Identification of new Sialic Acids Derived from Glycoprotein of Bovine Submandibular Gland

Improvements in the isolation procedure and the analytical equipment enabled the detection of seven novel sialic acids in bovine submandibular gland glycoprotein: N-acetyl-8-O-acetylneuraminic acid, N-acetyl-8,9-di-O-acetylneuraminic acid, N-acetyl-7,8,9-tri-O-acetylneuraminic acid, N-glycoloyl-7-O-acetylneuraminic acid, N-glycoloyl-7,9-di-O-acetylneuraminic acid, N-glycoloyl-8,9-di-O-acetylneuraminic acid, and N-glycoloyl-7,8,9-tri-O-acetylneuraminic acid. There are also indications for the presence of N-glycoloyl-8-O-acetylneuraminic acid. In addition, the sialic acids already known to occur in this tissue, namely N-acetylneuraminic acid, N-acetyl-7-O-acetylneuraminic acid, N-acetyl-9-O-acetylneuraminic acid, N-acetyl-7,9-di-O-acetylneuraminic acid, N-glycoloylneuraminic acid, and N-glycoloyl-9-O-acetylneuraminic acid could be identified. Sialic acids were released from the mucin by mild acid hydrolysis and prefractionated on a Dowex 2X8 anion-exchange column (formate form) by elution with a 0-0.6 M gradient of formic acid. The four pools of sialic acids obtained in this way were each further fractionated by column chromatography on cellulose with n-butanol/n-propanol/water (1/2/1, v/v/v) as eluent. By this procedure, N-acetylneuraminic acid, N-acetyl-7-O-acetylneuraminic acid, N-acetyl-9-O-acetylneuraminic acid, N-glycoloylneuraminic acid, and N-glycoloyl-9-O-acetylneuraminic acid were obtained in pure form. The other sialic acids could be enriched sufficiently in different fractions for structural identification. Analyses of sialic acids were carried out by one-dimensional and two-dimensional thin-layer chromatography, gas-liquid chromatography, and gas-liquid chromatography-mass spectrometry.