Structure determination of complex Oligoglycoconjugates by Nuclear Magnetic Resonance Spectroscopy

We will develope general and robust strategies and methodologies for structure characterization of the complex oligosaccharides found mostly in bacterial cell walls. This will be (to our knowledge) the first systematic approach using cryo-detected NMR experiments. Use of this recently deployed technology enables application of experiments which for sensitivity reasons had to be excluded in previous approaches. Novel routes will also be attempted by using non-aqueous solvents like the polar aprotic solvents DMSO, DMF and HMPT and possibly super-critical CO2 or N2O to increase solubility of slow down the fast chemical exchange of hydroxyl protons observed in water. The samples on which we will develop the strategy and methodology will be obtained from prokaryotic organisms by a cooperating research group. In prokaryotic organisms cell walls protect the fragile lipid plasma membrane against mechanical, chemical and osmotic damage. In the cell wall of the Gram-positive bacteria, the major glycosylated compounds comprise of the surface layer (S-layer) glycoproteins, peptidoglycan, and the secondary cell wall polymers (SCWP). Since many of these organisms are patheogenic there is a great interest in factors affecting the stability of the cell wall as potential drug targets. In the practical work we focus our attention on the elucidation of the primary structure of an SCWP and the structure of the linker connecting the SCWP and peptidoglycan matrix that have been isolated from newly isolated thermophilic organism GS-5. In addition to liquid state NMR with cryo-detection we will use solid state NMR spectroscopy (in collaborative effort) and mass spectrometry.