Chemistry & Biochemistry Theses and Dissertations

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    (2023) Lamar, Chelsey; Nie, Zhihong; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The self-assembly of inorganic nanoparticles (NPs) has garnered considerable attention due to the potential for fabricating functional structures with unique collective properties. In recent years, polymers have emerged as valuable candidates in assisting the organization of NPs into complex architectures with multiple capabilities. Researchers have shown that polymer-grafted nanoparticles (PGNPs) facilitate the use of advanced nanostructures with tailored properties in biomedical applications. Although, continued exploration of the rational design and tailoring of PGNP assemblies is needed to expand our understanding before we can fully realize the potential of these structures in desired applications. My dissertation aims to investigate the fundamental aspects and elucidate the underlying mechanisms in the self-assembly of PGNPs for modern biomedical applications. A facile and versatile solution-based strategy was utilized to explore the individual self-assembly of PGNPs with anisotropic NPs and the co-assembly of binary PGNPs with distinct sizes. We focused on designing, characterizing, and exploring the optical properties of hierarchical assembly structures produced from inorganic NPs tethered with amphiphilic block copolymers (BCPs). Individual PNGPs with anisotropic NPs and binary mixtures of small and large PGNPs produce vesicle structures with well-defined packing arrangements. My work shows how key parameters, including polymer chain length, nanoparticle size, and concentration, influence the self-assembly behavior and the formation of vesicles in each system. Through a combination of experimental observations and theoretical considerations, I highlight the significance of polymer shell shape in dictating the self-assembly behavior of individual anisotropic PGNPs. Moreover, I demonstrate that elevated temperatures impacted the stability and optical responses of the vesicle structures. In co-assembly studies, my work describes the macroscopic segregation of PGNPs with different sizes in the vesicular membrane, which is attributed to the conformation entropy gain of the grafted copolymer ligands. This research will provide valuable insights into the self-assembly behavior and fundamental design of PGNP structures relevant to biomedical applications.
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    Photoredox-Active Tertiary N-Oxyammonium Reagents For Selective sp3 C-H Oxidative Functionalization
    (2023) Hitt, Michael James; Vedernikov, Andrei N; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The C(sp3)-H bond functionalization under photoredox catalysis has been a topic of active research over two last decades. Photoredox catalysis involves the use of light to access catalysts’ excited states allowing for facile single electron transfer (SET) with a hydrogen atom transfer (HAT) agent precursor. One prominent HAT agent precursor is quinuclidine (Q), of which the active radical is the electrophilic radical cation (Q●+) able to abstract a hydrogen atom from a C(sp3)-H bond, so allowing for the bond functionalization. Q has been used by researchers since 2015 in the reductive photoredox catalytic C(sp3)-H functionalization (Chapter 1). In this work we focus primarily on the design of novel N-acyloxyquinuclidinium reagents with the goal of the development of new reaction protocols for the selective oxidative C(sp3)-H bond functionalization that utilize our new reagents under photoredox catalysis. In Chapter 2 we present our new catalytic system that allows for the selective oxidative C(sp3)-H trifluoroacetoxylation of donors of 1o, 2o and 3o benzylic C-H bonds using N-trifluoroacetoxyquinuclidinium trifluoroacetate that can be conveniently generated in situ by mixing quinuclidine N-oxide and trifluoroacetic anhydride in DCM solutions. Under photoredox catalyst under blue LED light, this reagent allows for the unique high-yielding (up to >99%) selective oxidative trifluoroacetoxylation of various (functionalized) alkylarenes used as limiting reactants (22 examples overall, including a pharmaceutical-derived substrate). The proposed reaction mechanism involves Q●+ as a highly selective HAT agent and benzylic carbocations resulting from oxidative radical polar crossover of transient benzylic radicals. In Chapter 3 we introduce a series of isolable N-aroyloxyquinuclidinium tetrafluoroborates (Q-Bz) that allow for the preparation of N-alkylimides in a first of its kind Ritter-Mumm type three-component oxidative imidation of donors of benzylic and cycloalkane C(sp3)-H bonds. In this reaction carbonitriles serve as the source of an imide nitrogen atom and a solvent, whereas the third reaction component, Q-Bz, acts as the oxidant, the source of HAT agents and one of two acyl groups of the imide products. All three reaction components can be varied. 33 different N-alkylimides were prepared using (substituted) alkylarenes as limiting reagents, with product yields up to 94%, and cycloalkanes taken in 3-fold excess with respect to the oxidant. The proposed reaction mechanism involves either Q●+ or aroyloxy radicals as HAT agents, depending on the identity of the aroyl group. Chapter 4 discusses the first example of a Balz-Schiemann – type C(sp3)-H fluorination of alkylarenes and cycloalkanes using N-aroyloxyquinuclidinium tetrafluoroborates (Q-Bz), with tetrafluoroborate anion as the source of the fluorine atom of the resulting alkyl fluorides. The proposed reaction mechanism involves either Q●+ or aroyloxy radicals as HAT agents. Chapter 5 discusses the use of N-trifloxypyridinium salts for Minisci type cross dehydrogenative coupling (CDC) of alkane C-H bonds and pyridine C(sp2)-H bonds. Some limitations and possible future development of this chemistry are presented. Finally, Chapter 6 gives a summary of the results of this work and suggests future directions for the advancement of oxidative C(sp3)-H functionalization chemistry using various N-oxyammonium salts as HAT agent precursors, oxidants, and co-reagents.
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    New method for kinetic isotope effect measurements
    (2023) Kljaic, Teodora; Poulin, Myles B; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Kinetic isotope effect (KIE) measurements are a powerful tool to interrogate the microscopic steps in enzyme catalyzed reactions and can provide detailed information about transition state structures. However, the application of KIE measurements to study enzymatic reactions is not widely applied due to the tedious and complex analytical workflows required to measure KIEs with sufficient precision. In this thesis I described the development of a novel competitive KIE measurement method using MALDI-TOF-MS and the investigation of the transition state of glycosyltransferase enzyme BshA from B. subtilis. We developed a method for the direct measurement of competitive KIEs using a whole molecule matrix assisted laser desorption ionization (MALDI) time of flight (TOF) mass spectrometry (MS). This approach enabled quantitative measurements of both relative isotope abundance of an analyte and fractional conversion F in single measurements without the need for purification prior to analysis. The application of this MALDI-TOF MS approach has demonstrated the precision of KIE measurements comparable to those obtained using competitive radioisotope labelling, and NMR based approaches while requiring smaller amounts of stable isotope labelled substrates. Using two chemoenzymatic approaches, we then synthesized 5 substrates for the application of our method to investigate the transition state of BshA: UDP-GlcNAc (3.1), [1''-13C]UDP-GlcNAc (3.2), [2''-13C]UDP-GlcNAc (3.3), [13C6]UDP-GlcNAc (3.4) and [2''-2H]UDP-GlcNAc (3.5). Finally, we have begun to work on the synthesis of [1''-18O]UDP-GlcNAc and describe an approach to prepare this substrate that is currently underway in the lab. Application of the quantitative whole molecule MALDI-TOF MS approach enabled us to determine multiple competitive KIEs for the enzymatic reaction catalyzed by BshA. While previous studies suggested a front-face SNi (DNAN) TS for the conjugation of UDP-GlcNAc and L-malate, our KIE results show that a stepwise mechanism resulting in the formation of a discrete, though likely short lived, oxocarbenium ion intermediate is more likely. Our method be applied to study other glycosyltransferases whose mechanisms still remain to be elucidated and to design TS based inhibitors for enzymes involved in different bacterial infections. Future work on automation of this method would simplify the KIE measurement process and increase reproducibility making the measurement of KIEs for TS analysis a more experimentally accessible technique for the broader enzymology research community.
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    (2023) Myers, Tanner Matthew; Winkler, Wade C; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The synthesis and degradation of RNA is a fundamental and essential process for all life forms. It is imperative that cells utilize multiple mechanisms to modulate the lifetimes of RNA molecules to ultimately control protein synthesis, to maintain homeostasis or adapt to environmental challenges. One mechanism to directly alter the stability or half-life of an RNA is through the direct enzymatic activity of ribonucleases (RNases). Bacterial organisms encode for many different RNases that possess distinct functions in RNA metabolism. It is through the actions of multiple cellular RNases that a long RNA polymer corresponding to an mRNA, rRNA, tRNA, or sRNA can be fully degraded back into nucleotide monophosphate precursors. The nucleoside monophosphate precursors are recognized by kinases that ultimately recycle these molecules for use in the synthesis of other long RNA polymers. The processing of RNA polymers by endo- and exoribonucleases generates nucleoside monophosphates as well as short RNA oligonucleotides ranging from 2-6 nucleotides in length. Previously, a subset of enzymes broadly referred to as “nanoRNases” were found to process these short RNA fragments. Oligoribonuclease (Orn), NanoRNase A (NrnA), NanoRNase B (NrnB), and NanoRNase C (NrnC) were previously ascribed the function of indiscriminately processing nanoRNA substrates. However, recent analyses of the evolutionarily related DnaQ-fold containing proteins Orn and NrnC have provided compelling evidence that some nanoRNase protein families possess distinct dinucleotide substrate length preferences (Kim et al., 2019; Lormand et al., 2021). To determine whether all nanoRNases are diribonucleotide-specific enzymes, we utilized a combination of in vitro and in vivo assays to conclusively elucidate the substrate specificity and intracellular roles of the DHH-DHHA1 family proteins NrnA and NrnB. Through an in vitro biochemical survey of many NrnA and NrnB protein homologs, including from organisms of varying degrees of relatedness, we have determined that there are many functional dissimilarities contained within the DHH-DHHA1 protein family. Furthermore, we have conducted a rigorous investigation into the biological and biochemical functions of NrnA and NrnB in the Gram-positive model organism Bacillus subtilis. These analyses have shown that B. subtilis NrnA and NrnB are not redundant in biochemical activities or intracellular functions, as previously believed. In fact, we have found that B. subtilis NrnA is a 5’-3’ exoribonuclease that degrades short RNAs 2-4 nucleotides in length during vegetative growth, while B. subtilis NrnB is specifically expressed within the developing forespore and functions as a 3’-5’ exoribonuclease that processes short RNA in addition to longer RNA substrates (>40-mers). Our collective data provide a strong basis for the subdivision of the DHH-DHHA1 protein family on the basis on their diverse substrate preferences and intracellular functions.
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    (2023) Lunde, Grace Henry; Wang, Lai-Xi; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Fucosylation is critical for molecular recognition events in the immune system, microbial interactions, and cancer metastasis. Terminal fucosylation (α1,2, α1,3, and α1,4) is characteristic of the histo-blood group antigens (ABO, H, and Lewis) and is expressed on many N- and O-glycans and most human milk oligosaccharides (HMOs). Core fucosylation (α1,6) exists primarily on the sixth position of the core GlcNAc of N-glycans and plays a profound role in modulating the biological functions of IgG antibodies. Due to their diverse biological properties, structurally well-defined fucosylated oligosaccharides and glycoconjugates are highly demanded for detailed structure-function relationship studies and translational applications. Three main approaches exist to meet this end: metabolic engineering, traditional chemical synthesis, and chemoenzymatic synthesis. Metabolic engineering has unmatched scalability but can suffer from limitations in many factors, including the availability of various sugar nucleotides, genetic instability, and inherent microheterogeneity due to the non-template-driven multiple-step assembly of glycans. Traditional chemical synthesis is ideal for producing a diverse array of pure targets due to its flexibility but may require prohibitively tedious multistep protocols. Alternatively, chemoenzymatic synthesis harnesses the precision of traditional chemical synthesis and marries it to the usually regio- and stereo-selective enzymatic transformations. Glycosyltransferases (GTs), the natural enzymes for synthesizing glycosidic bonds, are most widely applied in chemoenzymatic routes yet some glycosidases, which naturally hydrolyze glycosidic bonds, can be used in transglycosylation mode for chemoenzymatic synthesis. Generally, glycosidases can be easier to express, have a more relaxed acceptor substrate specificity, and utilize simpler synthetic donor substrates than GTs. However, glycosidases possess an inherent propensity toward product hydrolysis. This has led to the design of mutant glycosidases, known as glycosynthases and glycoligases, which have diminished hydrolysis and enhanced transglycosylation activity. A glycosynthase is a catalytic nucleophile mutant that inverts the anomeric configuration of the donor substrate (α→β or β→α) in glycosylation and a glycoligase is a mutant at the general acid/base residue that retains the anomeric configuration (α→α or β→β) in its catalytic glycosylation. Both require an activated glycosyl donor substrate with a suitable leaving group at the anomeric position. Readily synthesized glycosyl fluorides are frequently used. Given their superior stability in aqueous conditions, α-glycosyl fluorides are preferred over β-glycosyl fluorides. Therefore, the glycoligase approach is favored in the synthesis of α-glycosidic bonds, like the α-fucosides found in mammalian systems. Our group has successfully applied the glycoligase strategy for robust synthesis of core fucosylated (α1,6) N-glycans and glycoproteins. For my first project, I aimed to expand our fucoligase toolbox and designed an α1,3/4-fucoligase (AfcB E746A) for the synthesis of α1,3 and α1,4-fucosylated oligosaccharides. AfcB E746A very efficiently catalyzed the synthesis of the Lewis X (LeX) and A (LeA) trisaccharides and two HMOs: 3-fucosyllactose (3FL) and lacto-N-fucopentaose (LNFP) III with only slight excess (1.5 eq.) of the αFucF donor substrate. I concluded that AfcB E746A prefers to synthesize α1,3- over α1,4-fucosidic bonds, demonstrates a unique specificity for acceptors with a reducing end GlcNAc over glucose, and seems to require a free terminal Gal in its acceptor substrate. In my second project, I aimed to characterize the enzyme activity of two of our lab’s fucoligases, AfcB E746A and BfFucH E277G, in the synthesis of novel difucosylated tetrasaccharides. I concluded that AfcB E746A requires a free terminal galactose in the acceptor substrate given that AfcB E746A cannot efficiently utilize BfFucH E277G’s monofucosylated products. Alternatively, BfFucH E277G utilizes AfcB E746A’s monofucosylated products. BfFucH E277G synthesized several difucosylated tetrasaccharides, which were characterized by mass spectroscopy and one- and two-dimensional NMR analysis. I concluded that BfFucH E277G catalyzes the synthesis of an α1,3-fucosidic bond on the terminal galactose of 3FL and LeA to yield two novel HMO/Lewis antigen-like structures: 3’3-lactodifucotetraose (LDFT) and 3’-fucosyl-Lewis A (3’-FLeA), respectively. This study further elucidates BfFucH E277G’s unique acceptor substrate driven regioselectivity. In my first two projects, I meticulously characterized the transfucosylation activity of AfcB E746A and BfFucH E277G to provide insight into how these fucoligases may be integrated into synthetic schemes. Enzyme-catalyzed synthesis is an indispensable synthetic strategy given its reliability in substrate specificity and stereo- and regioselectivity. Pivoting from the study of fucoligases, in my third project I prepared highly pure and structurally well-defined IgG antibody glycoforms by chemoenzymatic remodeling with enzymes discovered and designed by our group. The objective of this work was to demonstrate our lab’s expertise in the chemoenzymatic remodeling of the IgG Fc N297 glycan. Furthermore, these antibodies will be applied in future experiments to more confidently characterize the effect of antibody core (α1,6) fucosylation on their binding affinity for Fcγ receptors (FcγRs) and demonstrate how the allosteric effect of immune complex formation contributes to this phenomenon. Core fucosylation of the N297 glycan reduces antibody-dependent cellular cytotoxicity (ADCC) by decreasing the antibody’s affinity for the FcγIIIa receptor. In fact, by removing core fucose, binding can be enhanced up to 50-fold, leading to improved ADCC and enhanced therapeutic efficacy. However, the current data is considerably disparate with enhancements ranging from 3- to 53-fold. These discrepancies may be attributed to glycoform heterogeneity and contamination with afucosylated glycoforms. Additionally, some studies have reported that, in the presence of core fucosylation, sialylation negatively impacts ADCC. This is accompanied by only a modest decrease in FcγRIIIa binding affinity which cannot solely account for the large difference in ADCC. This phenomenon may be explained by conformational allosteric cooperativity where a conformational change in Fab, upon antigen binding, is transmitted to the Fc to alter its affinity for the FcγRs. We hypothesize this phenomenon explains the discrepancy between FcγRIIIa binding affinity and the degree of ADCC activated by the sialylated and core fucosylated IgG antibodies. These detailed studies on the IgG-FcγRIIIa interaction are important for basic research and translational science. Exceedingly pure glycoforms are required for these experiments. My final project demonstrates the Wang group’s exceptional position in the field of basic antibody research and the indispensable nature of our work for the improved therapeutic application of monoclonal antibodies.
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    (2023) Li, Kungang; Lobb, Christopher; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    In this thesis, I discuss the fabrication and measurement of Al/AlOx/Al transmons that have electrodes with different superconducting gaps. With gap-engineering, the tunneling of single quasiparticle from the low-gap side to the high-gap side can be suppressed, hence increasing the relaxation time T1. The best gap-engineered device showed T1 exceeding 300 μs. Large T1 fluctuations in my devices were also observed. I proposed a mechanism for exploring the T1 fluctuation data and discuss the possible underlying cause of the T1 fluctuations. I first discuss the theory of the loss in gap-engineered transmons, with a focus on the loss from non-equilibrium quasiparticles. The model yields the quasiparticle-induced loss in transmons and its dependence on temperature. I also discuss how multiple Andreev reflection (MAR) effects might alter these conclusions, leading to a further reduction in T1. I then describe the design, fabrication and basic characterization of the transmon chip SKD102, which features two transmons – one with thin-film electrodes of pure Al and another that had one electrode made from oxygen-doped Al. I next examined T1 vs temperature and how the T1 fluctuations depended on temperature. I compare my results to a simple model and find reasonable agreement in transmons on chip SKD102, KL103 and KL109, which had different electrode and layer configurations. Finally, I analyze T1 fluctuations in different devices and as a function of temperature and propose a model to explain this behavior. Over the different devices, the T1 fluctuation magnitude roughly scaled as T13/2. With increasing temperature, T1 decreases due to a higher density of thermally generated quasiparticles. In contrast, for an individual device measured from 20mK to 250 mK, the fluctuation magnitude appears to be proportional to T1. I present a model of quasiparticle dissipation channels that reproduces both of these observed scaling relationships.
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    Versatile Strategies for Multifunctional Polyolefins
    (2023) Fischbach, Danyon Miles; Sita, Lawrence R; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Polyolefins have quickly become one of the world’s most utilized products since their discovery in the 1950s. With 350 million tons produced each year, it is clear that the use of polyolefins is not subsiding in the near future. Instead, it is imperative to develop novel materials that are more efficient than their current counterparts. As the function of a plastic is derived from its properties, creating polyolefins with designable and targetable attributes is a major priority. The Sita group has played a huge role in the development of ‘precision’ polyolefins. The techniques employed allow for the scalable synthesis of a plethora of polyolefins. To do this, input variables such as the monomer, tacticity, molar mass, and molar mass distribution are controlled in an organized manner to affect output properties such as crystallinity, elasticity, and tensile strength. The ability to create diverse plastics is necessary for the functions asked of them, however, the missing element in almost all polyolefin synthesis is chemical functionality. The inert nature of polyolefins leads to limited reactivity, therefore, reducing possible chemical reactions, such as recycling. The goal of this work is to increase the scope of functional polyolefins so that new materials with improved properties can be produced. The first step in adding functionality is choosing the proper functional group. A drawback to many polyolefin functionalities currently under study is that they have a very limited scope. Functional groups are designed and used individually, requiring different compounds for each target functionality. To overcome this obstacle, aryl functional groups were targeted in this report. Phenyl functionalities are known for undergoing a range of chemical transformations leading to a wide variety of possible materials. Described in this report, aryl-functionalized polyolefins were synthesized using three different techniques. Each method has been shown to later undergo post-synthetic transformations to yield new functional groups that can either be used as contact points for macromolecular building blocks or as chromophores for optical observation. The single use or combination of these techniques has led to polyolefin-based materials that may in fact lower the barrier for the next-generation of functional polyolefins.
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    Structural and Biophysical Explorations of Protein Degradation Tags
    (2023) Bonn, Steven Michael; Fushman, David; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Ubiquitin is a 76-amino acid, well-folded and highly stable protein that is highly conserved across all eukaryotes. It is a post-translational modifier of other proteins through itsattachment via an isopeptide bond to a substrate lysine sidechain. Multiple ubiquitin units can be stacked to form a polyubiquitin chain, with chain topologies and cellular outcomes varying based on which of ubiquitin’s lysine residues they are attached to. The most common and well-studied outcome of polyubiquitin attachment is degradation by the proteasome, a massive barrel-shaped protease complex responsible for general protein quality control as well as cell cycle progression. Proteasomes have been discovered in archaea and bacteria, and are controlled by the small archaeal modifier protein (SAMP) and the disordered prokaryotic ubiquitin-like protein (Pup), respectively. Recently, a second bacterial proteasome operon was discovered with a new putative signaling protein, ubiquitin bacterial (UBact). Here, the first investigation of the UBact proteasomal operon is presented. Using nuclear magnetic resonance (NMR) spectroscopy and a variety of biophysical techniques, UBact is demonstrated to be disordered in solution and interact with its putative proteasomal receptor. This sets the groundwork for further studies of the UBact system. Additionally, NMR is used to explore the activity and directionality of various deubiquitinase enzymes responsible for breaking down polyubiquitin chains, and for exploring small molecule binding to ubiquitin chains themselves. This likewise provides a groundwork for further studies of the ubiquitin system, whose dysregulation is responsible for many diseases and is an area of intense therapeutic development.
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    Experimental and Theoretical Characterization of Effective Interactions Near 132Sn
    (1987) Stone, Craig A.; Walters, William B.; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, MD)
    Experimental investigations have been undertaken to study the multiplet structure in six nuclei near 132Sn: 132,130Sb, 131,129,127Sb, and 132Te. Experiments were performed using ion beams of mass-separated fission products produced by the TRISTAN mass separator at Brookhaven National Laboratory. Extensive four-detector gamma-gamma coincidences, gamma-multiscaling and conversion-electron data have been collected. Ultralarge shell model calculations were performed using the VLADIMIR shell model code on the Cray/CDC 7600 supercomputer system at Lawrence Livermore National Laboratory. These calculations were designed to look at the performance of the Kallio-Kolltveit and Siemen's g-matiix potentials on the 1-3 quasiparticle nuclides in the gddsh model space. Results show that realistic potentials work well on nuclei near 132Sn but show problems with 129,130Sn and 131Sb which can not be accounted for by core-polarization corrections. Problems are shown to be due to the use of a potential derived with the Scott-Moszkowski separation metl1od. The separation distance was demonstrated to have a weak dependence on the principal quantum number but a strong dependence on the orbital angular momentum. This suggests the Kallio-Kolltveit potential is underestimating the strength of the h11/2 interactions in 129,130Sn and 131Sb.
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    Progress in Nitrogen Vacancy Nuclear Magnetic Resonance Detection
    (2023) Huckestein, Emma Kaye; Walsworth, Ronald L; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful analytic tool of use in the physics, chemistry, and biology disciplines, yet the resource costs to buy, maintain, and use the spectrometer limit the tool's accessibility and the limited sensitivity and spectral resolution limit its application space. In recent years, Nitrogen Vacancy (NV) centers have emerged as an alternative NMR sensor due to their atomic-scale resolution and minimal resource costs. However, NV-NMR similarly suffers from limited sensitivity and spectral resolution due to the technical challenges associated with increasing the applied magnetic field. In this work, the sensitivity of an existing NV-NMR setup is characterized to determine the experimental modifications necessary for measurements at higher magnetic fields (>0.5 T). As a consequence of this characterization, a coplanar waveguide integrated with a microfluidic channel is designed. Finally, metabolomics, particularly spheroids, are reviewed for a potential high-impact NV-NMR application given historically relevant sample concentration sensitivities.
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    (2023) Donahue, Thomas Connor; Wang, Lai-Xi; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Glycosylation is a prevalent post-translational modification referring to the attachment of glycans or sugars to proteins. Glycosylation has significant impacts on a protein’s structure and function, and the glycans themselves can also be recognized by carbohydrate-binding proteins to enact new functions that may improve the efficacy and half-life of protein-based therapeutics. However, the mammalian glycan biosynthesis pathway produces extraordinarily diverse and heterogenous glycans, making glycan function and recognition challenging to characterize. My research focuses on the development of tools to probe glycan function, evaluate protein-glycan interactions, and leverage the known glycan specificities of carbohydrate-binding proteins for therapeutic indications and includes three major research projects. The first project was to evaluate the immunogenicity of natural N-glycans aiming to raise N-glycan specific antibodies. For the purpose, a series of N-glycan-based immunogens were prepared from five common human N-glycan structures and chemically conjugated to a bacteriophage Qβ carrier protein as an adjuvant, and the conjugates were used as immunogens for immunization in mice. Analysis of the immune response revealed that most of the antibodies elicited by all N-glycan conjugates unexpectedly targeted the conserved chitobiose core, giving cross-reactive antibodies. Importantly, terminal sialylation and linker chemistry were found to have significant effects on the titer and specificity of the elicited antibodies. This study outlines significant challenges to raising selective N-glycan-specific antibodies and provides important guidelines for the further optimization of N-glycan-based immunogens towards the development of selective N-glycan-specific monoclonal antibodies as probes for studying glycan functions. The second project was focused on application of catanionic vesicles as synthetic scaffolds for the multivalent display of N-glycans to probe protein-glycan interactions. A general platformwas developed allowing for multivalent display of various N-glycans on catanionic vesicles. It was found that the N-glycan-coated vesicles had high affinities for several plant and mammalian lectins. Furthermore, vesicles were prepared that displayed more than one glycan structure simultaneously. These well-defined vesicles were employed to recapitulate diverse glycan-coated surfaces as mimics of the mammalian glycocalyx and provided valuable insights into lectin recognition of glycan ligands in such complex environments. Indeed, for the vesicles displaying two unique N-glycan structures simultaneously, the presence of an unrelated glycan structure was found to significantly impact lectin binding to its cognate glycan ligand. Thus, N-glycan-coated catanionic vesicles have great potential as tools for characterizing complex protein-glycan interactions and elucidating glycan function. The third project explored the chemoenzymatic method developed by our lab for constructing site-specific antibody-glycan conjugates as next-generation Lysosome Targeting- Antibody Chimeras (LYTACs). These site-specific conjugates were used in evaluating optimal glycan ligands for targeted lysosomal degradation of clinically relevant protein targets. Several natural and synthetic glycan ligands containing terminal galactose or N-acetylgalactosamine (GalNAc) were attached to monoclonal antibodies and evaluated in cell-based binding and protein-degradation assays. Interesting new trends in glycan ligand binding were discovered, and natural triantennary N-glycans were reported for the first time to be effective ligands for lysosomal delivery of target proteins. Antibody conjugates containing synthetic tri-GalNAc or natural triantennary N-glycan ligands were found to significantly degrade extracellular human PCSK9, a well-validated therapeutic target for treating high cholesterol. Additional experiments indicated that targeted degradation of PCSK9 may be a promising new therapeutic strategy for lowering cholesterol, and this strategy could easily be adapted for the targeted degradation of other extracellular disease-associated proteins. In summary, these studies present methodologies for producing diverse glycoconjugates as valuable tools for elucidating glycan function and intervening in disease.
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    (2023) King, David; Isaacs, Lyle D; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Supramolecular containers take advantage of non-covalent interactions to do a variety of tasks with high affinity. In particular, water-soluble containers are able to bind biologically relevant molecules to perform useful and interesting tasks. Chapter 1 introduces the field of supramolecular chemistry is introduced and establishes the ability of cucurbit[n]urils (CB[n]) to bind guests with high affinity. It also establishes the uses of water-soluble supramolecular containers, including new-generation pillar[n]arene sulfate (P[n]AS) hosts, in biologically relevant systems. Chapter 2 expands on previous attempts at finding high-affinity host-guest pairings by showing that triamantane amines and triamantane diamines are able to bind CB[8] with femtomolar dissociation constants. It also shows that these ultratight binding complexes can be found in competition measurements with slightly weaker ternary complexes, thus reducing the number of measurements needed and the error of those measurements. Chapter 3 shows the discriminatory power of P6AS towards various amino acids and amino acid amides, as well as their methylated derivatives. This discriminatory power is further explored by showing P6AS shows discriminatory power towards histone 3 peptide sequences that are methylation on either the lysine or arginine. This system was also modeled computationally to investigate the role of water in binding affinity. Chapter 4 expands on the use of P[n]AS in biologically relevant systems by measuring the binding constants and an assay to detect and differentiate various World Anti-Doping Agency (WADA) banned compounds in PBS. The same assay was then used to create a calibration curve in simulated urine for two compounds. In total, the proof-of-concept assay is able to detect Pseudo down to 31.8 μM concentrations.
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    (2023) He, Calvin Z; Hill, Wendell T.; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The viability of using relativistic Thomson scattering (RTS) as an intensity gauge of intense(I ≳ 2 × 1018 W/cm2) lasers is explored. Theory motivating measurement of the RTS second harmonic spectrum as well as angular distribution as possible diagnostics of the laser intensity are developed and presented. Experiments conducted at the Centro de Láseres Pulsados (CLPU) laser facility in Salamanca, Spain measuring the second harmonic spectrum radiated along E as well as the angular distribution along the E-k plane are described and their results summarized. We conclude that measurements of the second harmonic spectrum provide a means for determining the laser peak intensity via the corresponding Doppler shifted onset wavelength, provided that the intensity is in the range of ∼ 10^18 to ∼ 10^19 W/cm^2. Measurement of the angular distribution along the E-k plane, on the other hand, does not provide features that are useful for indicating laser intensity. Possible paths forward in the study of RTS are discussed.
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    Next-generation Mass Spectrometry With Multi-omics For Discoveries In Cell And Neurodevelopmental Biology
    (2022) Li, Jie; Nemes, Peter; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Understanding tissue formation advances our understanding of the causes of disease and the obtained knowledge can be potentially applied to develop personalized interventions. However, to explore the underlying mechanisms that govern tissue formation, there is a high and unmet need to develop new technologies to characterize different types of biomolecules from early-stage embryonic precursor cells and their descendent cells during development. This dissertation discusses new technological advancements to facilitate multi-omic (proteomic and metabolomic) analysis to explore cell-to-cell differences and uncover mechanisms underlying tissue formation. The work presented herein illustrates the development of in vivo microsampling and single-cell mass spectrometry (MS) to uncover cell heterogeneity among embryonic cells. Additionally, this dissertation work studies the biological role of metabolites in cell fate determination by exploring the mechanisms underlying metabolite-induced cell fate change. Moreover, this work introduces a novel technique called MagCar developed to track and isolate tissue-specific cells at later stages, which enables studying temporal molecular changes to gain new information about tissue formation.
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    (2023) Pawloski, Westley; Fushman, David; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The post-translational modification of proteins with ubiquitin (Ub) can induce a multitude of cellular signaling processes. Ubiquitination involves the attachment of the C-terminus of Ub to lysines on the substrate protein through an isopeptide linkage. This process is facilitated by a multitude of enzymes which work in concert to write and erase these linkages. The power of Ub signaling is that Ub itself can be modified by additional Ub units to generate polyubiquitin chains through any of the seven lysines or N-terminal amine, and each of these attachment points produces polyubiquitin (polyUb) chains with unique orientations of the internal Ub. This allows for K48 polyUb chains to mark a substrate for proteasomal degradation or K63 polyUb chains to trigger DNA repair and maintenance processes. The Ub signaling system is an amalgamation of post-translational modifications, enzymatic activity, and carefully curated protein-protein binding interactions for this small 76 amino acid protein. My work presented in this disseration involves harnessing the power of nuclear magnetic resonance (NMR) experimentation to observe interactions of multiple components of the Ub system with site-specific resolution and selective kinetics. To this end, I have implemented some standard and atypical NMR experiments to observe the potential for carbon dioxide carbamates to modulate the Ub signaling system. I have determined the kinetics of the enzymatic Ub-activating process, and this was extrapolated to understand how ubiquitiun-like proteins, which share a similar fold to Ub, are discriminated from erroneously taking the place of Ub. I have solved the solution structure of an unusual ubiquitin-like domain and explored how it interacts with Ub. Lastly, I will report on the implementation of an unnatural amino acid that is a photosensitive cross-linker and demonstrate that this technology can be used to detect novel ubiquitin-binding proteins.
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    Phase Transitions Affected by Molecular Interconversion
    (2023) Longo, Thomas; Anisimov, Mikhail; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Typically, pure substances may be found with only one gaseous or liquid state, while their solid state may exist in various polymorphic states. The existence of two distinct liquid forms in a single component substance is more unusual since liquids lack the long-range order common to crystals. Yet, the existence of multiple amorphous states in a single component substance, a phenomenon known as "liquid polyamorphism," has been observed or predicted in a wide variety of substances. In contrast to standard phase transitions, it has been suggested that polyamorphic liquid-liquid transitions are caused by the interconversion of molecular or supramolecular states. To investigate this phenomenon, a nonequilibrium thermodynamic model was developed to quantitatively describe the interplay between the dynamics of molecular interconversion and fluid-phase separation. The theory has been compared to a variety of interconverting systems, and has demonstrated a quantitative agreement with the results of Monte Carlo and Molecular Dynamics simulations. In this thesis, it is shown that there are two major effects of molecular interconversion on the thermodynamics and the kinetics of fluid-phase separation: if the system evolves to an equilibrium state, then the growth of one of the alternative phases may result in the destruction of phase coexistence - a phenomenon referred to as "phase amplification." It is demonstrated that depending on the experimental or simulation conditions, either phase separation or phase amplification would be observed. Previous studies of polyamorphic substances report conflicting observations of phase formation, which may be explained by the possibility of phase amplification occurring. Alternatively, if the system evolves to a nonequilibrium steady state, the phase domain growth could be restricted at a mesoscopic length scale. This phenomenon (referred to as "microphase separation") is one of the simplest examples of steady-state dissipative structures, and may be applicable to active matter systems, hydrodynamic instabilities, and bifurcations in chemical reactions, in which the nonequilibrium conditions could be imposed by an external flux of matter or energy.
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    (2023) McDonnell, Shannon Marie; Blough, Neil V; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Chromophoric dissolved organic matter (CDOM) is a large portion of the open ocean dissolved matter pool which contributes largely to ocean color. The composition and distribution of CDOM is essentially controlled by in-situ biological production, terrestrial inputs, photochemical degradation, and microbial consumption. Estuarine environments contain particularly diverse CDOM composition due to their large variety of inputs and shoreline land usage in addition to the mixing of freshwater and salt water. Developing a further understanding of CDOM variation and composition will help develop and improve satellite remote sensing algorithms, help us understand CDOM’s role in the global carbon, nitrogen, and oxygen cycles, and may help to prioritize in-situ sampling for water quality monitoring in areas of concern. The use of inherent optical properties combined with pH titration and chemical reduction with sodium borohydride (NaBH4), helps to probe the molecular composition of CDOM and its spatial variability. Detailed studies of CDOM from the Chesapeake Bay are limited with many studies only investigating the main channel of the Bay and neglecting the various tributaries. Also, there is a lack of studies which specifically probe the molecular composition of the CDOM samples. To address this, an in-depth analysis of the optical properties of CDOM and C18 extracted organic matter (C18-OM) from the Chesapeake Bay, focusing on various inputs, was performed. Chemical reduction with NaBH4 and pH titration were employed to probe the presence of specific functional groups and their contribution to overall optical properties, and how they vary between locations. Spectral slope (S300-700), E2:E3 absorption ratio, fluorescence intensity, and apparent quantum yield of fluorescence (AQY) were used to analyze 170 samples from various tributaries in the Chesapeake Bay. Overall, this study suggested 1) there may be multiple inputs of CDOM within the Chesapeake Bay 2) the Top of the Bay and central channel of the Bay are impacted by the heavy terrestrial input from the Susquehanna River 3) A lack of correlation between phytoplankton fluorescence and CDOM absorption suggest phytoplankton are not an immediate source of CDOM within the Chesapeake Bay and 4) removal of protein and phytoplankton fluorescence after sample filtration indicates these species must exist in aggregates >0.2 µm. Optical analysis combined with pH titration and NaBH4 reduction investigated the variation between 9 C18-OM extracts from various regions in the Chesapeake Bay and a humic material standard Suwannee River Fulvic Acid (SRFA). Additionally, this study investigated the validity of the Charge-Transfer (CT) model using the optical properties of model compounds. This study suggested 1) certain absorbing and emitting species are lost during C18 extraction but extracts are still representative of their CDOM 2) nearly identical optical responses to pH titration and NaBH4 reduction suggest similar chromophore content throughout the Chesapeake and 3) CT interactions leading to long wavelength absorption are more prevalent in Suwannee River Fulvic Acid (SRFA) than they are in the Chesapeake. To compare the molecular and optical properties of the Chesapeake Bay to other locales, these extracts were compared to extracts from the Delaware Bay (DEL), Equatorial Atlantic Ocean (EAO) and North Pacific Ocean (NPO) in addition to reference materials Suwannee River Fulvic Acid (SRFA), Pony Lake Fulvic Acid (PLFA), and Elliott Soil Humic Acid (ESHA). This study showed 1) composition of deprotonatable and reducible chromophores within the Chesapeake and Delaware Bays is nearly identical but different from the oceans 2) despite being estuaries and containing a mixture of fresh and ocean water, CDOM within both Bays looks terrestrially dominated 3) deep ocean extracts from the Atlantic and Pacific exhibit similar optical response to pH titration, NaBH4 reduction, and NaBH4 reduction combined with pH titration suggesting the similarity of deep ocean waters from both ocean basins.
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    (2023) Gutierrez Razo, Sandra Abigail; Fourkas, John T; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The goal of this project is to create thin films to improve resolution for 3-color lithography (3CL). Lithography is a technique that is used to pattern semiconductor chips. The current methods used to manufacture chips use deep and extreme ultraviolet light to create patterns on a photoresist. 3CL is an alternative that creates patterns using easily-accessible visible light instead of dangerous radiation that requires specialized and prohibitively expensive equipment. This work focuses on improving the resolution of the 3CL technique by using thin negative tone acrylate photoresist films. Modern microelectronic devices require semiconductor chips that have individual features less than 100 nm wide and patterns with features that pack closely together. The industry is moving to shorter wavelengths because feature size is directionally proportional to the wavelength of the light used. However, 3CL uses visible light, which has larger wavelengths than the desired feature size. One way to reduce the size of features is to shape and overlap the beams so that not all irradiated areas result in fabricated features. Two beams are used to excite the photoinitiator in the photoresist to initiate radical polymerization in the acrylate monomers. The third beam is used to deactivate the photoinitiator, thus inhibiting polymerization before it can occur. Another requirement for semiconductor chip patterns is high resolution, or closely packed features. To prevent unwanted polymerization between features in 3CL, and thereby increase resolution, initiation and deactivation should occur from different photoinitiator excited states. Therefore, a 3CL photoinitiator should have a long-lived chemically inactive excited state where either deactivation can relax it back down to the ground state, or further excitation can bring it to the chemically active excited state. We examine isopropylthioxanthone (ITX) and its excited states to probe for 3CL behavior. Deactivation limits the feature width, but the deactivated features in the bulk material are taller than their width and collapse. Thin films are employed to correct the aspect ratio and further improve resolution. This project focuses on ITX’s performance as a 3CL photoinitiator, the procedure to produce 40 nm thin films, and how polymerization and deactivation are different in thin film samples compared to the micron-thick bulk samples.
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    (2023) Kramer, Morgan; Vedernikov, Andrei N; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The use of platinum group metals for the activation and functionalization of C-H bonds has been a topic of substantial interest over the past 60 years. Specifically, platinum-based complexes represent a particularly promising avenue due to their ability to form air- and water-stable species that are capable of reacting with some of the most inert C-H bonds within organic substrates. Over the decades of research contributing to this field, platinum complexes have frequently been angled towards fundamental mechanistic analysis of homogeneous C-H bond activation. In turn, the development of homogenous PtII-based catalytic systems has remained underdeveloped for the practical applications in C-H bond functionalization and, in particular, deuteration of complex organic molecules, including pharmaceuticals. The latter direction is now attracting a significant interest by the pharmaceutical industry. In this work the kinetic and thermodynamic selectivity of our new catalyst, a Pt(II) sulfonated CNN-pincer complex 1.5, in the H/D exchange reaction between aromaticsubstrates and wet TFE-d1 was screened across thirty-four aromatic substrates with the catalysts TON up to 300 (Chapter 2). A kinetic preference of 1.5 for electron-rich C-H bonds and substrates was firmly established and a novel scale of Hammett-like σXM constants was introduced to characterize the reactivity of the substrates’ C(sp2)–H bonds in transition-metal-mediated C-H activation. To greatly enhance our PtII catalysts’ useful life, we used their rigid covalent immobilization to mesoporous silica nanoparticles (immobilized complex 3.5). The resulting robust material served as an efficient H/D exchange catalyst utilizing cheaper sources of exchangeable deuterium, AcOD-d4, and D2O, with the catalyst’s TON up to 1600 (Chapter 3). To understand our novel catalyst’s structure – activity relationship, a series of benzene fragment – R-substituted analogs of 1.5 (R = MeO, tBu, iPr, F, Cl, CF3) were synthesized and explored in the H/D exchange of a series of aromatic compounds (Chapter 4). Surprisingly, the complex 4.1-tBu (R = tBu) stood out as a most robust homogeneous catalyst compatible with AcOD-d4 and D2O at 120 oC as deuterium sources that can work under air. Thanks to this finding, the substrates scope for the H/D exchange with AcOD-d4 catalyzed by 4.1-tBu was expanded to include eight pharmaceuticals, some alkenes, with signs of engagement of some C(sp3)-H bond donors. A novel photo-induced (violet light) room temperature H/D exchange catalyzed by 4.1-OMe was discovered with a substantially different substrate selectivity, as compared to the thermal reaction at 80 oC. These observations may provide some important insight into the mechanism of PtII-mediated C-H activation. Finally, Chapter 5 summarizes the results of this work and suggests some future directions for this area of research.
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    Exploring Mechanisms and Predicting Reactivity of Transition Metal-Catalyzed and Photocatalyzed Radical and Polar Organic Transformations
    (2023) Martin, Robert Thompson; Davis, Jeffery; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The creation of protocols to form novel C-C and C-heteroatom bonds has been the primary goal of organic synthesis since its inception. Chemists have long harnessed both radical and polar reactivities, often as complementary paths to construct these bonds to yield more complex molecular architectures. However, compared to the development of synthetic protocols, development of mechanistic models and enriching of mechanistic understanding of many organic reactions has been limited. Computational studies into the mechanisms of organic transformations provide an avenue by which mechanisms of reactions can be better understood and new patterns of reactivity can be predicted. Herein, quantum-mechanical computational methods e.g., density functional theory (DFT) have been employed in the pursuit of understanding the mechanisms of a series of radical and polar reaction schemes. Specifically, DFT calculations were employed to understand the mechanism and origins of selectivity of two nickel(I)-catalyzed olefin functionalizations. These studies demonstrate a catalyst-control scheme by which selectivity can be induced by the steric properties of the catalyst (Chapter 1). Following this work, two photocatalytic transformations which yield difluorinated products were studied thoroughly with computations. First, a synthesis of difluorinated lactone derivatives revealed a long-lived radical intermediate and motivated mechanistic experiments to isolate this radical. Next, a synthesis of difluorinated oxindole derivatives demonstrated the ability of arenethiols to act as photocatalysts (Chapter 2). Then, computations were used to rigorously explore a copper-catalyzed reductive cross-coupling of imine and allenamides. Specifically, computations were employed to explore the mechanism of the transformation and the origins of stereoselectivity and the divergent formation of urea and diamine products (Chapter 3). Finally, two computational investigations into the mechanisms of transformations catalyzed by first-row transition metals are detailed. In particular, a nickel-catalyzed hydroarylation of gem-difluoroalkenes is explored computationally to determine the order of steps in the reaction. In addition, the mechanism of a cobalt(I)-catalyzed allylic substitution is considered to ascertain the nature of the transformation as either radical or polar (Chapter 4). Given the complexity of the mechanisms of these transformations, computational studies provide an alternative route to acquire useful mechanistic understanding that can support or explain observed experiments and suggest further mechanistic experiments that could provide stronger evidence for a given mechanistic proposal.