The thermodynamic properties of aqueous nitroxyl (HNO) and its anion (NO ⁻ ) have been revised to show that the ground state of NO ⁻ is triplet and that HNO in its singlet ground state has much lower acidity, pKa( ¹ HNO/ ³ NO ⁻ ) ≈ 11.4, than previously believed. These conclusions are in accord with the observed large differences between ¹ HNO and ³ NO ⁻ in their reactivities toward O ₂ and NO. Laser flash photolysis was used to generate ¹ HNO and ³ NO ⁻ by photochemical cleavage of trioxodinitrate (Angeli's anion). The spin-allowed addition of ³ O ₂ to ³ NO ⁻ produced peroxynitrite with nearly diffusion-controlled rate ( k = 2.7 × 10 ⁹ M ⁻¹ ⋅s ⁻¹ ). In contrast, the spin-forbidden addition of ³ O ₂ to ¹ HNO was not detected ( k ≪ 3 × 10 ⁵ M ⁻¹ ⋅s ⁻¹ ). Both ¹ HNO and ³ NO ⁻ reacted sequentially with two NO to generate N ₃ O \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*} as a long-lived intermediate; the rate laws of N ₃ O \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*} formation were linear in concentrations of NO and ¹ HNO ( k = 5.8 × 10 ⁶ M ⁻¹ ⋅s ⁻¹ ) or NO and ³ NO ⁻ ( k = 2.3 × 10 ⁹ M ⁻¹ ⋅s ⁻¹ ). Catalysis by the hydroxide ion was observed for the reactions of ¹ HNO with both O ₂ and NO. This effect is explicable by a spin-forbidden deprotonation by OH ⁻ ( k = 4.9 × 10 ⁴ M ⁻¹ ⋅s ⁻¹ ) of the relatively unreactive ¹ HNO into the extremely reactive ³ NO ⁻ . Dimerization of ¹ HNO to produce N ₂ O occurred much more slowly ( k = 8 × 10 ⁶ M ⁻¹ ⋅s ⁻¹ ) than previously suggested. The implications of these results for evaluating the biological roles of nitroxyl are discussed.

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 CO₂ 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 ) with sulfate radical anions (SO⨪₄) that, in turn, are derived from the photodissociation of persulfate anions (S₂O ) initiated by 308-nm XeCl excimer laser pulse excitation. The kinetics of the CO⨪₃ anion and neutral guanine radicals, G(−H)^⋅, 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) × 10⁷ m ⁻¹ s⁻¹. The decay of the CO⨪₃ anions and the formation of G(−H)^⋅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.

Purpose: The aim of this survey is to critically review the available information on one-electron oxidation reactions of nucleobases in cellular DNA with emphasis on damage induced through the transient generation of purine and pyrimidine radical cations. Since the indirect effect of ionizing radiation mediated by hydroxyl radical is predominant in cells, efforts have been made to selectively ionize bases using suitable one-electron oxidants that consist among others of high intensity UVC laser pulses. Thus, the main oxidation product in cellular DNA was found to be 8-oxo-7,8-dihydroguanine as a result of direct bi-photonic ionization of guanine bases and indirect formation of guanine radical cations through hole transfer reactions from other base radical cations. The formation of 8-oxo-7,8-dihydroguanine and other purine and pyrimidine degradation products was rationalized in terms of the initial generation of related radical cations followed by either hydration or deprotonation reactions in agreement with mechanistic pathways inferred from detailed mechanistic studies. The guanine radical cation has been shown to be implicated in three other nucleophilic additions that give rise to DNA-protein and DNA-DNA cross-links in model systems. Evidence was recently provided for the occurrence of these three reactions in cellular DNA. Conclusion: There is growing evidence that one-electron oxidation reactions of nucleobases whose mechanisms have been characterized in model studies involving aqueous solutions take place in a similar way in cells. It may also be pointed out that the above cross-linked lesions are only produced from the guanine radical cation and may be considered as diagnostic products of the direct effect of ionizing radiation.

The mechanistic aspects of hydration of guanine radical cations, G^•+ in double- and single-stranded oligonucleotides were investigated by direct time-resolved spectroscopic monitoring methods. The G^•+ radical one-electron oxidation products were generated by SO₄^•– radical anions derived from the photolysis of S₂O₈^2– anions by 308 nm laser pulses. In neutral aqueous solutions (pH 7.0), after the complete decay of SO₄^•– radicals (∼5 μs after the actinic laser flash) the transient absorbance of neutral guanine radicals, G(-H)^• with maximum at 312 nm, is dominant. The kinetics of decay of G(-H)^• radicals depend strongly on the DNA secondary structure. In double-stranded DNA, the G(-H)^• decay is biphasic with one component decaying with a lifetime of ∼2.2 ms and the other with a lifetime of ∼0.18 s. By contrast, in single-stranded DNA the G(-H)^• radicals decay monophasically with a ∼ 0.28 s lifetime. The ms decay component in double-stranded DNA is correlated with the enhancement of 8-oxo-7,8-dihydroguanine (8-oxoG) yields which are ∼7 greater than in single-stranded DNA. In double-stranded DNA, it is proposed that the G(-H)^• radicals retain radical cation character by sharing the N1-proton with the N3-site of C in the [G^•+:C] base pair. This [G(-H)^•:H⁺C ⇆ G^•+:C] equilibrium allows for the hydration of G^•+ followed by formation of 8-oxoG. By contrast, in single-stranded DNA, deprotonation of G^•+ and the irreversible escape of the proton into the aqueous phase competes more effectively with the hydration mechanism, thus diminishing the yield of 8-oxoG, as observed experimentally.

The carbonate radical anion is a biologically important one-electron oxidant that can directly abstract an electron from guanine, the most easily oxidizable DNA base. Oxidation of the 5'-d(CCTACGCTACC) sequence by photochemically generated CO3*- radicals in low steady-state concentrations relevant to biological processes results in the formation of spiroiminodihydantoin diastereomers and a previously unknown lesion. The latter was excised from the oxidized oligonucleotides by enzymatic digestion with nuclease P1 and alkaline phosphatase and identified by LC-MS/MS as an unusual intrastrand cross-link between guanine and thymine. In order to further characterize the structure of this lesion, 5'-d(GpCpT) was exposed to CO3*- radicals, and the cyclic nature of the 5'-d(G*pCpT*) cross-link in which the guanine C8-atom is bound to the thymine N3-atom was confirmed by LC-MS/MS, 1D and 2D NMR studies. The effect of bridging C bases on the cross-link formation was studied in the series of 5'-d(GpC(n)pT) and 5'-d(TpC(n)pG) sequences with n = 0, 1, 2 and 3. Formation of the G*-T* cross-links is most efficient in the case of 5'-d(GpCpT). Cross-link formation (n = 0) was also observed in double-stranded DNA molecules derived from the self-complementary 5'-d(TTACGTACGTAA) sequence following exposure to CO3*- radicals and enzymatic excision of the 5'-d(G*pT*) product.