Corradi Da Silva M L, Stubbs H J, Tamura T, Rice K G
Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, Ohio State University, Columbus 43210, USA.
Arch Biochem Biophys. 1995 Apr 20;318(2):465-75. doi: 10.1006/abbi.1995.1255.
A library of 15 N-linked oligosaccharide structures was prepared from ovalbumin and characterized using high-field NMR and mass spectrometry. The oligosaccharides were enzymatically released from ovalbumin glycopeptides, and the reducing ends were reacted with ammonium bicarbonate to form oligosaccharide-glycosylamines. These reacted with Boc-tyrosine-N-hydroxysuccinimide ester, resulting in a mixture of tyrosinamide-oligosaccharides. The Boc group was removed to expose an amine terminus which enhanced the resolution of tyrosinamide-oligosaccharides when chromatographed on reverse-phase HPLC. Ten major and five minor oligosaccharides were purified on a micromole scale and characterized using 1H NMR and FAB-MS. The structures include high-mannose, hybrid, and complex oligosaccharides possessing from two to five antenna, providing the most complete definition of ovalbumin N-linked oligosaccharides to date. The resulting library is well suited to biological studies due to the presence of a single terminal tyrosine residue on each oligosaccharide that allows radioiodination or the attachment of additional probes to these glycoconjugates prior to biological studies.
从卵清蛋白制备了一个包含15种N-连接寡糖结构的文库，并使用高场核磁共振和质谱对其进行了表征。寡糖通过酶法从卵清蛋白糖肽中释放出来，还原端与碳酸氢铵反应形成寡糖-糖基胺。这些与Boc-酪氨酸-N-羟基琥珀酰亚胺酯反应，生成酪氨酰胺-寡糖混合物。去除Boc基团以暴露胺端，这在反相高效液相色谱上进行色谱分析时提高了酪氨酰胺-寡糖的分辨率。以微摩尔规模纯化了10种主要寡糖和5种次要寡糖，并使用1H核磁共振和快原子轰击质谱对其进行了表征。这些结构包括具有两到五个天线的高甘露糖型、杂合型和复合型寡糖，提供了迄今为止对卵清蛋白N-连接寡糖最完整的定义。由于每个寡糖上存在单个末端酪氨酸残基，使得在生物学研究之前可以对这些糖缀合物进行放射性碘化或连接其他探针，因此所得文库非常适合生物学研究。
