SYNTHESIS OF ARYL AZIDE DERIVATIVES OF UDP-GLCNAC AND UDP-GALNAC AND THEIR USE FOR THE AFFINITY LABELING OF GLYCOSYLTRANSFERASES AND THE UDP-HEXNAC PYROPHOSPHORYLASE

The chemical synthesis and utilization of two photoaffinity analogs, I -125-labeled 5-[3-(p-azidosalicylamido)-1-propenyl]-UDP-GlcNAc and -UD P-GalNAc, is described. Starting with either UDP-GlcNAc or UDP-GalNAc, the synthesis involved the preparation of the 5-mercuri-UDP-HexNAc an d then attachment of an allylamine to the 5 position to give 5-(3-amin o)allyl-UDP-HexNAc. This was followed by acylation with N-hydroxysucci nimide p-aminosalicylic acid to form the final product, i.e., 5-[3-(p- azidosalicylamido)-1-propenyl]-UDP-GlcNAc or UDP-GalNAc. These product s could then be iodinated with chloramine T to give the I-125-derivati ves, Both the UDP-GlcNAc and the UDP-GalNAc derivatives reacted in a c oncentration-dependent manner with a highly purified UDP-HexNAc pyroph osphorylase, and both specifically labeled the subunit(s) of this prot ein. The labeling of the protein by the UDP-GlcNAc derivative was inhi bited in dose-dependent fashion by either unlabeled UDP-GlcNAc or unla beled UDP-GalNAc. Likewise, labeling with the UDP-GalNAc probe was blo cked by either UDP-GlcNAc or UDP-GalNAc. The UDP-GlcNAc probe also spe cifically labeled a partially purified preparation of GlcNAc transfera se I. (C) 1996 Academic Press, Inc.