Browsing by Author "Cuny, Gregory D."
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Item 1,4–Conjugate Addition with Pyrrole and Indole Enones & Propargylic Substitution using Boronic Acids as Nucleophiles with Gallium and Silver Catalysts(2021-08) Boylan, Amy; May, Jeremy A.; Gilbertson, Scott R.; Cuny, Gregory D.; Miljanić, Ognjen Š.; Guloy, Arnold M.This dissertation covers two projects: 1,4–conjugate addition on pyrrole and indole enones, and propargylic substitution using boronic acid as nucleophiles and silver/gallium catalysts. b-(2-indole)-enones and b-(2-pyrrole)-enones, both historically problematic substrate types with 1,4–conjugate addition reactions, were extensively examined. Analysis of isomer- related reaction rate trends showed that proximity of a heteroatom to the enone b-carbon was favorable to reaction rate and increased resonance electron donation also increased reaction rate. These enantioselective conjugate addition reaction were ultimately enhanced by using a less electron deficient catalyst and a base additive, ammonium carbonate. After many reactions and substrates were studied, this base was shown to have an advantage effect on enhancing the conjugate addition reaction but had an adverse effect on the starting material, which lead to other side reactions with this conflicted system. For the second project, three methods have been developed for nucleophilic propargylic substitutions that have been useful in forming tertiary carbons: (IPr)GaCl3/AgSbF6 with boronic acid at 23°C, AgSbF6 with boronic acid at 40°C, and GaCl3 with boronic acid at -78°C. A combination of AgSbF6 and IPrGaCl3 along with boronic acids at lower temperatures have been shown to decrease reaction times for the formation of nucleophilic substitution through the displacement of propargylic alcohols. GaCl3 with boronic acid at -78°C proved to be the best conditions for the formation of the quaternary carbon centers. An expanded substrate scope and nucleophile scope have been developed for the formation of the tertiary carbon centers and quaternary carbon centers using these three methodItem 1. SELF-SORTING OF DYNAMIC IMINE LIBRARIES UNDER CHEMICAL AND PHYSICAL STIMULI; 2. CHARACTERIZATION OF POROUS MATERIALS BASED ON A FLUORINATED TETRAZOLE(2016-05) Hsu, Chia-Wei 1984-; Miljanić, Ognjen Š.; Daugulis, Olafs; Baldelli, Steven; Cuny, Gregory D.; Thummel, Randolph P.Chapter 1 introduces the self-sorting processes of dynamic combinaotrial libraries (DCLs) under thermodynamic and kinetic control. Based on the concept of dynamic combinatorial chemistry (DCC), disulfide exchange, imino bond formation, transesterification, and metal-ligand interaction are widely utilized to perform the re-equilibration of molecules. Simplification of multicomponent systems and the manipulation toward the direction of equilibrium are reviewed. Chapter 2 of this dissertation describes an application of a new external stimulus—adsorption on the silica gel surface—to dynamic imine libraries constructed of as many as four aldehydes and four anilines. During the elution process, imines keep trading their components, resulting in the amplification of the least polar imine which travels down the silica gel-based column the fastest. This process is then iteratively repeated to isolate the next least-polar component in the remaining imine library, eventually simplifying the library from n2 into n members. In Chapter 3, we demonstrated iterative simplification of a dynamic imine library constructed from 10 aldehydes and 10 anilines using three orthogonal external stimuli. First, mild oxidation (using molecular iodine as an oxidant) was applied to oxidize the most electron rich imine with the rapid imine exchange. In the second self-sorting step, we obtained a halogenated [2×2] sub-system with bromo- and chloro-substituted imines from the imine library. Third and final step was a slow distillation of the residual imines from the column, leading to the amplification of the least volatile imine in 61% yield. Ultimately, this [10×10] imine library is simplified into two major products and one sub-system after iterative self-sorting. Chapter 4 will describe the synthesis and characterization of a porous material based on an extensively fluorinated aromatic tristetrazole. This molecule crystallizes in a porous structure which is held together through [N−H…H] hydrogen bonds and electrostatic interactions between C–F units in these molecules. This porous material is compositionally stable up to 270 °C and shows selective adsorption of CO2 over N2 (15:1 in mmol g−1) at 195 K and 760 mmHg.Item A CRISPR-Cas9 Repressor for Epigenetic Silencing of KRAS(2021-05) Liu, Jingwen; Guo, Bin; Cheng, Xiaodong; Cuny, Gregory D.; Wu, Tianfu; Zhang, RuiwenKRAS is one of the most frequently mutated oncogenes in cancers. KRAS mutations have been found in in nearly 30% of all human cancers. Despite decades of study, no effective approved anti-KRAS therapies are available. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein) system has a great potential for treating human genetic disease due to its genome editing efficiency. In this study, we designed a plasmid by introducing the epigenetic repressor transcriptional repressor histone deacetylase 1 (HDAC1) cDNAs into the pcDNA3.1-dCas9 plasmid. The catalytically dead Cas9 (dCas9) functioned as a DNA binding device, while HDAC1 acted as epigenetic repressor. We designed a panel of three CRISPR RNAs (crRNAs) covering 1500-bp range of the KRAS promoter and identified crRNA1 and crRNA2 for silencing K-Ras efficiently. The inhibition of K-Ras significantly inhibited cell growth, suppressed colony formation in soft agar, induced significant cell death, inhibited protein expression of K-Ras downstream targets and reduced cell migration and invasion in HCT-116 colon cancer cells and NCI-H358 lung cancer cells. In addition, we performed rescue experiments using mutated KRAS plasmid to overexpress K-Ras protein in NCI-H358. The results confirmed that the inhibitory effect of dCas9-HDAC1 system in NCI-H358 is through downregulation of K-Ras. The chromatin immunoprecipitation (ChIP) assay confirmed dCas9-HDAC1 modified histone acetylation on the KRAS promoter. Furthermore, we generated recombinant protein dCas9-HDAC1 in insect cells. The fusion protein was able to target the mutant KRAS promoter and suppress KRAS expression through epigenome editing as well. The suppression of K-Ras by the delivery of dCas9-HDAC1 protein system resulted in a significant inhibition of cell growth, induction of cell death and inhibition of colony formation in soft agar. Finally, we investigated RNA nanoparticle decorated exosomes as a delivery system for ribonucleoprotein (RNP) formed by dCas9-HDAC1 protein and gRNA. The RNA nanoparticle decorated exosomes were able to encapsulate RNP. The delivery system downregulated K-Ras level and inhibited cells proliferation in HCT116 cells. In summary, we have developed a novel strategy that combines CRISPR-Cas9 technology with HDAC1 epigenetic silencing to target cancers driven by KRAS mutations.Item A high-throughput screen to identify inhibitors of SOD1 transcription(Frontiers in Bioscience, 2014-07) Wright, Paul D.; Wightman, Nicholas; Huang, Mickey M.; Weiss, Alexandra; Sapp, Peter C.; Cuny, Gregory D.; Ivinson, Adrian J.; Glicksman, Marcie A.; Ferante, Robert J.; Matson, Wayne; Matson, Samantha; Brown, Robert H. Jr.Amyotrophic lateral sclerosis (ALS) is a fatal degenerative motor neuron disease. Approximately 20% of familial ALS cases are caused by mutations in the Cu/Zn superoxide dismutase (SOD1) gene. Rodents expressing mutant SOD1 transgenes develop progressive, fatal motor neuron disease and disease onset and progression is dependent on the level of SOD1. We investigated the possibility that a reduction in SOD1 protein may be of therapeutic benefit in ALS and screened 30,000 compounds for inhibition of SOD1 transcription. The most effective inhibitor identified was N-{4-[4-(4-methylbenzoyl)-1-piperazinyl]phenyl}-2-thiophenecarboxamide (Compound ID 7687685), which in PC12 cells showed an EC50 of 10.6 microM for inhibition of SOD1 expression and an LD50 >30 microM. This compound was subsequently shown to reduce endogenous SOD1 levels in HeLa cells and to exhibit a modest reduction of SOD1 protein levels in mouse spinal cord tissue. These data suggest that the efficacy of compound 7687685 as an inhibitor of SOD1 gene expression is not likely to be clinically useful, although the strategy reported could be applied broadly to screening for small molecule inhibitors of gene expression.Item A New Class of Small Molecule Inhibitor of BMP Signaling(PLoS One, 2013-04) Sanvitale, Caroline E.; Kerr, Georgina; Chaikuad, Apirat; Ramel, Marie-Christine; Mohedas, Augstin H.; Reichert, Sabine; Wang, You; Triffitt, James T.; Cuny, Gregory D.; Yu, Paul B.; Hill, Carolines S.; Bullock, Alex N.Growth factor signaling pathways are tightly regulated by phosphorylation and include many important kinase targets of interest for drug discovery. Small molecule inhibitors of the bone morphogenetic protein (BMP) receptor kinase ALK2 (ACVR1) are needed urgently to treat the progressively debilitating musculoskeletal disease fibrodysplasia ossificans progressiva (FOP). Dorsomorphin analogues, first identified in zebrafish, remain the only BMP inhibitor chemotype reported to date. By screening an assay panel of 250 recombinant human kinases we identified a highly selective 2-aminopyridine-based inhibitor K02288 with in vitro activity against ALK2 at low nanomolar concentrations similar to the current lead compound LDN-193189. K02288 specifically inhibited the BMP-induced Smad pathway without affecting TGF-? signaling and induced dorsalization of zebrafish embryos. Comparison of the crystal structures of ALK2 with K02288 and LDN-193189 revealed additional contacts in the K02288 complex affording improved shape complementarity and identified the exposed phenol group for further optimization of pharmacokinetics. The discovery of a new chemical series provides an independent pharmacological tool to investigate BMP signaling and offers multiple opportunities for pre-clinical development.Item A Novel Synthesis of Functionalized Oxazolidine-2,4-diones(2017-10-12) Duddupudi, Anantha; Vo, HienOxazolidinedione analogs have been found to be potent and selective Mineralocorticoid Receptor (MR) antagonists. The natural endogenous hormone Aldosterone activates MR leading to chronic kidney disease, hypertension and congestive heart failure via electrolyte imbalance.There is a significant need for the synthesis of Oxazolidinediones and related compounds. This motivated us to develop of a method for the synthesis of functionalized Oxazolidinediones that could be used in the preparation of pharmacologically active compounds.Item A Potential New Treatment Paradigm for Multiple Sclerosis(2017) Nikhar, Sameer; Degterev, Alexei; Cuny, Gregory D.This project was completed with contributions from Alexei Degterev from the Department of Developmental, Molecular and Chemical Biology, School of Medicine, Tufts University, Boston.Item AKAP12 Signalosome Regulates Cardiac β2-Adrenergic Receptor Signaling(2022-08) Qasim, Hanan; McConnell, Bradley K.; Bond, Richard A.; Schwartz, Robert J.; Cuny, Gregory D.; Li, NaHeart failure (HF) is a complex clinical syndrome, represented as an impairment in ventricular filling and myocardial blood ejection. As such, HF is one of the leading causes of death in the United States. An extensive network of intertwined intracellular signaling pathways regulates cardiac remodeling (a general determinant of HF). The ability of signalosomes (molecular signaling complexes) to compartmentalize several cellular pathways has been recently established. One example of these signalosomes is a family of scaffolding proteins known as, A Kinase Anchoring Proteins (AKAPs). In cardiomyocytes, the AKAP12 scaffolds a large signalosome that orchestrates spatiotemporal signaling through stabilizing pools of phosphatases and kinases. In addition, it is involved in regulating desensitization, downregulation, and recycling of β Adrenergic Receptors (βAR). Several AKAPs have been associated with cardiac function. AKAPs cardiac expression levels and intracellular distribution are altered under pathological conditions; however, AKAP12 cardiac roles are unclear. In this study, the critical roles of AKAP12 as a scaffold protein in mediating signaling downstream β2AR have been studied using overexpression and downregulation models. In vitro, on a molecular level, we showed that AKAP12 upregulation significantly reduced β2AR internalization and reduced intracellular cAMP levels downstream of the stimulated β2AR. Consequently, on a cellular level, we investigated how AKAP12 upregulation affected cardiomyocyte contractility. We observed that left ventricular cardiomyocytes overexpressing AKAP12 had significantly lower contractility as compared to cardiomyocytes expressing endogenous levels of AKAP12. In line with our in vitro data, we showed in vivo, significantly lower systolic cardiac function in the AKAP12 overexpression mice as compared to wild-type mice. Finally, we observed a significantly higher gene and protein expression of AKAP12 in patients with end-stage HF as compared to non-failing heart patients. Therefore, we conclude that AKAP12 plays a critical role in regulating cardiac contractility downstream β2AR and its upregulation deteriorates cardiac function.Item Akt Regulates TNF? Synthesis Downstream of RIP1 Kinase Activation during Necroptosis(PLoS One, 2013-03) McNamara, Colleen R.; Ahuja, Ruchita; Osafo-Addo, Awo D.; Barrows, Douglas; Kettenbach, Arminja; Skidan, Igor; Teng, Xin; Cuny, Gregory D.; Gerber, Scott A.; Degterev, AlexeiNecroptosis is a regulated form of necrotic cell death that has been implicated in the pathogenesis of various diseases including intestinal inflammation and systemic inflammatory response syndrome (SIRS). In this work, we investigated the signaling mechanisms controlled by the necroptosis mediator receptor interacting protein-1 (RIP1) kinase. We show that Akt kinase activity is critical for necroptosis in L929 cells and plays a key role in TNF? production. During necroptosis, Akt is activated in a RIP1 dependent fashion through its phosphorylation on Thr308. In L929 cells, this activation requires independent signaling inputs from both growth factors and RIP1. Akt controls necroptosis through downstream targeting of mammalian Target of Rapamycin complex 1 (mTORC1). Akt activity, mediated in part through mTORC1, links RIP1 to JNK activation and autocrine production of TNF?. In other cell types, such as mouse lung fibroblasts and macrophages, Akt exhibited control over necroptosis-associated TNF? production without contributing to cell death. Overall, our results provide new insights into the mechanism of necroptosis and the role of Akt kinase in both cell death and inflammatory regulation.Item Apratoxin D – A Formal Total Synthesis; Asymmetric Synthesis of all Four Stereoisomers of Mefloquine Hydrochloride; α-Alkylation and α-Arylation of N-alkoxyenamines and α-Epoxy-N-sulfonyl Hydrazones(2019-12) Elmore, Kyle Lee 1987-; Coltart, Don M.; May, Jeremy A.; Teets, Thomas S.; Do, Loi H.; Cuny, Gregory D.Apratoxin D, a potent inhibitor of cancer cell growth, particularly against H-460 human lung cancer cells, was synthesized for the first time by our lab in 2012. We endeavored to improve upon that synthetic route proposing an alternative strategy for a more flexible pathway to the polyketide fragment. Specifically, this allowed a modular process that could result in variable substituents at the C-37 carbon, which has been shown to be critical in the inhibitory activity of the apratoxins. In the first chapter, we present an effective strategy based off of an organocopper asymmetric allylation of α,ß-unsaturated-N-acyloxazolidinones, demonstrating good diastereoselectivity (9:1) and yield. Second, we present an asymmetric (er > 99:1) total synthesis of all four stereoisomers of mefloquine hydrochloride which are subsequently used for biological assays to evaluate its use as a TP53 readthrough enhancer. Mefloquine has shown effectiveness at enhancing TP53 readthrough alongside aminoglycoside G418, marking its potential in cancer treatment in cases of TP53 nonsense mutations. Finally, we present a new strategy for umpolung based functionalization of ketones and aldehydes through a copper catalyzed nucleophilic addition of Grignard reagents to N-alkoxyenamines. Classic enolate based α-functionalization of carbonyls is inherently limited due to the SN2 pathway of the transition state. Umpolung strategies allow access to a greater range of α-functionalized products. This was accomplished with as little as 30 mol % loading of CuBr•DMS with 77% yield, and in-situ removal of the 3,5-isoxazolidinone to reveal the ketone directly. Additionally, we expand upon our previously published umpolung strategy for α-arylation and α-alkylation of α-epoxy-N¬-sulfonyl hydrazones to aliphatic systems, demonstrating its effectiveness across a range of sp, sp2, sp3 hybridized Grignard nucleophiles with excellent diasteroselectivity (>25:1 syn:anti) and yield (64-89%). Classic enolate chemistry cannot produce the difficult to form α-substituted ß-hydroxy carbonyl compounds with α-quarternary centers. Our strategy can access these products with good diastereoselectivity (up to 9:1 syn:anti) and yield (36-80%). Additionally, we propose a mechanism for this reaction and support this with computational data accounting for the observed selectivity in both the unsubstituted and α-methyl substituted substrates.Item Azaaporphines: Creating A Novel Subclass of Aporphine Alkaloid Derivatives(2020-09-29) Martinez, AndreAporphine are a class of alkaloids that share a distinguishing rigid tetracyclic ring structure. While analogs of this class are found as natural products in a variety of different plant families, others are derived synthetically by researchers enticed by their broad biological properties. Properties ranging from being anti-cancer agents to treatment of advanced stage Parkinson’s disease. Hundreds of these alkaloids have been isolated and tested, however, there are no known examples of an aporphine which has a pyridine incorporated into its framework: an azaaporphine. After identifying two key intermediate compounds, we utilized a previously established reaction scheme to synthesize these stepping stones toward our final product. We have acquired and isolated these two intermediate compounds to construct the tetrahydroisoquinoline structure required for the final phenol arylation reaction that gives us an azaaporphine. Concurrently, we ran ligand similarity search within the Protein Data Base (PDB) to reveal potential biological targets of azaaporphines. We found that the enzyme phosphodiesterase 10A (PDE10A) has reported inhibitors similar to our compounds so we ran molecular docking simulations against it. This allowed us to determine binding affinity and several interactions between the enzyme and our compounds, revealing that PDE10A is a potential target of azaaporphines. In establishing the synthetic methodology of azaaporphine alkaloids, we are creating a novel sub-class of compounds with distinct properties and trailblazing a new pathway for others to explore and employ azaaporphines in new discoveries.Item BMP type I receptor inhibition reduces heterotopic ossification(Nature Medicine, 2010-03) Yu, Paul B.; Deng, Donna Y.; Lai, Carol S.; Hong, Charles C.; Cuny, Gregory D.; Bouxsein, Mary L.; Hong, Deborah W.; McManus, Patrick M.; Katagiri, Takenobu; Sachidanandan, Chetana; Kamiya, Nobuhiro; Tomokazu, Fukudu; Mishina, Yuji; Peterson, Randall T.; Bloch, Kenneth D.Fibrodysplasia ossificans progressiva (FOP) is a congenital disorder of progressive and widespread postnatal ossification of soft tissues1,2,3,4 and is without known effective treatments. Affected individuals harbor conserved mutations in the ACVR1 gene that are thought to cause constitutive activation of the bone morphogenetic protein (BMP) type I receptor, activin receptor-like kinase-2 (ALK2)5. Here we show that intramuscular expression in the mouse of an inducible transgene encoding constitutively active ALK2 (caALK2), resulting from a glutamine to aspartic acid change at amino acid position 207, leads to ectopic endochondral bone formation, joint fusion and functional impairment, thus phenocopying key aspects of human FOP. A selective inhibitor of BMP type I receptor kinases, LDN-193189 (ref. 6), inhibits activation of the BMP signaling effectors SMAD1, SMAD5 and SMAD8 in tissues expressing caALK2 induced by adenovirus specifying Cre (Ad.Cre). This treatment resulted in a reduction in ectopic ossification and functional impairment. In contrast to localized induction of caALK2 by Ad.Cre (which entails inflammation), global postnatal expression of caALK2 (induced without the use of Ad.Cre and thus without inflammation) does not lead to ectopic ossification. However, if in this context an inflammatory stimulus was provided with a control adenovirus, ectopic bone formation was induced. Like LDN-193189, corticosteroid inhibits ossification in Ad.Cre-injected mutant mice, suggesting caALK2 expression and an inflammatory milieu are both required for the development of ectopic ossification in this model. These results support the role of dysregulated ALK2 kinase activity in the pathogenesis of FOP and suggest that small molecule inhibition of BMP type I receptor activity may be useful in treating FOP and heterotopic ossification syndromes associated with excessive BMP signaling.Item Carboetomidate: A Pyrrole Analog of Etomidate Designed Not to Suppress Adrenocortical Function(Anesthesiology, 2011-03) Cotten, Joseph F.; Forman, Stuart A.; Laha, Joydev K.; Cuny, Gregory D.; Husain, Shaukat; Miller, Keith W.; Nguyen, Hieu H.; Kelly, Elizabeth W.; Stewart, Deirdre; Liu, Aiping; Raines, Douglas E.Background: Etomidate is a sedative-hypnotic that is often used in critically ill patients because it provides superior hemodynamic stability. However it also binds with high affinity to 11?-hydroxylase, potently suppressing synthesis of steroids by the adrenal gland that are necessary for survival. We report the results of studies to define the pharmacology of (R)-ethyl 1-(1-phenylethyl)-1H-pyrrole-2-carboxylate (carboetomidate), a pyrrole analogue of etomidate specifically designed not to bind with high affinity to 11?-hydroxylase. Methods: The hypnotic potency of carboetomidate was defined in tadpoles and rats using loss of righting reflex assays. Its ability to enhance wild-type ?1?2?2L and etomidate-insensitive mutant ?1?2(M286W)?2L human ?-aminobutyric acid type A receptor activities was assessed using electrophysiological techniques. Its potency for inhibiting in vitro cortisol synthesis was defined using a human adrenocortical cell assay. Its effects on in vivo hemodynamic and adrenocortical function were defined in rats. Results: Carboetomidate was a potent hypnotic in tadpoles and rats. It increased currents mediated by wild-type, but not etomidate-insensitive mutant ?-aminobutyric acid type A receptors. Carboetomidate was three orders of magnitude less potent an inhibitor of in vitro cortisol synthesis by adrenocortical cells than was etomidate. In rats, carboetomidate caused minimal hemodynamic changes and did not suppress adrenocortical function at hypnotic doses. Conclusions: Carboetomidate is an etomidate analogue that retains many of etomidate’s beneficial properties, but is dramatically less potent as an inhibitor of adrenocortical steroid synthesis. Carboetomidate is a promising new sedative-hypnotic for potential use in critically ill patients in whom adrenocortical suppression is undesirable.Item Carbon−Hydrogen Bond Functionalization using Removable Monodentate Directing Groups(2020-08) kwak, sehun; Daugulis, Olafs; Gilbertson, Scott R.; Cai, Chengzhi; Chen, Tai-Yen; Cuny, Gregory D.Chapter 1: 3,5-Dimethylpyrazole was employed as a mono-dentate directing group for palladium-catalyzed ortho-sp2 C−H arylation with aryl iodides. The reaction shows good functional group tolerance and outstanding selectivity for mono-ortho-arylation. Ozonolysis of ortho-arylated arylpyrazoles gave acylated biphenylamines that were further arylated to afford unsymmetrically substituted 2,6-diarylacetanilides. Chapter 2: N-Aminopyridinium ylides were used as mono-dentate directing groups for copper-promoted C−H/N−H coupling of sp2 C−H bonds with pyrazoles, imidazoles, and sulfonamides. Reactions proceed in fluorinated alcohol solvents at elevated temperatures and require use of 1.3−3 equiv of copper(II) acetate. This appears to be the first method for copper-promoted C−H/N−H coupling directed by a removable monodentate auxiliary in absence of added ligands. Chapter 3: N-Aminopyridinium ylides are competent monodentate directing groups for cobalt-catalyzed annulation of sp2 C−H bonds with internal alkynes. Pyridine moiety in ylide serves as an internal oxidant and is cleaved during the reaction. The annulation reactions possess excellent compatibility with heterocyclic substrates, tolerating furan, thiophene, pyridine, pyrrole, pyrazole, and indole functionalities.Item Carboxylic Acid Synthesis from Carbon Dioxide via First-Row Transition-Metal Catalysis and Copper-Promoted C(SP3)–H Functionalization(2015-12) Tran, Hung Vu 1982-; Daugulis, Olafs; May, Jeremy A.; Thummel, Randolph P.; Guloy, Arnold M.; Cuny, Gregory D.A general method for direct synthesis of aromatic carboxylic acids from aryl iodides and carbon dioxide was developed in our research group. The method uses copper(I) iodide as a catalyst in presence of inexpensive bidentate amine ligands, at mild temperatures under atmospheric pressure of carbon dioxide. The mild reaction conditions allow unparalleled reaction scope with excellent functional group tolerance. The compatible functional groups include bromide, chloride, fluoride, ester, ether, and carbonyl functional groups. Moreover, aryl iodides bearing unprotected amino and hydroxyl groups were also carboxylated for the first time. The methods works well even for hindered aryl iodides. A reaction mechanism was proposed based on the identification of copper(0) species involvement. Additionally, a method for cobalt-catalyzed direct carboxylation of allyl chlorides with carbon dioxide has been developed. The reactions work well at very mild conditions allowing highly selective synthesis of branched β,γ-unsaturated carboxylic acids. Furthermore, a method for copper-mediated oxidative cross-coupled annulation of non-activated C(sp3)–H bonds with terminal alkynes has been developed. The reaction uses a removable auxiliary and an inexpensive copper promoter in the presence of base and silver salt oxidant. This approach is highly useful for 5-methylene-pyrrolidin-2-one synthesis. Moreover, the reaction conditions for copper-catalyzed nitronate and enolate homodimerizations under an oxygen atmosphere were optimized. The control experiments were performed to confirm the role of catalyst and oxygen.Item Characterization of the 8-hydroxyquinoline scaffold for inhibitors of West Nile virus serine protease(Antiviral Research, 2013-04) Ezgimen, Manolya; Lai, Huiguo; Mueller, Niklaus H.; Lee, Kyungae; Cuny, Gregory D.; Ostrov, David A.; Padmanabhan, RadhakrishnanWest Nile virus (WNV) is a mosquito-borne member of flaviviruses that causes significant morbidity and mortality especially among children. There is currently no approved vaccine or antiviral therapeutic for human use. In a previous study, we described compounds containing the 8-hydroxyquinoline (8-HQ) scaffold as inhibitors of WNV serine protease (NS2B/NS3pro) in a high throughput screen (HTS) using the purified WNV NS2B/NS3pro as the target. In this study, we analyzed potencies of some commercially available as well as chemically synthesized derivatives of 8-HQ by biochemical assays. An insight into the contribution of various substitutions of 8-HQ moiety for inhibition of the protease activity was revealed. Most importantly, the substitution of the N1 of the 8-HQ ring by –CH– in compound 26 significantly reduced the inhibition of the viral protease by this naphthalen-1-ol derivative. The kinetic constant (Ki) for the most potent 8-HQ inhibitor (compound 14) with an IC50 value of 2.01 ± 0.08 ?M using the tetra-peptide substrate was determined to be 5.8 ?M. This compound inhibits the WNV NS2B/NS3pro by a competitive mode of inhibition which is supported by molecular modeling.Item Columnar Organization of Partially Fluorinated Arylene Ethynylene Macrocycles(2022-12-01) Karki, Sumitra; Miljanić, Ognjen Š.; Daugulis, Olafs; Lee, T. Randall; Zastrow, Melissa L.; Cuny, Gregory D.Porous molecular crystals have been proving themselves as an excellent emerging class of porous material not just in the solid state but also in liquid forms, as a porous liquid. They are composed of discrete molecules which hold the intrinsic pores or create extrinsic pores via weak non-covalent intermolecular interactions. Arylene ethynylene macrocycle belongs to the class porous molecular crystal, and they can be designed to be shape persistent as well as of different shape, sizes, and functionalities. In addition, these macrocyclic compounds self-aggregate via [π···π] interaction to form higher ordered material. This dissertation presents the syntheses of partially fluorinated arylene ethynylene macrocycles and their crystallographic study to understand the influence of fluoroarene rings on their self-aggregation properties. Chapter 1 introduces porous molecular crystal and their emerging development into porous liquids, as well as the arylene ethynylene macrocycle and their self-aggregation and liquid crystalline properties. Chapter 2 includes the synthesis of diamond-shaped partially fluorinated macrocycles with alternating arene and fluoroarene bridged by ethynylene units. This chapter presents the role of π-interactions between the fluoroarene and the alkyne moieties to direct the columnar organization of the macrocycles. Chapter 3 presents the designed pentagonally and hexagonally partially fluorinated macrocycles and their crystallographic studies to understand the intermolecular interactions leading to the formation of higher ordered material. Chapter 4 presents the idea and design of the partially fluorinated arylene ethynylene macrocyclic compound to create porous liquid crystals.Item Design and Synthesis of Proline-based NHC & Phosphine Bidentate Ligands and Their Applications in Asymmetrical Catalysis, Diastereoselective Synthesis of 1,3-disubstituted Isoindolines & Sultams via Brønsted Acid Catalysis(2018-08) Tao, Ye 1990-; Gilbertson, Scott R.; May, Jeremy A.; Thummel, Randolph P.; Xu, Shoujun; Cuny, Gregory D.Proline-based chiral NHC-oxazoline, NHC-phosphine, phosphine-sulfoxide and chiral NHC-pyridine bidentate ligands were designed and synthesized in Chapters 1 & 2. These ligands were complexed with Rh and Pd and utilized as catalysts for various reactions. A wide range of reactivities and selectivities were obtained. The Brønsted acid (Tf2NH) catalyzed intramolecular hydroamidation of terminal alkynes was described in Chapter 3. The combination of Et3SiH and Tf2NH provide cis-disubstituted isoindolines and sultams in high yield and high diastereoselectivity.Item Design, Synthesis, and Biological Evaluation of Novel Hetero-Bivalent Ligands Targeting Kinases(2020-05) Kedika, Samanthreddy; Udugamasooriya, D. Gomika; Cuny, Gregory D.; Ruan, Ke-He; Briggs, James M.; Young, Damian W.Hetero-bivalent ligands consist of two copies of monomers that are connected through a linker. They show improved affinity towards their target through an avidity effect and can also enhance specificity, as part of the binding pocket may be absent in a similar target. This strategy was successful in yielding potent and specific kinase inhibitors by simultaneously targeting ATP and secondary peptide binding pockets. We innovatively apply this concept in two different kinase systems where we link low-affinity ATP binding pocket targeted small molecule PP2 with natural peptides that recognize secondary binding sites to produce potent and or specific hetero-bivalent ligands. In the first application, we targeted Extra-cellular Regulated Kinase5 (ERK5) that is known to play key roles in maintaining cancer stem cell signaling in a variety of cancers. The conventional ATP binding site inhibitors have not yet yielded expected levels of anti-cancer effects, due to complexities in converting ERK5 activation into Cancer Stem Cell (CSC) biological effects. Therefore, we hypothesized that designing a hetero-bivalent ligand, which simultaneously blocks a unique regulatory peptide interaction involved in upstream ERK5 kinase activation, and the conventional ATP binding pocket, produces stronger CSC biological effects. The active hetero-bivalent ligand ERK5.1 (i). inhibited ERK5 activation and kinase activity (IC50~8.5 µM) simultaneously, in two independent assay systems (ii). inhibited CSC activities, such as colony formation, migration and cell proliferation (IC50~6.5 µM) In the second application, we expanded the idea of using peptide sequences belonging to the same protein as the non-ATP binding site moiety in hetero-bivalent ligand design. Typically, these secondary peptide sequences are derived from substrate peptides from interacting proteins or structure based or combinatorial methods. The use of an already existing natural peptide sequence of the same kinase that uniquely interacts with a binding pocket within the same kinase as the non-ATP binding moiety in hetero-bivalent ligand design has never been investigated. We selected EphA3 kinase as a model system because of known natural peptide sequence in the SAM domain linker region, which turns back and binds to the bottom of EphA3. We connected this unique sequence to a PP2 analogue. Using a combination of structure and combinatorial approaches, we optimized the much longer linker (57 Å) to yield potent hetero-bivalent ligand EPHB2.3 (Kd~250 nM). We also report our effort to the convert the linker part of EPHB2.3 to gain additional binding affinity.Item Designed inhibitors of insulin-degrading enzyme regulate the catabolism and activity of insulin(PLoS ONE, 5/7/2010) Leissring, Malcolm A.; Malito, Enrico; Hedouin, Sabrine; Reinstatler, Lael; Sahara, Tomoko; Abdul-Hay, Samer O.; Choudhry, Shakeel; Maharvi, Ghulam M.; Fauq, Abdul H.; Huzarska, Malwina A.; May, Philip S.; Choi, Sungwoon; Logan, Todd P.; Turk, Benjamin E.; Cantley, Lewis C.; Manolopoulou, Marika; Tang, Wei-Jen; Stein, Ross L.; Cuny, Gregory D.; Selkoe, Dennis J.Background- Insulin is a vital peptide hormone that is a central regulator of glucose homeostasis, and impairments in insulin signaling cause diabetes mellitus. In principle, it should be possible to enhance the activity of insulin by inhibiting its catabolism, which is mediated primarily by insulin-degrading enzyme (IDE), a structurally and evolutionarily distinctive zinc-metalloprotease. Despite interest in pharmacological inhibition of IDE as an attractive anti-diabetic approach dating to the 1950s, potent and selective inhibitors of IDE have not yet emerged. Methodology/Principal Findings- We used a rational design approach based on analysis of combinatorial peptide mixtures and focused compound libraries to develop novel peptide hydroxamic acid inhibitors of IDE. The resulting compounds are ?106 times more potent than existing inhibitors, non-toxic, and surprisingly selective for IDE vis-à-vis conventional zinc-metalloproteases. Crystallographic analysis of an IDE-inhibitor complex reveals a novel mode of inhibition based on stabilization of IDE's “closed,” inactive conformation. We show further that pharmacological inhibition of IDE potentiates insulin signaling by a mechanism involving reduced catabolism of internalized insulin. Conclusions/Significance The inhibitors we describe are the first to potently and selectively inhibit IDE or indeed any member of this atypical zinc-metalloprotease superfamily. The distinctive structure of IDE's active site, and the mode of action of our inhibitors, suggests that it may be possible to develop inhibitors that cross-react minimally with conventional zinc-metalloproteases. Significantly, our results reveal that insulin signaling is normally regulated by IDE activity not only extracellularly but also within cells, supporting the longstanding view that IDE inhibitors could hold therapeutic value for the treatment of diabetes.