Facile chemoenzymatic synthesis and purification of glycolipids Project Summary Carbohydrates and glycoconjugates play important roles in biological systems. However, they are very difficult to obtain by isolation from nature due to their structural complexity, the low abundance of some forms, general polarity of these compounds, and the presence of other compounds with similar properties. They are also challenging to synthesize despite various chemical, enzymatic, and chemoenzymatic methods that have been developed so far. Among diverse methods developed, chemoenzymatic methods have great advantages. They combine the flexibility of chemical synthesis of building blocks that can be used by enzymes, especially glycosyltransferases, for synthesizing biologically important glycans and glycoconjugates in a regio- and stereo-selective manner. A large array of enzymes have been characterized to allow the synthesis of desired targets, including those containing naturally occurring and non-natural modifications. However, product purification is a bottle-neck step. Chemoenzymatic methods have not been broadly used for producing glycolipids, a unique class of biomolecules that can be relatively easy to separate from other biomolecules by extraction. We plan to develop simple and convenient chemoenzymatic synthesis and facile purification methods for efficient production of complex bioactive glycolipids including those containing neutral and/or charged glycans with diverse lipid forms. The method will allow solution-phase synthesis of glycolipids using one-pot multienzyme (OPME) systems to achieve high selectivity and efficiency, and easy product purification by C18 cartridge-based solid-phase extraction (SPE). It is readily adaptable for automated synthesis. High efficiency of the enzymes that have been identified and will be discovered in the PI and the Co-PI's labs combining with easy isolation of the acceptor/products from other components will allow individual glycosylation reaction to be pushed to high yields with or without re-run of the reactions. As the variation of glycans and lipids are enormous, it is impractical to synthesize all interesting glycolipids in the current proposal. Instead, representative families and classes of glycolipids will be produced in the proposed project. Synthetic and purification protocols will be established. Convenient-to-store and easy-to-use enzymes and reagent kits will be assembled. These can be used by non-specialists for synthesizing, purification, and study of desired glycolipids of their interest in their own labs with a general research lab setting. Cross validation will be performed by different individuals in different labs. Protocols will be prepared and shared on a designated website and be included in the kits.