Novel Approaches for the synthesis of glycoprotein standards

项目摘要/摘要 几乎所有的细胞表面和分泌的蛋白质都被共价连接的碳水化合物部分和 这些所谓的糖蛋白上的糖链结构被认为是 蛋白质折叠、细胞信号传递、受精、胚胎发生、神经元发育、 荷尔蒙活性，以及细胞的增殖和它们组织成特定的组织。此外, 压倒性的数据支持糖基化与病原体识别、炎症、先天性 免疫反应、自身免疫性疾病和癌症的发展。 糖化组学是研究复合体结构与功能关系的一个新兴的综合性研究领域 碳水化合物。然而，由于缺乏明确定义的复合体，这一研究领域的进展受到了阻碍 低聚糖标准。在这方面，需要收集低聚糖来开发 用于指认低聚糖MS谱图的算法，用于制造微阵列，用于阐明 糖共轭生物合成途径，以及作为免疫原产生单抗以分离糖蛋白 和视觉化。 这项申请建议开发一种新的合成方法，它将使用合成核心 五糖在关键分支位置官能化，具有正交保护基，Fmoc， 列夫，特罗克和纳普。这些保护基团的选择性去除和平行的组合糖基化 通过一套精心挑选的糖基捐赠者，将以成本效益的方式提供大量 用于糖组学研究的多天线低聚糖标准。此外，化学方法将 用于制备由营养不良葡聚糖衍生的低聚糖。糖基化缺陷 与肌肉营养不良有关。营养不良多糖被一种不寻常的类型的 糖基化，即苏氨酸和丝氨酸的O-甘露糖基化和方法将被开发用于 化学合成法这类化合物的化学合成该方法的一个关键特点将是使用有限的 用于化学合成广泛的营养不良多糖的单糖构建块的数量- 衍生低聚糖。低聚糖将被用作串联质谱分析的标准， 作为糖基转移的底物，以阐明糖共轭生物合成的途径，并用于 制备免疫原，获得单抗。此外，合成的多糖将是 用于开发一种新的基于蛋白质无标记检测的糖链阵列技术 结合使用表面增强拉曼光谱。在这种方法中，新的无铜点击反应将是 用低聚糖配体修饰银纳米棒的固体表面。配基的重要性 将详细研究固体表面的密度、表面附着方法和性质。

Project Summary/Abstract Almost all cell surface and secreted proteins are modified by covalently-linked carbohydrate moieties and the glycan structures on these so-called glycoproteins have been implicated as essential mediators in processes such as protein folding, cell signaling, fertilization, embryogenesis, neuronal development, hormone activity, and the proliferation of cells and their organization into specific tissues. In addition, overwhelming data supports the relevance of glycosylation in pathogen recognition, inflammation, innate immune responses, and the development of autoimmune diseases and cancer. Glycomics is an emerging field of integrated research to study structure-function relationships of complex carbohydrates. Progress in this field of research is, however, hampered by a lack of well-defined complex oligosaccharide standards. In this respect, collections of oligosaccharides are need for the development of algorithms for the assignment of oligosaccharide MS spectra, for fabricating microarrays, for elucidating pathways of glycoconjugate biosynthesis, and as immunogens to produce MAB's for glycoprotein isolation and visualization. This application is proposing to develop a novel synthetic methodology that will employ a synthetic core pentasaccharide functionalized at key branching positions with the orthogonal protecting groups, Fmoc, Lev, Troc and Nap. Selective removal of these protecting groups and parallel combinatorial glycosylations with a set of carefully selected glycosyl donors will give, in a cost effective manner, access to large number of multi-antennary oligosaccharide standards for glycomics research. Furthermore, chemical methods will be developed for the preparation of oligosaccharides derived from dystroglycan. Defects in the glycosylation of dystroglycan have been implicated in muscle dystrophy. Dystroglycan is modified by an unusual type of glycosylation, namely O-mannosylation of threonine and serine and methods will be developed for the chemical synthesis of this class of compound. A key feature of the methodology will be the use of limited number of monosaccharide building blocks for the chemical synthesis of a wide range of dystroglycan- derived oligosaccharides. The oligosaccharides will be used as standards for tandem mass spectrometry, as substrates for glycosyl transferses to elucidate pathways of glycoconjugate biosynthesis, and for the preparation of immunogens to obtain monoclonal antibodies. In addition, the synthetic glycans will be employed for the development of a novel glycan array technology based on label-free detection of protein binding using surface enhanced Raman spectroscopy. In this approach, novel Cu-free click reactions will be employed to modify a solid surface of Ag-nanorods with oligosaccharide ligands. The importance of ligand density, surface attachment methodology, and nature of the solid surface will be investigated in detail.