Reducing-end modification of N-linked oligosaccharides with tyrosine.
The N-linked oligosaccharides from bovine fetuin were purified using newly developed preparative purification methodology. N-linked oligosaccharides were released from tryptic glycopeptides utilizing N-glycosidase F on the 5-g scale. Selective desialylation with neuraminidase from Clostridium perfringens resulted in the formation of a mono-sialyl-oligosaccharide and asialo-oligosaccharides. The reducing ends of the oligosaccharides were converted to the glycosylamine and reacted with the N-hydroxysuccinimide ester of Boctyrosine. The tyrosinated oligosaccharides were resolved into individual peaks on RP-HPLC and then characterized by proton NMR and FAB-MS. A single asialo-triantennary, an asialo-biantennary, and a mono-sialyl-triantennary oligosaccharide were recovered in good yield. Each product contained a single Boc-Tyr residue attached to the reducing-end GlcNAc residue through a beta-glycosylamide linkage. The procedure was utilized to isolate multi-micromole quantities of oligosaccharides from gram quantities of glycoprotein, thus providing a new route to purify large quantities of N-linked oligosaccharides which contain a terminal tyrosine residue. The tyrosinated oligosaccharides are valuable glycoconjugate ligands which contain a chromophore that absorbs at 280 nm and has sufficient hydrophobicity to facilitate RP-HPLC separations. Furthermore, this group can be deprotected by removal of Boc to reveal a primary amine suitable for further derivatization and can also be radioiodinated for tracking during biological experiments.
Tamura
T
T
Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, Ohio State University, Columbus 43210.
Wadhwa
M S
MS
Rice
K G
KG
D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D002240:Carbohydrate Sequence; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange; D005563:Formic Acid Esters; D006020:Glycopeptides; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000138:chemical synthesis / Q000302:isolation & purification*; D010084:Oxidation-Reduction; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D014443:Tyrosine; D000509:alpha-Fetoproteins / Q000032:analysis
D005563:Formic Acid Esters; D006020:Glycopeptides; D009844:Oligosaccharides; D000509:alpha-Fetoproteins; C000345:t-butyloxycarbonyl group; D014443:Tyrosine; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase