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Galectin-1: A Synthetic and Biological Study of a Tumor Target / Galectin-1: Eine Synthetische und Biologische Studie eines TumortargetsBertleff-Zieschang, Nadja Luisa January 2014 (has links) (PDF)
Galectin-1 (hGal-1) is overexpressed by numerous cancer types and previously conducted studies confirmed that the β-galactoside-binding protein mediates various molecular interactions associated with tumor growth, spread and survival. Upon interaction with carbohydrate-based binding epitopes of glycan structures on human cell surfaces galectin-1 induces proliferative, angiogenetic and migratory signals and modulates negative T cell regulation which essentially helps the tumor to evade the immune response. These findings attributed galectin-1 a pivotal role in tumor physiology and strongly suggest the protein as target for diagnostic and therapeutic applications.
Within the scope of this work a strategy was elaborated for designing tailor-made galectin-1 ligands by functionalizing selected hydroxyl groups of the natural binding partner N-acetyllactosamine (LacNAc) that are not involved in the sophisticated interplay between the disaccharide and the protein. Synthetic modifications intended to introduce chemical groups i) to address a potential binding site adjacent to the carbohydrate recognition domain (CRD) with extended hGal-1-ligand interactions, ii) to implement a tracer isotope for diagnostic detection and iii) to install a linker unit for immobilization on microarrays.
Resulting structures were investigated regarding their targeting ability towards galectin-1 by cocrystallization experiments, SPR and ITC studies. Potent binders were further probed for their diagnostic potential to trace elevated galectin-1 levels in microarray experiments and for an application in positron emission tomography (PET). / Galectin-1 (hGal-1) wird von zahlreichen Tumoren überexprimiert und frühere Studien bestätigten, dass das β-Galactosid-bindende Protein verschiedene molekulare Wechselwirkungen vermittelt, welche in direktem Zusammenhang mit Tumorwachstum, -ausbreitung und -überleben stehen. Durch die Wechselwirkung mit Kohlenhydrat-basierten Bindungsepitopen von Glykanstrukturen auf Zelloberflächen induziert Galectin-1 proliferative, angiogenetische und migratorische Signale und moduliert die negative Regulierung von T-Zellen, entscheidend für den Tumor, um der Immunantwort zu entkommen. Diese Beobachtungen schreiben Galectin-1 eine zentrale Rolle in der Tumorphysiologie zu, was dieses Protein zu einem attraktiven Target für diagnostische und therapeutische Anwendungen macht.
Im Rahmen dieser Arbeit wurde eine Synthesestrategie für das Design maßgeschneiderter Galectin-1 Liganden entwickelt, wobei ausgewählte Hydroxylgruppen des natürlichen Bindungspartners N-Acetyllactosamin (LacNAc), welche nicht an dem hochkomplexen Zusammenspiel zwischen Protein und Disaccharid beteiligt sind, funktionalisiert wurden. Synthetische Modifikationen wurden mit der Absicht eingeführt i) eine potentielle Bindungstasche in Nachbarschaft der Kohlenhydraterkennungsdomäne (CRD) zu adressieren, ii) einen Isotopenmarker für die diagnostische Detektion zu implementieren und iii) eine Brückeneinheit zu integrieren, welche einer späteren Immobilisierung auf Mikroarrays dient.
Resultierende Strukturen wurden mittels Kokristallisationsexperimenten, SPR- und ITC-Studien auf ihre Fähigkeit untersucht, Galectin-1 zu adressieren. Erfolgreich entwickelte Liganden wurden zudem auf ihr diagnostisches Potential getestet, erhöhte Galectin-1-Spiegel in Microarray-Experimenten zu detektieren und könnten zukünftig Einsatz in der Positronen-Emissions-Tomographie (PET) finden.
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Biologische Referenzstrukturen und Protokolloptimierung in der hochauflösenden Fluoreszenzmikroskopie mit dSTORM / Biological model structures and optimization of protocols in super-resolution fluorescence microscopy with dSTORMLöschberger, Anna January 2014 (has links) (PDF)
Die Lokalisationsmikroskopie ist eine neue, vielversprechende Methode der hochauflösenden Fluoreszenzmikroskopie. Sie ermöglicht detaillierte Einblicke in die Organisation und den strukturellen Aufbau von Zellen. Da die Vorbereitung der Proben und das Aufnehmen der Bilder im Vergleich zu herkömmlichen Methoden höhere Anforderungen stellt, mussten ihr Potential und ihre Zuverlässigkeit erst noch überzeugend gezeigt werden. Bis vor kurzem wurde das Auflösungsvermögen vor allem an Mikrotubuli gezeigt, deren filamentöse Struktur allerdings schon in konfokalen Bildern zu erkennen ist. Deswegen wurde in dieser Dissertation der Kernporenkomplex (NPC), dessen Struktur in der konventionellen Fluoreszenzmikroskopie nicht auflösbar ist, als Modellstruktur für die hochauflösende Fluoreszenzmikroskopie eingeführt.
Dazu wurden Kernporenkomplexe aus Kernhüllen von Xenopus laevis Oocyten mit dSTORM (direct stochastic optical reconstruction microscopy), einer Methode der Lokalisationsmikroskopie, hochaufgelöst. Damit konnte nun erstmals die Achtfachsymmetrie dieses Proteinkomplexes lichtmikroskopisch dargestellt werden. Desweiteren konnte der Zentralkanal mit einem Durchmesser von ca. 40 nm aufgelöst werden. Die Daten eigneten sich außerdem für eine automatisierte Bildanalyse nach dem sogenannten "particle averaging" - einer aus der Elektronenmikroskopie bekannten Methode, um eine Durchschnittsstruktur zu ermitteln.
Darüber hinaus wurden Zweifach-Färbungen von NPCs benutzt, um verschiedene Ansätze für Zweifarben-Aufnahmen mit dSTORM zu testen. Neben dem mittlerweile standardmäßig benutzten, sequentiellen Ansatz mit zwei spektral getrennten Farbstoffen, wurde auch ein simultaner Ansatz mit zwei spektral überlappenden Farbstoffen erfolgreich angewandt. Auch für 3D-Messungen mit den Ansätzen Biplane und Astigmatismus eignete sich die Markierung der Kernhülle. Hier wurden jedoch A6-Zellen benutzt und die Krümmung des Zellkerns über die gefärbten Kernporen dargestellt.
dSTORM-Messungen können nicht nur an fixierten, sondern auch in lebenden Zellen durchgeführt werden. Hierzu eignen sich vor allem sehr immobile Proteine, wie H2B oder Lamin C. Anhand von SNAP-Tag- und Halo-Tag-Konstrukten konnte gezeigt werden, dass sich kommerziell erhältliche, organische Farbstoffe auch in endogener zellulärer Umgebung schalten lassen, wodurch Lebendzell-Aufnahmen mit dSTORM möglich sind.
Ein weiterer Teil dieser Arbeit befasst sich mit korrelativen Aufnahmen aus dSTORM und Rasterelektronenmikroskopie (SEM). Hierzu wurden Xenopus laevis Kernhüllen zuerst mit dSTORM hochaufgelöst und danach für die EM präpariert. Anschließend wurden zugehörige Bereiche am Rasterelektronenmikroskop aufgenommen. Mit den erhaltenen korrelativen Bildern konnte gezeigt werden, dass sich dSTORM und SEM bei geeigneten Proben durchaus kombinieren lassen. Proteine können somit spezifisch markiert und im Rahmen ihrer strukturellen Umgebung mit nahezu molekularer Auflösung dargestellt werden.
Da hochwertige Aufnahmen eine ausgereifte Probenpräparation voraussetzen, darf deren Etablierung nicht zu kurz kommen. Unter dieser Prämisse wurde ein optimiertes Markierungsprotokoll mit dem Namen ClickOx entwickelt. Mit ClickOx bleibt bei der kupferkatalysierten Azid-Alkin-Cycloaddition die Feinstruktur von Aktinfilamenten, sowie die Fluoreszenz fluoreszierender Proteine, deutlich sichtbar erhalten. Während bei den klassischen Click-Protokollen auf Grund der Entstehung von reaktiven Sauerstoff-Spezies (ROS) feine zelluläre Strukturen, wie Aktinfilamente, angegriffen oder zerstört werden, schützt das neue Protokoll mit enzymatischem Sauerstoffentzug Proteine und somit Strukturen vor Reaktionen mit ROS. Das unterstreicht, wie wichtig es ist auch sogenannte "etablierte" Protokolle weiterzuentwickeln, denn bestimmte Nebeneffekte in Präparationen werden unter Umständen erstmals in der Hochauflösung sichtbar.
Ein weiterer Aspekt war die Untersuchung des Einflusses von D1 auf die Chromatinorganisation. Mit verschiedenen mikroskopischen Methoden konnten Hinweise auf eine mögliche DNA-Cross-Linking-Fähigkeit dieses Proteins gesammelt werden. Hier wurde die Einzelmolekülinformation der dSTORM-Filme genutzt, um unterschiedliche Grade von DNA- bzw. Chromatin-Akkumulation zu vergleichen. Die Ergebnisse deuten darauf hin, dass wildtypisches D1 DNA vernetzen kann. Dies erfolgt über die sogenannten AT-Haken-Motive. Sobald diese alle durch Mutation funktionsunfähig gemacht werden - wie bei der verwendeten R10xG-Mutante - lässt sich keine Akkumulation der DNA mehr beobachten. Neben der Chromatinaggregation durch D1-Expression konnte in FRAP-Experimenten gezeigt werden, dass nur die "echten" AT-Haken eine hohe Affinität zum Chromatin aufweisen, die sogenannten "potentiellen" hingegen nicht. / Localization microscopy is a new and promising imaging technique, which provides detailed insights into cellular organization and structural composition of cells with high spatial resolution. Due to the challenging preparation of samples and demanding imaging procedure, its potential and reliability had to be proven. Until recently the resolution has been shown mainly on microtubules, whose structure is already visible in confocal images. This thesis introduced the nuclear pore complex (NPC) as a more demanding model structure for super-resolution fluorescence microscopy as the structure of NPCs can not be resolved with conventional fluorescence microscopy.
For this purpose nuclear envelopes of Xenopus laevis oocytes were highly resolved with dSTORM (direct stochastic optical reconstruction microscopy). With this localization microscopy method it was further possible to resolve the eightfold symmetry of nuclear pore complexes with light microscopy for the first time. In addition the central channel could be resolved with a diameter of about 40 nm. Furthermore, the localizations were used for single particle averaging, a well known image analysis method from electron microscopy, to calculate an average structure.
Double staining of NPCs was used to check the potential of two-color imaging with dSTORM.
Beside the common way of sequential imaging with two clearly spectrally separated dyes, a spectral demixing approach with spectrally overlapping dyes was applied.
Labeling the nuclear envelope was also suitable for 3D measurements using two different approaches, i.e. biplane and astigmatism. In this case, labeled NPCs of Xenopus laevis A6-cells were used to illustrate the bending of the nucleus.
dSTORM can be applied not only in fixed but also in living cells. Immobile proteins such as H2B or lamin C are especially suitable for this approach. Using fusion proteins with SNAP-Tag or Halo-Tag, it was shown that photoswitching of commercially available organic dyes is possible in an endogenous cellular environment and thus enabeling dSTORM in living cells.
Another aspect of this work covers correlative microscopy using dSTORM and scanning electron microscopy (SEM). Therefor nuclear envelopes of Xenopus laevis were first imaged with dSTORM and then prepared for SEM. After that, corresponding areas were imaged with SEM. The resulting correlative images showed clearly that - assuming one has appropriate samples - dSTORM and SEM can be fairly combined. This way specifically labeled proteins can be imaged with nearly molecular resolution in the context of their structural environment.
Since the quality of localization microscopy strongly depends on sample preparation, ongoing developments of labeling protocols are required. On this premise an optimized labeling protocol called ClickOx was developed. ClickOx clearly preserves the fine structure of actin filaments and the fluorescence of fluorescent proteins when using copper-catalyzed azide-alkine-cycloaddition. Whereas fine cellular structures such as actin filaments are affected by reactive oxygen species (ROS) under standard clicking procedures, the new protocol, which contains an enzymatic oxygen scavenger, protects proteins and thus cellular structures from reactions with ROS. This demonstrates the importance of further developing even so called "well established" protocols, because some side effects may appear only in super-resolution.
Another aspect adressed the influence of D1 on chromatin organization. Hints for a possible DNA cross-linking ability of D1 were collected using different microscopic approaches. The single-molecule information of dSTORM measurements was used to analyse chromatin aggregation induced by D1 expression. The results indicate that wildtype D1 can cross-link DNA with its AT-hooks. Consequently the loss-of-function mutant R10xG is unable to aggregate chromatin. Furthermore FRAP experiments were performed to demonstrate that only "true" AT-hooks in D1 have a strong affinity to chromatin, but not the so called "potential" AT-hooks.
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Sulfur-functional polymers for biomedical applications / Schwefel-funktionale Polymere für biomedizinische AnwendungenKuhlmann, Matthias January 2015 (has links) (PDF)
Aim of this thesis was to combine the versatility of sulfur-chemistry, regarding redox-sensitivity as well as chemo- and site-specific conjugation, with multifunctionality of poly(glycidol)s as an alternative to poly(ethylene glycol).
First the homo- and copolymerizations of EEGE and AGE were performed with respect to molar-mass distribution and reaction kinetics. A detailed study was given, varying the polymerization parameters such as DP, counter ion, solvent and monomer influence. It can be concluded that in general the rates for all polymerizations are higher using K+, in contrast to Cs+, as counter ion for the active alkoxide species. Unfortunately, K+ as counter ion commonly leads to a reduced control over polymer dispersity. In this thesis it was shown that the broad molar-mass distributions might be reduced by adding the monomer in a step-wise manner. In experiments with a syringe pump, for continuously adding the monomer, a significant reduction of the dispersities could be found using K+ as counter ion.
In analogy to the oxyanionic polymerization of epoxides, the polymerization of episulfides via a thioanionic mechanism with various DPs was successful with thiols/DBU as initiator. In most experiments bimodality could be observed due to the dimerization, caused by oxidation processes by introduced oxygen during synthesis. Reducing this was successful by modifying the degassing procedure, e.g. repeated degassing cycles after each step, i.e. initiation, monomer addition and quenching. Unfortunately, it was not always possible to completely avoid the dimerization due to oxidation. Thiophenol, butanethiol, mercaptoethanol and dithiothreitol were used as thiol initiators, all being capable to initiate the polymerization. With the prediction and the narrow molar-mass distributions, the living character of the polymerization is therefore indicated.
Homo- and copolymers of poly(glycidol) were used to functionalize these polymers with side-chains bearing amines, thiols, carboxylic acids and cysteines. The cysteine side-chains were obtained using a newly synthesized thiol-functional thiazolidine. For this, cysteine was protected using a condensation reaction with acetone yielding a dimethyl-substituted thiazolidine. Protection of the ring-amine was obtained via a mixed-anhydride route using formic acid and acetic anhydride. The carboxylic acid of 2,2-dimethylthiazolidine-4-carboxylic acid was activated with CDI and cysteamine attached. The obtained crystalline mercaptothiazolidine was subjected to thiol-ene click chemistry with allyl-functional poly(glycidol). A systematic comparison of thermal- versus photo-initiation showed a much higher yield and reaction rate for the UV-light mediated thiol-ene synthesis with DMPA as photo-initiator. Hydrolysis of the protected thiazolidine-functionalities was obtained upon heating the samples for 5 d at 70 °C in 0.1 M HCl. Dialysis against acetic acid lead to cysteine-functional poly(glycidol)s, storable as the acetate salt even under non-inert atmosphere. An oxidative TNBSA assay was developed to quantify the cysteine-content without the influence of the thiol-functionality. A cooperation partner coupled C-terminal thioester peptides with the cysteine-functional poly(glycidol)s and showed the good accessibility and reactivity of the cysteines along the backbone. SDS-PAGE, HPLC and MALDI-ToF measurements confirmed the successful coupling. / Ziel der Arbeit war es die Vielseitigkeit der Schwefelchemie, hinsichtlich der Redoxsensitivität und chemo- und seitenspezifischer Konjugation, mit der Funktionalisierbarkeit von Poly(glycidol)en, als multifunktionale PEG-Alternative zu kombinieren.
Zunächst wurden die Homo- und Copolymerisationen von EEGE und AGE hinsichtlich der Molmassenverteilung und der Reaktionskinetik untersucht. Durch die Variation der Polymerisationsparameter, wie angestrebter Polymerisationsgrad, Gegenion, Lösungsmittel und Monomer, wurde der Einfluss dieser untersucht. Allgemein konnte gezeigt werden, dass die Polymerisationen schneller ablaufen, wenn K+, im Gegensatz zu Cs+, als Gegenion zum aktiven Alkoxidkettenende verwendet wird. Nachteilig bei der Verwendung von K+ als Gegenion ist der Kontrollverlust der Polymerisation, welcher mit einer Erhöhung der Dispersität einhergeht. Es konnte gezeigt werden, dass die Breite der Molmassenverteilung durch die Geschwindigkeit der Monomerzugabe kontrolliert werden kann. Tatsächlich konnte die Dispersität durch die Verwendung einer Spritzenpumpe verbessert werden, da das Monomer mit einer konstanten angepassten Flussrate hinzugefügt wurde.
Analog zur oxyanionischen Polymerisation von Epoxiden, war die Polymerisation von Episulfiden mittels thioanionischer Polymerisation ebenfalls möglich. Hierzu wurden verschiedene Polymerisationsgrade von EETGE und ATGE angestrebt und mittels Thiol/DBU als Initiator auch erreicht. In den meisten Fällen war jedoch eine Dimerisierung der Polymere zu beobachten, welche durch die Oxidation der aktiven Thiolatspezies verursacht wurde. Eine Möglichkeit zur Dimerisierungsunterdrückung war die wiederholte Durchführung von Entgasungszyklen nach jedem Arbeitsschritt, z.B. nach Zugabe des Initiators, des Monomers oder nach dem Quenchen. Trotz dieses experimentellen Aufwandes konnte nicht immer ein vollständiger Ausschluss der Dimerisierung erreicht werden. Thiophenol, Butanthiol, Mercaptoethanol und Dithiothreitol wurden als Thiolinitiatoren (in Kombination mit DBU) verwendet und waren alle in der Lage die Polymerisation zu starten. Die Kontrolle des Polymerisationsgrades und die enge Molmassenverteilung der Polymere verdeutlichen, dass die thioanionische Polymerisation ebenfalls lebend verläuft.
Glycidol Homo- und Copolymere wurden verwendet und die Seitenketten mit Amin-, Thiol-, Carbonsäure- und Cysteingruppen funktionalisiert. Die Cysteinseitenketten wurden durch ein neues thiolfunktionales Thiazolidin erhalten. Ausgehend von Cystein und Aceton wurde zunächst das Dimethyl-substituierte Thiazolidin erhalten, welches daraufhin am Ring-Amin mit Essigsäureanhydrid und Ameisensäure formyliert wurde. Die Carbonsäurefunktion des Thiazolidins wurde mittels CDI aktiviert und anschließend mit Cysteamin umgesetzt. Hierbei bildete sich das niedermolekulare kristalline thiolfunktionale Thiazolidin, welches mittels Thiol-En-Click Chemie an allyl-funktionales Poly(glycidol) geknüpft werden konnte. Eine systematische Untersuchungen der thermischen und UV-induzierten Thiol-En-Click Chemie zeigte, dass wesentlich höhere Umsätze und Geschwindigkeiten bei der photoinduzierten Reaktion erhalten werden. Mittels 0.1 M HCl konnte bei 70 °C innerhalb von 5 d die Hydrolyse der Thiazolidine im Anschluss erreicht werden. Nach der anschließenden Dialyse der Polymere gegen 0.1 M Essigsäure wurde erfolgreich das Acetatsalz der cysteine-funktionalen Poly(glycidol)e erhalten. Diese waren hinsichtlich der Thioloxidation unter atmosphärischen Bedingungen stabil. Ein oxidativer TNSBA-Assay wurde entwickelt, um die Menge der Cysteine zu quantifizieren und gleichzeitig den störenden Einfluss der Thiole zu unterbinden. Ein Kooperationspartner setzte die cysteinfunktionalisierten Poly(glycidol)e mit C-terminalen Thioestern um und konnte die gute Zugänglichkeit und Aktivität der Cysteine entlang des Polymerrückgrats nachweisen. SDS-PAGE, HPLC und MALDI-ToF Messungen bestätigten die erfolgreiche Konjugation im Anschluss.
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Enzymatic Mechanisms and Chemical Probes of the Myst Family of Histone AcetyltransferasesYang, Chao 01 August 2013 (has links)
As an important posttranslational modification, protein acetylation plays critical roles in many biological processes such as gene transcription, DNA damage repair, apoptosis and metabolism. The acetylation occurs on the ε-amino group of specific lysine residues, and is catalyzed by histone acetyltransferases (HATs). In cellular contexts, HATs are found to target hundreds and thousands of substrates including histone and nonhistone proteins. Lysine acetylation changes the microenvironment of protein and may potentially alter protein activity and protein-protein interaction. The goal of this dissertation project is to investigate the impact of lysine acetylation on the catalysis of MYST HATs, and to establish the strategy for labeling substrates of the MYST HATs at cellular level. To understand the regulatory mechanism of MYST HATs, a detailed study was carried out to investigate the active site lysine acetylation of two MYST HATs (MOF and Tip60). Autoradiography and immunoblotting data shows that mutation of active site lysine differentially affects the enzyme autoacetylation activity and the cognate substrate acetylation activity. In addition, deacetylated MOF and Tip60 were prepared by using the nonspecific lysine deacetylase Sirt1. Kinetic study demonstrated that the acetylation of the active site lysine on MYST HATs marginally modulates the HAT catalysis. This work provides new insights into the regulatory mechanism of MYST catalysis. In the second part of my work, we designed and synthesized a series of Ac-CoA analogs conjugated with alkynyl or azido functional groups. Meanwhile, the active site of the MOF was engineered to expand the cofactor binding capability. Fluorescence screening was carried out to characterize the enzyme activity to Ac-CoA analogs. MOF-I317A with all analogs and MOF-I317A/H273A–5HYCoA were identified and further applied in the labeling of the cognate histone H4 protein and HAT substrates in 293T cell lysate. Visualizing of the labeled substrate was achieved using the alkynyl or azido-tagged fluorescent reporters through the copper-catalyzed azide−alkyne cycloaddition. As expected, the histone H4 protein was successfully labeled by the active enzyme-cofactor pairs. More intriguingly, multiple protein bands in cell lysate were labeled and observed. This work provides a new versatile strategy in exploring the substrates of MYST HATs at the proteomic level.
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Evaluating the tension with a not-for-profit organization, when developing a business model for the maintenance of a sustainable profitable business ventureMosek, Linda. January 2007 (has links)
Thesis (PhD) -- Swinburne University of Technology, Australian Graduate School of Entrepreneurship, 2007. / [Submitted in partial fulfilment of the requirements of the degree of Doctor of Philosophy, Australian Graduate School of Entrepreneurship, Swinburne University of Technology, 2007]. Typescript. Includes bibliographical references (p. 206-214).
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The Synthesis of Linear and Nonlinear Photosensitive Organometallic Polymers Containing Mo-Mo Bonds: Evaluating the Effectiveness of Click ChemistryBrady, Sarah 03 October 2013 (has links)
This dissertation details the use of click chemistry to prepare linear and nonlinear polymers containing metal-metal bonds. The incorporation of metal-metal bonds into the polymer simplfies the degradation mechanism, allowing fundamental mechanistic studies of polymer degradation. Click chemistry offered a brand new route to explore the preparation of these useful but intricate metal-metal bond-containing polymers.
Chapter I discusses the utility of these types of polymers for mechanistic studies, the preparation of metal dimers with reactive functionalities, and the previous polymerization methods which have been explored. The need for a new polymerization strategy, such as click chemistry, is described. Chapter II explains the preparation of a new metal dimer click synthon, [(η5-C5H4(CH2)3OC(O)(CH2)2C≡CH)Mo(CO)3]2, and the necessary conditions needed to polymerize the synthon using click chemistry. A high molecular weight linear polymer was prepared, suggesting click chemistry is a viable route to nonlinear polymers.
Chapter III presents a second novel metal dimer click synthon, [(η5-C5H4(CH2)3N3Mo(CO)3]2, and attempts to use click chemistry to prepare a star polymer containing metal-metal bonds. A small amount of nonlinear polymer was prepared but several reactivity problems were also discovered and addressed. Due to these problems with click chemistry, Chapter IV details a brand new method for preparing asymmetric metal dimers. CpMo(CO)3-Mo(CO)3Cp(CH2)3CH=CH2 is the first reported example of an asymmetric dimer, and (CH3)3CSi(CH3)2O(CH2)3CpMo(CO)3-Mo(CO)3Cp(CH2)3OC(CH3)2OCH3 is the first example of a bifunctional asymmetric dimer.
Chapter V describes the synthesis of a different type of metal dimer, (CH3)2Si[(C5H5)Mo(CO)3]2, which is polymerized by thermal ring opening polymerization. The dimer did not polymerize as expected and yielded an interesting polymer which has both Mo-Mo single bonds and Mo≡Mo triple bonds. Finally, Chapter VI provides a summary of the work as well as an honest perspective of using click chemistry to prepare metal-metal bond-containing polymers.
This dissertation includes previously published and unpublished co-authored material. / 10000-01-01
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Planejamento, síntese e avaliação in vitro de híbridos 1,2,3-triazol-4-clorometilcumarinas com potencial atividade antioxidanteAlves, Anna Carolina Schneider January 2017 (has links)
Cumarinas são metabólitos secundários de plantas encontrados majoritariamente nas espécies das famílias Asteraceae, Rutaceae e Umbeliferae. Quimicamente, são compostos fenólicos, formados pela fusão de um benzeno e de um anel α-pirona, chamados de benzopironas. Elas apresentam diversas propriedades farmacológicas, associadas com baixa toxicidade. Nosso grupo de pesquisa sintetiza cumarinas pela reação de Pechmann, que ocorre através da condensação de um fenol com um β-cetoéster, na presença de um ácido de Bronsted ou Lewis. Um dos trabalhos mais recentes foi a síntese de 6-metil-4-clorometilcumarinas com um IC50 menor do que 1,6 μM para atividade antitripanocida. Em outro trabalho, um grupo de compostos de híbridos cumarina-triazol foi sintetizado e apresentou potencial atividade como agente antitumoral. Baseado nesses trabalhos, foi planejado a síntese de análogos da 6-metil-4-clorometilcumarina via condensação de Pechmann, com diferentes substituintes na posição 6, obtidos através das reações de click chemistry, no intuito de aumentar a atividade antioxidante desses compostos. Assim, para obter esses compostos, foi realizada uma condensação de Pechmann com hidroquinona e 4-cloroacetoacetato de etila. Após, uma eterificação de Williamson com brometo de propargila foi feita. Finalmente, a reação de click chemistry foi realizada sob irradiação de micro-ondas com diversas azidas previamente sintetizadas no laboratório, conduzindo à obtenção de diversos análogos da 6-metil-clorometilcumarina que foram avaliados quanto a viabilidade celular através ensaio do MTT (brometo de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazólio). Também foi testada a sua capacidade antioxidante pelo método do DCFH-DA (diacetato de 2’,7’ –diidroclorofluoresceína). Dessa maneira, sob as condições reacionais utilizadas neste trabalho, foi possível sintetizar 12 compostos inéditos com rendimentos entre 9 e 61%. Os ensaios biológicos preliminares indicaram que os compostos sintetizados apresentam potencial atividade antioxidante e algumas moléculas tiveram potencialidade como agente citotóxico. / Coumarins are secondary plant metabolites typically found in species of the Asteraceae, Rutaceae and Umbeliferae families that demonstrate diverse pharmacological properties associated with low toxicity to humans. Chemically, they are phenolic compounds characterized by the fusion of benzene with an α-pyrone ring, yielding the benzopyrone nucleus. Our research group usually synthesizes coumarins by the Pechmann reaction, through the condensation of phenols with β-ketoesters catalyzed by Bronsted or Lewis acids. One of the most recent works performed at our laboratory describes the synthesis of 6-methyl-4-chloromethylcoumarins with an IC50 of 1.6 μM concerning the anti-trypanocidal activity. Another work described the syntheses of coumarin-triazole hybrids with potential activity as anticancer agents. Based on the previous works, it was designed the synthesis of 4-chloromethylcoumarins via Pechmann condensation with several substituents at the position 6 of the coumarin ring through click chemistry reactions to improve their antioxidant activities. The synthesis of the coumarins started with Pechmann condensation using hydroquinone and ethyl 4-chloroaceacetate followed by functionalization of the phenolic hydroxyl with propargyl bromide via Williamsom ether synthesis. Subsequently, the click chemistry reactions were performed under microwave irradiation using different organic azides previously synthesized at our laboratory, yielding several 6-substituted-4-chloromethylcoumarin analogues which were evaluated for cell viability through MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Their antioxidant capacity was also tested by the DCFH-DA (2’,7’–diihydrochlorofluorescin diacetate) method. Therefore, under the reaction conditions used in this study, it was possible to synthesize 12 novel compounds with yields between 9 and 61%. Preliminary biological assays indicated that the compounds synthesized have potential antioxidant activity and some molecules had potential as an antitumor agent.
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Planejamento, síntese e avaliação in vitro de híbridos 1,2,3-triazol-4-clorometilcumarinas com potencial atividade antioxidanteAlves, Anna Carolina Schneider January 2017 (has links)
Cumarinas são metabólitos secundários de plantas encontrados majoritariamente nas espécies das famílias Asteraceae, Rutaceae e Umbeliferae. Quimicamente, são compostos fenólicos, formados pela fusão de um benzeno e de um anel α-pirona, chamados de benzopironas. Elas apresentam diversas propriedades farmacológicas, associadas com baixa toxicidade. Nosso grupo de pesquisa sintetiza cumarinas pela reação de Pechmann, que ocorre através da condensação de um fenol com um β-cetoéster, na presença de um ácido de Bronsted ou Lewis. Um dos trabalhos mais recentes foi a síntese de 6-metil-4-clorometilcumarinas com um IC50 menor do que 1,6 μM para atividade antitripanocida. Em outro trabalho, um grupo de compostos de híbridos cumarina-triazol foi sintetizado e apresentou potencial atividade como agente antitumoral. Baseado nesses trabalhos, foi planejado a síntese de análogos da 6-metil-4-clorometilcumarina via condensação de Pechmann, com diferentes substituintes na posição 6, obtidos através das reações de click chemistry, no intuito de aumentar a atividade antioxidante desses compostos. Assim, para obter esses compostos, foi realizada uma condensação de Pechmann com hidroquinona e 4-cloroacetoacetato de etila. Após, uma eterificação de Williamson com brometo de propargila foi feita. Finalmente, a reação de click chemistry foi realizada sob irradiação de micro-ondas com diversas azidas previamente sintetizadas no laboratório, conduzindo à obtenção de diversos análogos da 6-metil-clorometilcumarina que foram avaliados quanto a viabilidade celular através ensaio do MTT (brometo de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazólio). Também foi testada a sua capacidade antioxidante pelo método do DCFH-DA (diacetato de 2’,7’ –diidroclorofluoresceína). Dessa maneira, sob as condições reacionais utilizadas neste trabalho, foi possível sintetizar 12 compostos inéditos com rendimentos entre 9 e 61%. Os ensaios biológicos preliminares indicaram que os compostos sintetizados apresentam potencial atividade antioxidante e algumas moléculas tiveram potencialidade como agente citotóxico. / Coumarins are secondary plant metabolites typically found in species of the Asteraceae, Rutaceae and Umbeliferae families that demonstrate diverse pharmacological properties associated with low toxicity to humans. Chemically, they are phenolic compounds characterized by the fusion of benzene with an α-pyrone ring, yielding the benzopyrone nucleus. Our research group usually synthesizes coumarins by the Pechmann reaction, through the condensation of phenols with β-ketoesters catalyzed by Bronsted or Lewis acids. One of the most recent works performed at our laboratory describes the synthesis of 6-methyl-4-chloromethylcoumarins with an IC50 of 1.6 μM concerning the anti-trypanocidal activity. Another work described the syntheses of coumarin-triazole hybrids with potential activity as anticancer agents. Based on the previous works, it was designed the synthesis of 4-chloromethylcoumarins via Pechmann condensation with several substituents at the position 6 of the coumarin ring through click chemistry reactions to improve their antioxidant activities. The synthesis of the coumarins started with Pechmann condensation using hydroquinone and ethyl 4-chloroaceacetate followed by functionalization of the phenolic hydroxyl with propargyl bromide via Williamsom ether synthesis. Subsequently, the click chemistry reactions were performed under microwave irradiation using different organic azides previously synthesized at our laboratory, yielding several 6-substituted-4-chloromethylcoumarin analogues which were evaluated for cell viability through MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Their antioxidant capacity was also tested by the DCFH-DA (2’,7’–diihydrochlorofluorescin diacetate) method. Therefore, under the reaction conditions used in this study, it was possible to synthesize 12 novel compounds with yields between 9 and 61%. Preliminary biological assays indicated that the compounds synthesized have potential antioxidant activity and some molecules had potential as an antitumor agent.
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Síntise de 1,2,4-oxadiazóis ennantiomericamente enriquecidos e de novos glicoconjugados potencialmente ativosVersiani dos Anjos, Janaina 31 January 2008 (has links)
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Previous issue date: 2008 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Neo-glicoconjugados constituem uma classe de compostos em que os carboidratos estão
conjugados a outras moléculas de interesse biológico. Os 1,2,4-oxadiazóis são heterociclos
bastante conhecidos em química medicinal, apresentando várias atividades farmacológicas e
tendo sido considerados bioisósteros de ésteres e amidas. Este trabalho descreve a síntese de
oxadiazóis enantiomericamente enriquecidos através de biocatálise com Daucus carota, a fim
de preparar compostos glico-heterocíclicos; a síntese de novos neo-glicoconjugados contendo
heterociclos e aminoácidos através de catálise mediada por Pd(0) e click chemistry e a
avaliação da atividade biológica de alguns dos produtos sintetizados. A biorredução de
oxadiazóis-cetona com D. carota forneceu oxadiazóis enriquecidos enantiomericamente com
excessos enantioméricos variando de 52 a 72%. Os álcoois obtidos têm configuração absoluta
S, o que está de acordo com a regra de Prelog para as biorreduções utilizando este sistema
enzimático. A seguir, oxadiazóis com uma função fenol foram anexados na posição C-4 de
um carboidrato insaturado através de alquilação alílica mediada por Pd(0), de maneira
regiosseletiva. Estes glicosídeos insaturados foram transformados em manopiranosídeos
através de bis-hidroxilação seguida de bis-acetilação. Em outro momento, vários oxadiazóis,
derivados da uracila e aminoácidos foram anexados a carboidratos diversos através da
cicloadição 1,3-dipolar catalisada por Cu(I), fornecendo diversos glicoconjugados com um
espaçador 1,2,3-triazol. Os produtos foram obtidos de maneira regiosseletiva e os
glicoconjugados com a porção carboidrato insaturada foram bis-hidroxilados e bis-acetilados,
formando os respectivos manopiranosídeos. Alguns dos glicoconjugados tiveram suas
propriedades biológicas avaliadas. Nenhum dos compostos avaliados demonstrou atividade
antimicrobiana contra os microorganismos testados. Alguns produtos demonstraram ter
atividade citotóxica em linhagens de células cancerígenas HEp-2 e NCI-H292, com valores de
CI50 menores que 10 ug/mL. Estudos de microscopia eletrônica de varredura (MEV) foram
realizados nas células cancerígenas da linhagem HEp-2 e forneceram fortes indícios de que
estas substâncias ativariam a apoptose nas células estudadas
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Strain Promoted Click Chemistry of 8-Azidopurine and 5-Azidopyrimidine Nucleosides and Nucleotides with Cyclooctynes and Applications to Living Cell ImagingZayas, Jessica 10 June 2015 (has links)
The strain promoted azide alkyne cycloaddition (SPAAC) of azido nucleobase modified nucleosides and nucleotides with cyclooctynes to give fluorescent triazoles has been relatively unexplored. Thus, SPAAC between azido-nucleobases and various cyclooctynes in aqueous solution at ambient temperature resulted in the efficient formation (3 min - 2 h) of triazole products with inherent fluorescent properties. The 2- and 8-azidoadenine nucleosides reacted with fused cyclopropyl cyclooctyne, dibenzylcyclooctyne or monofluorocyclooctyne to produce click products functionalized with hydroxyl, amino, N-hydroxysuccinimide, or biotin moieties. The previously unexplored 5-azidouridine and labile 5-azido-2'-deoxyuridine were similarly converted to the analogous triazole products in quantitative yields in less than 5 minutes. The 8-azido-ATP quantitatively afforded the triazole product with fused cyclopropyl cyclooctyne (3 h). Addition of a triazole ring at the 2 or 8 position of adenine or 5-position of uracil induces fluorescent properties which were used for direct imaging with fluorescent microscopy in MCF-7 cancer cells without the need for traditional fluorogenic reporters. Fluorescent lifetime imaging microscopy of the click adducts in live cells were used to determine the lifetime of each fluorophore in the cellular nuclei demonstrating the potential utility of the synthesized triazole adducts for dynamic measuring and tracking of events inside single living cancer cells.
The SPAAC methodology developed has also been applied to study the cellular targets in protozoal parasite, Trichomonas vaginalis and bacteria, Pseudomonas aeruginosa. The 9-(2-deoxy-2-fluoro-β,D-arabino-furanosyl)adenine (arabino-F-Ado) was modified with an azido moiety at the C8 position for use in click chemistry. Tagging and subcellular localization studies using azido modified arabino-F-Ado could provide insight into the mechanism of action of arabino-F-Ado.
An activated analogue of S-adenosyl-L-methionine (SAM) with an EnYn group on the sulfur instead of a methyl group was prepared to study the transfer of the methyl group from SAM. I found that the EnYn group was transferred from SAM to a guanosine on tRNA by methytransferase Trm1. Thus, AdoEnYn is a competitive inhibitor of SAM and can be incorporated into tRNA in place of SAM.
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