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AroA catalyzes the sixth step of the shikimate biosynthetic pathway which produces aromatiG amino acids in plants and bacteria, but is absent in mammals. This makes AroA an attractive antimicrobial target. The transition state (TS) structures of AroA- and acid-catalyzed 5-eno/pyruvyl shikimate-3-phosphate (EPSP) hydrolysis were studied in atomic detail by kinetic isotope effect (KIE) measurement. Enzymes bind their transition states more tightly than any other species, so molecules that closely resemble the transition state would have a high affinity for the enzyme and be good inhibitors. Radiolabelled EPSPs were synthesized and a KIE measurement method was developed. Six KIEs were measured for both the AroA- and acid-catalyzed reactions. KIEs for the AroA reaction indicate a cationic TS structure. The acid-catalyzed reaction may employ a slightly different mechanism with an earlier TS. A computational TS model was found and its KIEs were calculated. It demonstrated good agreement with the experimental values at most positions. The model is being modified to improve the agreement with the experimental KIEs. This TS structure will be a good starting point for inhibitor design. All these efforts, hopefully, can make a positive contribution to the development of antimicrobial drugs. / Thesis / Master of Science (MSc)

<p> AlkA is a monofunctional DNA glycosylase from E.coli. This enzyme catalyzes the hydrolysis of the N-glycosidic bond, initiating the first step in the base excision repair pathway. This activity is crucial to the maintenance of the genetic code, as the persistence of DNA aberrations can have significant cellular consequences including mutation, and inhibition of DNA replication and transcription. This enzyme has a broad substrate specificity catalyzing the excision of various lesions (including alkylation, oxidation and deamination products) from DNA. While biochemical and structural studies have been carried out on AlkA; how this enzyme is able to recognize and excise a variety of structurally diverse lesions from DNA and the mechanism by which this excision occurs remains unknown. In this study we have shown that a stem-loop DNA structure containing a hypoxanthine bulge is an optimal substrate for TS analysis of AlkA-catalyzed N-glycoside hydrolysis. In addition, we have developed methods to synthesize radiolabeled deoxyinosine triphosphate (diTP) and incorporate this radiolabeled nucleotide into the stem-loop DNA structure. We have developed a facile method of purification for his-tagged AlkA and his-tagged AlkA containing a TEV protease recognition site (for removal of the his-tag), and have shown that these proteins display an activity similar to that of wild-type AlkA. The [1'-3H] KIE was measured using liquid scintillation in a proof-of-principle experiment. The observed value of 1.046 is indicative of either a relatively synchronous ANDN (SN2) TS or an early DN*AN (SN1) TS with oxacarbenium ion character in the sugar ring, but significant bond order to the leaving group base still remaining. Future work involves repeat measurements of the [1 '-3H] KIE to validate the accuracy of the measurement observed here, examination of commitment to catalysis and optimization of the hypoxanthine bulge substrate synthesis. Analysis of KIEs at additional sites on the hypoxanthine base and sugar ring will contribute to TS analysis of AlkA-catalyzed N-glycoside hydrolysis and help elucidate the mechanism of hydrolysis. </p> / Thesis / Master of Science (MSc)

Malaria continues to cause significant mortality in sub-Saharan Africa and elsewhere, and existing vector control measures are being threatened by growing resistance to pyrethroid insecticides.  With the goal of developing new human-safe, resistance-breaking insecticides we have explored several classes of acetylcholinesterase inhibitors.  In vitro assay studies have shown that trifluoromethyl ketones (TFK's) are potent inhibitors of An. gambiae AChE (AgAChE), that inhibit the enzyme by making a covalent adduct with the catalytic serine of the enzyme.  However research in the Carlier group has shown that trifluoromethyl ketones bearing benzene and pyrazole cores have shown very little toxicity to An. gambiae, perhaps due to hydration and rapid clearance. Focus was directed towards synthesis of oximes, oxime ethers, and hydrazones as potential prodrugs to prevent immediate hydration and reach the central nervous system.  The synthesis of various oximes, oxime ethers, and hydrazones has been shown to give cimpounds toxic to Anopheles gambiae within 3- to 4-fold of the toxicity of propoxur. However, thus far we have not been able to link the toxicity of these compounds to a cholinergic mechanism.  Pre-incubation studies suggest that significant hydrolysis of these compounds to TFKs does not occur or 22 h at pH 7.7 or 5.5.   Future work will be directed towards TFKs that have better pharmacokinetic properties.  Work will also be directed at synthesis of oxime and hydrazone TFK isosteres to determine the mechanism of action of these compounds. / Master of Science

The nitrate radical (NO3·) was first identified as early as the 1881, but its role in atmospheric oxidation has only been identified within recent decades. Due to its high one-electron reduction potential and its reactivity toward a diverse set of substrates, it dominates nighttime atmospheric oxidation and has since been the subject of much work. Despite this, studies on NO3· hydrogen atom transfer reactions have been somewhat neglected in favor of its more reactive oxidative pathways. The first section of the dissertation will highlight the role of substrate structure, solvent effects, and the presence of a polar transition state on NO3· hydrogen abstractions from alcohols, alkanes, and ethers. In this work the acquisition of absolute rate constants from previously unexamined substrates was analyzed alongside a curated list of common organic pollutants degraded through hydrogen atom abstraction. It was found that NO3· reacts with low selectivity through an early polarized transition state with a modest degree of charge transfer. Compared to the gas-phase, condensed-phase reactions experience rate enhancement—consistent with Kirkwood theory—as a result of the polarized transition state. These insights are then applied to abstractions by NO3· from carboxylic acids in the next section. It was found that the rate constants for abstraction of α-carbons were diminished through induction by the adjacent carbonyl compared to the activation seen for the aforementioned substrates. The deactivation of abstraction by the carbonyl was found to be dramatically reduced as the substrate's alkyl chain was lengthened and/or branched. This apparent change in mechanism coincides with hydrogen abstraction of the alkyl chain for sufficiently large carboxylic acids and rules out the possibility of concerted bond breaking elsewhere in the molecule. Finally, the dissertation will cover some additional projects related to the overall nature of the work including examination of the kinetics of radical clock systems when complexed with metal ions and the examination of a highly oxidative biosourced monomer. / Doctor of Philosophy / The nitrate radical (NO3·) was first identified as early as the 1881, but its role in the breakdown of atmospheric pollutants has only been identified within recent decades. Operating primarily at night, NO3· serves as a major atmospheric oxidant—it breaks down pollutants by reactions that involve the removal of electrons from those substrates. This chemistry is particularly important in understanding the consequences of an increasingly industrialized world and the subsequent short-term health and environmental implications. Geographically, these reactions will occur in large concentrations near locations that contribute greatly to atmospheric pollution, such as above coal-powered plants, heavily industrialized areas, above the canopy of large forests, and immediately behind the engines of airplanes as they move through the sky. The proximity of these locations to large population centers has caused the pollutants to greatly impact human health. These contaminants have been linked to several of the leading global causes of death, such as ischemic heart disease, stroke, and respiratory illnesses. The first section of the dissertation will focus on the role of pollutant structure, the medium in which the reaction occurs, and the development of a charged complex when NO3· reacts with alcohols, alkanes, and ethers. These substrates are often found as the result of incomplete combustion when burning fuel or as products of even more sustainable biodiesels. In this work the exact rate constants were found for substrates that were previously unexamined and compared with similar known reaction rates. It was found that NO3· has a low preference for what it reacts with and passes through a modestly charged complex early in the reaction. Compared to gaseous reactions, reactions in a liquid environment proceed faster due to the formation of a charged complex. This was then applied to reactions with carboxylic acids in the next section. Carboxylic acids are often found in large concentrations above the canopy of large forests resulting from the oxidation of isoprenes that are naturally released from broad-leaf trees. It was found that these reactions were slower than reactions with alkanes as the development of the charged complex was inhibited due to the presence of an adjacent dipole. When the carboxylic acid was longer and/or more branched, the formation of the charged complex was no longer inhibited as the reaction site moved further from the dipole. A change in reaction pathway was observed when the acids were sufficiently large. This ruled out the possibility of the reaction occurring simultaneously with a fracturing and rearrangement elsewhere. Finally, the dissertation will cover some additional projects that share some overlap with the work already described including the study of the rates of radical clock systems in the presence of metal ions and the study of naturally sourced monomers that are prone to losing electrons.

The timescale of chemical reactions in solid-state systems greatly exceeds what may be modeled by direct integration of Newton's equation of motion. This limitation spawned the development of many different methods such as (adaptive) kinetic Monte Carlo (A)KMC, (harmonic) transition state theory (H)TST, parallel replica dynamics (PRD), hyperdynamics (HD), and temperature accelerated dynamics. The focus of this thesis was to (1) implement many of these methods in a single open-source software package (2) develop standard benchmarks to compare their accuracy and computational cost and (3) develop new long timescale methods. The lack of a open-source package that implements long timescale methods makes it difficult to directly evaluate the quality of different approaches. It also impedes the development of new techniques. Due to these concerns we developed Eon, a program that implements several long timescale methods including PRD, HD, and AKMC as well as global optimization algorithms basin hopping, and minima hopping. Standard benchmarks to evaluate the performance of local geometry optimization; global optimization; and single-ended and double-ended saddle point searches were created. Using Eon and several other well known programs, the accuracy and performance of different algorithms was compared. Important to this work is a website where anyone may download the code to repeat any of the numerical experiments. A new method for long timescale simulations is also introduced: molecular dynamics saddle search adaptive kinetic Monte Carlo (AKMC-MDSS). AKMC-MDSS improves upon AKMC by using short high-temperature MD trajectories to locate the important low-temperature reaction mechanisms of interest. Most importantly, the use of MD enables the development of a proper stopping criterion for the AKMC simulation that ensures that the relevant reaction mechanisms at the low temperature have been found. Important to the simulation of any material is knowledge of the experimental structure. Extended x-ray absorption fine structure (EXAFS) is a technique often used to determine local atomic structure. We propose a technique to quantitatively measure the accuracy of the commonly used fitting models. This technique reveals that the fitting models interpreted nanoparticles as being significantly more ordered and of much shorter bond length than they really are. / text

In the technologically driven information age, the consumption of power is as vital to daily life as food and shelter. The generation of that power comes from a variety of sources of which coal is the predominant provider of electrical energy. Coal combustion is a well-known technology and the United States possesses the most abundant coal deposits on Earth, however, the drawback accompanying this process is the significant emissions which are released during combustion. Over the years, much effort has gone into reducing the emissions of majority constituent elements CO2, CO, NOx, SOx, etc. but it is only in the last decade or so that much attention has been given to the trace metals present within coal. Most of the work into examining these trace metals has been upon Hg and how it speciates within the flue gas in order to determine the most effective means of removal. In this study, the trace metals arsenic (As) and selenium (Se) will be investigated in a similar manner to evaluate the speciation of these elements. While previous experimental work has been performed in this area, it has been limited to thermodynamic studies which determine the speciation after equilibrium has been reached, this ignores the fact the residence times within the flue are often only several minutes during which time rapid quenching is taking place. This study takes a different approach by examining the speciation using computational chemistry which affords the advantage of being able to perform a kinetic study which is more useful in creating a flue gas model. Using ab initio the properties of various As and Se species can be evaluated compared to existing experimental data for validation. After which, a number of reactions may be selected and the structure of the transition state for each identified. Once the properties of the transition structure are known, the appropriate kinetic model, be it Transition State Theory, RRKM Theory, etc. can be applied and the rate constant determined. It is by the determination of these rate constants that the kinetic model of the flue gas can be improved and a more accurate depiction of the speciation of these race metals created.