Chemoenzymatic synthesis of an unnatural Manb1,4GlcNAc library using a glycoside phosphorylase with "reverse thiophosphorylase" activity

b-Mannosides are ubiquitous in nature, with diverse roles in many biological processes. Notably, Manb1,4GlcNAc a constituent of the core N-glycan in eukaryotes, was recently identified as a STING immune pathway activator, highlighting its potential for use in immunotherapy. Despite their biological significance, the synthesis of b-mannosidic linkages remains one of the major challenges in glycoscience. Here we present a chemoenzymatic strategy that affords a series of novel unnatural Manb1,4GlcNAc analogues using the b-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 -mannan-like glycans. We also pioneer a reverse thiophosphorylase enzymatic activity, favouring the synthesis of longer glycans by catalysing the formation of a phosphorylysis-stable thioglycoside linkage, an approach that may be generally applicable to other phosphorylases.