Synthesis of DNA oligonucleotides containing site-specifically incorporated O⁶-[4-oxo-4-(3-pyridyl)butyl]guanine and their reaction with O⁶-alkylguanine-DNA alkyltransferase

DNA pyridyloxobutylation occurs during the metabolic activation of the tobacco-specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN). This pathway contributes significantly to the carcinogenic and mutagenic activity of these nitrosamines. In general, the chemical structure of pyridyloxobutyl adducts are not well understood. Recently, an AGT reactive pyridyloxobutyl adduct was identified as O⁶-[4- oxo-4(3-pyridyl)butyl]guanine (O⁶-pobG). To better understand the importance of this adduct to the biological activity of pyridyloxobutylating agents, we developed a method for site-specifically incorporating O⁶-pobG into DNA oligonucleotides. They were synthesized using the phosphoramidite of the precursor 2'-deoxy-O⁶-{3-[2-(3-pyridyl)-1,3-dithian-2-yl]propyl}guanosine. The dithiane group was oxidatively removed with N-chlorosuccinimide in a final postoligomerization reaction to generate the desired product. Human AGT with a polyhistidine tag was able to repair the O⁶-pobG-containing DNA oligonucleotide, generating unmodified oligonucleotide. These results are consistent with an alkyl group transfer mechanism for the repair of O⁶-pobG by AGT.