No description available.

Thesis (M.Sc. (Chemistry)) - Rhodes University, 2008

This thesis concerns the electrochemical investigation of high-potential laccases. These multicopper oxidases are efficient electrocatalysts for the dioxygen reduction reaction. A method for stabilising laccase on a graphite electrode was established. The method involved modification of the graphite surface by diazonium coupling of a 2-anthracene molecule. A laccase ‘film’ adsorbed on this modified surface remained stable for over two months and, typically, the current density for dioxygen reduction was doubled compared to a laccase ‘film’ on an unmodified surface. Protein film voltammetry was used to investigate thermodynamic and kinetic aspects of the electrochemical behaviour of laccase. The effect of inhibitors on the magnitude of reduction current and the position of the wave (related to the overpotential for the reaction) was also studied. Fluoride, chloride and azide showed different modes of inhibition and inhibition constants ranged from micromolar for azide to millimolar for chloride. In cyclic voltammetry experiments it was only in the presence of high concentrations of the inhibitors fluoride, chloride and azide that a non-turnover signal, corresponding to a one electron transfer process, was revealed. The evidence suggested that the non-turnover signal arose from interfacial electron transfer between the electrode and the type 1 or ‘blue’ copper. Evaluation of the peak areas allowed determination of the catalytic rate constant, kcat, as 300 s–1, and the electroactive surface coverage as four pmol cm–2. The rate of interfacial electron transfer was rapid enough to not limit catalysis at high overpotentials. A spectroelectrochemical cell was designed to investigate the behaviour of the type 1 copper in the presence of inhibitors and at different pH values. The inhibitors fluoride, chloride and azide had little effect on the reduction potential of the type 1 copper, but at higher pH values the reduction potential of the type 1 copper was decreased.

Hydrogenases catalyse the interconversion of H₂ and H⁺. Protein Film Electrochemistry (PFE), a technique in which a redox enzyme is adsorbed directly onto an electrode, enables a detailed description of the catalytic function of these metalloenzymes to be obtained. Unlike small-molecule electrocatalysts, the hydrogenase active site is surrounded by a protein structure ensuring that it is relatively unperturbed by the electrode surface. In this thesis, PFE is used alongside mathematical modelling to explain differences between [NiFe]- and [FeFe]-hydrogenases, highlighting some important considerations for efficient, reversible electrocatalysis. This thesis probes the unusual reaction between [NiFe]-hydrogenases and cyanide. Through a detailed study utilising PFE, Electron Paramagnetic Resonance (EPR) and Attenuated Total Reflection Infrared spectroelectrochemistry (ATR-IR), it is demonstrated that cyanide promotes the formation of the inactive Ni-B state. Preferred formation of the Ni-B state over more slowly reactivating Unready states is considered an important characteristic of the O₂-tolerant class of [NiFe]-hydrogenases. The nature of the Ni-L state, commonly thought to be an artefact formed when a [NiFe]-hydrogenase is exposed to visible light, is probed via EPR and ATR-IR. In this thesis, the Ni-L state is shown to occur in samples of Hydrogenase-1 from Escherichia coli that have not been exposed to visible light, calling into question the true nature of this state. Finally, this thesis details the first study in which PFE is used to investigate the spontaneous incorporation of a synthetic active site mimic complex into apo-hydrogenase. Incorporation into apo-hydrogenase from Chlamydomonas reinhardtii and Clostridium pasteurianum is discussed, in both cases resulting in fully functional [FeFe]-hydrogenase, electrochemically indistinguishable from the native enzyme.

Nicotinamide cofactors in their reduced and oxidised forms are important redox agents in biology. Of about 3000 dehydrogenases available to date, many require these cofactors for their activity. Dehydrogenases are of interest to chemists as they offer asymmetric catalysis to yield chiral products. The requirement of dehydrogenases for nicotinamide cofactors necessitates research into finding the best way of recycling the oxidised or reduced forms of these cofactors. Electrocatalytic NAD(P)H oxidation and NAD(P)⁺ reduction on standard electrodes is problematic due to unwanted side reactions and high overpotential requirements, but in Nature efficient enzyme catalysts are available to facilitate these reactions. The focus of this Thesis, the Soluble Hydrogenase of R. eutropha (SH) is a multimeric bidirectional hydrogenase that couples H2 oxidation to the reduction of NAD⁺ to NADH. Protein Film Electrochemistry (PFE) has been employed to study NAD⁺-reducing catalytic moieties of the SH for the first time. It is shown that SH subunits on an electrode are able to catalyse NADH oxidation and NAD⁺ reduction efficiently with minimal overpotential, which is significant because in vivo, NAD(H) cycling is coupled to 2H⁺/H₂ cycling and these reactions are closely spaced in potential. Substrate affinities and inhibition constants for the SH, determined using PFE are discussed in the context of the SH function and the related catalytic domains of respiratory Complex I. A range of molecules that are known to inhibit the related Complex I have been investigated for their ability to inhibit the SH moieties: the similarity between inhibition constants is consistent with structural and functional similarity between the SH and Complex I. The ability of the SH moieties to sustain NAD(H) catalysis in the presence of O₂ is also demonstrated and is consistent with the requirement for the SH to function under aerobic conditions and to reactivate the inactivated hydrogenase moiety by supplying low potential electrons from NADH. Engineered variants of the SH, designed to enhance the affinity towards NADP⁺, were investigated for the first time, using PFE. Electrochemical characterisation of the variants is presented and results are discussed alongside findings on the wild type SH. The variants are shown to exhibit NADP⁺ reduction, and to have higher affinity towards NADP⁺ than the wild type SH. The first efficient NADP⁺ reduction and NADPH oxidation is observed for one of the variants on a graphite electrode and the best variant showed a K_M of 1.7 mM for NADP⁺. This Thesis also provides evidence for the ability of moieties of the SH to be used in cofactor regeneration systems. Two novel systems are demonstrated. The first involves H₂ driven NADH recycling based on the NAD⁺-reducing moiety of the SH immobilised on graphite particles together with a hydrogenase or platinum, with electrons from H₂ passed from the hydrogenase through the graphite to the NAD⁺-reducing moiety. The second involves an electrode modified with the NAD⁺-reducing moiety of the SH, and is demonstrated as an electrochemical NADH recycling system coupled with NADH-dependent pyruvate reduction to lactate by lactate dehydrogenase. The ability of variants of the SH to catalyse NADP⁺ reduction suggests that it may also be possible to use these systems for recycling NADPH for catalysis of important biotransformation reactions by NADPH-dependent dehydrogenases.

This thesis describes the combination of non-stabilized nucleophiles and prochiral/racemic electrophiles in transition metal catalyzed asymmetric transformations. These enantioselective reactions have tremendous potential for the formation of chiral building blocks and new structural motifs that can be found in a variety of natural products and their derivatives. The first part of the thesis focuses on the synthetic approach towards anti-cancer active diterpenoid structures. The two key steps involve a Cu-catalyzed asymmetric conjugate addition of alkylzirconocenes to enones and an intramolecular oxidative cyclisation. Particular investigations into the cyclisation are made with organocatalysis, transition metal catalysis and electrochemistry for the formation of these tricyclic scaffolds. In the second part this work builds on the Rh-catalyzed asymmetric Suzuki-Miyaura coupling of benzeneboronic acids and cyclic allyl chlorides, which has been developed in our group. Here, the main point is to use more challenging coupling partners, such as heteroaromatic boronic acids, which are coupled to racemic cyclic allyl halides. The utility of this method is demonstrated by performing further transformations and an asymmetric synthesis of the natural product (+)- isoanabasine. The last chapter describes the development of a new asymmetric Hiyama coupling of arylsiloxanes with racemic cyclic allyl chloride. Attempts are made to generate substrates that are not accessible via the asymmetric Suzuki - Miyaura reaction. After extensive optimisation a variety of arylsiloxanes is generated and tested with the best conditions to prove its utility in comparison to the asymmetric Suzuki-Miyaura coupling.

No description available.

<p> Brønsted acid zeolites can be synthesized in a wide range of topologies, each characterized by diverse void sizes, shapes, and micropore connectivity. The location of Brønsted acid sites (H+-sites) within microporous voids of different size and shape, and the relative proximity of H+-sites influences their reactivity. Additionally, the diffusion of reactant and product molecules through a given zeolite topology depends on micropore size, tortuosity, and connectivity. The coupled influences of reaction kinetics and intrazeolite reactant and product diffusion govern rates and selectivity for a plethora of zeolite-catalyzed reactions and underlie the well-established effects of “shape-selectivity”. The independent effects of reaction and diffusion on rates and selectivity for a given reaction are often obfuscated by concomitant changes in the zeolite properties governing diffusion (e.g., crystallite size) and reactivity (e.g., H+-site density or proximity) in zeolite materials synthesized with conventional methods. Herein, we develop synthetic methods to decouple H+-site density, proximity and crystallite size in medium-pore, 10-membered ring (10-MR) zeolites, and evaluate the independent effects of these material properties on the kinetic and transport phenomena that govern propene oligomerization catalysis. </p> <p>Among synthetic methods to influence H+-site proximity in zeolites, varying the charge-density and ratio of structure directing agent (SDA) cations that compensate anionic charges in frameworks at Al centers has been reported to influence H+-site proximity in MFI and CHA zeolites of fixed H+-site density. Changes in H+-site proximity can be evaluated using Co2+ cations to selectively titrate and quantify subsets of proximal H+-sites (H+-site pairs); conditions to perform such titrations were identified for MEL zeolites. The fraction of paired H+-sites changed concurrently with changes in framework Al content in MEL zeolites synthesized using a single organic SDA (OSDA), tetrabutylammonium hydroxide (TBA+). Synthesis of MEL with mixtures of TBA+ and Na+ as an inorganic SDA (ISDA), at fixed total SDA and Al content, allowed the fraction of paired H+-sites to be systematically varied in MEL zeolites of fixed H+-site density, reflecting changes in the location and quantity of charge-balancing SDAs with Na+/TBA+ ratio. The energetic favorability of SDA occlusion in MEL was also evaluated with density functional theory (DFT). In contrast to MEL, occluded SDA content in TON zeolites crystallized with varied OSDA (1,6-diaminohexane, or 1,8-diamooctane) and K+ content, at fixed total SDA content, was invariant with K+/OSDA ratio, reflecting a different mechanism of SDA occlusion in TON. These findings provide an approach to influence H+-site pairs in 10-MR zeolites of fixed H+-site density and demonstrate the dependence of SDA occlusion on zeolite topology.</p> <p>The independent influences of H+-site and crystallite properties on rates and selectivity of propene oligomerization to heavier alkenes in a representative medium-pore zeolite topology (MFI) were explored by interrogating suites of samples crystallized with independently varied H+-site density (0.3–5.7 H+/u.c.), proximity, and crystallite size (0.03–2.65 μm) over a wide range of reaction conditions (483–523 K, 7–615 kPa C3H6). Dimerization rates (per H+) decreased with increasing crystallite size among MFI materials synthesized with fixed H+-site density (0.3 or 1.3 H+/u.c.), revealing the strong and ubiquitous influence of intrazeolite diffusion limitations on measured dimerization rates. Weisz-Prater criterion analyses, in conjunction with dimerization rate transients upon step-changes in reaction conditions, indicate that these intrazeolite diffusion limitations arise from a product-derived organic phase occluded within zeolitic micropores during propene oligomerization catalysis, which restricts intrazeolite diffusion by lowering the effective diffusivities of propene and product alkenes. This occluded organic phase becomes heavier in composition at higher propene pressures and lower reaction temperatures, which favor chain growth over β-scission, resulting in more severe intrazeolite diffusional constraints. The composition of the occluded organic phase was also found to depend on H+-site density in MFI zeolites. Rate constants (per H+) of dimerization and trimerization were higher on MFI samples of dilute H+-site density, resulting in faster growth of heavier oligomer products and consequently lower effective diffusivities compared to MFI samples of higher H+-site density. The convoluted influences of reaction and diffusion on measured propene oligomerization rates result in apparent reaction orders that deviate from the first-order dependence of rates on propene pressure expected in the limit of strict kinetic control. Accounting for the coupled influences of reaction and diffusion on propene oligomerization rates and the influence of H+-site density on intrazeolite diffusion, rationalizes contradictory conclusions among prior reports about the dependence of oligomerization rates on H+-site density, proximity, and crystallite size, which did not identify or consider the influences of intrazeolite diffusion in their interpretations of rate data. </p> <p>Finally, we explore the consequences of zeolite pore size and connectivity for reactivity and intrazeolite diffusion during propene oligomerization by interrogating H-zeolites of different topologies. Intrazeolite diffusional constraints are imposed by an occluded organic phase and influence dimerization rates among medium-pore zeolite topologies (MFI, MEL, TON), but such constraints are alleviated on large-pore zeolite topologies (FAU, MOR, *BEA), reflecting the slower growth and faster diffusion of heavy oligomer products in large-pore zeolites. Among medium-pore zeolites, the composition of the occluded organic phase, and consequently the effective diffusivities of propene and product alkenes, is influenced by void size. Analysis of product selectivity on zeolites of different pore size and connectivity (TON, MOR, MFI) reveals that TON restricts the growth of heavier oligomer products, resulting in effective diffusivities that are higher on TON compared to MFI, and are relatively invariant with propene pressure and H+-site density. Together, the findings herein demonstrate the ability of slow-diffusing products to impose intrazeolite diffusional constraints on other products during alkene oligomerization catalysis, and reveal the critical influence of reaction conditions, H+-site density, and micropore size on the composition of this occluded organic phase, and consequently intrazeolite diffusional constraints. Ultimately, this work demonstrates how kinetic studies performed on well-defined zeolite materials can reveal important changes in reaction and diffusion phenomena, which are otherwise inextricably convoluted, and provides a framework through which such effects can be assessed for other zeolite-catalyzed molecular chain-growth reactions. </p>

The use of H2 as an energy carrier has in recent years been identified as a promising future solution to the current energy crisis. Hydrogenases are metalloenzymes found in many microorganisms and are used to catalyse the reversible inter-conversion of protons and H2. These enzymes and their synthetic analogues have been recognised as a way to facilitate the use of H2 as a fuel. A major challenge to the future use of these catalysts is their reactions with small molecule inhibitors, such as oxygen and carbon monoxide. Detailed understanding of the structure and catalytic mechanism of these highly efficient catalysts is vital for the design of bio-inspired functional analogues for use in technological applications. In this thesis electrochemical studies of three [FeFe]-hydrogenases are presented, performed using the technique of protein film electrochemistry. The strong potential dependence of the reaction of these hydrogenases with carbon monoxide and formaldehyde is characterised and rationalised. These studies provide compelling evidence for the formation of a previously ambiguous super-reduced state of [FeFe]-hydrogenase at low potential. This state is shown to be active and stable, and it is proposed that this state is involved in catalytic H2 production. This super-reduced state is shown to have a high affinity for the reversible binding of formaldehyde, but a very low affinity for both carbon monoxide and oxygen. Activation of carbon monoxide inhibited [FeFe]-hydrogenase can be very rapidly induced by the application of a sufficiently reducing potential. These enzymes, considered to be oxygen sensitive, are shown to be extremely tolerant to irreversible oxygen damage at very reducing potentials where the super-reduced state is formed.