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

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Applications of α-Chloroaldehydes toward the synthesis of natural products

Author: 
Date created: 
2012-04-20
Abstract: 

The stereochemical complexity evident in agrochemicals, pharmaceuticals, and natural products continues to provide motivation for the development of novel synthetic methods. In the last decade, research in the field of asymmetric organocatalysis has provided new tools for the functionalisation of the carbon atoms adjacent to a carbonyl. This thesis describes the use of asymmetric organocatalysis for the ɑ-chlorination of aldehydes and the subsequent use of these compounds as building-blocks for the preparation of a variety of oxygen-containing heterocycles. With the successful preparation of optically enriched ɑ-chloroaldehydes, these linchpin compounds were elaborated into trans-epoxides, substituted-tetrahydrofuranols, and carbohydrate analogues. In particular, this thesis describes the addition of organolithium reagents and lithium enolates to optically pure ɑ-chloroaldehydes to afford 1,2-anti-chlorohydrins in a highly diastereoselective manner. Further elaboration of these compounds allowed access to a variety of trans-epoxides, in an approach that is flexible and concise. Following the discovery of a novel silver-mediated cyclisation process substituted-tetrahydrofuranols were afforded from the 1,2-anti-chlorohydrins and the trans-epoxides. It was also realised that chloropolyols undergo regio- and stereoselective cyclisation by simply heating them in water to provide highly functionalised tetrahydrofuranols, including carbohydrate analogues. Together, these methods allowed access to a wide-range of stereochemically diverse oxygen-containing scaffolds. The methodologies described within this thesis were further applied to the synthesis of an array of biologically active natural products. To demonstrate the utility of

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

Synthesis of flexible, fluorous 'ponytail' modified triarylphosphines for utilization in fluorous biphase systems

Peer reviewed: 
No, item is not peer reviewed.
Date created: 
2012-02-22
Abstract: 

The synthesis of several triarylphosphines incorporating poly(hexafluoropropylene oxide) [pHFPO] is presented. Hydrogenation of 2-cyclohexen-1-one using a fluorinated Wilkinson’s catalyst analogue prepared from one of these ligands was investigated under fluorous biphasic conditions. A 0.5 % catalyst load was recycled through 6 cycles by exploiting the facile catalyst recovery afforded by the thermoregulated miscibility of the perfluorinated solvent with normal organic solvents. Catalytic performance was examined under biphasic as well as monophasic conditions using tris(4-diphenylphosphino) pHFPO methylene benzoate rhodium chloride, a fluorous analogue of Wilkinson’s catalyst. The partitioning behavior was analyzed for the free ligand, ligand oxide and rhodium-bound ligand. This represents the first example of a polyhexafluoropropylene oxide-modified triarylphosphine-based catalyst.

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

Flavin semiquinone stability and evolution of function in photolyases and cryptochromes

Date created: 
2012-01-27
Abstract: 

This thesis examines the mechanism under which Photolyases (PLs) and Cryptochromes (CRYs) tune their semiquinone stability and reactivity and correlate these properties with function. The work is supported by experiments in site-directed mutagenesis, oxidation kinetics, isotope effects, electrochemistry and in vivo cellular functional assays. I present the first evaluation of both flavin redox potentials for PL, which establish, counter to existing models, that its semiquinone radical is thermodynamically destabilized. Investigations of the N5-proximal Asn-to-Asp mutant decreases the driving force for oxidation but alleviates the kinetic barrier. Analysis of additional mutants demonstrates that the modulation at the N5-proximal residue is context dependent. These results support a regulatory mechanism that exploits proton-coupled electron transfer and changes in protein conformation to adjust the kinetic barriers to redox reactions of the semiquinone in PLs and CRYs. I also provide evidence for the biological relevance of redox tuning by correlating semiquinone stability to in vivo DNA repair by a series of PL mutants exhibiting a varying range of stability. Together, these investigations provide molecular-level rationale for the evolution of function in PLs and CRYs. My results also underscore two emerging general themes in biological electron transfer, the significance of proton transfer and kinetic mechanisms of redox regulation.

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

Mapping the enzyme specificities of intestinal maltase-glucoamylase and sucrase-isomaltase

Author: 
Date created: 
2012-01-20
Abstract: 

In humans, maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) are the small intestinal glucosidases responsible for catalyzing the last glucose-releasing step in starch digestion. MGAM and SI are each composed of duplicated catalytic domains, N-terminal membrane domains (ntMGAM and ntSI) and C-terminal luminal domains (ctMGAM and ctSI). They display complementary substrate specificities for the mixture of short, linear and branched oligosaccharide substrates that typically make up terminal starch-digestion products. As they are involved in the breakdown of dietary starch and sugars into glucose, regulating their activities with α-glucosidase inhibitors is an attractive approach to control blood glucose levels for the prevention and treatment of type-2 diabetes.This thesis work deals with mapping (determination of selectivity and specificity) of MGAM and SI with synthetic inhibitors. The syntheses and enzymatic evaluation of sulfonium-ion glucosidase inhibitors, with potent inhibitory activities against intestinal glucosidases are the main topics of this thesis. First, an alternative route for the synthesis of kotalanol, a naturally-occurring sulfonium-ion glucosidase inhibitor isolated from Salacia reticulata, and its 6'-epimer are described, and the inhibitory activities of these compounds against ntMGAM are reported. Second, the total syntheses of de-O-sulfonated ponkoranol, another naturally-occurring sulfonium-ion glucosidase inhibitor isolated from the same species, its 5'-epimer, and their selenium analogues are described. The synthetic route is also extended to obtain 3'-O-methylponkoranol. The inhibitory activities of these latter compounds against the four human intestinal glucosidase enzymes, ntMGAM, ctMGAM, ntSI, and ctSI are examined. Finally, from the structural studies of ntMGAM, it was postulated that ctMGAM might have an extended binding site compared to ntMGAM, which favours binding of longer inhibitors such as acarbose (an antidiabetic drug that is currently in use for the treatment of type-2 diabetes). Based on this difference, the syntheses of candidate inhibitors containing maltose extensions at 3'- and 5'- of de-O-sulfonated ponkoranol are described. The inhibition of maltose hydrolysis suggests that selective inhibition of one enzyme unite over the others is possible despite relatively small structural changes in the inhibitor. This panel of inhibitors can now be used to turn off certain enzymes while probing the action of others with respect to starch digestion.

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

Insight into O-GlcNAc protein modification using chemical and biochemical tools

Date created: 
2010-05-10
Abstract: 

Within higher eukaryotes, hundreds of nucleocytoplasmic proteins are modified by an N-acetylglucosamine (GlcNAc) residue. This O-GlcNAc modification is dynamic, a property imparted by the actions of two enzymes: O-GlcNAc transferase catalyzes the installation of GlcNAc onto specific serine and threonine residues of target proteins via a -glycosidic linkage, while O-GlcNAcase removes the modification. A leading hypothesis for the cellular role(s) of the O-GlcNAc modification is that an interplay between O-GlcNAc and phosphorylation exists to fine-tune cellular signaling pathways. In particular, elevated O-GlcNAc levels are thought to prevent phosphorylation of key signaling molecules in the insulin signaling cascade and cause insulin resistance. A key to many of these studies is the ability to modulate O-GlcNAc levels on proteins using two previously described inhibitors of O-GlcNAcase. These two inhibitors, however, also inhibit other targets and so it was unclear if results obtained using these compounds were an effect of modulating O-GlcNAc levels or, alternatively, an off-target effect of these inhibitors. With the goal of developing a selective O-GlcNAcase inhibitor, a series of biochemical studies provided evidence that O-GlcNAcase uses a catalytic mechanism involving substrate-assisted catalysis. Through these studies, a compound called NAG-thiazoline was found to be a potent inhibitor of O-GlcNAcase. By chemical modification of NAG-thiazoline, an inhibitor termed NButGT was generated that is selective for O-GlcNAcase over functionally-related enzymes yet retains high potency. NButGT functions within cells as well as in vivo within rodents to elevate O-GlcNAc levels. Surprisingly, neither NButGT nor another new inhibitor cause insulin resistance, strongly suggesting that off-target effects of previously used inhibitors are a concern. More surprising is that cultured cells and animals treated long-term with high doses of NButGT show no adverse effects. These results are challenging to reconcile with the leading hypothesis for the role of O-GlcNAc. Therefore, a new hypothesis was formed whereby O-GlcNAc may be acting as a protein chaperone. Preliminary experiments examining the biophysical properties imparted to a protein by O-GlcNAc support this new hypothesis. These new chemical tools and this newly formed hypothesis should enable exciting future research aimed at understanding the functional role of the O-GlcNAc modification.

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

Mechanistic investigation of families of retaining glycosyl hydrolases

Author: 
Date created: 
2012-04-24
Abstract: 

Enzymes that catalyze the removal of carbohydrate linkages from biological molecules are called glycosyl hydrolases (GH). These enzymes have been catagorized into more than 130 different families. Family-4 glycosyl hydrolases catalyze hydrolysis via an unusual oxidation-reduction mechanism that requires NAD+, Mn2+ and reducing conditions for catalytic activity, a situation that contrasts the classical nucleophilic substitution mechanism of most glycosyl hydrolases. Most of this thesis is directed towards detailed mechanistic evaluations of the GH4 alpha-galactosidase from Citrobacter freundii and the GH109 alpha-N-acetylgalactosaminidase, which has a bound active site NAD+, from Elizabethkingia meningosepticum. Experimentally, this involved cloning the gene, expressing and purifying the recombinant protein, synthesizing substrates, measuring pH-rate profiles, Brønsted parameters and deuterium kinetic isotope effects (KIEs). The MelA alpha-galactosidase (GH4, C. freundii) possesses a substrate specificity that is limited to non-phosphorylated alpha-D-galactosides. The measured primary deuterium KIEs show that the first irreversible step involves a concerted oxidation of the C-3 hydroxyl group and proton abstraction at C-2. Subsequent elimination of the leaving group is kinetically silent. The GH109 alpha-N-acetylgalactosaminidase (E. meningosepticum) displayed a significant degree of substrate promiscuity as it catalyzed the hydrolysis of aryl 2-acetamido-2-deoxy-alpha-D-galactosides and aryl alpha-D-galactosides efficiently. Notably, Brønsted analysis on both aryl 2-acetamido-2-deoxy-D-galactosides and aryl alpha-D-galactosides suggests that leaving group departure is not rate limiting. The measured deuterium KIEs kinetic isotope effects using the "non-natural" phenyl alpha-D-galactoside substrate revealed that both C-3 oxidation and C-2 proton abstraction are kinetically significant. A bicyclo[4.1.0]heptyl mimic of an aryl alpha-D-galactopyranoside, (1R,2S,3S,4R,5S,6S)-5-(3,5-difluorophenoxy)-1-(hydroxymethyl)bicyclo[4.1.0]heptan-2,3,4-triol, was made and shown to be an irreversible inhibitor of the GH36 alpha-galactosidase from Thermotogamaritima. In contrast, the diastereomeric L-altro isomer failed to inactivate the enzyme, while the analogous cyclohexene was shown to be a tight-binding competitive inhibitor. The measured pH-rate profiles for inhibition and reactivation as well as the corresponding catalytic and inhibitory proficiencies suggested that inhibition results from the formation of an oxacarbenium ion in the enzymatic site that is trapped rapidly by an active site enzymatic residue.

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

An investigation of redox-active isoindoline-based ligands and their coordination complexes

Date created: 
2012-02-21
Abstract: 

The structural and electronic properties of metal complexes with isoindoline-based redox-active ligands and the ability of these ligands to stabilize unusual oxidation states was investigated. Two types of redox-active ligands were examined: phthalocyanines (Pc) and bis(2-pyridylimino)isoindolines also known as lobsterates (Lb). In contrast to the ancillary or spectator ligands more commonly used in coordination chemistry, redox-active ligands can exist in a range of oxidation states. Additionally, the chemical inertness of the Pc ligand could be used to develop new catalysts where the Pc ligand can stabilize a highly reactive metal centre and also act as an electron reservoir in a catalytic cycle. With this in mind, the reduction of trichloro(phthalocyanato)niobium(V) was performed with various reducing agents in order to study the bond activating ability of the reduced species and to determine the crystal structure of the reduced products. C–O bond activation of ether-type solvents suggests that the Pc ligand is able to withstand bond activation from a highly reduced niobium centre while, at the same time, allowing the niobium centre to activate the C–O bonds of the solvent. NbPc complexes containing Pc(4–) ligands were structurally characterized. A reduced MgPc complex containing a Pc(3–) ligand was also structurally characterized. A report of gold(II) Pc and the ability of Pc to stabilize gold in an unusual, paramagnetic +2 oxidation state was re-investigated. Attempts to synthesize soluble versions of AuPc as well as the original AuPc did not produce the desired gold(II) products and only diamagnetic gold(III) Pcs were obtained. An unprecedented, ring- contracted gold(III) Pc analogue was discovered and structurally characterized in the process of these studies. A series of three new 5,6-substituted Lb ligands were synthesized and the redox properties of these ligands in copper(II) acetate complexes were evaluated using cyclic voltammetry. The reductions of the copper acetate Lb complexes were not chemically reversible. The 2,6-diisopropylphenoxy-substituted Lb copper acetate complexes formed acetate-bridged 1-D chains. Superconducting quantum interference device magnetometry suggests that the Cu centres are very weakly antiferromagnetically coupled.

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

Microfluidic microarray for pathogenic DNA Analysis: single-base-pair-mismatch discrimination, and modeling/simulation of centrifugal flows and dynamic hybridization

Author: 
Date created: 
2012-01-16
Abstract: 

In the development of surface-based biosensors, the combination of microfluidic technology with the DNA microarray chip has been realized with the intersection method. The method shows the advantages of less sample usage, multiple sample capability, enhanced hybridization signals, and reduced assay time. In this thesis, line arrays of DNA probes were printed on a glass chip through the microfluidic method. Target microchannels orthogonally intersected with these line arrays, and complementary DNA molecules, upon hybridization, were retained at the intersections as rectangular spots. Detection was achieved through the read-out of the fluorescent labels on the targets. The high surface-to-volume ratio in microchannels of nanolitre volume enhanced the detection sensitivity as compared to that obtained with the bulk solution method. The spot shape is more regular than that from the pin-spotted microarray, which is an advantage for subsequent image processing. For diagnostic purposes, PCR products amplified from the genomic DNA of fungal pathogens were detected with this microfluidic intersection method. Moreover, with the aid of gold nanoparticles, two 260-bp DNA sequences with single base-pair difference were discriminated from each other at room temperature for the first time. In addition to the pressure-driven flows used in rectangular chips, a centrifugal-pumping method was employed to drive liquid movement in a CD-like microchip. Connections from electrodes or tubing were avoided, resulting in the ease of conducting parallel hybridizations in multi-channels. For example, up to 100 samples can be analysed simultaneously with the CD-like microchip. The centrifugal-pumping flow in the spiral channel was modeled mathematically and simulated numerically by the computational flow dynamics (CFD) method. It was found that the mathematical results and simulation results are consistent with the experimental findings. Other than the flow study, the kinetics of microfluidic DNA hybridization was also studied and simulated. The effects of probe coverage, channel depths, assay conditions, and sample delivery rate on DNA hybridization were investigated. The variation of signal intensity inside a hybridization spot and from spots to spots was also discussed and compared with the experimental findings. The proposed method provides a way to optimize both chip design and experimental conditions during surface-based biosensor assays.

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

Proton transport in the short side chain perfluorosulfonic ionomer membranes

Author: 
Peer reviewed: 
No, item is not peer reviewed.
Date created: 
2011-12-05
Abstract: 

The influence of ion exchange capacity (IEC) on the water sorption properties of high IEC, short side chain (SSC) perfluorosulfonic acid (PFSA) ionomer membranes, and the relationships between water content, proton conductivity, proton mobility, water permeation, and oxygen diffusion were investigated. Transport properties of SSC ionomer membranes were compared with a series of long side chain (LSC) PFSA membranes. At 25 °C, fully-hydrated SSC ionomer membranes were characterized as possessing higher water contents, moderate λ values, high analytical acid concentrations, and moderate conductivity than LSC PFSA membranes; but lower than anticipated effective proton mobility. Complementary measurements of water permeability and oxygen diffusion also revealed lower than expected values given their much higher water contents than LSC analogues. Potential benefits afforded by reducing the side chain length of PFSA ionomer membranes, such as increased crystallinity, higher IEC, and high hydrated acid concentration were diminished by a less-developed, disorganized hydrophilic percolation network, which provides a motivation for future improvements of transport properties for this class of material.

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

Immobilization of hairpin deoxyribose nucleic acid aptamers on gold: surface folding dictated analyte-binding performance and electron transfer kinetics

Author: 
Date created: 
2011-11-28
Abstract: 

The performance of hairpin aptamer-based electrochemical sensors is highly dependent on the structural nature of the DNA aptamer monolayer immobilized on the electrode surface. It was discovered in this study that an increase of the surface density of aptamers on the electrode does not improve the sensor’s sensitivity, but in fact diminishes its response to the analyte. The electron transfer kinetics between the redox centre (methylene blue) and the gold electrode strongly depend on the surface density (and thus the folding state) of the hairpin DNA aptamers, and shows a unique two-stage behaviour, i.e., a relatively fast process (irrespective of the surface density) at low surface density followed by a sharp decrease in the apparent rate constants (when the surface density increases).

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