Glycosylium Ions in\nSuperacid Mimic the Transition\nState of Enzyme Reactions

The\nhydrolysis of glycosides is a biochemical transformation\nthat\noccurs in all living organisms, catalyzed by a broad group of enzymes,\nincluding glycoside hydrolases. These enzymes cleave the glycosidic\nbond via a transition state with substantial oxocarbenium ion character,\nresulting in short-lived oxocarbenium ion-like species. While such\ntransient species have been inferred through theoretical studies and\nkinetic isotope effect measurements, their direct spectroscopic characterization\nremains challenging. In this study, we exploit a superacid environment\nto generate, accumulate, and fully characterize nonprotected 2-deoxy\nglycosyl cations in the d-glucopyranose, d-galactopyranose,\nand l-arabinofuranose series using low-temperature NMR spectroscopy,\nsupported by DFT calculations. Additionally, QM/MM MD simulations\nreveal that the properties of these glycosyl cations in superacid\nclosely resemble those within the active sites of glycosidase enzymes,\nparticularly in terms of conformation and anomeric charge distribution.\nThese findings highlight a parallel between the stabilizing effect\nof counterions in superacid media and the network of multidentate\nnoncovalent interactions within glycosidase active sites, which stabilize\ntransition states with pronounced oxocarbenium ion character.