Novel chemoenzymatic approaches for the synthesis of complex glycans

 描述(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修饰，这些碳水化合物被认为是蛋白质折叠、细胞信号、受精、胚胎发生、神经元发育、细胞增殖及其组织成特定组织等过程的基本中介。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。糖科学的进展受阻于缺乏明确定义的复杂寡糖标准，这些标准是制造下一代微阵列所需的，开发分析方案以确定分离的多糖的确切结构，阐明糖共轭生物合成的途径，以及作为免疫原产生用于糖蛋白分离和可视化的单抗。本申请建议进一步开发一种新的化学酶方法，通过利用核心低聚糖来制备高度复杂的不对称取代N-糖链的文库，该核心低聚糖在关键分支位置被正交保护基团修饰，以允许通过化学糖基化选择性地连接独特的糖部分。选择附件的方式是，所得到的去保护化合物的天线可以被糖基转移酶唯一地延伸，以得到大量不对称的多天线多糖。该方法将被用于合成一组在人类上呼吸道上皮细胞上发现的具有代表性的复杂多糖。这些化合物将用于确定来自不同流感病毒株的一系列血凝素的葡聚糖特异性。我们将确定支化、乳糖胺部分的延伸和最小表位的多价呈现将以何种方式影响HA结合。此外，我们建议将我们的化学酶法扩展到制备含硫酸酯的不对称N-葡聚糖。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-内皮细胞黏附有关。将在不同的天线上制备一个带有一个或多个6-磺酸SLeX表位的双、三和四天线硫化低聚糖的文库。磺基转移酶的底物特性将被用来以区域选择性的方式安装硫酸酯。这些化合物将被用来揭示最小表位的空间排列和多价性对L-选择素介导的免疫细胞结合和生物活性的重要性。最后，将开发一种化学酶法制备人乳寡糖的方法。这些化合物在结构上往往非常复杂，涉及广泛的生物过程，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测以抑制细菌和病毒细胞的黏附和进入。

 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.