Reexamination of the pyridylamination used for fluorescence labeling of oligosaccharides and its application to glycoproteins.
The pyridylamination reaction of sugar chains from glycoproteins was re-investigated to raise the total yield of pyridylamino sugars. Sugar chains of glycoproteins were released by hydrazinolysis, and free amino groups were N-acetylated. The sugar chains were coupled with 2-aminopyridine, and the pyridylamino derivatives thus obtained were purified by Sephadex G-15 column chromatography and analyzed by high performance liquid chromatography. In this study, conditions for the coupling reaction (temperature, time, pH, concentration of 2-aminopyridine, and amount of the reducing reagent) were re-investigated. Under the conditions established in the present study, the total recovery of pyridylamino derivatives of sugar chains of glycoproteins was about 70%, and 0.15 nmol of a glycoprotein was enough to detect the pyridylamino derivatives of sugar chains. The stability of sialyl and fucosyl linkages under the present conditions was also studied.
D000107:Acetylation; D000586:Amination; D000631:Aminopyridines; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D006023:Glycoproteins / Q000302:isolation & purification*; D009844:Oligosaccharides / Q000302:isolation & purification*; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence; D013696:Temperature
D000631:Aminopyridines; D006023:Glycoproteins; D009844:Oligosaccharides; C032439:alpha-aminopyridine