Convergent Chemoenzymatic Synthesis of Glycopeptides and Glycoproteins

糖肽和糖蛋白的聚合化学酶合成

• 批准号: 7483366
• 负责人: LAI-XI WANG
• 金额: $ 13.73万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7483366
• 关键词: Address; Affect; Antibodies; Biological; Biological Models; Cell Adhesion; Cell Line; Chemicals; Class; Complex; Development; Endoglycosidases; Engineering; Enzymes; Eukaryota; Eukaryotic Cell; Evaluation; Event; Evolution; Facility Construction Funding Category; Fc domain; Fucose; Genetic; Glycopeptides; Glycoproteins; Goals; HIV Envelope Protein gp120; HIV-1; Half-Life; Heterogeneity; Human; Hydrolysis; Immune response; Immunoglobulin G; Infection; Knowledge; Link; Methodology; Methods; Modification; Monosaccharides; Mutagenesis; N acetylglucosaminidase; Neoplasm Metastasis; Nuclear; Oligosaccharides; Peptides; Play; Polysaccharides; Post-Translational Protein Processing; Proteins; Published Comment; Range; Recombinants; Research; Research Personnel; Rest; Risk; Role; Route; Score; Screening procedure; Serum; Source; Structure; Structure-Activity Relationship; Study Section; Therapeutic; Time; Writing; base; endo-alpha-sialidase; glycosylation; glycosyltransferase; in vivo; mutant; novel; pathogen; protein structure; protein structure function; ribonuclease B; sugar; tumor

DESCRIPTION (provided by applicant): The proposed research focuses on the development of efficient and widely applicable chemoenzymatic methods for synthesizing N-glycopeptides and N-glycoproteins of biomedical significance. N-linked glycosylation is a ubiquitous posttranslational modification of proteins in eukaryotes. Glycoproteins play important roles in many biological events such as cell adhesion, tumor metastasis, pathogen infection, and immune response. The covalently linked oligosaccharides can profoundly affect proteins' structure, function, and their serum half-life. However, homogeneous glycoproteins with structural defined oligosaccharides are difficult to obtain from natural or recombinant sources, since they are typically produced as a mixture of heterogeneous glycoforms. To obtain homogeneous materials for structural/biological studies and for biomedical applications, we propose to systematically explore the potential of endo-?-N-acetylglucosaminidases (ENGases), a special class of endoglycosidases, for constructing N-glycopeptides and N-glycoproteins. The biggest advantage is that some ENGases are able to transfer an intact oligosaccharide to a GlcNAc-containing peptide or protein in a single step without the need of any protecting groups, thus providing a highly convergent route to large glycopeptides and glycoproteins. But the method has hitherto suffered with low transglycosylation yield, the risk of product hydrolysis, and the limitation to the use of only natural N-glycans as donor substrates that themselves are difficult to prepare. Our preliminary studies have shown that the use of sugar oxazolines (the transition state mimics) as donor substrates not only expanded the substrate availability, but also resulted in a high-yield transglycosylation to form large glycopeptides and homogeneous glycoproteins. This proposal intends to systematically explore the scope and limitation of the novel methodology through pursuing five specific aims: 1) synthesis and examination of a range of oligosaccharide oxazolines as donor substrates for the enzymatic transglycosylation; 2) evaluation of novel acceptor substrates for synthesizing complex fucose-containing N-glycopeptides and for detecting O-GlcNAc glycosylation; 3) total synthesis of large HIV-1 gp120 fragments for structural and functional studies; 4) exploitation of the methodology for synthesizing natural and tailor-made homogeneous glycoproteins; and 5) synthesis and functional studies of homogeneous glycoforms of human antibody IgG-Fc. The knowledge gained from the proposed research will eventually facilitate the development of glycoprotein-based therapeutics.

描述(申请人提供)：建议的研究集中于开发高效和广泛适用的化学酶方法来合成具有生物医学意义的N-糖肽和N-糖蛋白。N-糖基化是真核生物中普遍存在的蛋白质翻译后修饰。糖蛋白在许多生物学事件中发挥重要作用，如细胞黏附、肿瘤转移、病原体感染和免疫反应。共价连接的寡糖可以深刻地影响蛋白质的结构、功能及其血清半衰期。然而，具有结构定义的寡糖的均一糖蛋白很难从自然来源或重组来源获得，因为它们通常是以不同的糖形混合物的形式产生的。为了获得用于结构/生物学研究和生物医学应用的均质材料，我们建议系统地探索内切糖苷酶(ENGase)在构建N-糖肽和N-糖蛋白方面的潜力。最大的优点是，一些ENGase能够在不需要任何保护基团的情况下，一步将完整的寡糖转移到含有GlcNAc的多肽或蛋白质上，从而提供了一条高度收敛的路线，形成大的糖肽和糖蛋白。但到目前为止，该方法存在转糖基化产率低、产物水解的风险以及仅使用天然N-葡聚糖作为供体底物的限制，而这些底物本身很难制备。我们的初步研究表明，使用糖恶唑啉(过渡态模拟物)作为供体底物不仅扩大了底物的利用率，而且还导致了高产率的糖基化反应，形成了大的糖肽和均一的糖蛋白。这项建议旨在通过追求五个具体目标来系统地探索新方法的范围和局限性：1)合成和检测一系列作为酶转糖反应供体底物的低聚恶唑类化合物；2)评价用于合成复杂的岩藻糖N-糖肽和检测O-GlcNAc糖基化的新型受体底物；3)全合成用于结构和功能研究的HIV-1 gp120大片段；4)开发合成天然和定制均一糖蛋白的方法；以及5)人抗体IgG-Fc均一糖形式的合成和功能研究。从拟议的研究中获得的知识最终将促进以糖蛋白为基础的疗法的发展。