Hijacking a biosynthetic pathway yields a glycosyltransferase inhibitor within cells

Feeding of modified carbohydrates has previously led to metabolic incorporation of these compounds into cellular glycans. Now the strategic use of a thiol analog that can be converted into an activated sugar but not incorporated into glycans provides a potent intracellular glycosyltransferase inhibitor. Glycosyltransferases are ubiquitous enzymes that catalyze the assembly of glycoconjugates throughout all kingdoms of nature. A long-standing problem is the rational design of probes that can be used to manipulate glycosyltransferase activity in cells and tissues. Here we describe the rational design and synthesis of a nucleotide sugar analog that inhibits, with high potency both in vitro and in cells, the human glycosyltransferase responsible for the reversible post-translational modification of nucleocytoplasmic proteins with O -linked N -acetylglucosamine residues ( O -GlcNAc). We show that the enzymes of the hexosamine biosynthetic pathway can transform, both in vitro and in cells, a synthetic carbohydrate precursor into the nucleotide sugar analog. Treatment of cells with the precursor lowers O -GlcNAc in a targeted manner with a single-digit micromolar EC 50 . This approach to inhibition of glycosyltransferases should be applicable to other members of this superfamily of enzymes and enable their manipulation in a biological setting.