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。