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Toluene dioxygenase (TDO) is a Rieske type non-haem enzyme found within the soil bacteria, Pseudomonas putida (P. putida) and is responsible for catalysing the enantioselective cis-dihydroxylation of aromatic substrates. This remarkable trait of the TDO enzyme has enabled its application in the preparation of over 400 novel cis-diol bioproducts using whole cell biocatalysis. Recently, a new and synthetically useful bioproduct, (4S,5S)-4,5-dihydroxy-3-iodocyclohex-2-en-1-one, has been identified from the TDO catalysed biotransformation of 3-iodophenol. The purpose of this project was to demonstrate the synthetic application of this new bioproduct in the synthesis of natural product analogues, which possess anti-proliferative activity against different cancer cell lines. This was achieved by developing methodology towards a key enone building block, which was successfully applied in the synthesis of an analogue of the natural product incarviditone. Furthermore, a robust computational docking model was developed using the Goldâ„¢ software, which showed significant correlation between the predicted docking outcome and the experimentally observed results for a series of monocyclic substrates. The docking model was also used to rationalise the major and minor binding modes of the 3-iodophenol substrate in the TDO enzyme active site. The project also sought to develop an alternative synthetic methodology using butan-1,2-diacetal (BDA) protected (-)-quinic acid, which would provide the same stereochemical outcome as the newly discovered bioproduct. This was achieved despite considerable synthetic challenges encountered when trying to control the diastereoselectivity of a key conjugate addition reaction. The antipodal compounds of the natural product analogues obtained using the 3-iodophenol bioproduct and BDA protected (-)-quinic acid were also synthesised using acetonide protected (-)-quinic acid. By reacting the enantiomers of a key hydroxyenone intermediate obtained using the two pathways, a novel heterodimerisation was reported to afford an analogue of the natural product, incarvilleatone. Finally, all the biological activities of the synthesised natural product analogues were evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay: the results showed significant differences between the enantiomers of the incarviditone natural product analogues.

The Streptomyces metabolite 2-crotonyloxymethyl-(4R,5R,6R)-4,5,6-trihydroxycyclohex-2-enone (COTC), the antheminones and the carvotacetone derivatives are all bioactive natural products, whose structure is based on the α oxymethyl-a,β-cyclohexenone moiety. Both COTC and antheminone A have been shown to exhibit cytotoxic and cancerostatic activity with low toxicity. The potent biological activity of these natural products has instigated numerous investigations into the synthesis of novel analogues in an attempt to determine the key structural features necessary for optimum bioactivity. The synthesis of a small library of novel anti-tumour agents which are structurally related to the natural products COTC and antheminone A is described, using the chiral pool material (-)-quinic acid as a starting material. At the outset, the aim of this project was to develop and optimise copper-mediated conjugate addition reactions and rhodium catalysed conjugate addition reactions of organoboron reagents to functionalised cyclic enones and subsequently, to apply the methodologies to the synthesis of the novel analogues. A range of novel mono-hydroxylated analogues bearing aryl side chains were prepared and their antiproliferative activity was assessed towards the A549 non-small cell cancer cell line. The biological assays revealed important structure-activity relationships and the most bioactive compound of this series had an IC50 value of 1.2 µM. In addition, the design and synthesis of a new class of GSH-activated prodrugs is described. These novel compounds are activated by GSH leading to intracellular release of an NQO1 inhibitor. The most potent compound of this new class of compounds had an IC50 value of 710 nm.

The natural product 2 crotonyloxymethyl-(4R,5R,6R)-4,5,6-trihydroxy-cyclohex-2-enone (COTC) was isolated from the microorganism Streptomyces griseosporeus in 1975. It was shown to exhibit 'cytotoxic and cancerostatic activity'. The simplified synthetic analogue 2-crotonyl-oxymethyl-cyclohex-2-enone (COMC) has been shown to exhibit potent anti tumour activity against murine and human tumours in cell culture. For several years, the Whitehead research group at the University of Manchester have focused on the synthesis of COTC and COMC analogues in an attempt to produce compounds with enhanced cytotoxicity. In this thesis, the syntheses of several polyfunctionalised cyclohexenones are described. These compounds are analogues of COTC and COMC which also bear structural resemblance to the antheminone and carvotacetone natural products. Initially, the syntheses of six novel compounds from the chiral pool starting material (-)-quinic acid are described. The first four synthetic steps of each sequence were carried out by slight modification of procedures previously reported by the Whitehead research group. As part of the synthetic strategy, the diastereoselective conjugate addition of carbon nucleophiles to several polyfunctionalised cyclohexenones was investigated. The cytotoxicity of four of the synthetic analogues towards A549 non small cell lung cancer cells was investigated by use of an MTT assay. Two of the analogues were found to be more cytotoxic then COMC. The most effective synthetic analogue had an IC50 value of 2.2 μM. This analogue was more cytotoxic than similar molecules that had previously been synthesised by members of the Whitehead research group. Based on the results of the MTT assay, another two analogues were designed and their synthesis from (-)-quinic acid is described. The cytotoxicity of these analogues has yet to be assessed. In summary, the general synthetic strategies developed in this thesis will provide easy access to new analogues of the natural products, enabling the development of new cytotoxic compounds.

Neurospora crassa, a model filamentous fungus, contains the quinic acid (qa) gene cluster, allowing the organism to effectively metabolize quinic acid as an alternative carbon source. In the presence of quinic acid, the qa gene cluster is significantly unregulated, showing an increased expression of both regulatory proteins as well as the structural proteins required for the utilization of quinic acid as an energy source. Of these structural genes, qa-2, qa-3, and qa-4 encode for the enzymes required for the conversion of quinic acid to protocathechuic acid while qa-y encodes for a quinate permease and the function of qa-x remains unknown.The present study aims to elucidate the expression pattern of qa-y, the quinate permease-producing gene required for efficient uptake and utilization of quinic acid in N. crassa, as well as qa-1F, the activator protein of the qa gene cluster, at differing times of quinic acid exposure. Quantitative real-time PCR (RT-qPCR) using reverse transcriptase was first used to determine Histone-3 as the optimal housekeeping reference gene. This gene was then used to accurately quantify the expression of both qa-y and qa-1F at differing times of exposure to quinic acid (0min, 15min, 30min, 1hr, 2hr, and 3hr).The expression of qa-y was observed to steadily increase for the first 30 minutes, showing up to a 17-fold change in expression after only 30 minutes following introduction of quinic acid. A prompt response of the qa-1F gene was also observed. At the 15-minute time point, a 3-fold increase in expression of the qa-1F occurred, presumably to initiate activation of transcription of the genes of the qa gene cluster, including itself. When combined, these data allow for a comprehensive representation of the timing and method of regulation of the qa gene cluster in N. crassa.

Neurospora crassa, like most fungi, is very flexible metabolically. When a preferred carbon source, such as dextrose, is unavailable, N. crassa has the ability to metabolize quinic acid. To do this, the quinic acid gene cluster is up-regulated by the qa-1F activator. This study uses a mutant form of N. crassa in which the qa-1F gene is knocked out. The protein profiles of N. crassa wild-type and qa-1F knockout when grown on both dextrose and quinic acid were analyzed and compared. The wild-type and knockout strains were first grown on 2% dextrose and then shifted either to fresh dextrose or 0.3% quinic acid. The proteins from these tissues were then extracted, quantitated, and separated using two-dimensional gel electrophoresis (2DGE). The 2DGE gels were then analyzed using PDQUESTTM. Cross-conditional comparisons were made and protein spots unique to each condition were identified. These gel comparisons show that, when grown on a preferred carbon source, nearly twice as many proteins are up-regulated than when grown on quinic acid. Also, the qa-1F knockout protein profiles had far fewer protein spots than their wild-type counterparts for both carbon sources.Protein spots were then selected, excised, and sent to the Ohio State University for mass spectrometry and bioinformatic analysis. Two proteins affected by the presence of qa-1F when grown on quinic acid were identified as hypothetical proteins NCU 04072 and NCU 08332 likely be a catechol dioxygenase and a translational protein SH3-like protein, respectively.