No description available.

L’ACC Oxydase est une enzyme à Fe(II) non-hémique impliquée dans la biosynthèse de l’éthylène chez les plantes. Notre compréhension du mécanisme ainsi le rôle des différents cofacteurs nécessite l’obtention des données structurales. Une structure cristallographique a été publiée montrant la partie C-terminale (C-term) éloignée du site actif. Ce n’est pas la conformation active car la partie C-term est essentielle à l’activité. Un modèle structural a été construit dans lequel la partie C-term est tournée vers le site actif. Différentes conformations semblent donc possibles. Le marquage de spin couplé à la spectroscopie RPE est une technique puissante pour sonder la dynamique structurale des protéines. Elle implique la liaison de nitroxydes sur des cystéines. Il est possible d’analyser la mobilité des sondes pour obtenir des informations sur leur environnement local. Par l’utilisation de techniques de RPE avancées, des mesures de distances entre deux sondes sont possibles. Des mutants portant une ou deux cystéines ont été conçus. La dynamique des mutants marqués a été étudiée in vitro par RPE. Par RPE impulsionnelle, des distances ont été mesurées pour l’ACCO en présence de différentes combinaisons de cofacteurs. Les distances expérimentales ont été comparées à celles prédites à partir des structures cristallographiques et du modèle structural et aussi à celles obtenues par des calculs de dynamique moléculaire. Pour cibler d’autres positions sur l’ACCO, l’introduction d’un acide aminé non naturel a été réalisée avec succès permettant d’obtenir de premières données structurales. Des données structurales préliminaires par RPE in cell sont également présentées / ACC Oxidase is a nonheme iron(II) containing enzyme involved in the biosynthesis of ethylene in plants. ACCO reaction mechanism and the role of the various cofactors are not well understood and structural and dynamic data are still required. A crystallographic structure has been reported showing the C-terminal part (C-term) away from the active site. This is not the active conformation as it has been shown that the C-term is essential. Later, a structural model has been proposed in which the C-term is folded towards the active site. Different conformations can be hypothesized. A technique well suited to monitor protein dynamics is site-directed spin labeling followed by EPR spectroscopy. It relies on the insertion of a nitroxide derivative on cysteines. Using this approach, it is possible to analyze the mobility of the label in order to obtain information on its local environment. Moreover using advanced EPR techniques, it is possible to acquire interspin distances between two incorporated probes. Mutants bearing one or two cysteines at desirable positions were designed. The dynamics of labeled mutants were studied in vitro using continuous wave EPR. By pulsed EPR, distances were recorded for ACCO in presence of different combinations of cofactors. The experimental distances were compared to the predicted ones obtained from the crystallographic and model structures, and also to the calculated ones obtained by molecular dynamic simulations. A successful introduction of an unnatural amino acid onto the sequence of ACCO was performed, allowing to obtain earliest results. The achievement of preliminary structural data by in cell EPR are also presented

No description available.

No description available.

Use of a mixed triisobutylaluminum/1,1-diphenylhexyllithium intiator enabled the anionic polymerization of methyl methacrylate at room temperature, resulting in narrow molecular weight distributions and syndiorich structures. Polymerizations were controlled above Al:Li = 2, and control significantly decreased at elevated temperatures above 25 °C. A significant increase in Tg with increasing control of syndiotacticity demonstrated the ability to tailor polymer properties using this technique. Analysis with MALDI-TOF/TOF spectroscopy revealed the dominance of a back-biting side reaction at elevated temperatures. Hydroxy-functional random and block copolymers of n-butyl acrylate (nBA) and 2-hydroxyethyl acrylate were synthesized using nitroxide mediated polymerization. Controlled polymerization was demonstrated, resulting in narrow polydispersities and linear molecular weight vs. conversion plots. In situ FTIR spectroscopy monitored the polymerizations and revealed pseudo first order rate kinetics for random copolymerizations. Protection of the hydroxyl using trimethylsilyl chloride alleviated isolation issues of amphiphilic polymer products. For the first time zwitterion-containing copolymers were electrospun to form nanoscale fibers with diameters as low as 100 nm. Free radical copolymerization of nBA and sulfobetaine methacrylamide produced zwitterionic copolymers with 6-13 mol % betaine. Dynamic mechanical analysis revealed a rubbery plateau and biphasic morphology similar to ionomers. Electrospinning from chloroform/ethanol solutions (80/20 v/v) at 2-7 wt % afforded polymeric fibers at viscosities below 0.02 Pa™s, which is the lowest viscosity observed for fiber formation in our laboratories. We hypothesized that intermolecular interactions rather than chain entanglements dominated the electrospinning process. Solution rheology of zwitterionic copolymers containing 6 and 9 mol % sulfobetaine methacrylate functionality revealed two concentration regimes with a boundary at ~1.5 – 2.0 wt %, regardless of molecular weight. This transition occurred at an order of magnitude lower specific viscosity than the entanglement concentration (Ce) for poly(nBA), and correlated to the onset of fiber formation in electrospinning. Comparison to existing models for polymer solution dynamics showed closest agreement to Rubinstein's theory for associating polymers, in support of our hypothesis that zwitterionic interactions dominate solution dynamics. The effect of ionic liquid (IL) uptake on mechanical properties and morphology of zwitterionic copolymers was explored using 1-ethyl-3-methylimidazolium ethylsulfate (EMIm ES). Dynamic mechanical analysis and impedance spectroscopy revealed a significant change in properties above a critical uptake of ~10 wt % IL. X-ray scattering revealed a significant swelling of the ionic domains at 15 wt % IL, with a 0.3 nm-1 shift in the ionomer peak to lower scattering vector. Results indicated the water-miscible IL preferentially swelled ionic domains of zwitterionic copolymers. / Ph. D.

High molecular weight star-shaped polystyrenes were prepared via the coupling of 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) terminated polystyrene oligomers with divinylbenzene (DVB) in m-xylene at 138 °C. Linear polystyrene oligomers (Mn = 19,300 g/mol, Mw/Mn = 1.10) were synthesized in bulk styrene using benzoyl peroxide in the presence of TEMPO at approximately 130 °C. In situ mid-infrared spectroscopy was successfully utilized to follow initiation, monomer conversion, and polymer formation. Real-time data allowed for the determination of apparent rate constants of 2.1E-5 s⁻¹ at 132 °C and 1.2E-5 s⁻¹ at 126 °C from the profile of the decaying styrene vinyl carbon-hydrogen (=CH₂) absorbance at 907 cm⁻¹. Coupling of the TEMPO terminated oligomers under optimum conditions resulted in a compact and dense product with a number average molecular weight exceeding 300,000 g/mol (Mw/Mn = 3.03) after 24 h, suggesting the formation of relatively well-defined star-shaped polymers. Synthetic factors that affected the molecular weight, yield, and composition of maleic anhydride (MAH), norbornene (Nb), and tert-butyl 5-norbornene-2-carboxylate (NbTBE) terpolymers were investigated. Pseudo first order kinetic analysis using in situ FTIR indicated that the observed rate of reaction was a strong function of the Nb/NbTBE ratio with a maximum of 6.7E-5 s⁻¹ for a 50/0/50 Nb/NbTBE/MAH monomer ratio and a minimum of 1.1E-5 s⁻¹ for a 0/50/50 Nb/NbTBE/MAH ratio. Polymer yields were also observed to be a function of the Nb/NbTBE ratio and also decreased with increasing NbTBE. Calculated work of adhesion values (Wadh) values were observed to increase as the content of NbTBE was increased. 193 nm photoresist formulations incorporating polymers with high NbTBE content showed increased imaging performance using 193 nm light and successfully produced sharp and defined features as small as 110 nm, which was demonstrated via scanning electron microscopy (SEM). Additional functionality was introduced via the copolymerization of MAH with several norbornene (Nb) derivatives that were synthesized from facile Diels-Alder cycloaddition reactions of cyclopentadiene with a-olefins containing electron withdrawing groups. Subsequent hydrolysis of the anhydride offered further versatility and provided an avenue to introduce aqueous base solubility into Nb/MAH copolymers. / Ph. D.

The imidazole ring offers great potential for a variety of applications including gene delivery vectors, ionic liquids, electromechanical actuators, and novel monomers and polymers. The imidazole ring provides a unique building block for these applications due to its thermal stability, aromatic nature, ability to form ionic salts, and ease of functionalization. Free radical polymerization of 1-vinylimidazole (1-VIm) and free radical copolymerizations with methyl methacrylate (MMA) and n-butyl acrylate (nBA) afforded homopolymers and copolymers with tunable solution and thermal properties. Aqueous SEC provided reproducible and reliable molecular weights for poly(1-VIm) in the absence of polymer aggregates. Analysis of the thermal properties revealed ideal random copolymers with MMA and non-ideal copolymers with nBA. Small angle X-ray scattering determined that the spacing between ionic groups remained constant with increased nonionic comonomer incorporation while the spacing between adjacent polymer backbones increased. Functionalization of 1-VIm with varying length alkyl halides and polymerization prepared a series of imidazolium homopolymers. Anion exchange reactions controlled the thermal and solution properties, and the bromide counteranion quantitatively exchanged to tetrafluoroborate (BF4), trifluoromethanesulfonate (TfO), and bis(trifluoromethanesulfonyl)imide (Tf2N). Thermogravimetric analysis revealed that thermal stability increased with decreased alkyl substituent length and larger counteranion size, and differential scanning calorimetry determined that glass transition temperature (Tg) decreased with increased alkyl substituent length and larger counteranion size. Electrochemical impedance spectroscopy determined the ionic conductivities of the imidazolium homopolymers, and analysis using the Vogel-Fulcher-Tammann equation revealed that the activation energy of ion conduction increased as alkyl substituent length increased. Polymer morphology determined using X-ray scattering also influenced the ionic conductivity. As the alkyl substituent length increased, the spacing between adjacent polymer backbones increased, which decreased the ionic conductivity due to the ion-hopping mechanism of ion conduction. Unsuccessful attempts to control the radical polymerization of 1-VIm led to the investigation of 1-(4-vinylbenzyl)imidazole (VBIm), which is a styrenic-based monomer with excellent propagating radical stability. Triblock copolymers incorporating VBIm monomer into a soft random copolymer center block and reinforcing, hard segment outer blocks provided a template for tuning the properties of the ionomer membranes for electroactive devices. Analysis of the morphology and mechanical properties using small angle X-ray scattering and dynamic mechanical analysis determined microphase separation and optimal mechanical properties for electromechanical transducer fabrication. Testing electromechanical transducers revealed superior performance relative to the benchmark Nafion®. Optimization of triblock copolymer design criteria through varying the comonomer ratio of VBIm and nBA in the soft center block, quaternization reactions, and ionic liquid introduction influenced mechanical properties and ionic conductivity. Higher percentages of VBIm and quaternization of VBIm in the random central block increased Tg and ionic conductivity. IL selectively incorporated into the imidazole-containing phases with no leakage observed for ionic systems, reduced the center block Tg, and increased ionic conductivity. Controlling charge density along poly(1-VIm) through well-defined alkylation reactions with 1-bromobutane provided a potential vector for nonviral gene delivery and polyanion binding. Analysis of DNA and heparin binding using gel electrophoresis revealed a decrease in N/P ratio with increased alkylation percentage. Dynamic light scattering indicated an increase in zeta potential with increasing alkylation percentages, and relatively uniform polyplex sizes in aqueous media. The MTT assay developed cytotoxicity profiles with little toxicity prior to 83% alkylation. Finally, the luciferase expression assay revealed inefficient nucleic acid delivery to multiple cell types. Synthesis of poly(1-VIm) vectors with glutathione conjugates provided an avenue for simultaneous therapeutic gene and anti-oxidant delivery in vitro. Cytotoxicity assays of cells pretreated with glutathione-conjugated poly(1-VIm) prior to oxidative stress showed that higher glutathione conjugation levels improved cell viability. / Ph. D.

Multifunctional monomers on the basis of acryl- and methacryl derivatives were synthesized and different protective groups were used. After polymerization the protective groups were removed by different methods. Various initiators for the NMP of the monomers were synthesized and the reaction conditions were optimized. The results showed that NMP was not a suitable method for multifunctional acryl- and methacryl derivatives to achieve well-defined homopolymers, although it was successful for control of polymerization of styrene and block copolymerization of multifunctional acryl- and methacryl derivatives with alkoxyamine terminated polystyrene. The ATRP of multifunctional acrylates and methacrylates has been successfully performed, as well as the block copolymerization of multifunctional acrylates and methacrylates. Relatively low polydispersities of the corresponding polymers (PD=1.18-1.36) and reasonably high rates of polymerization could be achieved when Me6TREN and PMDETA were used as ligands. However, the ATRP of multifunctional acrylamides and methacrylamides failed. The RAFT-polymerization of styrene, acrylamide and acrylate using BDTB as a CTA and AIBN as an initiator afforded polymers with narrow molecular weight distribution (PD=1.13-1.26). A kinetic investigation and the further synthesis of block copolymers using dithioester-terminated homopolymers as macroCTAs showed that the RAFT polymerization of acrylamide M9b proceeded in a living manner. However, BDTB does not control the reaction of methacrylic monomers, such as methacrylates and methacrylamides. The bulk phase behavior of the block copolymers were examined by means of DSC and the surface behaviors of block copolymers as thin layers were examined with AFM. Two-phase transitions in the block copolymers were observed clearly by DSC, indicative of the appearance of phase separations, which were seen in an AFM image. In conclusion, multifunctional acryl- and methacryl derivatives failed to achieve well-defined homopolymers by NMP. However, this method was successful for block copolymerization of multifunctional acryl- and methacryl derivatives with alkoxyamine terminated polystyrene. Multifunctional acrylates and methacrylates were successfully homopolymerized and block copolymerized by ATRP. Multifunctional acrylates and acrylamides were suitable for homopolymerization and block copolymerization by the RAFT process. Thus far, it is difficult to homopolymerize multifunctional methacrylamides in controlled way.

No description available.

The function of biomacromolecules is controlled by their structure and conformational flexibility. Investigating the structure of biologically important macromolecules can, therefore, yield information that could explain their complex biological function. In addition to X ray crystallography and nuclear magnetic resonance (NMR) methods, pulsed electron paramagnetic resonance (EPR) methods, in particular the pulsed electron electron double resonance (PELDOR) technique has, during the last decade, become a valuable tool for structural determination of macromolecules. Long range distance constraints obtained from pulsed EPR measurements, make it possible to carry out structural refinements on structures from NMR and X ray methods. In addition, EPR yields distance distributions that give information about structural flexibility. The use of EPR for structural studies of biomacromolecules requires in most cases site specific incorporation of paramagnetic centres known as spin labelling. To date, spin labelling nucleic acids has required complex spin labelling chemistry. The first application of a site directed and noncovalent spin labelling method for distance measurements on DNA is described. It is demonstrated that noncovalent spin labelling with a rigid spin label can afford detailed information on internal DNA dynamics using PELDOR. Furthermore, it is shown that noncovalent spin labelling can be used to study DNA protein complexes. PELDOR can also yield information about spin label orientation. Therefore, spin labels with limited flexibility can be used to measure the relative orientation of the spin labelled sites. Although information on orientation can be obtained from 9.7 GHz PELDOR measurements in selected applications, measurements at 97 GHz or higher, increases orientation selection. It is shown that PELDOR measurements on semi rigid and rigid nitroxide biradicals using a home built high power 97 GHz EPR spectrometer (Hiper) and model based simulations yield quantitative information on spin label orientations and dynamics. The most widely used spin labels for EPR studies on biomacromolecules are the aminoxyl (nitroxide) radicals. The major drawbacks of nitroxide spin labels include low sensitivity for distance measurements, fast spin spin relaxation in solution and limited stability in reducing environments. Carbon centered triarylmethyl (trityl) radicals have properties that could eliminate some of the limitations of nitroxide spin labels. To evaluate the use of trityl spin labels for nanometer distance measurements, models systems with trityl and nitroxide spin labels were measured using PELDOR and Double Quantum Coherence (DQC). This study shows that trityl spin labels yield reliable information on interlabel distances and dynamics, establishing the trityl radical as a viable spin label for structural studies on biomacromolecules.