Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1994. / Vita. Abstract. Includes bibliographical references (leaves 147-156). Also available via the Internet.

Several series of novel diamido, diamino and diimino ligands containing different spacers and heterocyclic donors have been synthesised. The spacers include the flexible biphenyl, the rigid 1,1 O-phenanthroline and various acyclic moieties, while the heterocyclic donors comprise pyridine, imidazole or benzimidazole groups. These ligands have been designed to complex copper and act as biomimetic models of the active site of the enzyme, tyrosinase, and their complexes with copper, cobalt, nickel and platinum have been analysed using microanalytical, IR, UV-Visible and cyclic voltammetric techniques. Attempted reduction of the biphenyl-based diimino ligands resulted in an unexpected intramolecular cyclisation affording azepine derivatives, the structures of which were elucidated with the aid of single crystal X-ray analysis of cobalt and nickel complexes. Computer modelling methods have been used to explore the conformational options of the copper complexes, and to assess the accessibility of the dinuclear copper site to substrate molecules. Computer modelling has also been used, in conjunction with the available analytical data, to visualise the possible structures of selected ligands and complexes. The copper complexes, although predominantly polymeric, were evaluated as biomimetic catalysts using 3,5-di-t-butylphenol and 3,5-di-t-butylcatechol as substrates. Some of the complexes clearly displayed biomimetic potential, exhibiting both phenolase and catecholase activity.

Ergothioneine and ovothiol A are naturally occurring thiol-histidine derivatives. Both of them are suggested to be beneficial to human health. Ergothioneine has anti-inflammation and anti-oxidative properties and ovothiol has anti-proliferative activities. Recently, ergothioneine has been suggested to be also linked to lifespan longevity. For these reasons, there is a need to investigate the mechanisms of ergothioneine and ovothiol biosynthesis is appealing. My thesis work has addressed this gap in knowledge, focusing on the mechanistic investigations of two C-S bond formation enzymes: EanB in anaerobic ergothioneine biosynthesis, and OvoA in ovothiol biosynthesis. Chapter 1 provides an overview of sulfur-containing metabolites, including the metabolism, potential biological functions, and biosynthesis of several key sulfur containing natural products. Chapter 2 contains my initial investigations into EanB catalysis, namely the original sulfur source for this enzyme. We demonstrated that the polysulfide (HSSnSR) is the direct sulfur source in EanB catalysis. With the discovery of the unique sulfur source, we then probed how EanB uses polysulfide for catalysis. A few reaction intermediate states were successfully characterized by X-ray crystallography and the proposed reaction mechanisms were further evaluated by QM/MM calculation. In Chapter 3, we evaluated the involvement of a proposed carbene intermediate involved in EanB catalysis by the deuterium exchange experiments with hercynine. In addition, using 3,5-difluoro-tyrosine containing EanB produced through amber suppressor method, we have also kinetically characterize the deuterium-exchange reaction. Chapter 4 reports the biochemical characterization of an OvoA homolog, OvoAMtht, from a mesophilic organism. OvoAMtht has dual activities: sulfoxide synthase and cysteine dioxygenase. In addition, I have demonstrated that both substrates and the active site iron’s secondary coordination shell residues exert exquisite control to OvoAMtht dual activities, which makes OvoAMtht an excellent system for future structure-function relationship studies for this class of enzymes. In summary, my thesis has laid the foundation for future detailed mechanistic investigations of the C-S bond formation reactions in both anaerobic ergothioneine biosynthetic and ovothiol aerobic biosynthetic pathways.

Development of selective artificial metalloenzymes by combining the biological concepts for selective recognition with those of transition metal catalysis has received much attention during the last decade. Targeting covalent incorporation of organometallic catalysts into proteins, we explored site-selective covalent coupling of phosphane and N–containing ligands. The successful approach for incorporation of phosphane ligands we report herein consists of site-specific covalent coupling of a maleimide functionalized hydrazide into proteins, followed by coupling of aldehyde functionalized phosphanes via a hydrazone linkage. Site selective incorporation of N–containing ligands was obtained by coupling maleimide functionalized N–ligands to proteins via Michael addition to the maleimide double bond. These two methods can be easily applied to virtually any protein displaying a single reactive cysteine and allows a wide range of possibilities in terms of cofactor design. Site-specific covalent incorporation of transition metal complexes of phosphane ligands into proteins was successfully obtained. The success of the approach is influenced by several factors like the metal precursor, the phosphane type and the protein scaffold. Metal complexes of 5–maleimido–1,10–phenanthroline modified proteins were formed in situ, via addition of a metal precursor to the phenanthroline modified proteins or by coupling preformed metal complexes to proteins via Michael addition of the thiol group from a cysteine residue to the maleimide double bond of the N-ligand. These successful coupling methods enable the use of a wide range of protein structures as templates for the preparation of artificial transition metalloenzymes, which opens the way to full exploration of the power of selective molecular recognition of proteins in transition metal catalysis.

This thesis describes the design, synthesis and application of artificial metalloenzymes for transition metal catalysed reactions not performed by natural enzymes. Unique cysteine containing protein templates were covalently modified with transition metal ligand complexes that generate catalytic activity, which allows for the use of virtually any protein template. SCP-2L was selected as template for the linear hydrophobic tunnel that traverses the protein, which has high affinity for linear aliphatic molecules. The use of catalysts based on this protein to induce increased activity in the biphasic hydroformylation of linear α-olefins is investigated in this work. For this purpose, unique cysteine containing mutants of SCP-2L were modified with phosphine ligands by application of a novel bioconjugation procedure. Application of rhodium adducts of the phosphine modified protein constructs led to up to a 100 fold increase of the turn over numbers was measured compared to a Rh/TPPTS model system which is used in industry. Furthermore, good selectivity towards the linear product was observed. If it can be confirmed that the found catalytic results truly are the result of substrate encapsulation by the protein scaffold, this system represents the first rationally designed artificial metalloenzyme which exploits the shape selectivity of the protein scaffold to direct the outcome of a catalytic reaction. In addition, a study was performed for the development of enantioselective artificial metalloenzymes. Nitrogen ligands were covalently introduced in SCP-2L and the obtained conjugates were applied in the copper catalysed Diels-Alder and Michael addition reaction. A promising 25% ee was found for the Diels-Alder reaction between azachalcone and cyclopentadiene using one of the created constructs. Further development of these catalyst systems with the use of both synthetic (e.g. optimisation of ligand structure) and biomolecular tools (e.g. optimisation of protein environment) for optimisation can lead to very efficient and enantioselective conversions in the future.

Thesis (MSc (Biochemistry))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: This study: 1. describes the preparation of a methanol extract of Sutherlandia frutescens and the HPLC fractionation of the methanol extract. 2. investigates the influence of S. frutescens on the binding properties of mitochondrial cytochrome 11 -hydroxylase (CYP11B1) to deoxycorticosterone (DOC) and deoxycortisol, demonstrating that methanol extracts of S. frutescens inhibit the Type I substrate-induced difference spectra. 3. investigates the influence of S. frutescens on the catalytic activity of CYP11B1 expressed in COS1 cells, demonstrating that the methanol extract of S. frutescens inhibits the conversion of DOC and deoxycortisol. 4. describes the sequential extraction of the methanol extract of S. frutescens using organic solvents and the inhibition of the conversion of DOC by CYP11B1 expressed in COS1 cells in the presence of these extracts. 5. describes the inhibition of the binding of DOC to CYP11B1 in ovine adrenal mitochondria, and the conversion of DOC by CYP11B1 expressed in COS1 cells by these fractions. 6. identifies the presence of the flavonoid compounds, orientin vitexin and rutin, in S. frutescens. 7. investigates the influence of the flavonoid compounds on the binding of DOC to CYP11B1 and on the catalytic activity of DOC by CYP11B1 expressed in COS1 cells. 8. identifies the presence of the triterpenoid, sutherlandioside A (SU1), in S. frutescens extracts and investigates its effect on the binding of DOC to CYP11B1. / AFRIKAANSE OPSOMMING: Hierdie studie beskryf: 1. die voorbereiding van ‘n metanol ekstraksie van Sutherlandia frutescens en die HPLC fraksionering van die metanol ekstrakte. 2. ‘n ondersoek na die invloed van S. frutescens op die bindingseienskappe van sitochroom P450 11 -hidroksilase (CYP11B1) in skaap bynier mitochondria en demonstreer dat S. frutescens metanol ekstrakte die vorming van steroïed-geinduseerde tipe I verskil spektra van deoksiekortisol en deoksikortikosteroon (DOC) inhibeer. 3. ‘n ondersoek na die invloed van S. frutescens op die katalitiese aktiwiteit van CYP11B1 in COS1 selle en demonstreer die inhibisie van DOC en deoksikortisol omsetting na hul produkte deur die methanol ekstrakte. 4. die opeenvolgende ekstraksie van methanol extrakte van S. frutescens met organiese oplosmiddels en beskryf die inhibisie van die CYP11B1 gekataliseerde omsetting van DOC in COS1selle in die teenwoordigheid van die ekstrakte. 5. die inhibeerende effek op die binding van DOC aan CYP11B1 in skaap bynier mitochondria en die inhibisie van die CYP11B1 gekataliseerde omsetting van DOC in COS1selle. 6. die identifisering van flavonoïed verbindings, orientin vitexin en rutin in S. frutescens. 7. ‘n ondersoek na die invloed van die flavonoïed verbindings op die binding van DOC aan CYP11B1 en op die katalitiese aktiwiteit van CYP11B1 in COS1 selle. 8. die indentifisering van die triterpenoïed, sutherlandiosied A (SU1), in S. frutescens en ondersoek die invloed van SU1 op die binding van DOC aan CYP11B1.

Thesis (MSc)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: This study: I. Investigates the influence of S. frutescens on the binding properties of cytochrome P450-dependent enzymes in ovine adrenocortical mitochondria and microsomes, demonstrating that S. frutescens extracts elicit difference spectra and inhibit the Type 1 difference spectra induced by natural steroids. II. Indicates inhibition by S. frutescens extracts of the catalytic activity of cytochrome P450-dependent-17a-hydroylase and cytochrome P450-dependentsteroid- 21-hydroxylase enzymes in ovine adrenocortical microsomes. III. Describes an assay determining the inhibitory effects of S. frutescens, in COS L cells, on individual cytochrome P450-dependent enzymes - ovine, baboon and human cytochrome P450-dependent- L7a-hydroylase, and bovine cytochrome P450-dependent-21-hydroxylase enzymes. IV. Demonstrates that the inhibition of elevated plasma glucocorticoid levels in rats exposed to chronic immobilization stress could possibly be attributed to the influence of hydrophilic and hydrophobic compounds in S. frutescens on cytochromes P450-depndent enzymes. / AFRIKAANSE OPSOMMING: Hierdie studie: I. Ondersoek die invloed van S. frutescens op die bindingseienskappe van sitochroom P450-afhanklike ensieme in skaapbynier mitokondriale en - mikrosomale preparate en toon aan dat komponente in S. frutescens ekstrakte verskil spektra induseer en tipe I verskil spektra van natuurlike steroïede inhibeer. II. Dui die inhiberende effek van S. frutescens ekstrakte op die katalitiese aktiwiteit van sitochroom P450-afhanklike-17a-hidroksilase en sitochroom P450- afhanklike-steroïed- 21-hidroksilase ensieme in skaapbyniermikrosome aan. III. Beskryf 'n tegniek om die inhiberende effek van S. frutescens op individuele sitochroom P450-afhankilike ensieme - bobbejaan, skaap en mens sitochroom P450-afhanklike-17a-hidroksilase, en bees sitochroom P450-afhanklike-steroïed- 21-hidroksilase - in COS 1 selle te bepaal. IV. Demonstreer dat inhibisie van verhoogde glukokortikoïed plasma konsentrasies waargeneem in rotte blootgestel aan kroniese immobiliserende stress, moontlik toegeskryf kan word aan die effek van S. frutescens op die sitochroom P450- afhanklike ensieme.

Selective oxidations are important for the functionalization of compounds in organic synthesis and chemical industry. Using O2 as a terminal e- acceptor would be ideal because it is cheap and environmentally friendly, but aerobic oxidations are often prone to unselective free radical autoxidation. Recently developed palladium catalysts use O2 as a selective multi-electron oxidant for various organic transformations. Although these methods are powerful and sophisticated, the lower cost of base metals makes them attractive as potential alternatives. The challenge is to develop methods for effecting multi-electron transformations at metals that typically prefer one electron changes. To this end, the development of manganese(III) complexes containing redox-active ligands as catalysts for selective oxidase-type oxidation of organic substrates was pursued. Bis(tetrabromocatecholato) manganese(III) complexes were shown to aerobically oxidize catechols to form quinones and H2O2. This reactivity was extended to other alcohol and amine substrates. In these reactions, the non-innocent tetrabromocatecholate ligands may impart a multi-electron character to the metal. To directly probe the intermediacy of ligand-centered radicals in catalytic turnover, a series of structurally similar manganese(III) complexes with aminophenol-derived ligands were prepared and characterized. The capacity of these ligands to undergo low-energy redox changes, allowed for isolation of an electron transfer series spanning two redox states without a change in oxidation state at the metal center. The ligand-centered redox events were a key feature in aerobic homocoupling of Grignard reagents.

Thesis (M.S.)--University of North Carolina at Wilmington, 2003. / Includes bibliographical references (leaves : 72-75).

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 163-191).