Mitochondrial cytochrome c (Cc) is a small haem protein, primarily involved in electron transfer, but is an example of an extreme multifunctional protein with roles in several stages of apoptosis through interaction with phospholipids and Apaf-1. The switch in function is associated with an interaction with cardiolipin (CL) and gain of peroxidase activity. Mutations in the Cc gene lead to Thrombocytopenia 4, a disease associated with enhanced apoptotic activity. The Y48H variant is found in the 40-57 W-loop, the lowest free-energy foldon. A 1.36 Å resolution X-ray structure of the Y48H variant reveals minimal structural changes to that of the wild-type protein and G41S variant (also associated with Thrombocytopenia 4). Despite this, the intrinsic peroxidase activity is enhanced, implying that a penta-coordinate haem species is more prevalent in the Y48H variant, corroborated through determination of a Met80 off-rate of > 125 s-1 compared with ~ 6 s-1 for the wild-type protein. Heteronuclear NMR experiments with the ferric Y48H variant have revealed heighted dynamics relative to the WT protein in the 40-57 and 71-85 W-loops, the latter being the loop containing the Met80 ligand, suggesting communication between these two substructures. When these results are considered in relation to the G41S variant, a dynamic picture emerges in which heightened dynamics in key substructures of the Cc fold facilitate an increased population of a penta-coordinate, peroxidase active species of the order wild-type < G41S < Y48H. Studying the alkaline transition through pH jump stopped-flow 2 spectroscopy has revealed that not only is the pK695 is reduced by ~ 1 pH unit compared to that of the wild-type protein, but also the pK of the trigger is lowered. In contrast, variants in the 71-85 W-loop do not alter the pK of the trigger but are nevertheless associated with enhanced peroxidase activity relative to the wild-type protein, due to enhanced local dynamics within the haem crevice.

The overall objective of this study was to prepare and optimise solid base catalysts for liquid phase reactions, based on silica support materials functionalised with alkyl amine groups. The study included an investigation of the effect of the pore volume, surface area and the surface chemical characteristics of the silica support on the catalyst performance. The role of surface silanol groups remaining on the silica surface after functionalisation working cooperatively with basic amine groups was also studied. A series of silica gel supports, together with a commercially-available ordered mesoporous silica, SBA-15, were functionalized with tethered aminopropyl groups. Activities were measured in the nitroaldol reaction between nitromethane and benzaldehyde to afford nitrostyrene, and the Knoevenagel condensation reaction between benzaldehyde and ethyl cyanoacetate to form ethyl-2-cyano-3-phenylacrylate. The results showed large variations in optimum activity depending on the support material, and its porosity in particular. All support materials exhibited an optimum catalyst loading which depended on support surface area, generally coinciding with complete coverage of the surface with a single layer of catalytic groups, based on a nominal surface area occupied by the three tethering groups linking each propylamine group to the surface. Further experiments were performed with catalytic groups tethered by one and by two (rather than three) groups, which occupy less space on the support surface. In addition, the possible role of silanol groups was investigated by capping free silanols with non-polar methyl groups using trialkoxymethylsilane. This was found to invariably reduce the activity of the supported amine groups. Whether the role of free silanol groups was through an acid-base cooperative catalytic process or whether it was due to the hydrophilicity they impart to the catalyst surface was investigated by altering the polarity of the reaction solvent/solution. The conclusion was that the role of free silanol groups is to provide mildly acidic groups in a cooperative mechanism rather than simply through control of surface hydrophilicity/hydrophobicity.

No description available.

Fluorinated compounds make up a large proportion of the output from both the pharmaceutical and agrochemical industries. However, there are still a limited number of methodologies involving the synthesis of such molecules. Six-membered nitrogen containing heterocycles such as pyridines and lactams are key compounds for medicinal chemistry. Therefore, there is much interest into the synthesis of fluorinated nitrogen heterocycle libraries which can be utilised effectively for numerous substrate screens. This thesis aims to produce a novel route for the generation of a host of fluorinated pyridines, lactams and piperidines. Work on non-fluorinated analogues has lead to a rapid two-pot process for the production of dihydropyridones, a potential intermediate for the above targets, starting from common aldehydes. This methodology has been utilised in the synthesis of fluorinated lactams through an efficient ring-closing metathesis protocol of vinyl fluoride compounds. A variety of aldehydes, including aromatic, aliphatic and heterocyclic examples, could be converted in 6 efficient steps into novel fluorinated δ-lactams. The final hydrogenation occurred on the opposite face to the R groups giving a single diasteromer in all cases. Taking inspiration from the δ-lactams series, a synthetic route was designed and implemented to grant access to an array of fluorinated polysubstituted pyrroles. The final step involved a novel addition-aromatisation reaction which allowed late-stage variation of the resulting fluorinated pyrroles. Installation of a hydride, n-butyl, phenyl and allyl moieties could be achieved, all in high yields of 75-93%. Finally the synthesis of a number of interesting benzoxazole containing compounds was undertaken to produce a range of novel fluorinated poly(ADP-ribose) polymerase (PARP) inhibitors, yielding a group of potential anti-cancer agents.

Efficient energy storage holds the key to reducing waste energy and enabling the use of advanced handheld electronic devices, hydrid electric vehicles and residential energy storage. Recently, Li-ion batteries have been identified and employed as energy storage devices due to their high gravimetric and volumetric energy densities, in comparison to previous technologies. However, more research is required to enhance the efficiency of Li-ion batteries by discovering electrodes with larger electrochemical discharge capacities, while maintaining electrochemical stability. The aims of this study are to develop new microwave-assisted synthesis routes to nanostructured insertion cathodes, which harbor a greater affinity for lithium extraction and insertion than bulk materials. Subsequent to this, state-of-the-art synchrotron based techniques have been employed to understand structural and dynamic behaviour of nanostructured cathode materials during battery cell operation. In this study, microwave-assisted routes to a-LiFePO4, VO2(B), V3O7, H2V3O8 and V4O6(OH)4 have all been developed. Muon spin relaxation has shown that the presence of b-LiFePO4 has a detrimental effect on the lithium diffusion properties of a-LiFePO4, in agreement with first principles calculations. For the first time, a-LiFePO4 nanostructures have been obtained by employing a deep eutectic solvent reaction media showing near theoretical capacity (162 mAh g–1). Studies on VO2(B) have shown that the discharge capacity obtained is linked to the synthesis method. Electrochemical studies of H2V3O8 nanowires have shown outstanding discharge capacities (323 mAh g–1 at 100 mA g–1) and rate capability (180 mAh g–1 at 1 A g–1). The electrochemcial properties of V4O6(OH)4 have been investigated for the first time and show a promising discharge capacity of (180 mAh g–1). Lastly, in situ X-ray absorption spectroscopy has been utilised to track the evolution of the oxidation states in a-LiFePO4, VO2(B) and H2V3O8, and has shown these can all be observed dynamically.

This thesis is composed of two independent research projects. The first major project discussed is electrophilic halogenation using inorganic halides in the presence of oxidant. This includes the conversion of tertiary propargyl alcohols in to α,α-dihaloketones. In addition, the oxidative bromination of a range of alkylbenzene derivatives using the inexpensive oxidant Oxone and sodium bromide is described with up to 4 C-H bonds being functionalised in this process. The second part of this thesis focuses on using silacyclobutanes in our aim to access new siliconcontaining chemical space. Silacyclobutanes are useful in organic synthesis because of their exciting reactivity based on their high Lewis acidity and ring strain. We describe our efforts at developing catalytic conditions for the Pd-mediated dimerisation of silacyclobutanes, as well as our preliminary results on the nickel-catalysed enantioselective ring expansion of benzosilacyclobutane with aldehydes. After Tamao-Fleming oxidation, this reaction produces extremely useful chiral benzylic alcohols.

Composition-structure-property relationships were studied in glasses of the type used to produce glass lonomer dental cements. These materials are currently being developed as a bone cement in joint replacement surgery. Initially, a simple quaternary (25i02-Al203-CaO-CaF2) glass was produced and was found to undergo minimal fluorine loss. This results in a glass whose composition is reproducible between batches. This composition, however, was too reactive to form a glass ionomer cement. A glass-ceramic approach was pursued to deactivate the glass and following heat treatments, cements were produced whose properties varied depending upon the precise heat treatment used. The heat treatments above the glass transition temperature resulted in amorphous phase separation, probably by a spinodal decomposition mechanism, followed by a two stage crystallization of calcium fluoride (fluorite) and anorthite. The anorthite phase crystallized from fluorite nuclei and, in effect, crystallization occurred by a bulk nucleation mechanism. The glass will also undergo significant crystallization to fluorite below the glass transition temperature, with surface nucleation the dominant mechanism. This explains the deactivation of ionomer glasses used commercially to control the setting properties of dental glass ionomer cements. The structural role of fluorine in these glasses was also investigated. It seems that fluorine is present in the glass network, rather than existing as a discreet entity bonded to a modifying cation. There is evidence that the fluoride ions are bonded only to aluminium sites and act to disrupt network connectivity. A commercial glass was examined and was found to be clearly phase separated. This glass was found to crystallize to apatite and mullite. Apatite is the mineral phase of bone and enamel and this may explain the excellent biocornpatability of glass ionomer cements. Glasses containing sodium and phosphate ions have also been produced. The sodium containing glasses were all found to crystallize from the melt to calcium fluoride. The phosphate containing glasses formed cements without any subsequent modification, and following a simple heat treatment, all of the phosphate containing glasses were found again to crystallize initially to an apatite phase. Selected compositions additionally crystallized to mullite. These glass-ceramics were tougher than the base glasses and this is attributed to the presence of interlocking needles of the two phases. Some of the glasses were easily castable to produce optically clear glasses, and in addition the glasses underwent bulk nucleation via prior amorphous phase separation, to give needles of apatite and mullite. These glass-ceramics thus show tremendous potential for use as a bloactive bone substitute material or as a castable crown.

An ever increasing incidence of diabetes has resulted in an epidemic of chronic wounds. Few therapies effectively promote wound healing, and thus, health care budgets are faced with an unsustainable burden and patients with poor quality of life. Recently, the isoleucine-glycine-aspartic acid (IGD) tripeptide motif of Migration Stimulating Factor (MSF) has been found to be responsible for the wound healing properties of this protein. Modelling studies of the IGD motif of MSF have lead to the design and synthesis of a benzodiazepinone core, which displays wound healing properties in vitro and in vivo. This thesis discusses the optimisation of the synthetic route to this exciting IGD peptidomimetic and investigations into the activity and the method of action of this molecule through scratch assays and qRT-PCR studies. Analogues of this benzodiazepinone with varying synthetic handles have been developed and used to tag the active bicyclic core with desired technologies. In one such example, BODIPY tagging and confocal microscopy allowed the investigation of fibroblast uptake of the BODIPY IGD peptidomimetic. An IGD peptidomimetic with a triethylene glycol synthetic handle has been developed for increased hydrophilicity and is envisioned to be used in the development of a therapeutic hydrogel.

Oligonucleotides are synthesised almost exclusively via the solid-supported phosphoramidite method. However popular this method may be, the expensive reagents used in large excess during the synthesis as well as the large amounts of organic and aqueous solvents and purification steps makes the scale-up of oligonucleotide synthesis costly and possibly harmful to the environment. The therapeutic use of anti-sense oligonucleotides (ASOs) is hindered by their susceptibility to nuclease catalysed hydrolysis and to overcome this problem ASOs have been modified commonly by the introduction of a phosphorothioate backbone. This research aims to provide a better understanding of some of the more problematic stages of the synthesis cycle, the formation of the sulfurizing agent and sulfurisation of inter-nucleotide phosphite linkages, in order to make this method more sustainable and efficient. The investigation of the activation, alcoholysis and hydrolysis of the phosphoramidites 2´-methoxy-5´-O-DMT-uridine 3´-CE phosphoramidite (UAm) and di-tert-butyl N,N-di-isopropyl phosphoramidite (DBAm) using several tetrazole activators found that complete conversion of the phosphoramidite UAm to products required an excess of activator and that this was due to the generation of di-isopropyl amine during coupling. Conductivity measurements show that the amine deprotonates the acidic activator and that the ammonium and tetrazolide ions that are subsequently formed strongly ion pair (Kip = 6540 M-1) removing free activator from solution. The tetrazole-catalysed reaction of phosphoramidites with oxygen nucleophiles was found to be first order with respect to phosphoramidite and activator and the nucleophilic displacement of the di-isopropyl amine group by the tetrazoyl group at phosphorus is rate-limiting. Investigation into the 3-picoline-catalysed ageing of the sulfur transfer reagent phenylacetyl disulfide (PADS) has shown that the process is overall second order and is proportional to the concentration of PADS and 3-picoline. Deuterium exchange experiments show that ageing proceeds via abstraction of the methylene CH2 protons of PADS via an E1cB-type decomposition of the PADS molecule generating a disulfide anion and a ketene by-product which was trapped using an intra-molecular [2+2]-cycloaddition reaction. Mass spectrometry data shows that disulfide anions act as nucleophiles with PADS molecules to generate polysulfides which are the active sulfur transfer reagents in aged PADS solutions. Using pyridines that are less basic than 3-picoline causes the rate of degradation of PADS to become slower, indicating the possibility that the rate-limiting step of this process is the generation of the disulfide anion. The rate of sulfurisation of phosphites by both ‘fresh’ and ‘aged’ PADS in the presence of 3-picoline was found to be first order with respect to phosphite, PADS and 3-picoline at low concentrations of each. However, the rate of the reaction becomes independent of base when using aged PADS in the presence of high 3-picoline concentration. Brönsted correlations for the sulfurisation of alkyl phosphites using fresh PADS give a βnuc value of 0.51, consistent with a mechanism involving nucleophilic attack by the phosphite on the disulfide bond of PADS to generate a phosphonium ion intermediate. This degrades to the phosphorothioate product via a base-catalysed mechanism which has been confirmed by removal of the methylene protons from the PADS molecule. Comparison of the βnuc values seen when altering the pKa of the pyridine catalyst used shows that the rate of the reaction of fresh PADS is much more sensitive to the pKa of the pyridine than is aged PADS (βnuc = 0.43 and 0.26 for fresh and aged PADS respectively). This suggests that in the case of aged PADS, the phosphite attacks the sulfur atom adjacent to the carboxylate group in the polysulfide chain. This generates a phosphonium intermediate which can be broken down via a much more facile S-S bond fission, as opposed to the C-S bond fission as seen in when using fresh PADS.

Healthcare associated infections are a major concern within the health services as they inflict a significant financial burdens and time constraints on the healthcare system. Effective skin antisepsis prior to incision of the skin, for example, during surgery, is essential in preventing subsequent infection. Current evidence-based guidelines recommend the use of 2 % (w/v) chlorhexidine digluconate (CHG), preferably in 70 % (v/v) isopropyl alcohol (IPA) prior to incision of the skin. However, many antimicrobial agents poorly permeate into the skin and microorganisms residing in the deeper layers and around hair follicles, may survive the procedure and cause infection. Lipid-based nanocarriers are promising drug delivery system with the potential to improve chemical stability, control drug release and alter drug pharmacokinetics. In present study, the ability of lipid-based nanocarriers to enhance the skin retention of antimicrobial agents was accessed. The solid lipid nanoparticles (SLNs) and nanoemulsions (NEs) of triclosan (TSN) and chlorhexidine digluconate (CHG) were prepared and compared based on their physicochemical parameter and better skin retention properties. SLNs of TSN was prepared using glyceryl behenate (GB) and glyceryl palmitostearate (GP) solid lipids, while NEs of TSN and CHG were prepared using eucalyptus oil (EO) and olive oil (OO) with combination of surfactants Tween® 80 and Span® 80. Characterisation and optimisation of SLN and NE formulations to find better skin retention ability is described with various other studies within this thesis. Skin permeation of TSN and CHG was subsequently investigated by in vitro Franz diffusion model using artificial membrane and full thickness porcine ear skin and the penetration profile were determined by differential stripping technique to quantify the amount of drug retained within skin. In both SLN and NE formulations, no detectable level of TSN and CHG was found in receiver medium through full thickness porcine ear skin in 24 h, which is advantageous for topical drug delivery system. SLNs prepared with GP, as solid lipid was able to produce smaller size formulation along with better skin penetration compared with GB-SLNs formulation. SLNs and NEs of TSN was analysed and compared for enhanced skin retention properties. The results demonstrated a significantly enhanced skin penetration of TSN for NE formulations compared to SLNs, which might be due to difference in composition and physical state of lipids and physicochemical parameter of formulations. In case of CHG-loaded NEs, the results demonstrated EO show better skin penetration compared to OO formulations due to its skin penetration enhancing property, which might be beneficial for skin antisepsis prior to invasive procedure to reduce the microorganisms on and within the skin. However, further studies are required to study antibacterial effects of nanoformulations against various skin microorganisms, to analyse skin permeation and retention ability of prepared nanoformulations in in vivo diffusion studies and further studies to analyse toxicity and skin tolerance of EO alone or in combination with antimicrobial agents.