SYNTHESIS OF MULTIPLY-LABELED [N-15(3),C-13(1)]-8-OXO SUBSTITUTED PURINE-BASES AND THEIR CORRESPONDING 2'-DEOXYNUCLEOSIDES

Stable isotope-labeled analogues of oxidatively modified purine bases are required as internal standards for accurate quantitation of free r adical induced damage in DNA using the isotope-dilution GC/MS techniqu e. For this reason, we report on a facile and expedient method to synt hesize the isotope-labeled oxidized DNA bases 8-oxoguanine (8-oxo-Gua, 5a) and 8-oxoadenine (8-oxo-Ade, 5b). Both routes have in common the introduction of two exocyclic N-15 isotopes simultaneously by halogen displacement of chlorine-substituted pyrimidines,vith [N-15]- benzylam ine. Debenzylation is achieved by either catalytic hydrogenation or tr eatment with aluminium chloride in benzene. An additional isotope is i ncorporated by nitrosation with N-15- labeled sodium nitrite. Cyclocon densation of the triamines with C-13-labeled urea then affords 5a and 5b in overall yields of 34% and 27%, respectively, and each with four isotope labels and at least 99 atom % excess. A further one-step enzym e catalyzed coupling of the C8 adducted purines with 2'-deoxyribose fu rnishes the isotope-labeled 2'-deoxynucleosides 2'-deoxy-7,8-dihydro-8 -oxoguanosine (8-oxo-dGuo) and 2'-deoxy-7,8-dihydro-8-oxoadenosine (8- oxo-dAdo).