Streamlining the chemoenzymatic synthesis of asymmetrical glycans of biological importance

项目摘要 几乎所有的细胞表面和分泌蛋白质都被共价连接的碳水化合物部分修饰， 这些所谓的聚糖被认为是诸如蛋白质折叠、细胞分裂、细胞分裂和细胞凋亡等过程的必需介质。 信号传导、受精、胚胎发生、神经元发育和细胞增殖及其 组织成特定的组织。此外，压倒性的数据支持病原体中糖基化的相关性， 识别，炎症，先天免疫反应和自身免疫性疾病的发展， 癌糖科学的进展受到缺乏明确定义的复合寡糖标准的阻碍 这是需要制造下一代的微阵列，为发展分析 确定分离聚糖的确切结构的方案，用于阐明糖缀合物的途径 生物合成，并作为免疫原以产生用于糖蛋白分离和可视化的MAB。 在本申请中，我们提出开发新的合成策略，可以容易地提供大的文库， 对称和不对称N-聚糖。新的方法将利用现有的 材料，并将充分标准化，许多实验室，包括合成服务单位，可以 采用这些方法。此外，新方法的合成原理可以很容易地应用于 制备其他类别的聚糖，如O-连接聚糖和人乳低聚糖（HMO）。 新方法将采用对称的双触角聚糖，可以很容易地从蛋黄中分离出来。 将开发创新的酶促转化以使该聚糖去对称化。此外，委员会认为， 重组N-乙酰葡糖胺转移酶（MGAT）将用于将双触角聚糖转化为三触角聚糖。 和四触角结构。在后一种转化中，化学修饰的UDP-GlcNAc供体将是 用于暂时防止臂被酶修饰。使用最新开发的技术， 表达重组哺乳动物糖基转移酶将是新方法的关键特征。到 为了验证方法的耐用性，将其应用于制备聚糖衍生物库， 来自人类上呼吸道上皮细胞。由此产生的聚糖将是有价值的下一个发展 生成聚糖微阵列，以探测这种细胞类型背景下的碳水化合物-蛋白质相互作用。的 通过开发能够 容易地提供被硫酸酯修饰的高度复杂的不对称聚糖。自动化平台 将进一步提高速度的化学酶合成使用新的捕获和释放 战略布局注意力将集中在离子交换和镍介导的组氨酸结合事件，用于捕获 标记的低聚糖。Chemspeed的多通道液体处理机器人配备了容积式 分配系统和冻干机将被用作自动化工具。后一种方法首先将 用于制备O-连接聚糖和人乳低聚糖。

Project Summary Almost all cell surface and secreted proteins are modified by covalently-linked carbohydrate moieties, and these so called glycans 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. In this application, we propose to develop novel synthetic strategies that can readily provide large libraries of symmetrical and asymmetrical N-glycans. The new methodologies will make use of readily available starting materials and will be sufficiently standardized that many laboratories, including synthesis service units, can adopt these methods. Furthermore, the synthetic principles of the new approaches can easily be applied to the preparation of other classes of glycans such as O-linked glycans and human milk oligosaccharides (HMOs). The new method will employ a symmetrical biantennary glycan that can easily be isolated from egg yolk. Innovative enzymatic transformations will be developed to desymmetrize this glycan. Furthermore, recombinant N-acetylglucosaminyltransferases (MGAT's) will be used to convert a bi-antennary glycan into tri- and tetra-antennary structures. In the latter transformations, chemically modified UDP-GlcNAc donors will be used to temporarily prevent an arm from enzymatic modification. The use of recently developed technology to express recombinant mammalian glycosyltransferases will be a key feature of the new methodology. To validate the robustness of the methodology, it will be applied to the preparation of a library of glycans derived from human upper airway epithelial cells. The resulting glycans will be valuable for the development of the next generation of glycan microarray to probe carbohydrate–protein interactions in the context of this cell type. The scope of the chemoenzymatic methodology will be further extended by the development of methods that can easily provide highly complex asymmetrical glycans that are modified by sulfate esters. An automation platform will be developed to further increase the speed of chemoenzymatic synthesis using novel capture and release strategies. Attention will focus on ion exchange and nickel-mediated histidine binding events for capture of tagged oligosaccharides. A multi-channel liquid handling robot from Chemspeed equipped with a volumetric dispensing system and lyophilizer will be employed as an automation tool. The latter methodology will, at first, be employed for the preparation of O-linked glycans and human milk oligosaccharides.