Chemistry

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Open Access
Selective Chemical Reactions for Nucleic Acid Sequencing and DNA-Encoded Library Synthesis
(2023-03-28) Mahdavi Amiri, Yasaman; Hili, Ryan
Part One: The ability to map methylation sites in the human genome and epitranscriptome has transformed our understanding of how these modifications govern and influence a host of cellular processes and human diseases. Amongst the most widely studied methylations is N6-methyladenine, known as 6mA in DNA and m6A in RNA. While traditional methods to sequence these modifications have depended on antibody pulldowns, chemistry-based approaches are often less sequence dependent, can work on either DNA or RNA, and thus can provide a robust, inexpensive, and universal sequencing approach. In part one of this thesis, the first chemistry-based single-nucleotide resolution sequencing method for the detection of N6-adenine methylation sites in DNA and RNA is presented. This method takes advantage of the chemoselective deamination of unmodified adenines under acidic nitrite conditions, resulting in a (d)A to (d)G transition, while leaving methylated adenine sites unaffected. As changes in N6-adenine methylation of RNA and DNA have been implicated in a range of human diseases, especially cancers, the method has been rapidly adopted by researchers globally as an affordable and straightforward sequencing approach to assist in understanding the role and impact of the epigenome and epitranscriptome on human health. The ability of this method to detect other nucleotide modifications was also evaluated and described. Part Two: DNA-encoded libraries (DELs) comprise millions to billions of small-molecules covalently linked to a unique DNA barcode that can be read using standard next-generation sequencing (NGS). This technology has revolutionized the field of drug discovery as a method to rapidly identify small molecules that can serve as novel leads for drug development. The success of a drug discovery campaign involving a DEL depends on the chemical diversity presented within the DEL; methods that can generate DELs with new molecular architectures and with greater chemical diversity are critically needed to advance drug discovery efforts both within industry and academia. To this end, the use of photoredox chemistry as a facile method to generate DELs with drug-like properties is presented as part two of this thesis. An efficient approach for the photoredox-catalysed hydroaminoalkylation between on-DNA secondary N-substituted (hetero)arylamines and vinylarenes is explored as a method to generate DELs with known bioactive architectures. The developed reaction proceeds efficiently with a broad and well-explored substrate scope, working best with electron poor to neutral vinylarenes. This method is well suited for the construction of DELs enabling an expansion of drug-like chemical space.
Open Access
Determining the Sampling Rates for a New Nylon Passive Sampler to Estimate the Atmospheric Concentrations of Nitric Acid and Perfluoroalkyl Acids Pollutants
(2023-03-28) Carmichael, Lindy; Young, Cora; VandenBoer, Trevor C.
Atmospheric pollutants such as gaseous nitric acid (HNO3) and perfluoroalkyl acids (PFAAs) are emitted or formed in the atmosphere as a result of anthropogenic activities. These acids could pose risks to organisms and the environment. The PFAAs are organofluorine chemicals that have been producing scientific and regulatory interest because of their persistence in the environment, toxicity, and bioaccumulation potential. They are detected in water, the atmosphere, human organs, and wildlife. There are scarce atmospheric measurements of PFAAs because of their low atmospheric concentrations. Nylon passive samplers are selective for sampling of atmospheric acids. They are well-validated for HNO3 but have never been used to collect PFAAs. The Nylasorb nylon filter, previously validated for HNO3 was discontinued and a new nylon filter was purchased from a different manufacturer. The new nylon filter has been characterized in this work for the monitoring of HNO3 and determination of PFAAs in the atmosphere.
Open Access
Diversification of 4-Alkylpyridines: Mining for Reactivity with Alkylidene Dihydropyridines
(2023-03-28) Doan, Brian Anh-Tuan; Orellana, Arturo
Pyridines are valued structures in pharmaceutical development. Using a soft enolization approach, we can diversify alkyl pyridines under mild conditions via alkylidene dihydropyridines (ADHPs). Recent work in our group has demonstrated the utility of ADHPs in palladium-catalyzed reactions. However, the fundamental reactivity of ADHPs remains largely unexplored, with only scattered reports in the literature. We seek to further explore the reactivity of these electron-rich intermediates in different contexts, as new transformations can provide useful synthetic tools for pharmaceutical discovery. Herein we describe our investigations in this area, including the development of two new synthetic methods. Specifically, we describe the oxidation of ADHPs to the corresponding pyridylic ketones under mild conditions, and the iridium-catalyzed asymmetric alkylation of alkylidene dihydropyridines branch-allylated alkyl pyridines.
Open Access
Design of Singlet Fission Chromophores through the Introduction of N-Oxyl Fragments.
(2022-12-14) Dylan Shangrow James; Zeng, Tao
Singlet fission is a highly desired phenomenon in photovoltaics. In the fission process, one short-lived singlet exciton splits to two long-lived triplet excitons. Generating a larger number of longer-lived excitons, singlet fission has the potential to enhance the photoelectric conversion efficiency. The exploitation of this phenomenon in the photovoltaic industry is however impacted by the small pool of existing singlet fission chromophores. Here, we report on the design of novel singlet fission chromophores through the substitution of N-oxyl fragments within the anthracene framework. The substantial diradical character brought on by the N-oxyl fragments located at specific positions on the anthracene pristine structure together with structural reorganization induced by excitation allows for a handful of chromophores to satisfy the thermodynamic requirements of singlet fission.
Open Access
Electrophoretic Separations for Continuous Flow Synthesis
(2022-12-14) Ivanov, Nikita; Krylov, Sergey N.
The continuous-flow synthesis field has grown considerably in the last several decades. Converting a reaction from its batch synthesis to a continuous-flow alternative offers a long list of potential improvements. The latest advances make it possible for reactions to take place “on the chip’, where microscopic channels are used to propagate and mix different reactants. Continuously propagating discrete volumes of reactants inside a small capillary has the advantage of improved mass and heat transfers. These transport phenomena directly affect the kinetics and thermodynamics of the reaction, two factors that influence reaction yield over time. Having higher yields means that more product is made in a similar or lesser time frame, which can potentially lower the production cost of a pharmaceutical product, resulting in a monetary advantage for the early adopter. Another positive aspect of continuous flow chemistry is safety. By miniaturizing a reactor, we gain another level of control over the system. Since flow-reactor volumes are microscopic, the enthalpy of exothermic reactions can be easily dissipated. This is extremely important when reactions of interest possess high enthalpic contribution, especially if they are self-accelerating decomposition reactions. Since no reaction can ever achieve a hundred percent yield, a product purification mechanism is required at the end of each synthesis step. It runs naturally that a continuous-flow synthesis system should feed into a continuous-flow separation compliment without breaking the fundamental continuity concept. Up to this day, this remains the most problematic area of continuous-flow chemistry. Available continuous separation methods are either pseudo-continuous (simulated moving bed chromatography, SMBC) or severely limited in the number of concurrently separated analytes (Continuous liquid-liquid extraction, CLLE). The real solution to this problem are molecular stream separation (MSS) platforms. MSS approach allows for multiple analytes to be separated and analyzed simultaneously without disturbing the synthesis platform's continuous nature. Only two major MSS branches exist today, continuous-flow electrophoresis (CFE) and continuous annular chromatography (CAC). Although CAC has always been developed with the organic synthesis in mind, CFE has historically been reserved for water-soluble biological analytes such as DNA or proteins. By adapting CFE to the world of organic chemistry, we open the door to the field of electrophoretic separations for continuous-flow synthesis. The following manuscript will touch on the subject of fundamental engineering challenges imposed by the project and will serve to summarize our latest efforts at transforming CFE into a simple yet comprehensive platform for continuous chemistry separations.
Open Access
Studying the Structural Dynamics and Aptamer-Ligand Interactions in the Cocaine-Binding Aptamer through Fluorescence Spectroscopy
(2022-12-14) Shoara, Amin Aron; Johnson, Philip E.
Since its first report in 1990, aptamers have been utilized in biosensor modeling technologies. One of the most important advantages of using aptamers is the structural flexibility and thermal stability of nucleic acids. These structural merits enable aptamers to be linked on solid surfaces, attached to chemical labels, or extended to build nanostructures for advanced therapeutic and diagnostic modeling purposes. The cocaine-binding aptamer was originally selected through a systematic evolution of ligands by exponential enrichment (SELEX) method to select cocaine molecules from cocaine metabolites in biological solutions. However, the aptamer showed binding to quinine and other antimalaria drugs tighter than its original ligand, cocaine. Work presented in this dissertation demonstrate how the cocaine-binding aptamer can be exploited as a model system for the structural analysis of aptamers using biophysical techniques including fluorometry methods. The results discussed in this study demonstrate how intrinsic fluorescence of ligands was exploited for aptamer-ligand binding and thermal stability analyses. Furthermore, photoisomerization of stilbene coupled with ligand-induced binding mechanism of the cocaine-binding aptamer were employed for the development of the Photochrome Aptamer Switch Assay. This research aims to gain insight into how aptamers interact with their ligands by utilizing the fluorescence properties of the ligands. Investigating the binding mechanisms of aptamers is essential in sensing technology since biosensors yield greater analytical sensitivity upon ligand-induced structural changes.
Open Access
The Synthesis and Reactivity of Carbamoyl Fluorides
(2022-12-14) Tiburcio, Tristan Redondo; Le, Christine
Chapter 1 Herein we report a protocol for the synthesis of carbamoyl fluorides from secondary amines using inexpensive, and commercially available starting materials. This method employs the use of a difluorocarbene generated from the thermolysis of (triphenylphosphonio)difluoroacetate. This becomes oxidized by 4-methylpyridine N-oxide, generating difluorophosgene, which serves as the key intermediate to make the carbamoyl fluorides from secondary amine starting materials. This method allows access to carbamoyl fluorides with a vast functional group tolerance, including Lewis-basic heterocycles, alkenes, and alkynes. Chapter 2 We report a base-free, facile cross-coupling reaction of carbamoyl fluorides to silylated nucleophiles. This reaction utilizes an affordable and Earth-abundant nickel transition metal catalyst, with a common phenanthroline ligand (both of which see an exceptionally low loading), to synthesize ureas, carbamates, and alkynamides in moderate to excellent yields. The transmetallation step of the purported catalytic cycle generates a fluorosilane, providing the thermodynamic driving force for the reaction to progress, and circumventing the requirement of base.
Open Access
Neutral Bidendate Guanidine-Based Zinc Complexes for the Ring Opening Polymerization of Lactide
(2022-12-14) Khan, Brandon Sharaz; Lavoie, Gino G.
Herein is reported the synthesis and structure elucidation of three novel zinc complexes bearing neutral bidentate guanidine ligands for the ring opening polymerization (ROP) of lactide to PLA. Ligand and complex structures were obtained computationally and leveraged against single-crystal studies to develop a method for predicting solid state structures of future complexes. The catalysts were tested for ring opening polymerization of lactide to PLA. The most active complex displayed a rate constant for propagation, kp, of 26 M-1h-1. The catalyst showed some stereoselectivity with a heterotactic bias in the polymer (Pr = 0.60). The polymer number average molecular weight was determined to be ~4700 g mol-1. The end analysis of the MALDI-TOF mass spectrum suggested water activation for the polymerization. The polydispersity index of the polymer was determined by gel permeation chromatography to be 1.10, indicating a well-behaved polymerization.
Open Access
Developing an Automated Nitrous Acid (HONO) Platform to Detect Emerging Pollutants in a Commercial and Domestic Environment
(2022-08-08) Lao, Melodie; Vandenboer, Trevor C.
Nitrous acid (HONO) is an emerging household pollutant linked to adverse health effects, with levels reported higher indoors than outdoors. Under low light conditions, HONO can readily photolyze to form hydroxyl radicals, impacting our indoor air quality by generating harmful secondary pollutants. Thus, HONO formation processes must be well understood to improve indoor air quality. This work presents two new instruments: a HONO calibration source and an automated indoor HONO platform. The calibration source generates low HONO calibration mixing ratios from low ppt to tens of ppb, minimizing measurement uncertainty and identifying interferences for other HONO-detecting instruments. The source is integrated into an automated HONO platform, custom-built to perform modulated measurements of NOx and HONO in indoor air quality applications. The platform was deployed in a commercial kitchen to validate its measurements and investigate indoor chemical HONO processes in a setting that has not been previously measured at high-time resolution.
Open Access
Studying Aptamer-Ligand Interactions and Dynamics in the Cocaine-Binding Aptamer through NMR Spectroscopy
(2022-08-08) Churcher, Zachary Robert; Johnson, Philip E.
Aptamers are oligonucleotide molecules with applications in biosensors, analytical chemistry, and therapeutics. They are often to 10 to 100 nucleotides in length and can be selected to bind a wide range of targets from ions to cells. The cocaine-binding aptamer was selected in 2000 by Milan Stojanovic to bind cocaine but not benzoylecgonine and ecgonine methyl ester, two common cocaine metabolites. Since its development the cocaine-binding aptamer has been found to bind to quinine with an affinity ~50 times stronger than to cocaine. The cocaine-binding aptamer has been used to help test and develop new biosensing systems. This research hopes to further the understanding of how the cocaine-binding aptamer interacts with its ligands in the hope that these interactions could be used to help understand other aptamer-ligand systems. Using nuclear magnetic resonance (NMR) spectroscopy the base pair dynamics of the cocaine-binding aptamer was investigated as a function of ligand binding. This study was later expanded to include the effects of temperature and buffer composition to determine thermodynamic parameters of base pair dissociation. The results showed a general reduction in dynamics with ligand binding in the aptamer at the ligand-binding site, but little change elsewhere in the aptamer. The binding of additional ligands to the cocaine-binding aptamer was characterized by NMR spectroscopy. Levamisole is an anti-parasitic worm medication and one of the most common adulterants found in cocaine. Levamisole was found to bind the cocaine-binding aptamer with a weak affinity at the same site as cocaine. The binding of the cocaine-binding aptamer to a set of three-way junction-binding dyes was also investigated. These dyes share a common structure but bind to the aptamer with a range of affinities. These dyes also bound at the same site as cocaine and quinine, with the dyes being able to be displaced by cocaine or quinine depending on their affinity. Finally, the binding of ochratoxin A to the ochratoxin A-binding aptamer was investigated using NMR. This aptamer had not been previously studied using NMR, was found to fold tightly in response to its ligand, and NMR proton assignments were obtained.
Open Access
Sensing and Imaging Biomolecules with Plasmonic Nanoparticle Assemblies Coupled with Darkfield Microscopy
(2022-08-08) Le, Nguyen Hoang; Chen, Jennifer I-Ling
Noble metal nanoparticles exhibit unique optical properties arising from the resonant oscillations of their conduction electrons with light. This phenomenon is called localized surface plasmon resonance (LSPR). The LSPR frequency is extremely sensitive to the size, shape, refractive index at the metal-dielectric interface, and other nearby metal nanoparticles. In an assembly of proximal nanoparticles, the LSPR of individual particles can couple to yield enhanced light scattering and large spectral shifts, which are useful for many applications including diagnostics and sensing. This dissertation presents a complex nanostructure comprising a core gold nanoparticle surrounded by multiple satellite gold nanoparticles for biosensing application. Chapter 2 introduces the fabrication and characterization of the core-satellite assemblies via a layer-by-layer process. Using ATP-aptamer as the linker, we demonstrated the detection of ATP based on the disassembly of the nanostructure, which can be readily captured by darkfield microscopy. The detection limit, dynamic range, and sensitivity can be tuned by controlling the size of the assembly. We found that the aptamer-linked nanoparticle assemblies were selective to only ATP, and not other adenine-containing compounds. Additionally, sensing of ATP in buffer and in bulk cell lysates was demonstrated. Chapter 3 presents the methodology for detecting ATP directly from lysed cells, down to the single-cell level without the need for purification or extraction. The intracellular ATP levels of two ovarian cancer cell lines were quantified to elucidate the differences and cellular distribution, and the potential of the stick-and-peel platform for mapping the microenvironment of 2D heterogeneous surfaces was demonstrated. In chapter 4, the optical properties of nanoparticle assemblies were tuned by changing the morphology of the nano building block, where the incorporation of gold nanoshells as satellites led to an extended redshift of LSPR to a much longer wavelength compared with using solid gold nanoparticles as satellites. This tunability in the LSPR of the assemblies allows for color-based analysis and color-coding of the plasmonic sensors. Lastly, Chapter 5 outlines the development of a multiplexed assay using the nanoparticle assemblies. Two types of assemblies, targeting either ATP or a nucleic acid (DNA-210), were fabricated with different DNA linkers in the same sensing area. The multiplexing was demonstrated by the selective disassembly process. Moreover, the ability to tune the optical properties of nanostructures using different morphologies was integrated; two different morphology of nanostructures, i.e. solid-solid and solid-shell nanostructures, for two targets, ATP and DNA-210, respectively, were fabricated. Based on a difference in scattered color, two types of biosensors among thousands of nanoparticle assemblies can be easily identified. Finally, we demonstrated duplex detection based on the change in the scattering intensity and the color read-out. Reflecting on the contributions of our work, this dissertation advances the fundamental knowledge and practical design of chip-based sensing platforms comprising complex plasmonic nanostructures. The work contributes to the sensing field by addressing some of the challenges in point-of-care or point-of-need measurement applications and provides an alternative bioanalytical tool for single-cell based analysis.
Open Access
Development, Validation, and Application of Methods for High Time-Response Measurement of Gaseous Atmospheric Chlorinated Species
(2022-03-03) Furlani, Teles Carlo; Young, Cora
Halogenated compounds that participate in catalytic cycles in the atmosphere can influence the fate of chemicals, including ozone, methane, and volatile organic compounds (VOCs). These halogen radicals, in particular atomic chlorine (Cl), can deplete ozone and will react rapidly with VOCs. Reliable, sensitive, and widely available hydrogen chloride (HCl) measurements are important for understanding Cl initiated oxidation in many regions of the troposphere. We configured a commercial HCl cavity ring-down spectrometer (CRDS) for sampling HCl in the ambient atmosphere and developed validation techniques to characterize the measurement uncertainties. The HCl analyzer was used to make continuous HCl measurements in the polluted marine boundary layer during the Halifax Fog and Air Quality Study (HaliFAQS). Bimodal HCl features in the high irradiance days indicated two photochemical processes; (1) morning time photolysis of Cl precursors, and (2) midday formation of nitric acid followed by acid displacement onto chloride (Cl) containing aerosols. A box model used measured HCl to estimate nitryl chloride mixing ratios at sunrise and assessed the contribution of photolabile Cl precursors to radical formation. Total gaseous chlorine (TClg) measurements can illuminate unknown sources of Cl to the atmosphere. Techniques for measuring TClg have been limited to offline analysis of extracted filters and do not provide suitable temporal information on fast atmospheric process. The utility of this novel TClg measurement technique will be crucial to future estimates and assessments of chlorinated compounds and their impact on air quality, climate, and health.
Open Access
Estimating Methane Emissions in Canada Using Atmospheric Observations from Earth to Space
(2022-03-03) Baray, Sabour Ahmad; Gordon, Mark; McLaren, Robert
Methane is a significant greenhouse gas with 25–32 times the global warming potential of carbon dioxide. Global sources and sinks of methane are understood to be 550 ± 60 Tg a-1. The possible causes of changing decadal trends in atmospheric methane concentrations since the 1990's is not well understood, since this requires a precision in global emissions quantification better than 20 Tg a-1. Atmospheric observations at the local, regional, or national scale can provide "top-down" constraints on emissions to verify "bottom-up" emissions that may not be well characterized. Cavity ring down spectroscopy (CRDS) instruments deliver highly precise in-situ measurements of methane, with 1 Hz precision better than 2 ppb. A comprehensive aircraft campaign in the Athabasca Oil Sands Region of Alberta (AOSR) in summer 2013, led by Environment and Climate Change Canada (ECCC), deployed a CRDS alongside a suite of instrumentation to measure atmospheric pollutants and meteorological parameters. These observations allowed for the comprehensive identification and quantification of methane emissions from unconventional oil extraction. Emissions estimates were 48% higher than those reported in the national greenhouse gas inventory. A series of lower cost follow up campaigns in 2014 and 2017 using a CRDS instrument mobilized with a vehicle allowed for cold season monitoring of emissions and select quantification where atmospheric parameters were favorable, showing continued discrepancies with inventory reporting. To estimate emissions across Canada at the national scale, methane measurements from ECCC long-term monitoring stations over 2010-2015 were utilized in conjunction with satellite remote sensing observations from the Greenhouse Gas Observing Satellite (GOSAT) operated by the Japanese Aerospace Agency (JAXA). These atmospheric observations were assimilated in the GEOS-Chem chemical transport model to constrain emissions using a Bayesian inverse modelling methodology. Results showed 42% higher emissions from anthropogenic sources and 21% lower emissions from natural sources, which are mostly wetlands, when compared to the prior estimate. Through the combinations of all studies presented herein, approximately 2–4 Tg a-1 of methane emissions in Canada were reallocated for the year of 2013, where 1–3 Tg a-1 was added to anthropogenic sources and 2–4 Tg a-1 was deducted from natural sources, which is substantial relative to the anthropogenic inventory in Canada which is 4–5 Tg a-1. This reallocation is 0.4–0.8% of the entire global budget of 550 Tg a-1, where only a ~3% change in the source-sink balance can cause the observed trends in atmospheric methane. These results show that atmospheric observations from surface, aircraft and satellites are critical for constraining the methane budget in Canada, and improvements are necessary to these types of atmospheric observations over the world to constrain the methane cycle within the precision needed to understand decadal trends.
Open Access
Crystallization Studies of the TraF Protein from the F Plasmid of Escherichia coli
(2022-03-03) Samari-Kermani, Naveed; Audette, Gerald F.
Crystallization of suitable crystals for diffraction analysis is a major hurdle in structure-function studies of proteins using X-ray crystallography. This is especially true when the protein may be generally hydrophobic, membrane associated or contain regions of increased flexibility. The objective of the research outlined in this thesis was to determine a more amenable set of crystallization conditions for the protein TraF from the F plasmid for X-ray diffraction and structure solution. The F plasmid of Escherichia coli utilizes a conjugative type IV secretion system (T4SS) by which the transmission of genes in bacteria occurs contributing to the current issue of antibiotic resistance. The TraF protein of this system is a periplasmic protein with a dynamic hydrophobic N-terminal tail which interacts directly with TraH and a C-terminal thioredoxin-like domain. TraF is hypothesized to chaperone and interact with several other proteins of this system. Although its exact function remains unclear, TraF is essential in in the F plasmids T4SS as a lack of function abolishes gene transfer through this system. Modification of upstream conditions, including expression and purification of a GST-tagged variant of TraF, as well as modification of buffer conditions including the detergent NP-40 allowed for an increased solubility and concentration of the protein for crystallization trials. The traditional screening process with GST-TraF resulted in two conditions that showed birefringent structures which did not yield any better results when optimized. Optimization of the original reported TraF purification and crystallization conditions did double the protein concentration, however resulted in a lack of miscibility between the protein stock and the reservoir solution during crystallization. Furthermore, a head-to-head crystallization screening was conducted incorporating 5-amino-2,4,6-triiodoisophthalic (I3C) in the crystallization drop to advance structure solution using experimental phasing methods. The head-to-head experiment led to multiple crystallization conditions that should be optimized and further assessed while validating the novel process of incorporating I3C in the screen formation process.
Open Access
Thermodynamic and Kinetic Analysis of Aptamer-Ligand Interactions Using Isothermal Titration Calorimetry
(2022-03-03) Dawood, Nusaibah Esmael; Johnson, Philip E.
Aptamers are short single-stranded DNA or RNA oligonucleotide sequences capable of binding to a broad range of target molecules with high affinity and specificity. They can interact with a large variety of targets such as small molecules, ions, enzymes, and proteins employing all kinds of non-covalent interactions. Isothermal Titration Calorimetry was employed to explore the binding behaviour of aptamer-ligand interactions using the cocaine-binding aptamer as a model system. This dissertation is an assembly of two distinct research projects; In the first part, a bifunctional cocaine and deoxycholic acid-binding aptamer was constructed from individual cocaine-binding aptamer variants and the binding affinity and thermodynamics were measured using isothermal titration calorimetry. We show that the bifunctional aptamer binds its ligands with positive cooperativity, having a Hill coefficient of 1.2 -1.5, whether the ligands are added individually or as an equimolar mixture. A mechanism where dynamics at one ligand-binding site is affected by the presence of the ligand at the other is proposed to account for the cooperative binding. The next chapter highlights the use of kinITC to extract binding rates from ITC experiments. Determining the binding thermodynamics and kinetics between aptamers and ligands is important for understanding their recognition mechanisms as well as providing a framework for developing biosensors. One aptamer that has been particularly well studied is the cocaine-binding aptamer. There has been a lot of research conducted on the thermodynamics for the cocaine-binding aptamer however, one feature not reported yet is the binding kinetics. Here, we measure the kinetics of quinine binding to two sequence variants of the cocaine-binding aptamer, one with a short stem-one (MN19) and the other with a long stem-one (MN4). When stem-one of the aptamer is six base pairs long, the aptamer retains its secondary structure in its free and bound forms. When the length is shortened to three base pairs, such as for MN19, the aptamer is loosely folded in its unbound state and undergoes structural changes upon binding quinine. Both binding kinetics and thermodynamic data were acquired as a function of different temperatures. A 1:1 binding model was applied to extract binding rates for both variants of the cocaine-binding aptamer. Arrhenius plots were derived to compare the activation energies for MN4 to that of MN19 since MN19 exhibits ligand-dependent conformational changes. The values obtained for the transition state enthalpy barrier were averaged to be 5.1 kcal mol^(-1) for MN4 and -0.8 kcal mol^(-1) for MN19. These results suggest that MN19 has an energy barrier that is almost negligible under standard conditions. Aptamers are short single-stranded DNA or RNA oligonucleotide sequences capable of binding to a broad range of target molecules with high affinity and specificity. They can interact with a large variety of targets such as small molecules, ions, enzymes, and proteins employing all kinds of non-covalent interactions. Isothermal Titration Calorimetry was employed to explore the binding behaviour of aptamer-ligand interactions using the cocaine-binding aptamer as a model system. This dissertation is an assembly of two distinct research projects; In the first part, a bifunctional cocaine and deoxycholic acid-binding aptamer was constructed from individual cocaine-binding aptamer variants and the binding affinity and thermodynamics were measured using isothermal titration calorimetry. We show that the bifunctional aptamer binds its ligands with positive cooperativity, having a Hill coefficient of 1.2 -1.5, whether the ligands are added individually or as an equimolar mixture. A mechanism where dynamics at one ligand-binding site is affected by the presence of the ligand at the other is proposed to account for the cooperative binding. The next chapter highlights the use of kinITC to extract binding rates from ITC experiments. Determining the binding thermodynamics and kinetics between aptamers and ligands is important for understanding their recognition mechanisms as well as providing a framework for developing biosensors. One aptamer that has been particularly well studied is the cocaine-binding aptamer. There has been a lot of research conducted on the thermodynamics for the cocaine-binding aptamer however, one feature not reported yet is the binding kinetics. Here, we measure the kinetics of quinine binding to two sequence variants of the cocaine-binding aptamer, one with a short stem-one (MN19) and the other with a long stem-one (MN4). When stem-one of the aptamer is six base pairs long, the aptamer retains its secondary structure in its free and bound forms. When the length is shortened to three base pairs, such as for MN19, the aptamer is loosely folded in its unbound state and undergoes structural changes upon binding quinine. Both binding kinetics and thermodynamic data were acquired as a function of different temperatures. A 1:1 binding model was applied to extract binding rates for both variants of the cocaine-binding aptamer. Arrhenius plots were derived to compare the activation energies for MN4 to that of MN19 since MN19 exhibits ligand-dependent conformational changes. The values obtained for the transition state enthalpy barrier were averaged to be 5.1 kcal mol^(-1) for MN4 and -0.8 kcal mol^(-1) for MN19. These results suggest that MN19 has an energy barrier that is almost negligible under standard conditions.
Open Access
Probing the Impact of Solvent on Lewis Acid Catalysis via Fluorescent Lewis Adducts
(2021-11-15) Laturski, Amy Elizabeth; Baumgartner, Thomas
Over the years, various multiparameter methods have been developed to measure the strength of a Lewis acid. However, a major challenge for these measurements lies in the complexity that arises from variables, such as solvent and other fundamental interactions, as well as perturbations of Lewis acids as their reaction environment changes. Herein, we evaluate the impact of solvent effects on the Fluorescent Lewis Adduct (FLA) method using a series of representative Lewis acids. The solution-state nature of the FLA method offers the ability to correlate Lewis Acid Units (LAUs) obtained from the FLA measurement with reactivity. The binding of a Lewis acid in various solvents quantitatively reveals a dichotomy between both polarity and donicity of the solvent. While not strictly separable, as solvent polarity increases observed LAU values increase; however, as solvent donicity increases observed LAU values decrease. This dichotomy was confirmed by titration data and catalytic Diels-Alder cycloaddition and hydrosilylation reactions, illustrating that solvation effects can be appropriately gauged by a LAU value determined from the FLA method.
Open Access
Insights into Genipin Dye Mechanisms and Colour Origins
(2021-11-15) Jeeva, Fiona; Caputo, Christopher
Genipin is a natural compound that forms colours when reacted with primary amines. Curiously, it contains a large amount of uncertainty surrounding its dye structure. It was previously believed that genipin reacts with primary amines to first form a red intermediate dye that polymerizes to form a blue dye when exposed to oxygen. We have determined that the dye colour can be altered by reacting genipin with aromatic amines, producing green dyes instead of blue dyes. In addition, we have found that these dyes are not polymers at all, and their characteristic blue colour is instead the result of a persistent radical, supported by NMR spectroscopy, MS, and EPR spectroscopy. Furthermore, we have shown that the oxidation of the allylic alcohol functionality can form the oxetane derivative via a 2,3-epoxyalcohol rearrangement that can alter the redox properties of the dye, and thereby halting the dye formation at its red intermediate.
Open Access
Functionalization of 4-Substituted Pyridines for Enantioselective Synthesis
(2021-07-06) Hunter, Isabelle Anne Carriere; Garcia, Josue Arturo Orellana
Enantioselective synthesis using nitrogen containing heterocycles, such as pyridines, is extremely useful in drug discovery. The prevalence of pyridines in the drug market is well-documented and a mild, enantioselective method to functionalize pyridines would allow faster access to more structurally diverse pyridines, improving efficiency in the search of drug candidates. The Orellana group has developed a strategy for selective allylation of 4-substituted pyridines using mild conditions, which results in broad functional group tolerance. Reported herein are ways to expand the allyl fragment to larger linear and cyclic fragments, and attempts at the development of an enantioselective variant using palladium catalyzed decarboxylative allylation. The pathway for reductive elimination was also discovered through mechanistic experiments which revealed more of this palladium catalyzed decarboxylative allylation mechanism. Furthermore, a new method was investigated for arylation of the pyridylic position using a high-valent copper catalysis.
Open Access
Development of New and Modification of Existing Methods for the Primary Screening of Aptamers and Antibodies by Means of Capillary Electrophoresis
(2021-03-08) Beloborodov, Stanislav; Krylov, Sergey N.
Discovery of new drugs starts with screening a library of drug candidates (ligands) for their ability to bind targets. Such a screening can be conducted by a variety of analytical techniques; however, Capillary Electrophoresis (CE) is the the most preferably one. CE provides homogenous separation of the species, generates minor amount of waste and can serve both analytical and preparative purposes. Sometimes, CE-based screening approaches fail due to insufficient separation of target-ligand complexes (TL) from non-binding ligands or due to the inability to collect the complexes. The first problem affects all types of ligands, but the second one is especially acute for the oligonucleotides, since oligonucleotide binders are often required to be collected for the further amplification and sequencing. To address this problem, I developed a mathematical model that allows precise prediction of elution times of protein-oligonucleotide complexes, which guarantees their successful collection. Such a predictor is especially useful for the cases when the complexes are present in the undetectably small amount. The developed model significantly advances the existing screening approaches, such as NECEEM (Non-equilibrium Capillary Electrophoresis of Equilibrium Mixtures) and Ideal Filter CE (IFCE). The problem of inefficient separation of TL complexes from the initial targets and ligands is especially acute for the field of CE-based immunoassays. Particularly, for the assays that study virus-antibody interactions. The existing assays provide a minor (almost negligible) separation of virus-antibody complexes from the source molecules and allow screening mixtures of antibodies and spherical viruses only. In order to address these problems, I developed an assay for screening bivalent antibodies for their ability to bind another common type of viruses, which are rod-like viruses. The developed assay was able to provide a baseline separation of virus-antibody complexes from free viruses and antibodies. In addition to that, my work resulted in the discovery of the previously unknown phenomenon of abnormally high electrophoretic mobility of the complexes between rod-like virions and bivalent antibodies. I explained this phenomenon and built a simple mathematical model for the prediction of the number of cross-linked virions in the complexes.
Open Access
Palladium-Catalyzed Functionalization of 4-Alkylpyridines: Pyridlyic Dehydrogenation & Mechanistic Investigation of Pyridylic Allylation
(2021-03-08) Wasfy, Nour Moataz Ahmed F.; Garcia, Josue Arturo Orellana
Pyridines are important frameworks in drug development, valued for their ubiquitous presence in biologically active compounds. As such, we have taken a keen interest in developing mild methods for pyridylic functionalization suited for drug discovery. By implementing a soft enolization approach we are able to effect pyridylic functionalization under mild conditions not achieved by traditional pyridylic activation strategies. Employing alkylidene dihydropyridines (ADHPs) as intermediates in palladium-catalysis, the group developed a mild and practical pyridylic allylation method with broad functional group tolerance. In this report, we extend this reactivity to induce pyridylic dehydrogenation as a direct and reliable approach to accessing 4-alkenyl pyridines. Additionally, we detail mechanistic investigation of the allylation analogue conducted to aid our efforts in achieving an enantioselective variant of the transformation.