A Continuous Flow-Based Approach to Automated Microbial Oligosaccharide Synthesis.

项目摘要 对用于生产定义明确的寡糖的有效方法的需要继续呈现主要的 微生物糖生物学领域的瓶颈。尽管自动化低聚糖合成器已经被 大多数依赖于固相合成，这可能限制可用的合成化学和规模 给他们.此外，现有的寡糖合成自动化方法几乎集中在 这些方法专门用于糖基化反应，并且不解决糖基化反应的耗时和繁琐的过程。 将单糖原料转化为完全取代的糖基供体以备偶合。难以置信的 微生物聚糖合成所需的构建模块的数量也使得将每一个可能的模块保持在 库存不可能所有这些问题都可以通过开发自动化连续流来解决 平台连续流反应可以比多步分批工艺更容易地自动化， 从而提供更大的批次间再现性。通过适当选择条件， 将几个反应浓缩到一次反应中。该提案的目的是建立一个平台， 自动化连续流基低聚糖技术，能够自动化每一步， 寡糖合成，从按需供体/受体生产到这些较大分子的组装 进入目标结构。我们将通过以下具体目标来实现这一目标。具体目标1将 检查糖基化准备单糖的自动化生产。通过叠加多重反应 这种方法将系统集成到一个单一的运行中，并使用开源MechWolf软件设计和控制系统， 将允许以更快的速度从商业原料构建这些重要的中间体。 比目前可能的时间更长。这将包括开发快速化学-酶促合成 否则难以获得非酮糖（9-碳）酸碳水化合物结构单元， 有几种致病微生物此外，MechWolf计划将提供一种开源化学品， 任何受保护单糖生产的最佳条件储存库，以确保批次间 这些构建模块的可重复性和按需访问。Specific Aim 2将把这项技术扩展到 低聚糖的自动化生产。连续流合成的灵活性和模块化特性将 允许构建糖苷键，其在现有平台上不是简单的， 即使有酶也是很少的。作为原理证明，该系统将用于构建几个囊 与ESKAPE病原体鲍曼不动杆菌相关的多糖;然而，这些 技术和概念可用于构建任何寡糖。总的来说， 通过这项研究开发的技术将导致一种快速，可靠，可重复和负担得起的方法 用于自动化低聚糖生产，最大限度地减少了对人工优化和干预的需求。

Project Summary The need for efficient methods for the production of well-defined oligosaccharides continues to present a major bottleneck in the field of microbial glycobiology. Although automated oligosaccharide synthesizers have been developed, most rely on solid-phase synthesis, which can limit the chemistry and scale of synthesis available to them. Furthermore, existing automated approaches to oligosaccharide synthesis have focused almost exclusively on glycosylation reactions, and do not address the time-consuming and tedious process of converting monosaccharide feedstocks into fully-substituted glycosyl donors ready for coupling. The incredible number of building blocks required for microbial glycan synthesis also makes keeping every possible block in stock impossible. All these issues could be addressed by the development of automated continuous flow platforms. Continuous flow reactions can be more easily automated than multi-step batch processes and thereby provide greater batch-to-batch reproducibility. Through proper selection of conditions it is also possible to telescope several reactions into a single run. The objective of this proposal is to generate platform technologies for automated continuous flow-based oligosaccharide that is capable of automating every step of oligosaccharide synthesis, from on-demand donor/acceptor production to assembly of these larger molecules into target structures. We will achieve this by pursuing the following Specific Aims. Specific Aim 1 will examine the automated production of glycosylation ready monosaccharides. By telescoping multiple reactions into a single run and designing and controlling the system with open-source MechWolf software, this approach will allow for the construction of these important intermediates from commercial feedstock in much more rapid timescales than is currently possible. This will include developing rapid chemo-enzymatic syntheses of otherwise difficult to access nonulosonic (9-carbon) acid carbohydrate building blocks commonly associated with several pathogenic microbes. In addition, the MechWolf program will provide an open-source chemical repository for optimal conditions for the production of any protected monosaccharide to ensure batch-to-batch reproducibility and on-demand access of these building blocks. Specific Aim 2 will extend this technology to the automated production of oligosaccharides. The flexible and modular nature of continuous flow synthesis will allow for the construction of glycosidic linkages that are not trivial to make on existing platforms and for which few if any enzymes are available. As proof of principle, the system will be used to construct several capsular polysaccharides associated with the ESKAPE pathogen Acinetobacter baumannii; however, these technologies and concepts could be used for the construction of any oligosaccharide. Taken together, the technologies developed through this research will lead to a rapid, robust, reproducible, and affordable method for automated oligosaccharide production with minimal need for human optimization and intervention.