Back in 1894 Lord Kelvin coined the term "chiral" in order to refer to molecules whose mirror images were not superimposable with themselves. Over the years, research has demonstrated the important role that chiral molecules play in life, chemistry, and biology as well as their importance in the development of new drugs and technologies. The efforts to understand chiral systems have been mainly driven by spectroscopic methods that leverage on the opposite responses that enantiomers have to linear or circularly polarized light of both handedness. More specifically, Electronic Circular Dichroism (ECD) which measures the differences in linear absorption of left and right circularly polarized light has been the method par excellence for the spectroscopic characterization of chiral compounds. Unfortunately, the fact that ECD is based on linear absorption severely limits the use of this method in the near to far UV region. This is mainly due to the interferences generated by the strong linear absorption of common organic solvents and buffers in this portion of the light spectrum. Nevertheless, the fact remains that many chiral biomolecules of interest related to deceases like Alzheimer and Parkinson, exhibit most of their linear absorption in the near to far UV region where ECD cannot be employed for their study. Therefore, it has become an urgent necessity to develop spectroscopic methods to study chiral molecules that can circumvent the limitations of ECD at shorter wavelengths. In order to overcome the existent limitations in linear chiral spectroscopy, the nonlinear equivalent of ECD arises as a promising alternative, i.e. Two-Photon Circular Dichroism (TPCD). Although, this phenomenon was theoretically predicted in 1975, it was not until 2008, with the introduction of the double-L scan, that a reliable and versatile method for the measurement of TPCD was introduced. The high sensitivity of this method is based on the use of "twin" pulses that allow accounting for fluctuations in the excitation source that prevented the experimental realization of the measurement. The first measurement of a full TPCD spectrum was performed on BINOL enantiomers and the results were supported and discussed with the help of theoretical calculations. After that seminal work, we embarked in expanding the understanding of the structure-property relationship of TPCD by performing, systematically, a series of theoretical-experimental studies in chiral biaryl derivatives and compounds with helical chirality. In Chapter 2 we present the theoretical-experimental study of the effect of the π-electron delocalization curvature on the TPCD of molecules with axial chirality. The targeted molecules for this part of our investigation were S-BINOL, S-VANOL, and S-VAPOL. Our findings revealed that an increase in the TPCD signal, within this series of compounds, was related to the curvature of the π–electron delocalization. The contributions of the different transition moments to the two-photon rotatory strength support our outcomes. Then, in Chapter 3 we introduce the development of the Fragment-Recombination Approach (FRA) for the calculation of the TPCD spectra of large molecules. This simple but powerful method is based on the additivity of the TPCD signal, and is subject to a strict conditional fragmentation approach. FRA-TPCD is demonstrated, theoretically, in two hypothetical molecular systems from the biaryl derivatives family. Afterward, in Chapter 4 we show the first experimental demonstration of FRA-TPCD through the conformational analysis of an axially-chiral Salen ligand in solution (AXF-155). The FRA-TPCD spectra calculated for the different isomers of AXF-155 allowed narrowing the number of possible isomers of this complex molecule in THF solution to only two. This represents a significant improvement from previously reported results using ECD. Subsequently, in Chapter 5 we present the study of the effect of intramolecular charge transfer (ICT) in S-BINAP, an axially dissymmetric diphosphine ligand with strong ICT. The evaluation of the performance of two different exchange-correlation functional (XCF) confirmed that in order to properly predict the theoretical TPCD spectrum of a molecule exhibiting strong ICT, it is required to use an XCF such as CAM-B3LYP. In addition, our findings revealed the importance of considering an adequate number of excited states in order to be able to fully reproduce the experimental TPCD spectrum, thus avoiding wrong assignments of theoretical transitions to experimental spectral features. Finally, and expanding on our previous study, in Chapter 6 we investigated the effect of the nature of ICT on two hexahelicene derivatives. Our investigation demonstrated that the TPCD signal of chiral molecules with strong ICT does not only depend on the strength of this effect but on its nature, i.e. extension of the π–electronic delocalization increasing beyond (EXO-ICT) or within (ENDO-ICT) the helicene core. In summary, with the results presented in this thesis we closed a first loop in the understanding of the structure-property relationship of TPCD. In the future, we expect to deepen in our knowledge of the structure-property relationship of this phenomenon by studying further helicene derivatives with donor-acceptor motif, and through the application of FRA-TPCD to the conformational analysis of amino acids in peptides. We foresee numerous applications of TPCD for the study of optically active molecules with implications in biology, medicine, and the drug and food industry, and applications in nanotechnology, asymmetric catalysis and photonics.

The induced circular dichroism (ICD) of several chiral nitrosamines and various chiral reagents has been investigated. The interaction is attributed to a 1:1 hydrogen bonded complex between the NO group of the nitrosamine and the hydroxyl groups of alcohols and polyols, or the amino group of amines. Only those chiral reagents possessing large differences in size of the groups about the hydrogen bonding site contributed to CD anomalies. The acyclic 2-octanols did not give observable Cotton effects, presumably due to the similarity in size of the methyl and methylene groups and rotational freedom of the acyclic system. The signs of the Cotton effects could be correlated with the absolute configuration of the sterically hindered alcohols and amines. Only the alpha, axial hydrogens of conformationally biased, heterocyclic nitrosamines were found to undergo selective hydrogen-deuterium exchange, suggesting that this feature is critical for nitrosamine carcinogenicity.

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Os calcógenos de európio (EuX, onde X representa O, S, Se ou Te) possuem propriedades magneto-ópticas únicas e interessantes, devido ao enorme magnetismo gerado dos elétrons na camada f do átomo pertencente a família dos terras raras, tornando estes materiais atraentes para aplicações na spintrônica (eletrônica baseada nos transporte de spins e não de carga). Neste trabalho investigamos em baixa temperatura o espectro de absorção utilizando luz circularmente polarizada na região próxima do limiar da banda para o telureto de európio EuTe e o seleneto de európio EuSe em alto campo magnético no ordenamento ferromagnético dos spins de Eu^{2+} da rede cristalina. As amostras crescidas por epitaxia por feixe molecular apresentaram um dicroísmo circular magnético intenso no espectro de absorção para a configuração de Faraday. O par de linhas estreitas observadas estão separadas de aproximadamente 200 meV para o EuTe e 300 meV para o EuSe. Em seguida, formulamos um modelo teórico para a interpretação deste espectro de absorção no arcabouço do modelo de transições eletrônicas entre o estado fundamental 4f^{7}({8}^S_{7/2}) e o estado excitado formado dos estados do caroço remanescente 4f^{6}({7}^F_{J=0...6}) mais o estado em que o elétron se encontra na banda de condução 5d(t_{2g}), resultando em uma excelente concordancia qualitativa e quantitativa com o experimento. / Europium chalcogenides (EuX, where X stands for O, S, Se or Te) have very interesting and unique magneto-optical properties, due to the huge magnetism that arises from the electrons in the f?shell of the rare earth element and which makes them attractive for spintronics applications ( spin transport electronics or spin basedelectronics) In this work we investigate the band-edge optical absorption in high magnetic fields in the Faraday geometry for EuTe and EuSe in the ferromagnetic order attained at low temperatures. In thin layers grown by molecular beam epitaxy, an intense magnetic circular dichroism were observed. The doublet of absorption lines showed a separation by about 200meV in EuTe and 300meV in EuSe. Next, we developed a theoretical model for the interpretation of the absorption spectrum, based in the framework of the model of an electronic transition from a localized ground state 4f^{7}({8}^S_{7/2)) to an excited state formed by the core states 4f^{6}({7}^F_{J=0...6}) and the electron extended state in the 5d(t_{2g}) conduction band, yielding an excellent qualitative and quantitavie agreement with experiment.

Understanding the local conformations of DNA at the level of individual nucleic acid bases is important for the study of the mechanism of DNA sequence-dependent behavior. Here we apply linear absorption, circular dichroism (CD), and fluorescence spectroscopy to study the DNA local base conformation using 6-methyl Isoxanthopterin (6-MI) labeled DNA. We interpret excitation–emission peak shift (EES) measurements of the 6-MI, both as a ribonucleotide monophosphate in solution and as a site-specific substituent for guanine in various DNA constructs, by implementing a simple two-state model. We show that the spectroscopic properties of the 6-MI probe in DNA can be used to obtain detailed information about local base conformations and conformational heterogeneity and fluctuations. Based on these findings, we apply a simple theoretical model to calculate CD of 6-MI substituted DNA constructs. We find that our model can be used to extract basesequence- dependent information about the local conformation of the 6-MI probe as modulated by the local base or base-pair environment. We next apply 6-MI to probe the ligand insertion of small molecules to duplex DNA, further extending the potential of 6- MI as a useful reporter of local nucleic acid base conformation. These studies served to establish a new level of sophistication in qualitatively analyzing 6-MI structural behavior in terms of local base stacking and unstacking conformations. v This dissertation contains previously published and unpublished co-authored material.

Neste trabalho, utilizando técnicas magneto-ópticas, estudamos tempos de relaxação spin-rede (T1) do estado fundamental de centros \'F\' e, cristais de halogenetos alcalinos (KCl-KBr). Descrevemos um sistema semi-automático para medidas ópticas de T1, capaz de medir tempos de relação curtos (~1mS), baseado na medida do Dicroísmo Circular magnético (DCM) que apresentam aqueles sais quando portadores de centros paramagnéticos. Obtivemos a dependência de T1 com o campo magnético H (até 65 Kgauss), bem como os espectros de DCM para diferentes concentrações nas matrizes mistas. Uma teoria desenvolvida por Panepucci e Mollenauer (1) para matrizes puras, foi adaptadas para explicar a relaxação spin-rede nos cristais mistos. Os resultados obtidos para o processo direto (T~2.0 K), confrontados com auqela teoria, mostram que o mecanismo de relaxação dominante até 25 KGauss continua sendo a modulação por fônons da interação hiperfins entre o elétron \'F\' e os núcleos vizinhos. / Using magneto-optic techniques we have studied the ground state spin- lattice relaxation times (T1) of \'F\' centers in mixed Alkali Halide cristals (KCl-KBr). We describe a computer assisted system to optically measure short relaxation times (~1mS). The technique is based on the measurement of the Magnetic Circular Dicroism (MCD) presented by F centers. We obtained the T1 magnetic field dependency at 2 K up to 65 kGauss), as well as the MCD spectra for different relative concentration at the mixed matrices. The theory developed by Panepucci and Mollenauer for F centers spin-lattice relaxation in pure matrices was modified to explain the behavior of T1 in mixed cristais. The Direct Process results (T~2.0 K) compared against that theory shows that the main relaxation mecanism, Up to 25 kGauss, continues to be phonon modulation of the hyperfine interaction between \'F\' electrons and surrounding nuclei.

Circular dichroism (CD) spectra were obtained for bacteriophage T4 lysozyme and three of its mutants in the presence and absence of colloidal silica nanoparticles. Mutant lysozymes were produced by substitution of the isoleucine at position 3 with tryptophan, cysteine and leucine. Each substitution resulted in an altered structural stability, quantified by a difference in free energy of unfolding from the wild type. CD spectra recorded in the absence of colloidal silica agreed with x-ray diffraction data in that the mutants and wild type showed similar secondary structures. CD spectra of protein-nanoparticle complexes recorded after contact for 90 minutes showed significant differences from those recorded in the absence of nanoparticles, and these differences varied among the proteins. The percentage of a-helix lost in these proteins upon adsorption to silica nanoparticles was also recorded as a function of time by CD. For a 1:2 protein to particle mixture, different kinetic behaviors were observed among the proteins. The more unstable the protein, the greater the rate and extent of secondary structure loss upon adsorption. For a 1:1 protein to particle mixture, only results recorded with the tryptophan mutant were significantly different from the other variants. The kinetic data recorded for the 1:2 protein to particle ratio was evaluated using two different protein adsorption models. Both models allow proteins at an interface to exist in two different states: state 1 molecules retain their native conformation, while state 2 molecules lose a certain amount of their native secondary structure and occupy more surface area than state 1 molecules. The main difference between these two models is that one allows state 2 molecules to be adsorbed directly from solution, while the other requires that state 2 molecules be generated by surface-induced conversion of state 1 molecules. The former model showed a better fit to the data than the latter from a least squares comparison. Both models indicated that proteins of lower thermal stability have a greater tendency to adopt state 2 on silica. / Graduation date: 1997

Graduation date: 1994

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 228-251).

To understand protein stability and the mechanism of protein folding, it is essential that we gain a better understanding of the ensemble of conformations that make up the denatured state of a protein. The primary goal of the research described here was to see what we might learn about the denatured state using fluorescence. To this end, tryptophan was introduced at five sites in Ribonuclease Sa (RNase Sa): D1W, Y52W, Y55W, T76W, and Y81W. The fluorescent properties of the denatured states of these five proteins were studied and compared to the fluorescent properties of eight model compounds: N-acetyl-tryptophan-amide (NATA), N-acetyl-Ala-Trp-Ala-amide (AWA), N-acetyl-Ala-Ala-Trp-Ala-Ala-amide (AAWAA), and five pentapeptides based on the sequence around the original tryptophan substitutions in RNase Sa. Regardless of the denaturant, λmax for the proteins and model compounds differed very little, 349.3 ± 1.2 nm. However, significant differences were observed in the fluorescence intensity at λmax (IF), suggesting that IF is more sensitive to the immediate environment than λmax. The differences in IF are due in part to quenching by neighboring side chains. More importantly, IF was always significantly greater in the protein than in its corresponding pentapeptide, indicating that the protein exerts an effect on the tryptophan, which cannot be mimicked by the pentapeptide models. Acrylamide and iodide quenching experiments were also performed on the model compounds and proteins. Significant differences in the Stern-Volmer quenching constant (KSV) were also observed between the proteins and between the proteins and their corresponding pentapeptides. Importantly, the KSV for the protein was always less than in its corresponding pentapeptide. These data along with the IF data show that non-local structure in the unfolded state influences tryptophan fluorescence and accessibility. In summary, these and our other studies show that fluorescence can be used to gain a better understanding of the denatured states of proteins.