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

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• 作者：Ramm ; Michael ; Wolfender ; Jean-Luc ; Queiroz ; Emerson Ferreira ; Hostettmann ; Kurt ; Hamburger ; Matthias
• 关键词：Nuclear magnetic resonance spectrometry ; Nucleotide sugars ; Carbohydrates ; Guanosine diphosphate&ndash ; mannose ; Guanosine diphosphate&ndash ; rhamnose
• 刊名：Journal of Chromatography A
• 出版年：2004
• 出版时间：April 23, 2004
• 年：2004
• 卷：1034
• 期：1-2
• 页码：139-148
• 全文大小：269 K

文摘

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.