Tamura T, Wadhwa M S, Rice K G
Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, Ohio State University, Columbus 43210.
Anal Biochem. 1994 Feb 1;216(2):335-44. doi: 10.1006/abio.1994.1050.
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.
利用新开发的制备纯化方法对牛胎球蛋白的N-连接寡糖进行了纯化。在5克规模上，利用N-糖苷酶F从胰蛋白酶糖肽中释放出N-连接寡糖。用产气荚膜梭菌神经氨酸酶进行选择性脱唾液酸处理，形成了单唾液酸寡糖和去唾液酸寡糖。将寡糖的还原端转化为糖基胺，并与对羟基苯丙氨酸的N-羟基琥珀酰亚胺酯反应。酪氨酸化寡糖在反相高效液相色谱上分离成单个峰，然后通过质子核磁共振和快原子轰击质谱进行表征。以良好的产率回收了一种单去唾液酸三触角、一种去唾液酸双触角和一种单唾液酸三触角寡糖。每个产物都含有一个通过β-糖基酰胺键连接到还原端N-乙酰葡糖胺残基上的单个Boc-酪氨酸残基。该方法用于从克量的糖蛋白中分离多微摩尔量的寡糖，从而提供了一条纯化大量含有末端酪氨酸残基的N-连接寡糖的新途径。酪氨酸化寡糖是有价值的糖缀合物配体，其含有在280nm处吸收的发色团，并且具有足够的疏水性以促进反相高效液相色谱分离。此外，该基团可以通过去除Boc进行脱保护，以暴露出适合进一步衍生化的伯胺，并且还可以进行放射性碘化以在生物学实验中进行追踪。
