Preparative purification of tetraantennary oligosaccharides from human asialyl orosomucoid

An approach to isolate micromole quantities of tetraantennary oligosaccharides from human orosomucoid is presented. The N-linked oligosaccharides from 500 mg of the glycoprotein were released enzymatically, desialylated, and isolated free of protein using ion exchange chromatography. The pooled oligosaccharides were converted into oligosaccharide glycosylamines by reaction with ammonium bicarbonate then coupled to BOC-tyrosine to prepare tyrosinamide oligosaccharides. These were resolved on semipreparative RP-HPLC to recover micromole quantities of six purified tyrosinamide oligosaccharides. The oligosaccharide structures were elucidated by a combination of high-field proton NMR and matrix-assisted time of flight mass spectrometry and included biantennary, triantennary, monofucosylated triantennary, tetraantennary, monofucosylated tetraantennary, and a tetraantennary containing a single polylactosamine extension. Edman degradation was utilized to reverse the tyrosinamide oligosaccharide derivatization leading to the generation of reducing oligosaccharides. These were used to characterize the elution profile of asialyl orosomucoid oligosaccharides on high pH anion exchange chromatography. This application of tyrosinamide derivatization has allowed for the first time the complete resolution of the complex oligosaccharide mixture from orosomucoid on a semipreparative scale in a single chromatogram and provide the first NMR characterization of polylactosamine tetraantennary oligosaccharide from this substrate. This study demonstrates the broad utility of the tyrosinamide derivatization to develop oligosaccharide libraries useful for probing the biological functions of glycosylation.