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The development of a procedure for the one-pot synthesis of glycosyl benzoates directly from unprotected sugars in aqueous media using 2-chloro-1,3-dimethylimidazolium chloride (DMC), thiobenzoic acid, and triethylamine is reported. These glycosyl donors are excellent substrates for wild-type and mutant glycosidases. β-Glucosyl benzoate was hydrolysed by the GH1 β-glucosidase derived from Halothermothrix orenii ( Hor GH1). Subsequent use of this substrate in thioligase-mediated glycosylation of p -nitrothiophenol demonstrated their superiority as donors compared to their p -nitrophenol counterparts with excellent conversions. Using a series of arene nucleophiles, we also demonstrate good to excellent conversions (up to 94%) of β-glucosyl benzoate to the corresponding p -nitrophenyl- and thioglycosides.

In order to expand the toolbox of enzymes available for thioglycoside synthesis, we describe here the first example of an extremophilic glycosyl hydrolase from Halothermothrix orenii ( Hor GH1) engineered towards thioglycosynthase activity with a novel combination of mutations. Using the triple mutant, Hor GH1 M299R/E166A/E354G, a range of thioglycosides from glycosyl fluoride donors and aromatic thiols could be synthesised with exquisite stereoselectivity and good to excellent conversions (61-93%).