Regio/stereochem defined, diol epoxide adducted ras

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DESCRIPTION: This proposal is a request for support for a beginning investigator's program in the area of chemical carcinogenesis by polycyclic aromatic hydrocarbons (PAHs). PAHs, by virtue of being ubiquitous environmental pollutant, pose a cancer threat to humans. Many members of this class undergo metabolic activation to four reactive diol epoxides. In the presence of DNA, these metabolites bind largely to purine residues forming covalent lesions. Formation of covalent diolepoxide-DNA adducts is implicated as the first step in the biological cascade that ultimately leads to mutagenesis and tumorigenesis. Although structural features o f the hydrocarbon metabolites associated with high carcinogenic activity have been known for quite some time, recent studies have become focused on answering questions on DNA adduct structure and biology subsequent to DNA binding by these metabolites. Of the four metabolically produced isomeric diol epoxides of any hydrocarbon, only one isomer exhibits high tumorigenic activity. However, all four diol epoxides alkylate DNA producing a total of 16 adducts. Thus a question that is central to the overall understanding of chemical carcinogenesis lies in determining how isomeric DNA adducts affect local DNA structure and enzymatic replication of repair. In order to address this question, it then becomes necessary to have a set of probes for physical, biochemical and biological experimentation. To date all 16 adducts of any PAH are not available for comprehensive, comparative studies. The PI proposes rational, highly diastereoselective syntheses of all 16 epoxide adducts of two structurally different PAHs benzo[a]pyrene and benzo[c]phenanthrene, within uniform, biologically important DNA sequence contexts. The differences in PAH structure provide a test for the generality of the syntheses, while uniformity of sequence context provides for comparisons and generalizations of structural and biological effects. For this study, the PI has chosen the human ras sequences (N-ras protooncogene codons 60-62 for A modification at codon 61 and c-Ki-ras protooncogene codons 11-13 for G modification at codon 12), both of which are purine rich. This study will therefore provide 16 N-ras and 16c-Ki-ras oligomers with two stereoisomers of two PAH diol epoxides. In and of itself, this is a nontrivial, previously accomplished task. The PI will then undertake evaluation of thermal denaturation, UV and CD properties of the PAH modified duplexes with normal targets and targets having mismatched bases opposite the lesion. The latter is important because it is believed that misincorporation opposite an adduct could be critical to the mutagenic event. Since structural information can be derived from NMR studies, the syntheses are designed to produce sufficient materials for this. The PI will also collaborate with other groups and the topics to be addressed are (a) NMR analyses, (b) possibly x-ray structures, (c) mutation analyses, (d) DNA repair, and (e) fluorescence studies.