Dissertations / Theses on the topic 'Binding and catalysis'

Due to their intimate roles in survival, longevity as well as pathogenesis via “epigenetic” and “metabolic” regulatory mechanisms, sirtuins have gained considerable interest toward undertaking detailed biochemical/biophysical studies. The present study was designed to ascertain the mechanistic details of ligand binding and catalysis in selected sirtuin isozymes (viz., SIRT1 and SIRT5) from the point of view of designing isozyme selective inhibitors as potential therapeutics. By screening of the in-house synthesized compounds, two barbiturate derivatives were identified as the SIRT5 selective inhibitors. These, along with some of known inhibitors of SIRT1 and SIRT5, namely, MH5-75, nicotinamide, suramin were investigated by a combination of spectroscopic, kinetic, and thermodynamic techniques. The influence of the sirtuin inhibitors in modulating the structural features of the enzymes were ascertained by CD spectroscopic, lifetime fluorescence, and thermal denaturation studies using wild-type and selected site-specific mutant enzymes. The experimental data revealed that the substrate selectivity and inhibitory features in SIRT5 were manifested via the mutual cooperation between Y102 and R105 residues of the enzyme, and the overall catalytic feature of the enzyme was modulated by changes in the protein structure. Whereas the stoichiometry of SIRT1 to suramin remained invariant as 1:1, that of SIRT5 to suramin increased from 1:1 to 2:1 upon increase in the molar ratio of the enzyme to the ligand. A comparative account of the experimental data presented herein sheds light on the structural-functional differences between SIRT1 and SIRT5, leading to the design of isozyme selective inhibitors as therapeutic tools for the treatment of sirtuin associated diseases.
NIH (GM110367)
NSF (DMR1306154)

Anion-binding catalysis by dual hydrogen-bond donors such as ureas and squaramides has been demonstrated as a powerful strategy for the development of highly enantioselective transformations involving prochiral cationic intermediates, such as iminium ions, oxocarbenium ions, carbenium ions, and episulfonium ions. The research described in this dissertation explores the ability of dual H-bond donor catalysts to engage chiral cationic intermediates and to induce enantioselectivity in transformations involving such intermediates. In Chapters 1, we provide an overview of the progress and challenges in the development of enantioselective halo- and seleno-functionalization reactions, which proceed via three-membered ring cationic halonium or seleniranium ions. In Chapter 2, we report a highly enantioselective selenocyclization reaction that is promoted by the combination of a chiral squaramide catalyst, a mineral acid, and an achiral Lewis base. Mechanistic studies reveal that the enantioselectivity originates from the dynamic kinetic resolution of seleniranium ions through anion-binding catalysis. Chapter 3 details our discovery of a squaramide-catalyzed enantioselective iodoisocyanation reaction, which represents a rare example in asymmetric intermolecular halofunctionalization of simple olefins. Kinetic studies reveal that [I(NCO)2]–1 anion is the counterion of iodonium intermediate and the dual H-bond donor catalyst aggregates in the resting state. Hammett analysis indicates that the degree of stabilization by catalyst to the iodonium intermediate accounts for both catalysis and enantioselectivity. The reactions developed in Chapter 2 and 3 have therefore extended anion-binding catalysis to reactions involving chiral and stereochemically labile halonium and seleniranium cations. Knowledge learned in these studies will provide valuable guidance to the development of asymmetric transformations involving other chiral cationic intermediates.
Chemistry and Chemical Biology

Chemistry
Ph.D.
This thesis describes the synthesis of functionalized spiroligomers and their applications in metal binding, metal-mediated catalysis, and organocatalysis. By synthesizing a family of functionalized bis-amino acids achieved from reductive alkylation, the Schafmeister group has developed access to highly functionalized and shape programmable structures named “spiroligomers.” The rigid backbones of spiroligomers are good at organizing the orientations of functional groups on their side chains. This property enables them as promising candidates for catalysts. Firstly we synthesized a few spiroligomer dimers presenting metal-binding groups such as terpys and bipys. With the right orientation of metal binding groups controlled by adjusting the stereocenter of the spiroligomer, macrocyclic “square” complexes with metals were obtained. The crystal structures of these intriguing complexes were solved. This work rendered the first structurally, spectroscopically and electronically characterized metal-spiroligomer complexes as well as the first crystal structure of spiroligomer. Secondly, the question of whether metal-binding spiroligomers are able to catalyze certain reactions became our major concern. We developed a binuclear copper catalyst that could accelerate a phosphate ester rearrangement, and that demonstrated that when the two copper binding terpyridine groups were best able to approach each other, they accelerated the rearrangement more than 1,000 times faster than the background reaction. Other molecules that did not properly organize the two copper atoms demonstrate considerably slower reaction rates. At last, catalysts based on spiroligomers without metals are also of interests. By displaying two hydrophobic groups in various directions on a monomeric spiroligomer (also can be regarded as a proline derivative), we observed variable activities and enantioselectivities in the catalysis of asymmetric Michael addition (up to 94% ee at -40 °C for one organocatalyst).
Temple University--Theses

Pseudouridine is the most common RNA modification found in all forms of life. The exact role pseudouridines play in the cell is still relatively unknown. However, its extensive incorporation in functionally important areas of the ribosome and the fitness advantage provided to cells by pseudouridines implies that its presence is important for the cell. The enzymes responsible for this modification, pseudouridine synthases, vary greatly in substrate recognition mechanisms, but all enzymes supposedly share a universally conserved catalytic mechanism. Here, I analyze the kinetic mechanisms of pseudouridylation utilized by the exemplary pseudouridine synthase RluA in order to compare it with the previously determined rate of pseudouridylation of the pseudouridine synthase TruB. My results demonstrate that RluA has the same uniformly slow catalytic step as previously determined for TruB and TruA. This constitutes the first step towards identifying the catalytic mechanism of the pseudouridine synthase family. Additionally, it was my aim to identify the major determinants for RNA binding by pseudouridine synthases. By measuring the dissociation constants (KD) for substrate and product tRNA by nitrocellulose filtration assays, I showed that both tRNA species could bind with similar affinities. These binding studies also revealed that TruB’s interaction with the isolated T-arm is the major contact site contributing to the affinity of the enzyme to RNA. Finally, a new contact between tRNA and TruB’s PUA domain was identified which was not observed in the crystal structure. In summary, my results provide new insight into the common catalytic step of pseudouridine synthases and the specific interactions contributing to substrate binding by the enzyme TruB. These results will enable future studies on the kinetic mechanism of pseudouridine synthases, in particular the kinetics of substrate and product binding and release, as well as on the chemical mechanism of pseudouridine formation.
xi, 122 leaves : ill. (some col.) ; 29 cm

To investigate the structural determinants of NitN specificity on short aliphatic amide substrates by analyzing binding and interactions of these molecules with the NitN binding pocket. To probe the catalytic role of the two active site glutamate residues (Glu61 and Glu139) using NitN as a model enzyme. To monitor the activity, interactions and reactivity of the WT NitN and the Glu61 and Glu139 NitN mutants with ACR.

2010/2011
Protein scaffolds are stable structures capable to recognize and bind, in different conditions, small guest molecules. They are proteins with known conformation which can be used and modified for the construction of variants. The goal of this work is the identification of peptide-based artificial receptors or catalysts. To this purpose, we have considered two different protein motifs to generate new scaffolds: a synthetic E/K coiled-coil domain, and one of the binding sites of the natural protein Human serum albumin. In order to generate stable peptide hosts, we developed peptide libraries to be selected for both properties, adopting two different approaches: RANDOM mutagenesis and SITE-DIRECTED mutagenesis.
XXIV Ciclo
1985

Bidentate ligands are a commonly used class of ligands in catalysis that generate highly-active and selective catalysts. Such bidentate ligands, however, often suffer from synthetic challenges, which can be alleviated by the use of simpler monodentate ligands that assemble through non-covalent interactions to mimic the structure of bidentate ligands at the metal center. To produce a strongly assembled catalyst complex, the Hamilton receptor motif was utilized. Hamilton receptors form six hydrogen bonds with complementary guests and have binding affinities for barbiturates of up to 104 M-1 in CDCl3. Complete bifurcation of the Hamilton scaffold produces a modular ligand structure that allows for modification of either end of the supramolecular ligand structure. Similarly, the barbiturate guest can be synthetically altered creating both chiral guests and guests with differing amounts of steric bulk. Both experimental titration data and density functional theory calculations show that steric bulk discourages binding of the guest while a pre-organized host encourages guest inclusion. Electronic effects on the bifurcated Hamilton system were studied by varying the electron donating or withdrawing ability of the benzamide moiety on the host molecule. Electron withdrawing moieties produce more acidic amide hydrogens on the host which are able to participate in stronger hydrogen bonds with the guest resulting in a stronger host-guest complex. The effects of substitutions on the barbiturate guest were examined as well, and increased steric bulk on the guest resulted in decreased affinities with the host. The bifurcated Hamilton receptor ligands were examined in the palladium-catalyzed Heck reaction of iodobenzene with butyl acrylate. Pd2(OAc)4 was used as a control and all reaction yields with the diphenylphosphine ligand-stabilized Pd were greater than or equal to those obtained with Pd2(OAc)4 alone. The reaction rates did not correlate with the determined binding constants, suggesting that phosphine substitution on the guest plays a larger role than affinity of the complex for the guest. Reaction temperatures were varied, and at lower temperatures the yields increased implying that the strength of the hydrogen bonds between the metal complex and the guest does play a secondary role in the catalysis. This dissertation includes previously published co-authored material.

Ion-binding organocatalysis is an emerging field that has the potential to control the stereochemical outcome of any transformation that goes via charged intermediates. The aim of this project was to explore how this concept could be applied to an asymmetric allylation reaction. Chapter 2 of this thesis discusses anion-binding catalysis and investigates a chiral cooperative thiourea catalyst that could bind to fluoride to control allylation using an allylsilane. Optimization using a non-chiral thiourea (Schreiner’s catalyst) demonstrated that the reaction proceeded in high yield with TBAT onto an N-benzoylhydrazone. A chiral cooperative thiourea catalyst library was then synthesized but unfortunately, although the allylation using these catalysts proceeded in excellent yield, the product was isolated as the racemate (Scheme 1). Scheme 1: Anion-binding catalysis gave allylated products in high yields but gave no stereocontrol. Chapter 3 examines a chiral quaternary ammonium fluoride as an example of chiral cation-directed catalysis. We hypothesized that an allylsilane activated by fluoride would generate an allyl anion species that would associate with the chiral quaternary ammonium cation through electrostatic interactions. Extensive optimization found that the allylation reaction proceeded in good yield in chloroform at reflux with N-benzoylhydrazones. Different fluoride catalysts were prepared using an ion-exchange resin, and cinchonidine-derived catalysts performed the best. This methodology was extended to a phase-transfer catalyzed process, where solid cesium fluoride exchanged with chloride in situ, removing the need to synthesize and isolate ammonium fluoride catalysts (Scheme 2). Scheme 2: Cation-directed asymmetric allylation. In Chapter 4 cation-directed asymmetric catalysis was extended to an intramolecular allylation reaction. Substrate synthesis was attempted by cross metathesis but the reaction was capricious and yields were low. Intramolecular allylation with these materials gave promising results (Scheme 3) but a lack of material prevented optimization. Scheme 3: Intramolecular allylation results.

Thesis advisor: Kian L. Tan
Thesis advisor: Shih-Yuan Liu
Chapter 1. In the Tan laboratory, we developed synthetic methods to control reaction selectivity (regio-, stereo-, and site-selectivity) using scaffolding catalysis. Our strategy utilizes directing groups that induce intramolecularity through the formation of a labile covalent bond between the substrate and a binding site in a catalytic system. In the first part, we described site-selective functionalization of various carbohydrates and complex polyhydroxylated molecules which contain cis-1,2-diol motif using a chiral organic scaffold. In the second part, meta-selective C–H functionalization of arenes was demonstrated. High meta-selectivity was achieved by the use of a nitrile-based silyl tether which is cleavable and recyclable. Chapter 2. In the Liu laboratory, we focuses on studies of boron-nitrogen containing heterocycles. In this chapter, synthesis of 1,2-azaborines and their binding study with T4 lysozyme mutants were described. Specifically, we directly compared binding of NH-containing 1,2-azaborines and their carbonaceous analogs to probe hydrogen bonding interaction between the NH group of azaborine and a carbonyl oxygen of protein residue. Structural and thermodynamic analysis provided us the first evidence of H-bonding of azaborines with a biological macromolecule. Chapter 3. Described are the synthesis of regioisomers of ethyl-substituted 1,2-azaborines and their binding thermodynamics to T4 lysozyme mutants. To access the azaborine ligands used in the binding study, we developed synthetic methods for regioselective functionalization of six positions of 1,2-azaborines. Isothermal titration calorimetry experiments showed differences in binding free energy for regioisomers to the L99A T4 lysozyme. This result could originate from electronic differences of the isosteric ligands inducing dipole-dipole interaction between ligand and surrounding protein residues or it may be from local dipolar interactions. Chapter 4. A general method for late-stage N-functionalization of 1,2-azaborines is described to afford libraries of BN-containing complex molecules. The chemical transformations include electrophilic substitution reactions, N–C(sp2) bond forming reactions under Buchwald-Hartwig amination conditions, and N–C(sp) bond forming reactions using copper-catalyzed N-alkynylation. As applications in materials science and medicinal chemistry, synthesis of the first parental BN isostere of trans-stilbene and lisdexamfetamine derivative is described utilizing the methodology developed in this work
Thesis (PhD) — Boston College, 2017
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry

The binding of carboxy-arabinitol bisphosphate (CABP), carboxy-arabinitol 1-phosphate (CA1P), carboxy-ribitol bisphosphate (CRBP), to carbamylated sites or xylulose bisphosphate (XuBP) and ribulose bisphosphate (RuBP) to decarbamylated sites on ribulose bisphosphate carboxylase/oxygenase (rubisco) exhibits negative cooperativity. The binding of ligands to decarbamylated sites was highly pH-dependent between 7.5 and 8.5. Lower pH enhanced binding affinity. The binding of ligands to carbamylated sites was pH-independent. A binding model for negative cooperative interactions among catalytic sites is proposed based on the observations and the crystallographic structure of rubisco. Fully activated, purified rubisco slowly loses its activity during in vitro catalysis after exposure to RuBP. This time-dependent kinetics is termed as "fallover". Two different fallover patterns were demonstrated during CO₂ fixation, one with little loss of activator CO₂ at pH 8.5 and the second with loss of activator CO₂ at pH 7.5. The two inhibitors being produced during fallover were isolated by high performance anion exchange chromatography (HPAE) and identified as XuBP and 3-ketoarabinitol 1,5-bisphosphate by pulsed amperometric detection (PAD) either directly or after reduction by NaBH₄. Because of the weak binding affinity of XuBP to catalytic sites of rubisco at pH 8.5, there was little loss of activator CO₂ during fallover. 3-keto-arabinitol-P₂ which binds to carbamylated rubisco sites became the major inhibitor at that pH. However, at pH 7.5, the binding of XuBP to decarbamylated sites can cause a shift in equilibrium with loss of carbamylated sites even in the presence of excess CO₂ and Mg²⁺, resulting in the loss of activator CO₂ during fallover. XuBP was isolated and identified from celery leaves by HPAE-PAD. A new method was developed for the determination of the substrate specificity of rubisco. It is based on the specific ¹⁴C-labeling of 3-phosphoglycerate (PGA) from the carboxylase reaction and its dilution from the oxygenase reaction. Therefore, the ratio of carboxylation to oxygenation can be measured directly by determining the specific radioactivity of PGA produced from both reactions with HPAE-PAD separation of the total PGA and scintillation counting of ¹⁴C-labeled PGA.

Red blood cell hemolysis in sickle cell disease (SCD) releases free hemoglobin. Extracellular hemoglobin and its degradation products, free heme and iron, are highly toxic due to oxidative stress induction and decrease in nitric oxide availability. We propose an approach that helps to eliminate extracellular hemoglobin toxicity in SCD by employing a bacterial protein system that evolved to extract heme from extracellular hemoglobin. NEAr heme Transporter (NEAT) domains from iron-regulated surface determinant proteins from Staphylococcus aureus specifically bind free heme as well as facilitate its extraction from hemoglobin. We demonstrate that a purified NEAT domain fused with human haptoglobin beta-chain is able to remove heme from hemoglobin and reduce heme content and peroxidase activity of hemoglobin. We further use molecular dynamics (MD) simulations to resolve molecular pathway of heme transfer from hemoglobin to NEAT, and to elucidate molecular mechanism of such heme transferring process. Our study is the first of its kind, in which simulations are employed to characterize the process of heme leaving hemoglobin and subsequent rebinding with a NEAT domain. Our MD results highlight important amino acid residues that facilitate heme transfer and will guide further studies for the selection of best NEAT candidate to attenuate free hemoglobin toxicity.

The cation–π interaction has been long-established to play an important role in molecular recognition, supramolecular chemistry, and molecular biology. In contrast, its potential application in small-molecule catalysis, especially as a selectivity-determining factor in asymmetric synthesis has been overlooked until very recently. This dissertation begins with an extensive literature review on the state-of-the-art research on the application of cation–π interactions in non-enzymatic catalysis of organic and organometallic transformations. The research in this field has been largely inspired and guided by the related biosynthetic systems incorporating the same type of interactions.

Chemistry and Chemical Biology

Histone deacetylases are an important class of enzymes that catalyze the hydrolysis of acetyl-L-lysine side chains in histone and non-histone proteins to yield L-lysine and acetate, effecting the epigenetic regulation of gene expression. In addition to the active site pocket, the enzyme harbors an internal cavity for the release of acetate by-product. To probe the role of highly conserved amino acid residues lining this exit tunnel, site-directed alanine substitutions were made at tyrosine-18, tyrosine-20 and histidine-42 positions. These mutants were characterized by various biochemical and biophysical techniques to define the effect of mutations on ligand binding and catalysis of the enzyme. The mutations altered the catalytic activity of HDAC8 significantly. Y18A mutation dramatically impaired the structural-functional aspects of the enzymatic reaction. Our data reveal that there is long range communication between the exit tunnel residues and the active site pocket of HDAC8, presumably regulating the overall catalysis of the enzyme.

The clcD gene encoding dienelactone hydrolase (DLH) is part of the clc gene cluster for the utilization of the B-ketoadipate pathway intermediate chlorocatechol. The roles that individual amino acids residues play in catalysis and binding of the enzyme were investigated. Using PCR a 1.9 kbp clcD fragment was amplified and subcloned yielding a 821 bp BamHi to ZscoRI subclone in the plasmid pUC19.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008.
Additional advisors: Houston Byrd, Chris Lawson, Sadanandan Velu, Charles Watkins. Description based on contents viewed Feb. 9, 2009; title from PDF t.p. Includes bibliographical references.

Thesis (Ph. D.)--School of Biological Sciences. University of Missouri--Kansas City, 2007.
"A dissertation in molecular biology and biochemistry and cell biology and biophysics." Advisor: Henry M. Miziorko. Typescript. Vita. Title from "catalog record" of the print edition Description based on contents viewed Sept. 12, 2008. Includes bibliographical references (leaves 109-126). Online version of the print edition.

Cations are essential for ribonucleic acids (RNA), as they neutralize the negatively charged phosphate backbone. Divalent metals play important roles in the folding and function of RNA. The relationship between RNA and divalent cations magnesium (Mg(II)) and iron (Fe(II)) has been investigated. Mg(II) is involved in tertiary interactions of many large RNAs, and necessary for ribozyme activity. The influence of Mg(II) on RNA secondary and tertiary structure is investigated experimentally. Mg(II) binding to A-form RNA is accompanied by changes in CD spectra, indicating that Mg-RNA interactions influence the helical structure of RNA duplexes and helical regions of unfolded RNAs. Quantum mechanics calculations are used to probe the energetics of Mg(II)-chelation with phosphate oxygen atoms of nucleic acids. We identify the specific forces that contribute to stability of Mg(II)-chelation complexes in RNA. Fe(II) can serve as a substitute for Mg(II) in RNA folding and function. Fe(II) was abundant on early earth, it is plausible that RNA folding and function was mediated by Fe(II) instead of, or in combination with, Mg(II) in the anoxic environment of early earth. We have investigated oxidoreductase catalytic activity observed in RNA when in combination with Fe(II). This activity, only observed in the presence of Fe(II) and absence of Mg(II)appears to be a resurrection of ancient RNA capabilities that were extinguished upon the depletion of Fe(II) from the environment during the rise of oxygen after the great oxidation event. Finally, metal-ion based cleavage of RNA is used to identify the binding sites of Mg(II) and Fe(II). We observe that both metals cleave RNA in similar positions, providing further support for Fe(II) as a substitute for Mg(II) in RNA.

A highly enantioselective acylation of silyl ketene acetals with acyl fluorides was developed to generate useful α,α-disubstituted butyrolactone products in high yield and excellent enantioselectivities. This transformation is promoted by an arylpyrrolidino thiourea catalyst and 4-pyrrolidinopyridine and represents the first example of enantioselective thiourea anion-binding catalysis with fluoride. Mechanistic investigations revealed both catalysts to be necessary for reaction to occur, suggesting the thiourea aids in the generation of the key N-acylpyridinium/fluoride ion pair. The outstanding hydrogen-bond-accepting ability of fluoride is likely important in this regard. In addition, a strong dependence on both the N-acylpyridinium counteranion and the substituents on the silicon group of the silyl ketene acetal were observed, highlighting the importance of the silicon-fluoride interaction. A cyclic oligomeric (salen)Co–OTf complex was used to catalyze a highly enantioselective addition of phenyl carbamate to meso-epoxides to afford protected trans-1,2-amino alcohols. The use of an oligomeric (salen)Co–OTf complex as the catalyst and aryl carbamates as nucleophiles was crucial to the development of this reaction protocol. This method is amenable to large-scale synthesis due to the low catalyst loadings and high concentration used, its operational simplicity, and the use of inexpensive, commercially-available starting materials. To demonstrate the synthetic utility of this protocol, optically pure trans-2-aminocyclohexanol hydrochloride and trans-2-aminocyclopentanol hydrochloride were prepared on a multigram scale using only 0.5 and 1 mol% catalyst loading, respectively.
Chemistry and Chemical Biology

During the last decades gold nanoparticles have proven to be an attractive scaffold for the realization of inorganic-organic hybrid systems. This occurs by exploiting the stable interaction between gold surfaces and different classes of functional groups (like amines, phosphonates and -in particular- thiols) in order to prepare self-assembled monolayers.[1] This kind of system has been used in a wide range of applications in nanomedicine, diagnostics and nanotechnology. It has also become an attractive platform for the development of innovative, nanoparticle-based catalysts. This is motivated by different features of the gold nanoparticles, such as the ease and versatility of preparation, the possibility to retrieve the catalyst from solution and the high surface to volume ratio of gold colloids.[2] In a recent contribution by Prins et al.[3] a model of artificial nuclease, based on TACN-Zn(II)-bearing 2 nm diameter nanoparticles, initially developed by Scrimin et al.[4], has been exploited as a sensing tool for the detection of proteolytic enzymes in solution. The sensing mechanism relies on the inhibition of the transphosphorylation reaction of the substrate HPNP (2-hydroxypropyl-4-nitrophenyl phosphate), which can be followed spectrophotometrically as the reporter molecule p-nitrophenate is released, due to the interaction between the nanoparticles and a negatively charged oligopeptide. The oligopeptide plays a double role in the system by acting both as a nanoparticle inhibitor (by blocking access to the substrate to the catalytic sites on the surface of the monolayer) and an enzymatic substrate. The cleavage of the oligopeptide allows for the regulation of the catalytic behavior of the system, depending on the presence of the specific enzyme, since the lesser charged fragments are not good inhibitors of the catalyst. The interest towards the improvement of this system has directed the research presented in this thesis, in particular focusing on the role and stability of the monolayer. These studies relied on the analysis of the catalytic parameters and the binding of oligoanions of structurally related monolayers supported on gold nanoparticles. The first step of this research project was aimed at the synthesis of three organic building blocks, in which the thiol moiety and the macrocycle are separated by alkyl chains of different lengths (6, 9, 12 carbon atoms). The preparation and characterization of three batches of gold nanoparticles, each coated with one of the thiols, demonstrated a similar behaviour for Au NPs C9TACN and Au NPs C12TACN, whereas Au NPs C6TACN showed a denser monolayer presumably due to a more efficient lateral interaction between thiols. The evaluation of the catalytic activity in this model reaction mediated by the three systems showed the similarity between Au NPs C9TACN and Au NPs C12TACN in terms of intrinsic catalytic activity (kcat) and interaction with the substrate (KM), in good agreement with published data referred to a monolayer composed of longer thiols.[5] Au NPs C6TACN exhibited a similar value of kcat, but displaying a KM value that was 3-fold higher, indicative of the weaker interaction between the nanoparticles and the substrate. In order to study this phenomenon in more detail, the interaction between gold nanoparticles and fluorescent oligoanion was studied. Exploiting the quenching of luminescence mediated by the gold cores, it was possible to observe differences in the saturation concentration of the monolayer for different fluorescent probes. A first probe (fATP), known for a predominantly electrostatic interaction with the surface of the monolayer[6], gave hardly any differences between the three nanosystems. However a different probe (MANT-AMP), that binds the monolayer as a result of both electrostatic and hydrophobic interaction[7], clear differences were observed between Au NPs C6TACN and Au NPs C9TACN. By working in absence of Zn(II) at basic pH, in order to keep the monolayer uncharged, we were able to observe the lack of interaction between MANT-AMP and Au NPs C6TACN, confirming that for this system the apolar part of the monolayer does not participate to the binding process as we observed in the binding to the catalytic substrate. This is in agreement with the higher KM values observed in the catalysis experiments. Nonetheless these experiments do not reveal whether this is a consequence of the different thickness of the monolayer or whether it’s due to the peculiar density of the Au NPs C6TACN’s monolayer. Next, the binding and catalytic performances of the three systems were assessed over a time span of four months in order to understand their long term stability. So far few studies have appreared in the literature that address this important issue. All the systems resulted stable in time, without displaying aggregation or a radical deterioration of the catalytic parameters. Au NPs C9TACN was the only system that shown no variation at all in the catalytic and binding behavior, thus emerging as as the most robust system for applications. Finally, the effect of the nanoparticle dimensions on the catalytic activity was investigated by studying 13 nm gold nanoparticles passivated with thiol C9TACN. The catalytic parameters were then confronted with those obtained for Au NPs C9TACN (2 nm). The results showed that the system retains catalytic activity, but with a neat loss both in kcat and KM, probably imputable to the uniformity of the monolayer and the lack of “hot catalytic spots” that may occur in small nanoparticles with extreme curvatures.
Nel corso dei decenni le nanoparticelle dâ€'oro si sono dimostrate supporti estremamente attraenti per la realizzazione di sistemi ibridi inorganici-organici, sfruttando l'€™interazione stabile che si crea tra superfici d'€™oro e particolari classi di molecole organiche (come ammine, fosfonati o tioli) per formare monostrati autoassemblati.[1] Questo tipo di sistemi è stato sfruttato ampiamente per una vasta gamma di applicazioni, tra cui spicca la realizzazione di catalizzatori organici nano-supportati. La motivazione dietro l’interesse verso le nanoparticelle d'€™oro per applicazioni catalitiche risiede nella versatilità  della realizzazione del monostrato, virtualmente applicabile ad ogni tipo di reazione catalizzata da molecole organiche, e dall'€™enorme rapporto superficie/volume dei colloidi d'™oro.[2] In un recente lavoro di Prins et al.[3], impiegando un sistema catalitico ideato da Scrimin et al. per l’idrolisi di fosfodiesteri[4], si utilizzano nanoparticelle d'oro di 2 nm di diametro che espongono complessi di 1,4,7-triazaciclononano-Zn(II) come effettori di uno strumento di sensing per enzimi proteolitici. Tale sistema si basa sull’inibizione della reazione di transfosforilazione di HPNP (2-idrossipropil-4-nitrofenil fosfato) promossa dalle nanoparticelle ed osservabile per via spettrofotometrica seguendo la liberazione della molecola reporter p-nitrofenato, dovuta all'€™interazione tra la nanoparticella d'€™oro e oligopeptidi carichi negativamente. L'oligopeptide agisce nel doppio ruolo di inibitore della nanoparticella e di substrato enzimatico, regolando il sistema dipendentemente dalla presenza o meno di un enzima in grado di degradarlo in frammenti a carica inferiore che non inibiscono efficacemente la nanoparticella. L’interesse verso il perfezionamento di tale sistema ha spinto allo studio discusso in questo elaborato di tesi, utilizzando la catalisi della transfosforilazione di HPNP e la capacità  di interazione con oligoanioni come strumenti diagnostici di monostrati organici analoghi. Lo studio ha portato alla sintesi di tre building blocks organici, in cui il residuo tiolico e il macrociclo sono separati da catene alchiliche di diverse lunghezze (6, 9, 12 atomi di carbonio). In prima analisi viene discussa la sintesi e caratterizzazione di nanoparticelle d’oro passivate con i tre tioli (C6TACN, C9TACN, C12TACN), in cui si evidenziano poche differenze in termini qualitativi tra Au NPs C9TACN e Au NPs C12TACN. Au NPs C6TACN dimostrano un monostrato più¹ densamente popolato, probabilmente dovuto ad interazione laterali reciproche più efficenti tra i tioli all'€™interno del monostrato. La valutazione della catalisi dei tre sistemi sulla reazione modello ha evidenziato una similitudine tra Au NPs C9TACN e Au NPs C12TACN in termini di attività  catalitica (kcat) ed interazione con il substrato (KM), senza discostarsi in maniera rilevante dai sistemi studiati nei lavori precedenti, dotati di un tiolo più lungo.[5] Au NPs C6TACN, nonostante mantenga pressochè inalterato kcat rispetto ai due sistemi analoghi, presenta un KM di circa tre volte più elevato, indice di interazione più debole del sistema col substrato. Al fine di studiare il processo di binding tra nanoparticella e oligoanione in maniera più ampia si è ricorso allo studio dell'™interazione delle nanoparticelle sintetizzate con oligoanioni fluorescenti, sfruttando il peculiare quenching della luminescenza mediato dai core d'€™oro per determinare la concentrazione di saturazione del monostrato.[6] L'€™utilizzo di un primo probe fluorescente (fATP), in cui l'interazione con la nanoparticella è mediato in larga misura da carica elettrostatica, ha evidenziato differenze minime tra i tre diversi campioni. Utilizzando un probe (MANT-AMP) in cui vi è un bilancio tra interazione elettrostatica (con la superfice del monostrato) ed idrofobica[7] (con la parte alchilica interna del monostrato) si sono evidenziate differenze sostanziali tra Au NPs C6TACN e Au NPs C9TACN. Tale esperimento è risultato in una assenza di quenching per Au NPs C12TACN, verificando l'™avvenuto binding con un esperimento di displacement indipendente. Il fenomeno è ritenuto imputabile ad un orientamento sfavorevole al quenching del probe nel monostrato. Lavorando in assenza di Zn(II) e a pH nettamente basico al fine di mantenere il monostrato privo di carica, si è potuto osservare una totale assenza di interazione tra MANT-AMP e Au NPs C6TACN, confermando come la differente natura del monostrato giochi un ruolo nell'€™interazione con molecole organiche che va al di là  dell'€™interazione elettrostatica, riscontrabile anche nell'interazione con il substrato catalitico. Questo effetto, seppur sicuramente attribuibile a differenze nella struttura del building block componente il monostrato, può essere dovuto sia al diverso spessore del monostrato sia alla particolare densità  del monostrato stesso per Au NPs C6TACN. Le performances catalitiche e di binding per oligoanioni dei tre sistemi sono state testate in un arco di tempo di 4 mesi per stabilire quale sistema mantenesse tali parametri nel tempo, allo scopo di ottenere informazioni sulla stabilità  dei tre sistemi. Tutti i sistemi si sono rivelati relativamente ben stabili nel tempo, senza dimostrare aggregazione o deterioramento consistente dei parametri osservati. Au NPs C9TACN, contrariamente ai sistemi analoghi, non ha dimostrato alcuna variazione apprezzabile nei valori osservati, confermandosi il sistema più robusto. In ultima analisi è stato preso in considerazione il ruolo delle dimensioni della nanoparticella nell'€™evento catalitico. La sintesi e caratterizzazione di un sistema composto da nanoparticelle di 13 nm di diametro e passivato da tiolo C9TACN ha permesso di confrontarne le performance catalitiche con quelle del sistema analogo utilizzato nello studio precedente. I risultati dimostrano come il sistema sia ancora cataliticamente attivo, con un netto peggioramento dell'attività  catalitica e del binding del substrato rispetto all'analogo supportato su nanoparticelle di diametro 2 nm. Questi risultati possono essere imputati alla mancanza di "œhot spot" catalitici presenti in nanoparticelle piccole e con curvature accentuate.

Dans un monde où les préoccupations environnementales sont de plus en plus présentes, il paraît primordial de mettre en place de nouvelles méthodologies de synthèse plus éco-compatibles. Cela constitue un moteur fondamental pour la conception de nouveaux objets moléculaires possédant des propriétés innovantes, conduisant à la conception de nouveaux matériaux, médicaments ou procédés chimiques. Dans la Nature, les polyols se révèlent comme étant des motifs essentiels pour expliquer l’activité biologique de nombreux produits naturels ou médicaments. Durant cette thèse, nous nous sommes attachés à préparer toute une gamme de polyols halogénés afin d'étudier l'influence de l'halogénation sur les propriétés supramoléculaires de ces molécules. La première partie de ces travaux a porté sur la synthèse énantiosélective de polyols comportant deux motifs halogénohydrines. Nous avons ensuite identifié l'importance de ce motif, permettant une augmentation notable des propriétés de reconnaissance supramoléculaire par établissement de réseaux de liaisons hydrogène. Ces nouveaux polyols se sont avérés être d'excellentes plateformes pour la coordination d'anions, pour la catalyse mais aussi pour la conception de matériaux non-covalents de type organogels. Dans la seconde partie, ces propriétés ont été renforcées via l'insertion de chaînes perfluorées. Ceci a été rendu possible grâce au développement d'une nouvelle réaction d'aldolisation bidirectionnelle énantiosélective catalysée au cuivre conduisant à des 1,3,5-triols perfluorés. Ces nouveaux polyols se sont révélés particulièrement efficaces pour la coordination sélective d’anions, notamment d’anions chiraux
In the actual context of green chemistry, it is of prime importance for organic chemists to develop new eco-compatible synthetic methodologies. These new approaches are fundamental to be able to modulate the molecular properties enabling the design of new materials, drugs or chemical processes. In Nature, 1,3-polyols are essential motifs for the biological activity of many natural products. During this PhD, we have thoroughly studied the influence of halogen insertion in polyols over supramolecular properties.The first part of our work has consisted in the synthesis of polyols possessing two different halogenohydrin motifs. We have then been able to highlight the importance of this motif triggering considerable increase of their supramolecular properties based on H-bonding networks (anion coordination, self-assembly, catalysis). In a second part, we have further strengthen the central H-bonding framework through insertion of lateral perfluorinated chains. The preparation of these new 1,3,5-triols scaffolds was possible thanks to the development of an original copper catalyzed bi-directional aldolization. These new perfluorinated triols were excellent platforms for selective anion binding notably chiral recognition as well as catalysis and for the elaboration of non-covalent materials such as organogelators

El trabajo de tesis doctoral ha consistido en el diseño y síntesis de diferentes receptores moleculares. En particular, de nuevos derivados calix[4]pirrolaril-extendidos con dos y cuatro grupos fosfonatocomo receptores ditópicos para la complejación de pares iónicos,importantes para la extracción de sales y para el transporte de iones a través de las membranas celulares. Asimismo, derivados de calix[4]pirrol han sido preparados para la inclusión de N-óxidos en conformación de alta energía.La importancia de la inclusiónha sido demostrada en la modificación dela reactividad químicatípica delN-óxidoenreacciones en las queel mismo seutilizacomo co-oxidante. Porúltimo, se presentan derivados de resorcin[4]areno con grupos etilo en la parte inferior y una pared móvil en la parte superior como potenciales ligandos para catálisis supramolecular.
The work developed during the PhD thesis deals mainly with the design and synthesis ofSupramolecular Hosts. In particular, we present new aryl-extended calix[4]pyrroleswith two or four phosphonate groups as heteroditopic receptors for the complexation of ion-pairs and that result important for the extraction of salts and for the transportof ions through membranes. Likewise, derivativesofcalix[4]pyrroles have been prepared for the selective inclusionofN-oxides in high energy conformation.The importance of the inclusion has been demonstrated by the change of the reactivity of the N-oxide in reactions in which it is commonly used as co-catalyst. Finally, we present derivatives ofresorcin[4]arenes with ethyl groups at the lower rim of thereceptor and a mobile wall in the upper rim as potential ligands for Supramolecular catalysis.

This thesis describes strategies for and examples ofcellulose fiber modification.The ability of an engineered biocatalyst, acellulose-binding module fused to theCandida antarcticalipase B, to catalyze ring-openingpolymerization of e-caprolactone in close proximity tocellulose fiber surfaces was explored. The water content in thesystem was found to regulate the polymer molecular weight,whereas the temperature primarily influenced the reaction rate.The hydrophobicity of the cellulose sample increased as aresult of the presence of surface-deposited polyester.

A two-step enzymatic method was also investigated. Here,Candida antarctica lipase B catalyzed the acylation ofxyloglucan oligosaccharides.The modified carbohydrates werethen incorporated into longer xyloglucan molecules through theaction of a xyloglucan endotransglycosylase. The modifiedxyloglucan chains were finally deposited on a cellulosesubstrate.

The action ofCandida antarcticalipase B was further investigated inthe copolymerization of e-caprolactone and D,L-lactide.Copolymerizations with different e-caprolactone-to-D,L-lactideratios were carried out. Initially, the polymerization wasslowed by the presence of D,L-lactide. During this stage,D,L-lactide was consumed more rapidly than ε-caprolactoneand the incorporation occurred dimer-wise with regard to thelactic acid units.

Morphological studies on wood fibers were conducted using asol-gel mineralization method. The replicas produced werestudied, without additional sample preparation, by electronmicroscopy and nitrogen adsorption. Information concerning thestructure and accessibility of the porous fiber wall wasobtained. Studies of never-dried kraft pulp casts revealedmicro-cavities and cellulose fibrils with mean widths of 4.7(±2) and 3.6 (±1) nm, respectively.

Finally, cationic catalysis by simple carboxylic acids wasstudied. L-Lactic acid was shown to catalyze the ring-openingpolymerization of ε-caprolactone in bulk at 120 °C.The reaction was initiated with methylß-D-glucopyranoside, sucrose or raffinose, which resultedin carbohydrate-functionalized polyesters. The regioselectivityof the acylation was well in agreement with the correspondinglipase-catalyzed reaction. The polymerization was alsoinitiated with a hexahydroxy-functional compound, whichresulted in a dendrimer-like star polymer. The L-lactic acidwas readily recycled, which made consecutive reactions usingthe same catalyst possible.

Keywords:Candida antarcticalipase B, cationic catalysis,cellulose-binding module, dendrimer, enzymatic polymerization,fiber modification, silica-cast replica, sol-gelmineralization, organocatalysis, xyloglucanendotransglycosylase

Thesis (Ph. D.)--Indiana University, Dept. of Chemistry, 2005.
Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0253. Adviser: Donald H. Burke. "Title from dissertation home page (viewed Feb. 9, 2007)."

Tetrahydrobiopterin (BH4) is a limiting cofactor for nitric oxide synthase (NOS) catalysed conversion of L-arginine to nitric oxide and citrulline. Content of BH4 in mammalian cells is regulated at many levels, but most important is de novo biosynthesis from GTP. GTP cyclohydrolase (GTPCH) is the rate-limiting enzyme for the de novo synthesis of BH4. While various immunostimulants, hormones and growth factors have been reported to increase GTPCH mRNA levels and intracellular biopterin (BH4 degradation product), it is not known whether these factors act at the level of GTPCH gene transcription. To test this I utilised 1, 3 and 6 kb 5'upstream GTPCH gene sequence in a secreted alkaline phosphatase reporter vector (SEAP). These constructs were stably transfected in PC-12 cells and rat aortic smooth muscle cells, and the cells were treated with various immunostimulants and growth factors in order to determine whether these factors could enhance GTPCH gene transcription. Intracellular biopterin levels were also measured to confirm that the upregulation of the SEAP-reporter correlated with a rise in biopterin. Our investigations conclude that transcriptional regulation of the GTPCH gene is indeed a major site for control of intracellular BH4 levels. In further experiments, we have characterised the binding of [3H]BH4 to endothelial NOS (eNOS) and examined influences of the substrate, arginine, on the BH4 binding. In addition we selected tetrahydropterins (that support NOS catalysis) and dihydropterins (that are catalytically incompetent) to determine the extent to which modifications of BH4 alter pterin binding affinity to eNOS. Dihydropterins are unable to support NOS catalysis. Studies showed for the first time that dihydropterins, but not tetrahydropterins, support superoxide generation by eNOS. We also have determined that eNOS may be able to produce NO in the absence of BH4 cofactor from the reaction intermediate hydroxyarginine. We have characterised this reaction and are able to provide a plausible mechanism for the NOx generation from eNOS in the absence of BH4 cofactor.

The current thesis deals with the ammonia oxidation on cobalt oxide catalyst at the molecular level. The catalytic oxidation of ammonia to NO is crucial in the industrial process of nitric acid production. Cobalt oxide catalysts are being used together with platinum gauzes to reduce the production cost and emission of greenhouse gas N2O. However, the fundamentals of ammonia oxidation on cobalt oxides are not known. This thesis aims to provide insights into our fundamental understanding of ammonia oxidation on Co3O4 surfaces. The performance of cobalt oxide catalysts in the oxidation of NH3 strongly depends upon the exposed surface terminations. Results indicate that different surfaces of Co3O4 behave markedly differently in oxidative reactions due to the difference in binding energy and O recombination energies and oxygen vacancy formation. Overall, NH3 oxidation follows stepwise dehydrogenative route (NH3* → NH2* → NH* → N*) on Co3O4 surfaces. Desorption of lattice products results in the formation of O vacancy sites opening the way for a Mars-van Krevelen mechanism. The successive dehydrogenation of ammonia preferably occurs on the surfaces exposing active lattice O sites. Removal of active lattice O sites from the Co3O4 surfaces in the form of products results in the surface reduction. If the rate of reduction is faster than that of re-oxidation, a CoO-like phase might form. The formation of CoO in Co3O4 catalysts during NH3 oxidation not only reduces the NH3 conversion but also alters the selectivity towards N2 rather than NO due to weak ability of lattice O at the CoO surface to assist the hydrogen abstraction process. A surface with a lower oxygen vacancy formation energy and a higher binding energy of hydrogen exhibits a higher activity towards ammonia oxidation to NO.