Facile chemoenzymatic synthesis and purification of glycolipids

糖脂的简易化学酶合成与纯化 项目摘要 碳水化合物和糖共轭化合物在生物系统中起着重要的作用。然而，他们很难做到 由于与自然隔绝，由于其结构复杂，某些形式丰度低，一般 这些化合物的极性，以及其他具有类似性质的化合物的存在。他们也是 尽管已经有了各种化学、酶和化学酶方法，但合成仍具有挑战性 到目前为止已经发展起来了。在各种方法中，化学酶法具有很大的优势。他们 结合了化学合成的灵活性，可以由酶来使用，特别是 糖基转移酶，用于在一个区域合成具有生物重要性的多糖和糖共轭化合物-和 立体选择方式。大量的酶已被表征为允许合成所需的 目标，包括那些包含自然发生和非自然修饰的目标。然而，产品 净化是一个瓶颈步骤。化学酶法还没有被广泛应用于生产 糖脂，一类独特的生物分子，可以相对容易地从其他生物分子中分离出来 拔牙。我们计划开发简便的化学酶法合成和简便的纯化方法。 用于高效生产复合生物活性糖脂，包括那些含有中性和/或带电的糖脂 具有多种脂类形式的多聚糖。该方法将允许使用一锅法进行糖脂的溶液相合成 多酶(OPME)系统，通过C18实现高选择性和高效率，并易于产物纯化 基于小柱的固相萃取(SPE)。它很容易适应于自动合成。高效率的 已经确定并将在PI和Co-PI的实验室中发现的酶结合Easy 将受体/产物与其他组分分离将允许单独的糖基化反应被推进 在有或没有反应重新进行的情况下获得高产率。由于糖类和脂类的变化很大，所以 在目前的方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 在拟议的项目中，将生产各种类型的糖脂。合成和提纯方案将是 已经成立了。将组装便于储存和易于使用的酶和试剂盒。这些可以是 非专业人士用于合成、提纯和研究他们自己感兴趣的所需糖脂 实验室具有一般的研究实验室环境。交叉验证将由不同的个人在不同的 实验室。协议将在指定的网站上编制和分享，并包括在工具包中。

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.