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Biocatalytic Production of Amino Carbohydrates through Oxidoreductase and Transaminase Cascades
(2019)
Plant-derived carbohydrates are an abundant renewable re- source. Transformation of carbohydrates into new products, in- cluding amine-functionalized building blocks for biomaterials applications, can lower reliance on fossil resources. Herein, bio- catalytic production routes to amino carbohydrates, including oligosaccharides, are demonstrated. In each case, two-step bio- catalysis was performed to functionalize d-galactose-contain- ing carbohydrates by employing the galactose oxidase from Fusarium graminearum or a pyranose dehydrogenase from
Agaricus bisporus followed by the w-transaminase from Chro- mobacterium violaceum (Cvi-w-TA). Formation of 6-amino-6- deoxy-d-galactose, 2-amino-2-deoxy-d-galactose, and 2-amino- 2-deoxy-6-aldo-d-galactose was confirmed by mass spectrome- try. The activity of Cvi-w-TA was highest towards 6-aldo-d-gal- actose, for which the highest yield of 6-amino-6-deoxy-d-galac- tose (67%) was achieved in reactions permitting simultaneous oxidation of d-galactose and transamination of the resulting 6- aldo-d-galactose.
The soluble blood protein beta2-glycoprotein I (beta2GPI; 326 aa, MW: 48 kDa, 5 domains) is one of the most abundant proteins in human serum and exhibits two main conformational states: the circular or closed conformation, where the first domain (DI) is bound to the last domain (DV) of the protein, and the linear or open conformation. The defined physiological function of beta2GPI is still unknown, though several roles in pro- and anticoagulation as well as oxidative stress protection were discovered. The open form is considered to play a crucial role in the systemic autoimmune disease antiphospholipid syndrome (APS), which is an acquired thrombophilia characterized by recurring thrombotic events and pregnancy morbidity. Beta2GPI constitutes the main antigen for APS autoantibodies which are supposed to bind a cryptic epitope within DI after a conformational change from closed to open form. However, the pathophysiological mechanism of APS is poorly understood. Therefore, investigating the structural dynamics of this protein in relation to its antigenicity is of high interest.
Post-translational modifications (PTM) of a target protein often show an impact on the formation of neoantigens, for instance in the autoimmune-mediated diseases type 1 diabetes mellitus, rheumatoid arthritis, or multiple sclerosis. Such modified antigens may lead to immune tolerance breakdown as they are unknown to the immune system, which therefore could mistakes self for non-self proteins. In this thesis, two frequently occurring PTM were introduced to beta2GPI and their impact on the protein conformation was studied by biophysical tools (i.e. atomic force microscopy (AFM) imaging, transmission electron microscopy (TEM) imaging, dynamic light scattering (DLS), and circular dichroism (CD) spectroscopy). In order to examine immunopathophysiological relevance of these PTM, additional insights were gained from ELISA which was used to examine binding of anti-DI autoantibodies purified from the blood of APS patients to the modified beta2GPI species.
A characteristic feature of beta2GPI is the high content of lysine residues. Previously, opening of beta2GPI was found to be triggered by a drastic shift in pH and salt concentration (pH 11.5 and 1.15 M NaCl), which results in reversible uncharging of the lysine residues. The aim of this study was to investigate the beta2GPI conformation after lysine acetylation as a model system, to elucidate the role of lysine residues on the conformational dynamics of this protein, and to examine anti-DI autoantibody binding to both the untreated as well as acetylated species.
A strategy to permanently open up the closed form under physiological conditions by chemical acetylation of lysine residues utilizing the sensitive acetylation agent acetic acid N-hydroxysuccinimide ester (NHS-Ac) was established. Complete and specific lysine acetylation was verified by quantification of primary amines exerting a fluoraldehyde o-phthaldialdehyde (OPA) reagent assay, as well as by native PAGE and western blot analysis with an anti-acetylated lysine antibody. Beta2GPI acetylation revealed a partial opening of beta2GPI molecules. Compared to untreated, i.e. native beta2GPI which exhibited 93% of the molecules in closed and 7% in open form, complete lysine residue acetylation generated 39% of beta2GPI in closed and 61% in open conformation as shown by AFM high-resolution imaging. pH 11.5-treated beta2GPI was used as a reference in the applied methods and revealed 38% of the protein in closed and 62% in open conformation. Thus, a significant shift in beta2GPI conformation occurred upon lysine residue acetylation as well as basic pH-treatment. The data indicate that lysine residue acetylation destabilizes the closed form, leading to a facilitated opening of the structure. The closed conformation might be predominantly stabilized by electrostatic interactions of lysine residues, which potentially control the conformational dynamics of this glycoprotein. ELISA confirmed that anti-DI autoantibodies do not bind to untreated (closed) beta2GPI. Although acetylated beta2GPI was shown to have a substantial portion of open proteins, no binding of anti-DI autoantibodies to the acetylated species was found either. Hence, acetylated lysine residues may disrupt the immunorelevant epitope in DI which prevents antibody binding. This finding reveals a new hint for epitope organization. However, further detailed epitope mapping has to be performed.
Beta2GPI carries two structural disulfide bonds per domain, whereas an additional disulfide bond Cys288/Cys326 is located at the C-terminus of DV near the putative contact interface of DI and DV in the closed conformation. It was previously shown that beta2GPI is a substrate of thiol oxidoreductases, including human thioredoxin-1 (Trx-1) generating different redox states of disulfide bond Cys288/Cys326, which might serve as a scavenger in oxidative stress protection in the blood stream. In APS patients, anti-DI antibody titers as well as an enhanced risk for thrombotic events are associated with an increase in the oxidized state of the protein. Hitherto, no structural study has been performed in order to prove a correlation of the redox state and the conformation of beta2GPI. Therefore, investigations of beta2GPI conformation in different redox states of disulfide bond Cys288/Cys326 were carried out. In addition, binding of anti-DI autoantibodies to the untreated (native) as well as reduced protein should be explored.
At first, cysteine residues of untreated, i.e. native beta2GPI were confirmed to be completely in oxidized state using Ellman’s reagent assay and the absence of binding of a thiol-specific agent. Statistical analyses of AFM images revealed that untreated beta2GPI was mainly in closed conformation (80% in closed and 20% in open conformation) in the respective system. In this study, an optimized protocol for enzymatic reduction of disulfide bond Cys288/Cys326 was established. The agent TCEP was used to reduce human Trx-1, which in turn enzymatically reduced beta2GPI. To block reoxidation of free thiols and to facilitate product analysis, cysteine residues of reduced beta2GPI were subsequently labeled with the sensitive and thiol-specific reagent 3-(N-maleimidopropionyl) biocytin (MPB), which carries a biotin function. During protocol establishment, complete and specific reduction of disulfide bond Cys288/Cys326 was confirmed utilizing SDS-PAGE, streptavidin western blot, mass spectrometry (MS) analyses, and a biotin quantification assay. Protocol improvements constituted a homogenous system with remarkable decrease of unspecifically reduced beta2GPI. Upon beta2GPI reduction, AFM imaging revealed no significant shift in protein conformation (75% in closed and 25% in open conformation). These results were qualitatively confirmed by TEM imaging. Therefore, reduction of beta2GPI disulfide bond Cys288/Cys326 did not result in a major conformational change of the protein. Upon in vitro reduction, the closed form is still the main conformation and a direct correlation of beta2GPI redox state and conformation must be refused. Furthermore, beta2GPI reduction led to a strong and statistically highly significant increase in anti-DI autoantibody binding compared to untreated beta2GPI. Thus, the reduced form might be the antigenic form of the protein. In contrast to previous knowledge, these findings suggest that anti-DI autoantibodies may also bind to the closed conformation under certain conditions. Hypothetically, reduction of beta2GPI could induce a minor structural change in DV that might facilitate the binding of APS autoantibodies.
Overall, this study reveals that PTM of beta2GPI may lead to a critical level of destabilization of the closed conformation (as in the case of acetylated beta2GPI) or significantly increase the binding of APS autoantibodies (as in the case of reduced beta2GPI), both of which could have a large impact on APS disease. However, further investigations are necessary to put these new findings in the context of APS immunopathophysiology.
Haloalkanes are serious environmental pollutants commonly employed as pesticides, herbicides, and chemical warfare agents. Although haloalkane production is performed mostly in the chemical industry, it also occurs naturally, mostly enzymatically (halide methyltransferases and haloperoxidases). Elimination of toxic haloalkanes is very important and using haloalkane dehalogenases is a promising and environmentally friendly way to achieve this.[53] Therefore, assays are needed for detecting dehalogenase activity either to find new enzymes or to generate laboratory-evolved variants. In this thesis, a new assay for dehalogenase activity was developed based on halide detection. In this assay halides, as dehalogenase products, are oxidized under mild conditions using the vanadium-dependent chloroperoxidase from Curvularia inaequalis, forming hypohalous acids that are detected using aminophenyl fluorescein.[53] This new halide oxidation assay is much more sensitive than previously known assays, with detection limits of 20 nM for bromide and 1 μM for chloride and iodide. Validation of the assay was done by comparison to a well-established GC-MS method in terms of determining the specific activities of two dehalogenases towards five common substrates (Figure 5).
The HOX assay was modified for iodide-specific detection by using two other dyes, o-phenylenediamine (OPD) and 3,3′,5,5′-tetramethylbenzidine (TBM), instead of APF. Also, selective bromide detection in the presence of the common contaminant chloride was achieved by using a bromoperoxidase. Since the assay relies on halide detection, it is possible to use it for other halide-producing enzymes (Section 8.1). For example, the TMB-modified version was used for screening of halide methyltransferase libraries towards various alkyl iodides.[166] Furthermore, the HOX assay was used to identify promiscuous dehalogenase activity of the epoxide hydrolase CorEH from Corynebacterium sp. C12.[105]
Moreover, studies showed that the HOX assay could be used with in-vitro synthesized protein. Selected dehalogenases, DhlA, DhaA, and DmmA, were synthesized in vitro and used in the assay; the product formation was also validated using GC-MS. In conclusion, the HOX assay can be used with purified protein, whole cells, or in vitro synthesized proteins.
The HOX assay application in microfluidic droplets was investigated since an ultra-high-throughput assay for haloalkane dehalogenases is needed. This investigation showed no leakage of reaction components and products in the short term (~24 h), based on tests done on water-in-oil droplets generated by microfluidic chips. Even though 20 μM droplets were not working, 70 μM droplets were successful for assay implementation. Since the Damborsky group in Brno (CZ) and the deMello group in Zürich (CH), have large dehalogenase libraries and more experience in microfluidics, respectively, we collaborated with these groups to finalize implementation of the assay in an ultrahigh-throughput format. Since the studies are ongoing, final results could not yet be shown in this thesis. However, it can be noted that the issue with 20 μm droplets has been sorted out since our collaborators in Brno noticed that the low fluorescence of the droplets is actually caused by excessive accumulation of fluorescein, which is self-quenching, resulting in low fluorescence once the concentration exceeds 1 μM. By lowering the APF concentration they could optimize the maximum amount of fluorescein formed, and a mutant library has now been successfully screened by our collaborators at the ETH. The last topic of the thesis was an investigation of converting an epoxide hydrolase into a haloalkane dehalogenase. These studies focused on increasing the minor dehalogenase activity of two previously identified epoxide hydrolase (Cif) variants. These Cif variants hardly led to soluble proteins, the PROSS algorithm was used to increase soluble expression. New variants of Cif were generated using a 3DM analysis and the PROSS[164] design. The activities of these variants were determined with the newly developed HOX assay in a whole-cell format. Cif23 E153N-H269D and the PROSS D7 E153N-H269D variant, were found being active against 1,2-dibromoethane. Since the determination of enzyme concentration was hard to measure due to the expression/purification problem, specific activities could not be determined. To solve this problem, a HiBiT-tag was added to the selected variants for determining soluble expression. However, the planned studies could not be completed because of a lack of time and will form the basis for a future study.
This thesis focuses on the establishment of biocatalytic cascade reactions for the production and detection of industrially relevant flavor and fragrance compounds for food and cosmetic products. To meet the consumer’s demand for those products to be natural, environmentally friendly biocatalytic manufacturing processes that operate GMO-free must be established. Thus, this thesis presents such pathways for the production of an industrially relevant long-chain hydroxy fatty acid and the important flavor and aroma compound raspberry ketone. Furthermore, a biosensor for aldehyde detection was implemented to facilitate screening for suitable biocatalysts that produce industrially relevant aldehydes that are widely applied in the flavor and fragrance industry.
The development of the two main types of diabetes mellitus, type 1 and type 2 (T1D, T2D), is closely associated with the formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in insulin-secreting pancreatic β-cells. In T1D, β-cell death
is triggered by proinflammatory cytokines, which mainly lead to the formation of ROS
in mitochondria and RNS in the cytosol. Pancreatic β-cells are extraordinarily sensitive
to oxidative stress due to their low glutathione peroxidase and catalase expression.
Thus, hydrogen peroxide (H2O2) cannot be detoxified, neither sufficiently, nor rapidly.
H2O2 itself is a rather weakly reactive ROS but can react in the Fenton reaction to form
highly reactive hydroxyl radicals (●OH), that can damage cells in a variety of ways and
induce cell death. The cell and its organelles are bounded by biological membranes
that differ in their permeability to H2O2. Aquaporins (AQPs) are water-transporting
transmembrane proteins, and some isoforms have been shown to facilitate a bidirectional transport of H2O2 across cellular membranes in addition to water. The role of
AQP8 was investigated in an insulin-producing cell model by stably overexpressing
AQP8 (AQP8↑) and by a CRISPR/Cas9-mediated AQP8 knockout. However, AQP8
proved to be an essential protein for the viability of the insulin-producing RINm5F cells, and so we established a tet-on-regulated AQP8 knockdown (AQP8 KD). Our results highlight that AQP8 is involved in H2O2 transport across the plasma and mitochondrial membranes, and that AQP8 expression gets upregulated by proinflammatory cytokines (in vitro) and in an acutely diabetic rat model (in vivo). Furthermore, it was shown that the increased proinflammatory cytokine toxicity is due to enhanced mitochondrial oxidative stress, because H2O2 cannot be efficiently transported in AQP8 KD cells and ●OH
are increasingly generated. Caspase activity then raises, and apoptosis is increasingly
induced coupled with a proportion of ferroptosis-mediated cell death because of a concomitant decrease in nitric oxide (NO●) concentration. In conclusion, AQP8 is localized in the plasma and mitochondrial membrane of insulin-producing RINm5F cells, where it is involved in H2O2 transport. In T1D, AQP8 plays an important role in the transport of H2O2 from the mitochondrial matrix to the cytosol so that the concentration is lowered in the mitochondria. This wider distribution of H2O2 may ease the inactivation of H2O2.
Ac(et)ylation is a post-translational modification present in all domains of life. First identified in mammals in histones to regulate RNA synthesis, today it is known that is regulates fundamental cellular processes also in bacteria: transcription, translation, metabolism, cell motility. Ac(et)ylation can occur at the ε-amino group of lysine side chains or at the α-amino group of a protein. Furthermore small molecules such as polyamines and antibiotics can be acetylated and deacetylated enzymatically at amino groups. While much research focused on N-(ε)-ac(et)ylation of lysine side chains, much less is known about the occurrence, the regulation and the physiological roles on N-(α)-ac(et)ylation of protein amino termini in bacteria. Lysine ac(et)ylation was shown to affect protein function by various mechanisms ranging from quenching of the positive charge, increasing the lysine side chains’ size affecting the protein surface complementarity, increasing the hydrophobicity and by interfering with other post-translational modifications. While N-(ε)-lysine ac(et)ylation was shown to be reversible, dynamically regulated by lysine acetyltransferases and lysine deacetylases, for N-(α)-ac(et)ylation only N-terminal acetyltransferases were identified and so far no deacetylases were discovered neither in bacteria nor in mammals. To this end, N-terminal ac(et)ylation is regarded as being irreversible. Besides enzymatic ac(et)ylation, recent data showed that ac(et)ylation of lysine side chains and of the proteins N-termini can also occur non-enzymatically by the high-energy molecules acetyl-coenzyme A and acetyl-phosphate. Acetyl-phosphate is supposed to be the key molecule that drives non-enzymatic ac(et)ylation in bacteria. Non-enzymatic ac(et)ylation can occur site-specifically with both, the protein primary sequence and the three dimensional structure affecting its efficiency. Ac(et)ylation is tightly controlled by the cellular metabolic state as acetyltransferases use ac(et)yl-CoA as donor molecule for the ac(et)ylation and sirtuin deacetylases use NAD+ as co-substrate for the deac(et)ylation. Moreover, the accumulation of ac(et)yl-CoA and acetyl-phosphate is dependent on the cellular metabolic state. This constitutes a feedback control mechanism as activities of many metabolic enzymes were shown to be regulated by lysine ac(et)ylation. Our knowledge on lysine ac(et)ylation significantly increased in the last decade predominantly due to the huge methodological advances that were made in fields such as mass-spectrometry, structural biology and synthetic biology. This also includes the identification of additional acylations occurring on lysine side chains with supposedly different regulatory potential. This review highlights recent advances in the research field. Our knowledge on enzymatic regulation of lysine ac(et)ylation will be summarized with a special focus on structural and mechanistic characterization of the enzymes, the mechanisms underlying non-enzymatic/chemical ac(et)ylation are explained, recent technological progress in the field are presented and selected examples highlighting the important physiological roles of lysine ac(et)ylation are summarized.
Die akute Pankreatitis ist durch eine vorzeitige Aktivierung von Verdauungsenzymen noch innerhalb der Azinuszellen gekennzeichnet. Die lysosomale Hydrolase Cathepsin B (CTSB) spielt hierbei eine entscheidende Rolle, indem sie Trypsinogen zu Trypsin aktiviert. Für die Trypsinogenaktivierung durch CTSB ist eine Co-Lokalisierung beider Enzyme innerhalb desselben subzellulären Kompartiments erforderlich. Ziel dieser Arbeit war es, die Regulation der CTSB-Aktivität durch den Cysteinprotease-Inhibitor Cystatin C im Verlauf der akuten und chronischen Pankreatitis näher zu untersuchen.
Subzelluläre Fraktionierungsexperimente zeigten eine deutliche Lokalisation von Cystatin C und aktiven Cathepsin B im sekretorischen Kompartiment muriner Azinuszellen. Immunofluoreszenzfärbungen zeigten ebenfalls, dass Cystatin C zusammen mit der pankreatischen Amylase im sekretorischen Kompartiment von Azinuszellen lokalisiert ist. Auch in humanen Probenmaterial konnten wir zeigen, dass Cystatin C im sekretorischen Kompartiment lokalisiert ist und auch sekretiert wird. Experimente mit rekombinanten Proteinen zeigten eine deutliche pH-abhängige inhibitorische Wirkung von Cystatin C auf Cathepsin B. Unter sauren pH Bedingungen dimerisiert Cystatin C und ist somit nicht mehr in der Lage die Aktivität von CTSB zu inhibieren. Weiterhin konnten wir zeigen, dass aktives Trypsin Cystatin C prozessiert. Bei dieser Spaltung entsteht ein Cystatin C-Fragment, welches nicht mehr in der Lage ist, CTSB zu inhibieren, sondern vielmehr die auto-inhibitorische Kapazität von Cathepsin B unterbindet und somit die Aktivität stabilisiert. Neben Cystatin C wird in Azinuszellen auch Cystatin B exprimiert, ein weiterer Inhibitor der Cystein-Proteasen. Im Gegensatz zu Cystatin C ist Cystatin B exklusiv im cytosolischen Kompartiment der Azinuszelle lokalisiert. Dies ist wahrscheinlich ein Schutzmechanismus, welcher die Zelle vor einer cytosolischen Cathepsin-Aktivität schützen soll. Die genetische Deletion von Cystatin C im Mausmodell der akuten Pankreatitis führte zu einer erhöhten Aktivität sekretorischer Proteasen in Azinuszellen, sowie im Gesamthomogenat und in subzellulären Fraktionen. Dementsprechend zeigte sich auch ein deutlich erhöhter Schweregrad in der akuten und chronischen Pankreatitis.
Unsere Experimente lassen vermuten, dass die Aktivität von Cathepsin B unter physiologischen Bedingungen durch Cystatin C unterbunden wird, um so eine verfrühte Aktivierung des Trypsinogens zu verhindern. Im Verlauf der Pankreatitis wird dieser protektive Mechanismus jedoch überwunden. Die Aktivität von Cathepsin B steigt deutlich in der schweren Zymogengranula-Fraktion an, trotz der Präsenz von Cystatin C.
Zusammenfassend lassen unsere Ergebnisse vermuten, dass prozessiertes (aktives) Cathepsin B selbst unter physiologischen Bedingungen im sekretorischen Kompartiment von Azinuszellen bereits vorhanden ist. Seine Aktivität wird dort durch Cystatin C inhibiert, wodurch eine vorzeitige, durch CTSB induzierte Trypsinogenaktivierung verhindert wird. Die Ansäuerung der sekretorischen Vesikel, wie bei der Pankreatitis, verringert die CTSB-Hemmung durch Cystatin C, während es gleichzeitig zu einer Cystatin C-Degradation durch Trypsin kommt. Dies ermöglicht eine verlängerte und pH-unempfindliche Protease-Aktivierung über CTSB in der Anfangsphase der Pankreatitis. Cystatin C spielt somit eine wesentliche Rolle für die Regulation der CTSB-Aktivität im sekretorischen Kompartiment von Azinuszellen und stellt damit einen entscheidenden pathophysiologisch relevanten Mechanismus für die akute und chronische Pankreatitis dar.
Abstract
Macroalgae species are fast growing and their polysaccharides are already used as food ingredient due to their properties as hydrocolloids or they have potential high value bioactivity. The degradation of these valuable polysaccharides to access the sugar components has remained mostly unexplored so far. One reason is the high structural complexity of algal polysaccharides, but also the need for suitable enzyme cocktails to obtain oligo‐ and monosaccharides. Among them, there are several rare sugars with high value. Recently, considerable progress was made in the discovery of highly specific carbohydrate‐active enzymes able to decompose complex marine carbohydrates such as carrageenan, laminarin, agar, porphyran and ulvan. This minireview summarizes these achievements and highlights potential applications of the now accessible abundant renewable resource of marine polysaccharides.
Abstract
The known Schiff base compound, (E)1‐benzyl‐3‐((4‐methoxyphenyl)imino)‐5‐methylindolin‐2‐one, was prepared as before by reacting 1‐benzyl‐5‐methylindoline‐2,3‐dione with 4‐methoxyaniline. The product was unambiguously characterized using elemental analysis, 1H and 13C‐NMR spectroscopy, and its new single‐crystal X‐ray structural analysis. Molecular orbital calculations were conducted in order to investigate the structures and relative stabilities of the (E) and (Z) isomers of 1‐benzyl‐3‐([4 methoxyphenyl]‐imino)‐5‐methylindolin‐2‐one. Specific attention was paid to the (E) isomer. The available crystallographic experimental data for the latter ensured also validation of the model structures computationally derived at the theoretical B3LYP/6‐31G(d,p) level.
Social arthropods such as termites, ants, and bees are among others the most successful animal groups on earth. However, social arthropods face an elevated risk of infections due to the dense colony structure, which facilitates pathogen transmission. An interesting hypothesis is that social arthropods are protected by chemical compounds produced by the arthropods themselves, microbial symbionts, or plants they associate with. Stegodyphus dumicola is an African social spider species, inhabiting communal silk nests. Because of the complex three-dimensional structure of the spider nest antimicrobial volatile organic compounds (VOCs) are a promising protection against pathogens, because of their ability to diffuse through air-filled pores. We analyzed the volatilomes of S. dumicola, their nests, and capture webs in three locations in Namibia and assessed their antimicrobial potential. Volatilomes were collected using polydimethylsiloxane (PDMS) tubes and analyzed using GC/Q-TOF. We showed the presence of 199 VOCs and tentatively identified 53 VOCs. More than 40% of the tentatively identified VOCs are known for their antimicrobial activity. Here, six VOCs were confirmed by analyzing pure compounds namely acetophenone, 1,3-benzothiazole, 1-decanal, 2-decanone, 1-tetradecene, and docosane and for five of these compounds the antimicrobial activity were proven. The nest and web volatilomes had many VOCs in common, whereas the spider volatilomes were more differentiated. Clear differences were identified between the volatilomes from the different sampling sites which is likely justified by differences in the microbiomes of the spiders and nests, the plants, and the different climatic conditions. The results indicate the potential relevance of the volatilomes for the ecological success of S. dumicola.
Scholz et al. developed an electrochemical assay to study the impact of reactive species on self-assembled monolayer (SAM). The aim of this thesis is to use this electrochemical assay with gold supported lipid bilayers instead of SAM to study the effect of reactive species on model membranes that mimic oxidative damage to the biological cell membrane. Here, three questions will be addressed: I) how specific substances such as lipophilic and hydrophilic antioxidants protect a membrane from oxidative damage, II) what are the lipid oxidation products after oxidative damage of the model membrane, and III) whether oxidative damage of the model membranes causes pore formation on lipid bilayer. Electrochemistry was first used to measure the oxidative damage over the entire lipid membrane. Then, mass spectroscopy was used to characterize how lipids as the molecular building blocks of the membrane, change when exposed to reactive species. Imaging the membrane with AFM showed how oxidative damage in the model membrane alters lipid self-assembly within the supported lipid bilayer in nanometer scale. In addition, cold physical plasma (CPP) was used to produce the biological relevant reactive species. This fundamental research demonstrates the great potential of supported lipid bilayers as model membranes and cold physical plasma as a source for the production of biologically relevant reactive species to study the effect of oxidative stress on cell membranes.
The target specificity of thioredoxin family proteins is determined by electrostatic compatibility
(2021)
The thioredoxin (Trx) family of proteins comprises many key enzymes in redox signaling, that catalyzes specific reversible redox reactions, e.g. dithiol-disulfide exchange reactions, (de-)glutathionylation, trans-nitrosylation, or peroxide reduction. With the analysis of a large number of proteins, as well as a certain redox couple in [article 1] and [article 4], we demonstrated that electrostatic complementarity is the major distinguishing feature that controls the specific interactions of Trxs with their target proteins. The primary aim of this work was to determine the importance of this specific interaction and the prediction, modulation, and engineering of functional redox interactions of Trx family proteins. To understand the role of electrostatic complementarity for the mammalian Trx1-TrxR complex, we generated more than 20 hTrx1 mutants and systematically engineered the electrostatic potential within and outside the contact area with TrxR [article 1]. The effects of these specific alterations distributed all over the protein surface were analyzed by enzyme kinetics, differential scanning fluorimetry (DSF), circular dichroism (CD) spectroscopy, and MD simulations. Trx family proteins have a broad and very distinct substrate specificity, which is a prerequisite for redox switching. In [article 4], we comprehensively compared the classification of various redoxins from all kingdoms of life based on their similarity in amino acid sequence, tertiary structure, and electrostatic properties. These similarities were then correlated to the existence of common interaction partners. Our analyses confirmed that the primary and tertiary structure similarities do not correlate to the target specificity of the proteins as thiol-disulfide oxidoreductases. However, we demonstrated that the electrostatic properties of the protein from both Trx or Grx subfamilies is the major determinant for their target specificity.
Although structurally very similar, CxxC/S-type or class I Grxs act as oxidoreductases and CGFS-type or class II Grxs act as FeS cluster transferases. In [article 3], we re-investigated the structural differences between the two main classes of Grxs to solve the mystery of the missing FeS transferase activity of the CxxC/S-type and the lack of oxidoreductase activity of the CGFS-type Grxs. The presence of a distinct loop structure adjacent to the active site is the major determinant of the Grx function. We confirmed that the function of Grxs can be switched from oxidoreductase to FeS cluster transferase by construction of a CxxC/S-type Grx with a CGFS-type Grx loop and vice versa. Results of several in vitro and in vivo assays together with the detailed structural analyses indicate that not a radically different substrate specificity accounts for the lack of activity, but rather slightly different modes of GSH binding, which is an essential nucleophile required in redox and iron homeostasis.
Various processes within the cell depend on GSH, including redox reactions, reversible posttranslational modifications, and iron metabolim. GSH is not only important in the export of FeS precursors from mitochondria, but it is also an essential cofactor for cluster binding in iron sulfur Grxs. In [article 2], we discussed the role of GSH and iron sulfur Grxs in iron metabolism, the physiological role of CGFS-type Grx interactions with BolA- like proteins, and the cluster transfer between Grxs and recipient proteins. The first well characterized physiological function of a Grx-BolA hetero complex is presented with the Grx3/4-Fra2-mediated regulation of iron homeostasis in yeast.
In synopsis, the results presented and discussed in these articles and the manuscript support the concept of electrostatic properties as the main determinant in substrate specificity towards functional predictions in Trx family proteins. The mathematical model presented here showed significantly accuracy and precision in function prediction. We are aware that our findings are focused on Trx family proteins as a particular family of proteins, but by using a machine learning strategy this mathematical model is being trained with numerous different protein models for better efficacy and accuracy, that may lead to new insights also in the specific interactions of other protein families. The new concept for the substrate specificity determinant doesn’t eliminate previously described aspects for molecular recognition, instead it reveals a deeper understanding of the protein-protein interaction. The 3D structural elements of a protein play a significant role in the specificity and function. We have been able to activate an inactive protein by replacing defined structural elements. Elimination of the loop structure from CGFS-type Grx5 transformed it from an FeS transferase into an oxidoreductase and the activity was further increased by modification of the active site. We believe that the present findings may be useful to investigate proteins in great detail regarding their function based on structure and electrostatic properties. Understanding the nature of the specific interactions may enable us to specifically modify the signal transduction pathways.
Abstract
White spot disease (WSD) is one of the most devastating viral infections of crustaceans caused by the white spot syndrome virus (WSSV). A conserved sequence WSSV131 in the DNA genome of WSSV was found to fold into a polymorphic G‐quadruplex structure. Supported by two mutant sequences with single G→T substitutions in the third G4 tract of WSSV131, circular dichroism and NMR spectroscopic analyses demonstrate folding of the wild‐type sequence into a three‐tetrad parallel topology comprising three propeller loops with a major 1 : 3 : 1 and a minor 1 : 2 : 2 loop length arrangement. A thermodynamic analysis of quadruplex formation by differential scanning calorimetry (DSC) indicates a thermodynamically more stable 1 : 3 : 1 loop isomer. DSC also revealed the formation of additional highly stable multimeric species with populations depending on potassium ion concentration.
Abstract
In the RNA world, the exchange of sequence patches between two RNAs is an intriguing evolutionary concept, allowing generation of new RNA molecules with novel functionality. Based on the hairpin ribozyme (HPR) with its unique cleavage‐ligation properties, we here demonstrate RNA supported RNA recombination as a possible scenario for the emergence of larger RNA molecules with more complex functionality. A HPR variant designed for the purpose of recombination is capable of cleaving two different RNA molecules, one being a hammerhead ribozyme (HHR) and the other an aptamer (A), and to subsequently recombine and ligate the resulting fragments to a hammerhead ribozyme that is allosterically controlled (HHA) by a cognate ligand. Two such recombination processes involving aptamers for either theophylline or flavine mononucleotide (FMN) are demonstrated with yields of functional recombination product of up to 34 %.
Die Pankreatitis ist eine relativ häufige gastrointestinale Erkrankung deren Pathomechanismus bisher nicht vollständig geklärt wurde. Besonders die Rolle des Immunsystems scheint einen wichtigen Einfluss auf den Verlauf dieser Erkrankung zu haben. Gut charakterisiert ist bereits die initiale lokale Immunantwort. Zerstörte Azinuszellen setzten DAMPs (engl. damage-associated molecular pattern) frei, die wiederum eine Infiltration von Zellen des angeborenen Immunsystems in das Pankreasgewebe induzieren und aktivieren. Zu diesen Zellen gehören Makrophagen und Neutrophile. T-Zellen, welche zum adaptiven Immunsystem gehören, wandern nicht in das Pankreas ein, sie werden jedoch systemisch aktiviert. Vor allem Th2-Zellen (T-Helferzellen Typ2) und Tregs (regulatorische T-Zellen) werden im Verlauf einer Pankreatitis induziert. In dieser Arbeit konnte gezeigt werden, dass Tregs während einer Pankreatitis nicht nur aktiviert werden, sondern ebenfalls eine höhere suppressive Kapazität besitzen.
Die genaue Rolle dieser antiinflammatorischen Immunantwort und im speziellen der Einfluss von Tregs sollte in dieser Arbeit mit Hilfe von DEREG Mäusen (engl. depletion of regulatory T cells) genauer charakterisiert werden. Durch gezielte Depletion von Tregs mittels DT (Diphtheria Toxin) kann die Auswirkung der Abwesenheit von Tregs im Pankreatitis-Mausmodell untersucht werden. Im akuten Modell kommt es zu einem systemischen Anstieg der T-Effektor-Immunantwort. Die Depletion von Tregs hat zudem eine Auswirkung auf den Schweregrad der Erkrankung. Unter Abwesenheit von Tregs sinkt im akuten Pankreatitis-Modell der pankreatische Schaden. Als eine mögliche Ursache konnte die Dysbalance der Treg/Th17 regulierten intestinalen Immunantwort identifiziert werden, welche zu einer Zerstörung der Darmbarriere führt und eine Translokation kommensaler Mikroorganismen ins nekrotische Pankreasgewebe initiiert.
Im chronischen Pankreatitis-Modell konnte gezeigt werden, dass die T-Zelldifferenzierung einen wichtigen Einfluss auf die Makrophagenpolarisation hat und dadurch den Verlauf der Chronifizierung der Pankreatitis mitbestimmt. Eine Depletion von Tregs in der chronischen Pankreatitis führt zu einer ungebremsten Th2-Antwort. Über die freigesetzten Zytokine, wie z.B. IL4, wird die Makrophagenpolarisation in Richtung der antiinflammatorischen Makrophagen verschoben. Diese Makrophagen induzieren über IL10 und TGFβ die Aktivierung ruhender PSCs (pankreatische Sternzelle) und regulieren somit Regenerationsprozesse. Kommt es zu einer Dysregulation dieser Makrophagenpolarisation, kann dieser Regenerationsprozess unkontrolliert erfolgen. Als Folge dessen kommt es nicht nur zu einer gesteigerten Aktivierung von PSCs, sondern auch zu einer exzessiven Kollagenproduktion, welche zu einer pathologische Fibrose führt. Die Ergebnisse dieser Arbeit zeigen deutlich, dass Tregs einen entscheidenden Einfluss auf die Gewebeumstrukturierung des Pankreas haben. Eine Depletion von Tregs im chronischen Pankreatitis-Modell induziert über die Aktivierung antiinflammatorischer Makrophagen eine Expression von PSCs. Diese unkontrollierte Induktion führt zu einer gesteigerten Kollagenproduktion und Bildung von fibrotischem Pankreasgewebe unter gleichzeitigem Verlust von Azinuszellen. Diese exzessive Gewebeumstrukturierung resultiert in einem Funktionsverlust des exokrinen Gewebes. Mäuse deren Tregs depletiert wurden verloren im chronischen Pankreatitis-Modell bereits nach 14 Tagen signifikant an Gewicht.
Weitere wichtige Faktoren, die im Regenerationsprozess eine Rolle spielen, sind Wachstumsfaktoren. Genexpressionsanalysen und histologische Färbungen verdeutlichen, dass Tregs die Induktion von Wachstumsfaktoren mitbestimmen.
Zusammengefasst bedeutet dies, dass Tregs im akuten Pankreatitis-Modell die T-Effektor-Immunantwort supprimieren und dadurch den Verlauf der Pankreatitis verschlechtern. Im chronischen Pankreatitis-Modell sorgen Tregs dahingegen für eine Balance der Makrophagenpolarisation, und regulieren den Remodeling-Prozess, indem sie z.B. die Bildung fibrotischem Gewebes limitieren.
ITN—VIROINF: Understanding (Harmful) Virus-Host Interactions by Linking Virology and Bioinformatics
(2021)
Analysis of bioactive lipids from different infection models during bacterial and viral infections
(2021)
Bioactive lipids or lipid mediators influence numerous processes like the reproduction, the bone turnover, the pain perception, the cardiovascular function and the immune system. Eicosanoids and oxylipins are parts of the immunomodulatory lipid mediators, which can be synthesized from polyunsaturated fatty acids (PUFAs) by enzymatic and non-enzymatic reactions. Typical members of eicosanoids are prostaglandins and leukotrienes. The properties of bioactive lipids include the activation of inflammatory reactions as well as the support of resolution. Like hormones, they act locally restricted and in low concentrations. Further bioactive lipids exist i.e. intermediates of the sphingolipid class. The biosynthesis of some of these compounds like the prostaglandins can be influenced by different drugs whereas for other groups of lipid selective inhibitors are still missing. Their impact on inflammatory processes and against chronic diseases has already been analyzed, while studies in context with infection are largely limited. Infection of the upper respiratory tract caused by viral and bacterial pathogens constitute a huge burden for the human healthcare. The main pathogens are the Influenza A virus (IAV), Staphylococcus aureus (S. aureus), Streptococcus pneumoniae (S. pneumoniae) and Streptococcus pyogenes (S. pyogenes). Besides mono-infection with one of these pathogens, frequently occurring bacto-viral co-infections exist, which negatively influence the etiopathology. The main task of the immune system is the detection and the elimination of pathogens, which can essentially be affected by lipid mediators. Their instability due to oxidizability, the existence of regioisomers and the low abundance of eicosanoids and other oxylipins are the main problems for their analytical measurement.
The mayor objective of this dissertation was the establishment of a suitable analytical method for selected lipid mediators and the detection of infection-related changes. The separation and detection was performed by using high-performance liquid chromatography (HPLC) coupled with triple quad mass spectrometry. This combination is called tandem mass spectrometry (MS/MS). The MS parameters were optimized for approximately 30 lipid mediators by use of chemical standards and the detection was achieved by dynamic multiple reaction monitoring (MRM). Furthermore, the spatial resolution of selected sphingolipids was analyzed in tissue samples using matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MS-Imaging). Concerning the HPLC-MS/MS detection, an MS method was established and optimized with standard compounds. Another crucial part of the establishment was the extraction of bioactive lipids from the different sampling materials. Whereas well tested protocols exist for the extraction and detection of lipid mediators, such protocols for MALDI-MS-Imaging are still limited due to the novelty of this measurement. Ultimately, robust and reproducible protocols for both techniques that were used for the analysis of a broad array of samples from infection experiments were established for both techniques. The analyses of infected cell culture, mice and pigs revealed infection-related perturbations of host lipid mediator levels. Depending on the scientific issue, the sample types cell pellets, lungs, spleens, livers, blood plasmas, pawns including bones or bronchoalveolar lavages were analyzed. For MALDI-MS-Imaging, the spatial distribution of sphingolipids in lung and spleen was detected.
The present dissertation includes four coherent research scopes, in which the pathogen impact on host-derived lipid mediators was detected with the above mentioned analytical methods. The infection models epithelial cells (article II), mouse (article III and IV) and pig (article I) – the latter as the most human like model - showed different aspects of the host-pathogen interaction. The analysis of samples from IAV infection for all three hosts revealed a couple of similarities for some oxylipins that were also described in human infections. Additionally, cell culture and mouse samples from mono-infections as well as co-infections with the pathogens S. aureus and S. pneumoniae were measured. In particular for the bacterial mono- and co-infections, these are the first published results with aspects of infection related changes of lipid mediators. The additional spatial resolution of the sphingolipid intermediates sphingosine 1-phosphate and ceramide 1-phosphate revealed important new insights into their tissue distribution and changes during co-infection.
Article I describes the IAV-specific oxylipin changes in the pig (german landrace) as infection model. Therefore, the sample types lung, spleen, blood plasma, and bronchoalveolar lavage from infected animals at different time points after infection were analyzed and compared with samples from uninfected pigs. Mainly in the lung and the spleen, increased amounts of certain lipid mediators were observed. These changes coincide well with already described alterations in humans and mice. Furthermore, the analysis of different sample material provided an overview about appropriate sample types. Surprisingly, many perturbations were detected in the spleen, which itself was uninfected. Based on the local reaction of lipid mediators, most studies concentrate on sample material with close contact to side of infection. Therefore, this dissertation reveals new insights into a form of systemic immune response. Besides the use of animals with a complex immune system for infection experiments, human bronchial epithelial cells (16HBE) were mono- and co-infected with the pathogens S. aureus, S. pneumoniae and IAV as described in article II. Such cells are the initial barrier for and first contact site with pathogens and thus the comprehension of this host-pathogen interaction is of essential importance. Most changes were detected during pneumococcal infection. Furthermore, the analyzed infections with bacterial pathogens differed from IAV infection by an increased synthesis of 5-hydroxyeicosatetraenoic acid (HETE). For further infections with the above mentioned pathogens, the mouse was used as an infection model. Besides infections affecting the respiratory tract, also the impact of an S. pyogenes infection in different mice strains was analyzed and described in article III. Infection-related changes in prostaglandins, which are involved in bone turnover in swollen pawns as well as enhanced amounts of sepsis- and arthritis-associated lipid mediators were detected, in case arthritis had been induced prior to infection. Furthermore, increased amounts of 20-HETE could be observed for such severe infections. An enhanced biosynthesis of 20-HETE was further confirmed in a high-pathogenic S. aureus LUG2012 infection in article IV for all examined sample types. In this last article of this dissertation, bacterial and viral infections in mice were analyzed similar to those described in article II. Mainly IAV-specific lipid mediator alterations were detected, which are in accordance with the findings of the infected pigs. The additional MALDI-MS-Imaging measurements revealed so far unknown accumulation of ceramide 1-phosphate in lung and spleen as well as enrichment in the red pulp of the spleen.
In summary, this dissertation provides substantial lipid mediator profiles for infections in three different model systems with selected bacterial and viral pathogens. The obtained data constitute a suitable basis for continuative research projects, in which the influence of single bioactive lipids on the course of infection could be examined in more detail.
Herein, we report the synthesis of a series of push–pull imines by considering cyclic diamino substituent at the C‐centre and fluoroaryl substituent at the N‐centre. This has been achieved by a selective aromatic nucleophilic substitution of different fluoroarenes by N‐H‐substituted N‐heterocyclic imines (NHIs) at ambient conditions without any additional reagent. Solid‐state molecular structure analysis reveals the elongation of the central C–N bond of the imine functionality, which is consistent with the push–pull nature of these imines. The push–pull nature of these imines is further validated by computational studies.
Entdeckung und Design promiskuitiver Acyltransferase‐Aktivität in Carboxylesterasen der Familie VIII
(2021)
Abstract
Promiscuous acyltransferase activity is the ability of certain hydrolases to preferentially catalyze acyl transfer over hydrolysis, even in bulk water. However, poor enantioselectivity, low transfer efficiency, significant product hydrolysis, and limited substrate scope represent considerable drawbacks for their application. By activity‐based screening of several hydrolases, we identified the family VIII carboxylesterase, EstCE1, as an unprecedentedly efficient acyltransferase. EstCE1 catalyzes the irreversible amidation and carbamoylation of amines in water, which enabled the synthesis of the drug moclobemide from methyl 4‐chlorobenzoate and 4‐(2‐aminoethyl)morpholine (ca. 20 % conversion). We solved the crystal structure of EstCE1 and detailed structure–function analysis revealed a three‐amino acid motif important for promiscuous acyltransferase activity. Introducing this motif into an esterase without acetyltransferase activity transformed a “hydrolase” into an “acyltransferase”.
Abstract
The 10–23 DNAzyme is an artificially developed Mg2+‐dependent catalytic oligonucleotide that can cleave an RNA substrate in a sequence‐specific fashion. In this study, new split 10–23 DNAzymes made of two nonfunctional fragments, one of which carries a boronic acid group at its 5′ end, while the other has a ribonucleotide at its 3′ end, were designed. Herein it is demonstrated that the addition of Mg2+ ions leads to assembly of the fragments, which in turn induces the formation of a new boronate internucleoside linkage that restores the DNAzyme activity. A systematic evaluation identified the best‐performing system. The results highlight key features for efficient control of DNAzyme activity through the formation of boronate linkages.
On the aqueous phase chemistry of atmospheric-pressure plasma jets for biomedical applications
(2021)
Cold atmospheric-pressure plasmas are candidate biomedical tools proposed for various applications, such as biological decontamination, cancer regression, and promotion of wound healing. Plasmas, which are in the fourth state of matter, can be generated using inert gases (e.g., argon, helium, ambient air) and different source concepts. Together with the applied parameters, the source design defines the chemical-physical characteristics of the resulting plasma, leading in turn to variable biochemical effects on biological matter. The medical effectiveness of cold plasmas has been proven in vitro and in vivo, also in clinical trials for wound healing in patients using two certified plasmas sources, the kINPen MED and the PlasmaDerm. However, molecular mechanisms leading to those effects are unclear. In the same way, it must be studied if the modulation of plasma properties could improve the specificity of biological effects. These findings are needed to define the concept of plasma dose to be optimized in targeting peculiar pathologic conditions. The present thesis consisting of five peer-reviewed publications has investigated these aspects of plasma research.
In the gaseous phase of cold plasmas, various components with biological activity are produced, such as radiation (e.g., vacuum UV, UV) and reactive species (e.g., •O, 1O2, •OH, •NO, •NO2, O3). As most gaseous species are short-lived, liquid compartments surrounding cells and molecular structures could mediate their transformation and/or the production of other aqueous species. For this reason, plasma-induced aqueous chemistry has been mainly investigated in this thesis. The reaction pathways of reactive oxygen and nitrogen species in liquid were analyzed by monitoring the oxidative modifications induced on tyrosine and cysteine, which are biological structures essential in cellular protein functioning. Liquid chromatography and mass spectrometry-based strategies have been elaborated to elucidate structural changes and characterize the oxidative pattern occurring on the tracers after treatment with plasmas.
As a first result, it could be shown that the oxidative pattern induced on tyrosine or cysteine variated qualitatively and quantitatively with the applied conditions, reflecting the action of differently produced/deposited species in liquid. Biologically relevant structures were identified and in part quantified (e.g., cystine, sulfonic acid, sulfinic acid, S-sulfonate, S-nitrosocysteine, nitrotyrosine, nitrosotyrosine). By using isotopically labeled oxygen or nitrogen in the gas plasma, or labeled oxygen in the target liquid, the incorporation of gaseous or aqueous species in the tracer’s structures was monitored via mass spectrometry. With this strategy, the reaction mechanisms involving gaseous oxygen and nitrogen species at the liquid interface were clarified, as well as the de novo production of reactive species in liquid. Short-lived gaseous oxygen species such as atomic and singlet oxygen (•O, 1O2), predominantly formed in conditions with oxygen in the plasma gas, were able to modify the cysteine structures in highly oxidized derivatives, such as cysteine sulfonic acid. Due to their half-life, however, their activity occurred mainly at the interface. Vacuum UV radiation and •O also led to the formation in liquid of hydroxyl radicals (•OH) and hydrogen peroxide (H2O2), due to water photolysis and homolysis. Water-derived species were responsible for the formation of reversible modifications, such as cysteine S-sulfonate, cystine, and cystine sulfoxides. Nitrosative modifications (e.g., S-nitrosocysteine, nitrosotyrosine, nitrotyrosine) could be observed only in conditions with both nitrogen and oxygen in the plasma gas, and further optimization occurred in presence of water molecules in the gas. In this case, the formation and action of peroxynitrite (ONOO-) in generating nitrotyrosine was proven by using a scavenger molecule for ONOO-.
Finally, the cysteine product pattern was applied as a tool to characterize and compare the overall chemistry generated in liquid by different plasma sources and applied parameters. These findings aim to support and contribute to the definition of plasma dose for plasma medicine, through the standardization, control, tuning, and optimization of plasma parameters and plasma liquid chemistry. These results may be applied in the future to improve the specificity and selectivity of the biological effects generated by the described atmospheric-pressure plasma jets.
Blood platelets are primary major players in the coagulation cascade, that act upon damage in blood vessels at the subendothelial surface. During this process, platelets change their shape, release granules and aggregate by cross-linking of integrin αIIbβ3 via fibrinogen. The heterodimeric transmembrane receptor integrin αIIbβ3 is highly expressed on platelets and its regulation is bidirectional. Inside-out signaling leads to increased affinity for ligands due to dramatic rearrangements in the integrin conformation changing from an inactive bent conformation to an extended, high-affinity conformation. The swing-out motion of the integrin head domain enables binding of ligands, e.g. fibrinogen, resulting in outside-in signaling guiding kinase activation, shape change, platelet aggregation and spreading, subsequently.
Agonists (e.g. thrombin) and other triggers (e.g. shear stress) promote the activity of platelets, making the study of specific proteins delicate. Therefore, this PhD thesis describes a biomimetic system used to study αIIbβ3 membrane receptors. Integrin αIIbβ3 was successfully reconstituted into liposomes and characterized by biophysical and molecular biological methods (e.g. dynamic light scattering, transmission electron microscopy, circular dichroism spectroscopy and flow cytometry). The fusion of liposomes to a solid substrate allows the analysis of potential activation triggers and interaction partners concerning their role in integrin αIIbβ3 activation in a lipid bilayer. Among others, quartz-crystal microbalance measurements show that divalent ions and clinically relevant drugs (e.g. unfractionated heparin and quinine), known to be involved in immune thrombocytopenia (ITP), are certainly candidates which induce integrin activation and minor changes in protein secondary structure. In addition, protein corona formation during contact of nanoparticles with blood components, such as fibrinogen, as well as their interaction with artificial platelet model membranes containing integrins were studied. Moreover, lipid environment can be strongly controlled as integrin activation is dependent on the ratio of liquid-ordered and disordered phases within the membrane. Eventually, by exclusion of disturbances of complex external and internal factors, the established system enables the interaction analysis of various substances with receptors under physiological conditions. In contrast, these disturbances are required to understand the complex machinery of cellular processes in vivo. Hence, an expression platform, on the basis of HEK293 cells, was established to study not only the interaction of integrin αIIbβ3 with cytoskeletal networks, but also the impact of mutations on integrin resulting in a disease-like phenotype. Mutations known to induce Glanzmann thrombasthenia (GT) symptoms, were introduced and led to different mechanical properties of integrin-expressing cells, especially during cell adhesion cells. Thereby, generation of biological and medically-relevant processes combined with the biophysical setup contribute to understand disease mechanisms as well as the action of therapeutic agents in diseases such as GT and ITP.
Promiscuous acyltransferases enable transesterification reactions in bulk water by preferentially catalyzing acyl transfer over hydrolysis. Until recently, only a small number of promiscuous acyltransferases have been described in the literature, exhibiting several limitations in terms of acyltransferase efficiency and applicability. This work focuses on the discovery of novel promiscuous acyltransferases and the engineering of promiscuous acyltransferases via rational design. Several promiscuous acyltransferases in the bacterial hormone-sensitive lipase family and family VIII carboxylesterases have been identified, demonstrating that promiscuous acyltransferase activity is not a rare phenomenon. Moreover, the efficiency and applicability of the enzymes could be improved via protein engineering in terms of acyltransferase activity, enantioselectivity, and substrate scope.
The aims of this thesis were the identification and development of whole-cell biocatalysts for the regio- and stereoselective hydroxylation of steroids, including hormones and bile acids by P450 monooxygenases. Steroids and their derivatives are applied as therapeutic agents. The chemical synthesis of such compounds depends on multi-step procedures, in a stereo- and regiospecific manner involving the protection and deprotection of functional groups and toxic reagents and intermediates. In this thesis, different P450 monooxygenases were investigated as ‘bio-based’ alternatives to chemical catalysts for the late-stage functionalization of steroids and bile acids and engineered by directed evolution procedures towards desired transformation activities. In Article I, the 16α-hydroxylation activity of the bovine CYP17A1 was enhanced by protein engineering to improve the transformation of progesterone into 16α-hydroxyprogesterone in Saccharomyces cerevisiae. Article II follows the same line of research and targets the selective synthesis of bile acid derivatives in Escherichia coli (E. coli) whole-cells. The P450 monooxygenase CYP107D1 (OleP) from Streptomyces antibioticus (S. antibioticus) was identified, which selectively hydroxylates bile acids like lithocholic acid (LCA) and deoxycholic acid (DCA) at the 6β-position, yielding murideoxycholic acid (MDCA), a gallstone solubilizing agent, and 3α-,6β-,12α-trihydroxy-5β-cholan-24-oic acid, respectively. The utilization of OleP as catalyst resulted in shorter synthesis routes for both compounds and additional in a higher yield for MDCA. Building on the results of Article II and the protein engineering approach from Article I, Article III deals with the switch of regioselectivity of the identified CYP107D1 from 6β- to 7β-hydroxylation to form the therapeutic agent ursodeoxycholic acid (UDCA) from LCA by direct hydroxylation. Following a rational protein engineering strategy, a variant with nearly perfect selectivity for UDCA formation was found. Until today, UDCA is either isolated from bile of catheterised farmed bears or produced semisynthetically through low-yielding multistep reactions starting from cholic acid (CA). Article III presents the first reported enzyme for the direct 7β-hydroxylation of LCA to UDCA.
Free radicals are known to induce significant structural and functional modifications to the cell membrane and its components. Biophysical quantification of such changes using single molecule studies highlight the role of these individual biomolecules. In this PhD work, we focus on nitric oxide radical and try to understand how they influence interaction of different biomolecules with lipid membranes by using biomimetic systems. In specific we try to answer how cell membrane permeability and bilayer thickness would be influenced by the nitric oxide radical with different phospholipids compositions (i.e. on planar supported lipid bilayers). Later we tested, interaction of transmembrane protein integrin αiibβ3 incorporated into the bilayer (i.e. nanodiscs) with nitric oxide. Finally, how to overcome the negative effects encountered by the phospholipids and proteins using biopolymer coated gold nanoparticles as delivery system. The study involved use of atomic force microscopy and quartz-crystal microbalance with dissipation as primary investigation tools complemented with other relevant biophysical and biochemical techniques.
This thesis deals with the process considerations and optimizations of a whole-cell enzyme cascade reaction for the synthesis of ɛ-caprolactone. The enzyme cascade synthesis of ɛ-caprolactone has been conceptualized and verified using a dehydrogenase and a monooxygenase. The advantage of this enzyme combination is the closed-loop co-factor regeneration. Dehydrogenase and monooxygenase expressed in discrete whole cells were applied in defined ratio to conceptualize the cascade reaction. This necessitates the use of separate co-factor regeneration system due to impermeability of the E. coli cell wall to the co-factor. Article I deal with the design and optimization of dehydrogenase and monooxygenase co-expression in a same E. coli cell. In Article II, the cascade reaction was upscaled and a fed-batch process was realized. Following which, the important reaction metrices were analyzed and optimized. Article III extends the two-enzyme cascade with a lipase. The use of lipase helps to overcome the product inhibition of monooxygenase by ɛ-caprolactone.
Unter promiskuitiver Acyltransferase-Aktivität versteht man die Eigenschaft bestimmter Hydrolasen, in wässriger Lösung bevorzugt Acyltransfer statt Hydrolyse zu katalysieren. Bis vor Kurzem waren nur wenige promiskuitive Acyltransferasen literaturbekannt. Dies führte zu der allgemeinen Annahme, dass diese Aktivität ein seltenes Phänomen in Hydrolasen ist. Diese Arbeit zeigt jedoch, dass promiskuitive Acyltransferase-Aktivität in der Familie der bakteriellen hormonsensitiven Lipasen und Carboxylesterasen der Familie VIII weit verbreitet ist. Detaillierte Struktur-Funktions-Analysen ermöglichen die sequenzbasierte Vorhersage und Optimierung der Acyltransferase-Aktivität in beiden Enzymfamilien. Insbesondere die Carboxylesterasen der Familie VIII überschreiten die Grenzen des bisher für möglich Gehaltenen, indem sie gute Enantioselektivität bei der kinetischen Racematspaltung sekundärer Alkohole zeigen und darüber hinaus die irreversible Bildung von Amiden und Carbamaten in Wasser katalysieren können. Die biokatalytische Acylierung von Zuckern in Wasser galt lange Zeit als unerreichtes Ziel der Biokatalyse. In dieser Arbeit wurde jedoch gezeigt, dass natürlich vorkommende und modifizierte Carboxylesterasen der Familie VIII die regioselektive Acetylierung von Glucose, Maltose und Maltotriose in Wasser mit hoher Effizienz katalysieren können.
Die akute Pankreatitis ist eine der häufigsten nicht malignen gastrointestinalen Erkrankungen, die zu Krankenhausaufenthalten führt. Sie ist als Selbstverdau des Pankreas durch seine eigenen Proteasen wie z.B. Trypsin, Elastase und Chymotrypsin definiert. Als Ursprung der Erkrankung wird die frühzeitige intrazelluläre Aktivierung dieser Verdauungsenzyme angesehen. Dies führt zum Zelltod der Azinuszellen und zur Schädigung des Gewebes.
Während der akuten Pankreatitis kommt es in 20% der Fälle zu einem schweren Verlauf der Erkrankung, der mit Organversagen in der Lunge und den Nieren assoziiert ist. Es ist bekannt, dass es zu einer Entzündungsreaktion kommt, bei der große Mengen an Zytokinen ausgeschüttet werden. Leukozyten infiltrieren das Pankreas und verstärken den Gewebeschaden. Es kommt zur Freisetzung von DAMPs, die das angeborene und adaptive Immunsystem aktivieren. Bislang ist nicht gut untersucht, wie das Immunsystem den schweren Verlauf der akuten Pankreatitis beeinflusst und es gibt wenig Theorien über den Organschaden in der Lunge und den Nieren.
In dieser Arbeit lag der Fokus auf dem Organschaden in Lunge und Niere und die Wirkung von Interleukin 33 (IL33) auf die Zellen des angeborenen Immunsystems und deren Einwanderung in verschiedene Organe während der schweren akuten Pankreatitis im Mausmodell. Die schwere akute Pankreatitis wurde mittels Gangligatur und einmaliger Gabe von Caerulein an Tag 2 nach Gangligatur induziert. An Tag 3 nach Induktion wurden die Mäuse getötet und die Organe wurden für weitere Analysen entnommen.
Am dritten Tag nach Induktion der Pankreatitis kam es zu einem Organschaden in der Lunge und den Nieren. In der Lunge fand sich eine Verdickung der Alveolarsepten und eine Verdichtung des Gewebes sowie eine Infiltration von Leukozyten und ein Ödem. In der Niere waren ebenfalls strukturelle Veränderungen zu finden und eine Infiltration von Leukozyten war zu beobachten. In durchflusszytometrischen Analysen der Lunge konnte beobachtet werden, dass CD11b+CD62L+ Monozyten während der akuten Pankreatitis signifikant anstiegen. Mittels RT-DC wurde gezeigt, dass diese Monozyten an Tag 3 signifikant an Größe zugenommen hatten. Mit einer CD11b Färbungen von Lungen und Nieren konnte die Infiltration durch Monozyten bestätigt werden. Unter einer Blockade von Monozyten durch systemische Gabe von anti-CCR2-Antikörpern verringerte sich die Schädigung in Lunge und Niere während der Pankreatitis signifikant.
Diese Daten legen nahe, dass der Organschaden in der schweren akuten Pankreatitis durch infiltrierende Monozyten verursacht wird, die über CD62L (L-Selektin) an die Gefäßwände binden und über ihre Größe Gefäße verstopfen, was in den Kapillaren zur Ischämie führt.
In vitro sezernierten Makrophagen, die mit CCK stimulierten Azinuszellen co-inkubiert wurden, IL33. Im Mausmodell wurde IL33 mittels sST2 blockiert, was die Schädigung des Pankreas in der Pankreatitis reduzierte. In IL33-depletierten Tieren fand sich im Vergleich zum Wildtyp ein geringerer Lungenschaden aber eine unveränderte Nierenschädigung. Somit scheint IL33 eine Rolle bei der Monozyten-vermittelten Organschädigung in der Pankreatitis zu spielen, die sich auf Grund von kompensatorischen Regulationsmechanismen im globalen IL33 Knock-out weniger gut belegen lässt als nach IL33 Inhibition. Die Hemmung von IL33 zur Behandlung der akuten Pankreatitis stellt somit ein vielversprechendes Therapieprinzip dar.
S-adenosyl-L-methionine- (SAM) dependent methyltransferases (MTs) catalyse methylation of halide ions and the C, O, N, S, Se, and As atoms of biomolecules ranging from biopolymers to small molecules. They display different chemo-, regio- and stereoselectivity according to their specific functions. This thesis focuses on the engineering of O-methyltransferases (OMTs) and halide methyltransferases (HMTs) through rational design and directed evolution to study their structure-function relationship and to explore their catalytic promiscuity. The influence of substrate binding residues on the substrate scope and regioselectivity of a plant OMT against various phenolic substrates (Article I) and flavonoids (Article II) has been investigated. Article III describes the directed evolution of an HMT for the biocatalytic synthesis of diverse SAM analogues. With the evolved HMT, regioselective alkylation of phenolic compounds and flavonoids, as well as the SAM analogue regeneration, were achieved through an HMT-MT cascade reaction.
Article I Specific residues expand the substrate scope and enhance the regioselectivity of a plant O-methyltransferase.
It was reported in literature that an isoeugenol 4-OMT (IeOMT) can be engineered to a caffeic acid 3-OMT (CaOMT) by replacing three consecutive residues. In this article, we investigated the effect of these residues on substrate preference and regioselectivity of IeOMT. The triple mutant T133M/A134N/T135Q and the respective single mutants were constructed and tested against a series of phenolic compounds. The variant T133M had a universal effect to improve enzymatic activities against all tested substrates while the mutant A134N had enhanced regioselectivity. The triple mutant T133M/A134N/T135Q benefits from these two mutations, which not only expanded the substrate scope, but also enhanced the regioselectivity of IeOMT. On the basis of this work, regiospecific methylated phenolics can be produced in high purity by different IeOMT variants.
Article II Influence of substrate binding residues on the substrate scope and regioselectivity of a plant O-methyltransferase against flavonoids
Flavonoid OMTs (FOMTs), isoflavonoid OMTs (IOMTs) and phenylpropanoid OMTs (POMTs) display different substrate preferences. Sequence comparison showed that the substrate binding residues at positions 322 and 326 are different between these OMT groups and might be critical for the substrate discrimination. Residues at positions 322 and 326 in IeOMT (a POMT) were mutated to the commonly presented residues in FOMT and IOMT. The introduced mutants, in cooperation with the variant T133M, have improved or brought novel activities and regioselectivity against the tested flavonoids eriodictyol, naringenin, luteolin, quercetin, and also the isoflavonoid genistein compared to the wild-type IeOMT. On the basis of this work, methylated flavonoids that are rare in nature were produced in high purity.
Article III Directed evolution of a halide methyltransferase enables biocatalytic synthesis of diverse SAM analogs
Biocatalytic alkylations to obtain chemo‐, regio‐ and stereoselectively alkylated compounds can be achieved by MTs with the supply of SAM analogues. It was recently discovered that SAM can be directly synthesized from S adenosyl-L homocysteine (SAH) and methyl iodide, catalysed by an HMT. To explore the promiscuity of HMT in the synthesis of SAM analogues, we performed directed evolution of the Arabidopsis thaliana HMT based on a sensitive, colorimetric iodide assay. The identified variant V140T displayed activities against ethyl‐, propyl‐, and allyl iodides to produce the corresponding SAM analogues. With this HMT variant, regioselective ethylation of luteolin and allylation of 3,4‐dihydroxybenzaldehyde, as well as the SAM analogue regeneration, were achieved through this HMT-MT one-pot cascade reaction.
Electrochemical characterisation of the redox behaviour of quinoide components in membrane models
(2020)
The leading idea of this thesis is to study the effects of (i) membrane composition and (ii) membrane environment (aqueous phases) on the redox properties of membrane-confined redox active compounds. For solutions, it is known since long, how strong solvents affect the redox properties of dissolved redox active species. However, for membranes this question has not yet been addressed, although it can be supposed that such effects may be important to understand the role of membrane-confined redox active compounds in biological systems. To interrogate this problem, a monolayer model was chosen. It consists of a lipid monolayer with embedded menaquinones on mercury electrodes. Since ion transfer across membranes is also a crucial question, in the first part of this project, 2,2-diphenyl-1-picrylhydrazyl (DPPH) was studied as a new redox probe for transferring anions and cation between an organic and an aqueous phase. The important findings of this thesis are: (i) accessing the ion pair equilibrium constant of anions and cations with DPPH redox probe as a model study using the three-phase electrochemistry, (ii) the redox potentials of menaquinone-4, -7, and -9 in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) monolayers and the acidity constants of menaquinones (MK’s) in membranes monolayer model, and (iii) the effects of membrane composition and the aqueous environment on the thermodynamics and kinetics of MK’s in membrane models.
The synthesis of several bioactive compounds and active pharmaceutical ingredients relies on the development of general and efficient methods to prepare optically pure amines. Transaminases are industrially relevant enzymes and are useful for synthesizing a large number of compounds that contain a chiral amine functionality. Although the immense potential associated to the use of these biocatalysts, the equilibrium position is often unfavorable for amine synthesis. The use of an excess of amine donor, compared to the ketone substrate, combined with selective removal of the formed product, can help in overcoming this limitation. This work mainly focused on broadening the application of membrane-based in situ product recovery (ISPR) techniques for the transaminase-catalyzed synthesis of chiral amines. The
overall work was designed around the implementation of amine donors, possessing considerably larger molecular ‘size’ compared to commonly used amine donors. To clearly
distinguish these molecules from traditional donor amines, we designate them as High Molecular Weigh amine donors. With a molecular weight between 400 and 1500 g/mol, in contrast to traditional donor amines, HMW amine donors enable a size-based separation between amine donor and amine product molecules. HMW amines, provided in excess for thermodynamic equilibrium shifting can thus be simply retained by a size-exclusion mechanism by commercial membranes, while the smaller product amines are permeated. Therefore, a selective recovery of the desired chiral amine product is possible. The implementation of ISPR techniques using HMW amine donors can theoretically lead to (i) equilibrium shifting, (ii) alleviation of product inhibition, and (iii) a highly pure product stream.
The feasibility of using HMW amine donors in aqueous, organic solvent and solvent-free media for the transaminase-catalyzed synthesis of 1-methyl-3-phenylpropylamine (MPPA) was proven in this thesis. The latter two approaches were investigated with the aim to achieve higher product concentrations. Along with that, we demonstrated two membrane-assisted ISPR proof of concepts. Specifically, nanofiltration was coupled with the enzymatic reaction performed in aqueous media (Article I), while liquid-liquid (L-L) extraction in a contactor was applied for transamination in organic solvent media (Article II). As an alternative to membrane-based strategies we also designed a spinning reactor concept for the integrated chiral amine synthesis (in organic solvent) and recovery (Article III).
G-quadruplexes (G4s) have been in the focus of research in the last decades for their regulatory roles in vivo and for their use in nano- and biotechnology. However, an understanding of the various factors that drive a particular quadruplex fold remains limited, challenging rational therapeutic targeting and design of these tetrahelical structures. In this regard, insights from modified G-quadruplexes may help to deepen our knowledge of G-quadruplex structure. In this dissertation, sugar-modified guanosine analogs are exploited for their altered conformational preferences regarding both glycosidic bond angle and sugar pucker by their incorporation into different syn positions of the G-core of a model G-quadruplex. Induced structural perturbations as characterized by NMR spectroscopy range from a local change in tetrad polarity to a complete refolding into an unusual structure with a V-shaped loop, a unique G4 structural element in the focus of this work. Detailed conformational analysis of the introduced G analogs and high-resolution structures of the modified quadruplexes reveal a complex interplay of glycosidic torsion angle, sugar pucker preferences and local interactions, which may all play a leading role in driving G4 folding.
Abstract
Methylation of free hydroxyl groups is an important modification for flavonoids. It not only greatly increases absorption and oral bioavailability of flavonoids, but also brings new biological activities. Flavonoid methylation is usually achieved by a specific group of plant O‐methyltransferases (OMTs) which typically exhibit high substrate specificity. Here we investigated the effect of several residues in the binding pocket of the Clarkia breweri isoeugenol OMT on the substrate scope and regioselectivity against flavonoids. The mutation T133M, identified as reported in our previous publication, increased the activity of the enzyme against several flavonoids, namely eriodictyol, naringenin, luteolin, quercetin and even the isoflavonoid genistein, while a reduced set of amino acids at positions 322 and 326 affected both, the activity and the regioselectivity of the methyltranferase. On the basis of this work, methylated flavonoids that are rare in nature were produced in high purity.
Abstract
Certain hydrolases preferentially catalyze acyl transfer over hydrolysis in an aqueous environment. However, the molecular and structural reasons for this phenomenon are still unclear. Herein, we provide evidence that acyltransferase activity in esterases highly correlates with the hydrophobicity of the substrate‐binding pocket. A hydrophobicity scoring system developed in this work allows accurate prediction of promiscuous acyltransferase activity solely from the amino acid sequence of the cap domain. This concept was experimentally verified by systematic investigation of several homologous esterases, leading to the discovery of five novel promiscuous acyltransferases. We also developed a simple yet versatile colorimetric assay for rapid characterization of novel acyltransferases. This study demonstrates that promiscuous acyltransferase activity is not as rare as previously thought and provides access to a vast number of novel acyltransferases with diverse substrate specificity and potential applications.
Abstract
Environmentally‐friendly processes for the manufacturing of valuable industrial compounds like ω‐hydroxy fatty acids (ω‐OHFAs) are highly desirable. Herein, we present such an approach by establishing a two‐step enzymatic cascade reaction for the production of 2,15,16‐trihydroxy hexadecanoic acid (THA). Starting with the easily accessible natural compound ustilagic acid (UA) that is secreted by the corn smut fungus Ustilago maydis, the recombinantly expressed esterase BS2 from Bacillus subtilis and the commercial β‐glucosidase from almonds were applied yielding 86 % product. Both hydrolases do not require expensive cofactors, making the process economically attractive. Additionally, no harmful solvents are required, so that the product THA can be labelled natural to be used in food and cosmetic products.
In modern-day organic synthesis, transitional metal catalysis has become an essential tool-kit to access the biologically significant complex organic scaffolds. The activation profile of these sophisticated catalytic systems in cross-coupling chemistry and ring-closing processes has been well appreciated and frequently employed by the scientific community.
The present thesis is describing the results of interdisciplinary research involving medicinal chemistry and transitional metal homogeneous catalysis. A molybdenum mediated process was employed to access 32 unprecedented heterocyclic fused poly sulfur ring containing pentathiepins in moderate to good yields as a part of medicinal chemistry. Biologically significant, such as quinoxaline, pyrazine, pyridine, nicotinamide, quinoline, imdazo-pyrazine, pyrrolo-pyrazine, purine, and pyridine sulfonamide scaffolds were functionalized with pentathiepin unit via multi-step organic synthesis. Essentially, the Sonogashira cross-coupling and(Et4N)2[MoO(S4)2] mediated ring-closing steps were commonly employed in all pentathiepin syntheses. The analytically pure samples were characterized by 1H, 13C, 19F-NMR, FTIR, ESI-MS, CHNS, and X-ray single-crystal diffraction analysis. Notably, all pentathiepins exhibited an ABX3 multiplet pattern between δ: 4.2-4.5 ppm with the integration of 2H for the ethoxy functional group's methylene protons substituted on the five-membered ring of pentathiepin, which was later considered as a fingerprint for pentathiepin formation. The mechanistic investigations via control experiments suggest that the tetra sulfur ring Mo(IV) precursor (Et4N)2[MoO(S4)2] is vital along with elemental sulfur for the pentathiepin formation, and the Mo(IV) complex regenerates in the reaction. Furthermore, For the first time, the GPx1 enzyme inhibitor properties of novel fused heterocyclic pentathiepins were established, where these probes exhibited 9-12 folds higher potency than mercaptosuccinic acid. Notably, <1 µM concentration of quinoxaline, pyrazine, and quinoline fused pentathiepins were potent enough to inhibit 50% of GPx1 enzyme activity. Additionally, cytotoxicity, antimicrobial and antifungal studies were conducted for all pentathiepins. In anticancer investigations, the IC50 concentrations for all pentathiepins were ranging between 0.22 to 4.7 µM.
The second half of the thesis introduces a novel water-soluble Pd/PTABS as a potent catalyst for C-X (X = N, O, and S) cross-coupling chloroheteroarenes and halonucleosides. The novel, mild and efficient Pd/PTABS catalytic system was successfully employed at low catalytic loadings (1 mol%) for the amination (C−N), etherification (C−O), and thioetherification (C−S) of chloroheteroarenes at ambient to moderate temperatures. The Pd/PTABS catalyst is well-tolerating various heterocyclic scaffolds, and under the optimized catalytic conditions, various secondary amines, electron-rich or electron-poor phenols, thiophenols, and alkylthiols, were efficiently employed as nucleophilic coupling partners. Notably, the catalyst offered tremendous regio and chemoselectivity with excellent temperature control. Besides, novel sulfones and sulfoximines were prepared from the thioethers obtained via Pd/PTABS. The catalyst was employed efficiently for synthesizing biologically significant known drugs or drug candidates such as alogliptin (anti-diabetic agent), XRK 469 (antitumor agent), and Imuran-Azathioprine (immunosuppressive) in competitive yields. Preliminary DFT investigations were performed, and based on the DFT analysis, the electropositive character of the phosphorous atom in quaternary ammonium salts of PTABS supports the heteroatom directed C−Cl activation hypothesis.
The aim of our research is a stereoselective synthesis development of 4-aminocyclohexanol by the application of a keto reductase (KRED) and an amine transaminase (ATA). 4-Aminocyclohexanol is a valuable precursor for active pharmaceutical ingredients, for example, lomibuvir (a HCV protease inhibitor), ambroxol (a secretolytic agent) and other bioactive molecules. Today, the trans-4-aminocyclohexanol is accessed via Ni-catalyzed synthetic procedure giving moderate yields. In our project we perform cis- and trans-4-aminocyclohexanol synthesis from 1,4-cyclohexanedione (a bio-based precursor) by an one-pot approach combining sequentially a KRED and an ATA as catalysts. For this, we envisaged two multistep enzymatic procedures. The route A would involve 4-hydroxycyclohexanone formation from 1,4-cyclohexanedione via a KRED-catalyzed monoreduction and a further transamination mediated by an ATA towards 4-aminocyclohexanol. The route B would consist of switching the steps of the previous sequential approach, that is, a monoamination of the diketone to yield 4-aminocyclohexanone, and the subsequent reduction of the remaining carbonyl group. Only route A turned out to be feasible, and we performed 4-aminocyclohexanol synthesis at the preparative scale in the sequential and tandem modes. Depending on the ATA, both isomers can be obtained.
Abstract
Biocatalysis has found numerous applications in various fields as an alternative to chemical catalysis. The use of enzymes in organic synthesis, especially to make chiral compounds for pharmaceuticals as well for the flavors and fragrance industry, are the most prominent examples. In addition, biocatalysts are used on a large scale to make specialty and even bulk chemicals. This review intends to give illustrative examples in this field with a special focus on scalable chemical production using enzymes. It also discusses the opportunities and limitations of enzymatic syntheses using distinct examples and provides an outlook on emerging enzyme classes.
Abstract
A N‐heterocyclic olefin (NHO), a terminal alkene selectively activates aromatic C−F bonds without the need of any additional catalyst. As a result, a straightforward methodology was developed for the formation of different fluoroaryl‐substituted alkenes in which the central carbon–carbon double bond is in a twisted geometry.