Chemistry
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Item Embargo Identification and Quantitation of Cannabinoid Solute-Column Interactions Using the Hydrophobic-Subtraction Model to Predict Separation in High-Performance Liquid Chromatography(East Carolina University, July 2024) Tunnell, Heather KayeThe Hydrophobic-Subtraction Model (HSM) has been widely used for identification of similar and orthogonal chromatography columns since its inception in 2002 but only had limited uses as a predictive tool. Recent refinement of the model has improved the prediction accuracy to a level that offers promise for use in high-performance liquid chromatography (HPLC) separation development. The HSM quantitatively describes the parameters affecting solute retention on HPLC columns including hydrophobic, steric, hydrogen-bond, cation exchange, and dipole interactions. To demonstrate the process, 16 cannabinoid compounds were chosen as the test solutes due to increasing interest and applications. The HSM was used to identify and quantify the various cannabinoid solute retention parameters based on actual retention of each solute on a set of calibration columns with previously quantified column retention parameters. The resulting solute parameters were used to predict relative retention of each cannabinoid on over 550 columns in the HSM column database. A sorting system was then developed to rank the columns. Overall, this Thesis demonstrates the application of the HSM to cannabinoid compounds starting with identification of the best columns for the separation of these solutes.Item Embargo A Catalytic Approach to Cyclopentenones: Unexpected Challenges in Synthesizing Pent-2,4-dienals(East Carolina University, 2023-05-04) English, Hunter WadeTransition metal catalysis has long been utilized to facilitate a number of chemical reactions outside the scope of traditional organic chemistry. Because of this, there has been a growing interest in the community in utilizing C-H bonds over C-X bonds as synthetic alternatives with better thermodynamic conditions (lower temperatures, pressures, energies, etc.) and forming C-C bonds. For example, rhodium-catalyzed hydroacylation can be used in the conversion of 4-pentenals to cyclopentanones. The mechanistic foundation of this reaction was applied to the synthesis of chiral 3-substituted indanones, unlocking a whole series of transformations towards the construction of biologically active molecules. The Morehead group recently proposed an alternate synthesis of cyclopentenones via a rhodium-catalyzed intramolecular hydroacylation reaction of penta-2,4-dienals by the following reaction sequence: 1. Vilsmeier-Haack Formylation to form 3-bromo-2-enals, 2. Suzuki Cross-Coupling with vinylboronates, and 3. Hydroacylation. Indeed, all reaction steps were performed successfully starting from acetophenone and allylboronic acid pinacol ester, resulting in 4-methyl-3-phenyl-2-cyclopenten-1-one. However, some surprising results were obtained along the way. It was found that when 3-bromo-3-phenyl-2-propenal underwent Suzuki Cross-Coupling, an unexpected mixture of pent-2,4-dienal isomers was obtained. To investigate this matter, a number of reaction conditions were examined in hopes of finding one with optimal yields and little to no isomerized pent-2,4-dienal product. In addition, a variety of cyclopentenones were synthesized to explore the steric effect in influencing the isomerization process. Some potential mechanistic details of this strange isomerization phenomenon along with results obtained from the synthesis will be reported and discussed.Item Embargo Towards Establishing Methods of Synthesis, Purification, and Structural Determination for Human Fibrinogen(East Carolina University, 2023-05-04) Kirby, Nicholas CarterFibrinogen is a prevalent blood-based glycoprotein. In its polymerizable form, fibrin, it serves as the structural component of blood clots and helps to incorporate red blood cells, macrophages, and fibroblasts to the wound-site. Fibrinogen plays important roles in inflammation and tissue regeneration, regulating growth factor receptor pathway activation, scar formation, and immune response, yet many questions remain about fibrinogen's structure-function link in these various pathophysiological states. Studies focused on improving expression systems for recombinant fibrinogen, and developing more rapid and robust techniques for purifying fibrinogen from complex media would provide a means by which to progress mutational studies of fibrinogen. Similarly, developing methods for high-resolution imaging of fibrinogen in its native, aqueous-based environment is necessary to establish proper structure-function relationships encompassing this complex 340 kDa glycoprotein. The work described in this thesis produced advancements in each of these crucial areas of fibrinogen research. Herein, chapter 2 reports, to our knowledge, the first methodology of transient recombinant human fibrinogen expression using suspension HEK Expi293TM cells. A highly selective and robust affinity-based approach was also developed for the rapid isolation of fibrinogen from a variety of complex media. Chapter 3 describes cryogenic electron microscopy and our process of obtaining a high-resolution, in-solution structure of human fibrinogen's D region. This structure is currently the highest resolution structure of any fibrinogen domain obtained in the native, solution state and further supports evidence for fibrinogen's intrinsic flexibility, expanding the textbook representations of fibrinogen as a rigid, rod-like structure. Lastly, future directions outlining fibrinogen's role during pregnancy and unique fibrinogen-specific protofibril-like structures are discussed. In an appendix, I have included figures from an illustrated review journal article, which I participated in as a co-first author.Item Open Access Development of Transient Recombinant Expression and Affinity Chromatography Systems for Human Fibrinogen(2022-01-19) Popovic, Grega; Kirby, Nicholas C.; Daub, Caroline E.; Offenbacher, Adam R.; Dement, Taylor C.; Belcher, Heather A.; Hudson, Nathan E.Item Open Access Crystal Structures of Non‑Oxidative Decarboxylases Reveal a New Mechanism of Action with a Catalytic Dyad and Structural Twists(2021) Zeug, Matthias; Iancu, Cristina V.; Choe, Jun-Yong; Markovic, Nebojsa; Tripp, Joanna; Oreb, MislavItem Open Access Subcritical Water Extraction of Salvia miltiorrhiza(2021-03-15) Kapalavavi, Brahmam; Doctor, Ninad; Yang, Yu; Zhang, BaohongItem Open Access Biophysical Investigation into the Protein Dynamics Governing the Allosteric Regulation of Plant and Animal 15-Lipoxygenases(East Carolina University, 2022-04-27) Roberts, DaniellaLipoxygenases (LOXs) are a family of enzymes found in plants, animals, fungi, and bacteria that catalyze the per-oxidation of polyunsaturated fatty acids. In plants, LOXs are involved in growth, development, and defense against pathogenic attacks. There are also multiple isoforms present in humans, which have contradictory roles in the body. Specifically, human 15-LOX isoforms, 15-LOX-1 and 15-LOX-2, are involved in both homeostasis and pro-inflammatory pathways. In order to selectively target the activity of these enzymes, research has turned to allosteric regulation, which is the focus of this Thesis. Previously, the allosteric regulation of a model plant 15-LOX, soybean lipoxygenase-1 (SLO), has been characterized using hydrogen-deuterium exchange mass spectrometry (HDX-MS), revealing that the addition of the allosteric effector, oleyl sulfate (OS), alters a specific region of the enzyme. Herein, we used a combination of thermodynamic and biophysical techniques such as isothermal titration calorimetry and differential scanning calorimetry to investigate the allosteric regulation of SLO by OS. We present data which supports that the allosteric regulation of SLO by OS does not induce oligomerization or large-scale conformational changes and that the allostery is dynamically driven. We also employed HDX-MS to study the dynamics of 15-LOX-1 compared to previously collected data of 15-LOX-2 to reveal structural differences between the two isozymes that may explain their altered catalytic behavior.Item Open Access DIRECT ELECTROCHEMICAL ANALYSIS OF THE REDOX ACTIVITY OF TRYPTOPHAN AND TYROSINE IN MODIFIED AZURINS: THE IMPACT OF THE PROTEIN ENVIRONMENT(East Carolina University, 2021-05-03) Tyson, Kris JanellProton-coupled electron transfer (PCET) is a biological process essential to life. It is imperative for respiration in animals as well as photosynthesis in plants. Long-range PCET is often facilitated by redox-active amino acids, such as tryptophan and tyrosine. While there are several examples in the literature for the involvement of these redox-active residues in PCET linked to biological catalysis, there has been a challenge in direct electrochemical efforts to resolve how the local protein environment controls PCET directionality. This thesis describes a protocol from which to directly test the reduction potentials of tyrosine and tryptophan radicals in a customizable protein environment. The model protein used for this study was azurin, a natural cupredoxin that natively contains two tyrosines and only one tryptophan, with the former mutated to phenylalanine to provide direct electrochemical detection of a single redox-active amino acid species. The reduction potentials of azurin with either tryptophan or tyrosine redox centers were monitored using the electrochemical technique square-wave voltammetry. Using this technique along with strategic protein engineering, it was found that the more solvent-exposed or polar mutants had a higher redox potential than those that were more solvent-excluded. These trends have biological importance as the difference in the reduction potentials between redox-active amino acid pairs is expected to control the thermodynamic driving force for PCET. This thesis details the impact of altering the surrounding protein environment, i.e. electrostatics, on the redox activity of tryptophan and tyrosine.Item Restricted EXPLORING STUDENT EXPERIENCES ACROSS A LINKED SEQUENCE COURSE-BASED UNDERGRADUATE RESEARCH EXPERIENCE(East Carolina University, 2020-11-23) Whiting, BrandonThis study reports the effect a linked Course-based Undergraduate Research Experience (CURE) has on a students' understanding of research and the associated experiences they encountered. CUREs offer an alternative path to authentic research which can increase student access and potentially increase the overall diversity in a laboratory setting. The institution in this study has recently developed a linked-CURE sequence that bridges a lower-division organic chemistry laboratory with an upper-division quantitative analysis laboratory. Focus groups consisting of 3-5 students each were conducted with students participating in two cohorts of the CURE sequence during the last week of the academic term. Through the analysis of survey and interview data three themes were developed: working towards a common goal, developing a deeper understanding of the science content, and addressing obstacles that were encountered.Item Open Access Transthyretin Amyloidosis: Proteolytic cleavage accelerates G53A TTR misfolding and aggregation(East Carolina University, 2020-06-22) Arreola, JenetteThe proteolytic cleavage of the peptide bond Lys48-Thr49 of the CD loop in TTR pathological mutants S52P and E51_S52dup was previously demonstrated to promote aggregation as a result of tetrameric structure destabilization. Type A and B fibrils were detected in vivo, suggesting alternative TTR misfolding and aggregation mechanisms. The main component of the fibrils was the residue 49-127 fragment. In the proceeding studies, the misfolding and aggregation of G53A TTR, whose mutation is nearby the K48-T49 peptide bond and also associated with TTR amyloidosis, was investigated in thepresence and absence of proteolytic agent, trypsin. Fragmented G53A TTR misfolded and aggregated via a similar mechanism as full-length TTR, but at a faster rate. Similar morphology was exhibited by fragmented and full-length G53A TTR oligomers, as revealed by TEM images, suggesting similar aggregation pathway. G53A TTR in the presence and absence of trypsin generated similar CD and FT-IR profiles, suggesting similar transthyretin morphology during the early and late stages of amyloidosis. Aggregation kinetics was accomplished by monitoring the optical density of G53A TTR in the presence and absence of trypsin and by comparing the ThT fluorescence signal produced. The differences in TTR solubility and ThT enhancement imply G53A TTR aggregation is promoted in the presence of trypsin. Oligomeric effects on mammalian cell line, SH-SY5Y, was probed employing the MTT assay, which determined G53A TTR was more toxic to the cells in the presence of trypsin.Item Open Access Platinum Catalyzed Synthesis of Alpha-Ketoester via C-H Functionalization(East Carolina University, 2019-12-16) Javed, ErmanAlpha-ketoesters have proven to be useful in a variety of fields. They have found wide spread applications in pharmaceuticals, photochemistry, and biology. Additionally, they are of great interest in synthetic chemistry and are frequently used as a precursor to many useful organic compounds including alpha-keto acids, alpha-hydroxy acids, and alpha-amino acids. Numerous methods have been reported for synthesizing alpha-ketoesters but they all amount to a few notable drawbacks. Herein reported is a potentially more effective transition metal catalyzed reaction to synthesize alpha-ketoester via C-H functionalization. A series of ligands with structural modifications have been designed, synthesized and acylated to shed light on the scope and limitations of the reaction. An inexpensive and readily accessible reagent namely ethyl chlorooxoacetate was employed as the acylating reagent. Reaction conditions were optimized by screening various solvents and catalysts. A variety of solvents were found useful in this reaction, including chlorobenzene, benzonitrile, toluene, and m-xylene although the best results were obtained when chlorobenzene was used. The reaction showed great tolerance to both electron withdrawing and donating groups on the phenyl ring however some electronic effects were observed and it was found that the presence of electron withdrawing group on the phenyl ring decelerated the acylation reaction. Experimental results of the acylation reaction will be reported and the mechanistic implications of these results will be discussed.Item Restricted Development of Green Subcritical Water Separation Technology(East Carolina University, 2019-08-19) Doctor, NinadTheoretically speaking, water under temperature and pressure conditions below its critical point is called subcritical water. However, the term “subcritical water” normally refers to hot water up to 374 C and pressure up to 218 atm. As the temperature increases, the hydrogen bonding between water molecules weakens, making water less polar at elevated temperatures. In chemical and pharmaceutical industry organic solvents are extensively used for extraction, chromatography, chemical analysis, and remediation of toxic chemicals. It requires a large amount of money not only for purchasing the expensive organic solvents but also properly disposing of their wastes. Subcritical water technology has shown a great potential in eliminating or minimizing the use of organic solvents in the processes mentioned above as it solely uses subcritical water as the solvent. The objective of this research is to further develop the green subcritical water separation technology. Specific aims include organic analyte stability in subcritical water, subcritical water extraction and analysis of active pharmaceutical ingredients (APIs) from medicinal herbs, subcritical water chromatographic separation of APIs from cold drugs, development of thermally stable stationary phase materials, and destruction of toxic polychlorinated biphenyls (PCBs) under subcritical water conditions. The stability of vanillin and coumarin in subcritical water was studied at 100-250 C. Vanillin was stable up to 60 min at all temperatures tested. While coumarin was stable at temperatures up to 150 C, it started to degrade at 200 and 250 C after heating for 60 min. Vanillin and coumarin were also extracted from vanilla beans and whole Tonka beans using subcritical water at 100-200 C. The vanillin quantity extracted increased from 10.7 mg per gram of vanilla beans (10.7 mg/g) at 100 C to 19.9 mg/g at 200 C. Similarly, the coumarin quantity extracted increased from 23.9 mg per gram of whole Tonka beans (23.9 mg/g) at 100 C to 36.8 mg/g at 200 C. Due to effectiveness, wide availability, and low side effects and costs, the use of herbal medicine has increased all around the world. Danshen (salvia miltiorrhiza) is a Chinese medicinal herb used to treat patients suffering from stroke, angina, chronic liver disease or other diseases. Active pharmaceutical ingredients including protocatechualdehyde, ferulic acid and caffeic acid were extracted from Dan Shen using water at 75 to 150 C. Our results show that the extraction efficiency for all three APIs is improved with increasing temperature. To eliminate the use of organic solvents in high-performance liquid chromatography (HPLC), subcritical water chromatography (SBWC) methods were developed for aspirin and metformin. SBWC separation of metformin HCL and aspirin were achieved at 95 C and 125 C, respectively. The recovery for both pharmaceutical active ingredients obtained by SBWC is 99% in comparing with the stated content of each drug. The relative standard deviation is less than 1% for SBWC assays developed in this work. This level of accuracy and precision achieved by SBWC is the same as that resulted by the traditional HPLC analysis also carried out in this work. For subcritical water chromatography separations, thermally stable stationary phases are critical. Since zeolite is superior to the traditional silica in thermal stability, a method was developed to synthesize organic-bonded zeolite through the substitution of silanol groups with C18. In FTIR-ATR experiment stretching band of CH2 and CH3 clearly revealed the presence of carbon chain after the modification. HPLC column was packed with C18 modified zeolite for the evaluation of chromatographic separation. Caffeine peak was detected at lower concentration at 272 nm wavelength. It also showed some separation between vanillin and coumarin at 284 nm. However, the HPLC column was unable to perform separation above 0.4 mL/min due to the small pore size, and it is one of the limitations of zeolite modified stationary phase. Lastly, the destruction of PCB-118, PCB-156 and PCB-180 congeners under subcritical conditions has been investigated. The percent degradation of PCBs was mostly improved by increasing the heating time, concentration of hydrogen peroxide and sodium hydroxide in water. All three PCB congeners investigated were completely destroyed after heating in 0.2% sodium hydroxide at 350 °C for an hour.Item Open Access Studying Collagen with PyrATS: Pyrene-Appended Trimeric Systems(East Carolina University, 2019-07-30) Keever, Jared MatthewCollagens are a family of triple-helical structural proteins that are ubiquitous in vertebrates. Improper folding of collagen can lead to disorders such as osteogenesis imperfecta, or "brittle bone disease." There is significant interest in understanding the factors that drive collagen folding and stability, but studying native collagens is difficult because they are hundreds of amino acids in length. This thesis describes a series of well-characterized (Pro-Hyp-Gly)7 model peptides which have been tagged at their N-termini with the fluorophore pyrene. When in close contact, pyrene units can form excimers that emit low-energy light. This allows for the study of several fundamental questions in collagen research using fluorescence spectroscopy, including concentration dependence, folding directionality, and local fraying, upon solutions that are significantly more dilute than those customarily used in circular dichroism (CD) experiments. Notably, for most of the peptides studied, there is agreement between the melting temperatures (Tm) obtained via fluorescence and CD techniques. In addition, the pyrene probes were found to provide a situational increase in thermal stability of triple helices.Item Restricted Elucidating the Mechanisms of Transthyretin Aggregation in Transthyretin Amyloidosis(East Carolina University, 2018-07-24) Dasari, Anvesh K. R.Accumulation of insoluble aggregates (amyloids) is a characteristic feature of many neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. Aggregation and deposition of Transthyretin (TTR) protein in tissues leads to transthyretin amyloidosis. Aggregation of wild-type (WT) TTR shown to affect the heart, causing senile systemic amyloidosis (SSA), while the two pathogenic mutants (V30M, and L55P) affect the peripheral nervous system. It is important to understand the misfolding/amyloid formation mechanism towards the development of therapeutic strategies. TTR is rich in the [beta]-sheet structure in which eight [beta]-strands are arranged in a [beta]-sandwich consisting of two [beta]-sheets (strands CBEF and DAGH). Previous solution NMR studies have shown that the amyloid precursor state of TTR is also rich in [beta]-sheets. Our solid-state NMR results indicated the presence of intact native-like [beta]-sheets in the amyloid state as well. But, the AB loop region appeared to be perturbed during the aggregation which could expose strand A for intermolecular associations. Solid-state NMR results of the mutant forms (V30M and L55P) also indicated the presence of native-like [beta]-sheets in the amyloid states but, with a distorted DA strand which further exposes strand A for more aggressive intermolecular interactions. We also reported the aggregation mechanism for early aggregates which are shown to be cytotoxic in the amyloid formation pathway. We also discovered that the hexamers of TTR are the building blocks of these early toxic aggregates.Item Restricted Using Fluorescent Probes to Study the Location and Interaction of Disaccharides within Lipid Bilayers(East Carolina University, 2016-04-26) Barker, Morgan ElizabethIt is well known that certain organisms, such as the tardigrade, are capable of surviving extreme conditions including dehydration, freezing and / or oxygen deficiency. The tardigrades ability to withstand such extreme conditions is associated with a decrease in its internal water content and the excess production of a disaccharide known as trehalose. Studies have focused on better understanding this organism survival mechanism in order to help aid in the preservation of biological cells. Researchers have attempted to replicate this phenomenon in vitro through a freeze-drying technique known as lyopreservation and have shown trehalose to be an effective lyoprotecting agent. However, lyopreservation is very technically challenging so some studies have focused on trehalose has a potential cryoprotectant. Cryopreservation involves storing hydrated cells or other materials at very low temperatures in a frozen state. Unfortunately, trehalose did not show effectiveness as a cryoprotecting agent. This may be due to trehalose being a highly hydrophilic molecule which does not penetrate into the membrane to offer protection during freezing. Our research aims to investigate other disaccharides with varying hydrophilicity as potential cryoprotectants for hydrated cells. In order to study the effectiveness of certain disaccharides as cryoprotectants, liposomes were used as a model membrane system. The location and interactions of both sucralose and sucrose within these membranes were monitored through fluorescence and ultra-violet visible spectrophotometry using two specific fluorophores. The first of these, diphenylhexatriene, is a hydrophobic probe quenched by water that exhibits strong fluorescence when incorporated into a cell membrane. The second, merocyanine 540, is a negatively charged cyanine dye located slightly above the glycerol backbone of the phospholipids and is sensitive to any structural variations within the membrane. The data obtained with each respective probe indicates that the packing efficiency and polarity of the membrane is decreased in the presence of sucralose, but not in the presence of sucrose. We hypothesize that this is due to different hydrophobic properties of the sugars allowing sucralose to penetrate further into the bilayer while sucrose does not. This study indicates that sucralose has a significant effect on both the packing efficiency and the polarity of the bilayer and thus its usefulness as a potential cryoprotectant for biological cells is of great interest and needs to be investigated.Item Open Access Wurster's crown ligands(2001-07-17) Sibert, John W.Wurster's crown ligands comprise a macrocyclic ligand such as a crown ether in which a hetero atom is substituted with a 1,4-phenylenediamine group. The phenylenediamine group is covalently bound to the macrocyclic ligand by one or both of the amine nitrogens, the amine nitrogen thereby substituting for the hetero atom of the macrocyclic ligand. The resulting compounds are redox active. Methods of making and using the compounds are also disclosed.Item Open Access Synthesis Of Non-Natural Fmoc-Protected Amino Acids To Provide Novel Fluorescent Anion Probes And Their Incorporation Into Synthetic Peptides(East Carolina University, 2015) Farrell, David PercyCystic fibrosis is a genetic disorder caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. CFTR serves to control the gradient of chloride and bicarbonate ions across the cellular membrane of epithelial tissues (e.g., mucosa, intestinal walls, and lungs). Excess chloride on the inside of epithelial cells causes mucus in the lungs to become very thick. This thickening is responsible for a patients' characteristic thick sputum, coughing, and trouble breathing. The thick mucus also creates an ideal environment for opportunistic bacteria like Pseudomonas aeruginosa and Mycobacterium tuberculosis to take residence and proliferate. Very little is known about the transmembrane structure of CFTR, but with current therapies a single mutation can shorten a person's life by about 50%. Many researchers have attempted to bypass the CFTR protein and synthesize anion shuttle transporters or transmembrane pores. The research presented here focuses on the synthesis of anion-responsive amino acids, for eventual incorporation into membrane-spanning peptides. Amino acids 2.1 and 2.2 were successfully synthesized from commercially available Fmoc-Glu-OtBu and Fmoc-Asp-OtBu through the conversion of an acid azide into an isocyanate via a Curtis Rearrangement. An N-substituted-4-amino-naphthalimide was chosen as the chromophore due to its ability to absorb and emit light at a significantly longer wavelength than those produced by naturally occurring amino acids. The fluorescence provides an indirect insight into the binding strength of the novel amino acids, and eventually designer peptides, to various biologically relevant anions. The fluorescence can also be used by a researcher to learn more about a peptides concentration, location, microenvironment, structure, and mechanism of action. The presence of a fluorenylmethyloxycarbonyl (Fmoc) protecting group allows a researcher to use standard solid phase peptide synthesis protocols to produce either a known peptide or any imaginable variation. Fluorescent amino acids are useful tools in grasping a better understanding of protein channelopathies such as cystic fibrosis. One day, the use of synthetic peptide ion channels will be an effective therapy to alleviate the many symptoms and even the causes of death that afflicts the many patients suffering from channelopathies today.Item Open Access Electrochemical Detection of Anti-Biofilm Activity Using Unnatural Amino Acid-Containing Antimicrobial Peptides(East Carolina University, 2015) Vinogradov, Sergey M.Bacterial infections are a significant health problem that can be detrimental to the human population. It is estimated that bacterial infections are responsible for billions of dollars’ worth of damages in the health care field alone, and numerous deaths annually. Bacterial infections can become so detrimental because they produce a structure called biofilm, which facilitates antibiotic resistance and is a major cause of chronic infections. In order to combat this threat, new anti-biofilm and antibiotic therapies are being developed and their efficiency must be tested. A series of antimicrobial peptides (AMP) containing unnatural Tic-Oic amino acids have been developed for this purpose. Traditional methods such as biological assays are the standard by which antibiotics are judged, but they have their drawbacks, such as the lengthy test times and the costs associated with it and the reagents. Electrochemical biosensors can remedy some of those drawbacks by offering speed and cost benefits. Electrochemical biosensors consisting of Layer-by-Layer (LbL) modified electrodes were constructed. These sensors were fabricated to test the anti-biofilm activity of the aforementioned unnatural amino acid-containing antimicrobial peptides against a model of Pseudomonas aeruginosa or against the bacteria itself. P. aeruginosa is a common biofilm producing bacteria. First, we employed alginate as one of the layers in our sensor as P. aeruginosa is known to produce this as its major biofilm component. We show that the penetration of the alginate layer by the AMP can be detected electrochemically utilizing a solution-phase redox active molecule that produces an increasing signal upon electrochemical reduction when the film becomes compromised. Biological assays are presented that provide some validation for the sensor, but elucidated a particular AMP as compared to the electrochemical alginate sensor. Based on this slight disagreement between our electrochemical model and the biological assay, we employed sensors that featured directly immobilized P. aeruginosa PAO1 on the electrode surface. These bacteria are electroactive, which negated the need for an external redox active molecule, and allowed the monitoring of anti-biofilm activity via a signal decrease over time. The P. aeruginosa sensor showed more agreement with the biological assay, highlighting the same AMP as active toward biofilm degredation at low (<1 mircoM) concentrations. Overall, these electrochemical biosensors utilizing models of and actual P. aeruginosa have opened up new avenues to test for effectiveness of potential anti-biofilm agents toward biofilm forming bacteria.Item Restricted Synthesis, structure, photophysics, and biological activity of platinum (II) complexes(East Carolina University, 2013) Vezzu, Dileep Atchyuth KumarThe synthesis, structure, and photophysical properties of luminescent platinum (II) complexes with different coordination patterns, (C^C*N^N), (N^C*N), (N^N*C) and (N^N^C) are reported, where "C^N or N^N" denotes a bidentate coordination to the platinum to form a five-membered metallacycle and "C*N" denotes a bidentate coordination to the platinum to form a six-membered metallacycle. Sixteen cyclometalated platinum complexes have been synthesized with different coordination patterns, which include six complexes with tridentate N^C*N cyclometalating ligands (13, 14, 15, 16, 17, and 18), five complexes with N^N*C cyclometalating ligands (19a, 19b, 20a, 20b, and 24), three complexes with N^N^C cyclometalating ligands (21a, 21b, and 29) and two complexes with tetradentate C^C*N^N cyclometalating ligands (33 and 34). The structures of the platinum complexes 13, 15, 16, 18, 19a, 19b, 20a, 21a, 21b, 24, 29, 33, and 39 were determined by single crystal X-ray diffraction. In platinum complexes with five-six membered metallacycle (15, 16, 18, 19a, 19b, 20a, 24, and 39), the square geometry of the complexes is improved when compared to that of platinum complexes with five-five membered metallacycle as the biting angle is increased. The tetradentate coordination (C^C*N^N), square planar geometry of complex 33 are revealed from its X-ray crystal structure. The DFT calculations have been carried out on complexes 13, 14, 15, 16, 17, 18, 33, and 34 to examine the molecular orbital character of the complexes, which are used in interpreting the electronic spectra of the complexes. The photophysical properties of the platinum complexes were studied and a majority of the complexes were highly emissive in solution at room temperature. Complex 13 exhibited the highest quantum yield (65%) among all of the complexes. Self quenching was not observed in a majority of the platinum complexes at lower concentrations. The cytotoxicity (IC₅₀) of the complexes in three lung cancerous cell lines and one prostate cancer cell line were determined by MTT assay. The toxic platinum complexes induced the cell death by triggering apoptosis. The interactions of the platinum complexes with plasmid and calf thymus DNA were studied. Activation of caspase -7, PARP, and p53 were also observed in RV1 and HCC827 cell lines when treated with platinum complexes. Complexes 35, 37 and 38 were more potent than the clinically approved drug, cis-platin.Item Open Access Identification of the Cu(II) Coordinating Residues in the Prion Protein by Metal-Catalyzed Oxidation Mass Spectrometry: Evidence for Multiple Isomers at Low Cu(II) Loadings(East Carolina University, 2008-09-02) Srikanth, Rapole; Wilson, Jonathan; Burns, Colin Sanderson; Vachet, Richard W.While the Cu(II) binding sites of the prion protein have been well studied under Cu-saturation conditions, the identity of the residues involved in coordinating Cu(II) at low stoichiometries and the order in which the binding sites load with Cu(II), remain unresolved. In this study, we have used two mass spectrometry based methods to gather insight into Cu(II)-prion binding under different stoichiometric loadings of Cu(II). The first method uses metal-catalyzed oxidation reactions to site specifically modify the residues bound to Cu(II) in solution, and the second method determines Cu binding sites based on the protection of His from modification by diethyl pyrocarbonate when this residue binds Cu(II) in solution. For both methods, the residues that are labeled by these reactions can then be unambiguously identified using tandem mass spectrometry. Upon applying these two complementary methods to a construct of the prion protein that contains residues 23-28 and 57-98, several noteworthy observations are made. Coordination of Cu(II) by multiple His imidazoles is found at 1:1 and 1:2 PrP:Cu(II) ratios. Notably, there appear to be four to seven isomers of this multiple histidine coordination mode in the 1:1 complex. Furthermore, our data clearly show that His96 is the dominant Cu(II) binding ligand, as in every isomer His96 is bound to Cu(II). The individual octarepeat binding sites begin to fill at ratios of 1:3 PrP:Cu(II) with no clear preference for the order in which they load with Cu(II), although the His77 octarepeat appears to saturate last. The existence of several ‘degenerate’ Cu binding modes at low PrP:Cu(II) ratios may allow it to more readily accept additional Cu(II) ions, thus allowing PrP to transition from a singly Cu(II) bound state to a multiply Cu(II) bound state as a function of cellular Cu(II) concentration. Originally published Biochemistry, Vol. 47, No. 35, Sep 2008