Proteins are chain molecules built from amino acids. The precise sequence of the 20 different types of amino acids in a protein chain defines into which structure a protein folds, and the three-dimensional structure in turn specifies the biological function of the protein. The reliable folding of proteins is a prerequisite for their robust function. Misfolding can lead to protein aggregates that cause severe diseases, such as Alzheimer's, Parkinson's, or the variant Creutzfeldt-Jakob disease. Small single-domain proteins often fold without experimentally detectable metastable intermediate states. The folding dynamics of these proteins is thought to be governed by a single transition-state barrier between the unfolded and the folded state. The transition state is highly instable and cannot be observed directly. However, mutations in which a single amino acid of the protein is substituted by another one can provide indirect access. The mutations slightly change the transition-state barrier and, thus, the folding and unfolding times of the protein. The central question is how to reconstruct the transition state from the observed changes in folding times. In this habilitation thesis, a novel method to extract structural information on transition states from mutational data is presented. The method is based on (i) the cooperativity of structural elements such as alpha-helices and beta-hairpins, and (ii) on splitting up mutation-induced free-energy changes into components for these elements. By fitting few parameters, the method reveals the degree of structure formation of alpha-helices and beta-hairpins in the transition state. In addition, it is shown in this thesis that the folding routes of small single-domain proteins are dominated by loop-closure dependencies between the structural elements. / Proteine sind Kettenmoleküle, die aus einzelnen Aminosäuren aufgebaut sind. Die genaue Sequenz der 20 verschiedenartigen Aminosäuren innerhalb der Proteinkette bestimmt dabei, in welche spezielle Struktur sich ein Protein faltet. Die dreidimensionale Struktur bestimmt wiederum die Funktion der Proteine. Doch nur korrekt gefaltet kann ein Protein seine Funktion erfüllen. Fehler bei der Faltung können zu Proteinaggregaten führen, die schwere Krankheiten wie Alzheimer, Parkinson oder das Creutzfeldt-Jakob-Syndrom hervorrufen. Viele kleine Proteine falten ohne experimentell beobachtbare metastabile Zwischenzustände. Entscheidend für die Faltungsdynamik dieser Proteine ist der Übergangszustand zwischen dem ungefalteten und gefalteten Zustand. Der Übergangszustand ist instabil und kann nicht direkt beobachtet werden. Einen indirekten Zugang ermöglichen jedoch Mutationen eines Proteins, bei denen einzelne Aminosäuren ausgetauscht werden. Die Mutationen verändern geringfügig die Übergangszustandsbarriere, und damit die Faltungs- und Entfaltungszeiten des Proteins. Die zentrale Frage ist, wie sich der Übergangszustand aus den beobachteten Änderungen der Faltungszeit rekonstruieren lässt. In dieser Habilitationsschrift wird eine neuartige Methode zur Rekonstruktion von Übergangszuständen aus Mutationsdaten vorgestellt. Die Methode beruht auf (i) der Kooperativität von Strukturelementen wie alpha-Helizes und beta-Haarnadeln, und (ii) der Aufspaltung von mutationsinduzierten Veränderungen der freien Energie in Komponenten für diese Strukturelemente. Die Modellierung der experimentellen Daten verrät, in welchem Grad alpha-Helizes and beta-Haarnadeln im Übergangszustand strukturiert sind. Zudem wird in dieser Habilitationsschrift gezeigt, dass die Faltungswege vieler kleiner Proteine durch Schleifenschließungsbeziehungen zwischen den Strukturelementen dominiert werden.

HIV-1 protease inhibitors are important in the most frequently used regimen for the treatment of HIV/AIDS, the highly active antiretroviral therapy (HAART). For patients with access to this treatment, an HIV infection is no longer lethal, but rather a manageable, chronic infection. However, the HIV-1 protease inhibitors are generally associated with serious shortcomings such as adverse events, development of drug resistance and poor pharmacokinetic properties. Most of the approved inhibitors suffer from high protein binding, rapid metabolism and/or low membrane permeability. In this project, novel HIV-1 protease inhibitors comprising a rarely used tertiary alcohol in the transition-state mimic were designed, synthesized and evaluated. The rationale behind the design was to achieve ‘masking’ of the tertiary alcohol by for example, intramolecular hydrogen bonding, which was believed could enhance transcellular transport. A reliable synthetic protocol was developed and a series of highly potent inhibitors was obtained exhibiting excellent membrane permeation properties in a Caco-2 cell assay. However, the cellular antiviral potencies of these compounds were low. In an attempt to improve the anti-HIV activity, microwave-accelerated, palladium-catalyzed cross-coupling reactions and aminocarbonylation of aryl bromide precursors were employed to produce P1'-extended test compounds. Inhibitors demonstrating up to six times higher antiviral effect were obtained, the best derivatives having para 3- or 4-pyridyl elongations in P1'. Fast metabolic degradation was observed in liver microsome homogenate, which is believed, at least partly, to be attributable to benzylic oxidation of the indanol P2 group of the inhibitors. To enable facile variation of the P2 side chain a new synthetic route was developed using an enantiomerically pure, benzyl-substituted epoxy carboxylic acid as the key intermediate. Cyclic and amino-acid-residue-derived P2 groups were evaluated, and inhibitors equipotent to the series containing an indanol moiety were produced.

The overall aim of this thesis is to compare different methods for calculation of Core-Level shifts in metallic alloys. The methods compared are the Initial State model, the Complete screening and the Transition state model. Core-level shifts can give information of chemical bonding and about the electronic structure in solids. The basic theory used is the so-called Density-Functional-Theory, in conjunction with the Local-Density Approximation and the Coherent-Potential- Approximation. The metallic alloys used are Silver-Palladium, Copper-Palladium, Copper-Gold and Copper-Platinum, all inface-centered-cubic configuration. The complete screening- and the transition-state model are found to be in better agreement with experimental results than those calculated with the initial state model. This is mainly due to the fact that the two former models includes final-state effects, whereas the last one do not. The screening parameters within the Coherent-Potential approximation are also investigated. It is found that the Screened-Impurity Model can extend the validity of the Coherent-Potential-Approximation and increase it's accuracy.

Orientador: Luiz Carlos Dias / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-20T22:05:16Z (GMT). No. of bitstreams: 1 Ferreira_MarcoAntonioBarbosa_D.pdf: 22008245 bytes, checksum: 306bdd05b22f9f07a0496a6e9806f940 (MD5) Previous issue date: 2012 / Resumo: SÍNTESE TOTAL DA (-)-GONIOTRIONINA: Foi desenvolvida uma rota sintética flexível, em uma abordagem modular, que permitiu a primeira síntese total da estrutura descrita para a goniotrionina (11), em 4% de rendimento global e em 17 etapas pela rota linear mais longa, a partir do glicidol quiral (R)-109. A nossa rota sintética fez uso de epóxidos quirais como blocos de construção, em uma estratégia envolvendo a abertura de epóxidos. As etapas chave incluem ciclização aeróbica de Mukaiyama para formação do anel THF-2,5-trans e reação aldólica de Mukaiyama 1,2-syn seletiva. Esta mesma abordagem sintética pode permitir a preparação de análogos e outros diastereoisômeros da (-)-goniotrionina (11). ESTUDO TEÓRICO DA INFLUÊNCIA ESTEREOELETRÔNICA NA SELETIVIDADE 1,5 EM REAÇÕES ALDÓLICAS ENVOLVENDO B-ALCOXI METILCETONAS. Nesta segunda parte do trabalho foi possível aprofundar o conhecimento sobre a origem da seletividade 1,5 em reações aldólicas envolvendo enolatos de boro de b-alcóxi metilcetonas, a partir do cálculo das estruturas eletrônicas dos estados de transição. Investigou-se a influência dos estereocentros em a, b, y e s em reações aldólicas, determinando os fatores decisivos que governam o senso de indução 1,5. Adicionalmente, foi proposto um modelo teórico que permitiu racionalizar a seletividade 1,5 em função das características estereoeletrônicas de cetonas com acentuado volume estéreo na posição beta com relação à carbonila / Abstract: TOTAL SYNTHESIS OF (-)-GONIOTRIONIN: We have accomplished the first total synthesis of the reported structure of goniotrionin (11) in 4% overall yield over a longest-linear sequence of 17 steps from glycidol (R)-109. Our synthetic route employed an epoxide-opening strategy, using chiral epoxides as building blocks. Key steps included a Mukaiyama aerobic oxidative cyclization and a 1,2-syn selective Mukaiyama aldol reaction. This synthesis employs highly flexible key couplings that allow for the preparation of analogs, including other diastereoisomers of goniotrionin (11). THEORETICAL STUDY OF THE STEREOELECTRONIC INFLUENCE IN 1,5 SELECTIVITY OF ALDOL REACTIONS INVOLVING b-ALKOXY METHYLKETONES: In the second part of this work was extended the knowledge about the origin of 1,5 selectivity in aldol reactions involving boron enolates of b-alkoxy methyl ketones, based on the calculation of electronic structures of the corresponding transition states. We investigated the influence of stereocenters at the a, b, y and s positions in aldol reactions, determining the key factors that govern the 1,5 induction. Additionally, we propose a theoretical model that racionalize the 1,5 selectivity depending on the stereoelectronic characteristics of b-bulky ketones / Doutorado / Quimica Organica / Doutor em Ciências

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Despite comprehensive studies addressing the chemical kinetics of butanol isomers, relevant uncertainties associated with the emissions of relevant pollutants persists. Also, a lack of chemistry knowledge of processes designed to produce biofuels limits their implementation at industrial scales. Therefore, the first objective of this thesis was to use cutting-edge kinetic theories to calculate rate constants of propen-2-ol, 1-pronenol, and vinyl alcohol keto-enol tautomerizations, which account for the production of the harmful carbonyl species. The second objective was to use the predictive capabilities of dynamic theories to reveal new chemistry of syngas oxy-combustion in supercritical CO2 and complexities of the zeolite dealumination, two processes involved in coal and biomass conversion. Rate constants computations considered transition state theory with variational effects, tunneling correction, and multistructural torsional anharmonicity. The study also included pressure effects by using and improving the system-specific quantum Rice-Ramsperger-Kassel/modified strong collision model. The atomistic simulations used ReaxFF force fields in hydrogen/oxygen/carbon monoxide/ carbon dioxide mixtures to represent the syngas system and an MFI zeolite with different water loading to model the dealumination. The results show that the studied assisted tautomerizations have much lower energy barriers than the unimolecular process. However, the “catalytic” effect is efficient only if the partner molecule is at high concentrations. Pressure effects are pronounced in the chemically activated tautomerizations, and the improved algorithm to compute pressure-dependent rate constants overcomes the initial difficulties associated with its application to C3 or larger molecules at temperatures above 800-1000 K. Reactive molecular dynamics simulations revealed the role of CO2 as an initiator in the syngas oxy-combustion and a new step involving the formation of formic acid. Those simulations for the zeolite dealumination process also showed that proton transfer, framework flexibility, and aluminum dislodging mediated by silicon reactions are complex dynamic phenomena determining the process. These aspects complement the dealumination theory uncovered so far and establish new paths in the study of water-zeolite interactions. Overall, the rate constants computed in this work reduce relevant uncertainties in the chemical kinetic mechanisms of alcohol oxidation, and the molecular dynamics simulations broaden the chemical knowledge of processes aimed at the utilization of alternative energy resources.

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<p>MurA catalyzes the first committed step of peptidoglycan biosynthesis and it is the target of the antibiotic fosfomycin. Due to a Cys-to-Asp substitution in the active site, MurAs from a number of pathogenic bacteria, including <em>Mycobacterium tuberculosis</em> and <em>Borrelia burgdorferi</em> (Lyme disease), are fosfomycin resistant. His-tagged <em>Borrelia burgdorferi</em> MurA (Bb_MurA) and its D116C mutant have been successfully expressed, purified and characterized. The <em>k</em>_cat value of wild-type Bb_MurA was 0.74 ± 0.01 s^-1. The D116C mutant’s <em>k</em>_cat decreased by 25-fold and was fosfomycin sensitive. The pH profiles of <em>k</em>_cat for both Bb_MurA and its mutant were characterized. There was little difference in p<em>K</em>_a1 values, but the p<em>K</em>_a2 value shifted from 7.4 ± 0.2 in wild-type enzyme to a value >11 in the mutant. This demonstrated that the p<em>K</em>_a2 of 7.4 was due to D116, and that it must be protonated for activity. Fosfomycin inactivation of Bb_MurA_H6(D116C) was time-dependent and only proceeded in the presence of UDP-GlcNAc. The dissociation constant, <em>K</em>_i, was 5.7 ± 0.4 µM and rate of covalent modification, <em>k</em>_inact, was 0.021 ± 0.003 s^-1.</p> <p>DAHP synthase catalyzes the first committed step in the shikimate pathway, and its catalysis has been proposed to proceed through two oxacarbenium ion intermediates. Pyruvate oxime, glyoxylate oxime and 4-imidazolecarboxylic acid have been evaluated as inhibitors of DAHP synthase. In the presence of glycerol 3-phosphate, the fitted <em>K</em>_i values of pyruvate oxime and glyoxylate oxime were 7.6 (± 0.9) × 10^-5 M and 7.4 (± 1.7) × 10^-5 M, respectively. 4-Imidazolecarboxylic acid’s inhibition was cooperative, and its binding was competitive with respect to PEP, and uncompetitive with respect to E4P. Its equilibrium dissociation constant was 3.0 (± 0.2) × 10^-3 M.</p> / Master of Science (MSc)

Organometallic reactions are a fundamental class of chemical transformations. The mechanisms of organometallic reactions are routinely modeled by calculating intermediates and transition-state structures on a potential energy surface with density functional theory (DFT). The translation of these calculated structures to a reaction mechanism is typically done under the umbrella of statistical transition state theory. This dissertation reports the use of DFT calculations and quasiclassical direct dynamics trajectories to explore the possibility of nonstatistical dynamic effects in organometallic reactions. Chapter 1 provides a brief review of potential energy surfaces, transition state theory, dynamics trajectories, and a review of previous dynamics studies of organometallic reactions. Chapter 2 reports dynamics trajectories of an organometallic β–hydride transfer reaction with Rh, Ir, and Co metal centers. This chapter was previously published as Dalton Trans. 2020, 49, 7747-7757. Chapters 3 reports the potential energy surface and structures for benzene reductive elimination for dimethyl silyl-bridged W and Mo metallocene complexes. Chapter 4 reports gas-phase and explicit solvent dynamics trajectories for this benzene reductive elimination reaction.