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Peroxynitrite-mediated oxidation of 8-oxoguanosine results in the formation of two product classes distinguished by the source of their incorporated oxygen atoms. The first product class consists of dehydroguanidinohydantoin (DGh), N-nitro-dehydroguanidinohydantoin (NO2-DGh), and 2,4,6-trioxo[1,3,5]triazinane-1-carboxamidine (CAC) with peroxynitrite as the exogenous O atom source, and the second includes spiroiminodihydantoin (Sp), guanidinohydantoin (Gh), and 4-hydroxy-2,5-dioxo-imidazolidine-4-carboxylic acid (HICA), with water serving as the exogenous O atom source. The first product class forms exclusively at high peroxynitrite fluxes, while the second forms exclusively at limiting peroxynitrite fluxes. At intermediate peroxynitrite fluxes, both sets of products are formed. At high fluxes, DGh was the major reaction product, and after several of the peroxynitrite-derived radicals were eliminated as the exogenous O atom source, the peroxynitrite anion emerged as the most likely candidate. On the other hand, at lower fluxes, either Gh or Sp was the major product, depending on the pH of the reaction mixture. At low and high pH, respectively, Gh and Sp were the major products, and the plot of pH vs ratio of Sp/(Sp+Gh) had an inflection at pH 5.8. Interestingly, the pH dependence for oxidation of 8-oxoGuo with CoCl2 and KHSO5 was identical to that for oxidation by peroxynitrite, indicating that the phenomenon arises due to characteristics of an 8-oxoGuo-derived rather than an oxidant-derived intermediate, since these two systems generate different reactive species. On the basis of these findings, a model in which 8-oxoGuo is oxidized to the bisimine intermediate, 1 is proposed. At high peroxynitrite fluxes, the reaction of 1 with ONOO- predominates over the reaction with H2O, leading exclusively to DGh, NO2-DGh, and CAC, while at limiting peroxynitrite concentrations, the reaction with H2O dominates, and Gh and Sp are formed exclusively. At intermediate peroxynitrite fluxes, the relative kinetics of the reaction between 1 and ONOO- or H2O are such that both product classes are formed. To explain the pH-dependent Gh and Sp yields, we propose that 5 has a pKa approximately 5.8 and that the differential reactivity of the protonated and deprotonated form of 5 leads to its partitioning into Gh and Sp, respectively.

Several products were previously identified from the reaction of 8-oxodG with bolus additions of peroxynitrite. Those experiments involved very high instantaneous concentrations of peroxynitrite and raised the possibility that the product spectrum may have differed from what might occur in vivo where much lower--and steadier--peroxynitrite levels would be expected. We consequently examined this issue by treating 2',3',5'-tri-O-acetyl-8-oxoGuo with infused authentic peroxynitrite and 3-morpholinosydnonimine and have found that under these conditions an additional product, not found under bolus addition conditions, was observed. Using tandem mass spectrometry, isotope labeling, and synthesis of a structural analogue, this compound was identified as 4-hydroxy-2,5-dioxo-imidazolidine-4-carboxylic acid (HICA). The mechanism of HICA formation is rationalized by water attack at C5 of a quininoid intermediate.

Peroxynitrite reacts with 2',3',5'-tri-O-acetyl-guanosine to yield a novel compound identified as 1-(2,3,5-tri-O-acetyl-beta-D-erythro-pentofuranosyl)-5-guanidino-4-nitroimidazole (6). This characterization was achieved using a combination of UV/vis spectroscopy and ESI-MS. Additionally, 1-(beta-D-erythro-pentofuranosyl)-5-guanidino-4-nitroimidazole (6a) was synthesized by an independent route, characterized by UV/vis spectroscopy, ESI-MS, and (1)H- and (13)C NMR, and shown to be identical to deacetylated 6. This product is extremely stable in aqueous solution at both pH extremes and is formed in significant yields. These characteristics suggest that this lesion may be useful as a specific biomarker of peroxynitrite-induced DNA damage. We also observed formation of 2',3',5'-tri-O-acetyl-8-nitroguanosine (2',3',5'-tri-O-acetyl-8-NO(2)()Guo), 2-amino-5-[(2,3,5-tri-O-acetyl-beta-D-erythro-pentofuranosyl)amino]-4H-imidazol-4-one (2',3',5'-tri-O-acetyl-Iz), and the peroxynitrite-induced oxidation products of 2',3',5'-tri-O-acetyl-8-oxoGuo. The formation of 6 and 2',3',5'-tri-O-acetyl-8-NO(2)()Guo was rationalized by a mechanism invoking formation of the guanine radical.

[reaction: see text]. The potent oxidant, peroxynitrite, will oxidize 8-oxo-7,8-dihydroguanosine to give several products. In the presence of a thiol agent, the major final product has been determined to be a spiroiminodihydantoin compound. Additionally, we have found that the spiroiminodihydantoin, and not the previously reported 4-hydroxy-8-oxo-4,8-dihydroguanosine, is the major final product formed during the methylene blue-mediated photooxidation of guanosine.

A novel nitration product, formed during the reaction of peroxynitrite with 2',3',5'-tri-O-acetyl-7,8-dihydro-8-oxoguanosine, has been characterized using a combination of UV/vis, CD, and NMR spectroscopy and mass spectrometry. This compound has been identified as N-nitro-N'-[1-(2,3, 5-tri-O-acetyl-beta-D-erythro-pentofuranosyl)-2, 4-dioxoimidazolidin-5-ylidene]guanidine (IV). Upon base hydrolysis, IV releases nitroguanidine (IVa) and an intermediate, 1-(2,3, 5-tri-O-acetyl-beta-D-erythro-pentofuranosyl)-5-iminoimidazolidine -2, 4-dione (IVb). This intermediate is ultimately hydrolyzed to the stable 3-(2,3,5-tri-O-acetyl-beta-D-erythro-pentofuranosyl)oxaluric acid (IVc). IV can be reduced by sodium borohydride to a pair of stable diastereomers (IV(red)()). The formation of this product is rationalized in terms of initial oxidation of 2',3', 5'-tri-O-acetyl-7,8-dihydro-8-oxoguanosine to a quinonoid diimine intermediate, 3. Nucleophilic attack at C5 of 3 by peroxynitrite leads to formation of a C5-oxyl radical species, 5, which then undergoes a series of rearrangements to yield an ylidene radical, 7. Combination of this radical species with nitrogen dioxide results in the formation of product IV.