Dirhodium complexes are effective catalysts in carbene transformations. These reactions involve chemistry between the catalyst and a carbene generated in situ. In order to better understand the rhodium-carbon bond, studies of rhodium acetate, Rh2(OAc)4, and a variety of functionalized benzonitriles were proposed. Diadducts of rhodium acetate with 4-nitrobenzonitrile, 4-aminobenzonitrile, 4-(dimethylamino)benzonitrile, and 3,5-dinitrobenzonitrile were successfully prepared and characterized by X-ray crystallography. IR and NMR spectroscopy were also employed for characterization purposes.

Bacterial 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (DAHPS) is an antimicrobial target. A transition state (TS) mimic inhibitor of DAHPS, DAHP oxime, was developed in our lab, with Ki = 1.5 μM. Despite being a potent inhibitor, some of DAHP oxime’s properties could hinder its efficacy in blocking bacterial growth. One problem was that DAHP oxime bound competitively with respect to the metal cofactor, Mn2+. Identifying the origin of this competition was crucial for in vivo success of DAHPS inhibitors, as the abundance of metal ions in living cells could hinder DAHP oxime’s effectiveness. Mutant enzymes and fragment-based inhibitors demonstrated that the competition originated in interactions involving the O4 hydroxyl of DAHP oxime and residues Asp326, and Cys61. This suggested improved inhibitor designs to avoid metal competition. Another drawback was the highly negatively charged nature of DAHP oxime and other DAHPS inhibitors. The charge hindered cell membrane penetration, and therefore its effectiveness in cells. Truncating DAHP oxime to a fragment reduced its hydrophilicity and charge, while the introduction of fluorine in the oxime’s α-position increased potency. DAHPS-specific bacterial growth inhibition was obtained with this fragment. DAHPS inhibition by high DAHP oxime concentrations resulted in a 15% residual activity. This residual activity would be sufficient for bacterial survival, so two derivatives, DAHP hydrazone and DAHP O-(2-fluoroethyl) oxime that showed complete inhibition were characterized. DAHP hydrazone was successfully co-crystallized with DAHPS. Its 100-fold higher potency relative to DAHP oxime was attributed to an extra water bound in the active site. The inhibitor bound in all four of the enzyme’s subunits. However, the observed subunit asymmetry showed that the subunits communicate with each other and that there is a significant energetic penalty to enforcing full subunit symmetry. / Thesis / Doctor of Philosophy (PhD) / Antibiotic resistance is imposing a growing disease burden on society that the United Nations calls “…a global crisis that we cannot ignore”. Many traditional antibiotics have now lost their efficacy as bacteria increasingly develop mechanisms of resistance. The development of new treatment options is therefore crucial. The first step towards this goal is the characterization of new antimicrobial targets. DAHPS is an enzyme that is exclusively expressed by bacteria, fungi and plants. Its absence in mammalian cells is expected to reduce side effects in humans. DAHPS inhibitors have previously been developed, but none of them has shown sufficient effectiveness in bacterial cell culture. Possible reasons for this failure were, among others, reduced inhibition in the presence of metal ions, low cell membrane penetration and incomplete enzyme inhibition. This thesis examines DAHPS’s properties and provides solutions on how to successfully inhibit this enzyme, to fight antibiotic resistance with new treatment options.

Betalaínas são pigmentos naturais derivados da L-tirosina encontrados em um número restrito de plantas e fungos. Sob o ponto de vista químico, betalaínas são iminas ou sais de imínio que se originam do acoplamento aldimínico entre o ácido betalâmico e aminas ou aminoácidos. Embora sejam conhecidas mais de 70 betalaínas naturais, os estudos sobre as relações entre a estrutura e as propriedades de betalaínas ainda são limitados. Esta Tese de Doutorado descreve a semissíntese e estudo da hidrólise e das propriedades fotofísicas e redox de três grupos de betalaínas não naturais: N-fenilbetalaínas (pBeets), N-metil-N-fenilbetalaínas (N-Me-pBeets) e N,N-difenilbetalaínas (dipBeets). O estudo de quatorze pBeets para substituidas sugere que a porção imina destes compostos está protonada em meio aquoso neutro e que substituintes atraentes de elétron na porção fenílica favorecem o deslocamento de carga do nitrogênio 2-piperideínico para a porção imínica. A constante de hidrólise aumenta linearmente com o aumento do valor de σp de Hammett do substituinte, dado o aumento da eletrofilificade do carbono aldimínico. Por outro lado, pBeets com substituintes atraentes de elétron são mais fluorescentes. A adição de metila ou fenila ao nitrogênio da imina cria um ambiente hidrofóbico que compromete o ataque da água e diminui a constante de velocidade de hidrólise provocando, contudo, uma diminuição na fluorescência. Os resultados foram racionalizados empregando-se espectroscopia de ressonância magnética nuclear, espectrofotometria e voltametria cíclica. / Betalains are natural pigments derived from L-tyrosine that are found in a restricted number of plants and fungi. From the chemical point of view, betalains are imines or iminium salts that originate from the aldimine coupling between betalamic acid and amines or amino acids. Although more than 70 natural betalains are known, studies on structure-property relationships of betalains are still limited. This doctoral thesis describes the semi-synthesis and study of the hydrolysis as well as photophysical and redox properties of three groups of non-natural betalaines: N-phenylbetalaines (pBeets), N-methyl-N-phenylbetalines (N-Me-pBeets) and N,N-diphenylbetalaines (dipBeets). The study of fourteen substituted pBeets suggests that the imine portion of these compounds is protonated in neutral aqueous medium and that attractive electron substituents on the phenyl portion favor the displacement of 2-piperidene nitrogen charge to the imine portion. The hydrolysis constant increases linearly with the increase of the Hammett\'s σp value of the substituent, given the increase in the electrophilicty of the aldimine carbon. On the other hand, pBeets with attractive electron substituents are more fluorescent. The addition of methyl or phenyl to the imine nitrogen creates a hydrophobic environment upon water attack and decreases the hydrolysis rate constant, however, causing a decrease in fluorescence. The results were rationalized using nuclear magnetic resonance spectroscopy, spectrophotometry and cyclic voltammetry.

Betalaínas são pigmentos naturais derivados da L-tirosina encontrados em um número restrito de plantas e fungos. Sob o ponto de vista químico, betalaínas são iminas ou sais de imínio que se originam do acoplamento aldimínico entre o ácido betalâmico e aminas ou aminoácidos. Embora sejam conhecidas mais de 70 betalaínas naturais, os estudos sobre as relações entre a estrutura e as propriedades de betalaínas ainda são limitados. Esta Tese de Doutorado descreve a semissíntese e estudo da hidrólise e das propriedades fotofísicas e redox de três grupos de betalaínas não naturais: N-fenilbetalaínas (pBeets), N-metil-N-fenilbetalaínas (N-Me-pBeets) e N,N-difenilbetalaínas (dipBeets). O estudo de quatorze pBeets para substituidas sugere que a porção imina destes compostos está protonada em meio aquoso neutro e que substituintes atraentes de elétron na porção fenílica favorecem o deslocamento de carga do nitrogênio 2-piperideínico para a porção imínica. A constante de hidrólise aumenta linearmente com o aumento do valor de σp de Hammett do substituinte, dado o aumento da eletrofilificade do carbono aldimínico. Por outro lado, pBeets com substituintes atraentes de elétron são mais fluorescentes. A adição de metila ou fenila ao nitrogênio da imina cria um ambiente hidrofóbico que compromete o ataque da água e diminui a constante de velocidade de hidrólise provocando, contudo, uma diminuição na fluorescência. Os resultados foram racionalizados empregando-se espectroscopia de ressonância magnética nuclear, espectrofotometria e voltametria cíclica. / Betalains are natural pigments derived from L-tyrosine that are found in a restricted number of plants and fungi. From the chemical point of view, betalains are imines or iminium salts that originate from the aldimine coupling between betalamic acid and amines or amino acids. Although more than 70 natural betalains are known, studies on structure-property relationships of betalains are still limited. This doctoral thesis describes the semi-synthesis and study of the hydrolysis as well as photophysical and redox properties of three groups of non-natural betalaines: N-phenylbetalaines (pBeets), N-methyl-N-phenylbetalines (N-Me-pBeets) and N,N-diphenylbetalaines (dipBeets). The study of fourteen substituted pBeets suggests that the imine portion of these compounds is protonated in neutral aqueous medium and that attractive electron substituents on the phenyl portion favor the displacement of 2-piperidene nitrogen charge to the imine portion. The hydrolysis constant increases linearly with the increase of the Hammett\'s σp value of the substituent, given the increase in the electrophilicty of the aldimine carbon. On the other hand, pBeets with attractive electron substituents are more fluorescent. The addition of methyl or phenyl to the imine nitrogen creates a hydrophobic environment upon water attack and decreases the hydrolysis rate constant, however, causing a decrease in fluorescence. The results were rationalized using nuclear magnetic resonance spectroscopy, spectrophotometry and cyclic voltammetry.

Grâce à l'introduction de deux groupes chimiques dynamiques distincts dans un réseau de polymères élastomères, un matériau auto-cicatrisant et recyclable a été synthétisé et caractérisé. La polycondensation d'un polybutadiène polyamine avec une uréidopyrimidinone fonctionnalisée par deux aldéhydes, a abouti à un matériau élastique fort et extensible, composé de liaisons croisées d'imines ainsi que de dimères et d'agrégats d'uréidopyrimidinones. La caractérisation physique a montré que ce matériau à double dynamique présente un comportement caoutchouteux à température ambiante, tandis que, à des températures élevées, les chimies supramoléculaires et covalentes réversibles sont activées, ce qui entraîne des propriétés vitrimères. Pour étudier de plus près le rôle des deux fragments dynamiques, le comportement des matériaux uniquement réticulés par des interactions supramoléculaires a été exploré. Dans ces matériaux, le comportement caoutchouteux aux températures d’usage est à nouveau observé, tandis qu’un état visqueux est observé à des températures élevées. Dans tous les cas, les matériaux dynamiques se sont auto-cicatrisés lors de l'exposition à la chaleur et sont recyclables par hydrolyse acide. / By the introduction of two distinct dynamic chemical groups into an elastomeric polymer network, a self-healing and soluble material was synthesised and characterised. The polycondensation of a polyamine polybutadiene with a novel dialdehyde-functionalised ureidopyrimidinone, resulted in a strong and stretchable elastic material, composed of imine cross-links as well as ureidopyrimidinone dimers and aggregates. Physical characterisation demonstrated that this double-dynamic material displays rubbery behaviour at ambient temperatures, while at elevated temperatures both supramolecular and reversible covalent chemistries are activated resulting in vitrimeric properties. To more closely investigate the role of both dynamic moieties, the behaviour of materials solely cross-linked by supramolecular interactions were studied. In these materials, rubbery behaviour at service temperatures is again observed, while viscous flow is observed at elevated temperatures. In all cases, the dynamic materials were self-healing on exposure to heat, and soluble by acid-catalysed hydrolysis.

L’organocatalyse permet l’activation de réactions chimiques diverses et représente une méthode complémentaire à la catalyse métallique ou à la catalyse par les enzymes. Cette stratégie peut être moins toxique et relativement plus accessible, en plus d’être fréquemment économique et écologique. En induisant une intramolécularité temporaire, il est possible de surmonter les pénalités entropiques inhérentes aux réactions intermoléculaires. Les travaux effectués par le groupe de recherche du Pr. Beauchemin ont jusqu’à date permis d’actualiser et d’améliorer des réactions d’hydroamination, d’hydratation d’amino nitriles et d’hydrolyse d’amides organophosphoniques grâce à l’organocatalyse aux aldéhydes simples. Dans cette optique, cette thèse étudie l’application de cette méthodologie dans des réactions d’hydrolyse. Dans un premier temps, les paramètres réactionnels favorisant l’hydrolyse d’α-amino esters furent investigués. Les problèmes de labilité de divers substrats en milieu aqueux furent abordés, de même que les difficultés à effectuer un « turnover » catalytique efficace. Dans un deuxième temps, la mono-hydrolyse de 1-amino diester phosphonique fut étudiée. Les esters d’acide 1-amino phosphoniques suscitent un intérêt en raison de leurs bioactivités diverses, notamment comme analogues d’acides aminés capables d’interactions biologiques avantageuses. Il fut possible d’optimiser plusieurs conditions réactionnelles, de même que d’étendre la portée de cette méthode à la synthèse de dérivés portant des groupements alkyles. Sur un autre ordre d’idées, les hétérocycles azotés font preuve de bioactivités importantes et variées. Conséquemment, leur emploi dans les domaines pharmaceutique et agrochimique est incontournable. Les cycles de type pyrazolone sont particulièrement présents dans des composés pharmaceutiques et dans des insecticides et des herbicides, tandis que les triazolium-olates présentes des activités biologiques intéressantes. Il devient ainsi essentiel de développer des voies polyvalentes, efficaces, peu dispendieuses et plus vertes pour leur synthèse. Des travaux antérieurs du groupe Beauchemin ont élaboré la génération in situ d’isocyanates et d’isothiocyanates N-substitués, des intermédiaires rares dans la littérature. Ils furent utilisés dans des réactions de cycloaddition avec des alcènes et des imines, pour former des azométhine imines. La deuxième partie de cette thèse s’intéresse à la dérivation de ces azométhine imines en dérivés de pyrazolone et en triazolium-olates grâce à une réaction d’isomérisation. Cette approche permet une déconnexion novatrice pour synthétiser directement des hétérocycles riches en azote complexes. L’optimisation de cette réaction fut mise au point et sa portée fut étudiée. Cette méthode s’avère peu dispendieuse, n’emploie aucun catalyseur métallique, ne requière pas de préfonctionnalisation et ne génère pas de sous-produits. Finalement, l’application de cette isomérisation à la résolution cinétique des azométhines imines fut tentée.

Chiral amine motifs are found in many bioactive compounds and therefore strategies for their direct asymmetric synthesis are of great interest. Alongside traditional chemical methods, biocatalysis serves as an important tool for the formation of these compounds that can confer the benefits of sustainable catalyst supply and mild reaction conditions. This thesis describes the application of imine reductase (IRED) biocatalysts for the asymmetric reduction of pre-formed imines and the reductive amination of carbonyl compounds to produce chiral amines. These enzymes are relatively recent additions to the toolbox of biocatalysts for chiral amine synthesis and therefore their scope and application is still very much being explored. The research carried out as part of this PhD is presented as a series of manuscripts that have either been published or are planned for submission to peer-reviewed journals. The choice of presenting this thesis in journal format was made because a considerable body of the candidate's PhD research has been published, with the rest planned for publication in the near future. Furthermore, the compiled review articles and research papers lend themselves to a clear thesis narrative and, combined, have taken considerable time and effort to prepare, equal to that of a traditional thesis format. The contents are organised as follows: Chapter 1: an introduction to biocatalysis and its impact on sustainable chemical manufacturing; Chapter 2: a review assessing the current state of the art in imine reductase biocatalysts; Chapter 3: a perspective on the design and implementation of biocatalytic cascades; Chapter 4: a research article on the application of IREDs in a biocatalytic cascade for the synthesis of chiral piperidine and pyrrolidine frameworks; Chapter 5: aims of the PhD project; Chapter 6: a research article on the discovery and investigation of a reductive aminase (RedAm) found within the IRED family; Chapter 7: a research article on the screening of a diverse set of novel IREDs for their ability to facilitate reductive amination; Chapter 8: a research article on the synthesis of complex bulky dibenz[c,e]azepine compounds using IRED and transaminase biocatalysts; Chapter 9: a summary and outlook; Chapter 10: manuscript supporting information further detailing experimental work; Appendix: list of other publications resulting from this doctoral research.

Philosophiae Doctor - PhD / Nitric oxide (NO) plays a key role as biological messenger in the biological system, however detection and quantification thereof has always posed significant problems. NOx is a principal constituent of air pollutants. There are seven oxides of nitrogen of which N2O, NO and NO2 are most important. NO is a free radical and reacts extremely fast with oxygen, peroxides and superoxides. It’s these reactions which are responsible for NO’s fleeting existence. The specific detection and quantification of NO still remains challenging. Most techniques rely on the measurements of secondary nitrite and nitrate species. Electrochemical techniques using ultra micro-electrode systems presented the possibility of direct detection of NO, offering a range of favourable characteristics; good selectivity towards NO, good sensitivity, fast response, long-term stability and ease of handling. Electrochemical detection of NO relies on the modification of electrode surfaces and exploiting the redox properties of NO. NO can either be oxidized or reduced electrochemically depending on the nature of the solution. Under cathodic current NO is reduced to nitrosyl, a highly unstable derivative of NO. These nitrosyls are subject to a serious of chemical reactions to eventually form nitrous oxide. Due to the interferences presented by the electrochemical reduction of NO, the electro oxidation of NO is therefore the methodology of choice for NO detection. The electrochemical oxidation of NO occurs at positive potentials around 800 mV vs. Ag/AgCl. However this potential range is not only favourable to NO oxidation but can lead to the oxidation of several other biological species. These interfering species are biologically present at concentrations higher than NO therefore selectivity is of the highest order when designing these electrode systems. Some nitric oxide sensors are limited in their sensitivity, stability and reproducibility. Direct electron transfer between redox proteins and conductive membrane layers has been scrutinized for years in an attempt to reproduce the mechanistic charge transfer processes for sensor application. However, literature reports have presented many arguments on the complexities associated with depositing these enzymes on electrode surfaces for the purpose of reproducing direct electron transfer at metalloprotein centres. The study sets out to design a material which could mimic the electrochemistry of denatured cytochrome-c. To achieve this it was imperative to design a polymer which could reproduce the electrochemistry of the ligands coordinated to the metal centre of the metalloprotein. A novel Schiff base was synthesized by cross-linking naphthalene to pyrrole to produce the monomer, N,N-bis((1H-pyrrol-2-yl)methylene)naphthalene-2,3-diamine). The monomer was electrodeposited on a screen print carbon electrode (SPCE) vs. Ag/AgCl and served as a supporting layer for denatured cytochrome-c. Cytochrome-c is classified as a metalloprotein. These metalloproteins possess metal centres which when denatured unfolds and allows access to the metal centre. Cytochrome-c was subjected to thermal denaturation which opened up the iron centre. The denatured metalloprotein was cross-linked to the ligand to reconstruct the heme centre environment. This was believed to facilitate the electrochemical activity of the system and allow for electrochemical analysis of these metalloproteins for sensor application. The redox behaviour of the sensors were modelled in phosphate buffer solution (PBS) with cyclic voltammetry. Electrochemical analysis reported the sensors to possess reversible electrochemistry with diffusion control characteristics. The sensor recorded a redox system in the negative potentials range. Following the establishment of the electrochemical profile of the sensor an attempt was made to produce a synthetic analogue of denatured cytochrome-c. Iron (II) was chelated to the monomer N,N-bis((1H-pyrrol-2-yl)methylene)naphthalene-2,3-diamine) to form an iron ligand complex. The complex was subjected to a series of characterization techniques which confirmed coordination to the metal centre. The iron ligand complex was electrodeposited on a SPCE over the potential window of -1 V and 1 V to model the electrochemical behaviour of the sensor. The material was found to be electroactive. Subsequent electrochemical analysis revealed the system to have electrochemical properties, analogous to that of the denatured cytochrome-c system. The sensor was applied in NO and NO2 studies and displayed an affinity towards NO. Based on extrapolated values it was postulated that the lower limit range for NO detection was in the range of 30 to 40 nM. The potentials recorded were lower than the reported oxidation potentials for nitric oxide. The sensor displayed stability and selectivity towards nitric oxide within a complex matrix. The complex matrix employed in this study was synthetic urine that was synthesised in the lab. The sensor displayed the capacity for linear range of NO detection with very low error margins. / 2021-09-01

Carbon nanotubes were dispersed and functionalized with polyethylene imine (PEI) before incorporation in a polyvinyl acetate matrix. The resulting samples exhibit air-stable N-type characteristics with electrical conductivities as great as 1600 S/m and thermopowers as high as 100 microV/K. Thermopowers and electrical conductivities correlate, in a reversal of the trend found in typical materials. This phenomenon is believed to be due to the increase in the number of tubes that are evenly coated in a better dispersed sample. Increasing the amount of PEI relative to the other constituents positively affects thermopower but not conductivity. Air exposure reduces both thermopower and conductivity, but a stable value is reached within seven days following film fabrication. The atmospheric effects on the electrical conductivity prove to be reversible. Oxygen is believed to be the primary contributor to the decay.

Nitrogen-containing molecules are ubiquitous in both natural products and pharmaceutical drugs, thus an efficient method for the formation of these motifs is of great importance. Hydroamination, that is the addition of an N-H bond across an unsaturated carbon-carbon bond of an alkene or alkyne, stands out as a potential approach to obtain such molecules. To date, most research in this area relies on transition-metal catalysis to enable such reactivity. In efforts directed towards metal-free alternatives, we have developed a simple, metal-free hydroamination of alkenes using hydrazides. Further investigation into the corresponding reactivity of alkynes with hydrazides has provided access to novel azomethine imine products. In Chapter 2, expansion of the substrate scope with respect to the intramolecular hydroamination of alkenes using hydrazides, as well as studies directed towards elucidation of the mechanism of this reaction will be presented. The intramolecular hydroamination of alkynes using hydrazides and methods to access and isolate the azomethine imine products formed will be discussed in Chapter 3.