Synthesis of 5-Azido-UDP-N-Acetylhexosamine Photoaffinity Analogs and Radiolabeled UDP-N-Acetylhexosamines

• Published in: Analytical biochemistry Vol. 258; no. 2; pp. 195 - 201
• Main Authors: Sunthankar, Prasanna, Pastuszak, Irena, Rooke, Agnieszka, Elbein, Alan D., van de Rijn, Ivo, Canfield, William M., Drake, Richard R.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.05.1998
• Subjects: Animals; Carbohydrate Conformation; Carbohydrate Sequence; Cattle; Cell Membrane - enzymology; Hexosamines - chemical synthesis; Hexosamines - chemistry; Kidney - enzymology; Liver - enzymology; Nucleotidyltransferases - chemistry; Phosphorus Radioisotopes; Photoaffinity Labels; Swine; Tritium; Uridine Diphosphate - analogs & derivatives; Uridine Diphosphate - chemical synthesis; Uridine Diphosphate - chemistry
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.1998.2600

Nuleotide sugar photoaffinity analogs have proven to be useful in the identification and characterization of glycosyltransferases. A radioenzymatic synthesis of [32P]5-azido-UDP-N-acetylglucosamine has been accomplished using 5-azido-UTP, [γ-32P]ATP, porcineN-acetylgalactosamine kinase, andEscherichia coliUDP-N-acetylglucosamine pyrophosphorylase, GlmU. This general enzymatic scheme was useful for the synthesis of [32P]5-azido-UDP-N-acetylgalactosamine and high-specific-activity [3H] or [32P]UDP-N-acetylhexosamines. A new chemical synthesis method for generating 5-azido-uridine compounds was also developed. [32P]5-Azido-UDP-N-acetylglucosamine was functionally characterized using different soluble and membrane-associated glycosyltransferases which utilize UDP-GlcNAc as a substrate. Site-specific photoincorporation was observed for partially purified GlmU and porcine UDP-GlcNAc pyrophosphorylase. The photoprobe also effectively photoincorporated into the α- and β-subunitsof purified bovine UDP-N-acetylglucosamine:lysosomal enzymeN-acetylglucosamine-1-phosphotransferase. Lastly, the photoprobe was also effective atphotolabelingStreptococcus pyogeneshyaluronate synthase in membrane preparations.