Novel Approaches for the synthesis of glycoprotein standards

描述（由申请人提供）：几乎所有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聚糖结构被认为是蛋白质折叠、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等过程中的必需介质。此外，大量数据支持糖基化在病原体识别、炎症、先天免疫应答以及自身免疫性疾病和癌症的发展中的相关性。糖组学是研究复杂碳水化合物结构与功能关系的新兴综合研究领域。然而，由于缺乏明确的复合寡糖标准，这一研究领域的进展受到阻碍。在这方面，需要寡糖的集合来开发用于寡糖MS谱的分配的算法，用于制造微阵列，用于阐明糖缀合物生物合成的途径，以及作为免疫原来产生用于糖蛋白分离和可视化的MAB。本申请提出开发一种新的合成方法，其将采用在关键分支位置用正交保护基团Fmoc、Lev、Troc和Nap官能化的合成核心五糖。这些保护基团的选择性去除和与一组仔细选择的糖基供体的平行组合糖基化将以具有成本效益的方式获得用于糖组学研究的大量多触角寡糖标准品。此外，将开发用于制备来自肌营养不良蛋白聚糖的寡糖的化学方法。肌营养不良蛋白聚糖的糖基化缺陷与肌营养不良有关。肌营养不良聚糖被一种不寻常的糖基化类型修饰，即苏氨酸和丝氨酸的O-甘露糖基化，并且将开发用于这类化合物的化学合成的方法。该方法的一个关键特征将是使用有限数量的单糖结构单元来化学合成广泛的肌营养不良聚糖衍生的寡糖。寡糖将用作串联质谱法的标准品，用作糖基转移的底物以阐明糖缀合物生物合成的途径，以及用于制备免疫原以获得单克隆抗体。此外，合成聚糖将用于开发一种新的聚糖阵列技术，该技术基于使用表面增强拉曼光谱法对蛋白质结合进行无标记检测。在这种方法中，将采用新的无Cu点击反应来用寡糖配体修饰Ag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。 公共卫生相关性：超过50%的人类蛋白质被复合聚糖（碳水化合物）修饰，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聚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.