Novel chemoenzymatic approaches for the synthesis of complex glycans

 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，所述碳水化合物部分被认为是蛋白质折叠、细胞信号传导、受精、胚胎发生、神经元发育和细胞增殖及其组织形成特定组织等过程的必需介质。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。糖科学的进展受到缺乏定义明确的复合寡糖标准品的阻碍，这些标准品是制造下一代微阵列、开发分析方案以确定分离的聚糖的确切结构、阐明糖缀合物生物合成途径以及作为免疫原以产生用于糖蛋白分离和可视化的MAB所需的。本申请提出进一步开发一种新的化学酶方法，以通过利用核心寡糖来制备高度复杂的不对称取代的N-聚糖的文库，所述核心寡糖在关键分支位置处被正交保护基团修饰，以允许通过化学糖基化选择性连接独特的糖部分。将以这样的方式选择附加物，使得所得脱保护化合物的触角可以通过糖基转移酶独特地延伸，以产生大量的不对称多触角聚糖。该方法将用于合成在人上呼吸道上皮细胞上发现的一组代表性复合聚糖。这些化合物将用于测定来自不同流感病毒株的一系列血凝素的聚糖特异性。我们将确定分支、乳糖胺部分延伸和最小表位的多价呈递将影响HA结合的方式。此外，我们建议扩大我们的化学酶的方法来制备不对称的N-聚糖含有硫酸酯。这些化合物与病毒-宿主细胞相互作用、淋巴细胞归巢和炎症部位的白细胞-内皮细胞粘附有关。将制备在不同触角上携带一个或多个6-磺基SLex表位的双触角、三触角和四触角硫酸化寡糖的文库。将利用磺基转移酶的底物特异性以区域选择性方式安装硫酸酯。这些化合物将用于揭示最小表位的空间排列和L-选择素介导的免疫细胞结合和生物活性的多价性的重要性。最后，将开发用于制备人乳低聚糖的化学酶法。这些化合物通常在结构上高度复杂，涉及广泛的生物过程，例如有益细菌的代谢底物、病原体受体的诱饵和免疫调节剂。新的合成化合物将被检查用于抑制细菌和病毒细胞粘附和进入。

 DESCRIPTION (provided by applicant): Almost all cell surface and secreted proteins are modified by covalently-linked carbohydrate moieties which have been implicated as essential mediators of processes such as protein folding, cell signaling, fertilization, embryogenesis, neuronal development, and the proliferation of cells and their organization into specific tissues. Also, overwhelming data supports the relevance of glycosylation in pathogen recognition, inflammation, innate immune responses, and the development of autoimmune diseases and cancer. Progress in glycoscience is hampered by a lack of well-defined complex oligosaccharide standards which are needed for the fabrication of the next generation of microarrays, for the development of analytical protocols to determine exact structures of isolated glycans, for the elucidation of pathways of glycoconjugate biosynthesis, and as immunogens to produce MABs for glycoprotein isolation and visualization. This application proposes to further develop a novel chemoenzymatic methodology to prepare libraries of highly complex asymmetrically substituted N-glycans by exploiting a core oligosaccharide that at key branching positions is modified by orthogonal protecting groups to allow selective attachment of unique saccharide moieties by chemical glycosylation. The appendages will be selected in such a way that the antenna of the resulting deprotected compounds can be uniquely extended by glycosyltransferases to give large numbers of asymmetrical multi-antennary glycans. The methodology will be employed to synthesize a representative set of complex glycans found on human upper airway epithelial cells. The compounds will be used to determine glycan specificities for a range of hemagglutinins from different strains of influenza virus. We will establish in which way branching, extension by lactosamine moieties, and multivalent presentation of the minimal epitope, will affect HA binding. In addition, we propose to expand our chemoenzymatic approach to the preparation of asymmetrical N-glycans containing sulfate esters. Such compounds have been implicated in viral-host cell interactions, lymphocyte homing, and leukocyte-endothelium adhesion at sites of inflammation. A library of bi-, tri-, and tetra-antennary sulfated oligosaccharides will be prepared bearing one or more 6-sulfo SLex epitopes on different antennae. Substrate specificities of sulfotransferases will be exploited to install the sulfate esters in a regioselective manner. The compounds will be used to uncover the importance of spatial arrangement of the minimal epitope and multivalency for L-selectin mediated immune cell binding and biological activity. Finally, a chemoenzymatic approach will be developed for the preparation of human milk oligosaccharides. These compounds, which are often highly complex in architecture, have been implicated in a wide range of biological processes such as metabolic substrates for beneficial bacteria, decoys for receptors of pathogens, and immune modulators. The novel synthetic compounds will be examined for inhibition of bacterial and viral cell adhesion and entry.