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Carbonate radical anions are potentially important oxidants of nucleic acids in physiological environments. However, the mechanisms of action are poorly understood, and the end products of oxidation of DNA by carbonate radicals have not been characterized. These oxidation pathways were explored in this work, starting from the laser pulse-induced generation of the primary radical species to the identification of the stable oxidative modifications (lesions). The cascade of events was initiated by utilizing 308 nm XeCl excimer laser pulses to generate carbonate radical anions on submicrosecond time scales. This laser flash photolysis method involved the photodissociation of persulfate to sulfate radical anions and the one electron oxidation of bicarbonate anions by the sulfate radicals to yield the carbonate radical anions. The latter were monitored by their characteristic transient absorption band at 600 nm. The rate constants of reactions of carbonate radicals with oligonucleotides increase in the ascending order:  5‘-d(CCATCCTACC) [(5.7 ± 0.6) × 10⁶ M^-1 s^-1] < 5‘-d(TATAACGTTATA), self-complementary duplex [(1.4 ± 0.2) × 10⁷ M^-1 s^-1] < 5‘-d(CCATCGCTACC [(2.4 ± 0.3) × 10⁷ M^-1 s^-1] < 5‘-d(CCATC[8-oxo-G]CTACC) [(3.2 ± 0.4) × 10⁸ M^-1 s^-1], where 8-oxo-G is 8-oxo-7,8-dihydroguanine, the product of a two electron oxidation of guanine. This remarkable enhancement of the rate constants is correlated with the presence of either G or 8-oxo-G bases in the oligonucleotides. The rate constant for the oxidation of G in a single-stranded oligonuclotide is faster by a factor of ~2 than in the double-stranded form. The site selective oxidation of G and 8-oxo-G residues by carbonate radicals results in the formation of unique end products, the diastereomeric spiroiminodihydantoin (Sp) lesions, the products of a four electron oxidation of guanine. These lesions, formed in high yields (40−60%), were isolated by reversed phase HPLC and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. These assignments were supported by the characteristic circular dichroism spectra of opposite signs of the two lesions. The oxidation of guanine to Sp diastereomers occurs, at least in part, via the formation of 8-oxo-G lesions as intermediates. The Sp lesions can be considered as the terminal products of the oxidation of G and 8-oxo-G in DNA by carbonate radical anions. The mechanistic aspects and biological implications of these site selective reactions in DNA initiated by carbonate radicals are discussed.

A simple photochemical approach is described for synthesizing site specific, stable 5-guanidino-4-nitroimidazole (NIm) adducts in single- and double-stranded oligodeoxynucleotides containing single and multiple guanine residues. The DNA sequences employed, 5‘-d(ACC CG1C G*2TC CG*3C G4CC) and 5‘-d(ACC CG1C G*2TC C), were a portion of exon 5 of the p53 tumor suppressor gene, including the codons 157 (G*2) and 158 (G*3) mutation hot spots in the former sequence with four Gs and the codon 157 (G*2) mutation hot spot in the latter sequence with two Gs. The nitration of oligodeoxynucleotides was initiated by the selective photodissociation of persulfate anions to sulfate radicals induced by UV laser pulses (308 nm). In aqueous solutions, of bicarbonate and nitrite anions, the sulfate radicals generate carbonate anion radicals and nitrogen dioxide radicals by one electron oxidation of the respective anions. The guanine residue in the oligodeoxynucleotide is oxidized by the carbonate anion radical to form the neutral guanine radical. While the nitrogen dioxide radicals do not react with any of the intact DNA bases, they readily combine with the guanine radicals at either the C8 or the C5 positions. The C8 addition generates the well-known 8-nitroguanine (8-nitro-G) lesions, whereas the C5 attack produces unstable adducts, which rapidly decompose to NIm lesions. The maximum yields of the nitro products (NIm + 8-nitro-G) were typically in the range of 20−40%, depending on the number of guanine residues in the sequence. The ratio of the NIm to 8-nitro-G lesions gradually decreases from 3.4 in the model compound, 2‘,3‘,5‘-tri-O-acetylguanosine, to 2.1−2.6 in the single-stranded oligodeoxynucleotides and to 0.8−1.1 in the duplexes. The adduct of the 5‘-d(ACC CG1C G*2TC C) oligodeoxynucleotide containing the NIm lesion in codon 157 (G2) was isolated in HPLC-pure form. The integrity of this adduct was established by a detailed analysis of exonuclease digestion ladders by matrix-assisted laser desorption ionization with time-of-flight detection MS techniques.

A novel photochemical approach is described for synthesizing site-specific 8-nitro-2‘-deoxyguanosine (8-nitro-dG) adducts DNA. The method is based on the bimolecular reaction of a neutral, deprotonated guanine radical [G(-H)^•] in DNA and nitrogen dioxide (^•NO2) radicals. This approach is illustrated using the single-stranded oligodeoxyribonucleotide 5‘-d(CCATCGCTACC) dissolved in an aqueous solution of nitrite and bicarbonate anions at pH 7.5. The photochemical synthesis was triggered by the selective photodissociation of persulfate anions to yield SO4^•- radical anions by either 308 nm XeCl excimer laser pulses or by a continuous irradiation with 290−340 nm light from a 1000 W Xe lamp. The sulfate radicals formed generate the CO3^•- and ^•NO2 radicals by one-electron oxidation of the bicarbonate and nitrite anions. In turn, the CO3^•- radicals site-selectively generate G(-H)^• radicals in DNA that combine with ^•NO2 to form 8-nitro-dG lesions in the oligonucleotide. The nitrated oligonucleotides were purified by reversed-phase HPLC techniques and are stable at 4 °C for at least 4 days, but depurinate at ambient temperatures of 23 °C at pH 7 with a half-life of ~20 h. The nature of the reaction and decomposition products were studied by a combination of ESI and MALDI-TOF mass spectrometric techniques.

Kinetic solvent isotope effects on the electron-transfer kinetics associated with the oxidation of guanine by a 2-aminopurine (2AP) neutral radical separated by 2 or 3 thymine, or 6 adenine residues on the same strand in 15-mer double-stranded oligonucleotides in H2O or D2O were measured. The evolution in time of the oxidized form of guanine, the neutral radical G(−H)^•, and the electron acceptor, the neutral 2AP(−H)^• radical, were followed directly by a spectroscopic laser pulse-induced transient absorption technique on a 1−200 μs time scale. About 70−100% of this one-electron-transfer reaction occurs on this relatively slow time scale in the three oligonucleotide duplexes studied. The rate constants of formation of G(−H)^• in the oligonucleotides are larger in H2O than in D2O by a factor of 1.3−1.7. This kinetic isotope effect suggests that the electron-transfer reaction from G to 2AP(−H)^• is coupled to a deprotonation of G^•+, and a protonation of 2AP^-, the primary products of the electron-transfer reaction. Thus, electron-transfer reactions occurring at a distance in these DNA duplexes can be considered in terms of proton-coupled electron-transfer reactions.

The carbonate radical anion (CO(3)) is believed to be an important intermediate oxidant derived from the oxidation of bicarbonate anions and nitrosoperoxocarboxylate anions (formed in the reaction of CO(2) with ONOO(-)) in cellular environments. Employing nanosecond laser flash photolysis methods, we show that the CO(3) anion can selectively oxidize guanines in the self-complementary oligonucleotide duplex d(AACGCGAATTCGCGTT) dissolved in air-equilibrated aqueous buffer solution (pH 7.5). In these time-resolved transient absorbance experiments, the CO(3) radicals are generated by one-electron oxidation of the bicarbonate anions (HCO(3)(-)) with sulfate radical anions (SO(4)) that, in turn, are derived from the photodissociation of persulfate anions (S(2)O(8)(2-)) initiated by 308-nm XeCl excimer laser pulse excitation. The kinetics of the CO(3) anion and neutral guanine radicals, G(-H)( small middle dot), arising from the rapid deprotonation of the guanine radical cation, are monitored via their transient absorption spectra (characteristic maxima at 600 and 315 nm, respectively) on time scales of microseconds to seconds. The bimolecular rate constant of oxidation of guanine in this oligonucleotide duplex by CO(3) is (1.9 +/- 0.2) x 10(7) m(-1) s(-1). The decay of the CO(3) anions and the formation of G(-H)( small middle dot) radicals are correlated with one another on the millisecond time scale, whereas the neutral guanine radicals decay on time scales of seconds. Alkali-labile guanine lesions are produced and are revealed by treatment of the irradiated oligonucleotides in hot piperidine solution. The DNA fragments thus formed are identified by a standard polyacrylamide gel electrophoresis assay, showing that strand cleavage occurs at the guanine sites only. The biological implications of these oxidative processes are discussed.

The redox reactions of guanine and its widely studied oxidation product, the 8-oxo-7,8-dihydro derivative, are of significant importance for understanding the mechanisms of oxidative damage in DNA. Employing 2‘-deoxyguanosine 5‘-monophospate (dGMP) and 8-oxo-7,8-dihydro-2‘-deoxyguanosine (8-oxo-dG) in neutral aqueous solutions as model systems, we have used nanosecond laser flash photolysis to demonstrate that neutral radicals, dGMP(−H)^•, derived by the one-electron oxidation and deprotonation of dGMP, can oxidize nitrite anions (NO2^-) to the nitrogen dioxide radical ^•NO2. In turn, we show that ^•NO2 can give rise to a one-electron oxidation of 8-oxo-G, but not of dGMP. The one-electron oxidation of dGMP was initiated by a radical cation generated by the laser pulse-induced photoionization of a pyrene derivative with enhanced water solubility, 7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene (BPT). The dGMP(−H)^• neutral radicals formed via deprotonation of the dGMP^•+ radical cations and identified by their characteristic transient absorption spectrum (λmax ~ 310 nm) oxidize nitrite anions with a rate constant of (2.6 ± 0.3) × 10⁶ M^-1 s^-1. The 8-oxo-dG is oxidized by ^•NO2 with a rate constant of (5.3 ± 0.5) × 10⁶ M^-1 s^-1. The 8-oxo-dG(−H)^• neutral radicals thus generated are clearly identified by their characteristic transient absorption spectra (λmax ~ 320 nm). The rate constant of 8-oxo-dG oxidation (k12) by the ^•NO2 one-electron oxidant (the ^•NO2/NO2^- redox potential, E° ≈ 1.04 V vs NHE) is lower than k12 for a series of oxidizing aromatic radical cations with known redox potentials. The k12 values for 8-oxo-dG oxidation by different aromatic radical cations derived from the photoionization of their parent compounds depend on the redox potentials of the latter, which were in the range of 0.8−1.6 V versus NHE. The magnitude of k12 gradually decreases from a value of 2.2 × 10⁹ M^-1 s^-1 (E° = 1.62 V) to 5.8 × 10⁸ M^-1 s^-1 (E° = 1.13 V) and eventually to 5 × 10⁷ M^-1 s^-1 (E° = 0.91 V). The implications of these results, including the possibility that the redox cycling of the ^•NO2/NO2^- species can be involved in the further oxidative damage of 8-oxo-dG in DNA in cellular environments, are discussed.

Oligonucleotide duplexes (15-mers) containing 8-oxo-7,8-dihydro-2‘-deoxyguanosine (8-oxo-dG) and the base analogue 2-aminopurine (2AP) separated from one another by zero, two, or four thymidine residues on the same strand, were synthesized. Selective two-photon excitation of 2AP with intense 308 nm XeCl excimer laser pulses (fwhm = 12 ns, ~80 mJ pulse^-1 cm^-2) gives rise to 2AP radical cations that, in turn, oxidize 8-oxo-dG at a distance. Transient absorption techniques allowed for the direct monitoring of the oxidant and product radical intermediates as a function of time on the 100 ns−1 ms time scale and revealed the existence of two-component oxidation kinetics. The evolution in time of the rapid component (<100 ns), attributed to the oxidation of 8-oxo-dG by the radical cation 2AP•⁺, was not resolved. However, this component was observed only in the case of zero or two intervening thymidine bases and was not evident when the 2AP and 8-oxo-dG residues were separated by four thymidines. A slower kinetic component (>100 ns) is attributed to the oxidation of 8-oxo-dG by the deprotonation product of 2AP•⁺, the neutral radical 2AP(−H)•. Only the slow, microsecond kinetic component was observed when the 8-oxo-dG and 2AP are separated by four thymidines. The rate constants of electron transfer are (3.8 ± 0.5) × 10⁴ s^-1 and (3.0 ± 0.5) × 10³ s^-1 in the case of two and four intervening thymidines, respectively. Because of the pKa properties of the donor/acceptor couples, this slow, 0.1−1000 μs time scale electron transfer at a distance from 8-oxo-dG to the 2AP(−H)• radicals, is likely to occur via a proton-coupled electron-transfer mechanism.

Photoionization of free 2-aminopurine (2AP) in aqueous solutions, or of 2AP residues in oligonucleotides, is observed upon excitation with 308 nm XeCl excimer laser pulses (fwhm = 12 ns, ≤100 mJ/cm²/pulse) and involves the consecutive absorption of two photons. The absorption of light by the normal DNA bases at this wavelength is negligible under the same conditions. The kinetics and transient absorption spectra of the hydrated electrons and of 2AP radicals resulting from the deprotonation of the 2AP radical cations formed initially, have been investigated using standard spectroscopic methods. The 2AP radicals in aqueous solutions reversibly oxidize 2‘-deoxyguanosine and guanosine 5‘-monophosphate but do not react with the other three DNA nucleosides or nucleotides. The efficiency of photoionization of 2AP decreases in the following order:  free 2AP > 2AP incorporated into single-stranded oligonucleotide > 2AP in double-stranded oligonucleotides. Photoionization of single 2AP residues incorporated into 18-mer single- and double-stranded oligonucleotides results in the oxidation of guanine residues in GG doublets. Employing gel electrophoresis methods, strand cleavage at these GG sites and at the site of the 2AP residue is observed after treatment of the irradiated oligonucleotides with hot piperidine. Using nanosecond transient absorption techniques, it is shown that the unimolecular oxidation of guanine by 2AP radicals at a distance can also be monitored directly in single-stranded oligonucleotides containing GG-doublets. It is found that increasing the number of nucleic acid bases between the 2AP radicals and the GG-doublet from 0 to 5 results in a decrease in the rate constant of guanine radical formation by a factor greater than 10³.

Two Ru(bpy)32+-viologen complexes, in which the components are linked by a hydrocarbon chain, were synthesized and characterized by PMR, visible, and UV spectrophotometry.

1.The break in the temperature dependence of the transmembrane electron transfer rate constant (in Arrhenius coordinates) corresponds to the phase transition of the lipid from the gel to the liquid crystalline state.2.The transmembrane electron transfer rate constant is not dependent on the structure of the electron carrier, the viologen.

Photoexcitation of a pyrene derivative covalently bound to double-stranded calf thymus DNA with 355 nm Nd:YAG laser pulses (fwhm = 24 ps, 50 mJ/cm²/pulse) results in the efficient two-photon-induced ionization of the pyrenyl residues. By use of nanosecond transient absorption techniques, it is shown that the excess electrons injected into the DNA can reduce methylviologen cations (MV²⁺) that are noncovalently bound to the DNA but not MV²⁺ in the outer aqueous solution phase. In double-stranded DNA, this reduction of MV²⁺ to MV^•+ occurs via two kinetic phases, a rapid one that is complete within ≤7 ns and a slower one (200−300 ns) due to the diffusive reduction of MV²⁺ by hydrated electrons. The appearance of the first, rapid reduction phase of MV²⁺ depends on the secondary structure of the DNA, since it is observed only in the double-stranded form but not in the denatured, single-stranded form. This rapid reduction phase is entirely eliminated upon the addition of magnesium ions, which displace the positively charged MV²⁺ cations from the double-stranded DNA molecules. By variation of the concentration of MV²⁺ cations at a constant distribution of covalently bound pyrenyl residues (60 base pairs per pyrenyl residue), a mean distance of migration of excess electrons in double-stranded DNA of ca. 40 Å is estimated.

The need for large-scale direct synthesis of stereochemically defined and site-specific benzo[a]pyrenediol epoxide−oligodeoxyribonucleotide adducts for detailed NMR and other biochemical and physicochemical studies has necessitated a better understanding of variables that lead to an enhancement of the reaction yields. It is shown that, in aqueous solution, the formation of noncovalent hydrophobic complexes between 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and the single-stranded oligonucleotide 5‘-d(CCATCGCTACC) precedes the covalent binding reaction of BPDE with the single deoxyguanosine residue. The yield of covalent reaction products (involving reaction of BPDE at the C10 position with the exocyclic amino group of the dG residue) increases with increasing DNA concentration and tends toward saturation at oligonucleotide single-strand concentrations above ~3 mM. The addition of NaCl (0.3 M) also tends to enhance the adduct reaction yields. However, organic solvents such as tetrahydrofuran in the reaction mixtures (10−40%) decrease the stabilities of the noncovalent complexes, which in turn leads to reductions in the yields of covalent BPDE−dG oligonucleotide adducts. The efficiencies of formation of hydrophobic complexes were probed by fluorescence and UV absorption techniques using the BPDE tetrol hydrolysis product 7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene as a model system.

1.Two photocatalytic systems for generation of a strong oxidant: the triple-charged tris-(2,2'-bipyridyl)osmium(III) cation [Os(bipy)33+] inside lipid bilayer vesicles were proposed.2.The quantum yield of formation of Os(bipy)33+ inside the vesicles corresponds to the quantum yield of irreversible oxidation of the photosensitizer, which attains 30% in the optimum conditions.

Photoexcitation of 2-aminopurine riboside (2APr, 2-amino-9-β-d-ribofuranosylpurine) and 2-aminopurine (2AP) in oxygenated aqueous buffer solutions (pH 7.0) with 308 nm XeCl excimer laser pulses (fwhm = 12 ns, ca. 70 mJ/pulse/cm²) results in the consecutive two-photon ionization of the aromatic 2APr (or 2AP) residues. In neutral solutions, the 2APr (or 2AP) radical cations rapidly deprotonate (<100 ns). The 2APr(−H)^• (or 2AP(−H)^• neutral radicals thus formed reversibly oxidize 2-deoxyguanosine 5‘-monophosphate (dGMP) on μs time scales, resulting in the formation of dGMP(−H)^• neutral radicals. Transient absorption measurements show that a remarkable solvent isotope effect is observed on the kinetics of oxidation of dGMP by 2APr(−H)^• (or 2AP(−H)^•) radicals in H2O and D2O solutions. In H2O, the rate constants of dGMP(−H)^• formation, as well as the rate constants of the reverse reaction of the 2APr (or 2AP) oxidation by dGMP(−H)^• is larger than in D2O by a factor of 1.5−2. This kinetic isotope effect indicates that the electron-transfer reaction from dGMP to 2APr(−H)^• (or to 2AP(−H)^•), and the reverse electron transfer from 2APr (or 2AP) to dGMP(−H)^•, is coupled to a deprotonation of the primary electron-transfer radical cation products, dGMP^•+ and 2APr^•+ (or 2AP^•+). Therefore, these reactions, involving redox equilibria between different nucleobases, can be considered in terms of proton-coupled electron-transfer reactions.