- Bayreuther Graduiertenschule für Mathematik und Naturwissenschaften (BayNAT) (18)
Dual-Responsive Polymer and Hybrid Systems: Applications for Gene Delivery and Hydrogels
- This thesis focused on the synthesis of functional materials based on water-soluble and responsive polymers, in particular poly((2-dimethylamino)ethyl methacrylate) (PDMAEMA). The dual-responsive behavior and polycationic character at physiological pH of PDMAEMA lead to outstanding properties and thus, to a versatile component for water-based applications. The main concept of the thesis was to combine the ability for gene delivery of PDMAEMA with the magnetic properties of iron oxide nanoparticles to enable an activity of the gene vector in an applied external magnetic field. Another point was to apply the dual-responsive behavior of PDMAEMA (temperature and pH) for physically cross-linked hydrogels.
Initial studies on magnetic dual-responsive gene vectors revealed a facile synthesis of PDMAEMA-grafted iron oxide nanoparticles utilizing dopamine as physically binding anchor group for the polymer chains. Here, a dopamine-based ATRP initiator was applied for the surface modification of the nanoparticles, which enabled a controlled polymerization technique via the “grafting-from” approach. Gene transfection experiments with CHO-K1 cells show that the transfection efficiency is significantly higher than for poly(ethyleneimine) (PEI), which is regarded as the “gold standard” among the polycationic gene vectors. Although the hybrid particles show a considerably high molecular weight (4.3 MDa), which should lead to a significant increase of the cytotoxicity as observed for linear PDMAEMA their cytotoxicity is remarkably low, lower than that of PEI. Thus, the excellent performance in gene delivery experiments can be attributed to the star-like architecture of the PDMAEMA. Moreover, the uptake of our superparamagnetic gene vector into the cells enables a magnetic cell separation by applying an external magnetic field.
However, due to the non-covalent bonds of dopamine to the iron oxide nanoparticles, the PDMAEMA chains undergo a detachment with time from the nanoparticle surface. This led to the synthesis of PDMAEMA-based magnetic core-shell-corona nanoparticles. Here, the iron oxide nanoparticles were covered with a thin silica shell in order to link the PDMAEMA chains covalently to the inorganic core via silane chemistry. This approach revealed stable dual-responsive hybrid nanoparticles with irreversible binding of the polymer chains and a high long-term stability in aqueous media. These hybrid star-like particles also show excellent gene delivery. The inter-polyelectrolyte complex formation between the PDMAEMA corona of the core-shell-corona particles and pDNA showed that the pDNA molecules are individually complexed with single nanoparticles at N/P ratios (polymer nitrogen / pDNA phosphorous) where the best transfection results are obtained. The magnetic cell separation was further improved by using a Magnetic Activated Cell Sorting system (MACSTM). The magnetically separated cells maintain a high transfection efficiency as well as viability and could even be further cultivated.
Another aspect of this thesis was to include PDMAEMA as stimuli-responsive block in a double switchable block copolymer-based hydrogel. For this purpose, we chose a physically cross-linked ABCBA pentablock terpolymer system, which was polymerized via sequential ATRP and consist of a water-soluble PEO middle block, two dual-responsive (temperature/pH) PDMAEMA B-blocks as well as two thermo-responsive poly(di(ethylene glycol) methyl ether methacrylate) (PDEGMA) A-blocks (PDEGMA-b-PDMAEMA-b-PEO-b-PDMAEMA-b-PDEGMA). The aggregation behavior in dilute solution was investigated via temperature-dependent Dynamic Light Scattering (DLS) revealing that both stimuli-responsive blocks can be triggered separately and the coil-to-globule transition temperatures of the stimuli-responsive blocks were found to be strongly dependent on the block lengths for low molecular weights. In concentrated solutions, however, rheology studies did not show a further change in the mechanical properties after gelation for the investigated ABCBA pentablock terpolymer compositions. As a result, the principle of our complex system points towards a successful synthesis of a dual-responsive ABCBA pentablock terpolymer hydrogel system, which may show two distinct phase transition even for the gel state, if longer block lengths of the outer A- and B-blocks would be applied.
Funktion und Einfluss der nicht-repetitiven, terminalen Domänen auf Speicherung und Assemblierung von Spinnenseidenproteinen
- Weibliche Radnetzspinnen sind in der Lage bis zu sechs verschiedene Spinnenseidenfasern mit unterschiedlichen mechanischen Eigenschaften herzustellen, die denen von syntheti-schen Hochleistungsfasern wie Kevlar oder Hochleistungsstahl zum Teil überlegen sind. Die am besten untersuchte Spinnenseide ist diejenige, die für die Speichen, den Rahmen sowie als Abseilfaden verwendet wird. Sie wird als dragline-Seide bezeichnet und ist hauptsächlich aus zwei Proteinklassen aufgebaut, die als MaSp1 und MaSp2(Major Ampullate Spidroin) bezeichnet werden. Die mehrere hundert Kilodalton großen Spidroine bestehen im Wesentlichen aus einer repetitiven, strukturell ungeordneten Kerndomäne, die von relativ kleinen, ca. 100-140 Aminosäuren langen nicht-repetitiven, globulären Domänen flankiert ist. Das Hauptunterscheidungsmerkmal zwischen MaSp1 und 2 ist der Prolingehalt der repetitiven Sequenzen wobei in MaSp2 viel (>10%) und MaSp1 (<0,4%) wenig Proline enthalten sind. In der dragline Seide der Gartenkreuzspinne Araneus diadematus sind zwei Spidroine, genannt ADF3 und 4, mit einem hohen Prolingehalt identifiziert wor-den, die beide deshalb zur Klasse der MaSp2-Proteine gehören. Die nicht-repetitiven terminalen Domänen sind globulär gefaltet und die Sequenzen sind über verschiedene Sei-denarten und Spezies stark konserviert.
Ziel dieser Arbeit war es den Einfluss der nicht-repetitiven amino- (NTD) und carboxyterminalen (CTD) Domänen auf das Lösungs- und Assemblierungsverhalten von rekombinanten Spinnenseidenproteinen auf Basis von ADF3 und 4 zu untersuchen. Der Fokus lag dabei zunächst auf der carboxyterminalen Domäne NR3 des Spinnenseidenproteins ADF3 aus A. diadematus. Die Struktur der Domäne wurde in Zusammenarbeit mit Dr. Franz Hagn (AG Kessler – TU München) mit Hilfe von NMR gelöst. Es bildet in Lösung ein parallel orientiertes, mittels Disulfidbrücke kovalent verknüpftes Dimer aus, zu dessen Struktur bisher keine Homologen bekannt sind. Es konnte gezeigt werden, dass die NR3-Domäne in Lösung die Bildung von sphärischen mizellartigen Assemblaten vermittelt, wie sie auch im natürlichen Spinntrakt beobachtet wurden. Eine weitere wichtige Rolle konnte bei der scherkraftinduzierten Assemblierung beobachtet werden, wo nur eADF3-Proteine mit der NR3-Domäne in der Lage sind ausgerichtete Faserbündel auszubilden.
In einem weiteren Projektteil wurde die Interaktion zwischen rekombinanten Varianten von ADF3 und 4 untersucht, da beide Proteine mit hoher Wahrscheinlichkeit auch in der Spinne in den gleichen Zellen produziert werden. Es konnte gezeigt werden, dass durch Co-Expression der entsprechenden Gene von eADF3-NR3 und eADF4-NR4 in E. coli ein NR3-NR4 vermitteltes Heterodimer entsteht. Dieses Heterodimer besitzt eine vergleichbare strukturelle Integrität wie die entsprechenden Homodimere. Das durch Phosphationen induzierte Assemblierungsverhalten des Heterodimers unterscheidet sich signifikant von dem der Homodimere. Während eADF3-NR3 sphärische Assemblate bildet und eADF4-NR4 mehrere hundert Nanometer lange Fibrillen, wie sie in ähnlicher Form in den natürlichen Fasern beobachtet wurden, lagert sich das Heterodimer zu einem Netzwerk aus im Vergleich dünneren und weniger langen Fibrillen zusammen. Diese Entdeckung ist unter Umständen wichtig für das Verständnis des natürlichen Spinnprozesses. Aus biotechnologischer Sicht stellt die quantitative Verknüpfung von Spinnenseidenproteinen mit unter-schiedlichen Eigenschaften über die carboxyterminalen Domänen einen interessanten Ansatz zur Generierung von neuen Materialien mit einstellbaren Eigenschaften dar.
Letztendlich wurden rekombinante eADF3 Spinnenseidenproteine, die sowohl amino- als auch carboxyterminale Domänen aufwiesen, näher charakterisiert. Die isolierte aminoterminale Domäne bildet bei Senkung des pH-Wertes von 7 auf 6 ein antiparalleles Dimer aus. Es konnte gezeigt werden, dass NTD-eADF3-Proteine, die nur die aminoterminale Domäne N1 enthielten, ebenfalls pH-abhängig Dimere bilden. Bei eADF3-Proteinen mit beiden terminalen Domänen, die per se als CTD-vermitteltes kovalentes Dimer vorliegen, ist die Ausbildung eines aminoterminal vermittelten intermolekularen Dimers-vom-Dimer hingegen nur schwach ausgebildet. Weitere Experimente deuten jedoch darauf hin, dass die NTD in einem intramolekularen Dimer vorliegen. Die Ergebnisse zeigen, dass die CTD einen starken Einfluss auf die Ausrichtung der repetitiven Kerndomäne hat und somit auch die NTD vermittelte intermolekulare Dimerisierung beeinflussen kann.
Die Erkenntnisse wurden verwendet um ein Modell über die molekularen Abläufe während des Spinnprozesses zu erstellen, dass sowohl die Speicherung der Proteine als auch die Assemblierung mit einschließt.
Janus Particles at Interfaces
- This thesis describes the synthesis and the characterization of both polymeric and hybrid Janus particles of well-defined size, shape and functionality and their high potential for applications in colloidal and material science.
Soft Janus particles, based on polystyrene-block-polybutadiene-block-poly(methyl methacrylate) (SBM) triblock terpolymers, represent a fascinating group of polymeric materials because their size, shape and functionality directly influences their adsorption behavior at liquid-liquid interfaces.
The adsorption behavior of Janus cylinders at liquid-liquid interfaces was studied using the pendant drop technique. The interfacial tension decreases with increasing Janus cylinder length and concentration. From the time evolution of the interfacial tension the characteristics of early and late stages of the Janus cylinder adsorption were specified. A series of TEM images of the liquid-liquid interface taken during the cylinder adsorption confirm these observations. Janus cylinders behave differently at the interfaces as compared to the block terpolymer precursor SBM and to cylinders of comparable sizes with a polybutadiene core and a homogeneous polystyrene shell.
Understanding the influence of particle size and architecture on the adsorption process is a very important criterion for an efficient industrial use of the Janus particles. To establish the effect of the Janus character together with the effect of particle shape on the interfacial activity and orientation of the Janus particles at an liquid-liquid interface, we present a combination of experimental and simulation data together with detailed studies elucidating the mechanisms governing the adsorption process of Janus spheres, Janus cylinders and Janus discs. These studies demonstrate that changes in the geometry of the particles strongly influence the stabilization of the liquid-liquid interface. As the shape changes from spheres to discs and cylinders, different adsorption kinetics, different packing behavior, different energy barriers and finally different equilibrium values for the interfacial tension can be found.
Another main point of this thesis was the synthesis of functional and/or stimuli-responsive hybrid core-shell-corona Janus particles based on inorganic colloids and the characterization of their unique properties and fascinating self-assembly behavior. The first step towards these Janus particles was to understand in detail the formation of core-shell-corona particles with a homogeneous corona, and then in a second step, to use our new knowledge to create hybrid core-shell-corona Janus particles.
We developed an easy and completely reproducible preparation and characterization of the solution behavior and functional properties of superparamagnetic and/or fluorescent, thermo-responsive inorganic/organic hybrid nanogels with an intermediate protective silica shell and an interactive polymer layer. These well-defined multifunctional nanogels were prepared via two consecutive encapsulation processes of superparamagnetic and/or fluorescent semiconductor nanocrystals with a silica layer and a crosslinked and responsive polymer poly(N-isopropylacrylamide) (PNIPAAm) corona. The precise adjustment of the conditions allows to achieve a reliable encapsulation and to either entrap several particles or single ones and to precisely tailor the thickness of the silica shell. Full functionality of the encapsulated nanocrystals is retained, but excellent wettability, biocompatibility, flexible surface chemistry, increased chemical stability are implemented together with a thermo-responsive polymer corona.
On the basis of our well-characterized core-shell particles we took advantage of the variable surface chemistry of the silica shell to combine the properties of the superparamagnetic core-shell nanoparticles with the catalytic character of nickel complexes in hybrid core-shell-corona nanoparticles forming heterogeneous nanocatalysts. In that way a heterogeneous catalyst was created for facile product separation in the catalytic conversion of olefins.
In the next level, an efficient strategy for the large-scale synthesis of well-defined hybrid Janus particles with a silica core (˂˂ 100 nm) and a stimuli-responsive PDMAEMA hemicorona was developed. The synthesis is based on a modified version of the Pickering emulsion technique in combination with surface-initiated atom transfer radical polymerization (ATRP) in a “grafting from” approach. First, 30 nm silica nanoparticles are immobilized at the interface of sub-micrometer sized droplets of poly(vinyl acetate). Since the nanoparticles are partially embedded, one hemisphere is protected. After the modification with an ATRP-initiator and the detachment of the modified silica particles, PDMAEMA was grafted from one hemisphere via ATRP. The obtained Janus nanoparticles are well-defined in size and shape and show stimuli-responsive structural changes depending on pH and temperature.
Structural and electronic properties of transition metal nanoalloys and magnetic compounds
- In transition metal clusters, potentially profitable technological applications and fascinating fundamental questions are closely connected. Bimetallic nanoalloys, e.g., have become increasingly popular as their performance in catalysis is often superior to their pure counterparts. Exemplary for this are gold-platinum (Au-Pt) nanoalloys that have been used as highly potent catalysts in electrocatalysis and in a variety of oxidation reactions. However, the mere existence of Au-Pt nanoalloys is astonishing, as Au and Pt cannot be mixed in bulk over a wide range of compositions. Furthermore, how a combination of Au and Pt in nanoalloys results in their special properties has not yet been determined conclusively.
It has been shown in empirical simulations and first-principles density functional theory (DFT) calculations that Au-Pt nanoalloys preferably arrange in a core-shell mixing pattern with Au forming a shell around a Pt core. This is in contradiction to many experimental studies that report the formation of solid solutions of Au and Pt. In the present work, this seeming discrepancy is addressed by simulating x-ray diffraction patterns that are experimentally used to characterize nanoalloys. It is shown that the interpretation of the diffraction patterns relies on questionable assumptions and therefore does not suffice as a definite characterization tool for Au-Pt nanoalloys.
To shed light on the special catalytic properties of Au-Pt nanoalloys under rather different experimental conditions, a thorough investigation of their electronic and structural properties has been carried out. It is found that features favorable for catalysis in Au-Pt nanoalloys emerge as a consequence of combining two fundamental properties: Pt contributes a high density of states close to the Fermi level, which promotes chemical activity. Au increases the structural flexibility of the Au-Pt system, which might be beneficial for the formation of active and element-specific binding sites as well as regeneration of the catalyst after the reaction.
Although DFT offers an attractive compromise between computational effort and accuracy for a theoretical description of Au-Pt nanoalloys, other transition metal compounds severely challenge existing DFT approximations. Manganese (Mn) doped silicon (Si) clusters represent an ideal model system to study the interaction of a single magnetic impurity with a semiconducting host both experimentally and theoretically. The transition from exohedral (lowly coordinated) to endohedral (highly coordinated) doping that occurs for Si clusters with more than ten atoms, is accompanied by complete quenching of the magnetic moment of Mn. We show that MnSi11+, the smallest endohedral cluster found in experiment, suffers strongly from a well-known general problem of most DFT approximations: the self-interaction error. Finally, a universal correlation between magnetic moment and the coordination of the Mn dopant is established that can be generalized to extended systems and suggests a route to stabilize the magnetic moment of bulk Mn-Si compounds.
Synthesis and Self-Assembly of Novel ABC Miktoarm Star Terpolymers
- A novel synthesis for ABC miktoarm star terpolymers and their self-assembly into complex superstructures in aqueous solution are described within this thesis. To this aim a modular route for such materials was developed, combining anionic polymerization and copper-catalyzed azide-alkyne cycloaddition. At the example of ABC miktoarm star terpolymers and an ABA’ miktoarm star copolymer containing a poly(N-methyl-2-vinypyridinium iodide) (P2VPq) segment, the counterion-mediated superstructure-formation of complex shaped aggregates was thoroughly investigated.
The key compound of the combinatorial synthesis is the newly synthesized 4-alkyne-substitued diphenylethylene derivative 1-[(4-(tert-butyldimethylsilyl)ethynyl)phenyl]-1-phenylethylene (“click-DPE”). This was applied in sequential anionic polymerization to prepare well-defined alkyne mid-functional diblock copolymers composed of polybutadiene (PB) as first and poly(tert-butyl methacrylate) (PtBMA), poly(2-vinylpyridine) (P2VP), or poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA) as second block. The alkyne-midfunctional diblock copolymers were afterwards conjugated with azido-functional polystyrenes (PS), poly(ethylene oxide) (PEO), PtBMA and PDMAEMA to successfully obtain different novel ABC miktoarm star terpolymers with narrow molecular weight distributions.
For an ABC miktoarm star terpolymer consisting of arms of PB, PtBMA and P2VP it was demonstrated that after quaternization with methyl iodide (yielding BVqT) and dialysis to water the nature of the counterion allows for manipulation of the obtained structures. The miktoarm star architecture together with iodide as counterion is essential for this directed self-assembly. Transformation of iodide to triiodide, via the addition of iodine before dialysis to water, decreases the hydrophilicity of the P2VPq corona and therefore induces the directed self-assembly of spherical micelles with a PB/PtBMA core, into cylinders, superstructures thereof and finally barrel-shaped aggregates of up to 1 µm with an internal lamellar fine structure. Based on their appearance in transmission electron micrographs these were termed “woodlouse” aggregates. The compact particles consist of alternating lamellae of a partially demixed PB/PtBMA phase and a swollen P2VPq phase.
The general applicability of this counterion-mediated hierarchical self-assembly was furthermore demonstrated by using two other miktoarm star systems. For three ABC miktoarm star terpolymers of different composition, consisting of PB, PS and P2VPq segments (BVqS), a dependence of the morphology on the fraction of the hydrophilic block was determined, in analogy to diblock copolymers. For long P2VPq blocks stacked lamellar/disk-like structures evolve from micellar building units. In contrast, a short P2VPq segment yields multilamellar vesicles via fusion of vesicular primary building blocks. The vesicle walls are supposed to consist of a lamellar structure with the PB phase in the centre, shielded from the P2VPq corona by thin PS layers. At the example of one BVqS miktoarm star terpolymer the successful formation of nanohybrids containing gold nanoparticles within the P2VPq phase is demonstrated.
In the second system the low-Tg PB segment was replaced by a second PS block of different length (SVqS’). Even though vesicles serve as initial building units, the triiodide-induced superstructure formation leads to anisotropic aggregation of deformed vesicles, rather than to the fusion into multilamellar vesicles. This is attributed to the two glassy PS core blocks which minimize the dynamics during self-assembly and allow only minor rearrangement of the aggregated structures. Similar to the “woodlouse” aggregates from BVqT, lamellar structured particles of elongated shape were obtained from SVqS’, despite vesicles serving as primary building units. Consequently, the presented triiodide-directed self-assembly into complex superstructures is not restricted to miktoarm star polymers containing a low-Tg segment, as the rearrangement processes take place during the dialysis process, where the organic co-solvent enables sufficient mobility of the core-forming blocks.
Besides the introduction of a novel synthetic approach for the construction of miktoarm star terpolymers and the synthetic advance of the alkyne-functionalized DPE, the presented triiodide-mediated superstructure formation represents an interesting concept for directed self-assembly processes.
Impact of time and spatial averages on the energy balance closure
- Secondary circulations are large and relatively stationary eddies, which are caused by the surface heterogeneity and normally reside away from the ground. They are believed to be the cause of the energy balance closure problem at the earth's surface, because their contribution to the turbulent fluxes is missed by a fixed eddy-covariance tower measurement that has a typical averaging time of 30 minutes. In this thesis, data from the LITFASS-2003 experiment was used to investigate the impact of time and spatial averages on the energy balance closure. This data consisted of many observations over a large heterogeneous landscape that could generate secondary circulations; some of which might be still near the earth's surface.
For the time average analysis, the averaging time was extended to increase the possibility that secondary circulations were picked up by the sensor. Two approaches, which were the modified ogive analysis and the block ensemble average, were applied to analyze the data from ground-based measurements. The modified ogive analysis requiring a steady state condition, could extend the averaging time up to a few hours and suggested that an averaging time of 30 minutes was still overall sufficient for the eddy-covariance measurement over low vegetation. The block ensemble average, on the contrary, did not require a steady state condition, but could extend the averaging time to several days. However, this approach could only improve the energy balance closure for some sites during specific periods, when secondary circulations existed in the vicinity of the sensor. Based on this approach, it was found that the near-surface secondary circulations mainly transported sensible heat, which led to an alternative energy balance correction by the buoyancy flux ratio approach, in which the attribution of the residual depended on the relative contribution of the sensible heat flux to the buoyancy flux. The fraction of the residual attributed to the sensible heat flux by this energy balance correction was larger than in the energy balance correction that preserved the Bowen ratio.
In the spatial average analysis, two energy balance correction approaches, the buoyancy flux ratio and the Bowen ratio approaches, were applied to the area-averaged fluxes (composite fluxes) in order to include contribution from secondary circulations. These composite fluxes were aggregated from multiple ground-based measurements. The energy balance corrected fluxes were validated against the spatial average fluxes, which were measured by an aircraft and a large aperture scintillometer (LAS). In this validation, the backward Lagrangian footprint model was used to estimate the source area of the measurement. It was found that both energy balance correction approaches did improve the agreement between time and spatial averages fluxes. This suggested that the contribution from secondary circulations could be properly accounted by the energy balance correction.
All findings in this thesis, therefore, depict that secondary circulations significantly transport energy in the atmospheric surface layer. The energy balance correction, accomplished by using either the Bowen ratio approach or the buoyancy flux ratio approach, is necessary to estimate the actual vertical transport of energy at the earth's surface.
Foamy Virus RNase H - Aktivität, Struktur und Funktion
Das für die Replikation des RNA-Genoms von Foamy Viren (FV) notwendige Enzym, die Protease-Reverse Transkriptase (PR-RT), beinhaltet die Protease-, die Polymerase- und RNase H-Domäne. Letztere ist für den Abbau der RNA im entstehenden RNA/DNA Hybrid verantwortlich. Während die FV PR-RT als Monomer vorliegt, besteht die HIV-1 RT aus einem p66/p51-Heterodimer. Erstaunlicherweise ist die isolierte HIV-1 RNase H im Vergleich zur z.B. E. coli oder separaten MoMLV RNase H nicht aktiv. Aus den Sequenzvergleichen verschiedener RNase H-Domänen ergibt sich, dass die Prototyp FV (PFV) RNase H im Gegensatz zur HIV-1 RNase H einen Sekundärstrukturbereich aufweist, bei dem es sich um die sogenannte C-Helix mit einer sich anschließenden basischen Schleife (basic protrusion) handelt. Da zu Beginn der Arbeit keine 3D-Struktur einer retroviralen RNase H mit basic protrusion bekannt war, sollte in dieser Arbeit die Struktur der PFV RNase H und die Funktion der basic protrusion bei der Substratbindung geklärt werden.
Die Tertiärstruktur der PFV RNase H-Domäne konnte mit NMR-Spektroskopie gelöst werden. Somit war es möglich, die basic protrusion einschließlich der C-Helix zu identifizieren. Die isolierte RNase H-Domäne zeigte in fluoreszenzbasierten Tests sowie in qualitativen RNase H-Versuchen mit radioaktiv markiertem Substrat Aktivität. Um die Funktion der C-Helix und der sich anschließenden basischen Schleife bei der Substratbindung zu analysieren, wurden NMR-Titrationsexperimente durchgeführt. Dafür wurde die PFV RNase H-Domäne zunächst durch den Austausch der zwei katalytisch wichtigen Reste Aspartat 599 und Histidin 724 zu Asparagin inaktiviert (RNase H-(D599N-H724N)), um den Abbau des Substrates während der Messungen zu vermeiden. Die Auswertung von [15N, 1H]-HSQC- und [1H, 15N, 1H]-NOESY-HSQC-Spektren erbrachte eine Übereinstimmung der Tertiärstrukturen der RNase H-(D599N-H724N) mit der wt RNase H.
Die NMR-Titrationsexperimente zeigten, dass die C-Helix in der PFV RNase H wie ein Lineal agiert, das die sich anschließende basische Schleife zum Substrat orientiert. Darüber hinaus besitzt die basic protrusion zusätzlich eine Reihe an positiv geladenen Resten, die gut lösungsmittelzugänglich sind und dadurch erste Kontakte mit dem Substrat ermöglichen. Damit bietet die basic protrusion eine Art Plattform für die Substratbindung. Der HIV-1 RNase H fehlt nicht nur die C-Helix, zusätzlich ist die sich anschließende Schleife vermutlich zu kurz, um das Substrat zu binden. Da diese Schleife außerdem nur über einen basischen Rest verfügt, ist wahrscheinlich auch die Gesamtaffinität dieses Bereichs für die Substratbindung zu gering. Strukturvergleiche der PFV RNase H mit der HIV-1 RT zeigen, dass die fehlende basic protrusion der HIV-1 RNase H durch eine Schleife aus der Verbindungs-Subdomäne der p66-Untereinheit kompensiert wird. Dieser Bereich könnte somit ein neuer Angriffspunkt für Inhibitoren in der antiretroviralen Therapie bei HIV 1 sein.
Um zukünftig weitere Strukturanalysen mit der PR-RT bzw. mit einzelnen Domänen durchführen zu können, wurden in einem weiteren Projekt verschiedene N- und C-terminale Deletionsvarianten der PR-RT des Simian Foamy Virus hergestellt. Durch Aktivitätstests mit diesen Deletionsvarianten konnten die Abgrenzungen für die PR-, die Polymerase-, die RNase H-Domäne und die Verbindungs-Subdomäne in der PR-RT identifiziert werden. Dabei zeigte sich, dass die Region H107-N143 C-terminal von der PR wichtig für die Funktion der Polymerase ist. Die Deletion der RNase H-Domäne und Verbindungs-Subdomäne führte zu einer drastischen Abnahme der Substrataffinität, Integrität und Polymerisationsfähigkeit des Enzyms. Trotzdem konnte eine minimale Polymerase-Domäne bestimmt werden (RT(107 454)), die ohne PR- und RNase H-Domäne sowie ohne die Verbindungs-Subdomäne in der Lage ist, zu polymerisieren. Für die Dimerisierung und damit Aktivierung der PR, die durch Bindung von zwei PR-RTs an das sog. PARM-Element (engl. protease activating RNA motif) auf der genomischen RNA geschieht, sind jedoch die RNase H-Domäne und die Verbindungs-Subdomäne unverzichtbar. Dadurch ist die RNase H nicht nur für die reverse Transkription essenziell; in FV stellt sie auch ein Regulationselement für die PR-Aktivierung dar und ist somit indirekt an der Prozessierung von Gag (Strukturproteine) und Pol (virale Enzyme) beteiligt.
Functional Triblock Terpolymers for Multicompartment Micelle and Janus Particle Synthesis
- This thesis describes the synthesis of ABC triblock terpolymers with functional moieties via living anionic polymerization, followed by Janus particle (JP) and multicompartment micelle (MCM) synthesis from the as prepared triblock terpolymers.
A synthesis method that can accomplish the tasks of the preparation of spherical as well as non-spherical JPs that are well-defined and in the nanometer size range is based on converting self-assembled triblock terpolymer bulk structures via selective cross-linking of the middle block. Until now such soft JPs were prepared mainly from polystyrene-block-polybutadiene-block-poly(methyl methacrylate) and polystyrene-block-polybutadiene-block-poly(tert-butyl methacrylate). However, these polymers do not offer many possibilities of chemical alterations and stimuli-responsive elements.
Therefore, potential new functional monomers for the use in JP synthesis from triblock terpolymer bulk structures were identified and their anionic polymerization examined, p-tert-butoxystyrene (tS) and 4-(dimethylaminomethyl)styrene (DMAMS). Polymers with low polydispersity indices could be prepared from both monomers in tetrahydrofuran (THF) with sec-butyllithium (sec-BuLi) as initiator.
Poly(p-tert-butoxystyrene) (PtS) was hydrolyzed to poly(p-hydroxystyrene) (PHS) which is water-soluble at high pH values, opening the possibility to prepare water-soluble JPs. The pH-responsive behavior of poly(4-(dimethylaminomethyl)styrene) could be confirmed and for the first time an LCST behavior was documented with cloudpoints of 59.3 °C at pH 7 and 28.5 °C at pH 8.
PtS was then used in the preparation of two triblock terpolymers, poly(tert-butoxystyrene)-block-polybutadiene-block-poly(tert-butyl methacrylate) (tSBT) and poly(tert-butoxystyrene)-block-polybutadiene-block-poly(2-(dimethylamino)ethyl methacrylate) (tSBD). tSBT exhibited a lamella-cylinder (lc) bulk morphology with polybutadiene (PB) spheres surrounded by alternating lamellae of PtS and poly(tert-butyl methacrylate) (PtBMA). However, the bulk structure of tSBD consisted of a symmetrical lamella-lamella pattern that is not suitable for JP synthesis.
From tSBT bulk material, three different types of non-spherical JPs could be obtained. Photo-cross-linking of the lamella-cylinder (lc)-morphology by co-casting a radical photo-initiator and UV exposure resulted in the expected Janus cylinders. When the bulk material was first swollen in acetonitrile and cross-linked by cold vulcanization, Janus sheets were obtained. Swelling in acetonitrile/decane emulsion lead to a new type of JPs, Janus ribbons. In both cases a phase transition had occurred; in case of the Janus sheets a thin PB layer had formed between the original PB cylinders, resulting in an undulated-lamella morphology. For the formation of Janus ribbons a connecting PB phase had formed in every second interspace along the major axis of the cylinders. Casting a tSBT film from tert-butanol, a non-solvent for PB, also enabled the synthesis of spherical JPs. This way, the importance and versatile application of swelling agents and cross-linking methods for the preparation of JPs from bulk structures was demonstrated. The obtained Janus cylinders were hydrolyzed to have one PHS and one poly(methacrylic acid) (PMAA) hemicylinder, resulting in water-soluble particles.
Further, solution structures of tSBD and tSBT triblock terpolymers were investigated. In water, tSBD formed core-corona micelles that exhibited pH-responsive and LCST behavior due to the responsive poly(2-(dimethylamino)ethyl methacrylate) corona. Employing the novel method of directed hierarchical self-assembly, “football” MCMs were obtained from tSBD whereas tSBT formed “clover” structures. Cross-linking of the B block in such MCMs and their subsequent dissolution in a solvent for all three blocks can be used to prepare spherical JPs. However, as tSBD MCMs existed in water, no sufficient cross-linking method could be found to cross-link the organic PB phase within the aqueous solution. For tSBT MCMs in ethanol the standard approach of adding a photo-initiator to the MCM solution followed by UV exposure was successfully employed and spherical JPs were obtained. These were again hydrolyzed to acquire water-soluble JPs. Apart from some isolated single Janus spheres, cryogenic transmission electron microscopy mainly revealed the formation of “clover” and “hamburger” oligomers possibly due to the slightly better solubility of PMAA. Here, the applicability of the concept of directed hierarchical self-assembly to create MCMs was demonstrated for two different triblock terpolymers and the corresponding method of solution-based JP synthesis was successfully conducted for tSBT.
"The New Chemistry" - Sustainable Catalysis with Alcohols
- Subject of the thesis are new iridium complexes stabilized by anionic P,N- or P,N,P-ligands. These complexes were used in homogeneous catalysis. Furthermore, mechanistic studies were performed to provide an insight into the catalytic cycles. Synthesis protocols for a multitude of different product classes have been developed.
The iridium complex 1, stabilized by a neutral P,N-ligand, reacts under basic conditions with 2-aminopyridines. By elimination of dipyridylamine the new catalyst species 2a was formed, which is more stable than catalyst 1.
Based on this finding eight new anionic P,N-ligands and the resulting iridium complexes were synthesized.
After optimization of the reaction conditions (solvent, base, temperature and catalyst loading) these catalysts were used in BH (borrowing hydrogen)/HA (hydrogen autotransfer) reactions. The selective monoalkylation of anilines with primary alcohols was investigated. In comparative experiments the superiority of the new class of catalysts versus the original catalyst 1 was clearly shown. Under mild reaction conditions (70 °C) the selectivity profile with respect to the monoalkylation has been preserved.
The catalytic protocol was subsequently extended to the alkylation of aromatic diamines. Therefore various diaminobenzenes were used as substrates. Also Dapsone®, an important drug in treatment of leprosy could be used as starting material. We succeeded in both symmetric and unsymmetric monoalkylations of diamines. Due to the selectivity profile of the catalyst regarding aromatic amines, also unprotected amino alcohols could be used as alkylating reagents.
By the use of tridentate P,N,P-ligands, a novel class of more stable catalysts compared to complexes 2a-9a, could be developed. Due to sealing the synthesis reactor with a semipermeable membrane, the retransfer of the “borrowed” hydrogen could be prevented and H2 is released. Dehydrogenation and condensation steps are now possible instead of BH/HA. By reacting secondary alcohols with β-amino alcohols, pyrroles were accessible.
After adapting the synthesis protocol to this new class of products the tolerance of functional groups was tested. Diversely functionalized alcohols were used. Under mild reaction conditions (90 °C) and very low catalyst loadings (down to 0.03 mol% iridium), a large number of novel pyrroles was accessible. Using this protocol 21 differently substituted α,α-pyrroles, 12 bicyclic pyrroles, symmetrically as well as non-symmetrically substituted oligopyrroles and three β-aminopyrroles were synthesized. The catalyst resting state was identified by NMR experiments and X-ray structure analysis to be an iridium trihydride. This trihydride is formed under catalytic conditions, by treatment of the pre-catalyst with alcohols or in hydrogen atmosphere.
In the final part of the work, a catalytic pyridine synthesis was developed. In this so far unknown heterocycle synthesis up to four different substituents could be introduced within a single reaction step. 2,6-, 2,5-, 2,4- and 2,3-substituted pyridines were synthesized selectively by using variously substituted primary or secondary alcohols and γ-amino alcohols. Furthermore, both the synthesis of bicyclic pyridines as well as the synthesis of pyridines that bear chiral substituents is possible.
Soil erosion and conservation potential of row crop farming in mountainous landscapes of South Korea
- Soils play an essential role for mankind because they provide fundamental ecosystem services required for human life, primarily for the production of food by providing the environment for plant growth. However, soils worldwide became highly threatened by human induced degradation, especially as a consequence of accelerated erosion by water during recent decades. In consideration of climate change and an increasing food demand of a rising population, there is an urgent need to conserve the soil resources by implementing effective erosion control measures for agricultural production. The effective implementation of those measures strongly depends on the specific conditions of particular regions and requires the analysis of the existing farming systems and their capability for erosion control.
Objective of this thesis is the analysis of the major agricultural practices applied for row crop cultivation in mountainous watersheds of South Korea with respect to water erosion and the identification of their conservation potential. Our first two studies analyze the subsurface flow processes, the runoff patterns, and the associated erosion rates of the widely applied plastic covered ridge-furrow system (plastic mulch), and our third study investigates the impact of herbicide applications on erosion associated with conventional and organic farming. To analyze the flow processes induced by the plastic mulch cultivation, we conducted four irrigation experiments on potato fields that represent a smooth surface, uncovered ridges, and plastic covered ridges with and without a developed crop canopy. With an automatic sprinkler, we irrigated small plots with a dye tracer solution of Brilliant Blue and potassium iodide, collected surface runoff, and excavated soil profiles to visualize the subsurface flow patterns, which were subsequently analyzed by image index functions. We found that the ridge-furrow system, especially when ridges are covered with plastic, decreased infiltration and generated high amounts of surface runoff, whereas a developed crop canopy increased infiltration due to interception and stem flow. The analyses of the subsurface flow patterns show that the plastic covered ridge-furrow system induces preferential infiltration in furrows and planting holes due to its topography and the impermeable covers, but that the impact on flow processes in the soils is relatively small compared to the impact on runoff generation. To identify the patterns of overland flow and the erosion rates associated with the plastic mulch system, we installed runoff collectors to monitor runoff and sediment transport of two potato fields with concave and convex topographies, and we applied the EROSION 3D model to compare the plastic covered ridge-furrow system to uncovered ridges and a smooth surface. We found that plastic mulch cultivation considerably increases soil erosion compared to uncovered ridges as a consequence of high amounts of surface runoff. Our results show that the ridge-furrow system concentrated overland flow on the concave field, resulting in severe gully erosion, but prevented flow accumulation and reduced erosion on the convex field, which demonstrates that the effect of this cultivation strategy is primarily controlled by the field topography and its orientation. To analyze the effects of conventional and organic farming on water erosion, we measured multiple vegetation parameters of crops and weeds of conventional and organic farms cultivating bean, potato, radish, and cabbage, and we simulated long-term soil loss rates with the Revised Universal Soil Loss Equation (RUSLE). We found that organic farming reduced erosion for radish, as a result of an increased weed biomass due to the absence of herbicides, but that it increased erosion for potato due to lower crop coverage, presumably as a consequence of crop-weed competition or herbivory associated with the absence of agricultural chemicals. Although we demonstrated that a developed weed cover in the furrows can potentially decrease the erosion risk for row crops, our results show that the average annual erosion rates of both farming systems exceed by far any tolerable soil loss.
In consideration of the generally high soil loss found in our studies, we conclude that the applied farming practices are not capable for effective erosion control and soil conservation in this region. However, based on our findings, we could identify possible modifications of those practices that can help to reduce the risk of erosion in the future. We recommend perforated plastic covers for ridges to reduce runoff generation, and the orientation of the ridge-furrow system along the contours or towards field edges to prevent flow accumulation and gully formation. Additionally, we suggest residue mulching of furrows to protect the soil surface from overland flow, and the cultivation of winter cover crops after harvest to maintain a better soil cover throughout the year.