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.

Automation of the chemical synthesis of human milk oligosaccharides Project Narrative The proposed research will deliver an automated approach to accelerate and broaden access to human milk oligosaccharides needed for analytical, infant formula, and biomedical applications. Machine-assisted synthesis ensures rigorous experimental design to obtain robust and unbiased results by significantly reducing variability, ensuring authentication of key chemical resources and improving reproducibility of experiments. Completely automated synthesis will enhance transparency, simplify the implementation of our protocols in other, even non- specialized, labs thereby increasing the availability of these molecules, and aid global efforts to develop carbohydrate-based therapeutics, dietary supplements, and analytical tools.