Oligosaccharides are of central importance in the development and maintenance of biological systems. They are found free in solution, as constituents of glycoproteins and glycolipids on the cell surface, and have been implicated in the mechanisms of action of diverse cellular processes such as inflammation, cell-cell recognition and adhesion, protein turnover, and bacterial infection. Oligosaccharides, or molecular mimics thereof, are becoming more frequent targets in drug design for the treatment of human illness and disease. The cellular functions of oligosaccharides are intimately related to their three-dimensional shapes or conformations, and knowledge of these 3D structures, both free in solution and in the bound state, is essential to deciphering the underlying factors that control recognition between oligosaccharides and specific cellular receptors. This proposal aims to develop new probes of oligosaccharide conformation based on NMR parameters involving 13C and 2H. Major objectives are to fully establish the structural dependencies of JCH and JCC scalar couplings using experimental and computational methods, and to apply these couplings to investigate O-glycoside linkage conformation in the range of biologically important 13C-labeled di-, tri-, and tetrasaccharides, either free in solution or tethered to oligopeptides. Scalar couplings involving 15N will also be studied. Residual 13C-1H and 13C-13C dipolar couplings and residual 2H quadrupolar couplings in oriented media, and 13C chemical shift anisotropy, will be investigated as additional conformational constraints in solution. Motional properties will be investigated using molecular dynamics methods and these data compared to experimental data in order to assist in the interpretation of the latter. Investigations will then be extended to larger, biologically relevant systems including a complex high-mannose N-glycan found on CD2, engineered N-glycans on IgG, and DNA aptamer-oligosaccharide complexes.
