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

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Studies Toward the Total Synthesis of Biselide A

Author: 
Date created: 
2015-05-12
Abstract: 

The use of natural products for medicinal purposes is a tradition dating back to ancient times. To this day bioactive natural products continue to inspire a large proportion of the new pharmaceuticals that are developed each year. Many natural products serve as drug leads and inspire the synthesis and development of more potent analogues. The biselides and haterumalides are two members of a class of recently described bioactive polyketide natural products that have been found in Okinawan ascidians. Many of the biselides and haterumalides exhibit anticancer activity, yet very little material is available from the natural sources. Of particular interest is biselide A, which is the only member of this class to demonstrate selective killing of human cancer cells over healthy cells. It has been proposed that C20 oxygenation in biselide A confers this selectivity and thus, derivatives with C20 oxygenation are also of pharmaceutical interest. While multiple syntheses of haterumalides have been published, biselide A has not yet been synthesized. This thesis highlights recent efforts towards a scalable total synthesis of biselide A. Three different approaches have been explored: the first incorporates a Horner-Wadsworth-Emmons reaction as a key step, and the second and third use a cross metathesis key step. While the Horner-Wadsworth-Emmons approach was ultimately unsuccessful, our successes in the cross metathesis approach should now yield access to this potentially important natural product.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Robert Britton
Department: 
Science: Chemistry
Thesis type: 
(Thesis) M.Sc.

Development of Assays and Irreversible Inhibitors for ATG4B: A Key Cysteine Protease Implicated in the Process of Autophagy

Author: 
Date created: 
2015-04-21
Abstract: 

Autophagy is a biological process responsible for the sequestration and degradation of cellular components for the purposes of maintaining homeostasis. ATG4B is a cysteine protease that plays a key role in the initiation of autophagy by cleaving the protein proLC3 revealing a C-terminal glycine residue and to form LC3-I. This glycine then becomes lipidated with phosphatidylethanolamine (PE) to form LC3-II which is inserted into partial protomembrane fragments in the Golgi which stimulates formation of autophagosomes. ATG4B also cleaves PE from the membrane bound LC3-II to reform and recycle LC3-I and to facilitate fusion of the autophagosomes with liposomes where the contents are subsequently digested producing energy and amino acids for protein synthesis. As cancer cells frequently “hijack” autophagy as a survival and resistance mechanism against therapy, autophagy has recently emerged as a potential therapeutic target for treating cancer. The development of two different assay methods for measuring ATG4B activity are reported. The first, LC/MS-based, assay monitors the cleavage of proLC3 to LC3-I based on the ratio of peak heights detected. This method has the added utility of being able to detect enzyme integrity throughout the reaction. The second, FRET-based, assay involves a novel YFP-LC3B-EmGFP doubly fluorescent protein substrate developed for the purposes of large scale screening of compound libraries. The FRET-based assay was successfully used to screen 5000 compounds in the commercial LOPAC and KD2 compound libraries with an overall hit rate of 0.6% and 0.5% respectively. Irreversible inhibitors of ATG4B were designed and synthesized based on information from the screening results as well as previous work on putative peptide substrates conducted by Dr. Nag Kumar (Young Lab, SFU). Inhibitors of the halo-methyl ketone type were developed and tested for inhibitory activity against ATG4B. Fluorescent analogues of these irreversible inhibitors were further developed and optimized for in cellulo potency. Kinetic analysis and digestion studies revealed the inhibitors were indeed active-site directed ATG4B inhibitors.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Robert Young
Department: 
Science:
Thesis type: 
(Thesis) Ph.D.

Gold Nanoparticle-Enhanced Detection of Single Nucleotide Polymorphisms in the NanoBioArray Chip

Author: 
Date created: 
2015-04-16
Abstract: 

In this thesis, we report the use of gold nanoparticles (AuNPs) to enhance the detection of single nucleotide polymorphism (SNPs) in the NanoBioArray (NBA) chip. A combination of gold nanoparticles (AuNPs) and nucleic acids has recently been used in many biosensing applications. However, there is a poor fundamental understanding of how gold nanoparticle surfaces influence the DNA hybridization process. Our kinetic analysis shows that in the presence of AuNP-ssDNA interactions, mechanisms of DNA hybridization and dehybridization are altered. Our proposed mechanisms include a shift of the rate-limiting step of hybridization from mismatch-insensitive to the mismatch-sensitive zipping step. Furthermore, the binding of gold nanoparticles to the single-stranded DNA segments (commonly known as bubbles) in the mismatched (MM) duplex DNAs, destabilize the duplexes and accelerates the dehybridization process. We employ these alterations in mechanisms, both of which disfavor the formation of MM duplexes, to enhance the detection of SNPs in the NBA chip. In this technique, we load the target DNAs on the surface of AuNPs (i.e. AuNP targets) and then introduce them to the surface-immobilized probes for DNA hybridization. Our results show that AuNP targets, in contrast to the targets free in the solution (free targets), were able to discriminate between the perfectly matched (PM) probes and the mismatched (MM) ones. Using AuNP targets, we developed a room-temperature method for detection of SNPs in the KRAS gene codon 12 in the NBA chip. Then, a novel wash method based on AuNPs was developed to preserve the DNA hybridization signals in CD-NBA chip while discriminating MM duplexes from PM duplexes. In this method, AuNPs are suspended in the wash buffers to preferentially destabilize the MM duplexes, in presence of the PM duplexes. Enjoying this targeted mechanism, AuNP wash method enhances specificity without compromising signal intensity. This method is simple and compatible with multiplexed DNA hybridization settings. The findings in this thesis can be used to enhance the reliability of DNA biosensors (e.g. DNA microarrays) and might lead to new applications in DNA biosensing.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Paul C. H. Li
Department: 
Science:
Thesis type: 
(Thesis) Ph.D.

Optimizing the Processability of Selenium Nanowires and Their Chemical Transformation into Polymer Coated Semiconductor Materials

Date created: 
2014-04-07
Abstract: 

This thesis seeks out to optimize the sonochemically-induced synthesis and the ease of handling high-aspect-ratio selenium nanowires. Understanding the selenium nanowire’s surface chemistry is crucial to controlling their dimensions during growth and to facilitate the manipulation of these materials. The surface chemistry of the nanowires was analyzed with a variety of surface sensitive techniques and electron microscopy. This knowledge of the surface chemistry of selenium nanowires was utilized to increase their colloidal stability. A stable dispersion of selenium nanowires improves the ease of handling and processing these materials for subsequent assembly or use in templated reactions. For example, surfactant stabilized nanowires enhanced their colloidal stability in media that are otherwise poor at stabilizing the nanowires and improved the uniformity of products from templated reactions on the nanowire surfaces. We also discovered that dispersions of selenium nanowires in a low dielectric constant solution could be organized by electrokinetic techniques into fibers that oriented along the electric field. We developed a general method for the assembly of the selenium nanowires into either macroscopic fibers or an array of fibers of various lengths over large areas. Isolated fibers of selenium nanowires could reversibly bend in response to electrostatic charges. These flexible selenium fibers also exhibited a photoconductive response when illuminated with white light. These properties of selenium nanowires can degrade over time as these nanowires are susceptible to oxidative damage, but we were able to demonstrate the first passivation of selenium nanowires with a thin layer of polystyrene. The thin layer of polystyrene was grafted onto the selenium surfaces by a surface-initiated atom transfer radical polymerization reaction. These encapsulated nanostructures demonstrate an enhanced resistance towards oxidative damage, such as corrosion. We were also able to synthesize polystyrene encapsulated copper selenide nanowires by a similar route in a template-engaged reaction in conjunction with a surface-initiated atom transfer radical polymerization reaction.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Byron D. Gates
Department: 
Science: Department of Chemistry
Thesis type: 
(Thesis) Ph.D.

Development of a neutron generator facility at Simon Fraser University

Date created: 
2015-04-27
Abstract: 

A new neutron generator facility at Simon Fraser University (SFU) utilizes a commercial deuterium-tritium neutron generator (Thermo Scientific P 385) to produce 14.2 MeV neutrons at a nominal rate of 3E8 neutrons/s. The facility will be used to produce radioisotopes to support a research program including nuclear structure studies and neutron activation analysis. As a prerequisite for regular operation of the facility and as a personnel safety consideration, dose rate predictions for the facility were implemented via the GEANT4 Monte-Carlo framework. Dose rate predictions were compared at two low neutron energy cutoffs: 5 keV and 1 meV, with the latter accounting for low energy thermal neutrons but requiring significantly more computation time. As the SFU facility geometry contains various openings through which thermal neutrons may penetrate, it was necessary to study their contribution to the overall dose rate. A radiation survey of the facility was performed as part of the commissioning process, consisting of a neutron flux measurement via copper foil activation and dose rate measurements throughout the facility via a 3He gas filled neutron detector (Thermo Scientific WENDI-2). When using the 1 meV low neutron energy cutoff to account for thermal neutrons in the dose rate predictions, the predictions and survey measurements agree to within 30% or better in most survey locations.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Krzysztof Starosta
Department: 
Science:
Thesis type: 
(Thesis) M.Sc.

Modeling of Water Sorption and Swelling in Polymer Electrolyte Membranes: Diagnostic Applications

Date created: 
2015-04-23
Abstract: 

The polymer electrolyte membrane (PEM) fulfills vital functions as separator, proton conductor, and electronic insulator in a polymer electrolyte fuel cell (PEFC). The well-studied and practically used solid polymer electrolyte membranes are perfluorosulfonic acid (PFSA) polymer membranes such as Nafion. These membranes offer high proton conductivity, high mechanical strength and good chemical stability. The efficiency of the chemical-to-electrical energy conversion in a PEFC critically depends on the ability of the PEM to transport protons from the anode to the cathode. Proton conductivity of the PEM is a key parameter to achieve high power density and performance. The main variable to characterize the state of a PEM and determine its transport properties is its water content. In particular, the proton conductivity is highly sensitive to the level of hydration. Membranes experience continuous stresses and consequently continuous loss of performance throughout their operational life. Chemical degradation alters the chemical structure of the PEM, which affects the water distribution in it. A consistent description of water sorption and swelling under conditions relevant for the PEFC operation lies at the heart of understanding transport properties, performance and degradation phenomena. This work expands on a previously developed poroelectroelastic model of water sorption in PEMs [Soft Matter 7, 5976 (2011)]. The theory relates the charge density at the pore walls to a microscopic swelling parameter. Extended to the water sorption equilibrium in a pore ensemble, the model reconciles microscopic swelling in a single pore with macroscopic swelling of the membrane. This work provides a generalized treatment of elastic effects in PEMs. Different deformation modes of polymeric pore walls are used to derive stress-strain relationships that determine the law of swelling. Moreover, this work enhances the diagnostic capabilities of the model; it provides the statistical pore size distribution as well as a statistical distribution of microscopic fluid and elastic pressures inside the PEM. Thereafter, the model is applied to different sets of water sorption data for PEMs that had undergone either hygrothermal aging or chemical degradation. The model-based analysis provides mechanistic explanations of structural changes and their impact on microscopic distributions of charge density and elastic properties in PEMs.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Michael Eikerling
Department: 
Science:
Thesis type: 
(Thesis) M.Sc.

Total synthesis of 5,5-spiroacetal natural products and studies toward the total synthesis of eleutherobin

Date created: 
2015-04-20
Abstract: 

A novel chlorohydrin-based spirocyclization reaction has been developed that complements contemporary approaches to spiroacetals. The discovery and optimization of a silver(I)-promoted cyclization of ketochlorohydrins enabled the synthesis of a wide range of 5,5-, 5,6-, and 5,7-spiroacetal compounds. In addition, this strategy was applied in the total syntheses of (+)-cephalosporolide E and (−)-cephalosporolide F, which were accomplished in six steps. Further extension of this methodology culminated in the first total synthesis and full stereochemical assignment of (+)-ascospiroketal A. Specifically, a silver(I)-promoted cyclization cascade was developed that effected both a spirocyclization as well as an oxetane rearrangement, and generated a quaternary all-carbon stereogenic centre with complete stereocontrol. This unique reaction sequence was a critical component of the eventual total synthesis of (+)-ascospiroketal A and several diastereomers that were made available in fourteen steps. An additional study detailing efforts geared toward the total synthesis of eleutherobin, an anti-cancer natural product, is also presented. By utilizing a cyclobutanone α-arylation/Haller-Bauer fragmentation/Friedel-Crafts acylation strategy, a tetralone intermediate for the synthesis of eleutherobin was made available on multi-gram-scale. Efforts to convert this material into the bicyclo[8.4.0]tetradecane core of eleutherobin are described including the identification of an intramolecular aza-Michael strategy that secured the carbon complement of eleutherobin.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Robert Britton
Department: 
Science: Chemistry
Thesis type: 
(Thesis) Ph.D.

Investigation of Protein-ligand Interactions by Molecular Dynamics and Saturation Transfer Difference NMR Spectroscopy

Author: 
Date created: 
2015-03-26
Abstract: 

Protein-ligand interactions form the molecular basis of many biological processes. The study of their interactions from a structural perspective can provide not only insights into the molecular recognition between the protein and the ligand but also clues to the design of better ligands that can serve to mediate the biological events. This thesis investigates such interactions for four proteins that are (potential) therapeutic targets. Techniques used in this thesis include molecular dynamics (MD) simulations, saturation transfer difference (STD) NMR spectroscopy, and complete relaxation and conformational exchange matrix (CORCEMA) analysis that calculates theoretical STD effects. MD simulations are employed to study the binding of two designed glycopeptides with SYA/J6, a monoclonal antibody specific for the O-polysaccharide of the Shigella flexneri Y bacterium, as well as the binding dynamics and strengths of a series of inhibitors against human lactate dehydrogenase A (LDHA), an enzyme implicated in the cell energy metabolism of various cancers. The computational results from both cases are consistent with experimental data, predicting that neither glycopeptide would bind to SYA/J6, and clarifying ambiguities in the binding modes of two well-known LDHA inhibitors. Furthermore, binding models of two inhibitors against the enzyme UDP-galactopyranose mutase (UGM), a potential target for the treatment of tuberculosis, and two substrates of UDP-N-acetylgalactopyranose mutase (UNGM), a potential target against diarrheal disease, are constructed by a protocol that combines MD, STD NMR, and CORCEMA calculations. The collective results indicate a unique binding mode for a UGM inhibitor and explain the bifunctionality of UNGM.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Gary Leach
Department: 
Science:
Thesis type: 
(Thesis) Ph.D.

An in-depth examination of the properties and behaviour of Au(III)-based [AuX2(CN)2]- (X = Cl, Br, I) as a coordination polymer building block

Date created: 
2014-11-27
Abstract: 

This thesis primarily focuses on the synthesis and characterization of [AuX2(CN)2]- (X = Cl, Br, I), a square planar Au(III)-based compound, and its use as a coordination polymer building block. K+, nBu4N+ and PPN+ salts of [AuX2(CN)2]- and the Au(I/III)-based double salts K3[Au(CN)2]2[AuBr2(CN)2]-H2O and K5[Au(CN)2]4[AuI2(CN)2]-2H2O were prepared and structurally characterized. All compounds exhibited many structure-defining weak interactions such as X-X, Au-X (X = Br, I) and Au(I)-Au(I) interactions, as well as a rare Au(I)-Au(III) interaction with an intermolecular distance of 3.58 A; no Cl-Cl interactions were observed. [AuX2(CN)2]- was introduced to solutions containing a variety of divalent metals (Mn, Fe, Co, Ni, Cu, Zn, Pb) and ancillary N-donating chelating ligands such as 1,10-phenanthroline or bridging ligands such as 4,4'-bipyridine. In most cases, an ionic material resulted, containing a metal cation coordinatively saturated by ligand and an unbound [AuX2(CN)2]- (X = Cl, Br) anion. In some cases, [AuX2(CN)2]- did bind, but in a pendant, non bridging fashion. With ethylenediamine and 4,4'-bipyridine ligands, and Ni(II) and Co(II) or Zn(II), respectively, an [AuBr2(CN)2]--bridged coordination polymer resulted; no [AuCl2(CN)2]--bridged materials were observed with added ancillary ligands. In the absence of ancillary ligand, several [AuX2(CN)2]--bridged (X = Cl, Br) formed; all reactions involving [AuI2(CN)2]- resulted in decomposition of the [AuI2(CN)2]- molecule. Exposing [AuX2(CN)2]- to temperatures above 80 C or UV light resulted in the reductive elimination of halogen, yielding [Au(CN)2]-. Kinetics (thermal and photochemical) studies revealed pseudo first-order rate kinetics with. Heating some [AuBr2(CN)2]--containing coordination polymers to 125 C converted them to analogous [Au(CN)2]--containing materials. The ligand-free Cu[AuX2(CN)2]2 materials exhibited a vapochromic response when exposed to H2O, DMF, DMSO, pyridine, 1,4-dioxane and ethyleneglycol; they also exhibited distinct IR and Raman vCN patterns. Using Raman analysis, these vCN patterns can be effectively used to detect and identify between DMF, DMSO and pyridine. A series of Cu(I) and Ag(I)-based coordination polymer materials containing bridging [AuX2(CN)2]-, [Au(CN)4]- and [Au(CN)2]- were synthesized and characterized. M[Au(CN)4] (M = Cu, Ag) exhibit negative thermal expansion with expansion coefficients between -11e-6 and -24e-6 K-1. M[Au(CN)2] are isomorphous to known AuCN, and exhibit turquoise (Ag) and red (Cu) emission. Incorporating pyridine, THT, PPh3 and Me2S in the Cu(I)/[Au(CN)2]- reaction yielded materials with distinct emissive properties; Cu(SMe2)[Au(CN)2] emits white light. Thus, Cu[Au(CN)2] shows potential as a sensor for P- or S-donor solvents.

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

Method Development for an Easy and Direct Quantitation of Protein Adsorption by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Date created: 
2014-11-20
Abstract: 

The current proteomic approach is to identify and analyze proteins of interest, which can potentially be disease biomarkers. However, these important proteins usually exist in very low concentration, making them vulnerable to sample loss by adsorption during any laboratory manipulations. A simple method, sodium dodecyl sulfate polyacrylamide gel electrophoresis coupled with direct protein adsorption analysis (SDS-PAGE/DPA), is presented here for the quantitation of adsorption-caused protein loss. No complicated steps and expensive equipment are involved; this method for protein study enables measurement of proteins adsorbed on vials at extremely low concentrations (in pg/μl). We used this method to characterize the effects of concentration, time, and volume on adsorption. We also applied this method to discover differential sample loss in protein mixtures and its utility in developing preventive strategies of adsorption. This method is further being employed for studying different aspects of adsorption-caused peptide loss.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Bingyun Sun
Department: 
Science:
Thesis type: 
(Thesis) M.Sc.