Gas-phase pyridylamination of saccharides: development and applications |
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Authors: | Nakakita Shin-ichi Sumiyoshi Wataru Miyanishi Nobumitsu Natsuka Shunji Hase Sumihiro Hirabayashi Jun |
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Affiliation: | Department of Functional Glycomics, Life Science Research Center, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan. |
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Abstract: | Pyridylamination is a versatile method for fluorescence labeling of oligosaccharides. The technique affords sensitive detection of saccharides with reducing termini and high-resolution separation by high-performance liquid chromatography. The conventional method, based on a liquid-phase reaction, has been extensively used in various aspects of glycobiology and glycotechnology. Unfortunately, the necessity for removing excess 2-aminopyridine makes the technique both laborious and time-consuming. Furthermore, removal of excess reagent can result in a significant loss of short saccharide components. In the present paper, we report an alternative methodology based on a "gas-phase" reaction, in which dried saccharides are reacted with vaporized 2-aminopyridine. The resultant Schiff base was also reduced in the gas phase within the same reaction microtube using a purpose-built device. The newly developed procedure was applied to both monosaccharide (GlcNAc) and oligosaccharides (isomalto-oligosaccharides) at quantitative yields with no requirement to remove excess reagent. The acid-labile sialyl linkages of alpha2-6-disialobiantennary oligosaccharides proved to be fully stable during the procedure. The developed method was also successfully applied to profiling N-linked oligosaccharides liberated from glycoproteins by hydrazinolysis and, thus, should contribute to various fields of glycomics. |
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