PROBING MICROTUBULES WITH TAXOL PHOTOAFFINITY ANALOGS

紫杉醇是从短叶红豆杉(Taxus brevifolia(Pacific Yew))中提取的一种复杂的二萜化合物。 作为一种极具潜力的抗肿瘤化疗药物，尤其是 治疗卵巢癌。它通过与微管结合来发挥作用 对他们来说是高度稳定的。虽然有一些关于生物多样性的研究 紫杉醇及其一些含有微管蛋白和微管的衍生物已经被 完成后，对其在微管蛋白中的结合位置或结构域一无所知。是这样的 为活性紫杉醇的进一步开发提供了有用的信息 衍生品。这项提议的总体目标是综合 紫杉醇的光亲和类似物及其用于鉴定紫杉醇中的多肽 紫杉醇结合部位。这项工作分为三个部分。我们计划 苯基叠氮、硝基苯肼和三氟乙基苯甲酰基的合成 紫杉醇的二氮杂衍生物。这些功能将放置在 巴卡菌素III和苯异丝氨酸侧链的不同位置 半个。如果我们发现，并不是所有建议的化合物都会被合成 在工作的早期就有用的。紫杉醇的衍生物将与 紫杉醇在微管组装反应中的活性 体外和体外培养的B16黑色素瘤细胞。主动者的绑定 还将测量化合物到微管和微管蛋白的含量。这个 具有良好活性和结合力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。五花八门 消化和分离程序以及多肽测序和 质谱学将用于鉴定含有共价键的多肽 绑定类比。

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.