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

项目摘要 对生产定义明确的低聚糖的有效方法的需求仍然是一个主要的 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 已开发的，大多数依赖于固相合成，这会限制可用合成的化学和规模 敬他们。此外，现有的自动化低聚糖合成方法几乎集中在 仅限于糖基化反应，而不涉及耗时和乏味的过程 将单糖原料转化为完全取代的糖基供体，准备偶联。不可思议的 微生物合成多糖所需的积木数量也使每一个可能的积木都保持在 股票是不可能的。所有这些问题都可以通过自动化连续流的开发来解决 站台。连续流动反应比多步骤间歇反应更容易实现自动化 从而提供更大的批次之间的重现性。通过适当地选择条件，也是可能的 把几个反应放在一次运行中。这项提议目标是生成平台 基于自动化连续流动的寡糖技术，能够自动化每一步 低聚糖的合成，从按需供体/受体的生产到这些大分子的组装 进入目标结构。我们将通过追求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。通过伸缩多个反应 将MechWolf开源软件设计和控制成单次运行的系统，这种方法 将允许以更快的速度从商业原料中构建这些重要的中间体 时间刻度比目前可能的更高。这将包括开发快速的化学-酶合成 否则难以获得通常伴随的非磺酸(9碳)酸性碳水化合物 有几种致病微生物。此外，MechWolf计划将提供一种开源化学物质 用于生产任何受保护的单糖的最佳条件的储存库，以确保批次到批次 这些构建块的可重复性和按需访问。特定目标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 允许在现有平台上构建不平凡的糖苷连接，并为此 几乎没有可用的酶。作为原理证明，该系统将用于构建几个胶囊 与ESKAPE病原体鲍曼不动杆菌相关的多糖；然而，这些 技术和概念可以用于构建任何低聚糖。总而言之， 通过这项研究开发的技术将导致一种快速、健壮、可重现和负担得起的方法 用于自动化低聚糖生产，最大限度地减少对人工优化和干预的需要。

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.