ABSTRACT
In connection with studies on lipopolysaccharide biosynthesis in respiratory pathogens we had a need to access potential biosynthetic intermediate sugar nucleotides. Herein we report the chemical synthesis of uridine 5'-diphospho 2,3-diacetamido-2,3-dideoxy-alpha-D-glucuronic acid (UDP-Glc-2,3-diNAcA) (1) from N-acetyl-D-glucosamine in 17 steps and approximately 9% overall yield. This compound has proved invaluable in the elucidation of biosynthetic pathways leading to the formation of 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid-containing polysaccharides.
Subject(s)
Bordetella parapertussis/metabolism , Lipopolysaccharides/biosynthesis , Pseudomonas aeruginosa/metabolism , Uridine Diphosphate Glucuronic Acid/analogs & derivatives , Bordetella parapertussis/pathogenicity , Carbohydrate Conformation , Humans , Lipopolysaccharides/chemistry , Pseudomonas aeruginosa/pathogenicity , Stereoisomerism , Uridine Diphosphate Glucuronic Acid/chemical synthesis , Uridine Diphosphate Glucuronic Acid/metabolismABSTRACT
The direct oxidation of UDP-alpha-d-glucose and UDP-N-acetyl-alpha-d-glucosamine to the corresponding uronic acids was explored using either TEMPO or platinum-catalysed oxidation with molecular oxygen. Whilst TEMPO-based procedures gave rise to substantial over-oxidation and/or degradation of UDP-glucose, oxidation of UDP-N-acetyl-glucosamine to UDP-N-acetyl-glucosaminuronic acid was achieved with >90% conversion and ca. 65% isolated yield using a platinum-catalysed procedure.
Subject(s)
Cyclic N-Oxides/chemistry , Nucleoside Diphosphate Sugars/chemistry , Platinum/chemistry , Uridine Diphosphate/analogs & derivatives , Uronic Acids/chemical synthesis , Catalysis , Oxidation-Reduction , Uridine Diphosphate/chemistry , Uridine Diphosphate Glucose/chemistry , Uridine Diphosphate Glucuronic Acid/chemical synthesis , Uridine Diphosphate N-Acetylglucosamine/chemistry , Uridine Diphosphate Sugars/chemical synthesisSubject(s)
Affinity Labels/chemical synthesis , Azides , Azides/chemical synthesis , Glycosyltransferases/metabolism , Uridine Diphosphate Glucose/analogs & derivatives , Uridine Diphosphate Glucuronic Acid/analogs & derivatives , Animals , Azides/metabolism , Binding Sites , Electrophoresis, Polyacrylamide Gel/methods , Fabaceae/enzymology , Glycosyltransferases/chemistry , Glycosyltransferases/isolation & purification , Indicators and Reagents , Isotope Labeling/methods , Liver/enzymology , Macromolecular Substances , Phosphorus Radioisotopes , Plants, Medicinal , Swine , Uridine Diphosphate Glucose/chemical synthesis , Uridine Diphosphate Glucose/metabolism , Uridine Diphosphate Glucuronic Acid/chemical synthesis , Uridine Diphosphate Glucuronic Acid/metabolismABSTRACT
A new active site-directed photoaffinity analogue, [beta-32P]5-azido-UDP-glucuronic acid (UDP-GlcA), was enzymatically synthesized from [beta-32P]5-N3UDP-Glc using UDP-glucose dehydrogenase. The product was characterized by its mobility on ion exchange and two thin-layer chromatographic systems, by its UV absorbance at 288 nm, and the loss of this absorbance after UV irradiation of the compound. Photoincorporation of [beta-32P]5-N3UDP-GlcA into bovine liver UDP-Glc dehydrogenase (EC 1.1.1.22) was saturable with an apparent Kd of 12.5 microM, and was inhibited by the known active-site effectors UDP-GlcA, UDP-Glc, and UDP-xylose. When human liver microsomes with known UDP-glucuronosyltransferase (EC 2.4.1.17) activities were photolabeled with [beta-32P]5-N3UDP-GlcA, major photolabeled bands of 35-37 and 50-54 kDa were detected. When rat liver microsomes from phenobarbital-injected rats were photolabeled with [beta-32P]5-N3UDP-GlcA, there was a marked increase in photoincorporation of a 51-kDa protein as compared with control animals. Evidence is presented which suggests that the photolabeled 51-54-kDa proteins in the liver microsomes from both tissues are UDP-glucuronosyltransferase and that [beta-32P]5-N3UDP-GlcA represents a new alternative approach in the study of UDP-glucuronosyltransferase and other UDP-GlcA-utilizing enzymes.