Alumni Dissertations and Theses

 
 

Alumni Dissertations and Theses

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  • RADICAL FORMATION AND FUNCTIONS IN MYCOBACTERIUM TUBERCULOSIS CATALASE-PEROXIDASE (KatG) AND IN CRYPTOCOCCUS NEOFORMANS MELANIN

    Author:
    Abdelahad Khajo
    Year of Dissertation:
    2012
    Program:
    Chemistry
    Advisor:
    Richard Magliozzo
    Abstract:

    The dual-function heme enzyme catalase-peroxidase, KatG, is found in many microorganisms but in the human pathogen M. tuberculosis it is the only catalase (2H2O2 → O2 + 2H2O) and plays an important role in protecting this organism against oxidative stress. The structural and functional origins of its high catalase activity are therefore of special interest. KatG also exhibits a broad-spectrum peroxidase activity (ROOH + 2AH → 2ROH + 2A·) and in M. tuberculosis it is responsible for peroxidative activation of the pro-drug isoniazid used to treat tuberculosis infection. Previous studies have shown that the catalase activity of KatG depends upon a unique adduct structure consisting of three conserved amino acids (Met255, Tyr229 and Trp107, M-Y-W) on the distal side of the heme pocket and that this adduct enables formation of a unique radical intermediate required for efficient turnover of H2O2. Here, EPR measurements along with site-directed mutagenesis and isotope labeling experiments allowed assignment of the `narrow doublet' radical signal found during catalase turnover to Tyr229 of the M-Y-W adduct, explained the operation of this radical in KatG using optical and stopped-flow spectrometry, and elucidated a function for this unique cofactor and a role for neighboring residues in the enzyme's catalase activity. Based on these findings, a new radical-dependent catalase reaction mechanism in KatG could be proposed. Previous studies also showed formation of other amino acid-based radicals in KatG upon reaction with alkyl peroxides. Gaining insights into the poorly-understood peroxidase reaction mechanism in KatG through identifying the location and the possible function of the `wide doublet' radical signal was among the goals of this thesis. Mutagenesis and rapid freeze-quench EPR experiments revealed the involvement of several residues in an electron transfer pathway including Tyr229, Tyr597, Tyr678, Trp90, Trp135, and Trp149. Other important aspects regarding the peroxidase function of KatG such as the electronic structure of its initial hypervalent heme species and the role of proximal Trp321 were studied. While details of the peroxidase reaction in KatG are far from being resolved, a mechanism was presented based on the available data. The second part of this work was devoted to studying another biologically-relevant free radical, which occurs in eumelanin from the fungus C. neoformans. With the application of EPR spectroscopy, UV-Vis spectrometry, chemical analyses, and other physico-chemical techniques to investigate how high-dose gamma irradiation under aqueous aerobic conditions interacts with the semiquinone radical and affects the physical and chemical properties of natural and synthetic melanins, the studies aimed to gain insights into a radioprotective role of melanin in fungal cells. Findings here revealed a shielding function for melanin in live fungal cells from high-dose ionizing radiation by attenuating the destructive effects of radiolysis-induced hydroxyl radicals and serving as a sacrificial barrier in the cell wall. Chemical analysis of irradiated samples showed that hydroxyl radical attack on susceptible sites in melanin subunits lead to C-C bond cleavage and the release of low molecular weight aldehydes.

  • BIMOLECULAR ENCOUNTERS: ACID-BASE AND/OR ELECTRON TRANSFER IN RUTHENIUM (II) COMPLEXES. EXPERIMENT AND THEORY.

    Author:
    Marta Kowalczyk
    Year of Dissertation:
    2013
    Program:
    Chemistry
    Advisor:
    Harry Gafney
    Abstract:

    This work correlates experimental and theoretical studies of ruthenium diimines, [Ru(bpy)2L]2+ where L = bpy, or dpp. Experimental research concentrates on excited state acid-base and electron transfer properties. Although the driving force for reduction of Fe(III) and Ag(I) are essentially equivalent, data presented here show that the bimolecular encounter between bis(2,2'-bipyridine)(2,3-bis(2-pyridyl)pyrazine)ruthenium(II), [Ru(bpy)2(dpp)]2+ in its dpp localized MLCT state, leads to coordination of Ag+, but electron transfer to Fe3+. Several redox reactions were performed between [Ru(bpy)2dpp]2+ and a strong oxidizing agents to analyze the spectroscopic signature of the oxidized form of the complex, [Ru(bpy)2dpp]3+ . The experiments confirm electron transfer between excited state of bis(2,2'-bipyridine(2,3-bis(2pyridyl)pyrazine)ruthenium(II), [Ru(bpy)2dpp]2+ and iron(III) in aqueous and buffered solutions. Electron transfer has been confirmed by trapping Fe(II), one of the products of excited state electron transfer. The efficiency of excited electron transfer is influenced by solvent cage, where redox pair products form by competition between back reaction and diffusion process. Two molecules of ruthenium complexes, [Ru(bpy)3]n+ and [Ru(bpy)2dpp]n+ , where n = 2+, 3+, have been investigated theoretically with a particular attention to their changes in electron density. Structural optimizations and energy calculations were performed by the Density Functional Theory (DFT) method in the Gaussian09 package including the effective core potentials (Los - Alamos ECP) for the ruthenium(II) ion. Electronic absorption spectra have been calculated and compared with experimental data. Computation of spectroscopic properties and electrochemical energies agree with experimental findings and provide a rationale for the spectroscopic properties of the oxidized form of [Ru(bpy)2dpp]3+. The possibility of proton-coupled electron transfer (PCET) reaction was investigated by examining the energetics and pKa's of all possible forms of [Ru(bpy)2dpp]2+, which suggested that the quenching by iron(III) is an electron transfer process. Theoretical electron transfer rates between iron(III) and excited state of [Ru(bpy)2dpp]2+ calculated with Marcus cross-relation are in good agreement with experimental findings.

  • SYNTHESIS AND CHARACTERIZATION OF CARBON NANOFILMS FOR CHEMICAL SENSING

    Author:
    Vivek Kumar
    Year of Dissertation:
    2012
    Program:
    Chemistry
    Advisor:
    Alexander Zaitsev
    Abstract:

    Carbon nanofilms obtained by high temperature graphitization of diamond surface in inert atmospheres or vacuum are modified by treatment in plasma of different precursor gases. At temperatures above 1000 oC, a stable conductive film of thickness between 10 - 100 nm and specific resistivity 10-3-10-4 ohm.m, depending upon the heating conditions and the growth atmosphere, is formed on diamond surface. A gray, thin film of high surface resistivity is obtained in high vacuum, while at low vacuum (below 10-4 mbar), a thick black film of low surface resistivity forms. It is observed that the exposure to plasma reduces the surface conductance of carbon nanofilms as result of a partial removal of carbon and the plasma-stimulated amorphization. The rate of the reduction of conductance and hence the etching ability of plasma depends on the type of precursor gas. Hydrogen reveals the strongest etching ability, followed by oxygen and argon, whereas SF6 is ineffective. The carbon nanofilms show significant sensitivity of their electrical conductance to temperature and exposure to the vapors of common organic compounds. The oxygen plasma treated films exhibit selective response to acetone and water vapors. The fast response and recovery of the conductance are the features of the carbon nanofilms. The plasma-treated carbon nanofilm on graphitized diamond surface is discussed as a promising sensing material for development of all-carbon chemical sensors, which may be suitable for biological and medical applications. An alternative approach of fabrication of temperature and chemical sensitive carbon nanofilms on insulating substrates is proposed. The films are obtained by direct deposition of sputtered carbon on highly polished quartz substrates followed by subsequent annealing at temperatures above 400 oC. It is observed that the as-deposited films are essentially amorphous, while the heating induces irreversible structural ordering and gradual conversion of amorphous carbon in disordered graphite. This evolution is confirmed by Raman spectroscopy and electrical measurements. The carbon nanofilms grown on diamond and deposited on quartz both show similar exponential dependence of their conductance on temperature, which is essentially different from the usual behavior of the thermally activated conduction and the conduction due to variable range hopping of charge carriers. The observed exponential dependence of conductance is explained by a model based on the thermally vibrating energy barriers. The as-grown nanofilms on diamond surface show a negative response (decrease in conductance) to the vapors of acetone, toluene and hexane, and a positive response (increase in conductance) to the water vapor. Sensitivity (relative change in conductance) to toluene is greater than to water, acetone, and hexane, in that order. Plasma exposure alters the sensitivity to positive for all the organic vapors. Overall, an increase in sensitivity is observed with the plasma exposure time. For acetone and water, an increased exponential dependence on vapor concentration is also observed. The exposure to oxygen plasma renders the carbon films on diamond selectively sensitive to acetone and water vapors. The hydrogen plasma exposure makes the films selectively sensitive to toluene vapor. It is found that the carbon nanofilms on quartz have p-type conductivity, as indicated by the opposite response to NO2 and NH3 analytes. NO2, a known electron acceptor, increases the conductance. NH3, a known electron donor, decreases the conductance. The phenomenological description of the chemical sensitivity of the carbon nanofilms σ = β/τ is proposed as a function of two main parameters: the time constant τ and the maximum relative change in conductance β. τ and β are described as the parameters related to the surface and bulk material properties of the films, respectively.

  • Strategies Towards Modular Syntheses of Fluorocarbons

    Author:
    Rakesh Kumar
    Year of Dissertation:
    2012
    Program:
    Chemistry
    Advisor:
    Rakesh Kumar
    Abstract:

    Synthesis of TMS-protected fluoropropargyl benzothiazolyl sulfone, a bifunctional fluoro-Julia-Kocienski reagent, was achieved by metalation-electrophilic fluorination of benzothiazolyl propargyl sulfone. Reactions of the fluoro-Julia-Kocienski reagent with carbonyl compounds produced fluoroenynes in high yields and with high E-selectivity. Olefinations proceeded with LHMDS, or the milder base, DBU. A one-pot deprotection-copper-catalyzed azide alkyne cycloaddition (CuAAC) to the alkyne moiety in the Julia-Kocienski reagent provided a new class of triazole-derived fluorinated and unfluorinated Julia-Kocienski reagents. Competitive experiments showed higher reactivity of the fluorinated reagent in the CuAAC than the protio analog. Computational analysis of electron densities in fluoro and protio propargyl sulfones showed that fluorine lowers electron density at the terminal alkynyl carbon, which can contribute to its higher reactivity. These triazole-derived "second generation" Julia-Kocienski reagents reacted smoothly with aldehydes and ketones, and olefinations could be tuned towards E- or Z-stereoselectivity by change in reaction conditions. The CuAAC-Julia olefination sequence was also used for the synthesis of triazole-derived analogs of biologically relevant combretastatin A-4. Biological testing of these compounds against HeLa cancer cell lines showed that two analogs displayed modest activity. Putative fjord region dihydrodiol and diol epoxide metabolites of 5-fluorobenzo[c]phenanthrene (5-FBcPh), arising by the oxidation of the angular ring remote to the fluorinated ring, were synthesized. A key step in the chemistry was fluoro-Julia-Kocienski olefination, followed by photocyclization. Deoxyadenosine adducts of the (+)-series 2 diol epoxide of 5-FBcPh were synthesized. Trans opening of this epoxide by azide, followed by reduction, furnished (+)-aminotriol. SNAr reaction of the aminotriol with silyl protected 6-fluoro-9-(2'-deoxy-β-D-ribofuranosyl)purine gave two diastereomeric deoxyadenosine adducts. Unequivocal stereochemical assignment to the individual adduct diastereomers came via conversion of the racemic trans dihydrodiol to the bis(-)-menthoxy acetate ester, and chromatographic separation of the diastereomers. Resolved enantiomers were converted to bis-p-(N,N)-dimethylaminobenzoates of a trans tetrahydrodiol. Absolute configuration was determined by CD spectroscopy. R,R-dihydrodiol enantiomer was converted to the R,S,S,R diol epoxide of 5-FBcPh, which was used for the synthesis of a single diastereomer of the 2'-deoxyadenosine adduct, whose CD spectrum and optical rotation were recorded. This allowed configurational assignment to the adenosine adducts, obtained from the racemic diol epoxides.

  • Synthesis of Analogs of Sphingophospholipids, Glycolipids, and Plasmalogen

    Author:
    Ravi Lankalapalli
    Year of Dissertation:
    2009
    Program:
    Chemistry
    Advisor:
    Robert Bittman
    Abstract:

    This dissertation presents the asymmetric syntheses of naturally occurring sphingoid bases and novel sphingolipid analogs, including (a) an unnatural sphingomyelin with a Δ4-cis double bond in the long-chain base (cis-SM), (b) L-threo-β-glucosyl and galactosyl-ceramides, (c) a photoactivatable analog of β-galactosyl sphingosine (psychosine), and (d) caged sphingosine 1-phosphate and ceramide 1-phosphate analogs. Also included in this dissertation is a novel synthesis of an unnatural analog of the glycerophospholipid plasmalogen with a trans-O-vinyl ether linkage at the sn-1 position of the glycerol backbone.

  • SYNTHESIS AND STRUCTURAL MODIFICATION OF THE MDMA ANTAGONIST NANTENINE: A NATURALLY OCCURRING APORPHINE ALKALOID

    Author:
    Onica Le Gendre
    Year of Dissertation:
    2010
    Program:
    Chemistry
    Advisor:
    Wayne Harding
    Abstract:

    MDMA ("Ecstasy") is a synthetic phenethylamine stimulant which is known to affect the re-uptake of serotonin, dopamine and nor-epinephrine in the brain. Adverse effects of "Ecstasy" in humans include development of hyperthermia, hallucinations, organ failure and in extreme cases, death. There is evidence that the behavioral and physiological effects of MDMA are mediated by α1-adrenergic and 5-HT2A receptors. Nantenine is a naturally occurring aporphine alkaloid which has been shown to block and reverse behavioral and physiological effects of MDMA in mice via antagonism of the aforementioned receptors. However, the relative role of these receptors in mediating the MDMA antagonizing effects of nantenine in vivo is unknown. The goal of this project is to explore different methods of synthesizing nantenine and nantenine derivatives. These compounds were subjected to in vitro testing for antagonistic activity at α1-adrenergic and 5-HT2A receptors and their ability to block/reverse MDMA-induced effects in mice through in vivo drug discrimination assays. This work provided insight into the relative role of these receptors in mediating the antagonistic activity of nantenine. Moreover, the results may provide the foundation for the design and development of potent and selective MDMA antagonists in the future. Here we present our synthetic studies as well as the results of biological evaluations of nantenine and analogues.

  • PHARMACOLOGICAL STUDIES OF OCTANAL RECOGNITION BY MAMMALIAN ODORANT RECEPTORS

    Author:
    Yadi Li
    Year of Dissertation:
    2013
    Program:
    Chemistry
    Advisor:
    Kevin Ryan
    Abstract:

    The sense of smell is initiated when small molecule odorants bind and activate specific subsets of olfactory receptors (ORs) expressed by olfactory sensory neurons (OSNs). The mammalian nose is able to distinguish thousands of chemical structures through these receptors. The molecular recognition strategies that ORs use to bind and distinguish among different odorants are poorly understood. Here we use drug design techniques to probe olfactory molecular recognition using a model odorant-receptor pair in vivo. Specifically, octanal analogs were designed and synthesized to probe the steric and electronic requirements of octanal recognition by the aldehyde-specific rat OR-I7 in OSNs. The testing of conformationally restricted octanal analogs (Chapter 1) indicates that OR-I7 distinguishes among aliphatic aldehydes according to their length and carbon chain conformation. A gauche conformation between C4 and C5 is proposed to be necessary to fill a small hydrophobic binding pocket 7 to 8 Å from the receptor-bound aldehyde with octanal's hydrophobic tail. In addition, small cycloalkyl groups at the tail enhance activation potency, leading to one analog that is more potent than octanal. Several pairs of stereoisomers and analogs derived from cyclohexylacetaldehyde (Chapter 2) were also designed and synthesized to study the active conformation of octanal at C4-C5 and C6-C7 positions. To study the possibility that OR-I7 and other aldehyde-specific olfactory receptors recognize, not the aldehyde, but its gem-diol hydrate, difluorooctanal, dimethyloctanal, octanol and octanal and were screened against more than 1,000 OSNs in vivo. 87 octanal-responsive OSNs were found. The response profiles of these cells support the hypothesis that for some ORs aldehydes function, by analogy to prodrugs, as pro-odorants, or "prodorants." The volatility of the aldehyde allows delivery to nasal mucous, where the aldehyde form is in equilibrium with the gem-diol, a form that has much richer H-bonding capacity. Through these studies, some molecular recognition details of octanal by ORs were obtained, which could contribute to the understanding of general odorant-receptor recognition strategy.

  • Synthetic Studies of Bioactive Nature Products: Azaspiracid-1 and Angelmicins

    Author:
    Jialiang Li
    Year of Dissertation:
    2013
    Program:
    Chemistry
    Advisor:
    David Mootoo
    Abstract:

    The trioxadispiroketal residue in the marine biotoxin azaspiracid-1, which exists in a configuration capable of a double anomeric effect, is believed to be the thermodynamically most stable bis-acetal diastereomer. In order to get insight into how structural factors affect this equilibrium, a simplified ABC trioxadispiroketal analog of azaspiracid-1 was synthesized and subjected to equilbration and computational studies. These results suggest that while a double anomeric effect may play a major role in the stability of the trioxadispiroketal configuration in the more complex natural product, the substituion pattern of the C ring is also a contributing factor. Angelmicins B (hibarimicin B) was first isolated from Microbispora rosea by Uehara and co-workers in 1993. It is a potent v-Src protein tyrosine kinase (PTK) inhibitor (IC50=23 μM), and also shows growth inhibiting and differentiation inducing activity on human myeloid leukemia (HL-60) cell lines (IC50=57 nM). The work reported in this part of the thesis presents a divergent synthetic strategy for the angelimicin family of anthraquinoid natural products, involving conversion of a central highly oxygenated decalin intermediate to the AB and A'B' subunits. The strategy centers on an intramolecular Diels-Alder (IMDA) reaction on a triene to provide the complex highly oxygenated decalin, which is elaborated to tricyclic AB and bicyclic A'B' subunits. The differentiating tact in the two syntheses is control of the Suárez radical fragmentation of lactol precursors by modulation of the structural rigidity of the substrate. A more flexible lactol gave the AB tricyclic framework, whereas a more rigid substrate led to the A'B' bicyclic precursor, presumably through divergent pathways from the radical produced in the initial fragmentation step. Given the symmetry with respect to the biaryl bond of HMP-Y1 (hibarimicin-mutant product Y1, a biosynthesis precursor of angelmicin B), a bi-directional synthesis is attempted. The bi-directional donors for Hauser annulation and Michael-Dieckmann condensation were synthesized from known 2,3,5-trimethoxytoluene in 27% and 42% yield, respectively. When they are applied to the annulation with cyclohex-2-enone, the Hauser annulation was unsuccessful while Michael-Dieckmann condensation gave 40% yield. However, the subsequent required aromatization of Michael-Dieckmann condensation product was unsuccessful.

  • Atomic and Molecular Low-n Rydberg States in Supercritical Fluids

    Author:
    Luxi Li
    Year of Dissertation:
    2009
    Program:
    Chemistry
    Advisor:
    Cherice Evans
    Abstract:

    The structure of low-n Rydberg states doped into supercritical fluids represents an important probe to investigate solvation effects, especially near the solvent (or perturber) critical point. We have investigated the solvation of excited atomic and molecular dopants in various perturbing fluids (both atomic and molecular). This systematic study was performed from low perturber number densities to the density of the triple point liquid, at both non-critical temperatures and on an isotherm near the critical isotherm. Dopant low-n Rydberg states were investigated using vacuum ultraviolet photoabsorption spectroscopy. The absorption spectra of these states were then simulated using a semi-classical statistical line shape function. With accurate line shape simulations, the perturber induced energy shift of the primary transition was obtained using a standard moment analysis. The moment analysis indicated that the dopant low-n Rydberg state energy blue shifts as a function of perturber number density without a significant temperature effect (except near the critical isotherm). A significant critical point effect was observed in all dopant/pertuber systems investigated here. This critical point effect is caused by a large increase in the dopant/perturber radial distribution function near the critical temperature of the perturber. Since the first perturber solvent shell shields the cationic core, the binding energy of the optical electron decreases. This acts to increase the dopant low-n Rydberg state excitation energy. However, the overall blue shift and critical point effect varies from atomic to molecular perturber systems due to the structure of the perturber. These differences are also discussed in more detail in this work.

  • A theoretical study of ionic liquids using analytical theory and molecular dynamics simulation

    Author:
    Hualin Li
    Year of Dissertation:
    2011
    Program:
    Chemistry
    Advisor:
    Mark Kobrak
    Abstract:

    In a series of theoretical analyses and simulation studies, we explore the relationship between ionic structure and liquid dynamics in room-temperature ionic liquids (ILs). As a framework, we define an ionic property we call the Charge Lever Moment (CLM) based on earlier theoretical work. We use the CLM to investigate ionic liquid dynamics and demonstrate a correlation between the CLM and IL viscosity. We extend this analysis using molecular dynamics simulation of a series of model molten salts with different intra-ionic charge distributions. These simulation clarify the relationship between charge distribution and ionic motion. We extend the analysis further with a thorough study using an instantaneous normal mode analysis in the model ILs, with the goal of investigating the roles of translational and rotational motion in IL dynamics.