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.
Well over 50% of human proteins are modified by complex glycans (carbohydrates) giving rise to glycoproteins. These glycoproteins have been implicated as essential mediators of health and disease. The proposed program will develop new methods for the chemical synthesis of well-defined glycans for use in biological and biomedical research.
|Chinoy, Zoeisha S; Friscourt, Frédéric; Capicciotti, Chantelle J et al. (2018) Chemoenzymatic Synthesis of Asymmetrical Multi-Antennary N-Glycans to Dissect Glycan-Mediated Interactions between Human Sperm and Oocytes. Chemistry 24:7970-7975|
|Liu, Lin; Prudden, Anthony R; Bosman, Gerlof P et al. (2017) Improved isolation and characterization procedure of sialylglycopeptide from egg yolk powder. Carbohydr Res 452:122-128|
|Prudden, Anthony R; Liu, Lin; Capicciotti, Chantelle J et al. (2017) Synthesis of asymmetrical multiantennary human milk oligosaccharides. Proc Natl Acad Sci U S A 114:6954-6959|
|Li, Xiuru; Martin, Sharon J H; Chinoy, Zoeisha S et al. (2016) Label-Free Detection of Glycan-Protein Interactions for Array Development by Surface-Enhanced Raman Spectroscopy (SERS). Chemistry 22:11180-11185|
|Li, Tiehai; Huang, Min; Liu, Lin et al. (2016) Divergent Chemoenzymatic Synthesis of Asymmetrical-Core-Fucosylated and Core-Unmodified N-Glycans. Chemistry 22:18742-18746|
|Prudden, Anthony R; Chinoy, Zoeisha S; Wolfert, Margreet A et al. (2014) A multifunctional anomeric linker for the chemoenzymatic synthesis of complex oligosaccharides. Chem Commun (Camb) 50:7132-5|
|Live, David; Wells, Lance; Boons, Geert-Jan (2013) Dissecting the molecular basis of the role of the O-mannosylation pathway in disease: ?-dystroglycan and forms of muscular dystrophy. Chembiochem 14:2392-402|
|Wang, Zhen; Chinoy, Zoeisha S; Ambre, Shailesh G et al. (2013) A general strategy for the chemoenzymatic synthesis of asymmetrically branched N-glycans. Science 341:379-83|
|Wolfert, Margreet A; Boons, Geert-Jan (2013) Adaptive immune activation: glycosylation does matter. Nat Chem Biol 9:776-84|
|Mo, Kai-For; Fang, Tao; Stalnaker, Stephanie H et al. (2011) Synthetic, structural, and biosynthetic studies of an unusual phospho-glycopeptide derived from ?-dystroglycan. J Am Chem Soc 133:14418-30|