The stereocontrolled and efficient syntheses of the aglycones of the potent anti-inflammatory pseudopterosins A-F and K-L have been achieved starting from convenient monoterpenic units and using a novel benzannulation protocol partially developed in or laboratories. For the synthesis of the K-L compounds a completely substrate-controlled stereoselective route was devised starting from commercial isopulegol using a sequence of epoxidation and Lewis acid-promoted oxirane opening followed by benzannulation and Friedel-Crafts type sulphone displacement to generate the tricyclic structure of 3.2.1.13. The route to pseudopterosins A-E started from (-)-citronellal and employed catalytic asymmetric reduction of , -unsaturated ester 8.2.1.5. After the formation of the aromatic ring, the second approach was convergent to the enantiomers of intermediates used in the first route. Oxidation with Fremy's salt led to the unstable aglycones of the natural compounds under mild conditions. (Fig. 3876A)

The low selectivity of chemotherapy is an ongoing problem in the treatment of cancer. Prodrugs that are activated in vivo provide a therapeutic advantage for selective cytotoxicity. Here we have designed redox-active compounds that are electrochemically reduced in hypoxic (poorly oxygenated) tissue, resulting in release of a nitrogen mustard cytotoxin. This thesis describes the synthesis of novel macrocyclic N-mustard drugs and the development of their Cu(II) complexes as hypoxia-selective prodrugs. (Fig. 3736A) The (2-trimethylsilyl)ethanesulfonyl (SES) protecting group is very versatile. It is removed under mild conditions using fluoride. The published synthesis of the sulfonyl chloride A gave variable yield and purity, but we have improved the conditions to give consistently pure material in high yield (70-86% overall) (Scheme 1). (Fig. 3736B) Triamines with carbon bridges longer than three are difficult to prepare, often requiring multistep syntheses. A route was developed to synthesise linear triamines, using the SES-amide B. This route produces these triamines in relatively high yields (60-80% overall), via simple reactions with little purification necessary (Scheme 2). A variation on the Richman-Atkins synthesis has been exploited to reach known an novel triazamacrocyclic compounds D (Scheme 3), in order to explore their structure-activity relationship as N-mustard drugs E (made as shown in Scheme 4). Eight novel macrocyclic N-mustards E were found to be potent DNA cross-linking agents (nM range) by Prof. John Hartley at University College London. Three of the novel triazamacrocycles D were assessed in vitro for anti-parasitic activity by Dr. Michael Barrett at the University of Glasgow. They showed moderate activity against Leishmania mexicana and Trypanosoma brucei. (Fig. 3736C) Water soluble Cu(II) complexes of cytotoxic macrocyclic nitrogen mustards have been prepared and their structures have been determined using X-ray crystallography by Dr. Louis Farrugia in this department. The redox behaviour and reduction potentials (Cu[II] to Cu[I]) of the complexes in phosphate buffer were assessed using cyclic voltammetry. The thermodynamic stabilities of the Cu(II) complexes in aqueous solution were analysed qualitatively using UV-Vis spectroscopy. The mustard complexes F and G showed irreversible redox behaviour and low thermodynamic stability, and were not hypoxia-selective but behaved as typical mustard drugs. The cyclen-based mustard complex H showed reversible redox behaviour and had high thermodynamic stability under aqueous conditions. H exhibited excellent hypoxia selectivity (the best so far in the lung tumour cell line tested) and is an attractive lead compound for further development of this novel approach to cancer chemotherapy (Fig. 3736D).

No description available.

Palmitoylation variant GPCR-G protein fusion proteins were created between the porcine u2A-adrenoceptor or the human 5-HT1A-serotonin receptor and the pertussis toxin resistant, Cys35lIle, form of the rat Go1u protein. These palmitoylation-variant fusions were transiently expressed in HEK293T cells prior to analysis of the regulation of palmitoylation and the functional consequences of palmitoylation for both the GPCR and G protein parts of the fusions. When the regulation of palmitoylation was studied for u2A-adrenoceptor-GoluCys35IIle fusion proteins, dynamic palmitoylation and depalmitoylation of both the Cys442residue of the u2A-adrenoceptor and the Cys ' residue of the GoluCys351Ile protein were found to occur. However, only the GOluCys351Ileprotein part of the fusion was found to undergo adrenaline-stimulated regulation of palmitoylation and the effect of adrenaline required G protein activation. Adrenaline regulation proceeded in a concentration-dependent manner correlating with agonist occupancy of the u2A-adrenoceptor. Such agonist effects were found to be, at least in part, due to agonist-stimulation of GOluCys351Ile protein depalmitoylation. The requirements for palmitoylation of the u2A-adrenoceptor and GoluCys351Ile protein elements of the u2A-adrenoceptor-GoluCys35IUe fusion proteins were subsequently assessed for various functional properties. Palmitoylation of neither the U2Aadrenoceptor nor the GoluCys351Ile protein parts of the fusion determined fusion protein expression levels, affinity for the agonist adrenaline, affinity for the antagonist RS- 79948-197, ability to bind or to hydrolyse GTP or their ability to influence the efficiency of RGS 16 protein to accelerate the GTPase reaction. In regulation of palmitoylation studies for 5-HTIA-receptor-GoluCys35IIle fusion proteins, dynamic palmitoylation of the Cys' residue of the GoluCys351Ue protein and the Cys417 residue of the 5-HTIA-receptor was observed as well as a lack of incorporation of palmitate into Cys420 of the 5-HT1A-receptor. Dynamic depalmitoylation was only observed for the Cys' residue of the GoluCys351Ile protein, not for the 5-HT1A-receptor. In the latter case, palmitate once incorporated appeared to remain stably attached. Both the 5-HT1A-receptor and the GoluCys351Ile protein parts of the fusion were found to undergo 8-0H-DPAT-stimulated regulation ofpalmitoylation. 8-0H-DPAT was able to regulate palmitoylation levels of both proteins in a concentration-dependent manner. For the regulation of GoluCys351Ile protein palmitoylation such agonist effects were found likely to be, at least in part, due to an agonist-stimulated rate of depalmitoylation. For the regulation of 5-HT1A-receptor palmitoylation such agonist-stimulated increases in observed palmitoylation levels were only attributable to the addition of palmitate, given that no depalmitoylation of the 5- HT1A-receptor could be detected. The requirements for palmitoylation of the 5-HT1A-receptor and GoluCys351Ile protein elements of the 5-HT1A-receptor-GoluCys351Ile fusion proteins were also assessed for a selection of functional properties. Similar to the results obtained with Go1uCys351Ile protein constrained to the uZA-adrenoceptor, the palmitoylation of the GoluCys351Ile protein did not determine fusion protein expression levels, their affinity for the antagonist WAYI00635, or their ability to bind GTP. Palmitoylation of 5-HT1Areceptor did not alter fusion protein expression levels or their affinity for the antagonist WAYI00635. However, in contrast, it did cause enhanced levels of GTP binding to the 5-HT1A-receptor-GoluCys351Ile fusion proteins. The results of this investigation suggest that there are different requirements for regulation of GPCR and G protein palmitoylation dependent on the GPCR-G protein fusion in question. These requirements may be responsible for the specific functional properties displayed by such fusions. The current study also demonstrates that GPCR-G protein fusion proteins can be successfully used as tools to study both the regulation of palmitoylation and the functional consequences of this modification.

The effective degree of micellar dissociation of cetyltrimethylammonium bromide (CTAB) micelles has been determined from electrochemical (EMF) measurements using a CTAB selective electrode. Thermodynamic parameters have been determined using the `Ion binding model' and show dominance of hydrophobic interactions in free CTAB micellization. The enthalpy contribution is insignificant. The binding of the surfactant CTAB onto the neutral watersoluble hydrophobic polymers: poly(propylene oxide) (PPO), poly(vinylmethylether) (PVME) and ethyl(hydroxyethyl) cellulose (EHEC) has also been investigated by the EMF technique. In all cases it has been shown that the surfactant binds to the polymer and the bound surfactant exists in the form of small aggregates. The EMF data has been used to investigate the characterization of the binding process. The formation of inclusion complexes between alkylpyridinium bromide (CPyBr) and alkyltrimethylammonium bromide (CIITAB) with a- and ß-cyclodextrins (CDs) have also been investigated by the EMF technique. In addition the thermodynamics of the inclusion on a- and ß-CDs by the surfactants : CTAB, CPyBr, alkylsulfates (COSO3Na) , and alkanesulfonates (CnSO3Na) have been carried out using an Omega isothermal titration calorimeter (ITC). Whereas ß-CD systems form predominantly 1: 1 complexes, a-CD form 1: 1 and a 2: 1 complexes with surfactants having long hydrophobic tails. For both a- and ß-CD the complexation constants increase as the chain length of the surfactant increases, showing that the driving force dominating the formation of these complexes is the hydrophobic effect. The 1: 1 ß- CD/surfactant complexation is accompanied by large increase in entropy. The 1: 1 a-CD/surfactant complexation and all 2: 1 complexation are accompanied by a large increase in enthalpy.

Transforming growth factor-P (TGF-p) plays a crucial anti-atherogenic role. TGF-p classically signals through the Smad pathway but is known to activate other signalling pathways such as the mitogen-activated protein kinase (MAPK) cascades. Foam cell formation is inhibited by TGF-p through the regulation of expression of genes involved in macrophage cholesterol homeostasis. However, the molecular mechanisms underlying this regulation are yet to be fully elucidated. Studying such mechanisms may lead to identification of novel avenues for treatment of this disease and was therefore the main focus of these studies. TGF-p regulates the stability of atherosclerotic plaques through the regulation of expression of genes encoding proteins involved in the turnover of the extracellular matrix (ECM). The ADAMTS proteases cleave proteoglycans within the ECM and have recently been hypothesised to have roles in atherosclerosis. Cytokine regulation of these proteases and elucidation of the molecular mechanisms behind this regulation may enhance understanding of the roles these proteases play in atherosclerosis with a view to identifying novel avenues for treatment of this disease and was therefore an additional focus of these studies.

Chapter I presents an overview of mass spectrometry on double focusing instruments. Special attention is paid to ionisation methods which may be of use in mixture analysis. In chapter II, following a brief introduction to chemometrics, results of the application of factor analysis to the problem of determining the components of a mixture from mass spectra of simple mixtures are given. After applying the technique to simulated data, the results of applying the technique to simple mixtures and to the deconvolution of overlapping GC/MC spectra are given. It is concluded that provided the pure compounds have 'unique' peaks in their mass spectra, and that the statistical variations in the intensities of peaks due to different mixtures are not correlated, then the technique will yield good results. Chapter III deals with the application of CI techniques and factor analysis to the analysis of engine oils. Attempts to use the factor analysis technique to yield a meaningful 'type' analysis of engine oil fractions was not successful. The use of proton transfer reagents and charge transfer reagents for CI spectra of engine oil fractions was also unsuccessful. It is concluded that the complexity of engine oil mixtures and the chemical similarity of the constituent molecules makes them difficult to differentiate by CI mass spectrometry. In the final chapter, details are gave of an investigation into the use of CI mass spectrometry and FAB mass spectrometry for the analysis of engine oil additives. The additives studied were calcium 2,2'-bis (4-alkylphenyl) sulphides. The FAB mass spectrometry of these sulphurised phenates gave poor results with glycerol, triethanolamine, triethanolamine / sodium sulphate or squalane used as FAB matrix. Of the CI techniques used, electron capture ionisation of the hydrolysed sample gave the most promising results. The molecular ion peak intensities were higher than those encountered in the EI spectra. In addition, the fragmentation in the EC spectra was simpler than the fragmentation in the EI spectrum. Metastable ion studies of the EI and EC spectra of the sulphurised phenols, using the technique of metastable mapping, gave some useful results, but the poor mass resolving power and the long run times were a major drawback. It is concluded that the technique of metastable mapping is of no use where mass resolution is important, and the use of inlet systems, such as an AGHIS, which allow long sample lifetimes is recommended. Where possible, the use of tandem mass spectrometry or Fourier transform mass spectrometry for metastable ion studies for mixture analysis, is also recommended.

The flux of metabolites within the cell is discussed. Two approaches were used in the study of this subject: 1. A preliminary investigation of the controlling factors of adenine metabolism in red blood cells was undertaken. 2. A study was made of adenylate cyclase in red blood cells and of the cyclic-AMP binding proteins of the bovine adrenal cortex. AMP deaminase was partially purified from human and sheep erythrocytes. This greatly facilitated the spectrophotometric assay of the enzyme’s activity. Some properties of this enzyme were studied. A method for examining the control of purine flux was developed by the use of radioisotopic tracers. The possibility of its use as a diagnostic assay for metabolic disorders is discussed. Adrenalin, which modifies the erythrocyte membrane, was tested for any effect it might have on the rate of transport of adenine into the red blood cell. The adrenal in or fluoride ion stimulated activities of adenylate cyclase were measured in turkey, rat and human red blood cells. The assay used was the saturation analysis procedure of Brown et al. (1971) using a cyclic-AMP binding protein from the bovine adrenal cortex. The cyclic-AMP binding moiety is the cyclic-AMP dependent protein kinase regulatory subunit. The crude preparation used in the assay for cyclic-AMP was fractionated by several methods and cyclic-AMP binding studies were carried out on the different fractions. Particular attention was paid to the interpretation of binding data. It was found that under different conditions certain preparations of the binding protein could produce either linear or curved binding plots ('bound' against 'bound/free’ ligand) The causes of this are discussed. The result of the binding study has mechanistic implications.

The crystallographic structure of vanadium carbide having a carbon-to-metal atom ratio close to 0.83 has been determined from electron diffraction and nuclear magnetic resonance (NMR) studies. Material of this composition occurs within the nominally cubic (rocksalt) phase field of the vanadium-carbon phase diagram, but in this investigation it is shown that the structure is modified substantially by an unusual type of ordering that is associated with the carbon sublattice. The ordered structure is based on two interpenetrating fcc lattices, only one of which (the metal lattice) is completely occupied. The other is only 83% filled, but the carbon atoms (and carbon vacancies) are distributed in an ordered manner on the available lattice sites. As a consequence of ordering, the electron diffraction patterns exhibit supplementary spots which may be analyzed to obtain information regarding the symmetry and size of the superlattice unit cell. Moreover, the (NMR) of the principal vanadium isotope, V51, exhibit spectra which provide information about the disposition of carbon atoms within the unit cell. In the proposed structure, which belongs to the trigonal space group F31, or its enantiomorph F32, the carbon atoms and carbon vacancies are arranged so that all the vanadium atoms have exactly five nearest neighbour carbon atoms. The observation that ordering in material of this composition leads to a distribution of atoms that is homogeneous on an atomic scale is consistent with the electronic structure of vanadium carbide as it is currently understood, and suggests that it is appropriate to refer to the ordered compound as V6C5 When the ordered compound is examined for extended periods of time in a 100 kV electron microscope, it is observed that the material becomes slowly disordered. Several possible explanations were considered for this effect, but it has been concluded that the disordering results from the displacement of carbon atoms by impinging electrons (i.e., by radiation damage). To determine the magnitude of the displacement threshold, studies have been made of the disordering rate under electron bombardment at energies from 33 keV to 100 keV, using a Faraday cup to measure superlattice spot intensities. The results have been compared with a theory for the damage process, from which it is concluded that the displacement energy of carbon atoms in V6C5 is 5.4 ev. This value is unexpectedly low in comparison with the values that have been reported for most other materials, but it is consistent with a simple model for the displacement mechanism which appears to account for the threshold energy in this, and a number of other solids also. According to this model, observable damage will occur in a solid only if the displaced atom has sufficient energy to (1) create a vacancy, (2) permit transferring the energy pulse for n interatomic distances through the lattice to a position at which the vacancy-interstitial pair is stable, and (3) form an interstitial at the terminal point of the disturbance. calculations show that for most solids, the threshold energy is determined principally by mechanisms (2) and (3), but for V6C5 only the first process is important since a large number of vacant sites are available in the carbon sublattice to accept a displaced atom with the expenditure of very little energy. The fact that V6C5 can be disordered in situ by electron microscope beam bombardment has presented an unusual opportunity to study the effect of electron channelling on radiation damage rates. It has long been a question whether significantly less damage occurs when a sample to be irradiated by energetic electrons is oriented for "anomalous transmission," a phenomenon which is similar to the Borrmann effect for x-rays and to channelling for ion beams , Experiments with ion beams have demonstrated that the damage rate in channelling orientations can be 10 times less than in -random orientations, but no analogous experiments have been performed previously for electron damage because of the small angular separation between the channelling and de-channelling orientations. The small divergence of the electron microscope beam, the ability to orient the crystal with great precision, and the fact that the full analytical capabilities of the electron microscope can be used to monitor the damage process in V6C5 have now made such an experiment possible. Finally, studies have been made of the re-ordering kinetics of radiation damaged V6C5 to determine the activation energy for carbon diffusion. These measurements, which have been made in a temperature range about 1000 o C below that for which previous values have been obtained, have important implications for the mechanical properties of V6C5 and provide an explanation for the extremely short time constant associated with the order-disorder transformation in this material.

In this thesis the direct synthesis of hydrogen peroxide (H2O2) from hydrogen and oxygen using gold-palladium supported catalysts was investigated. The direct route represents a greener and sustainable alternative to the current industrial manufacturing process. The main objective of this study was to achieve the industrial requirements of H2O2 yields and selectivity, which would make the direct process industrially viable. In order to reach the required target, two innovative approaches for the direct synthesis of H2O2 were examined. The first part of this thesis was dedicated to the development of a biphasic solvent system comprising an organic alcohol and water. The advantages of this system was highlighted and the effect of reaction variables (such as solvent composition, pressure, reagent ratio, temperature and reaction time) were evaluated using two different catalysts. The identification of two optimum conditions resulted in an important enhancement in the H2O2 yield for the two catalysts examined. By finely tuning the reaction conditions and using two different solvent systems ((i) decan-1-o1-water (ii) diisobutyl carbinol-water) H2O2 concentrations between ~ 0.30 and 28 wt. % were achieved. The second part of this thesis was dedicated to studying the direct gas phase synthesis of H2O2 in a continuous gas flow reactor. Two lab scale flow reactors were designed and built in situ: The first was for studying the direct gas phase synthesis of H2O2 at atmospheric pressure and the second for studying the reaction at pressures above atmospheric. The results demonstrate the direct gas phase synthesis of H2O2 was challenging and the absence of solvent seriously compromises the stability of the H2O2. Despite this, the results demonstrate by using gold-palladium nanoparticles and a mixture of hydrogen and oxygen it is possible to not only oxidise organic molecules in the gas phase but the synthesis rates were high enough to detect H2O2 as a product in a fixed bed gas phase reactor and a temporal analysis of products (TAP) reactor. This observation opens up the possibility of synthesising H2O2 directly in a gas phase reaction.