Theses/Dissertations - Chemistry & Biochemistry

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    Synthesis of boron, antimony, and bismuth diiminopyridine complexes and reactivity with tris(ortho-carboranyl)borane.
    (2023-08) Tidwell, John R., 1996-; Martin, Caleb D.
    Diiminopyridines (DIMPYs) are a unique ligand class capable of multi-site substitution to fine tune coordination. This ligand framework offers multiple binding modes and the potential for stabilization of abnormal oxidation states due to the ligand’s inherent redox activity. The utility of DIMPY metal complexes has garnered a multitude of attention as they are capable of facilitating polymerization reactions, selective hydrogenation, hydroboration, hydrosilylation, transfer hydrogenation, methanol reforming, carbene transfer, insertion chemistry (C-N, N-H, O-H), cyclopropanation, epoxidation, and various other named reactions. More than 50 elements across the periodic table are successfully synthesized bound to the pincer DIMPY ligand. The original work in this dissertation includes generating the first known examples of boron(bidentate), antimony(tridentate), and bismuth(tridentate) complexes. This completes group 13 of the periodic table in regard to complexation, reveals that row 2 elements can in fact bind to the DIMPY ligand, and provides further insight into potential complexes to explore the aforementioned catalytic processes and reactions. Boranes are desirable synthetic tools due to their ability to catalyze transformations, hydroboration reactions, and form frustrated Lewis pairs with increasing interest in regard to their design and stability. Tris(pentafluorophenyl)borane [B(C6F5)3] has been the benchmark Lewis acid and as history shifts toward a green approach, our lab generated a fluorine-free Lewis superacid, tris(ortho-carboranyl)borane. In exploration of its catalytic capabilities, we needed to understand the stability of our newly formed catalyst with E-H functional groups. We observed that with O-H and N-H bearing substrates protodeboronation is observed forming new bis(ortho-carboranyl)borane-based products whereas with S-H, P-H, and B-H substrates, no reaction was observed. This information is invaluable when considering catalyst screening and therefore is disclosed herein.
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    Synthesis and biological evaluation of structurally diverse indole-based analogues as inhibitors of tubulin polymerization and synthesis of drug-linker constructs cleavable by enzymes present in the tumor microenvironment.
    (2023-08) Ren, Wen, 1995-; Pinney, Kevin G.
    A sub-set of inhibitors of tubulin polymerization that bind to the colchicine site demonstrate dual functionality as both potent cytotoxins (antiproliferative agents) and effective vascular disrupting agents (VDAs). Our previous studies led to the discovery of a potent inhibitor of tubulin polymerization and cancer cell growth, OXi8006, and its water-soluble phosphate prodrug salt, OXi8007, which functioned as a promising VDA in mouse models of cancer. To expand our understanding of the relationship between structure and activity, we generated analogues that incorporated indole modifications and separately incorporated a piperazine motif. A number of compounds from these focused libraries showed robust inhibition of tubulin polymerization and potent cytotoxic effects against human cancer cells. The most promising of these molecules were further assessed for their efficacy to function as tumor-selective vascular disrupting agents (VDAs). Antibody-drug conjugates (ADCs) have emerged as a promising therapeutic approach to the treatment of various cancer types. Several small-molecule inhibitors of tubulin polymerization that demonstrate dual-functionality as highly potent antiproliferative agents and VDAs offer promise as next generation payloads. Further selective targeting is provided by betabodies, which are engineered proteins that selectively target accessible phosphatidylserine (PS). Two small-molecule dual-mechanism payloads, KGP18 and KGP156, were synthetically tethered to drug-linker constructs featuring protease-cleavable short peptides. The most promising drug-linker constructs will be conjugated to PS-targeting betabodies in future studies. The potent payloads, KGP18 and KGP156, are capable of inducing tumor damage through two complementary and distinct mechanisms: inducing irreversible damage to tumor-associated vasculature to promote tumor necrosis and inhibiting proliferation in tumor cells. Although PS-targeting betabodies are highly specific for tumors and tumor-associated vasculature, they are not efficiently internalized within cells. As a result, the dual-mechanism payloads are designed to be released from BDCs through cleavage of linkers by extracellular proteases, such as urokinase-type plasminogen activator (uPA) or plasmin, that are upregulated or activated in the tumor microenvironment. We have incorporated plasmin-targeted (D-Ile-L-Phe-L-Lys) and uPA-targeted (Gly-Gly-L-Arg) short peptide sequences within the constructs bearing KGP18 and separately KGP156 payloads.
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    Regulation, replication, and roadblocks : mechanistic and functional insights into E. coli helicase regulation and coupling in a dynamic replisome.
    (May 2023) Behrmann, Megan S., 1994-; Trakselis, Michael A.
    DNA replication is a process fundamental to life, and yet much about the mechanisms and proteins involved remain to be fully understood. Proximal to the replication fork is the helicase enzyme, responsible for separating duplex DNA into single strands. Because of high conservation of replicative enzymes across Domains, the bacterial helicase DnaB shares elements of both structural and functional homology to eukaryotic replicative helicases and is critical for coordination of fork activities. Regulation of the helicase is vital for fast, efficient, and faithful genome replication, but yet the mechanism for regulation of unwinding is still under debate. Mechanisms for helicase regulation that involve interactions with both DNA strands through a steric exclusion and wrapping (SEW) model and conformational shifts between dilated and constricted states have been examined in vitro. However, to better understand the mechanism in an active system and the cellular impact of helicase regulation, targeted genomic dnaB mutations were made using CRISPR-Cas9 to investigate the mechanism and downstream effects of helicase regulation in living organisms. We present data that reveals a dual regulation mechanism involving conformational changes within the hexamer and excluded strand effects and show that eliminating helicase regulation by these mechanisms leads to high genomic stress, mutations, and ultimately cell death. We investigated the downstream chromosomal architecture and confirm that dysregulated unwinding leads to daughter strand gaps that are prone to DNA damage and stimulate mutagenic repair. The homologous recombination protein, RecA, plays a critical role in processing the gaps and breaks left in the wake of rapid, uncontrolled unwinding at the fork. This work explores the genomic impacts of helicase dysregulation at and downstream of the replication fork, expanding current mechanistic understanding and relating it to functional helicase behavior in vivo.
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    Characterization of MCM8/9 in DNA repair.
    (May 2023) McKinzey, David Reno, 1995-; Trakselis, Michael A.
    MCM8 and MCM9 are recent additions to the minichromosomal maintenance family (MCM) of DNA helicases. While not directly implicated in replication, they show both a protective role in aiding the replication fork and assisting in various aspects of DNA repair. The previously uncharacterized C-terminal extension of MCM9 serves vital roles directing the MCM8/9 complex to the nucleus using a bipartite-like nuclear localization sequence (NLS) and promoting interacts with RAD51 through an identified BRC variant (BRCv) motif. Loss of MCM8/9 slows the overall replication speed resulting from more transient fork reversal, lack of protection of the nascent strand, and ultimately double strand breaks. Finally, enzymatic and structural characterization of MCM8/9 reveals a clear affinity for ssDNA containing substrates, stable protection from nucleases, DNA stimulated ATPase activity, and a preferred unwinding orientation. Using cryo-EM, a Walker B mutant version of the MCM8/9 complex shows the overall hexameric structure with the strong density for the C-terminal ATPase domain bound to nucleotide. Altogether this work shows the structure/function activity of the MCM8/9 complex for a more complete understanding of the importance and impact of this complex to maintain genomic integrity during DNA replication.
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    Heterotypic interactions between wild-type and mutant Cu, Zn superoxide dismutase : subunit swapping, metal migration, coaggregation, and cross seeding.
    (May 2023) Dashnaw, Chad M., 1996-; Shaw, Bryan Francis, 1976-
    Mutations in the Cu, Zn superoxide dismutase-1 (SOD1) gene accounts for approximately 10 % of familial amyotrophic lateral sclerosis (fALS). Interestingly, most SOD1 ALS-variants are dominantly inherited so that both homozygous and heterozygous individuals are affected. Furthermore, the dimeric nature of SOD1 allows for subunit exchange between wild-type (WT) and ALS-variant SOD1 creating a mixture of homo and heterodimeric SOD1. Despite this dominant inheritance, the heterotypic interactions between WT SOD1 and ALS-variant SOD1 remains largely unknown. In some cases (e.g., G85R, G93A, A4V), these heterotypic interactions seem to increase (or be required for) SOD1 toxicity and ALS pathogenesis. In this dissertation, I first investigate the heterodimerization of WT SOD1 and ALS-variant SOD1 in mismatched metalation states—where one subunit is metalated and the other is not—utilizing capillary electrophoresis (CE) and mass spectroscopy. Rates and free energies of subunit exchange were determined, and metal exchange was observed demonstrating that mutant or WT 4Zn-SOD1 could transfer Zn2+ to mutant or WT apo-SOD1. In the third chapter, I measure rates of SOD1 coaggregation between WT and five ALS-variants as well as the thermal stability of heterofibrils. Heterofibrils were observed to nucleate and propagate at slower rates compared to the respective homofibrils. Heterofibrils also showed increased thermal stability. Chapters four and five include projects dedicated to utilizing NHS esterStaudinger crosslinkers to measure the charge regulation in two proteins in a crowded environment as well as utilizing the lithophane data format as a tool for universal visualization to address the inaccessibility of STEM for people who are blind respectively.
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    Investigation of electron transfer and effects of post-translational modification in Cu, Zn superoxide dismutase.
    (May 2023) Zhang, Ao Yun, 1991-; Shaw, Bryan Francis, 1976-
    Mutations in the gene expressing the metalloenzyme copper, zinc superoxide dismutase (SOD1) are associated with the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Approximately 20% of SOD1 mutations are responsible for familial ALS and it is still unclear as to the exact mechanism as to how the mutated protein develops a toxic gain of function. The first part of this dissertation explores the electron transfer in SOD1 and how charge regulation of the protein is affected by the movement of an electron. The overall net charge of SOD1 showed no change during electron movement in the copper catalytic site, regardless of the oxidation state of copper, due to the protein experiencing complete charge regulation. The second part investigates how post-translational modification of SOD1, specifically cysteine oxidation, affected the heterodimerization of wild-type (WT) and mutant SOD1. The oxidation of cysteine 111 in WT was found to promote heterodimerization between WT and mutant SOD1, and computational simulations showed cysteine oxidation promoted heterodimerization via long range allosteric perturbations.
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    Total synthesis of (±)-caesalpinnone A and (-)-caesalpinflavan B, progress toward the total synthesis of (+)-alterbrassicicene C and (-)-alterbrassicicene B.
    (December 2022) Sims, Noah J., 1996-; Wood, John L. (John Louis)
    In 2017, caesalpinnone A was isolated alongside three congeners, caesalpinflavans A-C. Caesalpinnone A possesses an unprecedented oxa-bridged ring system and displays modest cytotoxicity against a variety of cancer cell lines including MFC-7. To explore the structural features and the aforementioned bioactivity, a synthesis of these natural products was developed. The latter entails an early stage Barluenga coupling to convergently cross-couple two hindered, functionalized arenes. The derived material was advanced by reduction, protecting group interchange, and a Claisen Schmidt condensation to furnish the remainder of the carbon skeleton. A late-stage allyl deprotection leads to a spontaneous, chemoselective oxa-Michael to complete the synthesis of (±)-caesalpinnone A in 7 steps from known materials. Alterbrassicicene C and B were isolated alongside a number of family members in 2019 from Alternaria brassicola. The alterbrassicicenes belong to the brassicene family, which is part of the even larger fusicoccane terpenoid family. Several investigations of the fusicoccane family have revealed that numerous members possess remarkable biological activity. Structurally, alterbrassicicene C manifests an unprecedented tetracyclic 5/6/6/5 ring system that features three quaternary centers. In developing an asymmetric synthesis of the alterbrassicicene, we designed a reductive enzymatic desymmetrization that effectively delivers a versatile early intermediate. The route employed to advance this latter intermediate features a lithium-halogen exchange and ring closing metathesis to assemble the 5/8/5 parent scaffold in only 7-steps from known materials. A silver promoted, oxiranium-stabilized rearrangement serves to transfer stereochemistry across the 8- membered ring. While this latter chemistry was employed in efforts to complete alterbrassicicene B, the transannular ethereal bridge this rearrangement produces can also serve as a precursor from which one can initiate an oxa-Michael/retro-oxa-Michael cascade to deliver the alterbrassicicene C oxygen/carbon scaffold.
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    Total synthesis of ent-plagiochianin B and development of a metathesis approach to aleutianamine.
    (December 2022) Jackson, Richard K., III, 1991-; Wood, John L. (John Louis)
    Isolated in 2018 from the Chinese liverwort Plagiochila duthiana, plagiochianin B features a novel 6/7/3 tricyclic core. The total synthesis of ent-plagiochianin B was achieved in 9 synthetic steps from a known cycloheptenone and 13 steps overall starting from (+)-3-carene. As anticipated, the inherent stereochemistry found in (+)-3-carene led to production of material that was enantiomeric to the natural product. Key features of the synthesis include the utilization of a 6p-azatriene electrocyclization and the subsequent discovery of a palladium catalyzed oxidative cleavage of a terminal olefin. The strained and heavily bridged pyrroloiminoquinone alkaloid aleutianamine was isolated in 2019 from the North Pacific sponge Latrunculia (Latrunculia) austini Samaai, Kelly & Gibbons, 2006 and exhibits cytotoxicity against a number of cancer cell lines. Noteworthy is aleutianamine’s IC 50 value of 25 nM against pancreatic cancer. Synthetic challenges posed by this natural product include the pyrroloiminoquinone core, an N,S-acetal, vinyl bromide containing 1,3-diene, and an iminium ion. Efforts to provide synthetic access to this biologically interesting alkaloid have been fruitful and several key checkpoints have been reached, including: the development of a scalable Larock-type indole synthesis for construction of the iminoquinone region, a model system study has validated the viability of a tandem metathesis reaction to forge a bromotetrahydrobenzo[b] thiophene moiety, and preparation of an intermediate that contains all necessary carbons for the natural product and is structurally poised for synthesis completion. The challenges, victories, and outlook of this synthetic campaign are herein described.
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    Understanding interactions of glyoxal and acetone at aqueous and ice interfaces using vibrational sum-frequency generation spectroscopy.
    (August 2022) Checinski, Camilla J., 1997-; Cyran, Jenée D.
    Persistent organic pollutants, particularly volatile organic compounds (VOCs) are common contaminants in the environment that undergo long range transport and are introduced into regions where they were not previously found. Glyoxal is well-studied and one of the most abundant oxygenated VOCs in the atmosphere, produced by the oxidation of aromatic hydrocarbons in the atmosphere and through isoprene and terpene reactions. Vibrational Sum-Frequency Generation (SFG) Spectroscopy and Attenuated Total Reflection (ATR) Fourier-Transform Infrared Spectroscopy have been utilized to obtain data of free OH and CH stretch vibrational bonds at the surface and in bulk for glyoxal and acetone in aqueous solution and on ice. Glyoxal can be studied using SFG to gain an understanding of the molecular structure and orientation on the surface of ice and water. Investigating molecular level details of glyoxal on aqueous interfaces can provide information for climate models and the impact of organic pollutants on the environment.
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    Measurement and kinetic analysis of complex ion-molecule reactions influenced by multiple electronic surfaces.
    (August 2022) Lewis, Tucker William Richard, 1994-; Bellert, Darrin Joseph, 1968-
    Energetically and temporally resolved gas phase studies of ion-molecule reactions, particularly those featuring open-shell transition metal ions, are fundamental to understanding complex chemical reactivity paradigms that can help shed light on the behavior of bulk and extended materials. Kinetic studies are particularly revealing as these elucidate mechanistic and dynamic features that would otherwise be impossible to determine. The unique single photon initiated dissociative rearrangement reactions (SPIDRR) technique measures the time dependence of product formation in ion-molecule reactions and is applied here to elucidate the mechanism of several ion-molecule systems. Moreover, the role of the transition metal /metal oxide ion’s electronic structure toward chemical reactivity is determined.
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    Development of a ring expansion approach toward (±)-Phyllantidine, ring expansion of tetramic acids to N-oxy-2,5-diketopiperazines, and total synthesis of (+)-Raistrickindole A.
    (August 2022) Jackson, Amy Catherine, 1995-; Wood, John L. (John Louis)
    In 2020 we reported a concise total synthesis of (±)-phyllantidine, a member of the securinega alkaloid family of natural products, which contains a unique oxazabicyclo[3.3.1]nonane core. Our strategy features a ring expansion of a substituted cyclopentanone to a cyclic hydroxamic acid, which allows facile installation of the embedded nitrogen-oxygen (N-O) bond. Herein we discuss the optimization process of the ring expansion reaction on a series of modified substrates, along with the observed regiochemical outcomes, leading up to the eventual completion of the natural product. Additionally, computational analyses of the mechanistic underpinnings aided us in fine-tuning both the ring expansion system and the reaction conditions, in order to obtain a single regioisomer of the ring expansion product. (±)-Phyllantidine was completed in 12-steps from known materials (9% overall yield), or 14-steps from commercially-available 1,4-cyclohexadiene. N-oxy-2,5-diketopiperazines (DKPs) are prevalent in many biologically-relevant natural products, and current methods of accessing this functionality are typically fraught with difficulty. To overcome this, we describe a ring expansion of tetramic acids (pyrrolidine-2,4-diones) to N-oxy-2,5-DKPs, inspired by a similar 2-step ring expansion strategy used in our total synthesis of (±)-phyllantidine: oxidation of a hydroxamic acid moiety to a acyloxy nitroso, followed by nucleophilic cleavage of the appended acetate to effect carbon-carbon bond migration into the nitroso component giving the ring-expanded product. This method allows for the facile and late-stage construction of the N-O bond and can thereby serve as a general method for accessing N-oxy-2,5-DKP-containing natural products. Herein we develop several model substrates with varying functionality around the tetramic acid ring, and describe the different outcomes regarding regioselectivity and overall yield. We report the successful ring expansion of a Bn-substituted DMB-protected tetramic acid using optimized conditions in the 2-step sequence, with the expectation to apply this chemistry toward the synthesis of natural products. (+)-Raistrickindole A was isolated in 2019 by Li and co-workers and shows modest activity against the hepatitis C virus. (+)-Raistrickindole A is embedded in a tryptophan/phenylalanine-derived 1,2-oxazine-containing tetraheterocycle, and features an N-oxy-2,5-DKP core, posing an attractive synthetic challenge. There are currently no reports to date of this natural product. Our synthetic efforts evolved through the attempted use of our developed ring expansion to synthesize the DKP core of the natural product, and eventually transformed into the successful utilization of a regio- and diastereoselective intermolecular nitroso Diels-Alder (NDA) reaction. We completed the first total synthesis of (+)-raistrickindole A in 9 steps from known materials, and we believe that this method can be used to synthesize other potentially pharmacologically-useful N-oxy-DKP/1,2-oxazine-containing- natural products.
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    Total synthesis of rameswaralide utilizing a pharmacophore directed retrosynthetic approach.
    (August 2022) Truax, Nathanyal J., 1995-; Romo, Daniel.
    A pharmacophore-directed retrosynthesis strategy was applied to rameswaralide which simultaneously allowed the total synthesis of rameswaralide and the identification of simplified analogs containing the common 5,5,6 and 5,5,7 skeleton present in several cembranoid and norcembranoids from Sinularia soft corals. The common cores not only serve as starting points for the synthesis of related natural products, but have also provided intriguing initial structure–activity relationships of intermediates en route to rameswaralide revealing differential and selective cytotoxicity. Our route intersects a versatile ⍺-bromo enone resulting in multiple possible endgame strategies, one of which lead to the first total synthesis of rameswaralide. Key steps include a kinetic resolution Diels–Alder lactonization organocascade delivering the common 5,5,6 core, followed by subsequent ring expansion to a 5,5,7 core serviceable for Stille cross coupling and an intra-molecular pyrrolidine catalyzed Michael addition providing access to rameswaralide.
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    Decontamination of metals from firefighter turnout gear and solubility determination of auranofin.
    (August 2022) Stolpman, Drew, 1995-; Solouki, Touradj, 1963-
    Heavy metals are one of the types of contaminants that firefighters are potentially repeatedly exposed to from prior fires when doffing gear if the gear is not effectively decontaminated. Some heavy metals can be acutely toxic, such as arsenic. Decontamination of gear is essential to reduce and stop continued exposure. Laundering methods can remove heavy metals from firefighter gear and minimize re-exposure. A nitric acid digest was used to extract heavy metals of samples of gear sections before and after laundering with a standard detergent and the Decon7 cleaning solution. Concentrations of arsenic (As), cadmium (Cd), chromium (Cr), lead (Pb), and antimony (Sb) in the acid digests were measured using inductively coupled plasma mass spectrometry (ICP MS). Chapter TWO presents the findings from multiplicate gear samples that showed that, on average, the standard detergent was less effective than Decon7 in removal of the studied metals. Metal-based drugs are increasingly developed for use in cancer treatment regimens. New compounds are typically characterized using mass spectrometry, x-ray crystallography, and nuclear magnetic resonance. A shake flask method uses partitioning to determine the polarity and estimate the solubility in water. However, this estimation is not a measurement of solubility and does not report the solubility of the compound in solutions that efficacy testing is done in, such as cell medium. Thus, dosages may be assigned above the solubility limit. The known metal to drug stoichiometry and concentrations of metal in solutions saturated with the compound can be used to determine the drug solubility in complex solutions, such as cell medium. Chapter THREE reports the solubility of auranofin, a well characterized drug, in water and cell medium. Chapter FIVE reports the solubility of synthesized gold(I) complexes in cell medium.
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    Investigating the chemistry of the 9-borataphenanthrene anion.
    (2021-07-12) Bartholome, Tyler A., 1996-; Martin, Caleb D.
    Boron-containing analogues of unsaturated hydrocarbons exhibit a diverse array of reactivity modes that can be exploited for the synthesis of other more complex boron-containing compounds. Examples of such analogues include boroles, analogous with the cyclopentadienyl cation; boratabenzenes, analogous with benzene; and borataalkenes, analogous with olefins. A dibenzo-fused borole, 9-phenyl-9-borafluorene, was shown to undergo insertion of one or two carbene units upon reaction with trimethylsilyldiazomethane to produce six- or seven-membered nonaromatic boracycles. The six-membered product was found to undergo deprotonation to form the first example of a 9-borataphenanthrene anion, which exhibits structural features characteristic of boratabenzenes and borataalkenes. Its boratabenzene-like character was demonstrated through η6 complexation with chromium(0), while its borataalkene-like character was demonstrated through protonation, C-methylation with iodomethane, B=C hydroboration with pinacolborane, and η2 complexation with gold(I). The B=C bond at the 9- and 10-positions of the 9-borataphenanthrene anion was also shown to undergo a broad range of hydrofunctionalization reactivity, including hydroalkylation, hydroarylation, hydroalkynylation, hydroamination, hydroalkoxylation, and hydration. The central BC5 ring was demonstrated to form η6 complexes with rhodium(I), iridium(I), and iron(II) centers. The wide array of boratabenzene- and borataalkene-like reactivity modes indicates the promising potential of 9-borataphenanthrene anions as reagents for inorganic and organic synthesis.
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    Targeted and non-targeted analysis of pesticides, algal toxins, and human pharmaceuticals in aquatic systems.
    (2021-06-14) Taylor, Raegyn B., 1994-; Chambliss, C. Kevin.
    It is well known that complex, chemical mixtures exist in the environment, particularly aquatic systems. Originally designed to treat illnesses, support crop production, and clean, among other uses, chemicals can enter water bodies and undergo chemical transformations via metabolic pathways or degradation. Additional threats to water quality include toxic metabolites produced by aquatic organisms themselves, such as harmful algal bloom toxins. To better understand the magnitude of environmental contamination, its effects, and treatment, analytical steps ranging from sampling techniques to mass spectral analysis and chemical identification must be optimized. The work presented here investigates environmental effects on passive sampling devices, toxin identification and degradation in harmful algal bloom species, and the temporal and spatial changes in pharmaceutical concentrations present in wastewater during a global pandemic. Findings from these studies improve our knowledge of environmental monitoring techniques and add impetus for developing better, comprehensive screening methods.
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    Synthesis of spongiolactone-based alkynyl proteomic probes, (+)-hypercalin C, and pharmacophore-directed retrosynthesis of ophiobolins.
    (2020-04-15) Tao, Yongfeng, 1989-; Romo, Daniel.
    The spongiolactones are β-lactone containing marine natural products with fused tricyclic β-lactone-containing ring system. We synthesized bis-epi-spongiolactone and its corresponding alkynyl probe. Our collaborators (Prof. Stephan Sieber, Technical University of Munich) discovered that the spongiolactone compounds likely exert their anticancer effects through a polypharmacology mechanism. We completed the first total synthesis of hypercalin C in 10 steps with an overall yield of 8%. The synthetic sequence featured a key Csp3 -Csp2 Suzuki coupling with an uncommon α-bromo enol ether coupling partner and a boronic monoester derived from carvone. In addition, we discovered an unexpected and conceptually novel C-H insertion Suzuki coupling process employing the α-bromo enol ether substrate. Several hypercalin C analogs were accessed and tested against two cancer cell lines for cytotoxicity. Some support for the proton shuttle hypothesis for hypercalin C was gathered through our studies. We completed the first two stages of the Pharmacophore-directed retrosynthesis (PDR) approach towards ophiobolins, and in particular the most potent member, ophiobolin A. In making the bicyclic analogs, we encountered synthetic challenges such as inherent 5-exo-trig cyclization and olefin isomerization, for which we adopted a defensive strategy by the careful refinement of designed intermediates. We developed an efficient cuprate addition desilylation diastereoselective protonation sequence to form the key C1-C2 bond while establishing the crucial C6 stereocenter that might have broader utility. We formed the 8-membered B ring by an RCM reaction. We developed a novel asymmetric allylation reaction for a particularly difficult substrate. We will synthesize the C ring building block and use it for the preparation of tricyclic analogs and some natural ophiobolins with our strategy developed to date for the bicyclic system (AB ring system). Our goal is to achieve the synthesis of certain ophiobolin A derivatives with simplified structures that retain the bioactivity of the natural product.
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    Expanding the reactive sulfur species metabolome : characterization of small oxoacids of sulfur and hydrogen sulfide within in vitro and in vivo models and use of the ionophore thiomaltol to target melanoma in a copper-dependent mechanism.
    (2022-02-03) Scrivner, Ottis, 1986-; Farmer, Patrick Joseph, 1957-
    Hydrogen sulfide (H2S) is an endogenously produced signaling molecule with vast regulatory function in cardiovascular physiology controlling vasodilation, amelioration of hypoxia, and blood pressure. While the enzyme systems responsible for hydrogen sulfide generation are well-characterized, the mechanism of hydrogen sulfide inducing physiological change is poorly understood. This work shows hydrogen sulfide (H2S) and oxoacids of sulfur (SOS) consisting of sulfenic acid (HSOH) and sulfoxylic acid (HOSOH) are generated endogenously in a variety of different cultured cell lines including human cancer and primary cell lines as well as bacterial and yeast cells. The methodology used involves a novel derivatization and LCMS metabolomics methodology, adapted from well-known protein trapping strategies. Steady-state measurements of both endogenous concentrations and efflux concentrations are reported as well as changes seen under hydrogen sulfide producing enzyme inhibition and hypoxia. Finally, the LCMS-derivatization methodology described for in vitro work was translated to measure these analytes in mutant mice models which confirmed the characterizations of these species in vivo. Chapter Four represents the culmination of a decade long project to investigate and characterize the toxicity of the copper ionophore thiomaltol (Htma) against human melanoma. While many strategies have been developed to target melanoma cancer, recent discoveries of copper regulation being correlated to highly upregulated signaling pathways like MAPK have resulted in many ionophore-related strategies to target melanoma in a copper-dependent fashion. Htma/Cu treatment appears to cause copper hyperaccumulation within lysosomes within a few hours with simultaneous proteasome inhibition leading to an apoptotic death response within 24 hours. Altogether, this work encourages further use of the LCMS strategy described to study biological sulfur metabolites in other models as well as the examination of copper trafficking protein networks under stress from copper ionophores.
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    Understanding the singlet oxygen reactivity of salarin C through synthesis of a simplified macrocyclic analogue; multicomponent, enantioselective organocatalytic synthesis of tetrahydropyridazinones.
    (2021-10-27) Jourdain, Roxane M., 1991-; Romo, Daniel.
    Natural products are a valuable source of inspiration for the discovery of new anticancer therapeutic agents. Sponges, especially, produce a wide variety of cytotoxic chemical defense agents. The macrolide salarin C is one of those cytotoxic sponge natural products, isolated from a Madagascar sponge. Of particular interest is the marked reactivity of its oxazole moiety with singlet oxygen that converts salarin C to its biologically inactive congener, salarin A. While this photocatalyzed rearrangement, i.e., Wasserman rearrangement, is known typically requiring UV irradiation and a photosensitizer to generate singlet oxygen, salarin C undergoes this transformation without the need for a photosensitizer. Through the synthesis of a macrocyclic model of salarin C, we determined that the macrocyclic conformation of the oxazole is the cause of the aerobic rearrangement. Secondly, a multicomponent reaction was developed towards the synthesis of tetrahydropyridazinones. Multicomponent reactions are extremely advantageous methodologies as they allow the formation of complex scaffolds from simple starting materials and have shown great utility in the synthesis of diverse libraries of compounds. We have applied our nucleophile-catalyzed acylammonium methodology to the synthesis of tetrahydropyridazinones using α,β-unsaturated acylammonium salts from commercially available acid chlorides, malonates and azodicarboxylates. While attempting the oxidation of the N-N bond of the derived tetrahydropyridazinone, an unusual rearrangement to a ketene acetal lactone was also observed. This methodology could be used to construct novel nitrogen-containing frameworks found in pharmaceuticals. Finally, outreach and education efforts were undertaken during this degree, notably the creation of two resources for nuclear magnetic resonance analysis, and the creation of posters, images, films, and 3D printed models for chemistry outreach during the Baylor Give Light campaign. Guidelines for creating similar illustrations and models are outlined.
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    Unveiling the orientation, conformations, and contacts of replicative hexameric DNA helicases that govern DNA replication dynamics.
    (2021-11-10) Perera, Himasha M., 1990-; Trakselis, Michael A.
    Replication of chromosomal DNA is an intricate process carefully orchestrated in all living organisms by a multiprotein molecular machine known as the replisome. The replicative DNA helicase motor protein is at the forefront of the replisome performing a vital process in DNA replication by unwinding duplex DNA at the replication fork. As a stable component that serves as the platform for the assembly of other replisome proteins, significant efforts have been made over the past several decades to characterize functional properties of this enzyme using model organisms. This collection of work describes our contributions to unveil the orientation, conformations, and contacts of replicative hexameric DNA helicases from two different model organisms, archaeal Saccharolobus solfataricus (Sso) and bacterial Escherichia coli (E. coli). DNA replication is controlled by helicase loading at replication origins, preferentially encircling one DNA strand prior to unwinding in a defined orientation. Using archaeal SsoMCM helicase we discovered that the active unwinding orientation of SsoMCM helicase places its N-terminal domain at the DNA replication fork. This work also explores how an interdisciplinary combination of biochemical, chemical, structural biology, and kinetic methods can provide greater insight into more thoroughly understanding active unwinding orientation of helicases in other organisms. DNA replication requires tight coordination between DNA unwinding and synthesis in the replisome. In E. coli replisome, this coordination is maintained by the interaction between E. coli DnaB helicase and Polymerase III holoenzyme (HE) that performs unwinding and synthesis respectively. Using site specific mutations in the DnaB helicase, we discovered specific helicase contacts that accelerates unwinding and decouple the replisome leading to impaired DNA synthesis. These results set the platform for future investigations of specific molecular contacts and kinetic control within the DnaB helicase that leads to replisome decoupling. Altogether, this work contributes to the fascination of understanding the role of replicative DNA helicases in DNA replication in detail which will undoubtedly be valuable in designing targeted therapeutic approaches for various diseases including cancer.
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    Design and activation of small organic molecules for localized immunosuppression.
    (2021-08-27) Plunk, Michael A., 1992-; Kane, Robert R.
    Since the implementation of the Edmonton Protocol in the early 2000s, many efforts have been made to further improve the long-term viability of transplanted islets in recipients with type 1 diabetes mellitus. These efforts have included suppression of both the innate and adaptive immune responses as well as the masking of pancreatic islets via encapsulation technologies. This dissertation details the synthesis and characterization of various prodrugs of small molecules designed to locally inhibit the immune system – either the innate or the adaptive response. Following successful synthesis and characterization, the prodrugs were then investigated for catalyzed activation using either exogenous nitroreductase (from e. coli) or palladium resins in aqueous buffer. These molecules were also looked at for biological relevance through the use of various in vitro studies. The experiments performed on the final prodrugs successfully demonstrated the safety and effectiveness of these prodrug strategies in vitro and have generated interest in further consideration for in vivo application.