Convergent Chemoenzymatic Synthesis of Glycopeptides and Glycoproteins

项目摘要/摘要 这项研究的目标是开发简便的化学酶合成方法。 具有生物医学意义的均一N-糖蛋白。功能糖组学研究中的一个主要问题 而糖蛋白的治疗应用是缺乏有效的方法来产生糖链定义的 糖蛋白。我们最近开发了一种利用转糖基化的化学酶方法。 一类内切糖苷酶(ENGase)的活性，使游离糖和ENGase能够“天然连接” GlcNAc标记的蛋白质形成具有天然糖苷连接的均一糖蛋白。我们发现 新的基于内切糖苷酶的突变体，即能够利用高活性多糖的糖合成酶 恶唑类化合物具有转糖基化活性，但产物缺乏水解性。糖合成酶催化的天然产物 连接允许糖和蛋白质部分的独立操作，并提供高度收敛的 糖蛋白组装的方法。该方法已成功地应用于一系列化合物的合成。 复杂的糖肽，如HIV-1糖肽抗原和CD52糖蛋白。它也一直是 重组人促红细胞生成素和重组糖蛋白的糖链重塑研究 治疗性抗体。在这一应用中，我们旨在改进化学酶法，扩大其 范围，并加快其作为合成均一糖形式的通用方法的应用 糖蛋白。为实现这些目标，将实现四个具体目标。前两个目标主要集中在 从细菌内糖苷酶和从一系列新的-岩藻糖苷酶产生新的内糖合成酶 -岩藻糖苷酶(-1，6，-1，3/1，4，和-1，2)-岩藻糖苷酶；第三个目标是开发一种大肠杆菌 共表达系统，以产生含有GlcNAc和Glc的蛋白质，将作为关键的前体 糖基化治疗蛋白的联合合成，目的是提高血清半衰期 治疗性蛋白质；第四个目的是建立一种溶酶体糖基化重塑的方法 含甘露糖-6-磷酸(M6P)寡糖的重组人-葡萄糖苷酶 以改善他们在酶替代疗法中的细胞摄取。圆满完成拟议中的 研究将显著扩大化学酶方法的范围；提供新的支持 向糖生物学和生物技术社区提供技术；并加快应用 提高治疗性蛋白质疗效的化学酶促方法。

Project Summary/Abstract The objectives of the proposed research are to develop facile chemoenzymatic methods for synthesizing homogeneous N-glycoproteins of biomedical significance. A major problem in functional glycomics studies and glycoprotein therapeutic applications is the lack of efficient methods to produce glycan-defined glycoproteins. We have recently developed a chemoenzymatic method that exploits the transglycosylation activity of a class of endoglycosidases (ENGases) that enables the “native ligation” between free glycan and GlcNAc-tagged protein to form homogeneous glycoproteins with native glycosidic linkage. We have discovered novel endoglycosidase-based mutants, the glycosynthases, that are capable of using highly active glycan oxazolines for transglycosylation but lack the product hydrolysis activity. The glycosynthase-catalyzed native ligation permits independent manipulations of the sugar and protein portions and provides a highly convergent approach to glycoprotein assembly. This method has been successfully applied for the synthesis of a series of complex glycopeptides such as the HIV-1 glycopeptide antigens and CD52 glycoproteins. It has also been explored for glycan remodeling of recombinant glycoproteins including human erythropoietin (EPO) and therapeutic antibodies. In this application, we aim at improving the chemoenzymatic method, expanding its scope, and speeding up its application as a general method for the synthesis of homogeneous glycoforms of glycoproteins. Four specific aims will be pursued to achieve the goals. The first two aims are focused on generating new endoglycosynthases from bacterial endoglycosidases and novel -fucoligases from an array of -fucosidases with distinct (1,6, 1,3/1,4, and 1,2)-fucosidic linkages; the third aim is to develop an E. coli co-expression system to produce GlcNAc- and Glc-containing proteins that will serve as the key precursor for a combined synthesis of glycosylated therapeutic proteins with a goal of enhancing the serum half-life of therapeutic proteins; and the fouth aim is to establish a method for glycosylation remodeling of lysosomal enzymes such as the recombinant human -glucosidase with mannose-6-phosphate (M6P) oligosaccharides for improving their cellular uptake in enzymatic replacement therapy. A successful completion of the proposed research will significantly expand the scope of the chemoenzymatic method; provide new enabling technologies to the community of glycobiology and biotechnology; and speed up the applications of the chemoenzymatic method for improving the efficacy of therapeutic proteins.