Site-specific modification of the human N-ras proto-oncogene with each diol epoxide metabolite of benzo[a]pyrene and thermal denaturation studies of the adducted duplexes

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Abstract
The central adenine residue (Å) of codon 61 in the human N-ras proto-oncogene, 5'-CGGÅCAAGAAG-3', has been modified with each enantiomer of the series 1 (DE-1, syn) and series 2 (DE-2, anti) benzo[a]pyrene diol epoxide through total chemical synthesis. The resulting DNA adducts correspond to a trans ring-opening of the oxiranes at the C-10 position of the hydrocarbon by the exocyclic amino group of the purine (the relative stereochemistry between the C-9 and C-10 substituents is trans). The synthesis involved coupling of 6-fluoro-9-(2-deoxy-β-D-erythro-pentofuranosyl)purine with the racemic aminotri-benzoates derived from each diol epoxide. The resulting pairs of diastereomeric adducts were converted to the 5'-O-DMT 3'-O-phosphoramidites and incorporated into the DNA sequence through a partially automated procedure. Resolution of the diastereomeric oligomers resulting from each diol epoxide enantiomer was conveniently achieved at the very end of the synthesis. This adds simplicity and efficiency to the preparation of alkylated oligomers through this route. Thermal denaturation of the modified duplexes with a complementary strand, as well as a partially complementary target containing a central apurinic site, has been evaluated. These studies indicate striking differences in the absorbance-temperature co-operativity when the 345 nm pyrene absorption is monitored. The results of such previously undescribed experiments provide a comparison of the physical properties of oligomers that differ in the arrangement of substituents in the hydrocarbon moiety, but are otherwise identical. We believe that such comparisons between diol epoxide-DNA adducts of the same hydrocarbon and those of different hydrocarbons will provide information about the orientation of the hydrocarbon moieties relative to the adjacent bases. Therefore, these results will be useful parameters in the evaluation of structure-activity relationships of diol epoxide-DNA lesions.

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