Chemistry

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Open Access
Synthesis of Electron Deficient N-heterocyclic-carbenes and Activity of Imidazol-2-imine Thioureate Ligand on Group 10 Transition Metals
(2014-07-09) Harkness, Michael Byron; Lavoie, Gino G
Previous work in the Lavoie group has resulted in the synthesis of new imidazol-2-imine ligands that have low activity towards ethylene polymerization due to their electron rich nature. In an attempt to alleviate this an electron poor naphthalene-1,4-dione-IMes imidazol-2-imine ligand (L) was synthesized in 90% yield (14a) and coordinated to titanium (LTiCpCl2, 15). The resulting complex successfully polymerizes ethylene at a rate of 75 kg PE mol catalyst–1 h–1. A separate study focused on the coordination of functionalized imidazol-2-imine thioureate ligands (L’) to late transition metal. Three complexes were synthesized. Ni(methylallyl)L’ (7), Pd(allyl)L’ (8), and NiL’2 (9) were isolated in 85, 85, and 61% yields, respectively and all spectroscopically characterized. The X-ray crystallographic structure of 9 shows the unexpected N^S binding mode for the ligand. Complexes 7 and 8 have sparing activity towards ethylene polymerization, neither having productivity above 2 kg PE mol–1 h–1.
Open Access
Effect of Saline and Non-Specific Insulin Binding on the Phase Behavior of Poly (Ethylene Glycol)-Grafted Phosphoethanolamine-Succinyl Model Membranes
(2014-07-09) Shahid, Muhammad Naeem; Tsoukanova, Valeria
Poly (ethylene glycol)-grafted membrane-mimetic surfaces bearing negatively charged phospholipid headgroups have gained significant attention due to their promising contributions in numerous biomedical applications. The conformational properties of PEG chains have been mainly studied at the air/water interface, which does not elucidate much about its behavior at the physiological pH ~ 7.4. In this contribution, binary mixtures of a phosphoethanolamine-Succinyl bearing C16 aliphatic chains, DPPE-Succinyl, and a PEG-phospholipid conjugate bearing a PEG chain of 2000 Da, DPPE-PEG2000, have been used as ideal models of bio-nonfouling membrane-mimetic surfaces. The effect of PBS with pH ~7.4 as well as each of its individual constituents including Na2HPO4, KCl, KH2PO4, and NaCl on the biophysical properties of model membrane was examined. Our findings suggest that saline and each of its individual constituents play a pivotal role in the phase and conformational behavior of PEG-grafted membrane models. Insulin as a model protein was then selected to further investigate the effect of phase and conformation behavior of PEG-grafted membrane models on protein/membrane interactions. The insulin/membrane interactions were quantified in terms of monolayer area expansion, ΔA, penetration area, Ap, as well as protein binding degree, χp. To the best of our knowledge, this study provides the first insight into mechanistic aspects of protein interactions with model negatively charged PEG-grafted membranes. This knowledge, may aid in understanding the in-vivo performance of advanced targeted therapeutic carriers.
Open Access
Nocturnal Measurements of HONO, NO2 and NO3 by Differential Optical Absorption Spectroscopy in Polluted Marine and Urban Atmospheres
(2014-07-09) Wojtal, Patryk Rajmund; McLaren, Robert
Nitrogen oxides are ubiquitous throughout the lower atmosphere and significantly affect the chemistry of the atmosphere, air quality, and climate. A data-set obtained using differential optical absorption spectroscopy (DOAS) was analyzed in order to quantify the NO3, HONO and NO2 concentrations at Saturna Island, and concentrations of N2O5 were calculated. Nocturnal measurements of NO3, NO2 and HONO were also performed using active-DOAS at York University. A method for calculating the lifetimes of NO3 without assuming a steady-state approximation was determined and non steady-state lifetimes of NO3 were calculated for both studies. The direct (via NO3) and indirect (via N2O5) rate loss constants of NO3 from the combined nocturnal reservoir (NO3+N2O5) were determined as a function of time of night. Measurements of HONO over the polluted open ocean were performed for the first time. Rapidly established steady-states of HONO were observed, persisting throughout the night until sunrise. During the steady-state period (d[HONO]/dt≈0), HONO was independent of the air mass source and NO2, leading to a zero-order HONO formation with respect to NO2, contrary to expectations. Potential reservoirs of HONO were explored and a conceptual model for HONO formation over aqueous surfaces was hypothesized. Subsequently, nocturnal measurements of HONO in the urban area were made at York University for a total of 242 nights. This urban data-set showed two types of HONO behavior. Firstly, a steady-state behavior was clearly observed for a subset of the data-set, similar to that observed in the aqueous environment at Saturna. Secondly, HONO concentrations were observed to highly correlate with NO2 for another subset of the data-set (d([HONO]/[NO2])/dt≈0), showing evidence of first-order behavior as expected for the accepted heterogeneous NO2 hydrolysis mechanism of HONO formation (2NO2 + H2O → HONO + HNO3). Steady-states of HONO were observed during atmospherically unstable nights, while HONO was strongly correlated with NO2 during stable nights. It was discovered that the main parameters distinguishing these two types of behavior were atmospheric stability and NO2 concentration.
Open Access
The Synthesis of Solar Cell Dyes
(2014-07-09) Hameed, Yasmeen Abdullah; Potvin, Pierre G.
In the recent years, solar energy has become of great interest due to the diminishing supply of the fossil and nuclear fuels and their environmental effects. Solar energy is the most abundant and accessible renewable energy source, and this led the researchers to trend toward the dye-sensitized solar cell (DSSC). The dye-sensitized solar cell is the best technique to convert the solar light to different applicable types of energy such as electrical energy. Ruthenium-based dyes have been widely used in dye-sensitized solar cells. The research proposal mainly focuses on an innovative approach to a new class of solar cell dyes, based on dicarboxylated terpyridine-type complexes. The research will focus on creating new ligands that will be used to attach the metal complexes, mainly Ru and Fe.
Open Access
Development of Microfluidic Devices for Studying Protein Conformational Dynamics via HDX/TRESI-MS
(2014-07-28) Rob, Tamanna; Wilson, Derek J.
Studies of protein conformational dynamics can lead to a deeper understanding of biological functions of proteins, protein/protein interactions, catalysis and allostery. Most of the conformers involved in biological pathways are short-lived. These conformers represent challenging analytical targets since they are weakly populated at equilibrium and demand ultra-rapid online analytical techniques. To address this demand, the development of new tools that will offer enhanced time and spatial resolution in measurements is essential. Towards that end, we took advantage of microfluidics to fabricate custom polymeric devices that would allow direct mass spectrometric measurements of protein dynamics. This dissertation presents successful development of new tools and demonstrates their practical applications in studying dynamics of weakly structured regions of proteins on the millisecond time scale, which is unattainable by conventional biophysical techniques. This thesis introduces a simple, rapid, inexpensive and easily modifiable fabrication protocol which enables the integration of multiple functionalities on a single platform that accommodates complete ‘bottom up’ continuous/pulse labelling Hydrogen-deuterium exchange (HDX) work flow. These integrated devices can be directly interfaced with mass spectrometry as custom electrospray ionization (ESI) sources to conduct site specific mass spectrometric protein conformational analysis. The continuous HDX labelling device enables structural analysis of ubiquitin and distinguishes loop dynamics in cytochrome c. The effect of ligand binding in conformational dynamics in a molten globule protein DAHP synthase was also monitored via continuous HDX labelling. Another analogous device, capable of performing millisecond HDX pulse labelling, reveals allosteric hot spots in TEM-1 which is an antibiotic resistant β-lactamase. Complementary experiments with multiple antibiotics compare the allosteric responses of TEM-1 upon inhibitor or substrate binding. The results exhibit an exciting correlation between allosteric dynamic responses and functionally relevant mutations at the periphery of the protein. This strong correlation demonstrates that this device can be applied in predicting sites of probable mutations. This dissertation successfully introduces integrated microfluidic devices that represent a promising tool for studying dynamics of weakly structured proteins, intrinsically disordered proteins and allostery.
Open Access
Synthesis, Characterization and Reactivity Study of Bis (Imino)-N-Heterocyclic Carbene Transition Metal Complexes
(2014-07-28) Al-Thagfi, Jameel Raddah; Lavoie, Gino G
Three generations of the first 1,3-bis(imino) N-heterocyclic carbene (NHC) ligand precursors were synthesized, isolated and characterized. The synthetic methodologies of the ligand precursors were controlled by the iminic carbon substituents. The corresponding complexes of Cr(III), Fe(II), Co(II), Pd(II), and Zn(II) were prepared from the in situ deprotonation of the NHC ligand precursors or from the related Cu(I) or Ag(I) adducts. The NHC ring fragment and iminic carbon substituents had a significant impact on the solid-state structure of these complexes in which mono-, bi- and tridentate coordination modes were observed. The catalytic activities of chromium, iron and cobalt complexes of 1,3-bis(imino) NHC ligands were evaluated in ethylene polymerization. The activities of chromium(III) complexes of imidazol-2-ylidene showed slightly enhanced activities with a relatively electron-poor phenyl group (compared to methyl) installed on the iminic carbons. These results suggest that a decrease in the electron-donating or an increase in the π-accepting capability of the ligand may produce more active olefin polymerization catalysts. The ligand scaffold was then modified by introducing a benzimidazole moiety to reduce σ-electron donating and increase the π-accepting ability of the ligand and this may lead to a more electropositive metal center. Although these ligands were designed as a tridentate ligand, such coordination mode could not be achieved in the transition metal complexes of imidazole-2-ylidene and benzimidazol-2-ylidene. Steric and electronic parameters perhaps prevent them from adopting this coordination fashion. The five-membered ring of the carbene was then replaced by a six-membered ring of pyrimidin-2-ylidene to achieve a tridentate coordination mode. DFT calculations were performed to assess the electronic properties of the bis(imino)-NHC ligands. The pyrimidin-2-ylidene and the benzimidazol-2-ylidene are predicted to be the best σ–donor and the best π-acceptor of these NHC ligands based on their energy of the highest occupied and the lowest unoccupied molecular orbitals, respectively.
Open Access
Structural Studies of the Receiver Domain of LytR from Staphylococcus Aureus and Interaction Studies of TraW and TrbC from the F Plasmid of Escherichia Coli
(2015-01-26) Shala, Agnesa; Audette, Gerald F.
The emergence of the multi-drug resistant bacteria and the evolutionary spread of the antibiotic resistance genes have become a great concern to human health. Bacteria use various systems in combination or alone in response to environmental stressors and stimuli to adapt to changing conditions. Such systems include the two-component regulatory system (TCS) and the type IV secretion system (T4SS). Microbes use TCSs to regulate important functions such as sporulation, chemotaxis, as well as autolysis that leads to biofilm formation. In addition, microbes utilize T4SSs to deliver DNA, protein substrates, toxin, and virulence factors, from a donor to a recipient cell. These systems are complex, versatile and have a significant impact to human health as they are a major driving force for infection and the spread of antibiotic resistance. The LytSR TCS from Staphylococcus aureus has been found to regulate murein hydrolase activity and autolysis by controlling the expression of the lrgAB and cidABC genes. LytS is predicted to autophosphorylate upon a membrane potential change, consequently transferring the phosphoryl group to the conserved Asp53 residue on the N-terminal domain of the response regulator LytR, leading to the transcription of lrgAB genes. In this study we present the X-ray crystal structure of the N-terminal domain of LytR in apo form and in complex with beryllium fluoride. The identification of dimerization interface residues can be utilized to predict modes of dimerization and therefore activation of the protein. A second project focuses on a representative of the conjugative T4SSs, is the F plasmid from Escherichia coli, which is responsible for the transfer of virulence and antibiotic resistance genes. Two proteins encoded by the F plasmid, TrbC and TraW, are unique to the F-like plasmids and are essential for the transfer of DNA from a donor cell to a recipient cell but their mechanism and function is not known. Interaction studies of TrbC and TraW, as well as crystallization experiments are reported. These studies suggest that the roles of TrbC and TraW are related, and that their interaction is important for the functional transfer of DNA in the conjugative process.
Open Access
Investigation of the Photoenhanced Reduction of Nitrogen Dioxide (NO2) on Organic Films and Above Soils as the Missing Source of Daytime Tropospheric Nitrous Acid (HONO)
(2015-01-26) Wall, Kristin; Harris, Geoffrey
Recent observed unpredictably high HONO daytime concentrations, demanding its ordinarily proposed heterogeneous source to proceed > 60 times faster at noon than during the night, prompted this study, concerning the effect of UV-A radiation on the uptake kinetics of gas-phase NO2 on various phenolic-containing organic films using a wetted-wall flow tube (WWFT) photoreactor. Experimental methods are discussed in detail. A time-dependent model for non-equilibrium conditions incorporating both the chemistry and diffusion for predicting a best-fit reaction probability, ɣbest-fit, as a function of the experimental parameters is detailed. Emphasis is placed on the kinetics of the photoenhanced NO2 uptake reaction under acidic conditions for humic acids (HA), a ubiquitous group of environmental compounds. The linear correlation of HONO production rate with [NO2]0 (k1 = 6.7×10-3 s-1) for commercial HA suggest NO2- particles only diffuse throughout the surface layer depth, as experimentally verified. In general, the pH-dependent results were qualitatively coherent with those of Stemmler et al. (2006), monitoring increases in the photoenhanced HONO formation with pH, owing to increases in reactivity in the reaction with NO2 with several carboxyl groups within the commercial HA with deprotonation, however centred on more authentic pH conditions of anaerobic humic soils (between pH 1.5 and 4.3). A value of krxn = 2.70×10-3 s-1 at pH 3 was obtained, indicative that there was no competition with the hydrolysis reaction, for the tested conditions. Assuming that ɣrxn was rate-limiting, ɣdiffusion was estimated to account for ~ 19% of the total uptake, consistent with the model results. The humification-generated bacteria likely functioned as multicellular aggregates on the acidic HA substrate, producing a biofilm containing numerous chromophoric sensitizer units capable of photochemically reducing NO2 to HONO, defending the observed exponential dependence of HONO yield on irradiance under the tested conditions. The datasets presented for the photoenhanced reaction of NO2 on acidic HA films provide a rather complete kinetic ‘picture’ of an important surface reaction (ɣbest-fit, max of 10-8 at pH 4 under the tested conditions). The scaling up assessments of the kinetic results for the small-scale photoreactor to that of both urban and rural scales are discussed.
Open Access
Studies of Protein Function by Liquid Chromatography-Mass Spectrometry
(2015-01-26) Declan Williams, Gwillym; Siu, K. W. Michael
Complete genomes of many organisms have been recorded using high throughput nucleic acid sequencing; however the intricate functions of the gene products remain largely undiscovered. As the most prominent technology for polypeptide sequence determination and quantification, mass spectrometry is used extensively to address questions regarding the behaviours of proteins in the biological context. The capacity of mass spectrometers to measure covalent modifications of amino acids is also of great value in the investigation of biological processes since specific reactive sites within proteins modulate their activity. Integration of liquid chromatography into mass spectrometry platforms has greatly improved sensitivity and throughput and is of particular benefit in the analysis of highly complex polypeptide mixtures. Studies of the behaviours of individual intracellular proteins in bacterial, plant and metazoan systems employing liquid chromatography-mass spectrometry are described herein. The phosphorylation of individual residues and resulting alterations in function were determined in GraR and VraR, two antibiotic resistance factors in Staphylococcus aureus. Three phosphosites of starch branching enzyme IIb which appear to regulate starch biosynthesis in Zea mays as well as kinases for which these sites are putative substrates were identified. Regulation of the multifunctional protein beta-catenin by p38 mitogen activated protein kinase was explored. Gene products with affinity for the highly conserved multifunctional protein beta-catenin, including several not previously known to interact, were identified from Rattus norvegicus smooth muscle cells. The specificity of peptide ion mass and peptide fragment ion mass relative to instrumental mass accuracy and the consequence for tandem-mass spectrometry-based quantification are explored in a separate chapter. In silico comparison of peptide ion/product ion mass pairs calculated from the human proteome revealed a range of mass redundancy from high to low simulated mass accuracy. Product ions from a single peptide sequence differ in their tendencies toward mass redundancy however no correlation between size and sequence specificity was apparent. This dissertation illustrates research into protein function conducted on three types of mass spectrometers and demonstrates some effects of liquid chromatographic and mass spectrometric performance on proteomic studies.
Open Access
Characterizing Protein Dynamics of Protein-Ligand Interactions by Hydrogen-Deuterium Exchange Mass Spectrometry
(2015-01-26) Resetca, Diana; Wilson, Derek J.
The study of protein-ligand and protein-protein interactions is of paramount importance to the understanding of their biological function. Whereas this area of research has been largely dominated by conventional structural biology techniques, such as NMR and X-ray crystallography, an emerging methodology that relies on the implementation of hydrogen deuterium exchange (HDX) powered by MS-based analysis holds the potential to greatly expand on our ability to probe the protein dynamics of fundamental biological processes. In this work, the entire HDX workflow for site-specific analysis of protein dynamics was integrated onto a concerted microfluidic device and applied to the interrogation of the dynamic changes that accompany protein-ligand interactions. This application is described for two model systems: the binding of glutathione (GSH) by Glutathione-S-Transferase (GST), and the binding of three novel salicylic acid-based inhibitors of Signal Transducer and Activator of Transcription 3 (STAT3) to its SH2 domain. This work extends the application of time-resolved electrospray ionization mass spectrometry (TRESI-MS) HDX to the study of protein ligand interaction dynamics and ligand-binding site mapping.
Open Access
Aryl-Substituted Imino-N-Heterocyclic Carbene Complexes Of Late Transition Metals: Synthesis and Reactivity Studies
(2015-01-26) Badaj, Anna Candace; Lavoie, Gino G
N-heterocyclic carbenes (NHCs) have found enormous success as ancillary ligands in catalysts in many areas of organometallic chemistry. Surprisingly, their use in olefin polymerization, until recently, was not widely explored. The focus of this thesis is to investigate the synthesis of a bidentate ligand scaffold that incorporates an NHC moiety and to study the ability of the resulting complexes to catalyze chemical transformations such as olefin polymerization. The synthesis and characterization of several N-aryl substituted imino-imidazolin-2-ylidene (C^ImineR) ligand precursors was achieved following one of two synthetic protocols. Coordination of these ligands was explored with Group 11 metals in order to develop synthetic protocols which were later extended to prepare complexes of ruthenium, cobalt, iron, nickel, palladium and zinc. The coordination of C^ImineR ligands to nickel using new copper carbene dimers as the transmetalating agent was established. All the nickel complexes were structurally characterized and the size of the iminic carbon substituent was found to have a profound impact on the bond angles and bond lengths around the metal center. However, when tested for ethylene polymerization activity at standard temperature and pressure, the nickel complexes were found to be inactive. With the discovery the nickel complexes of these C^ImineR ligands were inactive for ethylene polymerization, the research focus was extended to the diamagnetic palladium-methyl complexes in order to gain insight into their thermal stability and insertion chemistry. The nature of the iminic substituent profoundly affects the thermal stability of the neutral palladium complexes. While inactive for ethylene polymerization, these palladium methyl complexes react with CO and isocyanides to form various coordination and insertion products.
Open Access
Palladium (II) Mediated Transformation: Exploring Synthetic Methodology for the Synthesis of Cyclopentenones from Tertiary-Cyclobutanois
(2015-08-28) Nikolaev, Andrei; Orellana Garcia, Josue Arturo
A new method for the synthesis of ring-fused cyclopentenone is described. The method utilizes the built-in strain of tertiary cyclobutanol to drive the palladium(II)-catalyzed rearrangement to the corresponding ring-fused cyclopentanone followed by Saegusa-Ito reaction to furnish final cyclopentenone product. Extensive efforts to transform this reaction into a catalytic process by screening various palladium(II) sources, ligands, oxidants, bases, solvents and reaction temperatures failed. Interesting and unexpected results were however obtained while exploring copper(II) chloride and copper(II) bromide as potential oxidants.
Open Access
Investigation of Air Pollutants Near the Great Lakes in Ontario and Along the Coast of California
(2015-08-28) Nuaaman, Ibraheem M.; McLaren, Robert
This dissertation is of two parts. In the first part, carbonyls were measured in Ridgetown, Ontario using an automated HPLC-DNPH system during the BAQS-Met study in 2007. Median concentrations for formaldehyde, acetaldehyde, acetone, propanal, MEK and butanal were observed to be 1.5, 0.67, 1.9, 0.072, 0.22 and 0.041 ppb respectively. The highest carbonyl concentrations were observed with trans-boundary transport associated with airmasses that passed over Michigan and Illinois and could have originated from the Ohio Valley. Local ozone sensitivity was established using the HCHO/NO2 ratio and this area was found to be sensitive to both VOCs and NOx. Carbonyls accounted for 35% of O3 formation estimated using the MIR scale. Lake breeze events had no significant impact on carbonyl concentrations in this location. In the second part of this dissertation, aerosol concentrations and composition were obtained along the coast of California using a High-Resolution Time-of-Flight aerosol mass spectrometer (HR-AMS) onboard RV Atlantis during CalNex in 2010. Measured submicron aerosols were dominated by particulate sulfate and particulate organic matter (pOM), which accounted for 86% of all measured submicron aerosol mass. High aerosol concentrations were associated with land breezes from polluted cities while low concentrations were observed with sea breezes. For the first time, a method is proposed to establish non-refractory particulate chloride (NR p-Cl) in ambient air that is enriched with sea salt chloride. This method involves the subtraction of refractory chloride signal (RF HxCl+), which was estimated using the HxCl+ (total chloride signal) to Na35Cl+ ratio in artificially generated sea spray aerosols. Also for the first time, a method is proposed to determine percentage chloride depletion in ambient aerosols using HR-AMS measurements of Na+ and Na35Cl+. Using this proposed method, study-wide median chloride depletion in submicron aerosols was determined to be 78%. For NR p-Cl, increasing concentrations were observed with increasing RH and pH and decreasing temperature. Finally, pOM in sea spray aerosols was observed to be dominated by hydrocarbons (55%) and oxygenated hydrocarbon classes (37%); this pOM was found to be positively correlated with seawater DMS.
Open Access
Synthesis and Characterization of Organic Ligands and the Corresponding Ru Dyes for Dye-Sensitized Solar Cells
(2015-08-28) Bakhshaei, Baharak; Potvin, Pierre G.
Ru(II) complexes have been of great interest as dyes for Dye-Sensitized Solar Cells (DSSCs), where they absorb solar energy and inject an electron into the conduction band of a semiconductor to start the process. The requirements for efficient light capture and electron injection include having a wide absorption envelope, the presence of a surface-anchoring group and appropriate HOMO and LUMO levels. In this thesis: A novel bidentate ligand is introduced and the synthesis of its various derivatives are explored as well as the synthesis of homoleptic and heteroleptic Ru complexes followed by their characterization using 1H-NMR, 13C-NMR, ESI-MS, EA, FT-IR and UV-visible spectroscopy.
Open Access
Synthesis, Characterization and Reactivity Study of Bis (Imino)-N-Heterocyclic Carbene Transition Metal Complexes
(2015-08-28) Al-Thagfi, Jameel Raddah; Lavoie, Gino G.
Three generations of the first 1,3-bis(imino) N-heterocyclic carbene (NHC) ligand precursors were synthesized, isolated and characterized. The synthetic methodologies of the ligand precursors were controlled by the iminic carbon substituents. The corresponding complexes of Cr(III), Fe(II), Co(II), Pd(II), and Zn(II) were prepared from the in situ deprotonation of the NHC ligand precursors or from the related Cu(I) or Ag(I) adducts. The NHC ring fragment and iminic carbon substituents had a significant impact on the solid-state structure of these complexes in which mono-, bi- and tridentate coordination modes were observed. The catalytic activities of chromium, iron and cobalt complexes of 1,3-bis(imino) NHC ligands were evaluated in ethylene polymerization. The activities of chromium(III) complexes of imidazol-2-ylidene showed slightly enhanced activities with a relatively electron-poor phenyl group (compared to methyl) installed on the iminic carbons. These results suggest that a decrease in the electron-donating or an increase in the π-accepting capability of the ligand may produce more active olefin polymerization catalysts. The ligand scaffold was then modified by introducing a benzimidazole moiety to reduce σ-electron donating and increase the π-accepting ability of the ligand and this may lead to a more electropositive metal center. Although these ligands were designed as a tridentate ligand, such coordination mode could not be achieved in the transition metal complexes of imidazole-2-ylidene and benzimidazol-2-ylidene. Steric and electronic parameters perhaps prevent them from adopting this coordination fashion. The five-membered ring of the carbene was then replaced by a six-membered ring of pyrimidin-2-ylidene to achieve a tridentate coordination mode. DFT calculations were performed to assess the electronic properties of the bis(imino)-NHC ligands. The pyrimidin-2-ylidene and the benzimidazol-2-ylidene are predicted to be the best σ–donor and the best π-acceptor of these NHC ligands based on their energy of the highest occupied and the lowest unoccupied molecular orbitals, respectively.
Open Access
Ambient Ionization: Surface Analysis of Sexual Assault Evidence and 2-Dimensional Imaging of Whole-Body Zebra Fish (Danio Rerio) Using Desorption Electrospray Ionization
(2015-08-28) Chramow, Alexander; Ifa, Demian R.
Desorption electrospray ionization (DESI) is an ambient surface analysis technique capable of producing 2D and 3D images. The ionization mechanism utilizes a pneumatically assisted sprayer to deposit a charged solvent onto a sample surface. Subsequent impacting primary droplets produce ejecting surface secondary droplets containing desolved analytes, which are then detected by a mass analyzer. This thesis explores two fields of application of DESI, forensics and biological tissue analysis. The former involves the analysis of sexual assault evidence, in the form of condoms, lubricants, and their residues as a potential aid in convicting perpetrators. The latter focuses on investigating the potential use of the zebra fish (Danio rerio) as a model vertebrate organism for future toxicological and biological research. Whole-body 2D images were created, highlighting areas of interest such as, the brain, spinal cord, liver, and body fat.
Open Access
Adsorption and Self-assembly of Proteins at Chemically Modified Surfaces
(2015-08-28) Zahedijasbi, Shohreh; Morin, Sylvie
Our research is mostly focused on the subject of adsorption and self-assembly of proteins at modified surfaces. We employed atomic force microscopy (AFM) to study bovine serum albumin (BSA) and insulin adsorption at 1-decene- and methyl 10-undecenoate-modified Si(111) substrates. The results were compared to matrix-assisted laser desorption/ionization-mass spectroscopy (MALDI-MS) data previously published in the Morin group. The MALDI-MS spectra showed a big insulin signal, while BSA was not detected for 1 mg/mL BSA and 10% saturated insulin solutions. Our AFM images revealed considerable BSA adsorption, whereas it was not possible to observe insulin molecules. This clearly shows that in addition to the low quantity of the protein, a stronger interaction between the surface and the protein may result in a weak MALDI-MS signal and prevent quantitative analysis using this technique. Self-assembled monolayers on Au surfaces were used to investigate the oligomerization of truncated K122-4 (ΔK122-4) pilins into a new protein nanostructure. Employing AFM, we observed that at hydrophobic layers, with more than 10-30% accessible hydrophobic component, ΔK122-4 pilins oligomerize to a nanoweb. To differentiate the structure of ΔK122-4 nanoweb from the aggregation of the pilins at hydrophilic surfaces, electrochemical impedance spectroscopy was employed. Through equivalent circuit fitting, capacitance values of 3.7 ± 0.4 μF/cm2 and 2.5 ± 0.2 μF/cm2 were obtained for the aggregated pilins and the web pattern, respectively. Because of the higher average thickness of the former layer, this could support the presence of water in this layer. A dielectric constant (ε) of 5.1 ± 0.7 was obtained for the nanoweb. This low value could indicate a more compact and ordered assembly. Finally, conductive protein nanotube (PNTs) fabrication was performed by electroless copper deposition at ΔK122-4 PNTs in aqueous solution. The nanostructures were catalyzed by PdCl42-. At least 20% of the accessible amino acids of the PNTs have S-, N- and O-donor side chains which are able to form a complex with Pd(II). Metallization was verified using scanning electron microscopy (SEM).
Open Access
Characterization of Function and Role of FMTA in Staphylococcus Aureus
(2015-08-28) Rahman, Muhammad Masfiqur; Golemi-Kotra, Dasantila
Staphylococcus aureus is one of the major reasons for infectious mortality and morbidity. Due to extensive use of antibiotics, S. aureus has acquired resistance to almost all antibiotics. FmtA is one of the additional factors for methicillin resistance in S. aureus which interacts with wall teichoic acid (WTA) and localizes in the cell division septum. Previous studies showed that FmtA has very weak D-Ala-D-Ala carboxypeptidase (Cpase) activity. In lieu of these findings, we hypothesized that FmtA may require WTA as an activator ligand or protein-protein interactions to become fully functional as Cpase in the division septum. Here we show that WTA is not an activator ligand for Cpase activity of FmtA, but FmtA has an esterase activity on WTA. FmtA can remove D-Ala from D-alanyl ester in WTA. Additionally, we show serine and lysine from sequence motif Ser127-X-X-Lys130 (conserved among penicillin binding proteins (PBPs), β-lactamase and family VIII esterase) of FmtA are involved in this catalytic activity. Mutation studies suggest that both Ser-X-X-Lys motif in FmtA, located at the position S63 and S127 are involved in catalysis. Our results suggest that FmtA’s esterase enzymatic activity depends on both enzyme and substrate concentrations and high concentration of substrate (10 mg/mL WTA) can inhibit the reaction. Beside WTA, FmtA can also remove D-Ala from lipoteichoic acid (LTA). Results from in-vivo studies of WTA from fmtA deletion and fmtA-CM (conditionally over expressed) in S. aureus were in agreement with in-vitro esterase activity. In conclusion, WTA is not an activator ligand for FmtA and that FmtA shows esterase activity towards D-alanyl ester of WTA and LTA. Both SXXK motifs in FmtA are involved in catalysis and Serine and lysine from SXXK motif are important for esterase catalysis. To date, biological function of most esterase is unknown. To our knowledge, this is the first study to report esterase from S. aureus that has sequence similarity to PBPs and β-lactamase and very selective to its biological substrate WTA and LTA compare to synthetic substrate.
Open Access
A Comparative Study Performed on Two Ambient Ionization Techniques: Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) and Easy Ambient Sonic Spray Ionization Mass Spectrometry (EASI-MS)
(2015-08-28) Hamid, Tanam Sanjana; Ifa, Demian R.
Main project of my master’s thesis involved comparison between two sister methods: desorption electrospray ionization mass spectrometry (DESI−MS) and easy ambient ionization mass spectrometry (EASI−MS). These methods are compared in terms of spatial resolution, limits of detection and imaging capabilities. Their applicability in forensic science was further explored by comparing the MS/MS imaging capabilities. Under the same experimental conditions, we found that both DESI and EASI−MS have similar spray spot size resulting in similar spatial resolution. For the limits of detection (LOD) experiment, the LOD of DESI−MS was found to be lower than EASI−MS by an order of magnitude. When we compared the mass spectra and the chemical images of DESI and EASI, we found that both methods produce similar chemical specificity. When the spectra were carefully inspected, a reduction in signal intensity was observed for EASI−MS compared to DESI−MS demonstrating that the sensitivity of DESI−MS is higher than EASI−MS. Other projects involved characterization of zebra fish tissue system made up of bile salts, phospholipids and fatty acids. By mapping the spatial distribution of these ions, 2D chemical images of organs such as the stomach, the nervous system and the whole body zebra fish were created. I also studied if matrix can impact ionization of proteins by DESI−MS. The aim was to improve the poor desorption of proteins from the surface of DESI−MS. We concluded that the matrix neither increase nor decreases the ionization efficiency of proteins by DESI−MS.
Open Access
Evaluating the Utility of an Atmospheric Pressure Chemical Ionization Mass Spectrometer for Detecting Organic Peroxides During Beta-Pinene Ozonolysis Experiments
(2015-08-28) Jameer, Amanda Ophelia; Hastie, Donald R.
The intent of this study was to use a positive-ion atmospheric pressure chemical ionization mass spectrometer ((+) APCI-MS/MS) to detect organic peroxide products formed during β-pinene ozonolysis experiments. Detection was based on utilizing the neutral-loss scan (NLS) analysis mode of the APCI-MS/MS to observe ion signals exhibiting unique mass losses of 34 u. This mass loss was considered exclusive to protonated organic peroxides containing a hydroperoxy moiety. Given this observation, the NLS analysis mode was used to selectively detect organic peroxides formed during β-pinene ozonolysis experiments. Overall, six organic peroxide structures were proposed. Further product-ion scan analysis on the six proposed structures revealed additional unique mass losses of 32 u and 62 u that could be use in combination with 34 u NLS analysis to selectively detect organic peroxides. The results from this study can assist in ascertaining organic peroxide contribution to secondary organic aerosol for future ozonolysis experiments.