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

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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