Elucidation of oligosaccharide function by use of chemical synthesis of glycoprotein

我们发展了天冬酰胺连接唾液酸糖肽的简便合成方法。我们合成唾液酸糖肽的策略的典型特征是使用十一碳二唾液酸寡糖基Fmoc-天冬酰胺（Fmoc-Asn（CHO）-OH），除了NeuAc残基的苄基酯之外，其羟基上没有保护基团。我们的合成方法解决了活性氨基酸在肽链延伸过程中对糖羟基的酯化问题。当使用高浓度的Fmoc-氨基酸时，酯化反应明显发生，但当使用低浓度的反应物时，酯化反应几乎不发生。利用这些发现，我们研究了通过使用天然化学连接（NCL）合成具有两个复合型唾液酸寡糖的高分子唾液酸糖肽CTLA-4片段（113-150）。结果，我们成功地合成了在N-末端具有半胱氨酸残基的唾液酸糖肽（CTLA-4：129-150片段）和唾液酸糖肽硫酯（CTLA-4：113-128片段）。最后，将这些合成的唾液酸糖肽应用于NCL反应，并且该反应成功地提供了所需产物CTLA-4（113-150），其具有优异纯度的成熟和纯的复合型唾液酸寡糖。

We developed convenient synthetic method of asparagine-linked sialylglycopeptides. The typical feature of our strategy for the synthesis of a sialylglycopeptide is to employ undecadisialyloligosaccharyl Fmoc-asparagine (Fmoc-Asn(CHO)-OH) without protecting groups on its hydroxyl groups except for the benzyl ester of NeuAc residues. Our synthetic methodology solved the problem of esterification toward sugar hydroxyl groups by activated amino acid during the elongation of a peptide chain. When employing high concentrations of Fmoc-amino acid, esterification markedly occurred, but the esterification scarcely occurred when employing low concentrations of reactants. Taking advantage of these findings, we examined the synthesis of a high molecular sialylglycopeptide, CTLA-4 fragment (113-150) having two complex-type sialyloligosaccharides, by use of native chemical ligation (NCL). As a result, we succeeded in the synthesis of a sialylglycopeptide having a cysteine residue at the N-terminus (CTLA-4:129-150 fragment) and a sialylglycopeptide-thioester (CTLA-4:113-128 fragment). Finally, these synthesized sialylglycopeptides were applied to NCL reaction and the reaction successfully afforded a desired product, CTLA-4 (113-150), having mature and pure complex-type sialyloligosaccharides in excellent purity.