PROBING MICROTUBULES WITH TAXOL PHOTOAFFINITY ANALOGS

紫杉醇是一种来自短叶红豆杉（太平洋紫杉）的复杂二萜， 作为抗肿瘤化疗剂的优异潜力，特别是 对抗卵巢癌 它通过与微管结合而起作用， 超稳定性。 尽管一些关于 紫杉醇和它的一些衍生物与微管蛋白和微管已经被 尽管如此，关于它在微管蛋白中的结合位点或结构域仍是未知的。 等 这些信息对活性紫杉醇的进一步开发是有用的 衍生物. 本提案的总体目标是综合 紫杉醇的光亲和类似物，并使用它们来鉴定 紫杉醇结合位点。 这项工作分为三个部分。 我们计划 合成苯叠氮基、硝基苯叠氮基、三氟乙基苯甲酰基 紫杉醇的二氮杂环丙烷衍生物。 这些功能将放在 浆果赤霉素III和苯基异丝氨酸侧链的不同位置 部分。 并不是所有提出的化合物将被合成，如果我们发现， 在工作的早期有用的。 紫杉醇衍生物将与 紫杉醇在微管组装反应中的活性 在体外和在培养的B16黑素瘤细胞上。 活性物质的结合 还将测量化合物对微管和微管蛋白的作用。 的 具有良好活性和结合的类似物将用于标记微管蛋白。 这将需要合成放射性标记的类似物。 各种 消化和分离程序以及肽测序， 质谱法将用于鉴定含有共价键的肽 结合类似物

Taxol, a complex diterpene from Taxus brevifolia (Pacific Yew), has shown excellent potential as an anti-tumor chemotherapeutic agent, especially against ovarian cancer. It acts by binding to microtubules imparting hyperstability to them. Although some studies on the interaction of taxol and some of its derivatives with tubulin and microtubules have been done, nothing is known about its binding site or domain in tubulin. Such information is useful for the further development of active taxol derivatives. The overall aim of this proposal is to synthesize photoaffinity analogues of taxol and to use them to identify peptides in the taxol binding site. The work is divided into three parts. We plan to synthesize phenylazido, nitrophenylazido, and trifluoroethyl benzoyl diazirine derivatives of taxol. These functionalities will be placed at different positions of the baccatin III and phenylisoserine side chain moieties. Not all proposed compounds will be synthesized if we find useful ones early in the work. The taxol derivatives will be compared to taxol in regard to their activities in the microtubule assembly reaction in vitro and on B16 melanoma cells in culture. The binding of the active compounds to microtubules and tubulin will also be measured. The analogues with good activities and binding will be used to label tubulin. This will require the synthesis of radiolabeled analogues. Various digestion and separation procedures together with peptide sequencing and mass spectrometry will be used to identify peptides containing covalently bound analogue.