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NMR studies of glycoprotein ologosaccharide structures

Objective


New nuclear magnetic resonance (NMR) methods have been developed for the investigation of the solution conformation of glycoprotein oligosaccharide and the techniques have been applied to the most important N-linked oligosaccharides belonging to the family of oligomannoses.

The use of carbon-13 NMR spectroscopy has been explored on substrate at isotopic natural abundance in order to obtain supplementary experimental parameters directly correlated to the torsion angles Phi and Omega about the glycosidic linkage, namely 1-bond carbon hydrogen coupling constants and multiple bond carbon hydrogen coupling constants. Some new 2-dimensional and 1-dimensional proton detected NMR experiments were carried out which were specifically designed for carbohydrates. All the pulse sequences were successfully tested on model compounds and allowed the quantitative extraction of the interglycosidic heteronuclear long range coupling constants. The exploitation of selective NMR techniques such as soft shaped pulses or chemical shift selective filtration (CSSF) turned out to be a powerful tool to improve the sensitivity of the outlined methods. Advantages and drawbacks of 2-dimensional and 1-dimensional methods, with and without implementation of soft pulses were discussed. The validity of the proposed approach based on new proton detected 2-dimensional and 1-dimensional heteronuclear NMR experiments for conformational studies on oligosaccharides was confirmed by the measurements of 1-bond carbon hydrogen coupling constants and multiple bond hydrogen coupling constants on some members of the family of oligomannoses extracted from ribonuclease B, bovine tyroglobuline and soybean agglutinine. It was therefore demonstrated that modern heteronuclear techniques for the study of the conformational behaviour of complex oligosaccharides are successfully applicable to biologically relevant substrates. This opens new perspectives for the comprehension of the 3-dimensional structure of oligosaccharides and glycoproteins in solution.

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