Protein interactions are crucial for the survival of living organisms. The possibility of mapping the contact surfaces between proteins and their interacting partners is fundamental to understanding the mechanisms involved in the process. For these reasons techniques able to provide structural information on a short time scale and employing small amounts of material are sought after. The work reported in this thesis explores the use of carbene-based labelling in combination with mass spectrometry for protein footprinting and its applications in the study of protein structure and interactions. Studies on the efficiency and selectivity of a novel water-soluble photo-activated probe revealed its superior properties in comparison with diazirine-based reagents previously described for the same application. Using this methodology, the contact surface of the complex between lysozyme and NAG5, a carbohydrate substrate, was accurately mapped. The same technique was successfully employed to shed light on the structural change occurring to USP5, a large multi-domain deubiquitinating enzyme, upon its binding to diubiquitin. The use of carbene footprinting in combination with other biophysical techniques allowed to characterise the spatial arrangement of domains located at a module junction in the large multi-modular gladiolin polyketide synthase paving the way for future efforts by synthetic biologists to hijack the chemistry of this antibiotic-producing multiprotein enzyme to produce novel active compounds. The possibility of using carbene-based footprinting to gain insight into the structure of integral membrane proteins was also explored. The probe introduced in this work exhibited peculiar labelling properties when activated in the presence of a detergent-solubilised membrane protein. The reagent selectively reacted with portion of the protein in contact with detergent molecules showing potential to elucidate the quaternary structure of multimeric membrane proteins.

The stereochemistry of the loss of one enantiotopic C-2 hydrogen atom of malonyl-coenzyme A units during the transformations leading to pencillic acid was undertaken. Aspartic acid stereospecifically deuterated at C-3 via the formation of N-benzyl aspartic acid was attempted. The addition of benzylamine in 2H20 or dioxan across the double bond of maleic or fumaric acids was revealed to be non stereospecific and these results have been tentatively explained. (2S,3R)-[32H1]- and (2S,3S)-(2,3-2H2)-aspartic acids were derivatized to the corresponding deuterated diethyl N-p-toluenesulfonyl aspartates for vibrational infra red circular dichroism measurements. This technique enabled the determination of the absolute configuration at C-2 of [22H2)-aspartic acids. These stereospecifically deuterated aspartic acids were used for biological tracer experiments but no significant deuteration of penicillic acid was achieved. Malate was the next possible precursor to be used. The preparation of stereospecifically deuterated L-malic acid was undertaken via the reduction of cis- and trans-2,3-epoxysuccinate derivatives which had to be prepared enantiomerically pure. New chemical routes were devised for the preparation of diethyl trans- and cis-2,3-epoxysuccinate in high yield. The key intermediates were diethyl 2-tosyl tartrate for the synthesis of cis- and trans- isomers and its tert-butyldimethylsilyl ether for the preparation of diethyl cis-epoxysuccinate. The enzymatic kinetic resolution of diethyl cis- and trans-2,3-epoxysuccinates were attempted by ester hydrolysis but proved to be difficult due to their decomposition in aqueous medium. The transesterification of diethyl trans-2,3-epoxysuccinate with 1 - heptanol was catalyzed by lipases. Diheptyl (2S,3S)- and diethyl (2R,3R)-epoxysuccinates were obtained with an ee ≥ 97%. Diethyl cis-2,3-epoxysuccinate did not undergo transesterification with any of the enzymes tested. Nevertheless, ethyl isobutyl cis-(2,3)-epoxysuccinate was prepared indirectly in enantiomerically pure form. An enzymatic method involved a regioselective and/or stereoselective hydrolysis of racemic or enantiomerically pure diethyl 2-tosyl-tartrate by α-chymotrypsin. The resulting epoxide had an ee of ≥ 97%. Also, a chemical regioselective transesterification of enantiomerically pure diethyl 2-tosyl tartrate with isobutyl alcohol was achieved, leading to epoxides with ee values ranging from 71% to 94%. Thus, it was undoubtedly possible to obtain from these enantiomerically pure epoxides L-malic acid stereospecifically deuterated at C-3. In an attempt to understand the mode of action of pyruvate decarboxylase, an analytical method for estimating the enantiomeric composition of enzymatically produced samples of acetoin was required. Formation of the (S)-(-)-α-methoxy-α-(trifluoromethyl) phenyl acetate of acetoin was achieved using different coupling agents. The diastereomeric ratio could then be determined by HPLC, 1H and 19F NMR, but racemization was found to accompany ester formation.

This thesis is a feasibility study into the application of controlled polymerisation techniques to the production of polymers for use in the biomedical industry and was funded by Biocompatibles Ltd. UK. The focus has been polymerisation of functional methacrylates of interest to contact lens design and in particular the synthesis of polymers with terminal unsaturation (macromonomers). Monomers investigated include tris(trimethylsiloxy)-3-methacryloxy propylsilane (TR1S), 2-(methacryloxyethyl)-2’-(trimethylammoniumethyl)phosphate (HEMA-PC), 2-(2’-hydroxy-5’-methacrylyloxyethylphenyl)-2H-benzotriazole (NORBLOC) and 2-(2-hydroxy-3-/er/-butyl-5-vinylphenyl)-5-chloro-2H-benzotriazole (UVAM). The polymerisation techniques investigated are conventional chain transfer using mercaptans, catalytic chain transfer polymerisation and transition metal mediated living radical polymersiation (TMM-LRP). The application of these polymerisation techniques to the monomers discussed has been successful but not without certain difficulties. The production of macromonomers by the first two methods is relatively well documented however there are no reports of macromonomer production via TMM-LRP and in this thesis several methods of end group functionalisation have been demonstrated.

This thesis is concerned with three distinct but interrelated problems in inorganic photophysics, namely (i) the photosensitisation of an important alkylcobalt(III) complex, (ii) the photophysics and photoelectron transfer reactions of two bis(bipyridine) ruthenium(II) complexes and (iii) a preliminary study of a luminescent binuclear complex of platinum. The first two problems comprise the major part of the work and the literature survey is focused on these. The principal methods of investigation have been 347 nm laser flash photolysis (in emission and absorption), fluorimetry and quantum yield determination. Photosensitisation of Alkylcobalt(III) Complexes: While alkyl- cobalamins and their model compounds, the alkylcobaloximes are known to photodissociate in high yield under visible light irradiation, the multiplicity and energy of the photoreactive state have not been identified. Now acidopentammine complexes of cobalt(III) are known to be photosensitised both by organic triplet states and by the 'solar energy' complex [Ru(bpy)3]2+, indicating the dissociating state to have triplet character and to be of lower energy than signified by the absorption maxima. Accordingly we investigated the possibility of such sensitisers functioning in the case of the organocobalt(III) compounds both by measuring the kinetics of interaction between the donor triplet states and the alkylcobalt(III) compound by laser flash photolysis, which indicated that while donors with ET > 170 kJ mol-1 were quenched very effectively, those with low values of ET (< 110 kJ mol-1), such as rubrene and 6-carotene, where virtually without effect (kq < 107 dm3 mol-1 s-1). The detailed picture, covering a comprehensive range of triplet donors, enables a Wilkinson plot to be compiled and hence a clear picture of the energies of the active states of the alkylcobalt(III) complex. This work was extended to a range of inorganic donors of widely varying reduction potential from which it became clear that the principal process of quenching is energy- rather than electron- transfer (although the latter might participate in favoured cases). A comparative study was carried out on tris-(acetylacetonato) cobalt(III). The laser kinetic studies were augmented by a series of quantum yield experiments using sensitisers with Sj absorption well to the red of the band maxima of the CT transitions of the R-Co(III) species, but with very high values of φT. Photophysics and Photoelectron Transfer Reactions of Bis(polypyridyl) Ruthenium(11) Complexes: We examined the temperature dependence of the luminescence lifetimes of the complexes [Ru(bpy)2acac]+ and [Ru(bpy)2en]2+ from 77 to 300 K with the view of elucidating whether their comparatively very short lifetimes at ambient temperature are due to small activation barriers to deactivation, as shown by two groups for the well-known complex [Ru(bpy)3]2+, or to large pre-exponential terms. In fact the activation barriers are very much lower than for [Ru(bpy)3]2+ and the frequency terms are also somewhat lower, the overall behaviour is thus energy-controlled. However, sizeable solvent-isotope effects (up to ca. 2) are found indicating a considerable amount of CTTS character in the electronic transition responsible for the deactivation step. Studies of the quenching of these same Ru(II) complexes by a wide variety of electron acceptors, combined with electrochemical and lifetime measurements, indicates that whilst the behaviour of [Ru(bpy)2acac]+ follows Weller theory for excited- state electron-transfer very closely, as does [Ru(bpy)3]2+, the result for [Ru(bpy)2en]2+ indicates a generally much faster quenching than predicted for full electron-transfer, indicating the possibility of either non-radiative exciplex formation or an initial one electron oxidation of the ligand, ethylenediamine. Luminescence from a Binuclear Platinum Complex: The complex [Pt(PPh3)2]2 luminesces not only at low-temperature (77 K), t 660 nm in glassy solvents (as recorded before), but also at ambient temperatures at 'v 460—520 nm. The room temperature emission is strongly quenched by dioxygen, which indicates it to be spin-forbidden. Temperature studies indicate that there is no gradual shift in the emission band as the temperature is raised, but rather are two coexistent and distinct emissions at 460-520 and 660 nm.

No description available.

The subject, "Studies On Sialidases", is introduced In Chapter 1 by a review of the history, biological functions and properties of sialidases. The development of a rapid and sensitive (detection limit 10-6 units) assay system for sialidase, using tritiated fetuin labelled at C-8 of its N-acetylneuraminic acid (NANA) units, is discussed in Chapter 2. Included In this chapter, is the design and application of a radioimmunoassay for sialidases, in which antibodies specific for (NANA) have been obtained. These antibodies were raised in sheep challenged with BSA-colominic acid (the colominic acid was hydrolysed to a chain length of approximately 3 NANA units) conjugate. Further, an attempt has been made to determine the mole to mole ratio of NANA units per sialic acid-containing macromolecule. The extensive purification of the three sialidases is discussed in Chapter 3, using a double "affinity" column system and hydroxylapatite with which it was possible to separate the two forms of sialidases found in S. griseus and V. cholerae. The main body of the thesis (Chapter 4), reports on the work undertaken to resolve the controversy pertaining to which amino acids are, or are not, involved in the active centre of sialidases obtained from pathogenic (C. perfringens and V. cholerae) and non-pathogenic (S. griseus) sources. It was found, by chemical modification, that cysteine and lysine amino acid residues do not participate in the catalytic process, whereas on the other hand, arginine, tryptophan and carboxylic amino acid residues have been found to participate in or near the active centre, either by binding or by actual catalysis, during the catalytic process. Further, the involvement of arginine has been confirmed by differential labelling, isolation of the radiolabelled "active site peptide" and amino acid analysis, using phenyl(3H)glyoxal. The synthesis of the latter, via an inexpensive method, is elaborated upon as well. Preliminary chemical modification studies directed at the carbohydrate residues, covalently attached to these enzymes, reveal the possibility of these moieties participating in the maintenance of the three-dimensional structure of sialidase. With the aforegoing information, it is concluded that there is no difference between the active centres of sialidases obtained from pathogenic and non-pathogenic bacterial sources. Further, a catalytic mechanism involving the essential amino acid residues in or near the active site of the sialidases is speculated upon.

Vitamin B12 catalyses a variety of important reactions in metabolic pathways. Impairment of vitamin B12 by man leads to deficiency diseases e.g. pernicious anaemia. Many mechanistic possibilities have been suggested to account for the transformation of organic substrates to products in these enzymic reactions ranging from purely protein mediated reactions to the existence of discrete ionic or radical intermediates. A crucial point in these rearrangements is the possibility of discrete organocorrin intermediates. To investigate this possibility and to investigate reactions of alkyl groups attached to cobalt a series of alkylcobaloximes were studied. Cobaloximes were used as models for Vitamin on account of their similarity, relative ease of synthesis, and easily interpretable Spectroscopic data. The work described in this thesis explores reactions of substituted cyclopropylmethyl- and but-3-enyl- groups attached to cobalt. By use of 13C labelling experiments and kinetic studies on the rearrangement of cyclopropylmethyl- to but-3-enylcobaloxime the mechanism of this rearrangement has been postulated to be a unimolecular reaction involving a homoallylic transition state. 1-Methylbut-3-enylcobaloxime was found to equilibrate with the 2-methylbut-3-enyl isomer, the reaction presumably proceeding through the intermediacy of methylcyclopropylmethylcobaloximes. Both cis- and trans-isomers of the postulated 2-methylcyclopropylmethyl- intermediate were synthesised and it was shown by suitable kinetic study that the cyclopropanes were, indeed, plausible intermediates. (R)- and (S)-1-methylbut-3-enylcobaloximes were found to equilibrate stereospec- ifically with (S)- and (B)-2-methylbut-3-enylcobaloximes, respectively, under catalysis by TFA - and demonstrates the first stereospecific transformation of an organic ligand attached to a metal atom. Reactions of alkyl(pyridine)cobaloximes with TFA were explored and were shown to exhibit a general trend. TFA first protonates a dimethylglyoximato ligand and a subsequent molecule removes coordinated pyridine as pyridinium trifluoroacetate. Excess of TFA causes the eventual precipitation of a red crystalline complex characterised as a novel cis-cobaloxime by single crystal X-ray diffraction study.

No description available.

Furans are important heteroaromatic units that occur as subunits in various complex natural products, biologically active compounds and pharmaceuticals. Due to their pharmacophoric properties they find widespread application e.g. in the drug discovery process. In contrast to the classical condensation based-methods and metal-mediated approaches, organocatalytic methods for construction of furan have been relatively unexplored. The opening chapter details investigation undertaken into furan formation methodology developed within the Clark group. It was determined that the choice of the acid species was vital for proton transfer to ensure clean and effective conversion of the substrates into the desired furans. Studies were carried out using a chiral acid in an attempt to deliver the furan product in an enantioselective manner. Since the formation of a new stereocentre is achieved in this process, we investigated the potential development of a diastereoselective reaction using substrates bearing an existing stereocentre. The original organocatalytic furan synthesis using THT and ynenedione with nucleophiles was successfully expanded by designing a substrate with a tethered nucleophile that initiates a second cyclization to form polycyclic systems. Cyclohepta[b]furans are an important class of organic compounds found in many natural products, pharmaceuticals, bioactive compounds and functional materials. The development of efficient routes for their formation is therefore of great interest to the synthetic chemist. The second chapter details research undertaken towards a new methodology for the construction of cyclohepta[b]furans. Starting from a simple linear ynenedione the cascade reaction affords furans containing a fused bicyclic system which rearrange to cycloheptadienes. Since it has been observed that the cyclisation and rearrangement occurred successfully it was hypothesised that it may be possible to carry out furan formation followed by Cope rearrangement in a one-pot fashion without isolation of the furan intermediate.

Past and present techniques to remove toxic organic pollutants from industrial wastewaters have involved biological, oxidative and thermal treatment, but long biological degradation lifetimes and harmful emissions released via incineration type processes poses an environmental problem. Much of the new and emerging technologies have steered away from chemical treatment and progressed towards more sustainable and environmentally friendly processes. Wet air oxidation (WAO), being one of them, uses air as the oxidant mixed with the wastewater solution to oxidise the pollutants. This technology has evolved over the years to include catalysis (CWAO) which offers a greener and more cost effective form of industrial wastewater treatment. The majority of CWAO studies involve batch treatment in autoclave reactors, but this project’s aim was to make the treatment process continuous, using an active, stable heterogeneous catalyst in a trickle-flow reactor. Phenol was chosen as the model pollutant and the goal was to reduce its concentration from 1000 ppm to below the EPA limit under the least energyintensive conditions possible. The initial stages were made up of commissioning a reactor, followed by catalyst screening and optimisation, which included correlating activity with catalyst structure and composition. HPLC followed by UV detection was used to quantify phenol conversion, while a range of surface and bulk characterisation techniques were used to determine catalyst structure. Of the catalysts screened platinum supported by silicon carbide provided the most successful results in terms of conversion. SiC’s hydrophobic nature limits the wetting experienced during a CWAO reaction; a process that hinders oxygen activation. Doping with ceria improved the catalyst’s performance, allowing the metal loading to be reduced while maintaining high conversion of phenol. However when ruthenium was the active component, the more hydrophilic alumina was the preferred support. The reaction with ruthenium relies more on catalyst wetting as it is already in the oxide form. When tests were subsequently carried out on Pt/alumina catalysts, they confirmed the need to increase the hydrophobicity in order to achieve high activity. It is proposed that when the active sites are metallic, the optimum support surface is highly hydrophobic; whereas when metal oxide provides the active sites, the optimum support surface is hydrophilic. These findings explain how some of the catalytic components contribute towards CWAO’s reaction mechanism, and activity controlled, in a way not yet shown by previous publications.