Diastereoselective synthesis of glycosyl-1-thiophosphates as reporters for nucleotide diphosphate sugar metabolism

Glycosylation controls key physiological processes such as cell-cell recognition, signal transduction, and protein folding/trafficking within subcellular compartments. Glycans are often assembled by Leloir glycosyltransferase enzymes, which use nucleotide diphosphate monosaccharides (NDP-monosaccharides) as substrates. Cells produce NDP-monosaccharides through metabolic transformation of corresponding glycosyl-1-phosphates (G1Ps). Existing LC-MS-based approaches to study NDP-monosaccharide metabolism in living cells are limited due to the mass degeneracy of the epimeric physiological pyranoses that populate the NDP-monosaccharide pool. Technology that enables tracking of NDP-monosaccharide metabolism from chemically defined metabolic precursors would illuminate these challenging-to-study pathways that are central to immunity, signal transduction, and cellular homeostasis. To circumvent this challenge, we have developed a series of cell-permeable glycosyl-1- thio phosphates (G1TPs) in which a single oxygen atom is replaced with a sulphur atom at the phosphorus centre. This chalcogen substitution generated a mass-unique signature that enabled tracking of select NDP-monosaccharide metabolites. To generate G1TPs, we leveraged a cyclic thiophosphorochloridate reagent to selectively install a 1,3,2-dioxaphospholane group at the anomeric centre of per- O -acetyl-D-pyranoses. Our approach included anomeric stereocontrol, which provided access to both native (α) and non-physiological (β) diastereomers at C1. Next, we leveraged the intrinsic strain of the dioxaphospholane system to efficiently install an S -acetyl-2-thioethyl (SATE) group by direct ring opening with thioacetate. LC-MS analysis confirmed that the resultant per- O -Ac- and SATE-protected G1TPs were cell permeable and could be deprotected to free G1TPs once inside. Importantly, metabolomic analysis revealed that an archetypal G1TP – α-galactosyl-1-thiophosphate – was metabolized to the corresponding NDP-(S)-galactose in cellulo , whereas the β-counterpart was not. Finally, we demonstrated that α-galactosyl-1-thiophosphate G1TP was non-toxic, did not perturb native glycan biosynthesis, and did not impact gene transcription. Combined, these results establish G1TPs as biologically silent reporters for NDP-monosaccharide metabolism.