# Chemistry - Theses, Dissertations, and other Required Graduate Degree Essays

## A system-wide stable isotope labeling approach for connecting natural products to their biosynthetic gene clusters

Author:
Date created:
2021-08-19
Abstract:

Although the first bacterial genome sequence was published almost 20 years ago, there is still no generalizable method for automatically assigning natural products to their cognate biosynthetic gene clusters (BGCs). This thesis describes the development of a mass spectrometry-based parallel stable isotope labeling (SIL) platform, termed IsoAnalyst, which automatically associates metabolite stable isotope labeling patterns with BGC structure prediction in order to connect natural products to their cognate BGCs. The parallel SIL experiments were optimized for small scale and a custom tool written in Python was developed for the untargeted detection and interpretation of SIL labeling patterns. This approach was validated in the industrial production strains Saccharopolyspora erythraea and Amycolatopsis mediterranei demonstrating that the compounds erythromycin A and rifamycin SV respectively, could be associated with the proper BGCs based on the distribution of isotopomer labeling patterns. The method was further validated by connecting known biosynthetic intermediates of these compounds to their associated BGCs and the identification of various siderophores through a combination of SIL labeling patterns and MS/MS fragmentation data. Extension to environmental organisms using a sequenced Micromonospora sp. from our Actinobacterial isolate library led to the discovery of lobosamide D, a new member of the lobosamide family of natural products, and an update to the lobosamide BGC to include relevant tailoring enzymes. This discovery illustrates the power of the IsoAnalyst platform for identifying new compounds, linking molecules to BGCs, and generating new knowledge about biosynthesis.

Document type:
Thesis
File(s):
Supervisor(s):
Roger Linington
Department:
Science: Department of Chemistry
Thesis type:
(Thesis) Ph.D.

## The design, synthesis, and evaluation of radiopharmaceuticals for actinium-225

Author:
Date created:
2021-11-08
Abstract:

This thesis studies the design, synthesis, and optimization of radiopharmaceuticals for targeted alpha therapy applications with actinium-225 (225Ac). In Chapter 2, three novel radiopharmaceuticals - 2.1 (DOTA-CCZ-N-Me-α-CycMSH), 2.2 (Macropa-CCZ-N-Me- α-CycMSH), and 2.6 (Macropa-α-CycMSH) for malignant melanoma therapy were labeled with 225Ac and evaluated. All three radiopharmaceuticals exhibited excellent in vitro stability, while Macropa-CCZ-N-Me-α-CycMSH showed lower tumor uptake and moderate normal tissue uptake. In Chapter 3, a total of 5 diaza-18-crown-6 macrocyclic ligands (macropa, macropaquin, macroquin-SO3, macrohopo, and macrohopo’) as chelators for 225Ac were investigated. Two of the chelators (macrohopo and macrohopo’) which contain hydroxypyridinone pendant donor arms are novel and were synthesized/characterized in this work. Macropaquin was able to quantitively radiolabel at chelator concentrations as low as 10-6 M at ambient temperatures within one hour, while chelator macrohopo was unable to achieve 225Ac complexation under any conditions. This thesis showcases the complexity of radiopharmeticuals, in particular for 225Ac.

Document type:
Thesis
File(s):
Supervisor(s):
Caterina Ramogida
Department:
Science: Department of Chemistry
Thesis type:
(Thesis) M.Sc.

## Metalloporphyrins or vanadium (oxo) complexes and their applications to bond activation chemistry

Author:
Date created:
2021-05-28
Abstract:

Recent developments in new molecular designs, scaling relationships, and theoretical modeling of mechanisms have rapidly advanced the utility of metalloporphyrins as electrocatalysts in the activation of small molecules, in particular O2 and CO2. The development and improvement of electrocatalysts for the 4H+ /4e− reduction of O2 to H2O, and for the CO2 reduction reaction (CO2RR) to provide a route to turn a greenhouse gas into value-added products, are ongoing challenges. The addition of ancillary groups (e.g., hydrogen bonding, Brønsted acid/base) near the active site of metal-containing catalysts is an effective way to improve the selectivity and kinetics of the oxygen reduction reaction (ORR) and CO2RR. In this regard, iron porphyrins are among the most researched ORR/CO2RR catalysts. Closely related cobalt porphyrin catalysts can function closer to the O2/H2O thermodynamic potential and can reduce CO2 at a lower overpotentials, but they tend to be less selective and follow a different mechanism than iron porphyrins. Here, I explore strategies to extend the ideas about ancillary groups that have been developed for iron porphyrin ORR/CO2RR electrocatalysts to improve the performance of the corresponding cobalt complexes. I describe a series of porphyrin electrocatalysts for ORR/CO2RR that are modified versions of Co(5,10,15,20-tetraphenylporphyrin), where the 2-position of one of the phenyl groups contains -NH2, -N(CH3)2, and -N(CH3)3+. In Chapter 2, the presence of a cationic ancillary group gives rise to a catalyst that is selective for the conversion of O2 to H2O across a wide pH range. Electrostatic groups for cobalt porphyrins enhance the performance of 4e– reduction of O2 to H2O, while protic ancillary groups are important in the performance of iron porphyrin ORR catalysts. In Chapter 3 and 4, homogeneous and heterogeneous CO2RR are studied, respectively. For cobalt porphyrins, protic hydroxyphenyl ancillary groups aid in production of CO, while electrostatic stabilizers of CO2-bound intermediates seem to favor other CO2 reduction products. These studies show that the second sphere interactions for ancillary groups of metalloporphyrins must be carefully designed for different metals and different reactions. In addition, the photochemical activation of carbon-hydrogen bonds by vanadium-dioxo (VV(O)(O)) and vanadium-oxo-peroxo (VV(O)(O2)) diimine complexes is described in Chapter 5. The involvement of electronically excited V complexes as radical reaction initiators is described.

Document type:
Thesis
File(s):
Supervisor(s):
Jeffrey Warren
Department:
Science: Department of Chemistry
Thesis type:
(Thesis) Ph.D.

## Spectroscopic studies of neutron-rich 129Sn and 80Ge nuclei using the GRIFFIN spectrometer

Author:
Date created:
2021-07-30
Abstract:

Spectroscopic studies provide information about the structure of nuclei. Information that, in turn, serves to inform theoretical models and astrophysical studies.Recent work using the GRIFFIN spectrometer at TRIUMF has uncovered novel information in two isotopes, $^{129}$Sn and $^{80}$Ge, which lie close to the two magic nuclei: $^{132}$Sn and $^{78}$Ni, respectively. The work on $^{129}$Sn observed 31 transitions and 9 excited states for the first time, populated through the $\beta$-decay of $^{129}$In. Most notably, this experiment was the first to observe the $\beta$-decay of the $(29/2^{+})$ 1911-keV isomer of $^{129}$In. The work on $^{80}$Ge searched for a previously observed $0^{+}$ state at 639-keV, just below the $2^{+}$ 659-keV excited state, through the $\beta$-decay of $^{80}$Ga. The presence of the former was proposed as evidence for low-energy shape coexistence in $^{80}$Ge, in the region around doubly-magic $^{78}$Ni, though further signatures for the presence of this state were missing. The GRIFFIN experiment was unable to observe this state, despite using a superior detector system and observing higher statistics . The nonobservation, coupled with theoretical calculations performed, led to the conclusion that this $0^{+}$ 639-keV does not exist. Furthermore, the analysis observed, for the first time, 66 excited states and 157 $\gamma$-ray transitions, which were added to the known $^{80}$Ge level scheme. The data here presented will serve to improve upon existing nuclear structure theories and will inform future applications of nuclear science to fields such as astrophysics.

Document type:
Thesis
File(s):
Supervisor(s):
Corina Andreoiu
Department:
Science: Department of Chemistry
Thesis type:
(Thesis) Ph.D.

## Synthesis, structure and relaxor to ferroelectric crossover in the lead-strontium zirconate-titanate perovskite solid solution system

Author:
Date created:
2021-04-26
Abstract:

In order to investigate the structures and physical properties of intriguing relaxor ferroelectrics, a novel solid solution, (1-x)(Pb0.5Sr0.5)(Zr0.5Ti0.5)O3-xPbTiO3 (PSZT-PT), of complex perovskite structure was synthesized by solid-state reaction method. The ceramics of the PSZT-PT system were characterized by X-ray diffraction (XRD), dielectric spectroscopy, ferroelectric testing and transmission electron microscopy (TEM). Detailed structural analyses based on the refinements of XRD patterns by both the Pawley and Rietveld methods reveal a structural transformation from the cubic to tetragonal symmetry as the concentration of PT (x) increases, with a mixture of the two phases in the composition range of x ≈ 0.10 ~ 0.25. Subsequently, the dielectric properties of the PSZT-PT solid solution were characterized as a function of temperature at various frequencies (10 Hz to 1 MHz). The results indicate a crossover from typical relaxor behaviour to normal ferroelectric state with increasing concentration of PT. The characteristics of relaxor behaviour and its evolution were analyzed by the Vogel-Fulcher law and Curie-Weiss law in terms of the dielectric peak temperature (Tmax) shift with frequency, the freezing temperature (Tf) and the Burns temperature (TB). Furthermore, the measurements of polarization versus electric field demonstrate that, as the concentration of PT increases, the ferroelectric hysteresis loop of the PSZT-PT solid solution transforms from a narrow curve to a wide-open loop with enhanced remanent polarization and coercive field, confirming the relaxor to ferroelectric transformation. The domain structures of PSZT-PT were imaged and analyzed by means of TEM. The results show the presence of polar nano-domains in the relaxor composition, which transform into microscopic domains in the ferroelectric compound, revealing the transformation from the relaxor to ferroelectric state on the nano- to micro-scale. The mechanism of the crossover was illustrated from the effects of the substitution of ferroelectrically active lead titanate. Finally, the evolutions of the crystal structures and physical properties of the PSZT - PT solid solution are presented in the forms of structural and dielectric phase diagrams, providing a clear picture of the crossover from relaxor to ferroelectric state in this pseudo-binary system, and the phase diagram of the PbZrO3 - PbTiO3 - SrTiO3 ternary system is established.

Document type:
Thesis
File(s):
Supervisor(s):
Zuo-Guang Ye
Department:
Science: Department of Chemistry
Thesis type:
(Thesis) M.Sc.

## Stabilizing proton exchange membranes using Cerium(III) ions

Author:
Date created:
2021-10-05
Abstract:

Document type:
Thesis
File(s):
Supervisor(s):
Steven Holdcroft
Department:
Science: Department of Chemistry
Thesis type:
(Thesis) M.Sc.

## Studies of bacterial and insect cytochromes P450 in degradation of pesticides

Author:
Date created:
2021-08-16
Abstract:

Cytochromes P450 is a group of heme-containing enzymes with diverse catalytic activity that can be used for the biodegradation of environmental chemicals. Cytochrome P450cam (CYP101A1) from the soil bacterium Pseudomonas putida is known for hydroxylating camphor. Here, I have investigated the dehalogenation ability of two P450cam mutants, ES6 (G120S) and ES7 (V247F/D297N/K314E), in comparison to the wild-type (WT) enzyme. Six hexachlorinated persistent organic pollutants (POP), namely endosulfan (ES), ES diol, ES lactone, ES ether, ES sulfate and heptachlor, were tested since they are similarly structured to the native substrate. The mutated enzymes were capable of converting the selected substrates to phenols and o-quinones, which were detected using a colorimetric assay with 4-aminoantipyrine (4-AAP). Kinetic studies and statistical analysis were carried out and it was found that both ES6 and ES7 are significantly more active than the WT, with the highest activity noticed against ES ether and heptachlor. The western honey bee, Apis mellifera, is a vital pollinator of the ecosystem, however, its being threatened by the ectoparasitic mite, Varroa destructor. This pest is becoming immune towards commercially available pesticides, thus, new control agents have been previously synthesized that showed miticidal effects. Fortunately, insect cytochromes P450 are known to be responsible for the metabolism of such xenobiotics. Here, I have tested the ability of three potent dialkoxybenzene compounds, namely 1-allyloxy-4-propoxybenzene (3c{3,6}), 1,4-dipropoxybenzene (3c{3,3}) and 1,4-diallyloxybenzene (3c{6,6}), to get degraded by honey bee cytochromes P450. The formation of the dealkylated products was detected in abdomen extracts using a colorimetric assay with 4-aminoantipyrine (4-AAP). Kinetic studies and statistical analysis showed a downregulation of detectable P450 activity in the treated vs. the untreated extracts. Gas chromatography-mass spectrometry (GC-MS) quantitative assays were carried out and three dealkylated products were found, hydroquinone (HQ), 1-hydroxy-4-propoxybenzene (2c{3}) and 1-hydroxy-4-allyloxybenzene (2c{6}).

Document type:
Thesis
File(s):
Supervisor(s):
Erika Plettner
Department:
Science: Department of Chemistry
Thesis type:
(Thesis) M.Sc.

## Design, synthesis and use of chiral pheromone-based probes to study pheromone enantiomer discrimination in the pheromone binding proteins from the gypsy moth, Lymantria dispar

Author:
Date created:
2021-04-15
Abstract:

The gypsy moth is a widespread and harmful pest causing extensive damage to the Canada’s forest and orchard ecosystems. It uses (+)-disparlure as a sex pheromone. Discovery of the pheromone, including its absolute configuration, has enabled monitoring of gypsy moth populations. Disparlure of low enantiopurity is not attractive to the moths and, for this reason, enantiopure (+)-disparlure has been a synthetic target for many years. To access (+)-disparlure of high enantiopurity we have used a diastereoselective nucleophilic addition reaction with the enantiopure α-chloroaldehyde (2-chlorododecanal) that yields a stereocontrolled access to the 1,2-anti chlorohydrin core. The (+)-disparlure was prepared through a series of transformations that include a Mitsunobu inversion. We have successfully completed the synthesis of (+)-disparlure in 5 steps as compared to Iwaki’s first synthesis in 12 steps and Sharpless’s widely used synthesis in 6 steps. The same approach was used to produce 18-hydroxydisparlure enantiomers, which were coupled to a linker with an alkyne moiety at the end. The alkyne was then coupled to azide-based commercial fluorescent probes, to furnish fluorescent disparlure-based probes for physical studies. The gypsy moth has two different pheromone binding proteins, LdisPBP1 and LdisPBP2. Previously, our group has addressed the enantiomer selectivity of these two PBPs and found that PBP1 binds (-)-disparlure more strongly than (+)-disparlure, while PBP2 binds (+)-disparlure more strongly. Despite several binding assays, the interaction and discrimination of gypsy moth PBPs towards disparlure enantiomers are not fully understood due to lack of binding interaction and kinetic studies, which are technically demanding, due to the hydrophobicity of the pheromone. In this thesis, we have studied the binding interaction of deuterium-labelled (+)-disparlure and (-)-disparlure with LdisPBPs by 2H NMR spectroscopy. The results from NMR studies were correlated with the results from docking simulations of (+)-disparlure and (-)-disparlure bound to one internal site and multiple external sites of LdisPBP1 and LdisPBP2. These results indicated that (+)-disparlure and (-)-disparlure adopt different conformations and orientations in the binding pockets of LdisPBP1 and LdisPBP2. Most of the reported work on PBPs focuses on the pheromone binding affinities of PBPs. However, the pheromone-PBP interactions require more than half an hour to establish equilibrium, whereas male moths respond to female pheromones in milliseconds. Therefore, the interactions between pheromones and olfactory components such as PBPs and pheromone receptors may not be under thermodynamic control. In this thesis, we aimed to provide a dynamic perspective of pheromone-PBP interactions and to link these to the functions of PBPs. We have studied thermodynamic (Kd) and kinetic properties (kon and koff) of LdisPBPs-disparlure enantiomer interaction by fluorescence binding assays and kinetic experiments using fluorophore-tagged disparlure enantiomers. The result indicated that the binding preference of disparlure enantiomers to LdisPBPs. Based on the kinetic data of LdisPBPs with fluorophore-tagged disparlure enantiomers, we propose a kinetic model that includes a two-step binding process. Each of these two steps may contribute to a different function of the LdisPBPs.

Document type:
Thesis
File(s):
Supervisor(s):
Erika Plettner
Department:
Science: Department of Chemistry
Thesis type:
(Thesis) Ph.D.

## Development of chemical tools for the study of intracellular glycosylation

Author:
Date created:
2020-06-04
Abstract:

Intracellular protein O-glycosylation is an important post-translational modification in mammalian cells with critical regulatory functions relating to transcription, stress response, cell signalling, and cell cycle control. This process is controlled by two enzymes: O-GlcNAc transferase (OGT), which catalyzes the addition of a single O-linked N-acetylglucosamine (O-GlcNAc) to serine and threonine residues of proteins, and O-GlcNAcase (OGA), which hydrolyzes the glycosidic linkage on proteins. While notable progress has been made in the design and application of chemical tools for the study of OGA, methods and tool compounds to detect and modulate the activity of OGT remain limited. In this thesis, I describe the development of a fluorescent glycosyl donor analogue which is tolerated by OGT and transferred to peptides and proteins. This substrate was exploited to develop a convenient and direct in vitro activity assay, enabling the study of OGT catalysis in the presence of various substrates and inhibitors. After optimizing the assay for high-throughput screening, a collection of small molecule libraries encompassing approximately 64,000 compounds was screened, leading to the identification of a novel and selective inhibitor of OGT. This assay was further applied to the functional analysis of peptide-based inhibitors of OGT that were discovered through phage display and in vitro mRNA display technologies. These efforts enabled the development of chemical tools with potent nanomolar affinity for OGT. The in vitro assay was also used for the study of novel glycosylation activity by OGT towards cysteine-containing acceptor substrates, permitting a detailed kinetic analysis of this recently discovered phenomenon. Finally, I describe the biochemical characterization of human Hexosaminidase D (HexD), an intracellular glycoside hydrolase of unknown physiological function, and use this information in the design of potential selective inhibitors. The methods, results, and insights gained from this research should prove useful in advancing our understanding of intracellular glycosylation and for furthering the application of this knowledge for translational therapeutic benefit.

Document type:
Thesis
File(s):
Supervisor(s):
Department:
Science: Department of Chemistry
Thesis type:
(Thesis) Ph.D.

## The applications of interaction between light and chemical reactivity in molecular systems

Author:
Date created:
2021-06-30
Abstract:

This thesis presents a series of scientific examples that provide further insight into the connection of light and chemical reactivity for applications in catalysis, targeted drug delivery, and detection. Small organic molecules based on 1,2-dithienylethenes (DTEs) and noble metal-based nano-assemblies were incorporated into the system to impart control over their physical and chemical properties, and activity. The light was used as an external stimulus to initiate chemical reactions by causing either a structural, electronic, or chemical change in the chromophore or energy transformation (such as heat) in the system. In the first example (Chapter 2), light is employed to generate the variation in the activity of Karstedt’s catalyst by introducing a DTE-based inhibitor. It is demonstrated that the electronic changes that occur during the light-assisted isomerization of dithienylethene lead to changes in the binding strength of the inhibitor to the catalyst’s metal centre. This, in turn, leads to changes in the reaction progression of simple hydrosilylation reactions. In the second example (Chapter 3), a novel dual-mode (colourimetric and fluorescent) optical probe based on photoresponsive dithienylethene is developed for fast and user-friendly detection and consumption of hydrazine in the vapour and solution phase. The molecule reacts with hydrazine to induce an irreversible structural change in the 1,3,5-hexatriene system leading to deactivation of photochromic activity by quenching effects and simultaneously generating an emissive response. In the third example (Chapter 4), light is used as an external stimulus to indirectly gate the chemical reactivity for an on-demand release of therapeutics in aqueous environments by creating nanoassemblies based on gold nanoparticle core. The constructed nanoassemblies consist of the thermoresponsive Diels-Alder adducts of the molecules to be released close to gold nanoparticles enclosed in an amphiphilic polymer shell. The system uses the photothermal effect of gold nanoparticles to trigger thermo-responsive retro Diels-Alder reactions in the vicinity of nanoparticles to release therapeutic molecules in aqueous solutions.

Document type:
Thesis
File(s):
Supervisor(s):
Neil Branda
Department:
Science: Department of Chemistry
Thesis type:
(Thesis) Ph.D.