Dissertations / Theses on the topic 'Covalent approaches'

Aquesta tesi va dirigida específicament al desenvolupament de poliuretans (PU)s funcionalitzats en la cadena lateral (FPU)s, sintetitzats a partir de diols funcionals que provenen d’àcids grassos i dos diisocianats diferents: el diisocianat d’isoforona (IPDI) i el diisocianat d’hexametilé (HDI). Aquests nous FPUs presenten una amina terciària i grups alquil, al•lil, propargil o la combinació d'aquests en la cadena lateral. Posteriorment els FPUs es modifiquen mitjançant dos mecanismes de post-polimerització basats en enllaços covalents o en enllaços no covalents.En el primer cas, es duen a terme una sèrie de reaccions fotoiniciades d’acoplament tiol-é/í entre el grup al.lil i propargil que presenten els FPUs (formats a partir de IPDI), i tioglicerol. Els hidroxi-PUs obtinguts, exhibeixen una millora del seu caràcter hidrofílic. Alternativament, els FPUs que contenen només una amina terciaria com a grup funcional situat a la cadena lateral del PU, es barregen amb diferents àcids carboxílics per donar una reacció àcid base. Els supramoleculars PUs resultants (SPU)s es caracteritzen per espectroscòpia per a verificar la presència d'enllaços iònics d'hidrogen que uneixen les cadenes de PU mitjançant interaccions fisiques. A més, es demostra la correlació existent entre l'estructura química i les propietats tèrmiques i mecàniques dels materials sintetitzats. Aquests materials presenten prometedores propietats adaptatives. Per exemple, ressalten les bones propietats de regeneració i reciclatge/remodelació, degudes al caràcter reversible de les interaccions físiques. Addicionalment, aquests elastòmers posseeixen una inherent capacitat d’autoreparació, que en termes pràctics es podria veure com una millomillora de la seva sostenibilitat. Finalment, es sintetitzen xarxes de PU que tenen un doble caràcter estructural mitjançant enllaços iònics d'hidrogen dinàmics i entrecreuaments covalents. La variació de la densitat d’entrecreuament covalent introduït per a cada una d’aquestes xarxes, produeixun ajustament sistemàtic de les propietats mecàniques i de la sensibilitat del material a la calor. Aquesta preparació demostra una via simple i eficaç per a la fabricació de poliuretans multifuncionals i l’estratègia seguida també es pot estendre a l'exploració d'altres tipus d'interaccions covalents i no covalents en polímers
Esta tesis está dirigida específicamente al desarrollo de poliuretanos (PU)s funcionalizados en la cadena lateral (FPU)s, sintetizados a partir de dioles funcionales que provienen de ácidos grasos y dos diisocianats diferentes; el diisocianato de isoforona (IPDI) y el diisocianato de hexametileno (HDI). Estos nuevos FPUs presentan una amina terciaria y grupos alquilo, alilo, propargilo o la combinación de éstos en posiciones de cadena lateral. Posteriormente los FPUs se modifican mediante dos mecanismos de post-polimerización basados en enlaces covalentes o en enlaces no covalentes.En el primer caso, se llevan a cabo una serie de reacciones fotoiniciadas de acoplamiento tiol-eno/ino entre el grupo alilo y propargilo que presentan los FPUs (formados a partir de IPDI), y tioglicerol. Los hidroxi-PUs obtenidos, exhiben una mejora de su carácter hidrófilo. Alternativamente, los FPUs que contienen sólo una amina terciaria como grupo funcional situado en la cadena lateral del PU, se mezclan con diferentes ácidos carboxílicos mediante una reacción de ácido base. Los PUs supramoleculares resultantes (SPU)s se caracterizan por espectroscopia para verificar la presencia de enlaces iónicos de hidrógeno que unen las cadenas de PU formando interacciones físicas. Además, se demuestra la correlación existente entre la estructura química y las propiedades térmicas y mecánicas de los materiales sintetizados. Estos materiales presentan prometedoras propiedades adaptativas. Por ejemplo, resaltan las buenas propiedades de regeneración y reciclaje/remodelación, debidas al carácter reversible de las interacciones físicas. Adicionalmente, estos elastómeros poseen una inherente capacidad de autorautorreparación, que en términos prácticos se podría ver como una mejora de su sostenibilidad. Finalmente, se sintetizan redes de PU que tienen un doble carácter estructural mediante enlaces iónicos de hidrógeno dinámicos y entrecruzamientos covalentes. La variación de la densidad de entrecruzamiento covalente introducido para cada una de estas redes produce un ajuste sistemático de las propiedades mecánicas y la sensibilidad del material al calor. Esta preparación demuestra una vía simple y eficaz para la fabricación de poliuretanos multifuncionales.
This Thesis is addressed to the development of side-chain functionalized polyurethanes (FPU)s, with enhanced properties, made from fatty acid-based functional diols and two different diisocyanates; isophorone diisocyanate (IPDI) and hexamethylene diisocyanate (HDI). The novel FPUs present tertiary amine and alkyl, allyl, propargyl moieties or the combination of these, as side-chain positions groups. The FPUs were further modified via two post-polymerization mechanisms based on covalent or non-covalent bonds. In the first case, photoinitiated thiol-ene/yne coupling reaction between allyl, propargyl-functionalized PUs (based on IPDI) and thioglycerol was carried out. Obtained hydroxyl-PUs exhibit different thermal and mechanical properties in comparison with precursor PUs. Moreover, the incorporation of hydroxyl groups leads to PUs with enhanced hydrophilicity. Alternatively, the FPU (based on IPDI) containing only tertiary amine pendant group was mixed with different carboxylic acids in an acid-base reaction. Supramolecular ionic PUs were characterized by spectroscopic tools to verify the presence of ionic hydrogen bond as ionic interaction. Correlation between structure and thermal and mechanical properties was demonstrated. Samples show rapid thermal reversibility and recyclability thanks to the reversible bonds. In addition, elastomeric supramolecular PUs networks were prepared from HDI and aminodiol. The resulting materials exhibit some promising adaptive material properties such as effective energy dissipation upon deformation through unzipping the ionic hydrogen bonding network, combined with good shape-regeneration property and recycling/reshaping capability arising from their recoverable nature. More importantly, the resulting biobased elastomers possess the inherent self-healing ability, which can be seen as an upgrade of their sustainability.A novel thermo-reversible network is constructed by the thiol-ene functionalized polyurethane via dynamic ionic hydrogen bonds and covalent cross-links. By varying the covalent cross-linking density, the mechanical properties and the stimuli-responsive behaviour can be systematically tuned. This synthesis demonstrates a simple and effective pathway to fabricate multifunctional polyurethanes with desired functions.

S’ha estudiat la hidrofomilació d΄al·lens 1,1΄-disubstituïts catalitzada per rodi, emprant lligands de tipus bisfosfit amb un centre de regulació allunyat del centre catalític. L'ús de sals de metalls alcalins de BArF com a agents de regulació va fer augmentar la formació d'aldehids β,γ-insaturats (fins a un 60%), demostrant el principi de regulació supramolecular del sistema catalític. La hidroformilació d'octens terminals i interns catalitzada amb cobalt i en presència de Xantphos com lligand ha donat lloc a una alta selectivitat en la formació d'aldehids. El control de la regioselectividad mitjançant la temperatura de la reacció i la proporció constant entre els diferents regioisòmers dels aldehids ens ha portat a postular un procés tàndem d'isomerització-hidroformilació en les condicions optimitzades de reacció. A més, s'ha dut a terme la hidroformilació d'una mescla d'octens, obtenint-se els aldehids amb alta selectivitat i baixa incidència de reaccions secundàries, la qual cosa fa que sigui una ruta plausible a escala industrial. A més, l'ús de lligands regulats supramolecularment amb un centre de regulació allunyat del centre catalític, va permetre la hidroformilació enantioselectiva de aril vinil èters, amb un gran activitat catalítica, regioselectivitat i ràtio enantiomèric (fins a un 88% conversió, >99:1 b/l ràtio, 97:3 er). Estudis de complexació van revelar la formació d'espècies catalítiques inactives i la variació en la distribució de les espècies catalíticamente actives en funció de l'agent de regulació emprat. Finalment es van realitzar estudis cinètics sobre la reacció d'hidroformilació enantioselectiva de l'acetat de vinil emprant lligands bisfosfit regulats mitjançant interaccions supramoleculars. L'ús de l'Anàlisi de Normalització de Temps Variable va permetre esbrinar el pas limitant de la reacció, situant-lo en el començament del cicle catalític. Aquest mètode ha permès obtenir els perfils de reacció en absència dels períodes d'inducció de formació del catalitzador observats durant la reacció.
Se ha descrito la hidroformilación de alenos 1,1΄-disustitutidos catalizada por rodio empleando ligandos de tipo bisfosfito con un centro de regulación alejado del centro catalítico. El uso de sales de BArF de metales alcalinos como agentes de regulación aumentó la formación de aldehídos β,γ-insaturados (hasta un 60%), demostrando el principio de regulación supramolecular del sistema catalítico. La hidroformilación de octenos terminales e internos catalizada con cobalto y en presencia de Xantphos como ligando ha dado lugar a una alta selectividad en la formación de aldehídos. El control de la regioselectividad mediante la temperatura de la reacción y la proporción constante entre los diferentes regioisómeros de los aldehídos nos ha llevado a postular un proceso tándem de isomerización-hidroformilación bajo las condiciones optimizadas de reacción. Además, se ha llevado a cabo la hidroformilación de una mezcla de octenos, obteniéndose los aldehídos con alta selectividad y baja incidencia de reacciones secundarias, lo que la hace una ruta plausible a escala industrial. Además, el uso de ligandos regulados supramolecularmente con un centro de regulación alejado del centro catalítico permitió la hidroformilación enantioselectiva de aril vinil éteres, con una gran actividad catalítica, regioselectividad y ratio enantiomérico (hasta un 88% conversión, >99:1 b/l ratio, 97:3 er). Estudios de complejación revelaron la formación de especies catalíticas inactivas y la variación en la distribución de las especies catalíticamente activas en función del agente de regulación usado. Finalmente se realizaron estudios cinéticos sobre la reacción de hidroformilación enantioselectiva del acetato de vinilo empleando ligandos bisfosfito regulados mediante interacciones supramoleculares. El uso del Análisis de Normalización de Tiempo Variable permitió establecer el paso limitante de la reacción, situándolo en el comienzo del ciclo catalítico y ha permitido la obtención de los perfiles de reacción en ausencia de los periodos de inducción de formación el catalizador observados durante la reacción.
The rhodium-catalyzed hydroformylation of 1,1΄-disubstituted allenes was described employing bisphosphite ligands with a distal regulation site. The use of alkali metal BArF salts as regulation agents enhanced the formation of β,γ-unsaturated aldehydes (up to a 60% increase), proving the supramolecular regulation principle of the catalytic system. Cobalt-catalyzed hydroformylation of terminal and internal octenes in presence of Xantphos as ligand have displayed a high selectivity towards the formation of aldehydes. The control of the regioselectivity by the reaction temperature and the constant ratio in the different regioisomers led us to hypothesize that a tandem isomerization-hydroformylation transformation was taking place under optimized conditions. Furthermore, the hydroformylation of a mixture of octene isomers was reported with excellent aldehyde selectivity and diminished formation of side-products. Overall, the methodology may be suitable for industrial application. Moreover, the use of supramolecularly regulated ligands with distal regulation site enabled the rhodium-catalyzed enantioselective hydroformylation of aryl vinyl ethers, with high catalytic activity, regioselectivity and enantiomeric ratio (up to 88% conv, >99:1 b/l ratio and 97:3 er). Complexation studies revealed the formation of inactive catalytic species and a variation in the distribution of catalytic species based on the regulation agent used. Finally, kinetic studies of the rhodium-catalyzed enantioselective hydroformylation of vinyl acetate with a supramolecularly regulated bisphosphite complex were performed. The use of Variable Time Normalization Analysis allowed for the elucidation of the rate-determining step early in the catalytic cycle. Real kinetic profiles in the absence of catalyst induction periods observed during the reaction were also obtained.

To begin with, macromolecules consisting of poly(amido)amine dendrimers(PAMAM), polyglycidol, hyperbranched poly(amido)amine (HYPAM) were synthesized and characterized extensively. Porphyrins were also synthesized, characterized and modified in line with respective studies. Hereafter, surface modified TRIS PAMAM dendrimers and its analogs (hyperbranched polymers, hyperbranched PAMAMs) as potential drug delivery systems were studied with the use of model drugs (Ibuprofen and Porphyrin). Analogs of the model drugs were used to investigate the role of secondary interactions for high drug loading(s). UV-Vis Spectroscopy was utilized for studying and determining the maximum loading of the macromolecules under investigation. Further ahead, non-covalent approaches to improve dendrimer-protein binding were used by introducing amino acid chains as targeting groups on the dendrimer surface. Surface modified carboxylate PAMAM dendrimers were studied for their ability to bind with zinc metallated porphyrin. UV-Vis and Fluorescence Spectroscopy were used for protein binding studies. Lastly, Surface Crosslinked Micelles were synthesized and utilized as artificial blood mimics with an attempt to increase the half-life of encapsulated iron porphyrin acting as the heme mimic with the help of UV-Vis Spectroscopy.

Metabolism of xenobiotic drug molecules can result in the formation of metabolites which are more chemically reactive than the parent drug from which they are derived. These reactive species have the potential to covalently modify biological macromolecules if they are not detoxified. The formation of drug-protein adducts carries a potential risk of clinical toxicities and idiosyncratic adverse drug reactions which can, in severe cases, result in hospitalisation and even death. Current methods for the evaluation of the risk for a drug to cause adverse drug reactions due to drug-protein binding rely on risk factors such as quantitative covalent binding value, structure, dose etc. The objective of this project was to develop methods for the detection of reactive metabolites directly bound to proteins, which could be used in future evaluations of the mechanisms of binding of candidates in drug development. Three compounds known to produce reactive metabolites, acetaminophen, SB-648969 and amodiaquine, were used as tool substrates. In vitro incubations with human liver microsomes and individual cytochrome P450 enzymes (as Supersomes ) were used to produce reactive metabolite species and binding with the incubation proteins evaluated. Analysis of the intact proteins, peptides generated via trypsin digestion of the incubation protein, and amino acids generated via digestion with pronase were evaluated using a combination of LC/MS and LC-MS/MS. Reactive metabolite trapping experiments with glutathione were used to provide information about the likely structure of the bound species and the specificity of binding, and were useful in the development of sensitive targeted precursor ion scanning and multiple reaction monitoring methods. [14C] radiolabelled acetaminophen and SB-649868 were used to assess the quantitative levels of binding (<5% modification of protein in both cases). Radiodetection using accelerator mass spectrometry (AMS) was used to evaluate the stoichiometry of binding and aid the identification of adducted peptides through retention time comparison. Chemical and electrochemical methods were utilised to produce stable solutions of N-acetyl-p-benzoquinone imine (NAPQI) and amodiaquine quinone imine (AQQI), reactive metabolites of acetaminophen and amodiaquine, respectively, which were bound to selected proteins and used as chromatographic and mass spectrometric standards. These methods were used to successfully identify an acetaminophen-modified peptide (T56) of cytochrome P450 CYP2E1. No modified proteins were observed for the SB-649868 incubations, however, examination of the AMS chromatograms for the incubations with acetaminophen and SB-649868 revealed a difference in the stoichiometry of binding, with one modified peptide observed with acetaminophen, and several for the incubations with SB-649868. The detection and identification of drug-protein adducts remains extremely challenging due to the low levels of any adducts observed, which can be exacerbated by binding on multiple sites of a protein; however this project has demonstrated that sensitive and selective LC/MS methods can be successfully developed to identify drug-protein adducts.

Ce manuscrit aborde plusieurs interactions / réactions chimiques importantes se produisant dans la soluton en utilisant la calorimétrie par titrage isothermique (ITC) et la théorie de la densité fonctionnelle statique (DFT). Cette thèse porte son attention notamment sur : l'association de paires de Lewis (frustrées) ((F)LPs), la migration cis du groupe méthyle au sein du pentaméthylmanganèse induit par les phosphines, l'aminolyse de carbènes de Fischer, l'insertion d'alcynes dans des palladacycles, l'affinité de divers donneurs de Lewis à l’hexafluoroisopropanol. L'ITC s'est révélé être une technique expérimentale puissante pour obtenir des données thermochimiques fiables sur les systèmes étudiés. Les calculs statiques DFT-D ont montré une capacité d’estimation correcte des paramètres de réaction thermodynamique lorsque l’influence de la solvatation n’est pas significative. Autrement, lorsque l’influence du solvant est apparente, les calculs ne permettent pas de reproduire les résultats expérimentaux. En plus, les résultats expérimentaux et théoriques révèlent l’existence d’ensembles moléculaires plus grandes dans la solution de FLP, soulignant le rôle des interactions non covalentes
This manuscript adressed several important chemical interactions/reactions taking place in solutuon by using Isothermal Titration Calorimetry (ITC) and static Density Functional Theory (DFT). Namely, this thesis dealt with: association of (frustrated) Lewis pairs ((F)LPs), cis-migration of methyl group within pentamethylmanganese induced by phosphines, aminolysis of Fischer carbenes, insertion of alkynes into palladacycles, affinity of various Lewis donors to hexafluoroisopropanol. The ITC proved to be powerful experimental technique for obteining reliable thermochemical data of sutudied systems. The static DFT-D calculations showed capability for proper estiamtion of thermodynamic reaction parameters when an influence of solvation is not sighnificant. Otherwise, when the influence of solvent is not innocent, the calculations moslty failed to reproduce the experimantal results. In addition, Both the experimantal and therortical results revield existance of larger molecular clusters in solution of FLPs emphasising a role of non-covalent interactions

Polycomb-group (Pc-G) proteins regulate homeotic gene silencing associated with the repressive covalent histone modification, trimethylation of histone H3 lysine 27 (H3K27me3). Pc-G mediated silencing is believed to remodel chromatin, rendering target genes inaccessible to transcription factors. Pc-G mediated silencing might result in irreversible changes in chromatin structure, however, there has been little analysis addressing whether Pc-G mediated silencing is reversible. In this work we focused on CURLY LEAF (CLF), the first Pc-G homologue discovered in Arabidopsis. CLF mediated repression of the floral homeotic gene AGAMOUS (AG) was challenged during early and late leaf development. AG was activated by the late leaf promoter, revealing that Pc-G mediated silencing can be overcome in old leaves in the presence of CLF. AG was also activated in young leaf primordia, yet did not persist in older leaves, revealing that transient activation of a Pc-G target is not epigenetically stable. To address the mechanism of Pc-G action within an endogenous environment, the histone dynamics at the APETALA1 (AP1) locus were characterized by Chromatin Immunoprecipitation. Unexpectedly, we found that the activation of AP1 in leaves did not require the removal of H3K27me3, questioning whether H3K27me3 is sufficient to silence. The roles of CLF in leaf and flower development are masked due to partial redundancy with SWINGER (SWN). clf- swn- mutants form a callus-like mass on sterile-tissue culture with no distinguishable plant organs. The role of CLF in regulating flowering time in natural populations of A. thaliana was investigated by complementing clf- mutants with CLF alleles from two accessions. We found that natural variation in CLF did not affect flowering time. To dissect the roles of CLF and SWN in late leaf and flower development, two approaches were developed for targeted expression. Firstly, CLF was introduced into the LhG4/ pOp transactivation system to provide CLF during early plant development. For mosaic analysis, CLF was introduced into the CRE lox recombination system in order to create clf- sectors surrounded by CLF+ SWN+ and CLF+ swn- cells.

Thesis (MSc)--Stellenbosch University, 2003.
ENGLISH ABSTRACT: Advances in pharmacology, biochemistry and biotechnology are increasingly dependant upon affinity chromatography as a preferred separation technique for the purification and characterisation of specific biomolecules. In the past few years avidin-biotin technology has been widely and successfully used in the fields of medicine, pharmacy, biology and biochemistry. The avidin-biotin complex (ABC) has been used as a mediator for affinity chromatography, affinity cytochemistry, immunoassay, histopathology, bioaffinity sensors, erosslinking and immobilisation studies. The main reason for the popularity of the ABC and its growing usefulness in biotechnology is the exceptionally high affinity (1015 M-l) and stability of the noncovalent interaction between avidin and biotin. The use of the ABC is broadening as different biotin derivatives and avidin-containing conjugates are becoming commercially available. The aim of this work was to evaluate the usefulness of a plutonic" FI 08 and the ABC conjugate to effect affinity separation. Towards this aim, the adsorption of plutonic" F108 onto hydrophobic polysulphone membrane surfaces was studied. This information was used to determine the theoretical maximum amount of pluronic" FI08 that will adsorb onto a unit surface area of the membrane. It is known that the polypropylene oxide (PPO) centre block ofthe pluronic" F I08 surfactant molecule governs the concentration of pluronic" F I 08 molecules that will adsorb onto a given hydrophobic surface. If the maximum coating concentration of plutonic" FI08 is known, one can assume that the maximum coating concentration of any pluronic derivative, with the same PPO centre block size, will be the same. Adsorption studies were carried out, the Langmuir adsorption isotherm was determined, and subsequently the fractional coating was calculated. The end-groups of plutonic" FI08 were modified as follows and the substituted pluronic was adsorbed onto a membrane that was to act as the solid support matrix in the development of an affinity system: Amino pluronic was synthesised by first tosylating pluronic" FI08, followed by azidation with NaN3 then reduction with LiAI~. The synthesised amino pluronic was then biotinylated using N-hydroxysuccinimide biotin ester. The suitability of this synthetic route was first assessed on a model compound, 2-methoxyethylamine, and validated by NMR (Nuclear Magnetic Resonance) spectroscopy. The synthetic protocol was then used to derivatise the larger pluronic molecule. The affinity system was tested on two different hydrophobic surfaces: polystyrene and polysulphone membranes (PSMs). Avidin-conjugated horseradish peroxidase was obtained and used to interact with the immobilised biotin. The enzymatic reaction of the coupled peroxidase converted the substrate, 2, 2'-azino-di-(3-ethyl-benzthiazoline-6-sulphonic acid) (ABTS) to a coloured product. The colour developed is proportional to the amount of biotin that was immobilised on the hydrophobic surfaces studied. Non-covalent immobilisation of the synthesised biotin-pluronic molecule was successfully obtained onto the hydrophobic polystyrene as well as the polysulphone membrane surfaces.
AFRIKAANSE OPSOMMING: Vooruitgang in die farmakologie, biochemie en biotegnologie word al meer afhanklik van affiniteits chromatografie as die verkose tegniek vir die suiwering en karaterisering van spesifieke biomolekules. Oor die afgelope jare het die avidien-biotien tegnologie homself as baie bruikbaar bewys in die mediese, farmakologiese, biologiese en biochemiese velde. Toepassings waar die avidien-biotien kompleks betrokke was sluit in die toepassing as 'n mediator vir affiniteits chromatografie, affiniteits sitologie, immuno bepalings, histopatologie, bioaffiniteits sensors sowel as kruisbinding en immobiliserings studies en vele meer. Die hoofrede vir die gewildheid van die avidien-biotien kompleks en die groeiende bruikbaarheid in die biotegnologie is die buitengewone hoë affiniteit (l015 M-I ) en stabiliteit van die nie-kovalente interaksie tussen avidien en biotien. Die toepassingsveld van die avidien-biotien kompleks word wyer met die verskeidenheid biotien derivate en avidien-bevattende konjugate wat kommersiëel beskikbaar is. Die doel van die werk wat hier gedokumenteer word is om die bruikbaarheid van Plutonic" FI08 en die avidien-biotien kompleks, vir gebruik in 'n affiniteits chromatografie sisteem, te evalueer. Om hierdie doel te bereik is die adsorpsie van Pluronic" FI08 aan hidrofobiese polisulfoon membraan oppervlaktes bestudeer. Die eksperimentele data wat gegenireer is, is gebruik om die teoretiese maksimum hoeveelheid Pluronic wat per eenheids oppervlakte membraan adsorbeer te bepaal. Dit is reeds bekend dat die polipropileen (PPO) middel blok van die Pluronic emulgant die konsentrasie van die geadsorbeerde Pluronic molekules op 'n gegewe hidrofobiese oppervlakte bepaal. Indien die maksimum bedekkingskonsentrasie VIr maksimum oppervlakbedekking van Plutonic" FI08 bekend is, kan teoreties aanvaar word dat die bedekkingskonsentrasie vir enige Pluronic derivaat met dieselfde grootte PPO blok dieselfde sal wees. Adsorpsiestudies was uitgevoer om die Langmuir adsorpsie isoterm te bepaal. Daaropvolgend was die fraksionele bedekking bereken. Amino-pluronic was gesintetiseer deur die eindpunte van Pluronic te derivatiseer. Hierdie Pluronic derivaat was gevolglik geadsorbeer aan 'n membraan wat gedien het as die soliede oppervlakte vir die ontwikkeling van 'n affiniteits chromatografie sisteem. Amino-pluronic was gesintetiseer deur Pluronic eers te tosileer en daarna te asideer met NaN3 en laastens te reduseer met LiAI~. Die produk was gebiotinileer deur gebruik te maak van N-hidroksisuksinimied-biotien-ester. Die bruikbaarheid van hierdie sintetiese roete is eers bepaal deur van 'n model verbinding, 2-metoksiëtielamien, gebruik te maak en dit met behulp van KMR (Kern Magnetiese Resonans) spektroskopie te karakteriseer. Die affiniteits sisteem is getoets op twee verskillende hidrofobiese oppervlaktes naamlik polistireen en polisulfoon membraan oppervlaktes. Avidien gekonjugeerd met 'n peroksiedase ensiem is gebruik om met die geïmmobiliseerde biotien te assosieer. Die ensiematiese reaksie van die gekoppelde peroksiedase het die substraat 2, 2' -azino-di-(3-etiel-benzthiazolien-6-sulfoonsuur) (ABTS) omgesit na 'n gekleurde produk, waar dit teenwoordig is. 'n Reeks wasstappe is gebruik om die gemodifiseerde peroksidase ensiem wat nie aan die hidrofobiese oppervlakte gekoppel nie, weg te spoel. Hierdeur is die mate van binding aan die hirofobiese oppervlakte gekwantifiseer deur die kleur te kwantifiseer wat ontwikkelomdat die kleurontwikkeling direk proporsioneel is aan die hoeveelheid peroksidase wat nog aan die membraan gekoppel is. Nie-kovalente immobilisasie van die gesintetiseerde biotien-pluronic molekule is suksesvolop beide die hidrofobiese polistireen oppervlakte sowel as die polisulfoon membraan verkry.

This Thesis reports the synthesis, purification and characterisation of gold nanoparticles (NPs) functionalised with a monolayer of hydrazone ligands in order to perform post-synthetic manipulations of the NP-bound monolayer exploiting dynamic covalent chemistry. NP post-synthetic manipulation based on reversible non-covalent interactions between oligonucleotides represents a promising approach to achieve functionalisation and self-assembly for potential applications in biology and medicine. However, the stability of these nanosystems is ensured only in a narrow window of environmental conditions. On the other hand, irreversible covalent strategies potentially allow the full range of synthetic chemistry to be exploited but they provide poor control over the manipulation of the NP-bound monolayer and can only produce kinetically controlled amorphous NP aggregates. Dynamic covalent chemistry represents an interesting and an attractive alternative approach because it would combine the reversibility of non-covalent interactions with the stability of covalent bonds. By this way, ligand-functionalised NPs could be manipulated in order to introduce a large variety of molecular functionalities on the NP surface not only to subtly tune the NP physicochemical properties but also to access an entire range of novel nanomaterials.

This research explores the links between the structure and dissociation energetics of ionized non-covalent complexes. In chapter 3, a large series of similar non-covalent complexes were probed using electrospray tandem mass spectrometry (ESI-MS/MS) and RRKM modelling in order to identify any trends in the dissociation energetics based on charge state, overall size of the complex, or size of the substrate. Ion mobility spectrometry (IMS) in conjunction with molecular mechanics/molecular dynamics (MM/MD) was used to study the conformations of these non-covalent complexes in order to determine if the same trends identified in the energetics could be corroborated independently based on structure. The system of study consisted of varying lengths of the synthetic polymer, polymethylmethacrylate (PMMA) complexed with singly or doubly protonated diaminoalkanes (DAA) of varying length. The critical energies of dissociation (E0) increased as the length of the polymer increased and was not significantly affected by the length of the singly protonated DAA substrates. The E0 of dissociation of doubly protonated complexes was strongly influenced by the length of the DAA; longer DAA substrates had greater separation of charge which decreased coulombic repulsion within the complex resulting in higher E0 values. MM/MD low energy structures of all complexes were validated with experimental IMS measurements and showed that the arrangement between the polymer and DAA were similar for different singly protonated DAAs. When doubly protonated, the length of DAA was the most important factor in determining the overall structure of the complex. In chapter 4, a direct link is shown between the observed E0 dissociation energies and the molecular conformations for eight different peptide–saccharide complexes containing either a tri-saccharide (d-(+)-raffinose and d-panose) or tetra-saccharide (stachyose and maltotetraose) with a small peptide (FLEEL and FLEEV). The E0 values were highly related to the overall conformation adopted by the non-covalent complex in the gas phase. Complexes containing peptide FLEE(L/V) with the tri-saccharide raffinose or panose had similar E0 of dissociation (∼0.64 eV) and similar conformations based on MM/MD simulations and IMS drift times. Conversely, for complexes containing a FLEE(L/V) peptide with one of the isomeric tetra-saccharides; stachyose had a E0 ∼0.08 eV greater than maltotetraose. This difference of intermolecular interaction was also reflected by the IMS drift times; maltotetraose in complex with FLEEV or FLEEL had a 5.9% and 2.3% faster IMS drift time than stachyose respectively. This indicated that the molecular arrangement between maltotetraose and the peptides was more compact than the stachyose-peptide complexes. In chapter 5, RRKM modelling of breakdown diagrams is not possible when the reactant ion signal is overlapped by other isobaric species. Trimeric, non-covalent complexes that contained two PMMA molecules and a doubly protonated DAA, [(PMMAa)(DAA+2H)(PMMAb)]+2, have m/z signals that contain multiple different complexes having the same total number of polymer repeat units but differ in the length of the each polymer. In this situation, the applicability of using the simple kinetic method to gain insight into relative binding energies was explored. The major factors which determined the suitability of the kinetic method for this system were identified as the structural arrangement of the reactant ion complex, possible reverse activation barriers, and the evaluations of Δ(ΔS‡). MM/MD simulations coupled with IMS suggests that within the reactant ion, the DAA is almost equally shared between two PMMA oligomers and that the two PMMA oligomers interact predominately with the DAA, and not with each other. MS/MS of the trimeric reactant complexes proceeds by neutral loss of one polymer and is suggested to proceed with little or no reverse activation barrier based on the low coulombic repulsion factors. The IMS drift times of [(PMMAa)(DAA+2H)]+2 complexes that were generated directly by ESI-MS or by dissociation of a trimeric, [(PMMAa)(DAA+2H)(PMMAb)]+2 complex were found to be identical. This provides some evidence that Δ(ΔS‡) ≈ Δ(ΔS) and using a statistical mechanics approach, Δ(ΔS) ≈ 0. The effective temperature (Teff) variable in the kinetic method expression was found to decrease as a function of the size of the trimeric complex, suggesting that the population distribution of the dissociating ensemble of complexes narrows as size increases. Overall, when RRKM fitting is not possible, the simple kinetic method could provide relative energetic ranking of competing dissociations reactions however the Teff term contributed to the greatest uncertainty in obtaining absolute quantities. Fitting MS/MS breakdown diagrams of non-covalent complexes with multiple dissociation channels is difficult due to the number of total fitting variables. Building from the simple kinetic method, chapter 6 shows that the relationship between the natural logarithm of competing fragment ions and reciprocal collision energy yields a branching relationship that allows for the sign of Δ(ΔS‡) and Δ(E0) between the channels to be obtained. Furthermore, the relationships between the fitting variables of RRKM modelling are empirically related to the theoretical branching relationship characteristics. This allowed for the fitting variables of all dissociation channels to be expressed as a function of a single channel so that the theoretical branching relationship matches the experimental branching relationship. Using this method, RRKM fitting of a MS/MS breakdown diagram for APCI ionized anthracene determined the E0 and ∆S‡ was 4.69 ± 0.29 eV and -3 ± 17 J K-1; 4.21 ±0.29 eV and -19 ±15 J K-1; and 4.81 ± 0.29 eV and 36 ±22 J K-1 for hydrogen loss, acetylene loss and diacetylene loss respectively. With one exception, these values are within experimental error of the iPEPICO derived energetic values. In chapter 7, MS/MS of ammoniated triacylglycerides at multiple collision energies and computational analysis are used to explain the cause of uneven dissociation rates of the FAs from different positions on the glycerol backbone. The loss of sn-1 and sn-3 FAs are found to have lower activation energies than the loss of the sn-2 position FA, however the loss of the FA from the sn-2 position is more entropically favourable. Theoretical MS/MS breakdown curves were fit to experimental values using RRKM theory to estimate the E0 of dissociation of FAs from the three glycerol positions. The E0 values for cleavage from the sn-1 and sn-3 positions were found to be approximately 1.52 eV, while that for the sn-2 position was highly dependent on the identity of the FA at that position. Computational structures and energy analysis suggest that an important step in the dissociation of [TAG+NH4]+ is the loss of ammonia. In a model system, glyceryl tributyrate, the loss of NH3 produced two distinct [TAG+H]+ product structures sitting 148 kJ and 160 kJ in energy above the ammoniated structure. The [TAG+H]+ structure that leads to the loss of the sn-1(3) is 12 kJ lower than the [TAG+H]+ structure that leads to the loss of the sn-2 FA. From this, the loss of a neutral FA that follows sits only an additional 35–48 kJ above the [TAG+H]+ structures. In Chapter 8, singly deprotonated β-cyclodextrin monomers, [(β-CD-H+]-1, and doubly deprotonated dimers, [(β-CD)2-2H+]-2, are both present following ESI-MS and have the same monoisotopic m/z. Similar to chapter 5, this makes it difficult to generate an MS/MS breakdown diagrams that can be modelled with RRKM theory. IMS was used to mobility separate [(β-CD-H+]-1 and [(β-CD)2-2H+]-2 and was followed by MS/MS of the [(β-CycD)2-2H+]-2 ion. A second problem when generating a MS/MS breakdown diagram of non-covalent complexes that contain identical components is that the fragment ions could have an identical monoisotopic m/z as the reactant ion. MS/MS of [(β-CycD)2-2H+]-2 results in two [(β-CD-H+]-1 fragments. To overcome this, breakdown diagrams were then generated by monitoring the changes in the isotopic profile. The RRKM derived E0 for dissociation of [(β-CycD)2-H+]-1 and [(β-CycD)2-2H+]-2 were 1.85 ± 0.11eV and 1.79 ± 0.09eV, respectively, corresponding to a slight decrease in complex stability due to increased charge-charge repulsion in the dianion.

La réalisation de micro et nanomatériaux avec des propriétés contrôlées nécessite le développement de nouvelles voies d’élaboration dites ascendantes « ou Bottom-up ». Les travaux présentés dans ce manuscrit visent à maîtriser la formation d’agrégats de particules colloïdales et de cellules, avec des applications potentielles dans les domaines des biocapteurs, de la microélectronique, de l’optique et de l’ingénierie tissulaire. L’approche proposée pour assembler les particules en structures organisées se base sur l’emploi de la diélectrophorèse, reposant sur l’application d’un champ électrique non- uniforme. L’un des inconvénients de cette technique tient au caractère réversible des assemblages ainsi formés, l’annulation du champ électrique entrainant la redispersion des colloïdes. Afin d’apporter une solution à ce problème, il est possible de recourir à un couplage chimique pour maintenir la cohésion des structures formées dans le milieu liquide. Afin de mieux maîtriser le comportement diélectrophorétique et les réactions chimiques exploitées, des travaux de caractérisation électrique et chimique des particules manipulées ont été réalisés. D’autre part, différents types de microélectrodes ont été étudiées pour la génération du champ électrique nécessaire à l’assemblage. Une nouvelle filière technologique a été développée pour la réalisation de microélectrodes transparentes en ITO et leur intégration en système microfluidique, basée sur l’exploitation du pouvoir isolant d’une fine couche de PDMS micro-structurée. La méthode a été appliquée à la fabrication de microélectrodes « verticales » puis à la réalisation de matrices d’électrodes quadripolaires. Ces dernières ont permis d’obtenir des assemblages permanents de particules de polystyrène fluorescentes présentant des groupements carboxyliques en surface, en combinant l’emploi de la diélectrophorèse négative et l’utilisation d’un agent de couplage chimique (Jeffamine). Des agrégats de cellules HEK 293 ont également été réalisés par diélectrophorèse négative. Nous avons démontré qu’il était possible, sous certaines conditions, de préserver le caractère permanent des agrégats cellulaires après coupure du champ
The design of micro and nanomaterials with controlled properties requires the development of new bottom- up assembly approaches. The work presented in this manuscript aims to control the formation of aggregates of colloidal particles and cells, with potential applications in the fields of biosensors, microelectronics, optics and tissue engineering. The proposed approach for assembling colloids into organized structures is based on the use of dielectrophoresis, a phenomenon observed when polarizable particles are placed in a non-uniform electric field. One of the drawbacks of this technique is the reversibility of the assemblies thus formed, the cancellation of the electric field causing the redispersion of the colloids. As a solution to this problem, we proposed to use a coupling agent to maintain the cohesion of the structures formed in the liquid medium. In order to better control the dielectrophoretic behavior and the chemical reactions exploited, electrical and chemical characterizations of the manipulated particles were carried out. Moreover, different types of microelectrodes have been studied for the generation of the electric field required for the assembly. A new approach has been developed for the fabrication of transparent micro patterned ITO microelectrodes and their integration in microfluidic systems, based on the exploitation of a thin micro-structured PDMS membrane used as an insulating layer. The method has been applied to the fabrication of "vertical" microelectrodes and of quadrupolar electrode arrays. The latter were used to obtain permanent assemblies of carboxylic acid functionalized, fluorescent, polystyrene particles, by combining negative dielectrophoresis with the use of a chemical coupling agent (Jeffamine). HEK 293 cell aggregates were also produced by negative dielectrophoresis. We have demonstrated that it was possible, under certain conditions, to preserve the permanent character of the cell aggregates after field removal

La preparación de nuevos ligandos quirales P-OP (fosfina-fosfinitos y fosfina-fosfitos), fácilmente preparados con una estrategia sintética en dos etapas desde una aproximación covalente, es descrita en la presente Tesis Doctoral. El mejor catalizador de la serie ha demostrado tener propiedades catalíticas excelentes en la hidrogenación asimétrica catalizada por rodio de una amplia variedad de olefinas funcionalizadas. El resultado excelente y el diseño modular de los ligandos sintetizados hacen éstos muy atractivos para futuras aplicaciones.
La presente Tesis Doctoral describe también la preparación de nuevos ligandos quirales que pueden comportarse como catalizadores supramoleculares inspirados en el mecanismo de regulación alostérica de los enzimas.
A library of enantiomerically pure P-OP ligands (phosphine-phoshinites and phosphine-phosphites) straightforwardly available in two synthetic steps from enantiopure Sharpless epoxy ethers is reported in the present PhD. Thesis. The "lead" catalyst of the series has proven to have outstanding catalytic properties in the rhodium-catalysed asymmetric hydrogenation of a wide variety of functionalised alkenes. Their excellent performance and modular design makes them attractive for future applications.
This PhD. Thesis also reports the development of a practical route to chiral diphosphine ligands with supramolecular motifs, with potential for allosteric modulation, which we prepared for future catalytic studies.

The human 20S proteasome activity and malfunction has been related to numerous diseases and validated as a protein target for inhibition in the treatment of cancer, with three proteasome inhibitors approved as a drug. But these compounds could be improved, since usually the molecular mechanism of action is unknown. Thus, computational studies can clarify the mode of action of proteasome inhibitors, helping to understand the system and improve the inhibition process The present thesis is devoted to understand the mode of action of two classes of covalent inhibitors of the 20S proteasome, α,β-epoxyketones and γ-lactam-β-lactones. Molecular dynamics simulations with hybrid QM/MM potentials have been used to characterize the free energy landscape for the inhibition mechanism of these compounds and to provide the structures necessary to analyze and understand the inhibition process in the β5 active site of the proteasome, providing valuable knowledge to optimize the compounds into more efficient inhibitors.
Programa de Doctorat en Química Teòrica i Modelització Computacional

The goal of this work is to develop novel molecular systems, functionalization techniques, and data collection routines with which to study the binding of immobilized cognate binding partners. Our ultimate goal is the routine evaluation of thermodynamic parameters for immobilized systems through interpretation of the variation of the binary probability of binding as a function of soluble ligand concentration. The development of both data collection routines that minimize non-specific binding and functionalization techniques that produce stable ordered molecular systems on surfaces are of paramount importance towards achievement of this goal. Methodologies developed here will be applied to investigating the thermodynamics of multivalent systems.

In the first part of this work, the effect of contact force on molecular recognition force microscopy experiments was investigated. Increased contact forces (>250 pN) resulted in increased probabilities of binding and decreased blocking efficiencies for the cognate ligand-receptor pair lactose-G3. Increased contact force applied to two control systems with no known affinity, mannose-G3 and lactose-KDPG aldolase resulted in non-specific ruptures that were indistinguishable from those of specific lactose-G3 interactions. Thus, it is essential to design data collections routines that minimize contact forces to ensure that ruptures originate from specific, blockable interactions.

In the second part of this work we report the first example of the preparation of stable self assembled monolayers through hydrosilylation of a protected aminoalkene onto hydrogen-terminated silicon nitride AFM probes and subsequent conjugation with biomolecules for force microscopy studies. Our technique can be used as a general attachment technique for other molecular systems.

In the third part of this work we develop novel molecular systems for tethering oriented vancomycin and its cognate binding partner L-Lys-D-Ala-D-Ala to surfaces and AFM tips. Unbinding experiments demonstrated that traditional methods for forming low surface density amine layers (silanization with APTMS and etherification with ethanolamine) provided molecular constructs which displayed probabilities of binding that were too low and showed overall variability too high to use for probabilistic evaluation of thermodynamics parameters. Instability and heat-induced polymerization of APTMS layers on tips and surfaces also prohibited their utility. Formation of Alkyl SAMs on silicon provides a more reliable, stable molecular system anchored by Si-C bonds that facilitates attachment of vancomycin and is capable of withstanding prolonged exposure to heated organic and aqueous environments. It follows that covalent immobilization of KDADA to silicon nitride AFM tips via Si-C bonds using hydrosilylation chemistry will be similarly advantageous. These methods offer great promise for probabilistic evaluation of thermodynamic parameters characterizing immobilized binding partners and will permit unambiguous determination of the role of multivalency in ligand binding, using an experimental configuration in which intermolecular binding and aggregation are precluded.

Dissertation

Dissertação de Mestrado em Física apresentada à Faculdade de Ciências e Tecnologia
Non-covalent interactions have long been known to be responsible for significant mechanisms in biological structures, and have historically lacked a satisfying or intuitive quantum mechanical description. The recently developed NCI index method by Johnson et al [1] fills a gap that existed within topological methods for the visualization of non-covalent interactions [2]. Even following its implementation by some of the original authors on the NCIPLOT software [3, 4], the method itself has not gained much traction in the Physics community, despite its possible and pertinent use in visualizing and describing non-covalent interactions in solids, including novel materials in which this type of interaction is responsible for structural stabilization. The project described in this document aims to bridge this gap between the method and its potential users by implementing it on a significantly simple, fast, and easy to modify Python script which works for several types of systems and takes as input files of the universally known cube format, which can be output by most major quantum chemistry and electronic structure codes. The resulting code is tested on both simple systems and systems of significant interest.[1] Erin R. Johnson, Shahar Keinan, Paula Mori-Sánchez, Julia Contreras-García, Aron J. Cohen, and Weitao Yang. Revealing noncovalent interactions. Journal of the American Chemical Society, 132(18) 6498–6506, may 2010.[2] Christophe Narth, Zeina Maroun, Roberto A. Boto, Robin Chaudret, Marie Laure Bonnet, Jean Philip Piquemal, and Julia Contreras-García. A Complete NCI Perspective: From New Bonds to Reactivity. In Challenges and Advances in Computational Chemistry and Physics, volume 22, pages 491–527. 2016.[3] Julia Contreras-García, Erin R Johnson, Shahar Keinan, Robin Chaudret, Jean-Philip Piquemal, David N Beratan, and Weitao Yang. NCIPLOT: A Program for Plotting Noncovalent Interaction Regions. J. Chem. Theory Comput, 7:625–632, 2011.[4] Roberto A Boto, Francesca Peccati, Chaoyu Quan, Alessandra Carbone, Jean-Philip Piquemal, Yvon Maday, and Julia Contreras-Garcia. NCIPLOT4: Fast, Robust, and Quantitative Analysis of Noncovalent Interactions. J. Chem. Theory Comput, 16:0, 2020.
Há algum tempo se sabe que interações não-covalentes são responsáveis por mecanismos significativos em estruturas biológicas e, historicamente, careciam de uma descrição quântica intuitiva ou satisfatória. O método `NCI index' desenvolvido recentemente por Johnson et al [1] preenche uma lacuna que existia em métodos topológicos para a visualização de interações não-covalentes [2]. Mesmo depois da sua implementação por alguns dos seus autores originais no software NCIPLOT [3, 4], o método em si não ganhou muita visibilidade na comunidade de Física, apesar do seu uso possível e pertinente na visualização e descrição de interações não-covalentes em sólidos, incluindo novos materiais nos quais este tipo de interação é responsável pela estabilização estrutural. O projeto descrito neste documento visa preencher esta lacuna entre o método e seus potenciais usuários, implementando-o num `script' Python significativamente simples, rápido e fácil de modificar, que funciona para vários tipos de sistemas e toma como `input' ficheiros do formato universalmente conhecido `cube', que pode ser gerado pela maioria dos principais códigos de química quântica e estrutura eletrónica. O código resultante é testado tanto em sistemas simples como sistemas de interesse significativo.[1] Erin R. Johnson, Shahar Keinan, Paula Mori-Sánchez, Julia Contreras-García, Aron J. Cohen, and Weitao Yang. Revealing noncovalent interactions. Journal of the American Chemical Society, 132(18) 6498–6506, may 2010.[2] Christophe Narth, Zeina Maroun, Roberto A. Boto, Robin Chaudret, Marie Laure Bonnet, Jean Philip Piquemal, and Julia Contreras-García. A Complete NCI Perspective: From New Bonds to Reactivity. In Challenges and Advances in Computational Chemistry and Physics, volume 22, pages 491–527. 2016.[3] Julia Contreras-García, Erin R Johnson, Shahar Keinan, Robin Chaudret, Jean-Philip Piquemal, David N Beratan, and Weitao Yang. NCIPLOT: A Program for Plotting Noncovalent Interaction Regions. J. Chem. Theory Comput, 7:625–632, 2011.[4] Roberto A Boto, Francesca Peccati, Chaoyu Quan, Alessandra Carbone, Jean-Philip Piquemal, Yvon Maday, and Julia Contreras-Garcia. NCIPLOT4: Fast, Robust, and Quantitative Analysis of Noncovalent Interactions. J. Chem. Theory Comput, 16:0, 2020.