NMR studies of Carbohydrate Structure and Dynamics in Solution

Sammanfattning: Methods for studies of the structures of carbohydrates in solution and their dynamics are presented. Approaches employing nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), force-field computer simulations and chemical methods are described.First, methods for determinations of the structures of natural carbohydrates are described, exemplified by the galactofuranoside and glycerol residues and the phosphodiester moiety found in the O-antigenic polysaccharide from Escherichia coli O28. These structures are determined by the use of NMR spectroscopy, MS and chemical degradations/derivatizations.Subsequently, approaches for determination of the three-dimensional structures and dynamics of oligosaccharides in solution are presented. Carbon-13 NMR spin relaxation times and nuclear Overhauser effects (NOEs) were measured for the pentasaccharide lacto-N-fucopentaose-1 and for a vicinally disubstituted trisaccharide. Carbon relaxation data were used to describe the dynamics in terms of overall and internal correlation times and generalized order parameters according to the model-free approach. In addition, three-bond proton-carbon coupling constants, proton NOEs and transverse rotating-frame Overhauser effects (TROEs) across the glycosidic linkages were measured for the trisaccharide. The proton NOEs and TROEs were used to calculate proton-proton distances within and between the different sugar residues.Langevin dynamics and Metropolis Monte Carlo simulations were performed for the trisaccharide. Averaged proton-proton distances obtained from the simulations were compared to distances obtained from the NOE and TROE measurements. Three-bond proton-carbon coupling constants were calculated from the simulations and compared to the couplings determined by NMR. The comparisons of interglycosidic distances and coupling constants in many cases reveal poor agreement.A recent method for studies of biomolecular three-dimensional structure in solution was employed to the tetrasaccharide lacto-N-neotetraose (LNnT). It was dissolved in an aqueous dilute liquid crystalline medium and residual dipolar couplings were measured. The orientations of the interatomic vectors in the molecule were obtained from a structure generated by molecular modeling. The measured couplings showed good agreement with couplings calculated from the orientations of the vectors in the three-dimensional structure of LNnT.