Facile chemoenzymatic synthesis and purification of glycolipids

糖脂的简便化学酶法合成和纯化

• 批准号: 9167363
• 负责人: Xi Chen
• 金额: $ 70.25万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 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/9167363
• 关键词: Acylation; Animals; Binding; Binding Proteins; Biochemical Reaction; Biological; Biological Assay; Biological Process; Biological Sciences; Carbohydrates; Ceramides; Charge; Chemicals; Chloroform; Communities; Complex; Databases; Enzymes; Family; Fatty Acids; Glycobiology; Glycoconjugates; Glycolipids; Glycosphingolipids; Glycosylphosphatidylinositols; Individual; Laboratories; Lactosylceramides; Learning; Legal patent; Libraries; Link; Lipids; Lipopolysaccharides; Membrane Lipids; Methods; Modification; Monosaccharides; Nature; Neck; Oligosaccharides; One-Step dentin bonding system; Organic solvent product; Phase; Play; Polysaccharides; Preparation; Printing; Problem Solving; Production; Property; Protocols documentation; Reaction; Reagent; Research; Research Personnel; Role; Running; S Phase; Series; Sialic Acids; Solid; Solubility; Specialist; Sphingolipids; Structure; Surface; System; Thin Layer Chromatography; Validation; Variant; Western Blotting; aqueous; base; biological research; biological systems; carbohydrate structure; chemical synthesis; design; flexibility; glycoglycerolipid; glycosylation; glycosyltransferase; interest; physical property; programs; stem; tool; water solubility; web site

Facile chemoenzymatic synthesis and purification of glycolipids Project Summary Carbohydrates and glycoconjugates play important roles in biological systems. However, they are very difficult to obtain by isolation from nature due to their structural complexity, the low abundance of some forms, general polarity of these compounds, and the presence of other compounds with similar properties. They are also challenging to synthesize despite various chemical, enzymatic, and chemoenzymatic methods that have been developed so far. Among diverse methods developed, chemoenzymatic methods have great advantages. They combine the flexibility of chemical synthesis of building blocks that can be used by enzymes, especially glycosyltransferases, for synthesizing biologically important glycans and glycoconjugates in a regio- and stereo-selective manner. A large array of enzymes have been characterized to allow the synthesis of desired targets, including those containing naturally occurring and non-natural modifications. However, product purification is a bottle-neck step. Chemoenzymatic methods have not been broadly used for producing glycolipids, a unique class of biomolecules that can be relatively easy to separate from other biomolecules by extraction. We plan to develop simple and convenient chemoenzymatic synthesis and facile purification methods for efficient production of complex bioactive glycolipids including those containing neutral and/or charged glycans with diverse lipid forms. The method will allow solution-phase synthesis of glycolipids using one-pot multienzyme (OPME) systems to achieve high selectivity and efficiency, and easy product purification by C18 cartridge-based solid-phase extraction (SPE). It is readily adaptable for automated synthesis. High efficiency of the enzymes that have been identified and will be discovered in the PI and the Co-PI's labs combining with easy isolation of the acceptor/products from other components will allow individual glycosylation reaction to be pushed to high yields with or without re-run of the reactions. As the variation of glycans and lipids are enormous, it is impractical to synthesize all interesting glycolipids in the current proposal. Instead, representative families and classes of glycolipids will be produced in the proposed project. Synthetic and purification protocols will be established. Convenient-to-store and easy-to-use enzymes and reagent kits will be assembled. These can be used by non-specialists for synthesizing, purification, and study of desired glycolipids of their interest in their own labs with a general research lab setting. Cross validation will be performed by different individuals in different labs. Protocols will be prepared and shared on a designated website and be included in the kits.

糖脂的简便化学酶法合成和纯化 项目概要 碳水化合物和复合糖在生物系统中发挥着重要作用。然而，他们很难 由于其结构复杂，某些形式的丰度较低，一般是通过与自然界隔离而获得的 这些化合物的极性，以及具有类似性质的其他化合物的存在。他们也是 尽管已经采用了各种化学、酶法和化学酶法，但合成仍具有挑战性 发展至今。在开发的多种方法中，化学酶法具有很大的优势。他们 结合了化学合成可被酶使用的构建模块的灵活性，特别是 糖基转移酶，用于在区域和区域中合成具有生物学意义的聚糖和糖缀合物 立体选择性方式。大量酶已被表征为可以合成所需的 目标，包括那些含有自然发生和非自然修饰的目标。然而，产品 纯化是一个瓶颈步骤。化学酶法尚未广泛用于生产 糖脂是一类独特的生物分子，可以相对容易地通过以下方法与其他生物分子分离 萃取。我们计划开发简单方便的化学酶合成和易于纯化的方法 用于高效生产复杂的生物活性糖脂，包括那些含有中性和/或带电的糖脂 具有多种脂质形式的聚糖。该方法将允许使用一锅法溶液相合成糖脂 多酶 (OPME) 系统可实现高选择性和效率，并可通过 C18 轻松纯化产品 基于柱的固相萃取 (SPE)。它很容易适用于自动化合成。高效率 已鉴定并将在 PI 和 Co-PI 实验室中发现的酶，结合简单的 将受体/产物与其他组分分离将允许推动单独的糖基化反应 无论是否重新进行反应，都能达到高产率。由于聚糖和脂质的变化巨大， 在当前的提案中合成所有有趣的糖脂是不切实际的。相反，有代表性的家庭和 拟议项目将生产各类糖脂。合成和纯化方案将 已确立的。将组装方便储存和易于使用的酶和试剂盒。这些可以是 供非专家用于合成、纯化和研究他们自己感兴趣的所需糖脂 具有一般研究实验室设置的实验室。交叉验证将由不同的个人在不同的领域进行 实验室。方案将在指定网站上准备和共享，并包含在套件中。