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Title: "Assembly of Multi-Walled Carbon Nanotube Mats Through Covalent Cross-Linking".
Creator: Ventura, Darryl N., Kroto, Harold, Wang, Ben, Strouse, Geoffrey, Goldsby, Kenneth, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: This work describes a simple route for the production of carbon nanotube mats without high pressure processing or irradiation techniques that are generally used to produce Buckypaper. The Michael addition pathway was used to covalently cross-link thiol functionalized multi-walled carbon nanotubes with benzoquinone to produce high content nanotube mats of various thicknesses and diameters. The mats were characterized by a variety of techniques including X-ray photoelectron spectroscopy,...
Show moreThis work describes a simple route for the production of carbon nanotube mats without high pressure processing or irradiation techniques that are generally used to produce Buckypaper. The Michael addition pathway was used to covalently cross-link thiol functionalized multi-walled carbon nanotubes with benzoquinone to produce high content nanotube mats of various thicknesses and diameters. The mats were characterized by a variety of techniques including X-ray photoelectron spectroscopy, tensile strength measurements as well as qualitative structural analysis through scanning electron microscopy. It was found that the weight ratio for optimum cross-linking to be ca. 5:1 (benzoquinone:MWCNT-SH) and that the mat surface can be further functionalized with nanoparticles to form advanced carbon composite materials.
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Date Issued: 2009
Identifier: FSU_migr_etd-4582
Format: Thesis

Title: The 3D Solution Structure of the C Terminal Domain of Diphtheria Toxin Repressor: in the Free and Bound Forms.
Creator: Wylie, George P., Logan, Timothy M., Fajer, Piotr G., Blaber, Michael, Dalal, Naresh, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Diphtheria toxin repressor protein (DtxR) is a 226 amino acid protein that regulates the genes for iron uptake in Corynebacterium diphtheria and also regulates the Diphtheria toxin production. The known functions of this protein include binding divalent metals, dimerazation, and DNA binding. All these functions are accounted for by the N terminal domain of the protein. The C terminal domain was not well defined in early crystal structures but by 2000 both crystallography and NMR agreed that...
Show moreDiphtheria toxin repressor protein (DtxR) is a 226 amino acid protein that regulates the genes for iron uptake in Corynebacterium diphtheria and also regulates the Diphtheria toxin production. The known functions of this protein include binding divalent metals, dimerazation, and DNA binding. All these functions are accounted for by the N terminal domain of the protein. The C terminal domain was not well defined in early crystal structures but by 2000 both crystallography and NMR agreed that the C terminal domain has an SH3 like fold. This has led us to investigate the possible role of the C terminal domain as a "switch" for the activation of DtxR. We propose that the C terminal domain binds to the linker between the N and C terminal domains of this protein and stabilizes the monomeric form of DtxR. Once this region is released by the C terminal domain the N terminal domain most have some sort of "folding event" then metal is bound and dimerazation can take place. To investigate the mechanism of binding to this linker region by the C terminal domain two protein constructs were made one from residues D144-L226 and the other from D110-L226. The first construct would be the Free form and the second would be the bound form thus given us insight into the mechanism of binding. Here the 3D solution structures of these two domains and a comparison is presented.
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Date Issued: 2003
Identifier: FSU_migr_etd-0689
Format: Thesis

Title: 5-Endo-Dig Cyclization of a Carbon-Centered Radical and Utility of Cyclopentene Bromosulfone Product.
Creator: Abrams, Jason Nathaniel, Alabugin, Igor, Landing, William, Dudley, Gregory, Miller, Brian, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: The Baldwin rules provide a robust guideline for predicting the favorability of ring closure of reactive intermediates based upon stereoelectronic considerations. Our group was intrigued by the lack of examples of 5-endo-dig cyclizations with carbon-centered radicals, particularly because these reactions were suggested to be favorable according to the Baldwin rules and to our previous computational investigations using density functional analysis. We therefore set out to fill the gap in the...
Show moreThe Baldwin rules provide a robust guideline for predicting the favorability of ring closure of reactive intermediates based upon stereoelectronic considerations. Our group was intrigued by the lack of examples of 5-endo-dig cyclizations with carbon-centered radicals, particularly because these reactions were suggested to be favorable according to the Baldwin rules and to our previous computational investigations using density functional analysis. We therefore set out to fill the gap in the arsenal of radical C-C bond forming processes by using computational data to design a new radical process. The first part of this thesis describes our studies aimed at the discovery of the first efficient 5-endo-dig cyclization of a carbon-centered radical. This is followed by experimental design and synthesis of substrates and finally reaction conditions which yield products through this novel mode of cyclization. The second part of this thesis explores the synthetic utility of the cyclized cyclopentene bromosulfone products. First, background information for the preparation and utility of vinyl sulfones is provided. This is followed by our results for derivatization of the bromide functional group of our cyclopentene bromosulfone products. Proper design of substrates and reaction conditions has allowed the 5-endo-dig radical cyclizations to finally become an experimental reality after more than forty years since the original prediction. The cyclized products which are enriched with functionality have been transformed into a variety of other products, emphasizing the importance of this discovery.
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Date Issued: 2009
Identifier: FSU_migr_etd-0105
Format: Thesis

Title: ⁵⁵Mn NMR and Relaxation in Single Crystals of Mn₁₂-Ac and Analogs.
Creator: Harter, Andrew, Dalal, Naresh, Brooks, James, Marshall, Alan, Reyes, Arneil, Steinbock, Oliver, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: This dissertation presents the first single crystal 55Mn NMR characterization of three compounds related to Mn12-acetate [Mn12O12(O2CCH3)16(H2O)4]× 2CH3COOH-4H2O (henceforth Mn12-Ac) that have come to be known as Single-Molecule Magnets (SMMs). This study was undertaken because they exhibit novel phenomena such as quantum mechanical tunneling of their magnetization (QTM), the origin of which is still not fully understood, and also because they have the potential to form elements of magnetic...
Show moreThis dissertation presents the first single crystal 55Mn NMR characterization of three compounds related to Mn12-acetate [Mn12O12(O2CCH3)16(H2O)4]× 2CH3COOH-4H2O (henceforth Mn12-Ac) that have come to be known as Single-Molecule Magnets (SMMs). This study was undertaken because they exhibit novel phenomena such as quantum mechanical tunneling of their magnetization (QTM), the origin of which is still not fully understood, and also because they have the potential to form elements of magnetic memory storage at the molecular dimensions. The investigations herein involve studies related to both the bonding as well as spin-dynamics in these compounds to much higher precision than in earlier work. These experiments were made possible by the design of a high frequency goniometer probe and a 3He temperature facility. The first single crystal NMR of any Mn12-based molecule was conducted on [Mn12O12(O2CCH2Br)16(H2O)4]-4CH2Cl2 (Mn12-BrAc). Its 55Mn NMR spectrum, field dependence, angular dependence, and spin-lattice relaxation time (T1) measurements were conducted. Most importantly, data are presented that (a) confirm the alteration of the magnetic core of these molecules when the samples are crushed into powder (a practice used in earlier studies), (b) show the presence of transverse hyperfine fields at the nuclear site, and (c) do not yield any evidence of temperature independent relaxation below 1 K, suggesting that QTM is not the dominant relaxation mechanism at these temperatures, in contrast to earlier studies. Data from single crystals of Mn12-Ac, the most studied SMM, concur with previous x-ray findings in that isomers are present. Such detailed information was not obtainable with powder samples. T1-1 measurements over 400 mK – 1 K indicate the existence of an energy barrier, in this case ~1.65 K, which does not fit the current understanding of the electronic energy diagram. This value supports an earlier, yet unexplained observation of such a level by inelastic neutron scattering. [Mn12O12(O2CCH2But)16(MeOH)4]-MeOH (Mn12-t-Bu), arguably the most interesting SMM in terms of the structure of the NMR peaks, does appear to be a much cleaner sample than Mn12-Ac. Fine structure is noticed, however, in the Mn4+ peak, requiring either the addition of a quadrupole interaction or isomers to explain the splitting. The five resonances that make up the lower frequency Mn3+ group increase in width upon moving to higher frequency, a most unusual result which may also be explained by the presence of isomers. Finally, the bulky ligands contribute to this SMM having the longest relaxation time at low temperature, with no evidence for temperature independence down to 400 mK. Again, evidence was found for a barrier of 1 K. We thus arrive at three major conclusions important to the understanding of SMM systems: 1) Single crystals provide an order-of-magnitude higher spectral resolution than oriented powder samples, but also show that the powdered samples do not represent a statistical average of a crystal, 2) transverse hyperfine fields are present at the Mn4+ site, contradicting early models which predicted an isotropic hyperfine field, and 3) 55Mn spin-lattice times shows no evidence of temperature independent behavior for any of the molecules studied, in contrast to earlier experiments on powdered Mn12-Ac. This observation could be the most important one, as it may result in a reconsideration of the effective spin Hamiltonian for the electronic system if terms must be added to account for an energy level in between the ms = ±10 and ms = ±9 states, at about 1 – 2 K above the ground state
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Date Issued: 2006
Identifier: FSU_migr_etd-4224
Format: Thesis

Title: Access to Polysubstituted Heterocycles and Fluorescent Indicators from a Single Enamine Class.
Creator: Longstreet, Ashley Rose, McQuade, D. Tyler (David Tyler), Alamo, Ruﬁna G., Alabugin, Igor V., Shatruk, Mykhailo, Hanson, Kenneth G., Nowakowski, Richard S., Florida State...
Show moreLongstreet, Ashley Rose, McQuade, D. Tyler (David Tyler), Alamo, Ruﬁna G., Alabugin, Igor V., Shatruk, Mykhailo, Hanson, Kenneth G., Nowakowski, Richard S., Florida State University, College of Arts and Sciences, Department of Chemistry and Biochemistry
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Abstract/Description: Acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV) is an ongoing epidemic affecting millions of people worldwide, with the majority of those infected living in poverty. Today, multiple treatment options exist for anti-retroviral therapy (ART). However, access to ART for those living in low- to middle-income countries is hampered by the high costs of the medications. In an effort to increase the accessibility of one anti-viral, nevirapine, our group has...
Show moreAcquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV) is an ongoing epidemic affecting millions of people worldwide, with the majority of those infected living in poverty. Today, multiple treatment options exist for anti-retroviral therapy (ART). However, access to ART for those living in low- to middle-income countries is hampered by the high costs of the medications. In an effort to increase the accessibility of one anti-viral, nevirapine, our group has investigated alternative syntheses in order to lower the production cost. The majority of the nevirapine production cost is due to the polysubstituted pyridine building block, 2-chloro-3-amino-4-picoline (CAPIC), because the synthesis begins from an expensive ketone. To decrease the cost of the nevirapine process, the synthesis to CAPIC was redesigned to begin from an alkylidene malononitrile prepared from the inexpensive commodity chemicals, acetone and malononitrile. The alkylidene malononitrile is then treated with N,N-dimethylformamide dimethyl acetal (DMF-DMA) to afford an enamine intermediate. This enamine is readily cyclized under Pinner conditions with HCl to afford the polysubstituted 2-chloronicotinonitrile, a pyridine that could then be transformed into CAPIC in two steps. Unfortunately, the new proposed route to CAPIC was the low-yielding due to the dimerization of the alkylidene malononitrile during the enamine formation step. After testing a series of additives, we found that acetic anhydride in substoichiometric quantities enabled the enamine to be synthesized in quantitative yields. The acetic anhydride is presumed to be a unique acetate source that enhances the rate of the enamine formation to prevent the dimerization of alkylidene malononitrile. The method could also be applied to a number of other ylidenemalononitriles to synthesize a variety of mono- and disubstituted 2-halonicotinonitriles in moderate to high yields. To further lower the production costs to CAPIC, the newly revised synthesis from acetone to the 2-bromonicotinonitrile intermediate was implemented in a semi-continuous flow synthesis. The flow synthesis consists of first the Knoevenagel condensation between acetone and malononitrile in a basic aluminum oxide packed-bed column followed removal of any residual water by a 3 Å molecular sieve column to afford the alkylidene malononitrile. The alkylidene malononitrile is then met with a stream of DMF-DMA and acetic anhydride to form the enamine. Lastly, the enamine solution produced is added directly to a round-bottom containing acetic acid and HBr to perform the Pinner cyclization to the desired nicotinonitrile. With this process, the nicotinonitrile is produced in 69% overall yield within 2 h as opposed to over 24 h by the batch process. This process inspired our collaborators to develop a streamlined batch synthesis of the nicotinonitrile that will be implemented in an industrial setting. Overall, these improvements in the CAPIC process will enable at least a 75% decrease in the nevirapine production cost. Upon achieving a lower cost production to CAPIC, we investigated whether the proton in the Pinner cyclization could be replaced with an alternative electrophile to yield more diverse nicotinonitriles. After a deuterium incorporation experiment suggesting the mechanism of the Pinner cyclization would allow the addition of alternative electrophiles, we examined the cyclization with allyl bromide. While the original enamine only produced the desired allyl substituted nicotinonitrile in trace amounts, the ester analog of the enamine enabled a much more facile cyclization. However, the cyclization occurred with the ester to synthesize an allyl substituted α-pyrone. This prompted an investigation of the reactivity of the ester analog with other organohalides to synthesize a variety of α-pyrones. Lastly, three enamines were demonstrated to undergo a rapid amine exchange followed by a cyclization with primary amines to yield fluorescent products that were determined to be cyclic amidines by X-ray crystallography. A fluorescent "turn-on" effect is observed due to the amidines exhibiting emission intensities as high as 900 times greater than the starting enamines. The rate of cyclization is demonstrated to be substrate-dependent and simple structural variations to the enamine could both change the rate of the amine exchange/cyclization and emission wavelength. We further demonstrate the potential for one enamine to participate in biomolecule labeling applications.
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Date Issued: 2015
Identifier: FSU_migr_etd-9640
Format: Thesis

Title: Actinide Fluorides.
Creator: Chemey, Alexander Theodore, Albrecht-Schmitt, Thomas E., Tabor, Samuel L., Latturner, Susan, Hanson, Kenneth G., Florida State University, College of Arts and Sciences,...
Show moreChemey, Alexander Theodore, Albrecht-Schmitt, Thomas E., Tabor, Samuel L., Latturner, Susan, Hanson, Kenneth G., Florida State University, College of Arts and Sciences, Department of Chemistry and Biochemistry
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Abstract/Description: The periodic table of the elements, derived from numerous attempts in the 1800s to systematically examine the chemistry found in nature, has seen a stark evolution over the last century. The elements of the actinide series are the frontier of a radical discontinuity in periodicity, driven by a relativistic reorganization of the frontier orbitals. Characterization of the chemistry of these elements with simple model species enables detailed coordination between structural chemistry and...
Show moreThe periodic table of the elements, derived from numerous attempts in the 1800s to systematically examine the chemistry found in nature, has seen a stark evolution over the last century. The elements of the actinide series are the frontier of a radical discontinuity in periodicity, driven by a relativistic reorganization of the frontier orbitals. Characterization of the chemistry of these elements with simple model species enables detailed coordination between structural chemistry and theoretical characterization. Such examinations advance understanding of these relativistic shifts. With the ever-growing influence of these effects, periodic trends on the table the elements break down, and synthetic elements beyond uranium offer new opportunities to probe these divergent and nonlinear chemistries. The introductory chapter addresses the history of the synthesis and the initial chemical characterization of the transuranium elements, with a brief basic introduction given to discuss the reaction physics which will follow. These elements share a single common feature in that they all have large Z values, and thus have electronic structures that are significantly altered by both scalar relativistic effects and spin-orbit coupling. These effects scale nonlinearly with increasing Z and create unexpected deviations both across series and down groups of elements. The magnitude of these effects is large enough that orbital energies rearrange and mix in ways that complicate incomplete depictions that are based solely on electron repulsion. The second chapter discusses calculations with nuclear reaction codes to examine a potential production rate improvement that may be derived by super-heavy synthesis with reactions run in inverse kinematics. This result is particularly important for continuing the evolution of the periodic table, as increased production rates enable new spectroscopic analyses of electronic and nuclear structure that can, in future years, potentially rewrite our understanding of relativity at the most extreme scale heretofore produced. The third chapter discusses new structural, spectroscopic, and theoretical features of uranium fluorides that are relevant for fundamental uranium(IV) chemistry and actinide fluoride reactors. The simple system of tetraammonium octafluorouranate is employed to derive fundamental understanding of the uranium-fluorine interaction. The structure is composed of isolated molecules, enabling a clear look at the U4+ (f 2) species in the most ionic bonding environment possible, without the possibility of direct interactions or strong interactions through ligands between uranium centers. Characterization of single-crystals by x-ray diffraction, absorption spectroscopy, and magnetic analysis up to 45 T is interwoven with extensive theoretical treatment by CASSCF. The influence of different active spaces and representations of the structure is examined in the context of the experimental evidence. The Interacting Quantum Atoms method is used to examine the nature of the U-F bond, concluding that there is a non-negligible degree of covalent character (9% of the total bond energy) even with the most ionic simple anion of fluoride. Two new sodium uranium(IV) pentafluorides were synthesized from uranium dioxide, HF, and NaF under mild hydrothermal conditions. Although β-NaUF5·H2O crystallizes in the (lower) monoclinic crystal class, it possesses greater crystal lattice energy than the previously-known orthorhombic α-NaUF5·H2O. Trigonal β-NaUF5 possess significantly different bonding between [UF9]5- moieties than the α-phase, with higher symmetry and greater lattice energy than its orthorhombic structural isomer, which is most directly comparable in structure to Na3,4M2+/3+U6F30. Single-crystal absorption spectra of these compounds are reported and correlated. Simulated powder x-ray diffraction data are also reported and compared to address a (mis)identification of the NaUF5 series that dates back to the Manhattan Project. The final textual chapter extends the methods discussed in previous chapters to the actinide series, with new lithium plutonium fluorides analyzed and placed into the broader context of actinide fluorides, including a new evaluation of an analogous zirconium structure. The structure of Li4ZrF8 was refined from single crystal X-ray diffraction data. Alkali zirconium fluorides are important in nuclear-relevant technologies, and zirconium is commonly employed as an analogue for tetravalent f-block elements. This result is largely consistent with prior reports, but with significant improvements in precision. The similar reaction of 242PuO2 with HF and LiF under hydrothermal conditions results in the formation of Li4PuF8 and LiPuF5. These compounds were structurally characterized using single crystal X–ray diffraction and UV–vis–NIR absorption spectroscopy. The structure of Li4PuF8 consists of [PuF8]4‒ anions that adopt a non-bridged bicapped trigonal prismatic geometry with approximate C2v symmetry. In contrast, LiPuF5 forms a dense three–dimensional network constructed from [PuF9]5‒ units that are bridged by fluoride anions. The Pu4+ cations are found within tricapped trigonal prisms. Extensive theoretical analysis of electronic and bonding interactions is included with comparison between results derived from CASSCF at different levels of theory, QTAIM, IQA, NLMO, and WBO analyses. Covalent interactions in these compounds are examined and intra–molecular trends in covalent and electrostatic interactions are discussed.
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Date Issued: 2019
Identifier: 2019_Fall_Chemey_fsu_0071E_15478
Format: Thesis

Title: Activation Profiles and Regulatory Cascades of the Human Kallikrein-Related Peptidases.
Creator: Yoon, Hyesook, Blaber, Michael, Tang, Hengli, Miller, Brian, Steinbock, Oliver, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: This work describes the activation profiles and regulatory cascades of the human kallikrein-related peptidases. The human kallikrein (KLK)-related peptidases are the largest family of serine peptidases, comprising 15 members (KLK1–15) and with the majority (KLK4–15) being identified only within the last decade. Members of this family are associated with important diseased states (including cancer, inflammation, and neurodegeneration) and have been utilized or proposed as clinically important...
Show moreThis work describes the activation profiles and regulatory cascades of the human kallikrein-related peptidases. The human kallikrein (KLK)-related peptidases are the largest family of serine peptidases, comprising 15 members (KLK1–15) and with the majority (KLK4–15) being identified only within the last decade. Members of this family are associated with important diseased states (including cancer, inflammation, and neurodegeneration) and have been utilized or proposed as clinically important biomarkers or therapeutic targets of interest. All human KLKs are synthesized as prepro-forms that are proteolytically processed to secreted pro-forms via the removal of an amino-terminal secretion signal peptide. The secreted inactive pro-KLKs are then activated extracellularly to mature peptidases by specific proteolytic release of their amino-terminal propeptide. Although a key step in the regulation of KLK function, details regarding the activation of the human pro-KLKs are unknown, to a significant extent, but have been postulated to involve "activation cascades" with other KLKs and endopeptidases. To characterize more completely the KLK activation cascades, an individual KLK propeptides fused to the amino terminus of a soluble carrier protein have been expressed from Escherichia coli. The ability of 14 different mature KLKs to process the 15 different pro-KLK peptide sequences has been determined. Various autolytic and cross activation relationships identified using this system have subsequently been characterized using recombinant pro-KLK proteins. The results demonstrate the potential for extensive KLK activation cascades and, when combined with available data for the tissue-specific expression of the KLK family, permit the construction of specific regulatory cascades. One such tissue-specific cascade is proposed for the central nervous system. Much recent work has been devoted to elucidating the potential for activation cascades between members of the KLK family, with physiologically relevant KLK regulatory cascades now described in skin desquamation and semen liquefaction. Despite this expanding knowledge of KLK regulation, details regarding the potential for functional intersection of KLKs with other regulatory proteases are essentially unknown. To elucidate such interaction potential, the ability of proteases associated with thromostasis to hydrolyze the pro-peptide sequences of the KLK family have been characterized, using the previously described (chapter 2) pro-KLK fusion protein system. A subset of positive hydrolysis results were subsequently quantified with proteolytic assays using intact recombinant pro-KLK proteins. The results show that proteases of the thrombostasis family can efficiently activate specific pro-KLKs, uncovering for the first time the potential for important regulatory interactions between these two major protease families. Proteases of the thrombostasis system are activated under conditions of injury, inflammation, and tissue remodeling, such as occurs in various disease states, including cancer. Activation of KLKs by thrombostasis proteases is therefore relevant for KLK activity in such diseases states.
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Date Issued: 2008
Identifier: FSU_migr_etd-0712
Format: Thesis

Title: The Active Site Cysteine of Arginine Kinase: Structural and Functional Analysis of Partially Active Mutants.
Creator: Gattis, James L., Chapman, Michael, Ellington, W. Ross, Blaber, Michael, Cross, Timothy, Marshall, Alan, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Arginine kinase buffers cellular ATP levels by catalyzing reversible phosphoryl transfer between ATP and arginine. A conserved cysteine has long been thought important in catalysis. Here, cysteine 271 of horseshoe crab arginine kinase has been mutated to serine, alanine, asparagine, or aspartate. Catalytic turnover rates were 0.02-1.0% of wild type, but the activity of uncharged mutations could be partially rescued with chloride. Steady state binding constants were slightly increased, more so...
Show moreArginine kinase buffers cellular ATP levels by catalyzing reversible phosphoryl transfer between ATP and arginine. A conserved cysteine has long been thought important in catalysis. Here, cysteine 271 of horseshoe crab arginine kinase has been mutated to serine, alanine, asparagine, or aspartate. Catalytic turnover rates were 0.02-1.0% of wild type, but the activity of uncharged mutations could be partially rescued with chloride. Steady state binding constants were slightly increased, more so for phospho-L-arginine than ADP. Substrate binding synergy observed in many phosphagen kinases was reduced or eliminated in mutant enzymes. The crystallographic structure of the alanine mutant at 2.3Å resolution, determined as a transition state analog complex with arginine, nitrate, and MgADP, was nearly identical to wild-type. Enzyme–substrate interactions are maintained as in wild-type, and substrates remain at least roughly aligned for in-line phosphoryl transfer. Homology models with serine, asparagine, or aspartate replacing the active site cysteine similarly show only minor structural changes. Most striking, however, is the presence in the C271A mutant crystallographic structure of a chloride ion within 3.5Å of the non-reactive Nη substrate nitrogen, approximating the position of the sulfur in the wild-type's cysteine. Together the results contradict prevailing speculation that the cysteine mediates a substrate-induced conformational change, confirm that it is the thiolate form that is relevant to catalysis, and suggest that one of its roles is to help enhance the catalytic rate through electrostatic stabilization of the transition state.
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Date Issued: 2004
Identifier: FSU_migr_etd-0137
Format: Thesis

Title: Addition / C-C Bond Cleavage Reactions of Vinylogous Acyl Triflates and Their Application to Natural Product Synthesis.
Creator: Jones, David Mack, Dudley, Gregory B., Taylor, Kenneth, Saltiel, Jack, McQuade, D. Tyler, Goldsby, Kenneth, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: This dissertation describes the synthetic utility of tandem addition / C-C bond cleavage reactions of vinylogous acyl triflates. The first chapter provides background into carbon-carbon bond breaking reactions that have been applied in organic synthesis and the preliminary data that allowed for the original work presented here. Chapter 2 explains the significance as well as the prior syntheses of a commercially important moth pheromone, (Z)-6-heneicosen-11-one. The second chapter culminates...
Show moreThis dissertation describes the synthetic utility of tandem addition / C-C bond cleavage reactions of vinylogous acyl triflates. The first chapter provides background into carbon-carbon bond breaking reactions that have been applied in organic synthesis and the preliminary data that allowed for the original work presented here. Chapter 2 explains the significance as well as the prior syntheses of a commercially important moth pheromone, (Z)-6-heneicosen-11-one. The second chapter culminates in the synthesis of the sex attractant through a fragmentation reaction made possible by the direct extension of the initial nucleophile-triggered fragmentation studies to include the use of Grignard reagents. Chapter 3 describes the application of the fragmentation method, coupled to a benzannulation reaction, to afford penta- and hexasubstituted indanes. This two step sequence provides the basis for future work directed toward the synthesis of alcyopterosin A, a known cytotoxic agent with possible biological applications. The current difficulties pertaining to the treatment of melanoma are discussed in Chapter 4. Recently, an exciting natural product that provides promising activity against this horrible cancer was discovered. Palmerolide A has the ability to kill melanoma cells selectively at low concentrations. The fragmentation method developed in these laboratories provides entry into a key fragment. The Claisen-type condensation reaction of vinylogous acyl triflates was expanded to the synthesis of a novel beta-ketophosphine oxide olefinating reagent, which allowed for the rapid synthesis of the eastern hemisphere (C1-C15) of this exciting natural product. Optimization of the Claisen-type condensation reaction to provide the beta-ketophosphine oxide reagent, led to the optimal reduction of the number of equivalents of the nucleophile. Intrigued by this, these reactions were explored in more detail. The results of this investigation are described in Chapter 5. The reduction in the number of equivalents of nucleophile, a key feature in these reactions, may be attributed to the ability of the phosphorus atom to form of an oxaphosphetane-like intermediate. As a result, new, potentially useful, beta-ketophosphonates were synthesized.
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Date Issued: 2010
Identifier: FSU_migr_etd-3451
Format: Thesis

Title: Adeno-Associated Virus-2 and Its Primary Cellular Receptor-Cryo-EM Structure of a Heparin Complex.
Creator: O’Donnell, Jason, Li, Hong, Chapman, Michael, Taylor, Kenneth, Safron, Sanford, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Adeno-associated virus serotype 2 (AAV-2) is a leading candidate vector for gene therapy. Cell entry starts with attachment to a primary receptor, Heparan Sulfate Proteoglycan (HSPG) before binding to a co-receptor. Here, cryo-electron microscopy provides direct visualization of the virus–HSPG interactions. Single particle analysis was performed on AAV-2 complexed with a 17 kDa heparin fragment at 8.3 Å resolution. Heparin density covers the shoulder of spikes surrounding viral 3-fold...
Show moreAdeno-associated virus serotype 2 (AAV-2) is a leading candidate vector for gene therapy. Cell entry starts with attachment to a primary receptor, Heparan Sulfate Proteoglycan (HSPG) before binding to a co-receptor. Here, cryo-electron microscopy provides direct visualization of the virus–HSPG interactions. Single particle analysis was performed on AAV-2 complexed with a 17 kDa heparin fragment at 8.3 Å resolution. Heparin density covers the shoulder of spikes surrounding viral 3-fold symmetry axes. Previously implicated, positively charged residues R448/585, R451/588 and R350/487 from another subunit cluster at the center of the heparin footprint. The footprint is much more extensive than apparent through mutagenesis, including R347/484, K395/532 and K390/527 that are more conserved, but whose roles have been controversial. It also includes much of a region proposed as a co-receptor site, because prior studies had not revealed heparin interactions. Heparin density bridges over the viral 3-fold axes, indicating multi-valent attachment to symmetry-related binding sites.
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Date Issued: 2009
Identifier: FSU_migr_etd-2459
Format: Thesis

Title: Advanced Analytical Characterization of Molecular Changes Occurring in the Dissolved Organic Matter of Oxidized Landfill Leachate.
Creator: Osborne, Daniel M., Cooper, William T., Locke, Bruce R., Dorsey, John G., Safron, Sanford A., Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: The industrialization and growth of the world population has consequently led to the generation of larger quantities of both industrial and municipal wastes. Modern landfills are designed to sequester municipal solid wastes into a compact area and protect the surrounding environment from any hazardous materials. Precipitation, irrigated water, and the inherent moisture in the waste filter through the landfill absorbing inorganic and organic matter, and this resulting liquid waste is known as...
Show moreThe industrialization and growth of the world population has consequently led to the generation of larger quantities of both industrial and municipal wastes. Modern landfills are designed to sequester municipal solid wastes into a compact area and protect the surrounding environment from any hazardous materials. Precipitation, irrigated water, and the inherent moisture in the waste filter through the landfill absorbing inorganic and organic matter, and this resulting liquid waste is known as landfill leachate. As the water filters through the various layers of a waste material, pollutants are transferred to the leachate through a variety of different processes. Four groups of pollutants are present in varying compositions in all leachates, and they are: inorganic macrocomponents, heavy metals, xenobiotic organic carbons (XOCs), and dissolved organic matter (DOM). DOM is typically quantified by chemical oxygen demand (COD) or total organic carbon (TOC) measurements. As a landfill matures, the leachate that is produced becomes increasingly stabilized and biodegradability decreases, and the ratio of the biological oxygen demand over 5 days (BOD5) to the COD is used to quantify the biodegradability of a sample. Biological, physical and chemical methods can all be used to remove COD and increase BOD5/COD ratios, making the effluent more suitable for domestic wastewater treatment plants. Chemical oxidation of DOM with ozone can occur through direct molecular reactions or indirect pathways that result in the formation of hydroxyl radicals. Techniques where large concentrations of hydroxyl radicals are generated are known as advanced oxidation processes (AOPs), and one such AOP involves the photolysis of ozone with ultraviolet (UV) light. While COD and BOD5/COD are suitable methods for monitoring the effectiveness of landfill leachate treatment, they do not yield any molecular information regarding changes that occur to the complex DOM mixture during ozone or ozone/UV treatments. This work describes the use of ultraviolet/visible absorption spectroscopy (UV/Vis), excitation-emission matrix fluorescence spectroscopy (EEM), and size exclusion chromatography (SEC). These techniques were utilized to determine bulk properties of the DOM found in untreated landfill leachate and leachate treated using the following four reaction modes: aeration, aeration/UV, ozonation, and ozonation/UV. These reactions were carried out in a home-built bubble column reactor. The UV/Vis data yielded information about the relative size of the DOM components and the SEC experiments made it possible to calculate and compared average molecular weights and distribution. The EEM plots generated data about the types of fluorophores found in the DOM and how these fluorophores were affected by the given treatments. Ultrahigh resolution negative ion electrospray ionization Fourier transform-ion cyclotron resonance mass spectrometry (ESI FT-ICR MS) was used to determine molecular level information about the DOM components of both the raw and treated leachates. Spectral slope and absorbance ratios from the UV spectra of the treated leachates indicated that the ozone and ozone/UV treatments both reduced the relative size of the DOM components. The EEM plots showed that over 90% of DOM fluorescence was removed over the course of the two hour ozone and ozone/UV treatments. Also, integrated areas of fluorophores with the EEM plot suggested that aromatic, humic-like fluorophores may be preferentially oxidized during these treatments. Weight average molecular weights and polydispersities were lowered following both of the ozone methods, confirming that the DOM molecules were smaller after treatment and indicating that the size was also becoming more uniform. Molecular level information from the ESI FT-ICR MS experiments supported the findings from the other bulk property measurements. The average number of carbons in each molecule and the nominal mass were shown to decrease after oxidation. Double bond equivalence (DBE) and DBE-oxygen (DBE-O) distributions were lower for the treated samples. However, the shift in DBE-O was larger, suggesting that the oxidized DOM contained more oxygen, and elemental ratios supported these findings. Molecules that were less saturated and had lower O/C ratios were preferentially "removed" and DOM molecules with higher O/C ratios were "formed" during the both ozone reactions. Sulfur-containing molecules were also preferentially removed which may suggest that the sulfur moieties are on the edges of the DOM and not part of the backbone of the molecule.
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Date Issued: 2011
Identifier: FSU_migr_etd-5877
Format: Thesis

Title: Advances in Mechanistic Photochemistry: Dienes and Trienes.
Creator: Redwood, Christopher Evan, Saltiel, Jack, Cogan, Nicholas G., Alabugin, Igor V., Hilinski, Edwin F., Stagg, Scott, Florida State University, College of Arts and Sciences,...
Show moreRedwood, Christopher Evan, Saltiel, Jack, Cogan, Nicholas G., Alabugin, Igor V., Hilinski, Edwin F., Stagg, Scott, Florida State University, College of Arts and Sciences, Department of Chemistry and Biochemistry
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Abstract/Description: Found throughout nature, science, industry, and medicine, conjugated dienes and trienes are ubiquitous. While their behavior changes upon substitution, they are all built from the same fundamental units of either 1,3-butadiene or the 1,3,5-hexatrienes. These basic units undergo fascinatingly complex photochemistry, which is mimicked or changed upon substitution. Developing a proper understanding the photochemistry of these fundamental units, and the reasons for divergence from their basic...
Show moreFound throughout nature, science, industry, and medicine, conjugated dienes and trienes are ubiquitous. While their behavior changes upon substitution, they are all built from the same fundamental units of either 1,3-butadiene or the 1,3,5-hexatrienes. These basic units undergo fascinatingly complex photochemistry, which is mimicked or changed upon substitution. Developing a proper understanding the photochemistry of these fundamental units, and the reasons for divergence from their basic behavior upon substitution, enables their use in optical applications and provides evidence which can be used to advance modern molecular quantum mechanics. The compounds studied are the 1,4-diphenyl-1,3-butadienes, the isomers of Vitamin D3, and the 1,3,5-hexatrienes. These compounds undergo reaction upon excitation with ultraviolet light that causes bond breakage, reordering, and reorganization. The primary process studied herein is the cis-trans photoisomerization of double bonds. This process is described primarily through the application of the one bond twist, hula-twist, and bicycle pedal photoisomerization mechanisms. Significant debate focused on finding a unifying explanation to excited state cis-trans photoisomerization currently surrounds these mechanisms. The reader is encouraged to access the abstract of the electronic dissertation to view the movies modeled for these cis-trans photoisomerizations, which are described later on page 63 of the dissertation. This dissertation is split between three parts. First, the photoisomerization of the 1,4-diphenyl-1,3-butadienes was studied in ethanol to search for evidence of conical intersections, which are considered equivalent to transition states in photochemical reactions. Second, since the validity of the hula-twist mechanism has been questioned on many occasions in the literature, the prima facie evidence used for its justification, the photoisomerizations of Previtamin D3 in volume confining media, were reinvestigated using more powerful methods. To conclude this work, the photoisomerizations of the 1,3,5-hexatrienes were reexamined, as no experimental evidence currently accounts for the majority of their photochemical decay. Surprisingly, results provided prior to this work account for less than 10% of the hexatrienes excited state decay. Extracting the information contained in this dissertation required the development and application of unique and sophisticated spectral decomposition techniques for UV-Vis, fluorescence, fluorescence excitation, and 1H-NMR spectroscopies. The results of this work suggest that conical intersections exist close to the twisted excited singlet geometries of the 1,4-diphenyl-1,3-butadienes, show that the original experimental evidence for the hula-twist mechanism was based upon a misinterpretation of the photoisomerizations of Previtamin D3, and reveal that cis-trans photoisomerization accounts for nearly all of the excited state decay of the 1,3,5-hexatrienes in solution. The impact of the first study reconciles previous reports for the photoaddition of alcohol to the 1,4-diphenyl-1,3-butadienes under a general path of addition to a short lived phantom intermediate. This mechanism is likely general and extends to similar molecules which undergo photoaddition of alcohol in the singlet state. It additionally confirms that the twisted singlet intermediate of the 1,4-diphenyl-1,3-butadienes is zwitterionic in nature, and that the bicycle pedal photoisomerization mechanism occurs in a step-wise and not concerted manner, in certain circumstances. The second study raises concern regarding the validity of the conclusions from numerous studies which reported hula-twist products, and perhaps should be reconsidered as photoisomerizations from one bond twist or bicycle pedal precursors. The concluding study signifies need for renewed interest in the photochemistry of the 1,3,5-hexatrienes by theory. The latest theoretical studies on the matter are not in harmony with the recently obtained experimental evidence.
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Date Issued: 2016
Identifier: FSU_2016SP_Redwood_fsu_0071E_13189
Format: Thesis

Title: Adventures in Gold-Catalyzed Cascade Reactions and Rearrangements.
Creator: Vidhani, Dinesh V. (Dinesh Vinod), Krafft, Marie E., Houpt, Thomas A., Holton, Robert A., Dudley, Gregory B., Goldsby, Kenneth A., Department of Chemistry and Biochemistry,...
Show moreVidhani, Dinesh V. (Dinesh Vinod), Krafft, Marie E., Houpt, Thomas A., Holton, Robert A., Dudley, Gregory B., Goldsby, Kenneth A., Department of Chemistry and Biochemistry, Florida State University
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Abstract/Description: Gold-catalyzed organic transformations have been a hot topic of research in synthetic organic community over the last few years. Amazingly, most of the reactions can be performed under mild conditions using a catalytic amount of gold complexes. One of the widely reported reactions using gold catalysts is heterocyclization that involves an activation of a pi-system followed by a nucleophilic attack by a hetero atom. The Nazarov reaction is another important class of reactions which has gained...
Show moreGold-catalyzed organic transformations have been a hot topic of research in synthetic organic community over the last few years. Amazingly, most of the reactions can be performed under mild conditions using a catalytic amount of gold complexes. One of the widely reported reactions using gold catalysts is heterocyclization that involves an activation of a pi-system followed by a nucleophilic attack by a hetero atom. The Nazarov reaction is another important class of reactions which has gained considerable attention in the past few years. Mechanistic studies performed by the Frontier group helped to shed light on the Nazarov reaction of alpha, alpha'-activated substrates under catalytic conditions. We were able to combine the concept of gold-catalyzed furan formation with the Nazarov reaction of alpha-alkoxy substrates using gold(III) catalysts. Under mild reaction conditions, in the presence of gold(III) catalysts, alpha-alkoxy,alpha'-alkynyl divinyl ketones underwent a heterocyclization triggered Nazarov cascade reaction to give a synthetically important fused bicyclo compounds. A solvent dependent mechanistic dichotomy was observed while performing these reactions. Moreover, a computational study was undertaken to decipher the mechanism underlying the cascade process. The above study of the cascade heterocyclization-Nazarov reaction led to the discovery of coordinating preferences of the gold(I) cation in the presence of electronically different pi-systems. This result was in contrast to the commonly believed concept of gold(I) cation complexing to the alkyne moiety in the presence of other pi-systems. An exhaustive computational and experimental study suggested that our hypothesis was based on sound scientific principles. The coordinating preference of gold(I) to vinyl ethers, found during the above investigation, prompted us to examine the mechanism of the gold-catalyzed propargyl Claisen rearrangement reported by the Toste group. We were surprised to find that all the reactions worked extremely fast at room temperature in presence of various gold(I) catalysts. However, when reactions were performed at low catalyst concentration, we found that there was a significant substituent effect from the groups attached to the carbinol carbon. A further inquiry into the mechanism of this reaction led us to a breakthrough that changed the perception of the previously conceived mechanism. The reaction was catalyzed by two gold(I) cations as opposed to generally believed one gold(I) cation. An investigation into the electronic nature of gold(I) catalysts was undertaken using propargyl Claisen rearrangement as a probe. The electronic nature of gold(I) was explored as a function of reaction rate observed for different propargyl Claisen substrates. Different gold catalysts were studied through this reaction based on the nature of the ligand and anion attached on the gold. To our best knowledge, we are the first to take steps towards tuning gold(I) catalysts to perform functional group specific reactions.
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Date Issued: 2010
Identifier: FSU_migr_etd-4536
Format: Thesis

Title: The Afferent Circuitry of the Ventromedial Hypothalamus and Its Activation in Paternal Behavior of the Socially Monogamous Prairie Vole.
Creator: Rogers, Richard S., Department of Chemistry and Biochemistry
Abstract/Description: Paternal behavior is an interesting and important research topic due to its integral contribution to the fitness and well-being of multiple species, including humans. Although paternal behavior is well described in literature, attempts at neurobiological characterization have yielded conflicting results that fail to address brain region interconnectivity. This study was designed to evaluate the relationship between afferent VMH circuitry and the onset of paternal behavior, using the prairie...
Show morePaternal behavior is an interesting and important research topic due to its integral contribution to the fitness and well-being of multiple species, including humans. Although paternal behavior is well described in literature, attempts at neurobiological characterization have yielded conflicting results that fail to address brain region interconnectivity. This study was designed to evaluate the relationship between afferent VMH circuitry and the onset of paternal behavior, using the prairie vole (Microtus ochrogaster) model. Sexually naïve male prairie voles received injections of the retrograde neurotracer Fluoro-Gold (FG), into the VMH. Two weeks later, subjects were exposed to either conspecific pups, contained within a tea-ball, or an empty tea-ball (control) for 1 hr. Immunohistochemical labeling was conducted for both FG and the neuronal activity marker Egr-1, in order to evaluate neuronal and afferent pathway activation between the ventromedial hypothalamus (VMH) and the amygdala (AMYG), bed nucleus of the stria terminalis (BNST), lateral septum (LS) and ventral tegmental area (VTA). Similar to the pathway implicated in the onset of maternal behavior, the results of this study showed pup exposure-induced neuronal activation in the AMYG and BNST, particularly in the efferent pathways from these two brain areas to the VMH. This effect was not found in the LS and VTA projection neurons to the VMH. Together, the data suggests a brain region-specific neuronal activation by pup exposure in particular brain circuitry, implicating its possible involvement in paternal behavior.
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Date Issued: 2015
Identifier: FSU_migr_uhm-0545
Format: Thesis

Title: Allosteric Activation of Human Glucokinase.
Creator: Bowler, Joseph M., Miller, Brian G., Stefanovic, Branko, Logan, Timothy M., Yang, Wei, Zhu, Lei, Florida State University, College of Arts and Sciences, Department of Chemistry...
Show moreBowler, Joseph M., Miller, Brian G., Stefanovic, Branko, Logan, Timothy M., Yang, Wei, Zhu, Lei, Florida State University, College of Arts and Sciences, Department of Chemistry and Biochemistry
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Abstract/Description: The hexokinase family of enzymes catalyzes the ATP-dependent phosphorylation of glucose to generate glucose 6-phosphate and ADP. Consistent with highly evolved catalysts, hexokinases I-III possess high apparent substrate affinities (low Km) as a result of specificity optimization. Hexokinase IV, commonly known as glucokinase (GCK), has a low apparent affinity for its physiological substrate glucose and is not significantly inhibited by the reaction product. Interestingly, it displays...
Show moreThe hexokinase family of enzymes catalyzes the ATP-dependent phosphorylation of glucose to generate glucose 6-phosphate and ADP. Consistent with highly evolved catalysts, hexokinases I-III possess high apparent substrate affinities (low Km) as a result of specificity optimization. Hexokinase IV, commonly known as glucokinase (GCK), has a low apparent affinity for its physiological substrate glucose and is not significantly inhibited by the reaction product. Interestingly, it displays positively cooperative kinetic behavior while functioning exclusively as a monomer with a single glucose binding site. A lag in steady-state saturation curves places the inflection point in the range of physiological blood glucose levels, allowing activity to be highly tunable in this region. Fasting and fed states can therefore dictate the rate of glucose phosphorylation in the reaction that represents the rate-limiting factor of insulin release and glycogen synthesis. The most compelling evidence for the pivotal role of GCK in glucose homeostasis was the discovery of mutations in the glk gene that result in long-term glycemic complications. Established as a central regulator of blood glucose, efforts to target GCK in the treatment of diabetic disorders resulted in the development of glucokinase activators. Small-molecule activators of GCK bind to an allosteric site and increase the enzyme's apparent affinity for glucose (K0.5). Effects on turnover (kcat) and cooperativity (Hill coefficient) can vary widely, and these differences have remained largely uninvestigated. Conflicting kinetic models have been proposed in the literature that disagree as to whether glucose binding is pre-requisite to activator association. Our kinetic activity assays, obtained via stopped-flow, suggest that activators can indeed associate with some population of GCK in the absence of glucose. To investigate the differing effects on turnover by activators, we performed viscosity variation assays. We found that the kcat of GCK is partly dependent on some diffusion-limited process, such as product release. In the presence of different activators, whose effects on turnover varied from unchanged to augmented 30%, we observed a significant change in dependence for just one of the three compounds tested. Compound A increased kcat by 10% and showed a nearly complete dependence upon solvent viscosity, suggesting that the activator functions by accelerating some preceding event that makes product release the determinant for turnover. The precise in vivo regulation of GCK and the presence of its highly effective allosteric activation site have led some to speculate the existence of an endogenous activator. Such a molecule would likely provide a safe scaffold in the design of future GCK therapeutics. We tested a variety of biogenic compounds using linked-enzyme assays for activating properties. When these tests proved unsuccessful, we developed a methodology that would provide the ability to genetically select for activating cyclic peptides among a library of 106 unique members. We combined a proven genetic selection system devised in-house with a uniquely engineered plasmid construct that generates randomized cyclic peptides in vivo. The methodology was optimized to be sensitive to the presence of GCK activation. In addition, we describe alternative approaches and ongoing efforts on this front.
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Date Issued: 2014
Identifier: FSU_migr_etd-9147
Format: Thesis

Title: Alternative Electrophiles in the Morita−Baylis−Hillman Reaction.
Creator: Seibert, Kimberly A., Krafft, Marie E., Cronin, J. Joseph, Holton, Robert A., Dudley, Gregory B., Goldsby, Kenneth, Department of Chemistry and Biochemistry, Florida State...
Show moreSeibert, Kimberly A., Krafft, Marie E., Cronin, J. Joseph, Holton, Robert A., Dudley, Gregory B., Goldsby, Kenneth, Department of Chemistry and Biochemistry, Florida State University
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Abstract/Description: Discovering new carbon-carbon bond forming reactions is not only fundamental for the construction of organic molecular frameworks but is vital for the advancement of synthetic organic chemistry. The Morita–Baylis–Hillman reaction (MBH), dating back to both German and Japanese patents, is an organocatalytic three component reaction involving the coupling of the α-position of activated alkenes with carbon electrophiles under the catalytic influence of a nucleophilic species, providing a simple...
Show moreDiscovering new carbon-carbon bond forming reactions is not only fundamental for the construction of organic molecular frameworks but is vital for the advancement of synthetic organic chemistry. The Morita–Baylis–Hillman reaction (MBH), dating back to both German and Japanese patents, is an organocatalytic three component reaction involving the coupling of the α-position of activated alkenes with carbon electrophiles under the catalytic influence of a nucleophilic species, providing a simple and convenient method for the synthesis of densely functionalized molecules. The reaction mechanism is believed to proceed through a Michael type addition-elimination sequence in which the nucleophilic catalyst undergoes a reversible Michael-type nucleophilic addition to an activated alkene to form a zwitterionic enolate. Nucleophilic addition of this zwitterionic enolate to a carbonyl electrophile in an aldol fashion generated a second zwitterionic intermediate. Subsequent proton migration and release of the catalyst furnished the desired product. Although the Morita-Baylis-Hillman reaction was discovered in the late 1960's, it was not until the early 1980's when researchers began to study this reaction more thoroughly. Since then, the intermolecular Morita-Baylis-Hillman reaction has seen an exponential growth in terms of all three essential components and now encompasses a wide variety of activated alkenes, electrophiles and nucleophilic catalysts. However there has been significantly less research into the intramolecular Morita-Baylis-Hillman reaction. This, coupled with the fact that simple allylic electrophiles or unactivated alkyl halides have not been used as the electrophilic partner in this intriguing reaction, has influenced my research, which is aimed at extending the scope of the intramolecular Morita-Baylis-Hillman reaction with particular interest to using new electrophiles. Recently Krafft and Haxell reported a Morita-Baylis-Hillman reaction using allylic chlorides as the electrophilic partner. It occurred to us that a natural extension of this work was to explore the feasibility of the related cycloalkylation chemistry using sp3-hybridized electrophiles. We have successfully developed a novel, entirely organomediated, one-pot convenient method for the synthesis of cyclic enones through the use of an alternative electrophile in the Morita-Baylis-Hillman reaction. The method tolerates both alkyl- and aryl-enones with and without various substitution patterns on the tether to generate five- and six-membered rings in excellent yields. We have further expanded the method to proceed using only catalytic amounts of the tributylphosphine nucleophile with little to no reduction in yields. We have extended this reaction to include thioesters as viable activated alkenes providing the cyclized MBH adducts in good to excellent yields. We have further shown the thioester substrates to form the desired products when a catalytic amount of PBu3 is employed. Additionally, we have isolated for the first time a Morita-Baylis-Hillman intermediate exhibiting an unprecedented trans-geometry of the positively charged phosphorous and negatively charged oxygen. Our results suggested the interaction of the oxygen and phosphorous, which has been long understood to provide stabilization for the intermediate, is not a requirement for a successful MBH alkylation providing new insight into the Morita-Baylis-Hillman mechanism.
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Date Issued: 2007
Identifier: FSU_migr_etd-0281
Format: Thesis

Title: Analysis of the Inter- and Intraparticle Obstruction Factors in Size-Exclusion Chromatography.
Creator: Richard, Dustin Joseph, Striegel, Andre, Chella, Ravi, Logan, Timothy, Roper, Michael, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Size-exclusion chromatography (SEC) is a separation technique commonly used for size and molar mass analysis. The primary mechanism of separation by SEC is not by enthalpic interactions, like in such techniques as gas chromatography (GC) or reversed-phase liquid chromatography (RPLC), but instead by an entropic mechanism. This entropic mechanism of separation can be beneficial when used to determine and understand diffusion, or obstructed diffusion, during a chromatographic separation. The...
Show moreSize-exclusion chromatography (SEC) is a separation technique commonly used for size and molar mass analysis. The primary mechanism of separation by SEC is not by enthalpic interactions, like in such techniques as gas chromatography (GC) or reversed-phase liquid chromatography (RPLC), but instead by an entropic mechanism. This entropic mechanism of separation can be beneficial when used to determine and understand diffusion, or obstructed diffusion, during a chromatographic separation. The lack of enthalpic interactions allows for easier isolation of individual contributions of chromatographic peak broadening, which is primarily caused by analyte diffusion. Measuring changes in certain band broadening contributions under various conditions allows for the determination of the effective diffusion coefficient, or hindered diffusion due to the chromatographic media, of the analyte. It's these effective diffusion coefficients that allow for the measurement of how different the measured effective diffusion coefficient is to the diffusion of the analyte in an open or unobstructed environment. This difference in diffusion is known as the obstruction factor, γ. The capability to quantify and understand obstructed diffusion, and how it relates to band broadening, has its advantages. Currently, the most common method of determining molar mass distributions of large polydisperse analytes is done using a molar mass calibration curve. This type of calibration curve, as well as other types of calibrations, is vulnerable to small changes in band broadening due to the logarithmic dependence of molar mass to retention volume. Measuring or knowing an analyte's obstructed diffusion gives the system operator the ability to predict how much band broadening will be present, depending on system conditions or operator-controlled variables. This study investigates several types of obstruction factors. First, the intraparticle obstruction factor, γp, takes into account the obstruction that occurs as an analyte partitions in and out of the pores of chromatographic media. Second, the interparticle obstruction factor, γe, accounts for obstruction that occurs solely around the spherical particles present within most chromatographic media. And last, the "total" or combined obstruction factor, γt, represents the combined contribution to obstruction of both, γp and γe, as an analyte travels around and within a column's particles. These three types of obstruction are measured in different ways. The intraparticle obstruction factor was determined via a variable-flow-rate method that measures how much hindrance of diffusion occurs as it travels in and out of the particle's pores. By measuring the increase in peak broadening as the volumetric flow rate of the system was increased, we were able to determine γp. Several operator-controlled variables were changed to see what effect, if any, they had on γp, such as molar mass, analyte, and solvent, as well as several system parameters such as column particle size, pore size, and temperature. The results of the experiments show that γp is very small and in each case, except with change in temperature, that the intraparticle obstruction factors change depending on the variable of the system. The other two obstruction factors, interparticle and combined obstruction, are determined using a stop-flow method, which measures the change in longitudinal diffusion of an analyte over time. This measurement was performed by having a sample sit on a column for various lengths of time (from 1 hour to 72 hours). The resulting series of broadening peaks was then used to determine the effective diffusion coefficient. Depending on the column used, either γe or γt was determined. If an analyte is measured on a column that has pores too small for the analyte to enter, then only γe is measured, as it cannot enter the column's pores. Conversely, if the analyte is small enough to enter the column's pores, then the γt is measured due to the analyte's ability to diffuse not only within particles, but also around them. The results for γe were constant and comparable to other published results. Values for γt were also obtained and used to relate both the γe and γp to γt. This study provides not only information pertinent to SEC, but also the obstruction factor measurements provides a greater fundamental understanding of the diffusion and band broadening processes that happen in other forms of chromatography, such as GC or RPLC. Other scientific areas where diffusion plays an important role, such as biochemistry or the oil industry, can also benefit from the results of these experiments.
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Date Issued: 2011
Identifier: FSU_migr_etd-5930
Format: Thesis

Title: The Anion-Naphthalenediimide Interaction: from Non-Catenated and Complex Metal-Organic Frameworks to Heterodiptopic Receptors.
Creator: Hubley, Christian Thaddeus Nam Hoon, Saha, Sourav, Strouse, Geoffrey F., Landing, William M., Zhu, Lei, Miller, Brian G., Florida State University, College of Arts and Sciences,...
Show moreHubley, Christian Thaddeus Nam Hoon, Saha, Sourav, Strouse, Geoffrey F., Landing, William M., Zhu, Lei, Miller, Brian G., Florida State University, College of Arts and Sciences, Department of Chemistry and Biochemistry
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Abstract/Description: Non-covalent molecular interactions are a ubiquitous part of molecular interaction and the driving force behind macromolecules and host-guest binding. However, a more recent non-covalent molecular interaction, the cation-π interaction, has been recognized as another fundamental force behind macromolecules and host-guest binding. Along with this cation-π interaction, anion-π interactions have been demonstrated with an electron deficient aromatic ring and various anions. Naphthalenediimide (NDI...
Show moreNon-covalent molecular interactions are a ubiquitous part of molecular interaction and the driving force behind macromolecules and host-guest binding. However, a more recent non-covalent molecular interaction, the cation-π interaction, has been recognized as another fundamental force behind macromolecules and host-guest binding. Along with this cation-π interaction, anion-π interactions have been demonstrated with an electron deficient aromatic ring and various anions. Naphthalenediimide (NDI) represent a unique class of molecules that exhibit electron deficient aromatic rings suitable for anion-π interactions. Although the anion-pi interaction has been available for some time, there has been little known about the mechanism by which this interaction occurs. Recently published anion-pi interactions, through the anion-naphthalenediimide interactions, have given insight to the mechanism by which the phenomena occurs. With this recent insight, this research attempts to apply this knowledge to two areas of chemistry, metal-organic frameworks and ion-pair recognition chemistry. A metal-organic framework (MOF) is a material defined by its crystallinity and is composed of a metal ion, or metal clusters, connected to a rigid organic molecule, known as the organic linker or ligand. MOFs have gathered significant attention in the recent decade due to their unique properties such as extremely high surface area, ultra-low density, and others. One of the primary uses for MOFs is for gas storage and capture. Despite the many advancements of MOFs, their synthesis still poses challenges. One such challenge is catenation (interpenetration), which is the formation of subunits of MOFs within themselves. This catenation reduces the available space within a MOF. Several ways to prevent catenation have been demonstrated by using bulky ligands or templating agents. Inspired by this work, this research demonstrates how the anion-π, using perchlorate anion and NDI, can be used to direct the assembly of a two dimensional MOF without catenation. In addition, coordination complexes are prepared and give additional insight to the effects of solvents on coordination of ligands to metal. At the same time, more insight is gained from these coordination complexes and the interaction between the complex and anion. After displaying how the anion-NDI interaction can be applied to prevent the catenation of MOFs, this research investigates complex NDI ligands for the assembly of complex MOFs. MOFs have expanded into many other areas of chemistry and are no longer thought about for simple gas storage. Drug delivery, catalyst, sensors, and many other areas of chemistry are beginning to utilize MOFs. However, if these areas are to successfully apply MOFs, a simple MOF with little or no complex functionality will not work. Therefore, MOFs displaying complex functionality are needed. With complex MOFs in mind, this research set out to build MOFs displaying complex functionality, for sensing, redox potential, and other applications yet to be discovered. To impart complex functionality upon a MOF, one can use a complex ligand, metal ion, or insert materials into the MOFs. Here, research focus is on the synthesis of complex NDI ligands, by adding functionality to the core of the NDI, which then imparts complex functionality on the MOF or can help with retention of ions so that the MOF can possess functionality, such as a redox potential. This part of the research began with the synthesis bromine core-substitution of NDIs that allow for post-synthetic modification (PSM) of bromo-core-substituted MOFs with a variety of nucleophiles, thereby allowing access to a large variety of complex NDI based MOFs. This work is followed by the synthesis of the complex NDI ligand by functionalizing the core of the NDI with ethoxy functionality. Although the NDI is identified as viable candidate for the assembly of complex MOFs, another building block, tetrathiafulvalene (TTF), is also identified. TTF is a unique molecule with exceptional redox ability and is often employed as a building block in many supramolecular systems. In the attempt to address complex MOFs, the synthesis of ligands containing the TTF core is demonstrated and future work will allow for assembly of TTF ligand containing MOFs. Lastly, this research shifts direction back to the anion-NDI interaction in an attempt to apply the interaction to the area of chemistry known as ion-pair recognition. Due to the ubiquitous nature of ionic species in chemistry, biochemistry processes, and in the environment, capturing these ions has gained much attention from the chemistry community. Traditional means of capturing these ionic species involves capturing either the cation or the anion. However, a more recent strategy has been to capture both ionic species at once, using a heteroditopic receptor. The traditional heteroditopic receptor relies on well-known non-covalent interactions for capture. This research attempts to introduce the anion-π interaction, using naphthalenediimide, for the cooperative binding of both ionic species, in which the anion is captured through the anion-π interaction. In addition to cooperative binding using the anion-π interaction, recycling of the receptor is another aspect of this research, which is often an aspect ignored by the field due to the high energy barrier required to overcome. Currently, this research presents successful synthesis of heteroditopic receptors with indication of cooperative binding of both ionic species using the anion-π interaction. Future studies of these receptors will be done to determine their releasability.
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Date Issued: 2016
Identifier: FSU_FA2016_Hubley_fsu_0071E_13543
Format: Thesis

Title: Anionic Rearrangement of 2-Benzyloxypyridine Derivatives and a Synthetic Approach to Aldingenin B.
Creator: Yang, Jingyue, Dudley, Gregory B., Keller, Thomas C. S., Alabugin, Igor, Zhu, Lei, Shatruk, Michael, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: [1,2]-Anionic rearrangements are important tools for altering the complexity of molecules at hand. In Part I of this dissertation, an anionic rearrangement of 2-benzyloxypyridine is described. Pyridine-directed metallation of the benzylic carbon leads to 1,2 migration of pyridine via a postulated associative mechanism (addition / elimination). Several aryl pyridyl carbinols were obtained in high yields. A formal synthesis of carbinoxamine, an antihistamine drug used for the treatment of...
Show more[1,2]-Anionic rearrangements are important tools for altering the complexity of molecules at hand. In Part I of this dissertation, an anionic rearrangement of 2-benzyloxypyridine is described. Pyridine-directed metallation of the benzylic carbon leads to 1,2 migration of pyridine via a postulated associative mechanism (addition / elimination). Several aryl pyridyl carbinols were obtained in high yields. A formal synthesis of carbinoxamine, an antihistamine drug used for the treatment of seasonal allergies and hay fever, emerges from this methodology. As an update, the [1,2]-anionic rearrangement of benzyl 2-pyridyl ethers can also be accessed by a distinct and unusual mechanism: addition of alkyllithium reagents to α-(2-pyridyloxy)-styrene triggers anionic rearrangement to teriary pyridyl carbinols. This will be presented in Chapter 4 and the process is explained by invoking contraelectronic, pyridine-directed carbolithiation of the enol ether π-system. Part II of this dissertation is focused on a synthetic approach to aldingenin B. The synthesis of the tricyclic core of aldingenin B from a key internal alkyne was completed. Synthesis of alkynes by fragmentation is an on-going interest of the Dudley lab. One current goal is to apply our methodology in conjunction with an innovative oxidative alkyne ketalization to achieve a short and efficient synthesis of aldingenin B. The specific goal for this dissertation was to prepare a model alkyne by conventional methods and establish the feasibility of the oxidative alkyne ketalization. The preparation and selenium-mediated cyclo-ketalization of an alkyne-diol will be described as a model study for the synthesis of aldingenin B in Chapter 8. The oxidative cyclization is a simplifying transformation for aldingenin B, as it provides a convenient method for generating the tricyclic core of the natural product from a functionalized carbocycle. Some preliminary experiments to guide future efforts for completing the synthesis of aldingenin B will be presented in Chapter 9.
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Date Issued: 2011
Identifier: FSU_migr_etd-1017
Format: Thesis

Title: Anomalous Dispersion of Excitation Pulses in the 1,4−Cyclohexanedione Belousov−Zhabotinsky Reaction.
Creator: Hamik, Chad Thomas, Steinbock, Oliver, Dalal, Naresh, Stiegman, Albert E., Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: The formation of stable bound wave packets is studied in a modified Belousov-Zhabotinsky reaction. These densely stacked structures arise from an attractive interaction between oxidation pulses that is not known from the classical Belousov-Zhabotinsky system. The characteristic stacking period increases with the initial concentration of bromate but decreases with cyclohexanedione. Wave stacking can also induce cascades of bunching events in which internally dense but mutually well-segregated...
Show moreThe formation of stable bound wave packets is studied in a modified Belousov-Zhabotinsky reaction. These densely stacked structures arise from an attractive interaction between oxidation pulses that is not known from the classical Belousov-Zhabotinsky system. The characteristic stacking period increases with the initial concentration of bromate but decreases with cyclohexanedione. Wave stacking can also induce cascades of bunching events in which internally dense but mutually well-segregated wave clusters are formed. For different initial concentrations, the apparent merging of waves in front-to-back collisions is observed. All three modes of wave dynamics are analyzed in terms of their dispersion behavior. The dispersion relations proved to be anomalous in each case and revealed the existence of an attractor, which induces the formation of stable wave packets. The underlying mechanism has a pure reaction-diffusion character since wave propagation is not affected by fluid convection. At high initial concentrations of ferroin, complex relaxation kinetics which indicate the presence of at least two independent species that control the recovery and hence the dispersion behavior of the medium were detected. The stacking process creates either a traveling shock structure or a cascade of bunching events in which metastable wave packets are formed. The direction and the speed of the shock are explained in terms of a simple geometrical analysis. Experimental evidence for the corresponding instabilities in two-dimensional systems is presented. Here, wave stacking generates atypical structures in the collision of target patterns and wave bunching is accompanied by complex front deformations. Wave stacking and merging are also observed in thin reaction layers where they affect the evolution of target patterns. Additional results on the concentration dependences of the overall dynamics and pulse speeds are presented.
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Date Issued: 2003
Identifier: FSU_migr_etd-4307
Format: Thesis

Title: Application of Flow-Based Methods to Inorganic Materials Synthesis.
Creator: Miller, Levi Zane, Shatruk, Mykhailo, McQuade, D. Tyler (David Tyler), Alamo, Ruﬁna G., Stiegman, Albert E., Strouse, Geoffrey F., Roper, Michael Gabriel, Florida State...
Show moreMiller, Levi Zane, Shatruk, Mykhailo, McQuade, D. Tyler (David Tyler), Alamo, Ruﬁna G., Stiegman, Albert E., Strouse, Geoffrey F., Roper, Michael Gabriel, Florida State University, College of Arts and Sciences, Department of Chemistry and Biochemistry
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Abstract/Description: Controlling particularly reactive substances to achieve desired outcomes is a constant challenge in materials chemistry. Reactants and products consisting of main group and transition metal elements often exhibit extreme sensitivity to their environments. Therefore, it is desirable to develop new methods of synthesis and handling of the starting materials and resulting products in order to extend the chemical space available within this domain of science. Reactivity must be defined within the...
Show moreControlling particularly reactive substances to achieve desired outcomes is a constant challenge in materials chemistry. Reactants and products consisting of main group and transition metal elements often exhibit extreme sensitivity to their environments. Therefore, it is desirable to develop new methods of synthesis and handling of the starting materials and resulting products in order to extend the chemical space available within this domain of science. Reactivity must be defined within the context of this dissertation. Herein, 'reactive' is exceptional sensitivity to air and moisture leading to degredation of reactants or desired products. Reactivity may also correspond to the explosive or pyrophoric nature of reactants and products inevitably preventing their isolation and handling under ambient conditions. Several observations which are pertinent to the fundamental understanding of the reactivity of various metalorganic, orgnaometallic, and main group complexes are chronicled within this dissertation. A comparison is provided for two methods (batch and flow) that are typically used to perform and control reactions. Due to the prevalence flow chemistry within my work, emphasis will be placed upon flow-based methods. In chapter 1, a short primer on fluid dynamics relevant to materials chemistry will be provided to compare and contrast batch versus flow chemistry. Examples of flow chemistry applied to organic reactions are given, followed by examples of inorganic chemistry in flow which is much less developed. Finally, the overarching goals of this work are as follows: 1) Present the basics of fluid dynamics to provide a basis for the flow chemical approaches within this work. 2) Provide a discussion of current flow-based methods applied to organic and inorganic synthesis. 3) To detail and study the application of flow chemistry techniques to the synthesis of new and existing metal organic, organometallic, and main group compounds and materials. In chapter 2, a simplified droplet generator is introduced and utilized to yield hollow silica capsules from a liquid–liquid interfacial polymerization reaction. Further use of this simple droplet generator is examined for preparation of SiO2-TiO2 hybrid capsules along with a cartridge-based method to modify the capsule surface with additional TiO2. In chapter 3, our growing interest in reactive materals led to the discovery that alkali metal oxides can be trapped and crystallized using diethlyzinc. From this observation, a family of complexes were isolated and characterized. Chapter 3 will also incoporate flow-based synthesis of organozinc complexes. First, the continuous preparation of organozinc halides is established and then coupled directly to Negishi reactions for the production of desirable building blocks for active pharmaceutical ingredients. Second, a catridge-based method for the utilzation of pyrophoric solid reagent Na2(HZnEt2)2 is presented leading to a series of novel organozincates. In chapter 4, the traditional methods used to synthesize alkali metal polyphosphides are discussed. Our discovery of solution-phase methods which allow facile access to homoatomic polyanions of phosphorus which do not involve harsh reducing alkali metals or the white allotrope of the element is detailed. We then demonstrate a high-throughput continuous-flow approach for rapid generation of gram quantities of these soluble polyphosphide anions.
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Date Issued: 2015
Identifier: FSU_2015fall_Miller_fsu_0071E_12886
Format: Thesis

Title: Application of FT-ICR Mass Spectrometry in Hydrogen Deuterium Exchange and Lipidomics.
Creator: Liu, Peilu, Marshall, Alan G., Tang, Hengli, Dorsey, John G., Miller, Brian G, Florida State University, College of Arts and Sciences, Department of Chemistry and Biochemistry
Abstract/Description: High resolution mass spectrometry, especially Fourier transform ion cyclotron resonance mass spectrometry (FT ICR MS) is a widely practiced technique of choice in proteomics and lipidomics due to its high sensitivity, reproducibility and wide dynamic range. FT-ICR MS enables quick assignments of hundreds of peptides and lipids with extreme complexity. Chapter 1 introduces the fundamental of FT ICR phenomena for mass measurement and basic theories of LC-MS based hydrogen deuterium exchange ...
Show moreHigh resolution mass spectrometry, especially Fourier transform ion cyclotron resonance mass spectrometry (FT ICR MS) is a widely practiced technique of choice in proteomics and lipidomics due to its high sensitivity, reproducibility and wide dynamic range. FT-ICR MS enables quick assignments of hundreds of peptides and lipids with extreme complexity. Chapter 1 introduces the fundamental of FT ICR phenomena for mass measurement and basic theories of LC-MS based hydrogen deuterium exchange (HDX) technique for high order structure studies. Chapter 1 also introduces the application of mass spectrometry in lipidomics including lipid classification and MS analysis. Chapter 2 described the characterization of the binding interfaces in R2TP complex by hydrogen/deuterium exchange mass spectrometry. The two closely related AAA+ family ATPase Rvb1 and Rvb2 form a tight functional complex with two Hsp90 interactors: Pih1p and Tah1p. The R2TP complex involves in multiple biological processes including apoptosis, PIKK signaling, and RNA polymerase II assembly, and snoRNP biogenesis. The current lack of structural information on R2TP complex prevents a mechanistic understanding of many biological processes. By use of solution-phase HDX MS, we probed the contact surfaces on Pih1p-Tah1p upon Rvb1/2p binding. The present results demonstrate that Pih1p-Tah1p interacts with Rvb1/2p through N-terminal and IDR2 regions of Pih1p. Significantly, HDX also detected a rearrangement of residues 38–60 of Pih1p and 1–44 of Tah1p upon formation of the R2TP complex. Chapter 3 depicts the study of conformations of activated, disease-associated glucokinase variants by a comparative hydrogen/deuterium exchange mass spectrometry. Human glucokinase (GCK) acts as the body’s primary glucose sensor and plays a critical role in glucose homeostatic maintenance. Previous biochemical and biophysical studies suggest the existence of two activated variants. HDX results demonstrate that a disordered active site, which is folds upon binding of glucose, is protected from exchange in α helix variant. Additionally, α helix variant displays an increased level of exchange near enzyme’s hinge region. In contrast, β hairpin variant does not show substantial difference in global or local exchange relative to that of wild type GCK. The work elucidates the structural and dynamics origins of GCK’s unique kinetic cooperativity. Chapter 4 investigated the structure of an antibody with ‘Knob-into-hole’ mutations by HDX MS. Bispecific antibodies (BsAbs) have flourished in the biopharmaceutical industry for targeting two distinct antigens simultaneously. ‘Knob-into-hole’ approach is a way to manufacture bispecific antibodies. The applicability and advantage of ‘Knob-into-hole’ engineered bispecific antibody is vast. However, concerns about the conformational change and immunogenicity risks posed by the new approach has have been raised. To better understand the conformations and dynamics impacted by the ‘knob’ and ‘hole’ mutations, HDX MS is used to characterize peptide-level conformational changes of a ‘Knob-into-hole’ engineered antibody. The study shows that there is no significant structural alternation induced by ‘Knob-into-hole’ framework. In Chapter 5, the applicability of resolving HDX-derived isotopic fine structure by ultrahigh resolving power FT ICR mass spectrometry was discussed. In an HDX experiment, labeling protein with deuterium causes the deuterium incorporation, resulting in distributions of various combinations of 13C1H and 12C2H (Δm = 2.9 mDa). The isotopic fine structure typically cannot be used to evaluate deuteration level due to the difficulty of .resolving fine structures for all proteolytic peptides spanning wide mass range from HDX experiments. The introduction of hexapolar cell triples the observed resolving power on 14.5 tesla FT-ICR mass spectrometer, thus we successfully extend the capability of resolving isotopic fine structure to most of identified peptides. Additionally, a new method of analysis of isotopic fine structure-resolved HDX data was proposed to determine degrees of deuterium incorporation. Another research area I have worked on is characterization of polar lipids by LC coupled with FT-ICR mass spectrometry. Algae lipids contain long-chain saturated and polyunsaturated fatty acids. The lipids may be transesterified to generate biodiesel fuel. In Chapter 6, I compared polar lipid compositions for two microalgae, Nannochloropsis oculata and Haematococcus pluvialis, that are prospective lipid-rich feedstock candidates for an emerging biodiesel industry. Online nano liquid chromatography coupled with negative electrospray ionization 14.5 T Fourier transform ion cyclotron resonance mass spectrometry ((−) ESI FT-ICR MS) with newly modified ion optics provides ultrahigh mass accuracy and resolving power to identify hundreds of unique elemental compositions. Assignments are confirmed by isotopic fine structure for a polar lipid extract. Collision-induced-dissociation (CID) MS/MS provides additional structural information. H. pluvialis exhibits more highly polyunsaturated lipids than does N. oculata.
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Date Issued: 2018
Identifier: 2018_Su_Liu_fsu_0071E_14651
Format: Thesis

Title: Application of FT-ICR Mass Spectrometry in Study of Protein Modifications and Carbohydrates.
Creator: Wang, Xu, Marshall, Alan G., Blaber, Michael, Sang, Qing-xiang Amy, Logan, Timothy M., Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Proteins are organic compounds made of amino acids arranged in a linear chain and folded into a globular form. Protein synthesis follows the genetic code encoded on a sequence of gene. In the cell, shortly after protein biosynthesis, the amino acid residues in a protein are often chemically modified by post-translational modification, which alters the physical and chemical properties, folding, stability, activity, and ultimately, the function of the proteins. The protein post-translational...
Show moreProteins are organic compounds made of amino acids arranged in a linear chain and folded into a globular form. Protein synthesis follows the genetic code encoded on a sequence of gene. In the cell, shortly after protein biosynthesis, the amino acid residues in a protein are often chemically modified by post-translational modification, which alters the physical and chemical properties, folding, stability, activity, and ultimately, the function of the proteins. The protein post-translational modifications play a crucial role in generating the heterogeneity in proteins and also help in utilizing identical proteins for different cellular functions in different cell types. Also, the modifications play an important part in modifying the end product of protein expression and contribute towards biological processes and diseased conditions. Post-translational modifications may happen in several ways, such as glycoslyaiton, phosphorylation, acetylation, methylation, lipoylation, etc. In addition, protein engineering may produce some unnatural modifications on the proteins for some specific research. My research interests mainly focus on study protein glycosylation and phosphorylation. During my graduate study, I have developed many methods to accomplish several projects. Fourier transform ion cyclotron resonance mass spectrometer, FT-ICR MS, is particularly advantageous for protein modification analysis. High accurate mass collected from FT-ICR allows the reduction of precursor ion tolerances from the Dalton range to the order of few sub-parts per million, reducing the number of peptides initially considered by the peptide spectral matching algorithm. Chapter 1 introduces the theory of the instrument for mass measurement, ionization methods, MS-based strategies for protein modification analysis, and the biomolecule separation methods. Chapter 2 reviews phosphoproteomics and glycoproteomics by FT-ICR mass spectrometry. The contents of this review section cover: 1. background of phosphorylation and glycosylation; 2. sample preparation for mass spectrometry-enrichment and separation; 3. tandem mass methodology (CID, ECD, AI-ECD, ETD); 4. informatics. Many applications have been applied to study protein modifications. Chapter 3 presents an application utilizing top-down and bottom-up proteomics with 14.5 T FT-ICR mass spectrometry to study sites and extent of selenomethionine incorporation into recombinant Cas6 protein. Cas6 is a novel endoribonuclease found in Pyrococcus furiosus. it cleaves CRISPR(clustered regularly interspaced short palindromic repeats) RNAs within the repeat sequences to release individual invader targeting RNAs. Selenomethionine modified proteins are commonly used to improve x-ray crystallographic structure resolution by multi-wavelength anomalous diffraction (MAD) phasing. However, the specificity and extent of selenomethionine incorporation must first be assessed before crystallization. Bottom-up and top-down proteomics with a modified 14.5 T LTQ FT-ICR MS offer a quick, accurate, and robust method to locate and quantify selenomethionine incorporation after auxotrophic expression. Comparative analysis confirmed that all six methionines were fully replaced by selenomethionines in Se-Cas6 (Wang X. et al., Rapid Commun. Mass Spectrom., (2010), 24 (16), 2386-2392). A method applying LC ESI FT-ICR mass spectrometry for characterizing N-linked glycoproteins and assigning significant portion of glycopeptide along with composition/structural information of glycans is introduced in chapter 4. The method was first tested with a standard glycoprotein. Then it was applied to characterize N-linked glycoproteins in fetal bovine serum (FBS). N-linked glycans were enzymatically released from glycoproteins in FBS with PNGase F, followed by purification on a graphitized carbon cartridge SPE and separation on an TSK-Gel Amide80 column under hydrophilic interaction chromatography (HILIC) conditions. N-linked glycosylation sites were identified as well. To assign the glycopeptide compositions, experimentally determined glycan masses and glycosylation sites were applied. The masses of different combinations between glycan and peptide were searched in the mass measurements of glycopeptides. The uniquely identified masses were picked for assigning glycopeptides. In total, 137 unique glycopeptide compositions were assigned from 18 glycoproteins, and the glycan structures on most assigned glycopeptides were heterogeneous. High accurate mass measurements collected from FT-ICR MS provided confident identifications (Wang X. et al., Anal. Chem., (2010), 82 (15), 6542-6548). Chapter 5, talks about characterization chemotherapies effect(s) of 2-deoxy-D-glucose (2-DG) on global glycosylation in glioblastoma derived cancer cells and cancer stem cells. 2-DG is a stable glucose analogue in which hydroxyl group at the second position carbon is replaced by a hydrogen. In our study, we found that the toxicity of 2-DG is mainly via interfering with N-glycosylation of proteins, disrupting protein foldings, leading to ER stress and further inducing apoptotic cell death. As results of 2-DG, inhibition of glycolysis and ATP depletion are not fatal for cell viability. The findings we are presenting clearly address the mechanisms of how 2-DG targets and kills cancer cells and cancer stems cells as a chemotherapeutic agents, which may be used for supporting further clinical trials of 2-DG. A conference presentation has been accepted by the 15th anual scientific meeting of the society for neuro-oncology, 2010, Montreal, Canada. The manuscript of these results will soon be submitted to Nat. Med. Protein phosphorylation is a crucial event in most cellular processes. Since phosphorylation is always substoichiometric and the ionization efficiency of phosphopeptide is low during mass spectrometry analysis, selective enrichment will be required for large scale characterizing phosphorylation from complex biological samples. In chapter 6, an optimized phosphopeptide enrichment strategy consisted of calcium phosphate precipitation (CPP) coupling with TiO2 is addressed. The method was applied to study phosphorylations in androgen repressed cancer of the prostate (ARCaP) cells. Phosphopeptides in ARCaPs were analyzed on a hybrid LTQ FT-ICR mass spectrometer with LC MS/MS approach. After database search and stringent filtering, we generated a data set containing total of 385 high confident phosphoprotein identifications with overall mass error of 0.32 ± 0.6 ppm at peptide level. Chapter 7 talks about that the phosphorylation/dephosphorylation regulates MSP fiber protein 3 (MFP3) in MSP-based amoeboid motility of Ascaris sperm. The crawling movement of nematode sperm requires coordination of leading edge protrusion with cell body retraction, both of which are powered by modulation of a cytoskeleton based on major sperm protein (MSP) filaments. We used a cell-free in vitro motility system in which both protrusion and retraction can be reconstituted, to identify two proteins involved in cell body retraction. Protein phosphatase 2A (PP2A), a protein unique to nematode sperm that binds to the MSP filaments in the motility apparatus, targeted MSP fiber protein 3 (MFP3). Dephosphorylation of MFP3 caused its release from the cytoskeleton and generated filament disassembly. Our results suggest that interaction between PP2A and MFP3 leads to local disassembly of the MSP cytoskeleton at the base of the lamellipod in sperm that in turn pulls the trailing cell body forward (Yi K., Wang X. et al., Mol. Biol. Cell, (2009), 20 (14), 3200-3208). Chapter 8 presents comparative glycoproteomics data in a glioblastoma derived stem cell line, gCSC11, during differentiation. gCSC11 characterized by CD133 expression, grows as tumorsphere in culture and if subsequently implanted in nude mice brains, will recapitulate high grade glial tumors. Stimulation with a STAT3 inhibitor WP1193 or culturing in 10% FBS both led to loss of CD133 expression in gCSC11 cells, but differed in phenotype changes. STAT3 inhibitor treated gCSC11 cells underwent dissociation in culture and converted to progenitor cells if further treated with FBS. Glycoproteomics study revealed 33 differentially expressed glycoproteins, most of which has never been reported as glycosylated. ENO1 isoform alpha-enolase was preferentially expressed in native rather than in STAT3 or FBS treated gCSC11 cell. Preferential expression of alpha-enolase in gCSC11 glioblastoma stem cells might reflect the modulated metabolism or control the balance between self-renewal and differentiation via modulation of c-myc. Chapter 9 talks about phosphorylation sites characterization of galectin-1 and its potential secretion/regulation pathway. Galectin-1 is a multi-functional protein performing either intracellular or extracellular functions as the monomer or homodimer, and it works on different cell types to put either positive or negative effects on cell growth. It is involved in tumor transformation, cell cycle regulation, apoptosis, cell adhesion, migration and inflammation. Galectin-1 is produced in the cytosol and can either remain intracellular or be delivered to the outside of cells via a non-classical secretion pathway. Galectin-1 lacks recognizable secretion signal sequences and does not pass through the standard ER/Golgi pathway. Thus, galectin-1 secretion must operate by a novel mechanism distinct from classical vesicle-mediated exocytosis. Our primary data of phosphorylation on galectin-1 may indicate the mechanism of its secretion/regulation pathway. The appendixes include the papers I have published during my graduate study. Appendix A is the paper that covers the method development for characterizing N-linked glycans and glycopeptides by liquid chromatography electrospray ionization FT-ICR MS. Appendix B is a top-down mass spectrometry paper studying sites and extent of selenomethionine incorporation into recombinant Cas6. The publication in appendix C introduces a mechanism how MFP3 functions in cell body retraction, which was collaborated with Dr. Thomas M. Roberts in the biology department at Florida State University.
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Date Issued: 2010
Identifier: FSU_migr_etd-1245
Format: Thesis

Title: Application of FT-ICR Mass Spectrometry in Study of Proteomics, Petroleomics and Fragmentomics.
Creator: Mao, Yuan, Marshall, Alan G., Blaber, Michael, Dalal, Naresh S., Roper, Michael G., Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: With the advent of two "soft" ionization techniques in the late 1980s—electrospray ionization (ESI) and matrix assisted laser desorption/ionization (MALDI) for the routine and general formation of intact molecular ions—and continuing effort in the instrumentation development directed toward improving two key parameters of mass spectrometric performance—mass resolving power and mass accuracy, mass spectrometry has been an indispensable analytical technique for chemical and biochemical sample...
Show moreWith the advent of two "soft" ionization techniques in the late 1980s—electrospray ionization (ESI) and matrix assisted laser desorption/ionization (MALDI) for the routine and general formation of intact molecular ions—and continuing effort in the instrumentation development directed toward improving two key parameters of mass spectrometric performance—mass resolving power and mass accuracy, mass spectrometry has been an indispensable analytical technique for chemical and biochemical sample analysis, especially for highly complex mixture systems, e.g., proteomics, petroleomics, lipidomics, metabolomics, etc. The introduction of ESI and MALDI ionization techniques has extended the accessibility of mass spectrometry-based analysis from small volatile molecules to large non-volatile molecules, whereas mass accuracy and mass resolving power directly determine the usefulness of mass spectrometric experiments. Among the high resolution mass analyzers such as reflectron/multipass time of flight (TOF), orbitrap, and Fourier transform ion cyclotron resonance (FT-ICR), FT-ICR MS provides ten-fold higher mass resolution and mass measurement accuracy than other mass analyzers and has become the most powerful techniques that can deal with the complexity of various samples, e.g., it is possible to routinely achieve high mass resolving power of 400,000 (m/∆m50% ≈ 400,000, in which m is molecular mass and ∆m50% is the mass spectral peak width at half-maximum peak height) and mass accuracy (~ 100 ppb) up to 800 Da from high-field (≥9.4 T) FT-ICR MS, thus resolving > 40,000 different elemental compositions in a single mass spectrum and providing their unambiguous molecular formulas. Chapter 1 introduces the concept of mass resolving power, mass resolution and mass measurement accuracy, the principle of the FT-ICR instrument for mass measurement, ionization methods, factors that control the mass measurement accuracy of FT-ICR MS and utility of high mass accuracy for analysis of biological samples and complex mixtures. Chapter 2 describes the application of high mass accuracy for distinction of N-terminal and C-terminal electron capture dissociation/electron transfer dissociation (ECD/ETD) product ions of c and z⋅ based on their number of hydrogen plus nitrogen atoms determined by accurate mass measurement, and forms a basis for de novo peptide sequencing. The effect of mass accuracy (0.1-1 ppm error) on c/z⋅ overlap and unique elemental composition overlap is evaluated for a database of c/z⋅ product ions each based on all possible amino acid combinations and four subset databases containing the same c ions but with z⋅ ions determined by in silico digestion with trypsin, Glu-C, Lys-C, or chymotrypsin. High mass accuracy reduces both c/z⋅ overlap and unique elemental composition overlap. Of the four proteases, trypsin offers slightly better discrimination between N- and C-terminal ECD/ETD peptides. Interestingly, unique elemental composition overlap curves for c/c and z⋅/z⋅ peptide ions exhibit discontinuities at certain nominal masses for 0.1-1.0 ppm mass error. Also, the number of ECD/ETD product ion amino acid compositions as a function of nominal mass increases exponentially with mass, but with a superimposed modulation due to higher prevalence of certain elemental compositions. Chapter 3 presents an idea that at sufficiently high mass accuracy, it is possible to distinguish phosphorylated from unmodified peptides by mass measurement alone. We examine the feasibility of that idea, tested against a library of all possible in silico tryptic digest peptides from the human proteome database. The overlaps between in silico tryptic digest phosphopeptides generated from known phosphorylated proteins (1-12 sites) and all possible unmodified human peptides are considered for assumed mass error ranges of ±10, ±50, ±100, ±1,000, and ±10,000 ppb. We find that for mass error ±50 ppb, 95% of all phosphorylated human tryptic peptides can be distinguished from nonmodified peptides by accurate mass alone through the entire nominal mass range. We discuss the prospect of on-line LC MS/MS to identify phosphopeptide precursor ions in MS1 for selected dissociation in MS2 to identify the peptide and site(s) of phosphorylation. ETD and ECD are two similar fragmentation approaches, producing extensive and nonspecific fragmentation (c/z⋅ ions formed by cleavage of N-Cα backbone bond) while retaining thermally labile post-translational modifications. In chapter 4, we implemented dual electrospray ionization ETD on a custom-built 9.4 T FT-ICR MS. Two separate electrospray emitters are automatically switched for injection of positive (analyte) and negative (reagent) ions. Decarboxylated 2-(fluoranthene-8-carbonyl) benzoic acid is the ETD reagent anion. A linear octopole ion trap is the ETD ion/ion reaction chamber, and an RF voltage is applied to the front and back ion trap electrodes to confine both cations and reagent anions for ETD, after which the c- and z- type product ions are passed to the ICR cell for high resolution and mass accuracy analysis. Comparison of ETD and ECD spectra of standard peptides shows that ETD provides similar sequence coverage and fragmentation pattern to ECD. Chapter 5 describes a calibration procedure in which accurate masses of spacings from any two same type neighboring fragment ions differing by one amino acid residue are used to calibrate ECD and collision activated dissociation (CAD) MS/MS spectra of standard peptides with different molecular weights and charge states. High mass accuracy of tandem mass spectra is crucial for confident extraction and identification of spacings. FT-ICR mass spectrometry provides ultrahigh mass accuracy and resolving power and acquired MS/MS spectra with ppm mass accuracy level are routinely obtained when combined with external calibration by substance P fragments. Calibration by accurate masses of extracted spacings shows up to ~ 30% further reduction of rms mass error of MS/MS spectra on average compared with substance P MS/MS external calibration. ~ 25% improvement of c/z⋅ ion unambiguous distinction from ECD spectra based on valence parity rule increases the confidence of peptide sequencing. FT-ICR MS with ultrahigh resolving power and mass accuracy is essential to resolve and uniquely identify elemental compositions of thousands of components in complex organic mixtures, e.g., petroleum crude oils. To study how much resolving power and mass accuracy is necessary, in chapter 6 all possible closest mass doublets (0<∆m<45 mDa, ∆m is the mass difference of mass doublet) were counted for both electrospray ionization (ESI) and atmospheric pressure photoionization (APPI) absorption-mode spectra automatically by use of the algorithm written in LabWindows/CVI. As many as thousands of mass doublets with the mass difference less than 10 mDa (as low as ~ 0.70 mDa) were observed in APPI and ESI absorption-mode broadband mass spectra. Histograms of mass doublet distribution for APPI and ESI were plotted. In chapter 7 the effect of mass error (10 - 500 ppb) on elemental composition overlap is evaluated for ESI and APPI databases containing all possible elemental compositions with proper constraints, CcHhNnOoSs13Ccc34Sss, c, h unlimited, 0≤n<5, 0≤o<10, 0≤s≤3, 0≤cc<3 and 0≤ss<2 for even-electron ions (M+H)+ (or (M-H)-) in ESI and for both M+⋅ and (M+H)+ (or M-⋅ and (M-H)-) in APPI with nominal mass of 200-1200 Da. Number of element compositions of all possible components in complex mixtures is reduced by ~ 1100 on average for each class after applying 90% rule. High mass accuracy reduces elemental composition overlap and facilitates the unique identification of elemental compositions for components up to 1200 Da at mass errors of 200 ppb in ESI and 100 ppb in APPI. All possible theoretical mass doublets which may occur in petroleum crude oils are calculated based on elemental compositions of all possible components from ESI and APPI databases. Mass doublets with mass difference as low as 0.20 mDa, even smaller than mass of electron (0.548 mDa) and NO213C vs. C2H3S (0.71 mDa) (the smallest one currently observed in 9.4 T broadband absorption-mode ESI/APPI FT-ICR MS) are calculated and can only be resolved with higher-field FT-ICR MS (e.g., absorption-mode 21T). FTICR mass spectrometer coupled with ECD/ETD offers ultrahigh broadband mass resolving power (>105) and mass accuracy (<1 ppm) for detection of accurate precursor mass as well as the vast amount of isotopically resolved fragment ions required for protein identification and has become an increasing useful tool for top-down analysis. In chapter 8 we perform top-down ECD FT-ICR MS for structural analysis of an intact monoclonal antibody (IgG1-kappa (κ) isotype, ~148 kDa). Simultaneous ECD for all charge states (42+ to 58+) generates more extensive cleavages than ECD for an isolated single charge state. The cleavages are mainly localized in the variable domains of both heavy and light chains, the respective regions between the variable and constant domains in both chains, the region between heavy chain constant domains CH2 and CH3, and the disulfide bond (S-S) linked heavy chain constant domain CH3. The light chain yields mainly N-terminal fragment ions due to the protection of the inter-chain disulfide bond between light and heavy chain, and limited cleavage sites are observed in the variable domains for each chain where the S-S spans the polypeptide backbone. Only a few cleavages in the S-S linked light chain constant domain, hinge region, and heavy chain constant domains CH1 and CH2 are observed, leaving glycosylation uncharacterized. Top-down ECD with a custom-built 9.4 T FT-ICR MS for structural characterization of IgG1κ provides more extensive sequence coverage than top-down collision induced dissociation (CID) and ETD with time-of-flight but comparable sequence coverage with top-down ETD with orbitrap MS.
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Date Issued: 2013
Identifier: FSU_migr_etd-7489
Format: Thesis

Title: Application of Organic Synthesis to the Development of New Imaging Tools for Fluorescence Microscopy.
Creator: Macias Contreras, Miguel Angel, Zhu, Lei, Piekarewicz, Jorge, Hanson, Kenneth G., Miller, Brian G., Florida State University, College of Arts and Sciences, Department of...
Show moreMacias Contreras, Miguel Angel, Zhu, Lei, Piekarewicz, Jorge, Hanson, Kenneth G., Miller, Brian G., Florida State University, College of Arts and Sciences, Department of Chemistry and Biochemistry
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Abstract/Description: This dissertation consists of six chapters. Chapter one gives to the reader a brief introduction to several key concepts and backgrounds such as the relevance of zinc(II) ions in biology, the definition of small molecular probes and the development of protein tags, including the SNAP-tag technology, and bioorthogonal reactions for specific labeling of targets of interest. Chapter two is focused on the synthesis of small molecular probes for selective detection of zinc(II) ions. The rationale...
Show moreThis dissertation consists of six chapters. Chapter one gives to the reader a brief introduction to several key concepts and backgrounds such as the relevance of zinc(II) ions in biology, the definition of small molecular probes and the development of protein tags, including the SNAP-tag technology, and bioorthogonal reactions for specific labeling of targets of interest. Chapter two is focused on the synthesis of small molecular probes for selective detection of zinc(II) ions. The rationale behind their chemical structures involves (1) using a 7-dimethylaminocoumrin derivative as a fluorophore with good absorbance at 405 nm and (2) subtle modification of chemical structure to tune binding affinity. A family of three sensors was synthesized and their photophysical characterization was conducted in both organic and aqueous solutions. One of the sensors has a binding affinitity towards zinc(II) in the nanomolar range, which is relevant to certain organelles where the zinc(II) concentration is believed to be in that range. All sensors were used to image low and high concentration of zinc(II) ions in live HeLa cells. Chapter three shows how a small molecular probe was evolved into a substrate for the self-labeling enzyme SNAP-tag. The replacement of the benzyl ring in a O6-benzylguanine (BG) derivative with a pyridyl ring generated a new class of compounds, O6-(5-pyridylmethyl)guanine (5PG). 5PG derivatives were shown to be taken up by the enzyme, demonstrating its substrate ambiguity. A fluorescent 5PG derivative was employed to selectively label intracellular compartments such as nuclear envelope, actin fibers, lysosomes and Golgi apparatus. The pyridine ring was introduced with the purpose of forming part of a tetradentate ligand for zinc(II); thus, making the pyridine an integral part of a coordination motif and not only an spectator group. Such unique situation enabled the imaging of low and high concentrations of zinc at precise locations. Chapter four focusses on the development of SNAP-tag substrates for intracellular bioorthogonal labeling. A series of BG derivatives carrying reactive handles such as azides and strained alkenes was synthesized. The occurrence of strain promoted azide-alkyne cycloaddition (SPAAC) and inverse electron demand Diels-Alder (IED-DA) reaction inside cells was demonstrated using fluorescence microscopy. By introducing first a reactive handle via SNAP-tag, followed by the reaction with a suitable fluorophore, two intracellular targets –nuclear envelope and actin fibers –were specifically labeled. But the real novelty of this work is the concurrent dual labeling inside live HeLa cells. To demonstrate the feasibility of the simultaneous labeling of two protein targets the orthogonal pairs SNAP/CLIP and SPAAC/IED-DA were used. Two-color fluorescent microscopic images showed the orthogonality of the two pairs. In one experiment, the nuclear envelope was labeled with a green dye via CLIP-IED-DA and the actin fibers through SNAP-SPAAC. In a second experiment, the targets were reversed and the correct labeling was found, proving the specificity and flexibility of the method. Chapter five is divided in three sections and is intended to show the results that were not included in Chapters two, three and four but that were significant to the completion of the projects associated with those chapters. The first section is focused on the synthesis of a small molecular probe complementary to the ones described in Chapter two. After trying different synthetic strategies, such a compound could not be made. The second section describes the synthetic journey that was travelled to obtain a chelating azide containing 5PG derivative. Several strategies were followed but eventually it was shown that a bifunctional pyridine derivative carrying alcohol and acetal functionalities was the key intermediate needed to obtain the target compound. Two other BG derivatives with chelating azide functionality were also prepared. The third section focusses on the application of the azido-containing BG derivatives in the bioorthogonal labeling of live and fixed cells. The copper-catalyzed azide-alkyne cycloaddition (CuAAC) mediated labeling was not possible in living cells but it was made available in fixed cells. Fixed cells were labeled at concentrations as low 25 μM Cu(II) and with or without the use of an auxiliary ligand, showing the superiority of chelating azides versus regular azides. Chapter six concludes the work presented in the entire document.
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Date Issued: 2019
Identifier: 2019_Spring_MaciasContreras_fsu_0071E_14999
Format: Thesis

Title: Applications of 21 Tesla FT-ICR Top-down Proteomics in Clinical Research and Diagnosis.
Creator: He, Lidong, Marshall, Alan G., Tang, Hengli, Dorsey, John G., Hu, Yan-yan, Florida State University, College of Arts and Sciences, Department of Chemistry and Biochemistry
Abstract/Description: With recent progress in clinical proteomics, mass spectrometry (MS)-based methods have been widely implemented in diagnosis of diseases which offers high specificity that conventional clinical tests lack. The recent development of high resolution high mass accuracy mass spectrometry leads to full characterization of intact proteins (e.g., therapeutic monoclonal antibodies and endogenous protein biomarkers) in a top-down MS/MS fashion. Top-down MS/MS offers “birds’ eye” view of the proteins...
Show moreWith recent progress in clinical proteomics, mass spectrometry (MS)-based methods have been widely implemented in diagnosis of diseases which offers high specificity that conventional clinical tests lack. The recent development of high resolution high mass accuracy mass spectrometry leads to full characterization of intact proteins (e.g., therapeutic monoclonal antibodies and endogenous protein biomarkers) in a top-down MS/MS fashion. Top-down MS/MS offers “birds’ eye” view of the proteins and yields more confident protein sequence assignment and post-translational modifications localization. This dissertation describes the latest top-down applications in disease precision diagnosis that can potentially lead to future personalized treatment. Chapter 2 describes a pilot study for characterization of monoclonal antibodies by top-down and middle-down approaches with the advantages of fast sample preparation with minimal artifacts, ultrahigh mass accuracy, and extensive residue cleavages by use of 21 tesla FT-ICR MS/MS. The ultrahigh mass accuracy yields an rms error of 0.2–0.4 ppm for antibody light chain, heavy chain, heavy chain Fc/2, and Fd subunits. The corresponding sequence coverages are 81%, 38%, 72%, and 65% with MS/MS rms error ~4 ppm. Extension to a monoclonal antibody in human serum as a monoclonal gammopathy model yielded 53% sequence coverage from two nano-LC MS/MS runs. A blind analysis of five therapeutic monoclonal antibodies at clinically relevant concentrations in human serum resulted in correct identification of all five antibodies. Nano-LC 21 T FT-ICR MS/MS provides nonpareil mass resolution, mass accuracy, and sequence coverage for mAbs, and sets a benchmark for MS/MS analysis of multiple mAbs in serum. This is the first time that extensive cleavages for both variable and constant regions have been achieved for mAbs in a human serum background. Chapter 3 describes a novel protein de novo sequencing method given that top-down MS/MS complete sequence coverage is virtually impossible. To characterize the “AA sequence gap” between two adjacent fragments, the number of gap AA sequences with identical masses for di-, tri-, and tetra-AA gaps grows exponentially with increasing number of gap amino acids. If peptide fragment mass could be measured exactly (in practice, to 0.00001 Da), it would then be possible to define the overall atomic composition for the group of amino acids spanning a product ion gap 3-4 amino acids long. I show that de novo top-down/middle-down MS/MS can determine the germline sequence category for a given monoclonal antibody and further serve to identify its novel mutations. Chapter 4 applies my developed top-down protein de novo sequencing in characterization of serum monoclonal immunoglobulins from plasma cell disorders. The current five-year survival rate for systemic AL amyloidosis or multiple myeloma is below 50%, indicating the urgent need for better diagnosis methods and treatment plans. Unlike genomic testing, which requires bone marrow aspiration and may fail to identify all monoclonal immunoglobulins produced by the body, the present method requires only a blood draw. In addition, circulating monoclonal immunoglobulins spanning the entire population are analyzed and reflect the selection of germline sequence by B cells. The monoclonal immunoglobulin light chain FR2-CDR2-FR3 was sequenced by de novo MS/MS and 100% matched the gene sequencing result except for two amino acids with isomeric counterparts, enabling accurate germline sequence classification. This work represents the first application of top/middle-down MS/MS for de novo sequencing of endogenous monoclonal immunoglobulins with polyclonal immunoglobulins background. Chapter 5 is focused on top-down MS/MS diagnosis of hemoglobin disorders. Hemoglobinopathies and thalassemias are the most common genetically determined disorders. Current diagnosis methods include cation exchange high performance liquid chromatography and electrophoresis for screening whose results can be ambiguous because of limited resolving power, and expensive and laborious genetic testing is needed for confirmation. I developed a top-down MS/MS approach with the advantages of fast data acquisition (3 min), ultrahigh mass accuracy, and extensive residue cleavages by use of 21 tesla FT-ICR MS/MS for hemoglobin variant sequence de novo characterization and thalassemia diagnosis. With my developed generic approach for hemoglobin variant de novo sequencing, all eighteen hemoglobin variants were correctly identified in blind analysis which include the first characterization of homozygous hemoglobin Himeji variant. It is the first time that the abundance ratio between intact δ and β subunits (δ/β) is used for beta thalassemia (including beta thalassemia trait/major) screening. Therefore, 21 T FT-ICR MS sets the benchmark for top-down MS/MS analysis of hemoglobin variants and thalassemia.
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Date Issued: 2018
Identifier: 2018_Su_He_fsu_0071E_14616
Format: Thesis

Title: Applications of Advanced Magnetic Resonance Techniques to the Study of Molecule-Based Magnetic Materials.
Creator: Greer, Samuel Michael, Hill, S., Shatruk, Mykhailo, Xiong, Peng, Steinbock, Oliver, Florida State University, College of Arts and Sciences, Department of Chemistry and Biochemistry
Abstract/Description: The highly interdisciplinary study of molecular magnetism spans a wide array of topics, ranging from spintronics and quantum computing to enzyme function and MRI contrast agents. At the core of all these fields is the study of materials whose properties can be controlled through the rational design of molecules. The chemical tailoring of molecular magnetic properties can only be achieved by understanding the relationship between the physical and electronic structures. In this dissertation,...
Show moreThe highly interdisciplinary study of molecular magnetism spans a wide array of topics, ranging from spintronics and quantum computing to enzyme function and MRI contrast agents. At the core of all these fields is the study of materials whose properties can be controlled through the rational design of molecules. The chemical tailoring of molecular magnetic properties can only be achieved by understanding the relationship between the physical and electronic structures. In this dissertation, the interplay between structure and physical properties is probed using a variety of magnetic resonance techniques. In Chapter 1, we give a succinct overview of the various methods utilized in this dissertation. We first describe the experimental methods including electron paramagnetic resonance (EPR), 57Fe nuclear gamma resonance (Mössbauer) spectroscopy, electron double resonance detected nuclear magnetic resonance (ELDOR-NMR), and Fourier transform far-infrared (FTIR) spectroscopy. In addition to the introduction of each technique, we describe how the data is analyzed and what quantities may be extracted from each method. We also introduce the quantum chemical methods used to rationalize the spectroscopic parameters. In Chapter 2, we investigate a recently reported Fe-V triply bonded species, [V(iPrNPPh2)3FeI], using high frequency EPR (HFEPR), field- and temperature-dependent 57Fe Mössbauer spectroscopy, and high-field ELDOR-NMR. From the use of this suite of physical methods, we probe the electron spin distribution as well as the effects of spin-orbit coupling on the electronic structure. This is accomplished by measuring the effective g – factors as well as the Fe/V electro – nuclear hyperfine interaction tensors of the spin S = ½ ground state. We have rationalized these tensors in the context of ligand field theory supported by quantum chemical calculations. This combined theoretical and experimental analysis suggests that the S = ½ ground state originates from a single unpaired electron predominately localized on the Fe site. Chapter 3 describes a combined HFEPR and variable-field Mössbauer spectroscopic investigation of a pair of bimetallic compounds with Fe-Fe bonds, [Fe(iPrNPPh2)3FeR] (R = ≡NtBu and PMe3). Both of these compounds have high spin ground states, where R= PMe3 (S = 7/2) and the R= ≡NtBu displays (S = 5/2). The ligand set employed in this work encapsulates each Fe site in a different coordination environment. This results in polarized bonding orbitals which engender each nuclear site with unique hyperfine tensors as revealed by Mössbauer spectroscopy. Absent the metal-metal bond, the tris-amide bound site in both compounds is expected to be Fe(II). To gain insight into the local site electronic structure, we have concurrently studied a compound containing a single Fe(II) in a tris-amide site. Our spectroscopic studies have allowed us to assess the electronic structure via the determination of the zero field splitting parameters and 57Fe electronuclear-hyperfine tensors for the entire series. Through the insight gained in this study, we propose some strategies for the design of polymetallic single molecule magnets where the metal-metal interactions are mediated by the formation of covalent bonds between metal centers. Recently, a great deal of the work in molecular magnetism has moved away from polymetallic compounds and towards molecules containing only a single magnetic ion. A critical challenge in this endeavor is to ensure the preservation of orbital angular momentum in the groundstate. The stabilization of the ground state orbital moment generates the strong magnetic anisotropy which is often required for the design of magnetic materials. The presence of unquenched orbital angular momentum can be identified by significant shifts in the g-value away from the free ion value. In an initial report of a Ni(I) coordination complex, which was found to exhibit field-induced slow magnetic relaxation, no EPR signal was observed. Given the expectation that orbital angular momentum can shift the g-values beyond the range expected for a typical S= ½ system, we have reexamined this compound using multi-frequency EPR and field-dependent FTIR spectroscopy. Through a combined spectroscopic and theoretical effort, we have characterized the effect of first order spin-orbit coupling on the electronic structure. The final report, Chapter 5, examines an exciting new class of photomagnetic materials based on bisdithiazolyl radicals. These materials, and others with magnetic properties that can be modulated via optical excitation, offer enticing opportunities for the development of next generation technologies. The dimorphic system in this study crystallizes in two phases, one composed of diamagnetic dimers and the other of paramagnetic radicals. Here we report on the use of high-field electron paramagnetic resonance spectroscopy to characterize both the thermally- and light-induced transitions in the dimer phase. During the course of this study we show that signals originating from residual radical defects in the dimer phase can be differentiated from those arising from the radical phase.
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Date Issued: 2018
Identifier: 2018_Fall_Greer_fsu_0071E_14848
Format: Thesis

Title: Applications of High Frequency Electron Paramagnetic Resonance on Materials from Quantum to Classical Regime.
Creator: Wang, Zhenxing (EPR Specialist), Dalal, Naresh S., Brooks, James, Kroto, Harold, Strouse, Geoffrey F., Shatruk, Michael, Department of Chemistry and Biochemistry, Florida State...
Show moreWang, Zhenxing (EPR Specialist), Dalal, Naresh S., Brooks, James, Kroto, Harold, Strouse, Geoffrey F., Shatruk, Michael, Department of Chemistry and Biochemistry, Florida State University
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Abstract/Description: This dissertation has focused on studying the electron spin dynamics in the quantum and classical limit and, most importantly, at the quantum-classical boundary. We have successfully used high frequency electron paramagnetic resonance (HF-EPR) techniques to characterize six paramagnetic materials with increasing number of unpaired electrons and molecular sizes. The samples studied are Mn2+-doped CdSe Quantum Dots (Mn:CdSe QDs), Na20[Cu2Pd22PV12O60(OH)8] (Cu2), Na12[X2W18Cu3O66(H2O)3].32H2O (X...
Show moreThis dissertation has focused on studying the electron spin dynamics in the quantum and classical limit and, most importantly, at the quantum-classical boundary. We have successfully used high frequency electron paramagnetic resonance (HF-EPR) techniques to characterize six paramagnetic materials with increasing number of unpaired electrons and molecular sizes. The samples studied are Mn2+-doped CdSe Quantum Dots (Mn:CdSe QDs), Na20[Cu2Pd22PV12O60(OH)8] (Cu2), Na12[X2W18Cu3O66(H2O)3].32H2O (X = As, Sb) (Cu3), [Fe7O4(O2CPh)11(dmem)2].4MeCN (Fe7), [Mn7O4(pdpm)6(N3)4](ClO4)2 (Mn7) and Na34[Mn19(OH)12(SiW10O37)6].115H2O (Mn19). Our results have illustrated that four samples including Mn:CdSe QDs, Cu2, Cu3 and Fe7 can be perfectly described with quantum mechanics while sample Mn19 behaves like a typical classical system. Most interestingly, sample Mn7 (S = 29/2) straddles the interface between the classical and quantum mechanical spin descriptions. Chapter 1 gives the motivation, overview and organization of this dissertation. Chapter 2 describes synthetic details of the materials studied, introduction of two HF-EPR spectrometers, as well as the computer simulation programs employed in this undertaking. Chapter 3 summarizes the HF-EPR studies of Mn:CdSe QDs, the first application of HF-EPR to magnetic QDs. Chapter 4 presents the structure and magnetic characterization of an octahedrally coordinated Cu(II) pair, a very rare bonding for Cu(II) ions. Chapter 5 reports the coherent manipulation of electron spins in an antiferromagnetically coupled spin triangle {Cu3} impregnated in free standing nanoporous silicon (NS) by using 240 GHz microwave pulses. Chapter 6 discusses continuous wave (cw) and pulsed HF-EPR measurements on an Fe-based magnetic cluster: Fe7. Chapter 7 describes the HF-EPR characterization of a high spin (S) compound, Mn7, whose properties straddle the interface between the classical and quantum mechanical spin descriptions. Chapter 8 reports the structure and magnetic properties of a novel, unique, discrete polyanion comprising a cationic, planar Mn19 assembly incorporated in a 60-tungsto-6-silicate. Finally, chapter 9 summarizes the major results and conclusions of this dissertation.
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Date Issued: 2012
Identifier: FSU_migr_etd-5258
Format: Thesis

Title: Applications of Quantum Dots in Gene Therapy.
Creator: Barnes, Laura F., Strouse, Geoffrey, Logan, Timothy, Miller, Brian, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Gene therapy is a rising field and requires multifunctional delivery platforms in order to overcome the cellular barriers. Quantum dots (QDs) provide a optically fluorescent and biocompatible surface to act as a multifunctional delivery platform for gene therapy. The objective of this research is to manipulate the surface of quantum dots for use in gene therapy. The first goal was to make the QDs water soluble and therefore biocompatible. The second goal was to functionalize the surface of...
Show moreGene therapy is a rising field and requires multifunctional delivery platforms in order to overcome the cellular barriers. Quantum dots (QDs) provide a optically fluorescent and biocompatible surface to act as a multifunctional delivery platform for gene therapy. The objective of this research is to manipulate the surface of quantum dots for use in gene therapy. The first goal was to make the QDs water soluble and therefore biocompatible. The second goal was to functionalize the surface of the QDs with plasmid DNA for direct use in gene therapy. This approach uses chemoselective coupling chemistry between an InP/ZnS quantum dot (QD) and linker DNA (DNAlinker) to control the timing of protein expression. Linear DNA (lDNA), containing the CMV promoter and DsRed-Express gene, was condensed on the surface of the QD-DNAlinker. Optical and flow cytometry analysis of the DsRed-Express expression after transfection of the QD-lDNA into CHO cells shows a delayed protein expression for both coupling chemistries compared to naked lDNA. It is also clear that the protein expression form the QD-S-lDNA turns on quicker than the QD-NH-lDNA. We believe the protein expression delay is due to the site of coupling between the QD and DNAlinker and its affect on the lDNA packing strength. The S-DNAlinker is believed to couple by direct exchange at the vertices of the QD whereas the NH-DNAlinker couples through a condensation reaction to the facets. The delay in protein expression reflects the delayed exchange rate at the facets over the vertices. The ability to control the coupling chemistry and timing of release from the QD surface suggests a mechanism for dose control in transient gene therapeutics, and show QD delivery approaches are ideal candidates for multifunctional, targeted, drug carrying platforms that can simultaneously control dosing. The third goal of this research was to functionalize the surface of the QDs with the HIV cell penetrating peptide, TAT, and study its affects on QD internalization as well as toxicological affects within the cells. Tracking of the cellular uptake of these QDs by optical microscopy shows rapid, diffuse accumulation of both 10 % TAT and 100 % TAT passivated QDs throughout the cytosol of the cells. Toxicity studies were conducted by flow cytometry to investigate the effects of these materials on apoptosis, necrosis, and metabolic damage in Chinese Hamster Ovary (CHO) cells. These studies suggest toxic effects of the cell penetrating QDs are dependent on the amount of CAAKA-TAT used on the surface of the QD as well as the concentration of QD added. These observations aid in the use of QDs as self transfecting, nano delivery scaffolds for drug or gene therapy.
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Date Issued: 2010
Identifier: FSU_migr_etd-5467
Format: Thesis

Title: Arginine Kinase: A Crystallographic Investigation of Essential Substrate Structure.
Creator: Clark, Shawn A. (Shawn Adam), Chapman, Michael, Logan, Tim, Ellington, Ross, Stiegman, Al, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Phosphagen kinases are a family of enzymes that play a role in high-energy cells by buffering the ATP concentration. Arginine kinase (AK) catalyzes the magnesium-dependent reversible transphosphorylation between N-phospho-L-arginine (PArg) and ATP. Previous investigations of arginine kinase have suggested that precise substrate alignment may play an important role in this bimolecular reaction and that phosphagen specificity is mediated by an induced fit mechanism. In order to uncover the...
Show morePhosphagen kinases are a family of enzymes that play a role in high-energy cells by buffering the ATP concentration. Arginine kinase (AK) catalyzes the magnesium-dependent reversible transphosphorylation between N-phospho-L-arginine (PArg) and ATP. Previous investigations of arginine kinase have suggested that precise substrate alignment may play an important role in this bimolecular reaction and that phosphagen specificity is mediated by an induced fit mechanism. In order to uncover the mechanism of catalysis we present the crystallographic structures of arginine kinase with four arginine homologues: L-imino-ethyl-ornithine (ILO), L-citrulline (CIT), L-ornithine (ORN), and D-arginine (DARG) replacing the cognate arginine substrate. Each homologue was co-crystallized with MgADP- and nitrate as in the transition state analog complex (TSAC). The models were refined at 2.4Å, 2.0Å, 2.0Å and 2.8Å with Rfree values of 23%, 25%, 24% and 23%, respectively. These structures were used to study the basis of substrate specificity and the role of substrate alignment in catalysis. The non-cognate ligands bind and induce the substrate-bound enzyme conformation but are inactive. These data show that although AK is highly specific a number of different phosphagen mimics can bind, but are non-reactive. Comparison of these structures indicates that substrate specificity might be mediated by a mechanism that allows only the cognate substrate to be held rigidly in the proper orientation for efficient catalysis. These data also suggest that substrate alignment may play, at least, a role in catalysis. Here it is suggested that the induced fit movements of the enzyme most likely do not play a role in substrate discrimination but do play a role in substrate alignment. The structures also show that the guanidinium and α-amine moieties contribute little to substrate binding relative to the carboxylate, which appears to be key to substrate binding.
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Date Issued: 2006
Identifier: FSU_migr_etd-3596
Format: Thesis

Title: Assessment of Synthetic Matrix Metalloproteinase Inhibitors by Fluorogenic Substrate Assay.
Creator: Lively, Ty J., Department of Chemistry and Biochemistry
Abstract/Description: Matrix metalloproteinases (MMPs) are a family of metzincin enzymes that act as the principle regulators and remodelers of the extracellular matrix (ECM). While MMPs are involved in many normal biological processes, unregulated MMP activity has been linked to many detrimental diseases, including cancer, neurodegenerative diseases, stroke, and cardiovascular disease. To develop tools to investigate MMP functions and potential new therapeutics, matrix metalloproteinase inhibitors (MMPIs) have...
Show moreMatrix metalloproteinases (MMPs) are a family of metzincin enzymes that act as the principle regulators and remodelers of the extracellular matrix (ECM). While MMPs are involved in many normal biological processes, unregulated MMP activity has been linked to many detrimental diseases, including cancer, neurodegenerative diseases, stroke, and cardiovascular disease. To develop tools to investigate MMP functions and potential new therapeutics, matrix metalloproteinase inhibitors (MMPIs) have been designed, synthesized, and tested to regulate MMP activity. Inhibitor potencies were evaluated in terms of half maximal inhibitory concentrations (IC50 point) and apparent inhibition constants (Kiapp) for a series of YHJ cyclopentane and pyrolidine-based mercaptosulfonamide inhibitors using collagenase (MMPs-1), gelatinase A (MMP-2), matrilysin (MMP-7), and gelatinase B (MMP-9). MMPs with a shallow S1' binding pocket (MMP-1 and -7) were unable to distinguish between inhibitors showing low potency for nearly all synthetic analogs, the exception being GM6001. Conversely, potency levels of inhibitors tested with MMPs with an intermediate S1' pocket (MMP-2 and -9) varied among inhibitor. The most interesting variation occurred with YHJ-6-286 which was more than 30-fold more selective for MMP-2 than MMP-9, despite belonging to the same gelatinase class. To investigate the role stereoselectivity plays in enzyme inhibition, a dye-conjugate of inhibitor YHJ-7-52, YHJ-7-207, was tested for MMP-9. Results gathered suggest that the dye component of YHJ-7-207 produces a significant amount of steric hindrance as inhibition assays against MMP-9 revealed YHJ-7-207 having a larger IC50 point and Kiapp value than YHJ-7-52.
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Date Issued: 2015
Identifier: FSU_migr_uhm-0536
Format: Thesis

Title: Assessment of the Utility of Chemical Pretreatments for Estimating Carbon and Phosphorus Sequestration in Soils by 13C and 31P NMR Spectrscopy.
Creator: El-Rifai, Hasan M., Cooper, William T., Landing, William M., Dorsey, John G., Schlenoff, Joseph B., Safron, Sanford A., Department of Chemistry and Biochemistry, Florida State...
Show moreEl-Rifai, Hasan M., Cooper, William T., Landing, William M., Dorsey, John G., Schlenoff, Joseph B., Safron, Sanford A., Department of Chemistry and Biochemistry, Florida State University
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Abstract/Description: Sandy soils are a major forest resource in the southeastern U.S and intensive forest management is escalating; yet the effect of forest management on soil organic carbon (SOC) is not well documented. It is unclear to what degree root and aboveground litter inputs add to SOC; and if the relative importance of an input source changes with forest management. All these questions add to the ongoing discussion on the role of the soil in SOC storage and sequestration, as well as the impact of forest...
Show moreSandy soils are a major forest resource in the southeastern U.S and intensive forest management is escalating; yet the effect of forest management on soil organic carbon (SOC) is not well documented. It is unclear to what degree root and aboveground litter inputs add to SOC; and if the relative importance of an input source changes with forest management. All these questions add to the ongoing discussion on the role of the soil in SOC storage and sequestration, as well as the impact of forest management. Investigating the questions described above will allow us to better understand SOC sequestration and protection. In the first part of this work we have characterized labile and recalcitrant SOC pools from upland and wetland forests by acid hydrolysis (6 M HCl, 1 M HCl) and hot-water extraction. 13C nuclear magnetic resonance (NMR) spectroscopy was used to quantify the effect of acid hydrolysis and hot-water extraction on recalcitrant and labile carbon pools. Burial of phosphorus associated with organic matter has been reported as a major mechanistic sink for phosphorus in wetlands. Wetland soils tend to accumulate organic matter due to the production of detrital (plant) material from wetland biota and the suppressed rates of decomposition. Soil accretion rates for constructed wetlands are on the order of millimeters per year, although accretion rates in productive natural systems such as the Everglades have been reported as high as one centimeter per year or more. Current design of constructed wetlands for phosphorus removal is based upon this soil accretion. The rate of phosphorus accretion through this process is used to calculate the area needed to meet designed effluent criteria. However, although much of the phosphorus added to wetlands is retained within the system, this can serve as a phosphorus source to the water column for long periods of time, even after external loads are reduced. Wetlands are often used as `buffer zones' between agricultural areas and adjacent water bodies. The long-term effectiveness of these wetlands to retain and store phosphorus in stable forms depends upon interacting biogeochemical processes in water, detrital layers, and soil. Recognizing that detrital tissue and soil organic matter are the dominant components of wetlands, the breakdown of these materials and the release of nutrients have direct bearing on water quality and productivity of the ecosystem. Relationships developed between processes and physical and chemical properties of detrital plant tissue and soil organic matter can be incorporated into predictive models for extrapolation of results to other sites. The second phase of the research summarized in this dissertation addresses this important issue, and has several unique themes. First, the organic phosphorus forms in a range of wetland ecosystems and their relationships with soil physical and chemical properties have never been studied. We have addressed these issues here by employing state-of-the-art NMR techniques to provide insight into the forms of inorganic and organic phosphorus and organic carbon in recently accreted and native Everglades soils. Second, rarely have linkages been developed between organic carbon and organic phosphorus forms as they relate to their stability under a range of environmental perturbations, but this is of key importance for understanding the biogeochemistry of organic phosphorus in the environment. This research therefore provided information on the composition and stability of soil organic phosphorus and carbon in wetlands, again using primarily NMR techniques. Liquid chromatography and high resolution time-of-flight mass spectrometry were also used to validate the NMR studies.
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Date Issued: 2007
Identifier: FSU_migr_etd-0579
Format: Thesis

Title: Asymmetric Copper Catalyzed Addition to Activated Alkenes.
Creator: Lackey, Hershel, McQuade, Tyler D., Dudley, Greg B., Shatruk, Michael, Zhu, Lei, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: This thesis presents the synthesis of a chiral 6-membered NHC catalyst copper catalyst and its use in the β-borylation and allylic substitution reactions. The application of the catalyst in the known β-borylation reaction demonstrates that the catalyst can provide high yields, enatioselectivities, and activity comparable if not improved to traditional 5-membered NHCs and phosphine copper complexes. The 6-membered NHC copper complex was then used in a nucleophilic diboron addition to allylic...
Show moreThis thesis presents the synthesis of a chiral 6-membered NHC catalyst copper catalyst and its use in the β-borylation and allylic substitution reactions. The application of the catalyst in the known β-borylation reaction demonstrates that the catalyst can provide high yields, enatioselectivities, and activity comparable if not improved to traditional 5-membered NHCs and phosphine copper complexes. The 6-membered NHC copper complex was then used in a nucleophilic diboron addition to allylic aryl ethers where the catalyst demonstrated different chemoselectivity compared to 5-membered NHC and phosphine catalyst. Further investigation of this phenomenon led to development of a stereoconvergent, asymmetric reaction which could convert mixtures of E and Z isomers into a single enantiomer in high yield.
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Date Issued: 2011
Identifier: FSU_migr_etd-3325
Format: Thesis

Title: Asymmetric Michael Additions of Lithium Propionate Enolates to α,β-Unsaturated Esters: A Study Towards the Total Synthesis of Lonomycin A.
Creator: Jo, Sunjin, Holton, Robert A., Reeves, Robert H., Krafft, Marie E., Zakarian, Armen, Blaber, Michael, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: The Michael addition of lithium enolates to α,β unsaturated esters is considered to be one of the powerful and widely used C-C bond forming methods in organic synthesis. We investigated the Michael addition of lithium propionate enolates using the various chiral auxiliaries to α,β unsaturated esters, methyl 2-bromo-3-methoxy acrylate and dioxinones. Various chiral propionates were prepared from the optically pure and commercially available terpenes in several steps. Michael additions using...
Show moreThe Michael addition of lithium enolates to α,β unsaturated esters is considered to be one of the powerful and widely used C-C bond forming methods in organic synthesis. We investigated the Michael addition of lithium propionate enolates using the various chiral auxiliaries to α,β unsaturated esters, methyl 2-bromo-3-methoxy acrylate and dioxinones. Various chiral propionates were prepared from the optically pure and commercially available terpenes in several steps. Michael additions using the chiral auxiliary introduced moderate to high stereoselectivities, and the factors that influence the stereoselection were examined. These results are consistent with a chelated transition state. The stereochemistry of Michael adducts was determined by the Mosher's esterification and NMR experiments. Lonomycin A, which was isolated from Streptomyces ribosidificus in 1975, is a polycyclic ether constituted of six highly functionalized rings and 23 stereocenters. Our synthetic strategy relying on the asymmetric Michael addition of lithium propionate enolate to α,β unsaturated esters introduced stereoselectively the contiguous and alternating methyl and methoxy moieties. This attractive methodology gave an easy access to the crucial intermediates in the synthetic approach towards the right-half fragment of Lonomycin A.
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Date Issued: 2008
Identifier: FSU_migr_etd-3505
Format: Thesis

Title: Automated Analysis of Protein Side Chain Spectra.
Creator: Hart, Andrew, Department of Chemistry and Biochemistry
Abstract/Description: Manual Nuclear Magnetic Resonance (NMR) spectral analysis of proteins is a time intensive effort with methods often specific to each analysis. The method described in this thesis automates the resonance assignment of protein side chains using a TOCSY (Totally Correlated Spectroscopy) NMR experiment. The system under study is Ubiquitin (8.6 kDa). 54 of the 70 available amino acid side chains were identified by a single TOCSY spectrum in less than 5 min of local computer runtime using the...
Show moreManual Nuclear Magnetic Resonance (NMR) spectral analysis of proteins is a time intensive effort with methods often specific to each analysis. The method described in this thesis automates the resonance assignment of protein side chains using a TOCSY (Totally Correlated Spectroscopy) NMR experiment. The system under study is Ubiquitin (8.6 kDa). 54 of the 70 available amino acid side chains were identified by a single TOCSY spectrum in less than 5 min of local computer runtime using the algorithms described. Automation of spectral analysis can enhance reproducibility and create standards of spectral analysis.
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Date Issued: 2013
Identifier: FSU_migr_uhm-0244
Format: Thesis

Title: Autonomous and Guided Motion of Active Components at Interfaces.
Creator: Dhar, Prajnaparamita, Fischer, Thomas, Wiedenh¨over, Ingo, Steinbock, Oliver, Miller, Brian, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Nature presents active components in the form of proteins and molecular motors that are involved in locomotion and inter compartmental transport. Inspired by nature, this dissertation focuses on presenting experimental means to understand the motion of active components at interfaces. Active components are defined as components that either autonomously move or start to move in a viscous fluctuating environment. We investigate mechanisms by which energy may be converted into directed motion in...
Show moreNature presents active components in the form of proteins and molecular motors that are involved in locomotion and inter compartmental transport. Inspired by nature, this dissertation focuses on presenting experimental means to understand the motion of active components at interfaces. Active components are defined as components that either autonomously move or start to move in a viscous fluctuating environment. We investigate mechanisms by which energy may be converted into directed motion in case of synthetic nanomotors. Strategies for achieving guided motion along predetermined paths by overcoming fluctuations are explored. In addition we attempt to use dissipation in these systems to achieve guided motion at interfaces. Thus, this dissertation lays some of the foundations necessary for an understanding and smart design of synthetic motors.
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Date Issued: 2008
Identifier: FSU_migr_etd-0746
Format: Thesis

Title: Backbone Dynamics in an Intramolecular Prolylpeptide SH3 Complex from Diphtheria Toxin Repressor, DtxR.
Creator: Bhattacharya, Nilakshee, Logan, Timothy M., Zhou, Huan-Xian, Li, Hong, Steinbock, Oliver, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Diphtheria toxin repressor is a regulatory protein from Corynebacterium diphtheriae, the causal agent of Diphtheria. The diphtheria toxin repressor (DtxR) contains an SH3-like domain that forms an intramolecular complex with a proline-rich (Pr) peptide segment that serves to stabilize the inactive state of the repressor. During activation of DtxR by transition metals, this intramolecular complex must dissociate as the SH3 domain and Pr segment form different interactions in the active...
Show moreDiphtheria toxin repressor is a regulatory protein from Corynebacterium diphtheriae, the causal agent of Diphtheria. The diphtheria toxin repressor (DtxR) contains an SH3-like domain that forms an intramolecular complex with a proline-rich (Pr) peptide segment that serves to stabilize the inactive state of the repressor. During activation of DtxR by transition metals, this intramolecular complex must dissociate as the SH3 domain and Pr segment form different interactions in the active repressor. In this study we investigate the dynamics of this intramolecular complex using backbone amide nuclear spin relaxation rates determined experimentally using NMR spectroscopy and computed from molecular dynamics trajectories. The SH3 domain in the unbound and bound states showed typical dynamics in that the secondary structures were fairly ordered with high generalized order parameters and low effective correlation times while residues in the loops connecting b-strands exhibited reduced generalized order parameters and required additional motional terms to adequately model the relaxation rates. Residues forming the Pr segment also exhibited low order parameters with internal rotational correlation times on the order of 0.6 â 1 ns. Further analysis showed that the SH3 domain was rich in ms motions while the Pr segment was rich in motions on the 100s ms timescale. Molecular dynamics trajectories of PrSH3 and SH3 indicated structural rearrangements that might contribute to the observed relaxation rates and, together with the observed relaxation rate data, suggested that the Pr segment exhibits a binding â unbinding equilibrium. The intramolecular complex resisted any significant change in the binding affinity between the Pr segment and the SH3 domain due to mutations in the Pr segment. The results of this study provide key insights into the nature of the intramolecular complex and provide a better understanding of the biological role of the SH3 domain in regulating DtxR activity.
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Date Issued: 2007
Identifier: FSU_migr_etd-1408
Format: Thesis

Title: Batch Anion Exchange Separation: A Prefractionation Technique for Proteome Research and Its Applications on In Vivo Cancer Samples.
Creator: Sahab, Ziad Joseph, Sang, Qing-Xiang A., Deng, Wu-Min, Cooper, William T., Marshall, Alan G., Schlenoff, Joseph B., Department of Chemistry and Biochemistry, Florida State...
Show moreSahab, Ziad Joseph, Sang, Qing-Xiang A., Deng, Wu-Min, Cooper, William T., Marshall, Alan G., Schlenoff, Joseph B., Department of Chemistry and Biochemistry, Florida State University
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Abstract/Description: Two-dimensional gel electrophoresis (2-DE) is used to compare the protein profiles of different crude biological samples. Narrow pH range Immobilized pH Gradient (IPG) strips were designed to increase the resolution of these separations. To take full advantage of IPG strips, the ideal sample should be composed primarily of proteins that have isoelectric point (pI) values within the pH range of the IPG strip. Prefractionation of cell lysates from a human prostate cancer cell line cultured in...
Show moreTwo-dimensional gel electrophoresis (2-DE) is used to compare the protein profiles of different crude biological samples. Narrow pH range Immobilized pH Gradient (IPG) strips were designed to increase the resolution of these separations. To take full advantage of IPG strips, the ideal sample should be composed primarily of proteins that have isoelectric point (pI) values within the pH range of the IPG strip. Prefractionation of cell lysates from a human prostate cancer cell line cultured in the presence or absence of epigallocatechin-3-gallate was achieved in fewer than 30 minutes using an anion exchange resin and two expressly-designed buffers. The procedure was carried out in a centrifuge tube and standard instrumentation was used. The cell lysates were prefractionated into two fractions: proteins with pI values above 7 and between 4 and 7, respectively. The fractions were then analyzed by 2-DE, selecting appropriate pH ranges for the IPG strips, and the gels were compared with those of unprefractionated cell lysates. Protein loading capacity was optimized and resolution and visualization of the less abundant and differentially-expressed proteins were greatly improved. Furthermore, this technique was applied successfully to eliminate albumin from serum samples. Elimination of albumin, constituting more than 50% of total serum proteins, allowed for increasing protein loads on polyacrylamide gels. Visualization and resolution of lower-abundance proteins that were previously masked by albumin was achieved as well.
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Date Issued: 2005
Identifier: FSU_migr_etd-2111
Format: Thesis

Title: Biochemical Characterization of Human Matrix Metalloproteinases and Their Newly Designed Inhibitors Related to Stroke.
Creator: Cao, Qiang, Sang, Qing-Xiang Amy, Wang, Yan-Chang, Li, Hong, Alabugin, Igor, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Matrix metalloproteinases (MMPs), a family of enzymes known for their proteolytic activities on processing extracellular matrix substrates, may play an integral role in blood-brain barrier opening following an ischemic stroke. Several matrix metalloproteinases are proposed to play vital roles in early or late stages of blood brain barrier opening. Matrix metalloproteinase inhibitor (MMPI) has been showing beneficial effect in the treatment of the blood brain opening related to stroke. As old...
Show moreMatrix metalloproteinases (MMPs), a family of enzymes known for their proteolytic activities on processing extracellular matrix substrates, may play an integral role in blood-brain barrier opening following an ischemic stroke. Several matrix metalloproteinases are proposed to play vital roles in early or late stages of blood brain barrier opening. Matrix metalloproteinase inhibitor (MMPI) has been showing beneficial effect in the treatment of the blood brain opening related to stroke. As old generation matrix metalloproteinase inhibitor failed in oncology clinical trials, our collaborators, Drs. Martin A. Schwartz and Yonghao Jin, have designed and synthesized new biologically friendly mercaptosulfonamide inhibitors. Characterization and selection of effective matrix metalloproteinase inhibitors were performed by evaluating their stability, potency, and selectivity by enzymatic kinetics. According to dissociation constant related to enzyme and inhibitor binding, our data indicates that those inhibitors are capable of inhibiting MMP-2, -9, and membrane-type 1 MMP (MT1-MMP) effectively and selectively. Selected inhibitors were studied with cell wound healing assays in human microvascular endothelial cell model to investigate selected MMPI activities and impact on cell behavior. By blocking MMP activities in cell culture, our inhibitors were able to reduce human brain microvascular endothelial cell wound healing process. Protein expression patterns in cell culture were investigated with proteomics after inhibitor treatment. The reduced expressions of several proteins, which are related to cell division, cell adhesion and cell death, have been discovered. It is also verified the blocking function of our inhibitor in human brain microvascular endothelial cell wound healing assay. Overall, our newly designed matrix metalloproteinase inhibitor efficiently inhibits matrix metalloproteinase which carries intermediate or deep S1' pocket at protein and cellular level. Upon application of matrix metalloproteinase inhibitor, it has been implicated that blocking of matrix metalloproteinase activities are involved in the decreasing of other cell function modulators. The most potent and specific inhibitors have been selected as promising compounds, which have been further tested in animal models to evaluate their efficacy in the prevention of blood brain barrier opening associated with stroke by our collaborator Dr. Gary A. Rosenberg. This study is the first enzymological and cellular analysis of mercaptosulfonamide inhibitors.
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Date Issued: 2010
Identifier: FSU_migr_etd-4624
Format: Thesis

Title: Biomimetic Cascade Reactions Towards the Synthesis of Ciguatoxin.
Creator: Bou Hamdan, Farhan Ramez, Holton, Robert A., Keller, Laura L., Schwartz, Martin A., Krafft, Marie E., Logan, Timothy M., Department of Chemistry and Biochemistry, Florida State...
Show moreBou Hamdan, Farhan Ramez, Holton, Robert A., Keller, Laura L., Schwartz, Martin A., Krafft, Marie E., Logan, Timothy M., Department of Chemistry and Biochemistry, Florida State University
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Abstract/Description: Considerable attention has recently been given to the preparation of the ciguatoxins, the major group of toxins implicated with the onset of ciguatera fish poisoning (CFP) – a seafood-borne illness associated with the consumption of reef fish in tropical and subtropical areas. Ciguatoxins are characterized by a very complex polycyclic framework of 13 ether rings ranging from five to nine members. Previous studies directed towards the synthesis of the ciguatoxin skeleton have illustrated the...
Show moreConsiderable attention has recently been given to the preparation of the ciguatoxins, the major group of toxins implicated with the onset of ciguatera fish poisoning (CFP) – a seafood-borne illness associated with the consumption of reef fish in tropical and subtropical areas. Ciguatoxins are characterized by a very complex polycyclic framework of 13 ether rings ranging from five to nine members. Previous studies directed towards the synthesis of the ciguatoxin skeleton have illustrated the difficulty associated with the construction of medium rings, particularly eight and nine-membered. We envisaged that an electrophile-induced epoxy-alcohol cascade cyclization, previously developed in our laboratory and utilized in the synthesis of hemibrevetoxin-B, could be used for the construction of medium-ring ethers. As little was known about this cyclization cascade, an investigation has been launched to test the potential of this biomimetic approach in the construction of [6,8]-trans-fused bicyclic ethers and its applicability towards the synthesis of the HIJ rings of the ciguatoxins. This study has revealed that the construction of oxocanes via this epoxy-alcohol cascade cyclization methodology is feasible, and that molecular tethers facilitate the reaction. Several tethers including a benzene ring, a cis or trans-fused dioxolane were tested. Interestingly, the use of a dioxolane tether has demonstrated that a considerable level of diastereocontrol can be obtained. Moreover, the incorporation of the nucleophilic alcohol group within a cyclic structure has proven to have a large effect on the Baldwin vs. anti-Baldwin selectivity. A theoretical study regarding that aspect has further supported our findings. Finally, this work has resulted in the synthesis of an advanced tricylic intermediate of the ciguatoxin skeleton. It is one of the most efficient pathways reported so far, as it provides this advanced intermediate in 23 linear steps and 11% overall yield.
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Date Issued: 2007
Identifier: FSU_migr_etd-3506
Format: Thesis

Title: Biophysical and Biochemical Investigation of an Archaeal Box C/D SRNP: RNA-Protein Interactions of a Kink Turn RNA within the Functional Enzyme.
Creator: Moore, Terrie Luong, Li, Hong, Epstein, Lloyd M., Logan, Timothy M., Dorsey, John G., Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Box C/D snoRNPs catylze the specific 2'O-methylation of rRNA in important regions the ribosome, although the role of the modifications is unclear. Eukaryotic box C/D snoRNPs consists of a box C/D RNA and four proteins, Fibrillarin, Nop56, Nop58, and 15.5kD. Archaeal homologs are simiplified containing three proteins, L7Ae, Nop5p, and Fibrillarin with a box C/D RNA. The box C/D sequences are proposed to form the recently recognized kink turn structure which is found in many types of RNA. Most...
Show moreBox C/D snoRNPs catylze the specific 2'O-methylation of rRNA in important regions the ribosome, although the role of the modifications is unclear. Eukaryotic box C/D snoRNPs consists of a box C/D RNA and four proteins, Fibrillarin, Nop56, Nop58, and 15.5kD. Archaeal homologs are simiplified containing three proteins, L7Ae, Nop5p, and Fibrillarin with a box C/D RNA. The box C/D sequences are proposed to form the recently recognized kink turn structure which is found in many types of RNA. Most are associated with proteins and protein binding may nucleate the assembly of other proteins onto the RNA. Dissecting the structure and biochemical properties of box C/D snoRNPs may not only help in understanding the function of the modifications, but may also give insight into the role of kink turn RNAs in RNP assembly. An archaeal box C/D RNA embedded within the intron of pre-tRNATrp from Archaeglobus Fulgidus(AF) that guides two modifications in the tRNA was used as the model for the investigation of three complexes: L7Ae-box C/D RNA, L7Ae-box C'/D' RNA, and the entire box C/D sRNP. Extensive crystallization trials resulted in crystals for each complex. A crystal structure of the box C/D RNA-L7Ae complex was determined to 2.7Å and shows the box C/D sequences do form a kink turn. Detailed structural comparisons of the AF L7Ae-box C/D RNA complex with previously determined crystal structures of L7Ae homologs in complex with functionally distinct kink turn RNAs revealed a conserved RNA-protein interface suggesting a conformational "adaptability" of the kink turn RNAs in binding L7Ae homologs. NMR characterization of L7Ae-box C/D RNA and L7Ae-box C'/D' RNA interactions suggests a structural change in the RNAs upon binding L7Ae and the RNAs may be dynamic structures that do not form stable kink turns alone. The underlying differences in primary and secondary structures of the kink turns may lead to different tertiary structures and dynamic behavior in kink turn RNAs that may confer specificity of L7Ae homologs for different kink turn RNAs. These analyses provide a structural basis for interpreting the functional roles of the box C/D sequences in directing specific assembly of box C/D sRNPs.
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Date Issued: 2005
Identifier: FSU_migr_etd-2320
Format: Thesis

Title: Biophysical Characterization of a ssDNA Virus.
Creator: Morrison, Anneliese J., Department of Chemistry and Biochemistry
Abstract/Description: Viral capsids must assemble into stable structures that resist dissociation in extreme environments between hosts yet they simultaneously must be unstable enough to release their genome upon infection. The conflicting functions that the viral capsid must fulfill suggests that they exhibit an evolutionarily fine-tuned structure/function relationship that is not apparent in many other systems. Biophysical characterization of viral assembly and disassembly processes can aid in developing an...
Show moreViral capsids must assemble into stable structures that resist dissociation in extreme environments between hosts yet they simultaneously must be unstable enough to release their genome upon infection. The conflicting functions that the viral capsid must fulfill suggests that they exhibit an evolutionarily fine-tuned structure/function relationship that is not apparent in many other systems. Biophysical characterization of viral assembly and disassembly processes can aid in developing an understanding of the physical mechanisms that underlie the relationship between tightly linked phenotypes in complex protein systems. In this honor's thesis project, the dissociation process was biophysically characterized in an ssDNA bacteriophage. After the development of a PEG precipitation based purification method, intrinsic fluorescence spectroscopy, static light scattering, and plaque assays were used to develop a two-step model that describes the molecular events that occur during Microvirid bacteriophage capsid dissociation. At 57˚C using a scan rate of 1˚C/min, loss of 99% of viral activity is observed corresponding to loss of the major spike protein. At 69˚C transitions are seen in fluorescence and light scattering spectra that indicate a structural change is occurring. Plaque assays confirm that immediately after the structural transition occurs all viral activity is lost, indicating that this second step represents global capsid dissociation.
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Date Issued: 2014
Identifier: FSU_migr_uhm-0428
Format: Thesis

Title: Broadband Phase Correction of Fourier Transform Ion Cyclotron Resanonce Mass Spectra.
Creator: Xian, Feng, Marshall, Alan G., Hendrickson, Christopher L., Hill, Stephen, Dalal, Naresh S., Roper, Michael, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: It has been known for 35 years that phase correction of Fourier transform ion cyclotron resonance (FT-ICR) mass spectral data can in principle produce an absorption-mode spectrum with mass resolving power as much as a factor of 2 higher than conventional magnitude-mode display, an improvement otherwise requiring a (much more expensive) increase in magnetic field strength. However, temporally dispersed excitation followed by time-delayed detection results in steep quadratic variation of signal...
Show moreIt has been known for 35 years that phase correction of Fourier transform ion cyclotron resonance (FT-ICR) mass spectral data can in principle produce an absorption-mode spectrum with mass resolving power as much as a factor of 2 higher than conventional magnitude-mode display, an improvement otherwise requiring a (much more expensive) increase in magnetic field strength. However, temporally dispersed excitation followed by time-delayed detection results in steep quadratic variation of signal phase with frequency. We developed a robust, rapid, automated method to enable accurate broadband phase correction for all peaks in the mass spectrum. Low-pass digital filtering effectively eliminates the accompanying baseline roll. Experimental FT-ICR absorption-mode mass spectra exhibit at least 40% higher resolving power (and thus an increased number of resolved peaks) as well as higher mass accuracy relative to magnitude mode spectra, for more complete and more reliable elemental composition assignments for mixtures as complex as petroleum. Absorption-mode FT-ICR mass spectrum, which is produced by automatic broadband phase correction algorithm, demonstrates baseline distortion even with low-pass filter baseline correction. Significant baseline roll affects peaks picking algorithm and results in incorrect peak height measurement. Isotopic distribution in spectra presenting large baseline roll couldn't display correct information. Thus, identification and characterization of biomolecule become much more difficult. In Chapter 2, we designed a fast, robust and automated baseline correction process. Each minimum data point of reversed peak in absorption-mode spectrum has been collected as bases of baseline model, and then further linear interpolation and boxcar smoothing technique help to complete the baseline model. Finally, the baseline model is subtracted from original spectrum to produce a flat baseline. This algorithm has been experimentally proven to automatically flatten baseline of crude oil, environmental sample and biomolecule FT-ICR mass spectra. More peaks have been identified from absorption-mode spectrum with flat baseline without loss of mass accuracy. Isotopic distribution also demonstrates very accurate profile. Apodization function and zero-filling are two basic steps in data processing of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry and their effect on the conventional Fourier transform ion cyclotron resonance (FT-ICR) experimental and simulated magnitude-mode mass spectra and single-peak absorption-mode spectra are well known. In Chapter 4, we examine the effects of each of twelve apodization (window) functions and 0, 1, and 2 zero-fills for absorption-mode Fourier transform mass spectra peak height-to-noise ratio, mass measurement accuracy, and mass resolving power for dense FT-ICR mass spectra of petroleum. Half function windowing is best for resolving close absorption-mode doublets, whereas full function windowing is best for resolving magnitude-mode doublets. Absorption-mode offers significantly higher mass resolving power than magnitude-mode for any given windowing function. Half apodization increases absorption-mode mass accuracy, irrespective of the choice of window function. One (but not more than one) zero-fill improves mass accuracy for absorption-mode mass accuracy but not for magnitude-mode. Peak height-to-noise ratio for both absorption and magnitude spectra is improved by zero-filling. Although we have successfully demonstrated the automated phase correction method for complex Fourier transform ion cyclotron resonance (FT-ICR) mass spectrum, we can't express the exact quadratic phase function of frequency from calculated phase for discrete data point. In Chapter 5, we applied stationary phase method to excitation and detection signal and derived the accurate phase spectra for both the linear chirp excitation and detected FT-ICR signals analytically. Because phase spectrum of detected signal represents correct variation of accumulated phase with frequency, it could be directly used to recover the absorption-mode FT-ICR mass spectra. Also, the phase correction of FT-ICR mass spectra from stored waveform inverse Fourier transform (SWIFT) by phase spectrum has been experimentally described. The analytically phase correction results are compared to the previous results produced by automated phase correction method in terms of resolving power and mass measurement accuracy Except for phase correction based on mathematical calculation of accurate phase for different frequencies. Scientists have demonstrated that simultaneous excitation and detection (SED) enable Fourier deconvolution to provide broadband phase correction with no user interaction. However, the capacitive nulling technique which is applied in SED method for removing the saturated excitation signal in front of detected signal is not practical due to unstable capacitors. In Chapter 6, we describe a new data processing procedure to enable broadband phase correction of FT-ICR mass spectra by SED without any hardware modification. The resulting absorption-mode spectra yield improvement in resolving power as well as reduction in assignment errors relative to conventional magnitude-mode spectra. The Fourier deconvolution procedure has the additional benefit of correcting for spectral variation resulting from nonuniform power distribution over the excitation bandwidth and phasing spectra from different excitation waveforms (e.g., SWIFT with different magnitude modulations). Fourier transform mass spectrometry (FTMS) of the isolated isotopic distribution for a highly charged biomolecule produces time-domain signal containing large amplitude signal "beats" separated by extended periods of much lower signal magnitude. Signal-to-noise ratio for data sampled between beats is low, due to destructive interference of the signals induced by members of the isotopic distribution. Selective blanking of the data between beats has been used to increase spectral signal-to-noise ratio. However, blanking also eliminates signal components, and thus can potentially distort the resulting FT spectrum. In Chapter 7, we simulate the time-domain signal from a truncated isotopic distribution for a single charge state of an antibody. Comparison of the FT spectra produced with or without blanking and with or without added noise clearly show that blanking does not improve mass accuracy and introduces spurious peaks at both ends of the isotopic distribution (thereby making it more difficult to identify posttranslational modifications and/or adducts). Ergo, blanking should never be employed: it has no advantages and major disadvantages.
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Date Issued: 2012
Identifier: FSU_migr_etd-6074
Format: Thesis

Title: Calcium Dependency of Human Matrix Metalloproteinase-26 and Its Potential Contribution to Early-Stage Adenocarcinoma.
Creator: Lee, Seakwoo, Sang, Qing-Xiang Amy, Keller, Laura R., Li, Hong, Hilinski, Edwin F., Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Human matrix metalloproteinases-26 (MMP-26/endometase/matrilysin-2) is a newly identified MMP. MMP-26 has one high- and low-affinity calcium binding sites, C1 and C3, respectively, and loss of low-affinity Ca2+ induces tertiary structural changes resulting in dramatic decrease of enzymatic function without secondary structural change. Subsequent loss of high-affinity Ca2+ results in irreversible enzymatic deactivation without further tertiary and secondary structure change. No secondary...
Show moreHuman matrix metalloproteinases-26 (MMP-26/endometase/matrilysin-2) is a newly identified MMP. MMP-26 has one high- and low-affinity calcium binding sites, C1 and C3, respectively, and loss of low-affinity Ca2+ induces tertiary structural changes resulting in dramatic decrease of enzymatic function without secondary structural change. Subsequent loss of high-affinity Ca2+ results in irreversible enzymatic deactivation without further tertiary and secondary structure change. No secondary structural change is associated with the loss of high- or low-affinity Ca2+. Calcium titration revealed calcium dissociation constant (KD1) value of 63 nM at the high-affinity calcium binding site, and KD2 value of 120 mM at the low-affinity calcium binding site. Mutagenesis studies at the putative low-affinity calcium binding site revealed that a K189E mutation (C3 site) decreased KD2 value, resulting in 28 mM, while a V184D mutation (C2 site) increased KD2 value, resulting in 240 mM. K189E mutant acquires lower Ca2+ for active conformation, whereas V184D mutant acquires higher Ca2+. These lead to the conclusion that structure and function of MMP-26 may be regulated by the calcium concentration. Prostate cancer tissue analyses show the expressions of the MMP-26 and TIMP-4. Co-immunoprecipitation revealed a possible MMP-26/TIMP-4 complex formation. Disruption of the basal cell layer is necessary for high-grade prostatic intraepithelial neoplasia (HGPIN) to proceed toward cancer. Immunohistochemistry analysis of prostate cancer tissue slides shows that the expression levels of MMP-26 and TIMP-4 are highest in HGPIN. Moreover, immunohistochemical staining of serially sectioned prostate cancer tissue slides reveals similar patterns of staining for MMP-26 and TIMP-4 on adjacent sections. Therefore, MMP-26 and TIMP-4 are expressed in the HGPIN simultaneously. The highest levels of MMP-26 and TIMP-4 in HGPIN suggest that they might be biomarkers for the early detection of the prostate cancer.
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Date Issued: 2007
Identifier: FSU_migr_etd-3190
Format: Thesis

Title: Cancer Immune Evasion Mechanisms and the Role of Granzyme B in Tumor Progression.
Creator: Burjas Bou-Dargham, Mayassa J., Sang, Qing-Xiang, Zhang, Jinfeng, Zhu, Fanxiu, Logan, Timothy M., Bleiholder, Christian, Florida State University, College of Arts and Sciences,...
Show moreBurjas Bou-Dargham, Mayassa J., Sang, Qing-Xiang, Zhang, Jinfeng, Zhu, Fanxiu, Logan, Timothy M., Bleiholder, Christian, Florida State University, College of Arts and Sciences, Department of Chemistry and Biochemistry
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Abstract/Description: Major hallmarks of cancer include metastasis and evading the immune system. Despite cutting edge treatments developed in an era of extensive cancer research, immunotherapy has not been proven efficient enough in solid tumors, and metastasis still accounts for the majority of cancer deaths. The overall unsatisfactory response rates to immunotherapy are mainly due to the lack of biomarkers that can predict a patient’s response and the lack of a good understanding of the different immune cell...
Show moreMajor hallmarks of cancer include metastasis and evading the immune system. Despite cutting edge treatments developed in an era of extensive cancer research, immunotherapy has not been proven efficient enough in solid tumors, and metastasis still accounts for the majority of cancer deaths. The overall unsatisfactory response rates to immunotherapy are mainly due to the lack of biomarkers that can predict a patient’s response and the lack of a good understanding of the different immune cell infiltration trends observed in tumors. To address these gaps in breast and prostate cancer, RNA sequenced data for breast and prostate cancer samples were obtained from The Cancer Genome Atlas (TCGA) and analyzed to identify immune evasion mechanisms and understand immune cell infiltration. Breast and prostate cancer populations were each clustered into different immune evasion groups. Then biomarkers predictive of the identified clusters were identified and could be used as predictors of immune evasion and the corresponding immunotherapy options. In breast cancer, 77.4% of the clustered tumor specimens showed evasion through transforming growth factor-beta (TGF-β), 57.8% through decoy receptor 3 (DcR3), 48.0% through cytotoxic T-lymphocyte-associated protein 4 (CTLA4), and 34.3% through programmed cell death-1 (PD-1). Prostate cancer clustering showed immunologic ignorance in 89.77% of samples, upregulated CTLA4 in 58.8%, and upregulated DcR3 expression in 51.6%. However, in most clusters, there were different combinations of evasion mechanisms, which could explain the failure of immune monotherapy approaches. The immune profiling of breast cancer samples suggests that immunologically cold tumors are not only less immunogenic than hot tumors, but also have a high abundance of the pro-tumorigenic M2 macrophages and a stiff matrix, all of which can impede immune cell infiltration. Thus, M2 is a novel prognostic factor in breast cancer and a promising drug target. Epithelial-mesenchymal transition (EMT) is a critical early step in cancer metastasis. Further understanding of this process may shed light on how to stop the spreading of cancer cells. Androgen-repressed prostate cancer (ARCaP) cell lines representative of the epithelial (ARCaP-E) and mesenchymal (ARCaP-M) phenotypes were used and their secretome was investigated using proteomics approaches. High levels of proteins involved in bone remodeling and extracellular matrix degradation were detected in the ARCaP-M cells, indicative of a bone metastatic phenotype. LC-MS/MS analysis showed that the serine protease granzyme B (GZMB) was 800-fold higher in ARCaP-M conditioned media. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and Western blot further showed that GZMB was expressed and translated in ARCaP-M cells and the protein is only detected extracellularly. ARCaP-M cells with the GZMB gene knockdown using RNA interference showed a markedly reduced invasiveness as demonstrated by the Matrigel invasion assay. Our findings indicate a novel role for GZMB in prostate cancer invasion and extracellular matrix degradation.
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Date Issued: 2019
Identifier: 2019_Fall_BurjasBouDargham_fsu_0071E_15520
Format: Thesis

Title: Cascade Reactions for the Synthesis of Polycyclic Aromatic Hydrocarbons and Carbon Nanoribbons.
Creator: Byers, Philip M., Alabugin, Igor V., Alamo, Rufina, Strouse, Geoffrey, Saha, Sourav, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Radical and electrophile-promoted nucleophilic closure cascade reactions have been used for the synthesis of polycyclic aromatic hydrocarbons (PAHs) and carbon nanoribbons. Chapter 1 provides a discussion on radical cascade reactions, with a brief history and examples of well documented radical cascade reactions. Investigations into radical chain processes as well as redox processes are discussed. There is much development in these cascades currently due to the complex products that can be...
Show moreRadical and electrophile-promoted nucleophilic closure cascade reactions have been used for the synthesis of polycyclic aromatic hydrocarbons (PAHs) and carbon nanoribbons. Chapter 1 provides a discussion on radical cascade reactions, with a brief history and examples of well documented radical cascade reactions. Investigations into radical chain processes as well as redox processes are discussed. There is much development in these cascades currently due to the complex products that can be formed in a minimal amount of synthetic steps. Chapter 2 discusses the radical cascade that we developed, using a radical chain process. Selective radical generation in conjugated oligomeric o-aryleneethynylenes initiates a cascade which involves five fast radical cyclizations followed by aromatization via a 1,5-H shift to yield PAHs. Computation and 2D NMR studies were performed to determine the final cascade products. Chapter 3 discusses the growing trend in which gold catalysts are used to initiate cascade reactions through an electrophile-promoted nucleophilic closure mechanism. A number of current examples are also provided which demonstrate how gold catalyzed cascade reactions can efficiently synthesize complex PAH molecules. Chapeter 4 demonstrates our approach for the synthesis of fused benzofuran structures through gold catalysis. Through a gold catalyzed electrophile-promoted nucleophilic closure, PAHs containing benzofuran units with a polycyclic aromatic backbone were synthesized through a Sonogashira/5-endo-dig/6-endo-dig cascade. Depending on which mechanism is used, all exo cyclizations can be achieved (radical), or all endo cyclizations (electrophile-promoted nucleophilic closure). Starting materials for either cascade mechanism can be synthesized in a modular fashion, allowing for the systematic and controlled preparation of functionalized PAHs and carbon nanoribbons where, potentially, each of the peripheral aromatic rings can be different. The materials synthesized have future applications in the nano-materials and -electronics industries. Chapter 5 discusses future work in the field of gold catalyzed cascade reactions and provides conclusions on the work presented here within.
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Date Issued: 2013
Identifier: FSU_migr_etd-7737
Format: Thesis

Title: Cell Adhesion Behavior and Protein-Surface Interactions on Polyelectrolyte Multilayer Thin Films.
Creator: Surmaitis, Richard L., Schlenoff, Joseph B., Locke, Bruce R., Kennemur, Justin Glenn, Zhu, Lei, Florida State University, College of Arts and Sciences, Department of Chemistry...
Show moreSurmaitis, Richard L., Schlenoff, Joseph B., Locke, Bruce R., Kennemur, Justin Glenn, Zhu, Lei, Florida State University, College of Arts and Sciences, Department of Chemistry and Biochemistry
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Abstract/Description: Polyelectrolyte multilayer thin films have been used as coatings due to their biocompatibility for cell culture experiments. During layer-by-layer assembly of these multilayer thin films, the film is altered by adjusting the salt concentration, the rinsing time, and the temperature which changes the morphology of the film. Certain factors such as surface charge and the polymer effect the biocompatibility of the film and its ability to adsorb serum proteins. If cells adhere and proliferate on...
Show morePolyelectrolyte multilayer thin films have been used as coatings due to their biocompatibility for cell culture experiments. During layer-by-layer assembly of these multilayer thin films, the film is altered by adjusting the salt concentration, the rinsing time, and the temperature which changes the morphology of the film. Certain factors such as surface charge and the polymer effect the biocompatibility of the film and its ability to adsorb serum proteins. If cells adhere and proliferate on a film it is called 'cytophilic'. Conversely, if cells do not adhere to a film or undergo apoptosis on the film, the surface is called 'cytophobic'. Cell behavior was discussed in this dissertation for polyelectrolyte multilayers composed of the polycations; poly(diallyldimethylammonium), PDADMAC, and poly(allylammonium chloride), PAH, and polyanions; poly(4-styrenesulfonic acid), PSS, and poly(acrylic acid), PAA. The adhesion and proliferation of cells are dependent on the surface to which they attach. Aside from cell counting, cell "health" on surfaces is typically established by measuring the metabolic rate with dyes that participate in the metabolic pathway or using "live/dead" assays with combinations of membrane permeable/impermeable dyes. Whether cells are attached or not, and whether they are living or dead, provides an incomplete picture of cell health. In this dissertation, proliferation rates and net metabolism of 3T3 fibroblasts seeded on "biocompatible" ultrathin polyelectrolyte multilayer films and on control tissue culture plastic were compared. Cells adhered to, and proliferated on, both surfaces, which were shown to be nontoxic according to live/dead assays. However, adhesion was poorer on the multilayer surface, illustrated by diffuse organization of the actin cytoskeleton and less-developed focal adhesions. Proliferation was also slower on the multilayer. When normalized for the total number of cells, it was shown that cells on multilayers experienced a five-day burst of metabolic stress, after which the metabolic rate approached that of the control surface. This initial state of high stress had not been reported previously in studies of cell growth on multilayers, although the observation period for this system was usually a few days. The interaction of nanoparticles and surfaces with the complex array of proteins in physiological media is largely responsible for maintaining circulation in the bloodstream and biocompatibility in general. It is known that composition of the initial "soft" corona of exchangeable adsorbed proteins evolves to comprise a more tenaciously held "hard" corona. In this dissertation, the dependence of cell adhesion on a thin film of polyelectrolyte complex is connected to the "hardness" of the initial corona using albumin, the most prevalent protein in serum. The ease with which albumin can be displaced depended on the surface functional group - carboxylate or sulfonate, in particular aromatic sulfonate. Carboxylate permitted easier exchange of albumin, which presumably allowed the adsorption of proteins such as fibronectin, required for cell adhesion. Sulfonate held on to albumin more strongly, producing a persistent hard corona likely to remain biocompatible. The mechanism is thought to be related to the higher energy of interaction between sulfonate and amine than between carboxylate and amine.
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Date Issued: 2019
Identifier: 2019_Summer_Surmaitis_fsu_0071E_15315
Format: Thesis

Title: Characterization and Applications of Ph-Responsive Polyelectrolyte Complex and Multilayers.
Creator: Sui, Zhijie, Schlenoff, Joseph B., Alamo, Rufina G, Dorsey, John G., Rill, Randy L., Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Polyelectrolyte complexes (PECs) have received a growing interest since the early sixties. PECs have been used for large-scale industrial applications and have demonstrated enormous potentials in various fields such as coatings, binders and flocculants. Using the Layer-by-layer deposition technique, an ultrathin polyelectrolyte multilayer coating was first built in 1990 and soon both theoretical and practical interest in these coatings were growing exponentially. In the first part of this...
Show morePolyelectrolyte complexes (PECs) have received a growing interest since the early sixties. PECs have been used for large-scale industrial applications and have demonstrated enormous potentials in various fields such as coatings, binders and flocculants. Using the Layer-by-layer deposition technique, an ultrathin polyelectrolyte multilayer coating was first built in 1990 and soon both theoretical and practical interest in these coatings were growing exponentially. In the first part of this dissertation, studies were focused on the fundamental properties of polyelectrolyte multilayer and complex systems, such as the effect of molecular weight of polyelectrolytes and the effect of ionic strength on the multilayer buildup and the thermodynamics of the polyelectrolyte complexation. In the second part, a series of pH-tunable polyelectrolyte complexes and pH-responsive multilayers were designed and studied. Random copolymers composed of pH-independent "strongly" charged parts and pH-dependent "weakly" charged parts were introduced for making pH-tunable polyelectrolyte complex and pH-responsive multilayers. A systematic study of the pH induced change of multilayer configuration, including film decomposition, phase separation and surface charge rearrangement, was conducted. It was found, by varying the mole percent of the weakly charged segments in the multilayers, the outcome of external pH changes on the multilayers varied from total film decomposition, to forming microporous surface, and finally to yielding a surface-charge-tunable multilayer, which meant the ability of controlling the surface charge polarity and density via pH. The outcome was essentially due to the combination of two types of charge interaction, charge extrusion and charge expulsion. It was also demonstrated that, with the use of the pH-responsive polyelectrolyte multilayer coatings to modify the substrate surface, the adsorption and release of biomaterials, such as proteins, could be controlled by varying the surface charge property via simple pH switch. Potential applications of this finding were proposed in the dissertation.
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Date Issued: 2004
Identifier: FSU_migr_etd-0401
Format: Thesis

Title: Characterization of High Spin Molecular Magnets.
Creator: Stowe, Ashley Clinton, Dalal, Naresh S., Van Tol, Johan, Fajer, Piotr, Steinbock, Oliver, Stiegman, Albert, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: This dissertation describes the characterization of magnetic sandwich-type transition metal substituted polyoxotungstates and large Fe3+ containing clusters with large total spins. A series of isostructural copper trimer polyanions, [Cu3(H2O)3(α-XW9O33)2]n– (n = 12, X = AsIII, SeIII; n = 10, X = SeIV, TeIV), have been investigated to specifically understand the role of the diamagnetic hetero atom, which has a lone pair of electrons projecting into the magnetic plane, on the overall magnetic...
Show moreThis dissertation describes the characterization of magnetic sandwich-type transition metal substituted polyoxotungstates and large Fe3+ containing clusters with large total spins. A series of isostructural copper trimer polyanions, [Cu3(H2O)3(α-XW9O33)2]n– (n = 12, X = AsIII, SeIII; n = 10, X = SeIV, TeIV), have been investigated to specifically understand the role of the diamagnetic hetero atom, which has a lone pair of electrons projecting into the magnetic plane, on the overall magnetic properties. We have discovered that the magnetic parameters can be tuned by controlling the hetero atom which is incorporated into the polyanion, though the magnitude of change is small. Further, we have sought to understand the spin-frustration of (Cu2+) in general and we have made comparison to the copper tetramer, [Cu4K2(H2O)8(α-AsW9O33)2]8-. The (Cu2+)4 polyanion is again spin-frustrated, however, addition of the fourth unpaired electron into the magnetic system changes the spin arrangements. Both antiferromagnetic and ferromagnetic exchange couplings are observed and the ground state spin is determined to be S = 1. We also explored replacement of the transition metal substituted into the core of the polyanion "sandwich". Six Fe3+ ions are incorporated into [Fe6(OH)3(A-α-GeW9O34(OH)3)2]11- forming a Keggin dimer each containing three Fe3+ centers. High freqeuncy EPR experiments indicate a diamagnetic ground state spin though a broad paramagnetic transition is observed at higher temperature originating from an excited state. The second foci of this dissertation concerns the nature of anisotropy within large Fe3+-containing clusters with high spin ground states. An assumption has been made that the electron Zeeman parameter, the g tensor, is isotropic and equal to 2.00 for such species. We have investigated a series of similar complexes with very large ground state spins and nominal D,E, and higher order anisotropy. Employment of a variable frequency EPR approach has revealed considerable Zeeman anisotropy in these complexes such that the assumption that g = 2.00 must be reexamined for other such Fe3+-containing species.
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Date Issued: 2004
Identifier: FSU_migr_etd-1549
Format: Thesis

Title: Characterization of Interfacial Material Isolated from Petroleum Crude Oils by FT-ICR MS.
Creator: Clingenpeel, Amy, Marshall, Alan G. (Alan George), Landing, William M., Dorsey, John G., Dalal, Naresh S., Rodgers, Ryan P., Florida State University, College of Arts and...
Show moreClingenpeel, Amy, Marshall, Alan G. (Alan George), Landing, William M., Dorsey, John G., Dalal, Naresh S., Rodgers, Ryan P., Florida State University, College of Arts and Sciences, Department of Chemistry and Biochemistry
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Abstract/Description: As the global supply of easily accessible, light crude oil diminishes, production of heavy crude oils and offshore drilling for light crude oils will continue to increase. However, one problem that is often encountered during the production of these heavy and subsea crude oils is the formation of stable water-in-oil (W/O) or oil-in-water (O/W) emulsions. Although emulsions may be intentionally formed during the recovery of heavy crude oils, emulsions must be broken before the crude oil is...
Show moreAs the global supply of easily accessible, light crude oil diminishes, production of heavy crude oils and offshore drilling for light crude oils will continue to increase. However, one problem that is often encountered during the production of these heavy and subsea crude oils is the formation of stable water-in-oil (W/O) or oil-in-water (O/W) emulsions. Although emulsions may be intentionally formed during the recovery of heavy crude oils, emulsions must be broken before the crude oil is refined. If left untreated, these emulsions may contribute to severe corrosion issues (due to entrained salt) and equipment failure. In the case of subsea crude oils, stable W/O emulsions may be encountered if the crude oil enters the environment (e.g. Deepwater Horizon oil spill). The emulsions encountered here reduce the effectiveness of remediation efforts (e.g. skim boats used to remove crude oil from the environment) and slow recovery efforts. Therefore, a key objective in petroleum production is to understand what types of species contribute to stable emulsions in order to determine how to best treat such undesirable emulsions. Emulsion stability is controlled by the surface-active characteristics of a small fraction of compounds that exist or are added to the crude oil matrix. Collectively, these species generate a molecular layer at the oil/water interface which stabilizes the emulsion by resisting drainage and hence coalescence between dispersed droplets. Characterization of these compounds has been hindered by carryover issues from the whole crude oil. However, a novel method for the isolation of these surface-active compounds which minimizes carryover issues was recently introduced. Here, this "wet silica" method is used to isolate interfacially active material from petroleum crude oils. The method is modified (e.g. vary water loading, vary water pH) in order to understand what types of species partition to the oil/water interface under each condition through characterization by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). The method is also used to isolate IM from a variety of different crude oil samples (e.g. fractionated crude oil, weathered crude oils, etc.) to determine which fractions and types of compounds contribute most to stable emulsions. Chapter one presents background information on petroleum crude oils, petroleum emulsions, and the "wet silica" method necessary for understanding the data presented in chapters three through nine. Information on the techniques (electrospray ionization (ESI), FT-ICR MS) used to characterize isolated IM are later described in chapter two. Modifications to the "wet silica" method are described in chapters three and four: chapter three examines the role of water content adsorbed to silica gel for the selective isolation of interfacially active material from Athabasca bitumen while chapter four explores how varying water pH affects the composition of isolated IM from Athabasca bitumen. Chapters five and six examine how different IM fractions contribute to the overall Athabasca bitumen emulsion stability. Chapter five explores how isolated IM water-soluble organics differ in composition and stability characteristics compared to the oil-soluble IM fraction. Here, IM water-soluble organics are observed to generate stable emulsions. However, asphaltenes alone are generally believed to play the most significant role in emulsion stability. Therefore, in order to understand the contributions of asphaltenes to isolated IM, chapter six systematically explores the contributions of the asphaltene and maltene fractions of Athabasca bitumen to the IM isolated from the whole crude oil. Yet, previous studies have suggested that rearrangement occurs at the interface over time. Therefore, chapter seven characterizes the species that move to the oil/water interface once the most surface active Athabasca bitumen compounds are depleted. While differences are observed in the IM isolated from the different Athabasca bitumen fractions presented in chapters seven and eight, it is possible that low-molecular weight acids in the IM samples preferentially ionize and hinder the detection of high-molecular weight acids and asphaltene species in the IM fraction of these samples. In order to determine if low-molecular weight acids are preferentially ionized, chapter eight examines how the compositional space coverage of species isolated in 9 different IM acid fractions (separated by hydrophobicity) from the whole Athabasca bitumen compare. In contrast to the work performed on heavy crude oils in chapters three through eight, Chapter 9 characterizes IM isolated from a light crude oil. Here, IM is isolated from a variety of different Deepwater Horizon crude oils samples (i.e. whole crude, photochemically weathered, environmentally weathered) in order to gain insight into the degradation pathways (abiotic, biotic) of IM compounds in the environment. Finally, chapter ten details future work that will be performed to gain further insight into the structure and type of species which contribute most to emulsion stability under a range of conditions.
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Date Issued: 2015
Identifier: FSU_2016SP_Clingenpeel_fsu_0071E_12846
Format: Thesis

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