Abstract 1700: An improved synthesis and mutagenicity studies of O2-[4-(3-pyridyl-4-oxobut-1-yl]thymidine, the major adduct formed by tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in vivo

Abstract

Abstract Among the four pyridyloxobutyl (POB)-DNA adducts formed in the lung and liver of rats treated with tobacco specific nitrosamines [N′-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL)], the O2-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O2-POB-dThd) has been found to be the most persistent. It is an important biomarker to assess the human exposure to these carcinogens and also serves as a useful precursor for the synthesis of site-specifically modified oligomers required for mutagenicity studies. The only synthetic method reported for O2-POB-dThd requires repeated HPLC purifications and could only be used to prepare an analytical standard due to very low yield (0.4%). Our continued efforts have now led to the development of an efficient method for the total synthesis of O2-POB-dThd and its site-specifically adducted oligonucleotides. The synthesis of O2-POB-dThd was achieved by optimizing reaction between O2-5′-anhydrothymidine and sodium salt of 4-(1,3-dithian-2-yl)-4-(3-pyridyl)butan-1-ol, followed by deprotection of the dithiane group. The product was characterized by NMR (1H, 13C, and COSY spectra), MS, and HPLC analysis. This synthetic strategy provided for the first time a reliable method for a gram scale total synthesis of O2-POB-dThd that allowed for solid state site-specifically adducted oligomer synthesis. O2-POB-dThd was converted to its phosphoramidite and subsequently used for the synthesis of the oligomer by standard methods. The oligomer was characterized by MS and HPLC analysis. We examined the in vitro kinetics of the synthesized oligomer with the Klenow fragment of E. Coli DNA polymerase I with the exonuclease activity inactivated (Kf(exo-)) and human DNA polymerasesl eta, kappa, and iota). O2-POB-dThd was replicated in a mutagenic manner with with Kf(exo-)and human pol eta and was unreactive with pol kappa and iota. In summary, highly facile syntheses of O2-POB-dThd and its site-specifically adducted oligonucleotide are developed. The study will facilitate elucidation of mechanistic aspects and mutagenic potential of O2-POB-dThd adduct, and will provide further insight into the role it plays in inducing cancers in smokers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1700.