PHOTOAFFINITY LABELING STRATEGIES USING PURINE NUCLEIC ACID BASES
Denis I. Nilov
A Dissertation
Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
December 2011
Committee:
Dr. R. Marshall Wilson, Advisor
Dr. Robert M. McKay Graduate Faculty Representative
Dr. Thomas H. Kinstle
Dr. Alexander N. Tarnovsky
ii
ABSTRACT
Dr. R. Marshall Wilson, Advisor.
Photodecomposition of 8-azidoadenosine (24) and 8-azidoinosine (30) has been studied
by ultrafast transient absorption spectroscopy methods in different solvents. The formation of
two types of intermediates has been proposed for photodecomposition of 24 and 30: the
diiminoquinone 25 for 24 with an EDG on the purine ring, an iminonitriles 26 for 24-H, and 38 for 30 in the absence of an EDG on the purine ring. Thus, nitrenes 31, which is formed from
azide 24, undergoes proton reallocation to form iminoquinone 25. Iminoquinone 25 undergoes nucleophilic attack forming C2-adducts 33-Nuc, which aromatizes to 34-Nuc. Nitrenes 31-H and
31Bz5 afford to ring-opened iminonitriles 26-H and 26Bz5, respectively. Nucleophilic attack to
iminonitrile 26-H leads to the formation the same product as C2-adducts 33-Nuc, which
aromatizes. Azide 30 forms nitrene 36 which exclusively to rearranged to iminonitrile 38.
Iminonitrile 38Ac3 displays the spectroscopic behavior similar to 26-H in the μs-ms time
domains. A ring destruction product 35, 8-aminoadenosine, and adducts 34-IM and 34-OH have
been isolated. Small amounts of dimer-like structures and other products have been detected, but
have not been fully identified.
The azide 39 has been synthesized for the first time and its photodecomposition has
been studied. It has been proposed that in protic solvents a photolysis of azide 39 leads to singlet
nitrene 53s, which undergoes the proton transfer from N7 to N6 through the solvent-bridged
transition and forms iminoquinone 5. In aprotic media, singlet nitrene 53s undergoes ISC to
triple nitrene 53t. Nucleophilic attack on iminoquinone 5 leads to the formation of C2-adducts,
such as 56, that undergo aromatization to corresponding adducts 55-Nuc. Imidazole adduct 8 and iii product 2 and 3 have been isolated and characterized from the preparative irradiation of azide 39.
Additionally, some amounts of other photodecomposition products have been detected, but have not been fully identified.
Assignments of important intermediates based on experimental data have been supported by quantum chemical calculations using TD-DFT, CASSCF and CASSP2 methodologies. Azide 39 has been proposed as a candidate for a PAL reagent in biochemical studies of NA-protein and NA-NA interactions. iv
ACKNOWLEDGMENTS
At the beginning, I would like to thank my adviser, Professor R. Marshall Wilson. I am very grateful for sharing his knowledge, his support, encouragement, and guidance.
I want to express my appreciation Professor Alexander N. Tarnovsky for the great collaboration. The discussions and suggestions that he proved were highly useful. I thank
Kanykey Karabaeva and Andrey Mereshenko for acquisition of femtosecond spectroscopic data.
Also, I would like to thank Maxim Panov for acquisition of femtosecond spectroscopic data and his help in quantum chemical calculations.
I am very thankful to Professor Phil Castellano for opportunity to use instrumentation in his laboratories and Valentina Prusakova for her help in study of nanosecond spectroscopy technique.
I thank Professor Massimo Olivucci and Elena Laricheva for their helpful advices in quantum chemistry calculations.
I am very grateful to Professor Thomas H. Kinstle for his help and support during all this period. Also, I am very thankful for his sharing of knowledge of synthetic organic chemistry.
I thank my committee member Professor Robert Michael McKay for his time and patience.
I am grateful to Dr. Jedrzej Romanowicz and Dr. Larry Sallens for their help with mass spectroscopy and to Dr. D. Y.Chen for his help with NMR experiments.
I thank Dr. Pavel Kucheryavy and Ekaterina Mirzakulova for their help in electrochemistry experiments.
I also would like to thank personnel of Ohio Supercomputer Center for a possibility to support experimental data with theoretical calculations. v
I was pleased to work in laboratory with members of our research group Alexei
Shamaev, and Kanykey Karabaeva. Also, I am grateful to Dr. Dmitry Komarov, and Dr.
Valentyna Voskresenska, and Dr. Sergey Voskresensky. I would like to thank them for maintaining a productive and friendly atmosphere.
Finally, I would like to express my special gratitude to people who permanently supported me. I thank my dear friends, especially, Armen Ilikchyan, Alexander Krylov, Yuri
Strezhik, and Anna Parinova for their permanent encouragement, comprehension, and friendship.
I thank my dear parents for their love and support through all time. vi
TABLE OF CONTENTS
Page
CHAPTER I. BACKGROUND...... 1
1.1. Photoaffinity Labeling (PAL) ...... 1
1.2. Oxidation of Purine Bases via Formation of Quinoidal
and Iminoquinoidal Intermediates...... 5
1.3. Photochemistry of Aromatic Azide ...... 10
1.4. References ...... 19
CHAPTER II. EXPERIMENTAL METHODS ...... 30
2.1. Liquid Chromatography...... 30
2.2. UV-VIS Absorption Spectroscopy ...... 30
2.3. IR Spectroscopy...... 30
2.4. Mass Spectrometry ...... 31
2.5. NMR Spectroscopy...... 31
2.6. Femtosecond UV-VIS Time Resolved Absorption Spectroscopy ...... 31
2.7. Nanosecond UV-VIS Time Resolved Absorption Spectroscopy...... 33
2.8. Quantum Chemistry Calculations...... 34
2.9. References ...... 34
CHAPTER III. PHOTOCHEMISTRY OF 8-AZIDOADENOSINE
AND 8-AZIDOINOSINE...... 37
3.1. Introduction ...... 37
3.2. Photochemical Studies of 8-Azidoadenosine (24)...... 39
3.3. Synthesis and irradiation of 8-Azidoadenosine (24), vii
8-Azidoinosine (30), and Their Derivatives ...... 54
3.4. References ...... 60
CHAPTER IV. The Photochemistry of 6-azido-8-oxo-7,9-dihydropurine (39) ...... 62
4.1. Introduction ...... 62
4.2. Chemical Generation of Iminoquinone 5 via oxidation of 8-oxoadenosine (2). 64
4.3. Photochemical Studies of 6-Azido-8-oxo-7,9-dihydropurinoriboside (39)...... 68
4.4. Conclusions ...... 78
4.5. Synthesis of 6-azido-7,9-dihydro-8-oxopurine (39) and derivatives...... 78
4.6. References ...... 85
CONCLUSIONS ...... 87
SUPPLEMENTAL INFORMATION. HRMS SPECTRA. 1H AND 13C NMR SPECTRA. 88
viii
LIST OF SCHEMES
Scheme Page
1.1. Generally used PAL agents and their putative mechanisms...... 3
1.2. Stepwise oxidation of guanosine...... 7
1.3. Oxidation of 8-oxopurines via formation of quinones and iminoquinones
of purines and regioselectivity of nucleophile attack...... 8
1.4. Chemical oxidation of 8-oxoA (2) and 8-oxoI (3)...... 9
1.5. The donation of electron from aromatic ring system to p-π orbital of nitrene 11..... 12
1.6. Mechanism for photolysis of phenylazide...... 13
1.7. Formation of products from benzazirine 12 and dehydroazepine 13...... 14
1.8. Dimerization and reduction of triplet nitrene 11t...... 15
1.9. The chemistry of a fluorosubstitued aryl azide...... 15
1.10. Photolysis of p-(N,N-diethylamino)phenylazide...... 17
1.11. The chemistry of nitrenium ions...... 18
1.12. Photodecomposition of 21...... 18
1.13. Nucleophilic attack on nitrenium ion in ortho- positions to the nitrenium ion...... 19
3.1. The formation of diiminoquinone 24 and iminonitrile 26 in
photodecomposition of 8-azidoadenosine...... 37
3.2. Photolysis of 8-azidoadenosine in methanol...... 38
3.3. Opening of 5-membered ring...... 38
3.4. Azido-tetrazole isomerization for 8-azidopurines...... 39
3.5. The formation of diiminoquinone 25 and iminonitrile 26...... 42
3.6. Nucleophilic attack on diiminoquinone 25 and iminonitrile 26...... 42 ix
3.7. The formation of 8-amino-2-oxyadenosine (33-OH) in water...... 48
3.8. Preparative photolysis of 8-azidoadenosine (24)...... 49
3.9. The formation of iminoquinone 37 and iminonitrile 38...... 49
3.10. Nucleophilic attack on iminonitrile 38...... 51
4.1. Chemical and photochemical pathways of generation of iminoquinone 5...... 62
4.2. Thermolysis of 2-azidopyridine (43)...... 63
4.3. Photolysis of 3-azidoisoqunoline (42)...... 64
4.4. Thermolysis of 4-azido-2-phenylquinazoline (50)...... 64
4.5. Mechanism for the chemical oxidation of 8-oxoA (2)...... 65
4.6. The formation of C2-amines...... 67
4.7. Azido-tetrazine tautomerization for azide 39...... 68
4.8. Intermolecular and intramolecular mechanisms for proton transfer for 39...... 70
4.9. Mechanism for photolysis of azide 39Bz3...... 73
4.10. Imidazole attack on iminoquinone 5...... 74
4.11. Preparative irradiation of azides 39Ac3 and 39Bz3 ...... 75
4.12. Possible mechanism for the formation of over oxidation products...... 76
4.13. Hydrolysis of Iminoquinone 5...... 77 x
LIST OF FIGURES
Figure Page
1.1. General scheme for photoaffinity labeling (PAL)...... 1
1.2. An example of application of 4-azido-2-hydroxybenzamide PAL
in studying of intramolecular RNA interactions...... 4
1.3. Oxopurines: 8-oxoG (1); 8-oxoA (2); 8-oxoI (3). Purine ring numeration...... 5
1.4. Polar mesomeric structures of an organic azide...... 11
1.5. Low-lying spin states nitrene 11...... 12
1.6. Nitrenium ions...... 16
1.7. Singlet ground state phenyl nitrenium ion. Stabilization of positively charged
nitrenium nitrogen atom via conjugation with para- electron-donating group...... 17
2.1. Scheme of femtosecond setup, used in experiments...... 32
3.1. 8-Azidoinosine (30)...... 38
3.2. IR spectra of 8-azidoadenosine (24), 24Ac5, 30, and 30Ac3...... 40
3.3. Femtosecond and picosecond transient spectra of azide 24
in water solution at pH = 2.6 (A), 3.7 (B), and 9.2 (C) ...... 41
3.4. Acylated derivatives of azide 24...... 44
3.5. Femtosecond and picosecond transient spectra of azide 24 in nonprotic
solvents: 24Bz5 (A) in dichloromethane and 24Ac5 in methylcyclohexane (B)...... 45
3.6. Femtosecond and picosecond transient spectroscopy of azide 24
in methanol (A) and 2-propanol (B)...... 46
3.7. Nanosecond transient spectra of azide 24 in water at
pH = 3.1 (A), pH = 6.5 (B), and pH =11.5 (C)...... 47 xi
Figure Page
3.8. Nanosecond transient spectra of azide 24 in water in presence
of imidazole (1 M, A) and B: phenol (1 M, B) ...... 48
3.9. Femtosecond and picosecond transients of azide 30: in water at pH = 1.75 (A),
in water at pH = 7.5 (B), in methanol (C), and 30Ac3 in dichloromethane (D)...... 50
3.10. Nanosecond -millisecond transient spectra of azide 30Ac3 in methanol (A)
and in 1 M aqueous solution of imidazole (B)...... 51
4.1. Transient absorption spectra of oxidation of 8-oxoA (2) in MeOH with NBS...... 65
4.2. IR spectrum of azide 39Bz3...... 69
4.3. Femtosecond transient of azide 39Bz3 in MeOH and 2-PrOH...... 71
4.4. Transient absorbtion spectra of azide 39Bz3 in methylcyclohexane (A) and in
dichloromethane (B)...... 72
4.5. Nanosecond-millisecond transient absorption spectra of azide 39Bz3 in
pure methanol (A) and in 1M solution of imidazole in methanol (B)...... 73
4.6. Time-resolved emission spectra of azide 39Bz3...... 74
xii
LIST OF TABLES
Table Page
1.1. Yields of purine oxidation products...... 10
3.1. Calculated vertical excitations for intermediates 25Me, 26Me-H,
and 31Me in methanol...... 43
3.2. Time constants τ1 and τ2 for the reactions of reaction of purine
intermediates 25, 26-H, and 38 under various conditions...... 52
4.1 Calculated vertical excitations for intermediates 5 and N-bromoadenosines...... 66
4.2 Irradiation of azides 39Ac3 and 39Bz3 in MeOH...... 75
1
CHAPTER I. BACKGROUND
1.1. Photoaffinity Labeling (PAL)
Ligand Reversibly Specifically Bound + Macromolecule and Ligand PAL Macromolecules
Photo-initiated hν Cross-Linking
Separation Cutting Covalently Bound Macromolecule Cross-Linked and Ligand Fragment
Micro-level Macro-level Analysis Analysis
Figure 1.1. General scheme for photoaffinity labeling (PAL).
Photoaffinity labeling (PAL) plays an important role in biochemical structural studies of specific interaction sites of macromolecules.1-7 The first application of PAL agents for studying
of biomolecules was reported by Westheimer et al. about a half of century ago.8 From that time, 2
PAL has become one of the powerful tools in biochemistry, widely-applied for the determination
of structures of macromolecular complexes, functions and structures of membranes, and for the
studying of protein-NA (nucleic acid) and NA-NA interactions.9-17
The generalized scheme for the PAL experiment is shown on Figure 1.1. The PAL agent, a photosensitive functional group, is incorporated into a large ligand molecule. Labile complexes of specific macromolecules and ligands are exposed to light in order to generate a highly reactive species from the PAL label. This highly-reactive species reacts with any appropriate neighboring functional group and covalently binds the macromolecule to the ligand.
The resultant covalently bound complex can be analyzed by different methods either on a macro- level, to find which macromolecules bind preferably with the ligand, or on a micro-level after degradation to find the specific sites of macromolecule-ligand binding.4,6,9-11
The development of PAL agents, which have high-binding efficiency and selectively, is
one of the main goals in this research area. Several criteria for an effective photoaffinity labeling
agent have been described in the literature.1,2,7,18-19 To summarize, PAL agents should:
• be a small molecule which doesn’t affect the binding properties of the biological
molecule to which it is attached;
• become highly reactive when irradiated at a wavelength longer than 280 nm in
order to minimize photochemical damage of the biological sample;
• photochemically form a highly reactive intermediate that reacts with C-H bonds
and/or nucleophiles. Moreover, the lifetime of this intermediate should be long enough to
effectively target sites for covalent binding, but at the same time should be short enough to
form the covalent before the dissociation of the macromolecule-ligand complex; 3
• lead to the formation of a stable covalent bond between ligand and
macromolecule to produce a stable product which can be isolated, purified, and analyzed.
Benzophenones, diazo compounds, diazirines, diazonium salts, and azides have usually
been used as PAL agents.1,2,7,20-24 The putative mechanisms for PAL include the generation of nitrenes (from azides), carbenes (from diazo- and diazirines compounds), aryl cations (from diazonium salts), and biradicals (from enones) (Scheme 1.1).
Scheme 1.1. Generally used PAL agents and their putative mechanisms.
Macromolecule Macromolecule N3 N HN hν HN + -N2 Ligand Ligand Ligand Ligand
N CF CF Macromolecule CF N 3 3 3 hν
-N2 Ligand Ligand Ligand
O O hν
Ligand Ligand
OH HO Macromolecule
Ligand Ligand
4
The efficiencies and suitabilities of various PAL agents were compared in several
reviews.1,2,7,11,25 While ideal photoreactive molecules should satisfy all of the aforementioned requirements, none of the groups mentioned above does so. Nowadays, most of the PAL reagents are based on the chemistry of carbenes and nitrenes, which are isoelectronic and display very similar chemistry.23,24 To date, the most widely-used PAL agents are phenyl azides, since they have almost all the properties listed above and are easily synthesized or even commercially available.1-7,19,26,27
5' NH2 A-U A-U N N U-A C-G U-A O N N A-U U A A-U O P O O A-U O 100- A-U G-C -10 O OH A-U U-Aryl Azide A A-U C-G U G-C O U-A AU U-A A-U NH A-U U-A -20 O A U U-A N O G-C O P O G-C O Aryl U-A O U-A XL A U azide A-U O HN O A-U A-U 80- A-U OH U-A U-A -30 A-U G-C * 5' N3
Figure 1.2. An example of application of 4-azido-2-hydroxybenzamide PAL in studying of
intramolecular RNA interactions.28
The studies of NA-NA and NA-protein, protein-protein complexes, and protein-ligand interactions using of aryl azide derivatives as PAL agents resulted in hundreds of publications in 5 various medical and biochemical journals. One example is the photosensitized covalent bonding between RNA duplex strand and 2-azido-4-hydroxybenzamide in aqueous solution.28 This is an example of a cross-linking reaction that occurs when the photoaffinity labeling is carried out in an intramolecular fashion (Fig.1.2).29-33 The mechanism of this addition process was established by Platz, Weeks and coworkers.28 Progress in the development of new PAL agents and in the understanding of the processes of photoaffinity labeling in the last decade are among the factors that made it a major technique for studying molecular interactions in biological systems.
Therefore, it is important to determine the photochemical mechanisms of PAL agents in detail in order to design more effective reagents with increased labeling selectivity and efficiency.
1.2. Oxidation of Purine Bases via Formation of Quinoidal and Iminoquinoidal
Intermediates
O NH2 O H H H N 6 N N 7 5 1NH N NH O 8 O O 4 2 N9 N NH N N N N HO 3 2 HO HO O O O
OH OH OH OH OH OH 123
Figure 1.3. Oxopurines: 8-oxoG (1); 8-oxoA (2); 8-oxoI (3). Purine ring numeration.
One of the widely accepted principle sources of genetic damage of nucleic acids is oxidative damage.34-38 This process can lead to carcinogenesis, mutation, aging, and early cell death.39-41 Nucleic acids can undergo oxidative damage by different naturally occurring reactive 6
oxygen species, which involve hydrogen peroxide, singlet oxygen, and hydroxyl radical,39,42
upon exposure to high-energy UV and ionizing radiation,43 and directly by different oxidizing agents.44,46
Oxidative damage to purine bases can cause significant modification of DNA and
47-51 RNA. Purine bases have lower oxidation potentials compared to pyrimidines: EG =1.3 V and
52-55 EA = 1.4 V compared to EC = 1.6 V and ET = 1.7 V with respect to the NHE. Purines undergo
initial oxidation at the C8-position.47-51 These oxidized purines, 8-oxo-7,9-dihydoguanosine
(8-oxoG, 1), 8-oxo-7,9-dihydroadenosine (8-oxoA, 2), and 8-oxo-7,9-dihydroinosine (8-oxoI, 3)
have been detected in cells of living organism and in their metabolism products (Fig. 1.3).56-57
The presence of these oxidized bases in a living cell can be a potential threat when built
into a new nucleic acid chain, modified bases can cause mispairing in an existing strand58-60 or
miscoding during further replication of a nucleic acid chain.61,62 One of the natural protective
mechanisms from oxidative damage involves the selective phosphate bond cleavage of
8-oxoG (1) or 8-oxoA (2).63-65 Another repair mechanism is the enzymatic recognition and
replacement of 8-oxoG (1) and 8-oxoA (2) in nucleic acids strands.34,66-73
The replacement of 8-oxoG (1) with unmodified guanosines is well studied.34,70-72
However, similar enzymes, which replace of 8-oxoA (2) with unmodified adenosine, have been described in the literature only recently and have relatively low efficiency.73 Furthermore, it seems, that there is no repair system yet described for 8-oxoA:T lesions. 7
Scheme 1.2. Stepwise oxidation of guanosine.
O O O H N NH [O] N NH :O H N NH 2 O N N N N NH2 N NH2 [O] N NH2 R R R RC 1
[O] H N Nuc Nuc: O NH O Nuc O H N N H N N N N R NH2 O O + N N N NH2 N NH2 R R H Nuc 4 N O O N N HO R HN O R = NH2 OH OH
Oxidation processes of guanosine, the most easily oxidized nucleic acid base,74 have been widely-studied.75 The stepwise oxidation of guanosine by reactive oxygen species, typical
oxidizing agents,34,35 is outlined on Scheme 1.2. Under these oxidation conditions, guanosine
loses an electron forming the radical cation RC. This radical cation RC can undergo nucleophilic attack a C8-position and further oxidation and forming the adduct, 8-oxoG (1).44,76 Alternatively,
RC can oxidize a molecule of 8-oxoG (1),44,77 since the oxidative potential of unmodified
53,54 guanosine is higher than 1 (EG = 1.3 V vs E8-oxoG = 0.7 V, NHE). Further oxidation of 1 leads to the formation of purinequinones 4.78-82 The purinequinone 4 undergoes nucleophilic attack at 8
C5-position leading to thermally unstable adducts, which undergo destruction of purine ring and
formation of an abasic DNA/RNA site.76
In addition to the breakage of nucleic acid chains, the oxidation of guanosine might lead to cross-linking with proteins.76,78-84 In most cases, proteins became cross-linked with guanosine
at C8 and 8-oxoG (1) cross-linked at C5 positions through the amino groups of lysine and arginine residues.
Scheme 1.3. Oxidation of 8-oxopurines via formation of quinones and iminoquinones of
purines and regioselectivity of nucleophile attack.
NH2 NH H N N [O] N N O O N N N N R R :Nuc 2 5 HO O O O H R = N N NH [O] N OH OH O O N N N N :Nuc R R 6 3
The chemistry of oxidation of 8-oxoA (2) and 8-oxoI (3) has not been as well-studied as
the chemistry of 8-oxoG (1). However, oxidation of these compounds might form effective
cross-linking agents.85,86 The quinoidal intermediates, which are similar to 4, were recently
reported for the oxidation of 8-oxoA (2) and 8-oxoI (3) (Scheme 1.3).85,86 Iminoquinone 5 and
quinone 6 were proposed to play pivotal roles in these oxidation processes. These species
undergoes nucleophilic attack at the C2 posittion forming the corresponding adducts.
The chemical oxidation of 8-oxoA (2) at room temperature in methanolic NBS solution leads to the formation of 2-methoxy adduct 7 and in methanolic imidazole solution leads to the 9 formation of 2-imidazolyl adduct 8 (Scheme 1.4.). Also, adduct 8 has been isolated during the oxidation of 2 with NBS or Na2IrCl6 in water solution in the presence of imidazole as a nucleophile. 85,86
Scheme 1.4. Chemical oxidation of 8-oxoA (2) and 8-oxoI (3).
NH2 NH2 H H N N NBS N N O O N MeOH N N N OCH3 R 2 R 7 H NH2 N NH2 H H HO N N N N N O O O R = N N NBS, N N N R 2 MeOH R 8 OH OH N H O N O H H N NH N N NH O O Na IrCl N 2 6 N N H2O N N R 3 R 9 N
The reaction between 8-oxoI (3) and NBS under the same conditions as used for 2 does not lead to appreciable adduct formation. However, aqueous solutions of Na2IrCl6 oxidizes 3 producing 2-imidazolyl adduct 9 (Scheme 1.4). The same adduct is formed from chemical oxidation of 3 in water by sodium hypochlorite and under photochemical conditions by oxidation with peroxodisulfate anion in the presence of imidazole as nucleophile.85,86 Summarized data for oxidation of 2 and 3 are presented in Table 1.1.85,86 According to these results, 8-oxo-purines might be considered as potential candidates for cross-linking agent. However, the problem of overoxidation requires further investigation in this area of chemistry. 10
Table 1.1. Yields of purine oxidation products.
Purine Oxidizing Agent Solvent & Nucleophile Products and %Yeild
2 NBS CH3OH 7 triacetate, 6-acetamido,
6%a
b 2 NBS CH3OH, Imidazole 8 triTBDMS, 19%
2 NBS H2O, Imidazole 8, 23%
2 Na2IrCl6 H2O, Imidazole 8, 21%
3 NaOCl H2O, Imidazole 9, 40.5%
3 Na2S2O8, hν (254 nm) H2O, Imidazole 9, 35%
3 Na2IrCl6 H2O, Imidazole 9, 69%
a. Product isolated following treatment of crude reaction mixture with acetic anhydride.
b. Product isolated following treatment of crude reaction mixture with TBDMSCl.
1.3. Photochemistry of Aromatic Azides
Aryl Azides. In 1864, Peter Grieß reported the synthesis of phenylazide (10).87 This
event laid out the beginning for a new class of organic compounds named organic azides. The
intensive study of organic azides started in the 1950’s and 1960’s, and opened new applications
for these compounds not only in fundamental science (precursors for various other classes of 11
organic compound such as amine, triazene, azirine, aziridine etc.),88-90 but also in medicine and
industry (pharmaceutically active compounds, explosives, propellants, polymer cross-linkers,
blowing agents, reactive dyes, and rubber vulcanizations).90-97
As mentioned above, organic azides react via a variety of mechanisms and have a
variety of mesomeric structures. The polar mesomeric linear structures, which replace the
originally proposed cyclic triazirine structure for an organic azide in ground state is shown
on Fig 1.4.98-102
NNN NNN NNN NNN R R R R 12 3 abb' c
Figure 1.4. Polar mesomeric structures of an organic azide.
Furthermore, aryl azides have additional conjugation with aromatic system. According
to calculations for methyl azide, the azide group is almost linear (172.5°) and the R is positioned
at an angle of 115.2° with respect to the azide group.103 The established bond lengths correspond
to values of 1.5 for N1-N2 bond order and 2.5 for N2-N3 bond order. The N2-N3 bond was found
to be shorter in aryl azides.104 Thus, mesomeric structure determines the presence of
characteristic IR band of azide group at 2100-2200 cm-1.94 The mesomeric structures b and b’ provide justification for the reactivity of organic azides in 1,3-dipolar addition. The loss of molecular nitrogen molecule and the formation of a nitrene could be explained by mesomeric structures c.101
More than a century ago Tiemann proposed the formation of nitrenes as reactive intermediates derived from decomposition of azides.105 Nowadays, there is a wide range of
publications dedicated to the study of photochemistry of phenylazide and other simple aryl 12
azides.106-112 The chemistry of simple aryl nitrenes has been widely studied by time-resolved spectroscopy. The development of laser flash photolysis (LFP) and quantum chemical computation methods have made significant contributions to the understanding of mechanisms of photochemical processes of aryl nitrenes.111-115
1131 N N N N 11s11s 11s 11t CSSCSS OSS T
Ph N Ph N Ph N Ph N
Figure 1.5. Low-lying spin states nitrene 11.
Scheme 1.5. The donation of electron from aromatic ring system to p-π orbital of nitrene 11.
N N N N N N CSS: OSS or T:
The photodecomposition of phenylazide leads to the formation of phenylnitrene (11).
According to high-level calculations, molecular orbital of this species includes one sp hybrid orbital on the nitrogen atom, which is high in 2s character and occupied by a lone pair of electrons, two 2p orbitals, which are occupied by two nonbonding electrons. One of these two orbitals is p and in the plane of the aromatic ring, and the other is a p-π orbital. These two 2p orbitals are near-degenerated and produce 4 low-lying spin states, which are shown in Figure 1.5: 13
closed-shell singlet (CSS, 2 states), open-shell singlet (OSS), and the triplet state (T).114-116 In one of closed-shell singlet states, p-π orbital is unoccupied, and this state could stabilized by donation of an electron pair from the π-system of aromatic ring. However, the open-shell singlet state (OSS) was found to be the lowest energy singlet nitrene 11s based on computational work of Karney and Borden.115 A single electron from the aromatic ring system could be donated on p-
π orbital of nitrogen in this spin state. Thus, OSS and T of nitrene 11 represent biradical species
and the CSS of nitrene 11 can have a zwitterionic component (Scheme 1.5).
Scheme 1.6. Mechanism for photolysis of phenylazide.
1 N 0 hν N 1 Ph N3 Ph N Ph N 10 11s
165 Κ00 ps 6 -1 > 1 k = 3.2x10 s 0- ISC Τ 1 = T = 77K τ τ = 310 ns N N τ = 100 ps-1 ns 3 Ph N 11t 13 12
The mechanism of photodecomposition of phenylazide is shown on Scheme 1.6. Platz et
al. detected phenylnitrene (11s) spectroscopically for the first time as a band centered at λmax =
350 nm.112 As mentioned above, phenylazide (10) in its singlet excited state undergoes nitrogen
extrusion forming singlet phenylnitrene (11s). At the temperatures T > 165 K, singlet nitrene 11s rearranges to benzazirine 12 within 100 ps. Dehydroazepine 13 is derived from 12 in the time domain of 100-1000 ps.118 At low temperatures T< 165 K, nitrene 2s undergoes singlet-to-triplet
6 -1 119 intersystem crossing to nitrene 11t with kisc = 3.2×10 s at 77 K. 14
Scheme 1.7. Formation of products from benzazirine 12 and dehydroazepine 13.
H N N EtS NH EtS 2 EtSH
12 14
H N N NEt2 N NEt2 NEt2H
13 15
The chemistry in solution of benzazirine 12 and dehydroazepine 13, which were
originally proposed by Wentrup,120 in the presence of nucleophiles is shown on Scheme 1.7. In
spite of the facts, that 12 is the likely intermediate in the formation of 2-(ethylthio)aniline (14), reported for the photolysis of phenylazide (10) in ethanethiol,121 and supported by high-level
calculations, performed by Karney and Borden,115 no direct detection of benzazirine 12 has been reported.122,123
Time resolved UV-vis and IR spectroscopic measurements have been obtained for
-1 122,124-126 dehydroazepine 13 (λmax =340 nm, νmax = 1890 cm ). The lifetime for dehydroazepine
110 13 τ194K = 32 min has been reported. Dehydroazepine 13 is quenched by diethylamine (DEA)
6 -1 -1 118 with a rate constant of kDEA = 6.5x10 M s .
Triplet nitrene 10t, which is formed from 10s predominantly at low temperatures
(77 K) via intersystem crossing (ISC), displays biradical chemistry (Scheme 1.8). It undergoes
dimerization leading to azobenzene, 16, and reduction to aniline abstracting hydrogen atoms
from the solvent.127-129 Due to this chemistry triplet phenyl nitrene is of little value in
PAL studies.106 15
Scheme 1.8. Dimerization and reduction of triplet nitrene 11t.
Ph 3 Ph N NN + Ph NH2 Ph 10t 16
Phenylazide (1) is not an ideal candidate for the role of a PAL agent due to its relatively
low efficiency of photolabeling (< 30%).88 In order to discover which substituents on the
aromatic ring improved the photolabeling efficiency, many modified aryl azides have been
studied. The most promising aryl azide substituents seem to be carboxy, nitro, imino, and fluoro
substituents.
Scheme 1.9. The chemistry of a fluorosubstitued aryl azide.
3 F 1 F N F F N F N F N N
X F X F X F F F F
kFAr N F N F N N
F F F F
X F X F
As mentioned above, aryl azides substituted with fluoro groups have benefits as PAL agents. In particular, the activation energy barrier for the formation of dehydroazepines (the ring expansion) for fluorinated aryl nitrenes compared to their nonfluorinated analog is higher by about 5-6 kcal/mol.130 This higher barrier should increase the lifetimes of the fluorinated singlet
131,118 nitrenes (τFAr = 200-250 ns vs τAr = 0.1- 1 ns) This increase of the singlet lifetimes leads to 16
the reaction of fluorinated nitrenes with pyridine, secondary amines and hydrocarbons
(Scheme 1.9).131-137
Due to this photochemical behavior of fluorinated aryl nitrenes, described above,
modified fluorosubstitued aryl azides are widely-applied as PAL agents of biochemical systems.
The reported products of cross-linking correspond to adducts of nucleophile to dehydroazepines
and to products of direct nitrene insertion.
Photochemical Generation of Nitrenium Ions (NI) from Aryl Azides. One of the key
intermediates, which are highly-effective PAL agents, are nitrenium ions (NI). Nitrenium ions
(NI) were first introduced by Abramovitch about 50 years ago138 and are highly-reactive species
which have a positively-charged nitrogen atom with six electrons (Fig. 1.6).
N N N R H R' R R' R'
Figure 1.6. Nitrenium ions.
Depending on the ligands, these intermediates are similar to nitrenes, carbenium ions,
and carbenes, and have singlet or triplet multiplicity in their ground state.140 Nitrenium ions can
be generated thermally, electrochemically, and photochemically.139 A review of nitrenium ions,
which are generated by the first two pathways, is beyond of the scope the current work.
Quantum chemical calculation predicted that singlet ground state of phenyl nitrenium
ions should be lower in energy by 21 kcal/mol than the triplet and should have a planar geometry
(Fig. 1.7).141,142 The positive charge on the nitrogen atom could be delocalized into the π-system of aromatic ring by the donation of an electron pair from aromatic ring to p-π nonbonding p 17
orbital on nitrogen. Thus, the incorporation of electron-donating groups (EDG) into aromatic
ring, especially into para- position to the nitrene, should stabilize the singlet nitrene species.
H H H N N N
EDG EDG
Figure 1.7. Singlet ground state phenyl nitrenium ion. Stabilization of positively charged
nitrenium nitrogen atom via conjugation with para- electron-donating group.
Aryl azides substituted by para- electron-donating groups serve as high-yield precursors
for photogeneration of nitrenium ions (NI) as intermediates in PAL.143 The development of laser
flash photolysis techniques makes these species particularly useful for mechanistic study of these
reactions. The first reported attempt to observe these highly reactive species by LFP technique
was undertaken by Baetzold and Tong.144
Scheme 1.10. Photolysis of p-(N,N-diethylamino)phenylazide.
1 N3 N NH NH hν H
Et2N Et2N Et2N Et2N 17
The nitrenium ion intermediate was derived from azide 17 (Scheme 1.10) in an aqueous
solution of acetic acid, had absorbtion at λmax = 325 nm and a lifetime τ > 100 ms. Nevertheless,
the structure of this intermediate remained uncertain for a quarter of century. 18
Scheme 1.11. The chemistry of nitrenium ions.
1 N3 N NH NH O
hν H H2O H2O ps -N2 μs min EDG EDG EDG HO EDG HO EDG 18 19 20
Further development of LFP techniques allowed the direct detection these NI intermediates, involved in this reaction (Scheme 1.11).145-149 The singlet nitrene, derived from
aryl azide photodecomposition, is protonated by solvent to form aryl nitrenium ion 18 within the
ps time domain.147-149 The following attack of water on aryl nitrenium ion 18 leads to the
formation of ketimine 19 with rates constants corresponding to lifetimes of up hundreds of
microseconds.145 The further hydrolysis of ketimine 19 to enone 20 requires minutes. The same
mechanism was proposed for 4-azido-9-oxy-7H-furo[3,2-g]chromen-7-one (21) and its 9-alkoxy
derivatives (Scheme 1.12)150
Scheme 1.12. Photodecomposition of 21.
N3 NH NH
hν H2O
-N2 O O O O O O O O O OR OR O R = OMe, OEt, and H for 21
The addition of nucleophile to photogenerated NI can take place in ortho- positions as
well. Thus, it was recently reported that formation of adducts 22 and 23 occurred with different 19
nucleophiles such as secondary amines and alcohols during photolysis of 4-azido-N,N-diethyl-2-
nitroaniline (Scheme 1.13).19,151
Scheme 1.13. Nucleophilic attack on nitrenium ion in ortho- positions to the nitrenium ion.
N3 NH NH2 NH2 Nuc Nuc hν Nuc
NO2 -N2 NO2 NO2 NO2 NEt2 NEt2 NEt2 NEt2 22 23 minor major
Thus, the study of aryl azides, which form NI, as potential photoaffinity labeling (PAL) agents requires further development. The investigation of new potential PAL agents, which are devoid of some existing disadvantages, is one of the main objectives in the research described here. These new PAL agents, such 4-azido-N,N-diethyl-2-nitroaniline, should form stable covalent bond between the photoaffinity label and targeted molecule, which should survive during the further macromolecule disassembling and analysis. In addition, these PAL agents should not have the relatively slow rate of formation of cross-linking bond between targeted molecule and NI, because it might produce the incorrect information about sites of specific interactions of macromolecules which does not reflect the unmodified binding.
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1607. 29
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Vyas, S.; Winter, A. H.; Hadad, C. M. J. Am. Chem. Soc. 2009, 131, 11535–11547. 30
CHAPTER II. EXPERIMENTAL METHODS
2.1. Liquid Chromatography
Analytical TLC was performed using E.Merck aluminum-backed sheets of silica gel 60
F254. Preparative TLC was performed on Analtech 20×20 cm glass-backed 2 mm thick silica gel
60 F254 plates. HPLC analyses and separations were performed on Rainin HPLC system coupled with Agilent 8453 UV-Vis spectrometer. For analytical purposes a Waters C18
Spherisorb ODS1 column (4.6×250 mm) with flow rate 0.8 mL/min was used. Preparative separations were performed on a Dynamax C18 preparative column (60 Å, 21.2×250 mm) with flow rate 4 mL/min. Elution involved acetonitrile-water and methanol-water mixtures; concentration of water varied in the range 10-85%.1-3
2.2. UV-VIS Absorption Spectroscopy
UV-VIS absorbance spectra were recorded at 22 °C on an Agilent 8453 UV-Vis diode array spectrometer, using 10 mm or 1 mm path-length quartz cuvettes.3
2.3. IR Spectroscopy
IR spectra were recorded at 22 °C on Thermo 100 FTIR spectrometer attached diamond
IR200 spectrometer.3 31
2.4. Mass Spectrometry
ESI Q-TOF HRMS and FT-ICR HMRS spectra were recorded by Larry Sallens at the
mass spectrometry facility of University of Cincinnati.
MALDI spectra were obtained using Omniflex Bruker Daltonics MALDI Mass
Spectrometer.1,3
2.5. NMR Spectroscopy
One-dimensional 1H and 13C spectra were acquired on Bruker AM300 and 500
spectrometers at 23 °C. All chemical shifts are reported in ppm downfield (δ) referenced to
internal TMS. 13C spectra were acquired with broadband 1H decoupling. Abbreviations for
splitting patterns are as follows: br, broad; s, singlet; d, doublet, t, triplet; m, multiplet; etc.1-3
2.6. Femtosecond UV-VIS Time Resolved Absorption Spectroscopy
The femtosecond spectrometer, which was used in this work (Figure 2.6.1), is based on a Ti: Sapphire regenerative amplifier (Hurricane, Spectra Physics). It produces 800 nm pulses with a 1 kHz repetition rate. The energy per pulse is about 900 μJ, and a pulse width is 90 fs. The beam splitter divides the amplified beam into two beams with equal intensities. One of them is sent to the TOPAS-C (Light Conversion Lt.) optical parametrical amplifier, which generates 300 nm excitation pulses that go through the sample. The energy of excitation beam was 3 μJ per pulse at the sample position. 32
Figure 2.1. Scheme of femtosecond setup, used in experiments.a
a. The picture was provided by Dr. Tarnovsky and his group.
33
The second beam is sent to the optical delay line, and then focused onto the 3 mm thick
CaF2 window in order to produce white light continuum. After CaF2 window, this beam divides into two beams: reference and probe. Only the probe beam passes through the sample and after that along with the reference beam go to the monochromator/spectrograph (Spectra-Pro 2358,
Acton Research), and then register by the 512-pixel dual-diode array detector. The angle between the probe and pump beams is 8°. Polarization planes of the pump and probe beams were set at magic angle using a Berek compensator. The sample was measured in 0.2 mm thick flow cell. All measurements were performed at the temperature 22° C.
Femtosecond transient absorption spectra for azides 24, 30, and 39 were obtained by
Maxim Panov, Kanykey Karabaeva, and Andrew Mereshenko using the instrumentation available in the Dr. Tarnovsky’s facilities. Spectrometer information and scheme also was provided by Dr. Tarnovsky’s group.3
2.7. Nanosecond UV-VIS Time Resolved Absorption Spectroscopy
Nanosecond transient absorption spectra were collected on a Proteus Nanosecond
Transient Absorption spectrometer (Ultrafast Systems) equipped with a 150 W Xe arc lamp
(Newport), a Chromex monochromator (Bruker Optics) equipped with two diffraction gratings blazed for visible and near-IR dispersion, respectively, and Si photodiode detector (DET 10A,
Thorlabs) optically coupled to the exit slit of the monochromator. Excitation at 355 nm with a power of 5.8-6.0 mJ per pulse from a computer-controlled Nd:YAG laser from Opotek (Vibrant
LD 355 II) operating at 10 Hz was directed to the flow-cell with sample with an optical absorbance of studied azides 0.4-0.5 at the excitation wavelength. The analyzed data consist of
128 or 256 average shots. 34
Nanosecond transient absorption spectra for azides 24, 30, and 39 were recorded using
the instrumentation available in the Dr. Castellano’s facilities.
Spectrometer information was provided by Dr. Castellano’s group and
Dr. V. Voskresenska.4
2.8. Quantum Chemistry Calculations
All of the calculations are performed using Gaussian 20091 and MOLCAS 7.46 software at the Ohio Supercomputer Center. The model compounds and intermediates were optimized using Becke’s three-parameter hybrid exchange functional with Lee-Yang-Parr correlation functional (B3LYP)7 and Møller Plesset Perturbation Theory (MP2)8 methodologies with
6-31G(d,p) basis set. Vertical excitations of all species are obtained at optimized geometries
using time-dependent density functional theory (TD-DFT),9 Møller Plesset Perturbation Theory
(MP2)7 with cc-pVDZ basis set,10 and at the Complete Active Space Self-Consisted Field
(CASSCF)11 methodology with a scalar relativistic atomic natural orbital (ANO-RCC)12 basis set based on the use of Douglas-Kroll-Hess Hamiltonian and the subsequent second order perturbation theory (CASSP2)13 to take into account the dynamic electronic correlations.
CASSP2 calculations performed with MOLCAS 7.4 software were provided by
Dr. M. S. Panov.
2.9. References
1. Komarov, D. Y.; Nilov, D. I.; Wilson, R. M. “Oxidation of Adenosine and Inosine: The
Chemistry of Purine Iminoquinones and Quinones”, manuscript in preparation. 35
2. Komarov, D. Y. PhD Thesis, Bowling Green State University (USA), 2010.
3. Nilov, D.; Panov, M.; Karabaeva, K.; Mereshenko, A.; Wilson, R. M.;
Tarnovsky, A. N. “Iminoquinones of 8-Aminoadenosine, 8-Aminoinosine, and 8-Oxoadenosine
Generated from 8-Azidoadenosine, 8-Azidoinosine, and 6-Azido-8-oxopurine.” Manuscript in
preparation.
4. Voskresenska, V. PhD Thesis, Bowling Green State University (USA), 2010.
5. Gaussian 09, Revision A.1, Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.;
Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.;
Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.;
Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, Jr., J. A.; Peralta, J. E.;
Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi,
R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi,
M.; Rega, N.; Millam, N. J.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.;
Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.;
Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.;
Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö.; Foresman, J. B.; Ortiz, J. V.;
Cioslowski, J.; Fox, D. J. Gaussian, Inc., Wallingford CT, 2009.
6. MOLCAS 7.4: Aquilante, F.; De Vico, L.; Ferré, N.; Ghigo, G.; Malmqvist, P.;
Neogrády, P.; Pedersen, T. B.; Pitoňák, M.; Reiher, M.; Roos, B. O.; Serrano-Andrés, L.; Urban,
M.; Veryazov, V.; Lindh, R. J. of Comp. Chem. 2010, 31, 224–247.
7. Hehre, W. J.; Radom, L.; Schleyer, P. v. R.; Pople, J. A. Ab Initio Molecular Orbital
Theory; Wiley: New York, 1986. 36
8. Møller, C.; Plesset, M. S. Phys. Rev. 1934, 46, 618-622.
9. Olivucci, M. In Computational Photochemistry; Elsevier: Amsterdam, 2005, p 92-128.
10. Dunning, Jr, T. H. J. Chem. Phys. 1989, 90, 1007-1023.
11. Roos, B. O.; Taylor, P. R. Chem. Phys. 1980, 48, 157-173.
12. Roos, B. O.; Lindh, R.; Malmqvist, P. A.; Veryazov, V.; Widmark, P. O. Chem. Phys.
Lett. 2005, 409, 295-299.
13. Andersson, K.; Malmqvist, P. A.; Roos, B. O. J. Chem. Phys. 1992, 96, 1218-1226. 37
CHAPTER III. PHOTOCHEMISTRY OF 8-AZIDOADENOSINE AND
8-AZIDOINOSINE
3.1. Introduction
At this time, 8-azidoadenosine (24) plays an important role in the study of biological
interactions. This compound was proposed as a PAL agent about 30 years ago,1-4 and is widely- used for investigation of interaction in adenosine-contained cofactors-enzyme, NA-NA, and NA- protein systems.4-10 According to several works, the suggested mechanism for PAL with 8- azidoadenosine involves nitrenes as short-lived highly-reactive species which react directly with appropriate nearby targets.11-15 However, this type mechanism was revised in more recent work
and the participation of diiminoquinoidal intermediate 25 and iminonitrile 26 were proposed for
PAL process with the 8-azidoadenoisine (Scheme 3.1).9
Scheme 3.1. The formation of diiminoquinone 24 and iminonitrile 26 in photodecomposition
of 8-azidoadenosine.
NH2 NH2 NH N N N2 N N ROH N N N3 N HN N N N N N N R 24R R 25
NH2 N N N N N R 26 38
The irradiation of 8-azidoadenosine (24) by UV-light (c.a. 360 nm) in methanol leads to
the three major products: 8-aminoadenosine (a reduction product), 8-amino-2-methoxyadenoisne
(27, an addition product and its 6-N-formamide), and a rearrangement product 2,8-diamino-6- methoxypurinoriboside 28 (Scheme 3.2). The time-resolved UV-vis and IR spectroscopy was used to observe reactive intermediates. The intermediate, which had λmax = 460 nm was assigned
to diiminoquinone 25 based on relative lower energy absorbtion according to quantum chemical
calculations.9
Scheme 3.2. Photolysis of 8-azidoadenosine in methanol.
NH2 NH2 NHX OMe N N hν N N N N N N N3 H2N ++H2N H2N N N N N N N N OMe N NH2 R R R R 24 27 28 HO O R = X = H or CHO OH OH
Scheme 3.3. Opening of 5-membered ring. O
N NH hν N N 3 3 N N X -N X N 2 R 29 30 X = O, S, or NMe Figure 3.1. 8-Azidoinosine (30).
The possibility for the opening of five-membered ring in a purine and imidazole was
described in a number of works. It was reported, that nitrenes, derived from 2-azido- thiophenes,
pyrroles, and furans might undergo rearrangements after the formation of 29 upon photolysis 39
(Scheme 3.3) 16-20 Thus, either diiminoquinone 25 or iminonitrile 26 are possible intermediates in
the photodecomposition of 8-azidoadenosine (24).
In spite of the fact, that biological systems are studied with PAL in aqueous media, the
photochemistry of 8-azidoadenosine has not been studied mechanistically in water. The
investigation of photochemistry of aqueous solutions of 8-azidoadenosine might play an
important role in the development of PAL tools for biochemical studies.
Another PAL agent, based on purine bases, is 8-azidoinosine (30, Fig. 3.1).1 This
compound is not as widely-used as 8-azidoadenosine (24), since inosine occurs in biochemical
systems significantly less frequently than adenosine, and it seems that the photochemistry of 8-
azidoinosine (30) has not been studied to date.1,22-26
3.2. Photochemical Studies of 8-Azidoadenosine (24)
Scheme 3.4. Azido-tetrazole isomerization for 8-azidopurines.
NH NH O O 2 N 2 N N N N N N N N NH N NH N3 N N3 N N N N N N N N N R R R R 24 30
It must be noted at the outset that all of the 8-azidopurines 24 and 30 studied in this
work might undergo azide-tetrazole isomerization such as shown in Scheme 3.4.25,26 Therefore,
even though both isomers are photochemically reactive, for the molecules studied in this work,
the IR-spectra were obtained for 24, 24Ac5, 24Bz5, and 30, 30 Ac3 and in all cases showed a
substantial azide absorption bands at ca 2150 cm-1 (Fig. 3.2) 40
100 24
24Ac5 30 30Ac 80 3
60 Transmittance, %
40
350030002500200015001000-1 ν, cm
Figure 3.2. IR spectra of 8-azidoadenosine (24), 24Ac5, 30, and 30Ac3.
Photodecomposition of azide 24 has been studied previously by femtosecond transient spectroscopy.9 Transient absorption spectra of the photolysis of this azide in aqueous solution are
shown in Figure 3.3, and were found to be strongly pH dependent in the present work. At pH =
2.6, a band centered at λmax = 390 nm develops within τ ca. 500 fs, and remains for at least 1 ns.
In addition, absorption in the region 450-625 nm decays over about the same τ ca. 300 fs (Fig.
3.3A). In contrast, at pH = 9.2, the formation of the band centered at λmax = 390 nm was not
observed in aqueous solution of azide 24. Instead, a new band centered at λmax = 465 nm formed
with τ = 1.5 ps, and remained for at least 1 ns (Fig. 3.3C). Absorption in the 550-625 nm region
undergoes correlated decay with τ = 1.5 ps. Transient absorption spectra of azide 24, recorded at pH = 3.7, has a band centered at λmax = 440 nm (Fig. 3.3B). This band is broader compared with
the bands at pH = 2.6 and 9.2 and seems to be a combination of the 390 and 465 nm bands. 41
0.0030 0.0030 t20ps t1ns 0.0025 0.0025 A t10ps t800ps pH = 2.6 0.0020 0.0020 t1ps t500ps t0.5ps 0.0015 t100ps
0.0015 t0.3ps t200ps A 0.0010 Δ t50ps 0.0010 0.0005 0.0005 0.0000 350 400 450 500 550 600 650 700 350 400 450 500 550 600 650 700 0.0016 0.0016 t20ps t 1ns B t800ps pH = 3.7 t10ps 0.0012 0.0012 t1ps t500ps
A t0.5ps t200ps
Δ 0.0008 0.0008 t0.3ps t100ps t50ps 0.0004 0.0004 350 400 450 500 550 600 650 700 350 400 450 500 550 600 650 700 0.0025 0.0025 C t20ps t 1ns 0.0020 t10ps 0.0020 t800ps pH = 9.2 t1ps 0.0015 0.0015 t500ps t0.5ps
t200ps A
Δ 0.0010 0.0010 t0.3ps t100ps 0.0005 t50ps 0.0005 0.0000 350 400 450 500 550 600 650 700 350 400 450 500 550 600 650 700 Wavelength, nm Wavelength, nm
Figure 3.3. Femtosecond and picosecond transient spectra of azide 24 in water solution at pH =
2.6 (A), 3.7 (B), and 9.2 (C)
Clearly two quite different intermediates are being generated from azide 24 at different pH’s (Scheme 3.5). Under acidic conditions, the 6-amino group of 24 will be protonated, 24-H in
Scheme 3.5, and the nitrene, 31-H, will not be stabilized by the powerful electron-donating effect of this amino group. Under these conditions rapid fragmentation of the 5-membered ring might occur.9,16-19 In contrast, under basic conditions the nitrene will be stabilized by the electron-
donating effect of this amino group and becomes a powerful base, 32, which will lead to proton
reallocation to form 25 within about τ = 1.5 ps. Calculated absorption spectra support these
assignments (Table 3.1).9 42
Scheme 3.5. The formation of diiminoquinone 25 and iminonitrile 26.
NH 3 NH3 NH3 N hν N N N N N N3 N C N -N N N 2 N N N N R 24-H R 31-H R 26-H
NH 2 NH2 NH2 NH N N ~H N N hν N N N N N3 N N HN N -N2 N N N N N N N R R 24 R 31 R 31 25
Scheme 3.6. Nucleophilic attack on diiminoquinone 25 and iminonitrile 26.
NH2 NH2 NH2 H N N N N N N C C HN N H N N N N N N N Nuc H Nuc R 26 R R 32
H NH NH NH 2 N N N N N H N HN HN 2 N N Nuc N N N N 25 Nuc R R R 33
Thus, the ring-cleaved singlet iminonitrile 26-H (Scheme 3.5) is calculated
(Table 3.1) to have a major absorption band at 389 nm and this species might easily form within the 500 fs that are required for the full development of this band, Fig. 3.3A. In contrast, the diiminoquinone 25, is calculated to display absorption in the 563-430 nm region.9 The proton
reallocation necessary in transforming 31 to 25 in Scheme 3.5 will require somewhat longer 43 times than the aforementioned fragmentation of the five-membered ring, which is consistent with the 1.5 ps time required for formation of the 465 nm band.
Since there are apparently two alternative nitrene decay pathways, one via cleavage of the 5-membered ring and the other via diiminoquinone formation, there must be convergent product forming pathways, since the same products are formed from both types of intermediates.
Two such alternative pathways for nucleophile attack are outlined in Scheme 3.6. This same dichotomy has been discussed in detail previously,9 and little new can be added to that discussion except to note that these two pathways are regulated by the pH of the solution and other factors that alter the availability of electron density on the nitrene nitrogen.
Table 3.1. Calculated vertical excitations for intermediates 25Me, 26Me-H, and 31Me in
methanol.
Compound State λmax, nm Oscillator Strength Method
NH S2 498 0.0009 B3LYP/6-31G(d) N N HN S3 444 0.1756 B3LYP/6-31G(d) N N H3C S1 437 0.262542 CASPT2/cc-vPDZ 25Me
NH3 S2 396 0.0022 B3LYP/6-31G(d) N N N S3 389 0.1887 B3LYP/6-31G(d) N N H3C S6 280.72 0.1495 B3LYP/6-31G(d) 26Me-H
NH2 S5 416 0.106039 CASPT2/cc-vPDZ N N N S3 420.45 0.1355 B3LYP/6-31G(d) N N H C 3 31Me 44
O O O O
Ph N Ph H3C N CH3 N N N N N3 N3 Ph O N N H3C O N N O O O O
Ph O O Ph H3C O O CH3
O 24Bz O O 24Ac O 5 5
Figure 3.4. Acylated derivatives of azide 24.
Acylation of this 6-amino group has a similar effect to its protonation, and leads to
5-membered ring fragmentation in both 24Bz5 and 24Ac5, Figure 3.4. Thus, irradiation of 24Bz5 affords a band centered at λmax = 365 nm that decays into a band centered at λmax = 405 within τ =
3 ps (Fig. 3.5A). On the other hand, irradiation of 24Ac5 affords a band centered λmax = 375 nm
that undergoes little of no further change (Fig. 3.5B). These bands centered at λmax = 365 and 375
nm are assigned to corresponding nitrenes (31Bz5 and 31Ac5) and the mature bands in the 375-
405 region to the corresponding fragmented iminonitriles (26Bz5 and 26Ac5). Apparently even
acyl groups on the 6-amino group are sufficiently electron withdrawing to favor the
fragmentation pathway. 45
24Bz (1 mM), DCM 24Ac (1 mM), Methylcyclohexane A 5 B 5 t0.2ps 0.015 t0 ps 0.015 t0.2ps t0.3ps 0.010 t0.3ps 0.010 t0.5ps t0.8ps t0.5ps A A Δ Δ 0.005 t0.8ps 0.005 t1ps t1ps 0.000 0.000
350 400 450 500 550 600 650 700 350 400 450 500 550 600 650 700 0.015 0.015 t1ps t1ps t2ps t2ps 0.010 0.010 t3ps t3ps t6ps t6ps A A Δ Δ 0.005 t10ps 0.005 t10ps t20ps t20ps 0.000 0.000 350 400 450 500 550 600 650 700 350 400 450 500 550 600 650 700 0.015 0.015 t50ps t50ps t100ps t100ps 0.010 0.010 t200ps t200ps A A Δ Δ t500ps t500ps 0.005 t800ps 0.005 t800ps t1ns t1ns 0.000 0.000 350 400 450 500 550 600 650 700 350 400 450 500 550 600 650 700 Wavelength, nm Wavelength, nm
Figure 3.5. Femtosecond and picosecond transient spectra of azide 24 in nonprotic solvents:
24Bz5 (A) in dichloromethane and 24Ac5 in methylcyclohexane (B).
In recent work with electron-rich nitrenes, it has been observed that the rate of nitrene protonation was related to the structure of the proton source. Thus, protonation by water and methanol is ca. 4 times faster than protonation with 2-propanol.27 This same general trend is
observed with azide 24, Figure 3.6. Thus, in methanol, nitrene 31 (λmax = 460 nm) undergoes proton reallocation with τ = 1.5 ps, about the same rate as in water, but in 2-propanol a significantly longer reallocation time is observed, τ = 2.5 ps (Fig. 3). 46
A Azide 24 (1 mM), MeOH B Azide 24 (0.75 mM), 2-PrOH 0.005 0.005 t0.3ps t0.3ps 0.004 t0.5ps 0.004 t0.5ps t0.8ps 0.003 t0.8ps 0.003 t1ps A A t1ps Δ Δ 0.002 0.002 0.001 0.001
0.000 0.000 350 400 450 500 550 600 650 700 350 400 450 500 550 600 650 700 0.006 0.005 t1ps t1ps 0.005 0.004 t2ps t2ps 0.004 t3ps 0.003 t3ps 0.003 t6ps t6ps A A λ = 460 nm λ = 455 nm max1 Δ 0.002 max1 Δ t10ps t10ps 0.002 λ = 470 nm λ = 475 nm max2 max1 t20ps 0.001 t20ps 0.001 0.000 0.000 350 400 450 500 550 600 650 700 350 400 450 500 550 600 650 700 0.006 0.005 t50ps t50ps 0.005 0.004 t100ps t100ps 0.004 t200ps 0.003 t200ps A A
0.003 Δ t500ps Δ t500ps 0.002 0.002 t800ps t800ps 0.001 t1ns 0.001 t1ns 0.000 0.000 350 400 450 500 550 600 650 700 350 400 450 500 550 600 650 700 Wavelength, nm Wavelength, nm
Figure 3.6. Femtosecond and picosecond transient spectroscopy of azide 24
in methanol (A) and 2-propanol (B).
The reactions of iminoquinone 25 with various nucleophiles were studied by nanosecond transient absorption spectroscopy. Thus, azide 24 was irradiated in water at pH = 3.1, 6.5, and 11.5 (Fig. 3.7), and 24 in 0.1 M aqueous solutions of imidazole
(Fig. 3.8A), a model compound for hystidine, and in 0.1 M sodium phenoxide (Fig. 3.8B), a model compound for tyrosine. According to these spectra, quenching of iminoquinone 25 under neutral conditions occur in 100 µs (τ1), further aromatization of 32-OH is a relatively slow process (Fig. 3.7B, pH = 6.5) with τ2 > 35 ms. However, the aromatization is accelerated under 47
both acidic and basic conditions to τ2 c.a. 20 ms at pH = 3.1 and τ2 = 4.2 ms at pH 11.5. The reaction of 1 with 0.1 M aqueous sodium phenoxide takes place with τ = 3 ms and more rapidly with imidazole, τ2 = 900 µs. These nucleophilic substitution reactions all proceed through intermediates similar to 32 in Schemes 3.6 and 3.7.
A 32 μs pH = 3.1 32 μs B pH = 6.5 0.006 104 μs τ = 20 ms 250 μs 0.010 284 μs 0.005 1 ms 1 ms 5 ms 0.008 0.004 10 ms 2 ms 20 ms 0.006 5 ms A 0.003 A Δ
35 ms Δ 10 ms 0.002 0.004 20 ms 35 ms 0.001 0.002 τ μ 1 c.a. 100 s τ 2 > 35 ms 0.000 0.000 400 450 500 550 600 400 450 500 550 600 Wavelength, nm Wavelength, nm
C pH = 11.5 0.012 τ = 4.2 ms 0.010 32 μs 248 μs 0.008 1 ms 3 ms
A 0.006
Δ 5 ms 0.004 10 ms 20 ms 0.002 0.000 400 450 500 550 600 Wavelength, nm
Figure 3.7. Nanosecond transient spectra of azide 24 in water at pH = 3.1 (A), pH = 6.5 (B),
and pH =11.5 (C). 48
24 (1mM) and Imidazol (1M) in H O A 2 B 33 and PhONa in H2O 0.008 32 μs μ τ = 3 ms τ = 900 μs 21 s 2 500 μs 0.006 2 35 μs 1 ms 100 μs 0.006 2 ms 0.004 250 μs 3 ms 500 μs 5 ms ΔΑ A 0.004 1 ms Δ 10 ms 0.002 5 ms 0.002 0.000 0.000 400 450 500 550 600 400 450 500 550 600 Wavelength, nm Wavelength, nm
Figure 3.8. Nanosecond transient spectra of azide 24 in water in presence of
imidazole (1 M, A) and B: phenol (1 M, B).
Scheme 3.7. The formation of 8-amino-2-oxyadenosine (33-OH) in water.
NH NH2 NH2 N N N N H O N N HN 2 HN H N H 2 HO N N τ1 HO N N τ2 HO N N OH O O OH O
OH OH OH OH OH OH 25 32-OH 33-OH
λmax = 465 nm λmax = 405 nm
Preparative irradiation of azide 24 at 356 nm was carried out in water in the absence and
in the presence of imidazole (0.1 M). The analysis of these reaction mixtures indicated the
formation of reduction product, 8-aminoadenosine (34), dimerization products of undetermined
structure, purine-ring destruction product 35, water addition product 33-OH and, in presence of
imidazole, imidazole adduct 33-IM (Scheme 3.8). 49
Scheme 3.8. Preparative photolysis of 8-azidoadenosine (24).
NH2 NH2 NH2 hν = 356 nm N N N N N N N H N H N 3 H O 2 2 N N 2 N N N N OH R H R R 24N 34 33-OH HO N NH R = O 2 N NH2 OH OH N H N HN Dimerization 2 Products N N N NH R R N 33-IM 35
Scheme 3.9. The formation of iminoquinone 37 and iminonitrile 38.
O O O N N H NH NH N N N3 N N N -N N N 2 N N N R 30 R 36 R
~H XO O O R = O N N NH N OX OX C HN N N X = H N N N X = Ac for Ac R 3 R 38 37
It is interesting to compare the behavior of 8-azidoadenosine (24), with that of 8-
azidoinosine (30). The nitrene 36, generated from 30, is not conjugated to a powerful electron
donating group, and, therefore, might not undergo proton reallocation to form the iminoquinone
37, Scheme 3.9. Thus, irradiation of 30 in water, pH = 1.7-7.5, alcohols, or aprotic solvents, such
as dichloromethane, all lead to the rapid development of a 380 nm band characteristic of
fragmentation of the five-membered ring and formation of the iminonitrile 38 (Fig. 3.9). 50
A pH = 1.75 (HCl), Water, 1 mM B pH = 7.5, Water, (2 mMol) 0.004 0.004 t1ps t0.3ps 0.003 t0.8ps 0.003 t0.5ps t0.5ps t0.8ps 0.002 t0.3ps 0.002 A t1ps A Δ Δ 0.001 0.001 0.000 0.000 350 400 450 500 550 600 650 700 350 400 450 500 550 600 650 700 0.004 0.004
t20ps t1ps 0.003 t10ps 0.003 t2ps t6ps t3ps 0.002
A 0.002 t6ps
t3ps A Δ t2ps Δ t10ps 0.001 0.001 t1ps t20ps 0.000 0.000 350 400 450 500 550 600 650 700 0.004 350 400 450 500 550 600 650 700 0.004 t1ns t50ps 0.003 t800ps 0.003 t100ps t500ps A 0.002 t200ps A Δ t200ps Δ 0.002 t500ps 0.001 t100ps t800ps 0.001 t50ps t1ns 0.000 350 400 450 500 550 600 650 700 0.000 350 400 450 500 550 600 650 700 Wavelength, nm Wavelength, nm
C MeOH D DCM 0.009 0.004 t0.3ps t0ps 0.003 t0.2ps 0.006 t0.5ps t0.8ps t0.3ps 0.002 A t0.5ps t1ps A Δ 0.003 Δ t0.8ps 0.001 t1ps 0.000 0.000 350 400 450 500 550 600 650 700 350 400 450 500 550 600 650 700 0.009 0.004 t1ps t1ps 0.003 t2ps 0.006 t2ps t3ps t3ps 0.002
A t6ps A Δ
t6ps Δ 0.003 t10ps t10ps 0.001 t20ps 0.000 0.000
350 400 450 500 550 600 650 700 350 400 450 500 550 600 650 700 0.009 0.004 t50ps t50ps t100ps 0.003 0.006 t100ps t200ps t200ps A A Δ t500ps Δ 0.002 t500ps 0.003 t800ps t800ps 0.001 t1ns t1ns 0.000 0.000 350 400 450 500 550 600 650 700 350 400 450 500 550 600 650 700 Wavelength, nm Wavelength, nm
Figure 3.9. Femtosecond and picosecond transients of azide 30: in water at pH = 1.75 (A), in
water at pH = 7.5 (B), in methanol (C), and 30Ac3 in dichloromethane (D). 51
Scheme 3.10. Nucleophilic attack on iminonitrile 38.
O O O τ XO HH N τ N 2 N NH 1 NH NH R = O C HN H2N N H N N N N N N Nuc OX OX Nuc Nuc R R R X = H 38 X = Ac for Ac3
0.0225 0.015 30Ac in MeOH, Imidazole (1 M) 30Ac , in MeOH 3 3 A 32 μs B 2.5 μs 250 μs 10 μs 0.0150 1 ms 0.010 20 μs 2.5 ms 50 μs A A 75 μs
Δ 5 ms Δ 10 ms 150 μs 0.0075 0.005 20 ms 350 μs 35 ms
0.0000 0.000 400 450 500 550 600 400 450 500 550 600 Wavelength, nm Wavelength, nm
Figure 3.10. Nanosecond -millisecond transient spectra of azide 30Ac3 in methanol (A) and in 1
M aqueous solution of imidazole (B).
Since no proton reallocation patterns could be observed under any conditions examined, it would seem that irradiation of 30 affords exclusively fragmentation of the five-membered ring to form 38. It is worthy of note that during optimization of DFT and MP2 calculations of nitrene
37 the C8-N9 bond always broke to form the iminonitrile isomer 38. The most informative of
these transient experiments was that observed in water at pH = 1.75 and pH = 7.5 (Fig. 3.8A and 52
3.8B). The very broad absorption at 450-600 nm is assigned to the nitrene 37 that decays with
the same time constant as the growth of the iminonitrile 38 band at 380 nm (calc. λmax = 384 nm),
τ = 2.5 ps.
Table 3.2. Time constants τ1 and τ2 for the reactions of reaction of purine intermediates 25, 26-
H, and 38 under various conditions.a
Structure Conditions Time Constants
NH Aq. pH = 6.5 c.a 100 µs, τ1;25 ms, τ2 N N HN Aq. pH =11.5 4.2 ms, τ2 N N 25 PhONa 3 ms, τ2 H3C Imidazole 0.9 ms, τ2
Aq. pH = 3.1 25 ms, τ2 NH3 N N N N N 26-H CH3
O MeOH 15 ms, τ2 N NH N Imidazole 75 μs, τ2 N N 38 CH3
a The study of photochemistry of intermediates 25Me, 26Me-H, and 38Me in time domain
1ns -5 µs is very important. However, the observation in this time domain is limited by available
instrumentation.
This same pattern is observed for 30 and 30Ac3 in methanol (λmax = 380, τ = 5.5 ps,
Figure 3.9C) and dichloromethane (λmax = 378 nm, τ =3 ps, Figure 3.9D). Furthermore, the 53
iminonitrile 38Ac3 displays similar spectroscopic behavior in the μs-ms time domains as does the diiminoquinone 25 (Fig. 3.7 and 3.10). Thus, iminonitriles 26-H and 38Ac3 react with water and methanol with the expected relative rates τ = 20 ms and τ = 15 ms, respectively. Therefore, it seems that the iminonitriles are slightly better electrophiles than iminoquinones.
It is worthy of note that the absorption associated with the reaction of imidazole with the iminonitrile 38 is unusually broad and intense compared to other transient absorptions involving reactions with imidazole with iminoquinones (see Fig. 3.10B and 3.8A).
The extremely intense band signaling the reaction between imidazole and iminonitrile 38 would seem to be due to some sort of charge transfer absorption between the imidazole and the iminonitrile that precedes the nucleophilic attack by the imidazole.
The photodecomposition of 8-azidopurines may go through iminoquinone intermediates such as 25, and through iminonitrile intermediates, such as 26 and 38. The pathway followed depends upon the electron density on purine ring. Thus, the presence of EDG makes the formation of an iminoquinone more favorable; and in the absence of a powerful EDG, fragmentation of the 5-membered ring to form an iminonitrile becomes more favorable. 54
3.3. Synthesis and irradiation of 8-Azidoadenosine (24), 8-Azidoinosine (30),
and Their Derivatives
Synthesis of 8-Bromoadenosine.
NH2 NH2 N N Br2, AcONa N N Br HO N N H2O/AcOH HO N N O O
OH OH OH OH
The solution of 10.68 g (40 mmol) of (-)-adenosine in 50% aqueous acetic acid was added to the mixture of 400 mL water, 20 mL acetic acid, 34.4 g (0.4 mol) of anhydrous sodium acetate, and 2.44 mL (47.2 mmol) of bromine. The reaction mixture was stirred overnight during which time crystals formed. The excess bromine was removed by addition of 10% aqueous solution of sodium sulphite. The reaction mixture was basified to pH 7-8 with addition 10% aqueous solution of NaOH and chilled for 1 hour in ice. The resulting precipitate was collected by filtration, washed with water (2×200 mL), 100 mL of diethyl ether to yield 10.0 g (28.9 mmol, 72%) of 8-bromoadenosine. The product was used for the following reactions without further purification.1
1 H NMR (300 MHz, CDCl3) δ 8.12 (s, 1H), 7.60 (bs, 2H), 5.83 (d, J = 6.8 Hz, 1H), 5.47
(bs, 2H), 5.24 (bs, 1H), 4.19 (dd, J = 6 Hz, J’ = 6 Hz, 1H), 3.98 (dd, J = 2.1 Hz, J’ = 3.5 Hz, 1H),
3.68 (dd, J = 12.1, J’ = 3.9 Hz, 1H), 3.52 (dd, J = 12.0, J’ = 3.9 Hz, 1H). 55
Synthesis of 8-Azidoadenosine (24).2
NH2 NH2 N N N N Br NaN3 N3 HO N N HO N N O DMF O 24 OH OH OH OH
The suspension of 1.38 g (4 mmol) of 8-bromoadenosine and NaN3 (0.65 g, 10 mmol)
in 4 mL of anhydrous DMF was stirred for 48 h at 65°C under an argon atmosphere. The
reaction mixture was cooled room temperature and diluted with 50 mL of dichloromethane. The
resulting suspension was washed with CH2Cl2 (2×10 mL) and water (3×50 mL) to remove DMF and inorganic materials. The product 24 was isolated as tan crystals to yield 1 g (32.3 mmol,
81%).
1H NMR (500 MHz, DMSO) δ 8.09 (s, 1H), 7.35 (s, 2H), 5.65 (d, J = 6.7 Hz, 1H), 5.51
– 5.38 (m, 2H), 5.20 (d, J = 4.4 Hz, 1H), 4.91 (dd, J = 11.6, J’ = 5.9 Hz, 1H), 4.22 – 4.13 (m,
1H), 3.94 (dd, J = 6.4, J’ = 3.8 Hz, 1H), 3.67 (dt, J = 12.0, J’ = 3.9 Hz, 1H), 3.53 (ddd, J = 12.3,
J’ = 8.4, J’’ = 4.2 Hz, 1H).
Synthesis of 8-Azidoadenosine Pentaacetate (24Ac5).
O O
H3C N CH3 NH2 H3C O N N N N N3 N3 Ac2O O N N HO N N O O Pyridine
H3C O O CH3 OH OH O O 24 24Ac 5
A suspension of 1 g (3.25 mmol) of 8-azidoadenosine (24) in 10 mL of freshly
redistilled anhydrous pyridine was prepared at room temperature under an argon atmosphere. 56
Acetic anhydride, 3 g (29.4 mmol) was added slowly to a vigorously stirred suspension of 24 in
pyridine, and the reaction mixture was stirred overnight at 60 °C. Volatile components of
reaction mixture were removed under reduced pressure, and the residue redissolved in 25 mL of
CH2Cl2. Organic layer was washed with 5% aq. NaHCO3 (3×50 mL) and water (2×50 mL) and
dried with anhydrous MgSO4. The of solvent was removed under reduced pressure and the
residue separated by column chromatography on 20 g Merck 60 silica gel (CHCl3-EtOAc, 19-1)
to yield 1.15 g (2.41 mmol, 74%) of product 24Ac5 as a yellowish transparent glass.
1 H NMR (300 MHz, CDCl3) δ 8.85 (s, 1H), 7.28 (s, 1H), 6.23 – 6.14 (m, 1H), 6.07 (d, J
= 4.9 Hz, 1H), 5.82 (dd, J = 5.6 Hz, J’ = 5.6 Hz, 1H), 4.51 (dd, J = 11.4, J’ = 3.3 Hz, 1H), 4.37
(ddd, J = 17.3, J’ = 8.5, J’’ = 4.7 Hz, 2H), 2.38 (s, 6H), 2.17 (s, 3H), 2.12 (s, 3H), 2.09 (s, 3H).
9 Synthesis of 8-Azidoadenosine Pentabezoate (24Bz5).
O O
NH2 Ph N Ph N N N N N3 PhCOCl N3 HO N N Ph O N N O Pyridine O O OH OH Ph O O Ph
O O 24 24Bz5
A suspension of 1 g (3.25 mmol) of 8-azidoadenosine (24) in 10 mL of freshly
redistilled anhydrous pyridine was prepared at room temperature under an argon atmosphere.
Benzoyl chloride, 5 g (35 mmol) was added dropwise to a vigorously stirred suspension of 24 in
pyridine, and the reaction mixture was stirred overnight at 60 °C. Volatile components of
reaction mixture were removed under reduced pressure, and the rest was redissolved in 25 mL of
CH2Cl2. Organic layer was washed with 5% aq. NaHCO3 (3×100 mL) and water (2×50 mL) and
dried with anhydrous MgSO4. The rest of solvent was removed under reduced pressure and 57
separated by column chromatography on 25 g Merck 60 silica gel (CHCl3-EtOAc, 9-1) to yield
2.05 g (2.48 mmol, 76%) of product 24Bz5 as yellowish crystalline compound.
1 H NMR (300 MHz, CDCl3) δ 8.43 (s, 1H), 8.21 – 7.81 (m, 10H), 7.67 – 7.33 (m, 15H),
6.58 – 6.48 (m, 1H), 6.38 – 6.26 (m, 2H), 4.92 (dd, J = 12.0, J’ = 3.2 Hz, 1H), 4.77 (dd, J = 8.5,
J’ = 5.1 Hz, 1H), 4.67 (dd, J = 12.0, J’ = 4.7 Hz, 1H).
Synthesis of 8-Bromoinosine.28
NH2 O N N N NH Br NaNO2 Br HO N N HO N N O AcOH/H2O O
OH OH OH OH
A solution of 1 g (14.5 mmol) NaNO2 in 2 mL of water was added to a stirred solution
of 2 g (5.8 mmol) of 8-bromoadenosine in 100 mL of 95% aqueous acetic acid. The reaction
mixture was stirred overnight. The solvent was removed under reduced pressure and the oily
residue was recrystallized from 30 mL of 50% aqueous EtOH to yield 1.0 g (50%) of 8-
bromoinosine as colorless crystals.
1 H NMR (300 MHz, CDCl3) δ 12.60 (s, 1H), 8.11 (s, 1H), 5.81 (d, J = 6.4 Hz, 1H), 5.50
(s, 1H), 5.25 (s, 1H), 5.11 – 4.85 (m, 2H), 4.18 (s, 1H), 3.92 (dd, J = 8.0, J’ = 4.8 Hz, 1H), 3.64
(dd, J = 10.5, J’ = 5.7 Hz, 1H), 3.57 – 3.42 (m, 1H), 3.33 (s, 2H). 58
Synthesis of 8-Azidoinosine (30).28
O O
N NH N NH Br NaN3 N3 HO N N HO N N O DMF O 30 OH OH OH OH
The suspension of 1.38 g (4 mmol) of 8-bromoinosine and NaN3 (0.65 g, 10 mmol) in 4
mL of anhydrous DMF was stirred for 48 h at 65°C under an argon atmosphere. The reaction
mixture was cooled to room temperature and diluted with 50 mL of dichloromethane. The
resulting suspension was washed with CH2Cl2 (2×10 mL) and water (3×25 mL) to remove DMF and inorganic materials. The product 30 was isolated as tan crystals to yield 0.85 g (27.6 mmol,
68.9%)
1 H NMR (300 MHz, CDCl3) δ 8.01 (s, 1H), 5.63 (d, J = 6.4 Hz, 1H), 5.42 (bs, 1H), 4.82
(dd, J = 5.8 Hz, J’ = 5.8 Hz,1H), 4.14 (dd, J = 5.1, J’ = 3.1 Hz, 1H), 3.94 – 3.82 (m, 1H), 3.56
(ddd, J = 39.1, J’ = 11.9, J’’ = 4.7 Hz, 2H).
Synthesis of 8-Azidoinosine Triacetate (30Ac3).
O O H H H3C O N NH N NH N3 N3 Ac2O O N N HO N N O O Pyridine
H3C O O CH3 OH OH 30O 30Ac O 3
A suspension of 0.5 g (1.62 mmol) of 8-azidoinosine (30) in 5 mL of freshly redistilled
anhydrous pyridine was prepared at room temperature under an argon atmosphere. Acetic
anhydride, 1 g (9.8 mmol) was added slowly to a vigorously stirred suspension of 7, and the
reaction mixture was stirred overnight at 60 °C. Volatile components of reaction mixture were 59
removed under reduced pressure, and the residue redissolved in 15 mL of CH2Cl2. Organic layer was washed with 5% aq. NaHCO3 (3×25 mL) and water (2×25 mL) and dried with anhydrous
MgSO4. The of solvent was removed under reduced pressure and the residue separated by
column chromatography on 15 g Merck 60 silica gel (CHCl3-EtOAc, 19-1) to yield 0.5 g (1.15
mmol, 71%) of product 30Ac3 as a yellowish transparent glass.
1 H NMR (300 MHz, CDCl3) δ 13.45 (s, 1H), 8.13 (s, 1H), 7.28 (s, 1H), 6.11 – 6.03 (m,
1H), 5.98 (d, J = 4.8 Hz, 1H), 5.71 (t, J = 5.6 Hz, 1H), 5.31 (s, 1H), 4.48 (dd, J = 11.1, J’ = 2.9
Hz, 1H), 4.40 – 4.25 (m, 2H), 2.15 (s, 3H), 2.11 (s, 3H), 2.08 (s, 3H).
Preparative Irradiation of Azide 24 in 0.1 M Aqueous Solution ofIimidazole (See
Scheme 3.8 ). Azide 24 (300 mg, 0.97 mmol) was dissolved solution of imidazole (0.68 g, 10 mmol) in 100 mL of deaerated distilled water. The stirred solution was irradiated in a Rayonet
Photochemical Reactor at 356 nm for 3 hours. Reaction mixture was evaporated under reduced pressure to dryness. Excess of imidazole was sublimated under reduced pressure. The solid residue was prepared for further RP-HPLC analyses and separations on Rainin HPLC system coupled with Agilent 8453 UV-Vis spectrometer. For analytical purposes Waters C18 Spherisorb
ODS1 column (4.6×250mm) was used, whereas separations were performed on Dynamax C18 preparative column (60 Å, 21.2×250mm) C-18 reverse phase silicagel column (8×4×250mm) with system MeOH (30%) - H2O (70%).
+ (33-OH) HRMS calc. for C10H15N6O5 (M+H) m/Z = 299.11039, found 299.10996;
+ (33-IM) HRMS calc. for C13H17N8O4 (M+H) m/Z = 349.13673, found 349.13692;
+ (34) HRMS calc. for C10H15N6O4 (M+H) m/Z = 283.11493, found 283.11491;
+ (35) HRMS calc. for C6H14N3O4 m/Z = 192.09788, found 192.09788; 60
+ (dimer-like structures) HMRS calc. for C19H26N11O9 m/Z = 552.1909, found
5552.19101.
3.4. References
1. Cartwright I. L.,; Hutchinson D. W. Nucl. Acids Res. 1980, 7,1675-1691.
2. Marburg, S.; Jorn, D.; Tolman, R. L. J. Heterocyc. Chem. 1982, 19, 671-672.
3. Haley, B. E. Fed. Proc. 1983, 42, 2831-2836.
4. Potter, Robert L.; Haley, Boyd E. Meth. Enzymol. 1983, 91, (Enzyme Struct., Pt. I), 613-
633.
5. Meisenheimer, K. M., Koch, T. H. Crit. Rev. Biochem. Mol. Biol. 1999, 32, 101-140.
6. Cartwright, I. L., Hutchinson, D. W. Nucl. Acids Res. 1980, 8, 1675-1691.
7. Fleming, S. A. Tetrahedron 1995, 51, 12479-12520.
8. Bayley, H. Photogenerated Reagents in Biochemistry and Molecular Biology, Vol. 12,
Elsevier, New York. 1983.
9. Polshakov, D.; Rai, S.; Wilson, R. M.; Mack, E. T.; Vogel, M.; Krause, J. A.; Burdzinski,
G.; Platz, M. S. Biochem. 2005, 44, 11241-11253.
10. Hatanaka, Y., Nakayama, H., Kanaoka, Y. Rev. Heteroat. Chem. 1996, 14, 213-243.
11. Sylvers, L. A., Wower, J. Bioconjugate Chem. 1993, 4, 411-418.
12. Hanna, M. M., Bentsen, L., Lucido, M., Sapre, A. Meth. Mol. Biol. 1999, 118, 21-33.
13. Hanna, M. M. Meth. Enzymol. 1989,180, 383-409.
14. Karney, W. L., Borden, W. T. J. Am. Chem. Soc. 1997, 119, 1378-1387.
15. Gritsan, N. P., Zhu, Z., Hadad, C. M., Platz, M. S. J. Am. Chem. Soc. 1999, 121, 1202-
1207. 61
16. Gadosy, T. A., McClelland, R. A. J. Am. Chem. Soc. 1999, 121, 1459-1465.
17. Dehaen, W., Becher, J. Acta Chem. Scand. 1993, 47, 244-254.
18. Funicello, M., Spagnolo, P., Zanirato, P. Acta Chem. Scand. 1993, 47, 231-243.
19. Spinelli, D., and Zanirato, P. J. Chem. Soc., Perkin Trans. 2 1993, 1129-1133.
20. Sylvers, L. A., Wower, J. Bioconjugate Chem. 1993, 4, 411-418.
21. Walter, U. Eur. J. Biochem., 1981, 118, 339-346.
22. Schlichter, D. J.; Detre, J. A.; Aswad, D. W.; Chehrazi, B.; Greengard, P. Proc. Nat. Ac.
Sc. USA 1980, 77, 5537-41.
23. Casnellie, J. E.; Schlichter, D. J.; Walter, U.; Greengard, P. J. Biol. Chem. 1978, 253,
4771-4776.
24. Eccleston J. F. Biochem. 1981, 20, 6265-6272.
25. Claramunt, R. M.; Elguero, J.; Fruchier, A.; Nye, Martin J. Afinidad 1977, 34, 545-551.
26. Evans, R. A.; Wong, M. W.; Wentrup, C. J. Am. Chem. Soc. 1996, 118, 4009-4017.
27. Voskresenska, V.; Wilson, R. M.; Panov, M.; Tarnovsky, A. N.; Krause, J. A.; Vyas, S.;
Winter, A. H.; Hadad, C. M. J. Am. Chem. Soc. 2009, 131, 11547-11535.
28. Holmes, R. E.; Robinson, R. K. J. Am. Chem. Soc. 1964, 86, 1242-1245. 62
CHAPTER IV. THE PHOTOCHEMISTRY OF
6-AZIDO-8-OXO-7,9-DIHYDROPURINE (39)
4.1. Introduction
As mentioned above, the oxidation of 8-oxoA (2) probably involves the formation of iminoquinone 5 (See part 1.2). However, the oxidative generation of 5 and further kinetic of this species are difficult to study spectroscopically in vivo. An alternative mechanism for generation
5 is proposed in Scheme 4.1. Iminoquinone 5 might be generated photochemically via photodecomposition of azide 39. This compound can act as synthetic precursor for photogeneration of the intermediate 5, which might occur via normal oxidative damage to nucleic acids.
Scheme 4.1. Chemical and photochemical pathways of generation of iminoquinone 5.
NH2 N3 H NH H N N N N O N N O HO N O HO N N O hν N O HO N N O O -N2 OH OH 2 OH OH 39 OH OH 5 OXIDATION PHOTODECOMPOSITION Nuc NH NH2 N H N N N O O HO N N Nuc O HO N Ring Destruction O N Nuc Cleavage O OH OH OH OH Susceptible to further oxidation. 63
The chemistry of 2-azidopyridylnitrene (40) and its polycyclic analog 41, made from
3-azidoisoqunoline (42), has been studied by Wentrup (Scheme 4.2.and 4.3).5-7 Flash vacuum
thermolyses (FVT) have been applied to 2-azidopyridine (43), and formation of reactive
intermediates has been monitored in Ar matrices by ESR and IR spectroscopy. The nitrene 40
forms reduction product (2-aminopyridine), rearrangement intermediates ketimine 44, pyrroles
45, 46, and ring-opening intermediate 47 (Scheme 4.2).7 The photolysis of 3-azidoisoquinoline
(42) leads to the formation of nitrene 41, which is in equilibrium with azirene 48 (Scheme 4.3).6
The formation of the azirene 49 similar to 48 has been reported for the thermolysis of 4-azido-2- phenylquinazoline (50, Scheme 4.4).9 To date, it seems that photochemistry of 6-azidopurines
such as 39 has not been reported.
Scheme 4.2. Thermolysis of 2-azidopyridine (43).
CN N N N N CN N N N N 44 H H 45 46
N 3 Δ N N N N -N2 N C N C 43 40 47 N
NH2 C N N NH N N 64
Scheme 4.3. Photolysis of 3-azidoisoqunoline (42).
N N N N3 hν N N N N -N2 42 41
N N N N N N 48
H2N CN CN C NH N NH N
Scheme 4.4. Thermolysis of 4-azido-2-phenylquinazoline (50).
N3 N N N N Δ N N N -N N Ph 2 N Ph N Ph N Ph 50 49
4.2. Chemical Generation of Iminoquinone 5 via oxidation of 8-oxoadenosine (2)
The attempts to monitor of 25 formed via chemical oxidation of 8-aminoadenosine and of 5 via oxidation of 8-oxoadenosine (2) with NBS in methanol at -78° C by UV-vis spectroscopy were reported.1-3 During the oxidation of 2, the observed band, which was centered
2 at λmax = 380 nm, was assigned tentatively to iminoquinone 5. Later, it was found, that this 65
3 reaction rapidly gives rise to two absorption bands (Fig. 4.1, λmax = 335 and 380 nm). The 335
nm intermediate seems to decay to the 380 nm intermediate.
Oxidation of 8-oxoA in MeOH with NBS 1.00 1.00 20 sec 90 sec 0.75 30 sec 0.75 100 sec
A 40 sec 2 min Δ 0.50 50 sec 0.50 5 min 60 sec 10 min 0.25 70 sec 0.25 30 min 80 sec 0.00 0.00 300 350 400 450 500 300 350 400 450 500 Wavelength, nm Wavelength, nm
Figure 4.1. Transient absorption spectra of oxidation of 8-oxoA (2) in MeOH with NBS.
Scheme 4.5. Mechanism for the chemical oxidation of 8-oxoA (2).
NH NH2 Br NH2 2 NH2 H H N N N NH2 N NBS N N N N O O O O O CH OH H N N 3 N N N N N Nuc NH -75o C N R 2 R R Nuc R R 51 Br H + N H Overoxidation Products N N O -HBr HNuc N HO N 7: Nuc = -OCH O R 3 R= NH 8: Nuc = H Br NN N N OH OH N H O N N O N N R 5 N N R 66
However, the 380 nm band is identical to that of molecular bromine in methanol, persists for ca. 30 minutes at room temperature, and for quite long times in the cold (-75° C). The
335 nm band decays in ca. a minute and is attributed to one of the reactive intermediates leading to 7 and 8 (See Scheme 1.4). Several possible intermediates that might be considered for this
NBS reaction are shown in Scheme 4.5, and their calculated absorption properties are listed in
Table 4.1.
Table 4.1 Calculated vertical excitations for intermediates 5Me and N-bromoadenosines.
Compound State λmax, nm Oscillator strangth Method
NH S2 444.21 0.1097 B3LYP/6-31G(d) N N O S3 374.19 0.0017 B3LYP/6-31G(d) N N H3C 5Me
Br NH2 368.09 0.0171 B3LYP/6-31G(d) N N O N N H3C
NHBr 330.87 (E) 0.0529 B3LYP/6-31G(d) H N N O 345.23 (Z) 0.0601 B3LYP/6-31G(d) N N H3C
Therefore, the 380 nm band in Fig. 4.1 is assigned to traces of molecular bromine generated in this NBS reaction. Furthermore, sigma adducts related to 51 have been observed previously and found to aromatize in the µs and ms time domains.4 Therefore, based upon the 67
calculated values for the N-bromo- compounds listed in Table 4.1, the 335 nm transient would seem to be most reasonably assigned one or some combination of the N-bromo-8-oxoadenosines.
Many 8-oxoA (2) and 8-oxoI (3) adducts are susceptible to further oxidation, which
ultimately leads to the destruction of the purine nucleus, and the release of any cross-linked
molecules. While such further oxidation can be minimized by the photochemical generation of
the iminoquinone 5 from the azide 39, even this route suffers from adduct over oxidation which
will be discussed in this chapter below.
Scheme 4.6. The formation of C2-amines.
NH2 NH2 N N hν N N N3 H2N N R NH N N 2 N NR2 R R 24 33-NR2 N3 NH2 H H N N hν N N O O N R NH N N 2 N NR2 R R 39
This problem of oxidation of cross-linked molecules is a problem that requires further
study and evaluation. Further oxidation of amine adducts of 8-azidoadenosine (24) such as the
triamine 33-NR2 (Scheme 4.6) is a major problem, since these adducts are extremely susceptible
to oxidation even by atmospheric oxygen, which leads to ephemeral cross-links. Finally, the
azide 39 provides an excellent source for the photochemical generation of 5 in spectroscopic
studies of its chemistry and possible involvement in biological processes, and over oxidation is
more easily minimized in this photochemical route than in more conventional chemical oxidation
pathways. 68
4.3. Photochemical Studies of 6-Azido-8-oxo-7,9-dihydropurinoriboside (39)
A variety of azidopurines have been used in photoaffinity labeling studies, but
6-azido-8-oxo-7,9-dihydropurinoriboside (39) have never been applied in such studies. The azide
39 would seem to be the ideal tool for probing the fate/role of 8-oxoA (2) in biological processes leading to gene malfunction. One might argue that the 6-azido group of 39 might block its binding with functionalities in its intended binding site, altering of the usual Watson-Crick hydrogen bonding, or modifying the normal syn/anti base conformer distribution. In addition, it is well known that some azidopyrimidines undergo azide-tetrazine tautomerization such as shown for azide 39 in Scheme 4.7.10-12
Scheme 4.7. Azido-tetrazine tautomerization for azide 39.
N N N N N XO H H N X = H or N N O N N Ac for Ac O O R= 3 PhCO for Bz3 N N N N OX OX R R 39 52
-1 However, azide 39Bz3 displays an intense absorption at 2150 cm (Fig. 4.2). So its azide tautomer 39Bz3 is the major, if not the only, form present, and both tautomers, 39 and 52, might be expected to form nitrene precursors of 5 upon irradiation with UV light.13 69
100 39Bz 3 80
60
Transmittance, % 40
3500 3000 2500-1 2000 1500 1000 ν, cm
Figure 4.2. IR spectrum of azide 39Bz3.
Photodecomposition of azide 39 by UV-light has been studied in both protic and aprotic solvents. The assumed mechanisms for photodecomposition of azide 39 in protic solvents are shown in Scheme 4.8. Ring fragmentation does not seem to be a viable reaction pathway for nitrene 53. Instead, nitrene 53 is stabilized via proton reallocation via either protonation by intramolecular and intermolecular proton transfer. According to the intramolecular mechanism for protonation of nitrene 53, proton reallocation from N7 to N6 might occur via a 5-membered transition state and lead directly to the formation of iminoquinone 5. The rate of this process should not depend on the solvent acidity. Alternatively, the intermolecular mechanism requires the formation of the hydrogen-bounded complexes between nitrene 53 and solvent molecules such as the solvent-bridge shown in Scheme 4.8. Proton transfer from N7 to N6 of nitrene 53 via such bridge might be quite solvent dependent. 70
Scheme 4.8. Intermolecular and intramolecular mechanisms for proton transfer for 39.
N H H N N N N O N N3 O NH H N hν N N N N N N -N R 53 R N O 2 O N hν N N N R 39 or -N2 R 5 R OH
H N XO X = H or O N N R= Ac for Ac3 O PhCO for Bz OX OX 3 N N R 53
Ultrafast laser photolysis techniques have been used for the study of mechanisms of photodecomposition of azide 39. Femtosecond transient absorbtion spectra of azide 39Bz3 are
shown on Figure 4.3. Since the formation of 5 is dependent upon the proton source, ring
fragmentation seems unlikely and solvent-bridged proton transfer would seem to be the preferred
mechanism of proton reallocation. Thus, when 39Bz3 was irradiated in alcohol solvents, a band centered at λmax = 365 nm is initially formed and slowly shifts to a new band centered at λmax =
405 nm. In all cases, a broad band in the 550-625 nm region decays with the same time constant
as the growth of the 405 nm band. The 365 and 550-625 nm bands have been assigned to the
nitrene of 53Bz3 and the 405 nm band assigned to the iminoquinone 5Bz3. 71
MeOH, 1 mM i-PrOH 0.005 0.005 t0.2ps tM0.2ps 0.004 0.004 t0.3ps t0.2ps 0.003 t0.5ps 0.003 t0.3ps
A t0.5ps A t0.8ps
Δ 0.002 Δ 0.002 t1ps t0.8ps 0.001 0.001 t1ps 0.000 0.000 350 400 450 500 550 600 650 700 350 400 450 500 550 600 650 700 0.005 0.005
0.004 t1ps 0.004 t1ps t2ps t2ps 0.003 0.003 t3ps t3ps A A t6ps
Δ 0.002 t6ps
Δ 0.002 t10ps t10ps 0.001 0.001 t20ps t20ps 0.000 0.000
350 400 450 500 550 600 650 700 350 400 450 500 550 600 650 700 0.0075 0.008
t50ps t50ps 0.006 t100ps 0.0050 t100ps t200ps A
A t200ps
Δ 0.004 Δ t500ps t500ps 0.0025 t800ps t800ps 0.002 t1ns t1ns 0.0000 0.000 350 400 450 500 550 600 650 700 350 400 450 500 550 600 650 700 Wavelength, nm Wavelength, nm
Figure 4.3. Femtosecond transient of azide 39Bz3 in MeOH and 2-PrOH.
In methanol, the proton reallocation time (τ) is 90 ps and in 2-propanol 300 ps. These
observations indicate that the solvent plays an active role in proton reallocation and that the
direct 1,4-proton shift mechanism is not a viable route for the formation of the iminoquinone 5.
This alcohol mediated route is further supported by femtosecond/picosecond flashphotolysis data for azide 39Bz3 in aprotic solvents, Figure.4.4. In both methylcyclohexane
and dichloromethane, 39Bz3 affords bands at λmax = 345-360 nm and a broad band in the 550-625
nm region. The short wavelength band tends to slowly shift to the red to form a new band at ca.
360-390 nm with τ = 600 ps. While the long wavelength band remains stable. 72
Methylcyclohexane B DCM A 0.0100 0.002 t0ps tM0.2ps 0.0075 t0.2ps t0.2ps t0.3ps 0.001 t0.3ps 0.0050 t0.5ps
A t0.5ps A Δ t0.8ps Δ 0.000 t0.8ps 0.0025 t1ps t1ps 0.0000 -0.001 350 400 450 500 550 600 650 700 350 400 450 500 550 600 650 700 0.0075 0.002 t1ps t1ps t2ps t2ps 0.0050 t3ps t3ps t6ps 0.001 A t6ps A Δ 0.0025 t10ps Δ t10ps t20ps t20ps 0.0000 0.000
350 400 450 500 550 600 650 700 350 400 450 500 550 600 650 700 0.0075 0.002 t50ps t50ps t100ps t100ps 0.0050 t200ps t200ps A A Δ Δ t500ps 0.001 t500ps 0.0025 t800ps t800ps t1ns t1ns 0.0000 0.000 350 400 450 500 550 600 650 700 350 400 450 500 550 600 650 700 Wavelength, nm Wavelength, nm
Figure 4.4. Transient absorbtion spectra of azide 39Bz3 in methylcyclohexane (A) and in
dichloromethane (B).
These short wavelength bands centered at λmax = 350-360 nm, in methylcyclohexane and dichloromethane, are assigned to an initially formed singlet nitrene 53s (Scheme 4.9), which undergoes relatively slow ISC to a triplet state 53t having absorption shifted slightly to the red,
Fig. 4.4. The broad absorption in the region 550-625 nm apparently is associated with both singlet and triplet nitrenes. Triplet nitrenes such as 53t would be expected to react via abstraction of hydrogen atoms forming the radical 54, which in turn would ultimately undergo reduction to amine 2, which was isolated from preparative reactions of azide 39Bz3, Scheme 4.9. 73
Scheme 4.9. Mechanism for photolysis of azide 39Bz3.
N3 13N N NH H H H H N N hν N N N N N N O O O O -N N N 2 N N N N N N R 39Bz3 R 53sR 53tR 54 Singlet Triplet [H] NH NH2 NH 2 H H N N Nuc N N Ph O N N O O O O N N N O N N N Nuc R = R R 2 Ph O O Ph R 5 55-Nuc O O
Azide 39Bz in pure MeOH Azide 39Bz in MeOH with Imidazole A 3 B 0.015 3 0.0100 5 μs 50 μs 7.5 μs 0.0075 140 μs 0.010 10 μs 500 μs A 15 μs Δ 1 ms 0.0050 A 20 μs 5 ms Δ 0.005 30 μs 20 ms 90 μs 0.0025 35 ms
0.0000 0.000 400 450 500 550 600 400 450 500 550 600 Wavelengt, nm Wavelength, nm
Figure 4.5. Nanosecond-millisecond transient absorption spectra of azide 39Bz3 in pure
methanol (A) and in 1M solution of imidazole in methanol (B). 74
Scheme 4.10. Imidazole attack on iminoquinone 5.
NH NH2 NH2 H XO N N N N N N O O O O H R= N N N N N N N N OX OX R N R R 5 56 8 N X = H or N N Ac for Ac3 H PhCO for Bz3
0.10 0.068us 0.284us 0.08 0.5us 0.06
E 1.004us Δ 0.04 1.508us 0.02 2.012us 0.00 3.02us 400 450 500 550 600 5us Wavelength, nm
Figure 4.6. Time-resolved emission spectra of azide 39Bz3.
The nanosecond-millisecond transient spectroscopy for azide 39Bz3 is shown in pure methanol (Fig. 4.5A) and in 1 M solution of imidazole in methanol (Fig. 4.5B). Iminoquinone 5 is quenched by methanol within several µs. The following aromatization of corresponding adduct
55-OMe occurs with the τ > 30 ms. On the other hand, intermediate 56 (Scheme 4.5B) aromatizes to 7 with τ = 20 µs, which is the fastest aromatization observed in this work. This adduct formation is associated with an adduct band with λmax = 420 nm, but which display less 75
broadening than the imidazole adduct bands of other systems, 24Ac5 in Fig. 3.8A and 30Ac3 in
Fig. 3.10B (See Chapter III).
Scheme 4.11. Preparative irradiation of azides 39Ac3 and 39Bz3
N3 NH2 O NH2 H H H H NH N N hν N N N NH N N 2 O O O O O N N 254 nm N N N N N N N NH or R R R R R 39356 nm 2 3 8 N XO H for 2, 3, 7 O Over X = Ac for 2Ac3, 3Ac3, 7Ac3, 39Ac3 R= Oxidation PhCO for 2Bz , 3Bz , 7Bz , 39Bz Products 3 3 3 3 OX OX
Table 4.2 Irradiation of azides 39Ac3 and 39Bz3 in MeOH.
Azide Solven Conditions Nuc Product Distribution (%, Prot. Group)a t 2 3c 8 Unidentifiedb
39Bz3 MeOH 254 nm, 5 mmol/L, MeOH 30% 30% 40% 1 hour/mmol Bz3 Bz3
39Ac3 MeOH 254 nm, 5 mmol/L, Imidazole 10% 20% 50% 10% 1 hour/mmol Ac3 Ac3 Ac3
39Bz3 MeOH 356 nm, 5 mmol/L, Imidazole 0 30% 50% 20% 1 hour/mmol Bz3 Bz3
39Ac3 MeOH 356 nm, 5 mmol/L, Imidazole 20% 60% 20% 1 hour/mmol Ac3 Ac3
a. The sugar esters tend to hydrolyze partially during the course of the reaction due to
the basic alcoholic conditions of the imidazole reactions.
b. These products seem to constitute a complex mixture of over oxidation or coupling
products.
c. Water is necessary to form this product (Scheme 4.13). 76
Unfortunately, the photochemistry of 39 could not be studied in the time domain of
1ns-100 ns due to available instrumentation limitations and requires further investigation. In the region 100 ns- 5 µs products of photodecomposition of azide 39 exhibit strong emission. The time-resolved emission spectra of azide 39Bz3 have been recorded in methanol (Fig. 4.6). The observed lifetime τE for this emissive intermediates is c.a. 300 ns. This strong emission obstructs the study of the photodecomposition of azide in the time region 100 ns-5 µs.
Thus, azide 39 rapidly forms the iminoquinone 5 upon irradiation (100-300 ps) and 5 reacts with nucleophiles in several µs forming adducts 51. These adducts undergo aromatization much more slowly (μs to ms) to form 55-Nuc. So there is adequate time for 5 to probe suitable binding sites and react with nucleophiles in those sites. Thus, while one might question the viability of 39 as a mimic for the affinities of 8-oxoA (2), the iminoquinone 5 should be an ideal mimic for 8-oxoA (2).
Scheme 4.12. Possible mechanism for the formation of over oxidation products.
R N R N N N O O N N N N 5 H 2 N H H NH2 H NH NH N N Nuc N N Purine ring O O N destruction N Nuc N N Nuc R R 55-Nuc 77
Scheme 4.13. Hydrolysis of Iminoquinone 5.
NH H NOH O 2 H H O PhCOO N N 2 N N [H] N NH O O O O -NH R= N N N N 3 N N R R R PhCOO OOCPh 5Bz3 3Bz3
In order to determine the products of photodecomposition, the preparative irradiations with UV light (λ = 254 and 356 nm) of azide 39Ac3 and 39Bz3 were carried out in pure methanol
and in the presence of imidazole. Since methanol by itself is a poor nucleophile, the
photodecomposition of azide 39Ac3 in pure methanol leads to little, if any, adduct formation.
The major isolated products are reduction products, 8-oxoA (2) (Scheme 4.12), and the product
of its hydrolysis, 8-oxoI (3) (Scheme 4.11 and 4.13). However, photolysis of methanolic
solutions of 39Ac3 in the presence of the powerful nucleophile imidazole leads to formation of 8-
oxoA (2), 8-oxoI (3), and imidazole adduct 8Ac3 as major products. The formation of 3Ac3 and
3Bz3 is apparently due to the hydrolysis of the imino group in the iminoquinone 5Ac3 and 5Bz3,
respectively, either during the photoreaction or purification and isolation of the products as
outlined in Scheme 4.13. Some amount of ribosylurea and uncharacterized purine-ring
destruction products were detected by HPLC analysis in both cases: in pure methanol and in the
presence of imidazole. The formation of these products may be explained by oxidation reactions
of 55-Nuc by iminoquinone 5 as oxidizing agent (Scheme 4.12). 78
4.4. Conclusions
The photodecomposition of azide 39 proceeds through iminoquinone 5 even though there is no powerful EDG to activate nitrene 53 for protonation. This lack of nitrene activation causes protonation of nitrene 53 to occur much more slowly than in the case of the activated nitrene 31. On the other hand, once protonated, iminoquinone 5 is the most powerful electrophile studied in this work, and thus, is an effective cross-linking agent, which makes azide 39 a good candidate for consideration as a potential PAL agent.
4.5. Synthesis of 6-azido-7,9-dihydro-8-oxopurine (39) and derivatives
Synthesis of 8-Oxo-7, 8-dihydroadenosine (2).14
NH2 NH2 H N N OH N N Br HS O HO N N HO N N O Et3N, H2O O 2 OH OH OH OH
A mixture of 2 mL (30 mmol) of 2-mercapthoethanol, triehylamine (10 mL, 100 mmol), and 8-bromoadenosine (6.75 g, 20 mmol) in 100 mL of water was refluxed for 2 hours. The content of the flask was chilled and filtered thought celite to remove a white amorphous precipitate that formed during cooling. The filtrate was evaporated under reduced pressure, and the residue was recrystallized from 20 mL of 50% aqueous methanol. The product 2 was obtained as colorless crystals (3 g, 10 mmol, 50% yield).14 79
1H NMR (500 MHz, DMSO) δ 10.36 (s, 1H), 8.01 (s, 1H), 6.56 (s, 2H), 5.66 (d, J = 6.5
Hz, 1H), 5.23 (d, J = 6.2 Hz, 1H), 5.17 (bd, J = 4.0 Hz, 1H), 5.05 (d, J = 4.5 Hz, 1H), 4.85 (dd, J
= 11.7, J’ = 6.0 Hz, 1H), 4.11 (d, J = 2.9 Hz, 1H), 3.85 (dd, J = 7 Hz, J’ = 4.0 Hz, 1H), 3.60 (bd,
J = 12.0 Hz, 1H), 3.45 (ddd, J = 4.5 Hz, J’ = 7 Hz, J” = 12 Hz, 1H).
13C NMR (126 MHz, DMSO) δ 151.98, 151.11, 147.64, 146.95, 103.98, 86.09, 85.92,
71.40, 70.74, 62.82.
Synthesis of 8-Oxo-7,9-dihydroinosine (3).
NH2 O H H N N N NH O NaNO2 O HO N N HO N N O AcOH/H2O O 2 3 OH OH OH OH
A solution of 1 g (14.5 mmol) NaNO2 in 2 mL of water was added to a stirred solution
of 2 g (7 mmol) of 8-oxoA (2) in 100 mL of 95% aqueous acetic acid. The reaction mixture was
stirred overnight. The solvent was removed under reduced pressure and the oily residue was
recrystallized from 30 mL of 50% aqueous EtOH to yield 1.0 g of 8-oxoI (3) as colorless
crystals.
1H NMR (500 MHz, DMSO) δ 11.45 (bs, 1H), 8.01 (s, 1H), 5.67 (d, J = 6.0 Hz, 1H),
4.83 (dd, J = 5.6 Hz, J’ = 5.6 Hz, 1H), 4.13 (dd, J = 4 Hz, J’ = 4 Hz, 1H), 3.85 (d, J = 3.6 Hz,
1H), 3.60 (dd, J = 11.8 Hz, J’ = 4.1 Hz, 1H), 3.46 (dd, J = 11.8 Hz, J’ = 4.9 Hz, 1H).
13C NMR (126 MHz, DMSO) δ 152.23, 151.41, 145.36, 144.71, 109.01, 86.27, 85.61,
71.15, 70.78, 62.64. 80
Synthesis of 8-Oxoinosine Triacetate (3Ac3).
O O H H N NH N NH O Ac2O O HO N N H3C O N N O Pyridine O 3 O OH OH H3C O O CH3 O O 3Ac 3
A suspension of 5 g (17.6 mmol) of 8-oxoI (3) in 35 mL of freshly redistilled anhydrous
pyridine was prepared at room temperature under an argon atmosphere. Acetic anhydride, 10 g
(100 mmol) was added slowly to a vigorously stirred suspension of 8-oxoI (3) in pyridine, and
the reaction mixture was stirred overnight at 60 °C. Volatile components of the reaction mixture
were removed under reduced pressure, and the residue redissolved in 50 mL of CH2Cl2. Organic
layer was washed with 5% aq. NaHCO3 (3×100 mL) and water (2×100 mL) and dried with
anhydrous MgSO4. The solvent was removed under reduced pressure and the residue separated
by liquid chromatography on 75 g Merck 60 silica gel (CHCl3-EtOAc, 19-1) to yield 5 g (11.4 mmol 65%) of 3Ac3 as colorless glass.
1 H NMR (300 MHz, CDCl3) δ 12.72 (s, 1H), 11.57 (s, 1H), 8.02 (s, 1H), 6.00 (dd, J =
5.9, J’ = 4.7 Hz, 1H), 5.84 (d, J = 4.6 Hz, 1H), 5.55 (t, J = 5.9 Hz, 1H), 4.37 (dd, J = 11.8,
J’ = 3.6 Hz, 1H), 4.24 (dd, J = 5.7, J’ = 3.7 Hz, 1H), 4.11 (dd, J = 11.8, J’ = 5.7 Hz, 1H),
2.08 (s, 3H), 2.06 (s, 3H), 2.00 (s, 3H).
81
Synthesis of 6-Chloro-8-oxorurine Triacetate.
O Cl H H N NH N N O O SOCl2 H3C O N N H3C O N N O DMF(cat.) O O CH2Cl2 O H3C O O CH3 H3C O O CH3 O O O O 3Ac3
A suspension of 2 g (4.9 mmol) of 8-oxoinosine triacetate (3Ac3) in 10 mL of SOCl2
(0.14 mol) was prepared at room temperature under an argon atmosphere, and 50 µL of DMF were added. The reaction mixture was stirred for 48 h at 65 °C. The excess SOCl2 was removed
under reduced pressure and the oily residue redissolved in 20 mL of CH2Cl2. The organic layer
was washed subsequently with 5% aq. NaHCO3 solution (3×50 mL) and with water (3×50 mL)
and dried with anhydrous MgSO4. The solvent was removed under reduced pressure and the
crude product was purified by flash chromatography on 10 g Merck 60 silica gel to yield 1.2 g
(2.8 mmol, 57%) of 6-chloro-8-oxorurine triacetate (14Ac3) as a yellowish glass. This compound
was used for the next step without further purification.
Synthesis of 6-Azido-8-oxopurinoriboside Triacetate (39Ac3)
Cl N3 H H N N N N O O NaN3 H3C O N N H3C O N N O DMF O O O
H3C O O CH3 H3C O O CH3 O O O O 39Ac3
The solution of 0.5 g (1.15 mmol) of 6-chloro-8-oxopurine triacetate and 0.3 g (4.6
mmol) of NaN3 in 1 mL of anhydrous DMF were stirred for 48 h at 65°C under an argon 82
atmosphere. The reaction mixture was cooled to room temperature and diluted with 25 mL of
dichloromethane. The resulting suspension was washed with water (3×25 mL) to remove DMF
and inorganic materials. The organic layer was evaporated under reduced pressure to obtain a
crude oil that was purified by preparative thin-layer chromatography on silicagel plates
(2×250×250 mm) (CHCl3/EtOAc, 19/1). The product was isolated as colorless glass: 0.2 g (0.46
mmol 40 %).
1 H NMR (300 MHz, CDCl3) δ 9.68 (s, 1H), 8.49 (s, 1H), 6.15 (t, J = 4.7 Hz, 2H), 5.80
(t, J = 5.7 Hz, 1H), 4.49 (d, J = 3.0 Hz, 1H), 4.38 – 4.33 (m, 2H), 2.15 (s, 3H), 2.11 (s, 3H), 2.09
(s, 3H).
+ HRMS calc. for C16H18N7O8 (M+H), m/z calculated 436.121137, found 436.12114.
Synthesis of 8-oxoinosine tribenzoate. (3Bz3).
O O H H N NH N NH O O HO N N PhCOCl Ph O N N O O Pyridine O OH OH Ph O O Ph 3 O O 3Bz3
The suspension of 5 g (17.6 mmol) of 8-oxoI (3) in 35 mL of freshly redistilled
anhydrous pyridine was prepared at room temperature under an argon atmosphere. Benzoyl
chloride, 14 g (0.1 mol) was added slowly to the vigorously stirred suspension of 8-oxoI (3) in
pyridine, and the reaction mixture was stirred overnight at 60 °C. Volatile components of the
reaction mixture were removed under reduced pressure, and the rest was redissolved in 50 mL of
CH2Cl2. Organic layer was washed with 5% aq. NaHCO3 (3×100 mL), water (2×100 mL), and
dried with anhydrous MgSO4. The rest of solvent was removed under reduced pressure and the 83
residue was separated by liquid chromatography on 75 g Merck 60 silica gel (CHCl3-EtOAc, 9-
1) to yield 3Bz3 (7.3 g, 12.2 mmol, 72% theor) as colorless crystalline material.
1H NMR (500 MHz, DMSO) δ 12.70 (s, 1H), 11.64 (s, 1H), 8.03 – 7.86 (m, 7H), 7.69 –
7.62 (m, 3H), 7.53 – 7.41 (m, 6H), 6.42 (s, 1H), 6.16 (d, J = 7.2 Hz, 2H), 4.81 – 4.67 (m, 2H),
4.56 (dd, J = 12.1, 4.6 Hz, 1H).
13C NMR (126 MHz, DMSO) δ 165.88, 165.09, 151.68, 151.31, 145.56, 144.06, 134.45,
134.30, 133.95, 129.85, 129.79, 129.76, 129.28, 129.18, 129.14, 129.04, 129.02, 128.87, 109.31,
79.70, 79.44, 79.17, 78.67, 72.67, 70.81, 63.53.
Synthesis of 6-Chloro-8-oxopurine Tribenzoate.
O Cl H H N NH N N O O SOCl2 Ph O N N Ph O N N O DMF(cat.) O O CH2Cl2 O Ph O O Ph Ph O O Ph
O O O O 3Bz3
The suspension of 2 g (3.35 mmol) of 8-oxoI tribenzoate (3Bz3) in 10 mL of SOCl2 was mixed at room temperature under an argon atmosphere. The reaction mixture was stirred for 48 h at 65 °C. The excess SOCl2 was removed under reduced pressure and the oily residue was
redissolved in 20 mL of CH2Cl2. The organic layer was washed subsequently with 5% aq.
NaHCO3 solution (3×50 mL) and with water (3×50 mL) and the organic layer dried with
anhydrous MgSO4. The solvent was removed under reduced pressure and the crude product
purified by flash chromatography (20 g Merck 60 silica gel) to yield 1.2 g (1.95 mmol, 55%) of
6-chloro-8-oxorurine tribenzoate (14Bz3) as a yellowish glass. This material was used without
further purification. 84
Synthesis of 6-Azido-8-oxopurinoriboside Tribenzoate (39Bz3).
Cl N3 H H N N N N O O NaN3 Ph O N N Ph O N N O DMF O O O Ph O O Ph Ph O O Ph
O O O O 39Bz3
The solution of 1.3 g (2 mmol) of 6-chloro-8-oxopurine tribenzoate and NaN3 (0..65 g,
10 mmol) in 4 mL of anhydrous DMF was stirred for 48 h at 65°C under an argon atmosphere.
The reaction mixture was cooled to room temperature and diluted with 50 mL of dichloromethane. The resulting suspension was washed with water (3×50 mL) to remove DMF and inorganic materials. The organic layer was evaporated under reduced pressure to obtain a crude oil, which was purified by preparative thin-layer chromatography on silica gel plates
(2×250×250 mm) (CHCl3/EtOAc, 19/1). The product was isolated as colorless glass: 0.7 g
(53%).
1 H NMR (500 MHz, CDCl3) δ 8.32 (s, 1H), 8.09 (d, J = 7 Hz, 2H), 8.01 (d, J = 8.5 Hz,
2H), 7.96 (d, J = 8.5 Hz, 2H) , 7.57 (dd, J = 6.5 Hz, J’ = 6.5 Hz, 1H), 7.54 (dd, J = 7.5 Hz, J’ =
7.5 Hz, 1H), 7.52 (t, J = 7.5 Hz, 1H), 7.41 – 7.35 (c, 7H), 6.63 (dd, J = 6.0, J’ = 3.9 Hz, 1H),
6.40 (d, J = 3.9 Hz, 1H), 6.39 (dd, J = 6.3 Hz, J’ = 6.3 Hz, 1H), 4.89 (dd, J = 11.9, J’ = 3.6 Hz,
1H), 4.82 – 4.77 (c, 1H), 4.73 (dd, J = 11.9, 4.9 Hz, 1H).
13 C NMR (126 MHz, CDCl3) δ 171.94, 166.28, 165.34, 152.81, 151.34, 149.37, 142.20,
136.04, 133.70, 133.59, 133.18, 129.83, 129.64, 129.59, 128.86, 128.68, 128.50, 128.35, 128.32,
127.10, 109.98, 84.75, 79.71, 72.65, 71.12.
-1 νmax: 2300, 2150, 1740, 1660, 1525, 1370, 1225, 1050, 850, and 700 cm 85
λmax: 294 nm (39000).
+ HRMS calc. for C31H24N7O8 (M+H), m/z calc 622.16809, found 622.16799.
Irradiation of 39Ac3 and 39Bz3; general procedure. Solutions of azide 39Ac3 or 39Bz3
(c[39] = 5 mmol/L ) in methanol were irradiated in Rayonet Photochemical Reactor at 254 or
356 nm for 5 hour/mmol of the azide. Solvent was removed under reduced pressure and solid residue was prepared for the further RP-HPLC analyses and separations on Rainin HPLC system coupled with Agilent 8453 UV-Vis spectrometer. For analytical purposes Waters C18 Spherisorb
ODS1 column (4.6×250mm) was used, whereas preparative separations were performed on
Dynamax C18 preparative column (60 Å, 21.2×250mm) C-18 reverse phase silicagel column
(8×4×250mm) with system MeOH (30%) - H2O (70%).
+ 8Ac3 HRMS calc. for C19H22N7O8 m/Z = 476.15244, found 476.15257.
1 3Ac3 H NMR (300 MHz, CDCl3) δ 12.5 (bs, 1H), 11.60 (bs, 1H), 8.02 (s, 1H), 6.07 –
5.94 (m, 1H), 5.84 (d, J = 4.6 Hz, 1H), 5.55 (dd, J = 5.9, J’ = 5.9 Hz, 1H), 4.37 (dd, J = 11.8, J’
= 3.4 Hz, 1H), 4.25 (dd, J = 9.2, J’ = 5.5 Hz, 1H), 4.11 (dd, J = 11.7, J’ =5.6 Hz, 1H), 2.08 (s,
3H), 2.06 (s, 3H), 2.00 (s, 3H).
+ 3Ac3 HRMS calc. for C16H18N4O9Na m/Z = 433.0966, found 433.0966;
+ 2Bz3 HRMS calc. for C31H26N5O8 m/Z = 596.17759, found 596.1778;
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CONCLUSIONS
• The synthesis of 39Ac3 and 39Bz3 has been achieved for the first time.
• Irradiation of 39 affords adenosine iminoquinone 5 that reacts with nucleophiles at the 2-position to form adducts 55-Nuc with imidazole, amines, alcohols, and water.
• This new photochemical route to adenosine iminoquinone 5 opens the way for its application in a wide variety of photoaffinity labeling studies.
• Imidazole adducts 33-IM and 8 are the most easily studied adducts, since they seem to be the least susceptible to further oxidation.
• This azide photochemical route to adenosine iminoquinone also reduces problems with over-oxidation, and will make it possible to study this type of reaction in detail with many nucleophiles using transient spectroscopy.
• Nitrenes 31 and 36 derived from 8-azido purines 24 and 30 undergo ring-opening very rapidly in the absence of stabilizing EDG on the purine ring. In the presence of such stabilizing groups, 8-azidoadenosine (24), ring-opening is suppressed and reactive quinoidal intermediates are formed instead.
• The Nitrene 53 derived from 39 does not undergo ring opening, but is protonated relatively slowly, forming an iminoquinone at rates dependent on the proton source. 88
SUPPLEMENTAL INFORMATION. HRMS SPECTRA. 1H AND 13C NMR SPECTRA.
8-Aminoadenosine. 89
90
91
92
Dimer-like structure.
93
94
95
96
97
98
99 100 101