title: Rapid analysis of nucleotide-activated sugars by high-performance liquid chromatography coupled with diode-array detection, electrospray ionization mass spectrometry and nuclear magnetic resonance.
abstract: A generally applicable method for HPLC analysis of sugar nucleotides was established. Separation was achieved using ion-pair chromatography on a reversed-phase column. Ion-pair reagents were selected and various parameters optimized with respect to separation of 11 of the most important sugar nucleotides and compatibility with on-line detection by electrospray ionization MS and NMR. The method was applied to the on-line analysis of the GDP-D-mannose-4,6-dehydratase (Gmd) and GDP-4-keto-6-deoxy-D-mannose reductase (Rmd) catalyzed conversion of GDP-D-mannose to GDP-D-rhamnose. By LC-NMR, the intermediate product of the reaction was shown to be a mixture of GDP-4-keto-6-deoxy-D-mannose and GDP-3-keto-6-deoxy-D-mannose. Nucleotide co-factors of enzymatic reactions such as ATP and NADH did not interfere with the analysis of nucleotide-activated sugars.
authors.affiliation: Institute of Pharmacy, Friedrich-Schiller-University Jena, Semmelweisstrasse 10, D-07743 Jena, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Ramm
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Wolfender
authors.forename: Emerson Ferreira
authors.initials: EF
authors.lastname: Queiroz
authors.forename: Kurt
authors.initials: K
authors.lastname: Hostettmann
authors.forename: Matthias
authors.initials: M
authors.lastname: Hamburger
mesh_terms: D002241:Carbohydrates / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009711:Nucleotides / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D002241:Carbohydrates; D009711:Nucleotides