Chemistry & Biochemistry Doctoral Work

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4-Dimethylamino Pyridine (DMAP) Catalyst with Fluxional Chirality: Synthesis and Applications
(North Dakota State University, 2016) Ma, Gaoyuan
Organocatalysis using small organic molecules to catalyze organic transformations, has emerged as a powerful synthetic tool that is complementary to metal-catalyzed transformations and remarkably promote stereoselective synthesis. Our group has designed useful templates, ligands, and additives that use fluxional groups to control and/or enhance stereoselectivity in a variety of asymmetric transformations. A key feature of this strategy is that the size of the fluxional substituent can be varied readily. As an extension of this strategy we became interested in developing efficient and broadly applicable and adjustable 4-dimethylaminopyridine (DMAP) organocatalysts. In our design, we surmised that a fluxional group would be effective in relaying stereochemical information from the fixed chiral center to the catalytic center of DMAP. Presented herein the synthesis of novel fluxionally chiral DMAP catalysts and their application in the acylative kinetic resolution of secondary alcohols and axially chiral biaryls, dynamic kinetic resolution of chiral biaryls with low rotation barriers and allylic substitution reactions. In the beginning, a comprehensive study of the chiral relay concept in enantioselective transformations was reviewed and the historic and current story of the chiral relay concept is covered. The design and synthesis of fluxionally chiral 4-dimethylaminopyridine catalysts was introduced. The key issues addressed in this chapter include the design concept regarding a stereoselective fluxionally 4-dimethylaminopyridine catalyst and muti-step synthesis strategies developed for catalyst synthesis. The development of fluxionally chiral 4-dimethylaminopyridine catalysts in the acylative kinetic resolution studies of secondary alcohols as well as axially chiral biaryls is investigated. Six different secondary alcohols are resolved with good selectivity factors (6-37) and ten biaryl substrates are resolved with moderate to high selectivity factors (10-51). Dynamic kinetic resolution has more practical applications to organic synthesis than simple kinetic resolution. The dynamic kinetic resolution of atropisomeric biaryls using the novel fluxionally chiral 4-dimethylaminopyridine catalysts was explored and the corresponding acylated products were obtained with 11-80 %ee. The newly designed DMAP catalysts containing fluxional groups as a stereocontrol unit could also be effectively applied as a nucleophilic catalyst in asymmetric allylic aminations. A range of α-methylene-β-amino esters were obtained with good yields and selectivities (up to 72 %ee).
Achiral Templates in Asymmetric Catalysis: Applications in Construction of All Carbon Quaternary Centers
(North Dakota State University, 2018) Subramanian, Hariharaputhiran
Conjugated olefins are readily available and inexpensive starting materials and their functionalization offers a rapid access to many important building blocks for organic synthesis. The functionalization of these olefins by asymmetric catalytic methods for the formation of C-C and C-X bonds is an active area of research. Major advances in this field are not only triggered by the development of new catalysts but also by engineering of new acceptor olefins. In our lab we have successfully developed acceptor olefins appended to alkoxyimidazoles as a novel template. Using these templated acceptors, we demonstrated methodologies to construct all carbon-quaternary centers, one of the demanding tasks in synthetic methodology development. We have also made significant efforts towards understanding the solution structures of the intermediates involved in the catalytic asymmetric reactions developed in our lab. In chapter 1, the importance of templated acceptors in the field of chemical synthesis with special emphasis on acylimidazoles is reviewed. The versatility of the N-alkylimidazole templates is showcased by their utility in several organic transformations. In chapter 2, modes of activation of acceptor olefins by catalysts, need for templated acceptors and challenges associated with designing an asymmetric catalytic process is described. Our approach in designing novel acceptors based on imidazoles is also described. In chapter 3 the synthetic utility of these novel N-alkoxyimidazole based acceptors is shown by enantioselective construction all carbon quaternary centers by Lewis-acid catalysis. We also compare the effectiveness of other templates such as oxazolidinone and N-methylimidazoles in the Lewis-acidic catalyzed Friedel-Crafts alkylation reaction. The effect of various parameters such as Lewis acid, chiral ligands, temperature, additives and solvents on the conjugate addition of Friedel-Crafts nucleophiles are presented in this chapter. In chapter 4, our research efforts toward understanding solution structures of intermediates involved in catalytic asymmetric reactions are presented. A combination of Diffusion Ordered Spectroscopy (DOSY), heteronuclear NMR spectroscopy, mass spectrometry and X-ray crystallography has been used to study the solution structure of intermediates involved in asymmetric catalytic reactions. In chapter 5, a future outlook on templated chemistry developed in our laboratory is presented. Some preliminary results pertinent to future projects are presented.
Acyl Imidazole : A Promising Template for Asymmetric Lewis and Brønsted Acid Mediated 1,3-Dipolar Cycloadditions
(North Dakota State University, 2011) Rane, Digamber Sadanand
Construction of chiral complex molecules continues to be a challenge for organic chemists all over the world and to address this challenge numerous methodologies have been developed. 1,3-Dipolar cycloaddition reactions is one such simple and elegant method, which can be employed towards the construction of chiral heterocycles. The ability to construct multiple stereocenters in one operation is one of the salient features of dipolar cycloaddition reaction. Asymmetric dipolar cycloaddition via chiral Lewis or Bronsted acid catalyzed processes is aided by the development of various templates, which provide points of attachment for these catalyst. Application of acyl imidazoles as multifunctional templates has been investigated for Lewis and Bronsted acid catalyzed 1,3-dipolar cycloaddition of azomethine imines and nitrones. Chapter 1. A review of 1,3-dipolar cycloaddition towards to construction of chiral nitrogen containing heterocycles is discussed in this chapter. This chapter intends to provide the reader a current state of asymmetric 1,3-dipolar cycloaddition. Chapter 2. Development of exo and enantioselective Cu(II) catalyzed azomethine imine cycloaddition to pyrazolidinone acrylates is discussed in this chapter. The key issues approached in this chapter includes impact of metal geometry on diastereoselectivity as well as effect of N-l and C-5 substitution on enantioselectivity of cycloadducts. Investigation into the scope and limitation of azomethine imines and dipolarophiles has also been discussed. Chapter 3. This chapter introduces acyl imidazoles as multifunctional template for asymmetric azomethine imine cycloaddition. Limitation of substrate scope for azomethine imine cycloaddition encountered in the previous chapter has been resolved by the use of acyl imidazoles as templates. Synthesis of complementary diastereomers of azomethine imine cycloadducts via Lewis acid and Bronsted acid catalyzed reactions has been discussed in this chapter. Chapter 4. This chapter highlights the application of acyl imidazoles as template for first Bronsted acid catalyzed exo and enantioselective nitrone cycloaddition to electron deficient olefins. Study of appropriate chiral Bronsted acid and investigation of breadth and scope of nitrones and dipolarophiles has also been discussed here. Chapter 5. This chapter address one of the most challenging aspect of synthetic organic chemistry namely the construction of chiral quaternary stereocenters. This study highlights chiral Bronsted catalyzed nitrone cycloaddition to p,|3-disubstituted-a,P-unsaturated acyl imidazole leading to the formation of isoxazolidines with chiral quaternary stereocenter. This methodology is useful for the construction of chiral fluorinated heterocycles.
Adsorption Kinetics and Dynamics of Small Molecules on Graphene and Graphene oxide
(North Dakota State University, 2018) Sivapragasam, Nilushni
Graphene is an allotrope of carbon composed of sp2 hybridized carbon and arranged into a honeycomb lattice. Graphene is a mechanically strong material (200 times stronger than steel) and has high carrier mobility, high thermal conductivity, and high optical transparency. Owing to these outstanding properties graphene is used in many applications; often graphene is used on a support instead as a free-standing graphene. When graphene is utilized it can adsorb many molecules and this adsorption could be influenced by the support. Furthermore, comparing the adsorption of such molecules on the support alone and on the supported graphene (graphene on the support) could provide details on the transparency of graphene; transparency can be defined as the identical interactions of a molecule on a supported graphene and the respective support. Therefore, this dissertation focused on studying the adsorption kinetics and dynamics of selected molecules (water, benzene, n-alkane, and carbon dioxide) on two different types of graphene: chemical vapor deposited (CVD) and physical vapor deposited (PVD) graphene. In addition, the chemically inert graphene was functionalized with oxygen to produce graphene oxide and the reactivity of graphene oxide on carbon dioxide adsorption was studied. All the experiments were carried at ultrahigh vacuum conditions to ensure an atomically clean environment. The PVD graphene was synthesized on Ru(0001) and was further functionalized with oxygen to produce graphene oxide. The surface characterizations were carried out by various surface analytical techniques: Auger electron spectroscopy (AES), low energy electron diffraction (LEED), and X-ray photoelectron spectroscopy (XPS). The adsorption kinetics and dynamics were studied by thermal desorption spectroscopy (TDS) and molecular beam scattering techniques, respectively. Transparency of graphene, support effects, and the reactivity of graphene oxide were mapped. The studies clearly showed that the transparency of graphene depends on the polarizability of the molecule and the supports; the supports indeed influenced the adsorption of molecules on graphene. In addition, graphene oxide did not react with CO2 to produce any reaction products but it enhanced the CO2 adsorption.
Apoptosis-Inducing Factor Mediates Metabolism and Oxidative Stress Signaling through Functionally Uncoupled Mechanisms to the Benefit of Tumorigenesis
(North Dakota State University, 2019) Scott, Andrew
Cancer is one of the leading causes of death in the world, arising when cells accumulate genomic mutations that render them incapable of controlling their own proliferation and survival. While substantial improvements in patient outcome have been achieved over the years, completely eliminating tumorigenic cells remains a significant challenge, and some cancer types (such as pancreatic cancer) have not shown meaningfully improved patient survival rates despite our advancements in understanding cancer cell biology and treatment. Genetic targeting approaches to the treatment of cancer have yielded some success, but have shown rather disappointing results overall. In contrast to the diversity of genetic aberrations within tumor cells (making treatment difficult), cancers share common phenotypes that are more universally targetable. As cancers evolve within their biological environment to become aggressive, the net effects of their genetic alterations lead to 1) decreased sensitivity to cell death cues, 2) altered oxidative state and redox-dependent signaling, and 3) dramatically increased demand for energy production and biosynthesis (altered metabolism). The work of this study shows that in pancreatic cancer cells reversible metabolic adaptation alters oxidative stress sensitivity and triggers persistent drug resistance, demonstrating the interplay of the above three cellular activities. Further experimentation identifies the mitochondrial protein apoptosis-inducing factor (AIF) as an essential signaling node functioning at the convergence of all three of these processes. In response to cellular metabolic cues, AIF controls the ability of the mitochondria to produce energy required for tumorigenic growth, invasion, and survival while functioning as mitochondrial translation factor. Distinct from this pivotal metabolic role, AIF serves as a transmitter of extra-mitochondrial redox signaling cues regulating pro-tumor gene expression programs that are lethal without AIF. Finally, AIF exhibits involvement in atypical death pathways associated with the cell death regulators PGAM5 and XIAP. Remarkably, these AIF activities are functionally dissociable and demonstrate the multifunctional nature of AIF in control of tumorigenesis. Overall this study defines a variety of diverse AIF activities at the intersection of multiple tumorigenic phenotypes, furthering our understanding of how cancer cells converge upon different aggressive characteristics and revealing potential vulnerabilities for therapeutic intervention.
Atropisomeric Chromophores as Catalysts and Substrates for Asymmetric Light Induced Transformations
(North Dakota State University, 2016) Vallavoju, Nandini
Photochemical transformations hold a unique place as they can provide access to molecules with unique stereochemical and structurally complex scaffolds, thus serving as a complementary approach to thermal transformations. However, asymmetric photoreactions have been under-explored due to the challenges in controlling the nature of the excited state(s). Various elegant strategies have been developed by chemists to address this bottleneck and achieved varying degrees of success. This dissertation describes a novel and unique strategy that employs atropisomeric thioureas as organo-photocatalysts to perform desired chemical transformations. The motivation of the thesis is to develop an alternative strategy that does not depend on energy/electron transfer processes to initiate the photoreactions. This dissertation describes another unique strategy that employs atropisomeric chromophores where axial chirality in the reactant is transferred to point chirality in the photoproduct(s). This research explains about rotamers control in the ground state that allows stereospecific phototransformations in the excited state(s) thus leading to enantioenriched product(s). The chapter 1 introduces the fundamental differences between asymmetric photochemical reactions and conventional thermal method. Further, an overview of various methodologies developed towards asymmetric photochemical transformations are detailed. In chapter 2 and chapter 3, various thiourea-based organo-photocatalysts were developed for enantioselective intramolecular [2+2] photocycloaddition of coumarin derivatives. The atropisomeric thioureas were found to be efficient in promoting the photocycloaddition leading to the corresponding products with high enantioselectivity (77-96% ee) at low catalyst loading (1-10 mol%). The photocatalytic cycle is proposed to proceed by the mechanism of ‘energy sharing’ via the formation of both static and dynamic complexes (exciplex formation), which is promoted by hydrogen bonding. Chapter 4 describes the intermolecular [2+2]-photocycloaddition of coumarin with tetramethylethylene promoted by thiourea catalysts. The photocatalytic cycle of coumarin mediated by thioureas is proposed to proceed via a combination of minimized aggregation, enhanced intersystem crossing and altered excited state lifetime(s), which is promoted by hydrogen bonding. 5 describes the enantiospecific hydrogen abstraction of atropisomeric enone carboxamides leading to spiro-β-lactam photoproduct(s). Divergent photoreactivity was observed based on restricted bond rotation(s) in atropisomeric substrates, when compared to their achiral analogue. The hydrogen abstraction also proceeded efficiently under visible light sensitized irradiation.
Attempted Total Syntheses of a Novel Lycopodine-Class Alkaloid and (R)-Myricanol, Compounds for the Treatment of Alzheimer's Disease
(North Dakota State University, 2013) Ostlund, Anthony John
Alzheimer's Disease (AD) is a significant challenge to both the pharmaceutical world as well as the synthetic organic chemistry world. One of the most important problems in this field is the lack of a unified approach to the most biologically active class of natural products for the treatment of AD, the lycopodine alkaloids. Presented herein is an attempt at the total synthesis of a novel member of this family. The synthesis spans three complete generations of retrosynthesis and forward progress. In the ultimate effort, the carbon backbone of the entire lycopodine class was successfully synthesized using a 6 step process in 44% overall yield. Features of this synthesis are the formation of an intermediate aldehyde by tandem Horner-Wadsworth-Emmons olefination/Baylis-Hillman reaction, quantitative reduction resulting in a monoprotected diol, cationic Au(I) O-vinylation, and microwave assisted Claisen rearrangement. The final step towards the backbone was realized using a hydrozirconation/transmetalation sequence with addition to the aldehyde to make the penultimate allylic alcohol in 54% yield. Final studies on the elimation of the allylic alcohol to create the desired 1,3-diene functionality show limited access to this important precursor. Simultaneous model system studies have shown that an intramolecular, cationic Rh(I)-catalyzed ynamide Diels-Alder reaction is feasible to set the B and D rings of the final lycopodine core, although the reaction requires optimization. Model systems for the unique intramolecular allylation proposed in the retrosynthesis also show feasibility, however the intramolecular variant has not been fully explored. Additional studies towards the total synthesis (R)-myricanol are also presented as a continuance of efforts towards general approaches to AD combative compounds. Featured are straightforward methods to the two main segments of the natural product, including a three-step process to synthesize the southern half. Finally, a successful 2-step synthesis of (R)-convolutamydine E is used to showcase an In(0)-mediated allylation methodology.
Biochemical and Epidemiological Analysis of Mycobacterium Avium Subspecies Paratuberculosis and Investigation of its Relationship to Crohn's Disease in Humans
(North Dakota State University, 2011) Uzoigwe, Jacinta Chinwe
Background: Crohn's disease is a chronic inflammatory disease of the intestine in humans, with no known cause. Johne's disease is a chronic intestinal disease of ruminants caused by Mycobacterium avium suhspecies paratuberculosis (MAP), and has some features similar to Crohn's disease. Although MAP has been purported to play an etiologic role in Crohn's disease, this causal link is still under debate. Objective: The overall aim of this project is to analyze MAP strains from different hosts (cattle, sheep and humans) and regions in North Dakota by biochemical and epidemiological methods, in order to better understand the pathogenesis and epidemiology of MAP strains and the relationship between MAP and Crohn's disease. The specific aims of this research are the following: Aim 1. Investigate the epidemiological evidence for MAP as a cause of Crohn's disease. Aim 2. Conduct a comparative causality study to investigate whether MAP or other enteric pathogens cause Crohn's disease. Aim 3. Evaluate the occurrence of MAP infections in cattle in North Dakota, 1995-2005. Aim 4. Analyze MAP strains from symptomatic and asymptomatic cattle. Aim 5. Investigate the biochemical variations of rapid and slow growing MAP strains. Aim 6. Evaluate MAP strains from low shedders and high shedders. Methods: MAP isolates were analyzed by biochemical methods of gas chromatography, high performance liquid chromatography and mass spectrometry. In addition, extensive literature review was performed to (1) determine the epidemiologic causal link between MAP and Crohn' s disease and (2) determine whether MAP or other enteric pathogens cause Crohn's disease. Results: Results from our study indicated the availablity of epidemiologic evidence supporting the causal role of MAP in Crohn's disease. It was also demonstrated that MAP is the most implicated organism in the etiology of Crohn's disease when compared to other infectious agents. Investigation of the occurrence of MAP infection in North Dakota showed an increase in number of MAP cases reported, with seasonal trends. Biochemical typing of MAP strains from symptomatic and asymptomatic cattle indicated that the symptoms status of isolates was significantly associated with mass spectra patterns and shedder status (p < 0.05). However, the association between symptoms status and HPLC and GC patterns was not significant (p > 0.05). Investigation of biochemical variations of rapid and slow growing MAP strains showed associations between the biochemical variability of MAP strains and their growth rate and presence of symptoms in the source cattle. Evaluation of MAP strains of different shedding characteristics by univariate logistic regression revealed that the shedder status of isolates was significantly associated with growth rate of isolates, symptom status, and source regions, but not with mass spectra patterns of isolates. Conclusion: Overall, this study strengthens the theories of strain sharing, intraspecies and interspecies transmission, and supports an association between MAP and Crohn's disease. In addition, the understanding of the biochemical variation among MAP isolates will help in the future design of diagnostics, therapeutics and vaccines for Johne's and Crohn's diseases.
Biochemical and Functional Characterization of Rpa2 N-Terminal Phosphorylation during DNA Repair and Checkpoint Adaptation in Saccharomyces Cerevisiae
(North Dakota State University, 2018) Wilson, Timothy Michael
In response to DNA damage, a signaling cascade is activated within cells that promotes cell cycle arrest in order to provide adequate time for DNA repair to occur. Replication Factor A (RFA) is an essential, heterotrimeric complex that acts as a primary sensor of DNA damage, localizes to sites of and binds to broken DNA, and serves as a platform for several DNA damage repair proteins. RFA-coated ssDNA leads to activation of the sensor kinase Mec1. Activation of Mec1 leads directly to phosphorylation of the checkpoint regulator Rad53, which in turn activates several downstream effectors that halt cell growth and promote DNA repair. In response to a permanent, irreparable DNA lesion yeast cells undergo Rad53-dependent checkpoint arrest for approximately 12 hours. After this prolonged arrest, cell cycle restart occurs sometime between 12-15 hours, promoting mitosis despite the presence of an unrepaired lesion. This aberrant checkpoint exit is known as checkpoint adaptation. Checkpoint adaptation deficiency, or the inability to override the established Rad53-dependent checkpoint, can be conferred through deletion of non-essential but important genes involved in DNA damage repair (e.g., ku70, rdh54). The molecular mechanism(s) by which checkpoint adaptation occurs remains poorly understood; however, a single point mutation (K45E) in the Rfa1 subunit of the RFA complex is capable of rescuing adaptation deficiencies. During the DNA damage response, the RFA complex is known to be post-translationally modified through phosphorylation, ubiquitination, and sumoylation. Despite robust characterization of these modifications to RFA, the physiological role of phosphorylation within the N-terminal (NT) domain of the second subunit of the RFA complex, Rfa2, remains elusive. My studies using phospho-mimetic Rfa2 mutants demonstrates that phosphorylation of the NT is: 1) dispensable for activation of the Rad53-dependent checkpoint, 2) is readily detected during the temporal window within which checkpoint adaptation occurs and 3) promotes checkpoint adaptation in all adaptation-deficient mutations tested. Furthermore, analysis of Rfa2 phospho-mutants in sensor kinase deletions mec1 and tel1 reveal phosphorylation of the Rfa2 NT may directly impede Mec1 activity, thereby inhibiting maintenance of the Rad53-dependent checkpoint and providing a mechanism by which cells can escape an established DNA damage-dependent checkpoint.
Biological Effects of Vasoactive Intestinal Peptide/Pituitary Adenylate Cyclase Activating Polypeptide Receptor 1 (VPAC1) and VPAC2 on Chemotaxis and its Regulation of the Tumor Suppressor Ikaros in Leukemic T Cells
(North Dakota State University, 2012) Van der Steen, Travis
One in three children diagnosed with cancer has leukemia. Leukemia patients with mutations in the tumor suppressor transcription factor Ikaros (IK), an anti-leukemic factor that is critical for the development of blood cells; have a poor prognosis despite modern chemotherapy. There is, therefore, a critical need to understand the biology of IK. Research by our laboratory has identified a neurotransmitter, called vasoactive intestinal peptide (VIP), which blocks proliferation through one of its receptors (vasoactive intestinal peptide/pituitary adenylate cyclase activating polypeptide receptor 1(VPAC1)), but blocks apoptosis through a second inducible receptor (VPAC2), while both receptors have chemotactic properties in primary T cells. Some leukemia patients have reversed VIP receptor expression (low VPAC1; high VPAC2), and we hypothesizes that this contributes to a selective growth advantage. Understanding the biology by which VIP receptors regulate cellular growth (proliferation and apoptosis) and movement (chemotaxis) will be pivotal in establishing their signaling pathways as future drugs target candidates in the fight against leukemia. We hypothesized that VIP/VPAC1 signaling would alter the expression of IK protein and that VIP would direct cellular migration of both VPAC1 and VPAC2 expressing leukemic T cells. To test this hypothesis, we first asked whether VIP/VPAC1 signaling affects the expression and/or the phosphorylation profile of IK a transcription factor that regulates cellular growth. The second question was whether VPAC1 and/or VPAC2 signaling differentially control leukemic cell movement. By one- and two-dimensional polyacrylamide gel electrophoresis followed by Western blot analysis, we showed that VIP signaling suppresses IK expression and changes the isoelectric pools of IK protein in a human leukemia cell line. By using leukemia cells that only express VPAC1 or VPAC2 receptors; we demonstrated that VIP promoted cellular movement, but that this effect was controlled by different pathways elicited by VPAC1 versus VPAC2. Collectively, these data support the notion that the nervous system naturally contributes to normal blood cell function, but after leukemogenesis, the VIP signaling axis may exacerbate the leukemia phenotype.
Biophysical Studies of Ligand Binding to Human Histone Deacetylase-8
(North Dakota State University, 2014) Singh, Raushan Kumar
Due to an involvement in various patho-physiological conditions, human histone deacetylases (HDACs) are high priority drug targets for the treatment of several diseases, such as cancer, heart failure, neurodegeneration, etc. An effector (inhibitor/activator) of these enzymes has a great potential to alleviate the above disease conditions. In this regard, HDAC inhibitors – Zolinza and Istodax - have already been approved by the FDA for the treatment of T-cell lymphoma, aside from several other inhibitors which are in the advanced level of clinical trials. HDAC8 serves as a prototype to study structural-functional and catalytic features of human HDACs. In order to pursue the biophysical studies of the ligand-binding, HDAC8 was cloned, expressed, and purified from E. coli. A high-throughput screening (HTS) of an in-house library of small molecules was performed utilizing a trypsin-coupled in vitro HDAC8 assay to discover novel effectors of HDAC8, and the N-acetylthiourea and the thiopyridine derivatives were discovered as the isozyme-selective inhibitors and activators of HDAC8, respectively. In vitro HDAC8 assay utilizing a fluorogenic peptide as a substrate often produces artifactual results. Therefore, a substrate-independent HDAC8 assay was developed utilizing a fluorescent analog of a pan-HDAC-inhibitor. In view of the fact that the downstream cellular response of a drug is often dictated by the transient kinetic and thermodynamic parameters of its interaction with the target, the transient kinetics and thermodynamics of interaction of the selected HDAC8 inhibitor with the enzyme were thoroughly investigated. It was observed that the dissociation off-rate and/or the enthalpy of binding of an HDAC8 inhibitor to the enzyme could play a crucial role in determining its in vivo efficacy. A rationale has been presented that the above parameters of the ligand-protein interaction could be utilized for optimizing a drug candidate (HDAC8 inhibitor) in order to enhance its in vivo potency.
Characterization of RPA2 N-terminal Function in the DNA Damage Response in Saccharomyces Cerevisiae
(North Dakota State University, 2015) Ghospurkar, Padmaja Laxman
In response to DNA damage, two general but fundamental processes occur in the cell: (1) a DNA lesion is recognized and repaired, and (2) concomitantly, the cell halts the cell cycle to provide a window of opportunity for repair to occur. A key factor involved in the DNA damage response is the heterotrimeric protein complex Replication Protein A (RPA), which is not only essential for the repair of damaged DNA, but also is post-translationally modified on at least two of the three subunits in response to DNA damage by checkpoint kinases. Of particular interest is the 32-kDa subunit, called Rpa2, which is hyper-phosphorylated on its serine/threonine-rich N-terminus following DNA damage in human cells. This unstructured N-terminus is often referred to as the phosphorylation domain (PD) and is conserved amongst eukaryotic Rpa2 subunits, including Rfa2 in Saccharomyces cerevisiae. In this work we aim to characterize the function of Rfa2 N-terminus (Rfa2 NT) in DNA damage response and develop yeast as a tool to study human RPA. With the help of mutagenesis we developed Rfa2 NT extreme mutants, which showed that the phosphorylation of Rfa2 NT is dispensable in DNA damage response. However, the presence of Rfa2 NT is essential for cells to survive in stressed condition indicating an uncharacterized function. We further discovered seven S/T sites are responsible for the damage sensitive phenotype of Rfa2 NT extreme mutants. And the phosphorylation affects protein interaction of RFA complex. Although, the phosphorylation event of Rfa2 NT is dispensable in S. cerevisiae the cells have conserved the ability to phosphorylate Rfa2 N terminus. With the help Rfa2 NT fusion mutants we showed that S. cerevisiae could phosphorylate N terminus from seven different eukaryotic species. Hence, we successfully developed yeast as a tool to study Rpa2 phosphorylation amongst various eukaryotic species.
Delineating Signaling Mechanisms Involved in Lymphocyte Chemotaxis, Immune Homeostasis and Allergic Asthma
(North Dakota State University, 2015) Wanjara, Steve Biko
The vasoactive intestinal peptide (VIP) signaling axis constitutes VIP and its two G protein coupled receptors (GPCR) termed vasoactive intestinal/pituitary adenylate cyclase activating polypeptide (VPAC) 1 and 2. This signaling axis regulates numerous biological actions within the endocrine system, the nervous system and the immune system. Working as a gut hormone, VIP can increase cAMP signaling within beta-islet cells of the pancreas to impact insulin production. As a neurotransmitter, it acts as a master circadian regulator controlling light and dark cycling. Lastly, VIP regulates immune processes such as activation, chemotaxis, development and cytokine secretion. The focus of my doctoral research was to delineate VIP signaling mechanisms controlling immunity. We aimed at understanding: 1.) the molecular mechanism of VIP-induced T cell trafficking 2.) ability for VPAC2 signaling to regulate immune homeostasis and 3.) a phenotype of a B cell subset during asthma, an immune pathology devoid of VIP protein due to excessive protease activity. Methods employed utilized isolated primary mouse immune cells to measure a VIP-induced signaling pathway centered on the epidermal growth factor receptor (EGFR), a tyrosine kinase receptor, by qPCR and chemotaxis assays. Flow cytometry to enumerate immune cell numbers in VPAC2 deficient mice was done to accomplish aim 2. Lastly, using a published in vivo allergic asthma mouse model, we used qPCR, immunoblotting and flow cytometry analyses to measure expression of Hyaluronic acid binding proteins on B cells. Results from these studies revealed that VIP signaling in T cells is regulated by EGFR as inhibitors against its enzymatic activity abolished T cell movement towards VIP. Immune cell numbers were lowered as a consequence of VPAC2 deficiency, suggesting its involvement in homeostasis. Lastly, a unique B cell population homing to asthmatic lung secretes an anti-inflammatory mediator, TGF-beta, through the HA binding protein called RHAMM. Collectively, these data emphasize the importance of VIP signaling in the immune system controlling cell migration and homeostasis.
Designing Ru Catalysts for Selective C-H Bond Oxidation Reactions
(North Dakota State University, 2020) Herath, Hashini Nuradha Kumari
Typically, C-H bond oxidation proceeds via formation of high-valent metal oxo species. Attaining a high oxidation state of the metal complex is critical as it is the key step for many catalytic processes. Understanding ligand effects on structural and electronic changes of the metal complexes towards designing more robust catalysts is an effort of this dissertation. It will highlight initial attempts at developing novel ruthenium (Ru) catalysts and an analysis of their structural, electrochemical and spectroscopic properties. The catalytic behavior of the resulting complexes towards C-H bond hydroxylation reaction will also be shown. Chapter I introduces the background of C-H bond activation and hydroxylation reactions by Ru catalysts. Also, this chapter details the study of C-H bond hydroxylation mechanisms, reaction intermediates, the importance of C-H bond hydroxylation, electronic effects of ligands, and the pioneering work in this field. Chapter II describes development a of new Ru complex containing the pyridine alkoxide ligand, of general formula [Ru(tpy)(pyalk)Cl] (tpy = 2,2’:6’2”-terpyridine, pyalk = 2-(2′-pyridyl)-2-propanol). This chapter outlines the detailed synthesis, structural, electrochemical and spectroscopic properties of the complex by electrochemical techniques, UV Visible spectroscopy, NMR, mass spectrometry and X-ray crystallography. In order to overcome some limitations on project 1, new ruthenium complexes [Ru(MepyPO3H)(tpy)Cl] (1, tpy = 2,2’:6’2”-terpyridine , MepyPO3H = (pyridine-2-ylmethyl)phosphonic acid) and [Ru(bpyPO3H)(bpy)Cl] (2, bpy(PO3H2) = 2,2-bipyridine-6-phosphonic acid, bpy = 2,2-bipyridine) bearing phosphonate ligands were prepared and fully characterized. Catalytic properties of the complexes have been evaluated by testing their ability to catalyze C-H bond oxidation using a variety of sacrificial oxidants.
Development of Quantitative Lateral Flow Strip Biosensors for the Detection of Cancer Biomarkers
(North Dakota State University, 2019) Baryeh, Kwaku
The detection of cancer biomarkers is of great importance in oncology. Cancer biomarkers can provide diagnostic information which can aid disease screening and early diagnosis. Further, cancer biomarkers can help predict disease prognosis and response to therapy, and also help in the monitoring of disease. Thence, the accurate and sensitive detection of cancer biomarkers which may be present at very low concentrations is of great clinical importance. Traditionally, these biomarkers have been detected predominantly by Enzyme Linked Immunosorbent Assay. The traditional biomarker detection assays generally require multiple washing steps, long assay times, and have the need for trained expertise and expensive instrumentation. In this dissertation, Lateral Flow Strip Biosensors (LFSB) that provide rapid, low-cost and user-friendly screening of cancer biomarkers are discussed. The developed biosensors have the added advantages of being portable, sensitive and highly selective, which makes them ideal for routine cancer screening. Gold nanoparticles (GNP)-based Lateral Flow Strip Immunosensors (LFSI) that colorimetrically detected carbohydrate antigen (CA 19-9) and carcinoembryonic antigen (CEA) were developed for the screening of human plasma and pancreatic cyst fluid, respectively. Further, carbon nanotube based-LFSBs that targeted CA 19-9 and CEA were developed. The CNT-based LFSBs showed improved detection limits over the conventional GNP-based LFSB. A GNP-based LFSB was also developed for the detection of exosomes using an aptamer that targeted a cell surface protein, epithelial cell adhesion molecule (EpCAM). The developed assays showed good performance and were used for the screening of pancreatic cancer patient samples. Upon further development, the assays discussed in this dissertation could find application in the clinical screening and monitoring of cancer, especially in limited resource settings.
Differential Modulation of the Structural and Functional Characteristics of Human Matrix Metalloproteinase Isozymes upon Binding to Different Ligands
(North Dakota State University, 2010) Ganguly, Bratati
Matrix metalloproteinases (MMPs) are a family of Zn2 + -dependent, Ca2 + -containing endoproteinases involved in tissue remodeling and degradation of the extracellular matrix (ECM). Human MMP isozymes are known to be involved in the progression and metastasis of many diseases like cancer, Alzheimer's, and etc. The different nanoparticles (e.g. gold nanoparticles, liposomes, and charged quantum dots) used in this study provides insights into nanoparticle-induced differential modulation in the structural-functional characteristics of MMP 7, 9 and 10 for better therapeutic intervention. To demonstrate the relationship between the rigid and flexible surfaces on the differential modulation of functional and structural characteristics of MMP-7, polylysine (PLL) and cationic gold nanoparticles (Au-CNP) were selected as representative examples. These cationic nano-structures were expected to serve as "soft" (flexible) and ''rigid" (hard) ligands, respectively. Steady-state kinetic analysis demonstrated that PLL induces activation and inhibition of MMP-7 at stoichiometric and super-stoichiometric concentrations respectively. Circular Dichroism spectroscopy was used to confirm that binding of Au-CNP to MMP-7 induces denaturation of the protein. In pursuit of understanding the molecular origin of the intrinsic selectivity in binding of human MMP isozymes to differently charged lipid membranes, steady-state kinetic studies and intrinsic tryptophan quenching studies were carried out. Results demonstrated that differently charged lipid membranes bind to all three MMPs; phosphotidylserine (POPS) liposomes are selective for MMP-7. The bipolar distribution of negative and positive charges on the surface of this enzyme dictates the binding of liposomes and perturbation of catalytic activity. An attempt to explain the molecular rationale for alternative binding modes of differently charged quantum dots (QDs) to the three MMPs, steady-state tryptophan quenching, steady-state kinetics, and time-resolved fluorescence measurements were carried out. Differently charged QDs bind to all the three MMP isozymes. Enzyme activity of these MMPs was perturbed upon binding to cationic and anionic QDs. Binding of MMPs to the differently charged QDs is reversible and is mediated via electrostatic interactions. Analysis of time-resolved fluorescence data indicates that the protein expenences different micro-environments, due to different distribution of intrinsic tryptophan residues (buried and exposed) on MMP isozymes or the existence of two distinct conformations of the protein. Binding to charged QDs perturbs enzyme activity of MMPs either by restricting the access of the substrate to the active-site or through allosteric modulation. In order to develop new isozyme-selective inhibitors, small molecule inhibitors (SMis) were designed, synthesized and screened for MMP-7, 9 and 10. Results indicate that hydroxamates and carboxylates are preferred SMis. Binding preference is based on either the micro-environments of the active-site pockets.
Effect of Surface Ligands and Surface Defects on the Electronic and Optical Properties of Quantum Dots
(North Dakota State University, 2016) Dandu, Naveen Kumar
Quantum dots (QDs) have drawn a special attention since recent past due to their properties such as broad range absorption ability, size tunable narrow emission, high extinction coefficients, and charge carriers ability. Nonetheless, imbalanced surface passivation/defects leads to the appearance of surface trap states inside the band gap, affecting both radiative and non-radiative dynamics. Experimentally, it is difficult to explore the effect of surface states as they are optically inactive. However, computations provide valuable insights to these characteristics. We performed calculations using density functional theory (DFT) and time-dependent DFT (TDDFT) to provide our insights to such effects. Firstly, we performed DFT studies to understand the effect of QD- ligand interactions on their photophysical properties. Our studies on thiols passivated CdSe QDs showed that passivation of their surface by equilibrium concentration of neutral thiols and negatively charged thiolates is essential to achieve photoluminescence (PL) enhancement. Additionally, we investigated the effect of surface defects on photophysical properties of silicon QDs. Our results showed that defects introduce mid gap states inside band-gap. Absorption spectra showed the appearance of dark/semi-dark states at the first energy band, proving that the surface states quench PL efficiency in QDs. Secondly, we studied the effect of QD-QD interactions on their optoelectronic properties. In collaboration with the experimental group from Prof. Hobbies’ lab, we studied interactions between defective and non-defective QDs. Calculated Forster Resonance Energy Transfer rates suggest that all the trap states in a defective QD would be filled by the excited electrons from the non-defective QD and thus emission happens from the highest bright energy state. We proposed this as the reason for the experimental observation of the increased on-time blinking and overall enhancement of PL in these QDs. Furthermore, in collaboration with experimentalists from Los-Alamos National Lab, we have provided our insights into the chemical engineering of self-assembling of PbSe QDs into (100) directed 2D nanoplates. Our surface energy calculations on the oriented attachments revealed that 2D nanoplates grown in (100) are more feasible than 3D quantum dots. Overall, our calculations not only supported the experimental findings, but also provided solutions to questions raised by experimentalists.
Enhanced Sensitivity of Lateral Flow Strip Biosensors Based on Enyzmatic Reaction and Nanomaterials
(North Dakota State University, 2014) Xu, Hui
Ultrasensitive detection for trace amount of proteins plays pivotal role in the diagnosis of specific diseases in clinical application, basic discovery research and the improvement of proteomics. Recently, lateral flow strip biosensor (LFSB) has gained considerable attention for protein analysis. Compared with the traditional immunoassays, LFSB has several advantages: user-friendly format, short assay time (generally several minutes), less interference due to chromatographic separation, a relatively low cost, and no requirements for skilled technicians. This ideal technique is suitable for on-site testing by people who are untrained. Traditional gold nanoparticles (GNPs) based LFSB have been used for qualitative and semiquantitative analysis, the application of GNP-based LFSB is limited by its low sensitivity. In this dissertation, different nanomaterials and advanced detection technologies have been used to enhance the LFSB sensitivities. An ultrasensitive LFSB based on horseradish peroxidase (HRP)/GNP dual labels was developed for qualitative (Yes/No) and quantitative detection of protein. The LFSB signal was enhanced dramatically by introducing the second tracer (enzyme) on the GNP surface. The detection limit of LFSB was 100 times lower than that of GNP-based LFSB. A fluorescent LFSB based on enzyme tracers was developed for sensitive detection of proteins. Alkaline Phosphatase (ALP) was selected as a label to prepare the LFSB. The signal was from the fluorescent emission of the ELF-97 alcohol precipitate which was the product of ALP catalyzed dephosphorylation of ELF-97 phosphate. ALP-conjugated antibody (ALP-Ab) functionalized gold nanoparticles (GNPs) were used as labels for the development of a chemiluminescence-based quantitative LFSB. The use of detection and GNPs as enzyme carriers allowed accurate and sensitive analyte detection. GNP-decorated silica nanorods (GNP-SiNRs) were synthesized and employed as the labels for ultrasensitive detection of proteins on the LFSB. Owing to its biocompatibility and convenient surface modification, SiNRs were used as carriers to load numerous GNPs. The signal of the GNP-SiNR based LFSB was enhanced significantly compared to the GNP-based LFSB since more GNPs were captured through the sandwich-type immunoreactions.
Enzyme Behavior in Synthetic Materials and Structural Implications for Rational Design
(North Dakota State University, 2020) Farmakes, Jasmin Kaye
Combining enzymes with synthetic materials is the new frontier of biocatalysis, materials science, and protein engineering. Enzymes are biological macromolecule catalysts with incredible efficiency and specificity that are desirable for use in a variety of different fields. However, commercial applications have been limited by the stability and reusability of un-altered enzymes. An avenue for overcoming the challenges to harnessing enzyme power is to combine enzymes with materials to create an enzymatically-active material that has enhanced stability and activity. Unfortunately, the catalytic activity of the hybrid material is often lower than that of the enzyme alone. The activity of an enzyme is directly dependent on its structure and dynamics. Therefore, a deeper understanding of enzyme structure and dynamics upon incorporation into materials will provide the data necessary to rationally design enzymatically-active materials with the desired features. This dissertation explores the behavior of a model enzyme, T4 Lysozyme, with two different artificial material systems, metal-organic frameworks and polyethylene glycol. The underlying structural rationale for the behavior is probed using a variety of techniques, notably, Electron Spin Paramagnetic Resonance. Herein, the implications of structural alterations on activity and opportunities for exploitation are discussed. T4 Lysozyme is a perfect model for this study because it has a well characterized structure-activity relationship, thus providing a vast literature understanding which can be pulled from to verify and assist with interpretation of data. The structural basis of enzyme activity alteration in artificial materials can be used to rationally design systems with desired characteristics. After successfully demonstrating the tunability of proteins in artificial materials using T4L as a model, human Cu/Zn superoxide dismutase 1 was chosen for continuing studies due to its importance in diseased states. However, the superoxide dismutase mutant chosen is aggregation prone, which makes it difficult to express recombinantly in large amounts. Therefore, an efficient protocol for producing the superoxide dismutase protein was developed to set the stage for future studies.
Examining Thieno[3,4-b]pyrazine Through a Multifaceted Lens: From Extended Ring Functionalization to Ambipolar-Acceptor Copolymers
(North Dakota State University, 2022) Culver, Evan
A class of materials known as conjugated polymers (CPs) has been shown to integrate the physical properties of organic plastics such as low-weight, flexibility, and synthetic modularity with electronic semiconducting properties typically found in inorganic materials. While a variety of parameters determine the resultant material’s conductivity, a crucial factor is the bandgap (Eg). Specifically, thieno[3,4-b]pyrazine (TP) has found success in generating low Eg CPs (i.e. Eg < 1.5 eV), largely in part due to its ambipolar identity. Two strategies to achieve Eg values < 1 eV include extending the conjugation of TP through ring fusion and pairing TP with strong electron accepting moieties. The investigation into extended ring TPs as low bandgap homopolymers was initially pursued with the synthesis of poly(acenaphtho[1,2-b]thieno[3,4-e]pyrazine), a record setting low Eg homopolymer. Upon this realization of driving Eg¬ values down through ring fusion on the pyrazine portion of TP, additional analogues were considered with 2λ4δ2-dithieno[3,4-b:3’,4’-e]pyrazine as one of the most promising candidates due to its predicted Eg of 0.14 eV. Efforts into this research have produced a variety of precursors and analogues, adding to the family of TPs for further study. A second strategy for Eg reduction is through the pairing of electronically mismatched units known as donors and acceptors. While this does reduce Eg, the underlying principles of the cause is disputed. Thus, to further understand the interactions in these types of copolymer systems, a small molecule study was designed with a strong donor, a strong acceptor, and the ambipolar unit TP in which six possible dimer configurations were synthesized and analyzed to determine the extent of donor-acceptor interactions. Lastly, an investigation into TP-acceptor alternating copolymers was carried out by pairing TP with two acceptors of varying accepting strength and contributions to polymer solubility. Because of the atypical design of these copolymers, a relatively new cross-coupling method known as direct arylation polymerization was used in their synthesis. The optimization of which produced a CP with an Eg of 0.93 eV. These results thus provide evidence for a new design motif for low bandgap CPs that further refines our understanding of donor-acceptor relationships.

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