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DESIGN AND SYNTHESIS OF SQUARAMIDE -BASED MOLECULAR MACHINES
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Prof. Rajeev Muthyala
Artificial molecular machines are sought after in a wide variety of fields. They are useful in the construction of nanodevices (molecular valves, brakes, nanocars, rotors and ratchets), for ion transport and also for optical data storage. In an effort to develop new ion-transportand drug delivery strategies we became interested in designing molecular machines based on amide derivatives of squaric acid (squaramides). In this study, we first determined that secondary diaryl squaramides, which exist in the extended ZZ conformation, are excellent neutral receptors for biologically important anions such as chloride, carboxylate, and dihydrogen monophosphate. Next, we envisioned a molecular valve approach to regulate anion binding to squaramides via changes in the external environment (for example, a change in solvent polarity). We reasoned that in non-polar solvents, intramolecular hydrogen bonding between the carbonyl groups and the squaramide NHs would block anion binding (OFF state) while in a polar solvent disruption of intramolecular hydrogen bonding and reorientation of the carbonyl allows anion binding (ON state). Using ortho benzoyl substituted squaramides, we successfully applied the molecular valve approach to chloride binding. We subsequently studied the generality of the molecular valve approach with other ortho substituents such as secondary and tertiary amides, esters, and nitro groups. We found that the success of the molecular valve approach depends on whether, in a given solvent, intra-molecular hydrogen bonding is stronger or weaker relative to intermolecular hydrogen bonding with chloride ion. A significant effort was also spent on developing tertiary squaramide-based molecular machines for drug delivery. Initial studies revealed that simple (for example, N, N'-dimethyl derivatives) tertiary diaryl squaramide exhibited a preference for folded EE conformation regardless of solvent polarity. For our goal of transforming these squaramides to functional molecular machines, we decided to exploit the hydrophobic effect. In nonpolar solvents we anticipated that the tertiary squaramides would exhibit a preference for EE conformation while in aqueous medium we reasoned that the conformation would switch to ZZ driven by the hydrophobic effect. However we soon experienced major synthetic challenges. The literature procedures for the synthesis of these tertiary aryl squaramides routinely resulted in low yields with significant squaraine impurities. We therefore developed a novel copper-based method to synthesize symmetrical tertiary diaryl squaramides. Importantly, this method also enabled synthesis of unsymmetrical tertiary diaryl squaramides. Syntheses, conformational preferences, and our attempts at developing hydrophobically driven molecular machines will also be discussed in this dissertation.
ENERGETIC BASIS OF COILED COIL TOPOLOGY AND OLIGOMERIC STATE SPECIFICITY
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The coiled-coil protein oligomerization motif consists of two or more α-helices oriented parallel or antiparallel, which wrap around each other in a slight left-handed superhelical twist. The typical sequence of a coiled coil is characterized by a heptad repeat commonly denoted by the letters abcdefg, where residues in positions a and d are predominantly hydrophobic, while those in positions b, c, e, f, and g are usually charged or polar. Empirical rules have been established on the tendency of different core sequences to form a certain topology and oligomeric state but the physical forces behind this specificity are unclear. In this thesis we examine the ability of an effective energy function (EEF1.1) to discriminate the correct topology and oligomeric state for a given sequence using a molecular dynamics approach. We find that inclusion of entropic terms is necessary for discriminating the native structures from their misassembled counterparts. The decomposition of the effective energy into residue contributions yields theoretical values for the oligomeric propensity of different residue types at different heptad positions. We find that certain calculated residue propensities are general and consistent with existing rules, while other residue propensities are sequence context dependent. A variety of features contribute to the topological specificity of the motif, including electrostatics, side chain entropy change, steric matching, and the desolvation of hydrophobic side chains. Our results establish that the oligomeric state is dictated by similar rules in both parallel and antiparallel conformations but alignment of α-helices requires a broader set of both lateral and vertical interaction patterns. We found that the antiparallel topology can be directed by a/e' electrostatic attractions in the dimer, with e/e' and g/g' making minimal contributions. The antiparallel trimer topology is mainly the result of steric matching a/e' and d/g' side chain pairs in two antiparallel faces. The antiparallel tetramer is stabilized by similar interactions as the trimer in addition to b/e' electrostatics, which are only available in this oligomeric state. This work provides useful methodology and rules for designing coiled coils with a well defined and predictable three-dimensional structure.
Magnetic Janus Particles and Their Applications
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Magnetic properties are important since they enable the manipulation of particle behavior remotely and therefore provide the means to direct a particle’s orientation and translation. Magnetic Janus particles combine magnetic properties with anisotropy and thus are potential building blocks for complex structures that can be assembled from a particle suspension and can be directed through external fields. In this thesis, a method for the fabrication of three types of magnetic Janus particles with distinct magnetic properties is introduced, the assembly behavior of magnetic Janus particles in external magnetic and electric fields is systematically studied, and two potential applications of magnetic Janus particles are successfully tested. Janus particles with different magnetic properties are fabricated by varying the deposition rate of iron in an Ar/O2 atmosphere using physical vapor deposition (PVD). The extent of oxidation for each type of iron oxide is precisely controlled by the time it is exposed to the Ar/O2 atmosphere during deposition. Two of the three magnetic Janus particles produced show distinct assembly behavior into staggered and double chain structures, whereas the third shows no assembly behavior under an external magnetic field. The effect of the iron oxide cap thickness (≤ 50 nm) on the Janus particle assembly behavior is studied resulting in a deposition rate diagram that shows the relationship between the assembly behavior and the deposition rate. The cap materials for staggered chain, double chain, and no assembly behavior are assigned as Fe1-xO, Fe3O4, and Fe2O3, respectively, based on optical appearance and physical properties. The assignment is further confirmed by in-depth material characterization with scanning and transmission electron microscopy, atomic force microscopy, energy-dispersive X-ray spectroscopy, and X-ray photoelectron spectroscopy. The magnetic hardness of the iron oxides is tested using the magneto-optic Kerr effect. The assembly behavior of Fe3O4-capped Janus particles is studied in overlapping parallel and perpendicular AC electric and magnetic fields. The chains formed by Fe3O4-capped magnetic Janus particles show contraction behavior of ~30%, which suggests their application as an in situ viscometer. The chain contraction rate is found to depend on the viscosity of the liquid as well as the size of Janus particles and an in situ microviscometer is realized. Further, the magnetic dipole-dipole interactions of Fe1-xO and Fe3O4-capped Janus particles are studied by analyzing the particle-particle interaction force and energy during the process of Janus particle doublet formation. Using the magnetic particle interaction energy, the magnetization of each iron oxide cap is determined and found to be in excellent agreement with magnetization values obtained using standard SQUID measurements suggesting the application of magnetic Janus particles as a micro-magnetometer. In summary, three types of magnetic Janus particles with distinct magnetic properties have been fabricated and show versatile assembly behaviors that make them useful basic building blocks for complex structures and applications. For example, magnetic Janus particles can be used to measure the viscosity of a fluid or the magnetic property of a thin film cap material. It is likely that other interesting applications will emerge, when Janus particles of various sizes and/or patchy particles with magnetic properties are combined and explored.
Degradation of Three Trihaloalkyl Phosphates under Anoxic Condition in the Presence of Reduced Sulfue Species
Dickens Saint Hilaire
Year of Dissertation:
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Greener Syntheses of Metallic Nanoparticles and Zinc Oxide Nanopowders
Year of Dissertation:
In recent years, nanotechnology and nanomaterials synthesis have attracted a great deal of attention in the scientific community. Nanomaterials display size and morphology-related optical properties that differ from their bulk counterparts and therefore can be used for many applications in different fields such as biomedicine, electronics, antibacterial agents, and energy. Attempts to fabricate different morphologies of metallic and metal oxide nanoparticles (NPs) have successfully yielded attractive nanostructures such as particles, rods, helices, combs, tetra-pods, and flowers, all displaying properties mainly related to their enhanced surface area and/or aspect ratios. Most of the above mentioned nanomaterials productions have employed harsh synthetic routes such as high temperatures, low pressures, and the use of costly equipments. Here we show how a greener approach to nanomaterials synthesis is feasible with both minimization of aqueous precursors, energy and employment of a multi-block heater for temperature control. We present in this thesis several methods for the preparation of NPs of several materials that focus on minimizing the environmental impact of the synthesis itself. First, we describe the use of the toroidal form of plasmid DNA as a rigid narrowly dispersed bio-polymeric nanocavity, which mold the formation of disc-shaped nanoparticles of several types of metals. This approach exploits several properties of plasmid DNA: (a) DNA affinity for metal cations, (b) toroidal plasmid DNA structures which are favored by metal ionic binding, and (c) the ability to vary plasmid size. Herein, we present a complementary synthetic method based on a kinetic approach wherein the plasmid DNA acts as a template to initiate and control the formation of Au and other metallic NPs by incubation at elevated temperatures. Also reported herein is a simple, scalable hydrothermal method to make ZnO NPs that exploits temperature to precisely control the range of pH values of an organic amine buffer. The presence or absence of ethylenediaminetetraacetic acid in the tris(hydroxymethyl)aminomethane buffer further modulates the morphology of the ZnO nanomaterials since both compounds can serve as nucleating sites, and as stabilizing agents that prevents agglomeration.
Synthesis of Heteroatom Containing Aromatic Conjugated Polymers Using Acyclic Diene Metathesis (ADMET)
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This doctoral thesis describes the synthesis of heteroatom (B/Si/Ge/Sn) containing conjugated macromolecules via Acyclic Diene Metathesis (ADMET) polycondensation. The main objective was to obtain a library of macromolecules with unique optical properties based on different aromatic segments and heteroatoms. In chapter 2, the selective synthesis and characterization of a germanium containing macrocycle with two stilbene fluorophores is reported. The structure and size of the macrocycle were determined by 1H NMR, 13C NMR, GPC (polystyrene standards) and MALDI - TOF. The macrocycle features "all - trans" configuration at the vinylene bonds. The optical properties were studied by UV/Vis and fluorescence spectroscopy. The material emits in the blue region around 363 nm with a quantum efficiency of 0.4 relative to trans - stilbene. Theoretical calculations using B3LYP/6-31G** and Lanl2dz basis sets offered a better understanding of structural and electrooptical properties. They showed the presence of two important transitions in the absorption and the involvement of germanium orbital in the electronic conjugation with the stilbene segments. Chapter 3 outlines the extension of this aforementioned synthetic strategy to the group 14 element boron, in order to generate a new class of conjugated macromolecules - homopolymers based on boron (p2a, p2b) and co-polymers based on silicon and boron (p12a, p12b). The structures and these new systems were determined by 1H NMR, 13C NMR and correlation NMR spectroscopy. The molecular weights were determined by GPC using polystyrene standards. Both the homopolymers and copolymers have "all - trans" configuration around the internal vinylene bonds. The copolymers were found to be random. The optical properties showed that all the macromolecules absorbed in the range of 327 - 406 nm and emitted at 416 nm (p2a, p12a) and 494 nm (p12b). The quantum efficiencies of these macromolecules were in the range of 0.28 - 0.30. p12a was found to be a potential fluoride ion sensor with very high sensitivity due to a polymer co-operative effect. Two distinct emissions dominated the emission spectrum of p12b, investigation of which indicated possible intermolecular energy transfer. The thermal properties indicated higher stability of the copolymers compared to the homopolymers. Degradation of p12a followed a two-step process and p12b was found to be more stable than p12a. Chapter 4 of this thesis reports the synthesis of a library of homologous polymers based on Si, Ge, or Sn alternating with dithienylthiophene segments. The microstructures were analyzed by 1H NMR, 13C NMR and Correlation spectroscopy (HSQC, COSY). All the polymers p3a - c showed "all - trans" configuration at the vinylene bonds. The molecular weights were determined by GPC using polystyrene standards. No significant side products were observed under the ADMET conditions employed. Optical property analysis showed that the monomers were non-emissive whereas they absorb in the range of 263 - 264 nm. The polymers were highly fluorescent emitting in the range of 419 - 423nm. The quantum efficiencies were found to decrease from 0.18 to 0.11 from Si over Ge to Sn along with a small gradual blue shift of the emission maximum with the increase in size of the heteroatom. Thermogravimetric analysis indicated that Si based p3a showed the highest stability among the three homologous polymers. Chapter 5 is strongly related and involves alkyloxy homologous side - chain substituted systems. The microstructures of the macromolecular products were analyzed by 1H NMR, 13C NMR showing that these polymers also have an "all - trans" configuration around the vinylene bond. The molecular weights were determined by GPC using polystyrene standards. Alkyloxy substituted silicon containing stilbene polymers showed a strong red shift compared to their stilbene homologous without side chains, with an absorption maximum at 371 nm. The emission maximum was observed at 412 nm. The quantum yield was 0.50 - 0.52 indicating that, there is no photo - induced cis - trans isomerization.
Intelligent Nano/Microgels for Cell Scaffold and Drug Delivery System
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Stimulus-responsive polymer microgels swell and shrink reversibly upon exposure to various environmental stimuli such as change in pH, temperature, ionic strength or magnetic fields. Therefore, they become ideal candidates for biomaterial applications. For this work, we focus on the several intelligent microgels and their application on two areas: the cell scaffold and the drug delivery system. As for the cell scaffold, it can be realized by colloidal supra-structure microgels, which constructed by the thermo-driven gelation of the colloidal dispersion of poly(N-isopropylacrylamide-co-acrylamide) poly(NIPAM-co-AAm) microgels (Chapter 3). Such microgels exhibit a reversible and continuous volume transition in water with volume phase transition temperature (VPTT) 35 oC and remain partially swollen and soft under physiological conditions. More importantly, the size of the microgel particles can affect the sol-to-gel phase transition of the microgel dispersions, alter the syneresis degree of the constructed colloidal supra-structures, and tailor the cytocompatibility. The constructed colloidal supra-structure can be regarded as a model system for a new class of cell scaffolds. As for drug delivery system, Chapter 4 and Chapter 5 focus on the development of biocompatible microgels-based systems for delivering a traditional anti cancer drug curcumin. These thermo-responsive core-shell structure microgels are constructed from oligo(ethylene glycol) as a hydrophilic shell and hydrophobic biocompatible materials as core, such as poly(2-vinylanisole) and poly(4-allylanisole). The rationally designed core chain networks can effectively store the hydrophobic curcumin drug molecules via hydrophobic interactions, thus provide high drug loading capacity; while thermo-sensitive nonlinear poly(ethylene glycol) (PEG) gel shell can trigger the drug release by local temperature change, offering sustained drug release profiles. In Chapter 5, additionally embedded of magnetic Fe3O4 nanoparticles enable such hybrid nanogels to delivery pharmaceuticals to a specific site of the body by applying a gradient magnetic field. Chapter 6 investigated a class of well-defined glucose-sensitive microgels as an insulin drug release carrier, obtained via polymerization of 4-vinylphenylboronic acid (VPBA), 2-(dimethylamino) ethyl acrylate (DMAEA), and andoligo(ethylene glycol)methyl ether methacrylate (MEO5MA). The presence of MEO5MA monomer could retard the glucose-sensitive network from swelling because the rapid hydrogen bonding between the glucose molecules and the ether oxygens of the MEO5MA is prior to the glucose binding to the PBA groups. Therefore, the set point of glucose sensitivity of microgels could be adjusted possibly and result in potential biomedical applications. Compared to the non-imprinted copolymer microgels, the glucose imprinting of the microgels can create and rigidly retain more binding sites complementary to the shape of the target glucose molecule in the crosslinked polymer network, thus improve the sensitivity and selectivity of the microgels in response to the glucose level change. Additionally, the introduction of fluorescent Ag nanoparticles (NPs) to the microgels can realize the integration of optical glucose detection and self-regulated insulin delivery into a single nano-object.
Energy of the Quasi-free Electron in Atomic and Molecular Fluids
Year of Dissertation:
The ability to predict accurately the density dependent evolution of the conduction band energy of insulators has applications in the optimization of solvent choice and thermodynamic conditions for chemical reactions. However, directly investigating density dependent changes in the conduction band is experimentally difficult. Therefore, we have used field ionization of high-n dopant Rydberg states to determine the perturber induced shift of the dopant ionization energy Delta(ρP), where ρP is the perturber number density. Appropriate modeling allows the minimum of the conduction band energy V0(ρP) to be extracted from Delta(ρP). Field ionization requires the measurement of photoionization spectra of a dopant at two different electric field strengths. Thus, in this study, photoionization spectra of various dopants (i.e., CH3I, C2H5I, N,N-dimethylanaline, trimethylamine and triethylamine) were obtained under different electric field strengths in atomic (i.e., Ar, Kr, and Xe) and molecular (i.e., CH4, and C2H6) perturbers from low density to the density of the triple point liquid, at non-critical temperatures and on an isotherm near the perturber critical isotherm. At low perturber number density, a temperature dependence was observed in Delta(ρP), with |Delta(ρP)| increasing as the temperature decreases. This observation contradicts the prediction of the Fermi-Alekseev-Sobel'man model. Within the local Wigner-Seitz model developed by our group, the temperature behavior at low density arises from the ensemble average ion/perturber polarization energy P+(ρP and is caused by variations in the dopant/perturber radial distribution function. Moreover, a striking critical point effect in V0(ρP was observed in all of the perturbers investigated. This critical point effect is explained by the dramatic increase in the local density around a perturber particle near the critical point of the perturber. This local density increase, caused by an increase in the correlation length of the perturber, acts to confine the quasi-free electron, thereby increasing its kinetic energy. Various intermolecular potentials and integral methods necessary to calculate the radial distribution functions were studied and tested in order to achieve the best fit to the experimental Delta(ρP) in molecular perturbers.
Label-free Detection of Cancer Cells with Polysilicon Sensor Chips and Biomolecule-assisted Synthesis of Shape-controlled Nanoparticles
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Constant effort has been made for the detection of cancer cells. Recently, ovarian and kidney cancer cell lines have been shown to have higher cellular elasticity as compared to normal cells assessed by monitoring the degree of deformation under hyposmotic pressure. This method has been modified and applied to various cases. In cancer cells, the oncogenic mutant p53 (mtp53) protein is present at high levels and contributes to tumor growth and metastasis. Herein the influence of mtp53 on the mechanical property of breast cancer cells was assessed by monitoring the swelling ratio of cells with time using the impedance measurements. The depletion of mtp53 led to decrease of impedance variation, which is corresponding to the lower elasticity. All results suggest that electric probing for the extent of the mtp53 expression of breast cancer cells may serve as a meaningful fingerprint for the cancer diagnostics, and this outcome will also have an important clinical implication for the development of mtp53-based targeting for tumor detection and treatment. Meanwhile, the samples containing a mixture of red blood cells and cancer cells were examined by impedemetric detection. The results suggest that the cancer cells need to be enriched before applying impedance measurements to the samples with high concentration of red blood cells. Thus, dielectrophoresis (DEP) provides as a solution by taking most red blood cells apart from the cancer cells, which is supported by taking most red blood cells apart from the cancer cells, which is supported by experimental data. The DEP system will be integrated with microfluidic channels for DEP analysis in future. Biomolecule-assisted synthesis of shape-controlled nanoparticles is the other part of this dissertation. One project refers to the PbSe nanocrystal growth with the aid of peptide. Pb-binding TAR-1 peptides (ISLLHST) were covalently conjugated on a peptide nanotube substrate and the precursors of PbSe were incubated at room temperature. This resulted in the growth of highly crystalline PbSe nanocubes on this biomimetic cylindrical substrate. The growth mechanism to generate nanocubes occurs via the directed self-assembly of nanoparticles and then nanoparticle fusion. The peptide conformation and the cylindrical peptide nanotube substrate play important roles in the mesoscopic crystallization of PbSe nanocubes. The conformational change of the TAR-1 peptide on the nanotubes due to the change in the buffer seems to be responsible for aggregating intermediate nanoparticles in different directions for the directed fusion and mesoscopic crystallization of PbSe into the different shapes. The other project is the directional growth of LiFePO4 nanoribbons using glutathione and PVP as capping agents. The structure, size and morphology were investigated by XRD, TEM, AFM, SEM. The HRTEM result suggests that the nanoribbons are growing along , which is highly possible to enhance the rate capability, achieving the better battery performance.
SYNTHESIS OF NEW PORPHYRINOIDS FOR BIOMEDICAL AND MATERIALS APPLICATIONS
Year of Dissertation:
The facile synthesis of three non-hydrolysable thioglycosylated porphyrinoids is reported. Starting from meso perfluorophenylporphyrin (TPPF20), the non-hydrolysable thioglycosylated porphyrin (PGlc4), chlorin (CGlc4), isobacteriochlorin (IGlc4), and bacteriochlorin (BGlc4) can be made in 2-3 steps. The ability to append a wide range of targeting agents onto the perfluorophenyl moieties, the chemical stability, and the ability to fine-tune the photophysical properties of the chromophores make this a suitable platform for development of biochemical tags, diagnostics, or as photodynamic therapeutic agents. With reduction of one or two pyrrole double bonds, there is a red shift in the lowest energy absorption band and a significant increase in intensity. The fluorescence of these porphyrinoids is in the order PGlc4 = BGlc4 < CGlc4 4 and there is a corresponding decrease in the amount of triplet formed. Fluorescence micrographs of cells after treatment with these four porphyrinoids indicate they are taken up. The CGlc4 and IGlc4 may be dual function agents that can detect cancer by luminescence, and treat cancer by photodynamic therapy (PDT). Porphyrins appended with four rigid hydrogen bonding motifs on the meso positions were synthesized and self-assembled into a cofacial cage with four complementary bis- (decyl)melamine units in dry solvents, these hydrogen-bonded cages were analysed by diffusion-ordered spectroscopy (DOSY) in solution. The hydrocarbon chains on the melamine mediate the formation of nanofilms on surfaces as the solvent slowly evaporates. A water soluble zinc (II) phthalocyanine symmetrically appended with eight thioglucose units was synthesized from commercially available hexadecafluoro-phthalocyaninato zinc(II) by controlled nucleophilic substitution of the peripheral fluoro groups by thio-sugars. The photophysical properties and cancer cell uptake studies of this nonhydrolyzable thioglycosylated phthalocyanine are reported. The new compound has amphiphilic character, is chemically and photochemically stable, and can potentially be used as a photosensitizer in photodynamic therapy. A porphyrin bearing pyridyl groups at the meso positions was synthesized using 2,6-diacetamido-4-formylpyridine. A new method has been developed for the synthesis of the precursor aldehyde that avoid much of the problems associated with the earlier synthesis. With this porphyrin it is possible to build hetero-complementary rigid, multi-porphyrin supramolecular arrays via hydrogen bonds. For example, when using naphthalenediimide (NDI) units a checkerboard pattern is expected to be formed using this porphyrin as a donor and NDI as an acceptor where triple hydrogen bond is formed between the diimide and pyridyl units. Energy transfer can be studied through this hydrogen bonded supramolecular assembly. The synthesis of a triply bridged diporphyrin appended with six thioglucose units is reported. The electronic spectrum of this triply bridged porphyrin has enhanced intensity at low- energy wavelengths that reaches the near infrared region. The goal of this project is to create tumor targeting dyes that can be activated with red wavelengths of light that penetrate deeper into tissues. This new compound is amphiphilic in nature, chemically and photochemically stable, expected to have unusual photophysical and electrochemical properties, and can potentially be used as a photosensitizer in photodynamic therapy.