Synthesis of DNA oligonucleotides containing site-specifically incorporated O-6-[4-oxo-4-(3-pyridyl)butyl]guanine and their reaction with O-6-alkylguanine-DNA alkyltransferase

DNA pyridyloxobutylation occurs during the metabolic activation of the toba cco-specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone ( NNK) and N'-nitrosonornicotine (NNN). This pathway contributes significantl y to the carcinogenic and mutagenic activity of these nitrosamines. In gene ral, the chemical structure of pyridyloxobutyl adducts are not well underst ood. Recently, an AGT reactive pyridyloxobutyl adduct was identified as O-6 -[4-oxo-4-(3-pyridyl)butyl]guanine (O-6-pobG). To better understand the imp ortance of this adduct to the biological activity of pyridyloxobutylating a gents, we developed a method for site-specifically incorporating O-6-pobG i nto DNA oligonucleotides. They were synthesized using the phosphoramidite o f the precursor 2'-deoxy-O-6-{3-[2-(3-pyridyl)-1,3-dithian-2-yl]propyl}guan osine The dithiane group was oxidatively removed with N-chlorosuccinimide i n a final. postoligomerization reaction to generate the desired product. Hu man AGT with a polyhistidine tag was able to repair the O-6-pobG-containing DNA oligonucleotide, generating unmodified oligonucleotide. These results are consistent with an alkyl group transfer mechanism for the repair of O-6 -pobG by AGT.