The synthesis of mutagenic 2'-deoxycytidine adducts: Their incorporation into DNA and a study of their miscoding properties

1,3-Bis-(2-chloroethyl)nitrosourea (BCNU) and other haloethylnitrosourea are useful antitumor agents. The cytotoxic mechanism of BCNU has been extensively studied and it was found that the formation of DNA crosslinks and nucleoside adducts, namely 3,{dollar}Nsp4{dollar}-ethano-2{dollar}spprime{dollar}-deoxycytidine (Ethano-dC), 3-(2-hydroxyethyl)-2{dollar}spprime{dollar}-deoxycytidine (3HE-dC) and 3(2-hydroxyethyl)-2{dollar}spprime{dollar}-deoxyuridine (3HE-dU), play a key role in the expression of the biological activity of this compound. Closely related in chemical structure to these substances are several adducts that arise from the attack on DNA of the liver metabolites of vinyl chloride which is produced in large quantities and is widely used for the production of polymers, mainly PVC, by the chemical industry. Vinyl chloride is metabolized in vivo by the microsomal monooxygenases of the liver into chloroethylene oxide (CEO) and chloroacetaldyde (CAA), both of which are highly efficient mutagenic agents. They both react with DNA to form a variety of exocyclic nucleoside adducts, including 3,{dollar}Nsp4{dollar}-etheno-2{dollar}spprime{dollar}-deoxycytidine (Etheno-dC), {dollar}Nsp2{dollar},3-etheno-2{dollar}spprime{dollar}-deoxyguanosine and 1,{dollar}Nsp6{dollar}-etheno-2{dollar}spprime{dollar}-deoxyadenosine.; In order to understand fully the biological behavior of these nucleoside adducts, a number of adducts, namely 3,{dollar}Nsp4{dollar}-etheno-2{dollar}spprime{dollar}-deoxycytidine, 3,{dollar}Nsp4{dollar}-ethano-2{dollar}spprime{dollar}-deoxycytidine, 3-(2-hydroxyethyl)-2{dollar}spprime{dollar}-deoxycytidine and 3-(2-hydroxyethyl)-2{dollar}spprime{dollar}-deoxyuridine, 1,{dollar}Nsp6{dollar}-etheno-2{dollar}spprime{dollar}-deoxyadenosine, have been synthesized, and their structures and properties have been analyzed. 3,{dollar}Nsp4{dollar}-Etheno-2{dollar}spprime{dollar}-deoxycytidine, 3,{dollar}Nsp4{dollar}-ethano-2{dollar}spprime{dollar}-deoxycytidine and 3-(2-hydroxyethyl)-2{dollar}spprime{dollar}-deoxyuridine have been successfully incorporated into oligodeoxynucleotides at designated site using the phosphoramide method. The structures and sequences of these oligomers were confirmed by mass spectroscopy and by the enzyme degradation profile.; The miscoding properties of these adducts were then studied by the primer extension assay using the Klenow fragment of Polymerase I of E. coli as the polymerizing enzyme. Both the frequency of nucleoside insertion opposite the lesion and the rate of chain extension from the 3{dollar}spprime{dollar}-primer terminus were established by steady state kinetic analysis. Etheno-dC mainly induces both C {dollar}to{dollar} A and C {dollar}to{dollar} T misincorporation. 3HE-dU almost completely blocks primer extension, permitting only a small amount of dAMP and dCMP incorporation opposite the lesion. Ethano-dC blocks primer extension strongly.