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

• 批准号: 9533657
• 负责人: Geert-Jan Boons
• 金额: $ 66.99万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 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/9533657
• 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

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.

项目摘要 几乎所有细胞表面和分泌的蛋白质都通过共价连接的碳水化合物和 这些所谓的聚糖已被暗示是过程的必要介体，例如蛋白质折叠，细胞 信号传导，受精，胚胎发生，神经元发育以及细胞及其的增殖 组织成特定的组织。此外，压倒性数据支持糖基化在病原体中的相关性 识别，炎症，先天免疫反应以及自身免疫性疾病的发展和 癌症。由于缺乏明确定义的复杂寡糖标准，糖科的进展受到阻碍 制造下一代微阵列需要的是分析的开发 确定孤立聚糖的确切结构的协议，以阐明糖缀合物的途径 生物合成，作为免疫原子产生MAB，用于糖蛋白分离和可视化。 在此应用程序中，我们建议开发新的合成策略，这些策略可以很容易地提供大量的图书馆 对称和不对称的N-聚糖。新方法将利用随时可用的开始 材料，并且将足够标准化，许多实验室，包括合成服务单位，都可以 采用这些方法。此外，新方法的综合原理可以轻松地应用于 制备其他类别的聚糖，例如O连接的聚糖和人乳寡糖（HMOS）。 新方法将采用一种对称的双聚糖，可以轻松地从蛋黄中隔离。 将开发创新的酶转化，以使该聚糖对称化。此外， 重组N-乙酰葡萄糖氨基二氨基转移酶（MGAT）将用于将双静脉聚糖转化为三 和四局部结构。在后一种转换中，化学修改的UDP-GLCNAC供体将是 用于暂时防止手臂进行酶促修饰。使用最近开发的技术来 表达重组哺乳动物糖基转移酶将是新方法的关键特征。到 验证该方法的鲁棒性，它将应用于制备衍生的聚糖库 来自人类的上皮上皮细胞。由此产生的聚糖对于下一个的发展将是有价值的 在这种细胞类型的背景下，产生聚糖微阵列以探测碳氢化物 - 蛋​​白质相互作用。这 通过开发可以进一步扩展化学酶方法的范围 很容易提供高度复杂的不对称聚糖，这些聚糖是由硫酸盐酯修饰的。一个自动化平台 将开发以进一步提高化学酶合成的速度，并使用新颖的捕获和释放 策略。注意将集中于离子交换和镍介导的组氨酸结合事件，以捕获 标记的寡糖。来自配备体积的Chemspeed的多通道液体处理机器人 分配系统和冻干器将被聘为自动化工具。后来的方法首先将 用于制备O连锁的聚糖和人乳寡糖。