Investigating the structures of membrane proteins and their interactions with lipids remains challenging for well-established biophysical techniques. In this thesis the use of mass spectrometry (MS) and ion mobility (IM) spectrometry were explored for the interrogation of membrane proteins, their stoichiometry, stability and interactions with lipids. The techniques used were also applied to the identification of drug metabolites. In the first two chapters reviews of both mass spectrometry methods, and membrane protein biogenesis and membrane protein-lipid interactions are presented. The first challenge for studying membrane proteins by MS was to optimise solution conditions. A detergent screening strategy was developed for this purpose (Chapter 3). The various detergent environments studied revealed dramatic differences in mass spectral quality permitting investigation of membrane protein-lipid interactions. Changes were observed in the electrospray charging of membrane proteins and trends were established from an extensive collection of membrane proteins ejected from a wide variety of detergent environments. The physicochemical principles behind the MS of membrane proteins were deduced and are presented (Chapter 4). The results of these experiments led to a deeper understanding of the ionisation processes and the influence of detergent micelles on both charge state and release mechanisms. Experiments from a range of different micelles also allowed the influence of charge and its effects on the preservation of native-like membrane protein conformations to be monitored by IM-MS. By resolving lipid-protein interactions, and by monitoring the effects of lipid binding on the unfolding of three diverse membrane protein complexes, substantial differences in the selectivity of membrane proteins for different lipids were revealed (Chapter 5). Interestingly lipids that stabilised membrane proteins in the gas-phase were found to induce modifications in structure or function thus providing an approach to assess direct lipid contributions, and to rank order lipids based on their ability to modulate membrane proteins. Using the MS approaches developed here also enabled study of the diversity of oligomeric states of the mechanosensitive channel of large conductance (MscL) (Chapter 6). Results revealed that the oligomeric state of MscL is sensitive to deletions in its C-terminal domain and to its detergent-lipid environment. Additionally, a case study with GlakoSmithKline (GSK) was undertaken using IM-MS technology but in this case applied to the identification of drug metabolites (Chapter 7). The results showed that IM-MS and molecular modelling could inform on the identity of different drug metabolites and highlights the potential of this approach in understanding the structure of various drug metabolites.

This thesis firstly describes a synthesis of the natural product taurospongin A, a potent DNA polymerase beta inhibitor. Sharpless asymmetric dihydroxylation on olefin <b>E-1.60</b> followed by selective deoxygenation at C(2) via Barton‒McCombie reaction delivers the desired C(1)–C(10) carboxylic acid core. Subsequent esterification of the C(1)–C(10) fragment with C(1′)–C(25′) fatty acid furnishes the natural product in 13.5% yield. The structure of an unnamed natural product <b>2.14</b> isolated in 1974 is proven to be misassigned by previous studies within the Robertson group. As described in this thesis, two proposed structures A and B are obtained via total synthesis in order to reveal the identity of the natural product. A synthesis of key intermediate spirocycles <b>2.148</b> and <b>2.158</b> with desired trans- diol moiety is described by a dihydroxylation reaction on an electron deficient gamma-keto unsaturated acid with subsequent spirocyclisation reaction. Finally, methodology for generating high-value synthetic intermediates by an asymmetric, one-pot enzymatic di/polycarbonyl reduction is described. The concept of such methodology is first proven by the synthesis of (3R)-hydroxybutyl (3R)-hydroxybutanoate <b>3.20</b>. This thesis then describes substrate scope studies and corresponding stereochemical proof to provide more information about this methodology.

Kočová, K.: Natural compounds and their β-secretase inhibitory activity, Diploma thesis, Charles University in Prague, Faculty of Pharmacy in Hradci Králové, Department of Pharmaceutical Botany and Ecology, Hradec Králové 2015, 83 p. Data used in this Diploma Thesis have been taken from foreigner scientific literature. It provides the whole summary of natural compounds with β-secretase inhibitory activity. First chapter Alzheimer's disease briefly describes a definition, risk factors and symptomatology of the disease. Most of this chapter characterizes an etiopathology of the disease focused on amyloid theory. Finally there are also mentioned current approaches to therapy of Alzheimer's disease and those, which stay in clinical trials. Next part is dedicated to physiological functions and substrates of the β-secretase. This diploma thesis evaluates the current state of scientific research of synthetic inhibitors of β-secretase, describes the most important methods of testing inhibition and activity of β-secretase in vitro and outlines the issue of development of new inhibitors using in silico method. Main part of this work consists of the list of natural compounds with β- secretase inhibitory activity. This part is structured according to particular types of primary and secondary metabolites....

In 2006, the secondary metabolite leiodolide A was isolated from a newly discovered deep-sea sponge of the genus Leiodermatium. The 19-membered macrolide represented a new class of mixed polyketide, nonribosomal, peptide synthetase natural products. A total synthesis of leiodolide A is yet to be achieved and is of specific interest, not only for its complex structure and undefined stereochemistry, but also the potent cytotoxic properties it possesses, particularly towards leukaemia, non-small cell lung and ovarian cancers. A synthetic strategy for leiodolide A must be flexible to overcome the currently unresolved stereochemistry and a convergent route towards the synthesis of the molecule required three subunits. Following the earlier synthesis of the C21-C25 vinyl stannane fragment, this work describes the synthesis of the C1-C10 subunit in the both possible diastereomeric forms. The synthesis of the two required C13 epimers of the C11-C20 subunit is also detailed accompanied by an investigation into potential fragment coupling, in preparation for total synthesis.

The solvent-free (Cp*IrCl2)2 catalyzed N-alkylation of urea with benzyl alcohol has been studied. A variety of reaction conditions were studied and optimized to produce a high yield (82%) of N,N-dibenzylurea. A series of substituted benzyl alcohols were examined at the optimal reaction conditions. However, the preparation of substituted benzyl urea derivatives using conditions optimized for benzyl alcohol gave poor yields or intractable mixtures.

Orientador: Ian Castro-Gamboa / Coorientador: Rafael Teixeira Freire / Banca: Alberto José Cavalheiro / Banca: Letícia Veras Costa Lotufo / Banca: Alberto Colnago / Banca: Cristiano Soleo de Funari / Resumo: A descoberta de novos alvos farmacêuticos derivados de fontes naturais vem diminuindo ao longo das últimas décadas, impulsionando o uso de matrizes naturais novas e incomuns para a busca de metabólitos secundários bioativos. Dentre essas, a microbiota de plantas e os microrganismos marinhos demonstraram um grande potencial para fornecer novos metabólitos biologicamente ativos, produzindo uma alta diversidade de estruturas químicas encontradas em populações microbianas extensas e pouco exploradas. Convencionalmente, a triagem de metabólitos microbianos é realizada em monoculturas, na ausência de interações bióticas e abióticas. No entanto, a falta dessas interações encontradas na natureza limita a diversidade química que pode ser obtida por uma única cepa, permanecendo silenciadas diversos outros genes que codificam novos metabólitos secundários. Durante a última década, vários métodos têm sido desenvolvidos para compreender as condições sob as quais os genes cripticos são ativados. Dentre elas, as estratégias pós-genômicas revelaram um amplo potencial para o aumento da diversidade química, modificando diferentes níveis da maquinaria celular para uma regulação holística do metaboloma microbiano. Estas estratégias incluem co-cultivo, alimentação por metabólitos e One Strain Many Compounds (OSMAC) e têm sido utilizados com sucesso como uma alternativa rápida e barata para a indução de novos metabólitos. Apesar da eficácia das estratégias pós-genômicas na expansão do metaboloma m... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The discovery of new drug leads from natural sources has decreased over the last decades, emerging the use of new and uncommon matrices for the screening of bioactive secondary metabolites. Among those, the plant microbiota and marine microorganisms have demonstrated a great potential to provide new pharmaceutical leads, producing a high diversity of chemical structures encountered in little-explored and extensive microbial population. Conventionally, microbial metabolite screening is performed in monocultures, in the absence of biotic and abiotic interactions. However, the lack of these communications commonly found in nature seriously limit the chemical diversity that can be obtained by one single strain, remaining silenced many other genes encoded for new secondary metabolites. Over the last decade, several methods have been developed aiming to understand the conditions under which biosynthetic cryptic genes are activated. Among those, the post genomic strategies have revealed widespread potential for the enhancement of chemical diversity, modifying different levels of the cellular machinery for a comprehensive regulation of the microbial metabolome. These strategies include co-cultivation, biotransformation and One Strain Many Compounds (OSMAC) and have been successfully used as a fast and inexpensive alternative for the induction of new bioactive compounds. Notwithstanding these strategies for the expansion of the microbial metabolome, in the screening of these complex m... (Complete abstract click electronic access below) / Doutor

Thesis advisor: Marc L. Snapper / Intramolecular cycloadditions of cyclobutadiene provide rapid access to rigid polycyclic systems with high strain energies and unique molecular geometries. Further functionalization of these systems followed by strain-release fragmentation provides great opportunities to construct fused-medium-ring architecture, which are very common in natural products but challenging to achieve efficiently. An intermolecular cyclopropanation/acid-mediated rearrangement strategy has been previously developed to access the 5-7 bicyclic ring systems in a highly stereospecific manner. The application of this strategy is being studied for the synthesis of a biologically interesting guaiane natural product: torilin. In a complementary fashion, an intramolecular cyclopropanation/thermal rearrangement sequence is developed to access two different molecular frameworks of 5-7-5 tricyclic ring systems. A library of functionalized 5-7-5 tricyclic ring systems can be systematically built up from the same starting material for potential future use in high-throughput screening. / Thesis (PhD) — Boston College, 2012. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

The projects described in this dissertation are focused on compounds derived from the marine environment. Chapter 1 gives an introduction to the study of marine natural products to treat human ailments and a thorough review on compounds from lithistid sponges that have been isolated or synthesized since 2000. Chapter 2 describes the isolation and structure elucidation of two sesquiterpene substituted benzoquinone derivatives, petrosiquinones A and B, from a deep-water marine sponge from the Family Petrosiidae. Although initially purified following activity in a (Sb(B-catenin/Tcf4 assay they were later followed using tumor cell line cytotoxicity assays. Petrosiquinone A was the more active of the two compounds with moderate cytotoxicity in the DLD-1, PANC-1, and AsPC-1 cell lines. In Chapter 3, the isolation and structure elucidation of two new marine-derived macrolides, madeirolide A and B, isolated from a deep-water lithistid sponge of the genus Leiodermatium is described. / They were isolated using numerous chromatographic techniques and the structures were elucidated on the basis of 1D and 2D NMR spectra coupled with high resolution-mass spectrometry (HR-MS) data. Madeirolide A and B inhibited the growth of the fungal pathogen Candida albicans with minimum inhibitory concentrations (MIC) of 12.5 and 25 (So(Bg/mL, respectively, but were not cytotoxic in tumor cell assays under the conditions tested. Chapter 4 describes work performed to determine the molecular target of lasonolide A using affinity chromatography. The target of lasonolide A is of interest since lasonolide A is known to kill cancer cells in vitro through a unique mechanism. / This chapter highlights the research performed to create an affinity matrix with immobilized lasonolide. A target has not been confirmed but there are a number of interesting hits that are being pursued. In Chapter 5, a liquid chromatography-mass spectrometry (LC-MS) screening method was established in order to rapidly identify the metabolites from numerous collections of Lyngbya spp. obtained from Broward and Lee County, Florida sites that may help marine ecologists assess the effects of Lyngbya spp. blooms on the environment. A link between the metabolites produced and nutrients from both the algal tissue and water column was also explored. / by Priscilla L. Winder. / Thesis (Ph.D.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

Les alcaloïdes sont, en général, une famille de composés hétérocycliquesd'origine végétale qui intègrent des atomes d'azote dans leur structure complexe.Leur intérêt réside dans les activités biologiques intéressantes qu'ils présentent, etpour cette raison, ils sont largement utilisés par l'industrie pharmaceutique. Enparticulier, les anneaux de pyrrolidine et de pipéridine se trouvent comme basefondamentale dans un grand nombre de telles structures. D'autre part, l'un desprincipaux objectifs de la chimie synthétique est d'obtenir ces types de moléculesd'une manière asymétrique. Entre autres, la catalyse métallique s'est révélée trèsutile à cet effet. Par exemple, les ligands de type ferrocényle précédemmentdéveloppés dans notre groupe donnent naissance à des pyrrolidines énantiopuréesdensément substituées. De même, ces types de ligands ont été évalués dansdifférents types de réactions de cycloaddition, ce qui a entraîné la formationasymétrique de structures tricycliques d'intérêt biologique. D'autre part, une synthèseracémique collective de composés tétracycliques de la famille Securinega a étédéveloppée. Cette synthèse permet d'accéder à différents produits naturels de lafamille en raison de la polyvalence de l'intermédiaire lactone bicyclique. / Alkaloids are, in general, a family of heterocyclic compounds of vegetal originthat incorporate nitrogen atoms in their complex structure. Their interest lies in theoutstanding biological activities they present, and for this reason they are widely usedby the pharmaceutical industry. In particular, pyrrolidine and piperidine rings arefundamental scaffolds found in a large number of such structures. On the other hand,one of the main objectives of synthetic chemistry is to obtain these types ofmolecules in an asymmetric way. Among others, metal catalysis has proven to bevery useful for this purpose. For example, ferrocenyl proline ligands previouslydeveloped in our group gave rise to densely substituted enantiopure pyrrolidines.Likewise, these types of ligands have been evaluated in different types ofcycloaddition reactions, which have resulted in the asymmetric formation of tricyclicstructures of biological interest. In contrast, a collective racemic synthesis oftetracyclic compounds of the Securinega family has been developed. This synthesisprovides an access to different natural products of the family due to the divergencegiven by a bicyclic lactone intermediate.

Com o surgimento da era computacional com especial aplicação em química, as substâncias de origem naturais puderam ter suas informações armazenadas em bancos de dados. Desta forma, surge a oportunidade de se empregar bancos de dados de produtos naturais e de algumas ferramentas de quimioinformática como os estudos de Quantitative Structure-Retention Relationship (QSRR) para acelerar a identificação de substâncias em estudos metabolômicos. Este trabalho propôs o desenvolvimento de três estudos de QSRR, bem como a construção de um banco de dados (AsterDB) com estruturas químicas da família Asteraceae e informações a elas associadas (ex.: ocorrências botânicas e taxonômicas, atividade biológica, informações analíticas etc.) para auxiliar a desreplicação de substâncias em extratos vegetais. O primeiro estudo foi elaborado com 39 lactonas sesquiterpênicas (LST) analisadas em dois diferentes sistemas de solventes (MeOH-H2O 55:45 e MeCN-H2O 35:65), três grupos de descritores estruturais (2D-descr, 3D-1conf e 3D-weigh), dois diferentes conjuntos para treino e teste (26:13 e 29:10), quatro algoritmos para seleção de descritores (best first, linear forward - LFS, greedy stepwise e algoritmo genético - GA), três diferentes tamanhos de modelos (quatro, cinco e seis descritores) e dois métodos de modelagem (mínimos quadrados parciais - PLS e redes neurais artificiais - ANN). O segundo foi desenvolvido com 50 substâncias de diferentes classes químicas com intuito de avaliar as diferenças entre substâncias analisadas individualmente e em mistura em três diferentes equipamentos e dois métodos cromatográficos. O terceiro foi elaborado com 2.635 estruturas químicas com um teste externo comum a todos os modelos (25%, n = 656), três métodos de separação para teste e treino (partição baseada na resposta e baseada nos preditores 2D e 3D), três diferentes tamanhos de modelos selecionados por GA e dois métodos de modelagem (MLR e redes neurais feed-forward com regularização bayesiana - BRNN). O banco de dados AsterDB foi desenvolvido para ser preenchido de forma gradual e atualmente possui cerca de 2.000 estruturas químicas. O primeiro estudo de QSRR gerou bons modelos capazes de estimar o logaritmo do fator de retenção (logk) das LST com P2>0,81 para o sistema MeCN-H2O. O segundo estudo mostrou que não houve diferença estatística entre as substâncias analisadas individualmente e em mistura (p-valor>0,95) e que a correlação entre os dois métodos cromatográficos e equipamentos utilizados foi reprodutível (R>0,95). Estas análises mostraram que foi possível desenvolver modelos de QSRR para um método cromatográfico e equipamento e transpô-los para outro equipamento seguindo o uso de substâncias em comum. O terceiro estudo produziu modelos com boa capacidade de predição (P2>0,81) utilizando alta amplitude de espaço químico e rigor estatístico. Conclui-se que, estas informações podem ser utilizadas como uma plataforma piloto para análises de dados com objetivo de auxiliar na desreplicação de extratos de plantas em estudos metabolômicos / After the emergence of the computing era with special application in chemistry, all substances from natural sources might have their information stored in databases. Therefore, the opportunity arises to employ natural product databases and some chemoinformatic tools such as QSRR studies to speed up the identification of substances from metabolomic studies. This paper proposes the development of three QSRR studies as well as the building of a database (AsterDB) with chemical structures from the Asteraceae family and related information (i.e.: botanical and taxonomic occurrences, biological activity, analytical information, etc.) aiming to assist the dereplication of substances in plant extracts. The first study was carried out with 39 sesquiterpene lactones (STLs) analysed using two different solvent systems (MeOH-H2O 55:45 and MeCN-H2O 35:65), three groups of structural descriptors (2D-descr, 3D-1conf, and 3D-weigh), two different sets for training and testing (26:13 and 29:10), four algorithms for selection of descriptors (best first, LFS, greedy stepwise, and GA), three different model sizes (four, five, and six descriptors) and two modelling methods (PLS and ANN). The second study was developed with 50 compounds of different chemical classification in order to assess the differences between individual and mixed compounds analysed in three different equipments and two chromatographic methods. The third was elaborated with 2,635 chemical structures with a common external test to all models (25%, n = 656), three separation methods for testing- and training-set (based on response and on 2D and 3D predictors partitions), three different sizes of models selected by GA and two modelling methods (MLR and BrNN). The AsterDB database was developed to be populated gradually and currently, it has about 2,000 chemical structures. The first QSRR study generated good models, able to estimate the logarithm of the retention factor (logk) of STLs with P2>0.81 for the MeCN-H2O system. The second study showed that there was no statistical difference between the substances analysed individually and mixed (p-value>0.95) and the correlation between the two chromatographic methods and equipments used was reproducible (R>0.95). These analyses showed that it was possible to develop QSRR models for a chromatographic method and equipment and translate them into other equipment following the use of substances in common. The third study produced models with good predictive capacity (P2>0.81) using a high range of chemical space and statistical accuracy. In conclusion, this information can be used as a pilot platform for data analysis in order to assist in plant dereplication in metabolomics studies