Chemical synthesis of cross-link lesions found in nitrous acid treated DNA: a general method for the preparation of N2-substituted 2'-deoxyguanosines

Treatment of DNA with nitrous acid results in the formation of DNA-DNA cross-links. Two cross-link lesions have previously been isolated and their structures assigned based on spectroscopic data. The major lesion has been proposed to consist of two deoxyguanosine (dG) nucleosides sharing a common N2 atom (1), while the structure of the minor lesion has been proposed to consist of a common nitrogen atom linking C2 of a dG nucleoside to C6 of deoxyadenosine (2). The chemical synthesis of 1 and 2, utilizing a palladium-catalyzed coupling, is described herein. It is demonstrated that the spectroscopic properties of synthetic 1 are identical to that of lesion 1 obtained from nitrous acid cross-linked DNA, thus providing a proof of its structure. Comparison of the limited spectroscopic data available for lesion 2 originating from nitrous acid cross-linked DNA to synthetic 2 supports its structural assignment. The synthetic approach used for synthesis of 1 and 2 is shown to be a general method for the preparation of a variety of N2-substituted dG nucleosides in good yields.     

Electron attachment induced proton transfer in a DNA nucleoside pair: 2'-deoxyguanosine-2'-deoxycytidine

To elucidate electron attachment induced damage in the DNA double helix, electron attachment to the 2'-deoxyribonucleoside pair dG:dC has been studied with the reliably calibrated B3LYP/DZP++ theoretical approach. The exploration of the potential energy surface of the neutral and anionic dG:dC pairs predicts a positive electron affinity for dG:dC [0.83 eV for adiabatic electron affinity (EAad) and 0.16 eV for vertical electron affinity (VEA)]. The substantial increases in the electron affinity of dG:dC (by 0.50 eV for EAad and 0.23 eV for VEA) compared to those of the dC nucleoside suggest that electron attachment to DNA double helices should be energetically favored with respect to the single strands. Most importantly, electron attachment to the dC moiety in the dG:dC pair is found to be able to trigger the proton transfer in the dG:dC- pair, surprisingly resulting in the lower energy distonic anionic complex d(G-H)-:d(C+H).. The negative charge for the latter system is located on the base of dC in the dG:dC- pair, while it is transferred to d(G-H) in d(G-H)-:d(C+H)., accompanied by the proton transfer from N1(dG) to N3(dC). The low energy barrier (2.4 kcal/mol) for proton transfer from dG to dC- suggests that the distonic d(G-H)-:d(C+H). pair should be one of the important intermediates in the process of electron attachment to DNA double helices. The formation of the neutral nucleoside radical d(C+H). is predicted to be the direct result of electron attachment to the DNA double helices. Since the neutral radical d(C+H). nucleotide is the key element in the formation of this DNA lesion, electron attachment might be one of the important factors that trigger the formation of abasic sites in DNA double helices.     

Synthesis of Oligonucleotides Containing the N6-(2-Deoxy-α,β-d-erythropentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy⋅dG) Oxidative Damage Product Derived from 2′-Deoxyguanosine

N⁶-(2-Deoxy-α,β-d -erythropentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy⋅dG) is a major DNA lesion produced from 2′-deoxyguanosine under oxidizing conditions. Fapy ⋅ dG is produced from a common intermediate that leads to 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-OxodGuo), and in greater quantities in cells. The impact of Fapy ⋅ dG on DNA structure and function is much less well understood than that of 8-OxodGuo. This is largely due to the significantly greater difficulty in synthesizing oligonucleotides containing Fapy ⋅ dG than 8-OxodGuo. We describe a synthetic approach for preparing oligonucleotides containing Fapy ⋅ dG that will facilitate intensive studies of this lesion in DNA. A variety of oligonucleotides as long as 30 nucleotides are synthesized. We anticipate that the chemistry described herein will provide an impetus for a wide range of studies involving Fapy ⋅ dG.     

Synthesis of oligonucleotides containing Fapy.dG (N(6)-(2-deoxy-alpha,beta-D-erythropentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine) using a 5'-dimethoxytrityl dinucleotide phosphoramidite

Fapy.dG (N(6)()-(2-deoxy-alpha,beta-d-erythropentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine) is a modified purine lesion produced by a variety of DNA-damaging agents, which shows interesting biochemical properties. The previous method for synthesizing oligonucleotides containing Fapy.dG utilized a reverse dinucleotide phosphoramidite, which also required the synthesis of the appropriate reverse phosphoramidites. An improved method for synthesizing oligonucleotides containing Fapy.dG, which does not require reverse phosphoramidites, is described. Fapy.dG containing dinucleotide phosphoramidites containing 5'-thymidine (11a) or 5'-deoxycytidine (15) are prepared and employed in oligonucleotide synthesis. Oligonucleotide purity is assayed using the DNA repair enzyme formamidopyrimidine DNA glycosylase and by ESI-MS.     

Gas-phase studies of purine 3-methyladenine DNA glycosylase II (AlkA) substrates

3-Methyladenine DNA glycosylase II (AlkA) is an enzyme that cleaves a wide range of damaged bases from DNA. The gas-phase thermochemical properties (tautomerism, acidity, and proton affinity) have been measured and calculated for a series of AlkA purine substrates (7-methyladenine, 7-methylguanine, 3-methyladenine, 3-methylguanine, purine, 6-chloropurine, xanthine) that have not been heretofore examined. The damaged nucleobases are found to be more acidic than the normal nucleobases adenine and guanine. Because of this increased acidity, the damaged bases would be expected to be more easily cleaved from DNA by AlkA (their conjugate bases should be better leaving groups). We find that the gas-phase acidity correlates to the AlkA excision rates, which lends support to an AlkA mechanism wherein the enzyme provides a nonspecific active site, and nucleobase cleavage is dependent on the intrinsic N-glycosidic bond stability.     

Quantifying hydrogen-bonding strength: the measurement of 2hJNN couplings in self-assembled guanosines by solid-state 15N spin-echo MAS NMR

(2h)J(NN) hydrogen-bond mediated J couplings are measured in the solid state for two synthetic deoxyguanosine derivatives by (15)N MAS NMR spin-echo experiments. The use of rotor-synchronised Hahn-echo pulse train (RS-HEPT) (1)H decoupling, with a duty cycle of 6%, allows spin-echo durations out to 200 ms, hence enabling the accurate determination of J couplings as small as 3.8 Hz. A single-crystal X-ray diffraction structure exists for the shorter alkyl chain derivative dG(C(3))(2): the observation of significantly different (2h)J(NN) couplings, 6.2 +/- 0.4 and 7.4 +/- 0.4 Hz, for the two resolved N7 resonances is to be expected given the NH...N hydrogen-bonding distances of 2.91 and 2.83 A for the two distinct molecules in the asymmetric unit cell. For the longer alkyl chain derivative, dG(C(10))(2), for which there is no single-crystal diffraction structure, a (15)N refocused INADEQUATE spectrum (Pham et al., J. Am. Chem. Soc., 2005, 127, 16018-16019) has demonstrated the presence of N2-H...N7 intermolecular hydrogen-bonds indicative of a quartet-like structure. The (2h)J(NN) hydrogen-bond mediated J coupling of 5.9 +/- 0.2 Hz is at the lower end of the range (5.9-8.2 Hz) of (2h)J(NN) couplings determined from solution-state NMR of guanosine quartets in quadruplex DNA. A full discussion of the determination of error bars on the fitted parameters is given; specifically, error bars determined by a non-linear fitting (using the covariance matrix) or in a Monte-Carlo fashion are found to give effectively identical results.     

Tautomerization in gas-phase ion chemistry of isomeric C-8 deoxyguanosine adducts from phenol-induced DNA damage

Collision-induced dissociation (CID) of 8-(4'-hydroxyphenyl)-2'-deoxyguanosine and 8-(2'-hydroxyphenyl)-2'-deoxyguanosine was investigated using sequential tandem mass spectrometry. These adducts represent biomarkers of DNA damage linked to phenolic radicals and were investigated to gain insight into the effects of chemical structure of a C-8 modification on fragmentation pathways of modified 2'-deoxyguanosine (dG). CID in MS(2) of the deprotonated molecules of both the isomers generated the same product ion having the same m/z values. CID in MS(3) of the product ion at m/z 242 and CID in MS(4) experiments carried out on the selected product ions at m/z 225 and m/z 218 afford distinct fragmentation patterns. The conformational properties of isomeric product ions from CID showed that the ortho-isomers possess the unique ability to tautomerize through an intramolecular proton transfer between the phenolic OH group and the imine nitrogen (N7). Tautomerization of ortho-isomers to their keto-tautomers led to differences in their system of conjugated double bonds compared with either their enol-tautomer or the para-isomer. The charge redistribution through the N-7 site on the imidazole ring is a critical step in guanosine adduct fragmentation which is disrupted by the formation of the keto-tautomer. For this reason, different reaction pathways are observed for 8-(4'-hydroxyphenyl)-2'-deoxyguanosine and 8-(2'-hydroxyphenyl)-2'-deoxyguanosine. We present herein the dissociation and the gas-phase ion-molecule reactions for highly conjugated ions involved in the CID ion chemistry of the investigated adducts. These will be useful for those using tandem mass spectrometry for structural elucidation of C-8 modified dG adducts. This study demonstrates that the modification at the C-8 site of dG has the potential to significantly alter the reactivity of adducts. We also show the ability of tandem mass spectrometry to completely differentiate between the isomeric dG adducts investigated.     

Formation of a N2-dG:N2-dG carbinolamine DNA cross-link by the trans-4-hydroxynonenal-derived (6S,8R,11S) 1,N2-dG adduct

Michael addition of trans-4-hydroxynonenal (HNE) to deoxyguanosine yields diastereomeric 1,N(2)-dG adducts in DNA. When placed opposite dC in the 5'-CpG-3' sequence, the (6S,8R,11S) diastereomer forms a N(2)-dG:N(2)-dG interstrand cross-link [Wang, H.; Kozekov, I. D.; Harris, T. M.; Rizzo, C. J. J. Am. Chem. Soc.2003, 125, 5687-5700]. We refined its structure in 5'-d(G(1)C(2)T(3)A(4)G(5)C(6)X(7)A(8)G(9)T(10)C(11)C(12))-3'·5'-d(G(13)G(14)A(15)C(16)T(17)C(18)Y(19)C(20)T(21)A(22)G(23)C(24))-3' [X(7) is the dG adjacent to the C6 carbon of the cross-link or the α-carbon of the (6S,8R,11S) 1,N(2)-dG adduct, and Y(19) is the dG adjacent to the C8 carbon of the cross-link or the γ-carbon of the HNE-derived (6S,8R,11S) 1,N(2)-dG adduct; the cross-link is in the 5'-CpG-3' sequence]. Introduction of (13)C at the C8 carbon of the cross-link revealed one (13)C8→H8 correlation, indicating that the cross-link existed predominantly as a carbinolamine linkage. The H8 proton exhibited NOEs to Y(19) H1', C(20) H1', and C(20) H4', orienting it toward the complementary strand, consistent with the (6S,8R,11S) configuration. An NOE was also observed between the HNE H11 proton and Y(19) H1', orienting the former toward the complementary strand. Imine and pyrimidopurinone linkages were excluded by observation of the Y(19)N(2)H and X(7) N1H protons, respectively. A strong H8→H11 NOE and no (3)J((13)C→H) coupling for the (13)C8-O-C11-H11 eliminated the tetrahydrofuran species derived from the (6S,8R,11S) 1,N(2)-dG adduct. The (6S,8R,11S) carbinolamine linkage and the HNE side chain were located in the minor groove. The X(7)N(2) and Y(19)N(2) atoms were in the gauche conformation with respect to the linkage, maintaining Watson-Crick hydrogen bonds at the cross-linked base pairs. A solvated molecular dynamics simulation indicated that the anti conformation of the hydroxyl group with respect to C6 of the tether minimized steric interaction and predicted hydrogen bonds involving O8H with C(20)O(2) of the 5'-neighbor base pair G(5)·C(20) and O11H with C(18)O(2) of X(7)·C(18). These may, in part, explain the stability of this cross-link and the stereochemical preference for the (6S,8R,11S) configuration.     

Ochratoxin a forms a carbon-bonded c8-deoxyguanosine nucleoside adduct: implications for c8 reactivity by a phenolic radical

The ability of the carcinogenic fungal toxin Ochratoxin A (OTA, 1) to react with deoxyguanosine (dG) has been assessed using electrospray mass spectrometry and NMR. Photoexcitation of OTA (100 muM) in the presence of 50 mol equiv of dG led to the isolation and identification of the C8-deoxyguanosine nucleoside adduct 4. Importantly, the same adduct was formed upon oxidative activation of OTA using horseradish peroxidase (HRP)/H2O2 or the transition metals Fe(II) and Cu(II), as evidenced by mass spectrometry. Because the mutagenicity and subsequent carcinogenicity of OTA are believed to stem from oxidative DNA damage (strand scission and oxidative base products) and formation of guanine-specific DNA adducts, the adduct 4 confirms the ability of OTA to react covalently with dG and has important implications for the mechanism of action of OTA and other chlorophenolic toxins that undergo oxidation to yield phenoxyl radicals. The C8 position of dG is susceptible to radical attack, as was amply proven through formation of the hydroxyl radical-derived DNA lesion, 8-oxodeoxyguanosine. The adduct 4 is the first structurally characterized nucleoside adduct of a chlorophenolic toxin, and its formation has important implications for the mutagenicity of phenolic xenobiotics.     

Kinetic and thermodynamic analysis of the hydrolytic ring-opening of the malondialdehyde-deoxyguanosine adduct, 3-(2'-deoxy-beta-D-erythro-pentofuranosyl)- pyrimido[1,2-alpha]purin-10(3H)-one

3-(2'-Deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10(3H)-one (M1dG) is the major reaction product of deoxyguanosine with malondialdehyde or base propenals. M1dG undergoes hydrolytic ring-opening to N2-oxopropenyl-deoxyguanosine (N2OPdG) under basic conditions. We report that ring-opening of M1dG as a nucleoside or in oligonucleotides is a reversible second-order reaction with hydroxide ion. NMR and UV analysis revealed N2OPdG(-) to be the only product of M1dG ring-opening in basic solution. The rate constant for reaction of M1dG with hydroxide is 3.8 M(-1) s(-1), and the equilibrium constant is calculated to be 2.1 +/- 0.3 x 10(4) M(-1) at 25 degrees C. Equilibrium constants determined by spectroscopic analysis of the reaction end-point or by thermodynamic analysis of rate constants determined over a range of temperatures yielded a value 2.5 +/- 0.2 x 10(4) M(-1). Kinetic analysis of ring-opening of M1dG in oligonucleotides indicated the rate constant for ring-opening is decreased 10-fold compared to that in the nucleoside. Flanking purines or pyrimidines did not significantly alter the rate constants for ring-opening, but purines flanking M1dG enhanced the rate constant for the reverse reaction. A mechanism is proposed for ring-opening of M1dG under basic conditions and a role is proposed for duplex DNA in accelerating the rate of ring-opening of M1dG at neutral pH.     

Kinetics and mechanism of the general-acid-catalyzed ring-closure of the malondialdehyde-DNA adduct, N2-(3-oxo-1-propenyl)deoxyguanosine (N2OPdG-), to 3-(2'-Deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin- 10(3H)-one (M1dG)

3-(2'-Deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10(3H)-one (M1dG) is the major product of the reaction of deoxyguanosine with malondialdehyde (MDA). M1dG blocks replication by DNA polymerases in vitro and is mutagenic in vivo. M1dG reacts with hydroxide to form the N2-(3-oxo-1-propenyl)deoxyguanosine anion (N2OPdG-). This reaction is pH-dependent and reverses under neutral and acidic conditions to form M1dG. Here we describe the kinetics and mechanism of the ring-closure reaction in both the nucleoside and oligonucleotides. Kinetic analysis of absorbance and fluorescence changes demonstrates that ring-closure is biphasic, leading to the rapid formation of an intermediate that slowly converts to M1dG in a general-acid-catalyzed reaction. The dependence of the rate of the rapid phase on pH reveals the pKa for protonated N2OPdG is 6.9. One-dimensional 1H NMR and DQF-COSY experiments identified two distinct intermediates, N2OPdG-H and 8-hydroxy-6,7-propenodeoxyguanosine (HO-Prene-dG), that are formed upon acidification of N2OPdG-. Characterization of ring-closure in single-stranded and in melted duplex oligonucleotides shows M1dG formation is also acid-catalyzed in single-stranded oligonucleotides and that the denaturation of an oligonucleotide duplex enhances ring-closure. This work details the complexity of ring-closure in the nucleoside and oligonucleotides and provides new insight into the role of duplex DNA in catalyzing ring-opening and ring-closing of M1dG and N2OPdG.     

Computational and experimental evidence for the structural preference of phenolic C-8 purine adducts

The structural and spectral properties of (ortho and para) C8-aryl-purine adducts formed from carbon attachment by phenolic toxins were investigated through DFT calculations and UV-vis absorbance and emission studies. The global minima of both the deoxyadenosine (dA) and deoxyguanosine (dG) adducts adopted a syn conformation about the glycosidic bond due to the presence of an O5'-H...N3 hydrogen bond, where the anti minima are 20-30 kJ mol-1 higher in energy. While the nucleobase adducts are planar, the presence of the deoxyribose sugar induces a twist about the carbon-carbon bond connecting the phenol and nucleobase rings. ortho-Phenolic adducts are less twisted than the corresponding para adducts due to stabilization provided by an intramolecular O-H...N7 bond. Solvation calculations, in combination with UV-vis studies, demonstrate that the structural preference is solvent dependent, where solvents with hydrogen-bonding abilities disrupt the intramolecular O-H...N7 hydrogen bond such that a greater degree of twist is observed, and less polar solvents stabilize the planar structure. Indeed, the ratio of twisted to planar conformers is estimated to be as large as 50:50 in some aprotic solvents. Thus, the combined experimental and computational approach has provided a greater understanding of the structure of the ortho- and para-dA and dG C-bonded phenoxyl adducts as the first step to understanding the biological consequences of this form of DNA damage.     

Hydrogen bonding contributes to the selectivity of nucleotide incorporation opposite an oxidized abasic lesion

The ability of DNA polymerases to maintain the integrity of the genome even after it has been structurally altered is vital. There is considerable interest in determining the structural properties of the DNA template that polymerases recognize when determining which nucleotide to add to a nascent strand. Mechanistic, synthetic, and structural chemistries have been used to study how DNA polymerase activity is affected by size, shape, pi-stacking, and hydrogen bonds of the template molecules. Herein, we probe the structural aspects of abasic lesions that result in their distinct coding potential in Escherichia coli despite lacking a Watson-Crick base. In particular, we investigate why bypass of 2-deoxyribonolactone (L) results in significant amounts of dG incorporation opposite the lesion, whereas other abasic lesions (e.g., AP) adhere to the "A-rule". Experiments using synthetic analogues reveal that DNA polymerase V bypasses L and increased levels of dG incorporation result from a hydrogen bonding interaction between the carbonyl oxygen and dG. These results show that a DNA polymerase utilizes hydrogen bonding as one structural parameter when decoding an abasic lesion.     

Independent Generation and Time-Resolved Detection of 2′-Deoxyguanosin-N2-yl Radicals

Guanine radicals are important reactive intermediates in DNA damage. Hydroxyl radical (HO^.) has long been believed to react with 2′-deoxyguanosine (dG) generating 2′-deoxyguanosin-N1-yl radical (dG(N1-H)^.) via addition to the nucleobase π-system and subsequent dehydration. This basic tenet was challenged by an alternative mechanism, in which the major reaction of HO^. with dG was proposed to involve hydrogen atom abstraction from the N2-amine. The 2′-deoxyguanosin-N2-yl radical (dG(N2-H)^.) formed was proposed to rapidly tautomerize to dG(N1-H)^.. We report the first independent generation of dG(N2-H)^. in high yield via photolysis of 1 . dG(N2-H)^. is directly observed upon nanosecond laser flash photolysis (LFP) of 1 . The absorption spectrum of dG(N2-H)^. is corroborated by DFT studies, and anti- and syn-dG(N2-H)^. are resolved for the first time. The LFP experiments showed no evidence for tautomerization of dG(N2-H)^. to dG(N1-H)^. within hundreds of microseconds. This observation suggests that the generation of dG(N1-H)^. via dG(N2-H)^. following hydrogen atom abstraction from dG is unlikely to be a major pathway when HO^. reacts with dG.     

Can Copper(II) Mediate Hoogsteen Base‐Pairing in a Left‐Handed DNA Duplex? A Pulse EPR Study

Pulse EPR spectroscopy is used to investigate possible structural features of the copper(II) ion coordinated to poly(dG‐dC)?poly(dG‐dC) in a frozen aqueous solution, and the structural changes of the polynucleotide induced by the presence of the metal ion. Two different copper species were identified and their geometry explained by a molecular model. According to this model, one species is exclusively coordinated to a single guanine with the N7 nitrogen atom forming a coordinative bond with the copper. In the other species, a guanine and a cytosine form a ternary complex together with the copper ion. A copper crosslink between the N7 of guanine and N3 of cytosine is proposed as the most probable coordination site. Moreover, no evidence was found for an interaction of either copper species with a phosphate group or equatorial water molecules. In addition, circular dichroism (CD) spectroscopy showed that the DNA of the Cu^II‐poly(dG‐dC)?poly(dG‐dC) adducts resembles the left‐handed Z‐form. These results suggest that metal‐mediated Hoogsteen base pairing, as previously proposed for a right‐handed DNA duplex, can also occur in a double‐stranded left‐handed DNA.     

Independent Generation and Time‐Resolved Detection of 2′‐Deoxyguanosin‐N2‐yl Radicals

Guanine radicals are important reactive intermediates in DNA damage. Hydroxyl radical (HO^.) has long been believed to react with 2′‐deoxyguanosine (dG) generating 2′‐deoxyguanosin‐N1‐yl radical (dG(N1‐H)^.) via addition to the nucleobase π‐system and subsequent dehydration. This basic tenet was challenged by an alternative mechanism, in which the major reaction of HO^. with dG was proposed to involve hydrogen atom abstraction from the N2‐amine. The 2′‐deoxyguanosin‐N2‐yl radical (dG(N2‐H)^.) formed was proposed to rapidly tautomerize to dG(N1‐H)^.. We report the first independent generation of dG(N2‐H)^. in high yield via photolysis of 1 . dG(N2‐H)^. is directly observed upon nanosecond laser flash photolysis (LFP) of 1 . The absorption spectrum of dG(N2‐H)^. is corroborated by DFT studies, and anti‐ and syn‐dG(N2‐H)^. are resolved for the first time. The LFP experiments showed no evidence for tautomerization of dG(N2‐H)^. to dG(N1‐H)^. within hundreds of microseconds. This observation suggests that the generation of dG(N1‐H)^. via dG(N2‐H)^. following hydrogen atom abstraction from dG is unlikely to be a major pathway when HO^. reacts with dG.     

N(2)-phenyldeoxyguanosine: modulation of the chemical properties of deoxyguanosine toward one-electron oxidation in DNA

We have shown here that (1) substitution of an exocyclic amino group of dG is effective in modulating the chemical properties of dG toward one-electron oxidation and (2) decomposition of the guanine radical cation was effectively suppressed near dPhG. These results indicate that dPhG is a prototype of nucleosides functioning as an intrinsic antioxidant of duplex DNA toward one-electron oxidation.     

Identifying guanosine self assembly at natural isotopic abundance by high-resolution 1H and 13C solid-state NMR spectroscopy

By means of the (1)H chemical shifts and the proton-proton proximities as identified in (1)H double-quantum (DQ) combined rotation and multiple-pulse spectroscopy (CRAMPS) solid-state NMR correlation spectra, ribbon-like and quartet-like self-assembly can be identified for guanosine derivatives without isotopic labeling for which it was not possible to obtain single crystals suitable for diffraction. Specifically, characteristic spectral fingerprints are observed for dG(C10)(2) and dG(C3)(2) derivatives, for which quartet-like and ribbon-like self-assembly has been unambiguously identified by (15)N refocused INADEQUATE spectra in a previous study of (15)N-labeled derivatives (Pham, T. N.; et al. J. Am. Chem. Soc.2005, 127, 16018). The NH (1)H chemical shift is observed to be higher (13-15 ppm) for ribbon-like self-assembly as compared to 10-11 ppm for a quartet-like arrangement, corresponding to a change from NH···N to NH···O intermolecular hydrogen bonding. The order of the two NH(2)(1)H chemical shifts is also inverted, with the NH(2) proton closest in space to the NH proton having a higher or lower (1)H chemical shift than that of the other NH(2) proton for ribbon-like as opposed to quartet-like self-assembly. For the dG(C3)(2) derivative for which a single-crystal diffraction structure is available, the distinct resonances and DQ peaks are assigned by means of gauge-including projector-augmented wave (GIPAW) chemical shift calculations. In addition, (14)N-(1)H correlation spectra obtained at 850 MHz under fast (60 kHz) magic-angle spinning (MAS) confirm the assignment of the NH and NH(2) chemical shifts for the dG(C3)(2) derivative and allow longer range through-space N···H proximities to be identified, notably to the N7 nitrogens on the opposite hydrogen-bonding face.     

Fapy.dG instructs Klenow exo(-) to misincorporate deoxyadenosine

The effects of Fapy.dG (N-(2-deoxy-alpha,beta-d-erythropentofuranosyl)-N-(2,6-diamino-4-hydroxy-5-formamidopyrimidine)) on the activity of Klenow exo- have been determined by using oligonucleotide substrates containing the lesion at a defined site. Fapy.dG inhibits primer polymerization at two positions: nucleotide incorporation opposite the lesion and extension one nucleotide past the lesion. Klenow exo- is inhibited less by Fapy.dG than by its analogue, MeFapy.dG. Fapy.dG instructs the polymerase to misincorporate deoxyadenosine opposite itself 20 times more frequently than does dG. Extension of the primer containing the Fapy.dG:dA base pair is only slightly less efficient than when dC is opposite the lesion. Overall, Fapy.dG increases the probability that Klenow exo- will make a mistake during replication approximately 80-million fold compared to a template containing the native nucleotide, dG.     

Synthesis, DNA polymerase incorporation, and enzymatic phosphate hydrolysis of formamidopyrimidine nucleoside triphosphates

The nucleoside triphosphates of N6-(2-deoxy-alpha,beta-d-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy.dGTP) and its C-nucleoside analogue (beta-C-Fapy.dGTP) were synthesized. The lability of the formamide group required that nucleoside triphosphate formation be carried out using an umpolung strategy in which pyrophosphate was activated toward nucleophilic attack. The Klenow fragment of DNA polymerase I from Escherichia coli accepted Fapy.dGTP and beta-C-Fapy.dGTP as substrates much less efficiently than it did dGTP. Subsequent extension of a primer containing either modified nucleotide was less affected compared to when the native nucleotide is present at the 3'-terminus. The specificity constants are sufficiently large that nucleoside triphosphate incorporation could account for the level of Fapy.dG observed in cells if 1% of the dGTP pool is converted to Fapy.dGTP. Similarly, polymerase-mediated introduction of beta-C-Fapy.dG could be useful for incorporating useful amounts of this nonhydrolyzable analogue for use as an inhibitor of base excision repair. The kinetic viability of these processes is enhanced by inefficient hydrolysis of Fapy.dGTP and beta-C-Fapy.dGTP by MutT, the E. coli enzyme that releases pyrophosphate and the corresponding nucleoside monophosphate upon reaction with structurally related nucleoside triphosphates.