Physical Sciences and Mathematics Commons^™

Novel Near-Infrared Cyanine Dyes For Fluorescence Imaging In Biological Systems, Nilmi T. Fernando

Chemistry Dissertations

Heptamethine cyanine dyes are attractive compounds for imaging purposes in biomedical applications because of their chemical and photophysical properties exhibited in the near-infrared region. A series of meso amino-substituted heptamethine cyanine dyes with indolenine, benz[e]indolenine and benz[c,d]indolenine heterocyclic moieties were synthesized and their spectral properties including fluorescence quntum yield were investigated in ethanol and ethanol/water mixture. Upon substitution with amines, the absorption maxima of the dyes shifted to the lower wavelength region (~600 nm), showed larger Stokes shifts and stronger fluorescence which can be attributed to an excited state intramolecular charge transfer (ICT). High quantum yields …

Nucleic Acid Substrates: Investigation Of Structural And Dynamic Features That Influence Enzyme Activity, Christopher N. Johnson

Chemistry Dissertations

The previous view of DNA as a linear sequence of bases is evolving to consider structure, topology and dynamics. Sequences surrounding damage lesions have been shown to effect enzyme recognition and processing. Here we present an in depth investigation of subtle structural and dynamical features imparted to nucleic acid duplexes by a designed modification or damage lesions. Highly restrained solution structures were generated and validated utilizing a range of NMR techniques. This allowed for the characterization of multiple features of the nucleic acid duplex; such as base pairing, backbone torsion angles, deoxyribose sugar pucker, and intra and inter nucleotide proton …

Development Of Boronic Acid Flurescent Reporters, Boronic Acid-Modified Thymidine Triphosphates For Sensor Design And Antagonists Of Bacterial Quorum Sensing In Vibrio Harveyi, Yunfeng Cheng

Chemistry Dissertations

Carbohydrates are known to play important roles in a large number of physiological and pathological processes. Conceivably, “binders” of carbohydrates of biological importance could be used as diagnostic and therapeutic agents. Currently, lectins are the major available tools in research for carbohydrate recognition. However, the available lectins often have cross-reactivity issues, along with the high costs and stability issues. Therefore, there is a critical need to develop alternatives (lectin mimics). In this regard, there have been very active efforts in developing different “binders”, such as small molecule lectinmimics and aptamers. Among all the small molecule lectinbmimics developments, boronic acid stands …

I. Synthesis Of Anthraquinone Derivatives For Electron Transfer Studies In Dna. Ii. Characterization Of The Interaction Between Heme And Proteins., Yu Cao

Chemistry Dissertations

Anthraquinone (AQ) derivatives with relatively high reduction potentials have been synthesized to afford good candidates for electron transfer studies in DNA. Electron withdrawing groups on the anthraquinone ring gave derivatives with less negative reduction potentials. The anthraquinone imide (AQI) derivatives had reduction potentials less negative than AQ derivatives. The AQI ring system was subject to base-induced hydrolysis.

Water-soluble sulfonated tetraarylporphyrins have been studied in a wide variety of contexts. Herein, we report the first synthesis of a pentasulfonated porphyrin bearing an internal cyclic sulfone ring. Treatment of 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (TPPS4) with fuming H₂SO₄ gave a structure consistent with …

Tuning Calcium Bindging Affinities With Related Biological Functions Of Calmodulin And Designing Protein Based Contrast Agent, Jie Jiang

Chemistry Dissertations

Calmodulin (CaM) is a ubiquitous intracellular protein that regulates biological activities of numerous enzymes and ion channels. Upon responding Ca²⁺ concentration change, Ca²⁺- dependent CaM activates the hydrolyzation of cGMP by PDE and Ca²⁺ releasing channel activity of ryanodine receptor. In this dissertation, a series of CaM variants were engineered to enhance Ca²⁺ binding affinities by increasing the number of negative charged residues in individual EF-hand. The capability of shifting the biphasic Ca²⁺-activation profile of RyR1 is significantly altered by changing Ca²⁺ binding affinity of CaM at the C-terminal. This indicates that …

Mechanistic Studies Of Two Selected Flavin-Dependent Enzymes: Choline Oxidase And D-Arginine Dehydrogenase, Hongling Yuan

Chemistry Dissertations

Choline oxidase catalyzes the flavin-dependent, two-step oxidation of choline to glycine betaine via the formation of an aldehyde intermediate. The oxidation of choline includes two reductive half-reactions followed by oxidative half-reactions. In the first oxidation reaction, the alcohol substrate is activated to its alkoxide via proton abstraction and oxidized via transfer of a hydride from the alkoxide α-carbon to the N(5) atom of the enzyme-bound flavin. In the wild-type enzyme, proton and hydride transfers are mechanistically and kinetically uncoupled.

The role of Ser101 was investigated in this dissertation. Replacement of Ser101 with threonine, alanine, cysteine, or valine demonstrated the importance …

Development Of Inhibitors And Assay Methods For Histone Acetyltransferases, Jiang Wu

Chemistry Dissertations

Histone acetyltransferases (HATs) are important enzymes in transcriptional control and potential targets for chemotherapeutic intervention in malignant diseases. Among different HAT members, the yeast Esa1 and human Tip60 (the HIV-1 Tat interactive protein, 60KDa) play multiple roles in normal cellular processes including transcription, cell cycle and checkpoint machinery, double strand DNA break repair, apoptosis, and cell cycle progression. Tip60 is also implicated in several human diseases such as prostate cancer, and gastric cancer. These studies suggest that Tip60 is a potential therapeutic target for new cancer treatment. So, we designed experimental work to synthesize and investigate organic inhibitors of Tip60 …

Defining A Molecular Mechanism For Lead Toxicity Via Calcium-Binding Proteins, Michael Kirberger

Chemistry Dissertations

Essential metals like Ca²⁺ and Zn²⁺ play critical roles in biological processes through protein interactions. Conversely, non-essential metals (e.g., Gd³⁺ and Pb²⁺) also interact with proteins, often with toxic effects. Molecular metal toxicity is assumed to be due to ionic displacement, and studies have demonstrated that Pb²⁺ replaces Zn²⁺, Ca²⁺ and other essential metals in proteins. The focus of this work was to compare protein Ca²⁺ and Pb²⁺ -binding sites and to investigate a mechanism of Pb²⁺ toxicity in Ca²⁺-binding proteins, particularly the intracellular trigger protein …

Protein-Nucleic Acid Interactions In Nuclease And Polymerases, Abdur Rob

Chemistry Dissertations

DNA polymerase binds to the double stranded DNA and extends the primer strand by adding deoxyribonucletide to the 3’-end. Several reactions in the polymerase active site have been reported by Kornberg in addition to the polymerization. We observed DNA polymerase I can act as a pyrophosphatase and hydrolyze deoxyribonucletide. In performing the pyrophosphatase activity, DNA polymerase I requires to interact with RNA. RNA in general, was found to activate the DNA polymerase I as pyrophosphatase. This hydrolysis causes depletion of dNTP and inhibits DNA polymeration synthesis in vitro. In this RNA-dependent catalysis, DNA polymerase I catalyzes only dNTP but …

On The Biochemistry, Mechanism And Physiological Role Of Fungal Nitronate Monooxygenase, Kevin Francis

Chemistry Dissertations

Nitronate monooxygenase (E.C. 1.13.11.16), formerly known as 2-nitropropane dioxygenase (EC 1.13.11.32), is a flavin dependent enzyme that catalyzes the oxidation of nitronates to their corresponding carbonyl compounds and nitrite. Despite the fact that the enzyme was first isolated from Neurospora crassa 60 years ago, the biochemical and physiological properties of nitronate monooxygenase have remained largely elusive. This dissertation will present the work that established both the catalytic mechanism and physiological role of the fungal enzyme.

The biological and biochemical properties of propionate-3-nitronate, the recently discovered physiological substrate for nitronate monooxygenase, will be extensively reviewed. The nitronate is produced by a …

Sequence-Specific And Conformation-Specific Targeting Of Duplex And Quadruplex Dna Grooves With Small Molecules, Rupesh K. Nanjunda

Chemistry Dissertations

Small molecule mediated chemical intervention of biological processes using nucleic acid targets has proven extremely successful and is continually providing exciting new avenues for the development of anti-cancer agents and molecular probes. Among the alternative DNA confrormations, G-quadruplexes has certainly garnered much recognition due to increase in evidences supporting their involvement in diverse biological process. The grooves of the quadruplexes offer an alternate recognition site for ligand interactions with potentially higher selectivity than the traditional terminal stacking sites. DB832, a bifuryl-phenyl diamidine, was recently reported to selectively recognize human telomeric G-quadruplex, as a stacked species, with significant selectivity over duplex …

Rational Drug Design For Neglected Diseases: Implementation Of Computational Methods To Construct Predictive Devices And Examine Mechanisms, Catharine Jane Collar

Chemistry Dissertations

Over a billion individuals worldwide suffer from neglected diseases. This equates to approximately one-sixth of the human population. These infections are often endemic in remote tropical regions of impoverished populations where vectors can flourish and infected individuals cannot be effectively treated due to a lack of hospitals, medical equipment, drugs, and trained personnel. The few drugs that have been approved for the treatments of such illnesses are not widely used because they are riddled with inadequate implications of cost, safety, drug availability, administration, and resistance. Hence, there exists an eminent need for the design and development of improved new therapeutics. …

Design And Synthesis Of Novel Serotonin Receptor Ligands, Jeffrey D. Klenc

Chemistry Dissertations

Novel and potent ligands to the serotonin7 (5-HT7) receptor have been synthesized. The synthesized compounds include a set of substituted pyrimidines which show high affinity to the 5-HT7 receptor, synthesized by previously described methods [1,2] in high yield. Comparing the affinities of substituted pyrimidines to previously calculated models [3,4] yielded new hypotheses about the nature of interaction between the pyrimidine ligands and the 5-HT7 binding site. Several new series of compounds were synthesized by various methods to validate these hypotheses, including a conjugate addition to vinylpyrimidines [5]. These compounds include benzofurans, oximes, hydrazones, as well as a group of substituted …

Bead Modeling Of Transport Properties Of Macromolecules In Free Solution And In A Gel, Hongxia Pei

Chemistry Dissertations

On the bead modeling methodology, or BMM, a macromolecule is modeled as a rigid, non-overlapping bead array with arbitrary radii. The BMM approach was pioneered by Kirkwood and coworkers (Kirkwood, J.G., Macromolecules, E.P. Auer (Ed.), Gordon and Breach, New York, 1967; Kirkwood, J.G., Riseman, J., J. Chem. Phys., 1948, 16, 565) and applied to such transport properties as diffusion, sedimentation, and viscosity. With the availability of computers, a number of investigators extended the work to account for the detailed shape of biomolecules in the 1970s. A principle objective of my research has been to apply the BMM approach to more …

Design, Synthesis And Mechanistic Studies Of Small Molecule Inhibitors Of The Hypoxia Inducible Factor Pathway, Suazette Reid Mooring

Chemistry Dissertations

Cancer accounts for nearly one-quarter of deaths in the United States, exceeded only by heart diseases. In 2006, there were 559,888 cancer deaths in the US. Finding effective treatments for cancer is a major challenge among researchers. In solid tumor, hypoxia increases the progression of malignancy and metastasis by promoting angiogenesis. The transcription factor HIF-1 is responsible for the regulation of cellular processes, including glycolysis and angiogenesis. Clinical evidence has determined that expression of HIF-1 is strongly associated with poor patient prognosis. Also, activation of HIF-1 contributes to malignant behavior and therapeutic resistance. In view of these observations, there is …

Synthesis Of Fused Heterocyclic Diamidines For The Treatment Of Human African Trypanosomiasis And Fluorescence Studies Of Selected Diamidines, Jennifer Crystal Brown Barber

Chemistry Dissertations

A class of linear diamidines was synthesized for the evaluation as a treatment of Human African Trypanosomiasis. These fused heterocyclic compounds are thiazole[5,4-d]thiazoles and are of interest because the parent compound, 2,5-Bis(4-amidinophenyl)-thiazolo[5,4-d]thiazole HCl salt, which is also called DB 1929, has exhibited a low nanomolar IC50 value against Trypanosoma brucei rhodesiense and has shown selectivity for binding to the human telomere G-quadruplex over that of DNA duplex. A fluoro and a methoxy derivative have been synthesized and are currently undergoing testing for activity and binding affinity. In addition, fluorescence studies of selected diamidines were done to study the effect of …

On The Catalytic Roles Of His351, Asn510, And His466 In Choline Oxidase And The Kinetic Mechanism Of Pyranose 2-Oxidase, Kunchala Rungsrisuriyachai

Chemistry Dissertations

Choline oxidase (E.C. 1.1.3.17) from Arthrobacter globiformis catalyzes the four-electron oxidation of choline to glycine betaine (N,N,N-trimethylglycine) via two sequential, FAD-dependent reactions in which betaine aldehyde is formed as an enzyme-bound intermediate. In each oxidative half-reaction, molecular oxygen acts as electron acceptor and is converted into hydrogen peroxide. Biochemical, structural, and mechanistic studies on the wild-type and a number of mutant variants of choline oxidase have recently been carried out, allowing for the depiction of the mechanism of alcohol oxidation catalyzed by the enzyme. Catalysis by choline oxidase is initiated by the removal of the hydroxyl proton of alcohol substrate …

Roles Of Serine 101, Histidine 310 And Valine 464 In The Reaction Catalyzed By Choline Oxidase From Arthrobacter Globiformis, Steffan Finnegan

Chemistry Dissertations

The enzymatic oxidation of choline to glycine betaine is of interest because organisms accumulate glycine betaine intracellularly in response to stress conditions, as such it is of potential interest for the genetic engineering of crops that do not naturally possess efficient pathways for the synthesis of glycine betaine, and for the potential development of drugs that target the glycine betaine biosynthetic pathway in human pathogens. To date, one of the best characterized enzymes belonging to this pathway is the flavin-dependent choline oxidase from Arthrobacter globiformis. In this enzyme, choline oxidation proceeds through two reductive half-reactions and two oxidative half-reactions. In …

Design And Synthesis Of Boronic Acid-Modified Nucleotides For Fluorescent Sensing And Cell Imaging, Xiaochuan Yang

Chemistry Dissertations

With the rapidly increasing interest in the field of glycomics, which is the comprehensive study of the roles carbohydrates play in a living system, urgent need for developing quick and highly selective carbohydrate sensors is growing. The boronic acid group, with its electron-deficient structure (6 valence electrons with an open shell), acts as a Lewis acid with high intrinsic affinity towards Lewis bases such as fluoride, cyanide and hydroxyl groups. Specifically, formation of a 5- or 6- membered ring between the boronic acid moiety and a1,2- or 1,3-diol in aqueous solution has been fully explored as a strategy of carbohydrate …

Detection And Quantification Of Organophosphate Pesticides In Human Serum, Peter Kuklenyik

Chemistry Dissertations

The United States Environmental Agency permits the use of 39 organophosphate pesticides. Many of these pesticides are acutely toxic and have lasting effect on human health. Organophosphates quickly metabolize in the body, therefore currently human exposure is studied by measuring the metabolic products in urine. In this work a suite of analytical methods was developed to determine the presence of un-metabolized organophosphate pesticides in human serum. First mass spectroscopic detection methods were evaluated. Gas chromatograph coupled tandem mass spectrometer was used to compare the detection limits using chemical and electron impact ionization. Positive chemical ionization was selected, because it provided …

On The Preorganization Of The Active Site Of Choline Oxidase For Hydride Transfer And Tunneling Mechanism, Osbourne Quaye

Chemistry Dissertations

Choline oxidase catalyzes the two-step oxidation of choline to glycine betaine, one of limited osmoprotectants, with the formation of betaine aldehyde as an enzyme bound intermediate. Glycine betaine accumulates in the cytoplasm of plants and bacteria as a defensive mechanism to withstand hyperosmolarity and elevated temperatures. This makes the genetic engineering of relevant plants which lack the property of salt accumulation of economic interest, and the biosynthetic pathway of the osmolyte a potential drug target in microbial infections. The reaction of alcohol oxidation occurs via a hydride ion tunneling transfer from the substrate donor to a flavin acceptor within a …

Biochemical And Spectroscopic Characterization Of Tryptophan Oxygenation: Tryptophan 2, 3-Dioxygenase And Maug, Rong Fu

Chemistry Dissertations

TDO utilizes b-type heme as a cofactor to activate dioxygen and insert two oxygen atoms into free L-tryptophan. We revealed two unidentified enzymatic activities of ferric TDO from Ralstonia metallidurans, which are peroxide driven oxygenation and catalase-like activity. The stoichiometric titration suggests that two moles of H2O2 were required for the production of one mole of N-formylkynurenine. We have also observed monooxygenated-L-tryptophan. Three enzyme-based intermediates were sequentially detected in the peroxide oxidation of ferric TDO in the absence of L-Trp including compound I-type and compound ES-type Fe-oxo species. The Fe(IV) intermediates had an unusually large quadrupole splitting parameter of 1.76(2) …

Diagnosis And Inhibition Tools In Medicinal Chemistry, Senol Akay

Chemistry Dissertations

Cell surface saccharides are involved in a variety of essential biological events. Fluorescent sensors for saccharides can be used for detection, diagnosis, analysis and monitoring of pathological processes. The boronic acid functional group is known to bind strongly and reversibly to compounds with diol groups, which are commonly found on saccharides. Sensors that have been developed for the purpose of saccharide recognition have shown great potential. However, they are very hydrophobic and this lack of essential water-solubility makes them useful in biological applications. The first section of this dissertation details the process of developing water-soluble saccharide sensors that change fluorescent …

Metal-Assisted Hydrolysis Of Biological Molecules, Sarah Shealy Cepeda

Chemistry Dissertations

In Chapter I is a general description of novel metal complexes which hydrolytically cleave peptides, proteins, DNA, and other biological molecules. These reagents are becoming the more important as potential therapeutic agents. A panel of ligands was investigated for coordination to ZrIV and other metals in groups 4, 5, and 6 to effect the greatest degree of hydrolysis. Chapter II describes a ZrIV complex which is capable of hydrolyzing a 30 amino acid peptide, insulin chain B, with amino acid specificity. Oxidized insulin chain B peptide was hydrolyzed after only 4 h of treatment at pH 7.0 and 60 °C …

Synthesis, Structure And Function Studies Of Selenium And Tellurium Derivatized Nucleic Acids, Jia Sheng

Chemistry Dissertations

Nucleic acids play important roles in living systems by storing and transferring genetic information and directing protein synthesis. Recently, it was found that nucleic acids can catalyze chemical and biochemical reactions similar to protein enzymes. In addition, they can also serve as drug targets for the treatment of deadly diseases such as AIDS and cancers. As a result, the 3D structure study of nucleic acids and proteinnucleic acids complexes by X-ray crystallography has become one of the most active research areas. However, the two intrinsic bottlenecks of macromolecule X-ray crystallography, including crystallization and phase determination, have significantly limited its application …

Design Genetic Fluorescent Probes To Detect Protease Activity And Calcium-Dependent Protein-Protein Interactions In Living Cells, Ning Chen

Chemistry Dissertations

Proteases are essential for regulating a wide range of physiological and pathological processes. The imbalance of protease activation and inhibition will result in a number of major diseases including cancers, atherosclerosis, and neurodegenerative diseases. Although fluorescence resonance energy transfer (FRET)-based protease probes, a small molecular dye and other methods are powerful, they still have drawbacks or limitations for providing significant information about the dynamics and pattern of endogenous protease activation and inhibition in a single living cell or in vivo. Currently protease sensors capable of quantitatively measuring specific protease activity in real time and monitoring activation and inhibition of enzymatic …

The Design And Evaluation Of Boronic Acid Derivatives For The Recognition Of Cell Surface Carbohydrates For Medicinal Applications, Sandra Navonne Craig

Chemistry Dissertations

ABSTRACT Carbohydrates in various forms play vital roles in numerous critical biological processes including cell-cell adhesion and communication, embryo development, immune response, etc. Fluorescent sensors for such carbohydrates have a wide range of potential applications including glucose concentration determination, cell labeling and targeting based on carbohydrate biomarkers, as in vitro diagnostic tools, and biomarker-directed cellular imaging. Our group has been interested in the design and synthesis of multi-boronic acid compounds with well-defined three-dimensional scaffolding for the specific recognition of selected carbohydrate biomarkers. Aberrant expression of carbohydrate antigens such as sialyl Lewis X (sLex), sialyl Lewis A (sLea), Lewis X (Lex), …

Using Protein Design To Understand The Role Of Electrostatic Interactions On Calcium Binding Affinity And Molecular Recognition, Lisa Michelle Jones

Chemistry Dissertations

Calcium regulates many biological processes through interaction with proteins with different conformational, dynamic, and metal binding properties. Previous studies have shown that the electrostatic environment plays a key role in calcium binding affinity. In this research, we aim to dissect the contribution of the electrostatic environment to calcium binding affinity using protein design. Many natural calcium binding proteins undergo large conformational changes upon calcium binding which hampers the study of these proteins. In addition, cooperativity between multiple calcium binding sites makes it difficult to study site-specific binding affinity. The design of a single calcium binding site into a host system …

Determining The Site Specific Metal Binding And Structural Properties Of Ef-Hand Protein Using Grafting Approach, Hsiau-Wei Lee

Chemistry Dissertations

Calmodulin is an essential EF-hand protein with a helix-loop-helix calcium binding motif. Understanding Ca(II) dependent activation of calmodulin and other EF-hand proteins is limited by Ca(II)-induced conformational change, multiple and cooperative binding of Ca(II) ions, and interactions between the paired EF-hand motifs. The goal of this research project is to probe key determinants for calcium binding properties and pairing interactions at the site specific level using a grafting approach and high resolution NMR. An individual Ca(II) binding site of the EF-hand motifs of calmodulin was grafted into a non-calcium dependent protein, CD2, to bypass limitations associated with natural EF-hand proteins …

Rational Design Of Calcium Biosensors, April L. Ellis

Chemistry Dissertations

Understanding the temporal and spatial changes in calcium concentration has been a difficult endeavor for many years due to the relatively small changes in calcium concentration during messenging events, the rapid changes upon physiological messenging, and the unavailability of fast, efficient, and sensitive sensors to detect calcium changes. In addition, the key factors in calcium binding have yet to be determined due to the metal-metal interactions, cooperativity, and conformational change involved in calcium binding to natural calcium-binding proteins. To overcome these obstacles and to engineer calcium sensors for in vivo studies of calcium signaling events, calcium binding sites have been …