Almost all cell surface and secreted proteins are modified by covalently-linked carbohydrate moieties and the glycan structures on these so-called glycoproteins have been implicated as essential mediators in processes such as protein folding, cell signaling, fertilization, embryogenesis, neuronal development, hormone activity, and the proliferation of cells and their organization into specific tissues. In addition, overwhelming data supports the relevance of glycosylation in pathogen recognition, inflammation, innate immune responses, and the development of autoimmune diseases and cancer. Glycomics is an emerging field of integrated research to study structure-function relationships of complex carbohydrates. Progress in this field of research is, however, hampered by a lack of well-defined complex oligosaccharide standards. In this respect, collections of oligosaccharides are need for the development of algorithms for the assignment of oligosaccharide MS spectra, for fabricating microarrays, for elucidating pathways of glycoconjugate biosynthesis, and as immunogens to produce MAB's for glycoprotein isolation and visualization. This application is proposing to develop a novel synthetic methodology that will employ a synthetic core pentasaccharide functionalized at key branching positions with the orthogonal protecting groups, Fmoc, Lev, Troc and Nap. Selective removal of these protecting groups and parallel combinatorial glycosylations with a set of carefully selected glycosyl donors will give, in a cost effective manner, access to large number of multi-antennary oligosaccharide standards for glycomics research. Furthermore, chemical methods will be developed for the preparation of oligosaccharides derived from dystroglycan. Defects in the glycosylation of dystroglycan have been implicated in muscle dystrophy. Dystroglycan is modified by an unusual type of glycosylation, namely O-mannosylation of threonine and serine and methods will be developed for the chemical synthesis of this class of compound. A key feature of the methodology will be the use of limited number of monosaccharide building blocks for the chemical synthesis of a wide range of dystroglycan- derived oligosaccharides. The oligosaccharides will be used as standards for tandem mass spectrometry, as substrates for glycosyl transferses to elucidate pathways of glycoconjugate biosynthesis, and for the preparation of immunogens to obtain monoclonal antibodies. In addition, the synthetic glycans will be employed for the development of a novel glycan array technology based on label-free detection of protein binding using surface enhanced Raman spectroscopy. In this approach, novel Cu-free click reactions will be employed to modify a solid surface of Ag-nanorods with oligosaccharide ligands. The importance of ligand density, surface attachment methodology, and nature of the solid surface will be investigated in detail.
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 biological and biomedical research.
