Measurement of Internal Motions in Complex Oligosaccharides

Our research aims to study the conformations of complex carbohydrates and understand the degree of motion taking place within a timeframe of few milliseconds or less. This dissertation describes development and application of Nuclear Magnetic Resonance based methods using Residual Dipolar Coupling (RDC) measurement of weakly aligned molecules in liquid crystal media. We present a procedure that uses multivariate statistical techniques applied to multiple sets of RDC data to accurately quantify conformational space occupied by the macromolecule. The hexasaccharide Lacto–N–difuco–hexose–I (LNDFH–I) comprising of a tetrasaccharide epitope region (α–L–Fuc(1→2)β–D–Gal(1→3)(α–L–Fuc(1→4))β–D–GlcNAc) and a lactose tail region (β–D–GlcNAc1→3β–D–Gal1→4–D–Glc) was used as a model for the study. A liquid crystal concentration of 5% n–octyl penta ethylene glycol was found to be optimal to weakly align the oligosaccharide and measure the RDCs. To estimate the alignment tensors, at least five RDC values were measured for each pyranoside ring of the hexasaccharide, using one bond carbon — proton (13C, 1H) and long range proton — proton (1H, 1H) coupling experiments. The candidate conformers for this calculation were generated using sophisticated molecular dynamics (MD) simulation. Alignment tensors were determined using three different methods from which the dipolar couplings were back calculated. Multiple linear regression was applied to the calculated RDCs in order to find the sub-set of conformers which best fit the experimental data. The dihedral angles of the selected conformers were analyzed to understand the conformation. Results suggest relatively rigid conformation of the tetrasaccharide epitope of LNDFH–I. The average degree of motion for each glycosidic linkage within the tetrasaccharide was around ±7°. Three distinct families of conformers were observed for the β–D–GlcNAc1→3β–D–Gal linkage indicating flexibility. Large excursion of the dihedral angle was also observed for the lactose linkage at the reducing terminal. These results were validated using conformers generated from Monte Carlo simulation. The methodology was applied to the synthesized disaccharide (α–L–Col(1→2)β–D–Gal) and tetrasaccharide (α–L–Col(1→2)β–D–Gal(PO4−)(1→3)(α–L–Col(1→4))β–D–GlcNAc) components of the Vibrio cholerae O139 capsular polysaccharide. The results aided in further refinement of the conformational features of the tetrasaccharide as compared to previous studies. The disaccharide showed considerable flexibility with conformers in three distinct regions