BROWNSUGARS

The vast amount and diversity of carbohydrate-based structures present in nature requires a large group of enzymes responsible for their synthesis and degradation. This is notably the role of glycoside hydrolases (GHs) and glycosyltransferases (GTs), which achieve the synthesis and breakage of glycosidic bonds, respectively. Their myriad of biotechnological applications, ranging from biofuel production to drug design makes crucial the deciphering of their reaction mechanisms including the nature of the reactive intermediates involved at the atomic level. In general, approaches that are used to study these enzymes are either indirect (kinetic measurements) or theoretic (QM/MM, etc…). Bringing new methodologies into the filed would be a welcome advance in this dynamic field with high potential. The transition state (TS) of many GHs and GTs exhibit a strong cationic character at the level of the monosaccharide unit which glycosidic bond is either formed or broken. This cation, at the basis of most of the design of most glycosidase inhibitors, has been poorly scrutinized because of the extremely short lifetime of this type of ionic species that precludes its observation. In this project, we wish to apply a strategy combining superacid activation, NMR and DFT calculations, a previously winning strategy in the case of protected glycosyl donors, to unprotected monosaccharides to access the corresponding glycosyl cations in order to perform for the first time their study and characterization. These chemical species will then be compared to the supposed TS of a selection of GHs and GTs to evaluate the benefit of this methodology regarding the knowledge accumulated by other techniques. This strategy will be extended to known inhibitors of these enzymes in order to evaluate the relevance of these molecules as GHs and GTs TS mimics. Ultimately, this work should provide a useful tool not only to gain insights into enzymatic glycosyl transfer by GHs and GTs focusing on the cationic character of the TS, but also a tool allowing to improve the design of GH inhibitors in the future.