SYNTHESIS AND MECHANISM OF DNA--CROSSLINKING OF FR900482

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The primary objective of the proposed research is to study the mechanism of action of clinically significant new anti-tumor FR900482 (1a), FK973 (1b) and FR66979 (1c) (FK973, 1b was the first derivative to go to clinical trials). FK973 has been shown to cross-link double-stranded DNA and cross-link DNA to DNA-binding proteins in L1210 cells. Efforts will be directed at elucidating in complete mechanistic detail, the precise mechanism of the in vitro reductive activation of FR900482 that results in covalent modification of DNA. In the previous funding period, we have determined the sequence specificity of DNA cross-link formation by FR900482 and the natural reduction product FR66979 and have determined that reduction of these substances leads to the production of a mitosene derivative that preferentially cross-links DNA at 5'-dC-dG3' boxes. The interaction of FK973 with DNA complexed to various DNA-binding proteins will also be examined in detail. Synthetic DNA substrates will be constructed and incubated with their respective DNA-binding proteins in the presence of FK793 (or FR900482); subsequent enzymatic digestion of the cross-linked nucleotide-drug-amino acid adduct will be examined to isolate and characterize the structure of the covalent cross-link. Synthetic methodology developed during the first funding cycle will be utilized to complete a total synthesis of FR900482 (1a) and FR66979 (2); this technology will be applied to the synthesis of an isotopically-labeled form of the drug for use in elucidating the structure of the DNA-drug- protein cross-link. A new class of "latent" triggered mitosenes will be synthesized and utilized as potential new anti-cancer drugs and probes for the macromolecular cross-links.