Chemoenzymatic Synthesis LNT Series HMOs

Human milk oligosaccharides (HMOs) are complex glycans that are present at very high concentrations in human milk but not in infant formula. They play important roles in the early development of the infant digestive system, and improving the immune system and the ecological balance of newborns. Previous studies have shown that human milk oligosaccharides are structurally mimic epithelial cell surface carbohydrates and thus function as decoys to which pathogens can bind instead of the host, thereby preventing infection.Lacto-N-tetrasaccharide, lacto-N-trisaccharide are the core structures of predominant components of the human milk oligosaccharides, and its can be further modified by other processing enzymes such as fucotransferases and sialyltransferases to generate diverse fucosylated and sialylated derivatives. HMOs are believed to have beneficial biological properties, however, their specific biological roles are still not well understood. Thus, the sufficient amount of homomgeneous HMOs are demanded for the further biological function studies.It is well known that chemical synthesis strategy is a reliable method for obtaining structure defined oligosaccharides. In recent years, several chemical synthesis approaches have been developed for the synthesis human milk oligosaccharide, and its generally required various steps, harsh reaction conditions and the low overall yield. In order to get sufficient amount of HMOs, an highly efficient chemoenzymatic synthesis strategie was developed in this study. This study consists of two parts. Firstly, the conmon trisaccharide motif lacto-N-trisaccharide derivatives was prepared in a straight forward chemical synthesis. Then, an one-pot two-enzyme approach comprised a galactose kinase (GaLK) and a glycosidase (BiGaLNAcHexP) was introduced for the enzymatic elongation to generate lacto-N-tetrasaccharide and its derivatives.A concise chemical synthesis of lacto-N-trisaccharide intermediate was developed using widely available GlcNAc, lactose as the starting materials, and through, a series of highly efficient protecting group manipulation and glycosylation reactions including:acetylation of lactose, the anomeric acetyl deprotection and synthesis of propane azide glycosides, selectively exposed3’,4’hydroxyl groups of lactos acceptor, glycosylation with the activated N-acetyl glucosamine donor, the global deprotection and selective N-acetylation. In all the processes, the reaction conditions are mild, and some intermediates even no need to be purified. This chemical synthesis offers an invaluable approach for the large scale production of HMOs trisaccharide core structure. In the second part of this study, an one-pot two-enzyme approach was introduced for the glycoside chain elongation to build the core tetrasaccharide of HMOs.In summary, an highly efficient chemoenzymatic approach was developed for the large scale production of tri-and tetrasaccharide core structure of HMOs. These core structures can be invaluable intermediates for the generation most of HMOs when combined with other synthetic enzymes such as fucotransferases and sialyltransferases.