SYNTHESIS AND MECHANISM OF DNA--CROSSLINKING OF FR900482

建议的研究的主要目的是研究这种机制。 临床重要抗肿瘤新药FR900482(1a)、FK973的作用研究 (1b)和FR66979(1c)(FK973，1b是第一个导数 临床试验)。FK973已被证明是双链DNA的交联剂 并在L1210细胞中将DNA与DNA结合蛋白进行交联。努力将会 旨在阐明完整的机械细节，精确的 FR900482的体外还原活化作用机制 DNA的共价修饰。在上一个资助期，我们有 通过以下方法确定DNA交联形成的序列特异性 FR900482和天然还原产物FR66979，并测定了 这些物质的减少会导致有丝分裂素的产生 优先在5‘-DC-DG3’盒上使DNA交联的衍生物。这个 FK973与多种DNA结合蛋白络合DNA的相互作用 还将详细审查。合成的DNA底物将是 构建并与它们各自的DNA结合蛋白在 FK793(或FR900482)的存在；随后对 将对交联的核苷酸-药物-氨基酸加合物进行检测以分离 并表征共价交联物的结构。合成的 将利用在第一个供资周期内制定的方法 完成FR900482(1a)和FR66979(2)的全合成； 这项技术将应用于合成一种同位素标记的 用于阐明DNA结构的药物的形式-药物- 蛋白质交联剂。一类新的“潜伏”触发的有丝分裂将是 作为潜在的抗癌新药和探针的合成和利用 大分子的交联物。

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.