Alkylation of deoxyribonucleic acid by carcinogens dimethyl sulphate, ethyl methanesulphonate, N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea. Relative reactivity of the phosphodiester site thymidylyl(3′-5′)thymidine

Abstract

The ethyl phosphotriester of thymidylyl(3''-5'')thymidine, dTp(Et)dT, was identified as a product from reaction of salmon sperm DNA with N-ethyl-N-nitrosourea, by procedures parallel to those reported previously for the methyl homolog produced by N-methyl-N-nitrosourea. Enzymic degradation to yield alkyl phosphotriesters from DNA alkylated by these carcinogens and dimethyl sulfate and ethyl methanesulfonate was studied quantitatively, and the relative yields of the triesters dTp(Alk)dT were determined. The relative reactivity of the phosphodiester group dTpdT to each of the 4 carcinogens was obtained, and compared with that of DNA overall, or with that of the N-7 atom of guanine in DNA. Relative reactivity of the phosphodiester group was lowest towards dimethyl sulfate, the least electrophilic of the reagents used, and was highest toward N-ethyl-N-nitrosourea, the most electrophilic reagent. The nature of the alkyl group transferred also influenced reactivity of the phosphodiester site, since this site was relatively more reactive toward ethylation than would be predicted from the known Swain-Scott s values of the alkylating agents. The steric accessibility of the weakly nucleophilic phosphodiester group on the outside of the DNA macromolecule favors its reaction with ethylating, as opposed to methylating, reagents. Taking a value of the Swain-Scott nucleophilicity (n) of 2.5 for an average DNA nucleotide unit, a value of n of about 1 for the phosphodiester group was deduced, and this value was 2-3 units less than that for the N-7 atom of guanine in DNA. The reactivity of DNA overall was high toward the alkylnitrosoureas, despite their relatively low s values. This was ascribed to an electrostatic factor that favored reaction of the negatively charged polymer with alkyldiazonium cation intermediates.