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Rapid analysis of nucleotide-activated sugars by high-performance liquid chromatography coupled with diode-array detection, electrospray ionization mass spectrometry and nuclear magnetic resonance

Ramm, Michael
Queiroz, Emerson Ferreira
Hamburger, M.
Published in Journal of Chromatography. A. 2004, vol. 1034, no. 1-2, p. 139-148
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 I I 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. (C) 2004 Elsevier B.V. All rights reserved.
Stable URL https://archive-ouverte.unige.ch/unige:10834
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Deposited on : 2010-08-26

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