Formation of Carboxymethyl-Phosphotriester Adducts in DNA

Humans are exposed to endogenous and exogenous sources of <i>N</i>-nitroso compounds (NOCs). Metabolic activation of some endogenous NOCs can yield diazoacetate, which is known to induce the formation of carboxymethylated DNA adducts that are implicated in human gastrointestinal tumors. Although carboxymethylated nucleobase adducts have been investigated, no studies have assessed if carboxymethylation occurs on the phosphate backbone of DNA. In this study, we report the synthesis of a carboxymethyl phosphotriester (CM-PTE) phosphoramidite building block of thymidine and the preparation of oligodeoxyribonucleotides (ODNs) containing a site-specifically inserted CM-PTE. By employing liquid-chromatography-tandem mass spectrometry (LC-MS/MS) analysis, we also demonstrated the formation of CM-PTE adducts in calf thymus DNA treated with diazoacetate, where we identified a total of 16 CM-PTE products across all possible combinations of flanking nucleobases. Together, our findings laid the foundation for exploring the in vivo formation and biological consequences of the CM-PTE lesions.