Reactivity of haloketenes and halothioketenes with nucleobases:: Reactions in vitro with DNA

Ketenes are important and highly reactive intermediates. Thioketenes are formed by cysteine conjugate beta-lyase-dependent biotransformation of 1-halovinylcysteine S-conjugates which are metabolites of several halogenated olefins. Nucleic acid constituents react with haloketenes and halothioketenes in vitro. Thioketenes induce DNA strand breaks in incubations of 1,2-dichlorovinyl 2-nitrophenyl disulfide, a thioketene precursor, with pBr 322 plasmid DNA. After treatment of single-stranded or native calf thymus DNA with chlorothioketene generated by the hydrolysis of 1,2-dichlorovinyl 2-nitrophenyl disulfide, the formation of 3,N-4-thioacetylcytosine could be demonstrated. N-6-(Dichloroacetyl)adenine and N-4-(dichloroacetyl)cytosine, however, adducts formed by dichloroketene in vitro, are labile to hydrolysis. Therefore, the binding of this compound to DNA constituents in intact DNA is difficult to demonstrate. Substitution of one chlorine atom by fluorine allowed us to use F-19 NMR, as a tool to demonstrate the formation of adducts by dihaloketenes in intact DNA. N-6-(Chlorofluoroacetyl)adenine and N-4-(chlorofluoroacetyl)cytosine were synthesized (yields 77%, 15%, respectively) as references and characterized by LC/MS, H-1, C-13, and F-19 NMR, FT-IR, and elemental analysis. To demonstrate the ability of dihalogetenes to bind to DNA, poly-dA (1 mg) and calf thymus DNA (10 mg) were suspended in DMF and treated with different concentrations of chlorofluoroketene (50-200 mu mol). Analysis of the polymeric DNA by F-19 NMR showed one doublet at -137.2 ppm downfield from the reference (CFCl3). A doublet at -146.9 ppm, characteristic for chlorofluoroacetic acid, an expected product of DNA adduct hydrolysis, was not detected. These results demonstrate the formation of a stable adenine adduct by dihaloketenes in intact calf thymus DNA.