Reverse thiophosphorylase activity of a glycoside phosphorylase in the synthesis of an unnatural Manβ1,4GlcNAc library

beta-Mannosides are ubiquitous in nature, with diverse roles in many biological processes. Notably, Man beta 1,4GlcNAc a constituent of the core N-glycan in eukaryotes was recently identified as an immune activator, highlighting its potential for use in immunotherapy. Despite their biological significance, the synthesis of beta-mannosidic linkages remains one of the major challenges in glycoscience. Here we present a chemoenzymatic strategy that affords a series of novel unnatural Man beta 1,4GlcNAc analogues using the beta-1,4-d-mannosyl-N-acetyl-d-glucosamine phosphorylase, BT1033. We show that the presence of fluorine in the GlcNAc acceptor facilitates the formation of longer beta-mannan-like glycans. We also pioneer a "reverse thiophosphorylase" enzymatic activity, favouring the synthesis of longer glycans by catalysing the formation of a phosphorolysis-stable thioglycoside linkage, an approach that may be generally applicable to other phosphorylases. A carbohydrate phosphorylase is utilised in the synthesis of unnatural Man beta 1,4-GlcNAc and longer beta-mannan like glycans, including formation of phosphorolysis-stable thioglycoside linkages via novel "reverse thiophosphorylase" enzymatic activity.