The research work reported in this thesis is focused on the chiral analysis, quantification of enantiomeric composition, assignment of absolute configuration of molecules with chosen functional groups. The weak intra-molecular hydrogen bonding interactions are detected by exploiting several multinuclear and multi-dimensional techniques. Pulse sequences have been designed to manipulate the spin dynamics to derive specific information from the complex NMR spectra encountered in diverse situations. Broadly, the thesis can be classified in to three sections. The section I containing two chapters reports the introduction of new chiral auxiliaries and protocols developed for enantiomeric discrimination, measurement of enantiomeric contents, assignment of absolute configuration for molecules possessing specific functional groups using chiral solvating and derivatizing agents. The section II, reports NMR experimental evidence for the observation of the rare type of intramolecular hydrogen bonds involving organic fluorine in biologically important organic molecules, that are corroborated by extensive DFT based theoretical calculations. The section II also discusses the H/D exchange mechanism as a tool for quantification of HB strengths in organic building blocks. The section III reports the two different novel NMR methodologies designed for deriving information on the scalar interaction strengths in an orchestrated manner. The designed sequences are able to completely eradicate the axial peaks, prevents the evolution of unwanted couplings and also yields ultrahigh resolution in the direct dimension, permitting the accurate measurement of scalar couplings for a particular spin. The brief summary about each chapter is given below. Chapter 1 provides a general introduction to one and two dimensional NMR spectroscopy. The pedagogical approach has been followed to discuss the conceptual understanding of spin physics and the NMR spectral parameters. The basic introduction to chirality, existing approaches in the literature for discrimination of enantiomers and the assignment of absolute configuration of molecules with chosen functional groups and their limitations are briefly discussed. The brief introduction to hydrogen bond, experimental methods to obtain the qualitative information about the strengths of hydrogen bonds, and the theoretical approaches employed in the thesis to corroborate the NMR experimental findings have been provided. The mechanism of H/D exchange, the utilization of exchange rates to derive strengths of intra-molecular hydrogen bond in small molecules have also been discussed. This chapter builds the bridge for the rest of the chapters. Each of these topics are discussed at length in the corresponding chapters. Part I: NMR Chiral Analysis: Novel Protocols Chapter 2 discusses a simple mix and shake method for testing the enantiopurity of primary, secondary and tertiary chiral amines and their derivatives, amino alcohols. The protocol involves the in-situ formation of chiral ammonium borate salt from a mixture of C2 symmetric chiral BINOL, trialkoxyborane and chiral amines. The proposed concept has been convincingly demonstrated for the visualization of enantiomers of a large number of chiral and pro-chiral amines and amino alcohols. The protocol also permits the precise measurement of enantiomeric composition. The significant advantage of the protocol is that it can be performed directly in the NMR tube, without any physical purification. The structure of the borate complex responsible for the enantiodifferentiation of amines has also been established by employing multinuclear NMR techniques and DFT calculations. From DOSY and 11B NMR experiments it has been ascertained that there are only two possible complexes or entities which are responsible for differentiating enantiomers. From the combined utility of DFT calculations and the 11B NMR chemical shifts, the structure of the borate complex has been determined to be an amine-coordinated complex with the N atom of the amine. Chapter 3 discusses a simple chiral derivatizing protocol involving the coupling of 2-formylphenylboronic acid and an optically pure [1,1-binaphthalene]-2,2-diamine for the rapid and accurate determination of the enantiopurity of hydroxy acids and their derivatives, possessing one or two optically active centers. It is established that this protocol is not only rapid method for discrimination of enantiomers but also highly effective for assigning the absolute configuration of various chiral hydroxy acids and their derivatives. The developed protocol involves the coupling of 2-formylphenylboronic acid with (R)-[1,1-binaphthalene]-2,2-diamine, and 2-formylphenylboronic acid with (S)-[1,1-binaphthalene]-2,2-diamine as chiral derivatizing agents. The absence of aliphatic peaks from the derivatizing agent, large chemical shift separation between the discriminated peaks of diastereomers, and the systematic change in the direction of displacement of peaks for an enantiomer in a particular diastereomeric complex, permitted the unambiguous assignment of absolute configuration. Part II : Rare Type of Intramolecular Hydrogen Bonding In chapter 4 The rare occurrence of intramolecular hydrogen bonds of the type N–H˖˖˖F–C, in the derivatives of imides and hydrazides in a low polarity solvent, is convincingly established by employing multi-dimensional and multinuclear solution state NMR experiments. The observation of 1hJFH, 2hJFN, and 2hJFF of significant strengths, where the spin polarization is transmitted through space among the interacting NMR active nuclei, provided strong and conclusive evidence for the existence of intra-molecular hydrogen bonds. Solvent induced perturbations and the variable temperature NMR experiments unambiguously supported the presence of intramolecular hydrogen bond. The two dimensional HOESY and 15N–1H HSQC experiments reveals the existence of multiple conformers in some of the investigated molecules. The 1H DOSY experimental results discarded any possibility of self or cross-dimerization of the molecules. The results of DFT based calculations, viz., Quantum Theory of Atoms In Molecules (QTAIM) and Non Covalent Interaction (NCI), are in close agreement with the NMR experimental findings. In chapter 5 the rates of hydrogen/deuterium (H/D) exchange determined by 1H NMR spectra have been utilized to derive the strength of hydrogen bonds and to monitor the electronic effects in the site-specific halogen substituted benzamides and anilines. The theoretical fitting of the time dependent variation in the integral areas of 1H NMR resonances to the first order decay function permitted the determination of H/D exchange rate constants (k) and their precise half-lives (t1/2) with high degree of reproducibility. The comparative study also permitted the determination of relative strengths of hydrogen bonds and the contribution from electronic effects on the H/D exchange rates. Part III: Novel NMR Methodologies for the Precise Measurement of 1H-1H Couplings Chapter 6 describes two novel NMR methodologies developed for the precise measurement of 1H-1H couplings. Poor chemical shift dispersion and the pairwise interaction among the entire coupled network of protons results in the severely complex and overcrowded one dimensional 1H NMR spectra, hampering both the resonance assignments and the accurate determination of nJHH. The available two-dimensional selective refocusing (SERF) based experiments suffer from the evolution of magnetization from uncoupled protons as intense uninformative axial peaks. This creates ambiguity in the identification of peaks belonging to the coupled partners of a selectively excited proton, hindering the extraction of their interaction strengths. This challenge has been circumvented by designing two novel experimental technique, cited as “Clean-G-SERF” and “PS-Clean-G-SERF”. The Clean-G-SERF technique completely eradicates the axial peaks and suppresses the evolution of unwanted couplings while retaining only the couplings to the selectively excited proton. The method permits the accurate determination of spin-spin couplings even from a complex proton NMR spectrum in an orchestrated manner. The PS-Clean-G-SERF technique has been designed for the complete elimination of axial peaks and undesired couplings, with a blend of ultra-high resolution achieved by real time broad band homonuclear decoupling has been discussed in this chapter. The spin dynamics involved in both these pulse sequences have been discussed. The diverse applications of both these novel experiments have been demonstrated.

Abstract The subjects of this thesis are the dynamics of liquid crystals in external electric and magnetic fields as well as the magnetic properties of small molecules, both studied by liquid crystal nuclear magnetic resonance (LC NMR) spectroscopy. Director dynamics of a liquid crystal 5CB in external magnetic and electric fields was studied by deuterium NMR and spectral simulations. A new theory was developed to explain the peculiar oscillations observed in the experimental spectra collected during fast director rotation. A spectral simulation program based on this new theory was developed and the outcome of the simulations was compared with the experimental results to verify the tenability of the theory. In the studies on the properties of small solute molecules, LC NMR was utilised to obtain information about anisotropic nuclear magnetic interaction tensors. The nuclear magnetic shielding tensor was studied in methyl halides, the spin-spin coupling tensor in methyl mercury halides and the quadrupolar coupling tensor in deuterated benzenes. The effects of small-amplitude molecular motions and solvent interactions on the obtained parameters were considered in each case. Finally, the experimental results were compared to the corresponding computational NMR parameters calculated in parallel with the experimental work.

The initial aim of this project was to investigate ways of synthesising fused, spirocyclic and linked bicyclic amines. We built on methodology previously developed within our group, employing cyclic dienamides, prepared using the reductive cyclisation of bromoenynamides, as key structural building blocks for further annulation. In the course of investigating the reactivity of these cyclic dienamides, we discovered a new efficient and general route to their synthesis, by employing palladium- or ruthenium-catalysed enynamide cycloisomerisation. A wide range of attractive dienamide scaffolds were synthesised from simple enynamide precursors in rapid, high yielding and operationally simple reactions, underlining their potential utility as an atom-economical source of azacycles. Chiral enynamide substrates were used to generate 1,4-dienamides as a single diastereomer at the newly formed (quaternary) stereocentre. This relay of stereochemistry was exploited not only in the formation of monocyclic dienamides, but even in the formation of a spirocyclic product, and this bodes well for further stereocontrolled synthesis of polysubstituted azacycles. Finally the palladium- and ruthenium-catalysed cycloisomerisation of enynamides was discussed and investigated mechanistically, utilising ¹H NMR spectroscopy, timecourse and deuterium-labelling experiments.

This Thesis describes the synthesis, structural, photochemical and photophysical studies of modified retinal protonated Schiff bases in solution. Ultrafast laser spectroscopy, NMR and circular dichroism studies were employed to investigate speed, yield and selectivity of photoisomerisation in these chromophores. <b>Chapter 1</b> introduces relevant biological, photophysical and photochemical aspects of retinal protonated Schiff base photoisomerisation. It includes an overview of synthetic approaches to modified retinal synthesis pertinent to this this work. <b>Chapter 2</b> discuses the investigation of the hypothesis that twisting of the chromophore’s isomerising double bond is responsible for ultrafast photoisomerisation in the protein environment. In these studies it was discovered that addition of a methyl group to the retinal backbone in solution results in protein-like photophysics. <b>Chapter 3</b> presents photopysical and photochemical studies of modified all-trans retinal protonated Schiff bases that culminate in a qualitative model for the influence of electronic factors on photochemical and photophysical behaviour of these chromophores in solution. <b>Chapter 4</b> describes structural and photophysical investigations of 11-cis retinal protonated Schiff bases. NMR studies indicate conformational flexibility of the chromophores. The first synthetic solution-based chromophore to reach rhodopsins’s speed of photoisomerisation is described. <b>Chapter 5</b> presents an attempt to gain conformational information on retinal protonated Schiff bases using circular dichroism spectroscopy. Transfer of stereochemical information from the covalently attached stereogenic centre to the retinal backbone is demonstrated.

The bacteria Streptococcus pyogenes produces a multitude of proteins which interact with and alter the functions of the host immune system. Two such proteins, Endoglycosidase S (EndoS) and Immunoglobulin G-degrading enzyme from S. pyogenes (IdeS) are able to specifically alter the effector functions of immunoglobulin G (IgG). EndoS is a glycoside hydrolase which removes the conserved <i>N</i>-linked glycan from IgG Fc whereas IdeS is a cysteine protease that cleaves the exible protein hinge of IgG. The activity of both proteins results in the reduced ability of IgG to elicit immune responses through Fc receptor binding and complement activation. Amongst other applications, both EndoS and IdeS are actively being explored as new therapeutics for IgG-mediated autoimmune diseases. Given the therapeutic potential of EndoS and IdeS, experiments were designed to investigate the structural and functional characteristics of these enzymes in an effort to understand their specficity for and activity against IgG. Here, bioinformatic and biophysical characterisation of EndoS identified subdomains outside of the catalytic domain which contribute to glycoside hydrolase activity. The substrate specificity of EndoS was also explored and showed that EndoS hydrolyses a broad range of glycans from the IgG scaffold. EndoS was also shown to have activity against alternative glycoprotein substrates, however, this non-specific activity was negligible in the context of whole serum. The effect of EndoS-mediated deglycosylation on the structure of the IgG Fc domain was explored using both X-ray crystallography and small-angle X-ray scattering. Small angle X-ray scattering was also used to characterise both EndoS and IdeS in complex with IgG Fc. Solution-state models of each complex were produced providing preliminary data towards how these enzymes interact with IgG. Overall, the results presented here contribute to our understanding of these enzymes which is of importance as they go forward into clinical applications.

In type II diabetes, systemic metabolism is perturbed and on a cellular level the balance of fuel use is upset. More specifically, increased fatty acid use is seen alongside decreased glucose metabolism. This altered fuel use is mediated by changes in the activity and expression of multiple enzymes. One such enzyme within the glucose breakdown pathway is pyruvate dehydrogenase, whose activity is known to be reduced in the diabetic state. The field of real-time metabolic investigation has rapidly expanded over the past few years due to the invention of technology that has enabled the production of ¹³C labelled hyperpolarised compounds, which can generate high signal levels in magnetic resonance spectroscopy. This has provided the opportunity to measure real-time metabolism of injected hyperpolarised tracers both ex vivo and in vivo. This thesis aimed to develop the use of hyperpolarised compounds in vivo, to investigate the cardiac and hepatic metabolism of a diabetic rat model. We initially addressed the systemic nature of the disease by establishing a two-slice acquisition for obtaining cardiac and hepatic data during a single injection of hyperpolarised pyruvate. This was tested in the fed and fasted states before being used in the studies described in the subsequent chapters of this thesis. The value of hyperpolarised compounds in following metabolic modulation by drug treatment was explored in the next chapter. The effect on metabolism of two drugs targeted at pyruvate dehydrogenase, which differed in their isoform specificity, was investigated first in the perfused heart and subsequently in vivo, both in control and diabetic animals. Hyperpolarised magnetic resonance spectroscopy was combined with other established techniques to help both our understanding of the systemic changes that had occurred following treatment, and provide links between cardiac metabolism and function. The final chapter of this thesis explored the use of hyperpolarised ¹³C pyruvate to understand the effect of hypoxia on pyruvate dehydrogenase, firstly in healthy animals and subsequently in the diabetic, metabolically altered state. Understanding the combination of diabetes and hypoxia was interesting given the existence of several opposing metabolic effects seen in the two states. Overall this thesis has demonstrated developments in the use of hyperpolarised pyruvate that, when appropriately combined with other techniques, can yield valuable metabolic information, in terms of following disease progression, drug development, and understanding basic metabolism.

Les lichens, organismes symbiotiques associant un champignon et un partenaire photosynthétique (algue verte et/ou cyanobactérie), sont caractérisés par la biosynthèse de métabolites secondaires uniques dotés de bioactivités variées. Pour valoriser au mieux cette ressource privilégiée, des méthodes innovantes de spectrométrie de masse ont été développées dans le but de minimiser la préparation de l’échantillon et la durée des analyses. Deux techniques de spectrométrie de masse ont été évaluées en ce sens : le DART-MS et le LDI-MS. L’apport de chacune de ces deux méthodes a pu être établi sur un large panel de lichens, représentant une part importante de l’espace chimique couvert par ces organismes. Il a été démontré que des profils chimiques complets pouvaient être obtenus respectivement à partir de thalles lichéniques et d’extraits acétoniques totaux. Compte tenu de la très large utilisation de la CCM pour l’analyse chimique de lichens, les possibilités offertes par le couplage de la CCM à l’ionisation electrospray ont également été explorées. Une seconde partie de ces travaux avait pour but de cartographier la distribution des métabolites secondaires au sein du thalle lichénique. À ces fins, des analyses d’imagerie LDI ont été réalisées sur une coupe transversale d’un lichen crustacé modèle : Ophioparma ventosa. Ce lichen a été étudié en phytochimie pour identifier six napthopyranones à partir des apothécies dont quatre nouvelles structures. Les principaux métabolites de ce lichen ont pu être imagés par LDI-MSI avec une résolution spatiale de 50 μm environ. Une corrélation entre la distribution des molécules et leur rôle écologique présumé permet d’avancer des hypothèses d’écologie chimique. Des approches conjointes reliant histolocalisation et étude génétique des partenaires de la symbiose ont été entreprises. La recherche des gènes de la biosynthèse de la mycosporine sérinol chez les symbiontes isolés de Lichina pygmaea par microdissection capture laser a été initiée en ce sens. D’autres approches innovantes comme l’analyse cristallographique par diffraction de poudre par les rayons X sont également abordées dans ce document articulé autour de six publications issues de ce travail et de deux articles en cours de soumission. / Lichens are self-sustaining symbiotic partnerships comprising a fungus associated with a green alga and/or a cyanobacteria. This consortium produces unique secondary metabolites that are endowed with various biological activities. To harness this privileged chemodiversity, innovative mass spectrometry techniques were developed in the course of this study to accelerate the dereplicative holdup through both a minimal sample preparation and a decrease of the time of analysis. Two approaches were considered during this work: DART-MS and LDI-MS and their adequacy for lichen dereplication was assessed on a vast array of samples encompassing a wide range of metabolites. Both of them facilitated complete chemical profiles, respectively from unprocessed lichen material and crude acetone extracts. Since TLC still enjoys a wide-spread popularity among lichenologists, the advantages offered by TLC-ESI-MS hyphenation were evaluated as well. A second part of this manuscript focused on the histolocalization of lichen metabolites. For this purpose, LDI mass spectrometry imaging studies were undertaken on the crustose lichen Ophioparma ventosa. The phytochemical investigation of this species afforded the isolation of six naphthopyranones from its apothecia, four of them being new molecules. LDI-MSI revealed the distribution patterns of all the main metabolites of this lichen, reaching a spatial resolution of 50 μm. Most interestingly, the distribution pattern of imaged metabolites within the thallus is highly organized and is related to their ecological relevance. Joint strategies combining histolocalization and genetic investigation of lichen symbionts separated using laser capture microdissection were also considered. As such, an investigation of the biosynthesis of mycosporine serinol within Lichina pygmaea dissociated symbionts was initiated. Further analytical strategies such as X-ray powder diffraction are introduced in this thesis that contains six publications and two drafts to be submitted.

No description available.

Das Zytokin Interleukin-10 (IL-10) ist ein Schlüsselspieler in der Regulation des Immunsystems mit pro- und anti-inflammatorischen Funktionen. Es spielt eine wichtige Rolle bei der Terminierung und Unterdrückung einer Entzündungsantwort, die ansonsten zu einer bleibenden Schädigung des Gewebes führen kann. Eine Dysregulation von IL-10 ist mit verschiedenen Krankheitsbildern wie chronischen Entzündungen, Autoimmunerkrankungen und Krebs assoziiert. IL-10 wird von einem breiten Spektrum von Zelltypen, darunter hauptsächlich hämatopoetische Zellen, aber auch epitheliale und mesenchymale Zellen, gebildet und in den extrazellulären Raum freigesetzt, wo es mit Komponenten der extrazellulären Matrix in Kontakt kommt. Es ist bekannt, dass IL-10 an Glykosaminoglykane (GAGs) binden kann und dass diese Interaktion seine biologische Aktivität beeinflusst. GAGs sind eine Klasse linearer Polysaccharide der extrazellulären Matrix. Sie bestehen aus wiederholenden Disaccharideinheiten und haben einen hoch negativ geladenen Charakter, welcher durch einen hohen Grad an Sulfatierung in der Zuckerkette zustandekommt. Sie binden eine Vielzahl an Signalproteinen und regulieren deren biologische Funktionen, etwa indem sie Einfluss auf die Rezeptorbindung oder die räumliche Verteilung des Proteins im Gewebe nehmen. Die molekularen Mechanismen, wodurch GAGs die biologische Aktivität von IL-10 beeinflussen, sind bisher unbekannt. Insbesondere ist nichts über die strukturellen Grundlagen der Interaktion bekannt, die Voraussetzung für ihr funktionelles Verständnis sind. In dieser Arbeit wurden daher die Bindungseigenschaften von IL-10 und GAGs sowie der strukturelle Aufbau ihres molekularen Komplexes unter Verwendung von NMR-Spektroskopie in Lösung charakterisiert. Es wurde eine definierte GAG-Bindungsstelle in IL-10 identifiziert und die Bindungsepitope und Bindungsaffinitäten von GAGs bestimmt. Die Ergebnisse dieser Arbeit weisen auf eine wichtige Rolle, die GAGs in der Biologie von IL-10 spielen können, hin – etwa für seine Speicherung im Gewebe oder für die IL-10-Rezeptorbindung.

The appropriate lipid environment is crucial for the proper function of membrane proteins. There is a tremendous variety of lipid molecules in the membrane and so far it is often unclear which component of the lipid matrix is essential for the function of a respective protein. Lipid molecules and proteins mutually influence each other; parameters such as acyl chain order, membrane thickness, membrane elasticity, permeability, lipid-domain and annulus formation are strongly modulated by proteins. More recent data also indicates that the influence of proteins goes beyond a single annulus of next-neighbor boundary lipids. Therefore, a mesoscopic approach to membrane lipid-protein interactions in terms of elastic membrane deformations has been developed. Solid-state NMR has greatly contributed to the understanding of lipid-protein interactions and the modern view of biological membranes. Methods that detect the influence of proteins on the membrane as well as direct lipid-protein interactions have been developed and are reviewed here. Examples for solid-state NMR studies on the interaction of Ras proteins, the antimicrobial peptide protegrin-1, the G protein-coupled receptor rhodopsin, and the K+ channel KcsA are discussed.