分子进化α-L-鼠李糖苷酶及酶法合成抗肿瘤药物的鼠李糖苷研究

The synthesis of complex glycosides and their derivatives in larger quantities has been the bottleneck of the development of novel carbohydrate therapeutics. For more than one available reactive hydroxyl group of carbohydrates, the synthesis of stereo- and regio-specific glycosidic linkages by classical chemical synthesis strategy is laborious and very difficult for application, while some enzymes can catalyze the formation of a specific glycosidic bond in one step. The glycosyltransferases and the glycosidases are two kinds of enzymes which are used in the present for that purpose. The former needs the expensive sugar nucleoside as the glycosyl donor, while the latter uses simple sugar as the glycosyl donor. Thus, relative to the former, the latter cost remarkably reduced. The anticancer activity of natural rhamnoside, for example, solasodine glycosides that is a mixture of glycoalkaloids from the Devil’s Apple plant, is mainly attributed to the rhamnose residues. The anticancer activity of rhamnoside in vivo was found to be inhibited by rhamnose. RBL (rhamnose binding lectin) receptor with high level expression on the tumor cell surface could recognise and bind rhamnose residues of the natural rhamnosides to mediate the transportation, which provides a new strategy to design chemotherapy agents by introducing rhamnose into the structure to increase the cell uptake or cancer targeting effects. In this project, molecular evolution of a fungal α-L-rhamnosidase from Alternaria sp. L1 by directed evolution and site-saturation mutagenesis to enhance the rhamnosylation activity and widen the range of glycosyl acceptors will be carried out, and the anticancer drugs will be enzymatic rhamnosylated with the mutant enzymes. Then, biological function of the rhamnosylation products, including the ability of targeting to tumor cells and anti-tumor activity, will be examined. This research will firstly report an α-L-rhamnosidase from Alternaria sp. L1 with rhamnosylation activity and its molecular evolution in the world. Moreover, the mutant enzymes with high rhamnosylation activity and widening range of glycosyl acceptors will become the powerful tools for synthesis of rhamnoside derivatives. This work will promote the process of glycosylation and accelerate the development of carbohydrate therapeutics, especially provide scientific basis and technical foundation for the development of noval rhamnosylated anticancer drugs with the ability of targeting to tumor cells.

糖类物质的大量合成一直是新的糖类药物开发的首要瓶颈。糖分子的多个可反应羟基使得立体和区域特异糖苷键的化学合成步骤繁琐，而酶法合成能一步完成。糖苷酶法使用简单糖类为糖基供体进行合成，其成本显著低于需要糖核苷酸为供体的糖基转移酶法。天然的鼠李糖苷类抗肿瘤药物分子中，鼠李糖基团介导了药物与肿瘤细胞表面的鼠李糖识别受体特异结合，为靶向性抗肿瘤药物设计提供了依据。我们已筛选获得了一种真菌的α-L-鼠李糖苷酶，发现该酶能以鼠李糖为供体，催化糖基化反应，合成多种新的鼠李糖苷类化合物。本项目拟进行该酶的分子改造，提高糖基化活性，拓展糖基化受体底物特异性，进一步研究酶对现有抗肿瘤药物进行鼠李糖基化的能力，酶法合成抗肿瘤药物的鼠李糖苷，研究产物的肿瘤靶向性和抗肿瘤活性。本项目首次研究α-L-鼠李糖苷酶分子改造及其应用于抗肿瘤药物的鼠李糖苷合成，将为新的靶向性鼠李糖苷类抗肿瘤药物研发提供科学依据并奠定技术基础。

天然的鼠李糖苷类抗肿瘤药物分子中，鼠李糖基团介导了药物与肿瘤细胞表面高量存在的鼠李糖识别受体特异结合，为靶向性抗肿瘤药物设计提供了依据。本项目在进行了一种链格孢霉（Alternaria sp. L1）的α-L-鼠李糖苷酶（RhaL1）的非理性的随机突变及理性的定点突变分子改造，提高了酶的逆水解合成鼠李糖苷的活性。研究了酶对现有抗肿瘤药物进行鼠李糖基化的能力，酶法合成了抗肿瘤药物的4种鼠李糖苷鼠李糖基羟基脲（Rha-Hydrea）、3’-O-α-L-鼠李糖基5-氟-2'-脱氧脲核苷（Rha-FUDR-I）、5’-O-α-L-鼠李糖基-5-氟-2'-脱氧脲苷（Rha-FUDR-II）和5′-O-α-L-鼠李糖基-阿糖胞苷（Rha-Ara C），实现了糖苷酶法合成鼠李糖基化合物的目标，为复杂抗肿瘤药物的鼠李糖基化修饰提供了新的途径。进一步在证明了鼠李糖-罗丹明B荧光探针分子中的鼠李糖基通过两种肿瘤细胞细胞表面的鼠李糖凝集素介导了的高特异性吸附作用之后，研究证明了三种α-L-鼠李糖基抗肿瘤药物（前药）能够成功的释放原药进而发挥其抗肿瘤活性，为靶向抗肿瘤前药的设计和研发奠定了重要的基础，为新的鼠李糖苷类抗肿瘤药物的研发提供可靠的科学依据。之外，本项目还通过基因挖掘，首次发现了三种细菌源鼠李糖苷酶的逆水解合成鼠李糖苷的活性，拓宽了人们对鼠李糖苷酶的认识和理解。还发现了一株曲霉来源的鼠李糖苷酶能够催化阿糖胞苷和5-氟-2’-脱氧脲苷的鼠李糖基化，扩大了具有逆水解活性的α-L-鼠李糖苷酶库，为抗肿瘤药物的鼠李糖基化修饰提供了新的工具酶。最后，本研究还鉴定了一株放线菌的基因组中序列分析预测的dTDP-Rha合成基因，一釜酶法合成了dTDP-Rha、dUDP-Rha，为进一步开发放线菌源的鼠李糖基转移酶对具有抗肿瘤活性的天然产物的鼠李糖基化修饰奠定了基础。

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