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Item Open Access Precision 3D‐printed cell scaffolds mimicking native tissue composition and mechanics(2020) Erben, Amelie; Hörning, Marcel; Hartmann, Bastian; Becke, Tanja; Eisler, Stephan A.; Southan, Alexander; Cranz, Séverine; Hayden, Oliver; Kneidinger, Nikolaus; Königshoff, Melanie; Lindner, Michael; Tovar, Günter E. M.; Burgstaller, Gerald; Clausen‐Schaumann, Hauke; Sudhop, Stefanie; Heymann, MichaelCellular dynamics are modeled by the 3D architecture and mechanics of the extracellular matrix (ECM) and vice versa. These bidirectional cell‐ECM interactions are the basis for all vital tissues, many of which have been investigated in 2D environments over the last decades. Experimental approaches to mimic in vivo cell niches in 3D with the highest biological conformity and resolution can enable new insights into these cell‐ECM interactions including proliferation, differentiation, migration, and invasion assays. Here, two‐photon stereolithography is adopted to print up to mm‐sized high‐precision 3D cell scaffolds at micrometer resolution with defined mechanical properties from protein‐based resins, such as bovine serum albumin or gelatin methacryloyl. By modifying the manufacturing process including two‐pass printing or post‐print crosslinking, high precision scaffolds with varying Young's moduli ranging from 7‐300 kPa are printed and quantified through atomic force microscopy. The impact of varying scaffold topographies on the dynamics of colonizing cells is observed using mouse myoblast cells and a 3D‐lung microtissue replica colonized with primary human lung fibroblast. This approach will allow for a systematic investigation of single‐cell and tissue dynamics in response to defined mechanical and bio‐molecular cues and is ultimately scalable to full organs.Item Open Access Methacrylate‐based polymer foams with controllable pore sizes and controllable polydispersities via foamed emulsion templating(2020) Dabrowski, Miriam Lucia; Stubenrauch, CosimaThis study reports on a novel templating route, which uses foamed emulsions as templates for porous polymers. The concept is based on the generation of a monomer‐in‐water emulsion, which is subsequently foamed via microfluidics. The monomer of choice is 1,4‐butanediol dimethacrylate (1,4‐BDDMA). After polymerization of the foamed emulsion, one obtains open‐cell polymer foams with porous pore walls. Foamed emulsions and polymer foams are generated. It is shown that foamed emulsion templating in combination with microfluidics is well‐suited to synthesize 1) monodisperse poly(1,4‐BBDMA) foams with controllable pore sizes and 2) their polydisperse counterparts with controllable polydispersities. Monodisperse templates with different bubble sizes and thus polymer foams with different pore sizes ranging from about 100-400 μm in diameter are synthesized. Microfluidics is also used for the generation of polydisperse poly(1,4‐BDDMA) foams with polydispersities between 18% and 27% but the same mean pore sizes as the monodisperse ones, i.e., we have access to polymer foams that only differ in their polydispersity.Item Open Access The triple variant K170D/N174L/D239A compensates the destabilizing effect of variant K170D/N174L in β-hydroxyacid dehydrogenase (βHAD) from Arabidopsis thaliana(2020) Schelle, Luca S.; Stockinger, Peter; Pleiss, Jürgen; Nestl, Bettina M.Chiral amines are essential building blocks in biologically active compounds, fine chemicals, agrochemicals and pharmaceuticals. In the last ten years, various enzymes were identified as new biocatalysts for chiral amine synthesis. Promising enzymes for the synthesis of primary, secondary, and tertiary amines are NADPH-dependent imine reductases (IREDs). Bioinformatics analysis revealed that IREDs are closely related to β-hydroxyacid dehydrogenases (βHADs). In recent work, we engineered the βHAD from Arabidopsis thaliana (βHAD_At) into imine-reducing enzymes by a single amino acid exchange. The exchange of the proton-donor described lysine (K170) in βHAD_At by aspartic acid, the most common amino acid at this position in R-selective IREDs, led to a 12-fold increase in activity for the model substrate 2-methylpyrroline. At the same time, the activity for the natural substrate glyoxylic acid is reduced 885-fold, resulting in a total of 8200-fold change in catalytic activity through the exchange of an amino acid. At the same time, highly decreased soluble expression has been observed by exchanging asparagine at position 174 (N174) with leucine. We thus hypothesized, that the aspartic acid residue (D239) in near proximity to N174 will stabilize the underlying α-helix. Consequently, replacement of D239 with alanine should result in soluble expression of variants containing the N174 mutations. We generated variants K170D/D239A, as well as, and tested them on imine reduction of test substrates 2-methylpyrroline, 3,4-dihydroisoquinoline and 6-phenyl-2,3,4,5-tetrahydropyridine. Due to loss of essential cofactor and precipitation of purified proteins during purification procedure, activities of variants were determined using cell lysates. Notably, variants N174L/D239A and K170D/N174L/D239A demonstrated soluble expression and imine-reducing activities of up to 98 mU per mg of variant.Item Open Access Field evaporation and atom probe tomography of pure water tips(2020) Schwarz, T. M.; Weikum, E. M.; Meng, K.; Hadjixenophontos, E.; Dietrich, C. A.; Kästner, J.; Stender, P.; Schmitz, G.Measuring biological samples by atom probe tomography (APT) in their natural environment, i.e. aqueous solution, would take this analytical method, which is currently well established for metals, semi-conductive materials and non-metals, to a new level. It would give information about the 3D chemical structure of biological systems, which could enable unprecedented insights into biological systems and processes, such as virus protein interactions. For this future aim, we present as a first essential step the APT analysis of pure water (Milli-Q) which is the main component of biological systems. After Cryo-preparation, nanometric water tips are field evaporated with assistance by short laser pulses. The obtained data sets of several tens of millions of atoms reveal a complex evaporation behavior. Understanding the field evaporation process of water is fundamental for the measurement of more complex biological systems. For the identification of the individual signals in the mass spectrum, DFT calculations were performed to prove the stability of the detected molecules.Item Open Access Adjustable polystyrene nanoparticle templates for the production of mesoporous foams and ZnO inverse opals(2020) Abitaev, Karina; Qawasmi, Yaseen; Atanasova, Petia; Dargel, Carina; Bill, Joachim; Hellweg, Thomas; Sottmann, ThomasThe manifold applications of porous materials, such as in storage, separation, and catalysis, have led to an enormous interest in their cost-efficient preparation. A promising strategy to obtain porous materials with adjustable pore size and morphology is to use templates exhibiting the appropriate nanostructure. In this study, close-packed polystyrene (PS) nanoparticles, synthesized by emulsion polymerization, were used to produce porous PS and ZnO inverse opals. The size and distribution of the polystyrene nanoparticles, characterized by dynamic light scattering (DLS), small-angle neutron scattering (SANS), and scanning electron microscopy (SEM), were controlled via the concentration of sodium dodecyl sulfate (SDS). Systematic measurements of the water/styrene-interfacial tension show that the critical micelle concentration (CMC) of the ternary water–styrene–SDS system, which determines whether monodisperse or polydisperse PS particles are obtained, is considerably lower than that of the binary water–SDS system. The assemblies of close-packed PS nanoparticles obtained via drying were then studied by small-angle X-ray scattering (SAXS) and SEM. Both techniques prove that PS nanoparticles synthesized above the CMC result in a significantly unordered but denser packing of the particles. The polystyrene particles were subsequently used to produce porous polystyrene and ZnO inverse opals. While the former consists of micrometer-sized spherical pores surrounded by extended open-cellular regions of mesopores (Rpore ≈ 25 nm), the latter are made of ZnO-nanoparticles forming a structure of well-aligned interconnected pores.Item Open Access Porous polymers via emulsion templating : pore deformation during solidification cannot be explained by an osmotic transport!(2020) Koch, Lukas; Drenckhan, Wiebke; Stubenrauch, CosimaUsing microfluidics, we were able to synthesize monodisperse water-in-monomer emulsions with styrene and divinylbenzene (DVB) as monomers. When polymerizing and drying these emulsions, we found that the structure of the resulting macroporous polymer strongly depends on the type of initiator. With the oil-soluble azobisisobutyronitrile (AIBN), an open-cell structure with spherical pores was obtained. However, with the water-soluble potassium peroxydisulfate (KPS), a closed-cell structure with rhombic dodecahedron-shaped pores and thick, layered pore walls was formed. In the latter case, a yet unexplained mechanism counteracts the capillary pressure arising from surface minimization: the surface area of a rhombic dodecahedron is ~ 10% larger than that of a sphere. In our previous work, we suggested that the underlying mechanism may be osmotic transport of DVB from the plateau borders to the films. We argued that this transport also explains the layered pore walls, i.e., the formation of two outer poly-DVB-rich layers and one inner polystyrene-rich layer. In order to prove or disprove this mechanism, we carried out additional experiments. However, none of those experiments corroborated our hypothesis of osmotic transport! This study provides clear experimental evidence that our previously suggested mechanism via which spherical droplets become polyhedral pores is incorrect. We will describe (a) the rationale behind the additional experiments, (b) our expectations, and (c) our findings. Last but not least, we will discuss all of this in the light of the proposed osmotic transport.Item Open Access Fe-katalysierte Carben- und Nitren-Transferreaktionen in der Naturstoffsynthese(2020) Röske, Annika; Plietker, Bernd (Prof. Dr.)In der vorliegenden Arbeit wurden zunächst, aufbauend auf den Vorarbeiten von Alt und den Ergebnissen der eigenen Masterarbeit, eisenkatalysierte Carben-Transferreaktionen untersucht. Dabei gelang es mir verschieden substituierte Diazoverbindungen für die Insertion in unterschiedlich polare X H Bindungen unter Verwendung des nukleophilen Eisenkatalysators TBA[Fe] zu aktivieren (Schema 1). Thiole, Amine sowie Silane konnten erfolgreich umgesetzt und deren Substratspektrum erforscht werden. Thiolverbindungen zeigten sich in der Reaktion als besonders geeignet. Eine niedrige Katalysatorladung von 2.5 mol%, kurze Reaktionszeiten von einer Stunde und bis zu quantitative Ausbeuten mit hoher funktioneller Gruppentoleranz wurden erzielt. Amine zeigten geringere Reaktivität und ein limitiertes Substratspektrum, das sich auf sekundäre Amine begrenzt. Die längere Reaktionszeit konnte durch Mikrowellenstrahlung als alternative Energiequelle auf eine Stunde verkürzt werden. Erstmals konnte gezeigt werden, dass TBA[Fe] auch in der Si-H-Insertion Aktivität aufweist. Durch ausführliche mechanistische Studien mittels Hammett-Korrelation konnten Rückschlüsse auf die mechanistische Dichotomie der N H-Insertion und der S-H-Insertion gezogen werden.Das Konzept der TBA[Fe]-katalysierten Carben-Transferreaktion wurde auf aliphatische Azide als Nitren-Transferreagenzien übertragen. Dabei wurde keine C-H-Aminierung beobachtet, aber erfreulicherweise eine C-C-Aminierung von tertiären Alkylaziden. Bei Verwendung von Azidocyclobutanen konnte eine selektive ringexpandierende TBA[Fe]-Katalyse zu den korrespondierenden 3,4-Dihydro-2H-pyrrolderivaten entwickelt und optimiert werden. Die Reaktion zeichnet sich durch eine kurze Substratsynthese und geringe Katalysatorladung von 1.0 mol% aus. Die so dargestellten Strukturmotive finden sich in einer Vielzahl von Wirkstoffen wieder. Die Ergebnisse der Untersuchungen zum Substratspektrum und Reaktionsverfolgung via in-situ-FT-IR-Messung führten zur Postulierung eines Nitren-Transfermechanismus.Um die Anwendbarkeit der bereits von Alt entwickelten TBA[Fe]-katalysierten C-H Aminierung in komplexeren Molekülen zu untersuchen, wurden Studien zur Totalsynthese von Spirotryprostatin A und Spirotryprostatin B aus der Naturstoffklasse der 2,5-Diketopiperazine durchgeführt. Es konnte eine effiziente Synthese zum Schlüsselschritt-Substrat generiert werden.Item Open Access Melt-spinning of an intrinsically flame-retardant polyacrylonitrile copolymer(2020) König, Simon; Kreis, Philipp; Herbert, Christian; Wego, Andreas; Steinmann, Mark; Wang, Dongren; Frank, Erik; Buchmeiser, Michael R.Poly(acrylonitrile) (PAN) fibers have two essential drawbacks: they are usually processed by solution-spinning, which is inferior to melt spinning in terms of productivity and costs, and they are flammable in air. Here, we report on the synthesis and melt-spinning of an intrinsically flame-retardant PAN-copolymer with phosphorus-containing dimethylphosphonomethyl acrylate (DPA) as primary comonomer. Furthermore, the copolymerization parameters of the aqueous suspension polymerization of acrylonitrile (AN) and DPA were determined applying both the Fineman and Ross and Kelen and Tüdõs methods. For flame retardancy and melt-spinning tests, multiple PAN copolymers with different amounts of DPA and, in some cases, methyl acrylate (MA) have been synthesized. One of the synthesized PAN-copolymers has been melt-spun with propylene carbonate (PC) as plasticizer; the resulting PAN-fibers had a tenacity of 195 ± 40 MPa and a Young’s modulus of 5.2 ± 0.7 GPa. The flame-retardant properties have been determined by Limiting Oxygen Index (LOI) flame tests. The LOI value of the melt-spinnable PAN was 25.1; it therefore meets the flame retardancy criteria for many applications. In short, the reported method shows that the disadvantage of high comonomer content necessary for flame retardation can be turned into an advantage by enabling melt spinning.Item Open Access Umsetzung von CO2 und Ethen zu Na-Acrylat(2020) Weber, Sandra; Klemm, Elias (Prof. Dr.-Ing,)Die stoffliche Nutzung von CO2 als C1-Baustein in chemischen Endprodukten stellt einen zukunftsweisenden Forschungsansatz dar. Kohlenstoffhaltige Verbindungen wie Acrylsäure werden derzeit auf der Basis fossiler Rohstoffe hergestellt. Im Rahmen dieser Arbeit wird ein neuer Syntheseweg zur Herstellung von Natrium-Acrylat untersucht. Ziel ist es, aus günstigen Rohstoffen (CO2 und Ethen) ein wirtschaftlich interessantes Produkt (Na-Acrylat) zu synthetisieren. Die Umsetzung basiert auf einem Nickel-vermittelten Katalysezyklus, ausgehend von Kohlenstoffdioxid und Ethen. Innerhalb des Katalysezyklus werden unterschiedliche Elementarschritte durchlaufen, zu denen auch Gas-Flüssig-Reaktionen gehören. In der vorliegenden Arbeit wurden einzelne Schritte des Katalysezyklus reaktionstechnisch untersucht. Die systematische Untersuchung von Gas-Flüssig-Reaktionen stellte den Schwerpunkt der vorliegenden Arbeit dar.Item Open Access Darstellung und Reaktivität N-heterozyklischer Phosphenium-Carbonylkomplexe des Eisens und Chroms(2020) Feil, Christoph Matthias; Gudat, Dietrich (Prof. Dr.)