Novel Approaches for the synthesis of glycoprotein standards

描述（由申请人提供）：几乎所有的细胞表面和分泌蛋白都被共价连接的碳水化合物修饰，这些所谓的糖蛋白上的聚糖结构在蛋白质折叠、细胞信号传导、受精、胚胎发生、神经元发育、激素活性、细胞增殖及其组织成特定组织等过程中都是必不可少的介质。此外，大量数据支持糖基化在病原体识别、炎症、先天免疫反应、自身免疫性疾病和癌症发展中的相关性。糖组学是研究复杂碳水化合物结构与功能关系的一个新兴的综合研究领域。然而，由于缺乏明确定义的复杂寡糖标准，这一研究领域的进展受到阻碍。在这方面，低聚糖的收集需要用于开发低聚糖质谱分配算法，用于制造微阵列，用于阐明糖缀合物生物合成途径，以及作为免疫原生产用于糖蛋白分离和可视化的MAB。本申请提出了一种新的合成方法，该方法将使用在关键分支位置具有正交保护基团Fmoc， Lev， Troc和Nap的合成核心五糖。选择性地去除这些保护基团，并用一组精心挑选的糖基供体进行平行组合糖基化，将以经济有效的方式为糖组学研究提供大量的多天线寡糖标准。此外，化学方法将发展为制备低聚糖衍生的低聚糖。肌营养不良与糖基化缺陷有关。三聚糖聚糖是由一种不寻常的糖基化修饰，即苏氨酸和丝氨酸的o -甘露糖基化，并且将开发化学合成这类化合物的方法。该方法的一个关键特征将是使用有限数量的单糖构建块来化学合成范围广泛的三聚糖衍生的低聚糖。寡糖将用作串联质谱的标准，作为糖基转移的底物以阐明糖缀合物的生物合成途径，并用于制备免疫原以获得单克隆抗体。此外，合成的聚糖将用于开发一种基于表面增强拉曼光谱的无标记检测蛋白质结合的新型聚糖阵列技术。在这种方法中，新的无cu点击反应将被用于用低聚糖配体修饰银纳米棒的固体表面。配体密度、表面附着方法和固体表面性质的重要性将被详细研究。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Well over 50% of human proteins are modified by complex glycans (carbohydrates) giving rise to glycoproteins. These glycoproteins have been implicated as essential mediators of health and disease. The proposed program will develop new methods for the chemical synthesis of well-defined glycans for use biological and biomedical research.