Novel Approaches for the synthesis of glycoprotein standards

项目总结/摘要 几乎所有的细胞表面和分泌蛋白质都被共价连接的碳水化合物部分修饰， 这些所谓的糖蛋白上的聚糖结构被认为是 过程如蛋白质折叠、细胞信号传导、受精、胚胎发生、神经元发育， 激素活性和细胞增殖及其组织成特定组织。此外，本发明还提供了一种方法， 压倒性的数据支持糖基化在病原体识别、炎症、先天性免疫缺陷和免疫缺陷中的相关性。 免疫反应以及自身免疫性疾病和癌症的发展。 糖组学是一个新兴的综合性研究领域，它研究复杂的结构-功能关系， 碳水化合物然而，这一研究领域的进展受到缺乏明确定义的复合物的阻碍。 寡糖标准品。在这方面，低聚糖的收集是发展低聚糖的需要。 用于寡糖MS谱的分配、用于制造微阵列、用于阐明 糖缀合物生物合成途径，并作为免疫原产生MAB，用于糖蛋白分离 和可视化。 本申请提出开发一种新的合成方法， 在关键分支位置用正交保护基，Fmoc， 列夫，特洛克和纳普。选择性去除这些保护基团和平行组合糖基化 使用一组精心选择的糖基供体，将以成本有效的方式获得大量的 用于糖组学研究的多触角寡糖标准品。此外，化学方法将 被开发用于制备衍生自肌营养不良蛋白聚糖的寡糖。糖基化缺陷 与肌肉营养不良有关。肌营养不良聚糖被一种不寻常的 糖基化，即苏氨酸和丝氨酸的0-甘露糖基化，并且将开发用于糖基化的方法。 这类化合物的化学合成。该方法的一个主要特点是使用有限的 用于化学合成多种肌营养不良蛋白聚糖的单糖结构单元的数量- 衍生的寡糖。寡糖将用作串联质谱法的标准品， 作为糖基转移的底物，以阐明糖缀合物生物合成的途径， 制备免疫原，获得单克隆抗体。此外，合成聚糖将 用于开发基于蛋白质无标记检测的新型聚糖阵列技术 结合使用表面增强拉曼光谱。在这种方法中，新的无Cu点击反应将是 用于用寡糖配体修饰Ag纳米棒的固体表面。配体的重要性 密度，表面附着方法，和固体表面的性质将被详细研究。

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.