Glycosylations promoted by triflate-generating reagents are widespread synthetic methods for the construction of glycosidic scaffolds and glycoconjugates of biological and chemical interest. These processes are thought to proceed with the participation of a plethora of activated high energy intermediates such as the and glycosyl triflates, or even increasingly unstable glycosyl oxocarbenium-like species, among which only -glycosyl triflates have been well characterized under representative reaction conditions. Interestingly, the remaining less accessible intermediates, yet to be experimentally described, seem to be particularly relevant in -selective processes, involving weak acceptors. Herein, we report a detailed analysis of several paradigmatic and illustrative examples of such reactions, employing a combination of chemical, NMR, kinetic and theoretical approaches, culminating in the unprecedented detection and quantification of the true -glycosyl triflate intermediates within activated donor mixtures. This achievement was further employed as a stepping-stone for the characterization of the triflate anomerization dynamics, which along with the acceptor substitutions, govern the stereochemical outcome of the reaction. The obtained data conclusively show that, even for highly dissociative reactions involving -close ion pair (-CIP) species, the formation of the -glycoside is necessarily preceded by a bimolecular → triflate interconversion, which under certain circumstances does become the rate-limiting step. Overall, our results rule out the prevalence of the Curtin-Hammett fast-exchange assumption for most glycosylations and highlight the distinct reactivity properties of and glycosyl triflates against neutral and anionic acceptors.

Resistance to aminoglycoside antibiotics has had a profound impact on clinical practice. Despite their powerful bactericidal activity, aminoglycosides were one of the first groups of antibiotics to meet the challenge of resistance. The most prevalent source of clinically relevant resistance against these therapeutics is conferred by the enzymatic modification of the antibiotic. Therefore, a deeper knowledge of the aminoglycoside-modifying enzymes and their interactions with the antibiotics and solvent is of paramount importance in order to facilitate the design of more effective and potent inhibitors and/or novel semisynthetic aminoglycosides that are not susceptible to modifying enzymes.

A dynamic combinatorial analysis of carbohydrate/aromatic complexes clarifies the structural determinants and origins of these important interactions in water.

Thioglycosides are hydrolase‐resistant mimics of O‐linked glycosides that can serve as valuable probes for studying the role of glycosides in biological processes. The development of an efficient, enzyme‐mediated synthesis of thioglycosides, including S‐GlcNAcylated proteins, is reported, using a thioglycoligase derived from a GH20 hexosaminidase from Streptomyces plicatus in which the catalytic acid/base glutamate has been mutated to an alanine (SpHex E314A). This robust, easily‐prepared, engineered enzyme uses GlcNAc and GalNAc donors and couples them to a remarkably diverse set of thiol acceptors. Thioglycoligation using 3‐, 4‐, and 6‐thiosugar acceptors from a variety of sugar families produces S‐linked disaccharides in nearly quantitative yields. The set of possible thiol acceptors also includes cysteine‐containing peptides and proteins, rendering this mutant enzyme a promising catalyst for the production of thio analogues of biologically important GlcNAcylated peptides and proteins.

A dynamical combinatorial approach for the study of weak carbohydrate/aromatic interactions is presented. This methodology has been employed to dissect the subtle structure−stability relationships that govern facial selectivity in these supramolecular complexes.