基于非水相合成肝靶向核苷类药物的半乳糖苷酶分子改造研究

The galactosyl nucleoside analogs designed by biomimetic recognition feature, could be recongnized and specifically binding to asialoglycoprotein receptor (ASGP-R) of hepatic cell, and transported intracellular. Thus, galactosyl nucleosides exhibit the features of the high selectivity of liver targeting effect and low toxicity, and exploitation of new type of liver targeting disaccharide nucleosides analogues received much attention for the liver diseases therapy. However, the few sources and high cost of galactosidase, low efficiency and productivity of the reaction, especially the properties of region-selectivity, substrate recognition and different affinities of galactosidase limited the large-scale application. Under such circumstances, we explored the novel organic-solvent tolerant galactosidase for the galactosyl nucleosides preparation. We will investigate the mechanism of substrate recognition and regioselectivity of the galactosidase by the molecular dynamic and bioinformatics approaches, and analyze the proximity and orientation between the different nucleosides substrates with the key amino acid residues nearby the enzyme active center. This may give more comprehensively and accurately theoretical explanation of the mechanism of transgalactosylation reaction by galactosidase. Furthermore, we will put forward a proposal for rational design molecular alteration of the galactosidase for the nucleosides, and high efficient mutants of galactosidase could also be obtained by protein engineering. We further study the high efficient and low-cost synthesis of biotechnology of disaccharide nucleosides in non-aqueous system. This may promote the development and application of the liver targeting disaccharide nucleoside analogs.

基于无唾液酸糖蛋白受体（ASGP-R）生物分子特异性识别、结合并转运的功能设计肝脏靶向性核苷类药物，以肝靶向半乳糖基核苷类药物合成为目的来解析改造半乳糖苷酶分子。本项目针对目前生物酶法合成二糖核苷存在的酶源有限、底物识别亲和力低、底物溶解性差导致的产率低等问题，以自行筛选的耐有机溶剂半乳糖苷酶为对象，研究酶催化活性中心微环境与核苷类底物识别结合与糖基化反应区域选择性机制，阐释并丰富半乳糖苷酶的转糖基化反应理论；提出针对核苷类底物的半乳糖苷酶分子改造新策略，蛋白质工程手段获得定向合成二糖核苷类药物高糖基化活性、优越区域选择性的糖苷酶突变体，为肝靶向核苷类药物合成酶提供改造技术。同时将非水相催化理念引入酶法合成二糖核苷的催化体系，非水溶剂促进底物溶解性，抑制水解反应，调控酶微环境，调节糖基化选择性甚至反应程度，形成高效低成本的合成二糖核苷新型非水相生物催化技术，将推动肝靶向治疗药物的开发应用。

核苷类药物齐多夫定、肌苷、5-氟脱氧尿苷、利巴韦林、地西他滨、恩曲他滨、恩替卡韦等是重要的抗乙肝病毒、丙肝病毒、HIV病毒以及白血病等肿瘤疾病治疗药物。一些治疗效果显著的核苷类药物在临床使用往往剂量大、副作用较大、靶向性较弱；半乳糖基修饰的核苷类药物不仅具有等同生物活性，而且毒副作用更低（可降低约100倍），而且能增强靶向性。本项目在克隆表达获得的β-半乳糖苷酶基础上，表达策略优化实现了酶YZ06的高效可溶性表达，该酶能催化多种核苷类药物的高区域选择性地半乳糖基化修饰。同源建模构建了酶的三维结构模型，解析其与糖基供体乳糖、核苷类底物分子的识别微环境，并分析了该酶催化核苷类底物半乳糖基化反应的区域选择性机制。以提高半乳糖基核苷产物的合成产率等为目标，采用丙氨酸扫描、定点饱和突变等策略对酶分子进行定向进化，构建含约100突变体的酶分子突变库；筛选获得了5个较高效的酶突变体。探讨了亲水性有机溶剂及金属离子对酶活性及稳定性影响，亲水性有机溶剂调控产物的生成种类，并表征了其催化反应动力学参数。构建了半乳糖基核苷类产物合成的高效非水相催化体系3个；系首次报道亲水性有机溶剂调控半乳糖苷酶催化合成半乳糖基核苷类产物。该酶的多个突变体实现了10多种半乳糖基核苷产物的高浓度水平合成（大部分达10.5 g/L~16.0 g/L，最高达21.4 g/L），反应底物浓度是初始的10倍，合成产率最高可达43.9%。突破了现有的生物酶法制备半乳糖基核苷类产物浓度水平低、区域选择性较差等重大瓶颈问题，本项目的半乳糖苷酶筛选表达及其设计与定向改造、非水相生物催化提供了更丰富的实例。有助于低毒高效的抗病毒核苷类药物开发，相关的研究成果具备适用性和潜在的应用价值。本课题累计申请相关的国家发明专利7项（其中2项已获授权，其余均已获公开），共发表标注本项目资助的SCI论文12篇，并在国际会议上发表了2篇会议论文。

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