Synthesis and characterization of oligodeoxyribonucleotides containing a site-specifically incorporated N(6)-carboxymethyl-2′-deoxyadenosine or N(4)-carboxymethyl-2′-deoxycytidine

Humans are exposed to both endogenous and exogenous N-nitroso compounds (NOCs), and many NOCs can be metabolically activated to generate a highly reactive species, diazoacetate, which is capable of inducing carboxymethylation of nucleobases in DNA. Here we report, for the first time, the chemical syntheses of authentic N(6)-carboxymethyl-2′-deoxyadenosine (N(6)-CMdA) and N(4)-carboxymethyl-2′-deoxycytidine (N(4)-CMdC), liquid chromatography–ESI tandem MS confirmation of their formation in calf thymus DNA upon diazoacetate exposure, and the preparation of oligodeoxyribonucleotides containing a site-specifically incorporated N(6)-CMdA or N(4)-CMdC. Additionally, thermodynamic studies showed that the substitutions of a dA with N(6)-CMdA and dC with N(4)-CMdC in a 12-mer duplex increased Gibbs free energy for duplex formation at 25°C by 5.3 and 6.8 kcal/mol, respectively. Moreover, primer extension assay revealed that N(4)-CMdC was a stronger blockade to Klenow fragment-mediated primer extension than N(6)-CMdA. The polymerase displayed substantial frequency of misincorporation of dAMP opposite N(6)-CMdA and, to a lesser extent, misinsertion of dAMP and dTMP opposite N(4)-CMdC. The formation and the mutagenic potential of N(6)-CMdA and N(4)-CMdC suggest that these lesions may bear important implications in the etiology of NOC-induced tumor development.