SYNTHESIS AND CHARACTERIZATION OF COVALENT ADDUCTS DERIVED FROM THE BINDING OF BENZO[A]PYRENE DIOL EPOXIDE TO A -GGG- SEQUENCE IN A DEOXYOLIGONUCLEOTIDE

Direct synthesis and purification procedures are described for the pre paration of adducts derived from the covalent binding of xy-9S,10R-epo xy-7,8,9,10-tetrahydro-benzo[a]pyrene [(+)-anti-BPDE or (+)-BPDE 2] to each of the three guanine residues (trans-N-2-dG lesions) in the olig odeoxyribonucleotide d(CTATG(1)G(2)G(3)TATC). The positions of the mod ified Gs are defined by Maxam - Gilbert sequencing techniques. Six dif ferent oligonucleotides with one or two precisely positioned (+)-anti- BPDE residues are identified. The absorbance, circular dichroism and f luorescence characteristics are changed upon formation of duplexes wit h the complementary strands d(GATACCCATAG). In the doubly-modified oli gonucleotides, a broad, excimer-like long wavelength fluorescence emis sion band is observed with a maximum near 455 nm only if the two (+)-a nti-BPDE-modified Gs are adjacent to one another. The covalently attac hed (+)-anti-BPDE residues decrease the thermodynamic stabilities of t he duplexes; their melting points are markedly dependent on the positi on of the lesions, being highest with the (+)-anti-BPDE residue at G(1 ) (T-m = 40 degrees C, only 2 degrees C lower than in the case of the unmodified oligonucleotide) and lowest when it is situated at G(3) (T- m = 29 degrees C). The implications of these and other physical charac teristics are discussed. The facile synthesis of these or similar site -specific and stereochemically defined (+)-trans-anti-BPDE-N-2-dG lesi ons in runs of contiguous guanines in oligodeoxyribonucleotides of spe cified base sequence should be useful for the design of site-directed mutagenesis studies in vitro and in vivo.