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

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Chemical proteomics tools for probing carbohydrate processing enzymes

Author: 
Date created: 
2020-04-24
Abstract: 

The modification of proteins with O-linked N-acetylglucosamine (O-GlcNAc) is a nucleocytoplasmic modification that is present on over a thousand different protein targets. This post-translational modification is conserved among multicellular eukaryotes and has been found to play diverse physiological roles within cells. Remarkably, O- GlcNAc levels are globally controlled by only two enzymes; O-GlcNAcase (OGA) and O- GlcNAc transferase (OGT). How this modification is regulated on the large set of target substrates by just these two enzymes remains a topic of high interest within the field. In this thesis, I describe the development of a chemical proteomics method to interrogate OGA that help move the field toward elucidating the factors that regulate this enzyme. Specifically, I describe the creation of four distinct affinity-based probes that bind with high affinity to OGA and enable its precipitation at endogenous levels from tissues. These probes are designed to enable precipitation of OGA and its protein partners under gentle conditions followed by precipitation on streptavidin-coated beads. The disulfide linker that can be cleaved using gentle conditions enables release of OGA-containing protein complexes. Using these probes in a parallel series of experiments, I define a set of high confidence candidate OGA interacting proteins that are seen in multiple data sets from mass spectrometry-based proteomic analyses of the chemoproteomic precipitates obtained from bovine brain tissue. In addition, I detail targeted discovery of post- translational modifications on OGA from bovine brain tissue obtained using one of these new chemoproteomic probes. I envision this approach can ultimately be applied to identifying factors that regulate OGA activity within cells and provide a blueprint for robust chemoproteomics strategies that harness the use of multiple probes having distinct chemical structures.

Document type: 
Thesis
File(s): 
Supervisor(s): 
David Vocadlo
Leonard Foster
Department: 
Science: Department of Chemistry
Thesis type: 
(Thesis) M.Sc.

Developing tools to study carbohydrate processing enzymes in AmpC β-lactamase antibiotic resistance and OGA-based neurodegenerative research

Author: 
Date created: 
2020-12-18
Abstract: 

Carbohydrates are one of the four main classes of biological macromolecules in nature, alongside lipids, proteins, and nucleic acids. They serve in a wide range of cellular functions fundamental to the existence of biological organisms. These functions include but are not limited to cellular metabolism, energy production and storage, structural support, signaling, and recognition. The ubiquitous presence of carbohydrates in organisms has led to the study of their role in various maladies, such as neurodegenerative and infectious diseases. Deciphering the role of carbohydrates in these diseases allows for the possibility of developing treatments for associated diseases. In this manner, it is necessary to develop tools to exploit and test our understanding of these mechanisms. Existing methodologies may need to be adapted for application to larger scale experimental designs, such as those used in chronic dosing studies using preclinical animal models or high-throughput automated screening assays. This thesis describes improvements to previously published methods in the synthesis of one such chemical tool, Thiamet-G, a small molecule inhibitor used to study the carbohydrate processing enzyme O-GlcNAcase, which has been linked to neurodegenerative diseases including Alzheimer’s and Parkinson’s Disease. This thesis also seeks to apply the concepts developed during creation of a live cell assay towards creation of a new experimental approach suitable for large scale high through-put screening of compound libraries. Such an application would allow for the efficient pursuit of inhibitors of the bacterial protein AmpG - a transporter that is essential for inducible AmpC β-lactamase-driven antibiotic resistance.

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

Structure-property relationships in sterically-congested proton-conducting poly(phenylene)s: The impact of backbone linearity

Author: 
Date created: 
2020-04-09
Abstract: 

Decarbonatization of the World’s primary energy supply is becoming increasingly more important due to a rapidly changing climate. A hydrogen-based economy offers a potential means of zero-carbon energy production through the use of fuel cells and water electrolyzers. The development of robust, thermochemically-stable hydrocarbon-based proton exchange membrane materials that resist swelling for use in these devices represent a significant hurdle in their commercial adoption. In this thesis, the structure-property relationship of hydrocarbon-based sulfonated phenylated poly(phenylene) proton exchange membranes possessing either angled or linear backbone moieties is discussed. Polymers were synthesized using either bent (ortho or meta), or linear (para) biphenyl linkages and evaluated for differences in physical and electrochemical properties. Model compounds, structurally-analogous to the polymers, were prepared and characterized using spectroscopic and computational methods to elucidate structural differences and potential impacts on the properties of the respective polymers. A highly angled ortho biphenyl linkage resulted in a sterically hindered, rotationally-restricted molecule. When incorporated into a homo-polymer, the angled ortho biphenyl moiety was found to prevent membrane formation. The angled meta biphenyl-containing homo-polymer, while forming a membrane, exhibited a 74% increase in volumetric expansion, 31% reduction in tensile strength, and 72% reduction in the elongation at break when compared to the linear para biphenyl-containing analogue. The differences observed are attributed to a rotationally-restricted backbone in the angled biphenyl systems. Co-polymers containing a small fraction (≤5%) of the ortho or meta biphenyl linkage in an otherwise para biphenyl containing system were found to have a significantly lower degree of swelling than those containing solely para biphenyl linkages. Collectively, the work presented in this thesis suggests that incorporating angled biphenyl linkages into sulfonated phenylated poly(phenylene)s leads to highly rigid, inflexible backbones that prevents chain entanglement and the formation of free-standing membranes.

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

Evaluating the effects of 1-allyloxy-4-propoxybenzene on the parasitic mite Varroa destructor and synthesis of fluorescent probes to visualize the binding location(s) of the active compound in the mite

Author: 
Date created: 
2020-12-16
Abstract: 

Varroa destructor is a serious threat to the eastern honey bees, Apis melliferra. The available varroa controls are either ineffective due to resistance of varroa mites or require correct temperatures for efficacy and are labour intensive. To search new effective varroa control agents, 15 compounds were assesed for the acaricidal activities like paralysis and death, against varroa mites in laboratory bioassays. The data from structure-activity assays revealed that allyloxy and propoxy groups at the para position of a benzene-based structure, are necessary for the acaricidal activities tested. Compound 1-allyloxy-4-propoxybenzene, known as 3c{3,6}, showed the highest acaricidal activites from a group of 15 compounds tested . Activity of 3c{3,6} was the same as that of thymol, a widely used varroa control agent in structure-activity relationship studies. 3c{3,6} prevented the mites from staying on the abdomen of bees, which is a major feeding site of the mites. The active compound 3c{3,6} initially paralyzed the mites and eventually a high number of the mites were observed on the surface of the glass dishes used for bioassays. The varroa acetylcholine esterase (VdAChE) was not inhibited by 3c{3,6} and hence, we conclude that VdAChE is not a target of 3c{3,6}. Fluorescent probes in which the structure of 3c{3,6} was modified with a linker, and the linker was attached either to a fluorescein or a rhodamine fluorophore, were synthesized to visualise the target binding location(s) in the mites. All probes showed variable acaricidal activies against varroa mites. The fluorescein-containing compound, 6-FAM probe-2, had similar acaricidal activities as 3c{3,6}. The confocal images highlighted fluorescent signals in the regions of the central nervous system (CNS) in the mites, suggest that there may be a binding target of 3c{3,6} in the CNS. Compound 3c{3,6} could be a potential varroa control agent that could be used in combination with the current varroa controls in an integrated pest management (IPM) system.

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

Controlling redox processes in metal complexes and multifunctional materials

Date created: 
2020-12-17
Abstract: 

Transition metal complexes incorporating redox-active ligands have the potential to facilitate controlled multielectron chemistry, enabling their use in catalysis and energy storage applications. Moreover, the use of transition metal complexes containing redox-active ligands has been extended to two- (2D) and three-dimensional (3D) materials, such as supramolecular assemblies (i.e., metallacycles, molecular cages, or macrocycles) and metal-organic frameworks (MOFs) for catalytic, magnetic, electronic, and sensing applications. Salens (N2O2 bis(Schiff-base)-bis(phenolate) are an important class of redox-active ligands, and have been investigated in detail as they are able to stabilize both low and high metal oxidation states for the above-mentioned applications. The work in this thesis focuses on the synthesis and electronic structure elucidation of metal salen complexes in monomeric form, as discrete supramolecular assemblies and 3D MOFs. Structural and spectroscopic characterization of the neutral and oxidized species was completed using mass spectrometry, cyclic voltammetry, X-ray diffraction, NMR, UV-Vis-NIR, and EPR spectroscopies, as well as theoretical (DFT) calculations. Chapter 2 discusses the synthesis and electronic structure evaluation of a series of oxidized uranyl complexes, containing redox-active salen ligands with varying para-ring substituents (tBu, OMe, NMe2). Chapters 3 and 4 discuss the incorporation of a redox-active nickel salen complex equipped with pyridyl groups on the peripheral positions of the ligand framework into supramolecular structures via coordination-driven self-assembly. The self-assembly results in formation of a number of distinct metallacycles, affording di-, tetra-, and octa-ligand radical species. Finally, the design, synthesis, and incorporation of metal salen units into MOFs is discussed in Chapter 5. Preliminary assembly and oxidation experiments are presented as an opportunity to explore the redox-properties of salen complexes incorporated into a solid-state 3D framework. Overall, the work described in this thesis provides a pathway for salen ligand radical systems to be used in redox-controlled host-guest chemistry, catalysis, and sensing.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Tim Storr
Daniel B. Leznoff
Department: 
Science: Department of Chemistry
Thesis type: 
(Thesis) Ph.D.

Electrochemical pressure impedance spectroscopy as a diagnostic method for hydrogen-air polymer electrolyte fuel cells

Author: 
Date created: 
2020-12-08
Abstract: 

This work presents the implementation and analysis of electrochemical pressure impedance spectroscopy (EPIS) as an in situ diagnostic method for polymer electrolyte fuel cells (PEFCs). Inspired by electrochemical impedance spectroscopy (EIS), EPIS is an acoustic spectroscopic technique that analyses the response of the voltage signal to an applied pressure signal in the frequency domain. In EPIS, the cathode gas pressure is modulated as a sinusoidal wave and the voltage measured from the PEFC is monitored as the response signal. The EPIS measurements are sought as a means to probe cathode transport properties specifically as these processes are involving mass transport limitations during PEFC operation. The development of EPIS is sought to furnish tools to study, for example, water and oxygen transport in the MEA as a function of changes in its structure and its operating conditions. Flow channels are one of the core elements of PEFCs. To probe the contribution of the flow channel to an EPIS response, the flow channel is characterised by the response of the system as a function of correlations between pressure measured at the cathode inlet and outlet. The outlet pressure is excited as the input variable, and the response of the system is measured at the inlet, on the other side of the flow channels. An alternative way to characterise the flow channel is to apply a flow rate step excitation at the inlet of the gas flow stream and measure pressure at the flow channel inlet correspondingly. Both methods are discussed in this work. The experimental approach for EPIS requires an oscillating pressure signal in the form of a sinusoidal wave. In this work, oscillations are applied through the use of a: (i) mass flow controller (MFC) oscillation; (ii) back-pressure controller (BPC) oscillation; or (iii) acoustic speaker box (ASB) oscillation. The BPC method studies experimental parameters like the cathode stoichiometry ratio, cathode flow rate, oxygen partial pressure, and pressure oscillation amplitude. The MFC creates a pressure perturbation with a modulated flow and it probes the voltage change with respect to the perturbation in oxygen partial pressure and flow rate. The ASB method is proposed for future studies, and some theoretical basis for the ASB method is covered in the outlook section.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Byron Gates
Michael Eikerling
Department: 
Science: Department of Chemistry
Thesis type: 
(Thesis) M.Sc.

Unconventional silanization to create superhydrophobic substrates and their applications

Author: 
Date created: 
2020-03-10
Abstract: 

Superhydrophobic coatings have been developed to warrant waterproof properties of synthetic materials for diverse applications, such as outdoor clothing, construction materials, and µPAD. Commonly, superhydrophobicity is achieved by increasing the surface roughness and lowering surface tension. Previously, substrates (paper, glass, and polymers) treated with dilute solutions of organosilanes have reached hydrophobicity. However, achieving superhydrophobicity via such conventional silanization reaction without fluorine-based precursors and complex fabrication procedures remains as a challenge. The first work presented is that off-the-shelf laboratory filter papers can be treated into superhydrophobic with a binary solution of short- and long-chain organosilanes. SEM studies confirmed that it is the thickness rather than pore size of the cellulose filter paper governing the superhydrophobicity. The modified filter paper is chemically stable and mechanically durable; it can readily be patterned with UV/ozone treatment to create hydrophilic regions for colorimetric assays of various analytes. Compared to conventional cellulose filter paper, glass microfiber filters are ideal for preparing quantitative fluorometric assays, owing to their extremely low fluorescence background. It was discovered that superhydrophobicity can be achieved on glass microfiber filters by reacting with MTS. Moreover, a fluorometric assay for quantitative copper detection based on “click chemistry” with a customized smartphone app was showcased on patterned glass microfiber filter substrates. This work augments the potential of superhydrophobic glass microfiber filters for multiplex fluorescent assays with ultralow background and high signal-to-noise ratio. The most remarkable finding in this thesis is the development of a protocol that OTS stoichiometrically hydrolyzes and condensates to micro-to-nanoscale hierarchical siloxane aggregates dispersible in industrial solvents. The coating exhibited superior performance in cost, scalability, robustness, and particularly the capability of encapsulating other functional materials. The unconventional silanization reactions to create superhydrophobic surfaces reported in this thesis are beyond the sole purpose of analytical chemistry research, and can be extended to develop marketable daily products with complete waterproofing properties.

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

Modulating electronic structure and reactivity of Cr nitride complexes via xxidation

Author: 
Date created: 
2020-10-15
Abstract: 

Metal nitride complexes exhibit interesting structure and bonding properties that are invoked when discussing the reactivity of these systems. The nitride (N3-) moiety can be either nucleophilic or electrophilic based on a variety of factors such as metal identity, oxidation state, and nature of the ancillary ligands. Herein, the electronic tuning of Cr salen nitride complexes is investigated via modulation of phenolate para-R substituents of varying electron donating ability (R = CF3, tBu, NMe2) in order to influence reactivity. Salen ligands can exhibit non-innocent behavior, implying that redox processes can either be metal or ligand-based. This feature allows the ligand to help facilitate difficult substrate transformations uncommon to Earth-abundant first-row metals. Depending on the para-R group, the locus of oxidation in Cr nitride salen complexes (metal vs. ligand) can be influenced. The electronic structure of oxidized compounds is detailed, allowing for rationalization of nitride reactivity based on oxidation locus.

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

Modification of amyloid-beta peptide aggregation via photoactivation of Ru(II) polypyridyl complexes

Date created: 
2020-07-28
Abstract: 

Alzheimer’s disease (AD) is a chronic neurodegenerative disease characterized by progressive and irreversible damage to the brain. One of the hallmarks of the disease is the presence of both soluble and insoluble aggregates of the amyloid beta (Ab) peptide in the brain. In this work we investigate how photoactivation of three Ru(II) polypyridyl complexes [Ru(6,6’-dimethyl-2,2’-dipyridyl)2(2-thiophen-2-yl-1H-imidazo(4,5-f) (1,10)phenanthroline)] (Ru1), [Ru(6,6’-dimethyl-2,2’-dipyridyl)2(2-phenyl-1H-imidazo (4,5-f)(1,10)phenanthroline)] (Ru2), and [Ru(6,6’-dimethyl-2,2’-dipyridyl)2(2,2’-bipyridine)] (Ru3), alters the aggregation profile of the Ab peptide. Both Ru1 and Ru2 contain an extended planar (4,5-f)(1,10)phenanthroline ligand, as compared to a 2,2’-bipyridine ligand for Ru3, and we show that the presence of the phenanthroline ligand leads to a greater effect on peptide aggregation. The ability of photoactivated Ru1-3 to bind to the Ab peptide was evaluated by Nuclear Magnetic Resonance (NMR) which indicated the loss of the 6,6’-dimethyl-2,2’-bipyridyl (6,6’-dmb) ligand for all three complexes and the formation of a covalent bond with the Ab peptide via His residue shifts for Ru1 and Ru2. By comparison, no shift in His residues was observed for Ru3, or for the unactivated Ru1-3 samples. The influence of Ru1-3 on peptide aggregation was investigated using gel electrophoresis / Western blot, Transmission electron microscopy (TEM) and a Bicinchoninic acid assay (BCA assay). Upon photoactivation, the Ab aggregation was greatly enhanced in the presence of Ru1 and Ru2 relative to Ru3, in agreement with initial binding studies by 1H NMR. However, the three complexes resulted in a similar aggregate size distribution at 24 h, forming mostly insoluble amorphous aggregates. Excitingly, the complexes also changed Ab1-42 fibrils to amorphous aggregates upon photoactivation. The unactivated Ru1 and Ru2 complexes exhibited a much stronger binding affinity for Ab (via Tyr10 fluorescence) in comparison to Ru3, further indicating the important role of hydrophobic interactions between the Ru complexes and the insoluble fibrillar peptide aggregates. Overall, our results show that upon photoactivation the extended planar ligand of Ru1 and Ru2 promotes immediate covalent binding and formation of soluble high molecular weight Ab aggregates in comparison to Ru3, however similar aggregate size and morphology is observed after 24 h for all three Ru complexes.

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

Quantitative electroanalysis of host-guest binding at organized supramolecular interfaces

Author: 
Date created: 
2020-10-07
Abstract: 

As a young and important class of supramolecular host-guest chemistry, the macrocyclic cucurbit[n]uril (CB[n]) hosts consisting of one hydrophobic inner cavity and multiple carbonyl portals have shown dramatically increased research interests since 1980s, with tens of thousand publications focusing on their synthesis, distinct structural features, exceptional physical and chemical properties. More importantly, their excellent host-guest recognition behavior leads to their great application potentials in many fields, such as nanofabrication, biomedical/pharmaceutical science, analytical chemistry, catalytic chemistry, and adaptive chemistry, which have been explored extensively in the past two decades. Particularly, CB[7], an attractive member of CB[n] family, shows ultra-strong host guest binding ability towards small aromatic or ring-structured organic compounds, which is mainly attributed to its proper-sized inner cavity. As a representative, the host-guest complexes formed between CB[7] and various redox-active ferrocene (Fc) derivatives have extremely high binding affinities (109 to 1012 M-1), which have been employed as an alternative of natural binding pairs (e.g., antigen-antibody, biotin-avidin) for fabricating versatile functional molecular and biomolecular interfaces. In order to gain further understanding of this particular host-guest binding pair formed at molecular interfaces, in this thesis, based on both conventional cyclic voltammetry and advanced structural characterizations, the binding thermodynamics and kinetics were investigated on mixed ferrocenylundecanethiolate/octanethiolate self-assembled monolayers on gold as a highly-organized model system. The results show that the inclusion binding behavior of this host-guest pair, while significantly affected by the surface, still has satisfactory stability for practical application. In addition, the broad potential of this new interfacial Fc@CB[7] host-guest binding system is manifested as nanoscale probes for the distribution of Fc terminal groups on SAMs (as an indicative of their structural heterogeneity), as an environmental regulator of long-range electron transfer process, and as an electrochemical sensor for pharmaceutical drugs via competitive host-guest assay strategy. It is expected that this new interfacial host-guest binding system can be further explored for fabricating well-controlled, ratiometric electrochemical biosensors.

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