Browsing by Author "Crans, Debbie, committee member"

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Open Access
Antibacterial effects of sputter deposited silver-doped hydroxyapatite thin films
(Colorado State University. Libraries, 2011) Trujillo, Nathan Anthony, author; Popat, Ketul, advisor; Williams, John, advisor; Reynolds, Melissa, committee member; Crans, Debbie, committee member
Over recent years, researchers have studied innovative ways to increase the lifespan of orthopedic implants in order to meet the soaring demand of hip and knee replacements. Since many of these implants fail as a result of loosening, wear, and inflammation caused by repeated loading on the joints, coatings such as hydroxyapatite (HAp) on titanium with a unique topography have been shown to improve the interface between the implant and the natural tissue. Other serious problems with long-term or ideally permanent implants are bacterial colonization. It is important to prevent initial bacterial colonization as existing colonies have potential to become encased in an extracellular matrix polymer (biofilm) which is resistant to antibacterial agents. The following work considers the potential of etching using plasma based ion implantation and ion beam sputter deposition to produce hydroxyapatite thin films on etched titanium doped with silver as an antibacterial component. Plasma-based ion implantation was used to examine the effects of pre-etching on plain titanium. Topographical changes to the titanium samples were examined and compared via scanning electron microscopy. It was determined that plasma-based ion implantation at -700eV could etch titanium to produce similar topography as ion beam etching in a shorter processing time. Hydroxyapatite and silver-doped hydroxyapatite thin films were then sputter deposited on titanium substrates etched at -700eV. For silver-doped films, two concentrations of silver (~0.5wt% and ~1.5wt%) were used. Silver concentrations in the film were determined using energy dispersive x-ray spectroscopy. Film thicknesses were determined by measuring the surface profile using contact profilometry. Staphylococcus epidermidis (SE) and Pseudomonas aeruginosa (PA) adhesion studies were performed on plain titanium, titanium coated with hydroxyapatite, titanium coated with ~0.5 wt% silver-doped hydroxyapatite, and titanium coated with ~1.5wt% silver-doped hydroxyapatite. It was discovered during the study that the films were delaminating from the samples thus killing bacteria in suspension. Release studies performed in addition to adhesion confirmed that the silver-doped films prevented SE and PA bacterial growth in suspension. To prevent delamination, the films were annealed by heat treatment in air at a temperature of 600°C. X-ray diffraction confirmed the presence of a crystalline hydroxyapatite phase on each sample type. Films were immersed in PBS at 37°C and remained in incubation for four weeks to determine there was no delamination or silver leaching.
Embargo
Cryo-electron microscopy of cloneable inorganic nanoparticles
(Colorado State University. Libraries, 2024) Guilliams, Bradley Forrest, author; Ackerson, Christopher J., advisor; Sambur, Justin, committee member; Crans, Debbie, committee member; Stasevich, Tim, committee member
Our understanding of biology is best understood through direct, empirical measurements of biomacromolecules and biological systems. The functions of proteins are directly linked to both their structure and their intracellular organizations. Single particle cryo-electron microscopy has revolutionized modern structural biology by enabling the structural determination of proteins and protein-complexes in purified samples without the need to form large crystals as required by X-ray crystallography. With single particle cryo-EM, atomic and near-atomic resolution structures are now routine which offer insight into the functions of biomacromolecules. While these insights are invaluable, there is increasing momentum for integrative structural biology which aims to accomplish structural determination of biomacromolecules in their cellular, tissue, or organismal context. There remains a grand challenge in biological imaging where biological materials have low innate contrast. Cloneable contrast labels that impart contrast to discrete protein densities do not reliably exist for cryo-electron microscopy. In contrast, fluorescent proteins are reliable and routine for localizing fluorescent protein / protein of interest genetic fusions in visible-light microscopies. We have proposed and developed intracellularly synthesized inorganic nanoparticles called 'cloneable nanoparticles' as a solution to this grand challenge. Cloneable nanoparticles are inorganic nanoparticles, synthesized by a protein/peptide (or combination thereof) which controls and defines the properties of the inorganic nanoparticle. Here we have defined the cloneable nanoparticle paradigm and described the development of a cloneable selenium nanoparticle. Further, we show the application of the cloneable selenium nanoparticle as a cloneable contrast label for biological electron microscopy and correlative light-electron microscopy and detail progress towards adapting the cloneable selenium nanoparticle for use in cryo-electron tomography. With the aim to later expand cloneable nanoparticles to include a myriad of orthogonal cloneable contrast labels (analogous to different colored fluorophores), and to gain understanding about enzymatic nanoparticle synthesis, a single particle cryo-EM study is on-going. Lastly, we have shown the application of directed evolution for cloneable nanoparticles, suggesting that this is a viable path, alongside rational protein design, towards developing future cloneable nanoparticle cryo-electron microscopy labels.
Open Access
Engineering bacteriophage nanocarriers for targeted delivery of protein reagents to prostate cancer cells
(Colorado State University. Libraries, 2014) DePorter, Sandra M., author; McNaughton, Brian, advisor; Kennan, Alan, committee member; Crans, Debbie, committee member; Reynolds, Melissa, committee member; Di Pietro, Santiago, committee member
Proteinaceous reagents, including antibodies and synthetic proteins, have become some of the most effective reagents for targeted treatment and diagnosis of disease. The unique catalytic activity of some proteins and ability to bind disease-relevant receptors that can evade small molecule discovery, make these reagents well suited for use as therapeutic and bioimaging reagents. However, the large size and charge distribution of most proteins greatly inhibits their intracellular delivery to diseased cells, limiting targets to those displayed on the cell surface. In response to this challenge, we have developed a bacteriophage nanocarrier to deliver large payloads of proteinaceous cargo to the interior of prostate cancer cells. This reagent employs two distinct components: a genetically defined prostate cancer cell-selective protein transduction domain, and a biotinylation site on an orthogonal coat protein, which allows for complexation with streptavidin fusion proteins. Collectively, this approach permits targeted intracellular delivery of ~20 exogenous proteins per phage to human prostate cancer cells. This multifunctional technology offers a cell-selective solution to the challenges associated with delivering protein cargo to the interior of diseased cells and may lead to an expansion in the use of protein reagents.
Open Access
Establishing base-catalyzed halogen transfer as a general platform for C–H functionalization
(Colorado State University. Libraries, 2024) Bone, Kendelyn I., author; Bandar, Jeffrey, advisor; Crans, Debbie, committee member; Henry, Chuck, committee member; Belisle, John, committee member
In contrast to traditional multi-step routes, C–H functionalization offers a resource and time efficient route to desired products. Current methods for oxidative C–H functionalization are developed on three predominate reactivity platforms (1) hydrogen atom transfer, (2) single- electron transfer, and (3) C–H insertion. Despite their synthetic power, methods built on these platforms are restricted to similar bond conversions, substrates, and selectivities. Thus, there remains a strong demand for new mechanistic approaches to oxidative C–H functionalization that offer a departure from traditional reactivity. In efforts to address this need, new methods for oxidative coupling based on a base-catalyzed halogen transfer (X-transfer) reactivity platform are described herein. Chapter one provides an overview on the development of a X-transfer enabled direct C–H hydroxylation of mildly acidic N-heteroarenes and benzenes. Hydroxylated (hetero)arenes are valued in many industries as both key constituents of end products and diversifiable synthetic building blocks. Accordingly, the development of reactions that complement and address the limitations of existing methods for the introduction of aromatic hydroxyl groups is an important goal. To this end, this chapter discusses the development of a protocol that employs an alkoxide base to catalyze X-transfer from sacrificial 2-halothiophene oxidants to aryl substrates, forming SNAr-active intermediates that undergo nucleophilic hydroxylation. Key to this process is the use of 2-phenylethanol as an inexpensive hydroxide surrogate that, after aromatic substitution and ii rapid elimination, provides the hydroxylated arene and styrene byproduct. Use of simple 2-halothiophenes allows for C–H hydroxylation of 6-membered N-heteroarenes and 1,3-azole derivatives, while a rationally designed 2-halobenzothiophene oxidant extends the scope to electron-deficient benzene substrates. Mechanistic studies indicate that aromatic X-transfer is reversible, suggesting that the deprotonation, halogenation, and substitution steps operate in synergy, manifesting in unique selectivity trends that are not necessarily dependent on the most acidic aryl position. The utility of this method is further demonstrated through streamlined target molecule syntheses, examples of regioselectivity that contrast alternative C–H hydroxylation methods, and the scalable recycling of the thiophene oxidants. Chapter two describes the elaboration the X-transfer enabled C–H functionalization platform to encompass benzylic C(sp3)–H bonds. Thus, a benzylic C–H oxidative coupling reaction with alcohols that proceeds through a synergistic deprotonation, halogenation and substitution sequence is discussed. In contrast to existing radical-based pathways for C–H functionalization, this process is guided by C–H acidity trends. This gives rise to new synthetic capabilities, including the ability to functionalize diverse methyl(hetero)arenes, tolerance of oxidizable and nucleophilic functional groups, precision regioselectivity for polyalkylarenes and use of a double C–H etherification process to controllably oxidize methylarenes to benzaldehydes.
Open Access
Exploring the impacts of nanoconfinement using nuclear magnetic resonance (NMR) spectroscopy
(Colorado State University. Libraries, 2022) Miller, Samantha L., author; Levinger, Nancy, advisor; Krummel, Amber, committee member; Crans, Debbie, committee member; Graham, James, committee member
The chemical reactivity of molecules is typically studied under bulk aqueous conditions in the research laboratory. Although this standard may be appropriate for processes destined to be scaled up for industrial purposes, it ignores the fact that a great deal of the chemistry underlying physiological reactions occur in confined environments, like cellular organelles, protein pockets, or porous interfaces. The dissertation begins by describing the methodology for synthesizing size tunable reverse micelles, or surfactant enveloped nanodroplets. After physical perturbation, the ternary mixture of polar (usually aqueous), nonpolar, and amphiphilic surfactant self-assemble. Two small molecules, glucose and urea, were studied in these nano environments using a combination of analytical techniques including dynamic light scattering, differential scanning calorimetry, and molecular dynamics simulations that complemented the myriad nuclear magnetic resonance (NMR) spectroscopy studies. Quantification of single hydrogen exchange between glucose and water using exchange spectroscopy NMR in conjunction with custom MatLab code revealed that confinement of glucose and water within 8-10 nanometer reverse micelles slows the process of exchange by introducing a quantifiable energy barrier of ~75 kJ/mol. Deuterium NMR spectroscopy provided evidence for hydrogen tunneling below 283 K, a surprisingly high temperature for this phenomenon. The same robust methods of kinetic and structural analysis were used to characterize urea in water reverse micelles. Results showed that in addition to its well-known ability to denature proteins, urea can disrupt amphiphilic membranes and cause a ten-fold increase in the membrane surface area at low temperatures ~273 K as a result of this destabilization. Finally, the use of fluorine NMR spectroscopy demonstrated that the reverse micelle nanodroplet environments could achieve higher ionic strengths (~9.0 M) with simple divalent salts than possible in standard bulk solutions (~5.0 M). Together, these results presented compelling evidence that utilization of reverse micelle nanodroplets could provide alternative environments to facilitate previously inaccessible, novel conditions.
Open Access
Fluorinated materials synthesis and characterization for energy storage and energy conversion applications
(Colorado State University. Libraries, 2015) Bukovsky, Eric V., author; Strauss, Steven H., advisor; Ackerson, Christopher, committee member; Crans, Debbie, committee member; Barisas, B. George, committee member; Sutton, Sally, committee member
The synthesis and characterization of multiple fluorinated, p-block, cage, and organic compounds will be presented. The research effort is split up in to main topics, (i) fluorinated superweak anions based on B12 cages, and (ii) perfluoroalkylation of polycyclic aromatic hydrocarbon (PAH) and fullerene compounds. In the first three chapters, superweak anion research is presented; a new purification method for the synthetic intermediate K2B12F12, synthesis and thermal and physical characterization of highly purified (H3O)2B12F12·nH2O, Li2B12F12 and Na2B12F12 (synthesized from K2B12F12), and an HF-free, improved synthesis method and characterization of KB12F11NH3. Furthermore, the unanticipated, rapid fluorination of KB12H11NH3 in the presence of HF, contrary to, previously observed, slowed fluorination of K2B12H12 in the presence of HF, will also be described. Single crystal X-ray structures of three new isomers of C60(CF3)10 are discussed, and one putative isomer of C60(CF3)10 is confirmed along with comparisons of their crystal packing properties compared to 1,9-C60(cyclo-CF2(2-C6F4)), and industry-standard fullerene acceptor phenyl-C61-butyric acid methyl ester (PCBM). Discussion of how the structural and electrochemical data of the new C60(CF3)10 isomers and 1,9-C60(cyclo-CF2(2-C6F4)) agree with currently accepted literature will also be discussed. A new metal reactor design for the radical reactions of CF3I and polycyclic aromatic hydrocarbons (PAH) and fullerenes, and initial results will be discussed and compared to previous reaction methods. Single crystal X-ray structures of four separate compounds believed to be "trapped intermediates" formed from the radical substitution reaction isolated from radical reactions with CF3I using different PAHs and different reactions conditions will be discussed as well as the implications these trapped intermediates have on the proposed mechanism of CF3• radical substitution reactions. Crystal packing and nearest molecule analysis of five PAH(CF3)n will be compared to a single crystal X-ray structure of triphenylene with a C4F4 substitution. Insights into the structural effects of CF3 substitutions compared to the flat C4F4 substitutions, and, how those effects would translate into electronic communication in the solid state will be discussed. Finally, wet milling of metallurgical grade silicon in an attritor mill, under anaerobic and aerobic conditions with and without surface passivating additives to study the affects oxygen and additives can have on milled particle properties such as, crystallinity by powder X-ray diffraction, surface bonds by X-ray photoelectronspectroscopy, dynamic light scattering particle size, N2 gas uptake BET surface area and reactivity towards oxygen will be discussed. Under anaerobic conditions silicon was found to form Si–C bonds in the presence of dry- air-free heptane. Additionally, the extensive effect oxygen has on the comminution of silicon and the surprising result that, even in aerobic conditions, formation of Si–C bonds is observed. All of the research described in this dissertation has applications in one or multiple energy storage or energy conversion devices. The superweak anion salts as electrolyte salts in battery or fuel cell, C60(CF3)10 and 1,9-C60(cyclo-CF2(2-C6F4)), as electron acceptor materials in organic photovoltaic devices, and multiple PAH(CF3)n compounds as OLED active layer materials.
Open Access
Functionalization of pyridines and other azines via phosphorus ligand-coupling reactions
(Colorado State University. Libraries, 2019) Hilton, Michael C., author; McNally, Andrew, advisor; Reynolds, Melissa, committee member; Crans, Debbie, committee member; Montgomery, Tai, committee member
Nitrogen heterocycles are ubiquitous in pharmaceutical compounds with pyridine being one of the most frequently occurring examples. The discovery and development of new drugs rely heavily on our ability to modify these commonly occurring structures. The functionalization of pyridine has a long history but despite this, there remain some deficiencies in this area of synthesis. Reactions which expand upon the known methodologies are of tremendous value to medicinal chemists who frequently work with pyridines and similar azines. Chapter one will cover the relevance of pyridines in pharmaceuticals and will explain how structural features contribute to their presence in drugs. Conventional and newer methods to functionalize pyridine are also addressed. Chapter two will describe the work of the McNally lab in the development of heterocyclic phosphonium salts as reagents to selectively functionalize pyridines. An application of these salts is as precursors to form C−O bonds from alkoxide nucleophiles. Chapter three presents the development of a strategy to construct bis-heterobiaryls using phosphorus ligand-coupling. This method offers an alternative to the widely used metal-catalyzed approaches which often struggle in the synthesis of bis-heterobiaryls. Lastly, chapter four will expand upon this work showing a new approach to prepare bis-heterobiaryls using heteroaryl halides. This route enables easy access to 2,2'-bipyridines which are difficult to synthesize using conventional methods.
Open Access
Interaction of [VO₂(MA)₂]⁻ with model membranes: relevance to insulin enhancing effects of BMOV and its oxidized form
(Colorado State University. Libraries, 2010) Schoeberl, Samantha Kay, author; Roess, Deborah, advisor; Graham, James, committee member; Crans, Debbie, committee member
Anti-diabetic vanadium-containing compounds and salts reportedly have effects on the overall organization of the cytoskeleton and the plasma membrane of cells. For ligand-mediated signaling, appropriate cytoskeletal and membrane lipid organization is essential for down-stream signaling to occur. A number of vanadium-containing compounds and salts are of interest because of their effects on these important cellular structures. Promising results in regulating diabetic symptoms such as glucose and lipid metabolism have been shown to result from the use of various anti-diabetic vanadium drugs. Their effects on the cytoskeleton and plasma membrane are reviewed Chapter I. Due to the importance of membrane interactions of vanadium-containing compounds with insulin-enhancing activity in ligand-mediated signaling, two simple membrane model systems were used to investigate the interactions of an oxidized metabolite of bis(maltolato)oxovanadium(IV) with model lipid interfaces. Studies were carried out using multinuclear NMR spectroscopy with a focus on 1H NMR techniques. In Chapter II we demonstrate that there were slight changes in 1H NMR spectra indicating that this BMOV metabolite was able to penetrate the lipid interface. These findings are important in understanding the pharmacologic mechanism of action of this anti-diabetic compound in cells and intact animals.
Open Access
Investigating biosynthetic pathways of the Aspergillus genus through biomimetic total synthesis of secondary metabolites
(Colorado State University. Libraries, 2022) Benson, Brooke, author; Williams, Robert M., advisor; Kennan, Alan J., advisor; Paton, Robert, committee member; Crans, Debbie, committee member; Crick, Dean, committee member
The prenylated indole alkaloids are a class of secondary metabolites containing a unique bicyclo[2.2.2]diazaoctane core and a wide range of biological activity. This complex structure has prompted extensive investigation into the biochemical synthesis of these compounds. Currently, three disparate biochemical strategies are known to be used by producing fungi to construct the bicyclic core: (1) NADPH-dependent bifunctional reductase/Diels-Alderase-mediation in formation of the monooxopiperazines; (2) brevianamide assembly through cofactor-independent pinacolase resulting in spontaneous intramolecular Diels-Alder (IMDA) generation of the bicyclo[2.2.2]diazaoctane core; (3) Diels-Alderase mediated enantiodivergent generation of the dioxopiperazines via cytochrome P450 oxidation to achiral azadienes and successive enzyme-mediated stereoselective IMDA reaction. This work aimed to employ biomimetic total synthesis to aid in elucidation of the biosynthetic pathways in the Aspergillus genus, which utilizes the third strategy. This author reports the first total syntheses of 6-epi-Notoamides T10-12 and Notoamide T2, as well as an improved total synthesis of 6-epi-Notoamide T. Also reported are synthetic efforts towards 6-epi-Notoamide T9, Notoamide TI, and Citrinalin C.
Open Access
Kinetic, mechanistic, and active site studies of copper metal-organic framework catalyzed nitric oxide generation from S-nitrosoglutathione in water and blood plasma
(Colorado State University. Libraries, 2021) Tuttle, Robert Reeves, author; Reynolds, Melissa M., advisor; Finke, Richard, committee member; Crans, Debbie, committee member; Popat, Ketul, committee member
Catalytic generation of nitric oxide (NO) from endogenous sources by copper-based materials at the surfaces of implanted medical devices improves device performance by promoting vasodilation and inhibiting bacterial adhesion. Oxidation of the endogenous tripeptide S-Nitrosoglutathione (GSNO) to release NO is catalyzed by the copper-based metal-organic framework (MOF) H3[(Cu4Cl)3(BTTri)8] (CuBTTri) in the presence of glutathione (GSH). MOFs are solid-state, crystalline, porous materials composed of metal cation nodes and organic linkers forming three-dimensional structures. MOFs have generated interest as catalysts because of their unparalleled tunability via synthesis (compared to other solids), well-defined structures, coordinatively unsaturated metal sites, and high surface areas. Mechanistic insight into MOF catalysts promises to allow for the directed design of next-generation catalysts via leveraging synthetic tunability. However, because necessary studies to propose reliable reaction mechanisms are rarely reported for MOF catalysts, mechanistic understanding is lacking in the field. This Dissertation works toward a reaction mechanism of CuBTTri catalyzed GSNO to NO conversion in water in the presence of GSH. The strategies used to better understand this mechanism can also generate mechanistic knowledge in other MOF catalysis systems. Chapter I provides a discussion of NO release catalyzed by soluble and insoluble Cu-based species focusing on CuBTTri. Chapter I also introduces MOFs as catalysts and explains the requirements to propose a reliable reaction mechanism. Chapters II and III focus on the development of monitoring methods to quantify [GSNO], [GSH], and [glutathione disulfide] (the other main reaction product, GSSG) in real time in H2O and blood plasma. 1H nuclear magnetic resonance (NMR) and ultraviolet-visible (UV-VIS) spectroscopies can together effectively monitor the NO release reaction. The observation of an inverse dependence on added GSH for CuBTTri versus solvated Cu ions for NO generation shows that the two catalysts operate via different reaction mechanisms. Chapter III shows how the monitoring method in H2O reported in Chapter II can be extended to track the reaction in blood plasma. The observed GSNO to NO reaction stoichiometry is effectively identical in H2O and blood plasma, which indicates that the mechanism does not change in vivo versus the model biological solvent H2O. Hence, mechanistic findings in this dissertation for NO generation in water are likely biologically applicable. Chapter IV establishes the catalytically active Cu sites in CuBTTri for GSNO to NO conversion. Studies comparing the reaction rate (-d[GSNO]/dt) to particle size revealed that ~100% of the observed catalysis is caused by Cu atoms on the external surfaces of CuBTTri particles. Kinetic poisoning studies of CuBTTri particles with potassium cyanide (KCN) and 3,3',3''-phosphanetriyltris (benzenesulfonic acid) trisodium salt (TPPTS) showed that the active sites are kinetically uniform. Fourier transform infrared spectroscopic analysis of CN-poisoned CuBTTri detected Cu(CN)3 and Cu(CN) sites, which correspond to the idealized metal-terminated CuBTTri crystal structure. Size-selective kinetic poisoning studies of CuBTTri using TPPTS measured the active site density to be (1.3 ± 0.4)% of total Cu atoms in 600 ± 400 nm CuBTTri particles. Active site density was used to calculate a normalized turnover frequency for CuBTTri to make informed inter-catalyst comparisons. Chapter V presents the rate law and proposed mechanism for CuBTTri catalyzed GSNO to NO conversion. Four other competing, minimalistic mechanistic hypotheses were considered and disproven. The mechanism proposed is a CuII to formally CuIII redox mechanism with two proton-coupled electron transfer elementary steps. The proposed mechanism exhibits a derived rate law which matches the experimental rate law, has elementary steps which sum to the observed reaction stoichiometry, and provides a reasonable driving force for S-N bond homolysis in GSNO. Future computational and laboratory experiments suggested by the proposed mechanism promise to yield a level of mechanistic understanding for CuBTTri which has traditionally not been achievable for solid-state catalysts.
Open Access
Metal organic frameworks as heterogenous nitric oxide catalysts for use in the development of therapeutic polymer materials
(Colorado State University. Libraries, 2014) Harding, Jacqueline L., author; Reynolds, Melissa, advisor; Prieto, Amy, committee member; Crans, Debbie, committee member; Bailey, Travis, committee member; Worley, Deanna, committee member
Implantable polymeric medical devices are subject to surface biofouling due to the deposition of microbial agents and the accumulation of proteins at the material interface. Consequently, medical devices which are intended for beneficial functions can become a potentially fatal threat. As a result biofouling resistant materials are vigorously sought through the manipulation of material surface properties and by eluting therapeutics on the material surface. Nitric oxide (NO) is a bioactive agent generated by most nucleated cells in the human body and is known to mediate antimicrobial and antithrombus effects while maintain the capacity to promote the proliferation of healthy tissues. As such, the development of NO releasing biomaterials is known to reduce incidences of surface biofouling. However, current NO releasing materials are limited to short lifetimes of used based on limited capacity of exogenous NO which can be incorporated into the material. In order to circumvent this problem the goal of this research is to develop a biomaterial which generates NO from an endogenously supplied source. Metal organic frameworks (MOFs) were selected for investigation as heterogeneous catalysts for the generation of NO from bioavailable NO donors, S-nitrosothiols (RSNOS). MOFs were evaluated as NO catalysts based on their capacity to react with various RSNO substrates and their maintained structural integrity under reaction conditions. Presented herein is the successful demonstration of a Cu-MOF for the catalytic generation of NO from bioavailable RSNOs donors. However, the limited stability of this proof of principle MOF in aqueous solution prompted the development of a MOF-NO catalyst that is suitable for physiological applications through tuning the organic ligands used in the construction of the framework. Finally a two-fold demonstration of the feasibility towards designing composite MOF based biomaterials is presented as blended materials prepared via commercial manufacturing processes and via surface growth of MOFs on flexible polymeric substrates.
Open Access
Molecular basis of [PSI+] yeast prion nucleation
(Colorado State University. Libraries, 2013) Ben Musa, Zobaida A., author; Ross, Eric, advisor; Crans, Debbie, committee member; Zabel, Mark, committee member; Di Pietro, Santiago, committee member
Many fatal diseases arise from the conversion of soluble, functional proteins to insoluble misfolded amyloid aggregates. Amyloid fibers are characterized by filamentous morphology, protease resistance and cross]beta structure. Prions (infectious amyloids) are a specific subset of amyloid fibers, differing from other classes of amyloids by their infectivity. Prions are found in both mammals and yeasts, but there are differences between these two groups. Most yeast prions are characterized by the presence of large numbers of glutamine and asparagine (Q/N) residues, and some other common characteristics have been noted, including the presence of few hydrophobic and charged residues. Although, several attempts have been made with limited success to develop valuable systems to predict prion activity, there is no accurate algorithm that has the ability to predict the prion-forming proteins among the Q/N-rich protein group. In the yeast, it has been shown that amino acid composition, not primary sequence, drives prion activity. Recently, preliminary efforts to define the role of amino acid composition in prion formation have been examined. The fundamental question of this project is how, in yeast Q/N-rich prions, the sequence requirements for nucleation versus propagation differ, and how this information can be used in order to develop a precise prion prediction system. By answering this question we will be able to more accurately identify additional prions in both yeast and other organisms. Our long-term goal in the comprehensive studies of prion formation and propagation mechanisms is to apply this information to mammalian prion diseases. Consequently, we will be able to identify targets for therapeutic intervention to avoid, slow-down, or reverse the development of related diseases. The study determined that the amino acids required for prion formation differ from those required for prion propagation. Identifying the sequence feature for both activities is the first step towards mechanistic studies to examine how these sequences perform their function.
Open Access
New strategies to synthesize complex pyridines and tetrahydropyridines using main group chemistry
(Colorado State University. Libraries, 2021) Fricke, Patrick J., author; McNally, Andrew, advisor; Crans, Debbie, committee member; Chen, Eugene, committee member; Kipper, Matthew, committee member
Pyridine and piperidine are important molecular scaffolds in small molecule drug development in medicinal chemistry research. Because of their importance, methods to synthesize complex pyridines and piperidines are highly desirable. Chapter one discusses the importance of these scaffolds in the pharmaceutical industry along with the history of pyridine and piperidine synthesis and the challenges that still remain. Chapter two discusses the switching strategies for selective installation of phosphonium salts on polyazines. The methods include an acylation, base-mediate, phosphine mediate, and order-of-reagent addition strategies. Additionally, we demonstrate how these methods can be applied to medicinal chemistry research during structure-activity relationship studies by derivatizing the phosphonium salts. Chapter three presents a new strategy for selective pyridine alkylation at the 4-position of the pyridine ring. Using a triazine chloride activating group allows for 4-selective phosphonium ylide formation inside the pyridine ring. A Wittig olefination-rearomatization sequence with an aldehyde then furnishes the alkylated pyridine. This method offers an alternative strategy to conventionally used metal-catalyzed cross coupling and Minisci-type reactions. Chapter four describes a stepwise reduction method for the synthesis of dihydropyridine and tetrahydropyridine. Using N-Tf activation allows for a selective hydride reduction to the dihydropyridine, which can subsequently undergo hydrogenation to the tetrahydropyridine.
Open Access
Progress toward the total synthesis of stemocurtisine and asymmetric synthesis of endoperoxide anticancer agents via Brønsted acid cascade catalysis
(Colorado State University. Libraries, 2012) Rubush, David Michael, author; Rovis, Tomislav, advisor; Kennan, Alan, committee member; Crans, Debbie, committee member; Chen, Eugene, committee member; Kanatous, Shane, committee member
A viable route toward the pyrido-azepine core of stemocurtisine involving an N-heterocyclic carbene catalyzed Stetter reaction has been realized. The key steps involve a formal [3+2] cycloaddition of enones with isocyanoacetates and a catalytic asymmetric intramolecular Stetter reaction. Additionally, a diastereoselective intramolecular Stetter reaction was achieved to access highly substituted pyrrolidines. Asymmetric Brønsted acid catalyzed cascade reactions were also investigated. A diastereoselective acetalization/oxa-Micahel cascade has been developed to provide dioxolanes and oxazolidines using diphenylphosphinic acid as a catalyst. The enantioselective variant of this reaction was explored with minor success. The desymmetrization of p-peroxyquinols using a Brønsted acid catalyzed acetalization/oxa-Michael cascade was achieved in high yields and selectivities for a variety of aliphatic and aryl aldehydes. Mechanistic studies suggest that the reaction proceeds through a dynamic kinetic resolution of the peroxy-hemiacetal intermediate. The resulting 1,2,4-trioxane products were derivatized and show potent cytotoxicity toward specific cancer cells.
Open Access
Rotational motion and organization studies of cell membrane proteins
(Colorado State University. Libraries, 2016) Zhang, Dongmei, author; Barisas, B. George, advisor; Van Orden, Alan, committee member; Henry, Chuck, committee member; Roess, Deborah A., committee member; Crans, Debbie, committee member
Cell membranes are dynamic structures with complex organization. The complexity of the cell membrane arises from intrinsic membrane structure, membrane microdomains within the plasma membrane and the membrane cytoskeleton. Plasma membrane receptors are integral membrane proteins with diverse structures and functions which bind specific ligands to trigger cellular responses. Due to compartmentalization of the plasma membrane and the formation of membrane microdomains, receptors are distributed non-homogeneously in the cell membrane bilayer. Both lateral and rotational diffusion of membrane receptors reflects different kinds of intermolecular interactions within the plasma membrane environment. Understanding protein diffusion within the membrane is very important to further understanding biomolecular interactions in vivo during complex biological processes including receptor-mediated signaling. Rotational diffusion depends linearly on the in-membrane volume of the rotating proteins. Relative to lateral diffusion, rotational diffusion is a more sensitive probe of an individual molecule’s size and local environment. We have used asymmetric quantum dots (QD) to conduct imaging measurements of individual 2H3 cell Type I Fcε receptor rotation on timescales down to 10 msec per frame. We have also used time-tagged single photon counting measurements of individual QD to examine μsec timescales, although rapid timescales are limited by QD emission rates. In both approaches, decays of time-autocorrelation functions (TACF) for fluorescence polarization fluctuations extend into the millisecond timescale, as implied by time-resolved phosphorescence anisotropy results. Depending on instrumental parameters used in data analysis, polarization fluctuation TACFs can contain a contribution from the intensity fluctuation TACF arising from QD blinking. Such QD blinking feed-through is extremely sensitive to these analysis parameters which effectively change slightly from one measurement to another. We discuss approaches based on the necessary statistical independence of polarization and intensity fluctuations to guarantee removal of a blinking-based component from rotation measurements. Imaging results demonstrate a range of rotational behavior among individual molecules. Such slow motions, not observable previously, may occur with large signaling complexes, which are important targets of study in cell biology. These slow motions appear to be a property of the membrane itself, not of the receptor state. Our results may indicate that individual mesoscale membrane regions rotate or librate with respect to the overall cell surface. The luteinizing hormone receptor (LHR) is a seven transmembrane domain receptor and a member of the GPCR family. It is located on luteal cells, granulosa and theca cells in females. Understanding how these protein receptors function on the plasma membrane will lead to better understanding of mammalian reproduction. LHR becomes aggregated upon binding hCG when receptors are expressed at physiological numbers. Binding of hormone to LHR leads to activation of adenylate cyclase (AC) and an increase in intracellular cyclic AMP (cAMP). ICUE3 is an Epac-based cAMP sensor with two fluorophores, cyan fluorescent protein (CFP) and the YFP variant, cpVenus, and a membrane-targeting motif which can be palmitoylated. Upon binding cAMP, ICUE3 undergoes a conformational change that separates CFP and YFP, significantly reducing FRET and thus increasing the ratio of CFP to YFP fluorescence upon excitation with an arc lamp or 405nm laser source. Hence we have investigated hLHR signal transduction using the cyclic AMP reporter probe, ICUE3. A dual wavelength emission ratio (CFP/YFP) imaging method was used to detect a conformational change in ICUE3 upon binding cAMP. This technique is useful in understanding the sequence of intercellular events following hormone binding to receptor and in particular, the time course involved in signal transduction in a single cell. Our data suggested that CHO cells expressing ICUE3 and directly treated with different concentrations of cAMP with saponin can provide a dose-dependent relationship for changes in intracellular cAMP levels. Forskolin (50μM) causes maximal activation of the intracellular cAMP and an increase in the CFP/YFP emission ratio. In CHO cells expressing both ICUE3 and hLHR-mCherry, the CFP/YFP ratio increased in cells treated with forskolin and in hCG- treated cells. In flow cytometry studies, similar results were obtained when CHO cells expressed < 60k LHR-mCherry per cell. Our results indicate that ICUE3 can provide real time information on intracellular cAMP levels, and the ICUE3 is a reliable cAMP reporter can be used to examine various aspects of LH receptor-mediated signaling.
Open Access
Sources, sinks, and trends of ozone precursors and their impact on ozone in northern Colorado
(Colorado State University. Libraries, 2017) Abeleira, Andrew Joseph, author; Farmer, Delphine K., advisor; Henry, Charles, committee member; Fischer, Emily, committee member; Crans, Debbie, committee member
Ozone is a structurally simple molecule that plays immensely important roles in Earth's atmosphere. In the troposphere, ozone is vital in maintaining the oxidative capacity of the lower atmosphere. However, unlike the chemical structure, the formation and lifecycle of ozone in the troposphere is anything but simple. The role of ozone in severe air pollution episodes, and the negative human and ecosystem health impacts of ozone were first established in the United States during the "smog" pollution episodes of the early 1950s in the Los Angeles basin. Since then, understanding the formation and impacts of ozone has been an air quality research priority in the United States. The primary source of tropospheric ozone is the photochemically initiated oxidation of anthropogenic or biogenic volatile organic compounds in the presence of nitrogen oxides. The production of ozone relies on the interplay between two catalytic cycles that share initiation and termination reactions. The linkage of the ozone catalytic cycles, via those initiation and termination reactions, leads to the non-linear nature of the chemical production of ozone. The urbanization of the United States in the 1950s-1970s led to increased frequency of severe ozone events in urban areas from increased ozone precursor emissions – specifically emissions of NO and NO2 from automobiles and coal-fired electricity generating power plants. These high ozone events, coupled with results from ozone epidemiologic, exposure, and toxicology studies, prompted the U.S. Congress to establish the Clean Air Act of 1970. The Clean Air Act authorized the U.S. Environmental Protection Agency to establish the National Air Quality Standards for six criteria air pollutants – including ozone. The goal of this new standard was to systematically reduce ambient ozone concentrations by targeting major ozone precursor emission sources. Near 50 years later high ozone events are still occurring in densely populated urban and suburban regions in the United States. Herein, an in-depth study of the sources and sinks of ozone precursors, and the impact of precursor reductions on long-term ozone trends in Northern Colorado is presented. Chapter 1 of this dissertation provides relevant historical context regarding ozone in the United States, pertinent tropospheric ozone chemistry for urban and suburban regions, ozone precursor trends in the United States, and other important processes that affect regional and global ozone. Chapter 2 examines long-term (15 year) trends in ozone and ozone precursors in Northern Colorado with a focus on day of week ozone and NO2 trends that suggest Northern Colorado is transitioning from a NOx-saturated to peak ozone production region. Additionally, the impact of severe drought on the ozone/temperature relationship is addressed. Chapter 3 details the seasonal sources of a suite of volatile organic compounds measured during two 8-week periods in spring and summer 2015 at a ground site in Northern Colorado, and demonstrates the impact of drought on the local isoprene and reactive carbon budget. The reduction in isoprene emissions during drought is tied back to the suppression of the ozone/temperature relationship in the region. In the fourth chapter, the sources and sinks of alkyl nitrates, a key ozone precursor sink, are investigated using a simple sequential production-destruction reaction model. The final chapter highlights the need for long-term ozone and ozone precursor monitoring in Northern Colorado as population, energy demands, and ozone precursor emissions change.
Open Access
Synthesis, characterization, and investigation of nitric oxide donors for bacterial detection and antibacterial activity
(Colorado State University. Libraries, 2020) Hibbard, Hailey Althea Jane, author; Reynolds, Melissa M., advisor; Crans, Debbie, committee member; Bailey, Travis, committee member; Popat, Ketul, committee member
Antibiotic resistance is a critical problem, especially with the emergence of "superbugs," which are bacteria species that have become immune to most common antibiotics. To address this alarming issue, it is necessary to both prevent the overuse of current antimicrobial therapeutics, and to develop new, effective antibacterial treatments. By detecting when the source of a patient's infection is bacterial, and which species is causing the infection, antibiotics can be prescribed only when they are needed, and the most effective antibiotic can be chosen. The development of new antibiotics with new mechanisms of action are also a productive path to help solve the problem of antibiotic resistance. Bacteria that encounter antibiotics with novel mechanisms of action have not yet evolved pathways to resistance against the therapeutic, so these innovative compounds will be highly successful at eradicating infections. Nitric oxide is a small molecule with potent antibacterial activity due to its reactivity and ability to form reactive nitrogen species, which induces nitrosative stress in bacteria cells and cell death. The development of a new antibiotic incorporating nitric oxide could lead to a very powerful antibacterial agent. Combining the ideas of detecting bacterial infections with the antibiotic potential of nitric oxide into a multifunctional molecule would give the benefits of both detection of a bacterial infection, and potent antimicrobial action. The focus of this work is to develop a small molecule to both sense and kill bacteria. This goal is accomplished through the development of a dual-function small molecule to detect and kill bacteria through a colorimetric change and release of nitric oxide to kill bacteria. Another approach to achieve this goal is the synthesis of a molecule that bacteria can sense, an inactive drug until bacterial enzymes cause the release of nitric oxide to kill the bacteria. Nitric oxide release is localized to bacteria causing an infection, which can help prevent bacteria from developing resistance by avoiding unnecessary exposure. Toward the goal of addressing the problem of antibiotic resistance, a nitric oxide donor attached to a fluorescent compound is synthesized, creating a compound that can both detect and kill the deadly multi-drug resistant bacteria strain, Pseudomonas aeruginosa. Detection occurs through a bacterial enzyme-activated color change, showing a visible color change from blue to yellow under UV light. The synthesized compound spontaneously releases 853 µmol of nitric oxide/g at a 10 mM initial concentration of the compound. Antibacterial efficacy studies after exposing Pseudomonas aeruginosa to a 10 mM dose of the synthesized compound show a 65% reduction in bacteria after 24 hours. This work is the first instance of a small molecule dual-function material that can both detect and kill bacteria. To address the goal of developing a bacteria-specific nitric oxide releasing compound, novel nitroaromatic-protected piperazine diazeniumdiolate (nitric oxide donor) prodrugs are synthesized to release nitric oxide upon enzyme activation to kill bacteria. These prodrugs are activated by an enzyme in the nitroreductase family, which are found almost exclusively in bacteria, and reduces the nitroaromatic-protecting group of the synthesized compounds, catalyzing the release of nitric oxide. Experiments show that nitric oxide release from the synthesized compounds only occurs in the presence of a bacteria-derived nitroreductase enzyme, demonstrating the possibility of site-specific delivery of an antibacterial therapeutic. The amount of nitric oxide release is measured at concentrations of 0.01, 0.1, and 1 mM, and is well within known antibacterial levels at concentrations of 0.1 and 1 mM, reaching nitric oxide concentrations of up to 4.8 µM. The antibacterial activity of the compounds is demonstrated after exposure of the compounds to Escherichia coli, a nitroreductase-producing bacterial species and common infection forming species, leading to up to a 94% reduction in the number of viable bacteria after 24 hours at 1 mM concentrations of the prodrug. This study is the first example of an antibacterial diazeniumdiolate prodrug activated by a nitroreductase enzyme, and further demonstrates the possibilities of antibacterial prodrugs. Medical devices are a site where bacterial infections can develop, and these infections are often incredibly difficult to treat, sometimes requiring the removal of the device. Medical devices could be coated with an antibacterial material that releases antibiotics to prevent infections. To investigate the application of the nitroreductase enzyme-activated nitric oxide releasing prodrugs for antibacterial medical device coatings, the prodrugs are incorporated into polyvinyl chloride and polyurethane films to create antibacterial prodrug polymer composite materials. Characterization of nitric oxide release from the surface of the composite films is observed only after metabolism by a bacterial nitroreductase enzyme, demonstrating the prodrug nature of the polymer composite. Excitingly, antibacterial efficacy experiments resulted in a 66% reduction in Escherichia coli after exposure to the diazeniumdiolate-composite films. This work details the first example of an antibacterial enzyme-activated NO-releasing polymer. The development of these novel compounds and materials represents significant advances in research to develop new ways to detect and treat bacterial infections.
Open Access
The progress towards the total synthesis of (ent)-MPC1001
(Colorado State University. Libraries, 2011) Schuber, Paul, author; Williams, Robert M., advisor; Crans, Debbie, committee member; Finke, Richard, committee member; McNaughton, Brian, committee member; Slayden, Richard, committee member
Herein are my efforts toward the total synthesis of (ent)-MPC1001, beginning with the development of a novel asymmetric [1-3]-dipolar cycloaddition utilizing a vinyl silane and a chiral lactone template. The mechanism of the cycloaddition was investigated and the cyclized product can be elaborated in 6 steps to the A-B-C ring system of the MPC family of natural products. However, the key ring-closing metathesis reaction provided irreproducible results. Therefore, a macrolactonization was utilized to synthesize an advanced lactone derivative. Current research is focused on the elaboration of the lactone to the oxepin ring. Efforts were also focused on the development of a novel β-hydroxy-α-amino acid derivative to be used in the preparation of analogues of the natural product (ent)-MPC1001. The amino acid was efficiently prepared in six steps via a Mukaiyama aldol reaction by a chiral oxazinone and 3-bromo-4-methoxybenzaldehyde. With the dipole product and the β-hydroxyl-α-amino acid derivative in hand, efforts were focused on the coupling of the two components to afford the DKP. Research was also focused on the installation of the diaryl ether portion of (ent)-MPC1001 as well as an interesting dimerization reaction. The dimerization reaction can serve as a point of divergence to the aronotin family of natural products.
Open Access
Translocation of insulin receptors into plasma membrane microdomains in response to insulin and to insulin-enhancing vanadium and chromium compounds
(Colorado State University. Libraries, 2010) Al-Qatati, Abeer S. A., author; Roess, Deborah, advisor; Crans, Debbie, committee member; Graham, James, committee member; Anthony, Russ, committee member
We have examined the translocation of insulin receptors into specialized, cholesterol-enriched membrane microdomains called lipid rafts following treatment of RBL-2H3 cells with insulin, bis-maltolatooxovanadium (BMOV) and tris(pyridinecarbxylato) chromium(III) (Cr(pic)3). Isopycnic sucrose gradient ultracentrifugation was used to subfractionate membrane fragments and insulin receptors were identified within low or high buoyant density membrane fractions using insulin receptor-specific antibodies and western blotting. Single particle tracking methods were used to confirm the confinement of individual insulin receptors within small membrane compartments on intact, viable RBL-2H3 cells. We demonstrated that insulin receptors translocate into lipid rafts upon binding insulin or following exposure to BMOV or Cr(pic)3 Phosphorylated insulin receptors also appeared in membrane raft fragments in response to insulin and/or insulin-mimicking compounds. Extraction of cholesterol from lipid rafts disrupted these microdomains and caused a decrease in the number of unphosphorylated and phosphorylated insulin receptors within these compartments. In addition to their ability to induce translocation of insulin receptors into lipid rafts, BMOV and Cr(pic)3 caused an increase in the number of phosphorylated IRS-1 molecules within these membrane fragments. To determine why Cr(pic)3 and BMOV might affect the distribution of insulin receptors in non-raft and raft compartments, membrane fluidity was evaluated in Cr(pic)3 and BMOV treated cells. Fluidity, as suggested by a decrease in lipid packing, was increased following treating 2H3 cells with either BMOV or Cr(pic)3 These results suggest that changes in lipid packing resulting from exposure of cells to either Cr(pic)3 and BMOV may affect the distribution of receptors in non-raft and raft compartments. Increased receptor localization in rafts or small membrane compartments evaluated by single particle tracking studies, would result in increased likelihood of insulin receptor phosphorylation within these signaling platforms. Thus rafts may be an important membrane structures involved in cell signaling events mediated by insulin receptors.