Isoguanine Formation from Adenine

FULL PAPER

DOI: 10.1002/chem.201102415

Qianyi Cheng,[a] Jiande Gu,*[b] Katherine R. Compaan,[a] and Henry F. Schaefer, III*[a]

Abstract: Several possible mechanisms tautomer (isoG1 or isoG2). The local favored. The other option is indirect underlying isoguanine formation when and activation barriers for the two hydrogen transfer involving microsol-

OH radical attacks the C2 position of pathways are very similar. This evi- vation by one water molecule. The adenine (AC2) are investigated theoreti- dence suggests that the two pathways water lowers the reaction barrier by cally for the first time. Two steps are are competitive. After dehydrogena- over 20 kcalmol1, indicating that involved in this process. In the first tion, there are two possible routes for water-mediated hydrogen transfer is C step, one of two low-lying AC2···OH re- the second step of the reaction. One is much more favorable. Both A+ OH ! actant complexes is formed, leading to direct hydrogen transfer, via enol–keto isoG+ HC reactions are exothermic and

C2H2 bond cleavage. Between the two tautomerization, which has high local spontaneous. Among four isoguanine reactant complexes there is a small iso- barriers for both tautomers and is not tautomers, isoG1 has the lowest merization barrier, which lies well energy. Our findings explain why only below separated adenine plus OH radi- the N H and O H tautomers of isolated Keywords: adenine · nucleobases · 1 2 cal. The complex dissociates to free isoguanine and isoguanosine have been radicals · tautomerization molecular hydrogen and an isoguanine observed experimentally.

Introduction pair with a non-standard hydrogen-bonding pattern. They can formally only pair with each other, not with other nucle- Isoguanine is one of the components in the non-standard obases.[10] It is thought that this base pair makes ribonucleic isoguanine·isocytosine base pair, and it naturally occurs in acids more versatile as catalysts by adding diversity to the butterfly wings,[1,2] croton beans,[3,4] and mollusks.[5] It was structure,[11] and expands the genetic alphabet.[12,13] DNA first isolated by Cherbuliez and Bernhard in 1932 from the containing a string of isoguanine forms a stable parallel tet- croton bean.[3] Isoguanine exists as the aglycone fragment of raplex structure,[14] and it may contribute to an extracellular the glycoside 2-oxy-6-aminopurine-d-riboside.[3,4,6] Oxidation growth factor containing RNA.[15] of adenine to isoguanine was proposed based on studies of Isoguanine, isoguanine derivatives[16–19] and isoguanosine purine metabolism[7] and direct oxidation of adenine[8] in derivatives[10, 20–22] have been synthesized, and they are con- vivo, during Browns mechanistic studies of the conversion sidered to be involved in DNA lesions. These studies also of adenine to guanine.[9] A scheme involving the oxidation suggest that the formation of isoguanine from adenine plays of adenine at the C2 position was suggested by Bendich and an important role in A!T and A!C transversions in cellu- co-workers.[8] However, the detailed mechanism is unknown. lar DNA,[17–19] inducing mutations at various stages of In the standard Watson–Crick base pairing scheme, ade- cancer.[19] Thus, isoguanine is one important form of DNA nine (A) pairs with thymine (T) and guanine (G) pairs with damage produced by reactive oxygen species. Understanding cytosine (C) via hydrogen-bonding. These form the two nat- the detailed mechanism of formation of isoguanine from ad- ural base pairs in DNA that stabilize its double helix struc- enine could aid in studying DNA damage and understanding ture. However, isoguanine (2-hydroxyladenine or isoG) and human longevity. Reactions of OH radicals with adenine isocytosine (2-aminouracil or isoC) form an interesting base have been studied experimentally,[23–27] but most of the at-

tention has been focused on the C4,C5, and C8 positions. An C experimental investigation of direct OH addition at the C2 position was carried out by 60Co g-irradiating aqueous solu- [a] Dr. Q. Cheng, K. R. Compaan, Prof. Dr. H. F. Schaefer, III m m 3 tions containing 0.1–1.0 m adenine, 0.0–1.0 m Fe(CN)6 , Center for Computational Quantum Chemistry m University of Georgia, Athens, GA 30602 (USA) and 1.0 m phosphate (pH 7), which were saturated with [25] E-mail: [email protected] N2O. HPLC with electrochemical detection revealed no [b] Dr. J. Gu isoguanine. Taking the radiation dose and detection limit Drug Design & Discovery Center into account, they estimated that no more than 2% of the State Key Laboratory of Drug Research C OH radicals add at C2. However, one possible mechanism Shanghai Institute of Materia Medica of this rare reaction has been mentioned. It is analogous to Shanghai Institutes for Biological Sciences, CAS C C Shanghai 201203 (P. R. China) the conversion of A8-OH , which is formed by OH addition [25] E-mail: [email protected] directly at C8, to 8-OH-A by losing a hydrogen atom. The

Chem. Eur. J. 2012, 18, 4877 – 4886 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 4877 tautomerization following the formation of 2-OH-A (isogua- ies[46] of a wide range of electron affinities. In the present nine) is also of great interest. Keto (N1H, N3H)–enol (O2H) study, for purposes of reproducibility, there are 220 contract- tautomerism of 9-substituted isoguanosine has been exam- ed functions for the adenine molecule, 245 functions for ad- ined by UV absorption spectra at low pK1 in aqueous enine-OH complexes, 270 functions for adenine-2OH com- medium. N1H has a long wavelength absorption near plexes, and 239 functions for isoguanine molecule and its 310 nm, and the enol form absorbs around 270 nm.[28] tautomers. Neutral adenine[29] and five dehydrogenated adenine radi- Optimized geometries, harmonic vibrational frequen- cal derivatives[30] have been studied theoretically. However, cies,[47–49] and intrinsic reaction coordinates (IRC)[50–53] were there are few studies on adenine and OH radical reactions. computed with the QChem 3.2 package.[54] A 1998 study reported the relative energies of four hydroxy- lated adenine species.[31] In 2010, Cheng and co-workers ex- amined the dehydrogenation of adenine by COH using densi- Results and Discussion ty functional theory (DFT).[32] From their work, it is clear that OH radical attacking the C2 position is energetically fa- The structure and numbering Scheme for adenine are shown vorable, evidenced by formation of a low energy in Figure 1. Isoguanine N1H and N3H tautomers are also [32] A(C2)···OH complex. Based on the extremely high local shown in Figure 1, adopting numbering similar to adenine. C reaction barrier, dehydrogenation [(A-H) +H2O] of this complex is unlikely. Instead of dehydrogenation, the present mechanistic study reveals the possible formation of isoguanine from this adenine starting complex, as well as the tautomerization between several isoguanine tautomers. These results should give new insights into related biochemical ex- periments.

Theoretical Methods

The generalized gradient approximation exchange-correlation B3LYP functional was employed in this work, which is a combination of Beckes 3-parameter HF/DFT hybrid exchange functional (B3)[33] with the dynamical correlation functional of Lee, Yang, and Parr (LYP).[34] This method has been used in various DNA related computational studies, and has provided reasonable results for DNA bases, base pairs, and anions.[31,35–44] The B3LYP method is adopted along with double-z quali- ty basis sets with polarization and diffuse functions (denoted Figure 1. IUPAC numbering of atoms for adenine, similar numbering as DZP + +). The DZP+ + basis sets are generated by aug- adopted for isoguanine N1H and N3H tautomers. menting the Huzinaga–Dunning set of contracted double-z Gaussian functions with one set of p-type polarization functions for each H atom and one set of five d-type polarization functions for each first-row atom. Besides that, one even- Hydroxyl radical attacks adenine C2: When hydroxyl radical tempered diffuse s function was added to each H atom, attacks the C2–H2 region of adenine, it may be more favor- ACHTUNGRE while even-tempered s and p diffuse functions were centered able for the electron-rich oxygen to attack the C2 atom, on every heavy atom to complete the DZP + + basis. The which is more positively charged than the hydrogen. Seven even-tempered orbital exponents were determined accord- intermediate AC2···OH complexes, including three transition ing to the prescription of Lee:[45] states, are shown in Figure 2. The numbering schemes corre- spond to increasing energy, relative to separated A and OH 1 adiffuse ¼ 1=2 ða1=a2 þ a2=a3Þa1 ð1Þ radical. All energies are in kcalmol , with ZPVE corrected values in parentheses. A summary of the relative energies is where a1, a2, and a3 are the three smallest Gaussian orbital presented in Table 1. exponents of the s- or p-type primitive functions for a given Complexes 1 and 2 may be formed immediately in the atom (a1

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C2,N1C2 and C2N3, both increase by 0.12 in 1. Similar changes occur in 2 and TS1. All auxiliary geometric parameters in 1, 2, and TS1 remain nearly constant during the isomerization reaction. The exception is the OH radical hydrogen. In 1, it is point-

ing “up” (Figure 3), close to N1, forming a cis-structure.

However in 2, the hydrogen is pointing “down”, close to N3, forming a trans-conformer. The transition state (TS1)con-

necting 1 and 2 has the hydrogen pointing “out”, with O2-H

bisecting the purine plane. The 1 (A···O2Hcis)!2

(A···O2Htrans) isomerization process is mainly described by

the torsional angle tHO2C2H2 which varies from 92.38 in 1 to 133.78 in TS1 to 78.48 in 2, along with changes in the tor-

sional angle tHO2C2N1 (from 22.78 in 1 to 112.78 in TS1 to

167.18 in 2) and tHO2C2N3 (from 155.48 in 1 to 21.08 in TS1 to 34.68 in 2). Similar to our previous computational study,[32] when the

oxygen from the hydroxyl radical bonds directly to C2, the complex formed is much lower in energy than separated adenine and hydroxyl radical (Table 2). Complexes 1, 2 and TS1 lie 20.6 (17.6 with ZPVE), 19.7 (16.7) and 19.0 (16.3) kcalmol1 below separated reactants, respectively. The isomerization barrier for 1!TS1!2 is predicted to be 1.6 (1.2)

Table 2. Energies [DE, in kcal mol1, ZPVE corrected values in parentheses] of the transition state TS1 and intermediate 2 compared to 1. Also reported are reaction enthalpies [DH, in kcal mol1], entropies [DS,in cal mol1 K1], and Gibbs energies [DG, in kcalmol1] for 1!TS1!2. DE DH DS DG 1 0.0 (0.0)ACHTUNGRE 0.0 0.0 0.0 ACHTUNGRE Figure 2. Numbering of the seven adenine-OH radical complexes. Rela- TS1 1.6 (1.2) 0.7 2.0 0.1 ACHTUNGRE tive energies (in kcalmol1, ZPVE corrected values in parentheses) are 2 0.9 (0.8) 0.0 0.2 0.1 reported with respect to separated adenine and hydroxyl radical.

Table 1. Relative energies [E , in kcal mol1, ZPVE corrected values in parentheses] of seven structures with rel 1 respect to separated adenine plus OH radical. Dissociation energies with respect to separated isoguanine iso- kcalmol , and 0.7 (0.4) kcal 1 mers plus hydrogen atom [DE, in kcalmol1, ZPVE corrected values in parentheses]. mol for the reverse reaction. These energetics indicate that Description Erel DE DE A+ OHC isoG(NACHTUNGRE H)+HC isoG(NACHTUNGRE H)+HC 1 3 the AC2···OH complexes 1 and 2 A +OHC 0.0 (0.0)ACHTUNGRE are favorable for OH reaction C ACHTUNGRE ACHTUNGRE ACHTUNGRE 1 [A···O2Hcis] 20.6 (17.6) 24.6 (19.0) 25.0 (18.9) with adenine, and that the small C ACHTUNGRE ACHTUNGRE ACHTUNGRE 2 [A···O2Htrans] 19.7 (16.7) 23.7 (18.1) 24.1 (18.1) barrier will not stop OH rotat- ACHTUNGRE ACHTUNGRE C ACHTUNGRE ACHTUNGRE ACHTUNGRE 3 [isoG(O2Hcis)···H] 2.0 (4.3) 6.0 (5.7) 6.3 (5.6) ACHTUNGRE ACHTUNGRE C ACHTUNGRE ACHTUNGRE ACHTUNGRE ing along the C2O2 axis. 4 [isoG(O2Htrans)···H] 1.7 (4.1) 5.7 (5.5) 6.0 (5.4) ACHTUNGRE ACHTUNGRE ACHTUNGRE TS1 O2Hcis,trans 19.0 (16.3) 23.0 (17.7) 23.3 (17.7) ACHTUNGRE ACHTUNGRE ACHTUNGRE TS2 O2Hcis 5.7 (4.4) 1.7 (3.0) 1.3 (3.0) Dehydrogenation 1!TS2!3: ACHTUNGRE ACHTUNGRE ACHTUNGRE TS3 O2Htrans 6.0 (4.7) 2.0 (3.4) 1.7 (3.4) The optimized geometries of the reactant complex 1

(A···O2Hcis), transition state will take place and lead to the product complexes 3 or 4, re- (TS2), and product complex 3 are presented in Figure 4. In spectively (shown in Figures 4 and 5). this reaction, all the bond lengths in the purine system change minimally, by 0.01 to 0.03 , except those involving

Isomerization 1!TS1!2: Upon OH attack on adenine C2, C2. This is because H2 migrates far away from C2 ; the C2H2 aC2O2 single bond forms, which is 1.420 in 1 and bond length increases dramatically from 1.110 in 1,to 1.422 in 2. This change breaks the CN double bond and 1.712 in TS2, to 5.919 in the product complex 3.The causes alternating bond lengths in the purine ring to elon- N1C2,C2N3, and C2O2 bond distances all decrease by gate, while the other bonds shorten. The changes are less about 0.1 . The C2H2 bond change causes H2 to approach [32] than 0.05 compared to adenine. N C bonds involving the purine plane, while the dihedral angle tH2N3C4C5 decreases

Chem. Eur. J. 2012, 18, 4877 – 4886 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemeurj.org 4879 H. F. Schaefer III et al.

uct complex lies 2.0 (4.3) kcal mol1 below separated adenine plus hydroxyl radical.

Dehydrogenation 2!TS3!4: The optimized geometries of the reactant complex 2

(A···O2Htrans), transition state (TS3), and product complex 4 are shown in Figure 5. Similar

to the O2Hcis reaction pathway, the purine bond length changes

are small, except that the N1

C2,C2N3, and C2O2 bonds shorten by about 0.1 . As

before, the C2H2 distance elongates dramatically from 1.110 in 2, to 1.709 in TS3, to 4.750 in 4. The significant Figure 3. Optimized geometries for the two complexes (1 and 2) formed after hydroxyl radical attack on ade- distance change again causes H2 nine C2; and the isomerization transition state (TS1) connecting the two complexes. Bond lengths are in , angles are in degrees. Relative energies in black (in kcal mol1, ZPVE corrected values in parentheses) are to get closer to the purine given with respect to separated A +OH. Relative energies in red (in kcalmol1) are given with respect to the plane, the dihedral angle reactant complex 1. tH2N3C4C5 decreases from 37.08 in 2,to54.98 in TS4,to 102.58 in 4. At the same time,

O2 bends into the purine plane

with the dihedral tO2C2N1C6 changing from 130.0 to 170.7 to 179.98. The local barrier for this pathway is predicted to be 25.7 (21.5 with ZPVE) kcalmol1, which is only 0.6 (0.5) kcal 1 mol lower than the O2Hcis path. 4 is 18.0 (12.6) kcalmol1 higher in energy than the reactant complex 2, and 0.3 (0.2) kcalmol1 higher than 3. Simi- lar to 3, structure 4 is only 1.7 (4.1) kcalmol1 lower in energy than separated adenine and hydroxyl radical. The activation barrier for this reaction is 6.0 Figure 4. Optimized geometries for reactant complex (1), transition state (TS2), and product complex (3) for (4.7) kcalmol1, and only 0.3 the loss of atom H following formation of 1. Bond lengths are in , angles are in degrees. Relative energies in 1 black (in kcal mol1, ZPVE corrected values in parentheses) are given with respect to separated A +OH. Rela- (0.4) kcalmol higher than that 1 tive energies in red (in kcal mol ) are given with respect to the reactant complex. for the O2Hcis path. from 39.08 in 1,to55.98 in TS2,to99.18 in 3. At the

1 same time, O2 bends into the purine plane with tO2C2N1C6 Table 3. Energies [DE, in kcal mol , ZPVE corrected values in parenthe- changing from 132.0 to 170.3 to 179.98. ses] of the A···OH complexes 1, TS2, and 3. Also reported are reaction In Table 3, the local barrier for this reaction is predicted enthalpies [DH, in kcal mol1], entropies [DS, in calmol1 K1], and Gibbs 1 to be 26.3 (21.9 with ZPVE) kcalmol1, and the product energies [DG, in kcalmol ] for 1!TS2!3. complex is 18.7 (13.3) kcalmol1 higher in energy than the DE DH DS DG reactant complex. However, the activation barrier is only 1 0.0 (0.0)ACHTUNGRE 0.0 0.0 0.0 5.7 (4.4) kcalmol1 for this reaction pathway and the prod- TS2 26.3 (21.9)ACHTUNGRE 4.5 1.9 3.9 3 18.7 (13.3)ACHTUNGRE 5.3 3.2 6.2

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After loss of atom H2: In the isoguanine A···OH product complexes 3 and 4, the atom

designated H2 is loosely bound to the system through van der Waals forces. Thus, this hydrogen is easily abstracted from the system or may migrate to other regions in order to form a stronger bond. In this study, we only consider the formation of isoguanine and its tautomer following dehydrogenation. Two enol form isoguanine tautomers (isoG 1 and isoG2) will be produced after losing

the “free” hydrogen atom H2. Thus, isoG 1 and isoG2 will Figure 5. Optimized geometries for reactant complex (2), transition state (TS3), and product complex (4) for either undergo a direct enol– the loss of atom H following formation of 2. Bond lengths are in , angles are in degrees. Relative energies in keto tautomerization and form 1 black (in kcal mol , ZPVE corrected values in parentheses) are given with respect to separated A +OH. Rela- keto isoguanine tautomers, or tive energies in red (in kcal mol1) are given with respect to the reactant complex. react with a water to form an enol–water complex. This com-

Compared to the local barrier for C8-OH dehydrogena- plex then undergoes hydrogen transfer to produce a keto– tion (37.1 kcalmol1) from our previous study,[32] these two water complex. local barriers for losing hydrogen atom are much lower (26.3 kcalmol1 for the trans-pathway and 25.7 kcalmol1 Table 5. Standard enthalpy of activation [DH‡, in kcal mol1], stan- for the cis-pathway). Results are summarized in Table 4. dard entropy of activation [DS‡, in calmol1 K1], and ordered Gibbs energies of activation [DG‡, in kcal mol1] with respect to separated adenine plus OH radical at 298.18 K for the isoguanine formations [A+ 1 Table 4. Energies [DE, in kcal mol , ZPVE corrected values in parenthe- OHC!TS!isoG+ HC]. ses] of the A···OH complexes 2, TS3, and 4. Also reported are reaction ° ° ° enthalpies [DH, in kcal mol1], entropies [DS, in calmol1 K1], and Gibbs DH DS DG 1 energies [DG, in kcalmol ] for 2!TS3!4. TS1 (O2Hcis,trans) 1.4 34.4 11.7 DE DH DS DG TS2 (O2Hcis) 2.4 34.3 7.9 TS3 (O2Htrans) 2.4 34.8 8.0 2 0.0 (0.0)ACHTUNGRE 0.0 0.0 0.0 TS3 25.7 (21.5)ACHTUNGRE 4.4 2.6 3.7 4 18.0 (12.6)ACHTUNGRE 4.7 11.9 8.2 Direct enol–keto tautomerization: The six isoguanine tautomers are shown in Figure 6, numbered according to increas-

Thus, these two pathways are more likely than C8 dehydro- ing energy with respect to tautomer 1 (isoG1). The two tau- genation. However, previous investigation[25] showed no evi- tomerization pathways are shown in Figures 7 and 8. C dence of OH addition at C2-forming isoguanine. The low The optimized geometries of the reactant enol isoG 1 electron density at C2 and the electrophilic character of (O2H cis-isomer), transition state (isoGTS1), and product C OH, as well as these high local barriers, are quite insightful keto isoG3 (N1H isomer) are presented in Figure 7. In the in this respect. isoG1 tautomerization, the hydrogen migrates from O2 to ° 1 Standard enthalpies of activation (DH , in kcalmol ), N1. The O2H bond length increases from a normal OH standard entropies of activation (DS°, in calmol1 K1), and bond of 0.972 in the reactant, to 1.377 in isoG TS1,to ° 1 ordered Gibbs energies of activation (DG , in kcalmol ) 2.392 in the final keto N1H form. At the same time, the for the three transition states are shown in Table 5. Values N1H bond decreases dramatically from 2.222 in isoG 1, are reported with respect to separated adenine plus OH rad- to 1.261 in isoG TS1, to 1.016 in isoG 3. As the enol C C ical at 298.18 K for [A+ HO !TS!isoG +H ]. The enthal- transforms to keto, the C2O2 bond length decreases from a pies of activation for both reaction pathways are predicted CO single bond (1.354 in isoG 1) to a length between a to be 2.4 kcalmol1. The Gibbs energy of activation for single and a double bond (1.291 in isoG TS1) to a double 1 1 the cis-pathway, 7.9 kcalmol , is only 0.16 kcalmol lower bond (1.227 in product isoG 3). Simultaneously, the N1C2 than the trans-pathway. Such small differences indicate that bond increases from 1.349 to 1.402 to 1.459 . These the two pathway may be equally favorable for hydrogen dis- changes also lead to variation in the resonance structure of sociation from the A···OH complex. the rest of the six-membered ring (see Lewis structures in

Chem. Eur. J. 2012, 18, 4877 – 4886 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemeurj.org 4881 H. F. Schaefer III et al.

decrease in length as they become double bonds in the product isoG3. There are no major structural changes else- where in the five-membered ring. The local barrier for this reaction is predicted to be 36.1 (32.9 with ZPVE) kcalmol1 (Table 6), and isoG3 keto is 6.0 (5.7) kcalmol1 higher in energy than the reactant (isoG 1 enol form).

Table 6. Energies [DE, in kcal mol1, ZPVE corrected values in parentheses] of the three isoguanine isomers compared to isoG1. Also reported are reaction enthalpies [DH, in kcal mol1], entropies [DS, in cal m- ol1 K1], and Gibbs energies [DG, in kcalmol1] for isoG 1!isoGTS1! isoG3. DE DH DS DG isoG1 0.0 (0.0)ACHTUNGRE 0.0 0.0 0.0 isoGTS1 36.1 (32.9)ACHTUNGRE 3.3 0.3 3.4 isoG3 6.0 (5.7)ACHTUNGRE 0.2 0.4 0.4

When the other enol isoguanine isomer, isoG2 (O2H trans-isomer) undergoes enol–keto tautomerization, the hy-

drogen migrates from O2 to N3, instead of to N1 (see

Figure 8). Again, the O2H bond length increases from a normal OH bond 0.972 in the reactant, isoG2 to 1.389 in isoG TS2 to 2.446 in the final product, isoG4 keto. The

N3H bond decreases from 2.238 in the reactant enol to 1.261 in isoG TS2 to 1.014 in the product. Similar to the

isoG1!isoG3 tautomerization, the C2O2 bond changes Figure 6. Numbering of the six isoguanine tautomers. Relative energies from single to double bond character. However, the N3C2 (in kcalmol1, ZPVE corrected values in parentheses) are reported with bond length increases from 1.341 to 1.386 to 1.440 . These respect to lowest energy tautomer isoG1. changes also lead to alternating bond length changes in the rest of the six-membered ring, but do not interrupt the original resonance structure of isoG 2.

The N1C6 and C4C5 double bonds in the reactant remain double bonds in the product, albeit with shorter bond lengths. All three single bonds

(N1C2,N3C4 and C5C6) elongate and retain single bond character. The rest of the five- membered ring stays very much the same during tautomerization. The local barrier for this pathway is 38.8 (35.5) kcal mol1 (Table 7), which is 2.7 (2.7) kcalmol1 higher than the isoG1!isoG3 barrier. The product isoG 4 isomer is 6.0 Figure 7. Optimized geometries for the enol-keto tautomerization isoG1!isoG3. Included in the figure are (5.5) kcalmol1 higher in the isoG1 (O2H cis-isomer), transition state (isoGTS1), and product isoG3 (N1H isomer). Bond lengths are in . Relative energies (in kcalmol1, ZPVE corrected values in parentheses) are given with respect to isoG 1. energy than the reactant, isoG 2 enol. These findings explain why

only N1H and O2H tautomers Figure 7). All of the double bonds in the reactant become of isolated isoguanine and isoguanosine have been observed single bonds in the product, evidenced by increasing bond experimentally.[28,55] Since the cis-reactant complex (1) lies lengths. Two of the three single bonds (N3C4 and C5C6) lower in energy than the trans (2), and the isomerization

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isoG4. However, the main pur- pose of the present study is to elucidate the mechanism of isoguanine formation from adenine, so this slight difference in relative energies is inconse- quential. Both perturbation theory and DFT have inherent uncertainties larger than 0.5 kcalmol1. The reaction enthalpies, entropies and Gibbs energies for A+ OHC!isoG+ HC are presented in Table 8. They indicate that these reactions are exothermic and may happen simultaneously and spontaneously even at low temperatures.

Figure 8. Optimized geometries for the enol-keto tautomerization isoG2!isoG4. Included in the figure are Indirect enol–keto tautomeriza- the isoG2 (O2H trans-isomer), transition state (isoG TS2), and product isoG4 (N3H isomer). Bond lengths are in . Relative energies (in kcal mol1, ZPVE corrected values in parentheses) are given with respect to isoG2. tion: Microsolvation: Because direct hydrogen atom transfer

Table 7. Energies [DE, in kcal mol1, ZPVE corrected values in parenthe- Table 8. Energies [DE, in kcal mol1, ZPVE corrected values in parentheses] of the three isoguanine isomers compared to isoG2. Also reported ses] of the four isoguanine tautomers plus hydrogen atom, relative to sep- are reaction enthalpies [DH, in kcalmol1], entropies [DS,in arated adenine plus OH radical. Also reported are reaction enthalpies cal mol1 K1], and Gibbs energies [DG, in kcalmol1] for isoG2! [DH, in kcal mol1], entropies [DS, in cal mol1 K1], and Gibbs energies isoGTS2!isoG4. [DG, in kcalmol1] for A+OHC!isoG+HC. DE DH DS DG DE DH DS DG isoG2 0.0 (0.0)ACHTUNGRE 0.0 0.0 0.0 isoG1 1.9 (ACHTUNGRE4.3) 3.8 36.4 7.1 isoGTS2 38.8 (35.5)ACHTUNGRE 3.4 0.2 3.4 isoG2 1.7 (ACHTUNGRE4.2) 3.8 35.8 6.9 isoG4 6.0 (5.5)ACHTUNGRE 0.3 2.1 1.0 isoG3 4.0 (1.4)ACHTUNGRE 4.0 35.9 6.7 isoG4 4.3 (1.3)ACHTUNGRE 4.1 33.7 5.9 barrier is small, the reactant complex may exist mainly in the cis form. Both N1H and O2H tautomers lie along the cis from O2 to either N1 or N3 has a high local barrier, we inves- reaction pathway, and are thus preferentially formed over tigated the effects of microsolvation on isoguanine N1H and the other tautomers of isoguanine. N3H isomers by adding one water (Figures 10 and 11). The

Thermodynamics studies of duplex formation of dideoxy- six H2O···isoG complexes are presented in Figure 9, num- hexose-DNA[56] and pyranosyl-RNA[57,58] found isoguanine/ bered by increasing energy with respect to the lowest energy guanine Watson–Crick pairing, which provided evidence for complex, H2O–isoG1. the existence of isoG 4 (N3H) tautomer. Gas-phase ab initio Water and enol isoG1 form an enol–water reactant com- computations using second-order Møller-Plesset perturba- plex (H2O–isoG 1), following which hydrogen transfer tion [MP2/6-31G(d,p)]ACHTUNGRE method were consistent with experi- occurs, finally forming the keto–water product complex mental evidence for the keto and enol tautomers, and with (H2O–isoG3). The optimized geometries for H2O–isoG 1, our results. Roberts and co-workers computed relative ener- H2O–isoG TS1, and H2O–isoG3 in this hydrogen transfer gies of the four isoguanine tautomers; the isoG1 (O2H cis- process are shown in Figure 10. In the reactant complex, a isomer[59]) was found to be the lowest energy form, followed six-membered ring structure incorporating two hydrogen [59] 1 by isoG 2 (O2H trans-isomer ) (0.14 kcalmol ), isoG 4 bonds (N1···H and HO2···OH2O) is formed. The N1-C2 bond [59] 1 [59] (N3H form, 7.64 kcalmol ), and isoG3 (N1H form, length increases from 1.349 in isoG1 to 1.363 in H2O– 1 [59] 8.70 kcalmol ). In our study, isoG1 is also the lowest isoG1. The C2O2 and O2H bonds elongate and shorten, 1 energy isomer. IsoG 2 is less than 0.3 kcalmol higher in respectively. As the hydrogen bonded to O2 transfers to the ACHTUNGRE energy, in good agreement with the MP2/6-31G(d,p) result. oxygen of water, one of the water hydrogens migrates to N1.

The N1H and N3H isomers are both well separated from In this reaction, C2O2 changes from a single bond of length 1 isoG1 by 6.0 and 6.3 kcalmol . However, our ordering of 1.339 in H2O–isoG1, to 1.293 in H2O–isoG TS1,toa ACHTUNGRE isoG3 and isoG 4 differs from the MP2/6-31G(d,p) results; double bond (1.244 ) in H2O–isoG3. Thus, the resonance 1 isoG3 in this study is 0.3 kcalmol lower in energy than structure of the six-membered ring changes. The N3C2

Chem. Eur. J. 2012, 18, 4877 – 4886 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemeurj.org 4883 H. F. Schaefer III et al.

bond elongates to a single bond, N1C6 and C4C5 bond

lengths increase, and N3C4 and C5C6 bonds shorten in the product complex. The five-membered ring undergoes almost no change in bond lengths. The relative energies of the three complexes with respect

to H2O–isoG 1 are shown in Figure 10 and Table 9. The local barrier is predicted to be 12.7 (8.8 with ZPVE) kcalmol1, which is 23.4 (24.1) kcalmol1 lower than the local barrier

Table 9. Energies [DE, in kcal mol1, ZPVE corrected values in parenthe- ACHTUNGRE ses] of the three isoG(O2Hcis)···H2O complexes compared to reactant

complex H2O–isoG 1. Also reported are reaction enthalpies [DH, in kcal mol1], entropies [DS, in cal mol1 K1], and Gibbs energies [DG, in kcal 1 mol ] for H2O–isoG 1!H2O–isoGTS1!H2O–isoG3. DE DH DS DG ACHTUNGRE H2O–isoG 1 0.0 (0.0) 0.0 0.0 0.0 ACHTUNGRE H2O–isoG TS1 12.7 (8.8) 4.7 5.4 3.0 ACHTUNGRE H2O–isoG 3 2.4 (2.0) 0.2 1.0 0.5

for direct enol–keto tautomerization. H2O–isoG3 lies 2.4 1 (2.0) kcalmol above H2O–isoG 1. The relative energies, enthalpies, entropies, and Gibbs energies of activation for

the transition state of reaction isoG1+H2O!H2O–

isoGTS1!isoG3+ H2O are presented in Table 10. The activation energy is predicted to be 3.1 (1.7) kcalmol1 with respect to separated isoG 1 plus water. However, the enthalpies and entropies relative to separated reactants are both Figure 9. Numbering of the six isoG–H2O complexes. Relative energies negative for the transition state in the reaction. Therefore, (in kcalmol1, ZPVE corrected values in parentheses) are reported with the reaction will easily take place even at low temperature. respect to the lowest energy isomer H2O–isoG1. A similar process occurs when water attacks isoG2 (trans-

O2H isomer). The optimized geometries of the enol reactant

complex H2O–isoG2, transition

state H2O–isoGTS2, and keto

product complex H2O–isoG 4 are shown in Figure 11. The relative energies are given in Table 11. A six membered ring

(C2N3(HO)H2OHO2) forms with two hydrogen bonds (N3···H

and HO2···OH2O), where subscript H2O refers to atoms that are part of the water molecule. The

N1-C2 distance is almost un-

changed, but N3–C2 elongates from 1.341 in isoG2 to

1.353 in H2O–isoG2.The

O2–H distance increases from 0.989 to 1.208 to 1.768 from

H2O–isoG 2 to H2O–isoG TS2

to product complex H2O– isoG4. Simultaneously,

HO2···OH2O gets shorter, from Figure 10. Optimized geometries for the isoguanine O2H cis-isomer reaction with water. Included in the figure 1.804 to 1.231 to 0.994 , the are the reactant complex H O–isoG1, transition state (H O–isoGTS1), and product complex H O–isoG 3. 2 2 2 water OH bond elongates Bond lengths are in . Relative energies in black (in kcal mol1, ZPVE corrected values in parentheses) are 1 from 0.989 to 1.284 to 1.896 , given with respect to separated isoG1 plus H2O. Relative energies in red (in kcalmol ) are given with respect to H2O–isoG1. and N3···HH2O shortens from

4884 www.chemeurj.org 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Chem. Eur. J. 2012, 18, 4877 – 4886 Isoguanine Formation from Adenine FULL PAPER

compared to the direct trans- hydrogen transfer reaction. The product complex lies 3.0 (2.5) kcalmol1 higher in energy than the reactant complex. The relative energies, enthalpies, entropies and Gibbs energies of activation for the transition state of

isoG1+ H2O! H2O–

isoGTS1!isoG3+ H2O are presented in Table 12. The activation energy is predicted to be 3.8 (2.2) kcalmol1 relative to separated isoG1 plus water. However, the relative enthalpies and entropies are both negative with respect to separated reactants. Therefore, this Figure 11. Optimized geometries for the isoguanine O2H trans-isomer reaction with water. Included in the reaction will also easily take figure are the reactant complex H2O–isoG 2, transition state (H2O–isoGTS2), and product complex H2O– place. isoG4. Bond lengths are in . Relative energies in black (in kcal mol1, ZPVE corrected values in parenthe- 1 ses) are given with respect to separated isoG 2 plus H2O. Relative energies in red (in kcal mol ) are given with respect to H2O–isoG2.

Table 10. Energies [DE, in kcalmol1, ZPVE corrected values in paren- Table 12. Energies [DE, in kcalmol1, ZPVE corrected values in paren- ACHTUNGRE ACHTUNGRE theses] of the three H2O–isoG(O2Hcis)···H2O complexes compared to sep- theses] of the three H2O–isoG(O2Htrans)···H2O complexes compared to arated reactants isoG1 plus water. Also reported are reaction enthalpies separated reactants isoG2 plus water. Also reported are reaction enthal- [DH, in kcal mol1], entropies [DS, in cal mol1 K1], and Gibbs energies pies [DH, in kcal mol1], entropies [DS, in calmol1 K1], and Gibbs ener- 1 1 [DG, in kcal mol ] for isoG1 +H2O!H2O–isoG1!H2O–isoGTS1! gies [DG, in kcal mol ] for isoG 2+H2O!H2O–isoG2!H2O–

H2O–isoG 3!isoG3+H2O. isoGTS2!H2O–isoG 4!isoG4+H2O. DE DH DS DG DE DH DS DG ACHTUNGRE ACHTUNGRE H2O–isoG 1 9.6 (7.0) 1.8 32.9 11.6 H2O–isoG 2 9.9 (7.4) 1.8 32.3 11.4 [a] ACHTUNGRE [a] ACHTUNGRE H2O–isoG TS1 3.1 (1.7) 2.9 38.3 8.5 H2O–isoG TS2 3.8 (2.2) 3.0 37.4 8.2 ACHTUNGRE ACHTUNGRE H2O–isoG 3 7.2 (5.0) 1.5 31.9 11.0 H2O–isoG 4 6.9 (4.9) 1.5 30.5 10.6 [a] For the transition state we report barrier heights [DH°, in kcalmol1], [a] For the transition state we report barrier heights [DH°, in kcalmol1], barrier entropy changes [DS°, in calmol1 K1], and ordered Gibbs ener- barrier entropy changes [DS°, in calmol1 K1], and ordered Gibbs energies of activation [DG°, in kcalmol1]. gies of activation [DG°, in kcalmol1].

Table 11. Energies [DE, in kcalmol1, ZPVE corrected values in paren- ACHTUNGRE Conclusion theses] of the three isoG(O2Htrans)···H2O complexes compared to reactant complex H2O–isoG 2. Also reported are reaction enthalpies [DH, in kcal mol1], entropies [DS, in cal mol1 K1], and Gibbs energies [DG, in kcal The reaction pathways for hydroxyl radical attacking ade- 1 mol ] for H2O–isoG 2!H2O–isoGTS2!H2O–isoG4. nine C2 position have been investigated theoretically. The DE DH DS DG reactant complexes 1 and 2 both lie much lower in energy ACHTUNGRE than separated adenine and hydroxyl radical; 20.6 and H2O–isoG 2 0.0 (0.0) 0.0 0.0 0.0 ACHTUNGRE 1 H2O–isoG TS2 13.6 (9.6) 4.7 5.2 3.2 19.7 kcalmol , respectively. There is an isomerization ACHTUNGRE H2O–isoG 4 3.0 (2.5) 0.3 1.8 0.8 transition state TS1 connecting the two with a small inter- conversion barrier (1!TS1!2) of 1.6 kcalmol1. TS1 also lies below separated reactants, which indicates 1 and 2 are

1.879 to 1.221 to 1.028 . In the keto product complex amenable to formation when OH radical attacks adenine C2,

H2O–isoG 4, the C2–O2 single bond becomes a double bond, and one structure will easily isomerize to the other. 1 and 2 and the resonance structure of the six-membered ring will lead to cis and trans isomerization pathways respective- changes. The N1–C2 distance elongates to have single bond ly, then lose a hydrogen atom. Both pathways have transi- character, N1–C6 and C4–C5 shorten, N3–C4 and C5–C6 tion states (TS2 and TS3) which are higher in energy than lengthen, and the five-membered ring is nearly unchanged. separated adenine and hydroxyl radical; 5.7 and 6.0 kcal The local barrier is predicted to be 13.7 (9.6 with ZPVE) mol1, respectively. Both reactions A+OHC!isoG1/ kcalmol1, which is 1.1 (0.8) kcalmol1 higher than that of isoG2+ HC are found to be exothermic, and will take place the cis-reaction, but much lower [by 25.2 (24.9) kcalmol1] spontaneously even at low temperatures.

Chem. Eur. J. 2012, 18, 4877 – 4886 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemeurj.org 4885 H. F. Schaefer III et al.

After losing the hydrogen atom, two possible mechanisms [26] J. Llano, L. A. Eriksson, Phys. Chem. Chem. Phys. 2004, 6, 4707. have been considered for isoguanine tautomerization. The [27] S. Naumov, C. V. Sonntag, Radiat. Res. 2008, 169, 355. [28] J. Sepiol, Z. Kazimierczuk, D. Shugar, Z. Naturforsch. C 1976, 31, direct hydrogen transfer pathways have extremely high local 361. barriers for enol isomers isoG 1 and isoG 2 to transform to [29] S. Wang, H. F. Schaefer, J. Chem. Phys. 2006, 124, 044303. the keto forms isoG 3 and isoG4. However, adding one [30] F. A. Evangelista, A. Paul, H. F. Schaefer, J. Phys. Chem. A 2004, water to the reaction lowers the barriers by 23.4 and 108, 3565. 25.2 kcalmol1, respectively. Thermal energetic analysis sug- [31] S. Wetmore, R. Boyd, L. Eriksson, J. Phys. Chem. B 1998, 102, 10602. gests that this reaction may take place spontaneously even [32] Q. Cheng, J. Gu, K. R. Compaan, H. F. Schaefer, Chem. Eur. J. at low temperatures. 2010, 16, 11848. 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