Pyrimidine salvage pathways are essential to all cells. They provide a balance of RNA synthesis with the biosynthetic pathway in pyrimidine prototrophs and supply all the pyrimidine requirements in auxotrophs. While the pyrimidine biosynthetic pathway is found in almost all organisms and is nearly identical throughout nature, the salvage pathway often differs from species to species, with aspects of salvage seen in every organism. Thus significant taxonomic value may be ascribed to the salvage pathway. The pyrimidine salvage pathways were studied in 55 microorganisms. Nine different salvage motifs, grouped I-IX, were identified in this study based on the presence of different combinations of the following enzymes: cytidine deaminase (Cdd), cytosine deaminase (Cod), uridine phosphorylase (Udp), uracil phosphoribosyltransferase (Upp), uridine hydrolase (Udh), nucleoside hydrolase (Nuh), uridine/cytidine kinase (Udk), 5'-nucleotidase and CMP kinase (Cmk).

Ph.D., Faculty of Science, University of the Witwatersrand, 2011 / This thesis describes the application pteridine chemistry in various aspects of methodology development and natural product synthesis. The introductory chapter sets the scene by describing naturally occurring pteridines, their applications in biological systems, and recent synthetic strategies. Firstly, Sonogashira coupling reactions employing benzenesulfonyloxy-O-pteridine (27) and terminal alkynes to give various 6-substituted pteridines are described. This methodology allowed for the total synthesis of a natural occurring pteridine, Sepiapterin-C (46). Negishi coupling reactions involving benzenesulfonyloxy-O-pteridine (27) and various Znreagents are also reported. This methodology, representing the first Negishi coupling on a pteridine nucleus, allowed for the introduction of both aryl- and heteroaryl- substituents at the 6- position of the pteridine ring. The use of methanesulfonyloxy-O-pteridine (26) as a coupling partner is also described. Selective deprotection and hydrolysis of the formamidine protecting groups to give either the 6- substituted 2,4-diaminopterine or 2-amino-4-oxo-pteridine (pterin), is described. The synthesized structures are supported by NMR and mass spectral data and melting points where applicable. Novel compounds are verified by NMR spectroscopy, infrared and mass spectrometry.

La partie principale de ce manuscrit traite du développement de nouvelles méthodologies utilisant la substitution propargylique catalysée par des sels de fer(III), pour la formation de divers hétérocycles azotés (∆4-isoxazolines, isoxazoles, cis-acylaziridines et pyrimidines). En premier lieu, de nouvelles synthèses monotopes de ∆4-isoxazolines et d'isoxazoles diversement substitués impliquant des réactions de cyclisation catalysées par diverses espèces carbophiles ([Au], [Pd], [I+]) ont été développées. La fragilité de la liaison N-O des ∆4-isoxazolines a pu être ensuite exploitée pour conduire à la formation de cis-acylaziridines. De nouvelles voies d'accès aux (Z)-β-énaminones et aux pyrimidines trisubstituées ont été également développées. / The main part of this manuscript deals with the development of new methodologies using iron(III)-catalyzed propargylic substitution, for the synthesis of various nitrogen-containing heterocycles (Δ4-isoxazolines, isoxazoles, cis-acylaziridines and pyrimidines). Firstly, new one-pot syntheses of variously substituted Δ4-isoxazolines and isoxazoles involving cyclization reactions promoted by various carbophilic species ([Au], [Pd], [I+]) have been developed. The weakness of the Δ4-isoxazoline N-O bond has been then exploited, leading to the formation of cis-acylaziridines. New pathways to (Z)-β-enaminones and trisubstituted pyrimidines have also been developed.

Some compounds, analogous to those found in naturally occurring systems, are found to possess chemotherapeutic activity. Some, in the form of their nucleoside or nucleotide derivatives, are valuable antimetabolites in that they may block normal RNA or DNA polymerisation, or may be incorporated into nucleic acids to form fraudulent, but not necessarily defective, polymers. Modification of natural ring systems, with a view to promoting chemotherapeutic activity is therefore of considerable interest; variation in the position and nature of the modification or ring substituent having a marked effect on chemotherapeutic activity. It is the purpose of this thesis to suggest methods for the facile synthesis of various uracils, pyrazoles and pyrazolo [3,4-d] - pyrimidines with alkyl, aryl and glycosyl substituents such that the nature of the ring substituents is easily varied. To this end a number of ethoxymethylene reagents were prepared which, by reaction with primary amines and hydrazines, would give acyclic intermediates capable of easy cyclisation into the uracil, pyrazole and pyrazolo [3,4-d] pyrimidine ring systems. Variation in the nature of specific substituents being determined by the choice of amine or hydrazine, other substituents being varied by modification of the original reagent.

One current approach in the treatment of cancer is the inhibition of cyclin dependent kinase (CDK) enzymes with small molecules. Here the discovery and development of 2-anilino-4-(thiazol-5-yl)pyrimidine CDK inhibitors is described, including details of the design and successful synthesis of novel ring-constrained thiazolylpyrimidines. The structure-activity relationship (SAR) trends exhibited by this constrained thiazolylpyrimidine family of CDK inhibitors are presented and compared with those from an unconstrained series of analogues. One significant finding from this aspect of the project was that ring-constrained thiazolylpyrimidines in general inhibit CDK2-cyclin E with greater potency than the corresponding unconstrained forms. Furthermore, an X-ray crystal structure of 2-methyl-N-[3-nitrophenyl]-4,5-dihydrothiazolo[4,5-h]quinazolin-8-amine, a representative from the constrained thiazolylpyrimidine series, in complex with CDK2-cyclin A is reported; confirming the binding mode within the CDK2 ATP binding pocket. A further assessment of SARs through the synthesis of control compounds and an extended study into the synthesis of N-substituted derivatives is described. The identification of CDK inhibitors that possess a strong selectivity profile across the CDK family is important. For example, the identification of highly CDK4-selective inhibitors should enable researchers to study the biological role of this important enzyme and to enable a block of cell division in the G1 phase. Here synthetic attempts to prepare a potentially CDK4 selective inhibitor compound, namely 5-methyl-N8-[4-(piperazin-1-yl)phenyl]thiazolo[4,5-h]quinazoline-2,8-diamine, are described. This approach was inspired by SAR data published on a structurally related inhibitor, 8-cyclopentyl-5-methyl-2-[4-(piperazin-1-yl)phenylamino]pyrido[2,3-d]pyrimidin-7(8H)-one.

The regulatory gene for pyrimidine biosynthesis has been identified and designated pyrR. The pyrR gene product was purified to homogeneity and found to have a monomeric molecular mass of 19 kDa. The pyrR gene is located directly upstream of the pyrBC' genes in the pyrRBC' operon. Insertional mutagenesis of pyrR led to a 50- 70% decrease in the expression of pyrBC', pyrD, pyrE and pyrF while pyrC was unchanged. This suggests that PyrR is a positive activator. The upstream regions of the pyrD, pyrE and pyrF genes contain a common conserved 9 bp sequence to which the purified PyrR protein is proposed to bind. This consensus sequence is absent in pyrC but is present, as an imperfect inverted repeat separated by 11 bp, within the promoter region of pyrR. Gel retardation assays using upstream DNA fragments proved PyrR binds to the DNA of pyrD, pyrE, pyrF as well as pyrR. This suggests that expression of pyrR is autoregulated; moreover, a stable stem-loop structure was determined in the pyrR promoter region such that the SD sequence and the translation start codon for pyrR is sequestered. β-galactosidase activity from transcriptional pyrR::lacZ fusion assays, showed a two-fold in increase when expressed in a pyrR- strain compared to the isogenic pyrR+ strain. Thus, pyrR is negatively regulated while the other pyr genes (except pyrC) are positively activated by PyrR. That no regulation was seen for pyrC is in keeping with the recent discovery of a second functional pyrC that is not regulated in P. aeruginosa. Gel filtration chromatography shows the PyrR protein exists in a dynamic equilibrium, and it is proposed that PyrR functions as a monomer in activating pyrD, pyrE and pyrF and as a dimeric repressor for pyrR by binding to the inverted repeat. A related study discovered that the catabolite repression control (Crc) protein was indirectly involved in pyr gene regulation, and shown to negatively regulate expression of PyrR at the posttranscriptional level.

The ability to synthesize pyrimidine nucleotides is essential for most organisms. Pyrimidines are required for RNA and DNA synthesis, as well as cell wall synthesis and the metabolism of certain carbohydrates. Recent findings, however, indicate that the pyrimidine biosynthetic pathway and its intermediates maybe more important for bacterial metabolism than originally thought. Maksimova et al., 1994, reported that a P. putida M, pyrimidine auxotroph in the third step of the pathway, dihydroorotase (DHOase), failed to produce the siderophore pyoverdin. We created a PAO1 DHOase pyrimidine auxotroph to determine if this was also true for P. aeruginosa. Creation of this mutant was a two-step process, as P. aeruginosa has two pyrC genes (pyrC and pyrC2), both of which encode active DHOase enzymes. The pyrC gene was inactivated by gene replacement with a truncated form of the gene. Next, the pyrC2 gene was insertionally inactivated with the aacC1 gentamicin resistance gene, isolated from pCGMW. The resulting pyrimidine auxotroph produced significantly less pyoverdin than did the wild type. In addition, the mutant produced 40% less of the phenazine antibiotic, pyocyanin, than did the wild type. As both of these compounds have been reported to be vital to the virulence response of P. aeruginosa, we decided to test the ability of the DHOase mutant strain to produce other virulence factors as well. Here we report that a block in the conversion of carbamoyl aspartate (CAA) to dihydroorotate significantly impairs the ability of P. aeruginosa to affect virulence. We believe that the accumulation of CAA in the cell is the root cause of this observed defect. This research demonstrates a potential role for pyrimidine intermediates in the virulence response of P. aeruginosa and may lead to novel targets for chemotherapy against P. aeruginosa infections.

Previous research in our laboratory established that pyrB, pyrC or pyrD knock-out mutants in Pseudomonas aeruginosa required pyrimidines for growth. Each mutant was also discovered to be defective in the production of virulence factors. Moreover, the addition of exogenous uracil did not restore the mutant to wild type virulence levels. In an earlier study using non-pathogenic P. putida, mutants blocked in one of the first three enzymes of the pyrimidine pathway produced no pyoverdine pigment while mutants blocked in the fourth, fifth or sixth steps produced copious quantities of pigment, just like wild type P. putida. The present study explored the correlation between pyrimidine auxotrophy and pigment production in P. aeruginosa. Since the pigment pyoverdine is a siderophore it may also be considered a virulence factor. Other virulence factors tested included casein protease, elastase, hemolysin, swimming, swarming and twitching motilities, and iron binding capacity. In all cases, these virulence factors were significantly decreased in the pyrB, pyrC or pyrD mutants and even in the presence of uracil did not attain wild type levels. In order to complete this comprehensive study, pyrimidine mutants blocked in the fifth (pyrE) and sixth (pyrF) steps of the biosynthetic pathway were examined in P. aeruginosa. A third mutant, crc, was also studied because of its location within 80 base pairs of the pyrE gene on the P. aeruginosa chromosome and because of its importance for carbon source utilization. Production of the virulence factors listed above showed a significant decrease in the three mutant strains used in this study when compared with the wild type. This finding may be exploited for novel chemotherapy strategies for ameliorating P. aeruginosa infections in cystic fibrosis patients.

Thèse de doctorat : Chimie organique : Strasbourg 1 : 2006. / Titre provenant de l'écran-titre. Bibliogr. p. 179-184.

Thin coatings of photoresponsive, pyrimidine-terminated molecules, attached to gold or quartz substrates in contact with water, undego photodimerisation and wettability changes when irradiated with UV light at 280 and 240mm. Spin-casting and chemisorption techniques were used to prepare the thin films. / Thesis (PhDApSc)--University of South Australia, 2000.