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

项目摘要 地球上的每一个活细胞都被一层密集的聚糖所覆盖。这些复杂的结构对 在许多生物和疾病过程中的作用。功能研究和医学应用需要明确的 聚糖结构。虽然自动化肽和核酸合成已经成熟， 为了获得确定的标准品，聚糖和糖缀合物的合成仍然常常是费力的，耗时的， 消费，需要专业。到目前为止，只有少数几个平台被引入自动化化学品 聚糖的合成，但存在反应速率慢、选择性、效率和产率低的问题，特别是在 制备复合聚糖。一个成熟实用的自动化系统来合成复杂的聚糖是不 available.在过去的十年中，糖基转移酶（GT）催化的反应已经被广泛探索， 制备多种复合聚糖。具有完美的区域和立体选择性以及高转化率， 它们对于自动化是有吸引力的。挑战是分离中间体和中间体的繁琐的重复过程。 最终产品。这一点已经被肽和核酸的固相自动化技术很好地克服了 合成.但是当受体底物被固定时，GT通常活性低得多，导致缓慢的 转化率和低产量。捕捉和释放策略有可能解决这个问题。以这样 策略中，受体用官能团标记，并通过特异性结合被“捕获”在固相上。 基团和固相之间的相互作用。清理后，可以使用适当的 溶剂来破坏相互作用。已经采取了几项“抓了就放”的战略， 聚糖的酶促组装，但都遭受一个主要缺点：化学品，有机溶剂，高 必须引入一定浓度的盐等以从固相释放聚糖。因此， 通常不能在自动化平台上容易地实现，必须参与清理每个中间体， 下一轮反应和捕获-释放分离。这将大大复杂化和延长自动化 合成和降低效率。我们提出了一个简单的捕获和释放策略， 杂交（捕捉）和去杂交（释放）。在这种策略中，聚糖被方便地“捕获”， 任何GT反应混合物，并使用纯水“释放”。我们相信这种捕捉和释放的策略是 缺少解决实际自动化聚糖合成的难题。迭代酶组装已经广泛应用于 用于制备复合聚糖，并且许多液体处理系统是可商购的。于 由于当前技术的成功，这些可以很容易地集成，以产生实用且无成本的自动化 聚糖合成平台（未来工作）。

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).