Expedite Enzymatic Assembly of Glycans via DNA (de)Hybridization-Enabled Catch-and-Release

项目摘要 地球上的每个活细胞都覆盖着一层致密的葡聚糖。这些复杂的结构起着至关重要的作用 在许多生物和疾病过程中发挥作用。功能研究和医学应用需要明确的定义 糖链结构。虽然自动化的多肽和核酸合成已经成熟，并允许非专业人士 为了获得规定的标准，多糖和糖偶联物的合成通常仍然是费时费力的。 消费，并需要专业知识。到目前为止，只有几个平台被引入来实现化学自动化 多糖的合成，但存在反应速度慢、选择性低、效率低和产率低的缺点，特别是在 制备复杂的多聚糖。一个成熟而实用的合成复杂多糖的自动化系统并不是 可用。在过去的十年中，糖基转移酶(GTS)催化的反应被广泛探索以 准备各种复杂的葡聚糖。具有完美的区域和立体声选择性以及高转化率， 它们对自动化很有吸引力。挑战是单调乏味的重复分离中间体和 最终的产品。多肽和核酸的固相自动化已经很好地克服了这一点 综合。但当受体底物被固定时，GT的活性往往要低得多，导致反应缓慢 转化率和低收益率。捕捉和释放战略有可能解决这个问题。在这样的情况下 策略，接受者被标记上一个官能团，并通过特定的 基团与固相之间的相互作用。清理完成后，可以使用适当的 破坏相互作用的溶剂。已经引入了几种“即捕即放”战略，以加快 多聚糖的酶组装，但都有一个主要缺点：化学物质、有机溶剂、高 必须引入浓度的盐等，才能使多糖从固相中释放出来。因此，额外的步骤 往往不容易在自动化平台上实现，必须参与清理每一个中间件 下一轮反应和捕放分离。这将极大地复杂化并延长自动化时间 合成和降低效率。我们提出了一种由DNA实现的简单的捕获和释放策略 杂交(捕获)和去杂交(释放)。在这一策略中，可以很方便地用 任何GT反应混合物，并使用纯水释放。我们认为，这种即捕即放的策略是 找不到解决实际自动化多糖合成的难题。迭代酶组装已经被广泛应用 用于制备复杂的葡聚糖，许多液体处理系统在商业上都可以买到。在此之前 成功的现有技术，这些都可以很容易地集成在一起，产生实用和无成本的自动化 多糖合成平台(下一步工作)。

Project Summary Every living cell on the planet is covered by a dense layer of glycans. These complicated structures play critical roles in many biological and disease processes. Functional studies and medical applications require well-defined glycan structures. While automated peptide and nucleic acid syntheses have matured and allow non-specialists to access defined standards, the synthesis of glycans and glycoconjugates is still often laborious, time- consuming, and requires specialties. So far, only a few platforms have been introduced to automate chemical syntheses of glycans but suffer from slow reaction rates, low selectivity, efficiency, and yields, especially in preparing complex glycans. A mature and practical automated system to synthesize complex glycans is not available. In the past decade, glycosyltransferases (GTs)-catalyzed reactions have been widely explored to prepare diverse complex glycans. With perfect regio- and stereo-selectivity as well as high conversion rates, they are attractive for automation. The challenge is tedious repeated process of separating intermediates and the final product. This has been well overcome by solid-phase-based automation in peptide and nucleic acid synthesis. But GTs are often much less active when the acceptor substrate is immobilized, causing slow conversions and low yields. Catch-and-release strategies have the potential to solve the problem. In such strategy, acceptors are tagged with a functional group, and “captured” on solid phase through specific interactions between the group and the solid phase. After cleanup, they can be “released” using appropriate solvents to disrupt the interaction. Several “catch-and-release” strategies have been introduced to expedite enzymatic assembly of glycans, but all suffer from one major drawback: chemicals, organic solvents, high concentrations of salts, etc, must be introduced to release glycans from solid phase. Thus, extra steps which often cannot be easily realized on automated platforms have to be involved to clean up each intermediate for the next round reaction and catch-and-release separation. This will greatly complicate and prolong automated synthesis and decrease efficiency. We propose a simple catch-and-release strategy enabled by DNA hybridization (Catch) and dehybridization (Release). In this strategy, the glycan an conveniently “captured” with any GT reaction mixtures, and “released” using pure water. We believe this catch-and-release strategy is the missing puzzle to tackle practical automated glycan synthesis. Iterative enzymatic assembly is already widely employed to prepare complex glycans, and many liquid handling systems are commercially available. Upon the success of current technology, these can be readily integrated to generate a practical and costless automated platform for glycan synthesis (future work).