@misc{KiebistSchmidtkeSchrammetal., author = {Kiebist, Jan and Schmidtke, Kai-Uwe and Schramm, Marina and Hofrichter, Martin and K{\"o}nig, Rosalie and Quint, Stephan and Kohlmann, Johannes and Zuhse, Ralf and Ullrich, Ren{\´e} and Hofrichter, Martin and Scheibner, Katrin}, title = {Biocatalytic syntheses of antiplatelet metabolites of the thienopyridines clopidogrel and prasugrel using fungal peroxygenases}, series = {Journal of Fungi}, volume = {7}, journal = {Journal of Fungi}, number = {9}, issn = {2309-608X}, doi = {10.3390/jof7090752}, pages = {1 -- 17}, abstract = {Antithrombotic thienopyridines, such as clopidogrel and prasugrel, are prodrugs that undergo a metabolic two-step bioactivation for their pharmacological efficacy. In the first step, a thiolactone is formed, which is then converted by cytochrome P450-dependent oxidation via sulfenic acids to the active thiol metabolites. These metabolites are the active compounds that inhibit the platelet P2Y12 receptor and thereby prevent atherothrombotic events. Thus far, described biocatalytic and chemical synthesis approaches to obtain active thienopyridine metabolites are rather complex and suffer from low yields. In the present study, several unspecific peroxygenases (UPOs, EC 1.11.2.1) known to efficiently mimic P450 reactions in vitro—but requiring only hydroperoxide as oxidant—were tested for biocatalytic one-pot syntheses. In the course of the reaction optimization, various parameters such as pH and reductant, as well as organic solvent and amount were varied. The best results for the conversion of 1 mM thienopyridine were achieved using 2 U mL-1 of a UPO from agaric fungus Marasmius rotula (MroUPO) in a phosphate-buffered system (pH 7) containing 5 mM ascorbate, 2 mM h-1 H2O2 and 20\% acetone. The preparation of the active metabolite of clopidogrel was successful via a two-step oxidation with an overall yield of 25\%. In the case of prasugrel, a cascade of porcine liver esterase (PLE) and MroUPO was applied, resulting in a yield of 44\%. The two metabolites were isolated with high purity, and their structures were confirmed by MS and MS2 spectrometry as well as NMR spectroscopy. The findings broaden the scope of UPO applications again and demonstrate that they can be effectively used for the selective synthesis of metabolites and late-state diversification of organic molecules, circumventing complex multistage chemical syntheses and providing sufficient material for structural elucidation, reference material, or cellular assays.}, language = {en} } @misc{KlugeUllrichDolgeetal., author = {Kluge, Martin and Ullrich, Ren{\´e} and Dolge, Christoph and Scheibner, Katrin and Hofrichter, Martin}, title = {Hydroxylation of naphthalene by aromatic peroxygenase from Agrocybe aegerita proceeds via oxygen transfer from H2O2 and intermediary epoxidation}, series = {Applied Microbiology and Biotechnology}, volume = {81}, journal = {Applied Microbiology and Biotechnology}, number = {6}, issn = {0175-7598}, doi = {10.1007/s00253-008-1704-y}, pages = {1071 -- 1076}, abstract = {Agrocybe aegerita peroxidase/peroxygenase (AaP) is an extracellular fungal biocatalyst that selectively hydroxylates the aromatic ring of naphthalene. Under alkaline conditions, the reaction proceeds via the formation of an intermediary product with a molecular mass of 144 and a characteristic UV absorption spectrum (A max 210, 267, and 303 nm). The compound was semistable at pH 9 but spontaneously hydrolyzed under acidic conditions (pH <7) into 1-naphthol as major product and traces of 2-naphthol. Based on these findings and literature data, we propose naphthalene 1,2-oxide as the primary product of AaP-catalyzed oxygenation of naphthalene. Using 18O-labeled hydrogen peroxide, the origin of the oxygen atom transferred to naphthalene was proved to be the peroxide that acts both as oxidant (primary electron acceptor) and oxygen source.}, language = {en} } @misc{FriedrichGroebeKlugeetal., author = {Friedrich, Stephanie and Gr{\"o}be, Glenn and Kluge, Martin and Brinkmann, Tobias and Hofrichter, Martin and Scheibner, Katrin}, title = {Optimization of a biocatalytic process to gain (R)-1-phenylethanol by applying the software tool Sabento for ecological assessment during the early stages of development}, series = {Journal of Molecular Catalysis : B, Enzymatic}, journal = {Journal of Molecular Catalysis : B, Enzymatic}, number = {103}, issn = {1381-1177}, doi = {10.1016/j.molcatb.2013.10.002}, pages = {36 -- 40}, abstract = {Ecological assessment using the software tool Sabento was conducted to compare different processes to gain the fine chemical (R)-1-phenylethanol from ethylbenzene. The software was applied during the biocatalytic process development using the unspecific peroxygenase (EC .11.2.1) of the fungus Agrocybe aegerita. The process could be systematically improved with respect to the ecological performance during process development. Compared to a modern chemical process and a further biotechnological process, it now reaches the best environmental key indicator. The software tool Sabento proved to be well suited to work out the most important factors determining the ecological burdens in the early stages of process development.}, language = {en} } @misc{PorajKobielskaAtzrodtHollaetal., author = {Poraj-Kobielska, Marzena and Atzrodt, Jens and Holla, Wolfgang and Sandvoss, Martin and Gr{\"o}be, Glenn and Scheibner, Katrin and Hofrichter, Martin}, title = {Preparation of labeled human drug metabolites and drug-drug interaction-probes with fungal peroxygenases}, series = {Journal of Labelled Compounds and Radiopharmaceuticals}, volume = {56}, journal = {Journal of Labelled Compounds and Radiopharmaceuticals}, number = {9-10}, issn = {1099-1344}, doi = {10.1002/jlcr.3103}, pages = {513 -- 519}, abstract = {Enzymatic conversion of a drug can be an efficient alternative for the preparation of a complex metabolite compared with a multi-step chemical synthesis approach. Limitations exist for chemical methods for direct oxygen incorporation into organic molecules often suffering from low yields and unspecific oxidation and also for alternative whole-cell biotransformation processes, which require specific fermentation know-how. Stable oxygen-transferring biocatalysts such as unspecific peroxygenases (UPOs) could be an alternative for the synthesis of human drug metabolites and related stable isotope-labeled analogues. This work shows that UPOs can be used in combination with hydrogen/deuterium exchange for an efficient one-step process for the preparation of 4'-OH-diclofenac-d6. The scope of the reaction was investigated by screening of different peroxygenase subtypes for the transformation of selected deuterium-labeled substrates such as phenacetin-d3 or lidocaine-d3. Experiments with diclofenac-d7 revealed that the deuterium-labeling does not affect the kinetic parameters. By using the latter substrate and H2 (18) O2 as cosubstrate, it was possible to prepare a doubly isotope-labeled metabolite (4'-(18) OH-diclofenac-d6). UPOs offer certain practical advantages compared with P450 enzyme systems in terms of stability and ease of handling. Given these advantages, future work will expand the existing 'monooxygenation toolbox' of different fungal peroxygenases that mimic P450 in vitro reactions.}, language = {en} } @misc{KlugeUllrichScheibneretal., author = {Kluge, Martin and Ullrich, Ren{\´e} and Scheibner, Katrin and Hofrichter, Martin}, title = {Formation of naphthalene hydrates in the enzymatic conversion of 1,2-dihydronaphthalene by two fungal peroxygenases and subsequent naphthalene formation}, series = {Journal of Molecular Catalysis : B, Enzymatic}, journal = {Journal of Molecular Catalysis : B, Enzymatic}, number = {103}, issn = {1381-1177}, doi = {10.1016/j.molcatb.2013.08.017}, pages = {56 -- 60}, abstract = {The formation of naphthalene hydrates (i.e. 1- and 2-hydroxy-1,2-dihydronaphthalene) displays a new activity (besides epoxidation) in the enzymatic transformation of 1,2-dihydronaphthalene by two fungal unspecific peroxygenases (UPOs) accounting for 16-19\% of the overall turnover. These arene hydrates decayed into naphthalene that in turn was converted by UPOs into naphthols. The oxygen transferred during hydroxylation was shown to derive from hydrogen peroxide proving a true peroxygenation reaction.}, language = {en} } @misc{KlugeUllrichScheibneretal., author = {Kluge, Martin and Ullrich, Ren{\´e} and Scheibner, Katrin and Hofrichter, Martin}, title = {Stereoselective benzylic hydroxylation of alkylbenzenes and epoxidation of styrene derivatives catalyzed by the peroxygenase of Agrocybe aegerita}, series = {Green Chemistry}, journal = {Green Chemistry}, number = {2}, issn = {1463-9270}, pages = {440 -- 446}, abstract = {Here we report on the stereoselective benzylic hydroxylation and C1-C2 epoxidation of alkylbenzenes and styrene derivatives, respectively, by a heme-thiolate peroxygenase (EC 1.11.2.1) from the fungus Agrocybe aegerita. Benzylic hydroxylation led exclusively to the (R)-1-phenylalkanols. For (R)-1-phenylethanol, (R)-1-phenylpropanol and (R)-1-tetralol, the ee reached >99\%. For longer chain lengths, the enantiomeric excesses (ee) and total turnover numbers (TTN) decreased while the number of by-products, e.g. 1-phenylketones, increased. Epoxidation of straight chain and cyclic styrene derivatives gave a heterogeneous picture and resulted in moderate to excellent ee values and TTN: e.g., in the case of (1R,2S)-cis-[small beta]-methylstyrene oxide formation, an ee >99\% and a TTN of 110 000 was achieved. Hydroxylation and epoxidation were true peroxygenations, which was demonstrated by the incorporation of 18O from H218O2 into the products. The use of fed-batch devices and varying feeding strategies for the substrate and co-substrate turned out to be a suitable approach to optimize peroxygenase catalysis.}, language = {en} } @misc{KlugeUllrichScheibneretal., author = {Kluge, Martin and Ullrich, Ren{\´e} and Scheibner, Katrin and Hofrichter, Martin}, title = {Spectrophotometric assay for detection of aromatic hydroxylation catalyzed by fungal haloperoxidase-peroxygenase}, series = {Applied Microbiology and Biotechnology}, volume = {75}, journal = {Applied Microbiology and Biotechnology}, number = {6}, issn = {1432-0614}, doi = {10.1007/s00253-007-0942-8}, pages = {1473 -- 1478}, abstract = {Agrocybe aegerita peroxidase (AaP) is a versatile heme-thiolate protein that can act as a peroxygenase and catalyzes, among other reactions, the hydroxylation of aromatic rings. This paper reports a rapid and selective spectrophotometric method for directly detecting aromatic hydroxylation by AaP. The weakly activated aromatic compound naphthalene served as the substrate that was regioselectively converted into 1-naphthol in the presence of the co-substrate hydrogen peroxide. Formation of 1-naphthol was followed at 303 nm (ɛ 303 = 2,010 M-1 cm-1), and the apparent Michaelis-Menten (K m) and catalytic (k cat) constants for the reaction were estimated to be 320 μM and 166 s-1, respectively. This method will be useful in screening of fungi and other microorganisms for extracellular peroxygenase activities and in comparing and assessing different catalytic activities of haloperoxidase-peroxygenases.}, language = {en} } @misc{KiebistHollaHeidrichetal., author = {Kiebist, Jan and Holla, Wolfgang and Heidrich, Johannes and Poraj-Kobielska, Marzena and Sandvoss, Martin and Simonis, Reiner and Gr{\"o}be, Glenn and Atzrodt, Jens and Hofrichter, Martin and Scheibner, Katrin}, title = {One-pot synthesis of human metabolites of SAR548304 by fungal peroxygenases}, series = {Bioorganic \& Medicinal Chemistry}, volume = {23}, journal = {Bioorganic \& Medicinal Chemistry}, number = {15}, issn = {0968-0896}, doi = {10.1016/j.bmc.2015.06.035}, pages = {4324 -- 4332}, abstract = {Unspecific peroxygenases (UPOs, EC 1.11.2.1) have proved to be stable oxygen-transferring biocatalysts for H2O2-dependent transformation of pharmaceuticals. We have applied UPOs in a drug development program and consider the enzymatic approach in parallel to a conventional chemical synthesis of the human metabolites of the bile acid reabsorption inhibitor SAR548304. Chemical preparation of N,N-di-desmethyl metabolite was realized by a seven-step synthesis starting from a late precursor of SAR548304 and included among others palladium catalysis and laborious chromatographic purification with an overall yield of 27\%. The enzymatic approach revealed that the UPO of Marasmius rotula is particularly suitable for selective N-dealkylation of the drug and enabled us to prepare both human metabolites via one-pot conversion with an overall yield of 66\% N,N-di-desmethyl metabolite and 49\% of N-mono-desmethylated compound in two separated kinetic-controlled reactions.}, language = {en} } @misc{UllrichPorajKobielskaScholzeetal., author = {Ullrich, Ren{\´e} and Poraj-Kobielska, Marzena and Scholze, Steffi and Halbout, Claire and Sandvoss, Martin and Pecyna, Marek J. and Scheibner, Katrin and Hofrichter, Martin}, title = {Side chain removal from corticosteroids by unspecific peroxygenase}, series = {Journal of Inorganic Biochemistry}, volume = {183}, journal = {Journal of Inorganic Biochemistry}, issn = {1873-3344}, doi = {10.1016/j.jinorgbio.2018.03.011}, pages = {84 -- 93}, abstract = {Two unspecific peroxygenases (UPO, EC 1.11.2.1) from the basidiomycetous fungi Marasmius rotula and Marasmius wettsteinii oxidized steroids with hydroxyacetyl and hydroxyl functionalities at C17 - such as cortisone, Reichstein's substance S and prednisone - via stepwise oxygenation and final fission of the side chain. The sequential oxidation started with the hydroxylation of the terminal carbon (C21) leading to a stable geminal alcohol (e.g. cortisone 21-gem-diol) and proceeded via a second oxygenation resulting in the corresponding α-ketocarboxylic acid (e.g. cortisone 21-oic acid). The latter decomposed under formation of adrenosterone (4-androstene-3,11,17-trione) as well as formic acid and carbonic acid (that is in equilibrium with carbon dioxide); fission products comprising two carbon atoms such as glycolic acid or glyoxylic acid were not detected. Protein models based on the crystal structure data of MroUPO (Marasmius rotula unspecific peroxygenase) revealed that the bulky cortisone molecule suitably fits into the enzyme's access channel, which enables the heme iron to come in close contact to the carbons (C21, C20) of the steroidal side chain. ICP-MS analysis of purified MroUPO confirmed the presence of magnesium supposedly stabilizing the porphyrin ring system.}, language = {en} } @misc{PiontekStrittmatterUllrichetal., author = {Piontek, Klaus and Strittmatter, Eric and Ullrich, Ren{\´e} and Gr{\"o}be, Glenn and Pecyna, Marek J. and Kluge, Martin and Scheibner, Katrin and Hofrichter, Martin and Plattner, Dietmar A.}, title = {Structural basis of substrate conversion in a new aromatic peroxygenase: cytochrome P450 functionality with benefits}, series = {The Journal of Biological Chemistry}, journal = {The Journal of Biological Chemistry}, number = {288}, issn = {1083-351X}, doi = {10.1074/jbc.M113.514521}, pages = {34767 -- 34776}, abstract = {Aromatic peroxygenases (APOs) represent a unique oxidoreductase sub-subclass of heme proteins with peroxygenase and peroxidase activity and were thus recently assigned a distinct EC classification (EC 1.11.2.1). They catalyze, inter alia, oxyfunctionalization reactions of aromatic and aliphatic hydrocarbons with remarkable regio- and stereoselectivities. When compared with cytochrome P450, APOs appear to be the choice enzymes for oxyfunctionalizations in organic synthesis due to their independence from a cellular environment and their greater chemical versatility. Here, the first two crystal structures of a heavily glycosylated fungal aromatic peroxygenase (AaeAPO) are described. They reveal different pH-dependent ligand binding modes. We model the fitting of various substrates in AaeAPO, illustrating the way the enzyme oxygenates polycyclic aromatic hydrocarbons. Spatial restrictions by a phenylalanine pentad in the active-site environment govern substrate specificity in AaeAPO.}, language = {en} }