Reactivity of DNA guanyl radicals with phenolate anions

Guanine bases are the most easily oxidized sites in DNA. Electron-deficient guanine species are major intermediates produced in DNA by the direct effect of ionizing radiation (ionization of the DNA itself) because of preferential hole migration within DNA to guanine bases. By using thiocyanate ions to modify the indirect effect (ionization of the solvent), we are able to produce these single-electron-oxidized guanine radical species in dilute aqueous solutions of plasmid DNA where the direct effect is negligible. The guanyl radical species produce stable modified guanine products. They can be detected in the plasmid by converting them to strand breaks after incubation with a DNA repair enzyme. If a phenol is present during irradiation, the yield of modified guanines is decreased. The mechanism is reduction of the guanine radical species by the phenol. It is possible to derive a rate constant for the reaction of the phenol with the guanyl radical. The pH dependence shows that phenolate anions are more reactive than their conjugate acids, although the difference for guanyl radicals is smaller than with other single-electron-oxidizing agents. At physiological pH values, the reduction of a guanyl radical entails the transfer of a proton in addition to the electron. The relatively small dependence of the rate constant on the driving force implies that the electron cannot be transferred before the proton. These results emphasize the potential importance of acidic tyrosine residues and the intimate involvement of protons in DNA repair.     

Structure reactivity relationship in the reaction of DNA guanyl radicals with hydroxybenzoates

In DNA, guanine bases are the sites from which electrons are most easily removed. As a result of hole migration to this stable location on guanine, guanyl radicals are major intermediates in DNA damage produced by the direct effect of ionizing radiation (ionization of the DNA itself and not through the intermediacy of water radicals). We have modeled this process by employing gamma irradiation in the presence of thiocyanate ions, a method which also produces single electron oxidized guanyl radicals in plasmid DNA in aqueous solution. The stable products formed in DNA from these radicals are detected as strand breaks after incubation with the FPG protein. When a phenolic compound is present in the solution during gamma irradiation, the formation of guanyl radical species is decreased by electron donation from the phenol to the guanyl radical. We have quantified the rate of this reaction for four different phenolic compounds bearing carboxylate substituents as proton acceptors. A comparison of the rates of these reactions with the redox strengths of the phenolic compounds reveals that salicylate reacts ca. 10-fold faster than its structural analogs. This observation is consistent with a reaction mechanism involving a proton coupled electron transfer, because intra-molecular transfer of a proton from the phenolic hydroxyl group to the carboxylate group is possible only in salicylate, and is favored by the strong 6-membered ring intra-molecular hydrogen bond in this compound.     

Repair of guanyl radicals in plasmid DNA by electron transfer is coupled to proton transfer

By using gamma-irradiation in the presence of thiocyanate ions, we have generated guanyl radicals in plasmid DNA. These can be detected by using an Escherichia coli base excision repair endonuclease to convert their stable end products to strand breaks. The yield of enzyme-sensitive sites is strongly attenuated by the presence of micromolar concentrations of one of a series of singly substituted phenols, and it is possible to derive bimolecular rate constants for the reduction of DNA guanyl radicals by these phenols. More strongly reducing phenols were found to react more rapidly. This electron-transfer reaction also involves a proton transfer. By comparing the expected energetics of the reaction with the observed rate constants, the electron transfer is found to be mechanistically coupled with the proton transfer.     

Cytosine-gated hole creation and transfer in DNA in aqueous solution

The selenite radical, SeO3-, has been found to selectively produce the cytosyl radical upon one-electron oxidation of duplex DNA. This is at first a surprising result as SeO3- can only oxidize guanine of the DNA bases, implying that the transiently formed guanyl radical cation must transpose into the neutral cytosyl radical with loss of a proton. Back oxidation to produce the neutral guanyl radical, in competition with another fixation reaction, is observed.     

Triplet state mechanism for electron transfer oxidation of DNA

The interaction of anthraquinone-2-sulfonate with nucleotides and DNA in acetonitrile and acetonitrile water solvent mixture have been studied using KrF laser photolysis aimed at elucidation of the reaction mechanism. Laser spectroscopy directly demonstrates that the initial species from interaction of anthraquinone-2-sulfonate with nucleotides are radical cations of nucleotides and radical anion of anthraquinone-2-sulfonate. In addition, formation of ion pair from interaction of any of nucleotides with anthraquinone-2-sulfonate is synchronous with decay of triplet anthraquinone-2-sulfonate, which has provided dynamic evidence for initiation of electron transfer from DNA bases to triplet anthraquinone-2-sulfonate. Moreover, direct observation of stabilized DNA guanyl radical cation from interaction of anthraquinone-2-sulfonate with DNA has provided initial evidence for selective cleavage of DNA at guanine moiety. The solvent-separated ion pairs in acetonitrile have evidently dissociated into free ions, thereby enabling independent study of the behavior of guanyl radical cations and radical anion of anthraquinone-2-sulfonate.     

Reductive tert-Butylation of Anils by tert-Butylmercury Halides(1)

tert-Butyl radicals add to the carbon atom of benzylideneanilines to form anilino radicals, which are protonated in the presence of PTSA or NH(4)(+) in Me(2)SO. Reduction of the resulting aniline radical cations occurs readily by the ate complex, t-BuHgI(2)(-). In the absence of a proton donor, t-BuHgI will also transfer a hydrogen atom to the anilino radical to give the reductive alkylation product. Protonation can promote a free radical chain process involving electron transfer by substrate activation and/or by increasing the electron affinity of the intermediate radicals. Since the adduct radicals formed from benzylideneanilines are more easily protonated than the parent Schiff bases, PTSA but not NH(4)(+) demonstrates substrate activation, although both proton donors promote the free radical reaction.     

Direct chemical evidence for charge transfer between photoexcited 2-aminopurine and guanine in duplex DNA

Photoexcited 2-aminopurine (Ap*) is extensively exploited as a fluorescent base analogue in the study of DNA structure and dynamics. Quenching of Ap* in DNA is often attributed to stacking interactions between Ap* and DNA bases, despite compelling evidence indicating that charge transfer (CT) between Ap* and DNA bases contributes to quenching. Here we present direct chemical evidence that Ap* undergoes CT with guanine residues in duplex DNA, generating oxidative damage at a distance. Irradiation of Ap in DNA containing the modified guanine, cyclopropylguanosine (CPG), initiates hole transfer from Ap* followed by rapid ring opening of the CPG radical cation. Ring opening accelerates hole trapping to a much shorter time regime than for guanine radicals in DNA; consequently, trapping effectively competes with back electron transfer (BET) leading to permanent CT chemistry. Significantly, BET remains competitive, even with this much faster trapping reaction, consistent with measured kinetics of DNA-mediated CT. The distance dependence of BET is sharper than that of forward CT, leading to an inverted dependence of product yield on distance; at short distances product yield is inhibited by BET, while at longer distances trapping dominates, leading to permanent products. The distance dependence of product yield is distinct from forward CT, or charge injection. As with photoinduced charge transfer in other chemical and biological systems, rapid kinetics for charge injection into DNA need not be associated with a high yield of DNA damage products.     

Radical-based alkylation of guanine derivatives in aqueous medium

The radical-based alkylation of 8-bromoguanosine (1a) and 8-bromo-2'-deoxyguanosine (1b) at the C8 position has been investigated in aqueous solutions. Alkyl radicals were generated by scavenging of the primary species of γ-radiolysis by the alcohol substrate. These reactions result in the efficient formation of intermolecular C-C bonds in aqueous media, by using the reactivity of α-hydroxyalkyl radicals derived from alcohols with 1a and 1b. A mechanism for the formation of C8 guanine alkylated adducts has been proposed, based on the quantification of radiation chemical yields for the disappearance of starting material and the formation of all products. Two α-hydroxyalkyl radicals are needed to form an alkylated guanine, the first one adding to C8 followed by ejection of Br(-) with formation of guanyl adduct and the second one acting as reducing agent of the guanyl adduct.     

Methylation of DNA bases by methyl free radicals: mechanism of formation of C8-methylguanine

The C8-methylguanine (C8mG) lesions are reported to be produced in vivo due to methylation of guanine base of DNA by methyl free (·CH₃) radicals derived from the carcinogen 1,2-dimethylhydrazines and tert-butylhydroperoxide. It is believed that C8mG lesions can induce G to T and G to C transversion mutations and deletions. However, the mechanisms of reactions of ·CH₃ radicals with DNA bases leading to formation of C8mG and other methylated DNA bases and their biological implications are not properly understood. In the present contribution, we have carried out density functional theory (DFT) calculations to ascertain the various stable methylated derivatives of all the four DNA bases that are formed by the attack of ·CH₃ radicals on DNA bases as well as to understand the mechanism of formation of C8mG due to reaction of ·CH₃ radicals with the C8 site of guanine. Our calculations reveal that ·CH₃ radical would form stable methylated products at the C8 sites of purine bases (guanine and adenine) and at the C5 and C6 sites of pyrimidine bases (cytosine and thymine) by directly attacking to bases. The C8mG is the most stable. This is in agreement with experimental observation. Further, we have found that in absence of any external agents, the C8mG is formed preferably by direct addition of a ·CH₃ radical to the C8 site of guanine followed by abstraction of the H8 hydrogen atom by another ·CH₃ radical. The barrier energies for these two steps are found to be 18.16 (18.73) and 16.05 (18.54) kcal/mol, respectively, as determined at the M06-2X/6-311+G(d,p) level of theory in gas phase (aqueous media). Thus, the present study explains the mechanism of formation of C8mG.     

The Two Faces of the Guanyl Radical: Molecular Context and Behavior

The guanyl radical or neutral guanine radical G(-H)^• results from the loss of a hydrogen atom (H^•) or an electron/proton (e^–/H⁺) couple from the guanine structures (G). The guanyl radical exists in two tautomeric forms. As the modes of formation of the two tautomers, their relationship and reactivity at the nucleoside level are subjects of intense research and are discussed in a holistic manner, including time-resolved spectroscopies, product studies, and relevant theoretical calculations. Particular attention is given to the one-electron oxidation of the GC pair and the complex mechanism of the deprotonation vs. hydration step of GC^•+ pair. The role of the two G(-H)^• tautomers in single- and double-stranded oligonucleotides and the G-quadruplex, the supramolecular arrangement that attracts interest for its biological consequences, are considered. The importance of biomarkers of guanine DNA damage is also addressed.     

Highly oxidizing excited states of one-electron-oxidized guanine in DNA: wavelength and pH dependence

Excited states of one-electron-oxidized guanine in DNA are known to induce hole transfer to the sugar moiety and on deprotonation result in neutral sugar radicals that are precursors of DNA strand breaks. This work carried out in a homogeneous aqueous glass (7.5 M LiCl) at low temperatures (77-175 K) shows the extent of photoconversion of one-electron-oxidized guanine and the associated yields of individual sugar radicals are crucially controlled by the photon energy, protonation state, and strandedness of the oligomer. In addition to sugar radical formation, highly oxidizing excited states of one-electron-oxidized guanine are produced with 405 nm light at pH 5 and below that are able to oxidize chloride ion in the surrounding solution to form Cl(2)(?-) via an excited-state hole transfer process. Among the various DNA model systems studied in this work, the maximum amount of Cl(2)(?-) is produced with ds (double-stranded) DNA, where the one-electron-oxidized guanine exists in its cation radical form (G(?+):C). Thus, via excited-state hole transfer, the dsDNA is apparently able to protect itself from cation radical excited states by transfer of damage to the surrounding environment.     

Interactions of Amino Acids with Oxidized Guanine in the Gas Phase Associated with the Protection of Damaged DNA

Density functional theory calculations were employed to study the stabilization process of the guanine radical cation through amino acid interactions as well as to understand the protection mechanisms. On the basis of our calculations, several protection mechanisms are proposed in this work subject to the type of the amino acid. Our results indicate that a series of three‐electron bonds can be formed between the amino acids and the guanine radical cation which may serve as relay stations supporting hole transport. In the three‐electron‐bonded, π–π‐stacked, and H‐bonded modes, amino acids can protect guanine from oxidation or radiation damage by sharing the hole, while amino acids with reducing properties can repair the guanine radical cation through proton‐coupled electron transfer or electron transfer. Another important finding is that positively charged amino acids (ArgH⁺, LysH⁺, and HisH⁺) can inhibit ionization of guanine through raising its ionization potential. In this situation, a negative dissociation energy for hydrogen bonds in the hole‐trapped and positively charged amino acid–Guanine dimer is observed, which explains the low hole‐trapping efficiency. We hope that this work provides valuable information on how to protect DNA from oxidation‐ or radiation‐induced damages in biological systems.     

Formation of sugar radicals in RNA model systems and oligomers via excitation of guanine cation radical

In previous work, we have shown that photoexcitation of guanine cation radical (G*+) in frozen aqueous solutions of DNA and its model compounds at 143 K results in the formation of neutral sugar radicals with substantial yield. In this report, we present electron spin resonance (ESR) and theoretical (DFT) evidence regarding the formation of sugar radicals after photoexcitation of guanine cation radical (G*+) in frozen aqueous solutions of one-electron-oxidized RNA model compounds (nucleosides, nucleotides and oligomers) at 143 K. Specific sugar radicals C5'*, C3'* and C1'* were identified employing derivatives of Guo deuterated at specific sites in the sugar moiety, namely, C1'-, C2'-, C3'- and C5'-. These results suggest C2'* is not formed upon photoexcitation of G*+ in one-electron-oxidized Guo and deuterated Guo derivatives. Phosphate substitution at C5'- (i.e., in 5-GMP) hinders formation of C5'* via photoexcitation at 143 K but not at 77 K. For the RNA-oligomers studied, we observe on photoexcitation of oligomer-G*+ the formation of mainly C1'* and an unidentified radical with a ca. 28 G doublet. The hyperfine coupling constants of each of the possible sugar radicals were calculated employing the DFT B3LYP/6-31G* approach for comparison to experiment. This work shows that formation of specific neutral sugar radicals occurs via photoexcitation of guanine cation radical (G*+) in RNA systems but not by photoexcitation of its N1 deprotonated species (G(-H)*). Thus, our mechanism regarding neutral sugar formation via photoexcitation of base cation radicals in DNA appears to be valid for RNA systems as well.     

Photoinduced electron transfer in naphthalimide-pyridine systems: effect of proton transfer on charge recombination efficiencies

We studied the effect of proton-coupled electron transfer on lifetimes of the charge-separated radicals produced upon light irradiation of the thiomethyl-naphthalimide donor SMe-NI-H in the presence of nitro-cyano-pyridine acceptor (NO(2)-CN-PYR). The dynamics of electron and proton transfer were studied using femtosecond pump-probe spectroscopy in the UV/vis range. We find that the photoinduced electron transfer between excited SMe-NI-H and NO(2)-CN-PYR occurs with a rate of 1.1 × 10(9) s(-1) to produce radical ions SMe-NI-H(?+) and NO(2)-CN-PYR(?-). These initially produced radical ions in a solvent cage do not undergo a proton transfer, possibly due to unfavorable geometry between N-H proton of the naphthalimide and aromatic N-atom of the pyridine. Some of the radical ions in the solvent cage recombine with a rate of 2.3 × 10(10) s(-1), while some escape the solvent cage and recombine at a lower rate (k = 4.27 × 10(8) s(-1)). The radical ions that escape the solvent cage undergo proton transfer to produce neutral radicals SMe-NI(?) and NO(2)-CN-PYR-H(?). Because neutral radicals are not attracted to each other by electrostatic interactions, their recombination is slower that the recombination of the radical ions formed in model compounds that can undergo only electron transfer (SMe-NI-Me and NO(2)-CN-PYR, k = 1.2 × 10(9) s(-1)). The results of our study demonstrate that proton-coupled electron transfer can be used as an efficient method to achieve long-lived charge separation in light-driven processes.     

Electron Transfer through DNA in the Course of Radical-Induced Strand Cleavage

No benefit from base stacking is observed for rates of electron transfer in DNA. This conclusion was drawn from experiments with a new DNA assay in which a radical cationic site, generated by strand cleavage, can be reduced by the guanine bases in the same DNA (the electron transfer is indicated by arrows in the diagram). The distance dependence of this electron transfer step is determined by the chemical yield of the reduction product.     

Electron attachment to a hydrated DNA duplex: the dinucleoside phosphate deoxyguanylyl-3',5'-deoxycytidine

The minimal essential section of DNA helices, the dinucleoside phosphate deoxyguanylyl-3',5'-deoxycytidine dimer octahydrate, [dGpdC](2), has been constructed, fully optimized, and analyzed by using quantum chemical methods at the B3LYP/6-31+G(d,p) level of theory. Study of the electrons attached to [dGpdC](2) reveals that DNA double strands are capable of capturing low-energy electrons and forming electronically stable radical anions. The relatively large vertical electron affinity (VEA) predicted for [dGpdC](2) (0.38 eV) indicates that the cytosine bases are good electron captors in DNA double strands. The structure, charge distribution, and molecular orbital analysis for the fully optimized radical anion [dGpdC](2)(·-) suggest that the extra electron tends to be redistributed to one of the cytosine base moieties, in an electronically stable structure (with adiabatic electron affinity (AEA) 1.14 eV and vertical detachment energy (VDE) 2.20 eV). The structural features of the optimized radical anion [dGpdC](2)(·-) also suggest the probability of interstrand proton transfer. The interstrand proton transfer leads to a distonic radical anion [d(G-H)pdC:d(C+H)pdG](·-), which contains one deprotonated guanine anion and one protonated cytosine radical. This distonic radical anion is predicted to be more stable than [dGpdC](2)(·-). Therefore, experimental evidence for electron attachment to the DNA double helices should be related to [d(G-H)pdC:d(C+H)pdG](·-) complexes, for which the VDE might be as high as 2.7 eV (in dry conditions) to 3.3 eV (in fully hydrated conditions). Effects of the polarizable medium have been found to be important for increasing the electron capture ability of the dGpdC dimer. The ultimate AEA value for cytosine in DNA duplexes is predicted to be 2.03 eV in aqueous solution.     

Biphotonic Ionization of DNA: From Model Studies to Cell

Oxidation reactions triggered by low‐intensity UV photons represent a minor contribution with respect to the overwhelming pyrimidine base dimerization in both isolated and cellular DNA. The situation is totally different when DNA is exposed to high‐intensity UVC radiation under conditions where biphotonic ionization of the four main purine and pyrimidine bases becomes predominant at the expense of singlet excitation processes. The present review article provides a critical survey of the main chemical reactions of the base radical cations thus generated by one‐electron oxidation of nucleic acids in model systems and cells. These include oxidation of the bases with the predominant formation of 8‐oxo‐7,8‐dihydroguanine as the result of preferential hole transfer to guanine bases that act as sinks in isolated and cellular DNA. In addition to hydration, other nucleophilic addition reactions involving the guanine radical cation give rise to intra‐ and interstrand cross‐links together with DNA–protein cross‐links. Information is provided on the utilization of high‐intensity UV laser pulses as molecular biology tools for studying DNA conformational features, nucleic acid–protein interactions and nucleic acid reactivity through DNA–protein cross‐links and DNA footprinting experiments.     

Theoretical study of fast repair of DNA damage by cistanoside C and analogs: mechanism and docking

Experiments show that the natural substances phenylpropanoid glycosides (PPGs) extracted from pelicularis spicata are capable of repairing DNA damaged by oxygen radicals. Based on kinetic measurements and experiments on tumor cells, a theoretical study of the interaction between PPG molecules and isolated DNA bases, as well as a DNA fragment has been performed. An interaction mechanism reported early has been refined. The docking calculations performed using junction minimization of nucleic acids (JUMNA) software showed that the PPG molecules can be docked into the minor groove of DNA and form complexes with the geometry suitable for an electron transfer between guanine radical and the ligand. Such complexes can be formed without major distortions of DNA structure and are further stabilized by the interaction with the rhamnosyl side-groups.     

Antioxidant activity of trans-resveratrol toward hydroxyl and hydroperoxyl radicals: a quantum chemical and computational kinetics study

In this work, we have carried out a systematic study of the antioxidant activity of trans-resveratrol toward hydroxyl ((?)OH) and hydroperoxyl ((?)OOH) radicals in aqueous simulated media using density functional quantum chemistry and computational kinetics methods. All possible mechanisms have been considered: hydrogen atom transfer (HAT), proton-coupled electron transfer (PCET), sequential electron proton transfer (SEPT), and radical adduct formation (RAF). Rate constants have been calculated using conventional transition state theory in conjunction with the Collins-Kimball theory. Branching ratios for the different paths contributing to the overall reaction, at 298 K, are reported. For the global reactivity of trans-resveratrol toward (?)OH radicals, in water at physiological pH, the main mechanism of reaction is proposed to be the sequential electron proton transfer (SEPT). However, we show that trans-resveratrol always reacts with (?)OH radicals at a rate that is diffusion-controlled, independent of the reaction pathway. This explains why trans-resveratrol is an excellent but very unselective (?)OH radical scavenger that provides antioxidant protection to the cell. Reaction between trans-resveratrol and the hydroperoxyl radical occurs only by phenolic hydrogen abstraction. The total rate coefficient is predicted to be 1.42 × 10(5) M(-1) s(-1), which is much smaller than the ones for reactions of trans-resveratrol with (?)OH radicals, but still important. Since the (?)OOH half-life time is several orders larger than the one of the (?)OH radical, it should contribute significantly to trans-resveratrol oxidation in aqueous biological media. Thus, trans-resveratrol may act as an efficient (?)OOH, and also presumably (?)OOR, radical scavenger.