No description available.

No description available.

No description available.

Primary silanes of the type RSiH$ sb3$, where R = phenyl (Ph), benzyl (Bz) and n -hexyl, undergo catalytic dehydrogenative polymerisation in the presence of catalytic amounts of Cp$ sb2$TiMe$ sb2$ (Cp = $ eta sp5$-C$ sb5$H$ sb5)$ to give linear oligomers consisting of about 10 silicon atoms, regardless of the reaction conditions. When the amount of Cp$ sb2$TiMe$ sb2$ was increased, novel organometallic complexes of the type $ {$(Cp$ sb2$Ti)$ sb2( mu$-H)$( mu$-HSiRH)$ }$ and $ {$Cp$ sb2$Ti$( mu$-HSiRH)$ } sb2$ were isolated and characterised. X-ray crystal structural characterisation has confirmed the first Ti-H-Si-Ti bridged systems. Other titanocenes (Cp$ sbsp{2}{ prime}$TiMe$ sb2$(Cp$ sp prime$ = $ eta sp5$-C$ sb5$H$ sb4$Me), (Cp$ sb2$TiH) $ sb{ rm n}$, (Cp$ sb2$Ti) $ sb2$ and Cp$ sb2$Ti(CO)$ sb2)$ also polymerised PhSiH$ sb3,$ and in addition gave analogous complexes under the appropriate conditions. Furthermore PhSiH$ sb3$ undergoes polymerisation in the presence of CpCp$ sp *$TiMe$ sb2$ (Cp$ sp *$ = $ eta sp5$-C$ sb5$Me$ sb5),$ but not Cp$ sbsp{2}{ *}$TiMe$ sb2.$ Polymerisation of RSiH$ sb3$ (R = phenyl and benzyl) also occurs in the presence of Cp$ sb2$ZrMe$ sb2$ and complexes of the type $ {$Cp$ sb2$(SiHMeR)Zr$( mu$-H)$ sb2$Zr(SiH$ sb2$R)Cp$ sb2 }$ have been identified, and isolated (R = phenyl). (Cp$ sb2$ZrH$ sb2$) $ sb{ rm n}$ also polymerises PhSiH$ sb3,$ with the associated production of $ {$Cp$ sb2$Zr$( mu$-H)(SiH$ sb2$Ph)$ } sb2.$ The analogous complex was also observed during the reaction of Cp$ sb2$ZrMe$ sb2$ with BzSiH$ sb3.$ The reactions have been followed by $ sp1$H and $ sp{29}$Si NMR, and possible reaction paths are discussed.

No description available.

Nuclear magnetic resonance (NMR) yields remarkably detailed structural information about virtually any molecule. However, its application to non-equilibrium systems is hampered by a lack of sensitivity. To increase the amount of signal that can be obtained from a NMR experiment, various hyperpolarization schemes have been previously introduced. One such technique is dynamic nuclear polarization (DNP), which can enhance NMR sensitivity by several orders of magnitude. The work detailed here focuses on the development of methods utilizing DNP to study non-equilibrium systems such as chemical and biochemical reactions in real-time. To work with hyperpolarized samples, we have designed and constructed a rapid injection and mixing system. This system allows samples to be transported between superconducting magnets used for polarization and for NMR spectroscopy in less than two seconds. Rapid transport is essential for successful use of samples with short spin-lattice relaxation times. For the study of reactions under non-equilibrium conditions, the system provides the additional capability for samples to be mixed with a second, unpolarized reagent. A chromogenic trypsin catalyzed ester hydrolysis reaction was used to validate the DNP-NMR technique as a tool for kinetic analysis. It is shown that the DNP-NMR method agrees with the conventional UV method within the uncertainty of the measurement. Hyperpolarization in this modality presents both challenges and opportunities, each of which motivate the development of new NMR techniques. In addition to the determination of kinetics, DNP-NMR is amenable to mechanistic analysis of a reaction. We have developed a technique based on selective inversion of spin-polarization, which allows for mapping of atoms between reactant and product of a reaction. This scheme was applied to a Grignard reaction, demonstrating applicability to organic reactions. Signal averaging, as it is applied for conventional multi-dimensional correlation spectroscopy cannot always be applied easily when using hyperpolarized sample. For the rapid measurement of heteronuclear correlation spectra, we have developed a technique utilizing the differential scaling of scalar coupling under off-resonance irradiation. Although DNP-NMR yields spectra of outstanding quality even with small quantities of sample, peak intensities are not quantitative. It is nevertheless possible to compare peak multiplets obtained from fractionally isotope labeled samples. Using biosynthetically labeled lipids from E. Coli cells, we showed that the resulting labeling patterns reflect their biosynthetic pathways. As a final case-study employing several of these newly developed methods, the uronate isomerase catalyzed isomerization of glucuronate into fructuronate was studied. The ability to follow the reaction in real-time while directly observing all anomeric forms of the reactant and product permits the independent determination of kinetics for each anomeric form of substrate and product. This study revealed the anomeric specificity of the enzyme.

Reversible cis-trans isomerisation of a series of commercially interesting yellow azo dyes has been studied using the technique of Nuclear Magnetic Resonance (NMR) spectroscopy with in situ laser irradiation. Photostationary state (PSS) spectra of the azo dyes, provided by coupling laser irradiation into the sample within the probe of the NMR spectrometer, have allowed observation of azo cis isomer species that would otherwise elude detection and characterisation by NMR due to their rapid thermal decay times. The NMR results have been combined with geometry optimised structures obtained through ab initio (DFT) calculations in order to allow visualisation of the trans and cis isomer species, and explain NMR spectral features. In the majority of cases, these in vacuo calculated structures show a great deal of correlation with NMR observations of asymmetric, cis-trans isomerisation-induced chemical shift changes for protons adjacent to the azo bond. The cis isomer spectral pattern for substituted naphthyl group protons can hence be used as a diagnostic tool in determining the correct cis isomer conformation in molecules where more than one conformation may exist. In addition to the aforementioned characterisation studies, in situ irradiation has provided the opportunity to undertake a thorough investigation of the kinetics of photoand thermal isomerisation for the same yellow dye series. The results of these studies have been combined with previous work on similar systems to provide an extensive data set, and conduct analysis in a systematic fashion. Adding fibre-reactive groups, varying phenyl and chlorotriazine ring substituents, and altering naphthyl group sulfonation patterns have a profound effect on both the photochemical and thermal rates of isomerisation in these systems. In certain cases, the same structural calculations noted earlier have proved useful in rationalising the identified kinetic differences. The presence of phenyl substituents ortho to the azo bond has been shown to increase the rate of thermal cis isomer decay. Additionally, substituents bonded to a fibrereactive group distant from the azo bond have an appreciable effect on the barrier to thermal cis-trans isomerisation, but little effect on the photochemical characteristics of each isomer. Several sulfonated naphthyl group patterns have been studied, leading to an observation that sulfonate groups positioned ortho to the azo bond assist in retarding thermal isomerisation, with sulfonate groups in other positions having a much smaller effect. One particular molecule, a component of a currently available commercial dye, was studied for its interesting and previously unexplained behaviour, both photochemical and chemical. The dye demonstrated photoisomerisation at low concentrations only, with aggregation preventing formation of the cis isomer at higher concentrations. The trans isomer was found to undergo degradation to a product which did not photoisomerise. This product was identified as a benzotriazinium compound by multinuclear 2D correlation NMR spectroscopy, formed by a reversible cyclisation reaction involving the azo bond.

Two methods were used to produce exogenous lipid/bR complexes. A detergent method (Huang et al., 1980) reconstituted bR into DMPC or DMPG bilayers, free of all endogenous purple membrane lipids as shown by high resolution ³¹ P-NMR. A novel biological detergent-free method employed bovine liver non-specific lipid transfer protein (nsTP) to mediate addition of DMPC to purple membrane, while retaining 76 - 86% of the endogenous purple membrane phospholipids. The variations with temperature of 2H-NMR quadrupole splittings for the DMPC choline α- and β-methylene CD2 segments were similar to those for protein-free lipid (Gaily et al., 1975) implying that temperature dependent changes in segmental amplitudes of motion within the choline group are preserved in the presence of bR. Incorporation of small quantities of bR increased the amplitudes of segmental motion within the choline headgroup relative to that of pure lipid, but increasing the bR content induced an ordering effect. The choline α- and β-methylene segment quadrupole splittings showed a linear variation with protein content at constant temperature. This is consistent with freeze fracture electron microscopy data, which shows the bR particles to be dispersed at all lipid/protein ratios, when quenched from temperatures above the phase transition. Applying a fast two site exchange model to the ²H-NMR data, values between 12 and 15 were calculated for the number of boundary lipids for bR (26,000 M_r) in DMPC bilayers free of purple membrane lipids. From ESR data, for delipidated bR in DMPC and DMPG bilayers at temperatures above the phase transition, the number of boundary lipids calculated were 18 - 21, which is consistent with the bR being monomeric, as also observed in DMPC bilayers with all the purple membrane lipids retained (Cherry et al., 1978). The purple membrane lipids thus appear to mediate crystallization of the bR particles into a hexagonal lattice at temperatures slightly below the exogenous lipid phase transition.

A proton nuclear magnetic resonance spectrum of the brain in vivo contains peaks from every proton-containing molecule in the brain. Sensitivity limitations mean that only those molecules present at concentrations of at least a few millimolar are detectable in a reasonable period of time; this still leaves many important molecules such as amino acids and other small metabolites. Most of their resonance frequencies fall in the region between 1.0 and 4.5 p.p.m. A typical linewidth in vivo is about 0.05 p.p.m., so the number of distinct peaks observable is restricted. The use of two-dimensional NMR techniques such as COSY can spread peaks out into a second dimension enabling otherwise overlapping peaks to be resolved. This thesis describes the development, testing and application of two such 2D NMR pulse sequences, dubbed ISIS-COSY and ISIS-JRES. They are based on an existing magnetisation localisation sequence and excite detected magnetisation in a manner analogous to the high-resolution sequences COSY and 2D J-resolved spectroscopy. A method for quantifying the metabolites visible in an ISIS-COSY spectrum from their cross-peak intensities is described, and results presented from both control rat brains and those of animals treated with vigabatrin, an inhibitor of GABA-transaminase that has the effect of increasing brain γ-amino butyric acid (GABA) levels. Further applications mentioned are in the study of neutrophil-infiltrated rat brain and adaptation of the ISIS-COSY technique for human use.

The work described in this thesis is concerned with the development and application of real-time photo-CIDNP (Chemically Induced Dynamic Nuclear Polarisation) to the study of protein structure and folding. Chapters 1 and 2 introduce the protein folding problem, and its study by NMR, then go on to elucidate the mechanisms behind the photo-CIDNP phenomenon. Chapter 3 applies photo-CIDNP spectroscopy to the study of a small cytochrome protein. The difficulties of performing these experiments on chromophore-containing proteins are discussed. Chapter 4 begins with the development of a rapid mixing device for use in real-time NMR and CIDNP studies. Experiments used to characterise the device are presented. This chapter then goes on to describe CIDNP pulse labelling experiments, used to investigate the surface structure of some molten globule states of two a-lactalbumins. This chapter concludes with an application of the rapid mixing device to the real-time refolding of hen egg white lysozyme. Chapter 5 extends the work of the previous chapter, studying the real-time refolding of bovine pancreatic ribonuclease A. Refolding studies are performed from different denaturing conditions, and the effects of sample heating during the real-time CIDNP experiment are discussed. Chapter 6 describes the use of illumination during an NMR experiment to study the conformational changes in a plant blue light receptor protein, phototropin. The structural changes are characterised with 2-dimensional NMR spectroscopy and photo-CIDNP. The kinetics of the ground state recovery are also investigated by real-time NMR spectroscopy. Chapter 7 uses calculated hyperfine coupling constants and a radical pair diffusion model from the literature to simulate the nuclear polarisation obtained for the amino acid tryptophan. Comparisons are made between theory and experiment. Chapter 8 describes the structural characterisation of a homologous series of de novo peptides, designed for subsequent use in EPR experiments when derivatised with a suitable spin label.