Convergent Chemoenzymatic Synthesis of Glycopeptides and Glycoproteins

描述(申请人提供)：建议研究的目标是开发一种通用和高效的化学酶法来制备具有生物医学意义的各种均一的糖肽和糖蛋白。功能糖组学研究和糖蛋白治疗应用中的一个主要问题是缺乏有效的方法来生产由糖链定义的糖蛋白。我们最近开发了一种化学酶方法，该方法利用一类内切糖苷酶(ENGase)的反糖基化活性，使游离糖与GlcNAc标记的蛋白质之间的“天然连接”形成具有天然糖苷键的均一糖蛋白。我们发现我们创建的人工合成的葡聚糖恶唑啉和基于ENGase的糖合成酶是有效转糖基化的极好的配对。这种方法允许糖和蛋白质部分的独立操作，为糖蛋白组装提供了一种高度收敛和潜在的通用方法。在此次更新申请中，我们旨在通过追求以下四个具体目标来扩大化学酶法的范围，加快其应用速度。目标1是通过评估用于糖蛋白合成的新的酶、突变体和不同的供体和受体底物来扩展合成谱系。目的2是确定Endos和EndoF3及其与底物/底物类似物的复合物的晶体结构，这将为在分子水平上理解这些ENGase如何在水解和转糖基化过程中区别识别底物提供依据。目的3探索多肽和蛋白质直接糖基化的两步酶法策略，包括N-糖基转移酶(NGT)的研究，以直接将单糖引物引入多肽，然后通过ENG酶催化的转糖基化延长糖链。目的4是用人工合成的甘露糖6-磷酸多糖改造溶酶体酶，以提高酶替代疗法(ERT)的疗效。这项拟议的研究有望为化学酶合成提供重要的新工具，所获得的知识将加快糖蛋白的治疗应用。

DESCRIPTION (provided by applicant): The goal of the proposed research is to develop a general and highly efficient chemo enzymatic method for making diverse homogeneous glycopeptides and 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 chemo enzymatic method that exploits the trans glycosylation 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 found that synthetic glycan oxazoline and ENGase-based glycosynthase that we created is an excellent pair for an efficient transglycosylation. This method permits independent manipulations of the sugar and protein portions, providing a highly convergent and potentially general approach to glycoprotein assembly. In this renewal application, we aim at expanding the scope of the chemo enzymatic method and speeding up its application by pursuit of the following four specific aims. Aim 1 is to expand the synthetic repertoire by evaluating new enzymes, mutants, and distinct donor and acceptor substrates for glycoprotein synthesis. Aim 2 is to determine the crystal structures of EndoS and EndoF3, and their complexes with substrates/substrate analogs, which will provide a molecular level understanding of how these ENGases differentially recognize the substrates in hydrolysis and transglycosylation. Aim 3 is to explore a two-step enzymatic strategy for direct glycosylation of polypeptides and proteins, including the study of N-glycosyltransferase (NGT) for directly introducing a monosaccharide primer into polypeptide and the subsequent extension of the sugar chains via ENGase-catalyzed transglycosylation. Aim 4 is to remodel lysosomal enzymes with synthetic mannose 6-phosphate glycans aiming to enhance the therapeutic efficiency in enzyme replacement therapy (ERT). The proposed study is expected to provide important new tools in chemo enzymatic synthesis and the knowledge gained will speed up glycoprotein therapeutic applications.