Synthesis and characterization of site-specific and stereoisomeric fjord dibenzo[a,l]pyrene diol epoxide - N⁶-adenine adducts: Unusual thermal stabilization of modified DNA duplexes

The fjord polycyclic aromatic hydrocarbon compound dibenzo[a,l]pyrene (DB[a,l]P) is significantly more tumorigenic than the bay region benzo[a]pyrene in animal model systems. The molecular origins of the unusually strong genotoxic properties of DB[a,l]P and its fjord region diol epoxide metabolites are of great interest and are believed to be related to the structural characteristics of the DNA adducts formed. Site-specifically modified oligonucleotides were prepared by reacting the single adenine residue in 5′-d(CTCTCACTTCC) (I) with the racemic fjord diol epoxide r11,t12-dihydrodiol-t13,14-epoxide-11,12,13,14-tetrahydrodibenzo-[a,l]pyrene (anti-DB[a,l]PDE) in aqueous solutions. Four different oligonucleotides I with the single adenosine residues involving a covalent bond between the C14 position of DB[a,l]PDE and N⁶-dA are identified and purified. The CD spectra of the mononucleotide adducts are similar to those of Li et al. [Li et al. (1999) Chem. Res. Toxicol. 12, 758] who characterized DB[a,l]-PDE-N⁶-dA adducts by a combination of CD and NMR methods. The stereochemical properties of each of the four DB[a,l]PDE-modified oligonucleotides were assigned on the basis of a combination of empirical CD rules and other approaches and differ from those of Li et al. The thermal melting points, T_m, of the unmodified duplex of I with its complementary strand (IC), T_m = 43.8 ± 0.5 °C, were compared with the same duplexes containing stereoisomeric anti-DB[a,l]PDE-N⁶-dA lesions. The T_m of duplexes I·IC containing lesions with R absolute configurations at C14 of the DB[a,l]PDE residues are greater by 6-8 °C, while those with S configuration are lower by 6-10 °C. Similar effects are observed with adducts in the same sequence context derived from the fjord PAH anti-diol epoxides of benzo[g]chrysene, while duplexes containing lesions derived from benzo[c]phenanthrene diol epoxides with 1R and 1S configurations exhibit unchanged T_m values. In contrast, the T_m values of duplexes with lesions derived from the bay region benzo[a]pyrene diol epoxides (B[a]PDE) in the same sequence are lower by 12° (10R adducts) and by 19° (10S adducts). The greater thermal stabilities of duplexes with fjord PAH-N⁶-dA lesions relative to those with bay region B[a]PDE-N⁶-dA adducts, are correlated with lower susceptibilities of excision by human nucleotide excision repair enzymes [Buterin et al. (2000) Cancer Res. 60, 1849]. The implications of these relationships are discussed in terms of present knowledge of the conformations of fjord and bay region PAH diol epoxide-N⁶-dA lesions in double stranded DNA.