Title: Theory and application of optical spectroscopic methods for structural molecular studies Author: RNDr. Jana Hudecová Department / Institute: Institute of Organic Chemistry and Biochemistry Supervisor of the doctoral thesis: Prof. RNDr. Petr Bouř, DSc. Abstract: In the thesis, methods of the chiroptical spectroscopy (Raman optical activity, electronic and vibrational circular dichroism, circularly polarized luminescence) were utilized to obtain information on structure of chiral molecules. In four main projects, we focused on improving accuracy of quantum-chemical computations used for interpretation of experimental spectra by including anharmonic effects, solvent, molecular flexibility and dynamics. In the first project, the normal mode geometry optimization method was investigated and a suitable frequency limit providing realistic vibrational band broadening was found. Then the ability of harmonic and anharmonic computational approaches to describe the C-H stretching vibrations was explored for three terpene molecules and four spectroscopic methods. In the third project, we estimated the role of dispersion forces and different organic solvents for conformer equilibria and dynamics of cyclic dipeptides containing tryptophan. In the last project, circularly polarized luminiscence spectra, which were...

Title: Study of protein structure and dynamics by means of optical spectroscopy Author: Tomáš Pazderka Institute: Institute of Physics of Charles University Supervisor: RNDr. Vladimír Kopecký, Ph.D., Institute of Physics of Charles University Abstract: The aim of this thesis is to improve understanding of protein structure and dynamics and extend experimental setup and data processing for such stud- ies. We focus on the extension of experimental feasability of vibrational optical activity (VOA). We have demonstrated a usability of intensity calibration in the field of Raman optical activity. Advantages for measurements on multiple instru- ments and/or using different configurations have been shown. A new instrumental setup has been developed for microsampling measurements of vibrational circular dichroism spectra with a spatial resolution of 1 mm. Using this technique, spatial inhomogeneities in a sample of protein fibrils have been observed. Model com- pounds for amide nonplanarity have been investigated utilizing several methods of optical spectroscopy and key spectral features for determination of amide non- planarity and the absolute configuration have been identified. A comprehensive set of Raman spectra of proteinogenic amino acids has been measured. Sample concentration dependencies and consequent...

This diploma thesis studies magneto-optical (MO) response of epitaxial thin films of Co-doped ceria. Thin films were characterized by XPS, LEED, STM, spectroscopic ellipsometry and measurement of MO activity. The work focuses on studying MO response of the films depending on film thickness, cobalt concentration, oxidation state of cerium and chemical state of cobalt. Spectra of MO response consist of low energy region where the MO activity is mediated by transitions from defect induced states to conduction band and high energy region where a peak of MO activity appears which we attribute to transitions from valence band to conduction band. In this work we qualitatively explain the effects of the physico- chemical states of the thin films on the structure of the obtained MO spectra, mainly on the appearance of the MO activity in the low energy region, and on the changes of the position of the MO peak. Compared to other preparation methods the epitaxial thin films allow achieving a shift of the MO peak in the direction of higher photon energy.

This diploma thesis studies magneto-optical (MO) response of epitaxial thin films of Co-doped ceria. Thin films were characterized by XPS, LEED, STM, spectroscopic ellipsometry and measurement of MO activity. The work focuses on studying MO response of the films depending on film thickness, cobalt concentration, oxidation state of cerium and chemical state of cobalt. Spectra of MO response consist of low energy region where the MO activity is mediated by transitions from defect induced states to conduction band and high energy region where a peak of MO activity appears which we attribute to transitions from valence band to conduction band. In this work we qualitatively explain the effects of the physico- chemical states of the thin films on the structure of the obtained MO spectra, mainly on the appearance of the MO activity in the low energy region, and on the changes of the position of the MO peak. Compared to other preparation methods the epitaxial thin films allow achieving a shift of the MO peak in the direction of higher photon energy.

Heterocyclen mit einer a-Hydroxycarbonyl-Einheit wie 3-Hydroxy-[1,5]-benzothiazepin-4-one oder 3-Hydroxychroman-4-one sind von Interesse, da sie Bestandteil von Pharmazeutika (z. B. Diltiazem®) oder Naturstoffen (z.B. die Flavonoide) sind. Ringtransformationen von optisch aktiven Glycidaten mit Binucleophilen stellen eine nützliche Synthesemethode zur Darstellung solcher Produkte dar. In Abhängigkeit von der Position des Angriffes des Nucleophiles am Oxiranring (a- oder b-Angriff) werden zwei Produkttypen mit verschiedenen Ringgrößen gebildet. Der Ablauf der Reaktion hängt vom Binucleophil, von den Substituenten am Oxiran und von den Reaktionsbedingungen ab. 4-Hydroxypyrazolidin-3-one werden bei der Reaktion von Glycidaten mit Hydrazinen gebildet. Reaktionen mit 1-Amino-2-mercapto-Verbindungen geben ausschließlich [1,4]-Thiazin-3-one. Gelenkt durch die Substituenten am Oxiran reagieren Glycidate mit o-Phenylendiaminen entweder zu Tetrahydro-chinoxalin-2-onen oder zu 3-Hydroxy-[1,5]-benzodiazepin-2-onen. Bei der Bildung der Tetrahydro-chinoxalin-2-one aus unsymmetrisch substituierten o-Phenylendiaminen wird eine hohe Regioselektivität beobachtet. Epoxyamide, gebildet aus Glycidsäuren und o-Aminophenol, cyclisieren unter basischen Bedingungen zu 1,4-Benzoxazin-3-onen, während in Gegenwart einer Lewis-Säure 3-Hydroxy-[1,5]-oxazepin-4-one gebildet werden. Reaktionen von Oxiran-carbonsäureamiden mit o-lithiierten Phenolderivaten führen zu Epoxyketonen, die zu 2-Alkyliden-cumaranonen oder 3-Hydroxychroman-4-onen transformiert werden können. Ohne Inanspruchnahme der Carbonylgruppe sind [1,4]-Benzoxazine durch die Reaktion von Glycidaten mit o-Aminophenolen erhältlich. Alle Ringtransformationen verlaufen stereoselektiv, so daß enantiomerenreine Produkte erhalten werden. / Heterocycles with an a-hydroxycarbonyl moiety such as 3-hydroxy-[1,5]-benzothiazepin-4-ones or 3-hydroxychroman-4-ones are of interest because they are frequently encountered in pharmaceuticals (e.g. Diltiazem®) or natural products (e.g. flavonoids). Ring transformation of optically active oxirane carboxylic acid derivatives with ambident nucleophiles provide a useful synthetic method for such type of products. Depending on the position of attack on the oxirane ring by the nucleophile (a- or b-attack) two types of products with different ring sizes are formed. The mode of reaction depends on the type of binucleophile, the substituents at the oxirane, and the reaction conditions. 4-Hydroxypyrazolidin-3-ones are formed in the reaction of glycidates with hydrazines. Reaction of 1-amino-2-mercapto nucleophiles exclusively afforded [1,4]-thiazin-3-ones. Governed by the substituents at the oxirane on the reaction of glycidates with o-phenylendiamines result either in tetrahydroquinoxalin-2-ones or 3-hydroxy-[1,5]-benzodiazepin-2-ones. In the formation of tetrahydroquinoxalin-2-ones from asymmetrically substituted o-phenylendiamines, high stereoselectivity was observed. Epoxyamides, formed from glycidic acids and o-aminophenols, cyclize to [1,4]-benzoxazin-3-ones under basic conditions while 3-hydroxy-[1,5]-oxazepin-4-ones are formed in the presence of a Lewis acid. Reaction of oxirane carboxylic acid amides with o-lithiated phenol derivatives afforded epoxyketones that could be transformed to 2-alkyliden-coumaranones and 3-hydroxychroman-4-ones. Without the use of the carbonyl group [1,4]-benzoxazines are available in the reaction of glycidates with o-aminophenols. All ring transformations were highly stereoselective giving enantiomerically pure products.

Le travail de thèse effectué a pour objectif d'amener vers une meilleure compréhension des comportements électrique et optique des modules CPV, dans des conditions environnantes variables. La première partie est consacrée à l’étude de la performance des modules en conditions réelles de fonctionnement. Quatre technologies de module, toutes équipées de cellules triple-jonctions, mais de concentrateurs optiques différents, ont été testées en extérieur sur des périodes de un mois à deux ans. Les résultats montrent que la sensibilité à la température de lentille, la température de cellule et au spectre incident varie selon le type d'architecture optique. La sensibilité la plus importante à la température de lentille a été obtenue pour un dispositif sans optique secondaire. Le coefficient en température de la tension Voc a été calculé et varie entre les technologies. Enfin, les variations importantes de facteur de forme avec le spectre incident observées pour une technologie, mettent en évidence la nécessité d'étudier les phénomènes de non-uniformités d'irradiance sur la cellule. Dans une deuxième partie, le développement d’un banc de test en intérieur permettant de mesurer les performances électriques et optiques est présenté. Ce banc a pour objectif de permettre la reproduction des conditions réelles de fonctionnement des modules de façon contrôlée en intérieur. Un système d’imagerie est utilisé pour déterminer la distribution spatiale et spectrale d’irradiance sur la cellule. Associé à un traceur de courbes IV, il vise à caractériser les effets de flux non-uniformes sur la cellule. Le banc de mesure a pour avantage de découpler les paramètres d’études, telles que la température de la lentille et la température de la cellule, et permet ainsi de décorréler leurs effets respectifs sur l'ensemble optique-cellule, ce qui n’est que difficilement possible sur des mesures en extérieur. Le procédé de calibration et la validation du dispositif sont détaillés dans le manuscrit. Enfin, dans une dernière partie, le banc développé est utilisé pour caractériser trois différents dispositifs CPV : un sans optique secondaire, et deux avec des optiques secondaires différentes. Les impacts de la distance lentille-cellule et de la température de lentille sur les performances de la cellule sont quantifiés optiquement et électriquement. Les résultats montrent comment ces paramètres modifient la distribution de densités de courant des sous-cellules, et donc le comportement électrique du dispositif. Ils soulignent plus spécifiquement comment les non-uniformités spectrales et spatiales affectent les performances de la cellule pour les différents concentrateurs. Le dispositif sans optique secondaire montre une sensibilité importante à la température de la lentille et la distance optique primaire-cellule, qui se traduit par une perte de production d'énergie dans des conditions réelles de fonctionnement. / The goal of this doctoral thesis is to bring answers to a better understanding of the electrical and optical behavior of CPV modules, under different operating conditions. In the first part, a study on module performance under real conditions is presented. Using an outdoor automated test bench, the sensitivity of four different CPV module technologies to most operating conditions relevant to CPV systems has been studied, namely DNI, spectrum, cell and lens temperature and clearness of the sky. In order to isolate the influence of a single operation parameter, the analysis of outdoor monitoring data from one month to two years is performed. The results show how the optical design influences the sensitivity of the electrical parameters to the mentionned operating conditions. The effect of lens temperature on cell current has been found to be maximum for the CPV module without Secondary Optical Element. Also the $V_{oc}$ thermal coefficient was found to vary between module technologies. Finally, the important variations of the fill factor for one technology underlines the need of studying non-uniformities effects on the cell performance. According to the results observed outdoors, an indoor tool was developed in order to uncorrelate outdoor parameters. A test bench that measures multi-spectral irradiance profiles, through CMOS imaging and bandpass filters in conjunction with electrical $IV$ curves, is used as a mean to visualize and characterize the effects of chromatic aberrations and nonuniform flux profiles under controllable testing conditions. The bench allows decoupling the temperatures of the Primary Optical Element and cell allowing the analyze of their respective effects on optical and electrical performance. In varying the temperature of the Primary Optical Element, the effects on electrical efficiency, focal distance, spectral sensitivity, acceptance angle, or multi-junction current matching profiles can be quantified. Calibration procedures and validation process are detailed. Finally, the developed testbench is used for analyzing the behvaior of three different CPV devices : one without Secondary Optical Element, and two with different Secondary Optical Elements. The impacts of cell position and lens temperature on the cell performance are quantified optically and electrically. The results show how these parameters modify the current density distribution of the subcells, and hence the electrical behavior of the device. They underline more specifically how spectral and spatial non-uniformities affect the cell performance for the different devices. The device without SOE shows a strong sensitivity to lens temperature and POE-cell distance, that will correspond to a decrease of energy production under real conditions of operation.

Raman optical activity (ROA) is a powerful probe of the structure and behaviour of biomolecules in aqueous solution for a number of important problems in molecular biology. Although ROA is a very sensitive technique for studying biological samples, it is a very weak effect and the conditions of high concentration and long data collection time required limit its application for a wide range of biological samples. These limitations could possibly be overcome using the principle of surface enhanced Raman scattering (SERS). The combination of ROA with SERS in the form of surface enhanced ROA (SEROA) could be a solution for widening the application of ROA. In the last few years, the generation of reliable SEROA spectra of biomolecules has been problematic due to non-homogenous colloidal systems forming and low signal-to-noise ratios which complicated detection of the true SEROA signal from the analyte. L- and D-enantiomers give full or partially mirror image chiroptical spectra, this property of enantiomers can be employed to prove the chiroptical activity of the SEROA technique. In this thesis we employed a hydrophilic polycarbopol polymer as stabilising media which has led to the first report of mirror image SEROA bands for enantiomeric structures. This new technique of incorporating the hydrogel polymer as a means to stabilise the colloidal system has proven to be reliable in obtaining high quality SEROA spectra of D- and L-enantiomers of ribose and tryptophan. In an extension of the hydrogel-stabilised SEROA work, we also demonstrate that single nanoparticle plasmonic substrate such as silver silica nanotags can enhance the weak ROA effect. These dye tagged silica coated silver nanoparticles have enabled a chiral response to be transmitted from a chiral analyte to the plasmon resonance of an achiral metallic nanostructure. The measurement of mirror image SERROA bands for the two enantiomers of each of ribose and tryptophan was confirmed for this system. The generation of SEROA for both systems was achieved and confirmed SEROA as a new sensitive tool for analysis of biomolecular structure. In a related project, Raman and ROA spectra were measured for adenosine and seven of its derivative ribonucleotides. Both of these spectroscopic techniques are shown to be sensitive to the site and degree of phosphorylation, with a considerable number of marker bands being identified for these ribonucleotides. Moreover, the SERS studies of these ribonucleotides were also performed. The obtained SERS spectra were shown similar features that confirm these analytes interact with the surface in a similar manner, hence limiting the structural sensitivity of this method towards phosphate position. Short dipeptides such as diketopiperazine (DKP) have been investigated during the last decades as both natural and synthetic DKPs have a wide variety of biological activities. Raman and ROA spectra of linear and cyclic dialanine and diserine were measured to charecterize their solution structures. Density functional theory (DFT) calculations were carried out by a collaborator to assist in making vibrational band assignments. Considerable differences were observed between the ROA bands for the cyclic and linear forms of both dialanine and diserine that reflect large differences in the vibrational modes of the polypeptide backbone upon cyclicization. In this study, the ROA spectra of cyclic dialanine and diserine have been reported for the first time which demonstrated that ROA spectroscopy when utilised in combination with computational modelling clearly provides a potential tool for characterization of cyclic peptides.

Title: Theory and Applications of Optical Activity of Biomolecules Author: Monika Krupová Supervisor: prof. RNDr. Petr Bouř, DSc. Institutions: Faculty of Mathematics and Physics, Charles University, and Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic Abstract: This thesis describes how we used several chiroptical spectroscopic methods to study chiral molecules: vibrational circular dichroism (VCD), circularly polarized luminescence (CPL) and magnetic circular dichroism (MCD). VCD and induced lanthanide CPL were used to study the structure of amyloid protein fibrils. We found out that VCD is very sensitive to their structure and supramolecular chirality. It could be used to distinguish between various polymorphic fibrils. On the other hand, induced lanthanide CPL provided information on the local structure. VCD was also used to study the hydration polymorphism of nucleoside crystals. Due to the crystal packing, the VCD signal was strong and specific for different types of crystals. Finally, electronic structure of hydrated Ln3+ ions was studied by MCD. Molecular dynamics simulations together with crystal field theory (CFT) and multistate complete active space calculations with second order perturbation correction (MS-CASPT2) were used to interpret the spectra. CFT...

The primary purpose of this study was to investigate the combination of experimental and computational methods in the search for reproducible colloidal surface-enhanced Raman scattering of pharmaceutical compounds. In the search for optimal experimental conditions for colloidal surface-enhance Raman scattering, the amphipathic β-blocker propranolol was used as the target molecule. Fractional factorial designs of experiments were performed and a multiobjective evolutionary algorithm was used to find acceptable solutions, from the results, that were Pareto ranked. The multiobjective evolutionary algorithm suggested solutions outside of the fractional factorial design and the experiments were then performed in the laboratory. The results observed from the suggested solutions agreed with the solutions that were found on the Pareto front. One of the experimental conditions observed on the Pareto front was then used to determine the practical limit of detection of propranolol. The experimental conditions that were chosen for the limit of detection took into account reproducibility and enhancement, the two most important parameters for analytical detection using surface-enhanced Raman scattering. The principal conclusion to this study was that the combination of computational and experimental methods can reduce the need for experiments by > 96% and then selecting solutions from the Pareto front improved limit of detection by a factor of 24.5 when it was compared to the previously reported limit of detection for propranolol. Using the same experimental conditions that were used for the limit of detection, these experiments were extended to plasma spiked with propranolol in order to test detection of this pharmaceutical in biofluids. Concentrations of propranolol were prepared using plasma as the solvent and measured for detection using colloidal surface-enhanced Raman scattering. Detection was determined as <130 ng/mL, within physiological concentrations, previously achieved using separation techniques. The second part of this thesis also involved a combination of experimental and computational methods. Raman optical activity was utilized to investigate secondary structure of amino acids and diamino acid peptides in combination with density functional theory calculations. Amino acids are important biological molecules that have vital functions in the biological system. They have been recognized as neurotransmitters and implicated in neurodegenerative diseases. Raman and Raman optical activity experimental results were compared to determine site-specific acetylation, marker bands for constitutional isomers and identification of functional groups that interact with the solvent. The experimental spectra were then compared to those from the density functional theory calculations. The results indicated that; constitutional isomers cannot be distinguished from the Raman spectra but can be distinguished from the Raman optical activity spectra, site-specific acetylation can be identified from the Raman spectra, however, Raman optical activity provides more structural information in relation to acetylation. When the results were compared to the density functional theory calculations for the diamino acid peptides the results agreed reasonably well, however, agreement was not as good for the monoamino acids because diamino acid peptides support fewer conformations due to the peptide bond whereas monoamino acids can adopt a far greater number of conformations. Combined computational and experimental techniques have developed the ability to detect and characterize biomedical compounds, a significant move in the advancement of Raman spectroscopies.