RESUMEN
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.
Asunto(s)
Bordetella parapertussis/metabolismo , Lipopolisacáridos/biosíntesis , Pseudomonas aeruginosa/metabolismo , Uridina Difosfato Ácido Glucurónico/análogos & derivados , Bordetella parapertussis/patogenicidad , Conformación de Carbohidratos , Humanos , Lipopolisacáridos/química , Pseudomonas aeruginosa/patogenicidad , Estereoisomerismo , Uridina Difosfato Ácido Glucurónico/síntesis química , Uridina Difosfato Ácido Glucurónico/metabolismoRESUMEN
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.
Asunto(s)
Óxidos N-Cíclicos/química , Azúcares de Nucleósido Difosfato/química , Platino (Metal)/química , Uridina Difosfato/análogos & derivados , Ácidos Urónicos/síntesis química , Catálisis , Oxidación-Reducción , Uridina Difosfato/química , Uridina Difosfato Glucosa/química , Uridina Difosfato Ácido Glucurónico/síntesis química , Uridina Difosfato N-Acetilglucosamina/química , Azúcares de Uridina Difosfato/síntesis químicaAsunto(s)
Marcadores de Afinidad/síntesis química , Azidas , Azidas/síntesis química , Glicosiltransferasas/metabolismo , Uridina Difosfato Glucosa/análogos & derivados , Uridina Difosfato Ácido Glucurónico/análogos & derivados , Animales , Azidas/metabolismo , Sitios de Unión , Electroforesis en Gel de Poliacrilamida/métodos , Fabaceae/enzimología , Glicosiltransferasas/química , Glicosiltransferasas/aislamiento & purificación , Indicadores y Reactivos , Marcaje Isotópico/métodos , Hígado/enzimología , Sustancias Macromoleculares , Radioisótopos de Fósforo , Plantas Medicinales , Porcinos , Uridina Difosfato Glucosa/síntesis química , Uridina Difosfato Glucosa/metabolismo , Uridina Difosfato Ácido Glucurónico/síntesis química , Uridina Difosfato Ácido Glucurónico/metabolismoRESUMEN
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.
