Rapid analysis of nucleotide-activated sugars by high-performance liquid chromatography coupled with diode-array detection, electrospray ionization mass spectrometry and nuclear magnetic resonance.
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.
Ramm
Michael
M
Institute of Pharmacy, Friedrich-Schiller-University Jena, Semmelweisstrasse 10, D-07743 Jena, Germany.
Wolfender
Jean-Luc
JL
Queiroz
Emerson Ferreira
EF
Hostettmann
Kurt
K
Hamburger
Matthias
M
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*
D002241:Carbohydrates; D009711:Nucleotides