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

基于连续流的自动化微生物低聚糖合成方法。

• 批准号: 10455055
• 负责人: Clay Samuel Bennett
• 金额: $ 40.07万
• 依托单位: TUFTS UNIVERSITY MEDFORD
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10455055
• 关键词: Acids; Acinetobacter baumannii; Address; Area; Automation; Biological Phenomena; Biology; Biomedical Research; Carbohydrates; Carbon; Carbonic Acid; Catalysis; Chemicals; Chemistry; Complex; Computer software; Consumption; Coupling; Data; Detection; Development; Diagnostic; ESKAPE pathogens; Ensure; Enzymes; Glycobiology; Glycosides; Goals; Hour; Human; Human Microbiome; Individual; Infection; Intervention; Laboratories; Libraries; Methods; Mission; Modeling; Monitor; Monosaccharides; Nature; O Antigens; Oligosaccharides; Organism; Phase; Play; Polysaccharides; Process; Production; Public Health; Publishing; Raspberries; Reaction; Reproducibility; Research; Role; Route; Running; S phase; Solid; Structure; System; Technology; Time; United States National Institutes of Health; Work; base; design; diagnostic tool; flexibility; glycosylation; graphical user interface; microbial; novel therapeutics; open source; pathogen; pathogenic bacteria; pathogenic microbe; programs; rapid technique; repository; sugar; therapeutic development; tool

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.

项目摘要 对生产明确定义的寡糖生产的有效方法的需求继续呈现主要 微生物糖生物学领域的瓶颈。尽管自动寡糖合成器已经 开发的，大多数依赖于固相合成，这可以限制可用的化学和规模 给他们。此外，现有的自动化方法的寡糖合成方法几乎重点是 专门针对糖基化反应，并不解决时间耗时和繁琐的过程 将单糖原料转换为准备偶联的完全取代的糖基供体。令人难以置信的 微生物聚糖合成所需的构件数量也使每个可能的区块都保持在 不可能的股票。所有这些问题都可以通过自动连续流的发展来解决 平台。与多步批处理过程相比，连续流动反应更容易自动化 从而提供更大的批处理可重复性。通过适当的条件选择也可能 向望远镜进行几个反应。该建议的目的是生成平台 自动连续基于流动的寡糖的技术，能够自动化的每个步骤 寡糖合成，从点播供体/受体产生到这些较大分子的组装 进入目标结构。我们将通过追求以下特定目标来实现这一目标。具体目标1将 检查糖基化现成的单糖的自动产生。通过伸缩多个反应 通过开源机械狼软件进行单次运行，设计和控制系统，这种方法 将允许更快地从商业原料中构建这些重要的中间体 时间尺度比目前可能的时间。这将包括开发快速的化学酶合成 否则难以访问非硫酸（9-碳）酸碳水化合物构建块通常相关的 与几个致病性微生物。此外，Mechwolf计划将提供开源化学 用于生产任何受保护的单糖的最佳条件的存储库，以确保批处理 这些构建块的可重复性和按需访问。具体目标2将把这项技术扩展到 寡糖的自动产生。连续流合成的灵活和模块化性质将 允许在现有平台上建立并不重要的糖苷连接 很少有酶可用。作为原则的证明，该系统将用于构建几个囊 与Eskape病原体鲍曼尼的多糖相关的多糖；但是，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快速，健壮，可重复和负担得起的方法 对于自动化的寡糖产生，对人类优化和干预的需求最少。