An abstract is not provided. / Thesis / Master of Science (MSc)

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A series of studies are presented in which fluorine tagging reagents are used to analyze complex mixtures for compounds containing active hydrogen functional groups (e.g., hydroxyl, amine, thiol, and carboxylic groups). The existence of these derivatized functional groups is determined by utilizing a number of solution and solid-state nuclear magnetic resonance (NMR) techniques. In solution NMR studies p-fluorobenzoyl chloride was the fluorine tagging reagent of choice because of a large ¹⁹F chemical shift range for the different derivatized substrates (~10 ppm) and generally good reaction yields. Various classes of sterol and amino acid p-fluorobenzoyl derivatives were characterized on the basis of their ¹⁹F NMR isotropic chemical shifts. The presence (or absence) of hydroxyl, amine, and carboxylic acid functional groups in coal extract and pyrolysis products was also determined. The versatility of the p-fluorobenzoyl chloride as the fluorine tagging reagent in ¹⁹F NMR was enhanced by: a) enriching the carbonyl carbon of the acid chloride with labeled ¹³C isotope, thus synthesizing a dual ¹⁹F and ¹³C NMR sensitive reagent and b) using the reagent in conjunction with LC-NMR. The extension to either technique added another dimension to the NMR spectral data obtained from the p-fluorobenzoyl tagging reagent in solution NMR. Finally, preliminary data is presented illustrating how fluorine tagging reagents may be used to study functional groups (and atoms present in the immediate proximity of the group) existing on solid material utilizing solid-state NMR. Functional groups on the solid material are tagged with a fluorinated reagent. The sample is then analyzed using solid-state NMR with cross-polarization (CP), magic angle spinning (MAS), and high-power proton decoupling. The ¹⁹F dipolar coupling interactions, created by the presence of the fluorine tag, attenuate signals for these nuclei in the immediate proximity of the tagged site. A series of 1-adamantanol, steroid, and silica gel fluorinated derivatives show that the effects of the ¹⁹F dipolar interactions were modulated by complex anisotropic molecular motions (i.e., solid system with little motion, exhibiting greater signal attenuations due to ¹⁹F dipolar coupling). / Ph. D. / incomplete_metadata

Realizamos estudos, por espectroscopia de fluorescência de estado estacionário e resolvida no tempo, de dinâmica conformacional de três famílias de peptídeos: o terminal carboxílico do peptídeo neuroendócrino (7B2CT); o peptídeo estimulador do melanócito (alfa-MSH); e os peptídeos derivados da seqüência consensual de peptídeos que se ligam a heparina (peptídeo Cardin). Utilizamos os métodos de fluorescência baseados na supressão da emissão do fluoróforo presente no peptídeo, tanto por interação colisional com um supressor como por transferência de energia para um grupo aceitador presente no peptídeo. No segundo caso, dentro da hipótese de que diferentes conformações do peptídeo estão presentes durante o decaimento da fluorescência, supusemos que o decaimento da intensidade era modulado por uma função de distribuição de distâncias doador-aceitador, f(r). Foi desenvolvido um procedimento computacional usando o programa CONTIN, para recuperar, a partir dos dados de fluorescência resolvida no tempo, a distribuição de distância entre o doador e o aceitador presentes nos peptídeos. A metodologia se mostrou útil para obter informações quantitativas sobre a dinâmica conformacional dos peptídeos, sua dependência com o solvente e sua interação com outras moléculas presentes no meio. O peptídeo 7B2CT e seus análogos A12 e A18 foram estudados pela fluorescência da tirosina, presente na posição cinco na seqüência de aminoácidos. Não houve diferenças significativas no decaimento da intensidade, no decaimento da anisotropia e na supressão por iodeto entre os três peptídeos, que pudessem ser correlacionados com as diferenças observadas na inibição da pro-hormone convertase 2 (PC2). O peptídeo alfa-MSH e seu análogo mais potente [Nle(4),0-Phe(7)] alfa-MSH (NDP-alfa-MSH), que contém o resíduo fluorescente triptofano, foram modificados no seu terminal amino com o grupo ácido amino benzóico (Abz), e foram estudados por métodos de fluorescência. Observamos transferência de energia entre o resíduo triptofano, atuando como doador, e o aceitador o-Abz. Verificamos a uma ampla distribuição de distâncias Trp-o-Abz para o alfa-MSH em água, enquanto três populações de distâncias foram observadas para o análogo modificado NDP-alfa-MSH em água, indicando estados com conformações distintas para o petídeo sintético, comparado com o hormônio nativo. Medidas em trifluoroetanol resultaram em duas populações de distâncias, tanto para o hormônio nativo como para o hormônio análogo, refletindo a diminuição, induzida pelo solvente, das conformações disponíveis para os peptídeos. Os peptídeos Cardin foram estudados na presença de heprarina, de micelas de SDS e na mistura TFE/água. O grupo fluorescente Abz foi incorporado no terminal amino e o grupo aceitador etilenodiamina-N-[2,4- dinitrofenil] (Eddnp) foi incorporado ao terminal carboxílico dos peptídeos. A distribuição de distâncias entre as extremidades, recuperadas dos dados de fluorescência resolvida no tempo, indicaram a presença de conformações estendidas em solução aquosa e a ocorrência de conformações compactas na interação com heparina e SDS ou em soluções com TFE. As distâncias entre as extremidades recuperadas foram maiores que aquelas esperadas para osI peptídeos na conformação hélice alfa, sugerindo a ocorrência de estruturas dobradas além da hélice alfa. A metodologia da transferência de energia se mostrou útil para os estudos de dinâmica de peptídeos em solução. / Steady state and time-resolved fluorescence spectroscopy were used to study the conformational dynamics of three families of peptides: the carboxyl terminal of the neuroendocrine peptide (782Cl), the alpha melanocytestimulating hormone (alpha-MSH) and heparin-binding consensus sequence peptides (Cardin motif peptides). The fluorescence methods were based on quenching of the emission of a fluorophore present in the peptide, either by collisional interaction with a quencher, as by energy transfer to an acceptor group. In the second case, within the hypothesis that different peptide conformations are in equilibrium during the fluorescence decay, we supposed that the intensity decay was modulated by an acceptor-donor distance distribution function, f(r). A computational procedure was developed using the CONTIN program, to recover, from the time-resolved fluorescence data, the distance distribution between donor and acceptor groups attached to the peptides. The methodology proved to be useful to provide quantitative information about conformational dynamics of peptides, its dependency on the solvent and its interaction with other molecules present in the medium. The peptide 782CT and its analogs A12 and A18 were studied by examination of fluorescence from the tyrosine residue present in the position five of the amino acid sequence. There were no appreciable differences in the intensity decay, anisotropy decay and iodide quenching between the three peptides, that could be correlated to observed differences in the inhibition of the pro-hormane convertase 2 (PC2). The peptide hormone alpha-MSH and its more potent analog [Nle(4),0- Phe(7)]alpha-MSH (NDP-alpha-MSH), containing the fluorescent residue tryptophan, were labeled at the amino terminal with the amino benzoic acid (Abz) group, and were also examined by fluorescence methods. We observed energy transfer between the tryptophan residue acting as donor and Abz as acceptor, and verified a broad Trp-o-Abz distance distribution for alpha-MSH in aqueous medium, while three different distance populations could be identified for the labeled analog NDP-alpha-MSH in water, indicating distinct conformational states for the synthetic peptide, compared with the native hormone. Measurements in trifluoroethanol resulted in the recovery of two Abzlrp distance populations, both for the native and the analog hormones, reflecting the decrease, induced by the solvent, of the conformational states available to the peptides. The Cardin motif peptides were examined in the presence of heparin, SDS micelles and in TFE/water mixtures. The fluorescent group Abz was attached to the amino terminal and the acceptor group N-(2,4 dinitrophenyl)ethylenediamine (Eddnp) was bound to the carboxyl terminal of the peptides. The end-to-end distance distribution recovered from time-resolved fluorescence data indicated extend conformation in aqueous medium and the occurrence of compact conformations under interaction with heparin and SDS or in the medium containing TFE. The end-to-end distances recovered were lower than those expected for the peptides in alpha helix conformation, suggesting the occurrence of turned structures beyond the alpha helix. The energy transfer methodology shows to be useful to study conformational dynamics of peptides in solution.

Foi realizado um estudo por espectroscopia de ressonancia paramagnetica eletronica de solucoes de complexos formados por melanina sintetica, obtida por auto-oxidacao de l-dopa (dihidroxi-l-fenilalanina), e albumina de soro bovino (bsa). Investigaram-se os efeitos devidos a alteracoes de ph, de concentracao e conformacao da bsa, e presenca de O IND.2, tomando como base as variacoes das formas e intensidades das linhas espectrais dos radicais livres intrinsecos da melanina e dos induzidos por radiacao visivel-uv. Verificou-se uma dependencia da intensidade do sinal com o estado de protonacao do pigmento. Na presenca da albumina a inducao de radicais livres por luz foi maior, o que nao observou-se quando esta foi denaturada. A inducao e decaimento dos radicais fotoinduzidos foi monitorada por espectro de varredura temporal, observando-se uma cinetica que envolveu processos mais lentos com o aumento da concentracao da proteina. A partir de medidas de saturacao progressiva, verificou-se um comportamento de saturacao homogeneo para a melanina pura e nao homogeneo quando bsa e O IND.2 estavam presentes, acompanhados de variacoes nos tempos de relaxacao. Os resultados foram interpretados com base em um modelo que supoe a existencia de processos de transferencia de eletrons da melanina excitada para o O IND.2 e transferencia de carga entre o pigmento e a proteína. / Synthetic melanin (obtained by L-DOPA auto-oxidation) and melanin-bovine serum albumin complexes were studied by electron paramagnetic resonance spectroscopy (EPR). The melanin intrinsic and Iight-induced free radicals by visible-UV were studied. The dependence of the EPR line shapes on the pH value, albumin conformation and concentration, and O² presence was analyzed. It was found that the EPR signal intensity depended on the pigment protonation state. The light free radical induction in the melanin-albumin solutions was higher than in pure melanin solution. This effect was only observed when the protein was in its natural form. The induction and decay of the light-induced radicals were monitored by time-resolved experiments. A slower kinetic was observed with increasing of the albumin concentration. Progressive saturation measurements showed a homogeneous saturation behavior for pure melanin and inhomogeneous in the protein or O² presence, showing also changes in time relaxations. The results were interpreted assuming the existence of electron transfer processes from excited melanin to O² and charge exchange between the pigment and the protein.

Foi realizado um estudo por espectroscopia de ressonancia paramagnetica eletronica de solucoes de complexos formados por melanina sintetica, obtida por auto-oxidacao de l-dopa (dihidroxi-l-fenilalanina), e albumina de soro bovino (bsa). Investigaram-se os efeitos devidos a alteracoes de ph, de concentracao e conformacao da bsa, e presenca de O IND.2, tomando como base as variacoes das formas e intensidades das linhas espectrais dos radicais livres intrinsecos da melanina e dos induzidos por radiacao visivel-uv. Verificou-se uma dependencia da intensidade do sinal com o estado de protonacao do pigmento. Na presenca da albumina a inducao de radicais livres por luz foi maior, o que nao observou-se quando esta foi denaturada. A inducao e decaimento dos radicais fotoinduzidos foi monitorada por espectro de varredura temporal, observando-se uma cinetica que envolveu processos mais lentos com o aumento da concentracao da proteina. A partir de medidas de saturacao progressiva, verificou-se um comportamento de saturacao homogeneo para a melanina pura e nao homogeneo quando bsa e O IND.2 estavam presentes, acompanhados de variacoes nos tempos de relaxacao. Os resultados foram interpretados com base em um modelo que supoe a existencia de processos de transferencia de eletrons da melanina excitada para o O IND.2 e transferencia de carga entre o pigmento e a proteína. / Synthetic melanin (obtained by L-DOPA auto-oxidation) and melanin-bovine serum albumin complexes were studied by electron paramagnetic resonance spectroscopy (EPR). The melanin intrinsic and Iight-induced free radicals by visible-UV were studied. The dependence of the EPR line shapes on the pH value, albumin conformation and concentration, and O² presence was analyzed. It was found that the EPR signal intensity depended on the pigment protonation state. The light free radical induction in the melanin-albumin solutions was higher than in pure melanin solution. This effect was only observed when the protein was in its natural form. The induction and decay of the light-induced radicals were monitored by time-resolved experiments. A slower kinetic was observed with increasing of the albumin concentration. Progressive saturation measurements showed a homogeneous saturation behavior for pure melanin and inhomogeneous in the protein or O² presence, showing also changes in time relaxations. The results were interpreted assuming the existence of electron transfer processes from excited melanin to O² and charge exchange between the pigment and the protein.

The work presented in this thesis belongs to the domain of theoretical and experimental gas phase molecular spectroscopy. It consists of two parts dedicated to two relatively different aspects in this field. The first part presents a high resolution analysis of the ν5 fundamental band (NO2 in plane bending mode) positions and intensities of D14NO3 (deuterated nitric acid) in the 11 µm spectral region. For this study, we used an infrared spectrum of D14NO3 recorded in the 700−1400 cm−1 region on a Fourier transform spectrometer at Bergische Universität in Wuppertal (Germany). Our analysis demonstrates that the fundamental ν5 band centered at 887.657 cm−1 is strongly perturbed. Indeed, it proves that 5^1 and 7^1+9^1 energy levels of DNO3 are coupled through A and B type Coriolis resonances. The resonance scheme for the isotopologue D14NO3 therefore differs substantially from the schemes of H14NO3 and H15NO3 that feature dominantly Fermi type resonances. The second theme treated in this work is devoted to the lineshapes of pure rotational transitions of CH3F with the study of collisional broadening (collisions CH3F-CH3F and CH3F-He) of optical transitions and their Stark components. The microwave measurements were realised at ICT in Prague. Their analysis enabled to provide collisional parameters using various line profiles (Voigt, Rautian, Speed dependent) for the J, K → J + 1, K (K = 0, ..., J) transitions with J = 1 and J = 3 as well as for their various Stark components J, K, M → J + 1, K, M' (|M| = 0, ..., J ; |M − M'| = 0, 1). Moreover, a correct use of model based on Infinite Order Sudden approximation led to very satisfactory results of the observed line-mixing effects. The retrieved experimental results complete and extend the previous studies and provide the first successful demonstration of the ability of the IOS approximation to model line-mixing effects among Stark transitions

Fourier transform infrared emission spectra of MnH and MnD were observed in the ground <em>X</em>⁷&sigma;⁺ electronic state. The vibration-rotation bands from <em>v</em> = 1 to 0 to <em>v</em> = 3 to 2 for MnH, and from <em>v</em> = 1 to 0 to <em>v</em> = 4 to 3 for MnD were recorded at an instrumental resolution of 0. 0085 cm^-1. Spectroscopic constants were determined for each vibrational level and equilibrium constants were found from a Dunham-type fit. The equilibrium vibrational constant <em>&omega;_e</em> for MnH was found to be 1546. 84518(65) cm^-1, the equilibrium rotational constant <em>B_e</em> was found to be 5. 6856789(103) cm^-1 and the equilibrium bond distance <em>r_e</em> was determined to be 1. 7308601(47) Å. <br /><br /> New high resolution emission spectra of CoH and CoD molecules have been recorded in the 640 nm to 3. 5 <em>µ</em>m region using a Fourier transform spectrometer. Many bands were observed for the <em>A</em>^'3&phi;-<em>X</em>³&phi; electronic transition of CoH and CoD. In addition, a new [13. 3]4 electronic state was found by observing the [13. 3]4- <em>X</em>³&phi;₃ and [13. 3]4-<em>X</em>³&phi;₄ transitions in the spectrum of CoD. Analysis of the transitions with &delta;&omega; = 0, ±1 provided more accurate values of spin-orbit splittings between &omega; = 4 and &omega; = 3 components. The ground state for both molecules was fitted both to band and Dunham-type constants. The estimated band constants of the perturbed upper states were also obtained. <br /> <br /> The emission spectrum of gas-phase YbO has been investigated using a Fourier transform spectrometer. A total of 8 red-degraded bands in the range 9 800 ? 11 300 cm^-1 were recorded at a resolution of 0. 04 cm^-1. Because of the multiple isotopomers present in the spectra, only 3 bands were rotationally analyzed. Perturbations were identified in two of these bands and all 3 transitions were found to terminate at the <em>X</em>¹&sigma;⁺ ground electronic state. The electronic configurations that give rise to the observed states are discussed and molecular parameters for all of the analyzed bands are reported. <br /><br /> Electronic spectra of the previously unobserved EuH and EuD molecules were studied by means of Fourier transform spectroscopy and laser-induced fluorescence. The extreme complexity of these transitions made rotational assignments of EuH bands impossible. However, the spin-spin interaction constant, &lambda;, and Fermi contact parameter, <em>b</em>_F, in the ground <em>X</em>⁹&sigma;^- electronic state were estimated for the ¹⁵¹EuH and ¹⁵³EuH isotopologues. <br /><br /> Electronic spectra of SmH, SmCl, TmH and ErF molecules were recorded for the first time using Fourier transform spectrometer. The poor signal to noise ratio of the observed bands coupled with their complexity prevented a rotational analysis. The electronic states that may be involved in the observed transitions are discussed.