Every biological cell is surrounded by a membrane, which functions as a barrier to the environment and as a support matrix for membrane proteins. Membrane proteins facilitate the transport of manifold substrates across the membrane and are involved in fundamental cellular processes, such as signalling or energy generation to name a few. The key to the function of membrane proteins lies in their three dimensional structure, which can be determined by single crystal X-ray crystallography. However, membrane proteins are one of the most difficult protein classes to work with, which is reflected by the small number of available membrane protein structures. Protein crystallography requires milligram amounts of pure protein, which has to be expressed and purified to monodispersity to allow crystallisation. As membrane proteins have to be inserted into the membrane, recombinant expression yields are often low. In order to obtain enough protein for purification and crystallisation studies, the expression of membrane proteins requires screening for the best expression conditions. Purification of membrane proteins requires, due to their amphipathic character, the use of detergents to solubilise the membrane protein. The optimal combination of detergent and membrane protein is crucial for stability in aqueous solution in order to allow purification to monodispersity. Furthermore, the detergent has a high influence on the crystallisation of membrane proteins. An approach to overcome the challenges of membrane protein structural biology is to work in a high throughput (HTP) manner to increase the chances of success. The aim is to find the most promising targets out of a library of membrane proteins and in the presented work a small-scale HTP expression screen was developed in order to find the optimal expression conditions for each membrane protein from a target library of 12 E. coli inner membrane proteins. The targets were then expressed in the determined optimal conditions in sufficient amounts to allow purification. All membrane proteins were subjected to a purification pipeline, which employed a subset of parameters, that have proved to be the most successful to date in membrane protein purification for structural studies. Five membrane proteins were purified to monodispersity and were submitted to crystallisation trials. Crystals of two targets were obtained, which diffracted to 7 Å and 15 Å. Furthermore, the data collected on the expression and purification behaviour of the 12 membrane proteins, will help to optimise the starting parameters for the screening of future targets.

Asymmetric allylation of aldehydes with allyltrichlorosilane reagents, in recent years, has become a powerful synthetic tool towards the enantioselective construction of homoallylic alcohols. In general, the reaction displays good diastereocontrol. Thus, when the allylation is carried out in the presence of a Lewis base, the homoallylic alcohols anti-3 and syn-4 are stereospecifically obtained from the (E)-2 and (Z)-2 silanes, respectively, indicating that the reaction is likely to proceed via a cyclic, chair-like transistion state. Herein, the synthesis of isomerically pure allylsilanes 2, functionalised in the γ-position is reported. This has enabled the range of valuable synthetic intermediates available via the asymmetric allylation reaction of various aromatic aldehydes to be extended. The resultant homoallylic alcohols have two new stereogenic centres. These molecules can now undergo an intramolecular SN2 reaction to afford the corresponding vinyl epoxides 5 and 6 with retention of the relative stereochemistry. A variety of chiral Lewis bases, including pyridine N-oxides and phosphine oxides, were synthesised and screened for asymmetric induction. The most notable result was achieved using chiral phosphine oxide BINAPO, which produced the syn-homoallylic alcohol 4 in 50 % ee.

Reported herein is a conceptually new synthetic route to 1’-aryl C-ribofuranosides and their 2’,3’-didehydro 2’,3’-dideoxy (D4) analogues. We have successfully implemented a divergent synthetic route capable to reach two important, biologically significant groups of compounds. The first two strategic transformations are common for both families of target compounds (asymmetric allylic substitution, and ring-closing metathesis). D4 C nucleoside analogues are synthesised in a three step procedure, and 1’-aryl ribofuranoses are constructed in a four step procedure. The target compounds were prepared in an excellent enantio and diastereopurity, in good overall yields. The yield in the synthesis of 1’-aryl-2’,3’-didehydro-1’,2’,3’-trideoxyribofunanoses is up to 46% over all the reaction steps. The overall yield of the 1’-arylribofuranoses is up to 42%. All the strategic transformations rely on catalytic oranometallic reactions employing group 8a transition metals. All the reactions have been optimized with a view of maximal atom efficiency and environmental impact. In summary, our new methodology is perfectly suitable for the synthesis of 1’ arylribofuranoses, and their D4 analogues, bearing non-ortho-substituted aromatics and hereroaromatics, lacking coordinating (nitrogen) substituents or heteroatoms. In this point of view the most promising target application is the synthesis of lipophilic isosters of ribonucleosides for the RNA studies.

Surfactant activity is a property more commonly associated with small molecules than biological macromolecules. However, the significant advantages of improved biodegradability and biocompatibility that could be presented by natural surfactant proteins has elevated interest in a group of only a few proteins where intrinsic surfactant activity appears to be the primary function. Two examples of this group, ranaspumin-2 (Rsn-2) from the foam nests of the tungara frog and latherin, the surface active protein component of horse sweat, appear to be different from other surfactant proteins in the form of their activity. However, the exact molecular basis of this activity is poorly understood. This thesis describes work to rationalise surface activity and related properties in these unusual proteins. The properties of Rsn-2 and latherin including surface activity, interaction with lipid membranes and behaviour in solution were investigated to provide further insight into the characteristics that distinguish the surfactant proteins from both conventional surfactants and other proteins. A second protein component of the foam nests, ranaspumin-1 (Rsn-1), of previously unknown function was also found to be highly surface active and is proposed to function in a similar manner to Rsn-2. A model whereby Rsn-2 functions via a clamshell-like opening was tested through a combination of specialised NMR techniques and site-directed mutagenesis. The results identified features associated with surfactant activity, all of which were consistent with the model. The potential of Rsn-2 as a recombinant fusion partner for the production of functional surfactants or foams was proven by construction of a fluorescent conjugate. Solution state NMR was used to determine the structure of latherin. Information on the dynamic processes taking place in the molecule were derived by analysis of NMR relaxation data. The structure revealed is a super roll fold, similar to a single domain of the BPI-like proteins. A model is proposed whereby latherin recognises the air-water interface via three dynamic, hydrophobic loops at one end of its long cylindrical structure and then unfolds to expose its hydrophobic core at the air-water interface.

A fast and efficient synthetic route was developed for the synthesis of a novel class of enone-containing alpha-amino acid. An amino acid-derived beta-ketophosphonate ester was subjected to Horner-Wadsworth-Emmons conditions using a variety of aldehydes to produce a diverse library of alpha,beta-unsaturated amino acids. E-Configured enone-containing amino acids were also deprotected using a two-stage approach to give the parent alpha-amino acids. A minor modification to the route enabled the synthesis of Z-configured enones via the Still-Gennari reaction. A small library of Z-enones was produced using various aldehydes. Enone-functionalised alpha-amino acids were employed as substrates for an intramolecular cyclisation reaction to generate 6-substituted-4-oxopipecolic acids. A diastereoselective one-pot reductive amination/cyclisation strategy was developed to gain access to the anti-diastereomer of the chiral N-heterocycles. A small selection of 6-substituted-4-oxopipecolic acids was synthesised. 6-Substituted-4-oxopipecolic acids were also reduced diastereoselectively to generate 4- hydroxypipecolic acid analogues.

Industrial reactions can either be catalysed by acidic, basic or neutral supported catalysts. The work within this thesis includes two different projects of industrial interest, both of which are catalysed by basic and acidic supported catalysts. (1) Acrylonitrile is one of the top twenty large-volume commodity chemicals in the world. Nearly every person in the modern world owns something that is made of acrylonitrile. Currently acrylonitrile is synthesised industrially by the ammoxidation of propylene. During this process acetonitrile is produced as by product and is used commercially as a solvent. However, the production of acetonitrile is far greater than demand therefore considerable interest lies in the conversion of acetonitrile to acrylonitrile. In our studies the synthesis of acrylonitrile from methanol and acetonitrile was attempted using magnesium oxide and chromium-doped magnesium oxide catalysts. The catalysts were initially prepared by impregnation methods and then subsequently characterised. It was found that an impregnation of the magnesium hydroxide by chromium salt decreased the phase transformation temperature from magnesium hydroxide to magnesium oxide and yielded larger crystallite sizes. Using the chromium-doped magnesium oxide catalyst the reaction between acetonitrile and methanol gave 100% selectivity towards acrylonitrile. It is suggested that CrVI/V species play an important role in this reaction and act as a stabiliser for the acetonitrile carbanion. Further study showed that the main deactivation route was the reduction of the chromium from CrVI/V to lower oxidation states and the deposition of coke. It was found that over the course of a year the Cr/MgO catalyst significantly aged. Because the extent of ageing was so significant, it was decided to cease work on this project as it was of concern that the relationship between structure and activity would be difficult to rely on. (2) Hydrogen is one of the clean sources of energy which is currently obtained by the steam reforming of non-renewable fossil-fuel resources. However the rapid depletion of fossil-fuel resources has spurred further research into alternative and renewable H2 sources. Among the many different renewable sources available for H2 production, the steam reforming of bioethanol has attracted significant interest in recent years. However, crude bioethanol contains organic impurities which may deactivate the catalyst more rapidly than the pure ethanol. Therefore in the current project we have examined the tolerance of pure Al2O3 and Al2O3 supported noble metal (Rh, Ru and Pt) catalysts to the different impurities present in crude bioethanol. The direct use of crude bioethanol in the steam reforming reaction could result in a huge saving in capital expenditure for an industrial plant, as huge capital costs are associated with the distillation of the crude bioethanol. In the initial stage of the project, the Al2O3 and the noble metal impregnated Al2O3 catalysts were tested over a range of temperatures, under 20 barg pressures and a 5:1 steam to ethanol ratio. This was to determine the optimum temperature of reaction. A temperature of 500oC was found to be the optimum reaction temperature due to “hard” coke formation at higher temperatures over the Ru and Rh catalyst. The effect of the different impurities was examined by systematically adding 1mol.% of each impurity separately with respect to ethanol content in the water/ethanol mixture. The different noble metal catalysts showed similar tolerances towards the impurities. The addition of C3 alcohols significantly decreased the conversion of ethanol and increased the rate of catalyst deactivation. This deactivation of the catalyst in the presence of C3 alcohols was attributed to high olefin formation and incomplete decomposition of the C3 alcohols which deposited over the catalysts as coke. Separate propanal, propylamine and acetone addition to the water/ethanol mixture significantly increased the ethanol conversion and the activity of all the noble metal catalysts tested. It was found that the presence of these impurities in the ethanol significantly decreased the C2H4 in effluent mixture as these impurities blocked the acidic sites of the catalysts. The compound C2H4 was found to be the main route towards coke formation.

The research described herein focuses on the synthesis of chiral pinene-pyridine derivatives, and their application in asymmetric catalysis. Both transition metal catalysed and organocatalytic transformations were investigated. Chiral pyridine-phosphines based on α-pinene were synthesised and applied as efficient P,N-ligands for the enantioselective palladium(II)-catalysed Baeyer-Villiger oxidation of prochiral 3-substituted cyclobutanones to furnish chiral γ-butyrolactones in up to 81% ee. Complexes of these ligands with iridium can also promote asymmetric hydrogenation of olefins in up to 83% ee. Novel N,N’-dioxides were synthesised from α-pinene and a range of 2-pyridine-acetophenones by employing Kröhnke annulation reaction as the key cyclisation step. Although poor enantioselectivity was achieved (up to 12% ee), high reactivity of the catalysts is, however, promising.

The main focuses of this project have been investigations of a variety of hydrogen bonding systems for unusual behaviour such as disordered or migrating hydrogens/protons with both single crystal X-ray and neutron diffraction, crystallisation of a large number of molecular complexes of the chloranilic acid molecule, and examining the bifurcated hydrogen bond motif found in many of the chloranilic acid co-crystals discovered. The neutron single crystal diffraction instruments SXD and VIVALDI have been used to provide conclusive results in cases of suspected unusual hydrogen bond behaviour in molecular materials. 2,4-dihydroxybenzoic acid and its isomer 2,5-dihydroxybenzoic acid have been examined using X-ray and neutron diffraction to investigate possible disordered cooperative hydrogen bond systems. The energy difference of the three possible tautomers of 2,4-dihydroxybenzoic acid in different environments have been calculated in theoretical computations which concur with the neutron results. Single crystal neutron diffraction experiments were also carried out on isonicotinamidium formate, 2-iodoaniliium picrate, chloranilic acid 2,4-lutidine and malonic acid, where unusual behaviour in the hydrogen systems was also suspected. The molecular complexes of chloranilic acid with various pyridine-based molecules have been the main focus of the X-ray diffraction work of this thesis. Multiple crystallisations over a range of different conditions were set up for chloranilic acid with various series of molecules including lutidines (dimethylpyridines) and picolines (methylpyridines). This resulted in a large number of new crystal structures, determined by X-ray diffraction and all found to contain a bifurcated hydrogen bond motif producing two robust hydrogen-bonded supramolecular synthons. The investigation examines the bifurcated hydrogen bond interactions and the suitability of chloranilic acid complexes for crystal engineering. The two related supramolecular synthon units are discussed and difference Fourier maps and Hirshfeld surfaces used to examine the hydrogen bond architecture. Bromanilic acid co-crystals are also studied to examine the effect of the halogen in the crystal structures.

More than 11 million people worldwide are diagnosed with cancer every year. New cancer drugs are required that are more effective and selective. Nitrogen mustard alkylating agents crosslink DNA inhibiting transcription and replication. Use of the mustard pharmacophore as part of a macrocycle allows metal complexation and produces a prodrug. Hypoxic tumour cells have increased concentrations of reductase enzymes which could lead to reduction of the complex in situ and release of a cytotoxic drug. Human African Trypanosomiasis is commonly known as Sleeping Sickness and affects over 36 countries of sub-Saharan Africa. It is transmitted to humans by the tsetse fly which carries the parasitic subspecies Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. Any compounds synthesised would also be tested to assess their potential as anti-parasitic agents. Parker synthesised a range of polyazamacrocycles. Testing of compound A in vitro gave highly efficient DNA crosslinking activity. Copper complexes were formed of the macrocycles and B was found to be 24 times more toxic against hypoxic cells than oxic cells thus exploiting tumour hypoxia and creating a selective drug. Jones synthesised a range of oxaazamacrocycles such as C which when tested in vitro exhibited comparable cross-linking activity to the azamacrocycles although it proved impossible to synthesise the corresponding copper complexes. It was decided to vary the leaving group on the alkylating arms to see if the DNA crosslinking results could be improved. Eight carbamates and the corresponding copper complexes were synthesised. The R-groups were alkyl and aromatic. Anti-cancer DNA crosslinking and hypoxia selectivity results were disappointing however, a number of compounds displayed significant activity when tested against T. brucei. A range of thiaazamacrocycles would complete the set of heteroatom-containing macrocycles (N, O, S) and might produce good DNA crosslinking results. It might also be possible to synthesise the corresponding copper complexes producing prodrugs. Six thiaazamacrocycles were synthesised and 2-hydroxyethyl arms were attached. However it proved impossible to isolate the desired alkylating agents with the 2-chloroethyl arms. In the body, the p53 protein activates the transcription of specific genes. In healthy cells, the levels of p53 have to be kept to a minimum to allow the normal running of the cell, e.g. growth and replication. This function is carried out by the HDM2 protein, which forms an auto-regulatory feedback loop with p53. In some tumours, the p53 function is disrupted due to genetic mutations of p53. However other tumours possess ‘wild type’ p53 – this type of p53 has lost the ability to respond to oncogenic stress due to over-expression of HDM2. Drugs that inhibit HDM2 should cause stabilisation of p53 and induce apoptosis in cancer cells. A small library of 5-deazaflavins were synthesised and biologically tested producing some interesting biological results.

Abstract: Herein, we report the synthesis of new chiral hydroxamic acid ligands for the V-catalysed asymmetric epoxidation reaction in aqueous solution. During the course of this project, a wide range of allylic alcohols were epoxidised under mild reaction conditions. The target epoxides were isolated in good-to-excellent yields with up to 94% ee. To further develop the project, we also report that the range of substrates has been extended to unfunctionalised alkenes. These materials have been successfully converted to the corresponding allylic alcohols in the presence of SeO2 and acetic acid. Asymmetric epoxidation was then achieved under anhydrous conditions in good-to-moderate yields with up to 76% ee and in certain cases, the overall transformation can occur as a one-pot process. A brief investigation into the development of organocatalytic transfer hydrogenation mediated by chiral pyridines has also been carried out. In this project, a range of chiral quarternary ammonium salts have been prepared as precursors to the corresponding dihydropyridines. It was our initial intention to utilise these chiral dihydropyridines in the enantioselective reduction of imines. This would result in the formation of the desired chiral amine and a quaternary pyridinium salt, which could then be reduced to reform the dihydropyridine in a catalytic cycle. However, with all pyridinium salts formed we were unable to produce the target dihydropyridines and this project was consequently abandoned.