DIRECT SYNTHESIS AND CHARACTERIZATION OF SITE-SPECIFIC ADENOSYL ADDUCTS DERIVED FROM THE BINDING OF A 3,4-DIHYDROXY-1,2-EPOXYBENZO[C]PHENANTHRENE STEREOISOMER TO AN 11-MER OLIGODEOXYRIBONUCLEOTIDE

Site-specifically modified oligonucleotides were obtained in milligram quantities by reacting racemic -1,2t-epoxy-1,2,3,4-tetrahydrobenzo[c] phenanthrene (B[c]PhDE-2, or anti-B[c]PhDE) with the single deoxyadeno sine (dA) residue in the oligodeoxynucleotide d(CTCTCACTTCC). Enzyme d igestion of the covalently modified oligonucleotides with the exonucle ase spleen phosphodiesterase yielded covalently linked B[c]PhDE-N-6-de oxyadenosyl monophosphate (dAMP) adducts. Comparisons of the reverse p hase HPLC retention times and CD spectra of these B[c]PhDE-3'-dAMP mon onucleotide adducts, with those of standards derived from the reaction of the enantiomers (+)- and (-)-anti-B[c]PhDE with 3'-dAMP, show that two major oligonucleotide adducts (I and II) were obtained upon react ing racemic anti-B[c]PhDE with d(CTCTCACTTCC). In oligonucleotide addu ct I, the lesion is a (+)-trans-anti-B [c]PhDE-N-6-dA residue, and in oligonucleotide adduct II it is a (-)-trans-anti-B[c]PhDE-N-6-dA resid ue. These assignments were further confirmed using a standard P-32 pos tlabeling assay of B[c]PhDE-3'-dAMP mononucleotide adducts obtained fr om the digestion of oligonucleotides I and II by spleen phosphodiester ase. The melting points (T-m) of duplexes of modified oligonucleotides I and II and their natural complementary strands are not affected sig nificantly by the presence of the covalently bound benzo[c]phenanthren yl residues. Opposite stereoselective resistance to enzyme digestion b y the exonucleases snake venom phosphodiesterase and spleen phosphodie sterase is exhibited by the stereoisomeric (+)-trans- and (-)-trans-an ti-B[c]PhDE-modified oligonucleotide adducts I and II; these results a re consistent with the intercalative insertion of the benzo[c]phenanth renyl residues on the 5'-side of the modified dA residue in adduct I, and its insertion on the 3'-side of the dA residue in adduct II, as ob served in the duplexes by high resolution NMR techniques [Cosman et al . (1993) Biochemistry 32, 12488-12497, and Cosman et al., Biochemistry , in press].