Automated chemo-enzymatic synthesis of N-glycans for host-pathogen interactions

摘要 许多病毒通过与宿主细胞表面的糖链结合来启动感染。病毒的选择性 特定糖链的受体结合蛋白关键决定宿主的范围、组织和细胞的嗜性 和发病机制。对呼吸道病毒受体使用的详细了解对于 制定监测、预防和干预战略，以减轻未来大流行的风险 疫情爆发。由于缺乏多糖受体，呼吸道病毒对多糖受体的利用一直难以探测 用于结构-活性研究的适当的葡聚糖板。后者是由于在合成方面的限制 不允许制备大量生物相关多糖的方法。 在这个项目中，将开发化学酶方法，使其能够制备 广泛存在于人类及相关动物呼吸道中的N-葡聚糖。它基于一个 新的合成范例，我们创造了“走走停停的化学酶糖基化”。它使用 化学修饰的糖核苷酸供体，可被相关糖基转移酶利用来 给出特定残基暂时被阻止进一步酶修饰的产品。 在适当的合成阶段，可以去除封闭基以得到天然的葡聚糖。这个 通过开发一个可以执行的自动化平台，将提高多糖合成的速度 酶和化学操作。新的方法将被用来准备一系列 复杂的N-糖链，类似于呼吸道组织表达的结构，用于宿主-病原体研究。 在本申请中，收集的多糖将用于详细检查甲型流感受体的使用情况 病毒。这些多糖将被打印成微阵列，以探测人和动物的结合特异性 甲型流感病毒。选定的化合物将在动态结合分析中进行检查，以确定 血凝素(HA)和神经氨酸酶(NA)活性之间的相互作用。拟议的研究将 发现人类和动物IAV的独特特征，这将有助于实施监测， 预防和干预战略，以减轻未来大流行的风险。研究的结果将会 被利用来开发一种基于阵列的系统来抗原性表征IAV，这将大大 促进季节性流感疫苗接种的菌株选择。 1

SUMMARY Numerous viruses initiate infection by binding to cell surface glycans of the host. The selectivities of viral receptor binding proteins for specific glycans critically determine host range, tissue- and cell tropism and pathogenesis. A detailed understanding of receptor usage by respiratory viruses is critical for the development of surveillance, prevention and intervention strategies to mitigate risks of future pandemic outbreaks. Glycan receptor usage by respiratory viruses have been difficult to probe because of a lack of appropriate panels of glycans for structure-activity studies. The latter is due to limitations in synthetic methodologies that do not permit the preparation of large panels of biological relevant glycans. In this program, chemoenzymatic methodologies will be developed that make it possible to prepare a wide range of N-glycan found in the respiratory tract of human and relevant animals. It is based on a new synthetic paradigm, which we coined “Stop-and-Go-Chemoenzymatic Glycosylation”. It uses chemically modified sugar nucleotide donors that can be employed by relevant glycosyl transferases to give products in which particular residues are temporarily blocked from further enzymatic modification. At an appropriate stage of synthesis, the blocking group can be removed to give a natural glycan. The speed of glycan synthesis will be increased by developing an automation platform that can perform enzymatic and chemical manipulations. The new methodologies will be used to prepare a collection of complex N-glycans that resemble structures expressed by respiratory tissue for host-pathogen studies. In this application, the collection of glycans will used to examine in detail receptor usage of influenza A viruses. The glycans will be printed as a microarray to probe binding specificities of human and animal influenza A viruses. Selected compounds will be examined in dynamic binding assays to establish the interplay between hemagglutinin (HA) and neuraminidase (NA) activity. The proposed studies will uncover unique traits of human and animal IAVs, which will facilitate the implementation of surveillance, prevention and intervention strategies to mitigate risks of future pandemics. The result of the studies will be exploited to develop an array-based system to antigenically characterize IAVs, which will greatly facilitate strain selection for seasonal flu vaccination. 1