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The research presented in this study combines natural product chemistry with organic / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

The olefin metathesis reaction is a well established and powerful method for the synthesis of alkenes. This reaction can be further classified into the intermolecular process known as cross-metathesis and the intramolecular process known as ring-closing metathesis. The aim of these studies is the use of the two variants of the metathesis reaction for the development of new methods for the synthesis of heteroaromatic structures, in particular the synthesis of polysubstituted pyridines.

The aim of this thesis was to investigate the synthesis and the conformational analysis of fluorinated pyrrolidines. We focused on two strategies namely, the iodoamination and fluoroamination of fluorinated precursors. Iodoamination Our first approach for the synthesis of fluorinated pyrrolidines relied on the iodocyclisation of allylic fluorides bearing pendant nitrogen nucleophiles. These allylic fluorides were obtained by fluorodesilylation of suitably functionalised allylsilanes. After validation of this methodology, the scope and limitations of the iodoamination were investigated. Furthermore, we were able to probe the influence of the fluorine moiety on the level of diastereocontrol of the cyclisation. Fluoroamination The second route focused on a key reaction: an unprecedented electrophilic fluoroamination of an aminated allylsilane. From a mechanistic point of view, the presence of the silyl group act as a 1,2-dipole and activate the double bond towards electrophilic fluorination. This methodology required the initial screening of a silyl directing group that would promote electrophilic addition, without subsequent desilylation. Finally, we investigated the level of diastereocontrol displayed by these cyclisations as a function of the E/Z geometry of the starting aminated allylsilane. Conformational analysis Moreover the 3-fluoropyrrolidines obtained via iodoamination served to investigate the stereoelectronic influence of the fluorine gauche effect on ring conformations. Solid state single crystal X-ray analysis and solution phase NMR spectroscopy were used for this purpose. Due to complicated conformational analysis of saturated five-membered rings in solution, 1D 19F-1H heteronuclear nOe (HOESY) experiments have been optimised for applications to this type of small molecules. These have been employed to estimate 19F-1H internuclear distances and were combined with vicinal 3JFH and 3JHH scalar coupling constants in order to analyse the ring conformations.

The use of enabling technologies and continuous methods to enhance chemical synthesis is a vibrant area of research, gaining increasing attention from laboratories in academia and industry. Yet many prominent synthesis procedures have not changed for decades and require significant manual intervention from bench chemists, which may lead to the waste of both human and material resources. The research described herein details how chemistry has been bridged with engineering to address this issue in a world increasingly focussed on sustainability and efficiency. This thesis is divided into four chapters. The first describes the development of a novel Internet-based process control system which is applied to automate a cycling catalytic process. The capability of the system to conduct multi-dimensional self-optimisation processes is shown, where it is integrated with an on-line mass spectrometer and inline infrared spectrometer to drive optimisation against customisable multicomponent evaluation functions. Chapter 2 details the successful synthesis of the anti-cancer drug candidate AZ82. The control system is applied to assist with a number of these steps, including facilitating the integration between batch and flow processes on a single reactor platform. A new distillation unit to assist with downstream solvent switching is also described. In Chapter 3, the cloud-based nature of the control system is exploited by moving it to servers residing in Tokyo. The ability of the system to accelerate the drug development process is highlighted with the autonomous self-optimisation and synthesis of four active pharmaceutical ingredient targets: tramadol, lidocaine, bupropion and isoniazid. In the case of bupropion, the system maintained steady-state operation of a telescoped two-step process for an extended period. A researcher in Los Angeles was able to initiate and monitor all processes, via Japan, in real-time as they occurred in our laboratory in Cambridge, UK. Finally, Chapter 4 describes the development of a new parallel column supercritical fluid chromatography (SFC) unit that is capable of separating a multicomponent product stream continuously exiting a flow reactor. The versatility of the SFC unit is showcased with the telescoped synthesis of isoniazid in which all stages, including the SFC process itself, are managed by the control system without researcher intervention.

In humans and most eukaryotes, thymidylate synthase (TSase) serves as a key enzyme that catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) to synthesize deoxythymidine monophosphate (dTMP), a key component of DNA. The N5, N10- methylene-5,6,7,8-tetrahydrofolate (MTHF) serves as both the methylene donor and the hydride donor while generating dihydrofolate (H2folate) as the byproduct. However, in 2002, Myllykallio reported the discovery of flavin-dependent thymidylate synthase (FDTS) that also functions to maintain the dTMP pool, although the mechanism is different. Since then, considerable progress was made in characterizing this enzyme. It was found that structurally FDTS is substantially different from TSase both with respect to structure and with respect to the mechanistic pathway of catalysis. In the FDTS-catalyzed methylation of dUMP, MTHF serves only as the methylene donor, generating tetrahydrofolate (H4folate), unlike TSase, and FDTS utilizes NADPH as a reductant. Activity of the enzyme depends on the presence of the noncovalently bound prosthetic group, flavin adenine dinucleotide (FAD). Interestingly, the enzyme FDTS is present in several human pathogens that cause diseases including syphilis, tuberculosis, anthrax poisoning, typhus, botulism, peptic ulcers and more, but is absent in humans; thus, it poses an attractive target for antibiotics. In the modern world, antibiotic resistance is a menace; consequently, new targets for new antibiotics are being sought. Hence, elucidating the chemical mechanism of FDTS is of paramount interest, as we and others believe this could allow for rational design of drugs that selectively target these pathogens with minimal human toxicity. Although several chemical mechanisms for FDTS catalysis have been put forward, complete understanding has still not been achieved. One of the primary concerns was the role of FAD in catalysis, and we found – as described in Chapter II and III – that FAD is a methylene carrier rather than just a hydride donor, as previously postulated. Secondly, all mechanisms proposed so far predict the presence of a noncovalently bound putative exocyclic methylene intermediate (an isomer of dTMP) occurring in the catalytic pathway of FDTS. However, direct evidence to prove its existence was lacking. Recently, we have been able to synthesize this intermediate, as described in Chapter IV. As shown in Chapter V and VI, we used steady-state kinetics, isotopic substitution and NMR studies to test this intermediate with FDTS. We believe our findings will greatly improve the understanding of this enzyme and will impact drug design by government agencies, pharmaceutical companies, and academic laboratories.

Recent years have seen a dramatic expansion in synthetic and structural molybdenum - sulfur chemistry. The amplest binary Mo-S moiety in MO$ ion. The thromolybdates derived by sulfur substitution from the 0x0 analogue have interesting chemical properties. Although they have been studied extensively by synthetic inorganic chemists and bio-inorganic chemists as models for enzymes like introgenases, their reactivity towards organic substrates remamed unexplored.

The study of many of glycosyltransferases is limited due to an inadequate access to sugar nucleotides. Preparation of sugar nucleotides through the use of nucleotidylyltransferases with broad substrate specificities is gaining significant interest and offers high yields and stereospecificity. Physiologically, the glucose 1-phosphate thymidylyltransferase catalyzes the condensation of ?-D-glucose 1-phosphate and deoxythymidine triphosphate to yield deoxythymidine diphospho glucose. Exploiting and targeting these enzymes also has the potential of yielding new therapeutics. Cps2L is a thymidylyltransferase isolated from Streptococcus pneumoniae, with broad substrate flexibility. The substrate specificity of Cps2L was evaluated with new sugar 1-phosphate analogues to gain further insight into substrate and inhibitor requirements. Several sugar 1-phosphate analogues including sugar 1C-phosphonates (and analogues thereof), 2-deoxy-2-fluorosugar 1-phosphates, and glucopyranose 1- boranophosphates have been used to probe the sugar 1-phosphate modification tolerance of Cps2L. In addition, NMR spectroscopy was used to determine the anomeric stereochemistry of 2-deoxy-2-fluorosugars nucleotide products. For those substrates that were accepted by Cps2L, steady-state kinetic parameters were determined. The enzyme is able to almost equally form Michaelis complexes with different sugar substrates, whereas the turnover values for obtaining the corresponding sugar nucleotide were different. The evaluation of the substrate tolerance of Cps2L, as well as the synthesis of ?-D-glucose-1C-thiophosphonate, a difluoro and a bisphosphono analogue of ?-D-glucose 1C-phosphonate will be described.

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