Facile chemoenzymatic synthesis and purification of glycolipids

糖脂的简易化学酶法合成和纯化 项目摘要 碳水化合物和糖缀合物在生物系统中起着重要作用。然而，他们很难 由于其结构复杂，某些形式的丰度低，一般 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 尽管已经有各种化学、酶和化学酶方法， 发展至今。在已开发的各种方法中，化学酶法具有很大的优势。他们 联合收割机的灵活性化学合成的积木，可用于酶，特别是 糖基转移酶，用于合成生物学上重要的聚糖和糖缀合物， 立体选择性的方式。已经表征了大量的酶，以允许合成所需的酶。 目标，包括含有天然存在的和非天然修饰的那些。但产品 纯化是瓶颈步骤。化学酶法尚未广泛用于生产 糖脂是一类独特的生物分子，可以相对容易地与其他生物分子分离， 萃取我们计划开发简便的化学酶法合成和容易的纯化方法 为了有效生产复合生物活性糖脂， 具有不同脂质形式的聚糖。该方法将允许使用一锅法的糖脂的溶液相合成 多酶（OPME）系统，以实现高选择性和效率，并易于通过C18 固相萃取（SPE）。它很容易适用于自动化合成。效率高 已经确定的酶，并将在PI和Co-PI的实验室中发现， 受体/产物与其它组分的分离将允许单独的糖基化反应被推进 在反应重新进行或不重新进行的情况下达到高产率。由于聚糖和脂质的变化是巨大的， 在目前的提议中合成所有感兴趣的糖脂是不切实际的。相反，代表性的家庭和 将在拟议项目中生产各类糖脂。合成和纯化方案将是 确立了习将组装便于储存和易于使用的酶和试剂盒。这些可以 由非专业人员使用，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 labs.将在指定的网站上编写和分享方案，并将其纳入工具包。

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.