Research Lab - Marko Mihovilovic
- Research Topics
- Research at the chemistry-biology interface.
- Development and utilization of wild-type microorganisms and genetically engineered whole-cell systems as new tools in synthetic chemistry.
- Design of multi-step biocatalytic and chemo-enzymatic reaction cascades.
- Proliferation of the “green chemistry” concept with respect to sustainability, atom efficiency, and environmentally benign synthetic methods.
- Development new bioactive compounds for modification of cell differentiation
- Lead-optimization of natural-compound derived GABAA ligands
- Lead-optimization of natural-compound derived anti-inflammatory agents
- Continuous flow chemical processes.
- Utilization of chiral intermediates obtained via biocatalytic methods in natural compound total synthesis.
- Development of platform technologies for renewables valorization and circular economy.
- Exploitation of metal-assisted synthetic methods such as cross-coupling reactions (Negishi, Suzuki, Stille, Kumada), CH-activation, dircted ortho-lithiation, cyclization reactions (Zn-mediated [4+3] cycloaddition, Pauson-Khand cyclization), and metathesis techniques (ring-opening metathesis, ring-closing metathesis).
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MDM Concept TU Wien 2022
Mihovilovic Lab news and updates
Marko D. MihovilovicMarko was born in Steyr, Upper Austria, Austria in 1970. After the A-levels in Linz, he started to study Technical Chemistry / Organic Chemistry in 1988 at the Vienna University of Technology (VUT), Vienna, Austria. His diploma Thesis was entitled ‘Synthesis of Thieno[2,3-d]thiadiazole Derivatives’ and was supervised by Prof. Peter Stanetty. He started his PhD thesis in 1994 (‘Synthesis of Azasteroid Partial Structures as Potential Inhibitors of the Ergosterol Biosynthesis’) again in the group of Prof. Peter Stanetty and finished this work in June 1996. From 1994-1998 he was research assistant at the Institute of Organic Chemistry (IOC). He was then on two postdoctoral stays as Erwin Schrödinger Fellow of the FWF (Project no. J1471-CHEM (‘Designer Yeasts – New Bioreagents in enantioselective Synthesis’) with Prof. Margaret M. Kayser University of New Brunswick, Saint John, N.B., Canada and Prof. Jon D. Stewart University of Florida, Gainesville, Florida, USA. From 1999-2003 he was again university assistant at the Institute of Applied Synthetic Chemistry (IAS, former IOC), VUT. In November 2003 he received his Habilitation (venia docendi) in the field of Bioorganic Chemistry and was promoted to ‘University Dozent’ (Assistant Professor) at the IAS, VUT. From March 2004 till January 2014 he was Associate University Professor at the IAS, VUT. In May 2008 he declined the appointment to Full Professor in Bioorganic Chemistry at Johannes Kepler University Linz, Austria. From 2009 up to now he coordinates the Graduate School Program AB-Tec (Applied Bioscience Technology) at Vienna University of Technology. Since January 2013 he is the Head of Institute at the Institute of Applied Synthetic Chemistry (Vienna University of Technology, Vienna, Austria). In February 2014 he was appointed as Full Professor and Chair for Bioorganic Synthetic Chemistry. He is Dean of the Faculty for Technical Chemistry, TU Wien since 2020.
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From starch to wood adhesives: Within the BioSet project I am currently investigating and improving means of enzymatic oxidation of starch working under the supervision of Prof. Mihovilovic and Dr. Stanetty. The goal is to develop a competitive alternative to current chemical solutions which require the use of toxic reagents and are of waste-intensive nature. A close collaboration with industry partners and the interdisciplinary background between biochemistry, electrochemistry and synthetic chemistry provide a very interesting setting for my PhD thesis.
My research topic´s focus lies in the synthesis of modified and functionalized phosphatidylinositolphosphates (PIPs) for elucidation of peptide assemblies in monoamine transporters. Special attention is directed towards inositol desymmetrization/protection strategies and stereospecific assembly of functionalized diacylglycerols and phosphoramidites.
Katharina obtained her bachelor and diploma degree in technical chemistry at the TU Wien. She conducted her diploma thesis during a semester abroad at the Columbia University of New York under the supervision of Prof. Tomislav Rovis and the home co-supervision of Prof. Marko Mihovilovic. Her PhD research focuses on the design, synthesis and evaluation of novel photoswitchable monoamine transporter inhibitors in collaboration with computational and pharmacological groups at the University of Vienna and the Medical University of Vienna.
Dominik obtained his bachelor’s degree in chemistry at the TU Graz before he graduated with a master’s degree in Drug Discovery and Design at the VU University Amsterdam. In August 2019 he started his PhD studies in the BSC group at the TU Wien.
His research focus lies in the synthesis and labeling of ligands for the GABAA-receptor and other ion channels. The labeling of these tool compounds involves the introduction of radioisotopes (e.g., 3H, 14C) as well as photo-cross-linking units for photoaffinity labeling (PAL). The goal of this project is the development of strategies and methods to incorporate these labeling moieties into the compounds of interest while maintaining target affinity to obtain detailed insight into interactions of ligands with their protein targets. This information should facilitate making predictions with computaitonal models (e.g. prediction of binding modes) and ultimately help to design novel ligands for ion channels.
Mihovilovic Group projects
Publications in Scientific Journals
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.
The role of hydrogen bonding in the incommensurate modulation of myo-inositol camphor ketal
M. Bucko*, P. Gemeiner, T. Krajcovic, M. Hakarova, D. Chorvat, A. Chorvatova, I. Lacik, F. Rudroff, M.D. Mihovilovic
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  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  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.
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.
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—Hπ 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.
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.
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.
Publications in Scientific Journals
Cellular N-myristoyltransferases play a crucial picornavirus genus-specific role in viral assembly, virion maturation, and infectivity
I. Corbic Ramljak, J. Stanger, A. Real-Hohn, D. Dreier*, L. Wimmer*, M. Redlberger-Fritz, W. Fischl, K. Klingel, M.D. Mihovilovic, D. Blaas, H. Kowalski
PLOS Pathogens, 14(8) (2018), e1007203; 1 – 39.
In nearly all picornaviruses the precursor of the smallest capsid protein VP4 undergoes co-translational N-terminal myristoylation by host cell N-myristoyltransferases (NMTs). Curtailing this modification by mutation of the myristoylation signal in poliovirus has been shown to result in severe assembly defects and very little, if any, progeny virus production. Avoiding possible pleiotropic effects of such mutations, we here used pharmacological abrogation of myristoylation with the NMT inhibitor DDD85646, a pyrazole sulfonamide originally developed against trypanosomal NMT. Infection of HeLa cells with coxsackievirus B3 in the presence of this drug decreased VP0 acylation at least 100-fold, resulting in a defect both early and late in virus morphogenesis, which diminishes the yield of viral progeny by about 90%. Virus particles still produced consisted mainly of provirions containing RNA and uncleaved VP0 and, to a substantially lesser extent, of mature virions with cleaved VP0. This indicates an important role of myristoylation in the viral maturation cleavage. By electron microscopy, these RNA-filled particles were indistinguishable from virus produced under control conditions. Nevertheless, their specific infectivity decreased by about five hundred fold. Since host cell-attachment was not markedly impaired, their defect must lie in the inability to transfer their genomic RNA into the cytosol, likely at the level of endosomal pore formation. Strikingly, neither parechoviruses nor kobuviruses are affected by DDD85646, which appears to correlate with their native capsid containing only unprocessed VP0. Individual knockout of the genes encoding the two human NMT isozymes in haploid HAP1 cells further demonstrated the pivotal role for HsNMT1, with little contribution by HsNMT2, in the virus replication cycle. Our results also indicate that inhibition of NMT can possibly be exploited for controlling the infection by a wide spectrum of picornaviruses.
Electrochemical properties of halogenated benzylidenehydrazino-pyrazoles in various imidazolium-based ionic liquids
L. V Costea*, K. Bica, G. Fafilek, M.D. Mihovilovic
Monatshefte für Chemie, 149 (2018), 4; 823 – 831.
Indium- and Zinc-Mediated Acyloxyallylation of Protected and Unprotected Aldotetroses – Revealing a Pronounced Diastereodivergence and a Fundamental Difference in the Performance of the Mediating Metal
M. Draskovits, C. Stanetty*, I. Baxendale, M.D. Mihovilovic
Journal of Organic Chemistry, 83 (2018), 5; 2647 – 2659.
Magnolol dimer-derived fragments as PPARγ-selective probes
D. Dreier, M. Resetar, V. Temml, L. Rycek, N. Kratena, M. Schnürch, D. Schuster, V. Dirsch, M.D. Mihovilovic*
Organic & Biomolecular Chemistry, 16 (2018), 38; 7019 – 7028.
Partial agonists of the transcription factor PPARγ (peroxisome proliferator-activated receptor γ) have shown potential for the treatment of metabolic and inflammatory conditions and novel activators serve as valuable tool and lead compounds. Based on the natural product magnolol (I) and recent structural information of the ligand–target interaction we have previously developed magnolol dimer (II) which has been shown to have enhanced affinity towards PPARγ and improved selectivity over RXRα (retinoid X receptor α), PPARγ’s heterodimerization partner. In this contribution we report the synthesis and evaluation of three fragments of the dimeric lead compound by structural simplifications. Sesqui magnolol A and B (III and IV) were found to exhibit comparable activities to magnolol dimer (II) and selectivity over RXRα persisted. Computational studies suggest a common pharmacophore of the distinctive biphenyl motifs. Truncated magnolol dimer (V) on the other hand does not share this feature and was found to act as an antagonist.
Novel concurrent redox cascades of (R)- and (S)-carvones enables access to carvo-lactones with distinct regio- and enantioselectivity
N. Iqbal, J.D. Stewart, P. Macheroux, F. Rudroff*, M.D. Mihovilovic
Tetrahedron, 52 (2018), 74; 7389 – 7394.
Within this study, we investigated a one-pot enzymatic redox cascade composed of different enoate reductases (5 EREDs from diverse bacterial origins) and various Baeyer-Villiger monooxygenases (4 BVMOs) with complementary regioselectivity that enabled access to six out of eight carvo-lactone stereoisomers starting from readily available natural carvones. Applicability of this two-step cascade was demonstrated by preparative scale experiments yielding up to 76% of the desired chiral carvolactone.
Design and Synthesis of Novel Deuterated Ligands Functionally Selective for the γ Aminobutyric Acid Type A Receptor (GABAAR) α6 Subtype with Improved Metabolic Stability and Enhanced Bioavailability
D. Knutson, R. Kodali, B. Divovic, M. Treven, M. Stephen, N. Zahn, V. Dobricic, A. Huber, M. Meirelles, R. Verma, L. Wimmer, C. Witzigmann, L. Arnold, L. Ciou, M. Ernst, M.D. Mihovilovic, M. Savic, W. Sieghart, J. Cook
Journal of Medicinal Chemistry, 61 (2018), 2422 – 2446.
Recent reports indicate that α6β2/3γ2 GABAAR selective ligands may be important for the treatment of trigeminal activation-related pain and neuropsychiatric disorders with sensori-motor gating deficits. Based on 3 functionally α6β2/3γ2 GABAAR selective pyrazoloquinolinones, 42 novel analogs were synthesized, and their in vitro metabolic stability and cytotoxicity as well as their in vivo pharmacokinetics, basic behavioral pharmacology, and effects on locomotion were investigated. Incorporation of deuterium into the methoxy substituents of the ligands increased their duration of action via improved metabolic stability and bioavailability, while their selectivity for the GABAAR α6 subtype was retained. 8b was identified as the lead compound with a substantially improved pharmacokinetic profile. The ligands allosterically modulated diazepam insensitive α6β2/3γ2 GABAARs and were functionally silent at diazepam sensitive α1β2/3γ2 GABAARs, thus no sedation was detected. In addition, these analogs were not cytotoxic, which render them interesting candidates for treatment of CNS disorders mediated by GABAAR α6β2/3γ2 subtypes.
One-pot synthesis of triazines as potential agents affecting cell differentiation
T. Linder, M. Schnürch*, M.D. Mihovilovic
Monatshefte für Chemie, 149 (2018), 1257 – 1284.
This paper outlines the synthesis of a number of structural analogs of 3-[(4,6-diphenoxy-1,3,5-triazin-2-yl)amino]benzoic acid which represent compounds with potential cardiogenetic activity. A one-pot protocol was developed for swift functionalization of the 1,3,5-triazine core without the need of isolating intermediates. The developed route starts from readily available 2,4,6-trichloro-1,3,5-triazine, displacing the chlorine atoms sequentially by aryloxy, arylamino, or arylthio moieties to enable access to molecules with three different substituents of this type in good yields. To facilitate purification, tert-butyl, methyl, and ethyl ester derivatives of the target compounds were initially synthesized. The tert-butyl esters could be readily hydrolyzed to the desired compounds, while reduction of the methyl and ethyl esters gave the corresponding benzylic alcohols in high yields, thereby expanding the substrate scope for future relevant cell assays.
Biochemical characterization of an ulvan lyase from the marine flavobacterium Formosa agariphila KMM 3901T
L. Reisky, C. Stanetty, M.D. Mihovilovic, T. Schweder, J. Hehemann, U. Bornscheuer*
Applied Microbiology and Biotechnology, 102 (2018), 16; 6987 – 6996.
Combining chemo- and biocatalysis, opportunities and challenges
F. Rudroff, M.D. Mihovilovic, H. Gröger, R. Snajdrova, H. Iding, U. Bornscheuer*
Nature Catalysis, 1 (2018), 1; 12 – 33.
Stereoselective Synthesis of the Isomers of Notoincisol A: Assigment of the Absolute Configuration of this Natural Product and Biological Evaluation
L. Rycek, V. Ticli, J. Pyszkowski, S. Latkolik, X. Liu, A Atanasov, T. Steinacher, R. Bauer, D. Schuster, V. Dirsch, M. Schnürch, M. Ernst, M.D. Mihovilovic*
Journal of Natural Products, 81 (2018), 2419 – 2428.
Biocompatible metal-assisted C-C cross-coupling combined with biocatalytic chiral reductions in a concurrent tandem cascade
P. Schaaf, T. Bayer, M. Koley, M. Schnürch, U. Bornscheuer, F. Rudroff*, M.D. Mihovilovic*
Chemical Communications, 54 (2018), 12978 – 12981.
Easy Access to Enantiopure (S)- and (R)-Aryl Alkyl Alcohols by a Combination of Gold(III)-Catalyzed Alkyne Hydration and Enzymatic Reduction
P. Schaaf, V. Gojic, T. Bayer, F. Rudroff, M. Schnürch, M.D. Mihovilovic*
ChemCatChem, 10 (2018), 920 – 924.
Engineered Flumazenil Recognition Site Provides Mechanistic Insight Governing Benzodiazepine Modulation in GABAA Receptors
D. Siebert, K. Bampali, R. Puthenkalam, Z. Varagic, I. Sarto-Jackson, P. Scholze, W. Sieghart, M.D. Mihovilovic, M. Schnürch, M. Ernst*
ACS Chemical Biology, 13 (2018), 2040 – 2047.
The anxiolytic, anticonvulsant, muscle-relaxant, and sedative-hypnotic effects of benzodiazepine site ligands are mainly elicited by allosteric modulation of GABAA receptors via their extracellular αx+/γ2– (x = 1, 2, 3, 5) interfaces. In addition, a low affinity binding site at the homologous α+/β– interfaces was reported for some benzodiazepine site ligands. Classical benzodiazepines and pyrazoloquinolinones have been used as molecular probes to develop structure–activity relationship models for benzodiazepine site activity. Considering all possible α+/β– and α+/γ– interfaces, such ligands potentially interact with as many as 36 interfaces, giving rise to undesired side effects. Understanding the binding modes at their binding sites will enable rational strategies to design ligands with desired selectivity profiles. Here, we compared benzodiazepine site ligand interactions in the high affinity α1+/γ2– site with the homologous α1+/β3– site using a successive mutational approach. We incorporated key amino acids known to contribute to high affinity benzodiazepine binding of the γ2– subunit into the β3– subunit, resulting in a quadruple mutant β3(4mut) with high affinity flumazenil (Ro 15-1788) binding properties. Intriguingly, some benzodiazepine site ligands displayed positive allosteric modulation in the tested recombinant α1β3(4mut) constructs while diazepam remained inactive. Consequently, we performed in silico molecular docking in the wildtype receptor and the quadruple mutant. The results led to the conclusion that different benzodiazepine site ligands seem to use distinct binding modes, rather than a common binding mode. These findings provide structural hypotheses for the future optimization of both benzodiazepine site ligands, and ligands that interact with the homologous α+/β– sites.
SAR-Guided Scoring Function and Mutational Validation Reveal the Binding Mode of CGS-8216 at the α1+/γ2- Benzodiazepine Site
D. Siebert, M. Wieder, L. Schlener, P. Scholze, S. Boresch, T. Langer, M. Schnürch, M.D. Mihovilovic, L. Richter*, M. Ernst, G. Ecker
Journal of Chemical Information and Modeling, 58 (2018), 1682 – 1696.
The structural resolution of a bound ligand–receptor complex is a key asset to efficiently drive lead optimization in drug design. However, structural resolution of many drug targets still remains a challenging endeavor. In the absence of structural knowledge, scientists resort to structure–activity relationships (SARs) to promote compound development. In this study, we incorporated ligand-based knowledge to formulate a docking scoring function that evaluates binding poses for their agreement with a known SAR. We showcased this protocol by identifying the binding mode of the pyrazoloquinolinone (PQ) CGS-8216 at the benzodiazepine binding site of the GABAA receptor. Further evaluation of the final pose by molecular dynamics and free energy simulations revealed a close proximity between the pendent phenyl ring of the PQ and γ2D56, congruent with the low potency of carboxyphenyl analogues. Ultimately, we introduced the γ2D56A mutation and in fact observed a 10-fold potency increase in the carboxyphenyl analogue, providing experimental evidence in favor of our binding hypothesis.
Towards functional selectivity for α6β3γ2 GABAA receptors: a series of novel pyrazoloquinolinones
M. Treven, D. Siebert, R. Holzinger, K. Bampali, J. Fabjan, Z. Varagic, L. Wimmer, F. Steudle, P. Stolze, M. Schnürch, M.D. Mihovilovic, M. Ernst*
British Journal of Clinical Pharmacology, 175 (2018), 419 – 428.
GABAA Receptor Activity Modulating Piperine Analogs: In vitro Metabolic Stability, Metabolite Identification, CYP450 Reaction Phenotyping, and Protein Binding
V. Zabela*, T. Hettich*, G. Schlotterbeck*, L. Wimmer*, M.D. Mihovilovic*, F. Guillet*, B. Bouaita*, B. Shevchenko*, M. Hamburger*, M. Oufir*
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 1072 (2018), 379 – 389.
In a screening of natural products for allosteric modulators of GABAA receptors (γ-aminobutyric acid type A receptor), piperine was identified as a compound targeting a benzodiazepine-independent binding site. Given that piperine is also an activator of TRPV1 (transient receptor potential vanilloid type 1) receptors involved in pain signaling and thermoregulation, a series of piperine analogs were prepared in several cycles of structural optimization, with the aim of separating GABAA and TRPV1 activating properties. We here investigated the metabolism of piperine and selected analogs in view of further cycles of lead optimization.
Metabolic stability of the compounds was evaluated by incubation with pooled human liver microsomes, and metabolites were analyzed by UHPLC-Q-TOF-MS. CYP450 isoenzymes involved in metabolism of compounds were identified by reaction phenotyping with Silensomes™. Unbound fraction in whole blood was determined by rapid equilibrium dialysis.
Piperine was the metabolically most stable compound. Aliphatic hydroxylation, and N- and O-dealkylation were the major routes of oxidative metabolism. Piperine was exclusively metabolized by CYP1A2, whereas CYP2C9 contributed significantly in the oxidative metabolism of all analogs. Extensive binding to blood constituents was observed for all compounds.
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.