Publications
Total synthesis of [13C2]-labeled phytosiderophores of the mugineic and avenic acid families
Surveying the mugineic acid family: Ion mobility – quadrupole time-of-flight mass spectrometry (IM-QTOFMS) characterization and tandem mass spectrometry (LC-ESI-MS/MS) quantification of all eight naturally occurring phytosiderophores
Enzymatic reactions towards aldehydes: An overview
Lukas Schober, Hana Dobiašová, Valentina Jurkaš, Fabio Parmeggiani, Florian Rudroff, Margit Winkler*
Enlightening the Path to Protein Engineering: Chemoselective Turn-On Probes for High-Throughput Screening of Enzymatic Activity
Sebastian Hecko, Astrid Schiefer, Christoffel P. S. Badenhorst, Michael J. Fink, Marko D. Mihovilovic, Uwe T. Bornscheuer, and Florian Rudroff*
Chem. Rev. 2023, XXXX, XXX, XXX-XXX
Many successful stories in enzyme engineering are based on the creation of randomized diversity in large mutant libraries, containing millions to billions of enzyme variants. Methods that enabled their evaluation with high throughput are dominated by spectroscopic techniques due to their high speed and sensitivity. A large proportion of studies relies on fluorogenic substrates that mimic the chemical properties of the target or coupled enzymatic assays with an optical read-out that assesses the desired catalytic efficiency indirectly. The most reliable hits, however, are achieved by screening for conversions of the starting material to the desired product. For this purpose, functional group assays offer a general approach to achieve a fast, optical read-out. They use the chemoselectivity, differences in electronic and steric properties of various functional groups, to reduce the number of false-positive results and the analytical noise stemming from enzymatic background activities. This review summarizes the developments and use of functional group probes for chemoselective derivatizations, with a clear focus on screening for enzymatic activity in protein engineering.
Sterically Demanding Flexible Phosphoric Acids for Constructing Efficient and Multi-Purpose Asymmetric Organocatalysts
Fabian Scharinger, Ádám Márk Pálvölgyi, Melanie Weisz, Matthias Weil, Christian Stanetty, Michael Schnürch, Katharina Bica-Schröder*
Angew. Chem. Int. Ed. (2022) 61, e202202189
Herein, we present a novel approach for various asymmetric transformations of cyclic enones. The combination of readily accessible chiral diamines and sterically demanding flexible phosphoric acids resulted in a simple and highly tunable catalyst framework. The careful optimization of the catalyst components led to the identification of a particularly powerful and multi-purpose organocatalyst, which was successfully applied for asymmetric epoxidations, aziridinations, aza-Michael-initiated cyclizations, as well as for a novel Robinson-like Michael-initiated ring closure/aldol cyclization. High catalytic activities and excellent stereocontrol was observed for all four reaction types, indicating the excellent versatility of our catalytic system. Furthermore, a simple change in the diamine’s configuration provided easy access to both product antipodes in all cases.
Sterically Demanding Flexible Phosphoric Acids for Constructing Efficient and Multi-Purpose Asymmetric Organocatalysts
Fabian Scharinger, Ádám Márk Pálvölgyi, Melanie Weisz, Matthias Weil, Christian Stanetty, Michael Schnürch, Katharina Bica-Schröder*
Angew. Chem. Int. Ed. (2022) 61, e202202189
Herein, we present a novel approach for various asymmetric transformations of cyclic enones. The combination of readily accessible chiral diamines and sterically demanding flexible phosphoric acids resulted in a simple and highly tunable catalyst framework. The careful optimization of the catalyst components led to the identification of a particularly powerful and multi-purpose organocatalyst, which was successfully applied for asymmetric epoxidations, aziridinations, aza-Michael-initiated cyclizations, as well as for a novel Robinson-like Michael-initiated ring closure/aldol cyclization. High catalytic activities and excellent stereocontrol was observed for all four reaction types, indicating the excellent versatility of our catalytic system. Furthermore, a simple change in the diamine’s configuration provided easy access to both product antipodes in all cases.
Investigation of Leoligin Derivatives as NF-κΒ Inhibitory Agents
Thomas Linder, Eleni Papaplioura, Diyana Ogurlu, Sophie Geyrhofer, Scarlet Hummelbrunner, Daniel Schachner, Atanas G. Atanasov, Marko D. Mihovilovic, Verena M. Dirsch,* and Michael Schnürch*
Biomedicines, 2022, 10, 62.
The transcription factor NF-κB is an essential mediator of inflammation; thus, the identification of compounds that interfere with the NF-κB signaling pathway is an important topic. The natural products leoligin and 5-methoxyleoligin have served as a starting point for the development of NF-κB inhibitors. Using our modular total synthesis method of leoligin, modifications at two positions were undertaken and the effects of these modifications on the biological activity were investigated. The first modification concerned the ester functionality, where it was found that variations in this position have a significant influence, with bulky esters lacking Michael-acceptor properties being favored. Additionally, the substituents on the aryl group in position 2 of the tetrahydrofuran scaffold can vary to some extent, where it was found that a 3,4-dimethoxy and a 4-fluoro substitution pattern show comparable inhibitory efficiency.
Publications in Scientific Journals
A new carbohydrate-active oligosaccharide dehydratase is involved in the degradation of ulvan
Marcus Bäumgen, Theresa Dutschei, Daniel Bartosik, Christoph Suster, Lukas Reisky, Nadine Gerlach, Christian Stanetty, Marko D. Mihovilovic, Thomas Schweder, Jan-Hendrik Hehemann, Uwe T. Bornscheuer*
J. Biol. Chem. (2021) 297 (4) 101210
Marine algae catalyze half of all global photosynthetic production of carbohydrates. Owing to their fast growth rates, Ulva spp. rapidly produce substantial amounts of carbohydrate-rich biomass and represent an emerging renewable energy and carbon resource. Their major cell wall polysaccharide is the anionic carbohydrate ulvan. Here, we describe a new enzymatic degradation pathway of the marine bacterium Formosa agariphila for ulvan oligosaccharides involving unsaturated uronic acid at the nonreducing end linked to rhamnose-3-sulfate and glucuronic or iduronic acid (Δ-Rha3S-GlcA/IdoA-Rha3S). Notably, we discovered a new dehydratase (P29_PDnc) acting on the nonreducing end of ulvan oligosaccharides, i.e., GlcA/IdoA-Rha3S, forming the aforementioned unsaturated uronic acid residue. This residue represents the substrate for GH105 glycoside hydrolases, which complements the enzymatic degradation pathway including one ulvan lyase, one multimodular sulfatase, three glycoside hydrolases, and the dehydratase P29_PDnc, the latter being described for the first time. Our research thus shows that the oligosaccharide dehydratase is involved in the degradation of carboxylated polysaccharides into monosaccharides.
Biogenic colourants in the textile industry – a promising and sustainable alternative to synthetic dyes
Richard Fried, Ilinca Oprea, Karin Fleck and Florian Rudroff*
Green Chemistry 2021
Chemo-Enzymatic Cascade for the Generation of Fragrance Aldehydes
Catalysts 2021, 11(8), 932.
In this study, we present the synthesis of chiral fragrance aldehydes, which was tackled by a combination of chemo-catalysis and a multi-enzymatic in vivo cascade reaction and the development of a highly versatile high-throughput assay for the enzymatic reduction of carboxylic acids. We investigated a biocompatible metal-catalyzed synthesis for the preparation of α or β substituted cinnamic acid derivatives which were fed directly into the biocatalytic system. Subsequently, the target molecules were synthesized by an enzymatic cascade consisting of a carboxylate reduction, followed by the selective C-C double bond reduction catalyzed by appropriate enoate reductases. We investigated a biocompatible oxidative Heck protocol and combined it with cells expressing a carboxylic acid reductase from Neurospora crassa (NcCAR) and an ene reductase from Saccharomyces pastorianus for the production fragrance aldehydes.
A Kinetic Photometric Assay for the Quantification of the Open-Chain Content of Aldoses
H. Kalaus, A. Reichetseder, V. Scheibelreiter, F. Rudroff, C. Stanetty*, M.D. Mihovilovic
ACS Catalysis (2021), 11(5), 2831-2836.
Efficient Acylation of Sugars and Oligosaccharides in Aqueous Environment Using Engineered Acyltransferases
Godehard, Simon P.; Mueller, Henrik; Badenhorst, Christoffel P. S.; Stanetty, Christian; Suster, Christoph; Mihovilovic, Marko D.; Bornscheuer, Uwe T.*
ACS Catalysis (2021), 11(5), 2831-2836.
A major challenge for the enzymatic synthesis of sugar esters is the low solubility of sugars in anhydrous, often toxic, organic solvents. We overcame this limitation by using acyltransferases for efficient acetylation of sugars in water. Selective 6-O-acetylation of glucose, maltose, and maltotriose with conversions of up to 78% was achieved within 15 min using engineered acyltransferases (4 μM). Moreover, we identified EstA as a promiscuous acyltransferase preferentially acetylating sugars instead of hydrophobic acyl acceptors. This expands the applicability of promiscuous acyltransferases to sugar modifications and contributes to the understanding of how to adapt acyltransferases to hydrophilic substrates.
Biocatalysis in Green and Blue: Cyanobacteria
Jodlbauer, Julia; Rohr, Thomas; Spadiut, Oliver; Mihovilovic, Marko D.; Rudroff, Florian*
Trends in biotechnology (2021)
A major challenge for the enzymatic synthesis of sugar esters is the low solubility of sugars in anhydrous, often toxic, organic solvents. We overcame this limitation by using acyltransferases for efficient acetylation of sugars in water. Selective 6-O-acetylation of glucose, maltose, and maltotriose with conversions of up to 78% was achieved within 15 min using engineered acyltransferases (4 μM). Moreover, we identified EstA as a promiscuous acyltransferase preferentially acetylating sugars instead of hydrophobic acyl acceptors. This expands the applicability of promiscuous acyltransferases to sugar modifications and contributes to the understanding of how to adapt acyltransferases to hydrophilic substrates.
The role of hydrogen bonding in the incommensurate modulation of myo-inositol camphor ketal
Viktor Savic, Felix Eder, Christian Göb, Marko D. Mihovilovic, Christian Stanetty and Berthold Stögerd*
Acta Crystallographica Section B, 77 (2021), 83 – 92.
myo-Inositol-2,3-d-camphor ketal crystallizes as an incommensurate structure with the C2(0σ21/2) superspace group symmetry [σ2 = 0.1486 (3) at 100 K]. The bornane and myo-inositol moieties aggregate in distinct layers extending parallel to (001). The myo-inositol rings are connected by a complex hydrogen-bonding network extending in two dimensions, which is disordered in the basic structure and (mostly) ordered in the actual modulated structure. The domains of definition of the H atoms in internal space were derived by chemical reasoning and modeled with crenel functions. By tracing the hydrogen bonding, distinct chains, which are periodic in the [100] direction, are identified. These chains possess one of two possible orientations with respect to the hydrogen bonding. The incommensurate modulation is characterized by a non-periodic succession of the two chain orientations in the [010] direction. On heating, the σ2-component of the modulation wave vector decreases from σ2 = 0.1486 (3) at 100 K to σ2 = 0.1405 (6) at 430 K, which means that the periodicity of the modulation wave increases. No order-disorder phase transition was evidenced up to the melting point (with decomposition).
Publications in Scientific Journals
Immobilized cell physiology imaging and stabilization of enzyme cascade reaction using recombinant cells escherichia coli entrapped in polyelectrolyte complex beads by jet break-up encapsulator
M. Bucko*, P. Gemeiner, T. Krajcovic, M. Hakarova, D. Chorvat, A. Chorvatova, I. Lacik, F. Rudroff, M.D. Mihovilovic
A novel, high performance, and scalable immobilization protocol using a laminar jet break-up technique was developed for the production of polyelectrolyte complex beads with entrapped viable Escherichia coli cells expressing an enzyme cascade of alcohol dehydrogenase, enoate reductase, and cyclohexanone monooxygenase. A significant improvement of operational stability was achieved by cell immobilization, which was manifested as an almost two-fold higher summative product yield of 63% after five cascade reaction cycles as compared to the yield using free cells of 36% after the maximum achievable number of three cycles. Correspondingly, increased metabolic activity was observed by multimodal optical imaging in entrapped cells, which was in contrast to a complete suppression of cell metabolism in free cells after five reaction cycles. Additionally, a high density of cells entrapped in beads had a negligible effect on bead permeability for low molecular weight substrates and products of cascade reaction.
Keywords:
enzyme cascade reaction; immobilization; polyelectrolyte; multimodal optical imaging; biocatalysis; whole-cell biocatalyst
Structural features defining nf-κb inhibition by lignan-inspired benzofurans and benzothiophenes
T. Dao-Huy, S. Latkolik, J. Bräuer, A. Pfeil, H. Stuppner, M. Schnürch, V. Dirsch, M.D. Mihovilovic*
Biomolecules, 10 (2020), 1131; 1 – 20.
A silver‑coated copper wire as inexpensive drug eluting stent model: determination of the relative releasing properties of leoligin and derivatives
L. Czollner, E. Papaplioura, T. Linder, R. Liu, Y. Li, A Atanasov, V. Dirsch, M. Schnürch, M.D. Mihovilovic*
Monatshefte für Chemie, – (2020).
Cardiovascular diseases are overall the leading cause of mortality and morbidity worldwide. Therefore, treating and preventing coronary heart disease are of high scientific interest. Among several percutaneous coronary intervention procedures, coronary artery stenting displayed potent activity against restenosis, often observed using other invasive therapies. Nowadays, drug eluting stents’ superiority over bare metal stents is increasingly recognizable, since drug eluting stents are able to overcome problems encountered with bare metal stent technology. Within this study, we developed a novel method for performing drug-releasing experiments utilizing an affordable stent model made from a readily available silver-coated copper wire, which was further coated with poly(n-butyl methacrylate). Leoligin, previously reported to inhibit intimal hyperplasia and the regrowth of endothelial cells, was exploited along with several structural analogs in drug-releasing experiments. It was found that compounds exhibiting similar biological activity can have significantly different releasing properties, a crucial parameter to know for the selection of compounds for in vivo studies.
GABAa receptor ligands often interact with binding sites in the transmembrane domain and in the extracellular domain-can the promiscuity code be cracked?
M.T. Iorio, F. Vogel, F. Koniuszewski, P. Scholze, S. Rehman, X. Simeone, M. Schnürch, M.D. Mihovilovic, M. Ernst*
International Journal of Molecular Sciences, 21 (2020), 334.
Many allosteric binding sites that modulate gamma aminobutyric acid (GABA) effects have been described in heteropentameric GABA type A (GABAA) receptors, among them sites for benzodiazepines, pyrazoloquinolinones and etomidate. Diazepam not only binds at the high affinity extracellular “canonical” site, but also at sites in the transmembrane domain. Many ligands of the benzodiazepine binding site interact also with homologous sites in the extracellular domain, among them the pyrazoloquinolinones that exert modulation at extracellular α+/β− sites. Additional interaction of this chemotype with the sites for etomidate has also been described. We have recently described a new indole-based scaffold with pharmacophore features highly similar to pyrazoloquinolinones as a novel class of GABAA receptor modulators. Contrary to what the pharmacophore overlap suggests, the ligand presented here behaves very differently from the identically substituted pyrazoloquinolinone. Structural evidence demonstrates that small changes in pharmacophore features can induce radical changes in ligand binding properties. Analysis of published data reveals that many chemotypes display a strong tendency to interact promiscuously with binding sites in the transmembrane domain and others in the extracellular domain of the same receptor. Further structural investigations of this phenomenon should enable a more targeted path to less promiscuous ligands, potentially reducing side effect liabilities.
Characterization of a Structural Leoligin Analog as Farnesoid X Receptor Agonist and Modulator of Cholesterol Transport
A. Ladurner, T. Linder, L. Wang, V. Hiebl, D. Schuster, M. Schnürch, M.D. Mihovilovic, A Atanasov, V. Dirsch
Planta Medica, 86 (2020), 1097 – 1107.
The ligand-activated farnesoid X receptor is an emerging therapeutic target for the development of drugs against metabolic syndrome-related diseases. In this context, selective bile acid receptor modulators represent a novel concept for drug development. Selective bile acid receptor modulators act in a target gene- or tissue-specific way and are therefore considered less likely to elicit unwanted side effects. Based on leoligin, a lignan-type secondary plant metabolite from the alpine plant Leontopodium nivale ssp. alpinum, 168 synthesized structural analogs were screened in a farnesoid X receptor in silico pharmacophore-model. Fifty-six virtual hits were generated. These hits were tested in a cell-based farnesoid X receptor transactivation assay and yielded 7 farnesoid X receptor-activating compounds. The most active one being LT-141A, with an EC50 of 6 µM and an Emax of 4.1-fold. This analog did not activate the G protein-coupled bile acid receptor, TGR5, and the metabolic nuclear receptors retinoid X receptor α, liver X receptors α/β, and peroxisome proliferator-activated receptors β/γ. Investigation of different farnesoid X receptor target genes characterized LT-141A as selective bile acid receptor modulators. Functional studies revealed that LT-141A increased cholesterol efflux from THP-1-derived macrophages via enhanced ATP-binding cassette transporter 1 expression. Moreover, cholesterol uptake in differentiated Caco-2 cells was significantly decreased upon LT-141A treatment. In conclusion, the leoligin analog LT-141A selectively activates the nuclear receptor farnesoid X receptor and has an influence on cholesterol transport in 2 model systems.
Design and Synthesis of a Compound Library Exploiting 5-Methoxyleoligin as Potential Cholesterol Efflux Promoter
T. Linder, S. Geyrhofer, E. Papaplioura, L. Wang, A Atanasov, H. Stuppner, V. Dirsch, M. Schnürch, M.D. Mihovilovic*
Molecules, 25 (2020), 662; 1 – 13.
5-Methoxyleoligin and leoligin are natural occurring lignans derived from Edelweiss (Leontopodium nivale ssp. alpinum), displaying potent pro-angiogenic and pro-arteriogenic activity. Cholesterol efflux from macrophages is associated with reverse cholesterol transport which inhibits the development of cardiovascular disease. Within this study, we developed a modular and stereoselective total synthesis of 5-methoxyleoligin which can be readily used to prepare a novel compound library of related analogs. The target 5-methoxyleoligin was synthesized exploiting a recently disclosed modular route, which allows also rapid synthesis of analogous compounds. All obtained products were tested towards macrophage cholesterol efflux enhancement and the performance was compared to the parent compound leoligin. It was found that variation on the aryl moiety in 2-position of the furan ring allows optimization of the activity profile, whereas the ester-functionality does not tolerate significant alterations.
Investigation of a New Type I Baeyer-Villiger Monooxygenase from Amycolatopsis thermoflava Revealed High Thermodynamic but Limited Kinetic Stability
H. Mansouri Khosravi, M.D. Mihovilovic, F. Rudroff*
ChemBioChem, 21 (2020), 7; 971 – 977.
In silico screening: A Baeyer-Villiger monooxygenase (BVMO) from a thermophilic origin is identified through an in silico approach. There is a strong deviation between the thermodynamic and kinetic stabilities of the new BVMO, which might have a major impact for future enzyme discovery of BVMOs and their synthetic applications.
Straight Forward and Versatile Differentiation of the L-glycero and D-glycero-D-manno Heptose Scaffold
C. Suster, I. Baxendale, M.D. Mihovilovic, C. Stanetty*
Frontiers in Chemistry, 8 (2020), 625; 7 pages.
Bacterial lipopolysaccharides (LPS) are important bio-medical structures, playing a major role in the interaction with human immune systems. Their core regions, containing multiple units of L–glycero–D–manno heptoses (L,D-heptose), are highly conserved structurally (with O3 and O7 glycosidic bonds), making them an epitope of high interest for the potential development of new antibiotics and vaccines. Research in this field has always been restricted by the limited availability of the parent L,D-heptose as well as its biochemical epimeric precursor D–glycero–D–manno heptose (D,D-heptose). This problem of availability has recently been solved by us, through a rapid and efficient practical synthesis of L,D–manno-heptose peracetate demonstrated at scale. Herein we report an optimized, technically simple and versatile synthetic strategy for the differentiation of both the L–glycero and D–glycero–D–manno heptose scaffolds. Our approach is based on an orthoester methodology for the differentiation of all three positions of the sugar core using a O6, O7-tetraisopropyl disiloxyl (TIPDS) protecting group for the exocyclic positions. Furthermore, the regioselective opening toward 7-OH acceptors (6O-FTIPDS ethers) differentiates the exocyclic diol which has been demonstrated with a broader set of substrates and for both manno-heptoses for the first time.
Allosteric GABAA Receptor Modulators-A Review on the Most Recent Heterocyclic Chemotypes and Their Synthetic Accessibility
B. Vega Alanis, M.T. Iorio, L. Silva, K. Bampali, M. Ernst*, M. Schnürch*, M.D. Mihovilovic
Molecules, 25(4) (2020), 999; 1 – 47.
GABAA receptor modulators are structurally almost as diverse as their target protein. A plethora of heterocyclic scaffolds has been described as modulating this extremely important receptor family. Some made it into clinical trials and, even on the market, some were dismissed. This review focuses on the synthetic accessibility and potential for library synthesis of GABAA receptor modulators containing at least one heterocyclic scaffold, which were disclosed within the last 10 years.
6-[(tert-Butyldimethylsilyl)oxy]-3-ethenyl-7-methoxy-4-[(trimethylsilyl)ethynyl]naphtho[2,3-c]furan-1(3H)-one
M. Weil, T. Kremsmayr, M.D. Mihovilovic
IUCrData, 5 (2020), 5; x200224.
The tricyclic core in the title compound, C26H34O4Si2, shows disorder of the furan ring and deviates slightly from planarity, with the largest displacement from the least-squares plane [0.166 (2) Å] for the major disordered part of the methine C atom. To this C atom the likewise disordered vinyl group is attached, lying nearly perpendicular to the tricyclic core. In the crystal, mutual C—H
π interactions between the methine group of the furan ring and the central ring of the tricyclic core of an adjacent molecule lead to inversion-related dimers.
Immobilized cell physiology imaging and stabilization of enzyme cascade reaction using recombinant cells escherichia coli entrapped in polyelectrolyte complex beads by jet break-up encapsulator
M. Bucko*, P. Gemeiner, T. Krajcovic, M. Hakarova, D. Chorvat, A. Chorvatova, I. Lacik, F. Rudroff, M.D. Mihovilovic
A novel, high performance, and scalable immobilization protocol using a laminar jet break-up technique was developed for the production of polyelectrolyte complex beads with entrapped viable Escherichia coli cells expressing an enzyme cascade of alcohol dehydrogenase, enoate reductase, and cyclohexanone monooxygenase. A significant improvement of operational stability was achieved by cell immobilization, which was manifested as an almost two-fold higher summative product yield of 63% after five cascade reaction cycles as compared to the yield using free cells of 36% after the maximum achievable number of three cycles. Correspondingly, increased metabolic activity was observed by multimodal optical imaging in entrapped cells, which was in contrast to a complete suppression of cell metabolism in free cells after five reaction cycles. Additionally, a high density of cells entrapped in beads had a negligible effect on bead permeability for low molecular weight substrates and products of cascade reaction.
Keywords:
enzyme cascade reaction; immobilization; polyelectrolyte; multimodal optical imaging; biocatalysis; whole-cell biocatalyst
Amino Benzamidoxime (ABAO)-Based Assay to Identify Efficient Aldehyde-Producing Pichia pastoris Clones
Investigation of a New Type I Baeyer-Villiger Monooxygenase from Amycolatopsis thermoflava Revealed High Thermodynamic but Limited Kinetic Stability
Pyrazines: Synthesis and Industrial Application of these Valuable Flavor and Fragrance Compounds
Frederik B. Mortzfeld, Chiam Hashem, Kvetoslava Vranková, Margit Winkler, Florian Rudroff*
Publications in Scientific Journals
Structural features defining nf-κb inhibition by lignan-inspired benzofurans and benzothiophenes
T. Dao-Huy, S. Latkolik, J. Bräuer, A. Pfeil, H. Stuppner, M. Schnürch, V. Dirsch, M.D. Mihovilovic*
Biomolecules, 10 (2020), 1131; 1 – 20.
A silver‑coated copper wire as inexpensive drug eluting stent model: determination of the relative releasing properties of leoligin and derivatives
L. Czollner, E. Papaplioura, T. Linder, R. Liu, Y. Li, A Atanasov, V. Dirsch, M. Schnürch, M.D. Mihovilovic*
Monatshefte für Chemie, – (2020).
Cardiovascular diseases are overall the leading cause of mortality and morbidity worldwide. Therefore, treating and preventing coronary heart disease are of high scientific interest. Among several percutaneous coronary intervention procedures, coronary artery stenting displayed potent activity against restenosis, often observed using other invasive therapies. Nowadays, drug eluting stents’ superiority over bare metal stents is increasingly recognizable, since drug eluting stents are able to overcome problems encountered with bare metal stent technology. Within this study, we developed a novel method for performing drug-releasing experiments utilizing an affordable stent model made from a readily available silver-coated copper wire, which was further coated with poly(n-butyl methacrylate). Leoligin, previously reported to inhibit intimal hyperplasia and the regrowth of endothelial cells, was exploited along with several structural analogs in drug-releasing experiments. It was found that compounds exhibiting similar biological activity can have significantly different releasing properties, a crucial parameter to know for the selection of compounds for in vivo studies.
GABAa receptor ligands often interact with binding sites in the transmembrane domain and in the extracellular domain-can the promiscuity code be cracked?
M.T. Iorio, F. Vogel, F. Koniuszewski, P. Scholze, S. Rehman, X. Simeone, M. Schnürch, M.D. Mihovilovic, M. Ernst*
International Journal of Molecular Sciences, 21 (2020), 334.
Many allosteric binding sites that modulate gamma aminobutyric acid (GABA) effects have been described in heteropentameric GABA type A (GABAA) receptors, among them sites for benzodiazepines, pyrazoloquinolinones and etomidate. Diazepam not only binds at the high affinity extracellular “canonical” site, but also at sites in the transmembrane domain. Many ligands of the benzodiazepine binding site interact also with homologous sites in the extracellular domain, among them the pyrazoloquinolinones that exert modulation at extracellular α+/β− sites. Additional interaction of this chemotype with the sites for etomidate has also been described. We have recently described a new indole-based scaffold with pharmacophore features highly similar to pyrazoloquinolinones as a novel class of GABAA receptor modulators. Contrary to what the pharmacophore overlap suggests, the ligand presented here behaves very differently from the identically substituted pyrazoloquinolinone. Structural evidence demonstrates that small changes in pharmacophore features can induce radical changes in ligand binding properties. Analysis of published data reveals that many chemotypes display a strong tendency to interact promiscuously with binding sites in the transmembrane domain and others in the extracellular domain of the same receptor. Further structural investigations of this phenomenon should enable a more targeted path to less promiscuous ligands, potentially reducing side effect liabilities.
Characterization of a Structural Leoligin Analog as Farnesoid X Receptor Agonist and Modulator of Cholesterol Transport
A. Ladurner, T. Linder, L. Wang, V. Hiebl, D. Schuster, M. Schnürch, M.D. Mihovilovic, A Atanasov, V. Dirsch
Planta Medica, 86 (2020), 1097 – 1107.
The ligand-activated farnesoid X receptor is an emerging therapeutic target for the development of drugs against metabolic syndrome-related diseases. In this context, selective bile acid receptor modulators represent a novel concept for drug development. Selective bile acid receptor modulators act in a target gene- or tissue-specific way and are therefore considered less likely to elicit unwanted side effects. Based on leoligin, a lignan-type secondary plant metabolite from the alpine plant Leontopodium nivale ssp. alpinum, 168 synthesized structural analogs were screened in a farnesoid X receptor in silico pharmacophore-model. Fifty-six virtual hits were generated. These hits were tested in a cell-based farnesoid X receptor transactivation assay and yielded 7 farnesoid X receptor-activating compounds. The most active one being LT-141A, with an EC50 of 6 µM and an Emax of 4.1-fold. This analog did not activate the G protein-coupled bile acid receptor, TGR5, and the metabolic nuclear receptors retinoid X receptor α, liver X receptors α/β, and peroxisome proliferator-activated receptors β/γ. Investigation of different farnesoid X receptor target genes characterized LT-141A as selective bile acid receptor modulators. Functional studies revealed that LT-141A increased cholesterol efflux from THP-1-derived macrophages via enhanced ATP-binding cassette transporter 1 expression. Moreover, cholesterol uptake in differentiated Caco-2 cells was significantly decreased upon LT-141A treatment. In conclusion, the leoligin analog LT-141A selectively activates the nuclear receptor farnesoid X receptor and has an influence on cholesterol transport in 2 model systems.
Toward the Recovery of Platinum Group Metals from a Spent Automotive Catalyst with Supported Ionic Liquid Phases
O. Lanaridi, A Sahoo, A. Limbeck, S. Naghdi, D. Eder, E. Eitenberger, Z. Csendes, M. Schnürch, K. Schröder*
ACS Sustainable Chem. Eng. 2021, 9, 1, 375–386.
We present a novel approach for the separation and recovery of Pt and Pd leached from a spent automotive catalyst relying on conventional and polymerized supported ionic liquid phases (SILPs and polySILPs, respectively). A variety of parameters with possible effects on the separation behavior, namely, acidity and concentration of the platinum group metal (PGM) containing solution, as well as different SILP and polySILP loadings, were evaluated for the separation of PGMs in the presence of high concentrations of Al, Fe, Zn, and Ce. The polySILP material demonstrated the ability to separate the PGMs from major accompanying interferences in a single separation step, while problems arising from ionic liquid leaching in the case of SILPs could be avoided. Moreover, the use of supported ionic liquid phases allowed the drastic reduction of the amount of required ionic liquid compared to conventional liquid–liquid separation, while avoiding problems arising from emulsion formation. Subsequent stripping experiments lead to further purification of the PGMs and finally desorption from the solid material into a pure solution. Eventually, the concept of chemisorbed polySILPs provides a new and convenient approach for the recycling of platinum group metals.
Design and Synthesis of a Compound Library Exploiting 5-Methoxyleoligin as Potential Cholesterol Efflux Promoter
T. Linder, S. Geyrhofer, E. Papaplioura, L. Wang, A Atanasov, H. Stuppner, V. Dirsch, M. Schnürch, M.D. Mihovilovic*
Molecules, 25 (2020), 662; 1 – 13.
5-Methoxyleoligin and leoligin are natural occurring lignans derived from Edelweiss (Leontopodium nivale ssp. alpinum), displaying potent pro-angiogenic and pro-arteriogenic activity. Cholesterol efflux from macrophages is associated with reverse cholesterol transport which inhibits the development of cardiovascular disease. Within this study, we developed a modular and stereoselective total synthesis of 5-methoxyleoligin which can be readily used to prepare a novel compound library of related analogs. The target 5-methoxyleoligin was synthesized exploiting a recently disclosed modular route, which allows also rapid synthesis of analogous compounds. All obtained products were tested towards macrophage cholesterol efflux enhancement and the performance was compared to the parent compound leoligin. It was found that variation on the aryl moiety in 2-position of the furan ring allows optimization of the activity profile, whereas the ester-functionality does not tolerate significant alterations.
Carbamate-based P,O-ligands for asymmetric allylic alkylations
Á. Pálvölgyi, M. Schnürch, K. Schröder*
Tetrahedron, 76 (2020), 51; 1 – 8.
Herein we report the design and successful catalytic application of modified Trost-ligands in asymmetric allylic alkylation (AAA) reactions. A small set of carbamate-monophosphine P,O-ligands has been prepared in a straightforward two-step synthetic procedure. After optimization of the reaction conditions, high catalytic activities and excellent enantioselectivity up to >99% have been attained.
Counterion-Enhanced Pd/Enamine Catalysis: Direct Asymmetric α‑Allylation of Aldehydes with Allylic Alcohols by Chiral Amines and Achiral or Racemic Phosphoric Acids
Á. Pálvölgyi, J. Smith, M. Schnürch, K. Schröder*
Journal of Organic Chemistry, 86 (2020), 850 – 860.
We report a straightforward and efficient Pd/enamine catalytic procedure for the direct asymmetric α-allylation of branched aldehydes. The use of simple chiral amines and easily prepared achiral or racemic phosphoric acids, together with a suitable Pd-source resulted in a highly active and enantioselective catalyst system for the allylation of various α-branched aldehydes with different allylic alcohols. The reported procedure could provide an easy access to both product antipodes. Furthermore, two possible orthogonal derivatizations of the enantioenriched aldehydes were performed without any decrease in enantioselectivity.
Counterion Enhanced Organocatalysis: A Novel Approach for the Asymmetric Transfer Hydrogenation of Enones
F. Scharinger, Á. Pálvölgyi, V. Zeindlhofer, M. Schnürch, C. Schröder, K. Schröder*
ChemCatChem, 12 (2020), 14; 3776 – 3782.
We present a novel strategy for organocatalytic transfer hydrogenations relying on an ion‐paired catalyst of natural l‐amino acids as main source of chirality in combination with racemic, atropisomeric phosphoric acids as counteranion. The combination of a chiral cation with a structurally flexible anion resulted in a novel chiral framework for asymmetric transfer hydrogenations with enhanced selectivity through synergistic effects. The optimized catalytic system, in combination with a Hantzsch ester as hydrogen source for biomimetic transfer hydrogenation, enabled high enantioselectivity and excellent yields for a series of α,β‐unsaturated cyclohexenones under mild conditions. Moreover, owing to the use of readily available and chiral pool‐derived building blocks, it could be prepared in a straightforward and significantly cheaper way compared to the current state of the art.
Photocatalytic deaminative benzylation and alkylation of tetrahydroisoquinolines with N-alkylpyrydinium salts
D. Schönbauer, C. Sambiagio, T. Noël, M. Schnürch*
Beilstein Journal of Organic Chemistry, 16 (2020), 809 – 817.
A ruthenium-catalyzed photoredox coupling of substituted N-aryltetrahydroisoquinolines (THIQs) and different bench-stable pyridinium salts was successfully developed to give fast access to 1-benzyl-THIQs. Furthermore, secondary alkyl and allyl groups were also successfully introduced via the same method. Additionally, the typically applied N-phenyl group in the THIQ substrate could be replaced by the cleavable p-methoxyphenyl (PMP) group and successful N-deprotection was demonstrated.
Allosteric GABAA Receptor Modulators-A Review on the Most Recent Heterocyclic Chemotypes and Their Synthetic Accessibility
B. Vega Alanis, M.T. Iorio, L. Silva, K. Bampali, M. Ernst*, M. Schnürch*, M.D. Mihovilovic
Molecules, 25(4) (2020), 999; 1 – 47.
GABAA receptor modulators are structurally almost as diverse as their target protein. A plethora of heterocyclic scaffolds has been described as modulating this extremely important receptor family. Some made it into clinical trials and, even on the market, some were dismissed. This review focuses on the synthetic accessibility and potential for library synthesis of GABAA receptor modulators containing at least one heterocyclic scaffold, which were disclosed within the last 10 years.
6-[(tert-Butyldimethylsilyl)oxy]-3-ethenyl-7-methoxy-4-[(trimethylsilyl)ethynyl]naphtho[2,3-c]furan-1(3H)-one
M. Weil, T. Kremsmayr, M.D. Mihovilovic
IUCrData (2020). 5, x200224
π interactions between the methine group of the furan ring and the central ring of the tricyclic core of an adjacent molecule lead to inversion-related dimers.A Unified Approach to Phytosiderophore Natural Products
N. Kratena, T. Gökler, L. Maltrovsky, E. Oburger, C. Stanetty*
Chemistry – A European Journal, 26 (2020).
This work reports on the concise total synthesis of eight natural products of the mugineic acid and avenic acid families (phytosiderophores). An innovative „east‐to‐west“ assembly of the trimeric products resulted in a high degree of divergence enabling the formation of the final products in just 10 or 11 steps each with a minimum of overall synthetic effort. Chiral pool starting materials (l‐malic acid, threonines) were employed for the outer building blocks while the middle building blocks were accessed by diastereo‐ and enantioselective methods. A highlight of this work consists in the straightforward preparation of epimeric hydroxyazetidine amino acids, useful building blocks on their own, enabling the first synthesis of 3’’‐hydroxymugineic acid and 3’’‐hydroxy‐2’‐deoxymugineic acid.
Straight Forward and Versatile Differentiation of the L-glycero and D-glycero-D-manno Heptose Scaffold
C. Suster, I. Baxendale, M.D. Mihovilovic, C. Stanetty*
Frontiers in Chemistry, 8 (2020), 625; 7 pages.
Bacterial lipopolysaccharides (LPS) are important bio-medical structures, playing a major role in the interaction with human immune systems. Their core regions, containing multiple units of L–glycero–D–manno heptoses (L,D-heptose), are highly conserved structurally (with O3 and O7 glycosidic bonds), making them an epitope of high interest for the potential development of new antibiotics and vaccines. Research in this field has always been restricted by the limited availability of the parent L,D-heptose as well as its biochemical epimeric precursor D–glycero–D–manno heptose (D,D-heptose). This problem of availability has recently been solved by us, through a rapid and efficient practical synthesis of L,D–manno-heptose peracetate demonstrated at scale. Herein we report an optimized, technically simple and versatile synthetic strategy for the differentiation of both the L–glycero and D–glycero–D–manno heptose scaffolds. Our approach is based on an orthoester methodology for the differentiation of all three positions of the sugar core using a O6, O7-tetraisopropyl disiloxyl (TIPDS) protecting group for the exocyclic positions. Furthermore, the regioselective opening toward 7-OH acceptors (6O-FTIPDS ethers) differentiates the exocyclic diol which has been demonstrated with a broader set of substrates and for both manno-heptoses for the first time.
Contributions to Books
Book Title: Applied Biocatalysis : The Chemist’s Enzyme Toolbox
Synthesis of Six out of Eight Carvo-Lactone Stereoisomers via a Novel Concurrent Redox Cascade Starting from (R)- and (S)-Carvones
Pages 426 – 434.
Book Series: Topics in Heterocyclic Chemistry
Topics in Heterocyclic Chemistry – Vol. XXXI: Metalation of Azines and Diazines
M. Schnürch, M.D. Mihovilovic
Series editor: B. Maes; Springer-Verlag Berlin Heidelberg 2013, Berlin – Heidelberg, 2013, ISBN: 978-3-642-35021-4, 272 pages.
This chapter reviews the metalation of pyridazine, cinnoline, and phthalazine derivatives as well as the usage of the hereby formed organometallic compounds in subsequent reactions. The main topics under discussion are lithiation, magnesation, and zincation, the focus being on deprotonative metalation by properly chosen strong bases, e.g. lithiumdiisopropylamide or lithium 2,2,6,6-tetramethylpiperidide. A short discussion upon boronyl and stannyl derivatives of the three diazines under investigation is enclosed at the end of the chapter.
Book Title: Pd-Catalyzed Cross-Coupling Strategies in Thiazole Chemistry
Pd-catalyzed cross-coupling strategies in thiazole chemistry – synthesis of arylated thiazoles and bithiazole building blocks
M. Schnürch
Südwestdeutscher Verlag für Hochschulschriften, Saarbrücken, 2009, ISBN: 978-3-8381-1246-6; 264 pages.
Within this contribution the synthesis of anilino-substituted bithiazoles as potentially bioactive compounds is reported. The key chemical transformations in the applied synthetic strategy were Pd-catalyzed cross coupling reactions which were used for the formation of various bithiazoles. Different cross coupling methodologies were investigated. Starting from 2-thiazolamine the desired metal organyls were prepared in two steps. Besides the well documented Stille and Negishi reaction on thiazole derivatives, a new method could be established. After successful preparation of the first thiazoleboronic acid ester, Suzuki-Miyaura reactions were performed. Upon reaction with various bromo-thiazoles the desired bithiazole derivatives were obtained, whereby the Stille reaction gave the best results compared to the other two methods. Subsequently the chloro atom was used for a nucleophilic exchange reaction with aniline derivatives to give the target compounds.
Book Title: Applied Biocatalysis : The Chemist’s Enzyme Toolbox
Synthesis of Six out of Eight Carvo-Lactone Stereoisomers via a Novel Concurrent Redox Cascade Starting from (R)- and (S)-Carvones
Pages 426 – 434.
Publications in Scientific Journals
Methyl glycosides via Fischer glycosylation: translation from batch microwave to continuous flow processing
J. Aronow, C. Stanetty*, I. Baxendale, M.D. Mihovilovic
Monatshefte für Chemie, 150 (2019), 1; 11 – 19.
Intercepted dehomologation of aldoses by N-heterocyclic carbene catalysis – a novel transformation in carbohydrate chemistry
M. Draskovits, H. Kalaus, C. Stanetty*, M.D. Mihovilovic
Chemical Communications, 55 (2019), 81; 12144 – 12147.
The development of an N-heterocyclic carbene (NHC) catalysed intercepted dehomologation of aldoses is reported. The unique selectivity of NHCs for aldehydes is exploited in the complex context of reducing sugars. Examples of strong substrate governance for either intercepted dehomologation or a subsequent redox-lactonisation were identified and mechanistically understood. More importantly, it was shown that catalyst design allowed the tuning of the selectivity of the reaction with structurally unbiased starting materials towards either of the two scenarios.
Boosting photobioredox catalysis by morpholine electron donors under aerobic conditions
C. P. Goncalves*, H. Mansouri Khosravi, S. Pourmehdi, M. Abdellah, B. Fadiga, E. Bastos, J. Sá, M.D. Mihovilovic, F. Rudroff*
Catalysis Science & Technology, – (2019), 9; 2682 – 2688.
Light-driven reduction of flavins, e.g. FAD or FMN, by sacrificial electron donors emerged as a convenient method to promote biocatalytic transformations. However, flavin activation has been restricted to oxygen-free conditions to prevent enzyme deactivation caused by reactive oxygen species (ROS). Herein, we show that the photoreduction of FMN by morpholines, including 3-(N-morpholino)propanesulfonic acid (MOPS), lessens the deactivation of the enoate reductase XenB from Pseudomonas sp. during the stereoselective asymmetric enzymatic reduction of a model α,β-unsaturated diketone under aerobic conditions, leading to a 91% GC-yield and a stereoselectivity greater than 94%. The kinetic stability of the thermolabile XenB was increased by more than 20-fold in MOPS buffer compared to that in Tris-HCl buffer, and a pronounced positive effect on the transition midpoint temperature was observed. The reactive form of the FMN photocatalyst is stabilized by the formation of a 3[FMN˙−–MOPS˙+] ensemble, which reduces the formation of hydrogen peroxide and other ROS in the presence of oxygen. These results contribute to broaden the application of photobiocatalytic transformations using flavin-dependent reductases.
Morpholine-based buffers activate aerobic photobiocatalysis via spin correlated ion pair formation
Catalysis Science & Technology, – (2019), 9; 1365 – 1371.
The use of enzymes for synthetic applications is a powerful and environmentally-benign approach to increase molecular complexity. Oxidoreductases selectively introduce oxygen and hydrogen atoms into myriad substrates, catalyzing the synthesis of chemical and pharmaceutical building blocks for chemical production. However, broader application of this class of enzymes is limited by the requirements of expensive cofactors and low operational stability. Herein, we show that morpholine-based buffers, especially 3-(N-morpholino)propanesulfonic acid (MOPS), promote photoinduced flavoenzyme-catalyzed asymmetric redox transformations by regenerating the flavin cofactor via sacrificial electron donation and by increasing the operational stability of flavin-dependent oxidoreductases. The stabilization of the active forms of flavin by MOPS via formation of the spin correlated ion pair 3[flavin˙−–MOPS˙+] ensemble reduces the formation of hydrogen peroxide, circumventing the oxygen dilemma under aerobic conditions detrimental to fragile enzymes.
Variations on a scaffold – Novel GABAA receptor modulators
M.T. Iorio*, S. Rehman*, K. Bampali*, B. Stöger*, M. Schnürch*, M. Ernst*, M.D. Mihovilovic*
European Journal of Medicinal Chemistry, 180 (2019), 340 – 349.
Allosteric ligands of GABAA receptors exist in many different chemotypes owing to their great usefulness as therapeutics, with benzodiazepines being among the best known examples. Many allosteric binding sites have been described, among them a site at the extracellular interface between the alpha principal face and the beta complementary face (α+/β-). Pyrazoloquinolinones have been shown to bind at α+/β-binding sites of GABAA receptors, exerting chiefly positive allosteric modulation at this location. In order to further explore molecular determinants of this type of allosteric modulation, we synthesized a library of ligands based on the PQ pharmacophore employing a ring-chain bioisosteric approach. In this study we analyzed the structure-activity-relationship (SAR) of these novel ligands based on an azo-biaryl structural motif in α1β3 GABAA receptors, indicating interesting novel properties of the compound class.
Leoligin-inspired synthetic lignans with selectivity for cell-type and bioactivity relevant for cardiovascular disease
T. Linder, R. Liu, A Atanasov, S. Geyrhofer, S. Schwaiger, H. Stuppner, M. Schnürch, V. Dirsch, M.D. Mihovilovic*
Chemical Science, 10 (2019), 5815 – 5820.
Recently, a natural compound leoligin, a furan-type lignan, was discovered as an interesting hit compound with an anti-inflammatory pharmacological activity profile. We developed a modular and stereoselective approach for the synthesis of the edelweiss-derived lignan leoligin and used the synthetic route to rapidly prepare leoligin analogs even on the gram scale. Proof of concept of this approach together with cell-based bio-assays gained structural analogs with increased selectivity towards vascular smooth muscle versus endothelial cell proliferation inhibition, a major benefit in fighting vascular neointima formation. In addition, we identified the structural features of leoligin analogs that define their ability to inhibit the pro-inflammatory NF-κB pathway. Results are discussed in the context of structural modification of these novel synthetic lignans.
A marine bacterial enzymatic cascade degrades the algal polysaccharide ulvan
L. Reisky, A. Prechoux, M. Zühlke, M. Bäumgen, C. Robb, N. Gerlach, T. Roret, C. Stanetty, R. Larocque, M. Gurvan, T. Song, S. Markert, F. Unfried, M.D. Mihovilovic, A. Trautwein-Schult, D. Becher, T. Schweder*, U. Bornscheuer*, J. Hehemann*
Nature Chemical Biology, 15 (2019), 803 – 812.
Marine seaweeds increasingly grow into extensive algal blooms, which are detrimental to coastal ecosystems, tourism and aquaculture. However, algal biomass is also emerging as a sustainable raw material for the bioeconomy. The potential exploitation of algae is hindered by our limited knowledge of the microbial pathways—and hence the distinct biochemical functions of the enzymes involved—that convert algal polysaccharides into oligo- and monosaccharides. Understanding these processes would be essential, however, for applications such as the fermentation of algal biomass into bioethanol or other value-added compounds. Here, we describe the metabolic pathway that enables the marine flavobacterium Formosa agariphila to degrade ulvan, the main cell wall polysaccharide of bloom-forming Ulva species. The pathway involves 12 biochemically characterized carbohydrate-active enzymes, including two polysaccharide lyases, three sulfatases and seven glycoside hydrolases that sequentially break down ulvan into fermentable monosaccharides. This way, the enzymes turn a previously unexploited renewable into a valuable and ecologically sustainable bioresource.
Substrate-Independent High-Throughput Assay for the Quantification of Aldehydes
A. Ressmann, D. Schwendenwein, S. Leonhartsberger, M.D. Mihovilovic, U. Bornscheuer, M. Winkler*, F. Rudroff*
Advanced Synthesis & Catalysis, 361 (2019), 11; 2538 – 2543.
The selective and direct reduction of carboxylic acids into the corresponding aldehydes by chemical methods is still a challenging task in synthesis. Several reductive and oxidative chemical methods are known to produce aldehydes, but most of them require expensive reagents, special reaction conditions, are two‐step procedures and often lack chemoselectivity. Nature provides an elegant tool, so called carboxylic acid reductases (CARs) for the direct reduction of carboxylic acids to aldehydes. Discovery as well as engineering of novel CAR enzymes necessitates a robust, product selective high‐throughput assay (HTA). We report a simple and fast HTA that allows the substrate‐independent and chemoselective quantification of aldehydes (irrespective of their chemical structure) and is sensitive to the nM range. The HTA was validated by NMR and GC analyses and in microbial cells by reexamination of the substrate scope of CAR from Nocardia iowensis (CARNi). The results were fully consistent with reported data.
Random Mutagenesis-Driven Improvement of Carboxylate Reductase Activity using an Amino Benzamidoxime-Mediated High-Throughput Assay
D. Schwendenwein, A. Ressmann, M. Dörr, M. Höhne, U. Bornscheuer, M.D. Mihovilovic, F. Rudroff*, M. Winkler*
Advanced Synthesis & Catalysis, 361 (2019), 11; 2544 – 2549.
Carboxylic acid reductases (CARs) catalyze the direct adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH) dependent reduction of carboxylic acids to their corresponding aldehydes. The identification and improvement of CARs by protein engineering is, however, severely limited by the lack of fast and generic methods to quantify aldehydes. Within this study, we applied a convenient high‐throughput assay (HTA) based on amino benzamidoxime (ABAO) that allows the substrate‐independent and chemoselective quantification of aldehydes. Random mutagenesis of the well‐known CAR from Nocardia iowensis (CARNi) to improve its activity for sterically demanding 2‐substituted benzoic acid derivatives was conducted in a KM‐dependent fashion, and the HTA applied in the presence of microbial cells. The study identified a hot spot in the active site of CARNi that increased the affinity to 2‐methoxybenzoic acid 9‐fold upon mutation from glutamine to proline (Q283P). The catalytic performance of CARNiQ283P appeared to be significantly improved also for other substrates such as 2‐substituted (2‐Cl, 2‐Br) as well as 3‐ and 4‐substituted benzoic acids (3‐OMe, 4‐OMe), and even aliphatic octanoic acid.
Defined concatenated α6α1β3γ2 GABAA receptor constructs reveal dual action of pyrazoloquinolinone allosteric modulators
X. Simeone, M.T. Iorio, D. Siebert, S. Rehman, M. Schnürch, M.D. Mihovilovic, M. Ernst*
Bioorganic & Medicinal Chemistry, 27 (2019), 3167 – 3178.
Pyrazoloquinolinones (PQs) have been extensively studied as modulators of GABAA receptors with different subunit composition, exerting modulatory effects by binding at α+/β- interfaces of GABAA receptors. PQs with a substituent in position R7 have been reported to preferentially modulate α6- subunit containing GABAA receptors which are mostly expressed in the cerebellum but were also found in the olfactory bulb, in the cochlear nucleus, in the hippocampus and in the trigeminal sensory pathway. They are considered potentially interesting in the context of sensori-motor gating deficits, depressive-like behavior, migraine and orofacial pain. Here we explored the option to modify the lead ligands’ R7 position. In the compound series we observed two different patterns of allosteric modulation in recombinantly expressed α6β3γ2 receptors, namely monophasic and biphasic positive modulation. In the latter case the additional phase occurred in the nanomolar range, while all compounds displayed robust modulation in the micromolar range. Nanomolar, near silent binding has been reported to occur at benzodiazepine binding sites, but was not investigated at the diazepam insensitive α6+/γ2- interface. To clarify the mechanism underlying the biphasic effect we tested one of the compounds in concatenated receptors. In these constructs the subunits are covalently linked, allowing to form either the α6+/γ2- interface, or the α6+/β3- interface, to study the resulting modulation. With this approach we were able to ascribe the nanomolar modulation to the α6+/γ2- interface. While not all compounds display the nanomolar phase, the strong modulation at the α6+/β3 interface proved to be tolerant for all tested R7 groups. This provides the future option to introduce e.g. isotope labelled or fluorescent moieties or substituents that enhance solubility and bioavailability.
Boosting photobioredox catalysis by morpholine electron donors under aerobic conditions
C. P. Goncalves, H. Mansouri Khosravi, S. Pourmehdi, M. Abdellah, B. Fadiga, E. Bastos, J. Sá, M.D. Mihovilovic, F. Rudroff*
Morpholine-based buffers activate aerobic photobiocatalysis via spin correlated ion pair formation
L. Gonçalves*, H. Mansouri Khosravi, E. Bastos, M. Abdellah, B. Fadiga, F. Rudroff, M.D. Mihovilovic
Substrate-Independent High-Throughput Assay for the Quantification of Aldehydes
Advanced Synthesis & Catalysis, 361 (2019), 11; 2538 – 2543.
Random Mutagenesis-Driven Improvement of Carboxylate Reductase Activity using an Amino Benzamidoxime-Mediated High-Throughput Assay
Publications in Scientific Journals
Methyl glycosides via Fischer glycosylation: translation from batch microwave to continuous flow processing
J. Aronow, C. Stanetty*, I. Baxendale, M.D. Mihovilovic
Monatshefte für Chemie, 150 (2019), 1; 11 – 19.
Intercepted dehomologation of aldoses by N-heterocyclic carbene catalysis – a novel transformation in carbohydrate chemistry
M. Draskovits, H. Kalaus, C. Stanetty*, M.D. Mihovilovic
Chemical Communications, 55 (2019), 81; 12144 – 12147.
The development of an N-heterocyclic carbene (NHC) catalysed intercepted dehomologation of aldoses is reported. The unique selectivity of NHCs for aldehydes is exploited in the complex context of reducing sugars. Examples of strong substrate governance for either intercepted dehomologation or a subsequent redox-lactonisation were identified and mechanistically understood. More importantly, it was shown that catalyst design allowed the tuning of the selectivity of the reaction with structurally unbiased starting materials towards either of the two scenarios.
Boosting photobioredox catalysis by morpholine electron donors under aerobic conditions
C. P. Goncalves*, H. Mansouri Khosravi, S. Pourmehdi, M. Abdellah, B. Fadiga, E. Bastos, J. Sá, M.D. Mihovilovic, F. Rudroff*
Catalysis Science & Technology, – (2019), 9; 2682 – 2688.
Light-driven reduction of flavins, e.g. FAD or FMN, by sacrificial electron donors emerged as a convenient method to promote biocatalytic transformations. However, flavin activation has been restricted to oxygen-free conditions to prevent enzyme deactivation caused by reactive oxygen species (ROS). Herein, we show that the photoreduction of FMN by morpholines, including 3-(N-morpholino)propanesulfonic acid (MOPS), lessens the deactivation of the enoate reductase XenB from Pseudomonas sp. during the stereoselective asymmetric enzymatic reduction of a model α,β-unsaturated diketone under aerobic conditions, leading to a 91% GC-yield and a stereoselectivity greater than 94%. The kinetic stability of the thermolabile XenB was increased by more than 20-fold in MOPS buffer compared to that in Tris-HCl buffer, and a pronounced positive effect on the transition midpoint temperature was observed. The reactive form of the FMN photocatalyst is stabilized by the formation of a 3[FMN˙−–MOPS˙+] ensemble, which reduces the formation of hydrogen peroxide and other ROS in the presence of oxygen. These results contribute to broaden the application of photobiocatalytic transformations using flavin-dependent reductases.
Morpholine-based buffers activate aerobic photobiocatalysis via spin correlated ion pair formation
Catalysis Science & Technology, – (2019), 9; 1365 – 1371.
The use of enzymes for synthetic applications is a powerful and environmentally-benign approach to increase molecular complexity. Oxidoreductases selectively introduce oxygen and hydrogen atoms into myriad substrates, catalyzing the synthesis of chemical and pharmaceutical building blocks for chemical production. However, broader application of this class of enzymes is limited by the requirements of expensive cofactors and low operational stability. Herein, we show that morpholine-based buffers, especially 3-(N-morpholino)propanesulfonic acid (MOPS), promote photoinduced flavoenzyme-catalyzed asymmetric redox transformations by regenerating the flavin cofactor via sacrificial electron donation and by increasing the operational stability of flavin-dependent oxidoreductases. The stabilization of the active forms of flavin by MOPS via formation of the spin correlated ion pair 3[flavin˙−–MOPS˙+] ensemble reduces the formation of hydrogen peroxide, circumventing the oxygen dilemma under aerobic conditions detrimental to fragile enzymes.
Variations on a scaffold – Novel GABAA receptor modulators
M.T. Iorio*, S. Rehman*, K. Bampali*, B. Stöger*, M. Schnürch*, M. Ernst*, M.D. Mihovilovic*
European Journal of Medicinal Chemistry, 180 (2019), 340 – 349.
Allosteric ligands of GABAA receptors exist in many different chemotypes owing to their great usefulness as therapeutics, with benzodiazepines being among the best known examples. Many allosteric binding sites have been described, among them a site at the extracellular interface between the alpha principal face and the beta complementary face (α+/β-). Pyrazoloquinolinones have been shown to bind at α+/β-binding sites of GABAA receptors, exerting chiefly positive allosteric modulation at this location. In order to further explore molecular determinants of this type of allosteric modulation, we synthesized a library of ligands based on the PQ pharmacophore employing a ring-chain bioisosteric approach. In this study we analyzed the structure-activity-relationship (SAR) of these novel ligands based on an azo-biaryl structural motif in α1β3 GABAA receptors, indicating interesting novel properties of the compound class.
Leoligin-inspired synthetic lignans with selectivity for cell-type and bioactivity relevant for cardiovascular disease
T. Linder, R. Liu, A Atanasov, S. Geyrhofer, S. Schwaiger, H. Stuppner, M. Schnürch, V. Dirsch, M.D. Mihovilovic*
Chemical Science, 10 (2019), 5815 – 5820.
Recently, a natural compound leoligin, a furan-type lignan, was discovered as an interesting hit compound with an anti-inflammatory pharmacological activity profile. We developed a modular and stereoselective approach for the synthesis of the edelweiss-derived lignan leoligin and used the synthetic route to rapidly prepare leoligin analogs even on the gram scale. Proof of concept of this approach together with cell-based bio-assays gained structural analogs with increased selectivity towards vascular smooth muscle versus endothelial cell proliferation inhibition, a major benefit in fighting vascular neointima formation. In addition, we identified the structural features of leoligin analogs that define their ability to inhibit the pro-inflammatory NF-κB pathway. Results are discussed in the context of structural modification of these novel synthetic lignans.
A marine bacterial enzymatic cascade degrades the algal polysaccharide ulvan
L. Reisky, A. Prechoux, M. Zühlke, M. Bäumgen, C. Robb, N. Gerlach, T. Roret, C. Stanetty, R. Larocque, M. Gurvan, T. Song, S. Markert, F. Unfried, M.D. Mihovilovic, A. Trautwein-Schult, D. Becher, T. Schweder*, U. Bornscheuer*, J. Hehemann*
Nature Chemical Biology, 15 (2019), 803 – 812.
Marine seaweeds increasingly grow into extensive algal blooms, which are detrimental to coastal ecosystems, tourism and aquaculture. However, algal biomass is also emerging as a sustainable raw material for the bioeconomy. The potential exploitation of algae is hindered by our limited knowledge of the microbial pathways—and hence the distinct biochemical functions of the enzymes involved—that convert algal polysaccharides into oligo- and monosaccharides. Understanding these processes would be essential, however, for applications such as the fermentation of algal biomass into bioethanol or other value-added compounds. Here, we describe the metabolic pathway that enables the marine flavobacterium Formosa agariphila to degrade ulvan, the main cell wall polysaccharide of bloom-forming Ulva species. The pathway involves 12 biochemically characterized carbohydrate-active enzymes, including two polysaccharide lyases, three sulfatases and seven glycoside hydrolases that sequentially break down ulvan into fermentable monosaccharides. This way, the enzymes turn a previously unexploited renewable into a valuable and ecologically sustainable bioresource.
Substrate-Independent High-Throughput Assay for the Quantification of Aldehydes
A. Ressmann, D. Schwendenwein, S. Leonhartsberger, M.D. Mihovilovic, U. Bornscheuer, M. Winkler*, F. Rudroff*
Advanced Synthesis & Catalysis, 361 (2019), 11; 2538 – 2543.
The selective and direct reduction of carboxylic acids into the corresponding aldehydes by chemical methods is still a challenging task in synthesis. Several reductive and oxidative chemical methods are known to produce aldehydes, but most of them require expensive reagents, special reaction conditions, are two‐step procedures and often lack chemoselectivity. Nature provides an elegant tool, so called carboxylic acid reductases (CARs) for the direct reduction of carboxylic acids to aldehydes. Discovery as well as engineering of novel CAR enzymes necessitates a robust, product selective high‐throughput assay (HTA). We report a simple and fast HTA that allows the substrate‐independent and chemoselective quantification of aldehydes (irrespective of their chemical structure) and is sensitive to the nM range. The HTA was validated by NMR and GC analyses and in microbial cells by reexamination of the substrate scope of CAR from Nocardia iowensis (CARNi). The results were fully consistent with reported data.
Random Mutagenesis-Driven Improvement of Carboxylate Reductase Activity using an Amino Benzamidoxime-Mediated High-Throughput Assay
D. Schwendenwein, A. Ressmann, M. Dörr, M. Höhne, U. Bornscheuer, M.D. Mihovilovic, F. Rudroff*, M. Winkler*
Advanced Synthesis & Catalysis, 361 (2019), 11; 2544 – 2549.
Carboxylic acid reductases (CARs) catalyze the direct adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH) dependent reduction of carboxylic acids to their corresponding aldehydes. The identification and improvement of CARs by protein engineering is, however, severely limited by the lack of fast and generic methods to quantify aldehydes. Within this study, we applied a convenient high‐throughput assay (HTA) based on amino benzamidoxime (ABAO) that allows the substrate‐independent and chemoselective quantification of aldehydes. Random mutagenesis of the well‐known CAR from Nocardia iowensis (CARNi) to improve its activity for sterically demanding 2‐substituted benzoic acid derivatives was conducted in a KM‐dependent fashion, and the HTA applied in the presence of microbial cells. The study identified a hot spot in the active site of CARNi that increased the affinity to 2‐methoxybenzoic acid 9‐fold upon mutation from glutamine to proline (Q283P). The catalytic performance of CARNiQ283P appeared to be significantly improved also for other substrates such as 2‐substituted (2‐Cl, 2‐Br) as well as 3‐ and 4‐substituted benzoic acids (3‐OMe, 4‐OMe), and even aliphatic octanoic acid.
Defined concatenated α6α1β3γ2 GABAA receptor constructs reveal dual action of pyrazoloquinolinone allosteric modulators
X. Simeone, M.T. Iorio, D. Siebert, S. Rehman, M. Schnürch, M.D. Mihovilovic, M. Ernst*
Bioorganic & Medicinal Chemistry, 27 (2019), 3167 – 3178.
Pyrazoloquinolinones (PQs) have been extensively studied as modulators of GABAA receptors with different subunit composition, exerting modulatory effects by binding at α+/β- interfaces of GABAA receptors. PQs with a substituent in position R7 have been reported to preferentially modulate α6- subunit containing GABAA receptors which are mostly expressed in the cerebellum but were also found in the olfactory bulb, in the cochlear nucleus, in the hippocampus and in the trigeminal sensory pathway. They are considered potentially interesting in the context of sensori-motor gating deficits, depressive-like behavior, migraine and orofacial pain. Here we explored the option to modify the lead ligands’ R7 position. In the compound series we observed two different patterns of allosteric modulation in recombinantly expressed α6β3γ2 receptors, namely monophasic and biphasic positive modulation. In the latter case the additional phase occurred in the nanomolar range, while all compounds displayed robust modulation in the micromolar range. Nanomolar, near silent binding has been reported to occur at benzodiazepine binding sites, but was not investigated at the diazepam insensitive α6+/γ2- interface. To clarify the mechanism underlying the biphasic effect we tested one of the compounds in concatenated receptors. In these constructs the subunits are covalently linked, allowing to form either the α6+/γ2- interface, or the α6+/β3- interface, to study the resulting modulation. With this approach we were able to ascribe the nanomolar modulation to the α6+/γ2- interface. While not all compounds display the nanomolar phase, the strong modulation at the α6+/β3 interface proved to be tolerant for all tested R7 groups. This provides the future option to introduce e.g. isotope labelled or fluorescent moieties or substituents that enhance solubility and bioavailability.
Boosting photobioredox catalysis by morpholine electron donors under aerobic conditions
C. P. Goncalves, H. Mansouri Khosravi, S. Pourmehdi, M. Abdellah, B. Fadiga, E. Bastos, J. Sá, M.D. Mihovilovic, F. Rudroff*
Morpholine-based buffers activate aerobic photobiocatalysis via spin correlated ion pair formation
L. Gonçalves*, H. Mansouri Khosravi, E. Bastos, M. Abdellah, B. Fadiga, F. Rudroff, M.D. Mihovilovic
Substrate-Independent High-Throughput Assay for the Quantification of Aldehydes
Advanced Synthesis & Catalysis, 361 (2019), 11; 2538 – 2543.
Random Mutagenesis-Driven Improvement of Carboxylate Reductase Activity using an Amino Benzamidoxime-Mediated High-Throughput Assay
Publications in Scientific Journals
In vivo synthesis of polyhydroxylated compounds from a ‘hidden reservoir’ of toxic aldehyde species
T. Bayer, T. Wiesinger, S. Milker, M. Winkler, M.D. Mihovilovic, F. Rudroff*
ChemCatChem, 9 (2017), 2919 – 2923.
Synthetic enzyme cascades in living cells often lack efficiency owing to the formation of byproducts by endogenous enzymes or toxicity of the cascade intermediates. Highly reactive aldehyde species can trigger a metabolic stress response, and this leads to undesired side reactions and decreased yields. Owing to the metabolic background of Escherichia coli (E. coli), aldehydes may be irreversibly oxidized to carboxylic acids or reduced to the corresponding alcohols. Herein, we applied an approach to equilibrate the aldehyde concentration in vivo. We oxidized primary alcohols to the corresponding aldehydes by AlkJ, an alcohol dehydrogenase from Pseudomonas putida. Introduction of a carboxylic acid reductase from Nocardia iowensis allowed the target compound to be retrieved from the carboxylate sink. Further reduction of the aldehydes to alcohols by endogenous E. coli enzymes completed the equilibration between alcohols, aldehydes, and carboxylic acids. Thus, the aldehyde concentrations remained below nonviable concentrations. We demonstrated the concept on several primary alcohols, which reached the redox equilibrium within 6 h and persisted up to 24 h. Subsequent combination with a dihydroxyacetone‐dependent aldolase (Fsa1‐A129S, E. coli) demonstrated that the reactive aldehyde species were freely available and gave the aldol product, (3S,4R)‐1,3,4‐trihydroxy‐5‐phenylpentan‐2‐one, in 70 % yield within short reaction times.
Cloning and characterization of the Type I Baeyer-Villiger monooxygenase from Leptospira biflexa
R. Ceccoli, D. Bianchi, M. Fink, M.D. Mihovilovic, D. Rial*
AMB Express, 7 (2017), 87 – 99.
Linked magnolol dimer as a selective PPARγ agonist – Structure-based rational design, synthesis, and bioactivity evaluation
D. Dreier, S. Latkolik, L. Rycek, M. Schnürch, A. Dymáková, A Atanasov, A. Ladurner, E. Heiss, H. Stuppner, D. Schuster*, M.D. Mihovilovic, V. Dirsch*
Scientific Reports, 7 (2017), 13002; 1 – 10.
The nuclear receptors peroxisome proliferator-activated receptor γ (PPARγ) and its hetero-dimerization partner retinoid X receptor α (RXRα) are considered as drug targets in the treatment of diseases like the metabolic syndrome and diabetes mellitus type 2. Effort has been made to develop new agonists for PPARγ to obtain ligands with more favorable properties than currently used drugs. Magnolol was previously described as dual agonist of PPARγ and RXRα. Here we show the structure-based rational design of a linked magnolol dimer within the ligand binding domain of PPARγ and its synthesis. Furthermore, we evaluated its binding properties and functionality as a PPARγ agonist in vitro with the purified PPARγ ligand binding domain (LBD) and in a cell-based nuclear receptor transactivation model in HEK293 cells. We determined the synthesized magnolol dimer to bind with much higher affinity to the purified PPARγ ligand binding domain than magnolol (K i values of 5.03 and 64.42 nM, respectively). Regarding their potency to transactivate a PPARγ-dependent luciferase gene both compounds were equally effective. This is likely due to the PPARγ specificity of the newly designed magnolol dimer and lack of RXRα-driven transactivation activity by this dimeric compound.
Mutagenesis-Independent Stabilization of Class B Flavin Monooxygenases in Operation
C. P. Goncalves, D. Kracher, S. Milker, M. Fink*, F. Rudroff*, R. Ludwig*, A. Bommarius, M.D. Mihovilovic
Advanced Synthesis & Catalysis, 359 (2017), 2121 – 2131.
This paper describes the stabilization of flavin‐dependent monooxygenases under reaction conditions, using an engineered formulation of additives (the natural cofactors NADPH and FAD, and superoxide dismutase and catalase as catalytic antioxidants). This way, a 103‐ to 104‐fold increase of the half‐life was reached without resource‐intensive directed evolution or structure‐dependent protein engineering methods. The stabilized enzymes are highly valued for their synthetic potential in biotechnology and medicinal chemistry (enantioselective sulfur, nitrogen and Baeyer–Villiger oxidations; oxidative human metabolism), but widespread application was so far hindered by their notorious fragility. Our technology immediately enables their use, does not require structural knowledge of the biocatalyst, and creates a strong basis for the targeted development of improved variants by mutagenesis.
Cu(I)-catalyzed one-pot decarboxylation-alkynylation reactions on1,2,3,4-tetrahydroisoquinolines and one-pot synthesis oftriazolyl-1,2,3,4-tetrahydroisoquinolines
B. Gröll, P. Schaaf, M.D. Mihovilovic, M. Schnürch
Journal of Molecular Catalysis A: Chemical, 426 (2017), 398 – 406.
A facile and efficient method to introduce alkyne groups to the C-1 position of biologically interesting 1,2,3,4-tetrahydroisoquinolines via direct CH-functionalization is reported. Various alkynylated N-substituted 1,2,3,4-tetrahydroisoquinolines could be obtained by using copper(I)-chloride as catalyst, alkynoic acids as alkyne source and t-BuOOH as oxidant, in a one-pot two-step decarboxylation- alkynylation reaction in moderate to high yields. Furthermore, a one-pot protocol of a three-step decarboxylation-alkynylation-1,3-dipolar cycloaddition reaction leading to 1-triazolyl-tetrahydroisoquinolines was developed, a hitherto unknown reaction cascade.
Manipulating the Stereoselectivity of the Robust Baeyer-Villiger Monooxygenase TmCHMO by Directed Evolution
G. Li, M. Fürst, H. Mansouri Khosravi, A. Ressmann, A. Ilie, F. Rudroff, M.D. Mihovilovic*, M.W. Fraaije*, M.T Reetz*
Organic & Biomolecular Chemistry, 15 (2017), 9824 – 9829.
Baeyer–Villiger monooxygenases (BVMOs) and evolved mutants have been shown to be excellent biocatalysts in many stereoselective Baeyer–Villiger transformations, but industrial applications are rare which is partly due to the insufficient thermostability of BVMOs under operating conditions. In the present study, the substrate scope of the recently discovered thermally stable BVMO, TmCHMO from Thermocrispum municipale, was studied. This revealed that the wild-type (WT) enzyme catalyzes the oxidation of a variety of structurally different ketones with notable activity and enantioselectivity, including the desymmetrization of 4-methylcyclohexanone (99% ee, S). In order to induce the reversal of enantioselectivity of this reaction as well as the transformations of other substrates, directed evolution based on iterative saturation mutagenesis (ISM) was applied, leading to (R)-selectivity (94% ee) without affecting the thermostability of the biocatalyst.
Thiophene ring-fragmentation reactions: Principles and scale-up towards NLO materials
D. Lumpi*, J. Steindl, S. Steiner, V. Carl, P. Kautny, M. Schön, F. Glöcklhofer, B. Holzer, B. Stöger, E. Horkel, C. Hametner, G.A. Reider, M.D. Mihovilovic, J. Fröhlich
Tetrahedron, 73 (2017), 5; 472 – 480.
A systematic study on the thiophene ring-fragmentation (TRF) reaction, yielding the Z-isomer of ene-yne type compounds, is presented. The investigations focus on the origins and pathways of potential side-reactions, resulting in an advanced synthetic protocol featuring enhanced selectivity and efficiency. The fragmentation threshold temperatures as well as reaction kinetics have been investigated utilizing inline infrared spectroscopy revealing unexpected results particularly concerning the reaction order (zero-order process). With regard to safety, selectivity, and up-scaling a flow-chemistry procedure for the TRF reaction has been developed. Finally, the technological relevance of the ene-yne structural motif is extended by a new design concept for NLO-chromophores showing the highest second harmonic generation efficiencies reported for these scaffolds.
Kinetic modeling of an enzymatic redox cascade in vivo reveals cofactor-caused bottlenecks
S. Milker, M. Fink, N. Oberleitner, A. Ressmann, U. Bornscheuer, M.D. Mihovilovic, F. Rudroff*
ChemCatChem, 9 (2017), 3420 – 3427.
We describe the development of a kinetic model for the simulation and optimization of an in vivo redox cascade in E. coli in which a combination of an alcohol dehydrogenase, an enoate reductase, and a Baeyer–Villiger monooxygenase is used for the synthesis of lactones. The model was used to estimate the concentrations of active enzyme in the sequential biotransformations to identify bottlenecks together with their reasons and how to overcome them. We estimated adapted Michaelis–Menten parameters from in vitro experiments with isolated enzymes and used these values to simulate the change in the concentrations of intermediates and products during the in vivo cascade reactions. Remarkably, the model indicated that the fastest enzyme was rate‐determining because of the unexpectedly low concentration of the active form, which opens up reversible reaction channels towards byproducts. We also provide substantial experimental evidence that a low intracellular concentration of flavin and nicotinamide cofactors drastically decreased the performance of the in vivo cascade drastically.
Non-hazardous biocatalytic oxidation in Nylon-9 monomer synthesis on a 40-gram scale with efficient downstream processing
S. Milker, M. Fink, F. Rudroff, M.D. Mihovilovic
Biotechnology and Bioengineering, 8 (2017), 1670 – 1678.
This paper describes the development of a biocatalytic process on the multi-dozen gram scale for the synthesis of a precursor to Nylon-9, a specialty polyamide. Such materials are growing in demand, but their corresponding monomers are often difficult to synthesize, giving rise to biocatalytic approaches. Here, we implemented cyclopentadecanone monooxygenase as an Escherichia coli whole-cell biocatalyst in a defined medium, together with a substrate feeding-product removal concept, and an optimized downstream processing (DSP). A previously described hazardous peracid-mediated oxidation was thus replaced with a safe and scalable protocol, using aerial oxygen as oxidant, and water as reaction solvent. The engineered process converted 42 g (0.28 mol) starting material ketone to the corresponding lactone with an isolated yield of 70% (33 g), after highly efficient DSP with 95% recovery of the converted material, translating to a volumetric yield of 8 g pure product per liter. Biotechnol. Bioeng. 2017;114: 1670-1678. © 2017 Wiley Periodicals, Inc.
From waste to value – Direct utilization of limonene from orange peel in a biocatalytic cascade reaction towards chiral carvolactone
N. Oberleitner, A. Ressmann, K. Schröder, P. Gärtner, M.W. Fraaije, U. Bornscheuer, M.D. Mihovilovic, F. Rudroff*
Green Chemistry, 367 (2017), 19; 367 – 371.
In this proof of concept study we demonstrate direct utilization of limonene containing waste product orange peel as starting material for a biocatalytic cascade reaction. The product of this cascade is chiral carvolactone, a promising building block for thermoplastic polymers. Four different concepts were applied to augment limonene availability based on either water extraction solely, addition of extraction enhancers or biomass dissolution.
A fusion protein of an enoate reductase and a Baeyer-Villiger monooxygenase facilitates synthesis of chiral lactones
C. Peters, F. Rudroff, M.D. Mihovilovic, U. Bornscheuer
Biological Chemistry, 1 (2017), 398; 31 – 37.
Nature uses the advantages of fusion proteins for multi-step reactions to facilitate the metabolism in cells as the conversion of substrates through intermediates to the final product can take place more rapidly and with less side-product formation. In a similar fashion, also for enzyme cascade reactions, the fusion of biocatalysts involved can be advantageous. In the present study, we investigated fusion of an alcohol dehydrogenase (ADH), an enoate reductase (ERED) and a Baeyer-Villiger monooxygenase (BVMO) to enable the synthesis of (chiral) lactones starting from unsaturated alcohols as substrates. The domain order and various linkers were studied to find optimal conditions with respect to expression levels and enzymatic activities. Best results were achieved for the ERED xenobiotic reductase B (XenB) from Pseudomonas putida and the cyclohexanone monooxygenase (CHMO) from Acinetobacter sp., whereas none of the ADHs studied could be fused successfully. This fusion protein together with separately supplied ADH resulted in similar reaction rates in in vivo biocatalysis reactions. After 1.5 h we could detect 40% more dihydrocarvone lactone in in vivo reactions with the fusion protein and ADH then with the single enzymes.
First chemo-enzymatic synthesis of (R)-Taniguchi lactone and substrate profiles of CAMO and OTEMO, two new Baeyer-Villiger monooxygenases
F. Rudroff, M. Fink, R. Pydi, U. Bornscheuer, M.D. Mihovilovic
Monatshefte für Chemie, 148 (2017), 157 – 165.
This study investigates the substrate profile of cycloalkanone monooxygenase and 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-coenzyme A monooxygenase, two recently discovered enzymes of the Baeyer-Villiger monooxygenase family, used as whole-cell biocatalysts. Biooxidations of a diverse set of ketones were performed on analytical scale: desymmetrization of substituted prochiral cyclobutanones and cyclohexanones, regiodivergent oxidation of terpenones and bicyclic ketones, as well as kinetic resolution of racemic cycloketones. We demonstrated the applicability of the title enzymes in the enantioselective synthesis of (R)-(-)-Taniguchi lactone, a building block for the preparation of various natural product analogs such as ent-quinine.
Molecular tools for GABAA receptors: High affinity ligands for β1-containing subtypes
X. Simeone, D. Siebert, K. Bampali, Z. Varagic, M. Treven, S. Rehman, J. Pyszkowski, R. Holzinger, F. Steudle, P. Scholze, M.D. Mihovilovic, M. Schnürch, M. Ernst*
Scientific Reports, 7 (2017), 5674.
γ-Aminobutyric acid type A (GABAA) receptors are pentameric GABA-gated chloride channels that are, in mammalians, drawn from a repertoire of 19 different genes, namely α1-6, β1-3, γ1-3, δ, ε, θ, π and ρ1-3. The existence of this wide variety of subunits as well as their diverse assembly into different subunit compositions result in miscellaneous receptor subtypes. In combination with the large number of known and putative allosteric binding sites, this leads to a highly complex pharmacology. Recently, a novel binding site at extracellular α+/β− interfaces was described as the site of modulatory action of several pyrazoloquinolinones. In this study we report a highly potent ligand from this class of compounds with pronounced β1-selectivity that mainly lacks α-subunit selectivity. It constitutes the most potent β1-selective positive allosteric modulatory ligand with known binding site. In addition, a proof of concept pyrazoloquinolinone ligand lacking the additional high affinity interaction with the benzodiazepine binding site is presented. Ultimately, such ligands can be used as invaluable molecular tools for the detection of β1-containing receptor subtypes and the investigation of their abundance and distribution.
Cell Factory Design and Optimization for the Stereoselective Synthesis of Polyhydroxylated Compounds
T. Wiesinger, T. Bayer, S. Milker, M.D. Mihovilovic, F. Rudroff
ChemBioChem, 18 (2017), 1 – 9.
A synthetic cascade for the transformation of primary alcohols into polyhydroxylated compounds in Escherichia coli, through the in situ preparation of cytotoxic aldehyde intermediates and subsequent aldolase-mediated C-C bond formation, has been investigated. An enzymatic toolbox consisting of alcohol dehydrogenase AlkJ from Pseudomonas putida and the dihydroxyacetone-/hydroxyacetone-accepting aldolase variant Fsa1-A129S was applied. Pathway optimization was performed at the genetic and process levels. Three different arrangements of the alkJ and fsa1-A129S genes in operon, monocistronic, and pseudo-operon configuration were tested. The last of these proved to be most beneficial with regard to bacterial growth and protein expression levels. The optimized whole-cell catalyst, combined with a refined solid-phase extraction downstream purification protocol, provides diastereomerically pure carbohydrate derivatives that can be isolated in up to 91 % yield over two reaction steps.
In vivo synthesis of polyhydroxylated compounds from a ‘hidden reservoir’ of toxic aldehyde species
T. Bayer, T. Wiesinger, S. Milker, M. Winkler, M.D. Mihovilovic, F. Rudroff*
ChemCatChem, 9 (2017), 2919 – 2923.
Synthetic enzyme cascades in living cells often lack efficiency owing to the formation of byproducts by endogenous enzymes or toxicity of the cascade intermediates. Highly reactive aldehyde species can trigger a metabolic stress response, and this leads to undesired side reactions and decreased yields. Owing to the metabolic background of Escherichia coli (E. coli), aldehydes may be irreversibly oxidized to carboxylic acids or reduced to the corresponding alcohols. Herein, we applied an approach to equilibrate the aldehyde concentration in vivo. We oxidized primary alcohols to the corresponding aldehydes by AlkJ, an alcohol dehydrogenase from Pseudomonas putida. Introduction of a carboxylic acid reductase from Nocardia iowensis allowed the target compound to be retrieved from the carboxylate sink. Further reduction of the aldehydes to alcohols by endogenous E. coli enzymes completed the equilibration between alcohols, aldehydes, and carboxylic acids. Thus, the aldehyde concentrations remained below nonviable concentrations. We demonstrated the concept on several primary alcohols, which reached the redox equilibrium within 6 h and persisted up to 24 h. Subsequent combination with a dihydroxyacetone‐dependent aldolase (Fsa1‐A129S, E. coli) demonstrated that the reactive aldehyde species were freely available and gave the aldol product, (3S,4R)‐1,3,4‐trihydroxy‐5‐phenylpentan‐2‐one, in 70 % yield within short reaction times.
Mutagenesis‐Independent Stabilization of Class B Flavin Monooxygenases in Operation
C. P. Goncalves, D. Kracher, S. Milker, M. Fink*, F. Rudroff*, R. Ludwig*, A. Bommarius, M.D. Mihovilovic
Advanced Synthesis & Catalysis, 359 (2017), 2121 – 2131.
Nicotinamide adenine dinucleotide-dependent redox-neutral convergent cascade for lactonizations with Type II flavin-containing monooxygenase
L. Hunag, E. Romero, A. Ressmann, F. Rudroff, F. Hollmann, M.W. Fraaije, S. Kara*
Advanced Synthesis & Catalysis, 12 (2017), 2142 – 2148.
Manipulating the stereoselectivity of the thermostable Baeyer–Villiger monooxygenase TmCHMO by directed evolution
G. Li, M. Fürst, H. Mansouri Khosravi, A. Ressmann, A. Ilie, F. Rudroff, M.D. Mihovilovic*, M.W. Fraaije*, M.T Reetz*
Organic & Biomolecular Chemistry, 15 (2017), 9824 – 9829.
Kinetic modeling of an enzymatic redox cascade in vivo reveals cofactor-caused bottlenecks
S. Milker, M. Fink, N. Oberleitner, A. Ressmann, U. Bornscheuer, M.D. Mihovilovic, F. Rudroff*
ChemCatChem, 9 (2017), 3420 – 3427.
Non-hazardous biocatalytic oxidation in Nylon-9 monomer synthesis on a 40-gram scale with efficient downstream processing
S. Milker, M. Fink*, F. Rudroff, M.D. Mihovilovic
This paper describes the development of a biocatalytic process on the multi‐dozen gram scale for the synthesis of a precursor to Nylon‐9, a specialty polyamide. Such materials are growing in demand, but their corresponding monomers are often difficult to synthesize, giving rise to biocatalytic approaches. Here, we implemented cyclopentadecanone monooxygenase as an Escherichia coli whole‐cell biocatalyst in a defined medium, together with a substrate feeding–product removal concept, and an optimized downstream processing (DSP). A previously described hazardous peracid‐mediated oxidation was thus replaced with a safe and scalable protocol, using aerial oxygen as oxidant, and water as reaction solvent. The engineered process converted 42 g (0.28 mol) starting material ketone to the corresponding lactone with an isolated yield of 70% (33 g), after highly efficient DSP with 95% recovery of the converted material, translating to a volumetric yield of 8 g pure product per liter. Biotechnol. Bioeng. 2017;114: 1670–1678. © 2017 Wiley Periodicals, Inc.
Escherichia coli Fails to Efficiently Maintain the Activity of an Important Flavin Monooxygenase in Recombinant Overexpression
S. Milker, C. P. Goncalves, M. Fink*, F. Rudroff*
Frontiers in Microbiology, 8 (2017).
This paper describes the measurement and analysis of in vivo activity and stability of cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMO), a model Baeyer–Villiger monooxygenase, in the recombinant host Escherichia coli. This enzyme was often described as poorly stable in vitro, and has recently been found to deactivate rapidly in the absence of its essential cofactors and antioxidants. Its stability in vivo was scarcely studied, so far. Under conditions common for the overexpression of CHMO we investigated the ability of the host to support these properties using metabolomics. Our results showed that E. coli failed to provide the intracellular levels of cofactors required to functionally stabilize the enzyme, although the biocatalyst was produced in high concentration, and was invariably detected after protein synthesis had stopped. We thus infer that biotechnological applications of CHMO with this host relied on a residual activity of approximately 5-10%. Other microorganisms might offer a more efficient solution for recombinant production of CHMO and related enzymes.
From waste to value – Direct utilization of limonene from orange peel in a biocatalytic cascade reaction towards chiral carvolactone
N. Oberleitner, A. Ressmann, K. Schröder, P. Gärtner, M.W. Fraaije, U. Bornscheuer, M.D. Mihovilovic, F. Rudroff*:
A fusion protein of an enoate reductase and a Baeyer-Villiger monoxygenase facilitates synthesis of chiral lactones
C. Peters, F. Rudroff, M.D. Mihovilovic, U. Bornscheuer*
Biological Chemistry, 1 (2017), 398; 31 – 37.
First chemo-enzymatic synthesis of (R)-Taniguchi lactone and substrate profiles of CAMO and OTEMO, two new Baeyer-Villiger monooxygenases
F. Rudroff*, M. Fink, R. Pydi, U. Bornscheuer, M.D. Mihovilovic
Monatshefte für Chemie, 148 (2017), 157 – 165.
Four distinct types of E.C. 1.2.1.30 enzymes can catalyze the reduction of carboxylic acids to aldehydes
H. Stolterfohta, D. Schwendenwein, W. Sensen, F. Rudroff, M. Winkler
Journal of Biotechnology, 257 (2017), 222 – 232.
Cell Factory Design and Optimization for the Stereoselective Synthesis of Polyhydroxylated Compounds
T. Wiesinger, T. Bayer, S. Milker, M.D. Mihovilovic, F. Rudroff*
ChemBioChem, 18 (2017), 1 – 9.
- M. Abd El Hameid, M.D. Mihovilovic, H. B. El-Nassan: “Synthesis of Novel Pyrazolo[3,4-d]pyrimidine Derivatives as Potential Anti-breast Cancer Agents“; European Journal of Medicinal Chemistry, 57 (2012), 323 – 328.
- A. Alexander, D. Biedermann, M. Fink, M.D. Mihovilovic, T. Mattes: “Enantioselective Oxidation by a Cyclohexanone Monooxygenase from the Xenobiotic-Degrading Polaromonas sp. strain JS666“; Journal of Molecular Catalysis B Enzymatic, 78 (2012), 105 – 110.More information
- A Atanasov, B. Waltenberger, E. Pferschy-Wenzig, T. Linder, C. Wawrosch, P. Uhrin, V. Temml, L. Wang, S. Schwaiger, E. Heiss, J. Rollinger, D. Schuster, J. Breuss, V. Bochov, M.D. Mihovilovic, B. Kopp, R. Bauer, V. Dirsch, H. Stuppner: “Discovery and resupply of bioactive plant-derived natural products: a review“; Biotechnology Advances, 33 (2015), 1582 – 1614.
- B. C. Bayer, A. Khan, M. Mehmood, Z. Barber: “Effect of substrate on processing of multi-gun sputter deposited, near-stoichiometric Ni2MnGa thin films“; Thin Solid Films, 518 (2010), 2659.
- T. Bayer, S. Milker, T. Wiesinger, M.D. Mihovilovic, F. Rudroff: “Designer Microorganisms for Optimized Redox Cascade Reactions – Challenges and Future Perspectives“; Advanced Synthesis & Catalysis, 357 (2015), 1587 – 1618.More information
- D. Bianchi, R. Moran-Ramallal, N. Iqbal, F. Rudroff, M.D. Mihovilovic: “Enantiocomplementary access to carba-analogs of C-nucleoside derivatives by recombinant Baeyer-Villiger monooxygenases“; Bioorganic & Medicinal Chemistry Letters, 23 (2013), 2718 – 2720.More information
- G. Bolger, S. Roy, V. Zapolskii, D. Kaufmann, M. Schnürch, M.D. Mihovilovic, R. Nandy, W. Tegge: “Targeting aphA: a new high-throughput screening assay identifies compounds that reduce prime virulence factors of Vibrio cholerae“; Journal of Medical Microbiology, 65 (2016), 678 – 687.More information
- M. Bucko, P. Gemeiner, A. Schenkmayerova, T. Krajcovic, F. Rudroff, M.D. Mihovilovic: “Baeyer-Villiger oxidations: biotechnological approach“; Applied Microbiology and Biotechnology, 100 (2016), 15; 6585 – 6599.More information
- M. Bucko, A. Schenmayerova, P. Gemeiner, A. Vikartovska, M.D. Mihovilovic, I. Lacik: “Continuous testing system for Baeyer-Villiger biooxidation using recombinant Escherichia coli expressing cyclohexanone monooxygenase encapsulated in polyelectrolyte complex capsules“; Enzyme and Microbial Technology, 49 (2011), 284 – 288.More information
- M. Christakakou, M. Schön, M. Schnürch, M.D. Mihovilovic: “Arylation of pyridines via Suzuki-Miyaura cross-coupling and pyridine directed C-H activation using a continuous-flow approach“; Synlett, 2013 (2013), 24; 2411 – 2418.More information
- T. Dao-Huy, M. Haider, F. Glatz, M. Schnürch, M.D. Mihovilovic: “Direct arylation of benzo[b]furan and other benzo-fused heterocycles“; European Journal of Organic Chemistry, 2014 (2014), 8119 – 8125.
- T. Dao-Huy, B. Waldner, L. Wimmer, M. Schnürch, M.D. Mihovilovic: “Synthesis of endo- and exo-N-Protected 5-Arylated 2-Aminothiazoles through Direct Arylation: An Efficient Route to Cell Differentiation Accelerators“; European Journal of Organic Chemistry, 2015 (2015), 4765 – 4771.More information
- N. Dastbaravardeh, M. Christakakou, M. Haider, M. Schnürch: “Recent Advances in Palladium Catalyzed C(sp3)-H Activation for the formation of C-C and C-X bonds“; Synthesis, 46 (2014), 1421 – 1439.
- N. Dastbaravardeh, K. Kirchner, M. Schnürch, M.D. Mihovilovic: “Investigations and Substrate Scope Evaluation of Ruthenium-Catalyzed Direct sp3 Arylation of Benzylic Positions Directed by 3-Substituted Pyridines; Journal of Organic Chemistry“; Journal of Organic Chemistry, 78 (2013), 658 – 672.More information
- N. Dastbaravardeh, M. Schnürch, M.D. Mihovilovic: “Aryl Bromides and Aryl Chlorides for the Direct Arylation of Benzylic Amines Mediated by Ruthenium(II)“; European Journal of Organic Chemistry, 2013 (2013), 2878 – 2890.More information
- N. Dastbaravardeh, M. Schnürch, M.D. Mihovilovic: “Ruthenium(0)-Catalyzed sp3 C-H Bond Arylation of Benzylic Amines Using Arylboronates“; Organic Letters, 14 (2012), 7; 1930 – 1933.More information
- N. Dastbaravardeh, M. Schnürch, M.D. Mihovilovic: “Ruthenium(II)-Catalyzed sp3 C-H Bond Arylation of Benzylic Amines Using Aryl Halides“; Organic Letters, 14 (2012), 3792 – 3795.More information
- G. de Gonzalo, M.D. Mihovilovic, M.W. Fraaije: “Recent Developments in the Application of Baeyer-Villiger Monooxygenases as Biocatalysts“; ChemBioChem (invited), 11 (2010), 2208 – 2231.
- H. Dudek, M. Fink, A. Shivange, A. Dennig, M.D. Mihovilovic, U. Schwaneberg, M.W. Fraaije: “Extending the substrate scope of a Baeyer-Villiger monooxygenase by multiple-site mutagenesis“; Applied Microbiology and Biotechnology, 98 (2014), 4009 – 4020.
- T. Dvorak, L. Recnik, M. Schnürch, K. Mereiter, M.D. Mihovilovic, P. Stanetty: “Synthesis of substituted thieno[2,3-d]isothiazoles as potential plant activators“; ARKIVOC, 2013 (2013), iii; 245 – 265.
- D. Eigenmann, C. Dürig, E. Jähne, M. Smiesko, M. Culot, F. Gosselet, R. Cecchelli, H. Cederberg Helms, B. Brodin, L. Wimmer, M.D. Mihovilovic, M. Hamburger, M. Oufir: “In vitro blood-brain barrier permeability predictions for GABAA receptor modulating piperine analogs“; European Journal of Pharmaceutics and Biopharmaceutics, 103 (2016), 118 – 126.
- M. Fink, T. Fischer, F. Rudroff, H. Dudek, M.W. Fraaije, M.D. Mihovilovic: “Extensive substrate profiling of cyclopentadecanone monooxygenase as Baeyer-Villiger biocatalyst reveals novel regiodivergent oxidations“; Journal of Molecular Catalysis B Enzymatic, 73 (2011), 9 – 16.
- M. Fink, M.D. Mihovilovic: “Non-hazardous Baeyer-Villiger Oxidation of Levulinic Acid Derivatives: Sustainable Access to 3 Hydroxypropionates“; Chemical Communications, 51 (2015), 2874 – 2877.
- M. Fink, D.V. Rial, P. Kapitanova, A. Lengar, J. Rehdorf, Q. Cheng, F. Rudroff, M.D. Mihovilovic: “Quantitative Comparison of Chiral Catalysts Selectivity and Performance: A Generic Concept Illustrated with Cyclododecanone Monooxygenase as Baeyer-Villiger Biocatalyst“; Advanced Synthesis & Catalysis, 18 (2012), 354; 3491 – 3500.More information
- M. Fink, F. Rudroff, M.D. Mihovilovic: “Baeyer-Villiger monooxygenases in aroma compound synthesis“; Bioorganic & Medicinal Chemistry Letters, 21 (2011), 6135 – 6138.
- M. Fink, M. Schön, F. Rudroff, M. Schnürch, M.D. Mihovilovic: “Single Operation Stereoselective Synthesis of Aerangis Lactones: Combining Continuous Flow Hydrogenation and Biocatalysts in a Chemoenzymatic Sequence“; ChemCatChem, 5 (2013), 724 – 727.
- M. Fink, R. Snajdrova, A. Winninger, M.D. Mihovilovic: “Regio- and Stereoselective Synthesis of Chiral Nitrilolactones Using Baeyer-Villiger Monooxygenases“; Tetrahedron, 72 (2016), 7241 – 7248.
- T. Fischer, B. Cerra, M. Fink, F. Rudroff, E. Horkel, M.D. Mihovilovic: “First Total Synthesis of Piperenol B and Configuration Revision of the Enantiomers Piperenol B and Uvarirufol A“; European Journal of Organic Chemistry, 2015 (2015), 1464 – 1471.
- T. Fischer, H. Leisch, M.D. Mihovilovic: “Intramolecular Diels-Alder cyclization of biodihydroxylated benzoic acid derivatives towards novel heterocyclic scaffolds“; Monatshefte für Chemie, 141 (2010), 699 – 707.
- M. Geier, A. Braun, P. Fladischer, P. Stepniak, F. Rudroff, C. Hametner, M.D. Mihovilovic, A. Glieder: “Double site saturation mutagenesis of the human cytochrome P450 2D6 for steroid hydroxylation“; FEBS Journal, 280 (2013), 3094 – 3108.
- M. Ghobrial, K. Harhammer, M.D. Mihovilovic, M. Schnürch: “Facile, solvent and ligand free iron catalyzed direct functionalization of N-protected tetrahydroisoquinolines and isochroman“; Chemical Communications, 46 (2010), 8836 – 8838.
- M. Ghobrial, M. Schnürch, M.D. Mihovilovic: “Direct Functionalization of (Un)protected Tetrahydroisoquinoline and Isochroman under Iron and Copper Catalysis: Two Metals, Two Mechanisms“; Journal of Organic Chemistry, 76 (2011), 11; 8781 – 8793.
- M. Ghobrial, M. Schnürch, M.D. Mihovilovic: “Exploration of C-H and N-H- bond functionalization towards 1-(1,2-diarylindol-3-yl)tetrahydroisoquinolines“; Beilstein Journal of Organic Chemistry, 10 (2014), 2186 – 2199.
- B. Gröll, M.D. Mihovilovic, M. Schnürch: “Selective Ru(0)-catalyzed deuteration of electron-rich and electron-poor nitrogen-containing heterocycles“; Journal of Organic Chemistry, 77 (2012), 8; 4432 – 4437.More information
- J. Hämmerle, M. Schnürch, M.D. Mihovilovic, P. Stanetty: “A guideline for the arylation of positions 4 and 5 of thiazole via Pd-catalyzed cross-coupling reactions“; Tetrahedron, 66 (2010), 8051 – 8059.
- J. Hintersteiner, M. Haider, D. Luger, C. Schwarzer, G. Reznicek, W. Jäger, S. Khom, M.D. Mihovilovic, S. Hering: “Esters of Valerenic Acid as Potential Prodrugs“; European Journal of Pharmacology, 735 (2014), 123 – 131.
- N. Holmes, G. Akien, R. Savage, C. Stanetty, I. Baxendale, J. Blacker, B. Taylor, R. Woodward, R. Meadows, R. Bourne: “Online quantitative mass spectrometry for the rapid adaptive optimisation of automated flow reactors“; Reaction Chemistry & Engineering, 1 (2016), 1; 96 – 100.More information
- M. Hucik, M. Bucko, P. Gemeiner, V. Stefuca, A. Vikartovska, M.D. Mihovilovic, F. Rudroff, N. Iqbal, D. Chorvat, I. Lacik: “Encapsulation of recombinant E. coli expressing cyclopentanone monooxygenase in polyelectrolyte complex capsules for Baeyer-Villiger biooxidation of 8-oxabicyclo[3.2.1]oct-6-en-3-one“; Biotechnology Letters, 32 (2010), 675 – 680.
- N. Iqbal, F. Rudroff, A. Brige, J. van Beeumen, M.D. Mihovilovic: “Asymmetric Bioreduction of Activated Carbon-Carbon Double Bonds Using Shewanella Yellow Enzyme (SYE-4) as Novel Enoate Reductase“; Tetrahedron, 68 (2012), 37; 7619 – 7623.More information
- S. Khom, J. Hintersteiner, D. Luger, M. Haider, G. Pototschnig, M.D. Mihovilovic, C. Schwarzer, S. Hering: “Analysis of -Subunit-Dependent GABAA Receptor Modulation and Behavioral Effects of Valerenic Acid Derivatives“; Journal of Pharmacology and Experimental Therapeutics, 357 (2016), 580 – 590.
- A. Kolarovic, M. Schnürch, M.D. Mihovilovic: “Tandem Catalysis: From Alkynoic Acids and Aryl Iodides to 1,2,3-Triazoles in One Pot“; Journal of Organic Chemistry, 76 (2011), 8; 2613 – 2618.More information
- M. Koley, N. Dastbaravardeh, M. Schnürch, M.D. Mihovilovic: “Palladium(II)-Catalyzed Regioselective Ortho Arylation of sp2 C-H Bonds of N-Aryl-2-amino Pyridine Derivatives“; ChemCatChem, 4 (2012), 1345 – 1352.More information
- M. Koley, K. Kirchner, K. Mereiter: “Tris(acetonitrile-κN){2,6-bis[(diphenylphosphanyl)amino]-4-ethoxy-1,3,5-triazine-κ3P,N1,P’}iron(II) bis(tetrafluoridoborate) acetonitrile disolvate“; Acta Crystallographica Section E: Structure Reports, 67 (2011), m1842 – m1843.
- M. Koley, X. Koenig, K. Hilber, M. Schnürch, P. Stanetty, M.D. Mihovilovic: “Synthesis and screening of 2,6-diamino-substituted purine derivatives as potential cardiomyogenesis inducing agents“; ARKIVOC (invited), 6 (2011), 45 – 61.
- M. Koley, A. Mike, P. Heher, X. König, M. Schön, K. Hilber, M. Schnürch, G. Weitzer, M.D. Mihovilovic: “VUT-MK142 – A new cardiomyogenic small molecule promoting the differentiation of pre-cardiac mesoderm to cardiomyocytes“; MedChemComm, 4 (2013), 1189 – 1195.More information
- M. Koley, M. Schnürch, M.D. Mihovilovic: “Metal Assisted Synthesis of Mono and Diamino Substituted Pyridines“; Tetrahedron, 67 (2011), 23; 4169 – 4178.
- M. Koley, M. Schnürch, M.D. Mihovilovic: “Selective and Facile Palladium-Catalyzed Amination of 2-Fluoro-4-iodopyridine in the 4-Position under Microwave Conditions“; Synlett, 2010 (2010), 10; 1505 – 1510.
- M. Koley, L. Wimmer, M. Schnürch, M.D. Mihovilovic: “Pd(0) Catalyzed Cu(I) Thiophene-2-carboxylate Mediated Cross-Coupling of Heteroaromatic Thioethers and Boronic Acids – First Liebeskind-Srogl Reaction in Water“; Journal of Heterocyclic Chemistry, 50 (2013), 1368 – 1373.More information
- M. Koley, L. Wimmer, M. Schnürch, M.D. Mihovilovic: “Regioselective Syntheses of 2,3-Substituted Pyridines by Orthogonal Cross-Coupling Strategies“; European Journal of Organic Chemistry, 10 (2011), 1972 – 1979.
- F. Leipold, F. Rudroff, M.D. Mihovilovic, U. Bornscheuer: “The Steroid Monooxygenase from Rhodococcus rhodochrous – a Versatile Biocatalyst“; Tetrahedron – Asymmetry, 24 (2013), 1620 – 1624.More information
- C. Mair, L. Rongxia, A Atanasov, L. Wimmer, D. Nemetz-Fiedler, N. Sider, E. Heiss, M.D. Mihovilovic, V. Dirsch, J. Rollinger: “Piperine Congeners as Inhibitors of Vascular Smooth Muscle Cell Proliferation“; Planta Medica, 81 (2015), 1065 – 1074.
- F. Mayer, L. Wimmer, O. Dillon-Carter, J. Partilla, N. Burchardt, M.D. Mihovilovic, M. Baumann, H. Sitte: “Phase I Metabolites of Mephedrone Display Biological Acitivity as Substrates at Monoamine Transporters“; British Journal of Clinical Pharmacology, 173 (2016), 2657 – 2668.
- M.D. Mihovilovic: “A Heart in a Petri Dish“; Lab & More Orient, 2014 (2014), 20 – 23.
- M.D. Mihovilovic: “Das Herz in der Petrischale“; Labor & More (invited), 2013 (2013), 7; 10 – 14.
- M.D. Mihovilovic, M. Schnürch: “Ligand Assisted C-H Activation via Iron Catalysis“; ChemCatChem, 6 (2014), 2194 – 2196.
- A. Mike, X. Koenig, P. Heher, G. Wahl, L. Rubi, M. Schnürch, M.D. Mihovilovic, G. Weitzer, K. Hilber: “Small Molecule Cardiogenol C Upregulates Cardiac Markers and Induces Cardiac Functional Properties in Lineage-Committed Progenitor Cells“; Cellular Physiology and Biochemistry, 33 (2014), 205 – 221.
- J. Muschiol, C. Peters, N. Oberleitner, M.D. Mihovilovic, U. Bornscheuer, F. Rudroff: “Cascade catalysis – strategies and challenges en route to preparative synthetic biology“; Chemical Communications, 51 (2015), 5798 – 5811.
- A. Muskotal, V. Szabó, B. Toth, M.D. Mihovilovic, F. Vonderviszt: “Construction of a Xylanase A Variant Capable of Polymerization“; PLoS ONE, 6 (2011), 9 – 16.More information
- N. Oberleitner, C. Peters, J. Muschiol, M. Kadow, S. Saß, T. Bayer, P. Schaaf, N. Iqbal, F. Rudroff, M.D. Mihovilovic, U. Bornscheuer: “An Enzymatic Toolbox for Cascade Reactions: A Showcase for an In Vivo Redox Sequence in Asymmetric Synthesis“; ChemCatChem, 5 (2013), 3524 – 3528.
- N. Oberleitner, C. Peters, F. Rudroff, U. Bornscheuer, M.D. Mihovilovic: “In vitro characterization of an enzymatic redox cascade composed of an alcohol dehydrogenase, an enoate reductases and a Baeyer-Villiger monooxygenase“; Journal of Biotechnology, 192 (2014), 393 – 399.More information
- E. Oburger, B. Gruber, W Wanek, A. Watzinger, C. Stanetty, Y. Schindlegger, S. Hann, W. Schenkeveld, S. Krämer, M. Puschenreiter: “Microbial decomposition of 13C- labeled phytosiderophores in the rhizosphere of wheat: Mineralization dynamics and key microbial groups involved“; Soil Biology and Biochemistry, 98 (2016), 196 – 207.More information
- A. Oliveira, S. Dimopoulos, A. Busetto, S. Christen, R. Dechant, L. Falter, M. Chehreghani, S. Jozefczuk, C. Ludwig, F. Rudroff, J. Schulz, A. González, A. Soulard, D. Stracka, R. Aebersold, J. Buhmann, N. Hall, M. Peter, U. Sauer, J. Stelling: “Inferring causal metabolic signals that regulate the dynamic TORC1-dependent transcriptome“; Molecular Systems Biology, 4 (2015), 11; 802.More information
- C. Peters, R. Koelzsch, M. Kadow, L. Skalden, F. Rudroff, M.D. Mihovilovic, U. Bornscheuer: “Identification, Characterization, and Application of Three Enoate Reductases from Pseudomonas putida in In Vitro Enzyme Cascade Reactions“; ChemCatChem, 6 (2014), 1021 – 1027.More information
- R. Pollice, N. Dastbaravardeh, N. Marquise, M.D. Mihovilovic, M. Schnürch: “Mechanistic and kinetic studies of the direct alkylation of benzylic amines – A formal C(sp3)-H activation proceeds actually via a C(sp2)-H activation pathway“; ACS Catalysis, 5 (2015), 587 – 595.More information
- R. Pollice, M. Schnürch: “Expansion of the Concept of Nonlinear Effects in Catalytic Reactions Beyond Asymmetric Catalysis“; Chemistry – A European Journal, 22 (2016), 5637 – 5642.More information
- R. Pollice, M. Schnürch: “Investigations into the Kinetic Modeling of the Direct Alkylation of Benzylic Amines: Dissolution of K2CO3 Is Responsible for the Observation of an Induction Period“; Journal of Organic Chemistry, 80 (2015), 8268 – 8274.More information
- L. Recnik, A. El Hameid, M. Haider, M. Schnürch, M.D. Mihovilovic: “Selective Cross-Coupling Reactions on Imidazole towards Neurodazine Analogs“; Synthesis, 45 (2013), 1387 – 1405.
- J. Rehdorf, M.D. Mihovilovic, U. Bornscheuer: “Exploiting the Regioselectivity of Baeyer-Villiger Monooxygenases for the Formation of β-Amino Acids and β-Amino Alcohols“; Angewandte Chemie – International Edition, 49 (2010), 4506 – 4508.
- J. Rehdorf, M.D. Mihovilovic, M.W. Fraaije, U. Bornscheuer: “Enzymatic Synthesis of Enantiomerically Pure β-Amino Ketones, β-Amino Esters, and β-Amino Alcohols with Baeyer-Villiger Monooxygenases“; Chemistry – A European Journal, 2010 (2010), 16; 9525 – 9535.
- A. Riebel, M. Fink, M.D. Mihovilovic, M.W. Fraaije: “Type II Flavin-Containing Monooxygenases: A New Class of Biocatalysts that Harbors Baeyer-Villiger Monooxygenases with a Relaxed Coenzyme Specificity“; ChemCatChem, 6 (2014), 1112 – 1117.
- F. Rudroff, D. Bianchi, R. Moran-Ramallal, N. Iqbal, D. Dreier, M.D. Mihovilovic: “Synthesis of tetrahydrofuran-based natural products and their carba analogs via stereoselective enzyme mediated Baeyer-Villiger oxidation“; Tetrahedron, 72 (2016), 7212 – 7221.More information
- L. Rycek, R. Puthenkalam, M. Schnürch, M. Ernst, M.D. Mihovilovic: “Metal-Assisted Synthesis of Unsymmetrical Magnolol Analogs and their Biological Assessment as GABAA Receptor Ligands“; Bioorganic & Medicinal Chemistry Letters, 25 (2015), 400 – 403.More information
- J. Santos-Aberturas, J. Engel, J. Dickerhoff, M. Dorr, F. Rudroff, K. Weisz, U. Bornscheuer: “Exploration of the substrate promiscuity of biosynthetic tailoring enzymes as a new source of structural diversity for polyene macrolide antifungals“; ChemCatChem, 7 (2014), 3; 490 – 500.More information
- M. Schmid, B. Waldner, M. Schnürch, M.D. Mihovilovic, P. Stanetty: “Studying competitive lithiations at alpha-, ortho-, and benzylic positions in various N-protected aniline derivatives“; Tetrahedron, 67 (2011), 16; 2895 – 2904.More information
- M. Schnürch: “Exploiting the C-H bond in metal-catalyzed C-C bond-forming reactions“; ARKIVOC, 2015 (2015), 212 – 243.More information
- M. Schnürch, N. Dastbaravardeh, M. Ghobrial, B. Mrozek, M.D. Mihovilovic: “Functionalization of saturated and unsaturated heterocycles via transition metal catalyzed C-H activation reactions“; Current Organic Chemistry (invited), 15 (2011), 15; 2694 – 2730.More information
- M. Schnürch, J. Hämmerle, M.D. Mihovilovic, P. Stanetty: “A Systematic Study of Suzuki-Miyaura Cross-Coupling Reactions on Thiazoleboronic Esters in the 4- and 5-Position“; Synthesis, 2010 (2010), 5; 837 – 843.
- M. Schnürch, M.D. Mihovilovic: “4th Young Investigator Workshop“; Monatshefte für Chemie, 144 (2013), 4; 445.
- M. Schnürch, B. Waldner, K. Hilber, M.D. Mihovilovic: “Synthesis of 5-arylated N-arylthiazole-2-amines as potential skeletal muscle cell differentiation promoters“; Bioorganic & Medicinal Chemistry Letters, 21 (2011), 7; 2149 – 2154.More information
- A. Schöffmann, L. Wimmer, D. Goldmann, S. Khom, J. Hintersteiner, I. Baburin, T. Schwarz, M. Hintersteininger, S. Pakfeifer, M. Oufir, M. Hamburger, T. Erker, G. Ecker, M.D. Mihovilovic, S. Hering: “Efficient Modulation of γ-Aminobutyric Acid Type A (GABAA) Receptors by Piperine Derivatives“; Journal of Medicinal Chemistry, 57 (2014), 5602; 5602 – 5619.
- M. Schön, D. Dreier, M. Schnürch, M.D. Mihovilovic: “Library synthesis of cardiomyogenesis inducing compounds using an efficient two-step-one-flow process“; Monatshefte für Chemie, 147 (2016), 3; 523 – 532.More information
- M. Schön, M. Schnürch, M.D. Mihovilovic: “Application of continuous flow and alternative energy devices for 5-hydroxymethylfurfural production“; Molecular Diversity, 15 (2011), 639 – 643.More information
- M. Schwarz, N. Dastbaravardeh, K. Kirchner, M. Schnürch, M.D. Mihovilovic: “First selective direct mono-arylation of piperidines using ruthenium-catalyzed C-H activation“; Monatshefte für Chemie, 144 (2013), 539 – 552.
- D. Schwendenwein, G. Fiume, H. Weber, F. Rudroff, M. Winkler: “Selective Enzymatic Transformation to Aldehydes in vivo by Fungal Carboxylate Reductase from Neurospora crassa“; Advanced Synthesis & Catalysis, 21 (2016), 358; 3414 – 3421.More information
- C. Stanetty, I. Baxendale: “Large-Scale Synthesis of Crystalline 1,2,3,4,6,7-Hexa-O-acetyl-L-glycero-#A-D-manno-heptopyranose“; European Journal of Organic Chemistry, 2015 (2015), 12; 2718 – 2726.More information
- M. Steiger, A. Mach-Aigner, R. Gorsche, E. Rosenberg, M.D. Mihovilovic, R.L. Mach: “Synthesis of an antiviral drug precursor from chitin using a saprophyte as a whole-cell catalyst“; Microbial Cell Factories, 10 (2011), 9 pages.More information
- D. Stracka, S. Jozefczuk, F. Rudroff, U. Sauer, N. Hall: “Nitrogen source activates TOR (target of rapamycin) complex 1 via glutamine and independently of Gtr/Rag proteins“; Journal of Biological Chemistry, 289 (2014), 25010 – 25020.More information
- M. Sunnaker, E. Zamora-Sillero, A. Garcia de Lomana, F. Rudroff, U. Sauer, J. Stelling, A. Wagner: “Topological augmentation to infer hidden processes in biological systems“; Bioinformatics, 2 (2014), 30; 221 – 227.More information
- M. Turner, T. Linder, M. Schnürch, M.D. Mihovilovic, P. Stanetty: “Investigation of the regioselectivity of the Hurd-Mori reaction for the formation of bicyclic 1,2,3-thiadiazoles“; Tetrahedron, 66 (2010), 5472 – 5478.
- Z. Varagic, L. Wimmer, M. Schnürch, M.D. Mihovilovic, S. Huang, S. Rallapalli, J. Cook, P. Mirheydari, G. Ecker, W. Sieghart, M. Ernst: “Identification of novel positive allosteric modulators and null modulators at the GABAA receptor α + β – interface“; British Journal of Clinical Pharmacology, 169 (2013), 371 – 383.
- B. Waltenberger, A Atanasov, E. Heiss, D. Bernhard, J. Rollinger, J. Breuss, D. Schuster, R. Bauer, B. Kopp, C. Franz, V. Bochkov, M.D. Mihovilovic, V. Dirsch, H. Stuppner: “Drugs from nature targeting inflammation (DNTI) – A successful Austrian interdisciplinary network project“; Monatshefte für Chemie, 147 (2016), 479 – 491.
- L. Wang, A. Ladurner, S. Latkolik, S. Schwaiger, T. Linder, J. Hosek, V. Palme, N. Schilcher, O. Polansky, E. Heiss, H. Stangl, M.D. Mihovilovic, H. Stuppner, V. Dirsch, A Atanasov: “Leoligin, the Major Lignan from Edelweiss, Promotes Cholesterol Efflux from THP-1 Macrophages“; Journal of Natural Products, 79 (2016), 1651 – 1657.
- L. Wimmer, D. Schönbauer, P Pakfeifer, A. Schöffmann, S. Khom, S. Hering, M.D. Mihovilovic: “Developing Piperine towards TRPV1 and GABAA Receptor Ligands – Synthesis of Piperine Analogs via Heck-Coupling of Conjugated Dienes“; Organic & Biomolecular Chemistry, 13 (2015), 990 – 994.
- M.F. Zia, A. Vasko, Z. Riedl, C. Hametner, G. Hajos, K. Mereiter, M.D. Mihovilovic: “Biodihydroxylation of substituted quinolines and isoquinolines by recombinant whole-cell mediated biotransformations“; Tetrahedron, 72 (2016), 7348 – 7355.More information