Free-radical-induced formation of an 8,5′-cyclo-2′-deoxyguanosine moiety in deoxyribonucleic acid

Abstract

Isolation and identification of a novel .OH-induced product, namely an 8,5′-cyclo-2′-deoxyguanosine moiety, in DNA and 2′-deoxyguanosine are described. .OH radicals were generated in dilute aqueous solutions by gamma-irradiation. Analyses of 2′-deoxyguanosine and enzymic hydrolysates of DNA by gas chromatography-mass spectrometry (g.c.-m.s.) after trimethylsilylation showed the presence of 8,5-cyclo-2′-deoxyguanosine on the basis of its fragment ions. This product was isolated by h.p.l.c. Its u.v. and n.m.r. spectra taken were in agreement with the structure suggested by its mass spectrum. Exact masses of the typical ions from the mass spectrum of the trimethylsilyl derivative of this product were measured by high-resolution m.s. The values found were in excellent agreement with the theoretical mass derived from the suggested fragmentation patterns. Both (5′R)- and (5′S)-epimers of 8,5′-cyclo-2′-deoxyguanosine were observed. These two diastereomers were separated from each other by g.c. as well as by h.p.l.c. The assignment of the epimers was accomplished on the basis of the n.m.r. data. The formation of 8,5′-cyclo-2′-deoxyguanosine was suppressed by the presence of O2 in the solutions. The use of g.c.-m.s. with the selected-ion monitoring technique facilitated the detection of 8,5′-cyclo-2′-deoxyguanosine in DNA at radiation doses as low as 1 Gy. Its mechanism of formation probably involves hydrogen atom abstraction by .OH radicals from the C-5′ of the 2′-deoxyguanosine moiety followed by intramolecular cyclization with the formation of a covalent bond between the C-5′ and C-8 and subsequent oxidation of the resulting N-7-centred radical.