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.     

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.     

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.     

Laser ablation synthesis of selenium superoxide anion SeO4- via selenium trioxide photolysis. Time-of-flight mass spectrometry and ab initio calculations

Laser desorption/ionisation and laser ablation of solid selenium trioxide, as well as the gas-phase behaviour of selenium trioxide, were studied. Selenium trioxide undergoes photochemical decomposition and, from the mass spectra obtained by laser desorption/ionisation time-of-flight mass spectrometry (LDI-TOF-MS), the following species were identified: O-, O2-, O3-, SeO-, SeO2-, SeO3-, SeO4-, Se2O7-, Se3O11-, and Se4O14-. Formation of the selenium superoxide SeO4- anion is described in this work for the first time. In addition, low-abundance selenium species such as Se2O8H2-, Se3O11H-, and Se4O15H2- were also detected. The stoichiometry of all ions was confirmed via isotopic pattern modeling and/or post-source decay (PSD) analysis. Photolysis of selenium trioxide leads partly to ozone formation. It was found that the most likely mechanisms of selenium superoxide formation are oxidation of selenium trioxide with ozone and/or reactive oxygen radicals, or photolysis of selenium trioxide tetramer (SeO3)4. Therefore, ab initio calculations were performed to support the mass spectrometric evidence and to suggest probable geometries for selenium superoxide anion SeO4- and diselenium superoxide anion Se2O7-, as well as to provide insight into and/or predict possible formation pathways. It has been found that both cyclic and non-cyclic peroxide structures of SeO4- and Se2O7- ions are possible. In addition, the SeO4 structure was also calculated guided by thermodynamic considerations using Gaussian-2 methodology, and the inferred stability of the SeO4 neutral molecule was supported by ab initio calculations.     

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.     

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.     

PHOTOCHEMISTRY OF DNA CONTAINING IODINATED CYTOSINE*

Abstract— –Irradiation at 313 nm of compounds containing iodinated cytosine moieties results in the photolysis of iodine. Photolysis occurs with a quantum yield of 0·0224·024 for 5-iododeoxycytidine and 5-iododeoxycytidine monophosphate, and 0·004–0·008 for iodinated DNA as well as for iodinated polycytidylate. Photodegradation of the cytosine moiety occurs when air is present during irradiation, presumably due to the reaction of oxygen with the cytosyl radical formed when iodine is lost. This oxygen promoted photodegradation destroys the cytosine chromophore and is complete in the monomers but occurs to only a limited extent in the polymers. In the absence of oxygen or in the presence of ethanol, photodegradation is prevented and the loss of iodine leads exclusively to the formation of the cytosine chromophore. In DNA, the loss of iodine is accompanied by the formation of sugar damage and/or chain breaks. As measured by sedimentation in alkaline sucrose gradients, approximately one break is made for every six iodinqs lost in denatured DNA. The frequency of chain breakage per iodine photolyzed is reduced 2-fold in renatured DNA. Analysis in neutral gradients suggests that half of the breaks observed in alkali are alkali-labile bonds. Both ethanol and cysteamine reduce the number of chain breaks observed in alkali by ˜ 3-fold.     

Selenium dioxide oxidations of dialkyl-3H-azepines: the first synthesis of 2-azatropone from oxidation of 2, 5-Di-tert-butyl-3H-azepine

Oxidation reactions of 2,5- and 3,6-di-tert-butyl-3H-azepines (1 and 2) with selenium dioxide (SeO(2)) were performed. The oxidation of 1 with SeO(2) gave 3-tert-butyl-7,7-dimethyl-4-oxo-octa-2,5-dienal 3 in 36% yield, 4-tert-butyl-5-(3,3-dimethyl-2-oxo-butylidene)-1, 5-dihydro-pyrrol-2-one 4 in 13% yield, 2, 6-di-tert-butyl-2-pyridinecarbaldehyde 5 in 12% yield, and 4, 7-di-tert-butyl-2H-azepin-2-one (2-azatropone) 6 in 6% yield, respectively. Oxidation of 2 with SeO(2) gave 2, 2-dimethyl-1-[2-(5-tert-butyl)-pyridyl]propanol 7 in 55% yield, and 3,6-di-tert-butyl-2H-azepine 8 in 5% yield, respectively. We found that selenium dioxide oxidation of 1 affords 4-oxo-octa-2,5-dienal 3 by a new ring cleavage reaction of 1, and we described the first synthesis of 2-azatropone 6 from this oxidation of 1. In the case of 2, pyridylpropanol 7 was obtained as the major product. We now report in detail result of these oxidation reactions, which have led to the synthesis of a novel azatropone derivative.     

DNA cleavage by UVA irradiation of NADH with dioxygen via radical chain processes

Efficient DNA cleaving-activity is observed by UVA irradiation of an O(2)-saturated aqueous solution of NADH (beta-nicotinamide adenine dinucleotide, reduced form). No DNA cleavage has been observed without NADH under otherwise the same experimental conditions. In the presence of NADH, energy transfer from the triplet excited state of NADH ((3)NADH*) to O(2) occurs to produce singlet oxygen ((1)O(2)) that is detected by the phosphorescence emission at 1270 nm. No quenching of (1)O(2) by NADH was observed as indicated by no change in the intensity of phosphorescence emission of (1)O(2) at 1270 nm in the presence of various concentrations of NADH. In addition to the energy transfer, photoinduced electron transfer from (3)NADH* to O(2) occurs to produce NADH(*+) and O(2)(*-), both of which was observed by ESR. The quantum yield of the photochemical oxidation of NADH with O(2) increases linearly with increasing concentration of NADH but decreases with increasing the light intensity absorbed by NADH. Such unusual dependence of the quantum yield on concentration of NADH and the light intensity absorbed by NADH indicates that the photochemical oxidation of NADH with O(2) proceeds via radical chain processes. The O(2)(*-) produced in the photoinduced electron transfer is in the protonation equilibrium with HO(2)(*), which acts as a chain carrier for the radical chain oxidation of NADH with O(2) to produce NAD(+) and H(2)O(2), leading to the DNA cleavage.     

Direct observation of guanine radical cation deprotonation in duplex DNA using pulse radiolysis

The dynamics of one-electron oxidation of guanine (G) base mononucleotide and that in DNA have been investigated by pulse radiolysis. The radical cation (G+*) of deoxyguanosine (dG), produced by oxidation with SO(4)-*, rapidly deprotonates to form the neutral G radical (G(-H)*) with a rate constant of 1.8 x 10(7) s(-1) at pH 7.0, as judged from transient spectroscopy. With experiments using different double-stranded oligonucleotides containing G, GG, and GGG sequences, the absorbance increases at 625 nm, characteristic of formation of the G(-H)*, were found to consist of two phases. The rate constants of the faster ( approximately 1.3 x 10(7) s(-1)) and slower phases ( approximately 3.0 x 10(6) s(-1)) were similar for the different oligonucleotides. On the other hand, in the oligonucleotide containing G located at the 5'- and 3'-terminal positions, only the faster phase was seen. These results suggest that the lifetime of the radical cation of the G:C base pair (GC+*), depending on its location in the DNA chain, is longer than that of free dG. In addition, the absorption spectral intermediates showed that hole transport to a specific G site within a 12-13mer double-stranded oligonucleotide is complete within 50 ns; that is, the rate of hole transport over 20 A is >10(7) s(-1).     

Stable meso–meso-Linked 2NH-Corrole Radical Dimers as a Key Intermediate to Corrole Tape

While oxidation of 5,5′,15,15′-tetramesityl-10-10′-linked 3NH-corrole dimer with DDQ gave the corresponding triply linked 2NH-corrole tape, the use of an equimolar amount of p-chloranil as a milder oxidant resulted in the formation of a 10-10′-linked neutral 2NH-corrole radical dimer as a stable product. The stability of this peculiar product is ascribed largely to strong antiferromagnetic interaction of the two spins. Further oxidation of this diradical produced corrole tape, suggesting its involvement as a reaction intermediate to the corrole tape. Oxidation of 10-10′-linked bis-pyridine-coordinated Co^III corrole dimer with DDQ produced a cobalt corrole radical dimer and a doubly linked corrole dimer both as stable compounds bearing pyridine and cyanide axial ligands. This type of oxidative transformation involving neutral diradical intermediates is a unique reaction mechanism specific for corrole dimers.     

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.     

Effects of Micelle on Pyrazoles as Antioxidants in Radical-induced Oxidation of DNA

Effects of 4-(1,3-diphenyl-1H-pyrazol-5-yl)phenol(APP), 4-(1,5-diphenyl-1H-pyrazol-3-yl)phenol(BPP) and 4-(3,5-diphenyl-1H-pyrazol-1-yl)phenol(CPP) on 2,2'-azobis-(2-amidinopropane hydrochloride)(AAPH)-induced oxidation of DNA were measured in the presence of various concentrations of Triton X-100, cetyltrimethylammonium bromide(CTAB), or sodium dodecyl sulfate(SDS) in order to clarify the influence of neutral, cationic and anionic microenvironments on antioxidant capacities of APP, BPP and CPP. Although these surfactants can protect DNA against AAPH-induced oxidation, the pyrazoles in the presence of these surfactants functioned as prooxidants when the concentrations of Triton X-100 and CTAB increased. However, CPP exhibited antioxidant property with the increase of the concentration of CTAB. On the contrary, APP, BPP and CPP were antioxidants in the presence of various concentrations of SDS. The added surfactants resulted in the complication of the microenvironments around DNA, pyrazoles and peroxyl radical(ROO^·) derived from AAPH. The anionic charge of SDS was beneficial to enhancing the antioxidant effectiveness of these pyrazoles. It can be concluded that the charge property of surfactants markedly influenced the behavior of an antioxidant in AAPH-induced oxidation of DNA.     

Neutral histidine and photoinduced electron transfer in DNA photolyases

The two major UV-induced DNA lesions, the cyclobutane pyrimidine dimers (CPD) and (6-4) pyrimidine-pyrimidone photoproducts, can be repaired by the light-activated enzymes CPD and (6-4) photolyases, respectively. It is a long-standing question how the two classes of photolyases with alike molecular structure are capable of reversing the two chemically different DNA photoproducts. In both photolyases the repair reaction is initiated by photoinduced electron transfer from the hydroquinone-anion part of the flavin adenine dinucleotide (FADH(-)) cofactor to the photoproduct. Here, the state-of-the-art XMCQDPT2-CASSCF approach was employed to compute the excitation spectra of the respective active site models. It is found that protonation of His365 in the presence of the hydroquinone-anion electron donor causes spontaneous, as opposed to photoinduced, coupled proton and electron transfer to the (6-4) photoproduct. The resulting neutralized biradical, containing the neutral semiquinone and the N3'-protonated (6-4) photoproduct neutral radical, corresponds to the lowest energy electronic ground-state minimum. The high electron affinity of the N3'-protonated (6-4) photoproduct underlines this finding. Thus, it is anticipated that the (6-4) photoproduct repair is assisted by His365 in its neutral form, which is in contrast to the repair mechanisms proposed in the literature. The repair via hydroxyl group transfer assisted by neutral His365 is considered. The repair involves the 5'base radical anion of the (6-4) photoproduct which in terms of electronic structure is similar to the CPD radical anion. A unified model of the CPD and (6-4) photoproduct repair is proposed.     

A pulse radiolysis study of free radicals formed by one-electron oxidation of the antimalarial drug pyronaridine

Free radicals from one-electron oxidation of the antimalarial drug pyronaridine have been studied by pulse radiolysis. The results show that pyronaridine is readily oxidised to an intermediate semi-iminoquine radical by inorganic and organic free radicals, including those derived from tryptophan and acetaminophen. The pyronaridine radical is rapidly reduced by both ascorbate and caffeic acid. The results indicate that the one-electron reduction potential of the pyronaridine radical at neutral pH lies between those of acetaminophen (707 mV) and caffeic acid (534 mV). The pyronaridine radical decays to produce the iminoquinone, detected by electrospray mass spectrometry, in a second-order process that density functional theory (DFT) calculations (UB3LYP/6-31+G*) suggest is a disproportionation reaction. Important calculated dimensions of pyronaridine, its phenoxyl and aminyl radical, as well as the iminoquinone, are presented. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Studies of the Anodic Oxidation of 1,4-Diazabicyclo

The title compound (1) was studied at platinum and gold electrodes in acetonitrile. A reversible oxidation peak occurs at +0.30 V vs the standard potential for ferrocenium ion/ferrocene. This process is followed by a second irreversible anodic peak that is due to the oxidation of the initially formed radical cation to the dication. The principal ultimate product of the first oxidation, the conjugate acid of 1, is also oxidized over the range of potentials corresponding to the second anodic peak. The rate of disappearance of the radical cation of 1 has been determined by cyclic voltammetry. The results are best interpreted in terms of parallel pseudo-first-order decay (k(1) = 0.6 s(-)(1)) and second-order reactions. The first of these second-order reactions is either proton transfer from the radical cation to neutral 1 or hydrogen atom abstraction by the radical cation from neutral 1, reactions that give the same products (k(2) = 100 M(-)(1) s(-)(1)) and are kinetically indistinguishable. The other second-order reaction is the hydrogen-atom-transfer disproportionation of the radical cation giving the conjugate acid of 1 and the immonium ion (k(3) = 100 M(-)(1) s(-)(1)). Both second-order processes must be included to account for the results. The present results are thought to be the first experimental evidence for the occurrence of hydrogen-atom-transfer disproportionation of amine radical cations.     

Reaction mechanisms and structural characterization of the reactive intermediates observed after the photolysis of 3-(hydroxymethyl)benzophenone in acetonitrile, 2-propanol, and neutral and acidic aqueous solutions

Nanosecond time-resolved resonance Raman (ns-TR(3)) spectroscopy was employed to investigate the photoinduced reactions of 3-(hydroxymethyl)benzophenone (1) in acetonitrile, 2-propanol, and neutral and acidic aqueous solutions. Density functional theory calculations were utilized to help the interpretation of the experimental spectra. In acetonitrile, the neutral triplet state 1 [denoted here as (m-BPOH)(3)] was observed on the nanosecond to microsecond time scale. In 2-propanol this triplet state appeared to abstract a hydrogen atom from the solvent molecules to produce the aryphenyl ketyl radical of 1 (denoted here as ArPK of 1), and then this species underwent a cross-coupling reaction with the dimethylketyl radical (also formed from the hydrogen abstraction reaction) to form a long-lived light absorbing transient species that was tentatively identified to be mainly 2-(4-(hydroxy(3-(hydroxymethyl)phenyl)methylene)cyclohexa-2,5-dienyl)propan-2-ol. In 1:1 H(2)O:CH(3)CN aqueous solution at neutral pH, (m-BPOH)(3) reacted with water to produce the ArPK of 1 and then underwent further reaction to produce a long-lived light absorbing transient species. Three photochemical reactions appeared to take place after 266 nm photolysis of 1 in acidic aqueous solutions, a photoreduction reaction, an overall photohydration reaction, and a novel photoredox reaction. TR(3) experiments in 1:1 H(2)O:CH(3)CN aqueous solution at pH 2 detected a new triplet biradical species, which is associated with an unusual photoredox reaction. This reaction is observed to be the predominant reaction at pH 2 and seems to face competition from the overall photohydration reaction at pH 0.     

Isotope effects and the mechanism of allylic hydroxylation of alkenes with selenium dioxide

The mechanism of the allylic oxidation of 2-methyl-2-butene with selenium dioxide was explored by a combination of experimental and theoretical studies. A comparison of the experimental (13)C and (2)H kinetic isotope effects with predicted values shows that the observed isotope effects are consistent with an initial concerted ene step mediated by SeO(2). However, this comparison also does not rule out the involvement of a selenous ester in the ene reaction or a stepwise reaction involving reversible electrophilic addition of HSeO(2)(+) followed by rate-limiting proton abstraction. Becke3LYP calculations strongly favor SeO(2) over a selenous ester as the active oxidant, with the predicted barrier for reaction of 2-methyl-2-butene with SeO(2) being 21-24 kcal/mol lower than that for reaction with H(2)SeO(3). The possibility of a selenous ester being the active oxidant is also disfavored by the observation of oxidations in non-hydroxylic solvents. The involvement of HSeO(2)(+) does not appear consistent with a lack of dependence of the reaction on the basicity of the reaction mixture. A concerted ene reaction with SeO(2) as the active oxidant appears to be the major mechanistic pathway operative in these reactions.     

Near 0 eV electrons attach to nucleotides

To elucidate the mechanism of the nascent stage of DNA strand breakage by low-energy electrons, theoretical investigations of electron attachment to nucleotides have been performed by the reliably calibrated B3LYP/DZP++ approach (Chem. Rev. 2002, 102, 231). The 2'-deoxycytidine-3'-monophosphate (3'-dCMPH) and its phosphate-deprotonated anion (3'-dCMP(-)) have been selected herein as models. This investigation reveals that 3'-dCMPH is able to capture near 0 eV electrons to form a radical anion which has a lower energy than the corresponding neutral species in both the gas phase and aqueous solution. The excess electron density is primarily located on the base of the nucleotide radical anion. The electron detachment energy of this pyrimidine-based radical anion is high enough that subsequent phosphate-sugar C-O sigma bond breaking or glycosidic bond cleavage is feasible. Although the phosphate-centered radical anion of 3'-dCMPH is not stable in the gas phase, it may be stable in aqueous solution. However, an incident electron with kinetic energy less than 4 eV might not be able to effectively produce the phosphate-centered radical anion either in solution or in the gas phase. This research also suggests that the electron affinity of the nucleotides is independent of the counterion in aqueous solution.     

Electrochemical oxidation of several oxocarbon salts in N,N-dimethylformamide

The oxocarbon salts of croconic acid and its dicyanomethylene derivatives have been shown to undergo two consecutive reversible one-electron transfers in N,N-dimethylformamide to produce stable radical anions and the neutral croconates. Disproportion equilibrium constants were found to be quite small for all the crononate radical anions investigated. Following chemical reactions accompanied the second oxidation process of dicyanomethylene-substituted crononates. Substituent effects were shown to be ring position-independent and are discussed with respect to the unique resonance structure of the crononates.