Abstract
Oligonucleotide sequences synthesized with specifically positioned and structurally defined adducts of dihydrodiol epoxides (DE) of polycyclic aromatic hydrocarbons like benzo[a]pyrene (B[a]P) are useful tools to study in detail the solution structure of corresponding duplexes by NMR techniques as well as the interaction of a single adduct with DNA polymerases. Here we report the successful incorporation of trans-N 6-dA-B[a]PDE adducts derived from the syn- and anti-diastereomers of B[a]PDE into 18-mer oligonucleotides representing partial human Ha-ras sequences surrounding codon 61 (CAG). The key step in our approach is a nucleophilic displacement reaction of a deoxyinosine derivative activated at the 6-position by a sulfonate group with a racemic aminotriol providing a regio- and stereospecific synthesis of the N 6-dA adducts. The aminotriol precursors are prepared by direct aminolysis of the DE's or by a stereoselective opening of the oxirane ring with sodium azide followed by reduction. The fully protected diastereomeric trans-N 6-dA-B[a]PDE adducts were separated by preparative HPLC and subsequently transformed into the corresponding phosphoramidites. The synthesis of four 18-mers was performed on a DNA synthesizer incorporating in each sequence [d(5′-GGC·CA*G·GAG·GAG·TAC·AGC-3′)] a single dA adduct (A*) at Codon 61 using standard phosphoramidite chemistry. After extensive purification of the 18-mers by reverse phase HPLC and analysis by PAGE, the presence of the trans-N6 -dA-B[a]PDE adducts in the oligonucleotides was confirmed by fluorescence spectroscopy.