Universität Stuttgart
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Item Open Access Spectroscopic investigations of the magnetic anisotropy of lanthanide- and cobalt-based molecular nanomagnets(2016) Rechkemmer, Yvonne; Slageren, Joris van (Prof. Dr.)Single-molecule magnets are metal complexes exhibiting an energy barrier for spin reversal, leading to magnetic bistability and slow relaxation of the magnetization. Their potential for practical applications such as high-density magnetic data storage was recognized early on and with the goal of achieving high energy barriers, different kinds of single-molecule magnets have been synthesized. The quadratic dependence of the barrier height on the spin motivated chemists to synthesize metal complexes with very high total spins; however, with limited success. It was shown that high spins come along with low anisotropies and increased interest thus focused on the synthesis and investigation of (mononuclear) complexes of highly anisotropic metal centers, e.g. lanthanide or cobalt complexes. Although rather high energy barriers can be achieved in such systems, practical application remains problematic and has not been realized yet. Reasons are for example the lack of rational design criteria and the complex interplay of different magnetic relaxation pathways. The aim of this work was therefore the comprehensive magnetic and spectroscopic investigation of selected molecular lanthanide and cobalt compounds in order to obtain a deeper insight into the correlation of molecular and electronic structures as well as the corresponding magnetic properties. The applied spectroscopic methods included electron paramagnetic resonance spectroscopy, far-infrared spectroscopy and optical methods. Special emphasis was placed on magnetic circular dichroism (MCD) spectroscopy, which served as a main tool for electronic structure determination. However, since the MCD-spectrometer was not part of the available experimental equipment at the University of Stuttgart, its design, setup and characterization were the first part of this work. In the further course of this work MCD-spectroscopy was employed for the electronic structure determination of selected lanthanide and cobalt compounds. The studied lanthanide compounds were literature-known molecular tetra-carbonates of erbium (1-Er) and dysprosium (1-Dy). Detailed magnetometric studies showed that both 1-Er and 1-Dy are field-induced single-molecule magnets; however, 1-Er and 1-Dy show significant differences in their magnetic relaxation behavior. The magnetic studies were complemented by detailed spectroscopic investigations.The combination of far-infrared-, luminescence- and MCD-spectroscopy allowed for the experimental determination of 48 energy levels for 1-Er and 55 levels for 1-Dy, which built the foundation for the subsequent crystal field analysis and electronic structure determination. In addition, the results of EPR-spectroscopic studies were used for fine-tuning and verifying the respectively determined crystal field parameters. Calculating the magnetic dipole strengths for transitions between the relevant states led to a quantitative understanding of the magnetic relaxation pathways. Besides the investigation of lanthanide compounds, this thesis deals with two classes of cobalt complexes. The first class comprises mononuclear complexes in which one Co(II) ion is ligated by the nitrogen donors of two doubly deprotonated 1,2-bis(methanesulfonamido)-benzene-ligands. Rather acute N-Co-N bite angles indicate strong deviations from ideal tetrahedral symmetry. The static magnetic properties hint at very high energy barriers for spin reversal and with the help of far-infrared spectroscopy, largely negative axial zero-field splitting parameters were determined. The corresponding energy barriers belong to the highest ever reported for 3d-transition metal complexes and investigating the dynamic magnetic properties confirmed single-molecule magnet behavior. The unique magnetic properties were fully explained by analyzing spectroscopic results. The MCD-spectra showed intense signals that were assigned to spin-allowed d-d-transitions. Subsequent crystal field analysis revealed that the strong axial crystal field generated by the ligands leads to a large splitting of the electronic terms and thus in turn to a relatively small energy gap between the electronic ground state and the first excited state. The resulting increase in second-order spin-orbit coupling explains the high energy barriers observed in the studied complexes. The second class of cobalt compounds studied in this work included dimers of distorted octahedrally coordinated Co(II) ions bridged by symmetrical or asymmetrical quinone based bridging ligands. The main focus of investigation lay on the impact of the bridging ligand on the magnetic coupling between the cobalt centers. Thus, the magnetic properties of the complexes were studied with the help of static susceptibility and magnetization measurements and analyzed by means of different models. Depending on the bridging ligand, different signs for the exchange coupling constants were found. The varying signs can be explained by different relative contributions of possible exchange paths, influenced by the different substituents at the bridging ligands or slight geometry differences. The observations indicate that electron withdrawing substituents favor ferromagnetic couplings, which are preferred in the context of molecular magnetism. All in all, it can be concluded that this work provides a contribution to the deeper understanding of the features relevant for single-molecule magnets. The electronic structure determination for selected lanthanide and cobalt complexes applying advanced magnetometric and spectroscopic techniques not only led to an understanding of the static and dynamic magnetic properties but also allowed for the development of design criteria and new approaches for improved single-molecule magnets in the future.Item Open Access Enzymatic asymmetric dihydroxylation of alkenes(2016) Gally, Christine; Hauer, Bernhard (Prof. Dr.)The introduction of chirality into C=C double bonds is of special interest in organic synthesis. In particular, the catalytic asymmetric dihydroxylation (AD) of alkenes has attracted considerable attention due to the facile transformation of the chiral diol products into valuable derivatives. By chemical means, the metal-catalyzed AD of olefins provides both stereo- and regiospecific cis-diol moieties. Next to their toxicity, however, these metal catalysts can also lead to byproduct formation as a result of oxidative fission. In nature, Rieske non-heme iron oxygenases (ROs) represent promising biocatalysts for this reaction since they are the only enzymes known to catalyze the stereoselective formation of vicinal cis-diols in one step. ROs are key enzymes in the degradation of aromatic hydrocarbons and can target a wide variety of different arenes. Despite their broad substrate scope, limited data is available for the conversion of unnatural substrates by this class of enzymes. To explore their potential for alkene oxidation, three ROs were tested for the oxyfunctionalization of a set of structurally diverse olefins including linear and cyclic arene-substituted alkenes, cycloalkenes as well as several terpenes. Naphthalene- (NDO), benzene- (BDO) and cumene dioxygenases (CDO) from different Pseudomonas strains where selected as they are amongst the RO enzymes that have already been reported to catalyze the oxidation of a small number of olefins. The majority of compounds from the selected substrate panel could be converted by NDO, BDO or CDO and products were either isolated and identified by NMR analysis or using the authentic standards. Dependent on the substrate, allylic monohydroxylation was found in addition to the corresponding diol products, a reaction which is chemically still most reliably achieved by the use of SeO2 in stoichiometric amounts. However, having been evolved for the dihydroxylation of aromatic compounds, wild type ROs displayed low conversions (< 50%) and modest stereoselectivities (≤ 80% ee/de) for several of the tested olefins. To overcome these limitations, changes in the active site topology of RO catalysts were introduced. A single targeted point mutation that was identified based on sequence and structural comparisons with other members of the RO family proved to be sufficient to generate BDO and CDO variants displaying remarkable changes in regio- and stereoselectivity for various substrates. In particular biotransformations with CDO M232A gave excellent stereoselectivities (≥ 95% ee/de) and good activities (> 90%) also for linear alkenes, which have been reported to be challenging substrates for RO-catalyzed oxyfunctionalizations. Site-saturation mutagenesis at position 232 in CDO revealed a correlation between the steric demand of the amino acid side chain and its influence on regio- and/ or stereoselectivities for styrene and indene. While the wild type enzyme almost exclusively catalyzed the dihydroxylation of the aromatic ring, the regioselectivity was shifted with decreasing side chain size to the terminal vinyl group of styrene, yielding up to 96% of the alkene-1,2-diol. For cis-1,2-indandiol formation, enantiocomplementary enzymes could be generated, a fact further highlighting the importance of position 232 for the engineering of ROs. Moreover, site-saturation mutagenesis of additional residues in the substrate binding pocket of CDO (F278, I288, I336 and F378) identified further positions having an influence on selectivity and product formation for alkene oxidation. To proof the applicability of ROs for organic synthesis, semi-preparative scale biotransformations (70 mg) of selected substrates were performed with CDO M232A. Without further optimization of the reaction set-up, products were successfully isolated in > 30% yield. In addition, up-scaling of (R)-limonene hydroxylation to 4 L in a bioreactor with growing cells gave final isolated product titers of 0.4 g L-1 even though substrate volatility and product toxicity diminished the yield. In conclusion, these examples demonstrated that a single point mutation was sufficient to transform CDO wild type into an efficient catalyst, furthermore constituting the first example of the rational engineering of CDO and BDO enzymes for the oxyfunctionalization of a broad range of alkenes.Item Open Access Ion beam lithographic and multilayer fresnel zone plates for soft and hard X-rays: nanofabrication and characterization(2015) Keskinbora, Kahraman; Schütz, Gisela (Prof. Dr.)X-ray microscopy has become an important analytical characterization method for a plethora of applications in materials science, physics, chemistry and biology, thanks to the emergence of modern synchrotron radiation facilities. These facilities enable high brilliance, energy tunable, variable polarization X-rays which gives access to mass density, elemental, chemical, electronic and magnetic properties of materials. In the soft X-ray energies nearly all elements can be probed by spectromicroscopic methods. Another important property of synchrotron radiation is the time structure in the ns to ps range, which can be utilized for sophisticated time resolution studies. These opportunities can be combined with high spatial resolution which is determined by the focusing method and the optic. Focusing of X-rays has historically been a difficult task due to strong absorption and weak phase shift of X-rays within matter. The required phase shift of X-rays, which depends on the real part of the complex refractive index, differs from 1 (the vacuum refractive index) only on the order of 10^-2 to 10^-6 and conventional lenses do not work. One very successful X-ray optic is the Fresnel Zone Plate (FZP), a diffractive optic that act as a lens under certain conditions and can focus X-rays to nanometer sized spots. The resolution of the FZP depends on the width of the outermost zone and is highly correlated with the smallest feature that can be fabricated. Conventionally, the e-beam lithography (EBL) is used for production FZPs which could resolve up to 10 nm structures with serious limitations. One difficulty of EBL is its ever increasing complexity for many-step fabrication of smaller features or intricate geometries. Therefore, EBL is mostly constrained to planar, binary geometries with moderate efficiencies strongly decreasing with energy and not effective for hard X-rays. Special 3D geometries in the form of kinoform lenses can theoretically have 100 % focusing efficiencies. Attempts to approximate these geometries via EBL increased the number of process steps even further. The smallest FZP feature size even for low aspect ratios achievable via EBL is fundamentally limited due to the proximity effect which is the interaction and spread of electrons within the resist material. We addressed these issues by focusing our research on alternative FZP fabrication techniques as high-speed ion beam lithography (IBL), and gray scale ion lithography to realize efficient kinoforms. Another approach towards full-material multilayer FZPs with infinite aspect ratio was based on atomic layer deposition (ALD) with subsequent ion beam slicing. Each of these three methods targets specific challenges faced by the e-beam lithography based FZP fabrication techniques. All the fabricated FZPs were tested for their resolution and efficiency performances at a state of the art scanning transmission X-ray microscope at BESSY for soft X-rays and/or at optical test stations at ESRF and PETRA III for hard X-rays. Using IBL the rapid preparation of a 110 nm thick Au FZP with 50 µm diameter and 50 nm ∆r in less than 13 minutes is demonstrated. Employed for X-ray microscopy, the FZP clearly resolved 28.5 nm features with a cut-off of 24.3 nm at ~1120 eV. Additional process improvements were made towards smaller zones with higher zone quality. They allowed the preparation of a FZP with 30 nm outermost half-period remarkably, in about 8 min. This FZP was shown to clearly resolve 21 nm features on a multilayer test object with large room for improvement. This high through-put FZP production route is of special interest not only concerning the low cost and easy availability. A large array of these optical components is attractive, for experiments such as one-shot ultra-high brilliance FEL investigations due to the radiation damage or for instance for coded-aperture arrays for high-angle resolving X-ray astronomy. Towards fabrication of kinoforms for high efficiency X-ray focusing, we have performed various materials optimization studies in order to achieve a high surface quality optic. After various trials the materials were finally optimized and the fabricated lenses achieved more than 14 % absolute diffraction efficiency that is almost 90 % compared to the theoretical prediction. This confirms how closely we were able to replicate the ideal three dimensional surface relief structure for the first time. It was possible to carry out imaging with these lenses with half-pitch resolutions down to 60 nm. The kinoform lenses were tested at the soft X-ray range where a significant absorption is present in materials. These results also potentially pave the way for very high efficiency hard X-ray focusing which can in principle be utilized in laboratory based X-ray sources, X-ray astronomy and the new rising field of X-ray ptychography. To fabricate high resolution ML-FZPs, Al2O3/Ta2O5multilayers, deposited on a smooth glass optical fiber via atomic layer deposition using non-dedicated instruments were carefully cut-out, sliced and polished to a high quality surface finish using focused ion beams. Following the transfer of the slice to a TEM grid as holder the slices were polished to a high surface finish quality, also via a focused ion beam. Fabricated ML-FZPs were synchrotron tested using an in-house constructed 2-axis tilt stage specially designed for aligning ML-FZP with respect to the X-ray optical axis. The results showed that it was possible to resolve 21 nm features in direct imaging at 1200 eV and sub-30 nm focusing at 8 keV. This is the highest demonstrated resolving power for a multilayer type FZP, to date to the best of our knowledge. Results exhibit the potential for high-resolution hard X-ray focusing where this type of optics are especially efficient. For ultra-high resolution hard and soft X-ray imaging, with potentially achievable ∆r of a few nm is well below what can be achieved through any lithography method available today.Item Open Access In situ characterization of phase evolution in LiFePO4(2015) Ohmer, Nils; Maier, Joachim (Prof. Dr.)Among the candidates for electrodes in future Li-based batteries, LiFePO4 (LFP) is one of the most important and most frequently studied materials, undergoing a phase transformation upon delithiation to FePO4 (FP). In spite of the great scientific and practical interest in this material, there is still an extensive debate on the mechanism of this phase transformation and the underlying factors of influence. Within the framework of this thesis, first studies are carried out ex situ on multi-particle, full electrode LFP materials, being electrochemically cycled and analyzed at various states of charge by a combination of highly spatially resolved methods (high-resolution transmission electron microscopy and electron energy loss spectroscopy (HRTEM, EELS)) and integral measurement techniques (analyzing the X-ray diffraction and X-ray absorption near edge structure (XRD, XANES)). This combination of characterization techniques allows one to distinguish between the cycling behaviour of differently sized crystallites within the same electrode. It is found that for electrodes with hydrothermally grown LFP as active material, a particle size dependent cycling behaviour exists, with nanosized particles apparently not participating in the charging process at all. A turbostratic stacking of layers in these nanosized particles is found and identified to be responsible for sluggish lithium insertion and extraction. These higher dimensional defects prevent the small particles from participating in the charging process, most likely by disturbing the lithium diffusion along the 1-dimensional channels, as well as impair the transport along the other directions in the LFP host structure and thus blocking the lithium transport, resulting in a comparibly lower practical capacity during electrochemical cycling. To study the lithium exchange mechanism upon charging a LFP thin film cathode, an all-solid-state thin film battery cell with a lateral design concept is developed and realized by pulsed laser deposition (PLD) and thermal evaporation techniques. Using PLD and shadow masks LFP cathode, Li2O-V2O5-SiO2 (LVSO) electrolyte and LiAl anode thin films are deposited sequentially in a way that the Li transport pathway in the resulting battery is along the X-ray transparent commercial Si3N4 membrane substrate. This enables the usability of synchrotron-based energy resolved scanning transmission X-ray microscopy (STXM) with its high chemical and spatial resolution to perform in situ absorption measurements at the Fe L3 edge. Upon delithiation, a shift in the main absorption feature from 708 to 710 eV is used to fingerprint the change in the local state of charge, identifying areas containing Fe2+ (lithiated) and Fe3+ (delithiated), respectively. The initial lithiation process of a LFP thin film cathode material has been followed by in situ STXM, with a lateral resolution of 30 nm, during electrochemical charging of the thin film battery. The observed initial lithiation process does not follow the classical particle by particle mechanism, typical for multi-particle LFP cathodes, but instead a rather simultaneous, although inhomogeneous, lithiation is observed. The reason for this change in mechanism, compared to multi-particle powder electrodes, is found in mechanical interactions within the thin film upon lithiation, i.e. in the corresponding volume expansion and formation of high energy surfaces, changing the shape of the single-particle chemical potential to a monotone form upon lithiation. This has far-reaching consequences: not only the many-particle mechanism is changed to a concurrent lithiation, but also the single-particle mechanism is changed from a two-phase to a single-phase mechanism upon lithiation. Furthermore, a vanishing hysteresis loop and the disappearing of the memory effect is predicted. These findings are rather general and applicable to all kind of thin films of phase separating intercalation materials, undergoing a volume change upon lithium exchange. To fill the gap in literature on in situ observations of the (L)FP phase evolution on a single-particle level with appreciable space and time resolution, a micrometer-sized all-solid-state thin film battery is built with a defect-chemically well characterized LFP single crystal as cathode material with dimensions of 16x1x0.2 micrometer. Using STXM, the phase evolution along the fast (010) orientation is followed during in situ electrochemical (de)lithiation on a micro-meter scale with a lateral resolution of 30 nm and with minutes of time resolution. Furthermore, the STXM measurements performed on this sample are one of the few experiments ever taken on LFP materials with a well defined defect chemistry, even though fundamentally necessary for an overall understanding of the materials behaviour. This combination discloses not only the mechanism of LFP transformation on a single-particle level, but also the significance of elastic effects on the (de)lithiation process. Using a defect chemical analysis, the position of phase formation is found to be determined by the defect chemical situation, while the growth pattern of both LFP and FP is found to be dominated by elastic effects.Item Open Access Characterization and application of novel imine reductases(2016) Scheller, Philipp; Hauer, Bernhard (Prof. Dr.)Chiral amines are an ubiquitously distributed class of bioactive compounds, what turns them into preferred scaffolds for pharmaceuticals. The high chemical and enantiomerical purities required for such an application are ideally suited for biocatalysis as enzymatic methods routinely display high specificities. The established methods for chiral amine synthesis with lipases, ω-transaminases and amine oxidases, however have considerable limitations regarding their access to pharmaceutically relevant chiral secondary and tertiary amines. Recently the new enzyme class of imine reductases (IREDs) was described, offering an attractive extension to the currently used techniques as the preparation of imines by chemical methods in organic solvents is a well established and widely applicable method. As the number of IREDs known initially was limited to only three enzymes, this project started with a database search for the discovery of novel enzymes. For the first time it was shown that the IRED family is much larger than assumed and over 350 novel, putative IREDs were identified. A sequence analysis of the database members revealed (R)- type and (S)- type superfamilies and led after an update to the identification of IRED specific sequence motifs. These criteria allowed to define this new enzyme family on a sequence level and discriminate them from the closest related homologues. Based on the biochemical information about the three published IREDs and a conservation analysis of the database members, three new enzymes from Streptosporangium roseum DSM43021, Streptomyces turgidiscabies and Paenibacillus elgii were selected for characterization. The enzymes were shown to encode for functional IREDs with much higher activity than the previously known IREDs. By site directed mutagenesis the mechanism of the IREDs was probed and the importance of a conserved Tyr for catalysis of an (S)- type IRED shown, while the crucial role of the proposed Asp residue for catalysis in the (R)- type IREDs was questioned. The characterization of the new IREDs revealed their pH optima and confirmed the suspected dimerization. The thermostability of the IREDs was investigated and the selected (S)- type IRED identified as the most stable enzyme known to date. Further the activity in the presence of water miscible organic solvents was tested and high tolerance versus MeOH found. In biotransformations all IREDs showed high activity and a broad panel of cyclic imines was fully converted to piperidines and tetrahydroisoquinolines with enantioselectivities up to 99% ee. With purified IREDs kinetic constants for these substrates were recorded and their substrate preference investigated. This indicated a preference of the (S)- type IRED for more bulky substrates, compared to the (R)- type IREDs. After optimization of the reaction conditions, with purified IREDs also high activities and chemo- as well as enantioselectivities for very labile exocyclic imines were detected. The possibility to effectively reduce already low levels of such imines led to the application of one (R)- type IRED for the generation of novel C-N bonds by reductive aminations. The established methodology revealed the crucial influence, conditions that favor imine formation (high molar excess of the amine nucleophile and high pH) display on the conversion rates. Under optimized conditions, different carbonyls could efficiently be transformed with a variety of amines in the aqueous buffer system with moderate to good conversions into primary and secondary (chiral) amines with very high selectivities (ee up to 98%). Finally, an application of IREDs in cascade reactions to produce saturated N-heterocyclic compounds was envisioned. A microbial putrescine oxidase (PuO) was chosen to selectively oxidize polyamines to aminoaldehydes, thereby triggering their spontaneous cyclization to an imine. To target a broad range of heterocycles, PuO was characterized with a range of unnatural polyamines. The results indicated a narrow substrate scope and low activity for these compounds. To enhance the activity for such substrates directed evolution of PuO with epPCR was performed and led to the identification of a Glu residue, representing a hotspot for mutagenesis. This residue aligns to one of the multiple channels that lead into the deeply buried active site of PuO and it is located in the second shell around the active site. By site-saturation mutagenesis further mutants in the active site and this channel were generated and many mutants with smaller amino acids demonstrated the influence of this hotspot position to increase the activity of the enzyme. The best mutant exhibited considerably increased activities (up to 25-fold) for unnatural polyamines and also for natural polyamines the substrate spectrum was strongly shifted from putrescine towards longer polyamines like spermidine which is now transformed with a 10-fold increased catalytic efficiency (kcat/KM). The combination of both enzymes in purified form as well as in whole cells enabled the production of heterocyclic amines relying on the consecutive transformation of the substrate by both enzymes. While with the whole cell system only low amounts of the N-heterocyclic compounds were produced, the utilization of purified enzymes led in case of all three IREDs to high conversions of different polyamines into pyrrolines and piperidines.Item Open Access Einfluss von Platin-Abscheidungen auf die Membrandegradation in Polymerelektrolytbrennstoffzellen(2016) Helmly, Stefan; Friedrich, K. Andreas (Prof. Dr.)Item Open Access Validierung von Reaktionsmechanismen für biogene Kraftstoffkomponenten(Stuttgart : Deutsches Zentrum für Luft- und Raumfahrt, Institut für Verbrennungstechnik, 2016) Schuler, Dominik Friedemann; Zabel, Friedhelm (Prof. Dr.)In der vorliegenden Arbeit werden die Verbindungen Butanol, 2-Butanol, iso-Butanol und 2,5-Dimethylfuran bei Bedingungen, die für die technische Verbrennung relevant sind, untersucht (p=8.20 bar, T=1190-1520 K). Diese Verbindungen sind mögliche Komponenten zukünftiger Kraftstoffe. Die Experimente werden in Abwesenheit von Sauerstoff (Pyrolyse) in einem speziell für diese Untersuchungen konfigurierten Single-Pulse-Stoßrohr hinter reflektierten Stoßwellen durchgeführt. Die Experimente werden mit der Simulationsumgebung Chemkin II und kinetischen Reaktionsmodellen aus der Literatur simuliert, um die Produktverteilungen aus Experiment und Simulation gegenüber zu stellen. Zur besseren Charakterisierung der Unterschiede zwischen den Modellen werden Reaktionsfluss- und Störungsanalysen durchgeführt. Mit Hilfe dieser Daten kann Verbesserungspotential in den Mechanismen identifiziert werden und Ansätze für eine Optimierung können aufgezeigt werden. Bei der Pyrolyse der Butanolisomere wurden die Hauptprodukte Ethen, Ethin, Ethan, Methan und Propen quantifiziert. In geringeren Konzentrationen wurden die Produkte Ethanal, 1,3-Butadien, 1,3-Butadiin, 2-Buten, iso-Buten, Propen, Propin und Allen nachgewiesen. Bei den Simulationen zeigten sich einerseits teils erstaunlich gute Vorhersagen der experimentellen Daten, andererseits bei einigen Produktspezies auch eine große Diskrepanz der Vorhersagen der Mechanismen untereinander sowie zum Experiment. Bei der Pyrolyse von 2,5-Dimethylfuran wurden die Hauptprodukte Kohlenstoffmonoxid, Ethan, Ethen, Ethin und 1,3-Butenin nachgewiesen. Weitere quantifizierte Produkte sind Allen, Benzol, 1,3-Butadien, 1,3-Butadiin, 2-Buten, Butin, Cyclopentadien, 2-Methylfuran, Propen, Propin und Toluol. Die Mechanismen zeigen in den Hauptprodukten eine gute Übereinstimmung untereinander und mit den experimentell gewonnenen Daten. Bei den Nebenprodukten kommt es jedoch auf Grund der Unterschiede in den Mechanismen zu unterschiedlichen Vorhersagen. Neben den Untersuchungen im Single-Pulse-Stoßrohr wurde in einem weiteren Stoßrohr hinter reflektierten Stoßwellen zeitaufgelöst die Bildung von Kohlenstoffmonoxid bei der Pyrolyse von 2,5-Dimethylfuran gemessen. Kohlenstoffmonoxid wurde dabei mittels Absorptionsmessungen bei einer Wellenlänge von 151,0 nm detektiert. Das ebenfalls auf dieser Wellenlänge absorbierende Edukt 2,5-Dimethylfuran und das Produkt Ethin wurden bei der Vorhersage der Absorption berücksichtigt. Im Rahmen der Messgenauigkeit decken sich die gefundenen Absorptionen mit den von den Modellen vorhergesagten Werten, so dass die Messungen die Reaktionsmodelle unterstützen.Item Open Access Novel route to vanillin - an enzyme-catalyzed multi-step cascade synthesis(2016) Klaus, Tobias; Hauer, Bernhard (Prof. Dr.)The selective hydroxylation of aromatic compounds is one of the most challenging chemical reactions. As an alternative to traditional chemical catalysis, biocatalysis emerged during the past decades. Hence, in the present work, a number of biocatalysts was investigated with regard to the realization of a novel synthesis route to the valuable aromatic compound vanillin, starting from the simple low-cost aromatic substrate 3-methylanisole via the intermediate products 3-methoxybenzyl alcohol or 4-methylguaiacol and via vanillyl alcohol, as an example of consecutive enzyme-catalyzed oxidation reactions accomplished in a multi-enzymatic three-step cascade reaction. For this reason a preselected set of enzymes, namely the m-hydroxybenzoate hydroxylase MobA from Comamonas testosteroni GZ39 and the cytochrome P450 monooxygenases CYP116B3 from Rhodococcus ruber DSM 44319 and CYP102A1 from Bacillus megaterium ATCC 14581, was investigated towards the selective hydroxylation of the substrate 3-methylanisole. Beside the wild type enzymes, a variant of MobA, which was created by rational protein design, and an existing focused minimal mutant library of CYP102A1 were applied in initial biotransformation reactions, combined with an efficient cofactor recycling system. Though the wild type enzymes of CYP116B3 and CYP102A1 displayed only a basic level of activity towards 3-methylanisole, highly increased activity was detected for many of the CYP102A1 variants with a maximum of 59% total conversion for the double mutant F87V/A328L. With 3-methoxybenzyl alcohol and 4-methylguaiacol both intermediate compounds of the intended cascade synthesis were generated, though 4-methoxy-2-methylphenol was the main product in most of the reactions. However, none of the so far investigated variants accepted any of the intermediate compounds as substrate. As CYP116B3 was a good candidate for further protein engineering approaches, as a basic level of activity towards the substrate of interest was already present in the wild type enzyme, a focused mutant library of 20 single mutant variants of CYP116B3 was created based on literature, sequence and structure information in order to improve the enzymes activity and selectivity towards conversion of the substrate 3-methylanisole and in order to find variants for the conversion of 3-methoxybenzyl alcohol and/or 4-methylguaiacol. Therefore a homology model of the monooxygenase domain of CYP116B3 was generated. Though, compared to the wild type, variants with up to almost six time increased activity towards the model substrate 7-ethoxycoumarin were found, total activity towards 3-methylanisole was still much lower compared to the best CYP102A1 variants. In addition, none of the variants displayed appropriate conversion of the intermediate compounds 3-methoxybenzyl alcohol and 4-methylguaiacol. Moreover, additional mutation in the literature known amino acid position 437 of CYP102A1 variant F87V/A328L revealed no benefit towards conversion of any of the substrates, too. Molecular dynamics simulations of a CYP102A1 variant with 4-methylguaiacol as substrate revealed the bottleneck in the conversion of this compound. 4-Methylguaiacol was shown to be stabilized at the entrance of the substrate access channel by the polar amino acid residues R47 and Y51. Replacement of these residues by the hydrophobic residues leucine and phenylalanine, respectively, resulted in successful conversion of 4-methylguaiacol to vanillyl alcohol, the precursor of vanillin in the intended cascade synthesis. Though, as the yield of vanillyl alcohol synthesized from 4-methylguaiacol with CYP102A1 variants was rather low, a vanillyl alcohol oxidase from Penicillium simplicissimum and rationally designed variants thereof, described in literature, were investigated. As a result, not only vanillyl alcohol but also 4-methylguaiacol was converted in high yield to vanillin. Finally, a combination of the best 4-methylguaiacol producing variant, CYP102A1 variant A328L, with the best 4-methylguaiacol converting variant, VAO variant F454Y, in one reaction system both in vitro and in vivo yielded vanillin from 3-methylanisole with a maximal product formation of 2.0% and 1.1% vanillin, respectively. We demonstrated as a proof-of-principle the establishment of the proposed multi-enzymatic three-step cascade reaction pathway. Though further optimizations concerning increase of enzyme activity and improvement of enzyme selectivity are required, the above mentioned exemplary synthesis of vanillin illustrates the capability of biocatalysis.