2 kJmol-1 for cytosin-1-yl, 1.4 kJmol-1 for guanin- 9-yl and 2.9 kJmol-1 for thymin-1-yl. Only the overall effect of adenin-9-yl has a comparable magnitude in the α- and β-series (0.9 kJmol-1 in the β-anomers versus slightly stronger -1.7 kJmol-1 in the α-counterparts). The weakening of the anomeric effect in α-nucleosides implies that the configuration of the nucleobase at C1' presumably affects the ability of one of the lonepairs of the

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 76 O4' oxygen to be engaged in molecular orbital interactions with the σ∗ antibonding orbital. C1'-N1/N9 This may also suggest that the anomeric effect is the result of predominant molecular orbital interactions over electrostatic repulsions. One may possibly invoke a sp2 hybridization of the O4' 1 lonepairs orbitals, and an unfavourable orientation of the occupied lobe of the nsp2 (p-type) lonepair with respect to the σ∗ orbital in the α-series only (Fig 12). We have shown that in β- C1'-N1/9 nucleosides the pyrimidine nucleobases drive the two-state N S pseudorotational equilibrium toward N-type conformations more efficiently than the purine counterparts (Section 4.4). In the α- anomers, the ability of the nucleobase to stabilize S-type pseudorotamers is only slightly increased when the base is changed in the order guanin-9-yl, thymin-1-yl, adenin-9-yl, cytosin-1-yl. The fact that thymin-1-yl has an intermediate effect cannot be easily explained.

4.1.10 Weaker 3'-hydroxy gauche effect in α- compared with β-D-Ns The [HO3'-C3'-C4'-O4'] and [MeO3'-C3'-C4'-O4'] gauche effects drive the pseudorotational equilibrium of the pentofuranose moiety both in α- and β-nucleosides toward S-type conformations, however their magnitude is much reduced (by 2.4 and 2.0 kJmol-1, respectively) in the α-anomers compared with the β-counterparts (Table 5, cols 2 and 4). The fact that the nucleobase and the 3'- substituents do not influence the drive of the pseudorotational equilibrium in α- and β-nucleosides with the same efficiency is further evidenced by the results of the regression analysis (D), which has been performed using a dataset of 43 experimental ΔH°N values, including abasic sugars 12 - 16, β- D-ddNs 30 - 35, β-D-dNs (except 40), β-D-rNs, β-D-3'-dA (63) and β-L-dNs, α-D-ddNs 17 - 20, α- D-dNs 21 - 26 and α-L-dNs 27 - 29, basing upon the assumption that the magnitudes of the effects of all nucleobases as well as the gauche effects are independent of the configuration of the sugar moiety at C1' (for the effects of the nucleobase, we have only assigned the opposite sign in the α- series compared to the β-counterparts during the fitting procedure) (Table 5, col. 5). The multiple Pearson's correlation coefficient is 0.974 [0.993 for regression (B)]. The standard error of the estimates derived from regression (D) is twice higher (1.0 kJmol-1) than for those derived from regression (B) (0.5 kJmol-1). Whereas the estimates of the anomeric and gauche effects determined through the regression analyses (B) and (C) allow to reproduce the experimental ΔH°N values of β- D- and α-D-nucleosides, respectively, fairly accurately, some theoretical ∆H° values calculated on the basis of the estimates derived from regression (D) are much less accurate, and individual -1 residual errors between them and the corresponding experimental ΔH°N are -2.1 kJmol for α-D- ddA, 1.9 kJmol-1 for α-D-T, 1.7 kJmol-1 for α-L-T, 1.8 kJmol-1 for β-D-ddT, ±1.3 kJmol-1 for β-L- dA, β-D-dA and β-D-ddC.

4.1.11 Limitations of regression analysis to quantitate stereoelectronic effects For the regression analyses (A) - (E), the following approximations have been made: (i) The magnitude of the effect of each nucleobase has been assumed to be identical in β-D- ddNs, β-D-dNs and β-D-rNs (as well as in 63 for adenin-9-yl). This means that any potential modulation of the electron-withdrawing character of the glycosyl nitrogen and electron-donating ability of O4' by the presence of 2'- and/or 3'-OH(Me) substituents in β-D-dNs and β-D-rNs in

77 comparison with β-D-ddNs counterparts has been neglected. In the case of the glycosyl nitrogen, this hypothesis is supported by the fact that the change of the pKa value of the nucleobase in β-D- ddNs compared with the corresponding β-D-dNs and β-D-rNs is rather small (≤ 0.4 pD unit), as discussed in Section 4.8. (ii) It has also been assumed that 5'CH2OH and 5'CH2OMe exert the same (steric + gauche) effect upon the conformation of the sugar moiety in abasic sugars 12 - 16 and in all nucleosides, independently of their configuration at C1'. Any possible interaction (H-bonding or steric) between the nucleobase and 5'CH2OH or 5'CH2OMe (section 4.8) in β-D-nucleosides has been neglected.

N-type sugar S-type sugar Figure 12: Possible origin for the (A) weaker O4'-C1'-N1/9 anomeric effect N HOH2C σ* N HOH2C C1'-N9 O O in α-D-ddNs 17 - 20 (for the orbital N9 N N9 1 n 2 NH2 overlap in β-nucleosides, see Fig. 9, sp (p-type) N N N sections 2.8 & 4). Our interpretation

H NH2 H1' 1' is based on the fact that the anomeric 2 (B) nsp2 (s-type) 2 nsp2 (s-type) effect results from predominant nO4' O4' O4' C2' N9 σ* orbital mixing116 over C4' C1'-N9 β = 42ο C4' 1 1 1 C2' N n 2 nsp2 (p-type) ο 9 sp (p-type) β1 = 6 weaker electrostatic repulsions

2 (section 4.8). The O4' lonepairs (C) nsp3 2 N n 3 HOH2C sp HOH C σ*C1'-N9 N orbitals are represented using either 2 O O N N 2 1 9 N the sp [i.e. higher energy n 2 NH 9 sp (p- 1 2 1 n 3 N nsp3 sp N lonepair with predominant p- type) N

NH2 type character and lower energy H1' H (D) β = -108ο 1' 2 2 2 2 nsp3 n lonepair with nsp3 ο sp2(s-type) β2 = -144 3 O4' 1 O4' C2' predominant s-type character] or sp N9 nsp3 1 C ' C ' 1 2 n 3 4 4 [i.e. n 3 and n 3 lonepairs with sp C2' sp sp N9 the same energy] hybridization models123-126,129,130 (section 1.9). (A) & (B) Relative orientations of sp2-hybridized O4' lonepairs with respect to the

C1'-N9 bond in N- (P = 0°, Ψm = 40°) and S-type (P = 160°, Ψm = 40°) sugars of α-D-ddA. β1/2 were calculated 1 assuming a perfect trigonal symetry. Neither in N- (β1 ≈ 42°) nor in S-type (β1 ≈ 6°) sugars is the nsp2 (p-type) orbital antiperiplanar with the C1'-N9 bond, therefore the anomeric effect is at most weak (in favour of N-type forms!), and clearly much weaker than in the β-counterparts (i.e. β1 ≈ 162°, Panel (C) in Fig. 9). This model is consistent with our experimentally observed weaker anomeric effect in α-D-ddNs than in β-D-ddNs (Table 5). (C) & (D) Relative 3 2 orientations of sp -hybridized O4' lonepairs with respect to C1'-N9 bond in the same N- and S-type sugars. The nsp3 lonepair and C1'-N9 bond assume an antiperiplanar orientation (β1 ≈ -144°) in the S-type form, but they are perpendicular in N-type form. Therefore, if O4' lonepairs were sp3 hybridized, one would expect a stabilization of the S- over N-type form to a similar extent as the stabilization of N-type sugars in β-nucleosides, which is not consistent with our thermodynamic data for α- versus β-anomers.

(iii) The [HO3'-C3'-C4'-O4'] gauche effect has been attributed the same strength in β-D-dNs, β-D-rNs and abasic sugars 13 and 14. We have also assumed that the [HO2'-C2'-C1'-O4'] gauche effect has an equal strength in β-D-rNs, β-D-3'-dA (63) and abasic sugar 14, and that the [MeO3'-

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 78 C3'-C4'-O4'] gauche effect exerts the same influence upon the pentofuranose conformation in abasic sugars 15, 16 and adenosine derivatives 22, 23, 38 and 39. The implications of these assumptions are two-fold: (a) We have not taken into consideration any possible mutual compensation of different gauche effects in β-D-dNs, β-D-rNs and abasic sugars 13 - 16 in comparison with β-D-ddNs and 12, respectively. (b) The magnitude of the above gauche effects has been considered independent of the nature of the nature of the C1' substituent (either a hydrogen in abasic sugars 12 - 16 or a nucleobase in nucleosides).

4.2 Quantitation of the anomeric and gauche effects by pairwise comparisons Owing to the above simplifying approximations, the estimates obtained from our regression analyses can only be used for semi-quantitative purposes. In order to refine these estimates for a specific class of compounds (or even a single compound), we have also performed a set of pairwise comparisons between ∆H° of compounds that differ only in one structural feature, in order to assess the stereoelectronic contribution of that particular feature to the drive of the two-state N S pseudorotational equilibrium of the pentofuranose moiety in the compound(s) of interest. The strategy adopted to perform these pairwise comparisons is depicted in Fig 13, and the resulting estimates for the anomeric and gauche effects are compiled in Tables 6 - 8. In the following sections, all discussions are based upon the results of our pairwise comparisons.

4.3 Strengths of ∆H° and -T∆S° to ∆G°of pseudorotation of neutralnucleosides The rather limited experimental dataset of Altona et al.316,203 showed that the populations of N- and S-type pseudorotamers of β-D-dA and of other β -D-dNs in the N state remain virtually unchanged as the temperature increases from 291 K (75 % S) to 333 K (73 % S). They interpreted this result by assuming that the stabilization of S-type pseudorotamers in. these compounds does not have an enthalpic origin, but is instead the result of the larger entropy of the S-compared to the N- type pseudorotamers The comparison of ∆H° and -T∆S° contributions to ∆G° of the N S equilibrium in neutral 3 β-D-dNs and β-L-dNs, derived from our pseudorotational analyses of JHH over a wider temperature range (278 - 358 K) than that used by Altona et al., leads us to conclude that it is the enthalpy factor, not the entropy, that is mainly responsible for the overall conformational preference of sugar moieties in β-D-nucleosides. This conclusion is supported by the following observations: (i) For β-D/L-dA, 3'-OMe-β-D-dA, 3',5'-diOMe-β-D-dA, β-D-dImb, β-D/L-dG and β-D/L- T, ∆H° overrides -T∆S°, resulting in the overall stabilization of S-type sugars at 298 K (i.e. negative ∆G° value). (ii) For β-D/L-dC, β-D-dU and 5-F-β-D-dU, both ∆H° and -T∆S° contributions stabilize S- type conformations and have nearly the same magnitude. Therefore, in the N state of all β-D/L-dNs, -T∆S° is never found stronger than ∆H°.

79 OH OH o OH C-BP/N/D OH OMe ΔΔH 18 O o O O O o O ΔΔH 17 ΔΔH 1d

P/N P/N BP/N/D BP/N/D B B 17 - 20 HO HO OH MeO 22 MeO 23 21 & 24 - 26 56 - 62 o o o o o ΔΔH 30 ΔΔH ΔΔH ΔΔH 13 ΔΔH 14 o 15 16 ΔΔH 9 OH OH OH OH OMe O o O o O O o O ΔΔH 7 ΔΔH 8 ΔΔH 1a

12 HO 13 HO14 OH MeO 15MeO 16 o o o ΔΔH 4 o o ΔΔH 2 ΔΔH 3 o ΔΔH 5 ΔΔH 6 ΔΔH 12 P/N/D P/N/D P/N/D OMe B OH B OH BP/N/D OH B OH BP/N OMe BP/ O o O o o O o ΔH 1c ΔΔH 10 O ΔΔH 11 O ΔΔH 1b O

32 30, 31 & 33 - 35 HO HO OH MeO 37, 41 - 45 50 - 55 38 MeO 39 o o ΔΔH o ΔΔH 26 20 ΔΔH 23 OH BP/N/D OH BP/N/D OH BP/N/D OH BP/N/D OH BP/N/D O o O o O o O O ΔΔH 24 ΔΔH 27 ΔΔH 28

-1/-2 -1 -1 -1/-2HO PO OH HO3PO EtO3PO EtO3PO OH o 3 OH 64 - 68 69 - 7379 - 83 ΔΔH 29 74 - 78 63

Figure 13. Estimates for the anomeric and gauche effects in 12 - 83 from pairwise comparisons

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 80 Table 6. Estimates (∆∆H°) for the magnitudes of the effects of the nucleobases and various gauche effects that drive the sugar conformation in compounds 12 - 16 and in α- and β-N-nucleosides 17 - 63 from pairwise comparisonsa

Nucleobase A G Im C T U 5-F-U State P N P N D P N P N N D N D N D ∆∆H°1b 0.4 1.0 ------∆∆H°1c - - -0.2 0.9 0.9 ------∆∆H°1d 0.5 0.6 ------b ∆∆H°2 8.8 3.1 23.2 3.0 1.0 - - 9.2 6.2 5.0 3.1 5.3 - - - ∆∆H°3 3.4 0.2 6.2 1.3 -0.8 4.2 1.9 4.1 3.4 2.7 2.2 3.5 2.8 3.3 3.0 ∆∆H°4 -0.6 -4.8 5.0 -3.7 -8.0 - - 4.8 1.9 0.9 -0.6 1.6 -0.1 1.9 0.4 ∆∆H°5 3.2 0.0 ------∆∆H°6 3.2 0.6 ------∆∆H°10 -9.9 -7.4 -21.5 -6.2 -6.3 - - -9.6 -7.3 -6.8 -5.4 -6.3 - - - ∆∆H°11 0.5 -0.5 3.3 -0.5 -2.7 - - 5.2 3.0 2.7 1.7 2.6 1.6 3.1 1.9 ∆∆H°12 -0.7 -0.7 ------∆∆H°13 -2.1 -2.1 -9.1 -0.8 -0.8 - - -3.3 -3.3 -1.5 -0.9 - - - - ∆∆H°14 -0.9 -0.9 -6.2 -0.4 0.7 - - -3.0 -3.0 0.1 0.1 - - - - ∆∆H°15 -1.2 -1.8 ------∆∆H°16 -1.1 -1.6 ------∆∆H°17 -3.3 -3.3 -2.0 -4.1 -3.0 - - -4.2 -4.2 -2.9 -3.5 - - - - ∆∆H°18 -0.8 -1.4 ------∆∆H°19 -7.1 -5.7 ------a A, G, Im, C, T, U and 5-F-U represent adenin-9-yl, guanin-9-yl, imidazol-1-yl, cytosin-1-yl, thymin-1-yl, uracil-1-yl and 5-fluoro-uracil-1-yl, respectively. P, N and D denote -1 the protonated, neutral and deprotonated states of 17 - 63, respectively. All estimates (kJmol ) were calculated by subtracting ΔH° , ΔH°N or ΔH°D values (Table 1) of two -1 -1 -1 -1 compounds among 12 - 63, according to the strategy depicted in Fig 13. ∆∆H°1a (0.4 kJmol ), ∆∆H°7 (-4.5 kJmol ), ∆∆H°8 (4.5 kJmol ) and ∆∆H°9 (-0.5 kJmol ) are b -1 constant factors calculated by subtracting ΔH°N of abasic sugars 12 - 16. ∆∆H°2 was also calculated by subtracting ΔH°N of 12 from ΔH°N of β-D-ddI (36) (5.8 kJmol ).

(iii) In the N state of β-D-rT, β-D-U and 5-F-β-D-rU, opposing ∆H° (driving to the N) and - T∆S° nearly cancel each other, resulting in nearly unbiased equilibrium. However, in the N states of β-D-G and β-D-A, ∆H° (stabilizing S-type sugars) prevails over the opposing -T∆S°, and S-type sugars are favoured at 298 K. In the N state of β-D-C, ∆H° also prevails over counteracting -T∆S°, but it stabilizes N-type sugars at 298 K.

Table 7. Quantitation of various gauche effects (∆∆H°) driving the sugar conformation in mononucleotides 64 - 83 from pairwise comparisonsa

Nucleobase adenin-9-yl guanin-9-yl cystosin-1-yl thymin-1-yl uracil-1-yl ∆∆H°20 -8.9 -7.5 -9.8 -8.0 -8.4 ∆∆H°21 -9.0 -8.2 -10.2 -8.4 -8.4 ∆∆H°22 -1.5 -1.3 -2.5 -1.2 -2.1 ∆∆H°23 -1.6 -2.0 -2.9 -1.6 -2.1 ∆∆H°24 -0.1 -0.7 -0.4 -0.4 0.0 ∆∆H°25 -0.5 -1.2 -1.5 -2.2 -0.8 ∆∆H°26 -2.5 -2.5 -3.8 -3.8 -3.6 ∆∆H°27 -1.4 -1.0 2.1 0.5 1.1 ∆∆H°28 -2.0 -1.3 -2.3 -1.6 -2.8 ∆∆H°29 0.5 -0.4 4.0 1.7 3.9 a -1 All estimates (kJmol ) have been calculated by subtracting values ΔH°N (Table 2 & 3) of two compounds among β-D- ddNs 30 , 31 and 33 - 35, β-D-dNs 37 and 41 - 44, β-D-rNs 50 - 54 and mononucleotides 64 - 83 according to the nomenclature shown in Fig 13.

Table 8. Quantitation of the enthalpy (∆∆H°30) of the (steric + stereoelectronic) effect of the C-nucleobase, in each of its protonated (P), neutral (N) and deprotonated (D) states, driving the sugar conformation in C-nucleosides 64 - 83a

Compound P N D Formycin B (56) -0.9 -8.5 -9.2 Formycin A (57) -2.8 -8.5 -8.5 9-deaza-A (58) -7.8 -14.6 - Ψ-isoC (59) 3.8 -2.3 -8.3 Ψ-U (60) - 0.3 -4.9 1-Me-Ψ-U (61) - 1.2 -3.4 1,3-diMe-Ψ-U (62) - 1.6 -

a -1 -1 ∆∆H°30 (kJmol ) has been calculated by subtracting ΔH°N (0.4 kJmol , Table 2) of abasic sugar 14 from those of a particular C-nucleoside among 56 - 62.

(iv) The situation is far less complicated in neutral β-D-ddNs 30 - 36, since for all of them, ∆H° (driving to N) always outweighs the counteracting -T∆S° contribution, which results in a high preference for N-type pseudorotamers at room temperature. For abasic sugars 13, 15 and 16, the two-state N S pseudorotational equilibrium is driven toward S-type conformations mainly by

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 82 ∆H°, whereas for 12 ∆H° and -T∆S° contributions cancel each other. In contrast, in 14, the predominance of N-type conformations results from the cooperative drive of the prevailing - T∆S° contribution and the minor ∆H° term.

4.4 Nucleobase-dependent anomeric effects in neutral nucleosides

4.4.1 Effect of the 5'CH2OH versus 5'CH2OMe The strengthening of the overall effect of 5'CH2OMe moiety in abasic sugar 16 in comparison with 5'CH2OH in 15 is evident from the subtraction of the ΔH°N value of 15 from that -1 of 16 (i.e. ∆∆H°1a = 0.4 kJmol , Fig 13 and Table 6). The comparison of the effect of 5'CH2OMe in nucleosides 39 and 32 with the influence of 5'CH2OH in their counterparts, 38 and 31 supports the results of regression analysis (A) regarding the slightly stronger ∆H° with respect to (5'CH2OMe) ∆H° since ∆∆H° (1.0 kJmol-1) and ∆∆H° (0.9 kJmol-1) in neutral nucleosides are (5'CH2OMe) 1b 1c indeed slightly larger than ∆∆H°1a for abasic sugars.

4.4.2 The effect of the nucleobase is sugar-dependent In β-D-ddNs 30, 31 and 33 - 36, an estimate for the overall effect of the constituent nucleobases can be easily obtained by subtracting (∆∆H°2) the contribution of the 5'CH2OH effect

(i.e. reflected in the experimental ΔH°N value of 12) from experimental ΔH°N values of 30, 31 and 33 - 36 (Table 6). The comparison of ∆∆H°2 values shows that pyrimidines are able to shift the pseudorotational equilibrium in β-D-ddNs toward N-type conformations more efficiently than purines, as already suggested by the results of the regression analysis (A), since ∆∆H°2 increases in the order: guanin-9-yl (3.0 kJmol-1) ≈ adenin-9-yl (3.1 kJmol-1) << thymin-1-yl (5.0 kJmol-1) ≈ uracil-1-yl (5.3 kJmol-1) < cytosine-1-yl (6.2 kJmol-1) . In β-D-dNs 37 - 45, beside the effect of the nucleobase, both the influence of 5'CH2OH and the gauche effect of [HO3'-C3'-C4'-O4'] fragment contribute to the drive of the sugar conformation. Therefore, in order to obtain an estimate for the overall effect of the nucleobase in this series, it is necessary to subtract (∆∆H°3 in Table 6) from their experimental ΔH°N values an estimate for the combined effects of the 5'CH2OH and 3'-OH gauche effect. Such an estimate has been found in the -1 experimental ΔH°N value of abasic sugar 13 (-4.1 kJmol ). The comparison of ∆∆H°3 values for the nucleobases in β-D-dNs shows that, as in the β-D-ddNs series, pyrimidines exert a stronger -1 influence than for purines, i.e. ∆∆H°3 values are in the order: adenin-9-yl (0.2 kJmol ) < guanin-9- yl (1.3 kJmol-1) < imidazol-1-yl (1.9 kJmol-1) < thymin-1-yl (2.7 kJmol-1) < 5-fluorouracil-1-yl (3.3 kJmol-1) ≈ cytosin-1-yl (3.4 kJmol-1) ≈ uracil-1-yl (3.5 kJmol-1). The fact that the effect of imidazolyl-1-yl is slightly stronger than that of adenin-9-yl and guanin-9-yl is consistent with its reduced steric bulk in comparison with adenin-9-yl and guanin-9-yl. The subtractions of ΔH°N of -1 abasic sugar 15 from that of 3'-OMe-β-D-dA (38) (i.e. ∆∆H°5 = 0.0 kJmol ), on one hand, and of -1 ΔH°N of 16 from that of 3',5'-diOMe-β-D-dA (39), on the other (i.e. ∆∆H°6 = 0.6 kJmol ) shows that the effect of adenin-9-yl is the same in 38, 39 and β-D-dA (37) (0.2 kJmol-1, see above) itself.

(A) N-type sugar S-type sugar

NH2 N N H3' σ σ*C4'O4' C3'H3' N9 N HOH2C N HOH2C σ*C1'-N N O O 9 NH OH 2 H3' σ* σ C4'O4' 1n 2 C3'H3' 1 sp (p-type) nsp2 (p-type) N OH N (B) ______E6 ______E σ* 5 C1'-N9 σ*C4'O4' __ ΔE 1 E4 n 2 ΔE1 ΔE sp (p-type) σC3'H3' ______2 E ______3 E2 ΔE(AE) ______E1 ΔE(GE)

The relative energies of orbitals (E1 - E6) are based on their relative donor- acceptor abilities, and they are constantly modulated by the nature of each substituent at the sugar as well as on the aglycone in free, ionic and complexe state

Since nO4' is a better donor than σC3'H3' and σ*C4'O4' is a better acceptor than σ*C1'N9, therefore ΔE(GE) > ΔE(AE)

Figure 14. The interplay of the O4'-C1'-N9 anomeric effect and the [HO3'-C3'-C4'-O4'] gauche effect in β-D-dNs drives the N S equilibrium (the 2'-gauche effect in rNs is also a participant in this stereoelectronic interplay, but it is not shown here in 1 ∗ order to retain clarity in the diagram). (A) nsp2 →σ C1'N1/9 orbital overlap favours N-type sugars (i.e. anomeric effect), ∗ whereas σC3'H3' →σ C4'O4' interactions (i.e. [HO3'-C3'-C4'-O4'] gauche effect) stabilize S-type conformations. The differences in the electronic characters of the substituent at C1', C2' and C3' however decide the donor-aceptor capabilities of the occupied and vaccant orbitals (i.e. the interplay of the gauche and anomeric effects) (B) The energy difference between ∗ 1 ∗ σC3'H3' and σ C4'O4' (∆E1) is smaller (hence a better mixing of the orbitals) than that between nsp2 and σ C1'N1/9 (∆E2), therefore the gauche effect is more efficient than the anomeric effect This however is dictated on a case-to-case basis by the nature of a set of substituents at the endocyclic carbons that are present on the sugar ring in a specific nucleoside. The reason why we do not take 5'CH2OH into consideration is because it is viryually a free rotor where steric and gauche effect have been found to have negligible contribution to the drive of the sugar conformation (vide supra).

For each nucleobase, the comparison of ∆∆H°2 with ∆∆H°3 suggests that in β-D-dNs the effect of the nucleobase is much weaker than in the β-D-ddNs counterparts. The weaker anomeric effect in dNs compared to ddNs is explained as follows: (i) The electron-withdrawing character of 3'-OH reduces the electron density around O4', making O4' lonepair less available for n O4' →σ∗ interactions in β-D-dNs compared with β-D-ddNs (Fig 14). If the nucleobase is C1'N1/9 substituted for a hydrogen atom, no anomeric effect is operating, therefore the electron density around O4' is maximal and as a result σ →σ∗ interactions are weaker (because of higher C3'H3' C4'O4' difference between their energy levels) in abasic sugar 13 compared to β-D-dNs. Similarly as 3'-OH in dNs is substituted for 3'-phosphate, the 3'-gauche effect is enhanced. This means that the strength of the 3'-gauche effect increases as an electron-withdrawing group is placed at either C1' or C3' (Section 4.5). (ii) Conversely, σ →σ∗ interactions in β-D-dNs may result in the lowering of C3'H3' C4'O4' Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 84 the energy level of the n orbital. Since the efficiency of n →σ∗ interactions is inversely O4' O4' C1'N1/9 proportional to the difference between their energies, the anomeric effect is weakened in the dNs compared to ddNs. This means that the modulations of the gauche effect by the anomeric effect, and of the anomeric effect by the gauche effect are mutually interrelated and interdependent. In β-D-rNs, the sugar conformation is driven by the interplay of the following stereoelectronic effects: (a) the effect of the nucleobase, (b) the effect of 5'CH2OH, (c) the gauche effect of [HO2'-C2'-C1'-O4'], (d) the gauche effect of [HO3'-C3'-C4'-O4'] and (e) the gauche effect of [HO2'-C2'-C1'-N1/9]. The [HO3'-C3'-C2'-O2'H] gauche effect is negligible, since in N- and S- type sugars, O3'-C3' is gauche with respect to O2'-C2'. In order to quantitate the effect of the nucleobase in β-D-rNs, we subtracted from their ΔH°N values the contributions from the effects detailed in (b) - (e). The combined influence of 5'CH2OH and of the [HO3'-C3'-C4'-O4'] and [HO2'- C2'-C1'-O4'] gauche effects can be accounted for by subtracting (∆∆H°4) ΔH°N of 14 from that of a β-D-rN. ∆∆H°4 values increase in the following order: adenin-9-yl < guanin-9-yl < thymin-1-yl < uracil-1-yl ≈ 5-fluorouracil-1-yl ≈ cytosin-1-yl. In order to obtain an estimate for the overall effect of the nucleobase in each β-D-rN, it was then necessary to subtract the strength of the [O2'-C2'-C1'- N1/9] gauche effect from ∆∆H°4. Estimates for the strength of the [O2'-C2'-C1'-N1/9] gauche effect -1 (∆H°GE[O2'-C2'-C1'-N1/9]) in neutral β-D-rNs (Section 4.6) are as follows: -7.9 kJmol in β-D-A, - 6.7 kJmol-1 in β-D-G, -4.3 kJmol-1 in β-D-C, -4.1 kJmol-1 in β-D-rT and -3.7 kJmol-1 in β-D-U. The subtraction of ∆H°GE[O2'-C2'-C1'-N1/9] from ∆∆H°4 gives the effect of each nucleobase (AErN) in β-D-rNs (kJmol-1): 3.0 in β-D-G ≈ 3.1 in β-D-A < 5.0 in β-D-rT ≈ 5.3 in β-D-rU < 6.2 in β-D-C. The comparison of ∆∆H°2 and AErN values in the neutral state shows that the effect of a nucleobase is the same in β-D-ddNs and β-D-rNs, owing to the cancellation of the [HO2'-C2'-C1'-O4'] and [HO3'-C3'-C4'-O4'] gauche effects in the latter. The fact that the overall effect of the nucleobase in β-D-nucleosides is characteristic of its electronic and steric nature has three important implications in understanding structure-activity of nucleic acids: (i) The change of the electronic character of purine or pyrimidine heterocycles upon their protonation or deprotonation is expected to result into the modulation of the anomeric effect, which in turn should be reflected in the shift of bias of the two-state N S equilibrium toward more N- type sugars (upon protonation owing to the enhanced anomeric effect) or S-type pseudorotamers (upon deprotonation due to the weaker anomeric effect). Qualitative studies on the preferred conformation of the sugar moiety in various adenosine, guanosine and cytidine nucleosides and nucleotides as a function of pD, as discussed in Section 2.9 are consistent with this simple reasoning. We, on the other hand, have quantitated for the first time the actual magnitude of the modulation of the anomeric effect in α- and β-nucleosides upon the change of the pD of the aqueous solution (Sections 4-6). (ii) Divalent metal cations are essential in natural processes involving nucleic acids, such as the replication, transcription and translation of the genetic code479. In the presence of Mg2+ cofactor, the EcoRV restriction endonuclease is able to cleave DNA at one particular recognition

85 site with high specificity, whereas when Mg2+ is replaced by Mn2+, both the activity and specificity of the enzyme are dramatically reduced. Numerous studies have been undertaken to establish the sites of coordination of metal ions in nucleosides and nucleotides as well as the relative stabilities of the complexes247,391,393,480-495. Whereas hard metal ions such as Mg2+ mainly bind to the phosphate oxygen moiety of nucleotides, there is also spectroscopic evidence (NMR and UV) that many transition metal ions such as Mn2+, Co2+, Ni2+, Cu2+ coordinate to both the phosphate oxygens and the nucleobase nitrogens of adenosine and guanosine nucleotides either directly or via a coordinated water molecule487. At the other end of the affinity scale, softer Pt in anticancer drug cis- Pt(NH3)2Cl2 binds to DNA, by preferential coordination at N7 of the constituent adenin-9-yl and 393 2+ guanin-9-yl nucleobases . A slow titration with HgClO4 has shown that the binding of Hg to N3 of thymin-1-yl in the AT-tract of DNA causes the disruption of the Watson-Crick base-pairing, and is therefore responsible for a conformational transition from a B-type DNA to a new bulge- containing conformer. As in the case of protonation (Section 4.8), one expects that this metallation at N7 or N3 will enhance the strength of the anomeric effect of adenin-9-yl, guanin-9-yl or thymin- 1-yl which in turn should be reflected into an increased preference of the sugar moieties in the respective adenosine, guanosine and thymidine residues for N-type pseudorotamers. The results of recent studies on the conformational changes induced by the interaction of metal ions with the nucleobase and the phosphate moieties in guanin-9-yl nucleotides are discussed in Section 8.7. (iii) The overall effect of a N-nucleobase in a nucleoside upon the conformation of the constituent sugar moiety consists of the counteracting contributions of (i) stereoelectronic interactions within O4'-C1'-N1/9 fragment and (ii) the counteracting effect of the nucleobase. Therefore, to obtain an estimate for the stereoelectronic anomeric effect it is necessary to subtract from the overall effect of the nucleobase the steric component. Since it is possible to modulate the relative importance of both contributions by changing the electronic character of the nucleobase, it will be possible to engineer a nucleoside in which the nucleobase will steer the sugar conformation through its inherent steric effect alone, and such a nucleoside can therefore be used as a reference point further on (see our pD-dependent conformational studies on C-nucleosides in Section 6.1 which we used as the basis for the quantitation of the anomeric effect of adenin-9-yl in β-D-dA (37) and β-D-A (50) and of guanin-9-yl in β-D-dG (41) and β-D-G (51)).

4.5 The gauche effect of the 3'-substituent in neutral dNs

4.5.1 The influence of the C1'-substituent

The subtraction of ΔH°N of abasic sugar 12 from that of 13 yields an estimate for the -1 strength of the [HO3'-C3'-C4'-O4'] gauche effect in 13 (∆∆H°7 = -4.5 kJmol in Table 6). In order to compare the magnitude of the [HO3'-C3'-C4'-O4'] gauche effect in 13 and in β-D-dNs, we have also subtracted ΔH°N of each β-D-ddN 30, 31 and 33 - 35 from that of each β-D-dN counterpart 37 -1 and 41 - 44 (∆∆H°10 in Table 6). ∆∆H°10 values are within the range from -7.4 kJmol to -6.2 kJmol-1, showing that the [HO3'-C3'-C4'-O4'] gauche effect is much more efficient in β-D-dNs than ∗ in abasic sugar 13. This suggests that as O4' is involved in nO4' →σ C1'-N1/N9 stereoelectronic

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 86 interactions, the ability of O4'-C4' bond to adopt a gauche orientation with respect to O3'-C3' is much greater than in 13. It is however difficult to establish a straightforward quantitative correlation between the magnitude of the effect of the nucleobase and the corresponding strength of the [HO3'- -1 C3'-C4'-O4'] gauche effect in β-D-dNs 37 and 41 - 44. The small ∆∆H°9 value (-0.5 kJmol ) on the other hand, shows that the [MeO3'-C3'-C4'-O4'] gauche effect in 15 is only slightly more efficient than the [HO3'-C3'-C4'-O4'] gauche effect in 13. The conclusion remains the same in the β-D-dNs -1 series, as shown by the small ∆∆H°12 value (-0.7 kJmol ) reflecting the small additional stabilition of S-type conformations in 3'-OMe-β-D-dA (38) in comparison with β-D-dA (37).

4.5.2 3'-substituent electronegativity dictates 3'-gauche effect A comparative conformational analysis of 2'-deoxy-2'-substituted uridine235,199 and adenosine 206,417,418 derivatives has afforded linear relationships between the population of the N-type pseudorotamer and the electronegativity of the 2'-substituent (Section 2.11). Various hypotheses regarding the possible origin of the gauche effects in nucleos(t)ides have been discussed (See Section 1.12-1.14 for the general discussion about the gauche effect in any system). We have mentioned in above that substitution of 3'-OH in abasic sugar 15 and in β-D-dA (37) for 3'-OMe in 16 and 38 results in a slight increase in the preference of their sugar moieties for S-type conformations (as experimentally evidenced by the slightly negative ∆∆H°9 and ∆∆H°12 values, respectively, Table 6). This has been attributed to the modulation of the [MeO3'-C3'-C4'-O4'] gauche effect compared to [HO3'-C3'-C4'-O4'] gauche effect in the latter compared to the former. The next logical step consisted in addressing the following questions: (i) Is it possible to modulate the thermodynamics of the N S equilibrium in a particular 3'-substituted-β-D-ddN (X = 3'-substituent) simply by altering the nature of its 3'-substituent and conversely (ii) is it possible to predict quantitatively the strength of the [X3'-C3'-C4'-O4'] gauche effect driving the conformation of any 3'-substituted-β-D-ddN using a simple procedure? These questions have led us to perform a systematic comparative study26 on the preferred conformations of the pentofuranose moieties in a series of 3'-substituted-β-D-ddT derivatives [X = - H (34), NH2 (113), OH (43), OMe (114), NO2 (115), OPO3H (67), F (88)] with the same thymin-9- yl nucleobase. These 3'-substituted-β-D-ddT derivatives only differ in the nature of 3'-X, which varies from poorly (i.e. NH2 in 113) to strongly electronegative (i.e. F in 88). 3'-NH2 in 113 should be half-protonated at ca. pD 7.0 since its pKa is expected to be very similar to that of 2'-NH2 in the respective nucleoside (for instance, the pKa value of 2'-NH2 in 2'-NH2-β-D-dU has been reported to be 6.2496,497). 3 1 The pseudorotational analyses of temperature-dependent JHH extracted from H-NMR spectra of 88 and 113 - 115 and subsequent estimation of ΔH°N, ΔS°N and ΔG°N values of their N S equilibria have been performed using the same procedure as for β-D-ddT (34), β-D-T (43) and β-

D-TMP (67) (Section 3.1-3.7, Table 9 for ΔH°N, ΔS°N and ΔG°N values of 43, 67, 88 and 113 - 115.

From the initial comparison of ΔH°N, ΔS°N and ΔG°N values of 34, 43, 67 and 88, 113 - 115, the following conclusions can be drawn: (i) ΔH°N is the main determinant to the drive of the sugar conformation since it prevails over the counteracting (in 34, 43, 67 and 88) or cooperative (in all Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected]

87 other compounds) -ΔS°N contribution to ΔG°N. (ii) In β-D-ddT (34), the N S equilibrium is driven almost exclusively toward N-type conformations (16 % S at 278 K) by the cooperative effects of the nucleobase and of 5'CH2OH, owing to the absence of any electron-withdrawing substituent at C3'. (iii) In

τ contrast, the regularly r 5 o χ t 88 increasing stabilization of S- en 67 114 tu 4 ti 115 43 116 bs type pseudorotamers in the su 113 '- 3 3 114 (I) f 88 67 order 3'-NH2-β-D-T (113, 20 o 115 43 ty 116 (II) vi 113 ti 2 % at 278 K), β-D-T and β-D- ga ne o tr TMP (43 and 67, 64 % at 278 ec 1 el 88 67 114 116 113 up (III) K), 3'-OMe-β-D-T (114, 74 ro 115 43 G 0 % at 278 K), 3'-NO -β-D-T -12 -10 -8 -6 -4 -2 2 ΔHo (kJ mol-1) GE (115, 81 % at 278 K), 3'-F-β- Figure 15: Correlation plots of the group electronegativity of the 3'-X substituent in 3'-substituted-β-D-ddT derivatives [X = H (34), NH2 (113), OH (43), OMe D-T (88, 91 % at 278 K), can - (114), NO2 (115), OPO3H (67), F (88)] as a function of the strength (ΔH°GE) of be attributed unambiguously the [X3'-C3'-C4'-O4'] gauche effect. The group electronegativity has been 503,504 to the increasing preference expressed in the scales developed by Mullay (χMullay) [....., graph (I)], 505-508 ___ 502 for gauche orientation within Inamoto (ιInamoto) [ , graph (II)] and Marriott (χMarriott) [----, graph (III)]. All plots show straightlines with high Pearson's correlation [X3'-C3'-C4'-O4'] fragment, coefficients [R = -1.0, -0.98 and -0.96 for graphs (I) - (III), respectively]. In a which progressively prevails recent study42, we have determined the thermodynamics of the N S equilibrium [∆H° = 0.0 kJmol-1 (σ = 0.4) , ∆S° = 7.5 Jmol-1K-1 (σ = 1.5) and over the anomeric effect. ∆G° (298 K) = -2.3 kJmol-1 (σ = 0.6)] in 3'-OCF -β-D-ddT (116) using the 3 ΔH°N reflects the strength of procedure described in Section 3.1-3.7]. ΔH° (-5.4 kJmol-1) of the [OCF -C3'- GE 3 the combined influence of the C4'-O4'] gauche effect in 116 was subsequently calculated by subtracting ∆H° of β-D-ddT (34) from its own. From the equations of the straightlines (I) [χMullay (steric + stereoelectronic) = 2.63 - 0.19ΔH°GE], (II) [ιInamoto = 2.25 -0.08 ΔH°GE] and (III) [χMarriott = contributions of this gauche 0.26 - 0.022 ΔH° ], the group electronegativity of 3'-OCF in 116 has been GE 3 effect, increases in the order: determined: χ = 3.66 ± 0.05, ι = 2.64 ± 0.10 and χ = 0.38 Mullay Inamoto Marriott NH (113) < OH (43) < OMe ± 0.05. 2 - (114) < NO2 (115) < OPO3H (67) < F (88).

The [X3'-C3'-C4'-O4'] gauche effect (ΔH°GE) in 43, 67, 88 and 113 - 115 has been subsequently estimated by subtracting from their ΔH°N values that of β-D-ddT (34) (Table 9), which allows to account for the combined effects of thymin-9-yl and of 5'CH2OH groups in 43, 67, 88 and

113 - 115. It should be however noted that ΔH°GE values consist of three intractable components: (i) the stereoelectronic gauche effect, (ii) a term for the electrostatics and solvation, and (iii) the steric effect of the 3'-substituent. 498-500,501 We have examined the dependence of ΔH°GE on the group electronegativity of 3'-X 502 503,504 using the electronegativity scales developed by Marriott (χMarriott) , Mullay (χMullay) and 505-508 Inamoto (ιInamoto) and their coworkers. Marriott's electronegativity scale is based on ab initio calculations with the GAUSSIAN program298 (at HF/6-31G* level). The actual electronegativity of X in H-X corresponds to the atomic electron population on H in H-X, as determined from a Mulliken populations analysis of geometrically optimized H-X. Mullay's group electronegativities

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 88 are based on modified Slater effective nuclear charges, effective principal quantum numbers, fractional p characters, assuming charge conservation and electronegativity equalization within each bond in the group. Inamoto's scale ι is an inductive substituent parameter scale, which is derived from the corresponding group electronegativities showing a strong correlation with trans H,H coupling constant in monosubstituted ethene fragments. The plots of the ΔH°GE values as a function 502 503,504 505-508 of χMarriott , χMullay and ιInamoto gave all straight lines with high correlation coefficient (> 0.96) (Fig 15). From the equations of these simple correlation plots, we have - subsequently been able to estimate the group electronegativity of 3'-OPO3H in β-D-TMP (67), i.e. : χMarriott = 0.44, χMullay = 4.12 and ιInamoto = 2.8.

Table 9. The enthalpy and entropy contributionsa to the N S pseudorotational equilibrium of the pentofuranose moiety in 3'-substituted-β-D-ddT derivatives 43, 67, 88 and 113 – 115 (see ref 26)

Group electronegativity (χ or ι) of Compound d 3'-substituent Δ%S b c ΔH°N ΔS°N −ΤΔS°N ΔG°N 358-278 ΔH°GE Marriott Mullay Inamoto 3'-NH2-ddT (113) 2.6 -1.9 0.6 3.2 +5 -2.8 0.33 3.15 2.47

T (43) -1.8 -0.9 0.3 -1.5 -4 -7.2 0.43 3.97 2.79

3'-Ome-ddT(114) -2.1 1.1 -0.3 -2.4 -4 -7.5 0.44 4.03 2.82

3'-NO2-ddT (115) -2.4 3.7 -1.1 -3.5 -3 -7.8 0.4 4.08 2.75

TMP (67) -2.6 -4.3 1.3 -1.3 -3 -8.0 e e e 0.44 4.12 2.8

3'-F-ddT (88) -5.9 -2.3 0.7 -5.2 -6 -11.3 0.52 4.73 3.1

a -1 b In kJmol . ΔS°N and ΔG°N are given at 298K (see the methodology described in Section 3). Δ %S (358-278) indicates the change in the population of the S-type pseudorotamer as the temperature is raised from 278 K to 358 K, and shows the influence of both the gauche effect enthalpy and entropy contributions. At a particular temperature T, the population of the S-type species is calculated from the corresponding free energy values as follows: %S (T) = 100 * [exp T T c (- ΔG / RT)] / [exp (- ΔG / RT) +1]. ΔH°GE has been calculated by subtracting ΔH°N characterizing the N S d equilibrium of the sugar moiety in 43, 67, 88 and 113 - 115 from that obtained for β-D-ddT (34) (Table 2). The group electronegativities of the 3'-substituents NH2 (113), OH (43), MeO (114), NO2 (115) and F (88) are given e - according to ref. 502-508. For β-D-TMP (67), the group electronegativities of the OPO3H substituent have been back-calculated from its ΔH°GE value using the graphs shown in Fig 15.

S-type and the N-type pseudorotamer populations. Our NMR analysis of a set of 3′-substituted 26 thymidine derivatives [Scheme 1: for 1 – 7] , in which the gauche effect of 5′-CH2OH and the anomeric effect of the nucleobase remain as the constant factor, has clearly shown that the conformational preference for the S-type sugar conformation linearly increases with the increase of the strength of the electronegetivity of the 3′-substitutent. It has been envisioned that as the

89 electronegetivity of the 3′-substituent increases [H < NH2 < OCF3 < OH < OCH3 < NO2 < F] the C3′-H3′ becomes further polarised, the difference in the energy levels of the donor σ and C3′-H3′ acceptor σ* orbitals decreases, facilitating the σ σ* orbital mixing, thereby C4′-O4′ C3′-H3′ C4′-O4′ enhancing the 3′-gauche effect promoted S-type conformational preference.

We argued that since we already have a dependable experimental means for the estimation 298K ref of ΔG°NMR , we could challenge the theory with this by calculating the total energy difference

(ΔE) of N-type and S-type geometries (ΔES-N) of 1 – 7 (Figure 1) using high level ab initio calculations. The Gaussian calculations of both N- and S-type pseudorotamers of potential anti-HIV 2′,3′-dideoxy 3′-substituted thymidine derivatives 1 – 7 have been performed using systemetic variation of different basis functions at Hatree-Fock level. With the lower basis functions such as 6- 31G* or even with 6-311+G* basis sets (Table 1), we failed to observe any colinearity of the ab 298K initio calculated ΔES-N in the gas phase with our ΔG°NMR . This pilot study showed the necessity of introducing diffuse function with higher basis set in order to reduce the basis set superposition error6, which also illustrated the caveats of using a low-level ab initio method for structural calculation of flexible biomolecules. The use of higher basis set, such as HF/6-311++G**, showed a marked improvement of 298K correlation of ΔES-N with the experimental ΔG°NMR for 1 – 7 (Table 1). Thus a plot of ΔES-N (gas 298K phase HF/6-311++G**) as a function of ΔG°NMR showed a Pearson’s correlation coeffecient of 0.92 (Graph I in Figure 1A). In order to mimick the solvation behaviour of the experimental NMR analyses, the solution phase (ε = 78.0) ab initio calculations was performed at HF/6-311++G** level using Onsager solvation model. Remarkably, this gave even better correlation compared to the gas phase calculations as evidenced from the Graph II in Figure 1A, giving Pearson’s correlation coeffecient of 0.97, as a result of improved colinearity of the energies from the solution phase ab initio calculations with the experimental NMR data. Moreover, the single point ab initio calculations at B3LYP/6-311++G** level of theory in solution phase with Onsager solvation model have been performed using the optimised geometry at HF/6-311++G** level for 1 – 7 (Table 1).

The plot of ΔES-N calculated from this solution phase B3LYP/6-311++G**//HF/6-311++G** 298K calculation as a function of experimental ΔG°NMR gives the Pearson’s correlation coeffecient as 0.97 (Figure 1B). This straightforward correlation of our experimental NMR findings with the present theoretical ab initio calculations justifiably points to the following observations: (i) it is now 298K possible to predict the ΔG°NMR of nucleosides quite dependably by simply knowing the ab initio calculated ΔES-N; (ii) the substituent-dependent steric and stereoelectronic effects on the bias of the two-state1,2b-e N S equilibrium in nucleosides can also be easily predicted from the ab initio calculations provided a sufficiantly large basis set is used, and (iii) the comparison of Pearson’s correlation coeffecients in Figure 1 indicates the necessity of mimicking the solvation behaviour of the experimental NMR measurement condition in these high level ab initio calculations. It is quite likely that this correlation between the theory and the experiment would give much deeper insight into the molecular orbital basis of the role of the stereoelectronic forces in modulating the

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 90 conformation of nucleosides and nucleotides as well as shed light in their ubiquitous self-assembly process governing the chemistry of life in general.

4.5.3 Stronger gauche effect in nucleosides than in 1,2-difluoroethane In 1,2-difluoroethane, an energy stabilization of 2.4 -3.4 kJ/mol has been observed in favour of the gauche rotamer over the trans counterpart (section 1.13) but in 3'-substituted-2',3'- dideoxynucleosides we have found that the [X3'-C3'-C4'-O4'] gauche effect ranges from -2.8 kJ/mol for X = NH2 to -11.3 kJ/mol for X = F (Fig. 15, Table 9). This can be understood in terms of dihedral constraints imposed on the endocyclic torsions owing to the ring closure nature of the five- membered pentofuranose nucleosides in contrast to 1,2-difluoroethane where only constraint is imposed by favourable orbital overlap or bond-bending in the gauche compared to the trans rotamer.

4.6 The 2'-OH effect in ribonucleos(t)ides is nucleobase-dependent

The subtraction of ΔH°N of a particular β-D-dN 37 or 41 - 44 from that of its β-D-rN counterpart (50 - 54) yields an estimate (∆∆H°11, Table 6) for the combined strength of the [O2'- C2'-C1'-O4'] and [O2'-C2'-C1'-N1/9] gauche effects, i.e. the overall effect of 2'-OH in β-D-rNs in -1 comparison with β-D-dNs. ∆∆H°11 values are as follows (in kJmol ): adenin-9-yl (-0.5) = guanin- 9-yl (0.5) < uracil-1-yl (2.6) ≈ thymin-1-yl (2.7) ≈ cytosin-1-yl (3.0) ≈ 5-fluorouracil-1-yl (3.1). Thus, the ability of 2'-OH to drive the sugar conformation toward S-type pseudorotamers is greater in the purine β-D-rNs with respect to the pyrimidine β-D-rNs.

4 (113) 4 (113)

(I) (113) 0 0 (43) (34) (34)

(114) (34) ) ) N -4 N -4 (116) - - S (116) S (II) ( ( (116) E E Δ (43) Δ (115) -8 -8 (43) (115) (88) (114) (114) -12 -12 (115) (88) (88) -16 -16 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4

ΔGNMR ΔGNMR (A) B) 298K Figure 16. Panel (A) shows the plot of the free energy of the N S pseudorotational equilibrium ( ΔG°NMR , in kJ -1 mol ) as a function of ab initio calculated energy diffrenence between the S- and the N-type pseudorotamers (ΔES-N, in -1 kJ mol ) at HF/6-311++G** level for 2′,3′-dideoxy 3′-substituted (X) thymidine derivatives [with X = H (34), NH2 (113), OH (43), OCH3 (114), NO2 (115), OCF3 (116), F (88)], both in the gas phase [■, dotted line, graph I] as well as in the solution [, solid line, graph II] gives straight lines with slope = 1.03 (σ = 0.11), intercept = -1.22 (σ = 0.31) and R = 0.92 for I and slope = 1.50 (σ = 0.09), intercept = -4.92 (σ = 0.30) and R = 0.97 for II respectively. Panel (B) shows 298K -1 -1 the similar plot of ΔG°NMR (in kJ mol ) as a function ΔES-N (kJ mol ) derived from the single point solution phase DFT calculations at B3LYP/6-311++G**//HF/6-311++G** level, giving the straight line with slope = 1.41 (σ = 0.11), intercept = -4.17 (σ = 0.35) and R = 0.97. The fact that ∆ES-N for 116 is clearly smaller than that of 88 supports our experimental result of the reduced efficiency of the [3'-OCF3-C3'-C4'-O4'] gauche effect in the former in comparison with the [F3'-C3'-C4'-O4'] gauche effect in the latter.

2'-OH stabilizes N-type pseudorotamers in pyrimidine β-D-rNMPs 76 - 78 (by 1.7 - 4.0 -1 -1 kJmol , i.e. ∆∆H°29 in Table 7) and in β-D-rNMPEts 81 - 83 (by 0.5 - 2.1 kJmol , i.e. ∆∆H°27 in Table 7) in comparison with the 2'-deoxycounterparts β-D-dNMPs 66 - 68 and β-D-dNMPEts 71 - 73. In contrast, there is virtually no enthalpy stabilization of the S-type pseudorotamers in purine β- D-rNMPs 74 and 75 and in β-D-dNMPs 64 and 65, as shown by small ∆∆H°29 values (Table 7). Finally, in β-D-AMPEt (79) and β-D-GMPEt (80), 2'-OH even destabilizes N-type conformations in -1 comparison with β-D-dAMPEt (69) and β-D-dGMPEt (70) (by ∆∆H°27 = -1.4 and -1.0 kJmol , respectively). Thus, as in the ribonucleosides, the ability of 2'-OH to stabilize N-type pseudorotamers is much greater in the pyrimidine than in the purines series. -1 The subtraction (∆∆H°8 = 4.5 kJmol , Table 6) of ΔH°N of abasic sugar 13 from that of 14 yields an estimate for the combined influence of the [HO2'-C2'-C1'-O4'] and [HO2'-C2'-C3'-O3'] gauche effects, the later being negligible (because of gauche orientation in both N and S conformers). ∆∆H°8 has the same value, but the opposite sign, as ∆∆H°7, showing that the [HO2'- C2'-C1'-O4'] and [HO4'-C4'-C3'-O3'] gauche effects are cancelling each other, which is consistent with the results of regression (A) and also with the fact that the enhancement or reduction of electron-density of O4' owing to variable electronic character of the nucleobase will affect both [HO2'-C2'-C1'-O4'] and [HO3'-C3'-C4'-O4'] in a similar manner.

8 Figure 17. The correlation plot of the overall effect ) 81 52 76 (AEddN) of adenin-9-yl in β- -1 l 6 o 83 54 D-ddA (30), guanin-9-yl in β- m 77 78 J 82 53 k D-ddG (31), cytosin-1-yl in β- ( N d 4 D-ddC (33), thymin-1-yl in β- d 80 D-ddT (34) and uracil-1-yl in E 79 75 74 A β-D-ddU (35) as a function of 2 50 & 51 the overall effect of 2'-OH in β-D-rNs 50 - 54, β-D-rNMPs -4 -2 0 2 4 6 74 - 78 and β-D-rNMPEts 79 - 83 (in the N state). The effect of the C1'-aglycone in β-D-ddNs was estimated from ∆∆H°2 (Table 6 and Fig 13). The overall effect of 2'-OH was estimated from ∆∆H°11 for β-D-rNs, ∆∆H°29 for β-D-rNMPs and ∆∆H°27 for β-D-rNMPEts (Table 7 and Fig 13). The Pearson's corelation coefficient of the straight line is 0.87.

Therefore, if one assumes that the strengths of the [HO3'-C3'-C4'-O4'] and [HO2'-C2'-C1'-

O4'] gauche effects are identical but with the opposite signs (as suggested by the identical ΔH°N values of 12 and 14), then the nucleobase-dependent -∆∆H°10 values give estimates for the magnitudes of the [HO2'-C2'-C1'-O4'] gauche effect in β-D-rNs (Table 6). Subtraction of -∆∆H°10 from ∆∆H°11 affords the nucleobase-dependent strength of the [O2'-C2'-C1'-N1/9] gauche effect in β-D-rNs 50 - 54, i.e.: -7.9 kJmol-1 in β-D-A, -6.7 kJmol-1 in β-D-G, -4.3 kJmol-1 in β-D-C, -4.1 kJmol-1 in β-D-rT and -3.7 kJmol-1 in β-D-U, showing that in purine nucleosides this gauche effect is much more efficient than in the pyrimidine counterparts. The interdependency of the overall effect of 2'-OH in β-ribonucleosides and ribonucleotides is also evidenced by the fact that a plot of the effect of the nucleobase in β-D-ddNs 30 - 35 as a

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 92 function of ∆∆H°11 (for β-D-rNs 50 - 54), ∆∆H°27 (for β-D-rNMPEts 79 - 83) and ∆∆H°29 (for β- D-rNMPs 74 - 78) in Fig. 17 gives a straigthline with correlation coefficient of 0.87. As 2'-OH drives the pseudorotational equilibrium to the N, the strength of the anomeric effect of the nucleobase increases in a cooperative manner.

4.7 3'-gauche effect modulation by 2'-OH in ribonucleos(t)ide In ribonucleos(t)ides, 2'-OH influences the drive of the sugar conformation through the [O2'- C2'-C1'-O4'] and [O2'-C2'-C1'-N1/9] gauche effects. Unlike in β-D-dNs, in β-D-rNs a possible contribution of H-bonding142 interaction between 2'- and 3'-substituents to the preference for gauche orientation within [HO3'-C3'-C4'-O4'] fragment cannot be excluded. It has been for instance shown by 1H-NMR509,510 that there is an intramolecular water bridge between the vicinal 2'-OH and 3'- phosphate in cAMP in which the 2'-OH hydrogen accepts the lonepair of water oxygen. We have attempted to delineate27 the magnitudes of the gauche effects involving 2'-OH in β- D-A (50) in the N state by comparing the conformational preferences of its constituent pentofuranose sugar with those in β-D-ddA (30), β-D-dA (37), β-D-3'-dA (63), β-D-dAMP (64), β-

D-AMP (74) and abasic sugars 12 - 14 through a set of pairwise subtractions of the ΔH°N values of their N S equilibria (Table 6). The main results of this work are as follows: (i) As discussed in Section 4.6, in abasic sugars 13 and 14, the [HO3'-C3'-C4'-O4'] and [HO2'-C2'-C1'-O4'] gauche effects cancel each other, their magnitudes being respectively ∆∆H°7 = - -1 4.5 and ∆∆H°8 = 4.5 kJmol . -1 -1 (ii) The comparison of ∆∆H°10 (-7.4 kJmol ) and ∆∆H°19 (-5.7 kJmol ) shows that the strength of the [HO3'-C3'-C4'-O4'] gauche effect is reduced by 1.7 kJmol-1 in β-D-A in comparison with β-D-dA, which can be attributed to the effect of 2'-OH in the former. -1/-2 (iii) The strengths of the [O4'-C4'-C3'-O3'PO3H ] gauche effect in β-D-dAMP and β-D- -1 - AMP have been estimated by ∆∆H°20 (-8.9 kJmol ) for the former and by subtracting (-6.2 kJmol 1 - ) ΔH°N value of β-D-3'-dA from that of β-D-AMP, respectively. Thus the [O4'-C4'-C3'-O3'PO3H ] gauche effect is less efficient by 2.7 kJmol-1 in β-D-AMP than in β-D-dAMP, showing the effect of 2'-OH in the former. The fact that 2'-OH weakens the [O3'-C3'-C4'-O4'] gauche effect less efficiently in β-D-A than in β-D-AMP [compare (i) and (ii)] can be understood as a consequence of the greater freedom of the lonepair of O2' to participate in the [O2'-C2'-C1'-N9] and [O2'-C2'-C1'-O4'] gauche effects in β-D-AMP than in β-D-A, where it acts as a donor in the intramolecular hydrogen-bonding interaction between 2'-OH and 3'-OH511,512. Hydrogen bonding of 2'-OH with N3513 may induce the strengthening of the [O2'-C2'-C1'-N9] gauche effect in purine β-D-rNs. In A-type RNA, pseudorotamers are inherently stabilized when 2'-OH is involved in hydrogen bonding interaction with the nucleobase, and the substitution of 2'-OH in hammerhead ribozyme for NH2 or F changes the ratio of N- versus S-type pseudorotamers as well as influences the hydrogen bonding capabilities514-516 of the sugar.

(iv) The overall stereoelectronic effect of 2'-OH has first been estimated in β-D-3'-dA by -1 subtracting (-2.2 kJmol ) ΔH°N of β-D-ddA from its own. For β-D-A and β-D-AMP, the strength -1 -1 of the 2'-OH effect is reflected in the values of ∆∆H°11 (-0.5 kJmol ) and ∆∆H°29 (0.5 kJmol ), respectively. Thus the ability of the effect of 2'-OH to counteract the [O3'-C3'-C4'-O4'] gauche effect by stabilizing N-type conformations increases in the order: β-D-3'-dA < β-D-A < β-D-AMP. The 2'-OH stabilization of N-type conformations in β-D-AMP compared with β-D-A and β-D-3'-dA can be attributed either to a more efficient [O2'-C2'-C1'-O4'] gauche effect or to the weakening of the [O2'-C2'-C1'-N9] gauche effect.

4.8 Drive of pseudorotation in β-nucleosides by the nature of the nucleobase

4.8.1 The two-state N S equilibrium is evidenced by pKa values from ∆G° For all β-D-nucleosides, either in the 2',3'-dideoxy (30 - 34), 2'-deoxy (37 and 40 - 45) or ribo (50 - 55) series, the plots of ∆H°, -T∆S° and ∆G° values as a function of pD show that the bias of their two-state N S equilibrium can be successfully modulated by the protonation and/or deprotonation of the nucleobase (Fig. 11 and Table 2). The origin of this modulation will be discussed for each series in the next paragraph. (In β-L-nucleosides, similar trends are observed since the thermodynamics of the N S equilibrium in each of the D- and L-enantiomers are virtually the same owing to the mirror-image relationship, as discussed in Section 4.1(e)). The value of the pD at the inflection point of the plot of pD-dependent ∆G° values for each compound has been determined by fitting the experimental data to the Henderson-Hasselbach equation, as discussed in Section 3.7. These pKa(s) values are virtually identical to the known pKa(s) of the constituent nucleobases in 30 - 34, 37, 40 - 45 and 50 - 55, as found in the literature165,247,379,382-384 and to other estimates of pKa values independently derived by us from Hill plots of pD-dependent 1H chemical shifts of the anomeric and aromatic protons of the same nucleosides (± 0.4 pD unit, compare cols. 5 and 6, on one hand, cols. 10 and 11, on the other, in Table 2). This result unequivocally validates the two-state N S equilibrium model.

4.8.2 Identical pKas of the nucleobases in 2',3'-dideoxy, 2'-deoxy and ribo series 1 The pKa values of cytosin-1-yl and thymin-1-yl (determined from pD-dependent H chemical shifts) in β-D-ddC/T, β-D-dC/T and β-D-C/rT are the same (± 0.1 from the average values), showing the lack of influence of 2'- and/or 3'-OH on their electronic character (See Section 8.8 for the discussion on 3',5'-bisphosphate of guanosine). For adenin-9-yl and guanin-9-yl, the electron-withdrawing effect of 2'-/3'-OH is only partly reflected in the slight decrease of the pKa value in the order ddN > dN > rN (i.e. by 0.3 and 0.4 pD unit, respectively). This tendency should however be considered with caution, since the accuracy of the estimates of the pKa values determined from pD-dependent chemical shifts is ≈ 0.1 pD unit.

Table 10. The relative populations of syn versus anti rotamers around the glycosidic torsion in α-D-ddNs 17 - 20, β-D-ddNs 30 - 34, α-D-dNs 21 - 26, and β-D-dNs 37 - 39 and 41 - 43 at various pDs a from 1D-nOe difference experimentsb.

ηH1' (%) ηH6/H8 (%) (ηH1' + ηH6/H8) / 2 % syn Compound pD (H6/H8 irradiated) (H1' irradiated) rotamers α-D-ddA (17) 7.3 2.3 1.9 2.1 20 β-D-ddA (30) 7.0 2.3 2.4 2.4 22 α-D-ddG (18) 7.5 1.8 1.8 1.8 17 β-D-ddG (31) 2.1 1.4 1.5 1.5 14 7.4 1.9 2.4 2.2 21 11.5 2.8 2.8 2.8 26 5'-OMe-β-D-ddG (32) 2.1 1.6 - 1.6 15 6.6 1.7 1.4 1.6 21 11.7 1.3 1.3 1.3 12 α-D-ddC (19) 7.1 2.4 - 2.4 22 β-D-ddC (33) 7.0 1.5 - 1.5 14 α-D-ddT (20) 7.2 3.7 3.2 3.5 33 β-D-ddT (34) 7.0 2.1 2.0 2.1 20 α-D-dA (21) 2.1 2.5 2.8 2.7 25 6.5 5.4 4.0 4.7 44 3'-OMe-α-D-dA (22) 1.6 0.6 0.7 0.7 7 6.9 1.2 0.9 1.1 10 3',5'-diOMe-α-D-dA (23) 1.6 1.0 0.9 1.0 10 6.7 0.9 1.1 1.0 10 β-D-dA (37) 1.6 3.5 4.2 3.9 36 6.9 5.8 5.9 5.9 55 3'-OMe-β-D-dA (38) 1.6 4.1 4.2 4.2 39 7.0 5.5 6.5 6.0 56 3',5'-diOMe-β-D-dA (39) 2.2 2.4 2.4 2.4 22 7.3 1.4 1.2 1.3 12 α-D-dG (24) 1.8 3.8 2.3 3.1 29 7.3 4.7 2.3 3.5 33 11.6 6.1 7.7 6.9 65 β-D-dG (41) 1.8 3.5 3.1 3.3 31 7.7 3.8 4.0 3.9 36 11.1 4.5 4.6 4.6 43 α-D-dC (25) c 2.0 0.9 - 0.9 8 7.1 2.4 - 2.4 22 β-D-dC (42) d 7.3 2.8 2.8 2.8 26 α-D-T (26) 7.1 3.0 - 3.0 28 11.6 3.4 - 3.4 32 β-D-T (43) 6.7 3.7 3.0 3.4 32 11.7 3.1 2.4 2.8 26 a 1D-nOe difference experiments were performed in D2O [298 K except for all guanin-9-yl nucleosides (288 K) due to their decomposition above 288 K at acidic pDs]. b We used the method of Rosemeyer to calculate the population of syn rotamers around the glycosidic torsion from homonuclear 1H nOes517. c Selective saturation of H1' could not be performed at acidic pD for α-D-dC and at neutral pD for α-D-ddC and β-D-ddC due to near isochronous H1' and H5 . d Could not be measured in the acidic solution due to the fact that H1' and H5 in β-D-dC have the same chemical shift at pD < 3 at 298 K.

4.8.3 Anomeric effect in β-D-ddNs is modulated by the nature of the nucleobase o o o A comparative analysis of ΔH p , -T ΔSp and ΔGp values of 30 - 34 and of the corresponding o o o o o o o o ΔHN , -T ΔSN , ΔGN and ΔHD , -T ΔSD and ΔGD values (Table 2) shows that ΔH p and ΔHD prevail over o o the counteracting -T ΔSp and -T ΔSD which results into the overall stabilization of N-type sugars in o o ). the P and D states (i.e. positive ΔG p and ΔGD Protonation of the nucleobase in 30 - 34 steadily shifts the N S pseudorotational equilibrium toward more N-type conformation than in the N state. When the nucleobase is fully protonated, a plateau in thermodynamic values in the P state is reached o -1 [i.e. ΔΔGP−N (kJmol ) 1.5 (30), 3.8 (31), 3.2 (32), 1.1 (33)]. In contrast, deprotonation shifts the N S equilibrium toward more S-type sugars (with pseudoequatorially oriented nucleobase) than in o -1 the N state [i.e. ΔΔGD−N (kJmol ) = -1.1 (31), -0.3 (32) and -0.8 (34)]. We have compared the preferred conformation of the sugar moiety and of the nucleobase in β-D-ddG (31) and 5'-OMe-β- D-ddG (32) over the whole ≈ 2.0 - ≈ 12.0 pD range in order to examine whether 5'-OH and guanin- 9-yl, by forming an H-bond or guanin-9-yl alone, by adopting different orientations around the o o glycosyl torsion at different pDs, contribute to the above ΔΔGP−N and ΔΔGD−N values. ∆H° of 31 and 32 are nearly identical at any pD suggesting that 5'CH2OH and N3 in 31 do not form a hydrogen o o bond. Slightly greater ΔH N and ΔH N in 32 than in 31 can be attributed to the increased steric bulk 5'CH2OMe compared to 5'CH2OH. However, on the overall, N-type sugars are more preferred o at 298 K in 32 than 31 (see their Error! No topic specified. and ΔGD values), due to an entropy effect. o 1 [In the P state, slightly larger ΔH p for 31 than for 32 can be easily explained: (i) H-NMR spectra of 31 and 32 were recorded at pD 1.9 and 2.0, respectively, but not at lower pDs due to rapid decomposition. (ii) The standard deviations of ∆H°, ∆S° and ∆G° values at pD 1.9 for 31 (4.5 kJmol-1) and at pD 2.0 for 32 (3.6 kJmol-1) are higher than at other pDs, because 1H-NMR spectra could only be recorded in a narrow temperature range (274 - 303 K). Thus ΔH°, -TΔS° and ΔG° of 31 and 32 are inevitably less accurate in the P than in the N and D states.] Our 1D-nOe difference experiments in the P, N and D state of 31 and 32 show that guanin-9-yl assumes an anti orientation in both compounds at any pD and this preference is almost independent of the pD and of the nature of 5'-substituent. Moreover, the nucleobases in β-D-ddA (30), β-D-ddC (33) and β-D-ddT (34) adopt almost exclusively anti orientations (78 - 86 % at room temperature) at neutral pD (Table 10). The shift of the N S equilibrium in the protonated and deprotonated states of β-D-ddNs toward more N- and S-type sugars, respectively, compared with the N state is therefore neither the result of any interaction between 5'-OH and the nucleobase nor induced by a different orientation of the nucleobase around the glycosyl torsion at different pDs.

4.8.4 The orbital mixing as the origin of the O4'-C1'-N1/9 anomeric effect The modulation of the thermodynamics of the two-state N S equilibrium in β-D-ddNs 30 - 34 by the pD of the aqueous solution can be rationalized in terms of pD-dependent magnitude of the O4'-C1'-N1/9 anomeric effect: As the nucleobase becomes protonated, a partial positive charge is created at the glycosyl nitrogen. As a result, nO4' →σ∗C1'-N9 orbital mixing becomes more favourable because of the lowering of the antibonding σ* orbital (Section 2.8). This favourable

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 96 orbital mixing prompts the nucleobase to adopt a pseudoaxial orientation, which in turn is achieved in the N-type pseudorotamers. Conversely, as the nucleobase becomes deprotonated in the alkaline pD, the ability of the glycosyl nitrogen to participate in O4'-C1'-N1/9 stereoelectronic interactions is reduced, which is evident by the shift of N S equilibrium to more S-type (with pseudoequatorial aglycone) in comparison with the N state. Our interpretation is based on the assumption that the steric bulk of the nucleobase remains to be comparable in the P, D and N states. Of course we expect a change in the hydration chracteristics in the various cationic or anionic state vis-a-vis N state, but that cannot be estimated experimentally with the present state-of-the art. Our results also tend to suggest that the contribution of electrostatic repulsions (see Section 2.8, Fig 8) between the dipole of the pentofuranose moiety and the C1'-N1/9 dipole to the overall strength of the O4'-C1'- N1/9 anomeric effect in nucleosides and nucleotides is rather small in comparison with that of hyperconjugative interactions (Figs 8 and 9). Indeed, one can reasonably assume that the partial positive charge created at the glycosyl nitrogen upon protonation of the nucleobase in β-D-ddNs will weaken the C1'-N1/9 dipole, which in turn should lead to the reduction of the electrostatic repulsions with the O4' lonepair in the P compared with N state of 30 - 33. Therefore, if the origin of the O4'-C1'-N1/9 anomeric effect in nucleosides and nucleotides was mainly electrostatic, the preference of the nucleobase for pseudoaxial orientation in the N-type pseudorotamers should be much reduced in the P state than in the N state owing to the weakening of the anomeric effect in the former, but our results clearly show the opposite, i.e. a higher preference for the N-type sugars at acidic compared with neutral pDs. Similarly, upon deprotonation of the nucleobase in β-D-ddNs 31, 32 and 34, owing to the presumably higher electron-density at the glycosyl nitrogen, one may also suggest that dipole-dipole repulsions should be reinforced in comparison with the N or P state, and that the anomeric effect should be more efficient in the D compared with the N or P state, which is again in contradiction with the experimentally observed tendency of destabilization of N-type sugars in the alkaline compared with the neutral solution (see above, section 4.8). The strengthening of the effect of the nucleobase upon its protonation (or weakening upon o o o its deprotonation) can be estimated by directly subtracting ΔHN from ΔHp (or from ΔHD ). Thus o -1 ΔΔHP−N (kJmol ) = 3.0 (33, cytosin-1-yl) < 5.7 (30, adenin-9-yl) < 20.2 (31, guanin-9-yl) and 20.1 o -1 (32, guanin-9-yl) whereas ΔΔHD−N (kJmol ) = -2.0 (31, guanin-9-yl) and (32, guanin-9-yl) ≈ -1.9 ° (34, thymin-1-yl). The subtraction of ∆∆H 2 values in the P and N states (or N and D states) gives o o identical values as ΔΔHP−N and ΔΔHD−N . Thus purine nucleosides respond more to the protonation of their nucleobase than the pyrimidine counterparts, which is consistent with the fact that upon addition of one equivalent trifluoroacetic acid the chemical shift of the glycosyl nitrogen is more affected in the purine than in the pyrimidine series (∆δ (downfield shift of N9) = 6.6 ppm in N1H+- adenosine and 6.7 ppm in N7H+-guanosine401,403 >> ∆δ (downfield shift of N1) = 1.1 ppm in N3H+-cytosine404).

4.8.5 No reverse anomeric effect in pentofuranosyl nucleosides If the reverse anomeric effect were playing any role in the drive of the sugar conformation in pentofuranosyl β-nucleosides at acidic pD, one should observe an increase in the population of S- type pseudorotamers, in which the nucleobase adopts a pseudoequatorial orientation, in the acidic compared to the neutral pD. Our results show the opposite trends, therefore the pD-dependent conformational preferences of β-D-nucleosides can be attributed to the resulting modulation of the anomeric effect, not a reverse anomeric effect, as suggested in the case of imidazolium or pyridinium derivatives of pyranose (Section 1.7).

4.8.6 Variable tunablity of anomeric effect in β-D-ddNs, β-D-dNs and β-D-rNs In protonated β-D-dA, 3'-OMe-β-D-dA and 3',5'-diOMe-β-D-dA, and deprotonated β-D-dU and 5-F-β-D-dU, both ∆H° and -T∆S° stabilize S-type conformations either with the same magnitude or slightly stronger ∆H°. In the D state of β-D-dG and β-D-T, ∆H° overrides the counteracting -T∆S°, resulting in the overall stabilization of S-type sugars at 298 K. Only in protonated β-D-dC, -T∆S° is slightly stronger than ∆H° and determines the drive of the N S equilibrium toward S-type sugars, whereas in protonated β-D-dImb and β-D-dG, ∆H° (driving to N) and -T∆S° (driving to S) nearly cancel each other, therefore N and S-type sugars have almost the same population at 298 K. The balance of ∆H° and -T∆S° terms of the N S in β-D-rNs is also dictated by the nature of the nucleobase and the pD: In the D state of β-D-U and 5-F-β-D-rU, opposing ∆H° (driving to the N) and -T∆S° nearly cancel each other, resulting in nearly unbiased equilibrium. However, in the D state of β-D-G, ∆H° (stabilizing S-type sugars) prevails over the opposing -T∆S°, and S-type sugars are favoured at 298 K. In the P state of β-D-G and β-D-C, ∆H° prevails over counteracting -T∆S°, stabilizing N-type sugars at 298 K. In the P state of β-D-A and D state of β-D-rT, small negative ∆H° and -T∆S° values favour slightly S-type conformations at 298 K. In agreement with our observations in the case of β-D-ddNs 30 - 34, as the nucleobase in β- D-dNs and β-D-rNs is protonated, the two-state N S equilibrium of the constituent sugar moieties o -1 is also shifted toward N-type conformations [ ΔΔGP−N (kJmol ) = 1.0 (37 and 38), 0.4 (39), 1.3 (40), 1.6 (41), 0.5 (42) for β-D-dNs and 1.3 (50), 3.0 (51), 0.4 (52) for β-D-rNs] and conversely, as it is o -1 deprotonated, S-type sugars are further stabilized in comparison with the N state ΔΔGD−N (kJmol ) = -1.0 (41), -0.3 (43 - 45) for β-D-dNs and -1.3 (51), -0.3 (53 and 55), -0.2 (54) for β-D-rNs]. The o o comparison of ΔΔGP−N and ΔΔGD−N values for each nucleobase in β-D-ddNs (30, 31, 33 and 34), β- D-dNs (37, 41 - 43) and β-D-rNs (50 - 53) shows that the change in the ratio of N- and S-type pseudorotamers in the P and D states in comparison with the N state is much reduced in the 2'- deoxy and ribo series than in the 2',3'-dideoxy counterparts. This is owing to the fact that both the overall entropy of the system and the enthalpy of the N S equilibrium are much less affected by the pD of the aqueous solution in (37, 41 - 43) compared with (30, 31, 33 and 34) and (50 - 53). At any pD, the nucleobase in β-D-dNs 37 - 39 and 41 - 43 adopts preferentially an anti orientation around the glycosyl torsion (except β-D-dA (37) and 3'-OMe-β-D-dA (38) at neutral pD

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 98 since ≈ 1:1 ratio of syn and anti rotamers was found, Table 10), as suggested by our 1D nOe difference experiments37. These observations let us propose that N3 (in purines) or O2 (in pyrimidines) does not form a hydrogen bond with 5'-OH. 37 - 39 all have rather similar ∆H°, -T∆S° and ∆G° values at any pD, in spite of the fact that in 39 the shift from less to more syn rotamers occurs in going from the N to the P state, not reverse. The fact that 37 and 39 behave in the same way also implies that in 37 no H-bond between 5'-OH and adenin-9-yl is responsible for the o o positive ΔΔGP−N and ΔΔHP−N values. The modulation of the overall stereoelectronic forces (i.e. anomeric effect + gauche effects) upon protonation and/or deprotonation of the constituent nucleobase in β-D-dNs and β-D-rNs is reflected in the change in the enthalpy of the N S equilibrium in the P and/or D compared with N o o state, which has been estimated by subtracting their ΔHN values from the corresponding ΔH p and o ΔHD values, respectively : o -1 ΔΔHP−N (kJmol ) = 0.7 (42, cytosin-1-yl) < 2.3 (40, imidazol-1-yl) ≈ 2.6 (39, adenin-1-yl) < 3.2 (37, 38, adenin-1-yl) < 4.9 (41, guanin-9-yl) in β-D-dNs and 2.9 (52, cytosin-1-yl) < 4.2 (50, o -1 adenin-1-yl) < 8.7 (51, guanin-9-yl), in β-D-rNs whereas ΔΔHD−N (kJmol )= -0.3 (45, 5-fluorouracil-1-yl) ≈ -0.5 (43, thymin-1-yl) ≈ -0.7 (44, uracil-1-yl) < -2.1 (41, guanin-9-yl) in β-D-dNs and -1.5 (55, 5-fluoro-uracil-1-yl) = -1.5 (53, thymin-1-yl) ≈ -1.7 (54, uracil-1-yl) < -4.3 (51, guanin-9- o yl) in β-D-rNs (for ΔΔHD−N , the < sign signifies a reduced ability of the deprotonated nucleobase to destabilize N-type conformations with respect to the N state). [Note that the same values can be derived from the subtraction of ∆∆H°3 values in the N state from those in the P and D states, respectively, for β-D-dNs and from the subtraction of ∆∆H°4 values in the N state from those in the P and D states, respectively, for β-D-rNs (Table 6, Fig 13)]. Thus, in all ddNs (Section 4.8), dNs and rNs, pyrimidine nucleobases are much less able to transmit the free-energy of its o protonation deprotonation equilibrium than the purine counterparts. The smaller value of ΔHP−N of 40 in comparison with those of 37 - 39 and 41 shows the effect of the electron-withdrawing character of the fused pyrimidine ring upon the imidazole moiety in the purine nucleosides. o o ΔΔHP−N and ΔΔHD−N increase in the following order: β-D-dNs < β-D-rNs << β-D-ddNs (Table 2). The reduced modulation in the dNs compared to the ddNs is presumably the result of the opposing influence of the [HO3'-C3'-C4'-O4'] gauche effect in the former, which is also in agreement with the fact that ∆∆H° values (reflecting the overall effect of the nucleobase in for β-D- 2 ddNs) are much larger than the corresponding ∆∆H° values (showing the effect of the base in β-D- 3 dNs) at any pD (Fig 13, Table 6). As O4' is involved in the [HO3'-C3'-C4'-O4'] gauche effect, its electrostatic potential is changed because of σC3'-H3' → σ∗C4'-O4' participation and its ability to be participate to O4'-C1'-N1/9 stereoelectronic n → σ∗ interactions is reduced. In Table 6, ∆∆H° values show that as the O4' C1'-N 10 nucleobase is protonated, the strength of the [HO3'-C3'-C4'-O4'] gauche effect in β-D-dNs increases, whereas it is reverse when the nucleobase is deprotonated. Again, this is explained by the fact that as the O4' becomes more positively charged owing to the enhanced nO4' → σ∗C1'-N interactions with the protonated aglycone, the energy level of σ∗C4'-O4'

99 is lowered (in comparison with the neutral state), which renders the σC3'-H3' → σ∗C4'-O4' participation more efficient in the protonated nucleosides. The modulation of the thermodynamics of N S equilibrium in β-D-rNs is much smaller than ddNs counterparts [the relative degree of pD-dependent flexibility follows the following order: β-D-dNs < β-D-rNs << β-D-ddNs, Table 2] because of the interplay of three gauche effects ([O3'- C3'-C4'-O4'], [O2'-C2'-C1'-O4'] and [O2'-C2'-C1'-N1/9]) in the former modulating the strength of the anomeric effect. Since the [O3'-C3'-C4'-O4'] and [O2'-C2'-C1'-O4'] gauche effects presumably cancel each other (compare ∆H° of 12 and 14 in Table 2), the greater flexibility of rNs than dNs as a function of pD may be attributed to the [O2'-C2'-C1'-N1/9] gauche effect. The magnitude of the 2'-OH effect in o o o β-D-rNs as a function of pD can be estimated by subtracting ΔH p (or ΔHN or ΔHD ) values of β-D- dNs counterparts (∆∆H° in Table 6) from those of β-D-rNs. The ability of 2'-OH to drive the 11 pentofuranose conformation toward N-type pseudorotamers increases as follows: adenin-9-yl = guanin-9-yl <<< uracil-1-yl ≈ thymin-1-yl ≈ cytosin-1-yl ≈ 5-fluorouracil-1-yl (in the N state), adenin-9-yl << guanin-9-yl << cytosin-1-yl (in the P state) and guanin-9-yl <<< uracil-1-yl ≈ thymin-1-yl ≈ 5-fluorouracil-1-yl (in the D state) (see section 4.6 for correlation of the anomeric effect and the overall 2'-OH effect in ribonucleosides and nucleotides, Table 6, Figs 13 and 17). Therefore, in purines 2'-OH stabilizes more S-type conformations than in the pyrimidines at any pD. This is owing to the reduced [O2'-C2'-C1'-N1(pyrimidine)] gauche effect in the former in comparison with the [O2'-C2'-C1'-N9(purine)] gauche effect in the latter. In going from the P to the N and D state, 2'-OH becomes steadily more efficient to drive the sugar conformation in rNs toward S-type conformations, which are reflected in the values of ∆∆H° (Table 6, Figs 13 and 17). 11

4.8.7 Correlation of the electronic nature of aglycone with pseudorotational state The monitoring of chemical shift of aromatic and anomeric protons is a dependable marker to assess the protonation deprotonation equilibrium, which when measured as a function of pH 518 yields the pKa . For each β-D-ddN 30 - 34, β-D-dN 37 and 40 - 45 and β-D-rN 50 - 55, we have plotted the change in the chemical shift of the aromatic proton(s) of the constituent nucleobase as a function of the ∆G° of the two-state N S equilibrium over the whole pD range (Fig 18) to examine if there is an interdependency. A straightline could be fitted through all experimental points of each correlation plot. The Pearson's correlation coefficients (R) are larger than 0.97 for all nucleosides except for β-D-T (43, R = 0.90) and β-D-U (54, R = 0.95) owing to the errors involved 3 in the determination of their pD-dependent ∆G° values as a result of the limited change in J over HH the 278 K - 358 K temperature range. This shows that the electronic changes that take place in the aglycone as a result of pD-dependent change in the protonation deprotonation equilibrium is indeed transmitted to alter the ∆G° of the two-state N S equilibrium over the whole pD range. Interestingly, this transmission of the tunable electronic character of the nucleobase to steer the sugar conformation in β-D-nucleosides takes place through pD-dependent change of the strength of

8.6 9.2 8.1 (B)(C) ) (A) ) ) 8.0 m 8.5 m 8.8 m p p p p p p 7.9 , 8.4 , , K K 8.4 K 8 8 8 7.8 8 8 9 2 8.3 2 2 ( ( ( 8 8.0 6 7.7 8 H δH 8.2 δH δ α-D-ddG (18) α-D-ddC (19) α-D-ddA (17) 7.6 7.6 8.1 -3 -2 -1 0 -3 -2 -1 -3 -2 -1 0 o ΔG o (kJ/mol) ΔG o (kJ/mol) ΔG (kJ/mol)

7.7 8.6 9.2 (E) ) (D) ) (F) 7.6 ) m m m p p p 8.8 p p 8.5 p , 7.5 , , K K K 8 8 9 8 8 8.4 2 7.4 8 2 ( (2 8.4 ( 6 8 H 8 H δ 7.3 δH δ 8.0 α-D-ddT (20) α-D-dA (21) α-D-dG (24) 7.2 8.3 -0.9 -0.6 -0.3 0.0 -5 -4 -3 -2 -1 -5 -4 -3 -2 o ΔG o (kJ/mol) ΔG o (kJ/mol) ΔG (kJ/mol)

8.2 8.7 (H) (I) ) (G) ) ) 8.6 8.1 m m m p p p p p 8.5 p , , , 8.0 K K K 8.4 8 8 7.6 8 9 9 9 2 7.9 2 2 ( ( ( 8.3 6 8 6 H δH 7.8 δH δ 8.2 α-D-dC (25) α-D-dT (26) β-D-ddA (30) 7.7 8.1 -4.4 -4.0 -3.6 -3.2 -2.0 -1.5 -1.0 123456 o ΔG o (kJ/mol) ΔG o (kJ/mol) ΔG (kJ/mol)

9.2 8.4 (K) ) 9.2 (J) ) ) 8.3 (L) m m 8.8 m p p p p p p 8.2 , 8.8 , , K K K 8 8.4 8 8.1 8 8 9 8 8.4 2 2 (2 ( ( 8.0 8 6 6 H 8.0 H δH 8.0 δ δ 7.9 β-D-ddG (31) 5'-OMe-β-D-ddG (32) β-D-ddC (33) 7.6 7.8 246 3456 23456 o o ΔG o (kJ/mol) ΔG (kJ/mol) ΔG (kJ/mol)

7.8 8.6 9.6 ) (M) (N) ) (O) m m p 7.7 ) p 8.8 p m 8.4 p , p ( p c K ( H 8 7.6 2 δ/ 8.0 9 δH b 2 / H ( 8 8.2 δ/ 8 7.5 δH a 7.2 δH δH β-D-ddT (34) β-D-dA (37) β-D-dImb (40) 7.4 8.0 6.4 234 -2 -1 -1 0 ΔG o (kJ/mol) ΔG o (kJ/mol) ΔG o (kJ/mol)

9.3 8.1 7.65 (P) (Q) 7.60 (R) 9.0 8.0 ) ) ) 8.7 m m 7.55 m p p p p 7.9 p (p 8.4 ( ( 7.50 6 6 8 H H δH 8.1 δ 7.8 δ 7.45 7.8 7.40 β-D-dG (41) 7.7 β-D-dC (42) β-D-T (43) 7.5 7.35 -3.2 -2.4 -1.6 -0.8 0.0 -1.2 -1.0 -0.8 -1.8 -1.6 -1.4 -1.2 o o ΔG o (kJ/mol) ΔG (kJ/mol) ΔG (kJ/mol)

Figure 18 (See the legend p. 116)

101

7.9 8.1 8.7 (S) (T) (U) 8.0 8.6 7.8 ) ) ) m 8.5 m m 7.9 p p p (p 8.4 p 7.7 p ( ( 7.8 2 6 6 H 8.3 H H δ/ δ δ 8 7.6 7.7 δH 8.2 β-D-dU (44) 7.6 5-F-β-D-dU (45) 8.1 β-D-A (50) 7.5 8.0 -1.5 -1.4 -1.3 -1.2 -1.1 -1.0 -1.6 -1.5 -1.4 -1.3 -1.2 -1.1 -2.1 -1.4 -0.7 0.0 ΔGo (kJ/mol) ΔGo (kJ/mol) ΔGo (kJ/mol) 8.2 9.0 (V) (W)(X) 7.6 ) ) ) m 8.0 m m p p p 8.5 p p (p ( ( 6 6 8 H H 7.5 δH δ 7.8 δ 8.0 β-D-G (51) β-D-C (52) β-D-rT (53) 7.6 7.4 -3 -2 -1 0 1 1.4 1.5 1.6 1.7 1.8 1.9 2.0 -0.4 -0.3 -0.2 -0.1 o o ΔGo (kJ/mol) ΔG (kJ/mol) ΔG (kJ/mol)

7.9 (Y) (Z) (a) Formycin B (56) 7.8 8.0 8.8 ) ) ) m m m p 7.7 p p p p p 8.4 ( ( ( 6 7.6 6 7.8 2 δH δH δH 7.5 β-D-U (54) 5-F-β-D-U (55) 8.0 7.4 7.6 0.0 0.1 0.2 0.3 0.4 0.1 0.2 0.3 0.4 0.5 0.6 -4 -3 -2 -1 ΔGo (kJ/mol) ΔGo (kJ/mol) ΔGo (kJ/mol)

8.5 8.6 (c)(d) 8.4 (b) 8.5 ) ) 8.4 ) 7.7 m 8.3 m m p p 8.3 p p p p ( 8.2 ( ( 8 8 8.2 6 H δH δH δ 8.1 8.1 8.0 Formycin A (57) 8.0 9-deaza-A (58) ψ-isoC (59) 7.9 7.9 7.6 -4.0 -3.6 -3.2 -2.8 -2.4 -2.0 -5 -4 -3 -4 -3 -2 -1 0 1 o ΔGo (kJ/mol) ΔGo (kJ/mol) ΔG (kJ/mol)

4.7 8.0 (e) (f) ) ) m m p p (p 4.6 (p 7.6 ' 1 6 δH δH

ψ-U (60) 1-Me-ψ-U (61) 4.5 7.2 -2 0 -2 -1 o ΔGo (kJ/mol) ΔG (kJ/mol)

Figure 18: Correlation plots of the chemical shifts of H2/H6/H8/H1' in α-D-ddNs 17 - 20 [Panels (A)-(D)], α-D-dNs 21, 24 - 26 [Panels (E)-(H)], β-D-ddNs 30 - 34 [Panels (I)-(M)], β-D-dNs 37 and 40 - 45 [Panels (N)-(T)], β-D-rNs 50 - 55 [Panels (U)-(Z)] and β-D-C-rNs 56 - 61 [Panels (a)-(f)] (at 298 K unless stated otherwise) as a function of pD- dependent ∆G° of their N S equilibrium. The Pearson's correlation coefficients (R) of the straight lines are > 0.96 for all nucleosides except β-D-T (R = 0.90), β-D-U (R = 0.95), α-D-ddA (R = 0.62), α-D-dA (infinite slope) and formycin B (R ≈ 0.92) owing to the errors involved in the determination of their pD-dependent ∆G° values as a result of the 3 limited change in JHH over the 278 K - 358 K range.

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 102 anomeric and gauche effects. This change of N S equilibrium further predisposes the conformation of the phosphate backbone as discussed in section 7. Thus the single-stranded nucleotide acts like a wire (Nucleotide wire)38,44.

5. Comparison of stereoelectronic effects in α- and β-D-nucleosides β-D-nucleosides are solely chosen by Nature as the ubiquitous building blocks for the storage of information in DNA and RNA. Only a few α-nucleosides are found in Nature519-522. The biological (e.g. antitumor, bacteriostatic, cytostatic) activities of some α-nucleosides have been reported523-530. Séquin531 has postulated that a double helix featuring stabilizing hydrogen bonds through Watson-Crick base pairing and base-base stacking as in the natural DNA helix can be formed between a chain of α-nucleotides and its complementary α-counterpart (chains of opposite polarity) or its complementary β-counterpart (chains of the same polarity). NMR experiments on α- hexadeoxyribonucleotides532,533 have subsequently confirmed Séquin's predictions. It has been shown recently that native β-anomeric sequences containing a single α-anomeric nucleotide (inserted via 3'-3' or 5'-5' phosphodiester linkage) form duplexes whose structures emulate canonical B-DNA534, with subtle differences in stability and local structure dictated by the nature of the nucleobase in the α-nucleotide. A possible relation between the natural selection of β- over α- nucleosides in DNA and a lack of conformational variability of the pentofuranose moieties in the latter has been proposed basing on the relative energies of various pseudorotamers in both series175. However, no experimental comparison of the thermodynamics of the N S equilibrium in α- versus β-nucleosides has supported this theoretical work. We have assessed36,37 for the first time how the interplay of anomeric and gauche effects in α-nucleosides dictates their sugar conformation. We have initially36 considered only α-D-ddNs, owing to the fact that only the nucleobase and 5'CH2OH drive their sugar conformation. We have subsequently37 turned our attention to α-D-dNs in order to analyze the impact of the presence of 3'- OH on the drive of their sugar conformation in comparison with the dideoxy counterparts. In these works, we have uniquely shown that the change of the configuration at C1' results in a drastic modulation of the strengths of stereoelectronic forces as well as of the pD-dependent flexibility of the sugar conformation in the α- versus β-nucleosides.

5.1 The relative magnitude of the anomeric and gauche effects in Neutral state

5.1.1 Anti orientation of the nucleobase in α/β-D-ddN and α/β-D-dN Our 1D-nOe difference experiments in the N state of α-D-ddNs 17 - 20 and α-D-dNs 21 - 26 show that except in the case of α-D-dA (44 % syn) and α-D-dG (65 % syn in the D state), the constituent nucleobases in all α-nucleosides compounds adopt preferentially (67 - 93 %) an anti orientation around the glycosyl torsion (at 298 K or 288 K) (Table 10). Additionally, the extent of the stabilization of anti over syn rotamers is nearly independent of the configuration of the sugar moiety at C1' in α- versus β-D-ddNs and in α- versus β-D-dNs. There are only a few exceptions: In 3'-O-Me-α-D-dA (22), adenyl-9-yl is almost exclusively anti whereas in 3'-OMe-β-D-dA (38) the Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected]

103 population of syn rotamers is higher at any pD, and for α-D-dG (24) (in the D state), in which guanin-9-yl adopts preferentially a syn orientation whereas in β-D-dG (41) the anti and syn rotamers are almost equipopulated. These results have three important implications: (i) The observed differences in the drive of the N S equilibrium as a function of temperature and change of pD (see below) in α-D-ddNs compared to their β-counterparts is not correlated with the preferred orientation of the nucleobase around the C1'-N1/9 bond, since it is independent of the configuration at C1'. (ii) In α-D-dNs, the anti orientation of the nucleobase prevents it from forming a hydrogen bond with 3'-OH. (iii) In spite of the fact that syn orientations of adenyl-9-yl are more stable in α-D- dA (21) than in 3'-OMe-α-D-dA (22) and in 3',5'-diOMe-α-D-dA (23) , the three compounds all have pD-independent ∆H° values (see below) which discards any effect of the orientation of the nucleobase in the former.

5.1.2 The balance of ∆H° and -T∆S° in neutral α- and β-D-N In neutral α-D-ddNs 17 - 20, α-D-dNs 21 - 23, 25, 26 and α-L-dNs 27 - 29, the ∆H° contribution (favouring S-type conformations) to the free-energy ∆G° of the two-state N S equilibrium prevails over the counteracting -T∆S°; Only in the case of α-D-ddG (18) they cooperate, with slightly stronger -T∆S° than ∆H°. However, the absolute contribution of -T∆S° to the free-energy ∆G° of the N S equilibrium in α-nucleosides is negligible (0.2 kJmol-1) only in the case of α-D-ddA (17). Unlike in dNs and rNs, where the bias of the N S equilibrium can be explained by the complex interplay of gauche and anomeric effects, the conformation of the sugar moiety in ddNs is almost exclusively driven by the overall effect of the nucleobase, the contribution of the 5'CH2OH group being minimal. Therefore, α-D-ddNs and β-D-ddNs constitute the systems of choice to examine whether the magnitude of the O4'-C1'-N1/9 anomeric effect is dictated by the configuration of the pentofuranosyl moiety at C1'. In the β-series (30 - 36), the effect of the nucleobase drives toward N-type sugars (and cooperates with the much smaller effect of 5'CH2OH), as evident from the positive ∆H° values (Table 2, Section 4). In α-D-ddNs 17 - 20, owing to the o change of configuration at C1', ΔHN values of the N S equilibrium stabilize S-type conformations, ∗ in agreement with the fact that nO4' →σ C1'-N1/N9 stereoelectronic interactions, favouring S- over N-type conformations, prevail over the counteracting steric effect of the nucleobase (driving to the o o N) as found in the β-anomers. Since ΔHN values are the main contribution to GN values, S-type o o sugars are stabilized at room temperature (vide supra). However, the comparison of ΔGN and ΔHN values of the N S equilibrium in β-D-ddNs 30, 31, 33 and 34 and in α-D-ddNs 17 - 20 shows that the magnitude of the free-energy and enthalpy stabilization of the N-type sugars in the former is ≈ 2 - 6 times and ≈ 2 - 8 times greater, respectively, than the stabilization of S-type conformers in the later, depending on the nature of the nucleobase. This means that the pentofuranose moiety in β-D- ddNs is more prone to adopt multiple conformations as a result of the change of the temperature than the α-counterparts. o o In α-D/L-dNs 21 - 29, ΔHN as well as the overall ΔGN stabilize more S-type conformations than in the parent α-D-ddNs (Table 2). This is in agreement with the observation that in the former both the effect of the nucleobase and the [O3'-C3'-C4'-O4'] gauche effect are expected to prefer S-

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 104 type pseudorotamers, whereas in the later, only the effect of the nucleobase is operating. Larger o negative ΔHN values for α-D/L-dNs 21 - 29 than for β-D/L-dNs 37 - 39, 41 - 43 and 46 - 49 are consistent with the fact that in the former both the [O3'-C3'-C4'-O4'] gauche effect and the effect of the nucleobase stabilize the S-type sugars whereas in the latter the gauche effect counteracts the effect of the nucleobase driving the conformation toward N-type geometries. However, the additional enthalpy or free-energy stabilization of S-type conformations in the α-series is less than -1 o or equal to -2.6 kJmol for almost all compounds. ΔHN of α-D-dC (25) appears to be the only exception, since its sugar conformation is driven much more efficiently toward S-type geometries than that of β-D-dC (42) (Table 2). In view of the above remarks and since both 5'CH2OH and the steric bulk of the nucleobase are conserved structural features in both α-and β-D-ddNs, it is reasonable to attribute the lack of flexibility of the sugar conformation in the former compared with the latter to much less efficient O4'-C1'-N1/9 anomeric effect in the α-series, owing to the change of configuration at C1'.

5.1.3 Weakening of 5'-substituent effect in α- compared with β-nucleosides The weakening of the 5'CH2OMe effect in the α- compared to the β-series is shown by the smaller value of ∆∆H°1d [for 3'-OMe-α-D-dA (22)] compared with ∆∆H°1b [for 3'-OMe-β-D-dA (38)].

5.1.4 3'-gauche effect weakens anomeric effect in α-D-dN compared to α-D-ddN The subtraction (∆∆H° ) of ΔHo of abasic sugar 12 from that of a particular α-D-ddN 17 - 13 N 20 provides an estimate for the magnitude of the overall effect of the nucleobase in α-D-ddNs. Thus the ability of the nucleobase to promote the stabilization of S-type conformations increases as follows (in kJmol-1): guanin-9-yl in 18 (-0.8) < thymin-1-yl in 20 (-1.5) < adenin-9-yl in 17 (-2.1) < cytosin-1-yl in 19 (-3.3). The subtraction (∆∆H° ) of ΔHo of 13 from that of an α-D-dN 21 or 24 - 14 N 26 gives the strength of the effect of the nucleobase in α-D-dNs, assuming that the magnitude of the [HO3'-C3'-C4'-O4'] gauche effect in α-D-dNs and in abasic sugar 13 is the same. The ability of ∆∆H° to stabilize S-type pseudorotamers increases as follows (in kJmol-1): thymin-1-yl (0.1) < 14 guanin-9-yl (-0.4) < adenin-9-yl (-0.9) < cytosin-1-yl (-3.0). Thus, upon substitution of H3" in α-D- ddN 17 - 20 for 3'-OH in α-D-dNs 21 and 24 - 26 the effect of each nucleobase is slightly reduced in the latter compared to the former. This may originate from the change of the electrostatic potential around O4' in the 2'-deoxy compared to the 2',3'-dideoxy counterparts, as it becomes also involved in the [HO3'-C3'-C4'-O4'] gauche effect in the former. A similar trend has also been noticed in going from β-D-ddNs to β-D-dNs. (Section 4). However the weakening of the effect of the nucleobase in the β-D-dNs (calculated by subtracting ∆∆H°3 from ∆∆H°2) is much more pronounced than in the α-D-dNs (subtraction of ∆∆H°14 from ∆∆H°13) [the weakening is: 2.9 kJmol-1 for β-D-dA compared to β-D-ddA but only by 1.2 kJmol-1 for α-D-dA compared α-D-ddA, 1.7 kJmol-1 for β-D-dG compared to β-D-ddG but only by 0.4 kJmol-1 for α-D-dG compared α-D- ddG, 2.8 kJmol-1 for β-D-dC compared to β-D-ddC but only by 0.3 kJmol-1 for α-D-dC compared

105

α-D-ddC, 2.3 kJmol-1 β-D-T compared to β-D-ddT but only 1.6 kJmol-1 for α-D-T compared α-D- ddT].

5.1.5 Weakening of the 3'-gauche effect in α-D/L-dN compared with β-D/L-dN

∆∆H°7 gives an estimate for the [HO3'-C3'-C4'-O4'] gauche effect in abasic sugar 2 (-4.5 kJmol-1) (Table 6). In order to assess the contribution of the [HO3'-C3'-C4'-O4'] gauche effect to the drive of the sugar conformation in α-D-dNs 21 and 24 - 26, we have subtracted (∆∆H°17) from their o -1 ΔH N values those of the parent α-D-ddNs 17 - 20. ∆∆H°17 is weakest in α-D-T (20) (-2.9 kJmol ) and strongest in α-D-dC (19) (-4.2 kJmol-1). Thus the ability of the gauche effect of [HO3'-C3'-C4'- O4'] to stabilize S-type conformations in α-D-dNs is of the same order of magnitude as in abasic sugar 2 but it is also much reduced than in the β-D-dNs counterparts (compare ∆∆H°17 and ∆∆H°10 in Table 6). A qualitative trend between the reduced magnitudes of the effect of the nucleobase and of the [HO3'-C3'-C4'-O4'] gauche effect in α- compared to β-D-dNs emerges from the comparison of ∆∆H°17 and ∆∆H°10, on one hand, of ∆∆H°14 and ∆∆H°3, on the other. The comparison of

∆∆H°18 and ∆∆H°12 values shows that both in α- and in β-nucleosides, the substitution of 3'-OH for 3'-OMe results in a similar small stabilization of S-type conformations.

5.2 The relative magnitude of the anomeric and gauche effects in the ionic states

5.2.1 Virtualy identical pKa values of the nucleobase in α- and β-nucleosides 1 The pKa values, derived from pD-dependent H chemical shifts (Section 3.7) of the constituent nucleobases in α-D-ddNs versus β-D-ddNs counterparts, on one hand, in α-D-dNs versus β-D-dNs, on the other, are almost identical (within ± 0.2 and ± 0.3 pD unit in the ddN and dN series, respectively) (Table 2). This suggests that the electronic character of the nucleobase remains unchanged as the configuration of the pentofuranose at C1' is inverted. It also implies that the influence of the configuration of the sugar moiety at C1' upon the magnitude of the O4'-C1'- N1/9 anomeric effect cannot be attributed to the modulation of the electronegativity of the glycosyl nitrogen in the α- compared to the β-series.

5.2.2 Predominant enthalpy over entropy in the ionic states of α-D-ddN and -dN As in the neutral solution, in the P state of α-D-ddA (17), α-D-ddG (18) and α-D-ddC (19), o ΔHp s responsible for the free-energy stabilization of S-type pseudorotamers, since it overrides the

o counteracting -T ΔSp term. In the D state, enthalpy and entropy have nearly the same strength, but whereas they counteract each other in α-D-ddT (resulting in nearly unbiased pseudorotational equilibrium), in α-D-ddG they both stabilize S-type sugars. In the P and D states, as in the N state, the enthalpy or free-energy stabilization of S-type conformers in α-D-ddNs is much reduced compared to the corresponding preference for N-type forms in the β-D-ddNs counterparts. Both in the P and D states of α-D-dNs 21 - 26, ∆H° prevails over the opposing -T∆S° as reflected in the overall preference for S- over N-type sugars at room temperature.

5.2.3 Weaker anomeric effect in α-D-ddN gives poorer flexibility

In going from the N to the P state of α-D-ddA (17) and α-D-ddC (19) and from the N to the D state of α-D-ddG (18), both ∆H° and -T∆S° contributions to ∆G° of their N S equilibria remain unchanged and the preference of their constituent sugar moieties for S-type conformations remains the same (Table 2). This means that the free-energy of their protonation deprotonation equilibrium is not transmitted to enhance (for 17 and 19) or weaken (for 18) the N-aglycone effect in the P state (D for 18) compared to the neutral pD. This is in sharp contrast with what has been found for the β-D-ddNs counterparts (Section 4.8). pD-dependent conformational preferences in α-D- o ddNs have only been observed upon protonation of guanin-9-yl in α-D-ddG (18) [ ΔΔGP−N = -1.5 -1 o -1 kJmol ] owing to the enhancement of the N-aglycone substituent effect [ ΔΔHP−N = -8.3 kJmol ] and o -1 upon deprotonation of thymin-1-yl ΔΔGD−N = 0.3 kJmol ] owing to the weakening N-aglycone o -1 substituent effect ΔΔHD−N = 0.6 kJmol ]. These ∆∆H° and ∆∆G° values are however much smaller than in the β-counterparts, confirming our above conclusion on relative ability of nucleobase in α- and β-anomers to steer the sugar conformation.

5.2.4 The interplay of pD-independent ∆H° and ∆S° in α-D-dN In all α-D-dNs [except α-D-dG (24)], protonation and deprotonation of the nucleobase has almost no effect on the enthalpy of the two-state N S equilibrium, showing that the efficiencies of the effect of the N-aglycone and of the [HO3'-C3'-C4'-O4'] gauche effect are insensitive to the pD. This result is in agreement with our finding for α-D-ddNs, and it is in sharp contrast with the situation in β-D-dNs, in which a clear pD-dependent modulation of the anomeric effect has been observed, as shown by non negligible ∆∆H° values (Section 4.8). However, whereas in α-D-dA (21) the preference for S-type conformations remains the same at all pDs, owing to the fact that the entropy term is a constant factor, in the pyrimidine nucleoside α-D-dC (25), we have observed an entropy stabilization of S-type conformers in the P compared to o -1 the N state [ (−TΔS)P−N = -1.5 kJmol ]. Conversely, in α-D-T (26), as thymin-9-yl becomes deprotonated, N-type pseudorotamers become less unstable than in the neural solution, which is also o -1 o o the result of an entropy effect [ (−TΔS)D−N = 0.7 kJmol ]. A comparison of ΔΔGP−N and ΔΔGD−N values for α-D-dC, α-D-T and their β-D-counterparts shows that in fact, the entropy in the former modulates more efficiently the bias of the two-state N S equilibrium than the combined pD- dependent enthalpy and entropy in the latter. o In α-D-dG (24), as guanin-9-yl is protonated, both the effect of the N-aglycone [ (−TΔS)P−N = - -1 o -1 6.2 kJmol ) and the counteracting -T∆S° contributions [ (−TΔS)P−N = 3.7 kJmol ] are stronger than o in the N state, and further stabilize S- and N-type sugars, respectively. On the overall, since (ΔH)P−N o > Δ (−TΔS)P−N , the population of S-type pseudorotamers is higher in the P state than in the N state. The strengthening of the N-aglycone effect in going from the N to the P state is however much weaker in α-D-dG (24) than in α-D-ddG (18), showing the effect of 3'-OH in the former (compare o their ΔΔHP−N values).

5.2.5 Poor correlation of the nature of aglycone with pseudorotation in α-D-N

107

The correlation plots of the pD-dependent 1H chemical shifts as a function of ∆G° of the N S equilibria in α-D-ddNs (17 - 20), α-D-dA (21), α-D-dG (24), α-D-dC (25) and α-D-T (26) all give straight lines (Fig 18, Panels (A) - (H)). The Pearson's correlation coefficients for the plots are above 0.96 for α-D-ddNs and above 0.99 for the corresponding α-D-dNs, except for α-D-ddA (17, R = 0.62) and α-D-dA (21), straight line with infinite slope) owing to virtually pD-independent ∆G° values. For α-D-ddNs, α-D-dA (21) and α-D-dG (24), the slopes of the plots are much larger than in the case of β-anomers, showing the less efficient transmission of the pD-tunable electronic character of the nucleobase to drive the sugar conformation. Conversely, the pD-dependent entropy alone is more efficient to drive the sugar conformation in α-D-dC (25) and α-D-T (26) than the combined pD-dependent enthalpy and entropy in β-D-dC (42) and β-D-T (43), since, in absolute value, the slopes of the correlation plots in the former are much smaller than those of the later.

6. Quantitation of the anomeric effects in C- and N-nucleosides We herein report on our initial24,25 and revised32,33 pD-dependent conformational studies on C-nucleosides 56 - 62. Basing on the results of these works, we have developed a new method for the estimation25,33 of the stereoelectronic n →σ∗ anomeric effect in β-D-A, β-D-dA, β-D-G O4' C1'-N9 and β-D-dG through a set of pairwise comparisons of ∆H° of their N S equilibria with those of purine C-nucleosides, which were used as reference points for the quantitation of the counteracting steric effect of the N-nucleobases.

6.1 The anomeric effect in C-nucleosides The interplay of nO4'→σ∗C1'-N9 interactions and of the counteracting steric effect determines the overall effect of a nucleobase upon the sugar conformation in an N-nucleoside. The relative importance of both contributions can be assessed by monitoring the magnitude of the enthalpy of the C1'-substituent effect (∆H°C1'-subst.) in comparison with ∆H° of all steric and stereoelectronic forces driving the N S equilibrium: If the nucleobase acts as a C-substituent, it preferentially takes up a pseudoequatorial orientation, which in turn shifts the N S equilibrium to the S (i.e. negative ∆H° value), whereas predominant stereoelectronic interactions stabilize N-type sugars in C1'-subst. which the nucleobase is pseudoaxial (i.e. positive ∆H° value). If both terms are of equal C1'-subst. strength and cancel each other, ∆H° is zero. C1'-subst. The strength of the (stereoelectronic) anomeric effect alone can only be estimated when the magnitude of the counteracting steric term is known. If one can engineer a nucleoside X in which the nucleobase (isosteric to that of the N-nucleoside in which we want to quantitate the anomeric effect) drives the N S equilibrium exclusively through its steric effect (stereoelectronic interactions being minimal), ∆H° of the N S equilibrium in X can be used as a reference point for the steric effect of the nucleobase in the N-nucleoside. Making use of this observation, we have seeked for a family of nucleosides in which the effect of the nucleobase exerts its influence as much as possible through its steric effect, while the opposing stereoelectronic component remains negligible. This has led us to turn our attention to C-nucleosides, since we expected that substitution of the glycosyl nitrogen in N-nucleosides for C1' in C-nucleosides would result in minimal Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 108 stereoelectronic interactions between O4' and the nucleobase. In the crystal structures of N- nucleosides, the O4'-C1' bond is shorter (by ≈ 0.03 Å) than the C4'-O4' bond, supporting the existence of the O4'-C1'-N1/9 anomeric effect (Section 2.8). In contrast, no such clear trend is observed in the crystal structures of C-nucleosides: in general the difference between O4'-C4' and O4'-C1' bond lengths is much reduced (∆ ≈ 0.01 Å) compared with N-nucleosides: For 4-thio-ψ- uridine271 (three molecular structures in the crystalline cell), ψ-isocytidine hydrochloride272, (α-D) epishowdomycin monohydrate273, 3'-deoxyformycin A hydrochloride274, 5'-chloro-3',5'-dideoxy β- L-formycin A monohydrate275, 1-benzyl-2'-deoxyshowdomycin276, oxoformycin B277, formycin A monohydrate278 and α-pseudouridine monohydrate279, C4'-O4' and C1'-O4' bonds have nearly the same length (∆ ≈ 0.01 Å), whereas C4'-O4' is longer than C1'-O4' in formycin A hydrobromide monohydrate280 (C4'-O4' = 1.47 Å, C1'-O4' = 1.41 Å), formycin B277 (C4'-O4' = 1.46 Å, C1'-O4' = 1.42 Å) and its hydrochloride281 (C4'-O4' = 1.46 Å, C1'-O4' = 1.43 Å). ψ-uridine crystallizes in the form of two structures282. In one of them C4'-O4' (1.45 Å) is longer than C1'-O4' (1.42 Å), but in the other it is the opposite (C4'-O4' = 1.42 Å, C1'-O4' = 1.44 Å). We have shown that in β-D-rNs 50 - 55, the modulation of the bias of the N S equilibrium by the pD of the D2O solution can be attributed to the resulting fine tuning of the effect of the nucleobase (or to the anomeric effect alone, assuming that the steric effect is a constant factor at any pD) and of the [HO2'-C2'-C1'-N1/9] gauche effect (Section 4.8). In ribo C-nucleosides 56 - 62, the [HO2'-C2'-C1'-N1/9] gauche effect is absent. Therefore, any differences observed in the conformational preferences of the sugar moiety in 56 - 62 at various pDs is owing to the different steric and electronic nature of their C1'-aglycones.

6.1.1 Effect of the C1'-pyrimidine aglycone on the conformation of the sugar C-nucleosides differ from natural N-nucleosides by the unique carbon-carbon link between C9 of purines or C5 of pyrimidines and C1' of the pentofuranose sugar. Many of them have been isolated as antibiotics, and have antiviral and/or anticancer activity166,535-540. Their presence in tRNAs is absolutely vital to the biochemical function541,542. N-nucleosides are known to carry the genetic information, but our quantitative and other qualitative studies have also shown that their conformation, and in turn, biological function, can be engineered by changing the nature of either the nucleobase, the C2'-C4' substituents or the electronic character of the endocyclic O4' atom upon their substitution. Very little is known on how the absence of a glycosyl nitrogen in C-nucleosides affects their conformation and to which extent the stereoelectronic partnership of their nucleobase and pentofuranose moieties is affected by this modification. It is necessary to examine carefully whether the absence of N1/9 in the C1'-aglycone really results in the complete cancellation of its stereoelectronic interaction with O4', as suggested by part of the above data on X-ray crystal structures of C-nucleosides. The conformational analysis Ψ-isocytidine (59), its hydrochloride (59b), 1-Me-Ψ-uridine 24 (61) and 1,3-diMe-Ψ-uridine (62) was initially performed in neat D2O solution using the methodology described in Section 3. The thermodynamics (Table 11) of the N S equilibria in 59, 59b, 61 and 62, suggest that: (i) Only in Ψ-isocytidine (59), ∆H°, driving to S-type sugars, prevails Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected]

109 clearly over the counteracting -T∆S°, therefore at 298 K, S-type sugars are preferred. (ii) In the hydrochloride (59b), ∆H° (driving to the N) and counteracting -T∆S° almost cancel each other, and the pseudorotational equilibrium is unbiased. (iii) In 1-Me-Ψ-uridine (61) and 1,3-diMe-Ψ-uridine (62), -T∆S° is the predominant factor driving the conformation to the S over counteracting ∆H°. We have estimated the effect of C1'-aglycones in 59, 59b, 61 and 62 by subtracting (i.e. ∆∆H°30 in Fig 13) ∆H° of 14 from their ∆H°, which gives (in kJmol-1): -2.5 (59) < 0.5 (61) < 1.6 (62) < 3.5 (59b). To calculate ∆∆H°30 the strength of the [O3'-C3'-C4'-O4'] and [O2'-C2'-C1'-O4'] gauche effects and of the 5'CH2OH substituent effect is assumed to be the same in C-nucleosides and in 14. Only isocytosin-5-yl in 59 adopts preferentially a pseudoequatorial orientation in S-type sugars, however this preference is rather small. The comparison of ∆∆H°30 values for 59, 61, 62 and 59b suggests that only in 59, isocytosin-5-yl really acts as a C-substituent to drive the sugar conformation toward S-type pseudorotamers. In all other cases, ∆∆H°30 is slightly positive, which indicates a drive toward N-type conformation, and suggests that the steric effect of the nucleobase (stabilizing S-type sugars) is not the predominant factor contributing to its overall effect. This prompts us to suggest the existence of stereoelectronic interactions (see below) between the pentofuranose sugar and the nucleobase, i.e. the n →σ∗ anomeric effect by analogy with what we found for the N- O4' C1'-C5(sp2) nucleosides counterparts (Section 4).

Table 11. The ΔH˚ and ΔS˚ of N S equilibrium in C-nucleosides 56 - 59, 61 and 62 from our a initial studies at a single pD in native D2O solution

Compounds ΔH˚ ΔS˚ -TΔS˚ ΔG298 %S278 b %S358 b ∆% S b (358-278 K) formycin (A) (57)c -7.7 (0.7) -14.7 (0.9) 4.4 -3.3 83 69 -14 formycin (B) (56)c -7.1 (0.6) -13.4 (0.8) 4.0 -3.1 81 68 -13 9-deaza-A (58)c -5.2 (1.2) -5.4 (2.0) 1.6 -3.6 83 75 -8 Ψ-isoC (59)d -2.1 (0.3) -3.0 (2.0) 0.9 -1.2 63 58 -5 Ψ-isoC hydrochloride (59b)d 3.9 (0.2) 12.5 (0.6) -3.7 0.2 45 55 +10 1-Me-Ψ-U (61)d 0.9 (0.2) 5.1 (0.8) -1.5 -0.6 55 58 +3 1,3-diMe-Ψ-U (62)d 2.0 (0.2) 7.9 (0.5) -2.4 -0.4 52 57 +5 a ΔH°, -TΔS° (at 298 K) and ΔG298 are given in kJ/mol, ΔS° in J/molK. b ∆% S (358-278 K) = %S358 - %S278 shows the change of population of South type sugar owing to the net result of ΔH° and -TΔS° contribution as a function of temperature c Data taken from ref. 25. d Data taken from ref.24.

The remarkable shift of the N S equilibrium of 59b with respect to that of 59 to the N upon protonation of isocytosin-5-yl is easily explained on the basis of the results of our ab initio calculations on 5-methyl-isocytosine and N1H+-5-methyl-isocytosine suggesting that C5=C6 has a higher double bond character (since it is shorter) in 59b than in 59. In 59, C5=C6 π-electrons will easily be delocalized in the pyrimidine ring, and much less accessible for an interaction with nO4' than in 59b, where the protonated guanidine skeleton results in the more localized C5=C6 π- ∗ electrons, leading to a stronger steroelectronic anomeric effect nO4' →σ C1'-C5(sp2) .

The main results of this work are as follows: (i) Pyrimidine C1'-aglycones in 59, 61 and 62 do not provide good reference points for the estimation of the stereoelectronic O4'-C1'-N1 anomeric effect in the pyrimidine N-nucleosides, since they do not act exclusively as C-substituents, as shown ∗ by negative ∆∆H°30 values, owing to non negligible nO4' →σ C1'-C5(sp2) stereoelectronic interactions. (ii) Protonation of isocytosin-5-yl in 59b results in the strengthening of these stereoelectrionic interactions, as in the case of the anomeric effect in N-nucleosides.

6.1.2 Effect of the C1'-purine aglycone on the drive of the sugar conformation Our initial conformational analyses25 on purine C-nucleosides formycin B (56), formycin A (57) and 9-deaza-A (58) have also been performed in D2O solution at neutral pD (see the next sections for the discussion of the protonation state of 9-deaza-A in this work). The resulting thermodynamics of their N S equilibria are presented in Table 11, and their comparison suggests the main following conclusions: (i) The pseudorotational equilibrium in 56 - 58 is driven toward the S-type conformation by the ∆H° term, which prevails over the counteracting -T∆S° contribution to the free-energy and therefore S-type puckered geometries are favoured at room temperature. (ii) The C1'-aglycone in 56 - 58, in sharp contrast with the situation in the pyrimidine C-nucleosides counterparts 59 and 61 - 62, acts as an efficient C-substituent pushing the pseudorotational equilibrium toward S-type conformations. In the purine series, under the present experimental * conditions, stereoelectronic interactions nO4' and the σ C1'-C9(sp2) orbital are rather inefficient in comparison with the counteracting steric effect of the C1'-aglycone (see the next paragraph for the discussion on the case of 9-deaza-A). The strength of the overall C1'-substituent effect upon the drive of the sugar conformation in 56 - 58 has been estimated by subtracting from their ∆H° values -1 -1 that of the abasic sugar 14, which yields: ∆∆H°30 = -8.1 kJmol (formycin A) ≈ -7.5 kJmol (formycin B) > -5.6 kJmol-1 (9-deaza-A). The ">" sign indicates the increased ability of the nucleobase to shift the pseudorotational equilibrium toward S-type conformation through their steric effect. Therefore, in this work, the C1'-aglycone in formycin B (or formycin A) is the most pseudoequatorially oriented among those of 56 - 58 and it represents the best reference point for the estimation of the steric effect of adenin-9-yl in β-D-dA (37) and β-D-A (50) or guanin-9-yl in β-D- dG (41) and β-D-G (51) (see below).

6.1.3 pD-tunable anomeric effect in C-nucleosides The thermodynamics of the N S equilibrium in formycin A (56), formycin B (57), 9- deaza-A (58), Ψ-isoC (59), Ψ-U (60), 1-Me-Ψ-U (61) and 1,3-diMe-Ψ-U (62) have been subsequently determined32 over part or the whole 0.5 - 12.0 pD range in order to examine whether, ∗ as suggested above, stereoelectronic nO4' →σ C1'-C5/9(sp2) interactions also participate to the drive of the conformation of the pentofuranose sugar, in a similar manner to the anomeric effect operating in N-nucleosides (Section 4). We argued, that if an anomeric effect would also exist in C- nucleosides, it should also be reflected in the shift of the bias of their two-state N S equilibria

111 toward more N- and S-type conformations owing to increased and decreased electron-withdrawing character of the nucleobase in the P and D states, respectively, compared with the N state.

6.1.4 Transmission of the nature of the C-aglycone drives the NS equilibrium in C- nucleosides In this revised study, we found that ∆H°, -T∆S° and ∆G° values of the N S equilibrium in all C-nucleosides 56 - 61, including the purine derivatives 56 - 58, are pD-dependent (Table 232, Fig. 11, Panel (F)). Just as in the case of β-D-rNs, the energetics of the protonation deprotonation equilibrium of the nucleobase are transmitted to steer the sugar conformation through the modulation of its overall effect. This means that our previous hypothesis of the minimal contribution of the anomeric effect to the drive of the sugar conformation in purine C-nucleosides 56 - 58 was an oversimplification. Assuming that the steric effect of the nucleobase is constant over the whole pD range, it is possible to attribute the pD-dependent conformational preferences of the ∗ pentofuranose sugar in 56 - 61 to the tuning of the nO4' →σ C1'-C5(sp2) stereoelectronic interactions. The experimental pD-dependent ∆H°, -T∆S° and ∆G° values for 56 - 61 have been fitted to the Henderson-Hasselbach equation to give the limiting values in the P, N and D states and the pKa of the constituent nucleobases, as described in Section 3. These pKa values are nearly identical to those found in the literature34,385-390, and to our independent estimates derived from pD-dependent chemical shifts of aromatic and anomeric protons (Table 2). The efficient communication of stereoelectronic information between constituent nucleobase and pentofuranose is further evidenced by the fact that the plots (Fig 18) of the chemical shifts of the aromatic protons as a function of pD- dependent ∆G° values all give straight lines with high correlation coefficients (> 0.96 except for formycin B, owing to the smaller change in ∆G° values in the alkaline pD range, Table 2). In each of the P, N and D states, the pentofuranose moieties in purine C-nucleosides 56 - 58 prefer more S- type conformations than in the pyrimidine counterparts, in agreement with what we found for β-D- dNs and β-D-rNs (Table 2).

6.1.5 Estimates for the thermodynamics of the N S equilibrium For formycin A (57) and formycin B (56), Ψ-isoC (59), 1-Me-Ψ-U (61) and 1,3-diMe-Ψ-U o o o 24,25 32 (62), almost identical ΔHN , -T ΔSN and ΔGN values were found in our earlier and latest studies -1 o [the largest difference is 1.0 kJmol for ΔHN of formycin B, which is within the standard deviation of both estimates]. For 9-deaza-A (58), the comparison of our earlier estimates (Table 11) and those from the updated study (Table 2) suggested that in the earlier work, the pD of the aqueous solution o o o was in the acidic range, since in the P state (pD ≤ 5.2), ΔHp , -T ΔSp and ΔGp are respectively: -7.4 (σ -1 o o o = 0.8), 3.7 (σ = 0.6) and -3.6 (σ = 0.4) kJmol whereas in the N state ΔHN , -T ΔSN and ΔGN are - 14.2 (σ = 1.5), 9.1 (σ = 1.1) and -5.0 (σ = 0.4) kJmol-1 (Table 2).The above standard deviations, taken at a single pD in the original publication32, are higher than the errors shown in Table 2. The -1 o o difference of 2 kJmol between ΔHp and -T ΔSp values of the earlier and latest work is within the sum of the standard deviations of the estimates.

6.1.6 Enhanced anomeric effect upon protonation of the aglycone

In the P state, formycin B (56), formycin A (57) and 9-deaza-A (58) prefer S-type o o o conformations, owing to cooperative ΔHp and -T ΔSp values (in 56), or to predominant ΔHp over the

o o counteracting -T ΔSp (in 57 and 58). In contrast, in the P state of protonated Ψ-isoC, ΔHp is slighlty

o stronger than the opposing -T ΔSp contribution, which results in the slight stabilization of the N-type conformers. Protonation of formycin B at N3387,388, formycin A at N3385,386, 9-deaza-A at N3 (as shown by our pD-dependent 13C chemical shifts in ref. 34) and of Ψ-isoC at N1389 shifts their N S equilibria toward more N-type forms until full protonation of the heterocycle is achieved, which is reflected in the plateau of ∆G°, ∆H° and -T∆S° values in the P state (Table 2, Fig. 11, Panel (F)). o The additional stabilization of N type sugars in the P compared to the N state is given by ΔΔGP−N -1 o values (in kJmol ): 2.0 (for 56), 1.4 (57), 1.4 (58) and 1.9 (59). The corresponding ΔΔHP−N values (kJmol-1) are as follows: 7.6 (56), 5.7 (57), 6.8 (58) and 6.1 (59). The change of the steric effect of o o the nucleobase in 56 - 59 upon protonation cannot account for these ΔΔHP−N and ΔΔGD−N values: As the nucleobase is protonated, its steric effect will presumably increase, therefore this should shift the pseudorotational equilibrium toward more S-type conformations (with pseudoequatorially oriented nucleobase), however we observe the opposite. This validates our hypothesis of the tuning of nO4' ∗ →σ C1'-C5(sp2) stereoelectronic interactions by the pD of the aqueous solution.

6.1.7 Weaker anomeric effect upon deprotonation of the aglycone Deprotonation of formycin A at N7 does not affect the bias of its N S equilibrium compared with the N state. However, deprotonation of Ψ-isoC (59), at N3, of Ψ-U (60) at N1 and N3, and of 1-Me-Ψ-U (61) at N3 results into the increased population of S-type sugars in comparison with the N state, until a plateau in the D state is reached when the nucleobase is fully deprotonated. The additional stabilization of S-type sugars in the D state compared to the N state is o -1 reflected in the ΔΔGD−N values (in kJmol ): -1.6 (for 59), -1.7 (60) and -0.8 (61). The o -1 corresponding ΔΔGP−N values (kJmol ) are as follows: -1.6 (59), -1.7 (60) and -0.8 (61).

6.1.8 Quantitation of the anomeric effect in C-nucleosides We have estimated the enthalpy of the stereoelectronic interactions operating in C- nucleosides 56 - 62 by subtracting ∆H° of abasic sugar 14 from their ∆H° values (∆∆H°30). The results of these subtractions are compiled in Table 8. The perusal of ∆∆H°30 values shows that as purine C-nucleosides 56 - 58 become protonated, the contribution of stereoelectronic interactions to the overall effect of the C1'-aglycone increases, whereas the effect of deprotonation is insignificant. For pyrimidine C-nucleosides 60 - 62 in the N state, the steric effect is overriden by the anomeric ∗ effect, whereas in the D state it is reverse. Finally, in protonated Ψ-isoC (59), nO4' →σ C1'-C5/9(sp2) interactions predominate, but as the base becomes neutral and deprotonated, their magnitude is steadily reduced and they become overriden by the counteracting anomeric effect.

6.1.9 Comparison of pD-induced flexibility in C- and N-nucleosides The extent of the stabilization of the N-type (or S-type) pseudorotamers in 56 - 61 upon o protonation (deprotonation) is dictated by the electronic character of the nucleobase: ΔΔGP−N values Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected]

113 are of the same order of magnitude in purine C-nucleosides 56 - 58 and in the N-counterparts β-D-A o is negligible for 56 and 57 whereas it is -1.3 kJmol-1 in β-D- (50) and β-D-G (51). However, ΔΔGD−N G (51). At the other end of the flexibility scale, o ΔΔGP−N for Ψ-isoC (59) is ≈ 5 times larger than for o for Ψ-U (60) is ≈ 8.5 times larger than for β-D-U (54). β-D-C (52) and ΔΔGD−N

6.1.10 Correlation of the effect of the aglycone and its electronic nature Formycin A (57) and 9-deaza-A (58) differ only in the nature of the fused five-membered ring. The more efficient deactivating pyrrazolo ring in the former compared with pyrrolo ring in the later is evident from the lower pKa value of the nucleobase in 57 with respect to 58, and results in ∗ stronger nO4' →σ C1'-C5(sp2) stereoelectronic interactions in 57 than in 58 (i.e. less negative ∆G° and ∆H°, Table 2). Although in the N state, the anomeric effect in formycin B (56) and formycin A (57) o and o values has the same strength (compare their ΔGN ΔHN ), in the P state the stabilization of the N3H+ charge is more efficient through delocalization in the amidine moiety in the later than in the pyrimidone ring in the former, therefore the fused pyrrazolo ring will be more electron-deficient in ∗ 56 than in 57 and conversely nO4' →σ C1'-C5(sp2) stereoelectronic interactions are stronger in 56 than o and o of formycin B and A are the same, they drive the sugar conformation in 57. Whereas ΔGN ΔHN in Ψ-U (60) more to the N than in Ψ-isoC (59), showing that replacing the amidine function for an amide function in the six-membered ring of 60 compared with 59 has much more effect on the sugar conformation than when this change is made in the remote fused pyrimidone ring of 56 compared with pyrimidine in 57, which are both not directly attached at C1'. The comparison of ∆G° and ∆H° of the N S equilibrium in Ψ-isoC (59) and Ψ-U (60) shows that they stabilize more S-type conformations in in the former than in the latter, owing to more electron-rich pyrimidine ring in 59 than in 60. Interestingly, the latest study from our laboratory40 on the preferred conformation of the constituent pentofuranose sugar in benzene, pyridine and pyrimidine C-nucleosides in aqueous solution has allowed to establish a clear qualitative correlation between the electronic character of the C-aglycone and the magnitude of the O4'-C1'-C(aglycone) anomeric effect (Table 12): As the electron-deficient character of the C-aglycone increases (i.e. in the benzene derivatives, in the order: anilino (119) < benzyl (117) < α-naphthyl (118)), the n →σ∗ stereoelectronic O4' C1'-C(aglycone,sp2) interactions are strengthened and counteract more and more efficiently the steric effect, as indicated by the more positive ∆H° for the drive of the two-state N S equilibrium toward N-type conformation. In the pyridine derivatives, it was shown that the effect of a substituent in the para position (with respect to the sugar moiety) upon the electronic character of the pyridine ring is negligible, since negative ∆H° values of the N S equilibrium in compounds 120 and 122 stabilize S-type pseudorotamers to the same extent as C1'-benzyl in C-nucleoside 117, owing to relatively weak nO4' ∗ →σ C1'-C(aglycone,sp2) orbital mixing. In contrast, it was also found that the inductive (-I) effect of the ortho substituent promotes a strong O4'-C1'-C(aglycone) anomeric effect, which cancels the counteracting steric effect, as evident from negligible ∆H° values for compounds 121 and 123.

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 114 6.2 Quantitation of anomeric effect in N-nucleosides using C-nucleoside as reference The drive of the N S equilibrium in β-D-rNs 50 - 55 toward S-type conformations is the result of the interplay of six steric and stereoelectronic forces: (i) The effect of 5'CH2OH, (ii) The stereoelectronic component to the overall effect of the nucleobase (i.e. n →σ∗ stereoelectronic O4' C1'-N9 interactions), (iii) The counteracting steric effect of the nucleobase, (iv) the gauche effect of [HO3'- C3'-C4'-O4'], (v) the gauche effect of [O2'-C2'-C1'-O4'] and (vi) the gauche effect of [O2'-C2'-C1'- N9] (Section 4). Therefore, the anomeric effect of adenin-9-yl in β-D-A (50) or guanin-9-yl in β-D- G (51) can be estimated using Eq 13: AE of adenin-9-yl in β-D-A (50) or guanin-9-yl in β-D-G (51) =

∆H°(β-D-A or β-D-G) - [∆H°(14) + ∆H°GE[O2'-C2'-C1'-N9] + (∆H°ref. C-nucl. - ∆H°(14))] .....Eq 13

In Eq 13, ∆H°GE[O2'-C2'-C1'-N9] represents the strength of the [O2'-C2'-C1'-N9] gauche effect in β-D-A

(50) or β-D-G (51). The subtraction of ∆H°(14) from the experimental ∆H° value of 50 or 51 [∆H°(β-

D-A or β-D-G)] gives an estimate for the resultant of the effect of the nucleobase (stereoelectronic + steric) and the ∆H°GE[O2'-C2'-C1'-N9] gauche effect in 50 or 51.

Our preliminary conformational study25 suggested that among purine C-nucleosides 56 - 58, the nucleobase in formycin B (56) and A (57) prefers more pseudoequatorial orientations in S-type conformations than 9-deaza-adenin-9-yl in 9-deaza-A (58), which was experimentally evidenced by the larger negative ∆H° values for 56 and 57 in comparison with 58 (Table 11). Therefore, we have initially quantitated25 the anomeric effect of adenin-9-yl in β-D-A (50) and of guanin-9-yl in β-D-G (51) in their N state using the average (-7.4 kJmol-1, Table 11) of the ∆H° values of the N S equilibrium in formycin B (56) and A (57) as ∆H°ref. C-nucl. in Eq 13. In that work, ∆H°GE[O2'-C2'-C1'- -1 N9] (-6.3 kJmol ) was derived from a regression analysis similar to regression (A), based on a total set of 30 compounds, including β-D-dAMP (64), β-D-AMP (74), β-D-dAMPEt (69), β-D-dGMP (65), β-D-GMP (75), β-D-dGMPEt (80), β-D-dCMP (66), β-D-CMP (76), β-D-dCMPEt (71), β-D- dUMP (68), β-D-UMP (78), β-D-TMP (67) and β-D-TMPEt (72). Eq 13 was therefore rewritten as Eq 13a: AE of adenin-9-yl in β-D-A (50) or guanin-9-yl in β-D-G (51) in the N state =

∆H°(β-D-A or β-D-G) - [0.4 - 6.3 -7.8] .....Eq 13a -1 -1 Using -4.6 kJmol and -3.2 kJmol as estimates for ∆H°(β-D-A or β-D-G) of β-D-A (50) and β-D-G (51) (these values are in agreement with ∆H° values in the N state in Table 2, within the error of the estimates), we found that the strength of the AE of adenin-9-yl in β-D-A (50) is slightly weaker (9.1 kJmol-1) than that of guanin-9-yl in β-D-G (51) (10.5 kJmol-1). In Eq 13a, we considered that formycin A and B in their N state constitute the best reference points for the quantitation of the steric effect of an N-nucleobase. However, as discussed in Section 6.1, we have recently experimentally evidenced32 that the extent of the stabilization of S-type conformations in 56 - 58 is dictated by the protonation state of the constituent nucleobase, therefore the most pseudoequatorially oriented nucleobase is 9-deaza-adenin-9-yl in the N state of 9-deaza-A

115

o = -14.2 kJmol-1, in the pD range from 8.8 to 12.0, (58) ( ΔHN Table 2), not those of formycin A and B in the N state. Taking this fact into account, we have recently revised the estimates from Eq 13a using Eq 13b: AE of adenin-9-yl in β-D-A (50) or guanin-9-yl in β-D-G (51) at a certain pD: pD = ∆H° - [∆H° + ∆H° + ( ΔHo - ∆H° )] .... Eq 13b pD(β-D-A or β-D-G) (14) GEpD[O2'-C2'-C1'-N9] N (58) (14)

NH2 Br

4 2 N 5 3 3 1 6 2 4 6 OH OH OH 1 OH 5 O O O O

OH OH OH OH OH OH OH OH 117 118 119 120

O H 6 1N HN 2 N O 5 1 3 5 6 1NH 4 3 6 OH 2 OH 5 4 OH 4 32 OH N F O NH O O O O

OH OH OH OH OH OH OH OH 121 122 123 124

Table 12: Dependence of the Thermodynamicsa of the Two State N S equilibrium upon the Electronic Naturea of the C-aglycone. Benzene- Pyridine- Pyrimidine- derivativesb derivativesb derivativesb (117) (118) (119) (120) (121) (122) (123) (59) (60) ΔH° -5.4 0.4 -7.3 -4.4 0.4 -5.6 -0.1 -1.8 0.6 (0.8) (0.6) (1.0) (0.6) (0.5) (0.9) (0.5) (0.3) (0.2) ΔS° -8.7 2.7 -13.1 -4.4 5.2 -7.4 0.7 -2.0 4.0 (2.7) (1.7) (1.0) (2.0) (1.7) (2.0) (1.7) (1.1) (1.1) -ΤΔS° 2.6 -0.8 3.9 1.3 -1.5 2.2 -0.2 0.4 -1.2 (0.8) (0.5) (0.8) (0.6) (0.5) (0.6) (0.5) (0.3) (0.3) ∆G298 -2.8 -0.4 -3.4 -3.1 -1.1 -3.4 -0.3 -1.4 -0.6 (0.5) (0.5) (0.4) (0.4) (0.1) (0.5) (0.2) (0.2) (0.1) %S298 76 53 80 78 60 80 53 63 57 Actual -5.8 0.0 -7.7 -4.8 0.0 -6.0 -0.5 -2.2 0.3 AE c a The ΔH°, -ΤΔS° (at 298 K) and ΔG298 are in kJ/mol. The standard deviations (σ) are in parentheses. For 59 (pD = 7.0) and 60 (pD = 6.9), data are taken from ref.32. b In this work, the steric contribution of the substituents in ΔH˚ could not be dissected because of the unavailability of the corresponding saturated system. c The actual anomeric effect (AE) was obtained by a simple subtraction (i.e. ∆∆H° ) of the ΔH° of the N S pseudorotational drive of 1-deoxy-β- 30 D-ribopentofuranose (14) (ΔH˚ = 0.4 kJ/mol)20 from the ΔH° of a specific C-nucleoside.

1.0 4 (A) (B) 0.5 2 1 1 1 1 H H 0 Δ 0.0 Δ Δ Δ -0.5 -2

-1.0 -4 024681012 024681012 pD pD

-7 -5 (C) (D) -8 -10 0 0 1 1 H H Δ Δ -15 Δ -9 Δ -20 -10 -25 024681012 024681012 pD pD

-7 -5 (F) 1 (E) 1 1 1 H H Δ -8 Δ Δ Δ -10 + + 0 0 1 1 H -9 H -15 Δ Δ Δ Δ -10 -20 024681012 024681012 pD pD

24 40 (G) 35 (H) 22 ) ) 30 A G ( ( 25 E20 E A A20 18 15

16 10 024681012 024681012 pD pD

19 22 (I) (J) 18 20 ) ) A G18 d 17 d ( ( E E16 A16 A 15 14

14 12 024681012 024681012 pD pD Figure 19. Estimates (kJmol-1) for gauche and anomeric effects driving the sugar conformation in β-D-dA (37), β-D- A (50), β-D-dG (41) and β-D-G (51) from pairwise comparisons (Fig 13, Table 6, Section 6.2). The pD-dependent strength of the 2'-OH effect (i.e. [HO2'-C2'-C1'-N9] + [HO2'-C2'-C1'-O4'] gauche effects) in β-D-A [Panel (A)] and β- D-G [Panel (B)] was estimated from ∆∆H°11. The pD-dependent strength of the [HO3'-C3'-C4'-O4'] gauche effect in β- D-dA [Panel (C)] and β-D-dG [Panel (D)] is reflected in ∆∆H°10 values. The strength of the pD-dependent [O2'-C2'- C1'-N9] gauche effect in β-D-A [Panel (E)] and β-D-G [Panel (F)] was calculated by adding the respective ∆∆H°10 and ∆∆H°11 at each pD. The modulation of the strength of the anomeric effect of adenin-9-yl in β-D-A and β-D-dA and of guanin-9-yl in β-D-G and β-D-dG by the pD of the aqueous solution is shown in Panels (G), (I), (H) and (J) respectively.

Eq 13b allows to calculate the AE in 50 and 51 as a function of pD. ∆H°pD(β-D-A or β-D-G) represents the enthalpy of the N S equilibrium of 50 or 51 at a certain pD in the range 0.6 - 12.0. ∆H° represents the magnitude of the [O2'-C2'-C1'-N9] gauche effect in 50 or 51 GEpD[O2'-C2'-C1'-N9] at a certain pD, which has been calculated using a procedure consisting of four steps: (i) We have

117 first determined the strength of the overall 2'-OH effect (i.e. [O2'-C2'-C1'-O4'] + [O2'-C2'-C1'-N9] gauche effects) in 50 and 51 by subtracting from their experimental ∆H° values at each pD in the 0.6 - 12.0 range those of their 2'-deoxy counterparts β-D-dA (37) and β-D-dG (40), respectively (∆∆H°11 in Fig 13 and Table 6, Fig 19 for the plot of the change of ∆∆H°11 as a function of pD). (ii) We have subsequently estimated the change in the strength of the [O3'-C3'-C4'-O4'] gauche effect in

β-D-dA (37) and β-D-dG (40) by subtracting from their experimental ∆H° values those from their 2',3'-dideoxy counterparts β-D-ddA (30) and β-D-ddG (31) (∆∆H°10 in Fig 13, Table 6 and Fig 19 for the plot at each pD). (iii) An estimate for the [O2'-C2'-C1'-O4'] gauche effect in 50 and 51 has been subsequently obtained from pD-dependent -∆∆H°10 values (Table 6, Fig 13 and Fig 19) assuming that it has an equal magnitude (and opposite sign) to the [O4'-C4'-C3'-O3'] gauche effect in β-D-dNs. (iv) By adding the pD-dependent values for ∆∆H°10 and ∆∆H°11, the actual magnitude of the [O2'-C2'-C1'-N9] gauche effect has been quantitated (Fig 19). On the basis of these estimates, Eq 13b has been used to give the magnitude of the stereoelectronic O4'-C1'-N9 anomeric effect of adenin-9-yl in 50 and of guanin-9-yl in 51 over the whole pD range from 0.6 to 12.0 (Fig 19). Thus, the AE varies from 23.4 to 17.7 kJmol-1 from pD 1.2 to 7.0 for adenosine (50) and changes from 37.5 to 15.6 kJmol-1 from pD 0.6 to 11.6 for guanosine (51). In β-D-dA (37) and β-D-dG (41), the drive of the N S equilibrium toward S-type conformations is the result of the interplay of only four stereoelectronic forces: (i) The effect of 5'CH2OH, (ii) The stereoelectronic component to the overall effect of the nucleobase (i.e. nO4' ∗ →σ C1'-N9 stereoelectronic interactions), (iii) The counteracting steric effect of the nucleobase, (iv) the gauche effect of [HO3'-C3'-C4'-O4'] fragment. In order to quantitate the stereoelectronic AE of adenin-9-yl in 37 and of guanin-9-yl in 41, (ii), it is therefore necessary to subtract from the ∆H° values of their two-state N S equilibrium the contributions from (i), (iii) and (iv), according to Eq 14: AE of 9-adeninyl in β-D-dA (37) or 9-guaninyl in β-D-dG (41) at a certain pD = ∆H°pD(β-D-dA or β- o D-dG) - [∆H°(13) + ( ΔHN (58) - ∆H°(14))]..... Eq 14 In Eq 14, ∆H°pD(β-D-dA or β-D-dG) represents the experimental ∆H° value of the N S equilibrium in β-D-dA (37) and β-D-dG (41) at the pD of interest, whereas ∆H°(13) and ∆H°(14) denote the experimental ∆H° value for abasic sugars 13 and 14, respectively. The subtraction of ∆H° value of 13 from ∆H°pD(β-D-dA or β-D-dG) (referred as by ∆∆H°3 in Table 6) gives an estimate for the pD-dependent overall effect of the nucleobase in 37 or 41. During this quantitation, the same reference point (9-deazaadenin-9-yl in 9-deaza-A (58) in the N state) has been used to estimate the steric effect of the nucleobase in 37 and 41. The AE of adenin-9-yl in β-D-dA (37) is weakened from 18.0 to 14.8 kJmol-1 in going from pD 0.9 to pD 7.0, whereas the AE of guanin-9-yl in β-D- dG (41) is reduced from 20.7 kJmol-1 to 13.8 kJmol-1 from pD 0.9 to pD 11.6.

An alternative strategy based on the cancellation of the stereoelectronic interactions between the cyclopentane ring and the nucleobase in a carbocylic nucleoside to estimate the anomeric effect of the nucleobase in N-nucleosides

In a recent work543, an alternative strategy has been developed to estimate the strength of the stereoelectronic anomeric effect in purine nucleosides. The thermodynamics of the two-state N S equilibrium (suggested by ab initio calculations) in the pyrazolo[4,3-c]pyridine-carbaribo-C- nucleoside (124) were estimated using our methodology. It was found that the cyclopentane ring in 124 adopts preferentially S-type conformation (∆H° = -11.6 kJmol-1) as does anionic 9- -1 32 -1 deazadenosine (∆H° = -14.2 kJmol ) . The steric effect (∆H°steric = -12.0 kJmol ) of the C1'- pyrazolo[4,3-c]pyridine aglycone in 124 was estimated by subtracting ∆H° of 14 from that of 124. The C-nucleobase in 124 was assumed to be isosteric to adenin-9-yl in β-D-ddA, β-D-dA and β-D- rA and to guanin-9-yl in β-D-ddG, β-D-dG and β-D-rG. Subtraction of ∆H° of 12 and of ∆H°steric from ∆H° of β-D-ddA and β-D-ddG yielded estimates for the anomeric effect (16.4 kJmol-1) of adenin-9-yl and guanin-9-yl in these ddNs. The anomeric effect of adenin-9-yl in β-D-dA (14.7 kJmol-1) and of guanin-9-yl in β-D-dG (16.4 kJmol-1) was subsequently estimated by subtracting ∆H° of 124 and the strength of the [HO3'-C3'-C4'-O4'] gauche effect from ∆H° of each dN. The anomeric effect of adenin-9-yl in β-D-A (13.9 kJmol-1) and of guanin-9-yl in β-D-G (15.3 kJmol-1) was calculated by subtracting ∆H° of 124 and the strength of the [HO2'-C2'-C1'-N9] gauche effect from ∆H° of each rN. These estimates are of the same order of magnitude as our own33, except for adenosine, where a difference of 3.8 kJmol-1 has been found. This means that the 9-deazaadenin-9- yl aglycone in the D state indeed takes up a maximal pseudoequatorial orientation in 57 and hence serves a correct reference point for the estimation of the steric of purine N-nucleobase.

7. The interdependency of the sugar and phosphate conformation The sugar moiety, the nucleobase and the phosphate backbone constitute the three structural elements making ploynucleotide chains. We have discussed in Sections 2 - 6 how in nucleosides the change of the electronic character (for instance upon the change of the pD of the aqueous solution or complexation with metal ions) and steric bulk of the substituents at C1' - C5' allows to engineer certain conformational preferences of the constituent pentofuranose sugar, as the result of tunable stereoelectronic gauche and anomeric effects. The phosphate backbone is not itself an isolated structural element, as suggested by the qualitative correlation between preferred orientation around the ε torsion and sugar puckering modes in mononucleotides and RNA trimers. We report the results of our investigations23,28 that have adressed the following fundamental questions: Does the sugar conformation dictate the phosphate backbone torsions? Is there any preferred phosphate torsion that steers the sugar conformation in a certain manner? Are there any correlated interdependencies of endocyclic sugar torsions with the preferred phosphate torsions? In order to further understand the forces that govern the stabilization of the tertiary structure of oligo- and polynucleotides, we have examined and dissected the nature of fundamental intranucleotidyl interactions that contribute to the drive of the sugar-phosphate backbone in DNA and RNA using simple model systems, i.e. nucleoside 3'-ethylphosphates, both in the 2'-deoxy (69 - 73) and ribo series (79 - 83) in which the effect of internucleotidyl base-base stacking on the drive of the sugar-phosphate backbone is completely eliminated.

119

7.1 Methods to assess the preferred conformation across the phosphate backbone 3 3 3 The dependence of JC2'P3', JC4'P3' and JH3'P3' coupling constants on the torsion angle ε (Fig. 20) is described by the following Karplus-type equations223,435,544-546 (Eqs 15a - 15c): 3J = 15.3 cos2(ε + 120)- 6.2 cos(ε + 120) + 1.5 ..... Eq 15a H3'P3' 3J = 9.1 cos2(ε)- 1.9 cos(ε) + 0.8 ..... Eq15b C4'P3' 3J = 9.1 cos2(ε - 120)- 1.9 cos(ε - 120) + 0.8 ..... Eq 15c C2'P3' In Eqs 15a - 15c, a perfect trigonal symmetry for the position of C2', C4' and H3' with respect to the C3'-O3' bond is assumed. In our interpretation23,28 of the experimentally measured 3J , H3'P3' 3J and 3J coupling constants for nucleosides 3'-monophosphates 64 - 73 and their 3'- C4'P3' C2'P3' ethylphosphates 74 - 83, we have considered a two-state εt ε- equilibrium for the following reasons: (i) ε+ rotamers are not found in crystal structures of nucleotides, suggesting that this state is forbidden435 on account of steric and electrostatic repulsions547-550 between O4' and phosphoryl + 3 223,435 oxygen. (ii) Additionally, for ε rotamers, one expects JH3'P3' ≈ 23 Hz , but our experimental values23,28 are ≈ 7 - 8 Hz, therefore it is likely that the population of ε+ rotamers is negligible. - 551 4 A rough estimate for the population of ε rotamers can also be obtained from JH2'P3' using - 4 4 4 Eq 16: x(ε ) = JH2'P (obs) / JH2'P (-) .... Eq 16, Where JH2'P (obs) represents the 4 4 experimental value of JH2'P coupling constant for the compound of interest and JH2'P (-) designates the corresponding coupling constant in a pure gauche- (ε-) conformation (i.e. 2.3 Hz551). 3 3 3 The experimental time-averaged JH5'P5', JH5"P5' and JC4'P5' coupling constants can be used to estimate the population of the staggered rotamers around the C5'-O5' bond via simple linear relationships435 (Eqs 17a - 17b): t 3 3 % β = 100 x [25.5 - ( JH5'P5' - JH5"P5')] / 20.5 .... Eq 17a t 3 % β = 100 x ( JC4'P5' - 0.73 / 10.27) .... Eq 17b When P5'-O5'-C5'-C4'-H4' lie in the same plane and form a W-type conformation551-553 [i.e. t + 3 (β , γ ) rotamer], the JH4'P5' coupling constant is approximately 3 Hz and can be used as a marker to recognize this conformer, since in all other cases the coupling constant will be much reduced. 3 3 + + - - JH4'H5' and JH4'H5" are translated into the populations of the staggered γ [x(γ )], γ [x(γ )] and γt [x(γt)] rotamers554 using Eqs 18a - 18d which have been parametrized on the basis of crystallographic data: 3 + - t JH4'H5' = 2.4 x(γ ) + 10.6 x(γ ) + 2.6 x(γ ) .... Eq 18a 3 + - t JH4'H5" = 1.3 x(γ ) + 3.8 x(γ ) + 10.5 x(γ ) .... Eq 18b x(γ+) + x(γ-) + x(γt) = 1 .... Eq 18c + 3 3 % γ = 100 x [13.3 - ( JH4'H5' + JH4'H5")] / 9.7 .... Eq 18d Using Eq. 19, the value of the torsion angle δ [C5'-C4'-C3'-O3'] can be derived from that of ν3 for each of the N- and S-type pseudorotamers, since ν3 is itself related to the respective PN, Ψm(N), 17 PS and Ψm(S) values via Eq. 6a. Finally, owing to the low natural abundance of O (0.04 %), the preferred α and ζ conformations in nucleotides cannot be determined from spin-spin coupling constants: δ ≈ ν3 + 120° .... Eq 19

7.2 No correlation of sugar and phosphate conformation in 2'-dN-3'-ethylphosphates We have elucidated33 the conformational preferences of the pentofuranose moieties in β-D- dNMPs (64 - 68) and their 3'-ethylphosphates (69 - 73) using the methodology described in Section 3. The thermodynamics of the N S equilibrium in 64 - 73 in the neutral solution are compiled in Table 3. In order to assess the preferred orientation around the ε torsion in β-D-dNMPEts 69 - 73 in the 278 K - 358 K range, we have developed the program epsilon, which allows to translate 3 3 3 experimental JC2'P3', JC4'P3' and JH3'P3' coupling constants into the geometries and relative populations of the rotamers engaged in the two-state εt ε- equilibrium. These vicinal coupling constants did not significantly change (≤ 0.5 Hz) as the temperature was raised from 278 K to 358 K, as the result of nearly temperature-independent mole fractions of the εt and ε- rotamers (≈ 1:1 ratio). Similarly, 3J and 3J were nearly the same over the whole temperature range, CH2P3' CH3P3' suggesting that the population of βt rotamers (≈ 50%) is not affected by the temperature. The comparative analysis of ∆H°, -T∆S° and ∆G° values of 64 - 73 in Table 3 leads to the main following conclusions: (i) In all cases, ∆H° prevails over the counteracting -T∆S° term and it is the main factor responsible for the overall (∆G°) stabilization of S-type pseudorotamers at 298 K. (ii) S- type pseudorotamers in β-D-dNMPs 64 - 68 are slightly more preferred than the β-D-dNs 37 and 41 - 44 counterparts as shown by more negative ∆G° values for the former in comparison with the - latter. This can be attributed to the slightly more electronegative 3'-OPO3H group in the latter with respect to 3'-OH in the former. The additional stabilization of S-type pseudorotamers in 64 - 68 -1/-2 through [ HO3PO3'-C3'-C4'-O4'] gauche effect is shown by the ∆∆H°22 values in Table 7 (Fig 13) which are slightly nucleobase-dependent and within the range from -2.5 to -1.2 kJmol-1. (iii) Similarly, S-type pseudorotamers are slightly more favoured in β-D-dNMPEts 69 - 73 than in β-D- - dNs 37 and 41 - 44. This stabilization throught the stronger [ EtO3PO3'-C3'-C4'-O4'] gauche effect -1/-2 is shown in the ∆∆H°23 values in Table 7 (Fig 13). (iv) For each nucleobase, the [ HO3PO3'-C3'- C4'-O4'] gauche effect in 64 - 68 has been estimated by subtracting from their experimental ∆H° -1 values those of the corresponding β-D-ddNs 30, 31 and 33 - 35, yielding: ∆∆H°20 (kJmol ) = -8.9 (adenin-9-yl), -7.5 (guanin-9-yl) = -9.8 (cytosin-1-yl), -8.0 (thymin-1-yl), -8.4 (uracil-1-yl). Thus -1 ∆∆H°20 varies by ≈ ± 1kJmol from the average value depending upon the nature of the - nucleobase. (v) Using the same strategy, we have quantitated the [ EtO3PO3'-C3'-C4'-O4'] gauche -1 effect in 69 - 73: ∆∆H°21 (kJmol ) = -9.0 (adenin-9-yl), -8.2 (guanin-9-yl) = -10.2 (cytosin-1-yl), - 8.4 (thymin-1-yl), -8.4 (uracil-1-yl). Thus the comparison of ∆∆H°20 with ∆∆H°21 values shows that -1/-2 - the stereoelectronic gauche effects within [ HO3PO3'-C3'-C4'-O4'] and [ EtO3PO3'-C3'-C4'-O4'] fragments operate with nearly the same magnitude in the 2'-deoxynucleotides 64 - 68 and 69 - 73. This is also proven by the negligible ∆∆H°24 values in Table 7. We have also estimated the magnitudes of the gauche and anomeric effects operating in all β- D/L-nucleosides (i.e. the dataset used for regression analysis (B))

121 as well as β-D-dNMPs 64 - 68 and β-D-dNMPEts 69 - 73 (Table 5, Section 4.1) from regression (E), which has been performed using a dataset consisting of 40 experimental ∆H° values assuming 5' identical strengths for Torsion angle β (P5'-O5'-C5'-C4') -1/-2 A the [ HO PO3'-C3'- PO3 3 H O H H5' H5" 5' H5" H N 5" 5 C4'-O4'] and [- O ' O P PO 1 3 3 EtO3PO3'-C3'-C4'-O4'] (n-1) C4' C4' C4' t - + gauche effects in 64 - OH β = 180º β = -60º β = +60º ------O 68 and 69 - 73. The O O Torsion angle γ (O5'-C5'-C4'-C3') α N estimates resulting from P NH γ− γ+ -O O O O O5' 5' 5' regression analysis (E), β H O C3' H O4' C N N 4' 4' 4' 3' O χ NH2 γt γ its correlation H H H5' H H5' H5" (n) 5' 5" 5" coefficient and the δ C O4' H4' ε 3' OH t - + standard error on the ------O γ = 180º γ = -60º γ = +60º O ζ estimates are virtually P C -O O Torsion angle ε (C4'-C3'-O3'-P) N the same as of H O H3' H3' 3' O3P PO3 regression (B). The (n+1) - ε average [3'-phosphate- C C C C C C O OH 2' 4' 2' 4' 2' 4' t ε+ ε PO3 C3'-C4'-O4'] gauche εt = 180º ε- = -60º ε+ = +60º 3' effect is about ≈ -8.3 kJmol-1, i.e. -2.0 kJmol- Figure 20. The description of the phosphate backbone conformation in 1 stronger than the nucleosides and nucleotides by the α [(n-1)O5'-P5'-O5'-C5'], β [P5'-O5'-C5'-C4'], γ [O5'-C5'-C4'-C3'], δ [C5'-C4'-C3'-O3'], ε [C4'-C3'-O3'-P3'] and ζ [C3'-O3'-P3'- [HO3'-C3'-C4'-O4'] O5'(n+1)] torsion angles. counterpart. Thus this work shows that: (i) there is no straightforward correlation between preferred orientation across C3'-O3' bond and most stable sugar puckering mode in the 2'-deoxynucleotides, (ii) the additional stabilization of S-type pseudorotamers in 64 - 68 and 69 - 73 with respect to β-D-dNs 37 and 41 - 44 is nearly the same owing to equally efficient 3'-gauche effects.

7.3 Interaction of 2'-OH with vicinal 3'-phosphate in ribonucleotides The thermodynamics28 of the N S equilibrium in β-D-rNMPs 74 - 78 and β-D-rNMPEts 3 79 - 83 based upon pseudorotational analyses of temperature-dependent JHH coupling constants are presented in Table 3. The pairwise comparison of ∆H° values of 74 - 83 with those of nucleosides and other 2'-deoxynucleotides leads to following conclusions: (i) For all ribonucleotides (except β- D-CMPEt (81), for which they are of equal strength and cancel each other), ∆H° contribution to the free-energy ∆G° of the N S equilibrium stabilizes S-type conformations over the counteracting entropy, which prefers N-type sugars. (ii) The N S equilibrium in β-D-rNMPs 74 - 78 is driven more toward S-type conformations than in the parent β-D-rNs 50 - 55, as evident from negative -1 ∆∆H˚25 values. ∆∆H˚25 (kJmol ) is in the range -1.2 < ∆∆H˚25 < -0.5 for purines and -2.2 < -1/-2 ∆∆H˚25 < -0.8 for pyrimidines. This can be attributed to the stronger [ HO3PO3'-C3'-C4'-O4']

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 122 gauche effect in β-D-rNMPs compared with the [HO3'-C3'-C4'-O4'] gauche effect in the former. (iii) In β-D-rNMPEts 79 - 83, S-type conformations are also more stable than in β-D-rNs 50 - 55 by -1 -1 ∆∆H˚26 = -2.5 kJmol for purines and ∆∆H˚26 ≈ -3.7 kJmol for pyrimidines, owing to the - stronger [ EtO3PO3'-C3'-C4'-O4'] gauche effect in 79 - 83 in comparison with the [HO3'-C3'-C4'- - O4'] gauche effect in 50 - 55. (iv) The [ EtO3PO3'-C3'-C4'-O4'] gauche effect in β-D-rNMPEts is -1 -1/-2 clearly stronger (by -2.8 to -1.3 kJmol ) than the [ HO3PO3'-C3'-C4'-O4'] gauche effect in β-D- rNMPs, as shown by ∆∆H˚28 values, whereas in the 2'-deoxy counterparts, they are the same (as shown by nearly identical ∆∆H˚20 and ∆∆H˚21). (v) The overall 2'-OH effect drives the sugar conformation toward S and N-type geometries in purine and pyrimidine β-D-rNMPEts, respectively, as shown by the comparison of their conformational preferences with the β-D-dNMPEts counterparts (i.e. ∆∆H˚27). In contrast, 2'-OH drives the conformation of the sugar moiety in β-D- rNMPs to more N in comparison with β-D-dNMPs, as shown by negligible or positive ∆∆H˚29 values. (vi) As the temperature is increased from 278 K to 358 K, the population of S-type conformers decreases and the population of εt rotamers in β-D-rNMPEts 79 - 83 increases in a coopeative 3 3 3 manner, as shown by the concomittant change in JC2'P3', JC4'P3' and JH3'P3' coupling constants.

Table 13.The thermodynamics of the two-state N S and εt ε- pseudorotational equilibria in β- D-rNMPEts 79 - 83 showing the identical values (within experimental error) for ΔG298 of N S pseudorotational and εt ε- equilibria (see Fig 21 for the correlation plot).

Estimation of the drive of εt ε- conformational equilibria derived from 3 3 temperature-dependent JHP and JCP 298 ΔHº a ΔSº a -TΔS˚ b %ε-278 c %ε-358 c Δ%ε- d ε ε ΔG β-D-AMPEt (79) -6.6 (0.9) -14 (3) 4.2 -2.4 76 63 -13 β-D-GMPEt (80) -5.8 (0.9) -12 (3) 3.6 -2.2 74 62 -12

β-D-CMPEt (81) -2.8 (0.9) -9 (4) 2.7 -0.1 53 46 -7 β-D-rTMPEt (82) -3.8 (0.8) -8 (3) 2.4 -1.4 66 58 -8

β-D-UMPEt (83) -3.3 (0.7) -7 (2) 2.1 -1.2 64 57 -7 a ΔH˚ (kJ mol-1) and ΔS˚ (J mol-1 K-1) are the average values (standard deviations are given in brackets) and were calculated from individual van't Hoff plots using populations of N and S pseudorotamers from several t individual PSEUROT analyses. ΔHºε and ΔSºε were calculated from 15 van't Hoff plots using populations of ε and ε- rotamers. The signs of thermodynamic parameters are arbitrarily chosen in such a way that the positive values indicate the drive of N S and εt ε- equilibria to N and εt, whereas the negative signs describe the drive to S and ε-, respectively. b -TΔS˚ (kJ mol-1) term is given at 298 K. c The population of the S and ε- conformers were calculated using the relation: %S (T) or %ε- (T) = 100 * [exp (- ΔGT/ RT)] / [exp (- ΔGT/ RT) +1]. d Δ%ε- = %ε-358 - %ε-278.

(vii) The interdependency of the conformation of the sugar and phosphate moieties in β-D- rNMPEts 79 - 83 is evidenced by the fact that the free-energies (∆G298) of the two-state N S and εt ε- equilibria are the same (Table 2 and Table 13), within the experimental error of our measurements and calculations (±0.5 kJ/mol). This is further evidenced by a simple correlation plot of the temperature-dependent populations of ε- rotamers as a function of the temperature-dependent

123 t - population of the S sugar pseudorotamers (Fig 21, Panels (A1) - (E1)) and of ∆G° of the ε ε equilibrium versus ∆G° of the N S equilibrium for all β-D-rNMPEts (Fig 21, Panels (A2) - (E2)), showing their straightforward correlation with the correlation coefficients between 0.79 and 0.98 (in pyrimidine nucleotides, the relatively smaller values for the correlation coefficients are the result of the limited change in ∆G° or xε- or xS as a function of temperature in comparison with the purines counterparts).

80 (A ) 76 (B ) (B ) (A ) 2 1 -1.5 2 76 1 -2.0 72 ) 72 ) − ε − ε − t / − t / -2.0 ε ε( -2.5 ε ε( 68 o o % G %68 G Δ Δ 64 -3.0 -2.5 64 60 β-D-AMPEt (79) β-D-AMPEt (79) β-D-GMPEt (80) β-D-GMPEt (80) -3.5 56 60 -3.0 64 68 72 76 80 84 -3.2 -2.8 -2.4 -2.0 -1.6 -1.2 60 64 68 72 76 -2.8 -2.4 -2.0 -1.6 -1.2 % S ΔGo % S ΔGo (N/S) (N/S)

72 56 (C ) (D ) (D ) (C ) 2 1 2 1 0.5 68 -1.0 ) ) − ε − ε − 52 t / − 64 t / ε ε( 0.0 ε ε( o o % G % G Δ 60 Δ -1.5 48 -0.5 56 β-D-CMPEt (81) β-D-CMPEt (81) β-D-rTMPEt (82) β-D-rTMPEt (82) 44 -1.0 -2.0 46 48 50 -0.5 0.0 0.5 52 56 60 64 -1.0 -0.5 % S ΔGo % S ΔGo (N/S) (N/S)

66 (E ) -0.6 (E ) 1 2 63 ) − ε − t / -0.9 ε (ε 60 o % G Δ 57 -1.2

54 β-D-UMPEt (83) β-D-UMPEt (83) -1.5 54 57 60 -0.8 -0.6 -0.4 % S ΔGo (N/S)

Figure 21. The correlation plots of the temperature-dependent population of the ε- phosphate backbone ° rotamers versus the population of S-type conformers (at 298 K) [Panels (A1) - (E1)] and of ∆G (εt/ε-) of t - ° ε ε equilibrium versus ∆G (N/S) of N S equilibrium [Panels (A2) - (E2)] in β-D-rNMPEts 79 - 83. All plots show straight lines (s = slope, i = intercept, R = Pearson's correlation coefficient): For β-D- AMPEt, s = 1.02, i = -5.78, R = 0.98 (A1) and s = 1.01, i = 0.62, R = 0.95 (A2), For β-D-GMPEt, s = 0.95, i = 4.10, R = 0.98 (B1) and s = 0.94, i = -0.25, R = 0.95 (B2), For β-D-CMPEt, s = 1.44, i = -19.9, R = 0.79 (C1) and s =1.39, i = -0.25, R = 0.80 (C2), For β-D-rTMPEt, s = 1.37, i = -16.73, R = 0.93 (D1) and s = 1.30, i = -0.30, R = 0.86 (D2), For β-D-UMPEt, s = 1.96, i = -49.5, R = 0.94 (E1) and s = 1.94, i = 0.12, R = 0.85 (E2).

(viii) The unique interaction of 2'-OH with the lonepair of the vicinal heteroatom (e.g. O3') is able to act as a molecular switch between (N,εt) (S,ε-) conformational equilibria in 79 - 83. An alternative H-bond directly between 2'-OH and the 3'-phosphate oxygen is ruled out because they are too far away from each other, even on rotation around α and ζ torsions. This 2'-OH....O3' interaction stabilizes the S and ε- conformers ("On-Off" switch) in a cooperative manner over N and εt, and it is experimentally evidenced by the fact that the difference in the chemical shifts of the CH2 protons of 3'-ethylphosphate in 79 - 83 is steadily reduced as the temperature is increased (until

both protons become isochronous) owing to the increase in the population of N,εt-type conformers in which no H-bond between 2'-OH and the vicinal phosphate occurs. The N S equilibrium is unbiased (ΔG298 = 0.1 kJ mol-1) for β-D-CMPEt (81) because of the strongly opposing anomeric effect. (ix) The in-line attack of 2'-OH to the vicinal phosphate in the RNA self-cleavage reaction10,555, giving a 2',3'-cyclic phosphate via a transient trigonal bipyramidal phosphorane as intermediate, requires a S,ε- conformational state246,556,557. This cleavage is more facile whith cytosin-1-yl as the nucleobase at the cleavage site than any other nucleobase558, presumably owing to a smaller activation energy barrier for N- to S-type sugar interconversion. We, on the other hand, have found that ∆G° of the (N,εt) (S,ε-) conformational equilibrium in pyrimidine β-D-rNMPEts is much reduced (≈ 0 kJmol-1 for β-D-CMPEt) than for the purine counterparts. (x)

Owing to the internucleotidyl stacking, the preferred conformational state in RNA-RNA559 or RNA- DNA560 duplex is (N,εt). However, when these stacking interactions are absent such as in the single stranded hairpin loop, (S,ε-) and gg C4'-C5' conformers are favoured, as in our model systems β-D- rNMPEts 79 - 83.

8. Application of stereoelectronic effects in oligonucleotides

8.1 Design of antisense oligonucleotides via the gauche engineering What is the structure of DNA-RNA hybrid? The target for the antisense strand is the RNA. The mechanism of antisense effects of the antisense NA involve either RNase H mediated cleavage of the RNA strand in the hybrid duplex duplex561, or the physical blocking of the translation machinary562. Much is understood about the RNase H promoted RNA excission from the hybrid DNA-RNA duplex than the physical blocking. In order to be able to optimally design the antisense strand using the RNase H promoted RNA excission of the DNA-RNA hybrid, it is important to understand first the structure of the DNA- RNA hybrid both in the solution and in the solid state. The summary of various studies performed in many labs can be divided into three categories, and they are as follows: (i) The RNA and DNA strands of the chimeric duplex are similar to the corresponding A-RNA and the B-DNA563,564. (ii) The RNA strand of the duplex is similar to A-RNA and the DNA strand is somewhat intermediate between A- and B-forms of DNA560,563,564. (iii) In one crystalline duplex, the conformation of both ribose and deoxyribose sugars were found to be in C3'-endo (i.e. N-type) conformation, but the sugar residues in the DNA strand underwent a conformational transition to C2'-endo (i.e. S-type) in solution565. The occurrence of all the above variations in the DNA-RNA hybrid clearly shows that the RNA sugars are invariably C3'-endo, but the DNA sugars are flexible and can indeed take up various conformations (O4'-endo to C1'-exo to C2'-endo, 72° < 180°).

Configuration-dependent drive of the sugar conformation The effects of covalently linked substituents at C2', C3' and C5' upon the conformation of the sugar moieties of the resulting nucleosides and the overall stability in modified oligonucleotides have been extensively reviewed46,49. It has been demonstrated first by Remin et al 405 that inversion Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected]

125 of configuration at C2' from ribo to ara-cytidine induces further stabilization of the S-type conformation (40% S for cytidine and 50% S for ara-C at 24°C in D2O at pH 7). This preference for S-type conformation is dramatically increased as the hydroxyl groups are ionized at pH ≥ 13 because of intramolecular 5'-OH...O2'- H-bond (45%S for cytidine and 90%S for ara-C). Consistent with this study, it has been found226 that the electronegative substituents such as 2'-F at C2' on the α-face (i.e. 2'F-2'-deoxyribo analog) enhances the preference of the sugar moiety for N-type pseudorotamers, whereas C2'-F in the β-face (i.e. 2'F-2'-deoxyara-analog) enhances the preference of the sugar moiety for S-type pseudorotamers, thereby showing that the gauche effect is indeed configuration-dependent.

The engineered conformational preference of nucleosides preorganizes the oligo conformation. It has been shown64 that two South thymidines in a 12mer duplex greatly stabilized the double helix, whereas two North thymidines destabilized it by inducing an A-B junction in the middle of the duplex. The introduction of the North nucleosides into oligo-DNA will induce an RNA-type geometry, and consequently improves its binding capability with the complementary target RNA (but not the RNase H cleavage property; see below!). Consequently, the incorporation of 2'F-2'-deoxyribonucleosides into a DNA strand458 stabilizes the DNA:RNA heteroduplex by ≈2°C per modification. The modified antisense oligodeoxynucleotide adopts an A-form conformation, as a result of the drive of the sugar conformation toward N-type pseudorotamers by the [F2'-C2'-C1'-O4'] gauche effect458,459.

The antisense NA-RNA hybrid should mimic the DNA-RNA hybrid conformation for good RNase H cleavage Consistent with the studies of Remin et al 405 that the ara-nucleosides have intrinsic preference for the South-type conformation compared to ribo counterpart, fluoroarabinonucleic acids (2'F-ANA)-RNA and arabinonucleic acid(ANA)-RNA duplexes showed a close CD resemblance566 to the native DNA-RNA counterpart (suggesting that 2'F-ANA and ANA have preorganized structures similar to DNA, and their hybrids with RNA have very similar A-like helical conformations as the native DNA-RNA hybrid), but their melting studies showed that the former had higher thermodynamic stability compared to latter. Consequently, the 2'F-ANA-RNA duplex was cleaved by RNase H much faster than the ANA-RNA duplex. The susceptibility of the 2'F-ANA-RNA hybrids to RNase H was found to be very similar to that observed for native DNA- RNA and DNA-thioate-RNA hybrids. This study showed that lower thermal stability of the ANA- RNA hybrids translated into poorer digestion by RNase H although they had similar A-type helix, whereas 2'F-ANA-RNA, native DNA-RNA and DNA-thioate-RNA have also similar conformation but higher stabilities than ANA-RNA duplex, therefore they all showed faster cleavage. In contradistinction, 2'F-2'-deoxyribonucleosides has a predominant N-type conformation as the native ribonucleosides, and therefore 2'F-RNA has a preorganized RNA-type structure, which forms A- type hybrid with a target RNA as the native RNA-RNA duplex, and, as expected, none of them serves as a substrate to RNase H.

The antisense NA-RNA heteroduplex is more efficiently stabilized by 2'F-2'-deoxyribo- than by 2'-O-methyl ribonucleosides567, which is consistent with the stronger gauche effect induced by the former, owing to the higher electronegativity of fluorine compared with hydroxy458. The presence of North 2'-fluoro-2'-deoxyguanosine in a modified hexadeoxynucleotide enables the transition between A-DNA conformation at low ionic strength and Z-DNA at high salt concentrations460. 2'-O-methyl-ribonucleosides567 when incorporated into an oligodeoxynucleotide improves its binding affinity with the complementary RNA strand, but incorporation of 2'-SMe-46,49 or 2'-amino-2'-deoxyriboucleosides497 into the antisense strand results in the destabilization of the resulting antisense-NA-RNA duplex because of the propensity of these modified nucleosides to adopt S-type conformations, owing to the poorer electronegativity of 2'-SMe or 2'-NH2 group comapred to 2'-OH.

Requirements for the design of the antisense strand for RNase H cleavage of the hybrid The above discussion point to two distinct requirements for the design of the antisense strand complementary to RNA target: (i) The antisense strand should mimic the preorganized conformation of a native DNA strand in order to form a hybrid duplex with a target RNA with a structure closely similar to the native DNA-RNA hybrid, and (ii) the affinity of this preorganized antisense strand to the native RNA should be at least as high as the native DNA-RNA strand in order to be a good substrate to RNase H. In contradistinction to these requirements, the necessity for the antisense strand to mimic the preorganized conformation of a native DNA strand is often overlooked. A major consideration is still given49 in the design of antisense strand is the increase of the thermal stability of the antisense NA-RNA hybrid. Despite this, the great majority of the sugar and backbone-modified nucleoside building blocks (for example, -S-CH2-O-CH2- or -CH2-NCH3- O-CH2- backbone) have a preferred N-type conformation, thereby their antisense oligo is expected to have a preferred RNA-type, not the DNA-type, preorganized structure. The increase of melting point observed with the hybrid of these antisense NA with RNA target is therefore presumably owing to RNA-RNA duplex type structure, which are not as good substrates for RNAse H as DNA- RNA duplex. Regarding the relative stability of various types of duplexes, it is noteworthy that it increases in the following order: DNA-DNA < DNA-RNA < RNA-RNA. These studies are consistent with our own studies568 with ten different 5'-fluorophore tethered DNA-RNA hybrids as substrates for RNAse H: We showed that among all these ten different 5'-tethered chromophores, 5'-phenazine and 5'-phenanthridene tethered DNA-RNA hybrids had the shortest half-life of digestion by RNase H because these antisense oligos showed higher thermal stability (5-10°C higher than the native counterpart) and least deviation of the global structure from the native heteroduplex.

Requirements for 3'-modification for engineering antisense DNA strand (the Gauche engineering) It has been shown by our lab in 199426 that the distinct conformational preferences observed for the pentofuranosyl moieties in various 3'-substituted (X)-2',3'-dideoxynucleosides [X = H, NH2,

127

-1/-2 OH, OMe, NO2, OPO3H and F] are closely related to the strength of the 3'-gauche effect exerted by the 3'-substituent, X. This work demonstrated that the magnitude of the enthalpy of the 3'-gauche effect increases with the increase of the electronegativity of the 3'-substituent. Thus, as the -1/-2 electronegativity of the 3'-substituent decreases [F > OPO3H > NO2 > OMe > OH > NH2], the -1/-2 preference for the North-type pseudorotamer increases [F < OPO3H < NO2 < Ome < OH < NH2]. Thus 3'-amino-2',3'-dideoxynucleosides have been shown to possess a preferred N-type conformation26,569 which indeed is conserved in the sugar puckers (66 of the 72 nucleotides per asymmetric unit adopting C3'-endo conformation) in the N3' → P5' phosphoramidate 12mer DNA duplex as evident by the A-type duplex conformation62. Moreover, the CD-spectra of the N3' P5' phosphoramidate 12mer DNA duplex are consistent with adoption of an A-RNA conformation570.

8.2 Fused nucleosides to engineer preorganized DNA structures We have seen above that the engineered conformational preference of the flexible pentofuranose ring in nucleosides is capable of preorganizing the conformation of the oligonucleotide duplex. This can be simply achieved by engineering the stereoelectronics of the nucleosides, namely the gauche and the anomeric effects as described in the earlier chapters. Clearly, such stereoelectronic engineering gives us the possibility to steer a specific conformation depending upon the conformation of the target strand. The challenge in the stereoelectronic engineering is to direct the sugar conformation to a specific low energy minima and stabilize it by overcoming the intrinsic temptation to fall into other energy minima since the interconversions between various pseudorotamers take place through a low energy barrier. Alternatively, one can introduce appropriate rigidity to the pentofuranose or to the cyclopentane system in a nucleoside analogue and trap it into one specific conformation. Introduction of these fused systems in oligos have produced preorganization and appropriate rigidity of the single strand, which has been found to play a critical role for the formation of stable duplexes. Several such systems have been so far designed, and the readers are directed to a recent review article49.

8.3 Enzyme recognition by fused carbocyclic nucleosides of fixed conformation Recently, a conformationally constrained 2E (N)-methano-carbocyclic AZT mimic [(N)- 4',6'-methano-carba-3'-azidothymidine-5'-triphosphate] was shown to be equipotent to AZT-5'- triphosphate in inhibiting the target enzyme, reverse transcriptase, whereas the antipodal 3E carbocyclic analogue [(S)-1',6'-methano-carba-3'-azidothymidine-5'-triphosphate] was inactive571. When carbocyclic nucleosides are incorporated into a modified oligodeoxynucleotide59,572, they are able to mimic the conformation of the neighbouring nucleotides owing to the relatively more flexible nature of the cyclopentane ring (see below). In contrast, the incorporation of the conformationally constrained 2E carbathymidine analogue [(N)-4',6'-methano carbathymidine] was able to force a conformational change in the oligonucleotide that resulted in an enhanced stability of DNA-RNA heteroduplexes573,574.

8.4 The conformational transmission in the self-cleaving Lariat-RNA

Small synthetic lariat RNAs 129a, 130a and 131a have been found557 to undergo site specific self-cleavage to give an acyclic branched-RNA with 2',3'-cyclic phosphate and a 5'-hydroxyl termini as in 129b, 130b and 131b, which are reminiscent of the products formed in some catalytic RNAs. Noteworthy is the fact that in these self-cleavage reactions of 129a, 130a and 131a one specific phosphodiester bond between G and C residues underwent intramolecular transesterification reaction amongst all other phosphates moieties in the molecule (akin to the splicing of pre-mRNA to give functional mRNA), suggesting that the self-cleaving transesterified G C phosphodiester has enormously different chemical reactivity in comparison with all other phosphates in the molecule. These lariat-RNAs 125, 126, 129a, 130a and 131a are much smaller than the natural catalytic RNAs such as the hammerhead ribozyme (k = ~1 min-1 at 37 °C), and their rate of the self-cleavage is also much slower (k = 0.25 x 10-4 min-1 for lariat hexamer 129a, and 0.16 x 10-3 min-1 for lariat heptamer 130a at 22 °C). We have shown that the trinucleotidyl loop in the tetrameric and pentameric lariat-RNAs245 126 is completely stable whereas the tetranucleotidyl or pentanucleotidyl loop in the hexameric 129a or heptameric 130a lariat-RNA RNA246,556,575 does indeed have the required local and global conformation promoting the self- cleavage. It has been also shown that simple 2' 5' or 3' 5'-linked cyclic RNAs, 127 and 128, respectively, are completely stable and their structures are considerably different from the self- cleaving lariat-RNAs such as 129a or 130a. In our search to explore the optimal structural requirement for the self-cleavage reaction of RNA, we showed that the unique 3'-ethylphosphate function in 131a (in which the branch-point adenosine has a 2' 5'-linked tetranucleotidyl loop and a 3'-ethylphosphate moiety, mimicking the 3'-tail of the lariat-hexamer 129a) is the key structural feature that orchestrates its self-cleavage reaction (k = 0.15 x 10-4 min-1 at 19 °C) compared to the stable 2' 5'-linked cyclic RNA 127. A comparative study of the temperature dependence of the N S equilibrium for the lariat-tetramer 131a and the 2' 5'-linked cyclic tetramer 127 shows that the A1 residue in the former is in 92% S-type conformation at 20 °C, whereas it is only in 55% S in the latter. This displacement of the N S pseudorotational equilibrium toward the S-type geometry is due to the enhanced gauche effect of the 3'-OPO3Et- group at the branch-point adenosine in 131a compared to 3'-OH group in 127. This 3'-OPO3Et- group promoted stabilization of the S geometry at the branch-point by ΔH ≈ 4 kcal.mol-1 in 131a is the conformational driving force promoting its unique self-cleavage reaction. The comparison of ΔH° and ΔS° of the N S pseudorotational equilibria in 131a and 127 (see Table 14) clearly shows the remarkable effect of the 3'-ethylphosphate group in 131a in being able to dictate the conformational changes from the sugar moiety of the branch-point adenosine to the entire molecule (conformational transmission). Thus the S conformation in A1, U2 and C6 sugar moieties is clearly thermodynamically more stabilized while it is considerably destabilized in G3 owing to the 3'-ethylphosphate group in 131a compared to 127. This is a clear experimental evidence of conformational transmission through a crankshaft motion originating from the gauche effeect exerted by the 3'- ethylphosphate moiety at the A1 sugar. It is interesting to note that the magnitude of enthalpy and entropy for the North to South transition of the A1 sugar in 127 is comparable to the enthalpy and entropy of transition between the A- and

129

B-form of the lariat hexamer556,557. This self-cleaving tetrameric lariat-RNA 131a to 131b is the smallest lariat-RNA molecule hitherto known to undergo the self-cleavage reaction, and hence it is the simplest model of the active cleavage site of the natural self-cleaving catalytic RNA.

8.5 The conformational transmission by dihydrouridine in RNA It has been recently shown576 that S-type conformations of the pentofuranose sugar in dihydrouridine (D) 3'-monophosphate are more stable (by ≈ 1.5 kcalmol-1) than in the corresponding β-D-UMP (78). This effect is amplified for the D residue (5.3 kcalmol-1) in the ApDpA trimer (compared to ApUpA), and is also further transmitted to the neighbouring 5'- terminal A (3.6 kcalmol-1). This observation is consistent with the fact that the C5-C6 saturation of the uracil-1-yl aglycone to the dihydrouracil-1-yl moiety reduces the electron-withdrawing character of the former, and hence weakens the n →σ* interactions of O4'-C1'-N1 moiety. This is consistent

NH2 NH2 N N 1 1 O A N A N - O N N O P O N N HO OH OH O HO O O HO O O O O- O O- HN N O P O P O O - O O O P O O O O 5 O O N O- P C O N O P 2 O O- U N 4 O P O O O C N - O HO O 4 - NH O U O P 2 H2N O O N O HO O 3 O O G HO O NH G3 O O O P N N N O N - HO 2 O O N N U N O HN N H2N 125 N O O H2N H H 126

NH NH2 1 2 O A N N N - N O A1 O - P O N HO O O N N HO N O P O O O HN O HN O N N O O O O OH O 2 O HO - U - U2 O O P P O O O O - - O O P O O P O O O O 3 3 HO O G HO O G O O O O O O P HO N P HO N - O N - O N O N 6 O O N 6 O C C N O N O N N H N N H N N 2 2 H2N 128 H H2N 127 H

NH2 NH2 O N N O N N - O - O P O N N P O N N O HO O O HO O HN O HN O N N O O O O O HO O HO O O O O O O - O - O - O HO - P O HO P O P - P O O- O P O P O O O O O O O O O -4 -1 N HO N HO k = 0.25 x 10 m O O O - O O O - O O P P N N o O N O N at 22 C O NH2 O NH2 O O NHO NH O HO O HO O O O OH O O O P O N O P N OHO N N O N - O N - O O N O N N N N O H N Cleavage N H N 2 H2N 2 H H2N site H 129b 129a

HN O O OH NH O N O- O N 2 HN O OH - O NH O P O N N O N 2 O O N P N O O O N N O N O O HO O - O O O - HO O O O O HO - P O - P O - O O - O O P O O- P O HO P O P O O O O -3 -1 O - P k = 0.16 x 10 m O P O O O N O O O HO O N HO O o O O HO N at 22 C HO O N O NH O 2 N NH O O 2 N N O O N O NH O O O O NHO O N O N O O - P O P HO N N - P N H2N O O O H N O P O O N O - 2 - O N O OH O N HO O HO O O N O N H2N H N H 2 H N Cleavage N O site O HN HN O O 130a 130b

NH2 O N - N O NH2 P O N N O N N HO O - O O P HN O O N N N O OH O O O O HN O O O - N P O O O O O- O O CH3CH2O P O P O- -4 -1 CH CH O O- O k = 0.15 x 10 m 3 2 P O - O O O P o at 19 C - O O O P O O HO O O O O HO O O P O O HO N N O OH O N - O P O N O - N N O O N O N N N O Cleavage H2N N N H2N H H2N site H2N H 131a 131b

131

Table 14: The thermodynamicsa of for the N S equilibrium in 127, 129a & 131a

Compound Residue ΔH° ΔS° -TΔS°b ΔG° (kcal.mol-1) (cal.K-1.mol-1) (kcal.mol-1) (kcal.mol-1) Lariat- A1 -5.8 ± 1.3 -15.9 ± 3.8 4.7 ± 1.1 -1.1 ± 2.4 3'-Et-Phos U2 -7.1 ± 2.2 -21.3 ± 6.4 6.3 ± 1.9 -0.8 ± 4.1 RNA G3 12.3 ± 2.8 36.2 ± 8.2 -10.6 ± 2.4 1.7 ± 5.2 131a C6 1.2 ± 0.8 3.2 ± 2.5 -0.9 ± 0.7 0.3 ± 1.6 3'-OH A1 -1.8 ± 0.5 -5.5 ± 1.7 1.6 ± 0.5 -0.2 ± 1.1 RNA U2 -1.9 ± 1.2 -2.6 ± 3.8 0.8 ± 1.1 -1.2 ± 2.3 127 G3 6.1 ± 1.1 17.0 ± 3.2 -5.0 ± 0.9 1.2 ± 2.0 C6 6.6 ± 1.1 18.7 ± 3.4 -5.5 ± 1.0 1.1 ± 2.1 Hexameric A1 -1.2 ± 1.1 -0.7 ± 3.6 0.2 ± 1.0 -1.0 ± 2.2 Lariat- U2 -2.2 ± 1.0 -4.3 ± 3.0 1.3 ± 0.9 -1.0 ± 1.9 RNA G3 7.3 ± 1.2 22.1 ± 3.9 -6.5 ± 1.1 0.8 ± 2.4 129a C6 5.0 ± 1.1 16.2 ± 3.4 -4.8 ± 1.0 0.3 ± 2.1 a 90% confidence. b The -TΔS term is computed for 293 K.

with our observation that the strength of the anomeric effect of an aglycone increases as it becomes more electron-withdrawing, and decreases as it becomes electron-rich. The transmission of the stabilization of S-type conformations from the D nucleotide moiety to the 5'-terminal A moiety in the ApDpA trimer most probably takes place through a crankshaft motion across the sugar- phosphate backbone. As it has been stated above, the earlier example of this type of stereoelectronic transmission through the crankshaft motion across the sugar-phosphate backbone has been documented for the tetranucleotidyl lariat-RNA loop, in which placement of a 3'-ethylphosphate moiety at the branch point not only stabilized the S-type conformation of the constituent sugar of the nucleotide at the branch-point but also affected the conformation of all four other loop nucleotides246,556,556. Similar modifications of the electronic nature of five most common aglycones, say by methylation or by any hypermodification (as found in tRNAs) or by complexation with any ligand such as metal ion, drug or polypeptide will profoundly affect the local structure of the DNA and RNA, which may itself locally alter the hydration pattern, H-bonding or electrostatics, and any one of these, in turn, may serve as a recognition signal for a specific interaction and function.

8.6 Preferential recognition of 3'-anthraniloyladenosine by Elongation Factor Tu The functional role of transfer RNA (tRNA) as adapter in translating the genetic code is well recognized. The protein biosynthesis occurs via stepwise manner – (i) Initiation, (ii) Chain elongation and (iii) Termination. The initiation step requires the assembly of a ribosome-mRNA- tRNA ternary complex. During this process, amino acids are linked to tRNA by aminoacyl-tRNA synthetases to form aminoacyl-transfer RNA (aa-tRNA). The binding of the aa-tRNA to the ribosome is catalysed by the elongation factor Tu (EF-Tu), a GTP binding protein. The EF-Tu, in its

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 132 active form (EF-Tu.GTP), can specifically recognize the aa-tRNAs from the non-aminoacylated tRNAs. This precise recognition process depends on the complimentary structural features of the aa- tRNA scaffold. From this viewpoint, the development of tRNA-mimics is an interesting approach to understand the functional aspects of this biological process. Small molecule can mimic only a part of a large tRNA. There are three such molecules, having tRNA mimicry, found in literatures – (i) antibiotic puromycin [(a) Welch, M.; Majerfeld, I.; Yarus, M. Biocehmistry 1997, 36, 6614. (b) Monroe, R. E.; Marcker, K. A. J. Mol. Biol. 1967, 25, 347], which corresponds to the 3'-end of a tyrosinylated tRNA and found to be a powerful inhibitor in protein biosynthesis. However, it failed to interact with EF-Tu or aaRS (ii) micro-tRNA, CCAOH trinucleotides, which has been shown to mimic tRNA for aaRS recognition and can even be aminoacylated [Jovine, L.; Djordjevic, S.; Rhodes, D. J. Mol. Biol. 2000, 301, 401] and (iii) 3'-O-anthraniloyladenosine (used in our present study, see below). The differential recognition of 3'-O-anthraniloyladenosine (having 2'-endo sugar conformation and puromycin (having 3'-endo sugar conformation) towards EF-Tu has enormous biological significance. Anthranilic acid charged yeast tRNAPhe or E. coli tRNAVal are able to form a stable complex with EF-Tu*GTP, hence the NH2 N N 2'- and 3'-O-anthraniloyladenosines N NH2 N ROH2C ROH C and their 5'-phosphate counterparts 2 N N O N O OH N HO have been conceived to be the smallest O O O O NH units that are capable to mimic H N 2 2 aminoacyl-tRNA577-582. Since the 3'- O-anthraniloyladenosine (134) also 132: R = H 134: R = H -1/-2 135: R = OPO H-1/-2 133: R = OPO3H 3 binds more efficiently to EF-Tu*GTP complex compared to its 2'-isomer (132), we delineated the stereoelectronic features that dictate the conformation of 3'-O- anthraniloyladenosine (134) and its 5'-phosphate (135) vis-a-vis their 2'-counterparts (132 and 133) as well as address how their structures and thermodynamic stabilizations are different from adenosine and 5'-AMP. It has been found43 that the electron-withdrawing anthraniloyl group exerts gauche effects of variable strengths depending upon whether it is at 2' or at 3' because of either participation or absence of the [O2'-C2'-C1'-N9] gauche effect, thereby steering the pseudorotation of the constituent sugar moiety either to the N-type or S-type conformation.

The 3'-O-anthraniloyladenosine 5'-phosphate has a relatively more stabilized S-type conformation (ΔG° = -4.6 kJ/mol) than 3'-O-anthraniloyladenosine itself (ΔG° = -3.9 kJ/mol), whereas the ΔG° for 2'-O-anthraniloyladenosine and its 5'-monophosphate are respectively -0.9 and -1.8 kJ/mol, suggesting that the 3'-gauche effect of 3'-O-anthraniloyl group is stronger than that of 2'-O- anthraniloyl in the drive of the sugar conformation. Since the EF-Tu can specifically recognize the aminoacylated-tRNA from the non-charged tRNA, we have assesssed the free-energy (ΔG°) for this recognition switch to be at least ≈ -2.9 kJ/mol by comparison of ΔG° of N S pseudorotational Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected]

133 equilibrium 3'-O-anthraniloyladenosine 5'-phosphate and 5'-AMP. The 3'-O-anthraniloyladenosine and its 5'-phosphate are much more flexible than the isomeric 2'-counterparts as evident from the temperature-dependent coupling constant analysis. The relative rate of the transacylation reaction of 2'(3')-O-anthraniloyladenosine and its 5'-phosphate is cooperatively dictated by the two-state N S pseudorotational equilibrium of the sugar, which in turn is controlled by a balance of the stereoelectronic 3'- and the 2'-gauche effects as well as by the pseudoaxial preference of the 3'-O or 2'-O-anthraniloyl group. The reason for the larger stabilization of the 2'-endo conformer for 3'-O- anthraniloyladenosine and its 5'-phosphate lies in the fact that the C3'-O3' bond takes up an optimal gauche orientation with respect to C4'-O4' bond dictating the pseudoaxial orientation of 3'- anthraniloyl residue, which can be achieved only in the S-type sugar conformation with adenin-9-yl and the 2'-OH groups in the pseudoequatorial geometry, compared to the preferred C3'-endo sugar with pseudoaxial aglycone and 2'-OH found in 3'-terminal adenosine moiety in the helical 3'-CCA end of uncharged tRNA.

8.7 Conformational changes in nucleotides induced by interaction with metal ions (i) Effect of Zn2+ or Hg2+ binding to the nucleobase on the sugar conformation in β-D-dG (41) It has been shown by us30 that protonation at N7 in β-D-dG (41) shifts the N S pseudorotational equilibrium toward more N by ≈ 15% with respect to the neutral state, which was ∗ attributed to the strengthening of the nO4' →σ C1'-N9 anomeric effect at acidic pD owing to the reduced electron-density in the imidazole ring compared with the neutral pD (Section 4.8). In contrast, it has been found395 that binding of Zn2+ with N7 of the constituent guanin-9-yl in β-D-2'- dG has a negligible effect on the population of the preferred S-type pseudorotamers. Similarly, upon addition of 0.2 equivalent Hg2+, only a slight shift (by ≈ 4 % unit) of the two-state N S pseudorotational equilibrium toward more N-type has been found. These results suggest that binding of Zn2+ or Hg2+ with N7 do not modulate efficiently the strength of the O4'-C1'-N9 O anomeric effect. This is in agreement with the O MeO O N N NH observation395 that the resulting perturbation P NH OH - O O N N NH (with respect to the uncomplexed/neutral N N O 2 O NH2 state, Section 2.9.3) of the electronic character

O O of the imidazole moiety is much reduced than HO P O- OMe when N7 becomes protonated: Indeed, the 136: MepdG 137: dGpMe 15N chemical shift of N7 in β-D-2'-dG changes much less upon its complexation with Zn2+ (∆δ15N(N7) = 3.4 ppm) than upon its protonation401 [∆δ15N(N7) = 46 ppm]. In the same study, it was also noted that hard Mg2+ did not form any stable complex with N7 of the guanin-9-yl nucleobase in β-D-2'-dG.

(ii) Conformational changes observed across the sugar-phosphate backbone and in the pentofuranose sugar in 5'- and 3'-methylmonophosphates of β-D-dG as the result of the interaction of the nucleobase/phosphate moiety with Mg2+, Zn2+ and Hg2+ As an extension of the above work, Polak and Plavec have recently shown583 that the preferential binding of Mg2+ with the phosphate oxygen atoms and C6=O carbonyl group in 5'- and 3'- Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 134 methylmonophosphates of β-D-dG (i.e. MepdG (136) and dGpMe (137), respectively) does not alter the bias of the N S pseudorotational equilibrium toward S-type conformations (At 298K, 68% S for MepdG and 75% S for dGpMe). On the other hand, the interactions of Zn2+ or Hg2+ with N7 resulted in the small shift of the sugar conformation in MepdG and dGpMe toward N (by ≈ 1 - 5%), as the result of the slight strengthening of the anomeric effect (vide supra and Section 4.8). It was also observed that the binding of the softer Zn2+ causes a larger shift of the syn anti equilibrium toward anti than complexation with hard Mg2+. Whereas the preferred orientation around the C4'-C5' (γ torsion angle, in dGpMe) and O5'-C5' (β torsion angle, in MepdG) remain virtually unchanged in the metal ion complexed state in comparison with the free state, interaction of hard Mg2+ or softer Zn2+ or Hg2+ resulted in a small shift of the εt ε- equilibrium toward εt rotamers. Thus, this work has qualitatively shown that the small change of the electronic character of the nucleobase in dGpMe upon the complexation of N7 with a soft metal ion resuts in relatively minor strengthening of the O4'-C1'-N9 anomeric effect pushing the sugar conformation toward slightly more N-type forms, and this additional H C H3C 3 preference for N- CH2 CH2 ε−1 O O O O O type sugars is in ζ−1 N P α NH P turn transmitted to -O O -O O β control the N N NH γ O χ 2 O conformation of the δ phosphate- ε H N NH R 3 3 R backbone by further O ζ O O O Pt stabilizing εt P α+1 P -O O N N -O O β+1 7 7 rotamers. H C H2C 2 CH CH3 3 (iii) The change of

138: EtpdGpEt (R = H) 139: cis-(NH3)2Pt(EtpdGpEt)2 142: EtpdRpEt (R = H) the electronic 140: EtpGpEt (R = OH) 141: cis-(NH ) Pt(EtpGpEt) 143: EtpRpEt (R = OH) 3 2 2 character upon Pt2+ binding to guanin-9-yl nucleotide is transmitted to drive the conformation of the local sugar-phosphate backbone The anticancer drug cisplatin interferes with replication and transcription processes to form crosslinks as major lesions by reacting with the N7 of guanines and adenines in DNA. The dynamic microstructure alteration as a result of cisplatin binding to N7 of guanines in DNA has been recently assessed45 through the multinuclear temperature-, pD- and concentration-dependent NMR study of the effect of Pt2+ complexation to 2'-deoxyguanosine 3',5'-bisethylphosphate (138) and its ribo analogue (140). Compounds 138 and 140 serve as models mimicking the central nucleotide moiety in a trinucleoside diphosphate in order to shed light on how the nature and strength of intramolecular stereoelectronic effects change as a result of Pt2+ binding in complete absence of

135 intramolecular base-base stacking interactions. The study revealed that the N S pseudorotational equilibrium shifts towards more N-type conformers by Δ∆G° = 2.0 kJmol-1 and 2.6 kJmol-1, respectively, thereby showing that free-energy of complexation is transmitted to drive the sugar conformation. The increase in the population of N-type conformers was rationalized with the strengthening of the anomeric effect in both 2'-deoxy and ribo nucleotides upon the formation of Pt- ∗ N7 bond which promotes nO4' →σ C1'-N9 orbital interactions due to the reduction of π-electron density in imidazole part of guanine.

The additional stabilization of N-type conformers in Pt2+ complexes of ribonucleotide is due to the tuning of gauche effect of [N9-C1'-C2'-O2'] fragment, which is absent in 2'-deoxyribo counterparts. The platination of N7 favors N1 deprotonation in 139 and 141 by ΔpKa of 0.7 and 0.9 units in comparison to parent nucleotides 138 and 140, respectively. The NS pseudorotational equilibrium in 138 – 141 showed classical sigmoidal dependence as a function of pH with pKa values at the inflection points. Upon N1 deprotonation, the N S pseudorotational equilibrium was shifted toward more S- -1 -1 -1 type conformations by Δ∆G°D-N = -0.3 kJmol in 138, -0.9 kJmol in 139, -0.4 kJmol in 140 and -2.5 kJmol-1 in 141 which showed that the thermodynamics of N1 deprotonation in guanin-9-yl nucleosides is transmitted to drive N S equilibrium towards S–type pseudorotamers. The anomeric effect is weakened upon N1 deprotonation due to the increased π-electron density in the imidazole part of the guanin-9-yl moieties and the population of S-type pseudorotamers increases. The Pt2+-complex bound to N7 has been found to promote the redistribution of the π-electronic density from anionic deprotonated N1 which results in the larger increase in the population of S- type conformers in 139 and 141 in comparison to parent nucleotides 138 and 140. The formation of Pt-N7 bond in bifunctional complexes 139 and 141 simultaneously causes a shift of syn anti equilibrium towards anti by 43 and 63 %, and the increase of the population of εt conformers by 20 and 32 % at 278K, respectively. Only minor conformational redistributions along β, γ, β+ and ε- torsion angles have been observed which suggests weak conformational cooperativity between the shift in the N S pseudorotational equilibrium towards N-type conformers and the conformational changes across phosphate torsion angles other than ε as a result of platination of guanin-9-yl. In comparison to nucleotide phosphodiesters, apurinic 3',5'-bisethylphosphate sugars 142 and 143 showed no interaction with Pt2+ and therefore no conformational changes. It is thus clear that any intermolecular interaction between nucleic acid and a ligand (other than soft metal ion as described above) is expected to produce effects similar to partial electron- deficiency as arising from the protonation of the aglycone (such as in the case of a lone-pair donating aglycone involved in H-bonding or through-space charge transmission in stacked complexes with intercalated drugs), or to partial electron-enrichment as arising from the deprotonation of the aglycone (such as ionization of the aglycone by the basic sites of polypeptides in general or a phosphate moiety in the vicinity or by a relatively hard metal ion).

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 136 8.8 RNA as molecular wire The intrinsic dynamics and architectural flexibility of nucleic acids resulting into specific function are the result of cooperative interplay of pentofuranose, nucleobase and phosphodiester moieties. We have shown in Sections 4 - 7 that the interplay of various stereoelectronic gauche and anomeric effects and associated steric effects energetically drives the sugar conformation, which in turn is dictated by the electronic nature of the aglycone and other substituents on the sugar ring.

O OCH 3 N H3CO NH − O P O − (144a) R = O P O N N H NH2 O R O H

NH2 O R O H O N O− H OH N P (144b) R = O OH N N H3CH2CO O O− P O NH2 H3CH2CO N North: 3'-endo-2'-exo (144c) R = South: 3'-exo-2'-endo N O 0° < P < 36° 144° < P < 190° Ψm = 38.6 ± 3° Ψm = 38.6 ± 3° (145) R = H

Schematic representation of the dynamic two-state North (N) South (S) pseudorotational

equilibrium [P = phase angle and Ψm = puckering amplitude] of the constituent β-D-pentofuranose moiety in gaunosine 3',5'-bisphosphate [MepGpEt (144a)], adenosine 3',5'-bisphosphate [MepApEt (144b)] and cytidine 3',5'-bisphosphate [MepCpEt (144c)], as well as their apurinic counterpart [Mep(ab)pEt (145)].

In our latest work44a,b, we have employed guanosine 3'-ethylphosphate, 5'-methylphophate [MepGpEt (144a)] and adenosine 3',5'-bisphosphate [MepApEt (144b)], as a model mimicking the central nucleotide moiety in a trinucleoside diphosphate in order to shed light on how the strength of intramolecular stereoelectronic effects is modulated by the change of protonation deprotonation equilibrium of the aglycone in the complete absence of any intramolecular base-base stacking (For our work on 2'-deoxy23 and ribonucleoside28 3'-ethylphosphates at the neutral pD, see Section 7). The work uniquely shows a complete interdependency of conformational preference of sugar and phosphate backbone in 144a and 144b as the protonation deprotonation equilibrium of the aglycone changes as a function of pD. Moreover, similar thermodynamic studies (only at two extreme pDs i.e. at the neutral and the protonated states) of cytosine 3',5'-bisphosphate [MepCpEt (144c)] have been performed (unpublished work) in order to compare with 144a and 144b to demonstrate the tunibility of nucleobases in 144a – c. In oligonucleotides the protonation of nucleobase directly affects their hydrogen-bonding capabilities and therefore induces a change in overall three-dimensional structure (Section 2.9). Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected]

137

In these studies we have shown that changing the electronic character of aglycone through protonation at basic site (N7 of guanin-9-yl, N1 of adenin-9-yl and N3 of cytosin-1-yl) not only modulates the shift of N S pseudorotational equilibrium of its constituate sugar by strengthening the anomeric effect but is also transmitted to the sugar-phosphate backbone to influence the conformational characteristics of backbone in 144a – c, which mimic the model of trinucleoside (3'- 5') diphosphate. This tunable transmission which has been observed, compared to the abasic counterpart [Mep(ab)pEt] 145, to be much stronger at the 3'-phosphate backbone compared to 5'- end justifies RNA as "molecular wire".

(i) pD-dependent shift of N S pseudorotational equlibrium In this endeavour, the mole fractions of N- and S-type conformers have been plotted as the function of temperature in the van't Hoff type analyses to obtain ΔH° and ΔS° and subsequently to o calculate the total change in free energy at 298 K ( ΔG298 K ) of the N S pseudorotational equlibrium at each of the seven pDs ranging from 6.7 to 1.0 (Table 15 and 16). As the medium becomes more acidic the anomeric effect becomes more strengthened (See Section 4.8) by enhancing the n → σ∗ orbital interaction owing to the reduced electron O4' C1'-N density at N9 and due to which the N S pseudorotational equilibrium for MepGpEt (144a) and MepApEt (144b) is gradually shifted towards N-type pseudorotamers [79% S (for 144a) and 76% S (for 144b) in the neutral state to 55% S (for 144a) and 67% S (for 144b) in the protonated state o -1 -1 respectively] as reflected from change of ΔG298 K from -3.3 kJ mol (for 144a) and –2.8 kJ mol (for 144b) at pD 6.7 to -0.1 kJ/mol and -1.7 kJ mol-1 (for 144b) at pD 1.0 [Tables 15 and 16]. The o plots of pD-dependent ΔG298 K values of the N S equilibrium in MepGpEt (144a) as well as MepApEt (144b) [Panel (B) in Fig 22] have the typical sigmoidal shapes, as found for β-D- 2',3'- dideoxy, 2'-deoxy, ribo N- or C-nucleosides and some α-D-2',3'-dideoxy and 2’-deoxy-nucleosides (Section 3.7, Fig 11). The value of the pD at the inflection point of the plots of pD-dependent o ΔG298 K of N S equilibrium in 144a and 144b were found to be 2.4 ± 0.1 and 3.8 ± 0.2 respectively, which are identical to the pKa of the constituent guanin-9-yl and adenine-9-yl nucleobases, as determined independently from the plots of pD-dependent H8 chemical shift for 144a and that of both H8 and H2 chemical shifts for 144b [Panel (A) in Fig 22] [see the legend of 3 Fig 22 for details, Eq. 12 and Section 3.7.4]. As a control experiment, the difference in JHH values between neutral and acidic pDs at 298K was found to be negligible in apurinic phosphodiester 145, showing that in the absence of aglycone, the N S equilibrium is unbiased and remains unchanged at all pDs compared to 144a and 144b.

3 Table 15. Pseudorotational analyses and EPSILON calculations using temperature-dependent JHH 3 a and JCP at seven different pDs (1.0-6.7), and determination of pD-dependent thermodynamics of the two-state N ←→ S and εt ε- equilibrium in MepGpEt (144a).

Thermodynamics of N ←→ S Thermodynamics of equilibrium εt ε- equilibrium o o pD ΔG298 K = ΔG298 K = %S %ε- ( / ) -R*298*ln (xN / xS) -R*298*ln xεt xε- 1.0b -0.0 (0.1) -b 0.3 (0.2) -b 1.6 -0.3 (0.2) 53 0.2 (0.2) 48 2.0 -0.7 (0.2) 58 0.0 (0.5) 50 2.4 -1.4 (0.2) 64 -1.2 (0.2) 62 2.7 -2.3 (0.2) 72 -1.7 (0.2) 66 3.0 -2.4 (0.2) 72 -1.8 (0.2) 67 6.7 -3.2 (0.2) 78 -2.0 (0.2) 69

a For the experimental procedure used to calculate the values, see Section 3 and ref. 44a. b 3 No calculations performed due to unavailability of JHH owing to spectral overlap. ΔG° (at 298 K) value have been extrapolated.

3 Table 16. Pseudorotational analyses and EPSILON calculations using temperature-dependent JHH 3 a and JCP at nine different pDs (1.0-7.9), and determination of pD-dependent thermodynamics of the two-state N ←→ S and εt ε- equilibrium in MepApEt (144b).

Thermodynamics of N ←→ S Thermodynamics of equilibrium εt ε- equilibrium o o pD ΔG298 K = ∆G (at 298 K) = %S %ε- -R*298*ln (x t/x -) -R*298*ln (xN / xS) ε ε 1.0 -1.7 (0.2) 67 -1.5 (0.2) 65 2.0 -2.0 (0.2) 69 -1.6 (0.2) 66 2.5 -2.0 (0.2) 69 -1.6 (0.2) 66 3.0 -2.1 (0.2) 70 -1.6 (0.2) 66 3.5 -2.2 (0.2) 71 -1.7 (0.2) 66 4.0 -2.4 (0.2) 73 -1.8 (0.2) 67 4.5 -2.7 (0.2) 75 -1.9 (0.2) 68 4.8b -2.8 (0.2) -b -2.0 (0.2) -b 7.9 -2.8 (0.2) 76 -1.9 (0.2) 68 a For the experimental procedure used to calculate the values, see Section 3 and ref. 44b. b 3 No calculations performed due to unavailability of JHH owing to spectral overlap. ΔG° (at 298 K) value have been extrapolated.

139

(ii) pD-dependent shift of the εt ε- equlibrium

3 3 3 The temperature dependent JH3'P3', JC4'P3', JC2'P3' have been used to calculate the bias of conformational equlibrium across C3'-O3' bond (ε torsion). Since the ε+ conformer is energetically forbidden and is not found in crystal data we have interpreted the experimental coupling constants in terms of the two-state εt ε- equilibrium (Section 7.1). Logarithm of ratio of the resulting mole fractions of ε- and εt were plotted as a function of temperature in a van't Hoff type analyses to obtain o ΔH° and ΔS° and subsequently to calculate the total change in free energy at 298 K ( ΔG298 K ) of εt ε- equlibrium at each of the seven pDs ranging from 6.7 to 1.0 (Table 15). 69% (for 144a) and 68% (for 144b) in neutral state to 48% (for 144a) and 65% (for 144b) in protonated state o -1 -1 respectively. The corresponding shift of ΔG298 K is from -2.1 kJ mol (for 144a) and -1.9 kJ mol (for 144b) in neutral state to +0.3 kJ mol-1 (for 144a) and -1.5 kJ mol-1 (for 144b) in protonated o t - state [Tables 15 and 16]. Interestingly, the plots of pD-dependent ΔG298 K values of the ε ε equlibrium in MepGpEt (144a) as well as in MepApEt (144b) also give a sigmoidal curve [Panel o (C) in Fig. 22], as found for the plot of the corresponding ΔG298 K values of the N S equilibrium of the constituent pentofuranose sugar. The values of pD at the inflection point (i.e. pD = 2.3 for 144a and pD = 3.7 for 144b) of the graphs shown Panel (C) in Fig 22, and is nearly identical (i.e. within the accuracy of the measurements) to the pKa values (2.4 for 144a and 3.9 for 144b respectively) of the guanin-9-yl nucleobase in MepGpEt. Thus, it can be concluded that the pD- dependent reorientation of the 3'-ethylphosphate moiety across the C3'-O3' bond (reflected o t - in ΔG298 K values of the ε ε equlibrium) is directly dictated by the pKa of the constituent C1’- aglycone in MepGpEt (144a) and MepApEt (144b).

(A) (B) (C) 9.2 0.6 0.6 δH8G of MepGpEt 0.0 9.0 MepGpEt 0.0 MepGpEt δH8A of MepApEt K K -0.6 MepApEt 8 MepApEt 8.8 8 9 δH2A of MepApEt 9 2 2 t -0.6 at -1.2 a 8.6 ) ) H S − ε δ / t / (N -1.8 (ε -1.2 8.4 o o G -2.4 G 8.2 Δ Δ -1.8 -3.0 8.0 -3.6 -2.4 012345678 012345678 012345678 pD pD pD Figure 22. Panel (A) shows the plots of H8 chemical shift of the constituent guanine-9-yl nucleobase (δH8G in ppm) in 144a as well as H8 and H2 chemical shift of the constituent Adenin-9-yl nucleobase (δH8A and δH2A respectively in ppm) in 144b, as a function of pD at 298K. Panels (B) and (C) show the plots of the experimental ΔG° (in kJ mol-1) of the N S equilibrium of the constituent pentofuranose moiety and that of εt ε− equilibrium of the 3'-ethylphosphate group in 144a and 144b respectively, as a function of pD at 298K. The following pKa values have been obtained: 2.4 ± 0.1 (for δH8 in 144a), 4.0 ± 0.1 and 3.9 ± 0.02 (for δH8 and δH2 respectively in 144b) in Panel (A); 2.4 ± 0.1 (for 144a) and 3.8 ± 0.2 (for 144b) in Panel (B); 2.3 ± 0.4 (for 144a) and 3.7 ± 0.2 (for 144b) in Panel (C). These results constitute the first experimental evidence for transmission of electronic information to steer the conformation of the 3'-phosphate as a result of change of electronic properties of the nucleobase (protonation/deprotonation) via the tuning of the sugar conformation in a nucleoside 3',5'-bisphosphates (such as in 144a and 144b) (see the correlation plots below under subsection (iii)). In the case of our reference compound, i.e. apurinic phosphodiester [Mep(ab)pEt

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 140 3 3 (145)] no change in JHP and JCP coupling constant values has been observed compared to that of purinic phophodiester 144a and 144b over the whole pD range which allows us to conclude that the conformation across C3'-O3' remains unchanged over the whole pD range in abasic compound (145) since there is no nucleobase to be protonated in 145.

(iii) The cooperative shift of the (N,εt) (S,ε-) equilibrium as the result of protonation of guanin-9- yl in MepGpEt (144a) and MepApEt (144b) are evidenced by [∆G°(N/S) vs ∆G°(ε t/ε-)] or [∆G°(N/S) or (ε t/ε-) vs δH8] correlation plots o The correlation plots of pD-dependent ΔG298 K values of the NS equilibrium of the o t - pentofuranose sugar as a function of pD-dependent ΔG298 K values of the ε ε equilibrium of the 3'-phosphate moieties in 144a and 144b give a straight line with a high Pearson correlation coefficient [R = 0.98 for both 144a and 144b, Panel (A) in Fig 23]. This means that as the constituent guanin-9-yl in 144a and adenine-9-yl in 144b are gradually protonated in the acidic medium, the modulation of the strength of the anomeric effect shifts the N S equilibrium toward N, which in turn dynamically shifts the εt ε- equilibrium toward more εt in comparison with the neutral pD.

(A) (B) (C) 2 1.2 1.2 MepGpEt δH8G of MepGpEt δH8G of MepGpEt 1 δH8A of MepApEt δH8A of MepApEt MepApEt K K K 8 0.0 δH2A of MepApEt 8 0.0 δH2A of MepApEt 9 0 98 9 2 2 2 t t at a a ) ) ) − /S -1 /S -1.2 /ε -1.2 (N (N t ε o o o ( G -2 G G Δ Δ -2.4 Δ -2.4 -3 -3.6 -4 -3.6 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 8.0 8.2 8.4 8.6 8.8 9.0 8.0 8.2 8.4 8.6 8.8 9.0 o ΔG t − δ δ (ε /ε ) at 298 K H H o -1 o Figure 23. Panel (A) shows the plot of ΔG298 K (in kJ mol ) of the N S pseudorotational equilibrium [ΔG (N/S) at 298 o -1 t − o K] as a function of the ΔG298 K (in kJ mol ) of ε ε equilibrium [ΔG (εt/ε−) at 298 K] of the 3'-ethylphosphate group at o -1 298 K for 144a and 144b; R = 0.98 (for 144a) and 0.98 (for 144b). Panels (B) shows the plot of ΔG298 K (in kJ mol ) of the N S pseudorotational equilibrium as a function of the chemical shift of the constituent guanine-9-yl nucleobase (δH8G in ppm) in 144a as well as H8 and H2 chemical shift of the constituent adenin-9-yl nucleobase (δH8A and δH2A respectively in ppm) in 144b at 298 K; R = 1.00 (for 144a), 0.94 and 0.98 (for δH8A and δH2A respectively in 144b). o -1 t − Panels (C) shows the plot of ΔG298 K (in kJ mol ) of the ε ε equilibrium as a function of the chemical shift of the constituent guanine-9-yl nucleobase (δH8G in ppm) in 1 as well as H8 and H2 chemical shift of the constituent adenin- 9-yl nucleobase (δH8A and δH2A respectively in ppm) in 2 at 298 K; R = 0.99 (for 144a), 0.94 and 0.97 (for δH8A and δH2A respectively in 144b).

An additional evidence for the transmission of the free-energy of the protonationdeprotonation equilibrium of the nucleobase to steer phosphate conformation through o the change of the sugar conformation moiety is that the plots of the pD-dependent ΔG298 K values o t - of the NS equilibrium [Panel (B) in Fig 23] or of the pD-dependent ΔG298 K values of the ε ε equilibrium [Panel (C) in Fig 23] in 144a as well as in 144b as a function of the pD-dependent chemical shift of aromatic protons (H8 in 144a as well as both H8 and H2 in 144b) give straight lines with high correlation coefficients (R ≥ 0.94). Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected]

141

(A) (B) 1n 2 H+ sp (p-type, O4') 1 σC3'H3' N n 2 σ*C4'O4' sp (p-type, O4') σ* C4'O4' N H 3' HOH2C N9 HOH2C N9 σ*C1'-N9 O t O H ε -H+ 3' O σ*C1'-N9 P + O +H σC3'H3' ζ- ε- -O - α O σ*O3'P3' O ζ- O 1 nsp2 (p-type, O3') 1 P nsp2 (p-type, O3') H3CH2C α- O -O

CH2CH3 Change of Electronic σ*O3'P3' Character of the Aglycone + N9 (N7H ) N9 2 nsp2 (s-type) 2 + nsp2 (s-type) Φ = 159o -H O ' O ' C ' (a) AE (O4'-C1'-N9) 4 +H+ 4 2 H ' Φ = 128ο 1 C ' C ' 1 4 4 n 2 1 C ' sp (p-type) ON n 2 2 OFF H ' sp (p-type) 1 n io σ H3' s C3'H3' σ s Φ = -101o C3'H3' H3' i + Φ = -45o O ' C ' -H m C ' 4 5 s 1 C2' n +H+ C1' O4' σ* a C2' C4'O4' r C ' T σ* 5 (b) GE (O4'-C4'-C3'-O3') C4'O4' ay H ' ON 4 OPO3CH2CH3 OFF H ' OPO CH CH w 4 3 2 3 e- 1 n n 2 sp (p-type) 1 o O o n 2 Φ = -156 sp (p-type) Φ = -154 O O -H+ C C3' O ' 3' O3' 2 3 2 + n 2 (c) AE (O3'-P3'-OEt) n 2 +H sp (s-type) sp (s-type) - O- O t - ε : ON OCH2CH3 ε : OFF OCH2CH3

Figure 24. The demonstration of single stranded RNA as molecular wires using the model MepGpEt (144a). Transmission of the free energy of the protonation deprotonation equilibrium of the guanin-9-yl group in 144a drives the sugar-phosphate conformations through three consecutive stereoelectronic tunings (Newman projections: a - c; AE = anomeric effect, GE = gauche effect). Appropriate orbital overlap and the energy difference between the donor and acceptor orbitals (see Fig 25), dictated by various sugar atoms and substituents, drive the sugar-phosphate conformation through the interplay of gauche and anomeric effects. All orbitals are shown by straight arrows. Smaller curved arrows show the preferred torsional orientation, whereas the larger curved arrows indicate the mixing of donor and acceptor orbitals. The energy stabilization through either GE or AE, manifested by the interaction between the participating donor and the acceptor orbitals, is directly proprtional to the square of the overlap between the donor and the acceptor orbitals (i.e. S2) as well as inversely proportional to their energy differences (stabilization by AE or GE = S2/ΔE). When the aglycone is protonated at N7 (Panel A), the N-type pseudorotamers are preferred owing to the strengthening of the 1 1 AE(O4'-C1'-N9), as shown by the overlap between the nsp2 orbital of one of the O4' lone pairs [ nsp2 (p-type, O4')] and the antibonding orbital of the C1'-N9 bond [σ*C1’-N9]. This is illustrated through a Newman projection (Panel 3a), 1 2 where O4' lone pair orbitals [preferentially the higher energy nsp2 (p-type), not the lower energy nsp2 (S-type)] and ∗ the nO4' (P-type) → σ C1'-N9 overlap stabilizes the N-type over the S-type sugars. For an N-type sugar, nO4' → ∗ 1 σ C1'-N9 interactions are possible owing to a near antiperiplanar orientation of nsp2 (p-type) with respect to the 1 σ*C1'-N9 bond [Φ( nsp2(p-type)-O4'-C1'-N9) ≈ 159°], which takes place when the aglycone is pseudoaxial. In contrast, they are much weaker when the aglycone is pseudoequatorial in the S-type sugars owing to relatively large Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 142 1 deviation from antiplanarity, as indicated by smaller Φ values [Φ( nsp2(p-type)-O4'-C1'-N9) ≈ 128°]. On the other hand, in the neutral state (Panel 3B) the S-type pseudorotamers are relatively preferred owing to the absence of an O4'- C1'-N9 anomeric effect as well as due to the stabilization through the O3'-C3'-C4'-O4' gauche effect (i.e. the overlap of σC3'-H3' with σ*C4'-O4'. In the Newman projection (Panel 3b), the most efficient overlap of the best donor (σC3'-H3') and best acceptor orbitals (σ*C4'-O4') is indicated by a preference for the gauche orientation [smaller value for Φ(σC3'-H3'- C3'-C4'-σ*C4'-O4') ≈ -45°] in the S-type sugar over the trans conformer [higher value of Φ(σC3'-H3'-C3'-C4'-σ*C4'-O4') ≈ - 1 101°] in the N-type sugar. Thus, in the S-type conformation, a reduction of charge density at the O3' lone pair [ nsp2(p- type)] takes place owing to its maximal interaction with σ*C4'-O4' (presumably a combination of through-bond and - through-space effects are involved in this process), thereby weakening the O3'-P3'-OCH2CH3 anomeric effect in the S/ε ∗ state. Owing to the nO4' → σ C1'-N9 overlap (Panel 3A), the O4' lone pair is relatively more delocalized in the N-type conformation, which places the O3'-C3'-C4'-O4' fragment in the trans orientation, preventing the gauche effect from being fully operational (the reverse is true when the anomeric effect is weakened in the S-type conformation, Panel 3B). 1 Since the 3'-GE is not fully operational in the N-type sugar conformation, the charge density at the O3' lone pair [ nsp2 (p-type, O3')] is fully available to act as a donor and interact through the anomeric effect with the antibonding σ*P3'- t − − O(ester) orbital [AE(O3'-P3'-OCH2CH3], when C3'-O3' is in ε , O3'-P3' in ζ , and P3'-O5' in α conformations. The

Newman projection (Panel 3c) shows that the overlap between the O3' lone pair orbitals and the σ*P3'-O(ester) orbital [nO3' t - 1 → σ* P3'- O(ester) orbital mixing] stabilizes ε over ε . This is not only due to an antiperiplanar orientation of nsp2(p- 1 type) with respect to the P3'-O(ester) bond, as Φ( nsp2(p-type)-O3'-P3'-OCH2CH3) is nearly the same for the two cases, but largely owing to the greater electron density availible at nO3', arising from the absence of 3'-GE in the N-type sugar.

Therefore, these works constitute the first quantitative evidence for the transmission of the electronic character of the nucleobase to drive the 3'-phosphate conformation in a ribonucleoside 3',5'-bis-phosphate as the direct consequence of the concomittant modulation of the bias of the pseudorotational equilibrium of the constituent sugar moiety.

(iv) The mechanism of the transmission of anomeric effect of the nucleobase to 3’-gauche effect and further on to steer the 3’O-P-O(ester) anomeric effect In our earlier work on 2'-deoxy23 and ribonucleoside28 3'-ethylphosphates at the neutral pH (Section 7), we found a non-equivalent methylene protons of the 3'-ethyphosphate moiety, which turned out to be a temperature-dependent feature. At higher temperature, this non-equivalency disappeared and the methylene protons showed NMR time average chemical shifts with similar multiplicity as those of 2'-deoxy counterparts (owing to the absence of 2'-OH promoted hydrogen bonding). This observation demonstated that the nonequivalency of the methylene protons in ribonucleotides was owing to the 2'-OH promoted hydrogen bonding with the vicinal O3'. We attributed the concerted sugar-phosphate backbone conformational change solely to the 2'-OH effect, the strength of which was modulated by temperature (the “on-off switch”). In our work with pD-dependent conformational studies with MepGpEt (144a) and MepApEt (144b), we found similar temperature-dependent pattern and intensity of multiplicities of methylene protons of the 3'- ethylphosphate moiety at all pD as found for the neutral state of guanosine 3'-ethylphosphate (80) and adenosine 3'-ethylphosphate (79) respectively, thereby suggesting that the 2'-OH hydrogen- bonding remains the same in 144a and 144b over the whole pD range (pD 1.0 to 6.7) at room temperature (298 K). This means that all changes of free-energies observed at 298 K (Table 15) for 144a and 144b as a function of pD is attributed to the free-energy changes of the Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected]

143 protonationdeprotonation equilibrium of the aglycones (i.e. guanin-9-yl and adenin-9-yl) to drive the sugar-phosphate backbone in a concerted manner. As the pD-tunable change of the electronic character of the nucleobase tunes the strength of the anomeric effect, an increased preference of the ∗ N-type sugar conformation is imposed because of enhanced nO4'→σ C1'-N orbital interaction, which, in turn, affects the strength of the [O3'-C3'-C4'-O4'] gauche effect by retuning the energy ∗ levels of the donor and aceeptor orbitals in the σC3'-H → σ C4'-O4' interaction. The extent of σC3'-H ∗ → σ C4'-O4' participation influences the electron density at O3', which in turn modulates the O3'- P3'-O anomeric effect (a concerted transmission). Readers are directed to Fig 24 for a detailed discussion of the molecular orbital diagram based interpretation of the concerted pD-dependent change of sugar-phosphate backbone conformation (taking 144a as a model). We also suggest that the readers take a close look at Fig 25 for the corresponding energy diagram, which show that the energy levels of the orbitals involved in the anomeric and gauche effects are based on their relative acceptor/donor abilities in a purely qualitative manner. In Fig 25, various donor and acceptors orbitals are connected by the dotted lines to show their interdependent modulation and steering of different orbital mixing. This translates itself in terms of relative strength of gauche and anomeric effects and the preferred confomational states which make the RNA to act as a molecular wire. The electron flow is unidirectional from the nucleobase to the phosphate, which is relected by the fact that the hybrid orbital produced by the O3'-P3'-O(ester) anomeric effect is at a lower energy level than the corresponding hybrid orbital resulting from O4'-C1'-N anomeric effect. It is noteworthy that the proof of the one-way (i.e. from nucleobase to phosphate through sugar) transmission of electronic information in MepGpEt (144a) is evident by the fact that the pKa value of the guanin-9-yl aglycone is 2.4, which is the same as the 2',3'-dideoxuguanosine (pKa 2.5), 2'-deoxyguanosine (pKa 2.3) and guanosine (pKa 2.1) within the error limits of our experimentals. In fact, the same pKa value has been found for each nucleobase in corresponding nucleosides and nucleotides in either 2',3'-dideoxy, 2'-deoxy and ribo configuration as well as in MepGpEt (144a) and MepApEt (144b), as discussed in Section 4.8, show the minimal influence of the sbstituents at C2'/3'/5' on the electronic character of their constituent nucleobase at C1'. The final proof of the operation of O3'-P3'-O anomeric effect could only be experimentally obtained if we could only measure the ζ and α torsions across the 3'-phosphate backbone and the preferential O3'- P3'-O bond angle. (v) pD-dependent conformational change across β torsion is negligible The conformational equilibrium across the torsion angle β [C4'-C5'-O5'-P5'] for 144a has been 3 3 3 calculated by using temperature dependent JC4'P5' as well as the sum of JH5'P5' and JH5''P5' (Section 7.1). The analysis of both sets of data provided comparable results with discrepancies below 3%, which is within the accuracy of Karplus equation used, showing that the population of βt .

σ*C1'-N9 N σ*C4'O4' AN P A P1 σ*P3'-O(ester) 1 P 1 n 2 nsp2 (p-type, O4') D sp (p-type, O3') σC3'H3' N N CP BP 1 DP BN CN ΔE[O4'-C1'-N9 AE] ΔΔE(AE) ΔΔE (GE) Protonated State ΔE(GE) ΔE [O3'-P3'-O(ester) AE] Figure 25. The relative donor-acceptor abilities of various orbitals (i.e. the relative empirical energies) are modulated by the nature of each substituent at the sugar and of the phosphate backbone as well as on the aglycone in free, ionic and complex state. Since the electronic state of the aglycone modulates the sugar 1 1 conformation which in turn modulates the phosphate torsion, we have placed the n n sp2 (p-type, O3’) orbital at a relatively lower energy level than sp2 (p-type, O4’). As the O4’-C1’-N9 anomeric effect starts operating, the strength of the [O3’-C3’-C4’-O4’] gauche effect (σC3’-H3’ → σ*C4’-O4’) counteracts the anomeric effect owing to the relatively lower energy of σ*C4’-O4’ [AN becomes AP state when neutral nucleobase (N) becomes protonated (P)]. As the nucleobase in the (N) state takes up the (P) state in either MepGpEt (144a) or in MepApEt (144b), σ*C1’-N9 becomes a better acceptor and the O4’-C1’-N9 anomeric effect is strengthened, and that makes [O3’- C3’-C4’-O4’] gauche effect more effective [σC3’-H3’ (BN becomes BP state) → σ*C4’-O4’, i.e. more effective orbital mixing of BP with AP, see ΔΔH°10 in Fig 13 and Table 6). However, the anomeric effect is stronger than the gauche effect, therefore we se overall stabilization of more N-type sugars (ΔΔE(GE) < ΔΔE(AE), Table 15) in the P state compared to in N state. The O3’-P3’-O(ester) anomeric effect is weaker in the S-type pseudorotamers (at around neutral pH, i.e. in CN state) than in the N-type counterparts (at around acidic pH, i.e. in CP state) because the σC3’-H3’ orbital overlaps with the σ*C4’-O4’ (i.e. [O3’-C3’-C4’-O4’ gauche effect] is stronger in 1 n the former state), reducing the electron density at O3’ (B). This means that the sp2 (p-type, O3’) lonepair is relatively less available in S-type conformation (at neutral pH) to interact with the σ*P3’-O(ester) than in the N-type conformation (at acidic pH), which shows that the O3’-P3’-O(ester) anomeric effect is weaker in S-type conformation.

145 rotamer is pD independent. Hence, the transmission of the information on the change of the electronic character from nucleobase to 5'-phosphate ends at the γ torsion Similarly, the change of βt rotamer is pD independent and that of γ torsion is very much within the limit of error of the analyses in 144b (even less than 144a).

(vi) Conformational equilibrium across C1'-N9 (χ) bond The populations of the syn and anti conformations involved in the two-state syn anti conformational equilibrium across the glycosyl bond in 144a have been calculated from nOe enhancements experimentally measured at H1’ upon saturation of H8 using the semi-quantitative equation derived by Rosemeyer and Seela517. The change in population of anti conformer from neutral (6.7) to acidic (1.0) pDs is observed to be very small viz. 49% to 63% respectively. (vii) 1H and 31P Chemical shift correlations The correlation plot between the change in population of S-type sugar conformation owing to the stronger anomeric effect in 144a upon protonation at N7 of guanin-9-yl and the resultant pD- dependent H8 chemical shift has been shown in Panel (B) of Fig 26. Inspection of Panels (A) - (D) in Fig 26 also shows that the percentage conformational change in either 3'-sugar-phosphate backbone conformation (ε- conformer) or the 5'-end (population of γ+ conformer) in 144a has been nicely corroborated with the gradual downfield chemical shift of H8 of heterocyclic base due to protonation. However, the change of γ torsion as a function of pD is very much within the limit of error of the analyses in 144b (even less than 144a).

Figure 26. (A) The plot of percentage population of γ+ conformer (calculated w.r.t. original assignment, taking H5' downfield and H5" upfield) at 298K as a function of H8 chemical shift of guanin-9-yl in 144a at different pDs ranging from 1.6 to 6.7 showing the straight line (R = 0.91) with a slope of 7.37 (σ = 0.99) and an intercept of 11.88 (σ = 8.37). (B) The plot of %S at 298K as a

80 90 function of H8 chemical shift of (A) 85 (B) 78 guanin-9-yl in 144a at six pD 80 76 75 values ranging from 1.6 to 6.7 + γ 74 S 70 % % showing the straight line (R = 65 72 60 1.00) with a slope of -27.54 70 55 68 50 (σ = 0.39) and an intercept of 7.8 8.0 8.2 8.4 8.6 8.8 9.0 7.8 8.0 8.2 8.4 8.6 8.8 9.0 299.00 (σ = 3.31). (C) The plot of δH (ppm) δH (ppm) 8 8 percentage population of ε- 80 82 (D) conformer at 298K in 144a as a 75 (C) 81 70 function of H8 chemical shift of t 80 − ε 65 β its guanin-9-yl at six pDs ranging % %60 79 from 1.6 to 6.7 showing the 55 78 50 straight line (R = 0.98) with a 45 77 slope of -25.18 (σ = 1.43) and an 7.8 8.0 8.2 8.4 8.6 8.8 9.0 7.8 8.0 8.2 8.4 8.6 8.8 9.0 δH (ppm) δH 8 (ppm) 8 intercept of 273.27 (σ = 12.05). (D) The plot of percentage population of βt conformer in 144a at 298K as a function of H8 chemical shift of guanin-9-yl at six pDs ranging from 1.6 to 6.7 showing the straight line (R = 0.96) with a slope of -2.93 (σ = 0.25) and an intercept of 104.25 (σ = 2.07).

Thus the tranmission of the electronic character of the nucleobase to drive the phosphate backbone conformation via tuning of the pentofuranose conformation has been quantitatively established. Moreover, the plots in Panels (B) and (C) of Fig 23 and in Panels (A) and (B) of Fig 27 indicate a 31 direct correlation between P3' chemical shift respectively with both ∆G°298Κ (N S) and

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 146 t - ∆G°298Κ (ε ε ) over the entire pD range for 144a and 144b which can be considered as another proof of the transmission in terms of conformational energetics from base to phosphate backbone through nucleotidyl wire in our trinucleotide model system.

(A) (B)

-1.40 MepGpEt -1.40 MepGpEt MepApEt -1.45 -1.45 MepApEt

P -1.50 P -1.50 1 1 3δ δ3 -1.55 -1.55 -1.60 -1.60 -1.65 -1.65

-3.6 -3.0 -2.4 -1.8 -1.2 -0.6 0.0 -2.4 -1.8 -1.2 -0.6 0.0 0.6 o o ΔG ΔG t − (N/S) at 298 K (ε /ε ) at 298 K Figure 27. Panel (A) shows the plot of ΔG° (in kJ mol-1) of the N S pseudorotational equilibrium as a function of the phosphorous chemical shift (δ31P in ppm) for MepGpEt (144a) and MepApEt (144b) at 298 K; R = 0.97 (for 144a) and

0.96 (for 144b). Panel (B) shows the plot of ΔG° (in kJ mol-1) of the εt ε− equilibrium as a function of the phosphorous chemical shift (δ31P in ppm) for 144a and 144b at 298 K; R = 0.95 (for 144a) and 0.91 (for 144b).

(viii) Tunibility of aglycones and tranmission of the electronic character to drive the phosphate backbone conformation This aglycone dependent conformational transmission of sugar-phosphate backbone via pentofuranose depends upon the tunibility of aglycone vis-à-vis conformational modulation of sugar geometry. Our control studies with MepCpEt (144c) at neutral and protonated state showed that the relative conformational tunibility is in order MepGpEt (144a) > MepApEt (144b) > MepCpEt (144c) [Fig 28].

EtpGpMe (A) (B) (C) 2.5 2.3 4 1.2 EtpApMe 3.2 0.944 1 2 EtpCpMe -1 3 - l 0.9 l o1.5 m o 0.6 m2 m p 1.1 J 1 p 0.215 0.198 J 0.9 0.4 0.3 k1 k0.5 0.0 0 0 0 t − ΔΔδ (P-N) ΔΔG(P-N) (N/S) ΔΔG(P-N) (ε /ε )

Figure 28. The relative conformational tunibility of MepGpEt (144a), MepApEt (144b) and MepCpEt (144c) between neutral (N) and protonated (P) states at 298 K. Panel (A) shows the relative change of chemical shift of aromatic protons

[ΔΔδ (P-N)] in 144a – c; Panel (B) and (C) show the relative change of free energy of the N S pseudorotational -1 t − t − -1 equilibrium [ΔΔG°(P-N) (N/S), in kJ mol ] and that of ε ε equilibrium [ΔΔG°(P-N) (ε /ε ), in kJ mol ] in 144a – c. The relative order of tunibility is 144a > 144b > 144c

8.9 The importance of O4' in the self-organization of oligo-DNA In order to understand the implication of the endocylic oxygen (i.e. O4') in the origin of the stereoelectronic gauche and anomeric effects, we have determined584 the solution conformation of a 12mer oligo-DNA in which a specific sugar has been replaced by a carbocyclic analogue, Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected]

147 Aristeromycin (i.e. A6 residue). This Watson-Crick k1 Hoogsteen basepaired basepaired modified oligo-DNA, 5'- k Duplex (Ia) -1 Duplex (Ib) d(C1G2C3G4A5A6T7T8C9G10C11G12)-3'

(Duplex I) with the sugar moiety of A6 replaced by aristeromycin has been compared with the natural unmodified counterpart, the Dickerson-Drew dodecamer (5'-d(CGCGAATTCGCG)-3').

Table 16. Thermodynamics of duplex to single strand melting by UV spectroscopy for the native a and modified dodecamers from the plot of 1/Tm vs ln(concentration) at 8, 12, 16, 20 and 24 mM concentration (at least two measurements have been made at each concentration).

Native dodecamer Modified dodecamer (kcalmol-1) (kcalmol-1) ∆H° -82 (± 1) -79 (±1) -T∆S° -64 (±1) -63 (±1)

∆G°298 -18 (±1) -16 (±1) a See ref. 585 for an independent measurement.

Table 17. Thermodynamicsa of the exchange process between Hoogsteen and Watson-Crick basepaired duplexes.

kJmol-1 k−1 k1 (Ib) ⎯⎯→⎯ (Ia) (Ia) ⎯⎯→⎯ (Ib)

Ea 155 ±13 167 ±14 ∆Gθ 117 124 ∆Hθ‡ 153 ± 13 164 ± 14 -T∆Sθ‡ -36 -40 a The rate constants (k1 and k-1) for the two-state exchange process between Watson-Crick basepaired duplex (Ia) and Hoogsteen basepaired duplex (Ib) were calculated from the initial slope of build-up rates of NOESY and ROESY exchange crosspeaks at different mixing times at 7 different temperatures. The rate constants (k1 and k-1) were then used θ‡ in an Arrhenius plot to calculate the energy of activation (Ea) which in turn gives the enthalpy (∆H ) and enthropy (∆Sθ‡) contributions to the free energy of activation (∆Gθ) (see ref. 586 and 587).

The duplex (I) was found to exist in dynamic equilibrium between two different interconverting conformations, (Ia) and (Ib), with equilibrium constant K = k1 / k-1 = 0.56 ± 0.08 in the temperature range of 287 - 308K. Thus the free-energy of stabilization (ΔΔG°298) of Watson- Crick basepaired duplex (Ia) realtive to Hoggsteen basepaired duplex (Ib) is 1.4 kJmol-1, which is calculated from -RT* lnK. In the (Ia)-form, the duplex adopts a canonical B-type conformation where all basepairs are of the Watson-Crick type, whereas in the (Ib)-type structure, the basepairs formed between A6 and T7 are of the Hoogsteen type (the N1 of the adenin-9-yl base in aristeromycin is looking at the major groove), and the rest of the molecule however adopts a B-type conformation. The thermodynamics and the kinetics of the Watson-Crick basepaired (Ia) duplex Hoogsteen basepaired (Ib) duplex equilibrium have also been investigated, and are shown in Tables 16 and 17, respectively. It can be seen that the difference between the thermodynamics of the self- assembly of the modified DNA duplex and the native counterpart is negligible, well within the error

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 148 limits. This means that there is virtually no energy penalty for replacement of O4' by CH2, which is evident from the fact that the equilibrium populations of Watson-Crick and Hoogsteen duplexes are nearly the same. This suggests that there is no preference for furanose-based duplex over the cyclopentane-based duplex in energetic terms. However, the largest structural implication in the cyclopentane-based duplex (Ib) is the change of the local structure around the Hoogsteen basepaired junction compared to the Watson-Crick basepaired counterpart in that the N1 of the adenin-9-yl moieties in aristeromycin (i.e. A6) is now turned toward the major groove compared to N7 in the corresponding Watson-Crick basepaired duplex (Ia), which has considerable implication regarding the hydration behaviour as well as in the ligand binding capability of DNA duplex (Ib) comapred to (Ia), which is under investigation in the author's laboratory.

9. References

(1) Kirby, A. J. In The Anomeric Effect and Related Stereoelectronic Effects at Oxygen; Springer Verlag: Berlin, 1983; Vol. 15. (2) Deslongchamps, P. InStereoelectronic Effects in Organic Chemistry; Pergamon Press: Oxford, 1983. (3) Tvaroska, I.; Bleha, T. In Advances in Carbohydrate Chemistry and Biochemistry; R. S. Tipson and D. Horton, Ed.; Academic Press: San Diego, 1989; Vol. 47; pp 45. (4) Juaristi, E.; Cuevas, G. Tetrahedron 1992, 48, 5019. (5) Thatcher, G. R. J. In The Anomeric Effect and Associated Stereoelectronic Effects; G. R. J. Thatcher, Ed.; American Chemical Society: Washington, DC, 1993; Vol. 539; pp 6. (6) Graczyk, P. P.; Mikolajczyk, M. In Topics in Stereochemistry; Eliel, E. L. and Wilen, S.H., Ed.; John Wiley & Sons, Inc.: New York, 1994; Vol. 21; pp 159. (7) Perrin, C. L. Tetrahedron 1995, 51, 11901. (8) Taira, K.; Uebayasi, M.; Maeda, H.; Furukawa, K. Protein Engineering 1990, 3, 691. (9) Storer, J. W.; Uchimaru, T.; Tanabe, K.; Uebayasi, M.; Nishikawa, S.; Taira, K. J. Am. Chem. Soc. 1991, 113, 5216. (10) Zhou, D.-M.; Taira, K. Chem. Rev. 1998, 98, 991. (11) Bizzozero, S. A.; Dutler, H. Bioorg. Chem. 1981, 10, 46. (12) Post, C. B.; Karplus, M. J. Am. Chem. Soc. 1986, 108, 1317. (13) Taira, K. Bull. Chem. Soc. Jpn. 1987, 60, 1903. (14) Koole, L. H.; Buck, H. M.; Nyilas, A.; Chattopadhyaya, J. Can. J. Chem. 1987, 65, 2089. (15) Koole, L. H.; Buck, H. M.; Bazin, H.; Chattopadhyaya, J. Tetrahedron 1987, 43, 2989. (16) Koole, L. H.; Moody, H. M.; Buck, H. M.; Grouiller, A.; Essadiq, H.; Vial, J.-M.; Chattopadhyaya, J. Recl. Trav. Chim. Pays-Bas 1988, 107, 343. (17) Plavec, J.; Koole, L. H.; Sandström, A.; Chattopadhyaya, J. Tetrahedron 1991, 47, 7363. (18) Plavec, J.; Buet, V.; Grouiller, A.; Koole, L.; Chattopadhyaya, J. Tetrahedron 1991, 47, 5847. (19) Plavec, J.; Koole, L. H.; Chattopadhyaya, J. J. Biochem. Biophys. Meth. 1992, 25, 253. (20) Plavec, J.; Tong, W.; Chattopadhyaya, J. J. Am. Chem. Soc. 1993, 115, 9734. (21) Plavec, J.; Garg, N.; Chattopadhyaya, J. J. Chem. Soc. Chem. Comm. 1993, 1011. (22) Plavec, J.; Fabre-Buet, V.; Uteza, V.; Grouiller, A.; Chattopadhyaya, J. J. Biochem. Biophys. Meth. 1993, 26, 317. (23) Plavec, J.; Thibaudeau, C.; Viswanadham, G.; Sund, C.; Chattopadhyaya, J. J. Chem. Soc. Chem. Com. 1994, 781. (24) Thibaudeau, C.; Plavec, J.; Watanabe, K. A.; Chattopadhyaya, J. J. Chem. Soc. Chem. Com. 1994, 537. (25) Thibaudeau, C.; Plavec, J.; Chattopadhyaya, J. J. Am. Chem. Soc. 1994, 116, 8033. (26) Thibaudeau, C.; Plavec, J.; Garg, N.; Papchikhin, A.; Chattopadhyaya, J. J. Am. Chem. Soc. 1994, 116, 4038. (27) Plavec, J.; Thibaudeau, C.; Chattopadhyaya, J. J. Am. Chem. Soc. 1994, 116, 6558. (28) Plavec, J.; Thibaudeau, C.; Viswanadham, G.; Sund, C.; Sandström, A.; Chattopadhyaya, J. Tetrahedron 1995, 51, 11775. (29) Plavec, J.; Thibaudeau, C.; Chattopadhyaya, J. Pure Appl. Chem. 1996, 68, 2137. (30) Thibaudeau, C.; Plavec, J.; Chattopadhyaya, J. J. Org. Chem. 1996, 61, 266. (31) Chattopadhyaya, J. Nucleic Acids Res. Symposium Ser. 1996, 35, 111. (32) Luyten, I.; Thibaudeau, C.; Sandström, A.; Chattopadhyaya, J. Tetrahedron 1997, 53, 6433. (33) Luyten, I.; Thibaudeau, C.; Chattopadhyaya, J. J. Org. Chem. 1997, 62, 8800. Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected]

149 (34) Luyten, I.; Thibaudeau, C.; Chattopadhyaya, J. Tetrahedron 1997, 53, 6903. (35) Thibaudeau, C.; Chattopadhyaya, J. Nucleosides & Nucleotides 1997, 16, 523. (36) Thibaudeau, C.; Földesi, A.; Chattopadhyaya, J. Tetrahedron 1997, 53, 14043. (37) Thibaudeau, C.; Földesi, A.; Chattopadhyaya, J. Tetrahedron 1998, 54, 1867. (38) Thibaudeau, C.; Chattopadhyaya, J. Nucleosides & Nucleotides 1998, 17, 1589. (39) Thibaudeau, C.; Plavec, J.; Charropadhyaya, J. J. Org. Chem. 1998, 63, 4967. (40) Luyten, I.; Matulic-Adamic, J.; Beigelman, L.; Chattopadhyaya, J. Nucleosides & Nucleotides 1998, 17, 1605. (41) Thibaudeau, C.; Kumar, A.; Bekiroglu, S.; Matsuda, A.; Marquez, V. E.; Chattopadhyaya, J. J. Org. Chem. 1998, 63, 5447. (42) Thibaudeau, C.; Nishizono, N.; Sumita, Y.; Matsuda, A.; Chattopadhyaya, J. Nucleosides & Nucleotides, 18, 1035-1053 (1999). *(43) Acharya, P.; Nawrot, B.; Sprinzl, M.; Thibaudeau, C.; Chattopadhyaya, J. J. Chem. Soc. Perkin Trans. II, p1531-1536 (1999). *(44) (a) Acharya, P.; Trifonov, A.; Thibaudeau, C.; Földesi, A.; Chattopadhyaya, J. Angew Chem. Int. Ed. 38, 3645-3650 (1999); (b) Velikian, I., Acharya, P.; Trifonov, A.; Földesi, A.; Chattopadhyaya, J. J. Phys. Org. Chem.. 13, 300-305 (2000). *(45) Polak, M.; Plavec, J.; Trifonova, A.; Földesi, A.; Chattopadhyaya, J. JC.S. Perkin I, p2835-2843 (1999). (46) Griffey, R. H.; Lesnik, E.; Freier, S.; Sanghvi, Y. S.; Teng, K.; Kawasaki, A.; Guinosso, C.; Wheeler, P.; Mohan, V.; Cook, P. D. In Carbohydrate Modifications in Antisense Research; New Twists on Nucleic Acids; Structural Properties of Modified Nucleosides Incorporated into Oligonucleotides V.S. Sanghvi & P.D. Cook, Ed.; American Chemical Society: Washington DC, 1994; Vol. 580; pp 212, (47) de Mesmaeker, A.; Häner, R.; Martin, P.; Moser, H. E. Acc. Chem. Res. 1995, 28, 366. (48) Crooke, S. T. Med. Res. Rev. 1996, 16, 319. (49) Freier, S. M.; Altmann, K.-H. Nucleic Acids Res. 1997, 25, 4429. (50) Egli, M. Angew. Chem. Internat. Edit. 1996, 35, 1894. (51) Zong, G.; Matsukura, M. Int. J. Immunotherapy 1990, 6, 187. (52) Uhlmann, E.; Peyman, A. Chem. Rev. 1990, 90, 543. (53) Altmann, K.-H.; Bévierre, M.-O.; De Mesmaeker, A.; Moser, H. E. Bioorg. & Med. Chem. Lett. 1995, 5, 431. (54) Schmit, C.; Bévierre, M.-O.; De Mesmaeker, A.; Altmann, K.-H. Bioorg. & Med. Chem. Lett. 1994, 4, 1969. (55) Brown, S. C.; Thomson, S. A.; Veal, J. M.; Davis, D. G. Science 1994, 265, 777. (56) Betts, L.; Josey, J. A.; Veal, J. M.; Jordan, S. R. Science 1995, 270, 1838. (57) Lubini, P.; Zürcher, W.; Egli, M. Chem. & Biol. 1994, 1, 39. (58) Boggon, T. J.; Hancox, E. L.; McAuley-Hecht, K. E.; Connolly, B. A.; Hunter, W. N.; Brown, T.; Walker, R. T.; Leonard, G. A. Nucleic Acids Res. 1996, 24, 951. (59) Portmann, S.; Altmann, K.-H.; Reynes, N.; Egli, M. J. Am. Chem. Soc. 1997, 119, 2396. (60) Gao, X.; Jeffs, P. W. J. Biomol. NMR 1994, 4, 17. (61) Blommers, M. J. J.; Pieles, U.; de Mesmaeker, A. Nucleic Acids Res. 1994, 22, 4187. (62) Tereshko, V.; Gryaznov, S.; Egli, M. J. Am. Chem. Soc. 1998, 120, 269. (63) Berger, I.; Tereshko, V.; Ikeda, H.; Marquez, V. E.; Egli, M. Nucleic Acids Res. 1998, 26, 2473. (64) Ikeda, H.; Fernandez, R.; Wilk, A.; Barchi Jr., J. J.; Huang, X.; Marquez, V. E. Nucl. Acids Res. 1998, 26, 2237. (65) Edward, J. T. Chem. and Ind. 1955, 1102. (66) Lemieux, R. U.; Chu, P. In Abstracts of Papers, 133rd Meeting of the American Chemical Society,; American Chemical Society: San Francisco, CA, 1958; på 31N. (67) Lemieux, R. U. In Molecular Rearrangements, Pt. 2; de Mayo P.., Ed.; Interscience Publishers: New York, 1964; pp 709. (68) Biali, S. E. J. Org. Chem. 1992, 57, 2979. (69) Booth, H.; Everett, J. R. J. Chem. Soc. Perkin II 1980, 255. (70) Lemieux, R. U.; Kullnig, R. K.; Bernstein, H. J.; Schneider, W. G. J. Am. Chem. Soc. 1958, 80, 6098. (71) Lemieux, R. U. In Profiles, Pathways and Dreams; J. I. Seeman, Ed.; American Chemical Society: Washington DC, 1990; pp 89. (72) Franck, R. W. Tetrahedron 1983, 39, 3251. (73) Booth, G. E.; Ouellette, R. J. J. Org. Chem. 1966, 31, 544. (74) Anderson, C. B.; Sepp, D. T. J. Org. Chem. 1967, 32, 607. (75) Booth, H.; Khedhair, K. A.; Readshaw, S. A. Tetrahedron 1987, 43, 4699. (76) de Hoog, A. J.; Buys, H. R.; Altona, C.; Havinga, E. Tetrahedron 1969, 25, 3365. (77) Pierson, G. O.; Runquist, O. A. J. Org. Chem. 1968, 33, 2572. (78) Anderson, C. B.; Sepp, D. T. Chem. & Ind. (London) 1964, 2054. (79) de Hoog, A. J.; Havinga, E. Recl. Trav. Chim. Pays-Bas 1970, 89, 972. (80) El-Kafrawy, A.; Perraud, R. C. R. Acad. Sci. Ser. C. 1975, 280, 1219. (81) Barbry, D.; Couturier, D.; Ricard, G. J. Chem. Soc. Perkin II 1982, 249. (82) Eliel, E. L.; Hargrave, K. D.; Pietrusiewicz, K. M.; Manoharan, M. J. Am. Chem. Soc. 1982, 104, 3635. Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 150 (83) Eliel, E. L.; Giza, C. A. J. Org. Chem. 1968, 33, 3754. (84) Anderson, C. B.; Sepp, D. T. Tetrahedron 1968, 24, 1707. (85) Anderson, C. B.; Sepp, D. T. J. Org. Chem. 1968, 33, 3272. (86) Juaristi, E.; Cuevas, G. Tetrahedron Lett. 1992, 33, 2271. (87) Booth, H.; Dixon, J. M.; Khedhair, K. A. Tetrahedron 1992, 48, 6161. (88) Schneider, H.-J.; Hoppen, V. J. Org. Chem 1978, 43, 3866. (89) Eliel, E. L.; Allinger, N. L.; Angyal, S. J.; Morrison, G. A. In Conformational Analysis; Wiley: New York, 1965. (90) Jensen, F. R.; Bushweller, C. H.; Beck, B. H. J. Am. Chem. Soc. 1969, 91, 344. (91) Booth, H.; Grindley, T. B.; Khedhair, K. A. J. Chem. Soc. Chem. Comm. 1982, 1047. (92) Booth, H.; Khedhair, K. A. J. Chem. Soc. Chem. Comm. 1985, 467. (93) Eliel, E. L. Acc. Chem. Res. 1970, 3, 1. (94) Lemieux, R. U. Pure Appl. Chem. 1971, 25, 527. (95) Eliel, E. L. Angew. Chem. 1972, 84, 779. (96) Bailey, W. F.; Eliel, E. L. J. Am. Chem. Soc. 1974, 96, 1798. (97) Penner, G. H.; Schaefer, T.; Sebastian, R.; Wolfe, S. Can. J. Chem. 1987, 65, 1845. (98) Denmark, S. E.; Dappen, M. S.; Sear, N. L.; Jacobs, R. T. J. Am. Chem. Soc. 1990, 112, 3466. (99) Irwin, J. J.; Ha, T.-K.; Dunitz, J. D. Helv. Chim. Acta 1990, 73, 1805. (100) Luger, P.; Kothe, G.; Vangehr, K.; Paulsen, H.; Heiker, F. R. Carbohydr. Res. 1979, 68, 207. (101) Paulsen, H.; Luger, P.; Heiker, F. R. In The Anomeric Effect, Origin and Consequences; W.A. Szarek and D. Horton, Ed.; American Chemical Society: Washington DC, 1979; Vol. 87; pp 63. (102) Durette, P. L.; Horton, D. Carbohyd. Res. 1971, 18, 403. (103) Durette, P. L.; Horton, D. J. Org. Chem. 1971, 36, 2658. (104) Durette, P. L.; Horton, D. Carbohydr. Res. 1971, 18, 389. (105) Praly, J.-P.; Lemieux, R. U. Can. J. Chem. 1987, 65, 213. (106) Lemieux, R. U.; Morgan, A. R. Can. J. Chem. 1965, 43, 2205. (107) Paulsen, H.; Györgydeák, Z.; Friedmann, M. Chem. Ber. 1974, 107, 1590. (108) Hosie, L.; Marshall, P. J.; Sinnott, M. L. J. Chem. Soc. Perkin Trans II 1984, 1121. (109) Perrin, C. L. Pure Appl. Chem. 1995, 67, 719. (110) Vaino, A. R.; Chan, S. S. C.; Szarek, W. A.; Thatcher, G. R. J. J. Org. Chem. 1996, 61, 4514. (111) Perrin, C. L.; Armstrong, K. B. J. Am. Chem. Soc. 1993, 115, 6825. (112) Perrin, C. L.; Fabian, M. A.; Armstrong, K. B. J. Org. Chem. 1994, 59, 5246. (113) Finch, P.; Nagpurkar, A. G. Carbohydr. Res. 1976, 25, 275. (114) Fabian, M. A.; Perrin, C. L.; Sinnott, M. L. J. Am. Chem. Soc. 1994, 116, 8398. (115) Romers, C.; Altona, C.; Buys, H. R.; Havinga, E. In Topics in StereochemistryE.L. Eliel and N. L. Allinger, Ed.; Wiley Interscience: New York, 1969; Vol. 4; pp 39. (116) Lucken, E. A. C. J. Chem. Soc. 1959, 2954. (117) Perrin, C. L.; Armstrong, K. B.; Fabian, M. A. J. Am. Chem. Soc. 1994, 116, 715. (118) Wiberg, K. B.; Marquez, M. J. Am. Chem. Soc. 1994, 116, 2197. (119) Hutchins, R. O.; Kopp, L. D.; Eliel, E. L. J. Am. Chem. Soc. 1968, 90, 7174. (120) Box, V. G. S. Heterocycles 1990, 31, 1157. (121) Fuchs, B.; Schleifer, L.; Tartakovsky, E. Nouv. J. Chim. 1984, 8, 275. (122) Schleifer, L.; Senderowitz, H.; Aped, P.; Tartakovsky, E.; Fuchs, B. Carbohydr. Chem. 1990, 206, 21. (123) Sweigart, D. A. J. Chem. Educ. 1973, 50, 322. (124) David, S.; Eisenstein, O.; Hehre, W. J.; Salem, L.; Hoffmann, R. J. Am. Chem. Soc. 1973, 95, 3806. (125) Laing, M. J. Chem. Educ. 1987, 64, 124. (126) Brundle, C. R.; Turner, D. W. Proc. Roy. Soc. 1968, A307, 27. (127) Reed, A. E.; Schleyer, P. v. R. Inorg. Chem. 1988, 27, 3969. (128) Salzner, U. J. Org. Chem. 1995, 60, 986. (129) Cossé-Barbi, A.; Dubois, J.-E. J. Am. Chem. Soc. 1987, 109, 1503. (130) Cossé-Barbi, A.; Watson, D. G.; Dubois, J. E. Tetrahedron Lett. 1989, 30, 163. (131) Salzner, U.; von Ragué Schleyer, P. J. Org. Chem. 1994, 59, 2138. (132) Fuchs, B.; Ellencweig, A.; Tartakovsky, E.; Aped, P. Angew. Chem. Internat. Edit. 1986, 25, 287. (133) Kessler, H.; Möhrle, H.; Zimmermann, G. J. Org. Chem. 1977, 42, 66. (134) Wolfe, S. Acc. Chem. Res. 1972, 5, 102. (135) Phillips, L.; Wray, V. J. Chem. Soc. Chem. Comm. 1973, 90. (136) Brunck, T. K.; Weinhold, F. J. Am. Chem. Soc. 1979, 101, 1700. (137) Juaristi, E. J. Chem. Educ. 1979, 56, 438. (138) Zefirov, N. S.; Gurvich, L. G.; Shashkov, A. S.; Krimer, M. Z.; Vorob'eva, E. A. Tetrahedron 1976, 32, 1211. (139) Zefirov, N. S. Tetrahedron 1977, 33, 3193. (140) Zefirov, N. S.; Samoshin, V. V.; Subbotin, O. A.; Baranenkov, V. I.; Wolfe, S. Tetrahedron 1978, 34, 2953. (141) Wiberg, K. B.; Murcko, M. A.; Laidig, K. E.; MacDougall, P. J. J. Phys. Chem. 1990, 94, 6956. Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected]

151 (142) Murcko, M. A.; DiPaola, R. A. J. Am. Chem. Soc. 1992, 114, 10010. (143) Wiberg, K. B. Acc. Chem. Res. 1996, 29, 229. (144) Abe, A.; Inomata, K. J. Mol. Struct. 1991, 245, 399. (145) Craig, N. C.; Piper, L. G.; Wheeler, V. L. J. Phys. Chem. 1971, 75, 1453. (146) Kaloustian, M. K.; Dennis, N.; Mager, S.; Evans, S. A.; Alcudia, F.; Eliel, E. L. J. Am. Chem. Soc. 1976, 98, 956. (147) Eliel, E. L.; Juaristi, E. J. Am. Chem. Soc. 1978, 100, 6114. (148) Harris, W. C.; Holtzclaw, J. R.; Kalasinsky, V. F. J. Chem. Phys. 1977, 67, 3330. (149) Durig, J. R.; Liu, J.; Little, T. S.; Kalasinsky, V. F. J. Phys. Chem. 1992, 96, 8224. (150) Hirano, T.; Nonoyama, S.; Miyajima, T.; Kurita, Y.; Kawamura, T.; Sato, H. J. Chem. Soc. Chem. Comm. 1986, 606. (151) Fernholt, L.; Kveseth, K. Acta Chem. Scand. 1980, A34, 163. (152) van Schaick, E. J. M.; Geise, H. J.; Mijlhoff, F. C.; Renes, G. J. Mol. Struct. 1973, 16, 23. (153) Freisen, D.; Hedberg, K. J. Am. Chem. Soc. 1980, 102, 3987. (154) Dixon, D. A.; Smart, B. E. J. Phys. Chem. 1988, 92, 2729. (155) Kazerouni, M. R.; Hedberg, L.; Hedberg, K. J. Am. Chem. Soc. 1997, 119, 8324. (156) Zefirov, N. S. Zhur. Org. Khim. 1970, 6, 1761. (157) Zefirov, N. S.; Blagoveshchensky, V. S.; Kazimirchik, I. V.; Surova, N. S. Tetrahedron 1971, 27, 3111. (158) Hoffmann, R. Acc. Chem. Res. 1971, 4, 1. (159) Epiotis, N. D.; Sarkanen, S.; Bjorkquist, D.; Bjorkquist, L.; Yates, R. J. Am. Chem. Soc. 1974, 96, 4075. (160) Gleiter, R.; Kobayashi, M.; Zefirov, N. S.; Palyulin, V. A. Dokl. Chem. 1977, 235, 396. (161) Dionne, P.; St-Jacques, M. J. Am. Chem. Soc. 1987, 109, 2616. (162) Craig, N. C.; Chen, A.; Suh, K. H.; Klee, S.; Mellau, G. C.; Winnewisser, B. P.; Winnewisser, M. J. Am. Chem. Soc. 1997, 119, 4789. (163) Runtz, G. R.; Bader, R. F. W.; Messer, R. R. Can. J. Chem. 1977, 55, 3040. (164) Muir, M.; Baker, J. Molec. Phys. 1996, 89, 211. (165) (a) Saenger, W. In Principles of Nucleic Acid Structure; Springer Verlag: Berlin, 1988. (b) ibid, pp93-96 for anomeric effect across the 3'O-P-O-ester fragment in the sugar-phosphate backbone and references cited therein. (166) Watanabe, K. A. In Chemistry of Nucleosides and Nucleotides; L. B. Townsend, Ed.; Plenum Press: New York, 1994; Vol. 3; p 421. (167) IUPAC-IUB Eur. J. Biochem. 1983, 131, 9. (168) IUPAC-IUB Pure Appl. Chem. 1983, 55, 1273. (169) Angyal, S. J. In The Composition of Reducing Sugars in Solution, R.S. Tipson and D. Horton, Ed.; Academic Press: London, 1984; Vol. 42; p 15. (170) Squillacote, M.; Sheridan, R. S.; Chapman, O. L.; Anet, F. A. L. J. Am. Chem. Soc. 1975, 97, 3244. (171) Anderson, J. E. Top. Curr. Chem. 1974, 45, 139. (172) Kilpatrick, J. E.; Pitzer, K. S.; Spitzer, R. J. Am. Chem. Soc. 1947, 69, 2483. (173) Strauss, H. L. Ann. Rev. Phys. Chem. 1983, 34, 301. (174) de Leeuw, H. P. M.; Haasnoot, C. A. G.; Altona, C. Isr. J. Chem. 1980, 20, 108. (175) Swarna Latha, Y.; Yathindra, N. Biopolymers 1992, 32, 249. (176) Carreira, L. A.; Jiang, G. J.; Person, W. B.; Willis, J. N. J. J. Chem. Phys. 1972, 56, 1440. (177) Hall, L. D.; Steiner, P. R.; Pedersen, C. Can. J. Chem. 1970, 48, 1155. (178) Cremer, D.; Pople, J. A. J. Am. Chem. Soc. 1975, 97, 1354. (179) Geise, H. J.; Altona, C.; Romers, C. Tetrahedron Lett. 1967, 15, 1383. (180) Altona, C.; Geise, H. J.; Romers, C. Tetrahedron 1968, 24, 13. (181) Altona, C.; Sundaralingam, M. J. Am. Chem. Soc. 1972, 94, 8205. (182) Haasnoot, C. A. G.; de Leeuw, F. A. A. M.; de Leeuw, H. P. M.; Altona, C. Biopolymers 1981, 20, 1211. (183) Diez, E.; Esteban, A. L.; Bermejo, F. J.; Altona, C.; de Leeuw, F. A. A. M. J. Mol. Struct. 1984, 125, 49. (184) de Leeuw, F. A. A. M.; van Kampen, P. N.; Altona, C.; Diez, E.; Esteban, A. L. J. Mol. Struct. 1984, 125, 67. (185) Diez, E.; Esteban, A. L.; Bermejo, F. J.; Rico, M. J. Phys. Chem. 1980, 84, 3191. (186) Diez, E.; Esteban, A. L.; Guilleme, J.; Bermejo, F. L. J. Mol. Struct. 1981, 70, 61. (187) Davies, D. B.; Sadikot, H. Biopolymers 1983, 22, 1843. (188) Sarma, R. H.; Lee, C.-H.; Evans, F. E.; Yathindra, N.; Sundaralingam, M. J. Am. Chem. Soc. 1974, 96, 7337. (189) Lüdemann, H.-D.; Röder, O.; Westhof, E.; Goldammer, E. v.; Müller, A. Biophys. Struct. Mechanism 1975, 1, 121. (190) Rossi, C.; Pichi, M. P.; Tiezzi, E. Magn. Reson. Chem. 1990, 28, 348. (191) Davies, D. B.; Danyluk, S. S. Biochemistry 1974, 13, 4417. (192) Davies, D. B.; Danyluk, S. S. Biochemistry 1975, 14, 543. (193) Plach, H.; Westhof, E.; Lüdemann, H.-D.; Mengel, R. Eur. J. Biochem. 1977, 80, 295. (194) de Leeuw, H. P. M.; de Jager, J. R.; Koeners, H. J.; van Boom, J. H.; Altona, C. Eur. J. Biochem. 1977, 76, 209. (195) Westhof, E.; Plach, H.; Cuno, I.; Lüdemann, H.-D. Nucleic Acids Res. 1977, 4, 939. Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 152 (196) Ekiel, I.; Darzynkiewicz, E.; Dudycz, L.; Shugar, D. Biochemistry 1978, 17, 1530. (197) Klimke, G.; Cuno, I.; Lüdemann, H.-D.; Mengel, R.; Robins, M. J. Z. Naturforsch. 1979, 34c, 1075. (198) Ekiel, I.; Remin, M.; Darzynkiewicz, E.; Shugar, D. Biochim. Biophys. Acta 1979, 562, 177. (199) Guschlbauer, W.; Jankowski, K. Nucleic Acids Res. 1980, 8, 1421. (200) Guschlbauer, W. Biochim. Biophys. Acta 1980, 610, 47. (201) Klimke, G.; Cuno, I.; Lüdemann, H.-D.; Mengel, R.; Robins, M. J. Z. Naturforsch. 1980, 35c, 853. (202) Klimke, G.; Cuno, I.; Lüdemann, H.-D.; Mengel, R.; Robins, M. J. Z. Naturforsch. 1980, 35c, 865. (203) Haasnoot, C. A. G.; de Leeuw, F. A. A. M.; de Leeuw, H. P. M.; Altona, C. Org. Magn. Reson. 1981, 15, 43. (204) Lipnick, R. L.; Fissekis, J. D. Biochim. Biophys. Acta 1980, 608, 96. (205) de Leeuw, F. A. A. M.; Altona, C. J. Chem. Soc. Perkin II 1982, 375. (206) Uesugi, S.; Kaneyasu, T.; Imura, J.; Ikehara, M.; Cheng, D. M.; Kan, L.-S.; Ts'o, P. O. P. Biopolymers 1983, 22, 1189. (207) Yamamoto, Y.; Yokoyama, S.; Miyazawa, T.; Watanabe, K.; Higuchi, S. FEBS Lett. 1983, 157, 95. (208) Cheng, D. M.; Kan, L.-S.; Ts'O, P. O. P.; Uesugi, S.; Takatsuka, Y.; Ikehara, M. Biopolymers 1983, 22, 1427. (209) de Leeuw, F. A. A. M.; Altona, C. J. Comp. Chem. 1983, 4, 428. (210) Chiasson, J. B.; Jankowski, K.; Brostow, W. J. Bioelectricity 1983, 2, 93. (211) Westhof, E.; Sundaralingam, M. J. Am. Chem. Soc. 1983, 105, 970. (212) Birnbaum, G. I.; Brisson, J. R.; Krawczyk, S. H.; Townsend, L. B. J. Am. Chem. Soc. 1985, 107, 7635. (213) Rinkel, L. J.; Altona, C. J. Biomol. Struct. Dynamics 1987, 4, 621. (214) Orbons, L. P. M.; van der Marel, G. A.; van Boom, J. H.; Altona, C. Eur. J. Biochem. 1987, 170, 225. (215) Hosur, R. V.; Govil, G.; Miles, H. T. Magn. Reson. Chem. 1988, 26, 927. (216) Swapna, G. V. T.; Jagannadh, B.; Gurjar, M. K.; Kunwar, A. C. Biochem. Biophys. Res. Commun. 1989, 164, 1086. (217) Altona, C. In Theor. Biochem. & Mol. Biophys.; D. L. Beveridge and R. Lavery, Ed.; Adenine Press: 1990; Vol. 1 (DNA); pp 1. (218) Pieters, J. M. L.; de Vroom, E.; van der Marel, G. A.; van Boom, J. H.; Koning, T. M. G.; Kaptein, R.; Altona, C. Biochemistry 1990, 29, 788. (219) Jagannadh, B.; Reddy, D. V.; Kunwar, A. C. Biochem. Biophys. Res. Commun. 1991, 179, 386. (220) Everaert, D. H.; Peeters, O. M.; de Ranter, C. J.; Blaton, N. M.; van Aerschot, A.; Herdewijn, P. Antivir. Chem. Chemotherapy 1993, 4, 289. (221) Wijmenga, S. S.; Mooren, M. M. W.; Hilbers, C. W. In NMR of Macromolecules; G. C. K. Roberts, Ed.; Oxford University Press: Oxford, 1993; pp. 217-288. (222) van den Boogaart, J. E.; Kalinichenko, E. N.; Podkopaeva, T. L.; Mikhailopulo, I. A.; Altona, C. Eur. J. Biochem. 1994, 221, 759. (223) Mooren, M. M. W.; Wijmenga, S. S.; van der Marel, G. A.; van Boom, J. H.; Hilbers, C. W. Nucleic Acids Res. 1994, 22, 2658. (224) Verberckmoes, F.; Esmans, E. L. Spectrochim. Acta 1995, 51A, 153. (225) Rosemeyer, H.; Seela, F. J. Chem. Soc. Perkin Trans II 1997, 2341. (226) Barchi, J. J. J.; Jeong, L.-S.; Siddiqui, M. A.; Marquez, V. E. J. Biochem. Biophys. Meth. 1997, 34, 11. (227) de Leeuw, F. A. A. M.; Altona, C.; Kessler, H.; Bermel, W.; Friedrich, A.; Krack, G.; Hull, W. E. J. Am. Chem. Soc. 1983, 105, 2237. (228) Church, T. J.; Carmichael, I.; Serianni, A. S. J. Am. Chem. Soc. 1997, 119, 8946. (229) Haasnoot, C. A. G.; Liskamp, R. M. J.; van Dael, P. A. W.; Noordik, J. H.; Ottenheijm, H. C. J. J. Am. Chem. Soc. 1983, 105, 5406. (230) Pavelcik, F.; Luptakova, E. Collect. Czech. Chem. Commun. 1990, 55, 1427. (231) Varela, O.; Marino, C.; Cicero, D. An. Assoc. Quim. Argent. 1991, 79, 107. (232) Cremer, D.; Pople, J. A. J. Am. Chem. Soc. 1975, 97, 1358. (233) Pitzer, K. S.; Donath, W. E. J. Am. Chem. Soc. 1959, 81, 3213. (234) Hoffmann, R. A.; van Wijk, J.; Leeflang, B. R.; Kamerling, J. P.; Altona, C.; Vliegenthart, J. F. G. J. Am. Chem. Soc. 1992, 114, 3710. (235) Cushley, R. J.; Codington, J. F.; Fox, J. J. Can. J. Chem. 1968, 46, 1131. (236) Fang, K. N.; Kondo, N. S.; Miller, P. S.; Ts'o, P. O. P. J. Am. Chem. Soc. 1971, 93, 6647. (237) Schleich, T.; Blackburn, B. J.; Lapper, R. D.; Smith, I. C. P. Biochemistry 1972, 11, 137. (238) Tran-Dinh, S.; Cachaty, C. Biochim. Biophys. Acta 1973, 335, 1. (239) Allore, B. D.; Queen, A.; Blonski, W. J.; Hruska, F. E. Can. J. Chem. 1983, 61, 2397. (240) West, R. T.; Garza, L. A. I.; Winchester, W. R.; Walmsley, J. A. Nucleic Acids Res. 1994, 22, 5128. (241) Feigon, J.; Wang, A. H.-J.; van der Marel, G. A.; van Boom, J. H.; Rich, A. Nucleic Acids Res. 1984, 12, 1243. (242) Tran-Dinh, S.; Taboury, J.; Neumann, J.-M.; Huynh-Dinh, T.; Genissel, B.; Langlois d'Estaintot, B.; Igolen, J. Biochemistry 1984, 23, 1362. (243) Davis, P. W.; Hall, K.; Cruz, P.; Tinoco Jr., I.; Neilson, T. Nucleic Acids Res. 1986, 14, 1279. (244) Davis, P. W.; Adamiak, R. W.; Tinoco, I. J. Biopolymers 1990, 29, 109. Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected]

153 (245) Agback, P.; Sandström, A.; Yamakage, S.-i.; Sund, C.; Glemarec, C.; Chattopadhyaya, J. J. Biochem. Biophys. Meth. 1993, 27, 229. (246) Agback, P.; Glemarec, C.; Yin, L.; Sandström, A.; Plavec, J.; Sund, C.; Yamakage, S.-i.; Viswanadham, G.; Rousse, B.; Puri, N.; Chattopadhyaya, J. Tetrahedron Lett. 1993, 34, 3929. (247) Izatt, R. M.; Christensen, J. J.; Rytting, J. H. Chem. Rev. 1971, 71, 439. (248) Brahms, S.; Fritsch, V.; Brahms, J. G.; Westhof, E. J. Mol. Biol. 1992, 223, 455. (249) Westhof, E.; Sundaralingam, M. J. Am. Chem. Soc. 1980, 102, 1493. (250) Sasisekharan, V. Jerus. Symp. Quant. Chem. Biochem. 1973, 5, 247. (251) Coulter, C. L. J. Am. Chem. Soc. 1973, 95, 570. (252) Reddy, B. S.; Saenger, W. Acta Cryst. 1978, B34, 1520. (253) Saenger, W.; Eckstein, F. J. Am. Chem. Soc. 1970, 92, 4712. (254) Lee, C.-H.; Sarma, R. H. J. Am. Chem. Soc. 1976, 98, 3541. (255) Ku Chwang, A.; Sundaralingam, M. Acta Cryst. 1974, B30, 1233. (256) Coulter, C. L. Acta Cryst. 1969, B25, 2055. (257) Varughese, K. I.; Lu, C. T.; Kartha, G. J. Am. Chem. Soc. 1982, 104, 3398. (258) Lai, T. F.; Marsh, R. E. Acta Cryst. 1972, B28, 1982. (259) Shikata, K.; Ueki, T.; Mitsui, T. Acta Cryst. 1973, B29, 31. (260) Gurskaya, G. V.; Tsapkina, E. N.; Skaptasova, N. V.; Lindeman, A. A.; Struchkov, Y. T. Dokl Akad. Nauk. USSR 1986, 291, 854. (261) Birnbaum, G. I.; Giziewicz, J.; Gabe, E. J.; Lin, T.-S.; Prusoff, W. H. Can. J. Chem. 1987, 65, 2135. (262) Camerman, A.; Mastropaolo, D.; Camerman, N. Proc. Natl. Acad. Sci. USA 1987, 84, 8239. (263) van Roey, P.; Salerno, J. M.; Duax, W. L.; Chu, C. K.; Ahn, M. K.; Schinazi, R. F. J. Am. Chem. Soc. 1988, 110, 2277. (264) Parthasarathy, R.; Kim, H. Biochem. Biophys. Res. Commun. 1988, 152, 351. (265) Dyer, I.; Low, J. N.; Tollin, P.; Wilson, H. R.; Howie, R. A. Acta Cryst. 1988, C44, 767. (266) van Roey, P.; Schinazi, R. F. Antiviral Chem. & Chemotherapy 1990, 1, 93. (267) Camerman, N.; Mastropaolo, D.; Camerman, A. Proc. Natl. Acad. Sci. USA 1990, 87, 3534. (268) Chu, C. K.; Bhadti, V. S.; Doboszewski, B.; Gu, Z. P.; Kosugi, Y.; Pullaiah, K. C.; van Roey, P. J. Org. Chem. 1989, 54, 2217. (269) Birnbaum, G. I.; Lin, T.-S.; Prusoff, W. H. Biochem. Biophys. Res. Commun. 1988, 151, 608. (270) Koole, L. H.; Plavec, J.; Liu, H.; Vincent, B. R.; Dyson, M. R.; Coe, P. L.; Walker, R. T.; Hardy, G. W.; Rahim, S. G.; Chattopadhyaya, J. J. Am. Chem. Soc. 1992, 114, 9936. (271) Barnes, C. L.; Hawkinson, S. W.; Wigler, P. W. Acta Cryst. 1980, B36, 2299. (272) Birnbaum, G. I.; Watanabe, K. A.; Fox, J. J. Can. J. Chem. 1980, 58, 1633. (273) Barrett, A. G. M.; Broughton, H. B. J. Org. Chem. 1984, 49, 3673. (274) Mc Kenna, R.; Neidle, S.; Serafinowski, P. Acta Cryst. 1987, C43, 2358. (275) Mc Kenna, R.; Neidle, S.; Serafinowski, P. Acta Cryst. 1990, C46, 2448. (276) Mumper, M. W.; Aurenge, C.; Hosmane, R. S. J. Biomol. Struct. Dynamics 1994, 11, 1107. (277) Koyama, G.; Nakamura, H.; Umezawa, H.; Iitaka, Y. Acta Cryst. 1976, B32, 813. (278) Prusiner, P.; Brennan, T.; Sundaralingam, M. Biochemistry 1973, 12, 1196. (279) Rohrer, D.; Sundaralingam, M. J. Am. Chem. Soc. 1970, 92, 4950. (280) Koyama, G.; Umezawa, H.; Iitaka, Y. Acta Cryst. 1974, B30, 1511. (281) Singh, P.; Hodgson, D. J. Acta Cryst. 1977, B33, 3189. (282) Hempel, A.; Lane, B. G.; Camerman, N. Acta Cryst. 1997, C53, 1707. (283) Krol, M. C.; Huige, C. J. M.; Altona, C. J. Comp. Chem. 1990, 11, 765. (284) Marquez, V. E. In Advances in Antiviral Drug Design; E. de Clercq, Ed.; JAI Press, Inc.: Greenwich, CT, 1996; Vol. 2; pp 89. (285) Borthwick, A. D.; Biggadike, K. Tetrahedron 1992, 48, 571. (286) Marquez, V. E.; Lim, M.-I. Med. Res. Rev. 1986, 6, 1. (287) Altmann, K.-H.; Imwinkelried, R.; Kesselring, R.; Rihs, G. Tetrahedron Lett. 1994, 35, 7625. (288) Shealy, Y. F.; O'Dell, C. A. Tetrahedron Lett. 1969, 27, 2231. (289) Marumoto, R.; Yoshioka, Y.; Furukawa, Y.; Honjo, M. Chem. Pharm. Bull. 1976, 24, 2624. (290) Kinoshita, K.; Yaginuma, S.; Hayashi, M.; Nakatsu, K. Nucleosides & Nucleotides 1985, 4, 661. (291) Popescu, A.; Hörnfeldt, A.-B.; Gronowitz, S.; Johansson, N. G. Nucleosides & Nucleotides 1995, 14, 1639. (292) Siddiqui, M. A.; Ford, H. J.; George, C.; Marquez, V. E. Nucleosides & Nucleotides 1996, 15, 235. (293) Rodriguez, J. B.; Marquez, V. E.; Nicklaus, M. C.; Mitsuya, H.; Jr., B. J. J. Med. Chem. 1994, 37, 3389. (294) Kishi, T.; Muroi, M.; Kusaka, T.; Nishikawa, M.; Kamiya, K.; Mizuno, K. Chem. Pharm. Bull. 1972, 20, 940. (295) Kálman, A.; Koritsánszky, T.; Béres, J.; Sági, G. Nucleosides & Nucleotides 1990, 9, 235. (296) Biggadike, K.; Borthwick, A. D.; Exall, A. M.; Kirk, B. E.; Roberts, S. M.; Youds, P.; Slawin, A. M. Z.; Williams, D. J. J. Chem. Soc. Chem. Comm. 1987, 255. (297) Marumoto, R. AIDS-Iyakuhin Kaihatsu-Kenkyu-Hokuku (The Japan Health Sciences Foundation) 1991, 3. (298) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Gill, P. M. W.; Johnson, B. G.; Robb, M. A.; Cheeseman, J. R.; Keith, T.; Petersson, G. A.; Montgomery, J. A.; Raghavachari, K.; Al-Laham, M. A.; Zakrzewski, V. G.; Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 154 Ortiz, J. V.; Foresman, J. B.; Cioslowski, J.; Stefanov, B. B.; Nanayakkara, A.; Challacombe, M.; Peng, C. Y.; Ayala, P. Y.; Chen, W.; Wong, M. W.; Andres, J. L.; Replogle, E. S.; Gomperts, R.; Martin, R. L.; Fox, D. J.; Binkley, J. S.; Defrees, D. J.; Baker, J.; Stewart, J. P.; Head-Gordon, M.; Gonzalez, C.; Pople, J. A. Gaussian 94 Revision C.3; Gaussian, Inc.: Pittsburgh PA, 1995. (299) Govil, G.; Saran, A. J. Theor. Biol. 1971, 33, 399. (300) Lugovskoi, A. A.; Dashevskii, V. G. Mol. Biol. (USSR) 1972, 6, 354. (301) Lugovskoi, A. A.; Dashevskii, V. G.; Kitaigorodskii, A. I. Mol. Biol. (USSR) 1972, 6, 361. (302) Lugovskoi, A. A.; Dashevskii, V. G.; Kitaigorodskii, A. I. Mol. Biol. (USSR) 1972, 6, 494. (303) Levitt, M.; Warshel, A. J. Am. Chem. Soc. 1978, 100, 2607. (304) Olson, W. K.; Sussman, J. L. J. Am. Chem. Soc. 1982, 104, 270. (305) Olson, W. K. J. Am. Chem. Soc. 1982, 104, 278. (306) Pearlman, D. A.; Kim, S.-H. J. Biomol. Struct. Dynamics 1985, 3, 99. (307) Röder, O.; Lüdemann, H.-D.; von Goldammer, E. Eur. J. Biochem. 1975, 53, 517. (308) Plavec, J.; Roselt, P.; Földesi, A.; Chattopadhyaya, J. Mag. Res. Chem. 1998, 36, 732. (309) Jalluri, R. K.; Yuh, Y. H.; Taylor, E. W. In The Anomeric Effect and Associated Stereoelectronic Effects; G. R. J. Thatcher, Ed.; American Chemical Society: Washington, DC, 1993; Vol. 539; pp 277. (310) Allen, F. H.; Davies, J. E.; Galloy, J. J.; Johnson, O.; Kennard, O.; MacRae, C. F.; Mitchell, E. M.; Mitchell, G. F.; Smith, J. M.; Watson, D. G. J. Chem. Inf. Comput. Sci. 1991, 31, 187. (311) Lo, A.; Shefter, E.; Cochran, T. G. J. Pharm. Sc. 1975, 64, 1707. (312) Fox, J. J.; Shugar, D. Biochim. Biophys. Acta 1952, 9, 369. (313) Tran-Dinh, S.; Thiéry, J.; Guschlbauer, W.; Dunand, J.-J. Biochim. Biophys. Acta 1972, 281, 289. (314) Sarma, R. H.; Mynott, R. J. J. Am. Chem. Soc. 1973, 95, 1641. (315) Remin, M.; Shugar, D. J. Am. Chem. Soc. 1973, 95, 8146. (316) Altona, C.; Sundaralingam, S. J. Am. Chem. Soc. 1973, 95, 2333. (317) Tran-Dinh, S.; Guschlbauer, W. Nucleic Acids Res. 1975, 3, 873. (318) Hruska, F. E.; Wood, D. J.; Singh, H. Biochim. Biophys. Acta 1977, 474, 129. (319) Seela, F.; Becher, G.; Rosemeyer, H.; Reuter, H.; Kastner, G.; Mikhailopulo, I. A. Helv. Chim. Acta 1999, 82, 105. (320) Hillen, W.; Gassen, H. G. Biochim. Biophys. Acta 1978, 518, 7. (321) Egert, E.; Lindner, H. J.; Hillen, W.; Böhm, M. C. J. Am. Chem. Soc. 1980, 102, 3707. (322) Uhl, W.; Reiner, J.; Gassen, H. G. Nucleic Acids Res. 1983, 11, 1167. (323) O'Leary, D. J.; Kishi, Y. J. Org. Chem. 1994, 59, 6629. (324) Birnbaum, G. I.; Blonski, W. J. P.; Hruska, F. E. Can. J. Chem. 1983, 61, 2299. (325) Cadet, J.; Ducolomb, R.; Taieb, C. Tetrahedron Lett. 1975, 40, 3455. (326) Birnbaum, G. I.; Hruska, F. E.; Niemczura, W. P. J. Am. Chem. Soc. 1980, 102, 5586. (327) Bergström, D. E.; Zhang, P.; Toma, P. H.; Andrews, P. C.; Nichols, R. J. Am. Chem. Soc. 1995, 117, 1201. (328) Post, M. L.; Birnbaum, G. I.; Huber, C. P.; Shugar, D. Biochim. Biophys. Acta 1977, 479, 133. (329) Sundaralingam, M. J. Am. Chem. Soc. 1971, 93, 6644. (330) Rohrer, D. C.; Sundaralingam, M. J. Am. Chem. Soc. 1970, 92, 4956. (331) Remin, M.; Ekiel, I.; Shugar, D. Eur. J. Biochem. 1975, 53, 197. (332) Seto, H.; Otake, N.; Yonehara, H. Tetrahedron Lett. 1972, 38, 3991. (333) Ruzic-Toros, Z. Acta Cryst. 1979, B35, 1277. (334) Crowfoot Hodgkin, D.; Lindsey, J.; Sparks, R. A.; Trueblood, K. N.; White, J. G. Proc. Roy. Soc. 1962, A266, 494. (335) Brink-Shoemaker, C.; Cruickshank, D. W. J.; Crowfoot Hodgkin, D.; Kamper, M. J.; Pilling, D. Proc. Roy. Soc. 1964, A278, 1. (336) Hawkinson, S. W.; Coulter, C. L.; Greaves, M. L. Proc. Roy. Soc. 1970, A318, 143. (337) Savage, H. F. J.; Lindley, P. F.; Finney, J. L.; Timmins, P. A. Acta Cryst. 1987, B43, 280. (338) Lenhert, P. G. Proc. Roy. Soc. 1968, A303, 45. (339) Hamor, T. A.; O'Leary, M. K.; Walker, R. T. Acta Cryst. 1977, B33, 1218. (340) Shefter, E.; Kotick, M. P.; Bardos, J. J. Pharm. Sci. 1967, 56, 1293. (341) Ide, H.; Shimizu, H.; Kimura, Y.; Sakamoto, S.; Makino, K.; Glackin, M.; Wallace, S. S.; Nakamuta, H.; Sasaki, M.; Sugimoto, N. Biochemistry 1995, 34, 6947. (342) Gutowski, G. E.; Chaney, M. O.; Jones, N. D.; Hamill, R. L.; Davis, F. A.; Miller, R. D. Biochem. Biophys. Res. Commun. 1973, 51, 312. (343) Armstrong, V. W.; Dattagupta, J. K.; Eckstein, F.; Saenger, W. Nucleic Acids Res. 1976, 3, 1791. (344) Cline, S. J.; Hodgson, D. J. Biochim. Biophys. Acta 1979, 563, 540. (345) Post, M. L.; Huber, C. P.; Birnbaum, G. I.; Shugar, D. Can. J. Chem. 1981, 59, 238. (346) Piper, I. M.; MacLean, D. B.; Faggiani, R.; Lock, C. J. L.; Szarek, W. A. Can. J. Chem. 1985, 63, 2915. (347) Serianni, A.; Barker, R. J. Org. Chem. 1984, 49, 3292. (348) Serianni, A. S.; Chipman, D. M. J. Am. Chem. Soc. 1987, 109, 5297. (349) Kline, P. C.; Serianni, A. S. J. Am. Chem. Soc. 1990, 112, 7373. Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected]

155 (350) Raap, J.; van Boom, J. H.; van Lieshout, H. C.; Haasnoot, C. A. G. J. Am. Chem. Soc. 1988, 110, 2736. (351) Ellervik, U.; Magnusson, G. J. Am. Chem. Soc. 1994, 116, 2340. (352) Teng, K.; Cook, P. D. J. Org. Chem. 1994, 59, 278. (353) Cyr, N.; Perlin, A. S. Can. J. Chem. 1979, 57, 2504. (354) Le Doan, T.; Perrouault, L.; Praseuth, D.; Habhoub, N.; Decout, J.-L.; Thouong, N. T.; Lhomme, J.; Hélène, C. Nucleic Acids Res. 1987, 15, 7749. (355) Moser, H. E.; Dervan, P. B. Science 1987, 238, 645. (356) Povsic, T. J.; Dervan, P. B. J. Am. Chem. Soc. 1989, 111, 3059. (357) Xodo, L. E.; Manzini, G.; Quadrifoglio, F.; van der Marel, G. A.; van Boom, J. H. Nucleic Acids Res. 1991, 19, 5625. (358) Brown, T.; Leonard, G. A.; Booth, E. D.; Kneale, G. J. Mol. Biol. 1990, 212, 437. (359) Boulard, Y.; Cognet, J. A. H.; Gabarro-Arpa, J.; Le Bret, M.; Sowers, L. C.; Fazakerley, G. V. Nucleic Acids Res. 1992, 20, 1933. (360) Wang, C.; Gao, H.; Gaffney, B. L.; Jones, R. A. J. Am. Chem. Soc. 1991, 113, 5486. (361) Macaya, R. F.; Gilbert, D. E.; Malek, S.; Sinsheimer, J. S.; Feigon, J. Science 1991, 254, 270. (362) Gao, X.; Patel, D. J. J. Am. Chem. Soc. 1988, 110, 5178. (363) Dolinnaya, N. G.; Fresco, J. R. Proc. Natl. Acad. Sci. USA 1992, 89, 9242. (364) Dolinnaya, N. G.; Braswell, E. H.; Fossella, J. A.; Klump, H.; Fresco, J. R. Biochemistry 1993, 32, 10263. (365) Robinson, H.; van der Marel, G. A.; van Boom, J. H.; Wang, A. H.-J. Biochemistry 1992, 31, 10510. (366) Liu, K.; Miles, H. T.; Frazier, J.; Sasisekharan, V. Biochemistry 1993, 32, 11802. (367) Jaishree, T. N.; Wang, A. H.-J. Nucleic Acids Res. 1993, 21, 3839. (368) Gehring, K.; Leroy, J.-L.; Guéron, M. Nature 1993, 363, 561. (369) Leroy, J.-L.; Gehring, K.; Kettani, A.; Guéron, M. Biochemistry 1993, 32, 6019. (370) Garcia, B.; Palacios, J. C. Ber. Bunsenges. Phys. Chem. 1988, 92, 696. (371) Taylor, R.; Kennard, O. J. Mol. Struct. 1982, 78, 1. (372) Jardetzky, C. D.; Jardetzky, O. J. Am. Chem. Soc. 1960, 82, 222. (373) Angell, C. L. J. Chem. Soc. 1961, 504. (374) Tsuboi, M.; Kyogoku, Y.; Shimanouchi, T. Biochim. Biophys. Acta 1962, 55, 1. (375) Katritzky, A. R.; Waring, A. J. J. Chem. Soc. 1962, 1540. (376) Lord, R. C.; Thomas, G. J. J. Spectrochim. Acta 1967, 23A, 2551. (377) Clauwaert, J.; Stockx, J. Z. Naturforsch. 1968, 23B, 25. (378) Wagner, R.; von Philipsborn, W. Helv. Chim. Acta 1970, 53, 299. (379) Christensen, J. J.; Rytting, H.; Izatt, R. M. Biochemistry 1970, 9, 4907. (380) Poulter, C. D.; Anderson, R. B. Tetrahedron Lett. 1972, 36, 3823. (381) Dunn, D. B.; Hall, R. H. In Handbook of Biochemistry and Molecular Biology; G. D. Fasman, Ed.; C.R.C. Press: Cleveland, Ohio, 1975; Vol. I; pp 65. (382) Kremer, A. B.; Mikita, T.; Beardsley, G. P. Biochemistry 1987, 26, 391. (383) Wempen, I.; Fox, J. J. J. Am. Chem. Soc. 1964, 86, 2474. (384) Cushley, R. J.; Wempen, I.; Fox, J. J. J. Am. Chem. Soc. 1968, 90, 709. (385) Ward, D. C.; Reich, E.; Stryer, L. J. Biol. Chem. 1969, 244, 1228. (386) Cho, B. P.; McGregor, M. A. Nucleosides & Nucleotides 1994, 13, 481. (387) Koyama, G.; Umezawa, H. J. Antibiotics 1965, A18, 175. (388) Robins, R. K.; Townsend, L. B.; Cassidy, F.; Gerster, J. F.; Lewis, A. F.; Miller, R. L. J. Heterocycl. Chem. 1966, 3, 110. (389) Chu, C. K.; Wempen, I.; Watanabe, K. A.; Fox, J. J. J. Org. Chem. 1976, 41, 2793. (390) Chambers, R. W. Prog. Nucleic Acids Res. Mol. Biol. 1966, 5, 349. (391) Smith, R. M.; Martell, A. E.; Chen, Y. Pure Appl. Chem. 1991, 63, 1015. (392) Marzilli, L. G.; de Castro, B.; Solorzano, C. J. Am. Chem. Soc. 1982, 104, 461. (393) Marzilli, L. G.; Kline, T. P.; Live, D.; Zon, G. In Metal-DNA Chemistry; T. D. Tullius, Ed.; American Chemical Society: Washington DC, 1989; pp 119. (394) InInteractions of Metal Ions with Nucleotides, Nucleic Acids, and Their Constituents; A. Sigel and H. Sigel ed.; M. Dekker, Inc.: New York, 1996; Vol. 32 (Metal Ions in Biological Systems). (395) Polak, M.; Plavec, J. Nucleosides & Nucleotides 1998, 17, 2011. (396) Puglisi, J. D.; Wyatt, J. R.; Tinoco, I. J. Biochemistry 1990, 29, 4215. (397) Legault, P.; Pardi, A. J. Am. Chem. Soc. 1994, 116, 8390. (398) Legault, P.; Pardi, A. J. Am. Chem. Soc. 1997, 119, 6621. (399) Singleton, S. F.; Dervan, P. B. Biochemistry 1992, 31, 10995. (400) Tran-Dinh, S.; Guschlbauer, W.; Guéron, M. J. Am. Chem. Soc. 1972, 94, 7903. (401) Remaud, G.; Zhou, X.-x.; Welch, C. J.; Chattopadhyaya, J. Tetrahedron 1986, 42, 4057. (402) Schuster, I. I.; Roberts, J. D. J. Org. Chem. 1979, 44, 3864. (403) Remaud, G.; Welch, C.; Zhou, X.-x.; Chattopadhyaya, J. Nucleosides & Nucleotides 1988, 7, 167. (404) Markowski, V.; Sullivan, G. R.; Roberts, J. D. J. Am. Chem. Soc. 1977, 99, 714. (405) Remin, M.; Darzynkiewicz, E.; Dworak, A.; Shugar, D. J. Am. Chem. Soc. 1976, 98, 367. Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 156 (406) Froehler, B. C.; Wadwani, S.; Terhorst, T. J.; Gerrard, S. R. Tetrahedron Lett. 1992, 33, 5307. (407) Manoharan, M., unpublished results. (408) Ramzaeva, N.; Seela, F. Helv. Chim. Acta 1996, 79, 1549. (409) Seela, F.; Thomas, H. Helv. Chim. Acta 1995, 78, 94. (410) Buhr, C. A.; Wagner, R. W.; Grant, D.; Froehler, B. C. Nucleic Acids Res. 1996, 24, 2974. (411) Hashimoto, H.; Nelson, M. G.; Switzer, C. J. Am. Chem. Soc. 1993, 115, 7128. (412) Lesnik, E. (413) Gryaznov, S.; Schultz, R. G. Tetrahedron Lett. 1994, 35, 2489. (414) Sanghvi, Y. S.; Hoke, G. D.; Freier, S. M.; Zounes, M. C.; Gonzalez, C.; Cummins, L.; Sasmor, H.; Cook, P. D. Nucleic Acids Res. 1993, 21, 3197. (415) Dunkel, M.; Cook, P. D.; Acevedo, O. L. J. Heterocycl. Chem. 1993, 30, 1421. (416) Imazawa, M.; Ueda, T.; Ukita, T. Chem. Pharm. Bull. (Tokyo) 1975, 23, 604. (417) Uesugi, S.; Miki, H.; Ikehara, M.; Iwahashi, H.; Kyogoku, Y. Tetrahedron Lett. 1979, 42, 4073. (418) Ikehara, M. Heterocycles 1984, 21, 75. (419) Koole, L. H.; Wu, J.-C.; Neidle, S.; Chattopadhyaya, J. J. Am. Chem. Soc. 1992, 114, 2687. (420) Xi, Z.; Glemarec, C.; Chattopadhyaya, J. Tetrahedron 1993, 49, 7525. (421) Fraser, A.; Wheeler, P.; Dan Cook, P.; Sanghvi, Y. S. J. Heterocycl. Chem. 1993, 30, 1277. (422) Hossain, N.; Papchikhin, A.; Garg, N.; Fedorov, I.; Chattopadhyaya, J. Nucleosides & Nucleotides 1993, 12, 499. (423) Glemarec, C.; Reynolds, R. C.; Crooks, P. A.; Maddry, J. A.; Akhtar, M. S.; Montgomery, J. A.; Secrist, J. A. I.; Chattopadhyaya, J. Tetrahedron 1993, 49, 2287. (424) van Aerschot, A.; Everaert, D.; Balzarini, J.; Augustyns, K.; Jie, L.; Janssen, G.; Peeters, O.; Blaton, N.; de Ranter, C.; de Clerq, E.; Herdewijn, P. J. Med. Chem. 1990, 33, 1833. (425) Sanghvi, Y. S.; Vasseur, J.-J.; Debart, F.; Cook, P. D. Collect. Czech. Chem. Commun. 1993, 58, 158. (426) Thibaudeau, C.; Chattopadhyaya, J. unpublished result (427) de Leeuw, F. A. A. M.; van Wijk, J.; Altona, C. PSEUROT Version 5.4, Leiden University, Leiden, Netherlands 1992, (428) Hägele, G. DAISY-Program-Package for Simulation and Automated Analysis of High Resolution NMR Spectra on Bruker's Aspect X32 and Silicon Graphics Indy computers 1991-1996. (429) Altona, C.; Ippel, J. H.; Westra Hoekzema, A. J. A.; Erkelens, C.; Groesbeek, M.; Donders, L. A. Magn. Reson. Chem. 1989, 27, 564. (430) Altona, C.; Francke, R.; de Haan, R.; Ippel, J. H.; Daalmans, G. J.; Westra Hoekzema, A. J. A.; van Wijk, J. Magn. Reson. Chem. 1994, 32, 670. (431) Haasnoot, C. A. G.; De Leeuw, F. A. A. M.; Altona, C. Tetrahedron 1980, 36, 2783. (432) Harte, W. E. J.; Starrett, J. E. J.; Martin, J. C.; Mansuri, M. M. Biochem. Biophys. Res. Commun. 1991, 175, 298. (433) van Roey, P.; Salerno, J. M.; Chu, C. K.; Schinazi, R. F. Proc. Natl. Acad. Sci. USA 1989, 86, 3929. (434) Sanghvi, Y. S.; Hanna, N. B.; Larson, S. B.; Robins, R. K.; Revanker, G. R. Nucleosides & Nucleotides 1987, 6, 761. (435) Lankhorst, P. P.; Haasnoot, C. A. G.; Erkelens, C.; Altona, C. J. Biomol. Struct. Dynamics 1984, 1, 1387. (436) Imai, K.; Osawa, E. Magn. Reson. Chem. 1990, 28, 668. (437) For a recent review, see the contributions in Magn. Res. Chem. 1996, 34, S1-S186. (438) Karplus, M. J. Chem. Phys. 1959, 30, 11. (439) Huggins, M. L. J. Am. Chem. Soc. 1953, 75, 4123. (440) Karplus, M. J. Am. Chem. Soc. 1963, 85, 2870. (441) Barfield, M.; Smith, W. B. J. Am. Chem. Soc. 1992, 114, 1574. (442) Marshall, J. L.; Walter, S. R.; Barfield, M.; Marchand, A. P.; Marchand, N. W.; Segre, A. L. Tetrahedron 1976, 32, 537. (443) Jaworski, A.; Ekiel, I.; Shugar, D. J. Am. Chem. Soc. 1978, 100, 4357. (444) de Leeuw, F. A. A. M.; van Beuzekom, A. A.; Altona, C. J. Comp. Chem. 1983, 4, 438. (445) Diez, E.; San-Fabian, J.; Guilleme, J.; Altona, C.; Donders, L. A. Mol. Phys. 1989, 68, 49. (446) Donders, L. A.; de Leeuw, F. A. A. M.; Altona, C. Magn. Reson. Chem. 1989, 27, 556. (447) Plavec, J. Ph.D. Thesis, Department of Bioorganic Chemistry, Uppsala University, Sweden 1995. (448) Blandin, M.; Son, T.-D.; Catlin, J. C.; Guschlbauer, W. Biochim. Biophys. Acta 1974, 361, 249. (449) Blandin, M.; Jankowski, K. Rev. Latinoameric. Quim. 1983, 14, 45. (450) Huang, J.-T.; Chen, L.-C.; Wang, L.; Kim, M.-H.; Warshaw, J. A.; Armstrong, D.; Zhu, Q.-Y.; Chou, T.-C.; Watanabe, K. A.; Matulic-Adamic, J.; Su, T.-L.; Fox, J. J.; Polsky, B.; Baron, P. A.; Gold, J. W. M.; Hardy, W. D.; Zuckerman, E. J. Med. Chem. 1991, 34, 1640. (451) Heinemann, V.; Hertel, L. W.; Grindey, G. B.; Plunkett, W. Cancer Res. 1988, 48, 4024. (452) Brox, L. W.; LePage, G. A.; Hendler, S. S.; Shannahoff, D. H. Cancer Res. 1978, 34, 1838. (453) Tsuchiya, T. Adv. Carbohydr. Chem. Biochem. 1990, 48, 91.

157 (454) Marquez, V. E.; Lim, B. B.; Barchi, J. J. J.; Nicklaus, M. C. In Nucleosides and Nucleotides as Antitumor and Antiviral Agents; C. K. B. Chu, D.C., Ed.; Plenum Press: New York, 1993; pp 265. (455) Herdewijn, P.; Pauwels, R.; Baba, M.; Balzarini, J.; de Clerq, E. J. Med. Chem. 1987, 30, 2131. (456) Herdewijn, P.; van Aerschot, A.; Kerremans, L. Nucleosides & Nucleotides 1989, 8, 65. (457) Martin, J. A.; Brushnell, D. J.; Duncan, I. B.; Dunsdon, S. J.; Hall, M. J.; Machin, P. J.; Merret, J. H.; Parkes, K. E. B.; Roberts, N. A.; Thomas, G. J.; Galpin, S. A.; Kinchington, D. J. Med. Chem. 1990, 33, 2137. (458) Kawasaki, A. M.; Casper, M. D.; Freier, S. M.; Lesnik, E. A.; Zounes, M. C.; Cummins, L. L.; Gonzalez, C.; Cook, P. D. J. Med. Chem. 1993, 36, 831. (459) Monia, B. P.; Lesnik, E. A.; Gonzalez, C.; Lima, W. F.; McGee, D.; Guinosso, C. J.; Kawasaki, A. M.; Cook, P. D.; Freier, S. M. J. Biol. Chem. 1993, 268, 14514. (460) Fazakerley, G. V.; Uesugi, S.; Izumi, A.; Ikehara, M.; Guschlbauer, W. FEBS Lett. 1985, 182, 365. (461) Phillips, L.; Wray, V. J. Chem. Soc. (B) 1971, 1618. (462) Hall, L. D.; Manville, J. F. Chem. Comm. 1968, 37. (463) Foster, A. B.; Hems, R.; Hall, L. D. Can. J. Chem. 1970, 48, 3937. (464) Gopinathan, M. S.; Narasimhan, P. T. Molec. Phys. 1971, 21, 1141. (465) Govil, G. Molec. Phys. 1971, 21, 953. (466) Ihrig, A. M.; Smith, S. L. J. Am. Chem. Soc. 1970, 92, 759. (467) Williamson, K. L.; Li Hsu, Y.-F.; Hall, F. H.; Swager, S.; Coulter, M. S. J. Am. Chem. Soc. 1968, 90, 6717. (468) Williamson, K. L. W.; Li, Y.-F.; Hall, F. H.; Swager, S. J. Am. Chem. Soc. 1966, 88, 5678. (469) Berthelot, J.-P.; Jacquesy, J.-C.; Lesivalles, J. Bull. Soc. Chim. Fr. 1971, 5, 1896. (470) Lankin, D. C.; Chandrakumar, N. S.; Rao, S. N.; Spangler, D. P.; Snyder, J. P. J. Am. Chem. Soc. 1993, 115, 3356. (471) Emsley, J. W.; Phillips, L.; V., W. In Progress in Nuclear Magnetic Resonance Spectroscopy; J. W. Emsley, Feeney, J., Sutcliffe, L.H., Ed.; Pergamon Press: New York, 1977; Vol. 10(3/4); pp 229. (472) Abraham, R. J.; Cavalli, L. Molec. Phys. 1965, 9, 67. (473) Wilkinson, L. SYSTAT: The System for Statistics Systat Inc.: Evanston, IL, 1989. (474) Urata, H.; Shinohara, K.; Ogura, E.; Ueda, Y.; Akagi, M. J. Am. Chem. Soc. 1991, 113, 8174. (475) Urata, H.; Ogura, E.; Shinohara, K.; Ueda, Y.; Akagi, M. Nucl. Acids Res. 1992, 20, 3325. (476) Hashimoto, Y.; Iwanami, N.; Fujimori, S.; Shudo, K. J. Am. Chem. Soc. 1993, 115, 9883. (477) Doi, M.; Inoue, M.; Tomoo, K.; Ishida, T.; Ueda, Y.; Akagi, M.; Urata, H. J. Am. Chem. Soc. 1993, 115, 10432. (478) Joyce, G. F.; Visser, G. M.; van Boeckel, C. A. A.; van Boom, J. H.; Orgel, L. E.; van Westrenen, J. 1984, 310, 602. (479) Eichhorn, G. L. In Inorganic BiochemistryElsevier/North Holland: New York, 1973; pp 1210. (480) Buchanan, G. W.; Stothers, J. B. Can. J. Chem. 1982, 60, 787. (481) Dabrowiak, J. C.; Greenaway, F. T.; Longo, W. E.; van Husen, M.; Crooke, S. T. Biochim. Biophys. Acta 1978, 517, 517. (482) Buncel, E.; Boone, C.; Joly, H. Inorg. Chim. Acta 1986, 125, 167. (483) Shankar Nandi, U.; Wang, J. C.; Davidson, N. Biochemistry 1965, 4, 1687. (484) Kan, L. S.; Li, N. C. J. Am. Chem. Soc. 1970, 92, 4823. (485) Young, P. R.; Shankar Nandi, U.; Kallenbach, N. R. Biochemistry 1982, 21, 62. (486) Beveridge, S. J.; Walker, W. R. Aust. J. Chem. 1974, 27, 2563. (487) Sigel, H. In Metal-DNA Chemistry; T. D. Tullius, Ed.; American Chemical Society: Washington DC, 1989; pp 159. (488) Frøystein, N. Å.; Sletten, E. J. Am. Chem. Soc. 1994, 116, 3240. (489) Simpson, R. B. J. Am. Chem. Soc. 1964, 86, 2059. (490) Kajiwara, T.; Ito, T. J. Chem. Soc. Chem. Comm. 1994, 1773. (491) Kajiwara, M.; Sugiyama, M.; Kimura, E. J. Chem. Soc. Chem. Comm. 1994, 1747. (492) Chu, G. Y. H.; Mansy, S.; Duncan, R. E.; Tobias, R. S. J. Am. Chem. Soc. 1978, 100, 593. (493) Eichhorn, G. L.; Clark, P. J. Am. Chem. Soc. 1963, 85, 4020. (494) Gruenwedel, D. W.; Cruikshank, M. K. Biochemistry 1990, 29, 2110. (495) Sarker, M.; Chen, F.-M. Biophys. Chem. 1991, 40, 135. (496) Verheyden, J. P. H.; Wagner, D.; Moffatt, J. G. J. Org. Chem. 1971, 36, 250. (497) Aurup, H.; Tuschl, T.; Benseler, F.; Ludwig, J.; Eckstein, F. Nucleic Acids Res. 1994, 22, 20. (498) Wells, P. R. Prog. Phys. Org. Chem. 1968, 6, 111. (499) Bratsch, S. G. J. Chem. Educ. 1985, 62, 101. (500) Sanderson, R. T. J. Chem. Educ. 1988, 65, 112. (501) Bratsch, S. G. J. Chem. Educ. 1988, 65, 223. (502) Marriott, S.; Reynolds, W. F.; Taft, R. W.; Topsom, R. D. J. Am. Chem. Soc. 1984, 49, 959. (503) Mullay, J. J. Am. Chem. Soc. 1984, 106, 5842. (504) Mullay, J. J. Am. Chem. Soc. 1985, 107, 7271. (505) Inamoto, N.; Masuda, S. Chem. Lett. 1982, 1003. (506) Inamoto, N.; Masuda, S. Chem. Lett. 1982, 1007. Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 158 (507) Inamoto, N.; Masuda, S.; Tori, K.; Yoshimura, Y. Tetrahedron Lett. 1978, 46, 4547. (508) Inamoto, N.; Masuda, S. Tetrahedron Lett. 1977, 3287. (509) Bolton, P. H.; Kearns, D. R. Biochim. Biophys. Acta 1978, 517. (510) Bolton, P. H.; Kearns, D. R. J. Am. Chem. Soc. 1979, 101, 479. (511) Birnbaum, G. I.; Giziewicz, J.; Huber, C. P.; Shugar, D. J. Am. Chem. Soc. 1976, 98, 4640. (512) Ducruix, A.; Riche, C.; Pascard, C. Acta Cryst. 1976, B32, 2467. (513) Sundaralingam, M.; Rao, S. T. Int. J. Quantum Chemistry: Quantum Biology Symposium 1983, 10, 301. (514) Williams, D. M.; Pieken, W. A.; Eckstein, F. Proc. Natl. Acad. Sci. USA 1992, 89, 918. (515) Pieken, W. A.; Olsen, D. B.; Benseler, F.; Aurup, H.; Eckstein, F. Science 1991, 253, 314. (516) Kulinska, K.; Laaksonen, A. J. Biomol. Struct. Dynamics 1994, 11, 1307. (517) Rosemeyer, H.; Tóth, G.; Golankiewicz, B.; Kazimierczuk, Z.; Bourgeois, W.; Kretschmer, U.; Muth, H.-P.; Seela, F. J. Org. Chem. 1990, 55, 5784. (518) Sowers, L. C.; Fazakerley, G. V.; Kim, H.; Dalton, L.; Goodman, M. F. Biochemistry 1986, 25, 3983. (519) Kaplan, N. O.; Ciotti, M. M.; Stolzenbach, F. E.; Bachur, N. R. J. Am. Chem. Soc. 1955, 77, 813. (520) Suzuki, S.; Suzuki, K.; Imai, T.; Suziki, N.; Okuda, S. J. Biol. Chem. 1965, 240, 554. (521) Suzuki, K.; Nakano, H.; Suzuki, S. J. Biol. Chem. 1967, 242, 3319. (522) Dinglinger, F.; Renz, P. Hoppe-Seyler's Z. physiol. Chem. 1971, 362, 1157. (523) LePage, G. A.; Junga, I. G. Molec. Pharmacol. 1967, 2, 37. (524) LePage, G. A. Can. J. Biochem. 1968, 46, 655. (525) Perry, A.; LePage, G. A. Cancer Res. 1969, 29, 617. (526) Nakai, Y.; LePage, G. A. Cancer Res. 1972, 32, 2445. (527) Tamaoki, T.; LePage, G. A. Cancer res. 1975, 35, 1015. (528) Bobek, M.; Bloch, A. J. Med. Chem. 1975, 18, 784. (529) Falke, D.; Range, K.; Arendes, J.; Müller, W. E. G. Biochim. Biophys. Acta 1979, 563, 36. (530) Christensen, L. F.; Broom, A. D.; Robins, M. J.; Bloch, A. J. J. Med. Chem. 1972, 15, 735. (531) Séquin, U. Experientia 1973, 29, 1059. (532) Morvan, F.; Rayner, B.; Imbach, J.-L.; Chang, D.-K.; Lown, J. W. Nucleic Acids Res. 1987, 15, 4241. (533) Morvan, F.; Rayner, B.; Imbach, J.-L.; Lee, M.; Hartley, J. A.; Chang, D.-K.; Lown, J. W. Nucleic Acids Res. 1987, 15, 7027. (534) Aramini, J. M.; van de Sande, J. H.; Germann, M. W. Biochemistry 1997, 36, 9715. (535) Townsend, L. B. In Handbook of Biochemistry and Molecular Biology; G. D. Fasman, Ed.; C.R.C. Press: Cleaveland, Ohio, 1975; Vol. I; pp 271-401. (536) Hanessian, S.; Pernet, A. G. Adv. Carbohydr. Chem. Biochem. 1976, 33, 111. (537) Daves, G. D.; Cheng, C. C. Prog. Med. Chem. 1976, 13, 303. (538) Hacksell, U.; Daves, G. D. Prog. Med. Chem. 1985, 22, 1. (539) Daves, G. D. Acc. Chem. Res. 1990, 23, 201. (540) Burchenal, J. H.; Ciovacco, K.; Kalaher, K.; O'Toole, T.; Kiefner, R.; Dowling, M. D.; Chu, C. K.; Watanabe, K. A.; Wempen, I.; Fox, J. J. Cancer Res. 1976, 36, 1520. (541) Cortese, R.; Kammen, H. O.; Spengler, S. J.; Ames, B. N. J. Biol. Chem. 1974, 249, 1103. (542) Samuelsson, T.; Boren, T.; Johansen, T. I.; Lustig, F. J. Biol. Chem. 1988, 263, 13692. (543) Polak, M.; Mohar, B.; Kobe, J.; Plavec, J. J. Am. Chem. Soc. 1998, 120, 2508. (544) Davies, D. B.; Sadikot, H. Org. Magn. Reson. 1982, 20, 180. (545) Lankhorst, P. P.; Haasnoot, C. A. G.; Erkelens, C.; Westerink, H. P.; van der Marel, G. A.; van Boom, J. H.; Altona, C. Nucleic Acids Res. 1985, 13, 927. (546) Plavec, J.; Chattopadhyaya, J. Tetrahedron Lett. 1995, 36, 1949. (547) Dhingra, M. M.; Saran, A. Biopolymers 1982, 21, 859. (548) Sundaralingam, M. Biopolymers 1969, 7, 821. (549) Yokohama, S.; Inagaki, F.; Miyazawa, T. Biochemistry 1981, 20, 2981. (550) Damha, M. J.; Ogilvie, K. K. Biochemistry 1988, 27, 6403. (551) Blommers, M. J. J.; Nanz, D.; Zerbe, O. J. Biomol. NMR 1994, 4, 595. (552) Hruska, F. E.; Mak, A.; Singh, H.; Sugar, D. Can. J. Chem. 1973, 51, 1099. (553) Sarma, R. H.; Mynott, R. J.; Wood, D. J.; Hruska, F. E. J. Am. Chem. Soc. 1973, 95, 6457. (554) Haasnoot, C. A. G.; de Leeuw, F. A. A. M.; de Leeuw, H. P. M.; Altona, C. Recl. Trav. Chim. Pays-Bas 1979, 98, 576. (555) Uhlenbeck, O. C. Nature 1987, 328, 596. (556) Rousse, B.; Puri, N.; Viswanadham, G.; Agback, P.; Glemarec, C.; Sandström, A.; Sund, C.; Chattopadhyaya, J. Tetrahedron 1994, 50, 1777. (557) Rousse, B.; Sund, C.; Glemarec, C.; Sandström, A.; Agback, P.; Chattopadhyaya, J. Tetrahedron 1994, 50, 8711. (558) Ruffner, D. E.; Stormo, G. D.; Uhlenbeck, O. C. Biochemistry 1990, 529, 10695. (559) Varani, G.; Cheong, C.; Tinoco, I. Biochemistry 1991, 30, 3280.

159 (560) Maltseva, T. V.; Agback, P.; Repkova, M. N.; Venyaminova, A. G.; Ivanova, E. M.; Sandström, A.; Zarytova, V. F.; Chattopadhyaya, J. Nucleic Acids Res. 1994, 22, 5590. (561) Stein, C. A.; Subasinghe, C.; Shinozuka, K.; Cohen, J. S. Nucl. Acids Res. 1988, 16, 3209. (562) Knudsen, H.; Nielsen, P. E. Nucl. Acids Res. 1996, 20, 494. (563) Chou, S.-H.; Flynn, P.; Reid, B. Biochemistry 1989, 28, 2435. (564) Katahira, M.; Lee, S. J.; Kobayashi, Y.; Sugeta, H.; Kyogoku, Y.; Iwai, S.; Ohtsuka, E.; Benevides, J. M.; Thomas, G. J. J. J. Am. Chem. Soc. 1990, 112, 4508. (565) Wang, A. H.-J.; Fujii, S.; van Boom, J. H.; van der Marel, G. A.; van Boeckel, S. A. A.; Rich, A. Nature 1982, 299, 601. (566) Damha, M. J.; Wilds, C. J.; Noronha, A.; Brukner, I.; Borkow, G.; Arion, D.; Parniak, M. A. J. Am. Chem. Soc. 1998, 120, 12976. (567) Lesnik, E. A.; Guinosso, C. J.; Kawasaki, A. M.; Sasmor, H.; Zounes, M.; Cummins, L. L.; Ecker, D. J.; Cook, P. D.; Freier, S. M. Biochemistry 1993, 32, 7832. (568) Puri, N.; Chattopadhyaya, J. Tetrahedron 1999, submitted, (569) Ding, D.; Gryaznov, S. M.; Lloyd, D. H.; Chandrasekaran, S.; Yao, S.; Ratmeyer, L.; Pan, Y.; Wilson, W. D. Nucl. Acids Res. 1996, 24, 354. (570) Gryaznov, S. M.; Lloyd, D. H.; Chen, J.-H.; Schultz, R. G.; DeDionisio, L. A.; Ratmeyer, L.; Wilson, W. D. Proc. Natl. Acad. Sci. USA 1995, 92, 5798. (571) Marquez, V. E.; Ezzitouni, A.; Russ, P.; Siddiqui, M. A.; Ford, H. J.; Feldman, R. J.; Mitsuya, H.; George, C.; Barchi, J. J. J. J. Am. Chem. Soc. 1998, 120, 2780. (572) Denisov, A. Y.; Bekiroglu, S.; Maltseva, T. V.; Sandström, A.; Altmann, K.-H.; Egli, M.; Chattopadhyaya, J. J. Biochem. Biophys. Meth. 1998, 16, 569. (573) Altmann, K.-H.; Kesselring, R.; Francotte, E.; Rihs, G. Tetrahedron Lett. 1994, 35, 2331. (574) Marquez, V. E.; Siddiqui, M. A.; Ezzitouni, A.; Russ, P.; Wang, J.; Wagner, R. W.; Matteuci, M. D. J. Med. Chem. 1996, 39, 3739. (575) Agback, P.; Glemarec, C.; Sandström, A.; Yin, L.; Plavec, J.; Sund, C.; Yamakage, S.-i.; Viswanadham, G.; Rousse, B.; Puri, N.; Chattopadhyaya, J. In Proceedings of the 8th Conversations; Adenine Press, New York: 1994; pp 293. (576) Dalluge, J. J.; Hashizume, T.; Sopchik, A. E.; Closkey, J. A.; Davis, D. R. Nucleic Acids Res. 1996, 24, 1073. (577) Nawrot, B.; Milius, W.; Ejchart, A.; Limmer, S.; Sprinzl, M. Nucl. Acids Res. 1997, 25, 948. (578) Taiji, M.; Yokohama, S.; Miyazawa, T. J. Biochem. 1985, 98, 1447. (579) Sprinzl, M.; Kucharzewski, M.; Hobbs, J. B.; Cramer, F. Eur. J. Biochem. 1977, 78, 55. (580) Janiak, F.; Dell, V. A.; Abrahamson, J. K.; Watson, B. S.; Miller, D. L.; Johnson, A. E. Biochemistry 1990, 29, 4268. (581) Potts, R. O.; Ford, N. C. J.; Fournier, M. J. Biochemistry 1981, 20, 1653. (582) Limmer, S.; Vogtherr, M.; Nawrot, B.; Hillenbrand, R.; Sprinzl, M. Angew. Chem. Internat. Edit. 1997, 36, 2485. (583) Polak, M.; Plavec, J. Eur. J. Inorg. Chem. 1999, 547. (584) Isaksson, J.; Maltseva, T.; Agback, P.; Kumar, A.; Zamaratski, E.; Chattopadhyaya, J. 1999, Manuscript in preparation. (585) Fujiwara Nucl. Acids Res. Symposium Ser. 1997, 37, 51. (586) Overmars, F. Ph.D. Thesis Thesis, Leiden University, The Netherlands, 1997. (587) Mc Connell, H. M. J. Chem. Phys. 1958, 28, 430.

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 160 (10.4) Latest articles (1998 – 2002) on the aspects of Anomeric (AE) and Gauche (GE) Effects and discussions/comments on these works

(10.4.1) Review: Perrin, C. L. Acc. Chem. Res. 2002, 35, 28. A detailed discussion on the stabilities of product formed on hydrolysis of five- and seven- membered cyclic amides. Product studies showed that the substantial cleavage of the bond that is antiperiplannar to only one lone pair of electrons and syn to the other with transition-state stabilization of 2 kcal mol-1.

(10.4.2) NMR titration studies (Perrin, C. L. J. Am. Chem. Soc. 1999, 121, 6911) showed the absence of reverse AE (see also Kirby, A. J. J. Chem. Soc., Chem. Commun. 1998, 1695, Szarek, A. J. Org. Chem. 2001, 66, 1097, Pinto, B. M. J. Org. Chem. 2000, 65, 220) in cationic N-

(glycopyranosyl)imidazoles and their tetra-O-acetyl derivatives (ΔΔGβ-α is negative, thereby showing greater preference for the axial position of a protonated imidazolyl group than of an neutral group).

(10.4.3) The net effect (both steric and stereoelectronic) of substitution (X, where X = OH, O-alkyl, O-acetyl and F) in a hexapyranoside (J. Chem. Soc., Perkin Trans. 2, 2002, 337) indicates reduced reactivity (in terms of the rate of acid-catalysed hydrolysis) at the anomeric center compared to the parent tetrahydropyranyl acetal.

(10.4.4) Ab initio (MP2/6-31G*) studies of pseudorotation and conformational stabilities of pyrrolidine (Carballeria, L.; Perez-Juste, I. J. Chem. Soc., Perkin Trans. 2, 1998, 1339) showed that pseudorotation path is preferred for inter-conversion between the N-H axial and equatorial form with a barrier ~0.6 kcal mol-1 supporting the experimental microwave spectroscopy results.

(10.4.5) Computational studies (Senyurt, N.; Aviyente, V. J. Chem. Soc., Perkin Trans. 2, 1998, 1463) by ab initio (HF/6-31G*) of AE in 2-[(4-substituted phenyl)seleno]-1,3-dithianes with NO2, H, CH3, OCH3 and N(CH3)2 as substituents showed the preference for axial conformer with enhanced electron withdrawing groups. NBO analyses showed that delocalization involving the sulfur and selenium lone-pairs and the σ*C2-Se plays and important role in stabilizing the axial conformer.

(10.4.6) NMR and ab initio studies (Serianni, A. S. J. Am. Chem. Soc. 1997, 119, 8933) for 2- 4 deoxy-β-D-glycero-tetrofuranose showed that it favors the S-form in solution (89% T3, 11% E2). Protonated form showed exclusive preference for S-form (E3) with higher energy barrier than the neutral form.

(10.4.7) Conformational studies with constrained nucleosides (Lowary, T. L. J. Org. Chem. 2001) 0 showed that furanose ring conformation in all compounds is locked either into E3 or E. It has also been shown that a nucleoside containing a conformationally locked furanose ring does influence the conformation of adjacent neighbor.

(10.4.8) Comparison of conformatinal analysis by theoretical studies (at various level with ab initio and DFT methods) with that of experimentally measured by NMR for Methyl 3-O-Methyl-α-D- arabinofuranoside (Lowary, T. L. J. Am. Chem. Soc. 2001, 123, 8811) has been performed. Moreover, computational studies (at HF/6-31G* and B3LYP/6-31G*) of conformational analyses of Methyl-α-D-arabinofuranoside (Lowary, T. L. J. Am. Chem. Soc. 1999, 121, 9682) showed 3E as the 2 lowest energy N-type conformer and either E or E1 (depending upon the level of theory used) as lowest energy S-type conformer which is also the global minimum. Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected]

161

(10.4.9) Conformational analyses of carba sugar analogues of methyl α-D-arabinofuranosides and methyl β-D-arabinofuranosides have been reported (Lowary, T. L. J. Org. Chem. 2001, 66, 8961). It showed that furanose ring conformation in these compounds is biased towards N-type.

(10.4.10) The magnitude of the one-bond coupling constant between C1 and H1 in 2,3-anhydro-O- furanosides has been shown (Lowary, T. L. J. Org. Chem. 2001, 66, 4549) to be sensitive to the stereochemistry at the anomric center. A panel of 24 compounds was studied and in case where 1 anomeric hydrogen is trans to the epoxide moiety, JC1-H1 = 163 – 168 Hz; whereas for cis 1 orientation of this hydrogen with respect to the oxirane ring, JC1-H1 = 171 – 174 Hz. However, for 1 2,3-anhydro-S-glycosides, the JC1-H1 is insensitive to the C1 stereochemistry.

(10.4.11) NMR and molecular modeling studies of 8-Aza-3-deazaguanine (Plavec, J. J. Chem. Soc., Perkin Trans. 2, 2001, 1433) showed stabilization of N-type conformers by ΔΔHo of 3.1 kJ mol-1, which has been attributed to the strengthening of O4'-C1'-N9 anomeric effect (strength of 19.5 kJ mol-1).

(10.4.12) NMR and ab initio studies (Plavec, J. J. Chem. Soc., Perkin Trans. 2, 2000, 255) showed The S-C-N anomeric effect is stronger in purine than in pyrimidine 4'-thionucleosides (thymine < cytosine < guanine < adenine), which is opposite to natural 4'-oxonucleosides. However, the strength of AE in 4'-thionucleosides is weaker compared to that in natural nucleosides.

(10.4.13) Steric and stereoelectronic effects of 7- and 8-substituted 7-deaza-2'-deoxy-adenosine and -guanosine on the sugar pucker as well as conformation about C4'-C5' bond have been studied (Seela, F. J. Chem. Soc., Perkin Trans. 2, 1997, 2341): (i) higher electron-attracting effect of the substituents drives N/S equilibrium of the pentofuranose towards N-type conformation and (ii) higher electron-withdrawing effect of the 7-substituents, higher γ+ across C4'-C5'.

(10.4.14) Conformational analysis of 2-halocyclohexanones by NMR, theoretical and solvation studies (Yoshinaga, F. J. Chem. Soc., Perkin Trans. 2, 2002, 1494) showed axial conformation of 2- -1 fluoro compound in vapour phase is stabilized (ΔEeq – ax = 0.45 kcal mol ) whereas equatorial -1 conformer predominates for other haloketones (ΔEeq – ax = 1.05, 1.50 and 1.90 kcal mol for chloro, bromo and iodo compounds respectively).

(10.4.15) Detailed MO calculations of cyclohexane, 1,3-Dioxane, 1,3-Oxathiane and 1,3-Dithiane showed (Alabugin, I. V. J. Org. Chem. 2000, 65, 3910) the importance of hyperconjugative interaction (through the balance of three effects: σC-X → σ*C-Heq, σ*C-Heq → σC-X and nP → σ*C5-Heq interactions) to explain the relative elongation of equatorial C5-H bonds. The role of nP → σ*C5-Heq interaction is important in dioxane. In diathiane, distortion of the ring by long C – S bonds increases preferential overlap of σC5-Heq and σ*C-S orbitals.

(10.4.16) Recently a report has appeared (S.F. Wnuk, D.R. Companioni, V.Neschadimenko, and M.J. Morris, J. Org. Chem. (Web: http://dx.doi.org/10.1021/jo020428b) on the competition between steric and -fluorine effects, including the impact of fluorine regio- and stereochemistry, on radical deoxygenations of 2'(3')-O-phenoxythiocarbonyl (PTC) esters of fluoropentofuranosyl- adenine nucleosides. Thus it has been found that steric effects are decisive for determination of the stereoslectivity of transfer of deuterium from tributyltin hydride to fluoropentofuranosyl radicals generated from these adenine nucleoside derivatives. In all cases, deuterium abstraction occurs at the less hindered α face of the sugar ring trans to the heterocyclic base. However, this α face stereoselectivity is enhanced by the anti effect of a vicinal fluorine substituent with an arabino or xylo orientation (on the α face of the ring). A smaller anti effect is still apparent with a vicinal

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 162 fluorine on the α face (ribo orientations). Complex stereoelectronic/steric interactions might be involved with these furanose rings that have electronegative (F, O, N) substituents.

(10.5) The sensitivity of the RNase H discriminates the local structure changes owing to conformational transmission induced by 3'-endo sugar constrained nucleotides in the antisense strand of the antisense-RNA hybrid duplex. RNase H an ubiquitous enzyme cleaves the RNA in a DNA/RNA hybrid. Based on the dissociation constant determined by competitive inhibition analysis, the RNase H can bind to all duplexes, irrespective of their conformational preorganization [DNA/RNA > RNA/RNA > DNA/DNA]. The catalytic cleavage by the enzyme however demands a flexible hybrid duplex structure whose overall geometry should be close to an A type helix. For example, in the native DNA-RNA duplex, the conformation of the DNA strand is B type (all nucleotides are in O4’-endo conformation) whereas the RNA strand adopts a conformation, which is very similar to single stranded A-RNA type (all nucleotides are in C3’-endo conformation). It has emerged that the RNase H cleavage site retains this B-type-DNA/A-type RNA conformation in order to be substrate for cleavage reaction by RNase H. This conformational criteria has been so far difficult to achieve with the modified residues in the antisense strand at the cleavage site. We have thus witnessed the emergence of the gapmer or the mixmer strategies, which not only allows the cleavage of the complementary RNA strand, but also enhances the thermodynamic stability of the hybrid duplex.1 The tolerance of the modified nucleotide residues in the antisense strand in the above two strategies are very sensitive to the type of modifications used, both because of conformational preorganization and the intrinsic flexibility required of the hybrid duplex for catalytic cleavage by RNase H. Most of the North conformationally constraind nucleosides upon introduction (full modification or partial modification) to Antisense OligoNucleotide (AON) render their hybrid duplex with RNA (AON/RNA) to a rigid RNA/RNA type duplex, which were found to be insensitive towards the RNase H promoted cleavage, although the stability of the resulting hybrid duplexes are enhanced1. The reasons for this RNase H insensitivity to conformationally-constrained containing antisense complex with complementary RNA (both in the gapmer as well as in the mixmer strategies) is beginning to be understood. Remarkably, some conformational alterations brought about by the modifications is transmitted to the neighbouring nucleotides, which enzyme can sense, but spectroscopic techniques such as NMR or CD can not detect2. A slow consensus is however emerging as to how far the rigidity of the N-type conformationally constrained nucleotides in the AON can propagate to alter the conformational characters of the neighboring S-type residues in the hybrid duplex. This information is important because the alteration of S-type character in the neighbors to an N-type forces those nucleotides in the AON/RNA duplex to adopt RNA/RNA type conformation, which prevents it from the RNase H cleavage. The uniquness of RNase H promoted cleavage of AON/RNA hybrids holding varying number of conformationally constrained North-East (N/E)-type nucleotides, can be employed to address these issue which we have shown recently with the oxetane incorporated nucleotides in the antisense-RNA hybrid duplex 2. We have introduced single, double, triple N/E conformationaly constrained nucleoside, [1- (1',3'-O-anhydro-β-D-psico-furnosyl)thymine] (T), to a 15 mer AON and targeted to a 15 mer RNA. CD failed to detect any structural perturbances of the modified hybrids compared to the native counterpart; however, the RNase H cleavage pattern clearly showed that the local conformational changes spanning a total of 5 nucleotides including the modification towards the 5'-end of AON (3'- end of RNA). This was evident from the fact that the 5 nucleotide region of the RNA strand, beginning from the nucleotide opposite to the T modification, became completely inactive to the catalytic cleavage reaction by RNase H2. Since the site just after the 5 nucleotides were accessible for enzymatic cleavage (Figure 10), and the fact that the binding and cleavage sites are different for RNase H3, it is evident that the structurally altered duplex region was suitable for enzyme binding but not for cleavage. By suitably placing the just three T modifications we have shown that the

163 single cleavage site can be engineered in the 15mer AON/RNA hybrid duplex (Figure 1). This work clearly shows that owing to the N-type or N/E-type conformational constrain introduced at the oligonucleotide level, leads to microenvironmental conformational alterations in the neighboring 4 nucleotides toward the 5’-end. This conformational transmission can be effectively mapped by RNase H, when CD and NMR fails to show any conformational heterogeinity in the local structure. There is only limited information available regarding the tolerance of RNase H towards local conformational transmission of AONs holding other conformationally constrained nucleotides. A recent report4 outlined the RNase H tolerence of AON/RNA hybrids modified with North- conformationally constrained LNA monomers. Introduction of one LNA itself was found to alter global helical structure5 (detected by CD) and three LNA monomers were sufficient to create a RNA/RNA type AON/RNA hybrid duplex. None of these modifications are however reported to elicit any RNase H response, although considerable enhnacement in the thermodynamic stability was observed. Mixmer AONs with 3-5 deoxynucleotide gaps were found to be also insensitive towards RNAse H promoted cleavage once hybridized to RNA4. This clearly shows that microstructural alterations brought by the LNA modification propogates beyond 5 nucleotids in contradistinction to the oxetane modifications. An important question in this context is how far down the polynucleotide chain the conformational transmission propagates in the AON/RNA hybrid duplex2? Clearly, an appropriate answer should make it possible to use of the LNAs both as Tm enhancer and a substrate for RNAse H, as it has been achieved in a recent systematic study with a series of LNA incorporated AON- RNA duplexes in which the size of the deoxynucleotide gap (required for RNase H cleavage) in the mixmer as well as in the gapmer has been varied5. This study has shown that the gapmer/mixmer of LNA/RNA duplexes having 6 - 10 deoxynucleotide gaps was cleaved by RNase H. Quite expectedly2, it was also found that the maximum cleavage efficiency was observed as the size of the deoxynucleotide gap was increased to 10, presumably because it adopted a substantial degree of DNA/RNA type structure5. This shows that the conformational transmission of LNA stretches upto 7 nucleotides to make it conform to RNA/RNA type structure, which is resistant to RNase H, whereas for oxetane modifications, we have shown it stretches upto 5 nucleotides2 3 3 2’-O-methylnucleosides ( J1’2’ = 5.2 Hz, J3’4’ = 4.7 Hz,) show a slightly preferred N-type conformation in the North-South pseudorotational equilibrium. This slight preference for N-type conformation is attributed to the steric repulsion between the aglycone and the 2’-O-methyl group in the C2’-endo (South form)6. In the oligomer there would be additional steric clash of 2’-O-alkyl group with the 3’-phosphate, which drives it to more North conformation. Therefore, unlike the other North-constrained nucleotides (LNA, oxetane), the conformational influence by 2’-O-alkyl nucleotides, which can be detected by RNase H, to the neighbours are less pronounced, and it was found to be sequence dependent7. This is clearly visible in the RNase H digestion pattern of 2’-O- alkyl modified AON/RNA gapmer hybrids, where the microstructural alterations brought by the modification, which blocks the cleavage was found to vary from 3 to 5 nucleotides including the modification7. This means that the conformational transmission of the North-type 2’-O- alkylnucleotides in the AON/RNA/RNase H ternary complex is less pronounced than in other conformational constrained counterparts. Unlike the North constrained nucleotides, the conformational status of 2’-O-methylnucleosides and its analogs in the antisense strand or in the hybrid duplex with RNA can be envisioned as sensitive to both sequence make-up as well as whether they are in the complex form with RNase H or not. It has been recently shown that the modified AONs containing acyclic interresidue units (the 2’,3’-secouridine or a butanediol linker in the modified AONs containing 2’F-ANA) supports RNase H-promoted cleavage of complementary RNA (Damha et al., J. Am. Chem. Soc. 125, 654-661 (2003). These conformationally labile AONs shows some remarkable benefits in the enzymatic recognition of the AON/RNA hybrids, because of the flexible nature of their sugar-phosphate backbone conformation, which is consistent with our observation of the oxetane-modified AON/RNA recognition and cleavage by RNase H. It should be º noted that the introduction of a 2’,3’-secouridine moitey reduces the Tm by ca 10 C whereas a butanediol º linker reduces the Tm by ca 6 C.

12 8 10 11 (B) 7 5 6 7 9 1213 12 3 456 7 8 9101112131415 12 3 456 7 8 9101112131415 5'-r(G A A G A A A A A A U G A A G)-3' 5'-r(G A A G A A A A A A U G A A G)-3' 3'-d(C T T C T T T T T T A C T T C)-5' 3'-d(C T T C T T T T T T A C T T C)-5' AON (1) AON (4) 11 8 10 12 13

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 5'-r(G A A G A A A A A A U G A A G)-3' 5'-r(G A A G A A A A A A U G A A G)-3' 3'-d(C T T C T T T T T T A C T T C)-5' 3'-d(C T T C T T T T T T A C T T C)-5'

AON (2) AON (5) 1112 8 7 9

12 3 456 7 8 9101112131415 12 3 456 7 8 9101112131415 5'-r(G A A G A A A A A A U G A A G)-3' 5'-r(G A A G A A A A A A U G A A G)-3' 3'-d(C T T C T T T T T T A C T T C)-5' 3'-d(C T T C T T T T T T A C T T C)-5'

AON (3) AON (6)

Figure 10. (A) The PAGE analysis of RNase H hydrolysis of the hybrid duplexes, AON (1)- (6) / RNA. Time after the addition of the enzyme is shown on the top of each gel lane. The length and sequence of 5’-32P-labeled RNAs formed up on enzymatic cleavage are shown on the left and right side of the gel and it was deduced by comparing the migration of products with those oligonucleotides generated by parial digetion of the target RNA by Snake venom phosphodiesterase (SVPDE). (B) RNase H cleavage cleavage pattern of the hybrid duplexexs. Long and short arrows represents major and minor sits respectively (after 2h of incubation). Boxes represents the parts of the RNA sequence insensitive towards RNase H cleavage.

References:

1. (a) M. Manoharan, Biochim Biophys Acta., 1489, 117(1999). (b) P. Herdewijn, Biochim Biophys Acta., 1489, 167(1999). 2. (a) P. I.Pradeepkumar, E. Zamaratski, A. Földesi, J. Chattopadhyaya. Tetrahedron Lett., 41, 8601 (2000). (b) P. I. Pradeepkumar, E. Zamaratski, A. Földesi, J. Chattopadhyaya. J. Chem.

165 Soc., Perkin Trans. 2, 3, 402 (2001). (c) P. I. Pradeepkumar, J. Chattopadhyaya. J. Chem. Soc., Perkin Trans. 2, 11, 2074 (2001). 3. S. Kanaya, Enzymatic activity and protein stability of E. coli ribonuclease HI, in Ribonucleases H, Crouch, R. J., editors; INSERM: Paris, Chapter 1, pp. 1-37 (1998). 4. J. Kurreck, E. Wyszko, C. Gillen, V. A. Erdmann, Nucleic Acids Res., 30, 1911(2002). 5. K.Bodensgaard, M. Petersen, S.K. Singh, V.K. Rajwanshi, R. Kumar, J. Wengel, J.P. Jacobsen. Chem. Eur. J, 6, 2687 (2000). 6. G. Kawai, Y. Yamamoto, T. amimura, T. Masegi, M. Sekine, T. Hata, T. Iimori, T.Watanabe, T. Miyazawa, S. Yokoyama, Biochemistry, 31, 1040 (1992). 7. (a) H. Inoue, Y. Hayase, S. Iwai, E. Ohtuka., FEBS Lett., 215, 327 (1987). (b) Y-T.Yu, M-D. Shu, J. A. Steiz, RNA., 3, 324 (1997). (c) B.Larrouy, C. Boiziau, B.Saproat, J-J.Toulme, Nucleic Acids Res., 23, 3434 (1995).

(10.6) The influence of flouro substitution at the sugar moiety in modulating the furanose conformation of flourinated nucleosides.

The furanose conformation of a nucleoside is determined by interplay of stereoelectronic gauche and anomeric effects (vide infra) tuned by different sugar substituents. The strength of the gauche and anomeric effects is determined by the electronic nature of the nucleobase, and the electronegetivity, position, configuration of substituents in the furanose moiety. Thus highly electronegative flouro substitution in the sugar moiety can predominantly drive its overall North (N) South (S) equilibrium. The various flourinated nucleosides have therefore been designed and analysed for their ability to conformationally preorganize the furanosyl moiety in solution. Marquez et al. have performed a systematic study of various fluorinated dideoxy uridines by NMR and pesudorotational analysisis1. In 2'-flouro-2', 3'-didexy-ara-uridine (2'F-up, 1) and 3'- flouro-2', 3'-didexy uridine (3'F-down, 4) the F-gauche effect dominates over the opposing O4'-C1'- N anomeric effect, thereby the furanose ring adopts the South.type conformation in solution. However in the 2'F-down (2) and 3'F–up (3) dideoxy uridines, the F-gauche effect along with the anomeric effect plays in tandem to lock the sugar conformation in to the Nothern hemisphere (N- type) of the pseudorotational cycle. It is worth noting that the 2'F-down dideoxy uridine (2) exclusively prefers the N-type conformation at 25 oC with a pseudorotatonal phase angle of 19o. In the case of the 2'-flouro-2'-deoxyarabinoribofuranosyl thymine (2'-F up, 5) the gauche effect of O4'-C1'-C2'-F and F-C2'-C1'-N fragments prevails over the anomeric effect and the sugar adopts the C2'-endo/ C1'-exo conformation in solution.2,3 The 2'-flouro-2'-deoxyribofuranosyl thymine (2'F-down, 6) prefers the N-type sugar conformation due to the combined influence of gauche and anomeric effects. However, in the X-ray and NMR structure of the oligonucleotide hybrid duplexes containing the 2'F-up thymidine units showed the O4'-endo conformation for the furanose ring.4-6 This is explained on the basis of steric clash between the 2'F atom and the C6- carbon in the South sugar.

Chattopadhyaya et al, "Stereoelectronic Effects in Nucleosides & Nucleotides and their Structural Implications", Dept of Bioorganic Chemistry, Box 581, Uppsala University, S-75123 Uppsala, Sweden, Ver 160205 [email protected] 166 2', 3' monoflorinated dideoxyuridines O O O O NH NH NH NH HO N O N O O HO O HO N O N O O HO O H3' F H2' H' H2' H3' F H3' '' H3'' H 2 H3'' F 3 1 H3'' H2'' 4 F H2'' South -type North -type North -type South -type J = 5.2 Hz J = 11.3 Hz J = 1.15 Hz 3'-4' 3'-4' J3''-4' = 2.4 Hz 3'-4' J = 8.6 Hz J = 4.8 Hz J = 9.0 Hz 3''-4' 3''-4' J1'-2' = 2.1 Hz 1'-2' J = 3.3Hz J = 0.9 Hz J = 5.7 Hz 1'-2'' 1'-2' J1'-2'' = 8.2 Hz 1'-2''

o o o P = 131 P = 19 P = 27o P = 169 Barchi, J.J.; Jeong, L.K.; Siddiqui, M.A.; Marquez, V.E. J. Biochem. Biophys. Methods.1997, 34, 11.

2' monoflorinated thymidines O O

NH NH N O Ikeda, H.; Fernandez, R.; Wilk, A.; Barchi, J.J.; HO HO N O O O Huang, X.; Marquez, V.E. Nucleic. Acids. Res. F 1998, 26, 2237. 6 5 H2'' HO HO F Wilds, J.C.; Damha, M.J. Nucleic. Acids. Res. South -type North -type 2000, 28, 3625. J3'-4' = 4.5 Hz J3'-4' = 11.8 Hz J1'-2'' = 4 Hz 2' monoflorinated deoxy and dideoxy adenosines NH NH NH 2 NH2 2 2 N N N N N N N N N N N HO N HO N N HO N HO N O O O O F H3' F H3' 6 7 8 HO HO F H3'' 9 H3'' F South -type South -type North -type North -type J = 2.82 Hz J = 0 Hz o 1'-2' J1'-2' = 3.3Hz 1'-2' P = 130 o o P = 0 P = 130o P = 0 Ford, H.; Lan Mu, F.D.; Siddiqui, M.A.; Nicklaus, M.C.; Anderson, L.; Marquez, V.E.; Barchi, J.J. Biochemistry. 2000, 39, 2581. 2' monoflorinated deoxy 3-bromopyrazolo[3,4,-d] pyramidines Br O Br NH2 NH N N N N North -type N N NH North -type HO N NH2 HO O 2 O o F P = -2.1 11 F J = 7.5 Hz P = -2.1o 10 J3'-4' = 7.2 Hz 3'-4' J = 5.98 Hz HO J1'-2'' = 6.3 Hz HO 1'-2'' He, J.; Mikhailopulo, I.; Seela, F. J. Org. Chem. 2003, 68, 5519.

To unravel the structural preference of North-constrained deoxy and dideoxy 2'F-adenosine at the active site of adenosine deaminase, a detailed conformational analysis of various fluorinated adenosine analogues has been reported recently.7 In the case of 2'-flouro-2',3'-

167 dideoxyarabinofuranosyl adenosine (F-up, 8) and in the corresponding ribo analogue (F-down, 9) only O4'-C1'-C2'-F is operative along with the anomeric effect. In 8 this F-.gauche effect predominates over the anomeric effect and the sugar adopts the C1'-exo conformation (P =130o, 81% S-type). The assistance of anomeric effect in 9 locks the sugar conformation in to an exclusive N-type (P= 0o, 99% N-type). The situation is more complex in the case of 2'-flouro -2'- deoxyarabinofuranosyl adenosine (6, F-up) and 2'-flouro -2'-deoxyribofuranosyl adenosine (7, F- down) owing to the presence of 3'-OH. Here the additional O4'-C4'-C3'-O3' gauche effect plays a crucial role in dictating the sugar conformation. In 2'F-down compound 6 the 2'F -gauche effect and anomeric effect overcome the O4'-C4'-C3'-O3' gauche effect and the major pesudorotomer adapts the N-type conformation with 76% population. While in 2'F-up compound 7 the sugar adapts predominantly the C1'-exo pseudorotomer owing to the dominal influence of gauche over anomeric effect (P =130o, % of S-type = 64%) Sofar the studies on the stereoelectronic effects in nucleosides and nucleotides showed that 2' (or 2") and/or 3' (or 3") F/O mediated GE is stronger than AE. A recent report from Seela's group showed,8 for the first time, that the AE can be stronger than the F/O mediated GE in dictating the furanose conformation of the nucleosides. Thus, they have shown that they could overcome the strong GE of 2'-F substituent in the β-face (ara configuration) by the introduction of the extra nitrogen atom at the 6 position of a pyrimidine moity (6-azauracil-1-yl moiety) or at the 8 position of the purine moiety [3,4-d]pyrimidine- 1-yl aglycone (3-bromo or 3-H gave almost identical N/S population). The two nucleosides, 3- bromopyrazolo [3,4-d]pyrimidine-2'-deoxy-2"-flouro and its ara counterpart 10 and 11 showed exclusive N-type conformation in solution and solid state (with pseudorotational phase angle –2.10),8 which is unexpected in view of the earlier studies with 2'(β)-flouro substituted nucleosides. The strong electron * wihdrawing nature of nucleobase in these nuclosides enhances the nO4' → σ C1'-N1 interaction (anomeric effect) and it prevails over the gauche interactions results in the existence of C3'-endo (N-type) pseudorotomer in solution.

References: 1. Barchi, J.J.; Jeong, L.K.; Siddiqui, M.A.; Marquez, V.E. J. Biochem. Biophys. Methods.1997, 34: 11. 2. Ikeda, H.; Fernandez, R.; Wilk, A.; Barchi, J.J.; Huang, X.; Marquez, V.E. Nucleic. Acids. Res.1998, 26, 2237. 3. Wilds, J.C.; Damha, M.J. Nucleic. Acids. Res.2000, 28, 3625. 4. Berger, I.; Tereshko, V.; Ikeda, H.; Marquez, V.E.; Egli, M. Nucleic. Acids. Res.1998, 26, 2473. 5. Trempe. J.F.; Wilds, C.J.; Denisov, A.Y.; Pon, R.T.; Damha, M.J.; Gehring, K. J. Am. Chem. Soc.2001, 123, 4896. 6. Denisov, A.U.; Noronha, A.M.; Wilds, C.J. Trempe, J-F.; Pon, R.T.; Gehring, K.; Damha, M.J. Nucleic. Acids. Res. 2001, 29, 4284. 7. Ford, H.; Lan Mu, F.D.; Siddiqui, M.A.; Nicklaus, M.C.; Anderson, L.; Marquez, V.E.; Barchi, J.J. Biochemistry. 2000, 39, 2581. 8. He, J.; Mikhailopulo, I.; Seela, F. J. Org. Chem. 2003, 68, 5519.