A breakthrough to the microheterogeneity of the carbohydrate structure in glycoprotein by synthetic approach

碳水化合物对蛋白质起着多种重要作用，如细胞生长、分化和肿瘤转移。然而，这些功能的细节尚未在分子水平上被理解，这主要是由于碳水化合物部分的微观异质性。特别是，粘蛋白保留了密集的高异质性碳水化合物簇，这使得其功能分析变得困难。在本研究中，我们开发了一种方法来制备在不同氨基酸残基上携带不同碳水化合物的糖肽，以模拟天然糖蛋白的微异质性，这将为解决微异质性所带来的问题提供基础。首先，我们从MUC5AC的一致序列中合成了一个糖肽。固相合成过程中，在特定位置引入携带核心8碳水化合物的Fmoc-Ser和Fmoc-Thr，得到携带两种不同氨基酸残基碳水化合物的MUC5AC糖肽。在第二个例子中，研究人员开发了一种新的方法来研究携带一对独特唾液化的核心2 - o聚糖的糖肽。从白蛋白中提取的序列被分成两个较短的肽段。采用改进的Fmoc肽硫酯制备工艺，合成了携带一个核心2 - o聚糖的n端肽硫酯。采用常规的Fmoc法制备了携带核心2 - o聚糖的c端肽酰胺。N端和c端肽用不同的唾液酸转移酶处理，得到两种不同的唾液酸糖肽。然后用硫酯法将这两个片段浓缩成一个糖肽，该糖肽携带一对明显唾液化的核心2聚糖。在第三个例子中，开发了一种合成更大的粘蛋白糖肽模型的方法。MUC2重复单元的序列，在特定位置携带7个O-GalNAc片段，通过Fmoc肽硫酯制备的改进程序制备。然后用硫酯法重复凝聚6次，得到由141个氨基酸残基组成的糖蛋白，携带42个GalNAc基团。目前，这是用完全化学方法制备的最大的糖蛋白。该方法可用于合成在不同位置携带不同碳水化合物的粘蛋白模型。通过上述方法制备的合成的异质糖基化肽将有助于了解糖蛋白，特别是粘蛋白的微异质性。少

Carbohydrates on proteins play various essential roles, such as cell-growth, differentiation, and tumor metastasis. However, the details of these functions are not understood at a molecular level, which mainly arises from the microheterogeneity at the carbohydrate portions. Especially, mucins retain a dense cluster of highly heterogeneous carbohydrates, which makes its functional analysis difficult.In this research, we developed a method to prepare glycopeptides carrying different carbohydrates at distinct amino acid residues to mimic the microheterogeneity of the natural glycoproteins, which will provide a basis to solve the problem arising from the microheterogeniety. First, we synthesized a glycopeptide derived from the consensus sequence of MUC5AC. During the solid-phase synthesis, Fmoc-Ser and Fmoc-Thr carrying core 8 carbohydrate were introduced at specific positions, giving the MUC5AC glycopeptide carrying two carbohydrates at different amino acid residues. In the second example … More , a novel approach to the glycopeptide carrying a couple of distinctively sialylated core 2 O-glycan was developed. The sequence derived from the leukosialin was divided into two shorter peptide segments. The N-terminal peptide thioester carrying one core 2 O-glycan was synthesized using the modified procedure for the Fmoc peptide thioester preparation. C-terminal peptide amide carrying a core 2 O-glycan was prepared by the conventional Fmoc method. The N- and C-terminal peptide was treated with different sialyl transferases giving two differently sialylated glycopeptides. These two segments were then condensed by the thioester method to give a glycopeptide carrying a couple of distinctively sialylated core 2 glycan. In the third example, a method to synthesize larger glycopeptide models of mucins was developed. The sequence derived from the repeating unit of MUC2, carrying 7 O-GalNAc moieties at specific positions, was prepared by the modified procedure for the Fmoc peptide thioester preparation. This peptide was then repeatedly condensed 6 times by the thioester method to obtain glycoprotein composed of 141 amino acid residues carrying 42 GalNAc moieties. At present, this is the largest glycoprotein prepared by the fully chemical procedure. The method can be used to synthesize mucin models carrying different carbohydrates at distinct positions.The synthetic heterogeneously glycosylated peptides prepared by the above procedures will be useful to understand the microheterogeniety of glycoproteins, especially of mucins. Less