Convergent Chemoenzymatic Synthesis of Glycopeptides and Glycoproteins

描述（由申请人提供）：拟议研究的目标是开发一种通用且高效的化学酶方法来制备具有生物医学意义的多种均质糖肽和糖蛋白。功能性糖组学研究和糖蛋白治疗应用的一个主要问题是缺乏有效的方法来产生糖定义的糖蛋白。我们最近开发了一种化学酶法，利用一类内糖苷酶（ENGases）的反式糖基化活性，使游离聚糖和GlcNAc标记的蛋白质之间的“天然连接”形成具有天然糖苷连接的均匀糖蛋白。我们发现，合成的糖酰恶唑啉和基于engase的糖合酶是高效转糖基化的绝佳组合。这种方法允许对糖和蛋白质部分进行独立操作，为糖蛋白组装提供了一种高度收敛和潜在的通用方法。在这次更新申请中，我们的目标是通过追求以下四个具体目标来扩大化学酶法的范围并加快其应用。目的1是通过评估糖蛋白合成的新酶、突变体和不同的供体和受体底物来扩大合成库。目的2是确定EndoS和EndoF3的晶体结构，以及它们与底物/底物类似物的配合物，这将从分子水平上了解这些酶在水解和转糖基化过程中如何区分识别底物。目的3是探索多肽和蛋白质直接糖基化的两步酶学策略，包括研究n -糖基转移酶（NGT）直接将单糖引物引入多肽，以及随后通过engase催化的转糖基化延长糖链。目的4是用合成甘露糖6-磷酸聚糖改造溶酶体酶，以提高酶替代治疗（ERT）的疗效。该研究有望为化学酶合成提供重要的新工具，所获得的知识将加速糖蛋白的治疗应用。

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.