Amphiphile Kern-Schale-my-Netzwerke auf der Basis von Polyorganosiloxanen

Die Synthese funktionalisierter Polyorganosiloxan-µ-Netzwerke (Rh = 5 – 30 nm) gelingt durch Polycokondensation von Alkoxysilanen. Die entstehenden sphärischen Teilchen sind in unpolaren organischen Lösungsmitteln partikulär dispergierbar. Durch die sequentielle Zugabe der Silanmonomere können Kern-Schale-Partikel mit unterschiedlichen Teilchenarchitekturen realisiert werden. In der vorliegenden Arbeit wird p-Chlormethylphenyltrimethoxysilan als funktionalisiertes Monomer verwendet, um den µ-Netzwerken durch eine anschließende Quaternisierung der Chlorbenzylgruppen mit Dimethylaminoethanol amphiphile Eigenschaften zu verleihen. Durch den Kern-Schale-Aufbau der Partikel sind die hydrophilen Bereiche im Kugelinneren von der hydrophoben äußeren Schale separiert, was unerläßlich für die Verwendung der Partikel zur Verkapselung wasserlöslicher Substanzen ist.So können in den amphiphilen µ-Netzwerken beispielsweise wasserlösliche Farbstoffe verkapselt werden. Diese diffundieren sowohl aus Lösung als auch aus dem Festkörper in das geladene Partikelinnere und werden dort angereichert. Es wird eine Abhängigkeit der Farbstoffbeladung vom Quaternisierungsgrad gefunden, wobei die Anzahl an verkapselten Farbstoffmolekülen mit dem Quaternisierungsgrad zunimmt.Weiterhin können amphiphile µ-Gelpartikel auch als molekulare Nanoreaktoren zur Synthese von Edelmetallkolloiden verwendet werden, die in den Netzwerken topologisch gefangen sind. Hierzu werden zuerst Metallionen im Kugelinneren verkapselt und anschließend reduziert, wobei das Kolloidwachstum durch den wohldefinierten Reaktionsraum gesteuert wird. Neben Gold- und Palladiumkolloiden können auf diese Weise beispielsweise auch Silberkolloide in den Kernen von µ-Netzwerken hergestellt werden.

Photoreactive polyorganosiloxane nanoparticles and the fabrication of photocleavable microcapsules

In this thesis, we have presented the preparation of highly crosslinked spherical photoreactive colloidal particles of radius about 10 nm based on the monomer trimethoxysilane. These particles are labeled chemically with two different dye systems (coumarin, cinnamate) which are known to show reversible photodimerization. By analyzing the change in particle size upon UV irradiation with dynamic light scattering, we could demonstrate that the partially reversible photoreaction in principle can be utilized to control increase and decrease of colloidal clusters. Here, selection of the appropriate wavelengths during the irradiation employing suitable optical filters proved to be very important. Next, we showed how photocrosslinking of our nanoparticles within the micrometer-sized thin oil shell of water-oil-water emulsion droplets leads to a new species of optically addressable microcontainers. The inner water droplet of these emulsions may contain drugs, dyes or other water-soluble components, leading to filled containers. Thickness, mechanical stability and light resistance of the container walls can be controlled in a simple way by the amount and adjustable photoreactivity (= No. of labels/particle) of the nanoparticles. Importantly, the chemical bonds between the nanoparticles constituting the microcapsule shell can be cleaved photochemically by irradiation with uv light. An additional major advantage is that filling our microcapsules with water-soluble substrate molecules is extremely simple using a solution of the guest molecules as inner water phase of the W/O/W-emulsion. This optically controlled destruction of our microcontainers thus opens up a pathway to controlled release of the enclosed components as illustrated by the example of enclosed cyclodextrin molecules.

Design, synthesis and application of ultrastable rylene dyes for fluorescent labeling of biomolecules

Studies of organic fluorescent dyes are experiencing a renaissance related to the increasing demands posed by new microscopy techniques for high resolution and high sensitivity. While in the last decade single molecule equipment and methodology has significantly advanced and in some cases reached theoretical limits (e.g. detectors approaching unity quantum yields) unstable emission from chromophores and photobleaching become more and more the bottleneck of the advancement and spreading of single-molecule fluorescence studies. The main goal of this work was the synthesis of fluorophores that are water-soluble, highly fluorescent in an aqueous environment, have a reactive group for attachment to a biomolecule and posses exceptional photostability. An approach towards highly fluorescent, water-soluble and monofunctional perylene-3,4,9,10-tetracarboxdiimide and terrylene-3,4:11,12-tetra carboxidiimide chromophores was presented. A new synthetic strategy for the desymmetrization of perylenetetracarboximides was elaborated; water-solubility was accomplished by introducing sulfonyl substituents in the phenoxy ring. Two strategies have been followed relying on either non-specific or site specific labeling. For this purpose a series of new water-soluble monofunctional perylene and terrylene dyes, bearing amine or carboxy group were prepared. The reactivity and photophysical properties of these new chromophores were studied in aqueous medium. The most suitable chromophores were further derivatized with amine or thiol reactive groups, suitable for chemical modification of proteins. The performance of the new fluorescent probes was assessed by single molecule enzyme tracking, in this case phospholipase acting on phospholipid supported layers. Phospholipase-1 (PLA-1) was labeled with N-hydroxysuccinimide ester functionalized perylene and terrylene derivatives. The purification of the conjugates was accomplished by novel convenient procedure for the removal of unreacted dye from labeled enzymes, which involves capturing excess dye with a solid support. This novel strategy for purification of bioconjugates allows convenient and fast separation of labeled proteins without the need for performing time consuming chromatographic or electrophoretic purification steps. The outstanding photostability of the dyes and, associated therewith, the extended survival times under strong illumination conditions allow a complete characterization of enzyme action on its natural substrates and even connecting enzyme mobility to catalytic activity. For site-specific attachment of the rylene dyes to proteins the chromophores were functionalized with thioesters or nitrilotriacetic acid groups. This allowed attachment of the emitters to the N-terminus of proteins by native chemical ligation or complexation with His-tagged polypeptides at the N- or C-termini, respectively. The synthesis of a water-soluble perylenebis (dicarboximide) functionalized with a thioester group was presented. This chromophore exhibits an exceptional photostability and a functional unit for site-specific labeling of proteins. The suitability of the fluorophore as a covalent label was demonstrated via native chemical ligation with protein containing N-terminal cystein residue. We exploited also oligohisitidine sequences as recognition elements for site-selective labeling. The synthesis of a new water-soluble perylene chromophore, containing a nitrilotriacetic acid functional group was demonstrated, using solution-phase and solid-phase approaches. This chromophore combines the exceptional photophysical properties of the rylene dyes and a recognition unit for site-specific labeling of proteins. An important feature of the label is the unchanged emission of the dye upon complexation with nickel ions.