Browsing by Subject "Chemistry"

In the last two decades, the field of homogeneous gold catalysis has been

extremely active, growing at a rapid pace. Another rapidly-growing field—that of

computational chemistry—has often been applied to the investigation of various gold-

catalyzed reaction mechanisms. Unfortunately, a number of recent mechanistic studies

have utilized computational methods that have been shown to be inappropriate and

inaccurate in their description of gold chemistry. This work presents an overview of

available computational methods with a focus on the approximations and limitations

inherent in each, and offers a review of experimentally-characterized gold(I) complexes

and proposed mechanisms as compared with their computationally-modeled

counterparts. No aim is made to identify a “recommended” computational method for

investigations of gold catalysis; rather, discrepancies between experimentally and

computationally obtained values are highlighted, and the systematic errors between

different computational methods are discussed.

Metallosupramolecular polymers are increasingly of interest for functional and degradable polymeric materials. In these materials, the metal-ligand bonds often bear an external mechanical load, but little is yet understood about the nature of mechanically-triggered reactions of metal-ligand bonds and how that reactivity influences the mechanical limits of the material. This dissertation presents a poly(cyclooctene) polymer bearing 2,6-bis(1′-methyl-benzimidazolyl)pyridine (Mebip) ligands on sidechains, which provides easy incorporation into polymer backbones and sidechains, binding to a large variety of metal species, and facile synthesis with sites for future study substituent effects. This platform is employed in proof-of-concept studies comparing the crosslinking behavior of iron(II) trifluoromethanesulfonate and copper(II) trifluoromethanesulfonate. It was found through small molecule spectroscopic studies that both metal species bind in the desired 2:1 MeBip:metal stoichiometry for crosslinking. When these small molecule complexes are polymerized as crosslinkers in gel and solid networks, though the extent of crosslinking is found to be similar, the copper(II)-crosslinked networks exhibited a faster relaxation than the iron(II)-crosslinked networks. Further, under high strains, the copper(II)-crosslinked networks exhibited significantly higher extensibility. This work lays the foundation for further investigations of the effect of metal-ligand bonding on force-coupled properties of materials.

Biological electron transfer (ET) reactions are typically described in the framework of coherent two-state electron tunneling or multi-step hopping. Yet, these ET reactions may involve multiple redox cofactors in van der Waals contact with each other and with vibronic broadenings on the same scale as the energy gaps among the species. In this regime, fluctuations of the molecule and its medium can produce transient energy level matching among multiple electronic states. This transient degeneracy, or flickering electronic resonance among states, is found to support coherent (ballistic) charge transfer. Importantly, ET rates arising from a flickering resonance (FR) mechanism will decay exponentially with distance because the probability of energy matching multiple states is multiplicative. The distance dependence of FR transport thus mimics the exponential decay that is usually associated with electron tunneling, although FR transport involves real carrier population on the bridge and is not a tunneling phenomenon. Likely candidates for FR transport are macromolecules with ET groups in van der Waals contact: DNA, bacterial nanowires, multi-heme proteins, strongly coupled porphyrin arrays, and proteins with closely packed redox-active residues. The theory developed here is used to analyze DNA charge-transfer kinetics, and we find that charge transfer distances up to 3-4 bases may be accounted for with this mechanism. Thus, the observed rapid (exponential) distance dependence of DNA ET rates over distances of ≲10 Å does not necessarily prove a tunneling mechanism.

Molecular structures that direct charge transport in two or three dimensions could help to enable the development of molecule-based electrical switches and gates. As a step toward this goal, we use theory, modeling and simulation to explore DNA three-way junctions (TWJs). Molecular dynamics (MD) simulations and quantum calculations, indicate that DNA TWJs undergo dynamic interconversion among “well stacked” conformations on the time scale of nanoseconds, a feature that makes the junctions very different from linear DNA duplexes. The studies further indicate that this conformational gating would control charge flow through these TWJs, distinguishing them from conventional (larger size scale) gated devices. Simulations also find that structures with polyethylene glycol (PEG) linking groups (“extenders”) lock conformations that favor CT for 25 ns or more. The simulations explain the kinetics observed experimentally in TWJs and rationalize their transport properties compared to double-stranded DNA. Furthermore, we redesigned DNA TWJs that have equally coupled output pathways for charge. The TWJ was also designed to switch between the two conductive states in responsive to an applied electric field.

Computationally aided drug discovery confronts the problem to balance performance and computation cost. Earlier study reveals that a less-expensive docking approach is not reliable to estimate the protein-ligand affinity in exploring drug candidates targeting CARM1 (coactivator-associated arginine methyltransferase 1). However, more accurate binding free energy calculation based on molecular dynamics sampling is not affordable in a high throughput screening. A truncated MD method was developed and can be used to estimate the binding free energy with similar accuracy with the full-system MD methods, while reducing the computation cost ten fold. Thus, this truncated MD method is feasible in a high throughput screening towards drug discovery.

Recent work has shown that hyperpolarized magnetic resonance spectroscopy (HP-MRS) can trace in vivo metabolism of biomolecules and is therefore extremely promising for diagnostic imaging. The most severe challenge this technique faces is the short signal lifetime for hyperpolarization, which is dictated by the spin-lattice (T1) relaxation. In this thesis we show with theory, simulation and experiment that the long-lived nuclear spin states in chemically equivalent or near equivalent spin systems offer a solution to this problem. Spin polarization that has lifetime much longer than T1 (up to 70-fold) has been demonstrated with pulse sequence techniques that are compatible with clinical imaging settings. Multiple classes of molecules have been demonstrated to sustain such long-lived hyperpolarization.

Biofouling, the accumulation of biomolecules, cells, organisms and their deposits on submerged and implanted surfaces, is a ubiquitous problem across various human endeavors including maritime operations, medicine, food industries and biotechnology. Since several decades, there have been substantial research efforts towards developing various types of antifouling and fouling release approaches to control bioaccumulation on man-made surfaces. In this work we hypothesized, investigated and developed dynamic change of the surface area and topology of elastomers as a general approach for biofouling management. Further, we combined dynamic surface deformation of elastomers with other existing antifouling and fouling-release approaches to develop multifunctional, pro-active biofouling control strategies.

This research work was focused on developing fundamental, new and environment-friendly approaches for biofouling management with emphasis on marine model systems and applications, but which also provided fundamental insights into the control of infectious biofilms on biomedical devices. We used different methods (mechanical stretching, electrical-actuation and pneumatic-actuation) to generate dynamic deformation of elastomer surfaces. Our initial studies showed that dynamic surface deformation methods are effective in detaching laboratory grown bacterial biofilms and barnacles. Further systematic studies revealed that a threshold critical surface strain is required to debond a biofilm from the surface, and this critical strain is dependent on the biofilm mechanical properties including adhesion energy, thickness and modulus. To test the dynamic surface deformation approach in natural environment, we conducted field studies (at Beaufort, NC) in natural seawater using pneumatic-actuation of silicone elastomer. The field studies also confirmed that a critical substrate strain is needed to detach natural biofilm accumulated in seawater. Additionally, the results from the field studies suggested that substrate modulus also affect the critical strain needed to debond biofilms. To sum up, both the laboratory and the field studies proved that dynamic surface deformation approach can effectively detach various biofilms and barnacles, and therefore offers a non-toxic and environmental friendly approach for biofouling management.

Deformable elastomer systems used in our studies are easy to fabricate and can be used as complementary approach for existing commercial strategies for biofouling control. To this end, we aimed towards developed proactive multifunctional surfaces and proposed two different approaches: (i) modification of elastomers with antifouling polymers to produce multifunctional, and (ii) incorporation of silicone-oil additives into the elastomer to enhance fouling-release performance.

In approach (i), we modified poly(vinylmethylsiloxane) elastomer surfaces with zwitterionic polymers using thiol-ene click chemistry and controlled free radical polymerization. These surfaces exhibited both fouling resistance and triggered fouling-release functionalities. The zwitterionic polymers exhibited fouling resistance over short-term (∼hours) exposure to bacteria and barnacle cyprids. The biofilms that eventually accumulated over prolonged-exposure (∼days) were easily detached by applying mechanical strain to the elastomer substrate. In approach (ii), we incorporated silicone-oil additives in deformable elastomer and studied synergistic effect of silicone-oils and surface strain on barnacle detachment. We hypothesized that incorporation of silicone-oil additive reduces the amount of surface strain needed to detach barnacles. Our experimental results supported the above hypothesis and suggested that surface-action of silicone-oils plays a major role in decreasing the strain needed to detach barnacles. Further, we also examined the effect of change in substrate modulus and showed that stiffer substrates require lower amount of strain to detach barnacles.

In summary, this study shows that (1) dynamic surface deformation can be used as an effective, environmental friendly approach for biofouling control (2) stretchable elastomer surfaces modified with anti-fouling polymers provides a pro-active, dual-mode approach for biofouling control, and (3) incorporation of silicone-oils additives into stretchable elastomers improves the fouling-release performance of dynamic surface deformation technology. Dynamic surface deformation by itself and as a supplementary approach can be utilized biofouling management in biomedical, industrial and marine applications.

Perovskite photovoltaics has attracted tremendous attention recently due to the advance in the device performance. However, it is still challenging to effectively commercialize the perovskite technology due to several issues including current-voltage hysteresis, stability, complicated device architectures, etc. In this dissertation, we use the additive to tailor the properties of the functional layers in perovskite photovoltaic devices, aiming to engineer the interface, film morphology, carrier dynamics and film crystallization process. By using the additive engineering approaches, our goal is to achieve high-performance perovskite photovoltaics with reduced hysteresis, improved stability, versatile processing methods and simplified device architectures.

Perovskite solar cells usually employ p-i-n device architectures and TiO2 is a typical n-type semiconductor widely used in perovskite solar cells. However, perovskite/TiO2 interface is not preferable for the photo-excited carrier collection due to the energy band misalignment, conductivity mismatch, etc. In chapter 2, additive was used to tailor the properties of TiO2 and enable improved interface for perovskite solar cells. With Nb5+ as additive in TiO2, the conductivity of TiO2 and interface band alignment were simultaneously improved. Consequently, high-performance perovskite solar cells were successfully obtained with reduced hysteresis by using the Nb-TiO2.

In addition to the interface, we explored the impact of morphology and carrier dynamics of perovskite films on solar cell performance. In chapter 3, NH4SCN and PbI2 were used as additives to tune the morphology and charge carrier dynamics of perovskite films. Using NH4SCN additive could significantly enlarge the grain size of the polymorph perovskite films while using PbI2 additive could increase charge carrier lifetime of perovskite films. It was found that the open-circuit voltage and fill factor of perovskite photovoltaics were correlative with charge carrier lifetime while short-circuit current density of perovskite photovoltaics were correlative with grain sizes. Using both PbI2 and NH4SCN simultaneously could synergistically improve the quality of perovskite films and performance of perovskite solar cells.

Based on the understanding from chapter 3, a room-temperature process was developed to deposit high-quality perovskite films by using PbI2 and methylammonium thiocyanate (MASCN) as additives in chapter 4. Due to the synergistic effects of the additives, room-temperature-processed perovskite films with micron-size grains and microsecond-range carrier lifetime were successfully obtained for high-performance devices. More importantly, we established the correlation between the crystal grain size in resultant perovskite films and the precursor aggregate size in precursor solutions. The correlation suggested that the perovskite grain sizes from solution process depended on the precursor aggregate sizes.

Following the understanding built in chapter 3, we used the additive engineering method to impact the performance of ETL-free perovskite solar cells. In chapter 5, we found out that the photo-excited carrier injection at the interface was significantly inhibited without the assistance of an ETL, which would compromise the collection of the photo-excited carriers. By using PbI2 as additive to tune the carrier lifetimes in perovskite films, it was found out that increased carrier lifetimes in perovskite films could effectively counterbalance the inferior interface without ETLs and enabled high performance for ETL-free perovskite solar cells. By using perovskite with microsecond carrier lifetime, ETL-free perovskite solar cells were successfully realized with performance comparable to that of ETL-containing perovskite devices. Such results offer the opportunity for the perovskite devices with simplified device architecture.

Single-molecule spectroscopic (SMS) measurements have revolutionized biological science due to their ability to directly observe exactly one molecule in the crowd. This single molecule observation removes the ensemble average, revealing molecular heterogeneities. However, traditional SMS techniques fail to study single molecules in the solution phase for a long observation time, because the molecules rapidly diffuse away from the small focal spot. Accordingly, it is typically required to isolate the molecules from the native solution to study time-dependent dynamical behaviors, by either tethering the molecules to a stationary surface or confining the molecules in a small space based on physical principles. This isolation of molecules barricades in-situ and in-vivo single-molecule research. To address this gap, real-time feedback single particle tracking (RT-3D-SPT) has been developed, with the ability to directly monitor individual freely diffusing particles in the solution phase less disruptively. Real-time tracking is realized by estimating the particle’s position using photon information and applying active feedback to keep the particle in a small detection center. This set of techniques is largely divided into two classes, each with its limitations. The first class of RT-3D-SPT techniques spatially separates the emission of the particle using a set of detectors. The signal variation detected by these detectors can be used to estimate the particle’s position in real-time. The second class of RT-3D-SPT uses a spatiotemporally patterned laser excitation to illuminate the particle. The detected photon arrivals can therefore be used to estimate the particle’s position within the dynamic laser excitation pattern. A feedback control actuator such as a scanning mirror or a piezoelectric stage is driven to compensate for the particle’s diffusion in real-time, keeping the particle in the focal volume. However, both methods have limited ability to track dim and fast diffusing objects, such as single molecules. Moreover, very few of these optical configurations provide simultaneous contextualization in three dimensions yet fail to observe rapid processes happening in the surrounding of the tracked objects.In this dissertation, we present a new real-time 3D tracking and imaging method to directly observe fast biological and chemical processes. These processes include the rapid protein adsorption onto nanoparticles when the nanoparticles are exposed to biological fluids. This adsorbed protein layer, called the protein corona, alters nanoparticles’ biological identity and their fate in vivo. Therefore, it is important to understand this critical roadblock in the biological application of engineered nanoparticles. Herein, we first introduce the construction of this microscope, called 3D real-time ultrafast local microscopy (3D-RULM), with its ability to track fast diffusing and lowly emitting objects while rapidly imaging the surroundings concurrently. Next, we show this RT-3D-SPT method can be applied to quantitatively characterize individual nanoparticle protein corona in situ particle by particle, with single protein sensitivity at signal-to-background ratios down to 1%. Finally, to expand this method to smaller NP-protein systems, we have further implemented a Galvo mirror as a control actuator with a response time five times faster than the currently used piezoelectric stage, opening the possibility to study transient NP-protein interactions and other fast biological phenomena in situ and in vivo.

While a number of different proteomic, genomic, and computational approaches exist for the characterization of drug action, each of the experimental approaches developed to date has both strengths and weaknesses. Currently, there is no one "perfect" assay for drug mode-of-action studies. A protocol that could assay all the proteins in the proteome for both direct and indirect binding interactions of drugs would greatly facilitate studies of drug action. Recently, the SPROX (stability of proteins from rates of oxidation) technique was developed as a chemical modification- and mass spectrometry-based strategy for detecting protein-ligand interactions by monitoring the change in thermodynamic stability of proteins upon ligand binding. This is accomplished by monitoring the denaturant dependent oxidation of globally protected methionine residues. The SPROX technique has been interfaced with bottom-up proteomics methods to allow for the proteome-wide analysis of protein-ligand interactions. However, the strategy has been limited by the need to detect and quantify methionine containing peptides in the bottom-up proteomics experiment.

The work in this dissertation is focused on evaluating the current SPROX protocol, developing modifications to improve proteome coverage, and applying the SPROX platform to two different drug mode-of-action studies. Three main strategies were employed to improve protein coverage. First, a chemo-selective isolation of un-oxidized methionine containing peptides was employed to enrich for methionine containing peptides, and it was found to produce a ~2-fold improvement in proteomic coverage. Second, a pre-fractionation strategy involving the use of isoelectric focusing was employed to decrease sample complexity prior to LC-MS/MS analysis and it was found to generate a ~2-3 fold improvement in proteomic coverage, however when combined with the methionine enrichment strategy the improvement was ~6-fold as the benefits of both were additive. Third, a tryptophan modification strategy was developed that could ultimately expand the number of useful peptides in proteome-wide SPROX experiments to include those that contain tryptophan. Also, investigated was the use of several different mass spectrometer systems (including a bench-top quadrupole and orbitrap system and two different quadrupole time-of-flight systems) in the SPROX protocol. The results of these studies indicate that there is a significant advantage in proteome coverage when faster mass spectrometers are used. The use of high energy collision dissociation (HCD) in the orbitrap system was also more advantageous than the use of collision induced dissociation (CID) in the Q-ToF systems. Regardless of the mass spectrometer used, the major source of error in the SPROX experiment was found to be the random error associated with the LC-MS/MS analysis of isobaric mass tagged peptides. This random error was found to yield a false discovery rate of between 3 and 10% for "hit" peptides in the SPROX experiment.

The above improvements in the SPROX protocol were used in two protein-ligand binding experiments. One set of experiments involved studies on two small molecules with a specific anti-cancer phenotype in human colon cancer cells. These studies identified 17 proteins as potential "hits" of these two small molecules. After preliminary validation of these proteins, approximately 50% were eliminated as false positives and one protein, p80/nucleophosim, showed consistent data indicating a destabilizing interaction with both small molecules. The destabilization is indicative of an indirect interaction with the small molecules that would be mediated through a protein-protein interaction network. In another set of experiments the breast cancer drug, tamoxifen, and its main, active metabolite, 4-hydroxy tamoxifen, were assayed for binding to the proteins in a yeast cell lysate to better understand its adverse effects on yeast cells. The results of these studies identified ~80 proteins as potential "hits" of these two drugs. After preliminary validation of these proteins, approximately 30% were eliminated as false positives and one protein, SIS1, type II Hsp40, showed consistent data indicative of a direct binding interaction.

Recently, several mass spectrometry-based proteomics techniques have been developed for the large-scale analysis of thermodynamic measurements of protein stability. This has created the possibility of characterizing disease states via differential thermodynamic stability profiles. Described here is the application of the Stability of Proteins from Rates of Oxidation (SPROX) technique to characterize mouse models of disease. The mouse models studied here are of normal aging and two genetically induced Parkinson’s Disease (PD) models.

Thermodynamic stability profiles were generated for 809 proteins in brain cell lysates from C57BL/6 mice at age 6- (n=7) and 18-months (n=9). The biological variability of the protein stability measurements was low, and within the experimental error of the SPROX technique. Remarkably, the large majority of the 83 brain protein hits were destabilized in the old mice, and the hits were enriched in proteins that have slow turnover rates (p<0.07). Furthermore, 70% of the hits have been previously linked to aging or age-related disease.

One of the PD mouse models involved characterizing the protein interactions induced by mutated leuceine-rich repeat kinase 2 (LRRK2) at a pre-symptomatic time point (3 months old). The models used were a control, overexpressed wildtype LRRK2, and overexpressed R1441G mutated LRRK2 (n=2 for all models). Comparative analyses on thermodynamic stability profiles of ~470 proteins revealed relatively few differences. In fact, the observed hit rate in each comparative analysis was close to that associated with the biological variability of the mice. However, four protein hits, dihydropyrimidinase-related protein 2, eukaryotic translation initiation factor 4A2, Rap1 GTP-GDP dissociation stimulator 1 and myelin basic protein, were identified with consistent thermodynamic stability in multiple mice within a biological state and as hits in multiple comparisons suggesting they are the most likely to be true positives.

The second PD mouse model studied was one in which the human α-synuclein protein, containing the known PD mutation A53T, was overexpressed. To characterize the disease progression of PD induced by this mutation, mice were sacrificed at 1 month (n=4), 6 months (n=4) and when they became symptomatic at 10-16 months (n=3). Thermodynamic stability profiles were generated for >850 proteins at each time point. The relative stabilities of these proteins were assayed in a series of comparative analyses involving mice at the different time points and the normally aged mice from above. In total 244 peptides were found to be differentially stabilized during PD progression. A subset of 52 peptide hits was identified to be of particular interest. Of these 52 peptides 22 were identified with early disease progression, 5 peptides showed late disease progression, 5 peptides reported a gradual difference in stability over disease progression and 20 peptides indicated no disease progression trend. More than 90% of the 32 peptides indicating a trend in disease progression showed progression related destabilization.

The results of this thesis help validate the use of thermodynamic stability measurements to capture disease-related proteomic differences in mice. Furthermore, these results establish a new biophysical link between the hit proteins identified and their role in aging, LRRK2 protein interactions, and PD progression.

Melanin is a biological pigment that is ubiquitous in nature and generally produced within melanosomes, specialized organelles. Typically, melanin is categorized into two distinct classes, based on color and molecular precursor: eumelanin (brown-black) and pheomelanin (yellow-red). Whereas much is known regarding the molecular precursors to the two pigments, an understanding of their resulting molecular structure remains elusive. Despite this lack of knowledge, several functions are attributed to the pigments, including photoprotection and photosensitization. Epidemiological data for skin and ocular cancers have observed an increased incidence for increased relative concentrations of pheomelanin. Furthermore, eumelanin is generally identified as photoprotective and antioxidant, whereas pheomelanin is generally identified as photoreactive and pro-oxidant. This thesis describes the photophysical properties of the naturally-occuring melanin pigments and presents new insights into their roles within the context of skin and ocular cancers.

Photoemission electron microscopy provides a unique opportunity to probe the complex photoproperties of melanins contained within intact melanosomes isolated from tissues of bovine and human eyes. Photoionization threshold potentials characteristic of eumelanin and pheomelanin have been determined and are used to investigate the molecular architecture of the pigments within the melanosome. Furthermore, a novel approach to photoemission electron microscopy is used to obtain the first direct measurements of the absorption coefficients from intact melanosomes.

Human iridal stroma melanosomes are comprised of both eumelanin and pheomelanin in various ratios according to iris color; dark brown and blue-green iris melanosomes are characterized by a eumelanin:pheomelanin ratio of 14.8 and 1.3, respectively. Despite the significant difference in the overall pigment composition, a common eumelanin surface photoionization threshold is obtained for both melanosomes. This data indicates that within the melanosome, the phototoxic pheomelanin pigment is encased by eumelanin. This structure mitigates the adverse photochemical properties of pheomelanin. However, damage to the eumelanic exterior and or significant reduction in the amount of eumelanin present could compromise the protective ability of eumelanin, providing mechanisms for exposure of pheomelanin and consequently contributing to oxidative stress.

The absorption spectra of intact melanosomes of varying melanin compositions were determined over the spectral range from 244 to 310 nm. The absorption spectra of eumelanic melanosomes are similar regardless of monomer composition or embryonic origin. Furthermore, the absorption spectra of melanosomes containing a mixture of pigments were similar to those containing pure eumelanin, arguing that the absorption properties of the melanosome are maintained regardless of increased pheomelanin composition. Therefore, the correlation between epidemiological data and the eumelanin:pheomelanin ratio is not predicted to be a reflection of the melanosome's decreased ability to attenuate biologically relevant wavelengths, but instead is predicted to be a reflection of the different photoreactivities of the melanin pigments contained within.

The development of electronic textiles, which have many potential healthcare and consumer applications, is currently limited by a lack of energy storage that can be effectively incorporated into such devices while having sufficient energy density, power density, and durability to perform well. The overall goal of this work was to improve the energy density and potential for use in electronic textile applications of a nanostructured composite of few-walled carbon nanotubes, manganese oxide, and reduced graphene oxide. Two approaches towards improving the desired properties by controlling the macroscopic structure of the composite were pursued: one, to make fiber or wire-shaped electrodes via wet-spinning in aqueous chitosan solutions (10% acetic acid), and the other, to make composite films with controlled porous structures using nitrocellulose as a sacrificial filler material. Both approaches yielded the desired macroscopic structures. The composite fibers were non-conductive due to the insulating nature of manganese oxide and its positioning on the surface of the fibers. Composite fibers of few-walled carbon nanotubes and reduced graphene oxide made by the same method were found to have good volumetric capacity, rate capability, stability and flexibility. Nonintuitively, electrochemical performance of composite films declined with increasing porosity due to a decrease in conductivity, highlighting the importance of balancing the interplay between properties important to device performance when designing controlled structures of complex materials.

The phylum Apicomplexa encompasses multiple obligate intracellular parasites that pose significant burdens to human health including the causative agents of malaria, toxoplasmosis, and cryptosporidiosis which infect millions of humans and cause hundreds of thousands of deaths each year. During their complex life cycles, apicomplexan parasites coordinate the function of specific proteins to both evade the host immune system and thrive under stressful conditions. Notably, Toxoplasma gondii has been found to harbour multiple polyketide synthase (PKS) genes by bioinformatic analysis, suggesting they can produce secondary metabolite polyketides. While secondary metabolite biosynthetic gene clusters (BGCs) have been known in Apicomplexa for over two decades, limited studies on these enzymes have been completed to date and there have been no characterized products, leaving a void in our understanding of the role of these enzymes in parasite biology. Therefore, characterization of these proteins may aid in our ability to target these biosynthetic enzymes as sources of potential therapeutic candidates in Apicomplexa.While protists are underexplored for biosynthetic potential, research points to this kingdom as an untapped potential for new chemical space. T. gondii for instance possesses multiple putative PKS biosynthetic gene clusters (BGCs) however there have been no secondary metabolite products elucidated thus far. Therefore, our work explores a T. gondii PKS, TgPKS2, and investigates the architecture, predicted structures, and activity of multiple domains within this synthase. Subsequently, Chapters 2 and 3 describes our initial studies on TgPKS2 including hydrolysis activities of acyltransferase (AT) domains, mutagenesis studies, and a first of its kind self-acylation activity of acyl carrier protein (ACP) domains in a modular type I PKS. Chapter 4 further emphasizes the unique attributes of TgPKS2, delving into a never before characterized chain release mechanism, while Chapter 5 compares TgPKS2 transacylation activity to well-characterized bacterial and fungal systems. Combined, these chapters describe our work to biochemically explore TgPKS2, discover the role it plays within the T. gondii life cycle, and further our work to elucidate the metabolite(s) produced by this synthase. Altogether, this research lays the ground work for exploring other apicomplexan and eukaryotic polyketide synthases and significantly increases our knowledge of the biochemical properties of these unique proteins.

Natural products belonging to the lipid II-binding family act as potent antimicrobial agents by disrupting cell wall biosynthesis via sequestering the late-stage intermediate lipid II. However, the emergence of resistance mechanisms and poor bioavailability have hindered the utility of these molecules as promising therapeutic intervention strategies to combat pathogenic bacterial infections. Gaining a deeper understanding of structural components and biosynthetic pathways can lead to the creation of second-generation derivatives to improve bioactivity and pharmacological properties. To explore this superfamily, we have used bioanalytical, biochemical, synthetic, computational, and enzymatic approaches that have been applied to three distinct projects. The first includes efforts to characterize the relationship between structural feature and bioactivity for the lipid II-binding CDA (calcium dependent antibiotic), malacidin. Through a series of minimally complex analogs, we determined non-proteinogenic amino acids and the N-acyl fatty acid moiety are essential for bioactivity. For the second project, we investigated a conserved mechanism of action for phylogenetically-related natural products within the lasso peptide subfamily. This work led to the discovery of a novel class I lasso peptide, arcumycin, and we validated a conserved mechanism of action for Actinobacteria-produced lasso peptides in targeting lipid II biosynthesis. Our last project sought to elucidate the mechanism of lipoinitiation for the ramoplanin family of molecules. Through a series of bioactivity assays, we found the transfer to the acyl carrier protein (ACP) in a fatty acyl-AMP ligase (FAAL)-dependent manner determined the specificity of lipids selected in the biosynthetic process. Collectively, through each project we have gained a deeper understanding of the structural elements and biosynthetic pathways of lipid II-binding antimicrobials.

In boranophosphate (BP) nucleotides, a borane (BH3) group is substituted for a non-bridging phosphoryl oxygen of a normal phosphate group, resulting in a class of modified isoelectronic DNA and RNA mimics that can modulate the reading and writing of genetic information. 5'-(α-P-borano)nucleoside triphosphates (NTPαBs) are good substrates or inhibitors for many viral RNA polymerases and reverse transcriptases (RT) when compared with natural nucleoside 5'-triphosphates (NTPs). A practical aspect of this coding phenomenon employs T7 bacteriophage DNA-dependent RNA polymerase (DdRP) to synthesize BP-modified RNA utilizing NTPαBs as monomeric substrates. Additionally, the α-P-borano modification can be used to probe the catalytic phosphoryl transfer mechanism used by viral polymerases, and possibly enhance existing anti-viral chain terminating nucleotides.

The primary goal of this dissertation is to better understand the effects of NTPαBs on the activity of viral polymerases. In the last decade several NTPαBs have been shown to be efficient and selective substrates for wild-type (wt) and, to a greater extent, HIV and MMLV drug-resistant viral reverse transcriptases. More recently NS5B, the Hepatitis C viral RNA-dependent RNA polymerase (HCV RdRP), is a viable target for nucleotide-based inhibition studies. Due to the similarities between the active sites of HIV-RT and HCV NS5B, it is therefore relevant to investigate the substrate properties of this unique modification. We investigated, for the first time, the inhibition kinetics of HCV NS5BΔ55 RdRP by two newly synthesized NTPαB analogs: 2'-O-methyladenosine 5'-(α-P-borano) triphosphate (2'-OMe ATPαB, 9a) and 3'-deoxyadenosine 5'-(α-P-borano) triphosphate (3'-dATPαB, 9b) and the steady state incorporation kinetics of ATPαB (51a). Our results showed that:

(1) Rp-2'-OMe ATPαB (9a) and Rp-3'-dATPαB (9b) exhibited a 3.5- and 16-fold lower IC50 respectively when compared with natural phosphate controls, suggesting greater inhibitory potency.

(2) Additionally 9a and 9b demonstrated a 5- and 21-fold lower inhibition constant (Ki) respectively when compared with the natural phosphate. Both compounds retained the competitive inhibition behavior of their parent nucleotides.

(3) HCV NS5BΔ55 preferred the Rp isomer of ATPαB (Vmax/Km = 0.095) over the natural ATP substrate (Vmax/Km = 0.057). None of the Sp isomers were substrates for HCV NS5BΔ55. We further concluded that wild-type (wt) HCV NS5B seems to discriminate against 3'-deoxy NTPs via lost interactions between the 3'-OH on the ribose and the active site residues, or lost intramolecular hydrogen bonding interactions between the 3'-OH and the pyrophosphate leaving group during phosphoryl transfer. The overall implications of this proof of concept study are that existing viral RdRP inhibitors could be retro-fitted with the boranophosphate modification to possibly increase potency.

This dissertation also explored the synthesis of anti-HIV-RT boranophosphate nucleotides which act through a chain terminating or mutagenic mechanism. 2'-3'-didehydro-2'-3'-dideoxythymidine 5'-(α-P-borano)-diphosphate (D4TDPαB, 30) was synthesized and later stereoselectively phosphorylated to yield the Rp-form of D4TTPαB (31). This was tested as a substrate in two multi-drug resistant forms of HIV-RT. Additionally, the NTPαB analogue of the mutagenic 5-aza-5,6-dihydro-2'-deoxycytidine (KP-1212-TPαB, 16) was synthesized with the eventual goal of inducing error catastrophe during viral genomic replication.

Lastly we detail the extraction and purification of gemcitabine (dFdC) from Gemzar® drug mixture using a derivatization method that produced a protected form of gemcitabine nucleoside. This protected gemcitabine was then used to synthesize gemcitabine 5'-triphosphate (dFdCTP, 42).

The fracture of polymer networks is usually perceived macroscopically and is considered as a mechanical engineering problem. However, to advance a crack in a polymer network, lots of polymer strands that bridge the crack need to be broken, thus network fracture is molecular as well. In the past 80 years, scientists have been trying to build up quantitative connections between network fracture mechanics and the molecular details of the networks, but to date, there is still no well-accepted quantitative molecular model for network fracture. This is due to the lack of understanding of the molecular details (i.e., strand scission reaction, network topology, etc.) in polymer networks. Developments in polymer mechanochemistry and polymer physics open the door to understanding network fracture from the molecular level. With the concepts of polymer mechanochemistry and polymer physic, this dissertation investigates the correlations between bond/strand scission reaction and the network fracture mechanics theoretically and experimentally.The Lake-Thomas theory is the most well-known molecular theory of network fracture which connects the network critical tearing energy to the scission of polymer strands. Although it has been widely used to explain experimental data, the energy parameter in this theory does not capture the correct chemistry of strand scission and the physics of polymer networks. We provided a conceptual framework to modify the molecular energy parameter in the Lake-Thomas theory by considering the force-coupled reactivity of polymer strand scission reaction (SSR) and network connectivity. First, we consider the strand scission during crack propagation as a mechanochemical reaction of polymer strands, the kinetics of which is dictated by the force on the strands instead of the bond dissociation energy of the repeating monomers. By incorporating the data reported from the single-molecule force spectroscopy experiments, we found the elastic energy stored per bond when typical hydrocarbon polymers break is ca. 60 kJ ∙ mol-1, which is well below the typical carbon-carbon bond dissociation energy (ca. 350 kJ ∙ mol-1). This modification introduced the concept of strand scission reaction into the molecular fracture model of polymer networks and explained the underlying criteria of chain scission. Next, we consider the energy contribution of unbroken strands in the polymer networks during crack propagation. This modification includes not only the energy stored in the breaking network strands (bridging strands) but also the energy stored in the tree-like structure of the strands connecting the bridging strands to the network continuum, which remain intact as the crack propagates. We show that the tearing energy stored in each of the generations of this tree depends non-monotonically on the generation index due to the nonlinear elasticity of the stretched network strands. We further show that the energy required to break a single bridging strand is not necessarily dominated by the energy stored in the bridging strand itself but in the higher generations of the tree. To verify our theoretical modification of the Lake-Thomas theory, we designed and synthesized covalent polymer gels in which the macroscopic fracture “reaction” is controlled by mechanophores embedded within mechanically active network strands. The gels were prepared through the end-linking of azide-terminated tetra-arm PEG (Mn = 5 kDa) with different bis-alkyne linkers under identical conditions, except that the bis-alkyne was varied to include either a cis-diaryl or cis-dialkyl linked cyclobutane mechanophore that acts as a mechanochemical “weak link” through a force-coupled cycloreversion. A control network featuring a bis-alkyne without cyclobutane (non-mechanophore) was also synthesized. The networks show the same small strain elasticity and swelling, but they exhibit tearing energies that span a factor of 8 (3.4, 10.6, and 27.1 J ∙ m-2 for networks with cis-diaryl, cis-dialkyl cyclobutane mechanophores, and non-mechanophore control, respectively). The difference in fracture energy is well-aligned with the force-coupled scission kinetics of the mechanophores observed in single-molecule force spectroscopy (SMFS) experiments, implicating local resonance stabilization of a diradical transition state in the cycloreversion of cis-diaryl cyclobutane mechanophore as a key determinant of the relative ease with which its network is torn. The connection between macroscopic fracture and a small-molecule reaction mechanism suggests that the fracture of polymer networks is a chemical reaction. Further characterizations of the force-coupled kinetics of cis-diaryl and cis-dialkyl cyclobutane mechanophores with SMFS suggest opportunities for constructing quantitative correlations between strand scission reaction and network fracture mechanics. Although the tearing energy of polymer networks is highly dependent on the strand scission reaction of network strands, how the networks with mixed strand scission reactions behave remains unclear. Hence, the impact of mixed strand scission reaction is studied through the synthesis of networks with varied ratios of cis-diaryl cyclobutane mechanophore (“weak”) and non-mechanophore (“strong”) control linkers. The strands with mechanophore linkers are about 4 ~ 5 times weaker than the strands with non-mechanophore linkers according to SMFS experiments. Tearing energy versus strong linkers percentage demonstrate the existence of plateau regions for < 40% strong linker at 2 ~ 3 J∙m-2 and >75% strong linker at ~20 J∙m-2. These regions correspond somewhat closely to the expected Flory-Stockmayer percolation thresholds for a tetra-functional network, pc (strong) = 0.67 and pc (weak) = 0.33. From the classical point of view (e.g., Lake-Thomas theory), the crack propagation is usually assumed to be path-determined instead of reactivity-determined. These data suggest the path-determined mechanism, which predicts that the tearing energy should vary linearly with the average strength of the bonds, does not correctly capture the trend in tearing energy, and the reactivity-determined mechanism is likely to be correct. Ongoing experiments on the actual percolation threshold of either weak or strong chains in the networks would provide more understanding of the reactivity-determined mechanism. Our current work on end-linked networks suggests that the network is weak with incorporated weak mechanophores. However, incorporating weak mechanophores as crosslinkers in radically polymerized networks yields an opposite result: the network is tougher with incorporated weak mechanophores. A cis-diaryl cyclobutane-mechanophore is developed as a mechanochemically weak covalent crosslinker and incorporated into controlled radical-polymerized networks. The networks consist of crosslinkers that are mechanochemical weak and long primary chains that are mechanochemically strong. The activation force of the covalent crosslinker is estimated to be ca. 5 times weaker than the corresponding control crosslinkers and the polymer backbone bonds. However, the networks made from the weak crosslinkers are 2 ~ 9 times tougher than the networks made from the corresponding control crosslinkers (non-mechanophore) while the former exhibit the same small strain elasticity as the latter. By altering the degree of polymerization (DP) of primary chains while keeping the crosslinking density similar, we found that, with primary chains that have DP ≈ 1300 and larger, the networks made from the weak crosslinkers are 6 ~ 9 times tougher than that made from control crosslinkers. With primary chains that have DP ≈ 300, the tearing energies of these two types of networks have no significant difference. This suggests that having long enough primary chains is critical for the toughening effect. The underlying principles of this toughening effect are the weak crosslinkers can be activated before the primary chain breaks, and the inherent topological loopy structures of the network can be released as “stored-length”. Such change in topological structures of the networks after weak crosslinker activation can redistribute the load and toughen the networks.

The  first  chapter  introduces  the  background  about  energy  storage  and  lithium   ion  battery.  The  concepts  of  graphene,  carbon  nanotube,  and  carbon  aerogel  were   covered  as  well.  Then  powder-­based  metal  oxide-­carbon  composite  materials  and   binder-­free  CNT-­metal  oxide  films  for  lithium  storage  applications  were  further   elaborated.  Finally,  the  significance  of  our  research  was  summarized.  

The  second  chapter  is  about  freestanding  and  highly  conductive   Fe3O4/Graphene/CNT  film  as  lithium-­ion  battery  anodes.  Iron  oxide  is  intensively   studied  as  a  lithium-ion  battery  anode  material  due  to  its  high  theoretical  specific   capacity,  but  it  has  low  conductivity  and  poor  cycling  performance.  Herein,  we  present   the  design  of  freestanding  Fe3O4/graphene/Carbon  nanotube  film  via  in-­‐‑situ  growth  by   solvothermal  reaction,  vacuum  filtration  and  annealing  methods.  The  film  had  a  sheet   resistance  of  23  Ω/☐  and  a  BET  surface  area  of  132  m2/g.  The  synergistic  effect  of   graphene  and  CNTs  provide  a  flexible  matrix  to  accommodate  the  volume  change  of   metal  oxide  in  lithium  ion  batteries  application.  This  lightweight  film  was  tested  without   using  a  current  collector,  binder  and  conducting  additives,  eliminating  unnecessary   weight  in  the  overall  devices.  The  film  shows  excellent  cyclic  performances,  and  stable   rate  capability.  The  specific  capacity  retained  803  mAh/g  at  the  rate  of  200  mA/g  after  50   cycles.  This  method  demonstrated  a  promising  path  for  flexible  energy  storage  devices.

The  third  chapter  discusses  facile  synthesis  of  three‑dimensional  TiO2/carbon  co-­aerogel  nanostructures  and  their  applications  for  energy  storage.  In  the  field  of  energy   storage,  it  is  important  to  design  new  materials  and  understand  the  fundamental   principles  of  the  electrode  structure.  Facile  synthesis  of  TiO2/carbon  co-aerogel  material   via  a  sol-­gel  method  was  discussed.  This  new  material  was  composed  of  a  3-­D   interconnected  network  of  TiO2  and  carbon  aerogel.  TEM,  SEM,  XRD,  BET  SA,  and   electrochemistry  measurements  were  discussed.  With  an  operating  voltage  between  0.05   and  3.00  V,  the  discharge  capacity  was  ~400  mAh/g  at  168  mA/g  current  density.