Browsing by Author "Carlier, Paul R."

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    1,3-Disubstituted-tetrahydro-β-carbolines: A New Method for Stereochemical Assignment and Synthesis of Potential Antimalarial Agents
    Cagasova, Kristyna (Virginia Tech, 2021-06-21)
    Malaria is a serious mosquito-borne disease affecting the majority of Earth's southern hemisphere. While consistent efforts to curb malaria spread throughout 20th and early 21st century were largely successful, the recent rise in resistance to antimalarial treatments resulted in an increasing incidence rate and stalling mortality rate. This trend clearly signifies the need for the development of novel antimalarial agents able to circumvent current drug-resistance mechanisms. In 2014, in collaboration with Prof. Maria Belen Cassera from the University of Georgia, our group found that compound 1a (1R,3S-MMV008138), discovered from the publicly available Malaria Box, targets an essential biosynthetic pathway (MEP pathway) of malaria-causing parasite Plasmodium falciparum. Analogs of 1a synthesized in our laboratory were found effective against multi-resistant Dd2 strain of P. falciparum which, together with an absence of MEP pathway in humans, suggests that potent analogs of 1a may be safe and efficient antimalarial drug candidates. The initial bioassay studies determined that only one of four possible MMV008138 stereoisomers satisfactorily inhibits the target PfIspD enzyme. Thus a secure determination of stereochemistry in 1a analogs was of utmost importance to the structure-activity relationship studies performed in our group. The second chapter of this work discusses the validation of the previously known empirical stereoassignment method based on analysis of relative shift of 13C NMR resonances between cis and trans diastereomers and compares it to a new method based on 3JHH coupling constants developed in our laboratory. We demonstrate that the new method relying on the analysis of 1H-1H coupling is reliable over large samples of experimental data and suitable even when only a single diastereomer is produced in the synthetic process. Importantly, the origin of 3JHH coupling constants is well understood, unlike the source of relative differences in 13C NMR shifts observed in the older method. The empirical observations for both stereoassignment methods are supported by extensive density-functional theory calculations, which validate the new 1H-1H coupling-based assignment but do not provide a conclusive explanation for the origin of the 13C NMR-based method. In the third chapter, we discuss the replacement of the carboxylic acid moiety in 1a by alternative functional groups promising improved toxicity and bioavailability profile. The total synthesis of tetrazole (trans-23a) and phosphonic acid ((±)-62a) derivatives of 1a is discussed in detail. The tetrazole analog 23a was previously synthesized in the Carlier group as a diastereomeric mixture of cis and trans isomers (dr = 3:7), and it was tested for growth inhibition of multi-resistant P. falciparum with promising results. Later, the synthesis was revisited to obtain a stereochemically pure sample of trans-23a, which was expected to show improved potency compared to the original sample. Furthermore, the synthesis of pure trans-23a confirmed the accuracy of the previous assignment of cis and trans diastereomers in the mixture. Unfortunately, neither analog showed an improvement in potency relative to 1a.
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    Acetylcholinesterase of the sand fly, Phlebotomus papatasi (Scopoli): construction, expression and biochemical properties of the G119S orthologous mutant
    Temeyer, Kevin B.; Tong, Fan; Totrov, Maxim M.; Tuckow, Alexander P.; Chen, Qiao-Hong; Carlier, Paul R.; Pérez de León, Adalberto A.; Bloomquist, Jeffrey R. (2014-12-10)
    Background Phlebotomus papatasi vectors zoonotic cutaneous leishmaniasis. Previous expression of recombinant P. papatasi acetylcholinesterase (PpAChE1) revealed 85% amino acid sequence identity to mosquito AChE and identified synthetic carbamates that effectively inhibited PpAChE1 with improved specificity for arthropod AChEs compared to mammalian AChEs. We hypothesized that the G119S mutation causing high level resistance to organophosphate insecticides in mosquitoes may occur in PpAChE1 and may reduce sensitivity to inhibition. We report construction, expression, and biochemical properties of rPpAChE1 containing the G119S orthologous mutation. Methods Targeted mutagenesis introduced the G119S orthologous substitution in PpAChE1 cDNA. Recombinant PpAChE1 enzymes containing or lacking the G119S mutation were expressed in the baculoviral system. Biochemical assays were conducted to determine altered catalytic properties and inhibitor sensitivity resulting from the G119S substitution. A molecular homology model was constructed to examine the modeled structural interference with docking of inhibitors of different classes. Genetic tests were conducted to determine if the G119S orthologous codon existed in polymorphic form in a laboratory colony of P. papatasi. Results Recombinant PpAChE1 containing the G119S substitution exhibited altered biochemical properties, and reduced inhibition by compounds that bind to the acylation site on the enzyme (with the exception of eserine). Less resistance was directed against bivalent or peripheral site inhibitors, in good agreement with modeled inhibitor docking. Eserine appeared to be a special case capable of inhibition in the absence of covalent binding at the acylation site. Genetic tests did not detect the G119S mutation in a laboratory colony of P. papatasi but did reveal that the G119S codon existed in polymorphic form (GGA + GGC). Conclusions The finding of G119S codon polymorphism in a laboratory colony of P. papatasi suggests that a single nucleotide transversion (GGC → AGC) may readily occur, causing rapid development of resistance to organophosphate and phenyl-substituted carbamate insecticides under strong selection. Careful management of pesticide use in IPM programs is important to prevent or mitigate development and fixation of the G119S mutation in susceptible pest populations. Availability of recombinant AChEs enables identification of novel inhibitory ligands with improved efficacy and specificity for AChEs of arthropod pests.
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    Activation of diboron reagents: The development of mild conditions for the synthesis of unique organoboron compounds
    Thorpe, Steven Brandon (Virginia Tech, 2012-03-23)
    The first successful synthesis and isolation of a boronic acid was reported in 1860 by Frankland in the pursuit of novel organometallic compounds. For more than a century, further studies of boronic acids were sparsely published. Suzuki and Miyaura jumpstarted the field in 1979 with an innovative carbon-carbon bond forming reaction employing an organoboronic acid and a carbon halide under palladium catalysis. Indeed, the Nobel Prize in Chemistry was awarded to Professor Akira Suzuki, along with Professors Richard Heck and Ei-ichi Negishi, in 2010 for their important contributions in palladium-catalyzed cross-coupling chemistry. Over the last 30 years, reports on organoboron compounds have increased exponentially. This dissertation describes the author's contributions to the development of preparative methods for organoboronic acid derivatives using transition metal-catalyzed reactions of diboron reagents. A unique "mixed" diboron reagent was developed (PDIPA diboron) that contains sp2- and sp3-hybridized boron atoms, unambiguously confirmed by X-ray crystallography. PDIPA diboron is sufficiently activated internally through a dative-bonding amine to selectively transfer the sp2-hybridized boron regioselectively, in the presence of copper, to electron deficient alkenes including α,β-unsaturated ketones, esters, amides, aldehydes, and nitriles to provide the corresponding boratohomoenolates. A unique β,β-diboration of an α,β-acetylenic ketone was also discovered. The scope of PDIPA diboron reactions was then expanded to a set of substrates with a more complex structural backbone. Allenoates are α,β,γ-unsaturated esters with orthogonal pi systems, which pose several possible difficulties with the regioselectivity of addition, not to mention known isomerizations catalyzed by copper. However, we successfully installed the boron moiety regioselectively on the β-carbon of a variety of allenoates, providing a vinyl boronic ester, and also observed exclusive formation of the (Z)-isomer from racemic starting materials. The resulting vinyl boronic ester was then shown to be an excellent Suzuki-Miyaura cross-coupling partner, affording a diastereopure, trisubstituted alkene in quantitative yield. Commercially available bis(pinacolato)diboron has shown remarkable stability towards hydrolysis and autoxidation. Using this reagent, we developed a copper- and amine-catalyzed boration protocol performed entirely in water and open to air. Using only 1 mol% copper, extraordinary activity was observed. UV-Vis, 11B NMR, and solvent kinetic isotope experiments were employed to gain insight into the mechanism, which showed the possibility of autocatalysis. Attempts to control stereoselectivity were not successful, although these results were rationalized by a dynamic catalyst structure.
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    Amine compounds and inhibiting neurotransmitter reuptake
    (United States Patent and Trademark Office, 2007-05-08)
    The invention relates to amine compounds as well as methods and materials involved in modulating neurotransmitter reuptake. Specifically, the invention provides amine compounds, methods for synthesizing amine compounds, and methods for inhibiting neurotransmitter reuptake.
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    Amine compounds and inhibiting neurotransmitter reuptake
    (United States Patent and Trademark Office, 2004-03-02)
    The invention relates to amine compounds as well as methods and materials involved in modulating neurotransmitter reuptake. Specifically, the invention provides amine compounds, methods for synthesizing amine compounds, and methods for inhibiting neurotransmitter reuptake.
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    Amine compounds and inhibiting neurotransmitter reuptake
    (United States Patent and Trademark Office, 2005-07-05)
    The invention relates to amine compounds as well as methods and materials involved in modulating neurotransmitter reuptake. Specifically, the invention provides amine compounds, methods for synthesizing amine compounds, and methods for inhibiting neurotransmitter reuptake.
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    Analysis of Biogenic Amines by GC/FID and GC/MS
    Nakovich, Laura (Virginia Tech, 2003-07-16)
    Low levels of biogenic amines occur naturally, but high levels (FDA sets 50 ppm of histamine in fish as the maximum allowable level) can lead to scombroid poisoning. Amines in general are difficult to analyze by Gas Chromatography (GC) due to their lack of volatility and their interaction with the GC column, often leading to significant tailing and poor reproducibility. Biogenic amines need to be derivatized before both GC and HPLC analyses. The objective of this research was to develop a relatively fast, reproducible method to derivatize and quantitate biogenic amines in fish at trace levels using GC/FID. The derivatizing reagent used in the experiments was propyl chloroformate, useful for aqueous samples. To confirm the identity of six derivatized biogenic amines GC/MS was used. To our knowledge no reference spectra for these derivatives has been published. It was concluded that best results are obtained using a Cold-On-Column (C.O.C.) inlet with a short column (15 meters), thick film stationary phase (ZB-5, 1.00μm df), and with recommendations to cut 40 cm from the inlet end of the column every 25 injections when using C.O.C. Duplicate samples of Atlantic Salmon were analyzed on days 0, 3, and 5. Levels of histamine were below 50 ppm for days 0 and 3, but day 5 showed average levels of 160 pm (cadaverine), 1000 ppm (histamine), and 350 ppm (tyramine). Good precision of six amine stardards at 50 ppm was shown: heptylamine 5.2%, putrescine 5.6%, cadaverine 5.0%, histamine 9.9%, tyramine 5.1%, and spermidine 6.2% RSD.
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    Antimalarial Agents: New Mechanisms of  Actions for Old and New Drugs
    Ghavami, Maryam (Virginia Tech, 2018-06-29)
    Worldwide, malaria is one of the deadliest diseases. In 2016 it sickened 216 million people and caused 445,000 deaths. In order to control the spread of this deadly diseases to human, we can either target the mosquito vector (Anopheles gambiae) or the parasite (Plasmodium falciparum). Due to recent emergence of resistance to current insecticides and antimalarial drugs there is a pressing need to discover and develop new agents that engage new targets in these organisms. To circumvent the effect of resistance to pyrethroid insecticides on the efficacy of insecticide treated nets (ITNs), the use of acetylcholinesterase (AChE) inhibitors on ITNs has drawn attention. In the first project, we explored a small library of γ- substituted oxoisoxazole- 2(3H)-carboxamides and isoxazol-3-yl carbamates, and nitriles as AChE inhibitors targeting wild- type (G3) and resistant (Akron) An. gambiae mosquito. In total 23 compounds were synthesized and evaluated. Both carbamates and carboximides with a 2-cyclopropylethyl side chain (1-87a and 1-88a) were extremely toxic to Akron mosquitos, yet these compounds did not exhibit appreciable selectivity between human and An. gambiae AChE. Unfortunately, none of the nitriles showed appreciable toxicity to G3 strain of the mosquitoes, nor did they inhibit An. gambiae AChE. In the second project, conducted in collaboration with Professor Michael Klemba, we focused on the mode of action of an established antimalarial drug, Mefloquine (MQ). Dr. Klemba's recently developed amino acid efflux assay was used to determine the effect of MQ and its open-ring analogs on hemoglobin endocytosis and catabolism in P. falciparum-infected erythrocytes. In total 26 MQ analogs were synthesized and 18 were studied in depth to determine their potency to inhibit leucine (Leu) efflux and parasite growth (SYBR Green). An excellent correlation (R² = 0.98) over nearly 4 log units was seen for these 18 compounds in the two assays. These data are consistent with the hypothesis that the antimalarial action of these compounds principally derives from inhibition of hemoglobin endocytosis. After this observation, a number of photo-affinity probes were designed and synthesized in hopes of isolating the molecular target of MQ. These analogs are currently being used by Dr. Klemba in pull-down experiments. In the third project, conducted in collaboration with Professor Belen Cassera, we sought to optimize a new antimalarial drug lead that would circumvent current resistance mechanisms. In Plasmodium parasites, the methylerythritol phosphate (MEP) pathway is known to be essential for its growth. This pathway is absent in humans, presenting the opportunity to develop potentially safe and effective therapeutic candidates. Previous work in the Cassera and Carlier lab had established that MMV008138 was the only compound in the Malaria Box that targeted the MEP pathway and that it was (1R,3S)-configured. My research expanded previous efforts in the Carlier group and produced synthesis of 73 analogs of MMV008138 (3-21a'1) that were tested for growth inhibition. These analogs featured variation at the A-, B-, C- and D-ring. In the process, a novel Pictet-Spengler ring expansion reaction of ortho-substituted acetphenones was discovered. The ring-expanded products were identified by means of 1D and 2D NMR experiments, HRMS, and X-ray crystallography. Among the 73 analogs prepared, four compounds showed similar growth inhibition potency to the lead 3-21a'1. In particular, the methoxyamide 3-80a, and the fluorinated A-ring analogs 3-124a, 3-124c and 3-124d all showed excellent (500-700 nM) growth IC₅₀ values against P. falciparum. All four showed full rescue upon co-application of IPP (200 μM), confirming that they target the MEP pathway. ADME-Tox evaluation of these new analogs will soon be underway.
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    Antiproliferative Natural Products from the Madagascar Rainforest
    Hou, Yanpeng (Virginia Tech, 2009-09-09)
    As part of an International Cooperative Biodiversity Groups (ICBG) program and a continuing search for anticancer natural products from the Madagascar rainforest, twenty extracts from Madagascar were selected for investigation based on their antiproliferative activity. Bioassay-guided fractionation of five of the extracts yielded sixteen new compounds, and their structures were determined using a combination of 1D and 2D NMR experiments, including COSY, HSQC/HMQC, HMBC, and ROESY/NOESY sequences, mass spectrometry, and chemical conversion. In addition, ten known compounds were obtained from five of the extracts. Studies on the remaining extracts were suspended due to various reasons. A multi-step synthesis of the sesquiterpenoid, (7R*)-opposite-4(15)-ene-1beta,7-diol, was also described. The first chapter of this dissertation reviews the new compounds isolated from Malagasy plants and marine organism in the last two decades. Chapters II to VI discuss the isolation, structure elucidation and bioactivities of new compounds from Scutia myrtina, Cordyla madagascariensis ssp. madagascariensis, Elaeodendron alluaudianum, Cassipourea lanceolata, and Sclerocarya birrea subsp. caffra. Chapter VII describes the synthesis and bioactivity of the sesquiterpenoid,(7R*)-opposite-4(15)-ene-1beta,7-diol. The isolation of known compounds is discussed briefly in the last chapter.
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    Biological and biochemical characterization of the extracellular signal-regulated kinase 8  homolog (TbERK8) in Trypanosoma brucei
    Valenciano Murillo, Ana Lisa (Virginia Tech, 2016-05-02)
    Trypanosoma brucei species are vector-borne protozoan parasites that cause Human African typanosomiasis (HAT) and nagana in cattle. In humans, the diseases caused by these parasites are fatal if left untreated. Treatments for these diseases are complicated because the approved drugs for treatment are ineffective against the parasites and have many toxic side effects associated with their use. There is a clear need to identify new therapeutics that are less toxic and more effective against T. brucei. Our approach for identifying new therapies is to identify novel targets in the parasite that can be modulated by small molecules. The mitogen-activated protein kinases (MAPK) pathway is a three-tiered signaling cascade that regulates cell responses to stimuli and are involved in essential processes. MAPKs can regulate differentiation, virulence, apoptosis, cell cycle and gene expression, which makes MAPKs interesting drug targets in T. brucei. The extracellular-signal regulated kinase 8 homolog in T. brucei (TbERK8) is essential for survival in bloodstream form T. brucei. The work in this dissertation involves characterizing this T. brucei MAPK to better understand its biological function and identify small molecules that can inhibit its activity to kill the parasite. Here, we report that TbERK8 is an atypical MAPK kinase that is able to autophosphorylate and no upstream kinases that activate TbERK8 have been identified. We have demonstrated that TbERK8 is able to phosphorylate the proliferating cell nuclear antigen homolog in T. brucei (TbPCNA). This is in contrast to the reported function the human ERK8 and PCNA homologs that form a stable complex in normal breast cells which does not result in PCNA phosphorylation. We also report here that TbPCNA is phosphorylated on three residues localized to a unique insertion loop by TbERK8. TbPCNA is tightly regulated in the parasites such that either upregulating or downregulating its expression arrests T. brucei proliferation. Although, this mechanism of phosphorylation is unique to TbPCNA, the role that such phosphorylation has in regulating TbPCNA is not known. Finally, we have identified small molecules that can selectively inhibit either TbERK8 or HsERK8, demonstrating that TbERK8 can be selectively inhibited to kill the parasite. The unique properties of TbERK8 can be exploited by small molecules that can be developed into new parasite-specific therapies that kill T. brucei with fewer side effects to the patients.
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    Boron-Mediated Semireduction of Alkynoic Acid Derivatives
    Grams, Robert Justin (Virginia Tech, 2021-04-30)
    Organoboron compounds are commonly used precursors for a variety of reactions in organic synthesis as is exemplified by the Suzuki-Miyaura cross-coupling, which is ubiquitous in industry and academia. Additionally, the Chan-Evans-Lam cross-coupling, lithiation-homologation, allylboration, and many other reactions rely on boron to achieve otherwise difficult chemical transformations. Thus, developing novel methods towards the regio- and/or stereoselective installation of boron into organic molecules remain important for designing new drugs. Boron reagents are also useful in chemical transformations that do not ultimately install a boron moiety on the organic molecule. We have developed several methods that achieve the trans-selective borylation or semireduction of internal alkynes, a process dominated by transition metals and often results in incomplete (E)-stereoselectivity. This dissertation describes three novel uses of a diboron reagent or pinacolborane that reduce propiolic acid derivates selectively to (E)-alkenes and one method that installs pinacolatoboron yielding exclusively (E)-β-borylacrylamides. We investigated the trans-selective hydroboration of primary and secondary propiolamides as reports in the literature accomplish trans-hydroboration via transition metal catalysis, which are limited in substrate scope. We discovered that addition of n-butyllithium to propiolamide and pinacolborane exclusively yields (E)-β-borylacrylamides in good to excellent yield. During the reaction, deprotonation of the amide with a strong base generates an alkoxide that coordinates to pinacolborane and forms a boronate complex. Upon warming to room temperature, a hydride transfer is directed to the α-carbon generating a β-carbanion that subsequently captures boron. Workup protonates the amide, furnishing the (E)-β-borylacrylamide product. As a follow-up from the trans-hydroboration of propiolamides described above, we developed a complementary semireduction of primary and secondary propiolamides. In this reaction, addition of catalytic amounts of potassium tert-butoxide to propiolamides generates an alkoxide that coordinates to pinacolborane and rapidly produces (E)-cinnamamides in 35 – 96% yield and >99:1 E:Z stereoselectivity. This reaction effectively reduces an internal alkyne to afford a product with trans geometry. A deuterium labeling study provided mechanistic insight for the transformation suggesting that the β-proton in the products is derived from the amide nitrogen of the propiolamide. Further, we demonstrated the utility of this reaction by augmenting the total synthesis of FK866, a potent nicotinamide mononucleotide adenyltransferase (NMNAT) inhibitor, and isolated the cinnamamide product in good yield with >99:1 E:Z stereoselectivity. Using a similar strategy, we investigated the ability of bis(pinacolato)diboron and base to mediate the α-borylation of propiolic acids. We observed the formation of a semireduced product, i.e., cinnamic acid. In the presence of a crown ether and cesium carbonate, propiolic acid is deprotonated and activates bis(pinacolato)diboron. Subsequent α-borylation and β-carbon protonation affords a highly unstable α-boronic acid derivative that undergoes rapid protodeborylation yielding predominantly (E)-cinnamic acids. The dual role of the carboxylate in activating the diboron reagent and directing α-borylation was unprecedented and is the first transition metal-free method to reduce propiolic acids to (E)-cinnamic acids. In contrast with boron activation from in situ generated alkoxide, an alternative approach is using phosphine catalysts—a major interest in the development of transition metal-free methodology. As such, we investigated the ability of phosphines to mediate the borylation of primary and secondary propiolamides. Surprisingly, we observed an efficient semireduction of primary and secondary propiolamides to their respective (E)-cinnamamides in the presence of catalytic n-tributylphosphine and stoichiometric pinacolborane. We surveyed the scope and reactivity of substrates bearing N-substitutions, electron-rich or deficient aryls, and aliphatics using optimized reaction conditions and produced a library of (E)-cinnamamides in 31 – 98% yield with >99:1 E:Z stereoselectivity. Deuterium labeling studies suggest that hydrogens on the α- and β-carbon come from pinacolborane and the amide hydrogen, respectively. We also trapped and characterized a key intermediate using tris(pentafluorophenyl)borane that supports a mechanism wherein the phosphine catalyst activates the propiolamide by conjugate addition.
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    Chemical Reactivity and Regioselectivity of Trimetallic Nitride Endohedral Metallofullerenes
    Cai, Ting (Virginia Tech, 2008-03-26)
    Endohedral metallofullerenes (EMF) have attracted increasing attention during past decades for their potential applications in the fields of biomedicine and nanomaterials. Trimetallic nitride template endohedral metallofullerenes (TNT EMFs) are some of the most promising fullerene-based materials (e.g., as MRI and X-ray contrast agents) because of their high yields compared to classic endohedral metallofullerenes. This dissertation addresses the chemical reactivity and regioselectivity of TNT EMFs. Based on the extraordinarily high stability of TNT EMFs relative to empty cage fullerenes and classic endohedral metallofullerenes, macroscopic quantities of high purity TNT EMFs were obtained directly from crude soot in a single facile step by using a cyclopentadiene-functionalized resin to trap the more reactive species via Diels-Alder reactions, allowing the TNT EMFs to pass through. We also developed a support-free chemical separation method of TNT EMFs from Sc- and Lu-based soot extract that makes use of the differing solubilities of unreacted TNT EMFs versus 9-methylanthracene-derivatized empty cage fullerenes. The exohedral functionalization of metallofullerenes can fine-tune their chemical and physical properties. The first N-methylpyrrolidino derivatives of TNT EMFs (Ih Sc3N@C80 and Ih Er3N@C80) were synthesized via 1,3-dipolar cycloaddition of N-methylazomethine ylides (Prato reaction). The demonstration of planar symmetry in the N-methylpyrrolidino derivatives by 13C NMR spectroscopy suggested that the reaction exclusively took place at the 5,6-ring junction. However, both 5,6-ring and 6,6 ring junction adducts were obtained when Ih Sc3N@C80 reacted with N-triphenylmethyl-5-oxazolidinone, as characterized by NMR spectroscopy and X-ray crystallography. The kinetically favored 6,6-ring junction adduct was converted to the thermodynamic product, the 5,6-ring junction adduct, upon thermal equilibration. The synthesis of pyrrolidino derivatives was also extended to two other Sc-based TNT EMFs, D5h Sc3N@C80 and Sc3N@C78. The reactivity and regioselectivity of D5h Sc3N@C80 and Sc3N@C78 were demonstrated by NMR spectroscopy, X-ray crystallography and theoretical calculations. Another type of reaction, the Bingel-Hirsch cyclopropanation was carried out with D3h Sc3N@C78 for the first time, yielding a single Cs-symmetric monoadduct and a dominant C2v-symmetric diadduct for the first time. The symmetric diadduct clearly demonstrates the remarkable regioselectivity control exerted by the encapsulated Sc3N cluster. We employed a LUMO electron density surface computational approach to predict multiadduct docking sites on the ellipsoidal fullerene cage surface. We also successfully synthesized the first derivative of a non-IPR fullerene, the diethyl malonate derivative of Sc3N@C68 by a Bingel-Hirsch reaction. The reactivity and regioselectivity of Sc3N@C68 were investigated by NMR spectroscopy and theoretical calculations.
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    Click Chemistry on DNA and Targeting RNA structure with Peptide Boronic Acids
    Crumpton, Jason B. (Virginia Tech, 2012-04-20)
    The utilization of click chemistry to perform inter- and intramolecular ligation on DNA has become ubiquitous in the literature. Advances in copper (I) stabilizing ligands that prevent DNA degradation via redox pathways have provided nucleic acid researchers access to the efficiency and quantitative nature of the click reaction. The majority of ligation procedures in the literature are performed in solution after DNA assembly and modification with alkyne reporter groups. However, without specialty alkyne reagents that can be sequentially and selectively deprotected, the solution phase method requires that the click reaction be performed on all DNA-attached alkynes simultaneously. Therefore, the variability of the azide reagent is limited to a singular R group. However, performing the click reaction on DNA during synthetic elongation (immediately after each alkyne installation) allows for the possibility of performing multiple click reactions with variable azide reagents. Unfortunately, most solid phase click procedures require long reaction times or the utilization of microwave irradiation to accelerate the reaction. The development of methods for the ligation of azides to alkynes without the use of microwave irradiation on solid phase is potentially very useful. Herein, we report a simple, efficient, and robust solid phase synthetic method for the ligation of azido-diamondoids to the alkyne-modified phosphate backbone of DNA with click chemistry using [Cu(CH₃CN)₄]PF₆ without stabilizing ligand. Interestingly, it was found that as the size of diamondoid increased, a corresponding increase in melting temperature of hybridized duplexes was observed. The developed method has the potential to complement existing DNA ligation procedures for applications in biotechnology and diagnostics. Interest in peptides incorporating boronic acid moieties is increasing due to their potential as therapeutics/diagnostics for a variety of diseases such as cancer. The utility of peptide boronic acids may be expanded with access to vast libraries that can be deconvoluted rapidly and economically. Unfortunately, current detection protocols using mass spectrometry are laborious and confounded by boronic acid trimerization, which requires time consuming analysis of dehydration products. These issues are exacerbated when the peptide sequence is unknown, as with de novo sequencing, and especially when multiple boronic acid moieties are present. Thus, a rapid, reliable and simple method for peptide identification is of utmost importance. Herein, we report the identification and sequencing of linear and branched peptide boronic acids containing up to five boronic acid groups by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Protocols for preparation of pinacol boronic esters were adapted for efficient MALDI analysis of peptides. Additionally, a novel peptide boronic acid detection strategy was developed in which 2,5-dihydroxybenzoic acid (DHB) served as both matrix and derivatizing agent in a convenient, in situ, on-plate esterification. Finally, we demonstrate that DHB-modified peptide boronic acids from a single bead can be analyzed by MALDI-MSMS analysis, validating our approach for the identification and sequencing of branched peptide boronic acid libraries. It is well known that RNA ligands incorporating basic and intercalating moieties display high RNA affinity. Unfortunately, these ligands are also often plagued by promiscuous binding to off-target substrates. Due to the potential utility of RNA ligands in biology and medicine, it is imperative to elucidate RNA binders which display high specificity as well as affinity. Boronic acid peptides promise unique RNA binding motifs through the interaction between the empty p-orbital of boron and the 2'-hydroxyl group of RNA. Herein, we describe the incorporation of lysine and phenylalanine boronic acid analogues into a branched peptide combinatorial library in an effort to impart increased selectivity towards the HIV-1 Rev Response Element (RRE). We were able to easily select and deconvolute 6 resulting "hit" peptides from 65,536 unique library members by high throughput screening and de novo sequencing. Although we were unable to evaluate peptide selectivity towards RRE due to general insolubility in aqueous media, we demonstrated the efficient deconvolution of a branched peptide library that incorporates boronic acids.
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    Complex Heterocycles as Mitochondrial Uncouplers
    Murray, Jacob Hadley (Virginia Tech, 2021-04-30)
    Small molecule mitochondrial uncouplers are compounds that dissipate the proton motive force independent of ATP synthase that results in increased energy expenditure. Mild mitochondrial uncoupling has therapeutic potential in treating obesity, diabetes, neurological diseases, non-alcoholic steatohepatitis (NASH), and aging. Our group has previously reported the discovery of a small molecule mitochondrial uncoupler BAM15, which was efficacious in an obesity mouse model. Herein, we describe the design and synthesis of two scaffolds as well as their characterization as mitochondrial uncouplers through a series of in vitro and in vivo assays. Compounds that pass as bona fide mitochondrial uncouplers are administered in mice to determine pharmacokinetic properties and promising compounds are then tested in a mouse model of obesity. The first series of mitochondrial uncouplers are anilinopyrazines. By changing the substitution pattern and electronics on the aniline rings, our investigations reveal the importance of the proximity of aniline rings on the pyrazine core, with the 2,3-positions being crucial for uncoupling activity. We found that mitochondrial uncouplers 2.5g and 2.5l elicited a maximum oxygen consumption rate (OCR) of 260% and 343% with an EC50 of 2.5 and 5.9 µM, respectively. Utilizing the knowledge gained from the anilinopyrazine series, we designed a second novel chemical scaffold based on a related BAM15 analog 6-amino-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol. The new series of 6-amino-[1,2,5]oxadiazolo[3,4-b]pyridin-5-ol derivatives have a pyridine instead of pyrazine core that is decorated with aniline substituents. We found that derivatives with electron withdrawing groups (EWG) substitutions in the 2,5-position on the aniline ring exhibited the greatest uncoupling activity compared to other structural isomers. Strong EWGs CF3/OCF3/SO2CF3 were well tolerated and demonstrated the highest uncoupling activity compared to other EWGs. Our studies indicated that placement of the hydroxyl group in the 2-position of the pyridine moiety was crucial for uncoupling activity. Several of the most promising compounds tested in vitro were examined in vivo and found to have good oral bioavailability in mice with ranges in Cmax of 10-90 µM and t1/2 of 0.9 to >24 hours. We found that analogs that have F/OCF3/SO2CF3 groups on the 4-position exhibited the longest t1/2 compared to other structural isomers, suggesting that this position is a site of metabolic lability. Among the 51 derivatives tested, SHM20519115 demonstrated mild uncoupling activity with 48% BAM15 OCR and an EC50 of 17.1 µM in L6 myoblast cells. SHM20519115 was found to have good oral bioavailability with a Cmax of 57 µM and a t1/2 of 4.4 hours. Additionally, SHM20519115 had significant distribution in adipose tissue where it can promote mitochondrial uncoupling. In a mouse model of obesity, SHM20519115 prevented fat mass gain by 59% compared to the western diet (WD) control group. Importantly, weight loss did not alter lean mass or food intake. Further characterization demonstrated that SHM20519115 prevented glucose and insulin intolerance in mice. Taken together, our investigations support the utility of mitochondrial uncouplers for the treatment of obesity and other metabolic disorders.
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    Computational and Spectroscopic Determination of Lithiated Benzylic Nitriles in THF/HMPA Solution
    Harmon, Henry Jason (Virginia Tech, 2008-09-10)
    The synthetic utility of nitrile-stabilized carbanions as reactive intermediates for selective carbon-carbon bond formation has prompted numerous studies toward characterization of the solution structure of these nucleophiles. In hopes of eventually gaining a better understanding of the structural properties which may mediate reactivity and selectivity, researchers have designed elegant structure elucidation strategies. These studies have offered key advancements toward the characterization of these intermediates; however, contradictory evidence has hindered unambiguous structural determination—particularly for lithiated benzylic nitriles in low dielectric, ethereal media. Chapter 1 of this dissertation presents a review of the synthetic utility of metalated nitriles and the spectroscopic and computational techniques employed to characterize their solution structure. Also reviewed herein are the controversial determinations drawn from these efforts. The research and data which follow in Chapters 2 and 3 focus on resolution of the conflicting structural determinations drawn from multinuclear magnetic resonance (NMR) and vibrational (IR and Raman) spectroscopy. Employing a strategy to slow the lithium-nitrogen exchange rate in low dielectric media, new 7Li, 31P, and 15N NMR spectroscopic evidence (with support from computational modeling) lead us to amend our previous assessments and propose that lithiated arylacetonitriles adopt an aggregated triple-ion structure in THF/hexane with sub-stoichiometric HMPA. Due to the limitations of computer resources and the effect of non-linear scaling, theoretical modeling of aggregated and solvated lithiated benzylic nitriles became impractical at the 6-31+G(d) basis set. These limitations led to the use and comparative analysis of two alternative basis sets for the DFT analysis of lithiated benzylic nitrile derivatives' 6-31(+LiX)G(d) and 6-31â +â G(d). Defined upon the principal of resonance stabilization, these basis sets were constructed by application of varying levels of computational theory on a per-atom basis. By applying higher levels of theory only to the atoms most intimately involved in the electronic distribution, "accurate" replacement models for 6-31+G(d) structures were obtained with considerable savings in computational resources. This study in basis set economy is detailed fully within Chapters 4 and 5.
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    Computational Studies of Protonated Cyclic Ethers and Benzylic Organolithium Compounds
    Deora, Nipa (Virginia Tech, 2010-05-10)
    Protonated epoxides feature prominently in organic chemistry as reactive intermediates. Gas-phase calculations studying the structure and ring-opening energetics of protonated ethylene oxide, propylene oxide and 2-methyl-1,2-epoxypropane were performed at the B3LYP and MP2 levels (both with the 6-311++G** basis set). Structural analyses were performed for 10 protonated epoxides using B3LYP, MP2, and CCSD/6-311++G** calculations. Protonated 2-methyl-1,2-epoxypropane was the most problematic species studied, where relative to CCSD, B3LYP consistently overestimates the C2-O bond length. The difficulty for DFT methods in modeling the protonated isobutylene oxide is due to the weakness of this C2-O bond. Protonated epoxides featuring more symmetrical charge distribution and cyclic homologues featuring less ring strain are treated with greater accuracy by B3LYP. Ion-pair separation (IPS) of THF-solvated fluorenyl, diphenylmethyl, and trityl lithium was studied computationally. Minimum-energy equilibrium geometries of explicit mono, bis and tris-solvated contact ion pairs (CIPs) and tetrakis-sovlated solvent separated ion pair (SSIPs) were modeled at B3LYP/6-31G*. Associative transition structures linking the tris-solvated CIPs and tetrakis-solvated SIPs were also located. In vacuum, B3LYP/6-31G* ΔHIPS values are 6-8 kcal/mol less exothermic than the experimentally-determined values in THF solution. Incorporation of secondary solvation in the form of Onsager and PCM single-point calculations showed an increase in exothermicity of IPS. Application of a continuum solvation model (Onsager) during optimization at the B3LYP/6-31G* level of theory produced significant changes in the Cα-Li contact distances in the SSIPs. An increase in of ion pair separation exothermicity was observed upon using both PCM and Onsager solvation models, highlighting the importance of both explicit and implicit solvation in modeling of ion pair separation.
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    Cyclopentadiene-Maleimide Platform for Thermally Reversible Polymers
    Stegall, Jeremy Brent (Virginia Tech, 2014-12-04)
    This dissertation describes a new platform for the synthesis of thermally reversible polymers, based on Diels-Alder reactions of bis-cyclopentadienes (bis-CPDs) and bis-maleimides (bis-MIs), that meets two main objectives. First, the bis-CPD must resist characteristic self-coupling. Second, the CPD-MI adducts should undergo the retro-Diels-Alder (rDA) reaction (i.e., thermal depolymerization) in a temperature regime that is comparable or slightly higher than that of the freely reversible bis-furan/bis-MI polymers (rDA between 80 °C and 130 °C) but much lower than that of bis-CPD homopolymers (rDA above 160 °C). Structure-reactivity relationships gleaned from the literature and from related but as yet unpublished work in our own laboratories led to our main hypothesis that a CPD moiety bearing one sterically encumbering substituent such as isopropyl (𝑖Pr) or tert-butyl (𝑡Bu) and one electronwithdrawing substituent such as perfluoroaryl would have the desired reactivity and adduct stability in combination with an 𝑁-substituted maleimide. Synthetic considerations led to a bisCPD monomer design in which two alkylcyclopentadiene groups (alkyl = 𝑖Pr or 𝑡Bu) are connected by an octafluorobiphenylene linker. As an initial fundamental step (Chapter 3), 1-(nonafluorobiphenyl-4’-yl)-4-tertbutylcyclopentadiene (1) was synthesized to provide a monofunctional model for the proposed difunctional CPD monomer. Reactions of 1 and 𝑁-(4-fluorophenyl)maleimide (FMI) afforded up to five regio- and stereo-isomeric adducts (of fourteen possible). Variable-temperature reactivity studies combined with NMR spectroscopic analysis, X-ray crystallography, and computational modeling enabled product distributions to be understood according to a conventional kinetic-vs- iii thermodynamic framework. These studies also predicted the microstructure of polymers derived from the proposed bis-CPD monomer, which is structurally analogous to 1, and bis-MIs. Moreover, 1 does not undergo DA self-coupling under ordinary conditions (T < 180 °C). Thermolysis studies of the major adducts revealed that the rDA becomes observable on a laboratory timescale (hours) at about 140 °C, which is at the upper end of the temperature range reported for furan+MI adducts but well below that of CPD+CPD adducts. In contrast, adducts formed from either of the analogous monosubstituted cyclopentadienes (𝑡BuC₅H₅ and C₆F₅C₅H₅) do not undergo rDA below 180 °C. These results strongly support the proposed bis-CPD monomer design. In a second fundamental step (Chapter 4), the hypothesis that an electron-withdrawing CPD substituent would destabilize a CPD-MI adduct was further tested by reacting 𝑁-(4- fluorophenyl)maleimide with a series of triarylated cyclopentadienes (1,2,3-Ar₃C₅H₃ and 1,2,4- Ar₃C₅H₃, Ar = C₆F₅, C₆F₄CF₃, and Ar = C₅F₄N). The perfluorophenyl- and perfluorotolylsubstituted compounds were previously reported, but the perfluoropyridyl-substituted cyclopentadienes were prepared for this study using SNAr reactions of pentafluoropyridine and sodium cyclopentadienide. The least electron deficient cyclopentadiene in each series (Ar = C₆F₅) reacted the most quickly and with the highest ultimate equilibrium binding constant, confirming the electron-effects hypothesis as well as the underlying presumption that DA reactions of even relatively electron-poor CPDs with MI would behave according to normal-electron-demand principles. In the main section of this dissertation (Chapter 5) the proposed bis(cyclopentadiene)s reacted with a series of previously reported bis(maleimides) to form linear polymers having molecular weights (Mn) up to 40 kDa. Relationships among the length and flexibility of the bis-MI linker (C₆H₁₂, C₁₂H₂₄, C₆H₄OC₆H₄, and (C₂H₄O)₂), the identity of the CPD alkyl substitutent (CHMe₂, CMe₃ and CMe₂Ph) and the glass transition temperature (Tg) as measured by differential scanning calorimetry (DSC) were understood in terms of a general model of local segmental mobility and free volume. Solution thermolysis of a model polymer system (bis-MI linker = C₆H₁₂ (7), CPD alkyl substituent = 𝑡Bu) showed a rapid decrease in molecular weight at 160 °C as determined by size exclusion chromatography (SEC). Solution thermolysis in the presence of excess FMI (as a trap for free CPD moieties) revealed that the onset temperature for rDA on a laboratory time scale (hours) was as low as 120 °C. In the bulk, thermolysis above 250 °C under vacuum led to recovery of a small portion of the bis-CPD monomer, but bulk thermolysis at 200 °C did not reveal a change in molecular weight as determined by SEC. The current interpretation of these observations is that limited mobility in these glassy polymers prohibits retro-DA decoupling. These findings largely validate the main hypothesis of this dissertation.
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    Design and Syntheses of Potential Drugs Based on GABA(A) Receptor Pharmacophores
    Clement, Ella Chow (Virginia Tech, 2005-06-28)
    Numerous previous studies of GABAAR ligands have suggested that GABAAR agonists must be zwitterionic and feature an intercharge separation similar to that of GABA (approx. 4.7-6.0 Ã ). We have demonstrated that monomeric, homodimeric and heterodimeric non-zwitterionic GABA amides are partial, full, or superagonists at the murine GABAA receptor (GABAAR). The agonism of these GABA amides is comparable to that of THIP, as shown by in vitro assay results. The assay data indicate that the agonism of GABA amides is tether length-dependent. Optimum agonism is achieved with a tether length of four methylenes in GABA amide dimers and in GABA amides bearing pendant amide or amino groups. We have further investigated the structure-activity relationship for GABA amides on the GABAAR by performing structural modifications to both the superagonist 2c and the agonist 6c. Synergism and [3H]muscimol binding experiments show that 2c binds to the same sites as GABA. Structural modification of 2c demonstrated that partial rigidification of the tether eliminated agonism and caused ligands to behave as weak competitive antagonists. We have also investigated the agonism of four ZAPA derivatives in 36Cl- uptake functional assay. Two of them are found to be as potent as GABA. In our studies of 1,4-benzodiazepines, our goal was to synthesize three different subtypes of quaternary 1,4-benzodiazepines by use of the memory of chirality (MOC) strategy. Disappointingly, most of the deprotonation/alkylations failed, due to various reasons. The failure of the reactions of (S)-alanine-derived tetrahydro-1,4-benzodiazepin-3-ones was probably due to either the unexpected side reactions or the steric hindrance of enolate alkylation. In the case of tetrahydro-1,4-benzodiazepin-2-ones, computational studies suggested that steric hindrance by both the benzo ring and N4-allyl group might retard deprotonation at C3 by bulky bases like KHMDS or LDA. Finally, (S)-serine-derived 1,4-benzodiazepin-2-ones and their elimination products (ï ¡-methylene benzodiazepines) were prepared. These proved unreactive towards deprotonation/alkylations and conjugate additions, respectively. The low reactivity of the ï ¡-methylene benzodiazepines towards nucleophiles was attributed to highly delocalized LUMOs that failed to direct nucleophiles to the ï ¢-carbons.
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    Design and Synthesis of Novel Benzodiazepines
    MacQuarrie, Stephanie Lee (Virginia Tech, 2005-11-30)
    Bivalent drug design is an efficient strategy for increasing potency and selectivity of many drugs. We devised a strategy to prepare agonist-benzodiazepine heterodimers that could simultaneously bind to agonist and BZD sites of the GABAAR. We synthesized a benzodiazepine-MPEG model compound that relied on physiological GABA to elicit flux. We established that a tether at the N1 position of the BZD would not prevent binding to the receptor. However, coupling of GABA amides with long chain PEG tethers studied by another group member resulted in complete loss of agonist activity. We therefore ceased research in this particular area. 1,4-Benzodiazepin-2,5-diones display a wide range of pharmacological activities. Compounds containing the tricyclic proline-derived subtype have received attention as potent anxiolytic agents and as starting materials for anthramycin-inspired anticancer agents. More recently enantiopure (S)-proline-derived 1,4-benzodiazepin-2,5-diones have been recognized as selective α5 GABAA receptor ligands. Despite the impressive diversity of 1,4-benzodiazepine-2,5-diones prepared to date, enantiopure examples possessing a quaternary stereogenic center have been largely unexplored. "Memory of chirality" (MOC) is an emerging strategy for asymmetric synthesis. This technique enables the memory of a sole chiral center in the substrate to be retained in a process that destroys that center. We have used this technique to prepare a library of quaternary proline-derived, thioproline-derived and hydroxyproline-derived 1,4-benzodiazepin-2,5-diones, in high ee. We have developed an efficient synthetic method for preparing oxaproline-derived 1,4-benzodiazepin-2,5-diones in high yields, and by applying the MOC strategy we have prepared quaternary derivatives in acceptable %ee. We envision oxaproline-derived 1,4-benzodiazepin-2,5-diones may exhibit similar or more potent pharmacological properties than proline-derived 1,4-benzodiazepin-2,5-diones. Using density functional theory (DFT) methods, we modeled the formation of an enantiopure, dynamically chiral enolate intermediate and the slow racemization of the enolate on the alkylation reaction time scale.
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    Design and Synthesis of Orally Bioavailable Sphingosine Kinase 2 Selective Inhibitors
    Sibley, Christopher David (Virginia Tech, 2020-07-16)
    In humans, mammals, and perhaps all vertebrates, sphingolipids exist as a family of cellular signaling molecules and have been shown to be involved in a wide range of biological processes ranging from proliferation to apoptosis. As such, sphingolipid signaling has garnered the attention of numerous researchers as an attractive candidate for pharmacological manipulation. The synthetic pathway of one prominent sphingolipid, sphingosine 1-phosphate (S1P), has been implicated in a variety of disease states such as cancer, sickle cell disease, multiple sclerosis, and renal fibrosis. Formation of S1P is facilitated from the ATP dependent phosphorylation of sphingosine (Sph) through its generative enzyme's sphingosine kinase 1 and 2 (SphK1 and SphK2). Inhibition of SphK1 and SphK2 results in the manipulation of S1P levels, which has been shown to be therapeutic in various animal models of disease. While there are multiple examples of potent SphK1-selective and dual SphK1/2 inhibitors, SphK2-selective inhibitors are scarce. Herein, we describe the design, synthesis and biological testing of SphK2-selective inhibitors. We first describe the discovery that introducing a trifluoromethyl group onto the internal aryl ring of our inhibitor scaffold led to superior selectivity and potency towards SphK2. We demonstrate that the trifluoromethyl moiety is interacting with a previously unknown side cavity in the substrate binding site of SphK2 that is unique and could be exploited in the design of SphK2-selective inhibitors. The synthesis of 21 derivatives with various substituents spanning off the internal aryl ring was completed, therefore characterizing the preferred size and chemical nature of moieties positioned in that portion of the binding site. This work led to the development of the most potent SphK2-selective inhibitor known at the time. We then describe the transformation of our SphK2-selective inhibitors into an orally bioavailable drug. We explain how the guanidine functionality on our inhibitor scaffold hinders our compounds from being orally bioavailable. Consequently, a library of 24 derivatives with various modifications to the guanidine functionality was synthesized and evaluated for improved orally bioavailability. Highlighted in this work is the development of the most potent SphK2-selective inhibitor currently known 3.14 (SLS1081832), which displays a hSphK2 Ki of 82 nM and 122-fold selectivity for SphK2. Chemical modification and in vivo assessment of 3.14 (SLS1081832) prodrugs was explored.