Novel Approaches for the synthesis of glycoprotein standards

描述(申请人提供)：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修饰，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、受精、胚胎发生、神经元发育、激素活性以及细胞增殖和组织成特定组织等过程中的重要中介。此外，压倒性的数据支持糖基化在病原体识别、炎症、先天性免疫反应以及自身免疫性疾病和癌症的发展中的相关性。糖化组学是研究复杂碳水化合物结构-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低聚糖标准，这一研究领域的进展受到阻碍。在这方面，需要收集低聚糖来开发算法，用于指认低聚糖的MS光谱，用于制造微阵列，用于阐明糖共轭生物合成的途径，以及作为免疫原来产生用于糖蛋白分离和可视化的单抗。本申请建议开发一种新的合成方法，该方法将使用在关键分支位置官能化的合成核心五糖，其保护基团为Fmoc、Lev、Troc和Nap。这些保护基团的选择性去除和与一组精心选择的糖基供体的平行组合糖基化将以经济有效的方式获得大量用于糖组学研究的多天线低聚糖标准。此外，还将开发化学方法来制备由营养不良多糖衍生的低聚糖。营养不良糖链糖基化缺陷与肌肉营养不良有关。营养不良葡聚糖被一种不寻常的糖基化修饰，即苏氨酸和丝氨酸的O-甘露糖化，将开发出化学合成这类化合物的方法。该方法的一个关键特点是使用有限数量的单糖构建块来化学合成广泛的营养不良葡聚糖衍生的寡糖。这些低聚糖将被用作串联质谱仪的标准，作为糖基转移的底物以阐明糖共轭生物合成的途径，并用于制备免疫原以获得单抗。此外，合成的多糖将被用于开发一种新的基于表面增强拉曼光谱无标记检测蛋白质结合的多糖阵列技术。在这种方法中，我们将利用新的无铜点击反应来修饰带有低聚糖配体的银纳米棒的固体表面。将详细研究配体密度、表面附着方法和固体表面性质的重要性。 与公共卫生相关：超过50%的人类蛋白质被复杂的多糖(碳水化合物)修饰，从而产生糖蛋白。这些糖蛋白被认为是健康和疾病的重要介质。拟议的计划将开发用于生物和生物医学研究的明确定义的多糖的化学合成的新方法。

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.