This thesis describes the use of Nuclear Magnetic Resonance (NMR) for characterizing two host-pathogen interactions: The behavior of a regulatory RNA of the Hepatitis B virus (HBV) and the attachment of Helicobacter pylori (H. pylori) to the gastric mucosa. NMR is a powerful tool in biomedicine, because molecules ranging from small ligands to biomacromolecules can be studied with atomic resolution. Different NMR experiments are designed to determine structures, or to monitor interactions, folding, stability or motion. Paper I describes the analysis of the motions of a regulatory RNA of HBV. The NMR structure of the RNA had revealed before that several well-conserved nucleotides adopt multiple conformations. Therefore an analysis of possible underlying motions was undertaken using two different NMR techniques, one of which (off-resonance ROESY) was applied to nucleic acids for the first time. The observed motions suggest an explanation why the structurally poorly defined nucleotides are highly conserved. In paper II we improved the ROESY NMR experiment, which is used to measure internuclear distances for structure determination of medium-sized molecules. Using a small protein and an organometallic complex as examples, we demonstrated that the new EASY ROESY experiment yields clean spectra that can directly be integrated to derive interatomic distances. H. pylori, the bacterium involved in peptic ulcer disease and gastric cancer, survives in the harsh acidic environment of the stomach. It possesses many membrane proteins which mediate adherence, raising the question, if their activity is related to membrane composition. In paper III & IV we analyzed therefore the phospholipid composition of H. pylori membranes. In paper III, an advanced method for the analysis of the phospholipid composition of biological membranes was developed. The two-dimensional semi-constant-time 31P,1H-COSY experiment combines information from phosphorus and hydrogen atoms of phospholipids for their unambiguous identification. Furthermore, the high resolution of the two-dimensional experiment allows the quantification of phospholipids where conventional methods fail. In paper IV we applied the new experiment to analyze the lipid composition of whole H. pylori cells, their inner and outer membranes, and of vesicles shed by the bacterium. The goal of this study was to characterize the vesicles which are suggested to play a role in the inflammation process. We established that the outer membrane and the vesicles have similar phospholipid compositions, suggesting that the vesicles are largely derived from the outer membrane. The NMR results presented here elucidate details of molecular systems engaged in pathogenicity, as basis for therapeutic strategies against these pathogens.

Les liquides ioniques, associations de cations organiques et d'anions, sont des milieux structurés sur plusieurs nanomètres et présentent une ségrégation en domaines polaires et apolaires. Utilisés comme solvants de réactions catalytiques, ils peuvent de ce fait engendrer des phénomènes de solvatation spécifique. Ainsi des réactions d'échange d'ions dans certains systèmes catalytiques et des interactions de type p-cation avec les hydrocarbures insaturés ont été mises en évidence par RMN. Les conséquences de ces solvatations spécifiques sur des réactions d'hydrogénation ont été étudiées. Il ressort que plus l'interaction entre les liquides ioniques et les réactifs est grande, plus les réactions sont lentes. <br />Enfin la présence de microdomaines polaires et apolaires conduit à une solubilisation préférentielle des complexes organométalliques dans les poches apolaires ce qui permet d'utiliser ces milieux comme moules supramoléculaires et de contrôler la croissance cristalline des nanoparticules de ruthénium générées in situ en fonction de la longueur de la chaîne alkyl et de la température.

Taxol™, isolated from the bark of Taxus brevifolia in the late 1960s, and the semisynthetic analog Taxotere™ have proven clinical importance for the treatment of ovarian and breast cancer. Taxol™ exerts its biological effect by binding to polymerized tubulin and stabilizing the resulting microtubules. Studies aimed at understanding the biologically active conformation of taxol and its binding environment on β-tubulin are described. This knowledge is important because it could lead to the design of structurally less complicated drugs with better efficacy and better bioavailability. Moreover, the information can be extended to other natural products that possess microtubule-stabilizing properties similar to Taxol™. In this work, the synthesis of a triply labeled taxol analog is described as well as REDOR studies of this compound complexed to tubulin are in progress. Macrocyclic analogs of taxol have been prepared and their biological activities were evaluated. Chemical modeling of these analogs and their activities agrees with the hypothesis that Taxol™ adopts T-shaped conformation. Difficulties were encountered with the key ring-closing metathesis strategy, suggesting that a more flexible and efficient macrocyclization method will be needed to synthesize additional macrocyclic analogs. / Ph. D.