Studies of transalkylation of phosphotriesters in DNA: Reaction conditions and requirements on nucleophiles for determination of DNA adducts

Abstract

Reactive compounds form adducts at several sites in DNA. One of these sites, the phosphate groups, forms phosphotriesters (PTE) which are both chemically stable and little repaired. A measurement of PTE in DNA could therefore be advantageous for the determination of doses in vivo of mutagens/cancer initiators. In this paper, the possibilities of utilizing the weakly alkylating properties of PTE for the transfer of adducts to strong nucleophiles have been investigated. Model compounds, thymidine 3′-[thymidine 5′-(methyl phosphate)], TpMeT, and thymidine 3′-[thymidine 5′-(2-hydroxyethyl phosphate)], TpHOEtT, were incubated with thiosulfate, a relatively strong nucleophile and the formation of dealkylated model PTE, thymidine 3′-(thymidine 5′-phoshate), TpT, was followed by HPLC. Transalkylation to thiosulfate or aniline of methyl PTE in DNA alkylated by [ 3H] N-methyl- N-nitrosourea was demonstrated. The methyl groups transferred, forming methyl thiosulfate and N-methylaniline, respectively, were determined by HPLC. These experiments demonstrate that it is possible to transfer alkyls from DNA phosphate to nucleophiles. Kinetic aspects of the transalkylation and requirement on nucleophiles for a practically useful method for determination of DNA adducts are discussed. Constants of reaction rates are presented.