Involvement of proton transfer in the reductive repair of DNA guanyl radicals by aniline derivatives

The most easily oxidized sites in DNA are the guanine bases, and major intermediates produced by the direct effect of ionizing radiation (ionization of the DNA itself) are electron deficient guanine species. By means of a radiation chemical method (gamma-irradiation of aqueous thiocyanate), we are able to produce these guanyl radicals in dilute aqueous solutions of plasmid DNA where the direct effect would otherwise be negligible. Stable modified guanine products are formed from these radicals. They can be detected in the plasmid conversion to strand breaks after a post-irradiation incubation with a DNA base excision endonuclease enzyme. If aniline compounds are also present, the yield of modified guanines is strongly attenuated. The mechanism responsible for this effect is electron donation from the aniline compound to the guanyl radical, and it is possible to derive rate constants for this reaction. Aniline compounds bearing electron withdrawing groups (e.g., 4-CF3) were found to be less reactive than those bearing electron donating groups (e.g., 4-CH3). At physiological pH values, the reduction of a guanyl radical involves the transfer of a proton as well as of an electron. The mild dependence of the rate constant on the driving force suggests that the electron is not transferred before the proton. Although the source of the proton is unclear, our observations emphasize the importance of an accompanying proton transfer in the reductive repair of oxidative damage to guanine bases which are located in a biologically active double stranded plasmid DNA substrate.     

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.     

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.     

Role of the guanine N1 imino proton in the migration and reaction of radical cations in DNA oligomers

Oxidation of a guanine nucleobase to its radical cation in DNA oligomers causes an increase in the acidity of the N1 imino proton that may lead to its spontaneous transfer to N3 of the paired cytosine. This proton transfer is suspected of playing an important role in long-distance radical cation hopping in DNA and the decisive product-determining role in the reaction of the radical cation with H2O or O2. We prepared and investigated DNA oligomers in which certain deoxycytidines are replaced by 5-fluoro-2'-deoxycytidines (F5dC). The pKa of F5C was determined to be 1.7 units below that of dC, which causes proton transfer from the guanine radical cation to be thermodynamically unfavorable. Photoinitiated one-electron oxidation of the DNA by UV irradiation of a covalently attached anthraquinone derivative introduces a radical cation that hops throughout the oligomer and is trapped selectively at GG steps. The introduction of F5dC does not affect the efficiency of charge hopping, but it significantly reduces the amount of reaction at the GG sites, as revealed by subsequent reaction with formamidopyrimidine glycosylase. These findings suggest that transfer of the guanine radical cation N1 proton to cytosine does not play a significant role in long-range charge transfer, but this process does influence the reactions with H2O and/or O2.     

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.     

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.     

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.     

The flash-quench technique in protein-DNA electron transfer: reduction of the guanine radical by ferrocytochrome c

Electron transfer from a protein to oxidatively damaged DNA, specifically from ferrocytochrome c to the guanine radical, was examined using the flash-quench technique. Ru(phen)2dppz2+ (dppz = dipyridophenazine) was employed as the photosensitive intercalator, and ferricytochrome c (Fe3+ cyt c), as the oxidative quencher. Using transient absorption and time-resolved luminescence spectroscopies, we examined the electron-transfer reactions following photoexcitation of the ruthenium complex in the presence of poly(dA-dT) or poly(dG-dC). The luminescence-quenching titrations of excited Ru(phen)2dppz2+ by Fe3+ cyt c are nearly identical for the two DNA polymers. However, the spectral characteristics of the long-lived transient produced by the quenching depend strongly upon the DNA. For poly(dA-dT), the transient has a spectrum consistent with formation of a [Ru(phen)2dppz3+, Fe2+ cyt c] intermediate, indicating that the system regenerates itself via electron transfer from the protein to the Ru(III) metallointercalator for this polymer. For poly(dG-dC), however, the transient has the characteristics expected for an intermediate of Fe2+ cyt c and the neutral guanine radical. The characteristics of the transient formed with the GC polymer are consistent with rapid oxidation of guanine by the Ru(III) complex, followed by slow electron transfer from Fe2+ cyt c to the guanine radical. These experiments show that electron holes on DNA can be repaired by protein and demonstrate how the flash-quench technique can be used generally in studying electron transfer from proteins to guanine radicals in duplex DNA.     

双(萘-类硫脲盐)化合物的光化学反应

合成了一种带芳基的硫脲盐类化合物,对其在光照下的光化学和光物理行为进行了较详细的研究.发现该化合物在光照下,能通过光诱导的分子内电子转移,产生离子自由基和自由基,继而有可能经自由基的重合反应形成环状化合物,并引起所含芳基基团处于合适的易于出现很强激基缔合物的位置.为进一步搞清上述反应机制,工作中设计了相应的实验,包括:ESR的测定、加入稳定的氮氧自由基化合物以及加入β-CD来阻抑重合反应的进行等,以证明上述的看法.     

Studies on curcumin and curcuminoids. XXXIV. Photophysical properties of a symmetrical, non-substituted curcumin analogue

Curcumin is the main constituent of curry. In its ground state it shows chemo-preventive, chemo-therapeutic, anti-inflammatory and immune stimulating effects, and it is considered as a drug or drug model in the treatment of AIDS and cystic fibrosis. Further biological activity is induced in curcumin by light exposure: cytotoxicity is enhanced and photosensitized antibacterial effects are achieved. For the curcumin cis enol conformer, the fastest deactivation mechanism of the first excited singlet state is an excited-state intra-molecular proton transfer, which brings curcumin back to the ground state. This mechanism, as well as reketonization, interaction with the solvent and photodegradation, compete with the phototherapeutic action. The native compound curcumin carries phenolic hydroxyl and methoxy groups that influence the molecular charge distribution and hence the excited-state intra-molecular proton transfer rate in an unpredictable way. In this work we study static and time-resolved spectroscopic properties of a non-substituted curcuminoid that lacks both the phenolic hydroxyl and the phenolic methoxy groups. The photophysical properties of this compound are compared to those of native curcumin, in order to provide a rationale to the design of curcuminoids with molecular structures optimized for a photosensitizer.     

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.     

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.     

Carboxylates as proton-accepting groups in concerted proton-electron transfers. Electrochemistry of the 2,5-dicarboxylate 1,4-hydrobenzoquinone/2,5-dicarboxy 1,4-benzoquinone couple

Concerted proton and electron transfers (CPET) currently attract considerable theoretical and experimental attention, notably in view of their likely involvement in many enzymatic reactions. The role of carboxylate groups as proton-accepting sites in CPET reactions is explored by means of a cyclic voltammetric investigation of the 2,5-dicarboxy 1,4-benzoquinone/2,5-dicarboxylate 1,4-hydrobenzoquinone couple in a nonaqueous medium. The presence of carboxylate groups ortho to the phenol groups induces the removal of an electron to be coupled with the transfer of the phenolic proton to a carboxylate oxygen. The kinetics of the electrochemical reaction and the observation of a significant hydrogen/deuterium kinetic isotope effect unambiguously indicate that electron transfer and proton transfer are concerted, thus providing an additional demonstration of the role of carboxylate groups as proton-accepting sites in concerted proton-electron transfer reactions.     

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.     

Mechanisms of photosensitized DNA cleavage

Photosensitization may promote DNA damages such as nucleic acid oxidation or single strand breaks via three main pathways: hydroxyl radicals attack, electron transfer process or oxidation by singlet oxygen. While direct production of OH^. by photosensitization is rarely observed, the mechanism of DNA attack by OH^. is now well established on the basis of informations provided by water radiolysis experiments. Some dyes may also induce single strand breaks via an electron transfer occurring from a nucleobase to the sensitizer in the excited state. This process generates base radical cations identical to those arising from DNA photoionisation. These radicals may undergo deprotonation or dehydration to form the same neutral radicals as those produced by OH^. but with a slightly different pattern. In contrast, while many sensitizers produce singlet oxygen, the mechanism of DNA damages induced by this way is still unclear. In this case the guanine moiety in nucleosides or in DNA is selectively altered leading to the formation of 8 oxoG or 8 oxodG and FapyGua. The mechanism of single strand breaks formation by singlet oxygen is discussed in this overview.     

Synthesis and Characterization of Redox‐Active Charge‐Transfer Complexes with 2,3,5,6‐Tetracyanopyridine (TCNPy) for the Photogeneration of Pyridinium Radicals

The heteroaromatic polynitrile compound tetracyanopyridine (TCNPy) is introduced as a new electron acceptor for the formation of deeply colored charge‐transfer complexes. In MeCN, TCNPy is characterized by a quasireversible one‐electron‐reduction process at ?0.51 V (versus SCE). The tetracyanopyridine radical anion undergoes a secondary chemical reaction, which is assigned to a protonation step. TCNPy has been demonstrated to generate 1:1 complexes with various electron donors, including tetrathiafulvalene (TTF) and dihydroxybenzene derivatives, such as p‐hydroquinone and catechol. Visible‐ or NIR‐light‐induced excitation of the intense charge‐transfer bands of these compounds leads to a direct optical electron‐transfer process for the formation of the corresponding radical‐ion pairs. The presence of available electron donors that contain protic groups in close proximity to the TCNPy acceptor site opens up a new strategy for the photocontrolled generation of pyridinium radicals in a stepwise proton‐coupled electron‐transfer (PCET) sequence.     

ESR study of radical processes in irradiated DNA: The effect of the complexation with cis-dichloro diamino Pt(II)

The room temperature esr spectra of irradiated neat duplex hydrated, dehydrated and thermally denaturated DNA are interpreted in terms of the radicals formed by protonation of the thymine and cytosine anion radicals, by deprotonation of the guanine cation radicals, by addition of OH radicals at C8 of guanine and by loss of a methyl hydrogen from thymine. The complexation of DNA with cis-dichlorodiamino Pt(II) causes a drastic alteration of the radical distribution interpreted in terms of electron scavenging by the Pt(II) complex, a partial inhibition of the radical-cation chemistry of guanine, and enhancement of the reactivity of thymine toward hydrogen abstractions.     

Fast back electron transfer prevents guanine damage by photoexcited thionine bound to DNA

The phenothiazinium dye thionine has a high excited state reduction potential and is quenched by guanine on the femtosecond time scale. Here, we show by gel electrophoresis that irradiation of thionine with 599 nm light in the presence of an oligonucleotide duplex does not produce permanent DNA damage. Upon photoexcitation of thionine weakly associated with guanosine-5'-monophosphate, the reduced protonated thionine radical and neutral guanine radical are detected by transient absorption spectroscopy, indicating that the quenching of thionine by guanine occurs via an electron-transfer mechanism. The observation of radical formation without permanent guanine damage indicates that fast back electron transfer plays a critical role in governing the yield of damage by DNA-binding molecules.