Enzymatic Transglycosylation for N-Glycopeptide Synthesis

描述（由申请人提供）：拟定研究的目标是开发有效且广泛适用的化学酶法，用于合成具有生物医学意义的N-糖肽和N-糖蛋白。N-连接糖基化是真核生物中最常见的蛋白质翻译后修饰之一。糖蛋白在细胞粘附、肿瘤转移、病原体感染和免疫应答等许多生物学事件中起重要作用。然而，糖蛋白的结构异质性往往阻碍了对糖蛋白结构-功能关系的清晰理解。天然糖蛋白通常以糖型的混合物形式产生，其极难以纯形式分离。为了获得用于结构和生物学研究的均质材料，我们建议系统地探索一类特殊的内切糖基化酶，内切-β-N-乙酰氨基葡萄糖苷酶（ENGases），用于N-糖肽和N-糖蛋白合成的转糖基化活性。一些ENGase具有独特的能力，可以在单一步骤中将完整的寡糖转移到含GlcNAc的肽中，以区域和立体特异性的方式形成新的糖肽，从而为糖肽合成提供高度收敛的途径。但该方法存在转糖基化反应收率低、供体底物有限、产物易水解等问题。这些研究旨在通过探索新的底物和筛选新的酶突变体来解决这些问题。为实现这一目标，提出了三个具体目标。在具体目标1中，将合成并评价一系列寡糖供体底物，包括三种主要类型N-聚糖的糖恶唑啉（假定的过渡态模拟物）和对硝基苯基糖苷（基态但动力学有利的底物）。具体目标2是通过使用偶联酶测定和基于荧光共振能量转移（FRET）的测定筛选突变体文库来发现具有新的和/或增强的转糖基化活性的ENGase突变体。具体目标3集中于几个大的和复杂的HIV-1包膜糖蛋白片段的全合成，以及选定的糖蛋白的半合成。从长远来看，拟议的研究将有助于开发基于糖蛋白的药物。

DESCRIPTION (provided by applicant): The goal of the proposed research is to develop efficient and widely applicable chemoenzymatic methods for synthesizing N-glycopeptides and N-glycoproteins of biomedical significance. N-linked glycosylation is one of the most common posttranslational modifications of proteins in eukaryotes. Glycoproteins play important roles in many biological events such as cell adhesion, tumor metastasis, pathogen infection, and immune response. However, a clear understanding of the structure-function relationships of glycoproteins is often hampered by their structural heterogeneity. Natural glycoproteins are usually produced as a mixture of glycoforms that are extremely difficult to isolate in pure form. To obtain homogeneous materials for structural and biological studies, we propose to systematically explore the trans-glycosylation activity of a special class of endoglycosidases, the endo-beta-N-acetylglucosaminidases (ENGases), for N-glycopeptide and N-glycoprotein synthesis. Some ENGases possess unique ability to transfer an intact oligosaccharide to a GlcNAc-containing peptide in a single step to form a new glycopeptide in a regio- and stereo-specific manner, thus providing a highly convergent route to glycopeptide synthesis. But the method suffers with serious problems such as the low yield in transglycosylation, the limitation of donor substrates, and the product hydrolysis. The proposed studies intend to solve these problems by exploring new substrates and screening new enzyme mutants. Three specific aims are proposed to achieve the goal. In specific aim 1, a range of oligosaccharide donor substrates, including sugar oxazolines (presumed transition state mimics) and p-nitrophenyl glycosides (ground-state but kinetically favorable substrates) of three major types of N-glycans will be synthesized and evaluated. The specific aim 2 is to discover ENGase mutants with new and/or enhanced transglycosylation activity by screening mutant library, using the coupled enzyme assay and the fluorescent resonance energy transfer (FRET)-based assay. Specific aim 3 focuses on total synthesis of several large and complex HIV-1 envelope glycoprotein fragments, as well as semi- synthesis of selected glycoproteins. In the long term, the proposed studies will contribute to the development of glycoprotein-based drugs.