Coil-ring-coil block copolymers as building blocks of supramolecular hollow cylindrical brushes

This work describes the synthesis of a new class of rod-coil block copolymers, oligosubstituted shape persistent macrocycles, (coil-ring-coil block copolymers), and their behavior in solution and in the solid state.The coil-ring-coil block copolymers are formed by nanometer sized shape persistent macrocycles based on the phenyl-ethynyl backbone as rigid block and oligomers of polystyrene or polydimethylsiloxane as flexible blocks. The strategy that has been followed is to synthesize the macrocycles with an alcoholic functionality and the polymer carboxylic acids independently, and then bind them together by esterification. The ester bond is stable and relatively easy to form.The synthesis of the shape persistent macrocycles is based on two separate steps. In the first step the building blocks of the macrocycles are connected by Hagiara-Sogonaschira coupling to form an 'half-ring' as precursor, that contains two free acetylenes. In the second step the half-ring is cyclized by forming two sp-sp bonds via a copper-catalyzed Glaser coupling under pseudo-high-dilution conditions. The polystyrene carboxylic acid was prepared directly by siphoning the living anionic polymer chain into a THF solution, saturated with CO2, while the polydimethylsiloxane carboxylic acid was obtained by hydrosilylating an unsaturated benzylester with an Si-H terminated polydimethylsiloxane, and cleavage of the ester. The carbodiimide coupling was found to be the best way to connect macrocycles and polymers in high yield and high purity.The polystyrene-ring-polystyrene block copolymers are, depending on the molecular weight of the polystyrene, lyotropic liquid crystals in cyclohexane. The aggregation behavior of the copolymers in solution was investigated in more detail using several technique. As a result it can be concluded that the polystyrene-ring-polystyrene block copolymers can aggregate into hollow cylinder-like objects with an average length of 700 nm by a combination of shape complementary and demixing of rigid and flexible polymer parts. The resulting structure can be described as supramolecular hollow cylindrical brush.If the lyotropic solution of the polystyrene-ring-polystyrene block copolymers are dried, they remain birefringent indicating that the solid state has an ordered structure. The polydimethylsiloxane-ring-polydimethylsiloxane block copolymers are more or less fluid at room temperature, and are all birefringent (termotropic liquid crystals) as well. This is a prove that the copolymers are ordered in the fluid state. By a careful investigation using electron diffraction and wide-angle X-ray scattering, it has been possible to derive a model for the 3D-order of the copolymers. The data indicate a lamella structure for both type of copolymers. The macrocycles are arranged in a layer of columns. These crystalline layers are separated by amorphous layers which contain the polymers substituents.

Self-assembly of Tetraurea Calix[4]arenes

We have elaborated a multistep strategy to synthesize ABAB-type tetraureas. There are overall nine steps but they involve very simple chemistry. The sequence starts with a 1,3-dialkylation and this is the step in which a difference between distal phenolic units is introduced. The selective ipso-nitration in the next step is based on the difference in reactivity between free phenolic units and alkylated ones. The direct reaction of tetraamino calixarene with tolylisocyanate appears not to be an appropriate method to synthesize 1,3-ditolylurea calixarenes but can be used to get tetraureas of ABBB- and AABB-types in two steps with yields of about 60%. A complete regioselective dimerization was obtained with mono-loop derivatives in which two adjacent urea residues are covalently connected. As predicted/expected the loop prevents the formation of one regioisomer, and only the dimer in which the open-chain residue slips through the loop is formed. To synthesize mono-loop tetraureas 1,2-diBoc protected tetraamino calixarene was acylated with activated di-urethanes under high dilution conditions. Di-loop compounds were synthesized by two different ways. In the reaction of tetraamine and di-urethanes the yield is about 30-40%. The second method is based on the metathesis reaction within a suitable heterodimer. For this strategy, tetraurea derivatives with residues which have terminal double bonds were prepared. The exclusive formation of the heterodimer with tetratosylurea as template is the key point in this strategy. Metathesis followed by hydrogenation give exceptionally good yields (> 80%) of the loop compounds. All the NMR data for di-loop compounds confirm that the loops prevent the interaction of the urea residues which are connected and thus, as expected, the di-loop derivatives do not form homodimers. The heterodimer between di-loop compounds and tetratolylurea (open-chain tetraureas) was the only species observed for a 1:1 mixture in benzene or chloroform. The rational synthesis of bis-[2]catenanes was a consequence of the selective formation of one regioisomer of mono-loop derivatives and the exclusive formation of heterodimers by di-loop derivatives. The formation of interlocking-ring in the synthesis of bis-[2]catenanes is an additional evidence that one open-chain residue slips through the loop in mono- or di-loop derivatives. Exceptionally good yields in the synthesis of bis-[2]catenanes are due to the high preorganization in the dimer which undergoes the metathesis. This preorganization decreases the number of the wrong connections and favors the new connections to be formed. Although the procedure for working up the reaction mixture should be still improved, these results are promising. A C2-symmetrical bis-[2]catenane was successfully resolved by column chromatography using a chiral stationary phase. Thus it should be possible to separate a larger amount to obtain pure enantiomers for further studies.

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.

Influence of micro-site conditions and environmental gradients on tree-ring based climate reconstructions

Tree-ring chronologies are a powerful natural archive to reconstruct summer temperature variations of the late Holocene with an annual resolution. To develop these long-term proxy records tree-ring series are commonly extended back in time by combining samples from living trees with relict dead material preserved onshore or in lakes. Former studies showed that low frequency variations in such reconstructions can be biased if the relict and recent material is from different origins. A detailed analysis of the influence of various ecological (micro-) habitats representing the recent part is required to estimate potential errors in temperature estimates. The application of collective detrending methods, that comprise absolute growth rates, can produce errors in climate reconstructions and results in an underestimation of past temperatures. The appearance of these kind of micro-site effects is a wide-spread phenomenon that takes place all over Fennoscandia. Future research in this key region for dendroclimatology should take this issue into account. Especially the higher climate response at the lakeshore site is interesting to achieve smaller uncertainties when a tree-ring series is transformed to temperature anomalies. For new composite chronologies the main aim should be to minimize potential biases and this includes also micro-site effects.