A novel nitroimidazole compound formed during the reaction of peroxynitrite with 2',3',5'-tri-O-acetyl-guanosine.

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.

Spiroiminodihydantoin is the major product of the 8-oxo-7,8-dihydroguanosine reaction with peroxynitrite in the presence of thiols and guanosine photooxidation by methylene blue.

[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: N-nitro-N'-[1-(2,3,5-tri-O-acetyl-beta-D-erythro-pentofuranosyl)- 2, 4-dioxoimidazolidin-5-ylidene]guanidine.

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.

Peroxynitrite reaction products of 3',5'-di-O-acetyl-8-oxo-7, 8-dihydro-2'-deoxyguanosine.

Of the DNA bases, peroxynitrite (ONOO-) is most reactive toward 2'-deoxyguanosine (dGuo), but even more reactive with 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodGuo), requiring a 1,000-fold excess of dGuo to provide 50% protection against the reaction with 8-oxodGuo. Therefore, it seems reasonable that 8-oxodGuo is a potentially important target in DNA and that the structures of the reaction products with ONOO- should be characterized. Using 3', 5'-di-O-Ac-8-oxodGuo as a model compound, the reaction products with ONOO- have been isolated and identified under simulated physiological reaction conditions (phosphate/bicarbonate buffer at pH 7.2). The major reaction product, II, is unstable and undergoes base-mediated hydrolysis to 2,5-diaminoimidazol-4-one, IIa, and 3-(3, 5-di-O-Ac-2-deoxy-beta-D-erythro-pentofuranosyl)-5-iminoimidazolidine -2,4-dione, IIb. The latter compound further hydrolyzes to 3-(3, 5-di-O-Ac-2-deoxy-beta-D-erythro-pentofuranosyl)oxaluric acid, IIc. Other products include 3-(3, 5-di-O-Ac-2-deoxy-beta-D-erythro-pentofuranosyl)-2,4,6-trioxo-[1,3, 5]triazinane-1-carboxamidine, I, which further hydrolyzes to 1-(3, 5-di-O-Ac-2-deoxy-beta-D-erythro-pentofuranosyl)cyanuric acid, Ia. 1-(3,5-di-O-Ac-2-deoxy-beta-D-erythro-pentofuranosyl)parabanic acid, III, is a minor product that also may contribute to formation of IIc. The major products formed in these reactions are biologically uncharacterized but are similar to modified DNA bases that have been shown to be both premutagenic and blocks to DNA polymerization.

DNA damage in deoxynucleosides and oligonucleotides treated with peroxynitrite.

Peroxynitrite (ONOO-) is a powerful oxidizing agent that forms in a reaction of nitric oxide (NO*) and superoxide (O2-*). We have investigated ONOO--induced DNA damage using deoxynucleosides and oligonucleotides as model substrates, with particular attention paid to the oxidation of 8-oxodG by ONOO-. With regard to deoxynucleosides, ONOO- was found to have significant reactivity only with dG; dA, dC, and dT showed minimal reactivity. However, two of the major products of ONOO--induced oxidation of dG (8-oxodG and 8-nitroG) were both found to be significantly more reactive with ONOO- than with dG. In the context of an oligonucleotide, we observed a concentration-dependent oxidation of 8-oxodG to at least two types of products, one appearing at ONOO- concentrations of /=500 microM. We also examined the susceptibility of these oxidation products to repair by FaPy glycosylase, endonuclease III, uracil glycosylase, and MutY. FaPy glycosylase, which recognizes 8-oxoG as its primary substrate, was the only enzyme that exhibited an efficient reaction with 8-oxodG oxidation products at low ONOO- concentrations (/=500 microM either was not recognized or was poorly repaired by the enzymes. While processing of the lesions was inefficient with endonuclease III and not apparent with uracil glycosylase, the excision of A opposite an 8-oxoG lesion by the enzyme MutY was not affected by the reaction of 8-oxoG with ONOO-. In addition to demonstrating the complexity of ONOO- DNA damage chemistry, these results suggest that 8-oxodG may be a primary target of ONOO- in DNA.

Peroxynitrite-induced reactions of synthetic oligonucleotides containing 8-oxoguanine.

8-Oxoguanine (8-oxo-G) is one of the most common DNA lesions present in normal tissues due to exposure to reactive oxygen species. Studies at this and other laboratories suggest that 8-oxo-G is highly susceptible to secondary oxidation, making it a likely target for endogenous oxidizing agents, such as peroxynitrite (ONOO-). Synthetic oligonucleotides containing 8-oxoguanine were treated with ONOO-, and the reaction products were analyzed by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI--MS). CCACAACXCAAA, CCAAAGGXAGCAG, CCAAAXGGAGCAG, and TCCCGAGCGGCCAAAGGXAGCAG (X is 8-oxo-G) were found to readily react with peroxynitrite via the same transformations as those observed for free 8-oxo-2'-deoxyguanosine. The composition of the reaction mixtures was a function of ONOO- concentration and of the storage time after exposure. The oligonucleotide products isolated at low [ONOO-]/[DNA] ratios (<5) were tentatively assigned as containing 3a-hydroxy-5-imino-3,3a,4,5-tetrahydro-1H-imidazo[4, 5d]imidazol-2-one, 5-iminoimidazolidine-2,4-dione, and its hydrolytic product, oxaluric acid. At a [ONOO-]/[DNA] ratio of >10, 2,4,6-trioxo[1,3,5]triazinane-1-carboxamidine- and cyanuric acid-containing oligomers were the major products. The exact location of a modified base within a DNA sequence was determined using exonuclease digestion of oligonucleotide products followed by LC/ESI--MS analysis of the fragments. For all 8-oxo-G-containing oligomers, independent of the sequence, the reactions with ONOO- took place at the 8-oxo-G residues. These results suggest that 8-oxo-G, if present in DNA, is rapidly oxidized by peroxynitrite and that oxaluric acid is a likely secondary oxidation product of 8-oxo-G under physiological conditions.

The chemistry of DNA damage from nitric oxide and peroxynitrite.

Nitric oxide is a key participant in many physiological pathways; however, its reactivity gives it the potential to cause considerable damage to cells and tissues in its vicinity. Nitric oxide can react with DNA via multiple pathways. Once produced, subsequent conversion of nitric oxide to nitrous anhydride and/or peroxynitrite can lead to the nitrosative deamination of DNA bases such as guanine and cytosine. Complex oxidation chemistry can also occur causing DNA base and sugar oxidative modifications. This review describes the different mechanisms by which nitric oxide can damage DNA. First, the physiological significance of nitric oxide is discussed. Details of nitric oxide and peroxynitrite chemistry are then given. The final two sections outline the mechanisms underlying DNA damage induced by nitric oxide and peroxynitrite.