Automation of the chemical synthesis of human milk oligosaccharides

人乳低聚糖化学合成的自动化 项目摘要 碳水化合物构成了所有生物的基础，在自然界和医学中无处不在。 然而，化学或酶法合成碳水化合物的方法仍然很繁琐。The Rapid 糖科学研究的发展要求开发快速、高效和简单的糖链检测方法 综合。这项建议旨在通过重点推出负担得起的和 可访问的自动化平台，使专家和非专家都能够执行 多聚糖。 目前合成多糖的方法非常复杂，操作复杂，并且需要 重要的用户专有技术。相比之下，基于高效液相色谱设备的自动化 Demchenko和Stine介绍的(高效液相-A)代表了一种高度可获得的合成方法，因为 许多科学家已经掌握了高效液相色谱设备的操作知识，并且很容易接触到这些设备。自动化 合成不仅通过使用标准的高效液相组分传递所有试剂来提供操作简单性，而且 还可以方便地对每一步反应进行实时监控。这项建议旨在改进我们目前的高效液相色谱-A 通过引入一个完全自动合成多糖的通用平台进行设置。 我们的前两个目标致力于解决我们以前平台中的主要弱点，我们的第三个目标将 将这些改进应用于人乳低聚糖(HMO)的合成。我们将开发专门的 用于固相合成多糖的性能增强的整体载体(目标A)。我们还将 开发专门的糖基化反应，并为我们的系统配备新的模块，以增强 再循环的效率，提高糖基化的重复性，减少对多余积木的需要， 并导致合成所有步骤的完全自动化(目标B)。然后我们将演示这些技术有多好 战略调整可以应用于HMO的完全自动化、“一键操作”的生产， 它们在婴儿发育过程中作为益生素、抗菌剂和营养素发挥着关键作用(目标C)。 在完成建议的研究后，我们预期已达成一个可靠和简单的平台，以 全自动合成。有权使用标准高效液相色谱设备的调查人员应该能够 使用我们的方法自动合成葡聚糖。机器辅助合成确保严谨 实验设计，以获得可靠的结果，消除变异性，并准确地重现实验 由不同的用户多次访问。使用这个用户友好的自动化平台合成多糖将 加速许多科学学科的发现，并可显著影响技术、社会、 经济和公共卫生。

Automation of the chemical synthesis of human milk oligosaccharides Project Summary Carbohydrates form the basis of all living organisms and are ubiquitous both in nature and in medicine. However, methods for the chemical or enzymatic synthesis of carbohydrates remain cumbersome. The rapid growth of R&D in glycoscience demands the development of rapid, efficient and simple procedures for glycan synthesis. This proposal seeks to meet this demand by focusing on the introduction of an affordable and accessible automation platform that will enable both specialists and non-specialists to perform the synthesis of glycans. Current methods for the synthesis of glycans are highly sophisticated, operationally complex, and require significant user know-how. By contrast, high performance liquid chromatography equipment-based automation (HPLC-A) introduced by Demchenko and Stine represents a highly accessible method of synthesis because many scientists already have operational knowledge of, and easy access to, HPLC equipment. Automated synthesis offers not only operational simplicity by delivering all reagents using standard HPLC components, but also convenient real-time reaction monitoring of every step. This proposal aims to improve our current HPLC-A setup by introducing a universal platform for completely automated synthesis of glycans. Our first two aims are dedicated to addressing key weaknesses in our previous platform, and our third aim will apply these improvements to the synthesis of human-milk oligosaccharides (HMO). We will develop dedicated monolithic supports with enhanced properties for solid phase synthesis of glycans (Aim A). We will also develop a dedicated glycosylation reaction and accessorize our system with new modules that will enhance the efficiency of recirculation, improve reproducibility of glycosylations, reduce the need for excess building block, and lead to complete automation of all steps of the synthesis (Aim B). We will then demonstrate how well these strategic adjustments can be applied to the completely automated, “press of a button” production of HMO, which have crucial roles as prebiotics, antimicrobial agents, and nutrients in infant development (Aim C). Upon completion of the proposed studies, we expect to have achieved a reliable and simple platform for completely automated synthesis. Investigators with access to standard HPLC equipment should be able to perform automated synthesis of glycans using our methods. Machine-assisted synthesis ensures rigorous experimental design to obtain robust results, to eliminate variability, and to accurately reproduce experiments multiple times by different users. Synthesis of glycans using this user-friendly, automated platform will accelerate discovery in many scientific disciplines and can significantly impact technology, society, the economy, and public health.