Nuclear Magnetic Resonance Spectroscopic and Computational Investigations of Chloroperoxidase Catalyzed Regio- and Enantio-Selective Transformations

Chloroperoxidase (CPO) is the most versatile heme-containing enzyme that catalyzes a broad spectrum of reactions. The remarkable feature of this enzyme is the high regio- and enantio-selectivity exhibited in CPO-catalyzed oxidation reactions. The aim of this dissertation is to elucidate the structural basis for regio- and enantio-selective transformations and investigate the application of CPO in biodegradation of synthetic dyes. To unravel the mechanism of CPO-catalyzed regioselective oxidation of indole, the dissertation explored the structure of CPO-indole complex using paramagnetic relaxation and molecular modeling. The distances between the protons of indole and the heme iron revealed that the pyrrole ring of indole is oriented toward the heme with its 2-H pointing directly at the heme iron. This provides the first experimental and theoretical explanation for the "unexpected" regioselectivity of CPO-catalyzed indole oxidation. Furthermore, the residues including Leu 70, Phe 103, Ile 179, Val 182, Glu 183, and Phe 186 were found essential to the substrate binding to CPO. These results will serve as a lighthouse in guiding the design of CPO mutants with tailor-made activities for biotechnological applications. To understand the origin of the enantioselectivity of CPO-catalyzed oxidation reactions, the interactions of CPO with substrates such as 2-(methylthio)thiophene were investigated by nuclear magnetic resonance spectroscopy (NMR) and computational techniques. In particular, the enantioselectivity is partly explained by the binding orientation of substrates. In third facet of this dissertation, a green and efficient system for degradation of synthetic dyes was developed. Several commercial dyes such as orange G were tested in the CPO-H2O2-Cl- system, where degradation of these dyes was found very efficient. The presence of halide ions and acidic pH were found necessary to the decomposition of dyes. Significantly, the results revealed that this degradation of azo dyes involves a ferric hypochlorite intermediate of CPO (Fe-OCl), compound X.

Study of molecular reorientation and quantum rotational tunneling in tetramethylammonium selenate by H-1 NMR

H-1 NMR spin-lattice relaxation time measurements have been carried out in [(CH3)(4)N](2)SeO4 in the temperature range 389-6.6K to understand the possible phase transitions, internal motions and quantum rotational tunneling. A broad T, minimum observed around 280K is attributed to the simultaneous motions of CH3 and (CH3)(4)N groups. Magnetization recovery is found to be stretched exponential below 72 K with varying stretched exponent. Low-temperature T-1 behavior is interpreted in terms of methyl groups undergoing quantum rotational tunneling. (c) 2007 Elsevier Inc. All rights reserved.

Optimal pulse spacing for dynamical decoupling in the presence of a purely dephasing spin bath

Maintaining quantum coherence is a crucial requirement for quantum computation; hence protecting quantum systems against their irreversible corruption due to environmental noise is an important open problem. Dynamical decoupling (DD) is an effective method for reducing decoherence with a low control overhead. It also plays an important role in quantum metrology, where, for instance, it is employed in multiparameter estimation. While a sequence of equidistant control pulses the Carr-Purcell-Meiboom-Gill (CPMG) sequence] has been ubiquitously used for decoupling, Uhrig recently proposed that a nonequidistant pulse sequence the Uhrig dynamic decoupling (UDD) sequence] may enhance DD performance, especially for systems where the spectral density of the environment has a sharp frequency cutoff. On the other hand, equidistant sequences outperform UDD for soft cutoffs. The relative advantage provided by UDD for intermediate regimes is not clear. In this paper, we analyze the relative DD performance in this regime experimentally, using solid-state nuclear magnetic resonance. Our system qubits are C-13 nuclear spins and the environment consists of a H-1 nuclear spin bath whose spectral density is close to a normal (Gaussian) distribution. We find that in the presence of such a bath, the CPMG sequence outperforms the UDD sequence. An analogy between dynamical decoupling and interference effects in optics provides an intuitive explanation as to why the CPMG sequence performs better than any nonequidistant DD sequence in the presence of this kind of environmental noise.

Synthesis and characterization of high molecular weight polyaniline for organic electronic applications

High molecular weight polyaniline (PANI) was synthesized by a combined procedure incorporating various synthesis methods. Temperature and open circuit potential of the reaction mixture were collected to monitor the reaction progress. The polymer is characterized by various techniques including gel permeation chromatography, dynamic light scattering, infrared spectroscopy, solid-state nuclear magnetic resonance, and differential scanning calorimetry for elucidating the molecular architecture obtained by this method. As-synthesized PANI was found to possess high molecular weight, reduced branching, reduced cross-linking, and to predominantly consist of linear polymer chains. This polymer was also found to be more stable in solution form. JV characteristics of as-synthesized PANI films indicate a high current density which is due to increased free pathways and less traps for the charge transport to occur in PANI films. POLYM. ENG. SCI., 2012. (C) 2012 Society of Plastics Engineers

Structural Integration of Tellurium Oxide into Mixed-Network-Former Glasses: Connectivity Distribution in the System NaPO3–TeO2

FSodium phosphate tellurite glasses in the system (NaPO3)(x)(TeO2)(1-x) were prepared and structurally characterized by thermal analysis, vibrational spectroscopy, X-ray photoelectron spectroscopy (XPS) and a variety of complementary solid-state nuclear magnetic resonance (NMR) techniques. Unlike the situation in other mixed-network-former glasses, the interaction between the two network formers tellurium oxide and phosphorus oxide produces no new structural units, and no sharing of the network modifier Na2O takes place. The glass structure can be regarded as a network of interlinked metaphosphate-type P(2) tetrahedral and TeO4/2 antiprismotic units. The combined interpretation of the O 1s XPS data and the P-31 solid-state NMR spectra presents clear quantitative evidence for a nonstatistical connectivity distribution. Rather the formation of homootomic P-O-P and Te-O-Te linkages is favored over mixed P-O-Te connectivities. As a consequence of this chemical segregation effect, the spatial sodium distribution is not random, as also indicated by a detailed analysis of P-31/No-23 rotational echo double-resonance (REDOR) experiments. ACHTUNGTRENUNG(TeO2)1 x were prepared and structurally characterized by thermal analysis,vibrat ional spectroscopy,X-ray photoelectron spectroscopy (XPS) and a variety of complementary solid-state nuclear magnetic resonance (NMR) techniques. Unlike the situation in other mixed-network-former glasses,the interaction between the two network formers tellurium oxide and phosphorus oxide produces no new structural units,and no sharing of the network modifier Na2O takes place. The glass structure can be regarded as a network of interlinked metaphosphate-type P(2) tetrahedral and TeO4/2 antiprismatic units. The combined interpretation of the O 1s XPS data and the 31P solid-state NMR spectra presents clear quantitative evidence for a nonstatistical connectivity distribution. Rather,the formation of homoatomic P O P and Te O Te linkages is favored over mixed P O Te connectivities. As a consequence of this chemical segregation effect,the spatial sodium distribution is not random,as also indicated by a detailed analysis of 31P/23Na rotational echo double-resonance (REDOR) experiments.

Effect of main-chain rigidity on the phase transitional behavior of comblike polymers

The influence of the rigidity of polymer backbones on the side-chain crystallization and phase transition behavior was systematically investigated by a combination of differential scanning calorimetry (DSC), wide-angle X-ray diffraction (WAXD), Fourier transform infrared spectroscopy (FTIR), and high-resolution solid-state nuclear magnetic resonance spectroscopy (NMR). DSC investigation indicated that the crystallization number of alkyl carbon atoms of the side chains grafted onto the rigid polymer backbone, poly(p-benzamide) (PBA), is much lower than that of the alkyl carbon atoms of the side chains grafted onto the flexible polymer backbone, poly(ethyleneimine) (PEI), implying that the conformational state of the polymer backbones has a strong effect on the side-chain crystallization behavior in comblike polymers. WAXD and FTIR results proved that these two comblike polymers pack into hexagonal (PBA18C) and orthorhombic (PEI18C) crystals, respectively, depending on the adjusting ability of the polymer backbones for particular conformational states. It was also found that the presence of the crystalline-amorphous interphase (delta = 31.6 ppm) in PBA18C detected by solid-state C-13 NMR spectroscopy can be attributed to the rigid PBA backbone, which restricts the mobility of the alkyl side chains.

Synthesis and characterization of functionalized mesoporous silica by aerosol-assisted self-assembly

An efficient, productive, and low-cost aerosol-assisted self-assembly process has been developed to produce organically modified mesoporous silica particles via a direct co-condensation of silicate species and organosilicates that contain nonhydrolyzable functional groups in the presence of templating surfactant molecules. Different surfactants including cetyltrimethylammonium bromide, nonionic surfactant Brij-56, and triblock copolymer P123 have been used as the structure-directing agents. The organosilanes used in this study include tridecafluoro-1, 1,2,2-tetrahydrooctyltriethoxysilane, methytriethoxysilane, vinyltrimethoxysilane, and 3-(trimethoxysilyl)propyl methacrylate. X-ray diffraction and transmission electron microscopy studies indicate the formation of particles with various mesostructures. Fourier transform infrared and solid-state nuclear magnetic resonance spectra confirm the organic ligands are covalently bound to the surface of the silica framework. The porosity, pore size, and surface area of the particles were characterized using nitrogen adsorption and desorption measurements.

Unraveling the Structure and Function of G Protein-Coupled Receptors Through NMR Spectroscopy

G protein-coupled receptors (GPCRs) are a large superfamily of signaling proteins expressed on the plasma membrane. They are involved in a wide range of physiological processes and, therefore, are exploited as drug targets in a multitude of therapeutic areas. In this extent, knowledge of structural and functional properties of GPCRs may greatly facilitate rational design of modulator compounds. Solution and solid-state nuclear magnetic resonance (NMR) spectroscopy represents a powerful method to gather atomistic insights into protein structure and dynamics. In spite of the difficulties inherent the solution of the structure of membrane proteins through NMR, these methods have been successfully applied, sometimes in combination with molecular modeling, to the determination of the structure of GPCR fragments, the mapping of receptor-ligand interactions, and the study of the conformational changes associated with the activation of the receptors. In this review, we provide a summary of the NMR contributions to the study of the structure and function of GPCRs, also in light of the published crystal structures.

Novel microporous silicates and mesoporous MCM materials derivatised with inorganic and organometallic complexes

Os materiais microporosos e mesoporosos são potenciais catalisadores heterogéneos. Os zeólitos e outros materiais microporosos do tipo zeolítico tradicionais, têm átomos tetracoordenados no esqueleto. Nos últimos anos, um vasto número de titanossilicatos contendo Ti(IV) hexacoordenado e Si(IV) tetracoordenado, com estruturas tridimensionais, têm sido alvo de grande interesse. Um dos objectivos desta tese foi preparar silicatos microporosos, contendo átomos metálicos com número de coordenação superior a quatro, e possuindo quer novas estruturas quer propriedades físicas e químicas interessantes. Neste contexto, foi preparado um novo ítriossilicato de sódio, AV-1, análogo do raro mineral montregianite, Na4K2Y2Si16O38·10H2O. Este material é o primeiro sólido microporoso que contem quantidades estequiométricas de sódio (e ítrio) no esqueleto. Foi, também, sintetizado um silicato de cério, AV-5, análogo estrutural do mineral montregianite com potencial aplicação em optoelectrónica. Nesta tese é, ainda, descrita a síntese e caracterização estrutural de um silicato de cálcio hidratado, AV-2, análogo do raro mineral rhodesite (K2Ca4Na2Si16O38.12H2O). Na continuação do trabalho desenvolvido em Aveiro na síntese de novos titanossilicatos surgiu o interesse de preparar novos zirconossilicatos microporosos por síntese hidrotérmica. Foram preparados dois novos materiais análogos dos minerais petarasite Na5Zr2Si3O18(Cl,OH)·2H2O (AV-3) e kostylevite, K2Si3O9·H2O (AV-8). Foram, também, obtidos análogos sintéticos dos minerais parakeldyshite e wadeite, por calcinação a alta temperatura de AV-3 e de umbite sintética. A heterogeneização de complexos organometálicos na superfície de materiais mesoporosos do tipo M41S permite associar a grande actividade catalítica e a presença de sítios activos localizados típicos dos complexos organometálicos, com a robustez e fácil separação, características dos materiais mesoporosos siliciosos. Nesta dissertação relata-se a derivatização dos materiais MCM-41 e MCM-48 através da reacção de [SiMe2{(h5-C5H4)2}]Fe e [SiMe2{(h5-C5H4)2}]TiCl2 com os grupos silanol das superfícies mesoporosas. Os materiais MCMs derivatizados com ansa-titanoceno foram testados na epoxidação de cicloocteno a 323 K na presença de hidrogenoperóxido de t-butilo. Estudou-se a heterogeneização dos sais de complexos com ligação metal-metal [Mo2(MeCN)10][BF4]4, [Mo2(m-O2CMe)2(MeCN)6][BF4]2 e [Mo2(m- O2CMe)2(dppa)2(MeCN)2][BF4]2 via imobilização nos canais do MCM-41. A imobilização dos catalisadores homogéneos na superfície do MCM-41 envolve a saída dos ligandos nitrilo lábeis, preferencialmente em posição axial, através da reacção com os grupos Si-OH da sílica. Verificou-se que a ligação Mo-Mo se mantém intacta nos produtos finais. É provável que estes materiais sejam eficientes catalisadores heterogéneos em reacções de polimerização. As técnicas de caracterização utilizadas nesta tese foram a difracção de raios-X de pós, a microscopia electrónica de varrimento, a espectroscopia de ressonância magnética nuclear do estado sólido (núcleos 13C, 23Na e 29Si), as espectroscopias de Raman e infravermelho com transformadas de Fourier, as análises termogravimétricas e as análises de adsorção de água e azoto.

Post-synthetic modification of metal–organic frameworks

Post-synthetic modification (PSM) of metal-organic frameworks encompassing the chemical transformation of the linker present is a promising new route for engineering optical centres and tuning the light emission properties of materials, both in the visible and in the near infrared (NIR) spectral regions. Here, PSM of isoreticular metal-organic framework-3 (IRMOF-3) with ethyl oxalyl monochloride, ethyl acetoacetate, pentane-2,4-dione, 3-(2- hydroxyphenyl)-3-oxopropanal, 2-chloroacetic acid, glyoxylic acid, methyl vinyl ketone and diethyl (ethoxymethylene)malonate followed by chelation of trivalent lanthanide ions afforded intriguing near infrared (Nd3+) and visible (Eu3+, Tb3+) light emitters. IRMOF-3 was used as a case in point due to both its highly porous crystalline structure and the presence of non-coordinating amino groups on the benzenedicarboxylate (bdc) linker amenable to modification. The materials were characterised by elemental analysis, powder X-ray diffraction, optical, scanning and transmission electron microscopy, Fourier transform infrared spectroscopy, and liquid and solid-state nuclear magnetic resonance. The solid-state luminescence properties of Ln-modified-IRMOF-3 were investigated at room temperature. The presence of the bdc aromatic ring, β– diketonate and oxalate enhanced the Ln3+ sensitization via ligand-to-metal energy transfer (anthena effect). As far as photocalysis is concerned, we have synthesized metal−organic frameworks (Cr-MIL-125-AC, Ag-MIL-125-AC) by a green method (solid–vapors reactions). The resulting functionalized materials show a photocatalytic activity for methylene blue degradation up to 6.52 times larger than that of the commercial photocatalyst hombikat UV-100. These findings open the door for further research for improving the photocatalytic performance of metal-organic frameworks.

Production and characterization of Chitin-Glucan Complex by Komagataella pastoris

This thesis was focused on the production, extraction and characterization of chitin:β-glucan complex (CGC). In this process, glycerol byproduct from the biodiesel industry was used as carbon source. The selected CGC producing yeast was Komagataella pastoris (formerly known as Pichia pastoris), due the fact that to achieved high cell densities using as carbon source glycerol from the biodiesel industry. Firstly, a screening of K. pastoris strains was performed in shake flask assays, in order to select the strain of K. pastoris with better performance, in terms of growth, using glycerol as a carbon source. K. pastoris strain DSM 70877 achieved higher final cell densities (92-97 g/l), using pure glycerol (99%, w/v) and in glycerol from the biodiesel industry (86%, w/v), respectively, compared to DSM 70382 strain (74-82 g/l). Based on these shake flask assays results, the wild type DSM 70877 strain was selected to proceed for cultivation in a 2 l bioreactor, using glycerol byproduct (40 g/l), as sole carbon source. Biomass production by K. pastoris was performed under controlled temperature and pH (30.0 ºC and 5.0, respectively). More than 100 g/l biomass was obtained in less than 48 h. The yield of biomass on a glycerol basis was 0.55 g/g during the batch phase and 0.63 g/g during the fed-batch phase. In order to optimize the downstream process, by increasing extraction and purification efficiency of CGC from K. pastoris biomass, several assays were performed. It was found that extraction with 5 M NaOH at 65 ºC, during 2 hours, associated to neutralization with HCl, followed by successive washing steps with deionised water until conductivity of ≤20μS/cm, increased CGC purity. The obtained copolymer, CGCpure, had a chitin:glucan molar ratio of 25:75 mol% close to commercial CGC samples extracted from A. niger mycelium, kiOsmetine from Kitozyme (30:70 mol%). CGCpure was characterized by solid-state Nuclear Magnetic Resonance (NMR) spectroscopy and Differential Scanning Calorimetry (DCS), revealing a CGC with higher purity than a CGC commercial (kiOsmetine). In order to optimize CGC production, a set of batch cultivation experiments was performed to evaluate the effect of pH (3.5–6.5) and temperature (20–40 ºC) on the specific cell growth rate, CGC production and polymer composition. Statistical tools (response surface methodology and central composite design) were used. The CGC content in the biomass and the volumetric productivity (rp) were not significantly affected within the tested pH and temperature ranges. In contrast, the effect of pH and temperature on the CGC molar ratio was more pronounced. The highest chitin: β-glucan molar ratio (> 14:86) was obtained for the mid-range pH (4.5-5.8) and temperatures (26–33 ºC). The ability of K. pastoris to synthesize CGC with different molar ratios as a function of pH and temperature is a feature that can be exploited to obtain tailored polymer compositions.(...)

Extracting Ramachandran torsional angle distributions from 2D NMR data using Tikhonov regularization /

Solid state nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for studying structural and dynamical properties of disordered and partially ordered materials, such as glasses, polymers, liquid crystals, and biological materials. In particular, twodimensional( 2D) NMR methods such as ^^C-^^C correlation spectroscopy under the magicangle- spinning (MAS) conditions have been used to measure structural constraints on the secondary structure of proteins and polypeptides. Amyloid fibrils implicated in a broad class of diseases such as Alzheimer's are known to contain a particular repeating structural motif, called a /5-sheet. However, the details of such structures are poorly understood, primarily because the structural constraints extracted from the 2D NMR data in the form of the so-called Ramachandran (backbone torsion) angle distributions, g{^,'4)), are strongly model-dependent. Inverse theory methods are used to extract Ramachandran angle distributions from a set of 2D MAS and constant-time double-quantum-filtered dipolar recoupling (CTDQFD) data. This is a vastly underdetermined problem, and the stability of the inverse mapping is problematic. Tikhonov regularization is a well-known method of improving the stability of the inverse; in this work it is extended to use a new regularization functional based on the Laplacian rather than on the norm of the function itself. In this way, one makes use of the inherently two-dimensional nature of the underlying Ramachandran maps. In addition, a modification of the existing numerical procedure is performed, as appropriate for an underdetermined inverse problem. Stability of the algorithm with respect to the signal-to-noise (S/N) ratio is examined using a simulated data set. The results show excellent convergence to the true angle distribution function g{(j),ii) for the S/N ratio above 100.

Chelated fluoroboron cations : synthesis and multinuclear nmr studies

The preparation of chelated difluoroboron cations (DD)BF2+, where DD is a saturated polydentate tertiary-amine or polydentate aromatic ligand, has been systematically studied by using multinuclear solution and solid state nuclear magnetic resonance spectroscopy and fast atom bombardment mass spectrometry. Three new methods of synthesis of (DD)BF2+ cations are reported, and compared with the previous method of reacting a chelating donor with Et20.BF3. The methods most effective for aromatic donors such as 1,1O-phenanthroline are ineffective for saturated polydentate tertiary-amines like N,N,N' ,Nil ,Nil-pentamethyldiethylenetriamine. Polydentate tertiary-amine donors that form 5-membered rings upon bidentate chelation were found to chelate effectively when the BF2 source contained two leaving groups (a heavy halide and a Lewis base such as pyridine =pyr or isoxazole =ISOX), i.e., pyr.BF2X (X = CI or Br), ISOX.BF2X and (pyr)2BF2+. Those that would form 6membered rings upon chelation do not chelate by any of the four methods. Polydentate aromatic ligands chelate effectively when the BF2 source contained a weak Lewis base, e.g., ISOX.BF3, ISOX.BF2X and Et20.BF3. Bidentate chelation by polydentate tertiaryamine and aromatic donors leads to nmr parameters that are significantly different then their (D)2BF2+ relatives (D =monod~ntate t-amines or pyridines). The chelated haloboron cations (DD)BFCI+, and (DD)BFBr+ were generated from D.BFX2 adducts for all ligands that form BF2+ cations above. In addition, the (DD)BCI2+ and (DD)BBr2+ cations were formed from D.BX3 adducts by the chelating aromatic ligands, except for the aromatic ligand 1,8-bis(dimethylamino)naphthalene, which formed only the (DD)BF2+ cation, apparently due to its extreme steric hindrance. Chelation by a donor is a two-step reaction. For polydentate tertiary-amine ligands, the two rates appear to be very dependent on the two possible leaving groups on the central boron atom. The order of increasing ease of displacement for the donors was: pyr < Cl < Br < ISOX. The rate of chelation by polydentate aromatic ligands appears to be dependent on the displacement of the first ligand from the boron. The order of increasing ease of displacement for the donors was: pyr < CI < ISOX ~ Br < Et20.

A single-step procedure for the preparation of palladium nanoparticles and a phosphine-functionalized support as catalyst for Suzuki cross-coupling reactions

We here report the preparation of supported palladium nanoparticles (NPs) stabilized by pendant phosphine groups by reacting a palladium complex containing the ligand 2-(diphenylphosphino)benzaldehyde with an amino-functionalized silica surface The Pd nanocatalyst is active for Suzuki cross-coupling reaction avoiding any addition of other sources of phosphine ligands The Pd intermediates and Pd NPs were characterized by solid-state nuclear magnetic resonance and transmission electron microscopy techniques The synthetic method was also applied to prepare magnetically recoverable Pd NPs leading to a catalyst that could be reused for up to 10 recycles In summary we gathered the advantages of heterogeneous catalysis magnetic separation and enhanced catalytic activity of palladium promoted by phosphine ligands to synthesize a new catalyst for Suzuki cross-coupling reactions The Pd NP catalyst prepared on the phosphine-functionalized support was more active and selective than a similar Pd NP catalyst prepared on an amino-functionalized support (C) 2010 Elsevier Inc All rights reserved