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.

项目概要 对生产明确寡糖的有效方法的需求仍然是一个主要问题 微生物糖生物学领域的瓶颈。尽管自动化寡糖合成仪已 开发的，大多数依赖于固相合成，这会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于 专门针对糖基化反应，并且没有解决耗时且繁琐的过程 将单糖原料转化为完全取代的糖基供体，准备偶联。令人难以置信的 微生物聚糖合成所需的构建模块的数量也使得保留每个可能的模块 库存不可能。所有这些问题都可以通过自动化连续流的开发来解决 平台。连续流动反应比多步骤间歇过程更容易实现自动化， 从而提供更高的批次间再现性。通过适当选择条件也可以 将多个反应压缩到一次运行中。该提案的目标是创建平台 基于自动化连续流的寡糖技术，能够实现每一步的自动化 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 检查糖基化单糖的自动化生产。通过伸缩多个反应 这种方法将其集成到单次运行中，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中构建这些重要的中间体 比目前可能的时间尺度。这将包括开发快速化学酶合成 否则很难获得通常相关的非声波（9碳）酸性碳水化合物构建块 带有多种病原微生物。此外，MechWolf 计划将提供一种开源化学品 存储任何受保护单糖生产的最佳条件，以确保批次间的一致性 这些构建块的可重复性和按需访问。具体目标 2 将该技术扩展到 低聚糖的自动化生产。连续流合成的灵活和模块化性质将 允许构建糖苷键，这在现有平台上并非易事，并且为此 可用的酶（如果有）也很少。作为原理证明，该系统将用于构建多个胶囊 与 ESKAPE 病原体鲍曼不动杆菌相关的多糖；然而，这些 技术和概念可用于构建任何寡糖。综合起来， 通过这项研究开发的技术将带来一种快速、稳健、可重复且负担得起的方法 用于自动化寡糖生产，几乎不需要人工优化和干预。