title: Synthesis of 5-azido-UDP-N-acetylhexosamine photoaffinity analogs and radiolabeled UDP-N-acetylhexosamines.
abstract: 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, [gamma-32P]ATP, porcine N-acetylgalactosamine kinase, and Escherichia coli UDP-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 alpha- and beta-subunits of purified bovine UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase. Lastly, the photoprobe was also effective at photolabeling Streptococcus pyogenes hyaluronate synthase in membrane preparations.
authors.affiliation: Department of Biochemistry and Molecualr Biology, University of Arkansas for Medical Sciences, Little Rock 72205, USA.
authors.forename: P
authors.initials: P
authors.lastname: Sunthankar
authors.forename: I
authors.initials: I
authors.lastname: Pastuszak
authors.forename: A
authors.initials: A
authors.lastname: Rooke
authors.forename: A D
authors.initials: AD
authors.lastname: Elbein
authors.forename: I
authors.initials: I
authors.lastname: van de Rijn
authors.forename: W M
authors.initials: WM
authors.lastname: Canfield
authors.forename: R R
authors.initials: RR
authors.lastname: Drake
mesh_terms: D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D006595:Hexosamines / Q000138:chemical synthesis* / Q000737:chemistry; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D009713:Nucleotidyltransferases / Q000737:chemistry; D010761:Phosphorus Radioisotopes; D019920:Photoaffinity Labels; D013552:Swine; D014316:Tritium; D014530:Uridine Diphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry
chemical_list: C112928:5-azido-UDP-acetylhexosamine; D006595:Hexosamines; D010761:Phosphorus Radioisotopes; D019920:Photoaffinity Labels; D014316:Tritium; D014530:Uridine Diphosphate; D009713:Nucleotidyltransferases; C023226:UDPacetylglucosamine pyrophosphorylase