Design, synthesis and photochemical studies of stilbenoid dendrimers

Stilbenoid dendrimers with stilbene in the periphery and stilbene in periphery as well as core were synthesized by convergent approach except 2nd generation dendrimer with stilbene in the periphery as well as in core (D-5). All dendrimers were characterized by standard techniques such as 1H NMR, 13C NMR, MS and IR spectroscopy. The MALDI-TOF technique proved to be very helpful in the identification of the 2nd generation dendrimer (D-5) with a mass of 3231 a.m.u. The dendrimers were designed in such a way that an intramolecular photochemical CC bond formation was favored. As two stilbene units of the same molecule were close enough so they preferred an intramolecular cyclic process except for zero generation dendrimers. Apart from the cycloaddition, some E/Z isomerization and oligomer formation was also observed on irradiation. These processes were observed by 1H NMR and MALDI-TOF MS. The photochemical behavior was also studied by UV absorption spectroscopy. Irradiating by monochromatic light led to an initial E/Z isomerization and by prolonged irradiation, an irreversible cyclic structure was formed. The choice of the wavelength of incident light is very important as irradiation at 320 nm leads to a reversible E/Z isomerization and a non-reversible cyclobutane formation, but irradiation at 340 nm favors the one-way process E Z. The [2+2] cycloaddition of molecule Tm2De was also studied by irradiating thin films on a quartz surface. An AFM image was taken before irradiation, after 3 sec irradiation and after long irradiation (1 hour). AFM studies show that a short irradiation leads to a cyclic structure as formation of hills of about 20-30 nm on the surface. A prolonged irradiation leads to a CC cross linking which can be monitored on AFM images as disappearance of hills. The roughness goes back to an almost smooth surface. These results prove a very complex material transport, which accompanies the reaction in the surface region.

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.

Coarse-graining methods and polymer dynamics

This thesis studies molecular dynamics simulations on two levels of resolution: the detailed level of atomistic simulations, where the motion of explicit atoms in a many-particle system is considered, and the coarse-grained level, where the motion of superatoms composed of up to 10 atoms is modeled. While atomistic models are capable of describing material specific effects on small scales, the time and length scales they can cover are limited due to their computational costs. Polymer systems are typically characterized by effects on a broad range of length and time scales. Therefore it is often impossible to atomistically simulate processes, which determine macroscopic properties in polymer systems. Coarse-grained (CG) simulations extend the range of accessible time and length scales by three to four orders of magnitude. However, no standardized coarse-graining procedure has been established yet. Following the ideas of structure-based coarse-graining, a coarse-grained model for polystyrene is presented. Structure-based methods parameterize CG models to reproduce static properties of atomistic melts such as radial distribution functions between superatoms or other probability distributions for coarse-grained degrees of freedom. Two enhancements of the coarse-graining methodology are suggested. Correlations between local degrees of freedom are implicitly taken into account by additional potentials acting between neighboring superatoms in the polymer chain. This improves the reproduction of local chain conformations and allows the study of different tacticities of polystyrene. It also gives better control of the chain stiffness, which agrees perfectly with the atomistic model, and leads to a reproduction of experimental results for overall chain dimensions, such as the characteristic ratio, for all different tacticities. The second new aspect is the computationally cheap development of nonbonded CG potentials based on the sampling of pairs of oligomers in vacuum. Static properties of polymer melts are obtained as predictions of the CG model in contrast to other structure-based CG models, which are iteratively refined to reproduce reference melt structures. The dynamics of simulations at the two levels of resolution are compared. The time scales of dynamical processes in atomistic and coarse-grained simulations can be connected by a time scaling factor, which depends on several specific system properties as molecular weight, density, temperature, and other components in mixtures. In this thesis the influence of molecular weight in systems of oligomers and the situation in two-component mixtures is studied. For a system of small additives in a melt of long polymer chains the temperature dependence of the additive diffusion is predicted and compared to experiments.

Methods and strategies to identify the mode of action of phytoactive compounds

Small molecules affecting biological processes in plants are widely used in agricultural practice as herbicides or plant growth regulators and in basic plant sciences as probes to study the physiology of plants. Most of the compounds were identified in large screens by the agrochemical industry, as phytoactive natural products and more recently, novel phytoactive compounds originated from academic research by chemical screens performed to induce specific phenotypes of interest. The aim of the present PhD thesis is to evaluate different approaches used for the identification of the primary mode of action (MoA) of a phytoactive compound. Based on the methodologies used for MoA identification, three approaches are discerned: a phenotyping approach, an approach based on a genetic screen and a biochemical screening approach.rnFour scientific publications resulting from my work are presented as examples of how a phenotyping approach can successfully be applied to describe the plant MoA of different compounds in detail.rnI. A subgroup of cyanoacrylates has been discovered as plant growth inhibitors. A set of bioassays indicated a specific effect on cell division. Cytological investigations of the cell division process in plant cell cultures, studies of microtubule assembly with green fluorescent protein marker lines in vivo and cross resistant studies with Eleusine indica plants harbouring a mutation in alpha-tubulin, led to the description of alpha-tubulin as a target site of cyanoacrylates (Tresch et al., 2005).rnII. The MoA of the herbicide flamprop-m-methyl was not known so far. The studies described in Tresch et al. (2008) indicate a primary effect on cell division. Detailed studies unravelled a specific effect on mitotic microtubule figures, causing a block in cell division. In contrast to other inhibitors of microtubule rearrangement such as dinitroanilines, flamprop-m-methyl did not influence microtubule assembly in vitro. An influence of flamprop-m-methyl on a target within the cytoskeleton signalling network could be proposed (Tresch et al., 2008).rnIII. The herbicide endothall is a protein phosphatase inhibitor structurally related to the natural product cantharidin. Bioassay studies indicated a dominant effect on dark-growing cells that was unrelated to effects observed in the light. Cytological characterisation of the microtubule cytoskeleton in corn tissue and heterotrophic tobacco cells showed a specific effect of endothall on mitotic spindle formation and ultrastructure of the nucleus in combination with a decrease of the proliferation index. The observed effects are similar to those of other protein phosphatase inhibitors such as cantharidin and the structurally different okadaic acid. Additionally, the observed effects show similarities to knock-out lines of the TON1 pathway, a protein phosphatase-regulated signalling pathway. The data presented in Tresch et al. (2011) associate endothall’s known in vitro inhibition of protein phosphatases with in vivo-effects and suggest an interaction between endothall and the TON1 pathway.rnIV. Mefluidide as a plant growth regulator induces growth retardation and a specific phenotype indicating an inhibition of fatty acid biosynthesis. A test of the cuticle functionality suggested a defect in the biosynthesis of very-long-chain fatty acids (VLCFA) or waxes. Metabolic profiling studies showed similarities with different groups of VLCFA synthesis inhibitors. Detailed analyses of VLCFA composition in tissues of duckweed (Lemna paucicostata) indicated a specific inhibition of the known herbicide target 3 ketoacyl-CoA synthase (KCS). Inhibitor studies using a yeast expression system established for plant KCS proteins verified the potency of mefluidide as an inhibitor of plant KCS enzymes. It could be shown that the strength of inhibition varied for different KCS homologues. The Arabidopsis Cer6 protein, which induces a plant growth phenotype similar to mefluidide when knocked out, was one of the most sensitive KCS enzymes (Tresch et al., 2012).rnThe findings of my own work were combined with other publications reporting a successful identification of the MoA and primary target proteins of different compounds or compound classes.rnA revised three-tier approach for the MoA identification of phytoactive compounds is proposed. The approach consists of a 1st level aiming to address compound stability, uniformity of effects in different species, general cytotoxicity and the effect on common processes like transcription and translation. Based on these findings advanced studies can be defined to start the 2nd level of MoA characterisation, either with further phenotypic characterisation, starting a genetic screen or establishing a biochemical screen. At the 3rd level, enzyme assays or protein affinity studies should show the activity of the compound on the hypothesized target and should associate the in vitro effects with the in vivo profile of the compound.

Highly accurate quantum chemistry : spin-orbit splittings via multireference coupled-cluster methods and applications in heavy-atom main-group chemistry

Diese Dissertation demonstriert und verbessert die Vorhersagekraft der Coupled-Cluster-Theorie im Hinblick auf die hochgenaue Berechnung von Moleküleigenschaften. Die Demonstration erfolgt mittels Extrapolations- und Additivitätstechniken in der Single-Referenz-Coupled-Cluster-Theorie, mit deren Hilfe die Existenz und Struktur von bisher unbekannten Molekülen mit schweren Hauptgruppenelementen vorhergesagt wird. Vor allem am Beispiel von cyclischem SiS_2, einem dreiatomigen Molekül mit 16 Valenzelektronen, wird deutlich, dass die Vorhersagekraft der Theorie sich heutzutage auf Augenhöhe mit dem Experiment befindet: Theoretische Überlegungen initiierten eine experimentelle Suche nach diesem Molekül, was schließlich zu dessen Detektion und Charakterisierung mittels Rotationsspektroskopie führte. Die Vorhersagekraft der Coupled-Cluster-Theorie wird verbessert, indem eine Multireferenz-Coupled-Cluster-Methode für die Berechnung von Spin-Bahn-Aufspaltungen erster Ordnung in 2^Pi-Zuständen entwickelt wird. Der Fokus hierbei liegt auf Mukherjee's Variante der Multireferenz-Coupled-Cluster-Theorie, aber prinzipiell ist das vorgeschlagene Berechnungsschema auf alle Varianten anwendbar. Die erwünschte Genauigkeit beträgt 10 cm^-1. Sie wird mit der neuen Methode erreicht, wenn Ein- und Zweielektroneneffekte und bei schweren Elementen auch skalarrelativistische Effekte berücksichtigt werden. Die Methode eignet sich daher in Kombination mit Coupled-Cluster-basierten Extrapolations-und Additivitätsschemata dafür, hochgenaue thermochemische Daten zu berechnen.

Brand formative design - development and assessment of product design from a future, brand and consumer perspective

With this dissertation research we investigate intersections between design and marketing and in this respect, which factors do contribute that a product design becomes brand formative. We have developed a Brand Formative Design (BFD) framework, which investigates individual design features in a holistic, comparable, brand relevant, and consumer specific context. We discuss what kinds of characteristics contribute to BFD but also illuminate how they should be applied and examine: rnA holistic framework leading to Brand Formative Design. Identification and assessment of BFD Drivers. The dissection of products into three Distinctive Design Levels. The detection of surprising design preferences. The appropriate degree of scheme deviation with evolutionary design. Simulated BFD development processes with three different products and the integration of consumers. Future oriented objectification, comparability and assessment of design. Recommendations for the management of design in a brand specific context. Design is a product feature, which contributes significantly to the success of products. However, the development of new design contains challenges. Design can hardly be objectified; many people have an opinion concerning the attractiveness of new products but cannot formulate their future preferences. Product design is widely developed based on intuition, which can be difficult for the management of design. Here the concept of Brand Formative Design can provide a framework which contributes to structure, objectify, develop and assess new evolutionary design in brand and future relevant contexts, but also integrates consumers and their preferences without restricting creativity too much.