@misc{KinnePorajKobielskaUllrichetal., author = {Kinne, Matthias and Poraj-Kobielska, Marzena and Ullrich, Ren{\´e} and Nousiainen, Paula and Sipil{\"a}, Jussi and Scheibner, Katrin and Hammel, Kenneth E. and Hofrichter, Martin}, title = {Oxidative cleavage of non-phenolic b-O-4 lignin model dimers by an extracellular aromatic peroxygenase}, series = {Holzforschung}, volume = {65}, journal = {Holzforschung}, number = {5}, issn = {1437-434X}, doi = {10.1515/HF.2011.057}, pages = {673 -- 679}, abstract = {The extracellular aromatic peroxygenase of the agaric fungus Agrocybe aegerita catalyzed the H2O2-dependent cleavage of non-phenolic arylglycerol-b-aryl ethers (b-O-4 ethers). For instance 1-(3,4-dimethoxyphenyl)-2-(2-methoxy-phenoxy)pro- pane-1,3-diol, a recalcitrant dimeric lignin model compound that represents the major non-phenolic substructure in lignin, was selectively O-demethylated at the para-methoxy group to give formaldehyde and 1-(4-hydroxy-3-methoxyphenyl)- 2-(2-methoxyphenoxy)propane-1,3-diol. The phenol moiety of the latter compound was then enzymatically oxidized into phenoxy radicals and a quinoid cation, which initiated the autocatalytic cleavage of the dimer and the formation of monomers such as 2-methoxy-1,4-benzoquinone and phenoxyl-substituted propionic acid. The introduction of 18O from H218O2 and H218O at different positions into the products provided information about the routes of ether cleavage. Studies with a 14C-labeled lignin model dimer showed that more than 70\% of the intermediates formed were further coupled to form polymers with molecular masses above 10 kDa. The results indicate that fungal aromatic peroxyge- nases may be involved in the bioconversion of methoxylated plant ingredients originating from lignin or other sources.}, language = {en} } @misc{PorajKobielskaKinneUllrichetal., author = {Poraj-Kobielska, Marzena and Kinne, Matthias and Ullrich, Ren{\´e} and Scheibner, Katrin and Kayser, Gernot and Hammel, Kenneth E. and Hofrichter, Martin}, title = {Preparation of human drug metabolites using fungal peroxygenases}, series = {Biochemical Pharmacology}, volume = {82}, journal = {Biochemical Pharmacology}, number = {7}, issn = {1873-2968}, doi = {10.1016/j.bcp.2011.06.020}, pages = {789 -- 796}, abstract = {The synthesis of hydroxylated and O- or N-dealkylated human drug metabolites (HDMs) via selective monooxygenation remains a challenging task for synthetic organic chemists. Here we report that aromatic peroxygenases (APOs; EC 1.11.2.1) secreted by the agaric fungi Agrocybe aegerita and Coprinellus radians catalyzed the H₂O₂-dependent selective monooxygenation of diverse drugs, including acetanilide, dextrorphan, ibuprofen, naproxen, phenacetin, sildenafil and tolbutamide. Reactions included the hydroxylation of aromatic rings and aliphatic side chains, as well as O- and N-dealkylations and exhibited different regioselectivities depending on the particular APO used. At best, desired HDMs were obtained in yields greater than 80\% and with isomeric purities up to 99\%. Oxidations of tolbutamide, acetanilide and carbamazepine in the presence of H₂¹⁸O₂ resulted in almost complete incorporation of ¹⁸O into the corresponding products, thus establishing that these reactions are peroxygenations. The deethylation of phenacetin-d₁ showed an observed intramolecular deuterium isotope effect [(k(H)/k(D))(obs)] of 3.1±0.2, which is consistent with the existence of a cytochrome P450-like intermediate in the reaction cycle of APOs. Our results indicate that fungal peroxygenases may be useful biocatalytic tools to prepare pharmacologically relevant drug metabolites.}, language = {en} } @misc{KinnePorajKobielskaArandaetal., author = {Kinne, Matthias and Poraj-Kobielska, Marzena and Aranda, Elisabet and Ullrich, Ren{\´e} and Hammel, Kenneth E. and Scheibner, Katrin and Hofrichter, Martin}, title = {Regioselective preparation of 5-hydroxypropranolol and 4′-hydroxydiclofenac with a fungal peroxygenase}, series = {Bioorganic \& Medicinal Chemistry Letters}, volume = {19}, journal = {Bioorganic \& Medicinal Chemistry Letters}, number = {11}, issn = {1464-3405}, doi = {10.1016/j.bmcl.2009.04.015}, pages = {3085 -- 3087}, abstract = {An extracellular peroxygenase of Agrocybe aegerita catalyzed the H2O2-dependent hydroxylation of the multi-function beta-adrenergic blocker propranolol (1-naphthalen-1-yloxy-3-(propan-2-ylamino)propan-2-ol) and the non-steroidal anti-inflammatory drug diclofenac (2-[2-[(2,6-dichlorophenyl)amino]phenyl]acetic acid) to give the human drug metabolites 5-hydroxypropranolol (5-OHP) and 4′-hydroxydiclofenac (4′-OHD). The reactions proceeded regioselectively with high isomeric purity and gave the desired 5-OHP and 4′-OHD in yields up to 20\% and 65\%, respectively. 18O-labeling experiments showed that the phenolic hydroxyl groups in 5-OHP and 4′-OHD originated from H2O2, which establishes that the reaction is mechanistically a peroxygenation. Our results raise the possibility that fungal peroxygenases may be useful for versatile, cost-effective, and scalable syntheses of drug metabolites.}, 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{PorajKobielskaKinneUllrichetal., author = {Poraj-Kobielska, Marzena and Kinne, Matthias and Ullrich, Ren{\´e} and Scheibner, Katrin and Hofrichter, Martin}, title = {A spectrophotometric assay for the detection of fungal peroxygenases}, series = {Analytical Biochemistry}, volume = {421}, journal = {Analytical Biochemistry}, number = {1}, doi = {10.1016/j.ab.2011.10.009}, pages = {327 -- 329}, abstract = {Rapid and simple spectrophotometric methods are required for the unambiguous detection of recently discovered fungal peroxygenases in vivo and in vitro. This paper describes a peroxygenase-specific assay using 5-nitro-1,3-benzodioxole as substrate. The product, 4-nitrocatechol, produces a yellow color at pH 7, which can be followed over time at 425 nm (ε425 = 9,700 M-1 cm-1), and a red color when adjusted to pH >12, which can be measured in form of an end-point determination at 514 nm (ε514 = 11,400 M-1 cm-1). The assay is suitable for detecting peroxygenase activities in complex growth media and environmental samples as well as for high-throughput screenings.}, language = {en} } @misc{PorajKobielskaScheibnerGroebeetal., author = {Poraj-Kobielska, Marzena and Scheibner, Katrin and Gr{\"o}be, Glenn and Kiebist, Jan and Gr{\"u}n, Manfred and Ullrich, Ren{\´e} and Hofrichter, Martin}, title = {Verfahren zur Deacylierung von Corticoiden}, language = {de} } @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{PorajKobielskaPeterLeonhardtetal., author = {Poraj-Kobielska, Marzena and Peter, Sebastian and Leonhardt, Sabrina and Ullrich, Ren{\´e} and Scheibner, Katrin and Hofrichter, Martin}, title = {Immobilization of unspecific peroxygenases (EC 1.11.2.1) in PVA/PEG gel and hollow fiber modules}, series = {Biochemical Engineering Journal}, volume = {98}, journal = {Biochemical Engineering Journal}, issn = {1369-703X}, doi = {10.1016/j.bej.2015.02.037}, pages = {144 -- 150}, abstract = {The immobilization of enzymes has many advantages, such as higher stability, easier handling, and reuse of the catalyst. Here we report, for the first time, two effective methods for the immobilization of unspecific peroxygenase (UPO; EC 1.11.2.1). This biocatalyst type comprises heavily glycosylated heme-thiolate proteins that catalyze various biotechnologically relevant oxyfunctionalizations. Both the encapsulation in cryogel and the retention of the enzyme in hollow fiber modules were found to be efficient methods for their immobilization. After encapsulation, the enzyme still exhibited 60\% of its initial activity. Interestingly, we did not find differences in the kinetic parameters of free and immobilized UPOs. In long-term experiments, the conversion of the pharmaceutical diclofenac with immobilized UPOs in different reactor types yielded between 62 mg and 154 mg of the major human drug metabolite 4′-hydroxydiclofenac. The maximal total turnover number was about 60-fold higher compared to the free enzyme. A test over 5 months showed that storage of encapsulated UPOs in non-polar solvents (e.g., cyclohexane) helps to preserve the enzyme stability and increases their relative activity (by about ∼150\%, in the case of diclofenac hydroxylation). In addition to the hydrophilic substrate diclofenac, encapsulated UPOs also oxidized the hydrophobic model compound cyclohexane.}, 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} }