Streamlining the chemoenzymatic synthesis of asymmetrical glycans of biological importance

简化具有生物学重要性的不对称聚糖的化学酶合成

基本信息

批准号：
9752086
负责人：
Geert-Jan Boons
金额：
$ 30.02万
依托单位：
UNIVERSITY OF GEORGIA
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-01 至 2020-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9752086
关键词：
Address Adopted Anabolism Antigens Attention Autoimmune Diseases Automation Binding Biological Biological Process Biomedical Research Carbohydrates Cell Proliferation Cell surface Chemicals Complex Data Development Disease Egg Yolk Embryonic Development Enzymes Epithelial Cells Epitopes Esters Event Fertilization Freeze Drying Glycoconjugates Glycoproteins Health Histidine Human Human Milk Imagery Inflammation Innate Immune Response Ion Exchange Laboratories Libraries Link Liquid substance Malignant Neoplasms Mediating Mediator of activation protein Methodology Methods Modification N-Acetylglucosaminyltransferases Nickel Oligosaccharides Pathway interactions Plant Resins Polysaccharides Preparation Procedures Process Protein-Carbohydrate Interaction Proteins Protocols documentation Reaction Recombinants Reporting Robot Services Signal Transduction Solid Speed Standardization Structure Substrate Specificity System Technology Time Tissues Transferase Unspecified or Sulfate Ion Sulfates arm base biological research cell type chemical synthesis expectation glycosylation glycosyltransferase innovation method development neuron development new technology next generation novel novel strategies pathogen prevent programs protein folding skills sulfation sulfotransferase tool

项目摘要

Project Summary Almost all cell surface and secreted proteins are modified by covalently-linked carbohydrate moieties, and these so called glycans have been implicated as essential mediators of processes such as protein folding, cell signaling, fertilization, embryogenesis, neuronal development, and the proliferation of cells and their organization into specific tissues. Also, overwhelming data supports the relevance of glycosylation in pathogen recognition, inflammation, innate immune responses, and the development of autoimmune diseases and cancer. Progress in glycoscience is hampered by a lack of well-defined complex oligosaccharide standards which are needed for the fabrication of the next generation of microarrays, for the development of analytical protocols to determine exact structures of isolated glycans, for the elucidation of pathways of glycoconjugate biosynthesis, and as immunogens to produce MABs for glycoprotein isolation and visualization. In this application, we propose to develop novel synthetic strategies that can readily provide large libraries of symmetrical and asymmetrical N-glycans. The new methodologies will make use of readily available starting materials and will be sufficiently standardized that many laboratories, including synthesis service units, can adopt these methods. Furthermore, the synthetic principles of the new approaches can easily be applied to the preparation of other classes of glycans such as O-linked glycans and human milk oligosaccharides (HMOs). The new method will employ a symmetrical biantennary glycan that can easily be isolated from egg yolk. Innovative enzymatic transformations will be developed to desymmetrize this glycan. Furthermore, recombinant N-acetylglucosaminyltransferases (MGAT's) will be used to convert a bi-antennary glycan into tri- and tetra-antennary structures. In the latter transformations, chemically modified UDP-GlcNAc donors will be used to temporarily prevent an arm from enzymatic modification. The use of recently developed technology to express recombinant mammalian glycosyltransferases will be a key feature of the new methodology. To validate the robustness of the methodology, it will be applied to the preparation of a library of glycans derived from human upper airway epithelial cells. The resulting glycans will be valuable for the development of the next generation of glycan microarray to probe carbohydrate–protein interactions in the context of this cell type. The scope of the chemoenzymatic methodology will be further extended by the development of methods that can easily provide highly complex asymmetrical glycans that are modified by sulfate esters. An automation platform will be developed to further increase the speed of chemoenzymatic synthesis using novel capture and release strategies. Attention will focus on ion exchange and nickel-mediated histidine binding events for capture of tagged oligosaccharides. A multi-channel liquid handling robot from Chemspeed equipped with a volumetric dispensing system and lyophilizer will be employed as an automation tool. The latter methodology will, at first, be employed for the preparation of O-linked glycans and human milk oligosaccharides.

项目概要几乎所有细胞表面和分泌的蛋白质都被共价连接的碳水化合物部分修饰，并且这些所谓的聚糖被认为是蛋白质折叠、细胞信号传导、受精、胚胎发生、神经元发育以及细胞及其增殖此外，大量数据支持病原体中糖基化的相关性。识别、炎症、先天免疫反应以及自身免疫性疾病的发展缺乏明确的复杂寡糖标准阻碍了糖科学的进展。这是制造下一代微阵列、开发分析技术所需的确定分离聚糖的精确结构的方案，用于阐明糖复合物的途径生物合成，并作为免疫原生产用于糖蛋白分离和可视化的 MAB。在此应用中，我们建议开发新的合成策略，可以轻松提供大型库新方法将利用现成的起始原料。材料并将充分标准化，许多实验室，包括合成服务单位，可以此外，新方法的合成原理可以很容易地应用于其他类别聚糖的制备，例如 O-连接聚糖和人乳低聚糖 (HMO)。新方法将采用一种对称的双触角聚糖，可以很容易地从蛋黄中分离出来。将开发创新的酶促转化来使这种聚糖去对称化。重组 N-乙酰氨基葡萄糖转移酶 (MGAT) 将用于将双触角聚糖转化为三触角聚糖在后面的转化中，化学修饰的 UDP-GlcNAc 供体将是用于暂时防止手臂受到酶促修饰的使用最近开发的技术表达重组哺乳动物糖基转移酶将是新方法的一个关键特征。验证该方法的稳健性，将其应用于衍生聚糖文库的制备来自人类上呼吸道上皮细胞的聚糖对于下一代的开发具有重要价值。生成聚糖微阵列以探测该细胞类型背景下的碳水化合物-蛋白质相互作用。化学酶方法的范围将通过开发可进一步扩展的方法轻松提供经过硫酸酯修饰的高度复杂的不对称聚糖。将开发利用新型捕获和释放进一步提高化学酶合成的速度策略的注意力将集中在离子交换和镍介导的组氨酸结合事件上。 Chemspeed 的多通道液体处理机器人，配备容量测量装置。分配系统和冻干机将首先用作自动化工具。用于制备O-连接聚糖和人乳低聚糖。