Theses and Dissertations at Montana State University (MSU)
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Item Illuminating dynamic phenomena within organic microstructures with time resolved broadband microscopies(Montana State University - Bozeman, College of Letters & Science, 2024) Hollinbeck, Skyler Robert; Chairperson, Graduate Committee: Erik Grumstrup; This is a manuscript style paper that includes co-authored chapters.Materials derived from organic chromophore subunits are currently at the forefront of academic and industrial interest. This strong interest is driven in part by the tunability of their extant properties through engineering of both the intra-molecular and inter-molecular structure. The structure of organic materials affects optoelectronic properties because organic chromophores are sensitive to dipole-dipole and charge-transfer coupling interactions. This sensitivity presents both opportunities for tuning functional properties through designing specific packing geometries, and liabilities arising from the disruptive effects of structural disorder. Many organic materials are built from weak noncovalent interactions between chromophores, leading solid-state deposition, and crystallization to be susceptible to microscopic variations in environmental conditions. Structural heterogeneity is regularly intrinsic to organic materials, and even self-assembled systems of covalently linked chromophores exhibit defects. Ergo, in order to disentangle the effects of structural heterogeneity from the inherent properties of the material, the study of organic materials must be anchored with techniques that are capable of correlating optoelectronic properties and excited state evolution with microscale morphological characteristics. We have employed broadband pump-probe microscopies, in conjunction with steady-state and time resolved fluorescence techniques, to examine the effects of structure and coupling on excited state dynamics in solid-state organic microstructures. The study of perylene diimide (PDI) materials revealed that kinetically trapping PDI (KT-PDI) enhanced long-range ordering and led to distinct excited state evolution, delocalized charge-transfer states and long-lived charge separated species. In the MOF PCN-222, excitation energy dependent excited state behavior was observed. Pumping the first excited state (Q-band) led to immobile excited states that were relatively unaffected by local defect densities, whereas pumping the second excited state (Soret-band) led to mobile subdiffusive excited state species whose lifetimes are significantly impacted by morphologically correlated defect sites. Finally, we present progress made toward the construction and utilization of a frequency modulated-femtosecond stimulated Raman microscope, yielding spectra that resolve the location of photoinduced anion formation in KT-PDI. The work presented herein highlights broadband time-resolved microscopy as a potent tool for studying the structure-function relationship and photophysical behavior in molecular solids, deepening our understanding of how structural characteristics influence excited state evolution.Item Synthetic and mechanistic strategies to achieve unconventional site-selectivity in cross-couplings of dihalo-heteroarenes(Montana State University - Bozeman, College of Letters & Science, 2024) Norman, Jacob Patrick; Chairperson, Graduate Committee: Sharon Neufeldt; This is a manuscript style paper that includes co-authored chapters.Pd-catalyzed cross-couplings rank among the most powerful methods for constructing substituted biaryls, polyaryls, and heteroarenes. Frequently, di- or polyhalogenated (hetero)arenes are employed as starting materials in cross-couplings to access products with increased structural complexity via multiple cross-coupling or substitution steps. N-heteroarenes bearing multiple reactive handles--such as halides, are of particular interest as starting materials since their cross- coupled products can be medicinally relevant. Non-symmetrical dihalogenated N-heteroarenes typically exhibit a site-selectivity bias for C-X bonds which are adjacent to at least one heteroatom in Pd-catalyzed cross-couplings. However, some Pd catalysts--particularly those with hindered ligands, promote atypical selectivity at distal C-X bonds of 2,X-dichloropyridines and related heterocycles during the selectivity-determining oxidative addition step. This dissertation explores the mechanistic origins of these ligand trends and emphasizes the critical importance of Pd's ligation state--either mono (PdL) or bis (PdL 2), in controlling the site of oxidative addition. Ligation state is also relevant when selecting for the products of mono- vs difunctionalization in cross-couplings of dihalogenated substrates, since bisligated 14 e - Pd dissociates quickly from the monofunctionalized intermediate after an initial cross-coupling cycle, whereas monoligated 12 e - Pd is slow to dissociate and may "ring-walk" to the remaining reactive site(s). Additionally, this dissertation explores alternative methods to access minor regioisomers in cross-couplings of dichloro-azines. One approach involves ligand-free conditions where atypical site-selectivity at dichloropyridines and dichloropyrimidines arises from a change in Pd's speciation from mono- to multinuclearity. Another approach employs a thiolation/Liebeskind-Srogl arylation sequence to achieve site-selectivity which is orthogonal to that of Suzuki-Miyaura couplings.Item Investigating the metalloproteome of bacteria and archaea(Montana State University - Bozeman, College of Letters & Science, 2024) Larson, James Daniel; Chairperson, Graduate Committee: Brian Bothner; This is a manuscript style paper that includes co-authored chapters.Metalloproteins are proteins that rely on a bound metal for activity and comprise 30-50% of all proteins which are responsible for catalyzing imperative biological functions. Understanding the interplay between essential and toxic metals in the environment and the metalloproteins from an organism (metalloproteome) is important for a fundamental understanding of biology. A challenge in studying the metalloproteome is that standard proteomic methods disrupt protein-metal interactions, therefore losing information about protein- metal bonds required for metalloprotein function. One of the focuses of my work has been to develop a non-denaturing chromatographic technique that maintains these non-covalent interactions. My approach for investigating the native metalloproteome together with leading- edge mass spectrometry methods was used to characterize microbial responses to evolutionarily relevant environmental perturbations. Arsenic is a pervasive environmental carcinogen in which microorganisms have naturally evolved detoxification mechanisms. Using Escherichia coli strains containing or lacking the arsRBC arsenic detoxification locus, my research demonstrated that exposure to arsenic causes dramatic changes to the distribution of iron, copper, and magnesium. In addition, the native arsRBC operon regulates metal distribution beyond arsenic. Two specific stress responses are described. The first relies on ArsR and leads to differential regulation of TCA-cycle metalloenzymes. The second response is triggered independently of ArsR and increases expression of molybdenum cofactor and ISC [Fe-S] cluster biosynthetic enzymes. This work provides new insights into the metalloprotein response to arsenic and the regulatory role of ArsR and challenges the current understanding of [Fe-S] cluster biosynthesis during stress. Iron is an essential and plentiful metal, yet the most abundant iron mineral on Earth, pyrite (FeS2), was thought to be unavailable to anaerobic microorganisms. It has recently been shown that methanogenic archaea can meet their iron (and sulfur) demands solely from FeS2. This dissertation shows that Methanosarcina barkeri employs different metabolic strategies when grown under FeS2 or Fe(II) and HS- as the sole source of iron and sulfur which changes the native metalloproteome, metalloprotein complex stoichiometry, and [Fe-S] cluster and cysteine biosynthesis strategies. This work advances our understanding of primordial biology and the different mechanisms of iron and sulfur acquisition dictated by environmental sources of iron and sulfur.Item Characterization of osteoarthritis metabolism: a mass spectrometry based-approach(Montana State University - Bozeman, College of Letters & Science, 2024) Welhaven, Hope Diane Aloha; Co-chairs, Graduate Committee: Brian Bothner and Ronald K. June II; This is a manuscript style paper that includes co-authored chapters.Osteoarthritis (OA) effects 7% of the global population, equating to more than 500 million people worldwide, and is the leading cause of disability. Its multifaceted etiology includes risk factors ranging from genetics, to aging, obesity, sex, race, and joint injury. OA manifests differently across the patient population where symptom severity, rate of progression, response to treatment, pain perception, as well as others vary person to person posing significant challenges for effective management and prevention. At the cellular level, imbalanced matrix catabolism and anabolism contribute to the breakdown of cartilage, underlying bone, and other tissues affected by OA. Leveraging mass spectrometry-based techniques, particularly metabolomics, offers a promising avenue to dissect OA metabolism across musculoskeletal tissues, while considering individual patient-specific risk factors. Therefore, the goals of this research were to: (1) comprehensively characterize OA phenotypes and endotypes and (2) explore OA pathogenesis within the context of disease-associated risk factors. The first area of research focuses on profiling OA phenotypes and endotypes across disease development. These results provide clear evidence of OA-induced metabolic perturbations in OA cartilage and bone and elucidate mechanisms that shift as disease progresses. Several metabolites and pathways associated with lipid, amino acid, matrix, and vitamin metabolism were differentially regulated between healthy and OA tissues and within OA endotypes. The second area of research focuses on the impact of OA risk factors -- sex, injury, obesity, loading -- on the metabolism of circulatory fluids (i.e., serum, synovial fluid) and chondrocytes. Identification of metabolic indicators of disease, such as cervonyl carnitine, and metabolic pathways associated with these risk factors holds potential for improving screening, monitoring disease progression, and guiding preventative strategies. Overall, this work contributes to our current understanding of OA, its diverse metabolic landscape, risk factors and their interactions. Moreover, it lays the groundwork for personalized medicine by providing detailed insights into individualized phenotypic profiles, thereby advancing the prospect of tailored treatment strategies for OA individuals.Item The synthesis and characterization of fluorescently labeled, lactose-functionalized poly(amidoamine) (PAMAM) dendrimers(Montana State University - Bozeman, College of Letters & Science, 2024) Frometa, Magalee Rose; Chairperson, Graduate Committee: Mary J. CloningerCellular uptake of lactose-functionalized poly(amidoamine) dendrimers (PAMAM) has yet to be fully understood and deeply studied. Before sufficient cellular uptake studies can be made, optimization of the synthesis of the lactoside, and the coupling and purification of dye-tagged lactose-functionalized PAMAM had to be completed, as reported here. The synthesis of the requisite lactoside derivative for dendrimer functionalization was optimized. The coupling of the dye, Alexa Fluor 647, to the lactoside-functionalized PAMAM was performed in the presence of a sodium acetate buffer and utilized size separation methods to ensure purity. The structures of the lactoside derivatives and of lactose functionalized PAMAMs were confirmed via nuclear magnetic resonance (NMR) spectroscopy. The purity and degree of labeling (DOL) of the dye labeled, lactose-functionalized PAMAMs were determined with UV-vis. Results show high success of yield and purity resulting from the optimized procedure described in this study.Item Structural characterization of the Csa3/cA4 complex - a nexus for class 1 CRISPR-Cas immune response coordination & establishing a cure for highly efficient galectin expression(Montana State University - Bozeman, College of Letters & Science, 2024) Charbonneau, Alexander Anthony; Chairperson, Graduate Committee: C. Martin Lawrence; This is a manuscript style paper that includes co-authored chapters.Though Class I CRISPR-Cas systems, primarily Type I and Type III, are the most abundant CRISPR systems in archaea and bacteria, mechanisms driving their immune response regulation are not well understood. Csa3 family transcription factors, composed of N-terminal CARF and C-terminal winged helix-turn-helix domains, are frequently encoded within Type I CRISPR-Cas systems. Csa3 transcription factors are hypothesized to bind cyclic oligoadenylate (cOA) second messengers produced by Type III interference complexes, likely modulating their DNA-binding activity. Therefore, we investigated the interaction between Csa3a and cyclic tetra-adenylate (cA4). Isothermal titration microcalorimetry showed S. solfataricus Csa3a binds cA4 at biologically relevant concentrations in an entropically driven interaction. Ring nuclease assays revealed Csa3a lacks self-regulatory phosphodiesterase activity exhibited by other CARF domain proteins. We crystallized and solved the structure of the Csa3/cA4 complex, which revealed conserved motifs are responsible for cA4 binding and illuminated significant conformational changes induced by the interaction. We also identified an 18-bp palindromic motif, which we designated CAPPa, that is conserved in the 27 sequenced members of the order Sulfolobales, and shows synteny with Csa3a and acquisition genes in these genomes. We found Csa3a binds CAPPa in a nonspecific, cooperative, and cA4-independent manner. These characteristics suggest a more complex method of transcriptional regulation than previously hypothesized. However, the interaction between Csa3a and cA4 confirmed here signifies a nexus between Type I and Type III systems; we thus propose a model in which this interaction coordinates the two arms of an integrated immune system to mount a synergistic, highly orchestrated, adaptive immune response. We applied the workflow designed to produce significant protein quantities for crystallographic studies of Csa3a to the study of Homo sapiens galectin proteins, a family of beta-galactoside-binding proteins. Here, we identified a putative autoinhibitory mechanism affecting traditional IPTG-induction methods by characterizing IPTG-binding capabilities of galectins and quantifying basal protein expression over various IPTG concentrations. To bypass this predicted feedback loop, we employed a highly efficient and approachable autoinduction method, resulting in a 7-fold increase in protein expression. Much of this work was done in the context of a course-based undergraduate research experience with great success.Item Elucidating the impacts of structural heterogeneity on excited state dynamics in solution-processed materials(Montana State University - Bozeman, College of Letters & Science, 2024) Afrin, Sajia; Chairperson, Graduate Committee: Erik Grumstrup; This is a manuscript style paper that includes co-authored chapters.Solution-processed inorganic and organic semiconductors hold enormous promises due to their low manufacturing cost, scalability, and compatibility with flexible substrates. However, solution processing techniques do not require control over crystal growth, which can lead to structural defects within the crystal structure. The defects within solution-processed semiconductors can create significant challenges in optimizing device functionality; therefore, it is crucial to understand the impact of structural defects on photophysical properties. Traditional ensemble measurement techniques can conceal the effects of microscale structural defects on functional properties in the structure-averaged observation of solution-processed materials. The work presented in this dissertation employs time-resolved and spectrally resolved microscopy techniques to investigate the influence of structural heterogeneity on the photophysical properties of microscale solution-processed materials. Measurements collected across multiple discrete and highly crystalline domains of multiple classes of solution-processed materials have helped establish a relationship between the functionality and the local structure of these materials. Initially, the focus was on elucidating anisotropic carrier transport in lead halide perovskites by investigating lattice strain and energetic distribution in microcrystals. Later, the focus shifted towards characterizing and understanding the impact of structural defects on the excited state dynamics in another class of solution-processed material called metal-organic frameworks (MOFs). PCN-222 exhibited rapid exciton transport with time-averaged diffusion coefficients ranging from 0.27 to 1.0 cm2/s and subdiffusive behavior, showing transport slowing on the tens of ps time scale. Subdiffusivity indicated that excited states were rapidly transported through the porphyrin network of PCN-222 before being trapped. Moreover, the first transport measurements and transient absorption microscopic measurements in PCN-222 are reported here. Photoluminescence quenching and heterogeneous relaxation pathways were noted in regions with higher structural heterogeneity. Furthermore, the spectral evolution of porphyrinic PCN-222 MOF was investigated, which revealed excitation-dependent chromophore coupling in the MOF structure. Soret band excitation with enhanced coupling can create more mobile excited states, whereas Q band excitation with reduced coupling will generate fewer mobile excited states. Excitation-dependent chromophore coupling strongly dictates the transport and relaxation properties in MOF microstructures that also illustrate the impact of structural defects on the excited state transport and relaxation dynamics. A significant spectral shift has also been observed in microrods stemming from structural heterogeneity. These findings contribute to a deeper understanding of the impact of structural defects on the photophysical properties of solution-processed materials, facilitating the development of optimized semiconductor devices for various applications. The results reported in this dissertation will not only continue to aid in the characterization of MOFs but will also advance our understanding of excited state dynamics in a variety of solution-processed materials.Item Stereoselective allylic cyclizations and rearrangements(Montana State University - Bozeman, College of Letters & Science, 2022) Stankevich, Ksenia Sergeyevna; Chairperson, Graduate Committee: Sharon NeufeldtHerein, we aim to explore the unique reactivity of allyl groups in two different areas: synthesis of densely functionalized five-membered ring systems and mechanistic studies of Pd- catalyzed formation of complex quaternary nitriles. The first part addresses the paucity of methods available for the formation of highly substituted five-membered rings, which are a common motif in natural compounds and pharmaceuticals. We developed a method that provides access to cyclopentenols and methylene cyclopentenols via the union of the Claisen rearrangement and Sakurai allylation. In this instance, the Claisen rearrangement allows for the stereospecific generation of the carbon framework, whereas the intramolecular Sakurai allylation provides a stereoselective cyclization reaction. For 1,2,5-trisubstituted cyclopenten-1-ols this approach has proven to be highly general and stereoselective, furnishing a library of cyclized products in good and very good yields and >20:1 diastereomeric ratio. For 1,2,5-trisubstituted 3- methylene cyclopentan-1-ols, we have developed a stereodivergent method whereby the one-pot stepwise Claisen-Sakurai reaction provided anti-, anti- product and the cascade Claisen-Sakurai reaction furnished syn-, anti- product as a major diastereomer with good yield. In both cases reaction mechanism was investigated to uncover the origin of diastereoselectivity using density functional theory. The second part of this research covers investigating the mechanism of a Pd- catalyzed double rearrangement to form quaternary nitriles, which are molecules of synthetic interest. We studied the mechanism of recently developed highly complex auto-tandem catalytic double allylic rearrangement of N-alloc-N-allyl ynamides to complex quaternary nitriles using density functional theory. This reaction proceeds through two separate and distinct catalytic cycles with both decarboxylative Pd-pi-allyl and Pd(0)-promoted aza-Claisen rearrangements occurring. We discovered previously unreported concomitant decarboxylation/C-C bond formation, reversible C-N ionization and a Pd(0) catalyzed [3,3]-rearrangement along with its stepwise variant. These catalytic cycles are characterized by the highly dynamic nature of the catalyst systems with large degrees of conformational flexibility and a flat potential energy surface. Our studies have rationalized the reactivity observed and can be further developed into predictive models for ligand and catalyst screening.Item A computational study of a high-spin iron(I) complex for possible dinitrogen reduction to ammonia(Montana State University - Bozeman, College of Letters & Science, 2023) Pollock, Charlie Jeananne; Chairperson, Graduate Committee: Martin A. MosqueraA series of high-spin, low coordinate, paramagnetic iron complexes bearing a phenyltris((tert- butylthio)methyl)borate ligand were computationally modeled with density functional theory (DFT) and complete active space self-consistent field theory (CASSCF). The iron complexes examined in this research were inspired by nitrogenase, a naturally occurring, dinitrogen- fixating, iron-containing metalloenzyme. DFT and CASSCF offer a convenient way to explore reactions, complexes, and molecular orbitals without an immediate need to perform synthetic experiments. Our computational work can be used to guide synthetic efforts as well as urge future theoretical work in related research. DFT was utilized to compute two different thermodynamic properties: bond dissociation free energy (BDFE N-H) and Gibb's free energy. The conductor-like polarizable continuum model (CPCM) was applied to examine the solution phase of the system, and all BDFE and DeltaG values found were endothermic in tetrahydrofuran (THF). The methods, BP86 and BP86 ZORA, examined the gas phase of the system. The BDFE and DeltaG values calculated when using those two methods were largely inconsistent, which lead to the conclusion that the solution phase model is the most appropriate method for computing values of the dinitrogen complex ([Fe] 2(Mu-N 2)) and its related complexes. An N 2 vibrational mode was found (1915.30 cm -1) for [Fe] 2(Mu-N 2), which reflects a strongly coordinated dinitrogen bridge (Fe-N identical to N-Fe). Broken symmetry DFT (BSDFT) was used to examine the exchange coupling, which was found to have positive values (JAB =82.51 cm -1, 61.88 cm -1, 81.36 cm -1), and implied that [Fe] 2(Mu-N 2) is ferromagnetically coupled. Lastly, CASSCF and DFT were applied to plot and characterize certain molecular orbitals of [Fe] 2(Mu-N 2). The plotted and characterized molecular orbitals reflected moderate (DFT) to strong (CASSCF) covalent bonding between iron and dinitrogen. All this data reflected the synthetic plausibility of dinitrogen coordination to the bridged, Fe(I) complex ([Fe] 2(Mu-N 2)) that can be reduced through the dinitrogen cleavage mechanism.Item The expansion and optimization of ZN(II)-mediated intramolecular metalloamination and subsequent CU(I)-catalyzed functionalization for the construction of pyrrolidines and piperidines(Montana State University - Bozeman, College of Letters & Science, 2023) Frabitore, Christian Ames; Chairperson, Graduate Committee: Thomas S. Livinghouse; This is a manuscript style paper that includes co-authored chapters.Nitrogen-containing heterocycles (azacycles) are ubiquitous in pharmaceutical agents. Their ability to moderate and modulate the activity of drugs in the body make them especially powerful, and thus sought after, synthetic targets. While the synthesis of many popular azacycles has been greatly improved in recent years, the production of pyrrolidines and piperidines has not received as much attention despite their standing as the 1st and 5th most common azacycles in FDA-approved drugs. The intramolecular Zn(II)-mediated metalloamination/cyclization of N,Ndimethylhydrazinoalkenes provides structurally diverse pyrrolidines and piperidines with the added advantage of a subsequent functionalization step, efficiently building molecular complexity in one reaction sequence. Herein, this method is optimized and improved by the addition of a new hydrazone reduction method, the inclusion of 1-bromoalkynes in the functionalization step, and multiple key discoveries in the reagents used to effect these transformations. Furthermore, preliminary results adding N,N-dimethylhydrazinoallenes as substrates for this powerful method are presented.