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 EC50. 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.
2-Acetamido-2-deoxy-3,4,5,6-di-O-isopropylidine-aldehydo-D-glucose dimethyl acetal
2-Acetamido-2-deoxy-3,4-O-isopropylidine-aldehydo-D-glucose dimethyl acetal
2-Acetamido-6-O-benzoyl-2-deoxy-3,4-O-isopropylidene-aldehydo-D-glucose dimethyl acetal
2-Acetamido-6-O-benzoyl-2-deoxy-3,4-O-isopropylidine-5-O-mesyl-aldehydo-D-glucose dimethyl acetal
2-Acetamido-5,6-anhydro-2-deoxy-3,4-O-isopropylidene-aldehydo-L-idose dimethyl acetal
2-Acetamido-2,5,6-trideoxy-5,6-epithio-3,4-O-isopropylidine-aldehydo-D-glucose dimethyl acetal
2-Acetamido-6-O-acetyl-5-S-acetyl-2-deoxy-3,4-O-isopropylidine-5-thio-aldehydo-D-glucose dimethyl acetal
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The plasmid encoding GNK was a gift from M. Berger (Charite Universitatsmedizin Berlin) and S. Hinderlich (University of Applied Sciences, Berlin), and the plasmid encoding AGX1 was a gift from V. Piller (Centre de Recherche Scientifique, UPR 4301). Polyclonal antibody to OGA was a gift from G. Hart (John Hopkins University). EMEG32 cells were a gift from T. Mak (University of Toronto). We thank G. Davies (University of York) for the E. coli expression construct of BtGH84. T.M.G. is a Sir Henry Wellcome postdoctoral fellow and a Michael Smith for Health Research (MSFHR) trainee award holder. D.J.V. is a scholar of MSFHR and holds a Canada Research Chair in Chemical Glycobiology. We thank the Natural Sciences and Engineering Research Council of Canada and Simon Fraser University for funding support.
Supplementary Methods, Supplementary Schemes 1–4 and Supplementary Figures 1–15
About this article
Nature Chemical Biology (2017)