DE102015120587A1 - Method for the selective oxygenation of testosterone and testosterone-like steroids - Google Patents

Method for the selective oxygenation of testosterone and testosterone-like steroids Download PDF

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DE102015120587A1
DE102015120587A1 DE102015120587.4A DE102015120587A DE102015120587A1 DE 102015120587 A1 DE102015120587 A1 DE 102015120587A1 DE 102015120587 A DE102015120587 A DE 102015120587A DE 102015120587 A1 DE102015120587 A1 DE 102015120587A1
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dione
testosterone
steroids
epoxy
hydroxy
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Jan Kiebist
Katrin Scheibner
Kai-Uwe SCHMIDTKE
Tino Koncz
Martin Hofrichter
René Ullrich
Marzena Poraj-Kobielska
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Technische Universitaet Dresden
Brandenburgische Technische Universitaet Cottbus
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J1/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, not substituted in position 17 beta by a carbon atom, e.g. estrane, androstane
    • C07J1/0003—Androstane derivatives
    • C07J1/0018—Androstane derivatives substituted in position 17 beta, not substituted in position 17 alfa
    • C07J1/0022—Androstane derivatives substituted in position 17 beta, not substituted in position 17 alfa the substituent being an OH group free esterified or etherified
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J5/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond
    • C07J5/0007—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond not substituted in position 17 alfa
    • C07J5/0023—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond not substituted in position 17 alfa substituted in position 16
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J5/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond
    • C07J5/0046—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond substituted in position 17 alfa
    • C07J5/0061—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond substituted in position 17 alfa substituted in position 16
    • C07J5/0092—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond substituted in position 17 alfa substituted in position 16 by an OH group free esterified or etherified
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J7/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms
    • C07J7/0005—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms not substituted in position 21
    • C07J7/001—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms not substituted in position 21 substituted in position 20 by a keto group
    • C07J7/004—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms not substituted in position 21 substituted in position 20 by a keto group substituted in position 17 alfa
    • C07J7/005—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms not substituted in position 21 substituted in position 20 by a keto group substituted in position 17 alfa substituted in position 16
    • C07J7/006—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms not substituted in position 21 substituted in position 20 by a keto group substituted in position 17 alfa substituted in position 16 by a hydroxy group free esterified or etherified
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
    • C07J71/0005—Oxygen-containing hetero ring
    • C07J71/001—Oxiranes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P33/00—Preparation of steroids
    • C12P33/12—Acting on D ring
    • C12P33/14—Hydroxylating at 16 position
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
    • C12Y111/02—Oxidoreductases acting on a peroxide as acceptor (1.11) with H2O2 as acceptor, one oxygen atom of which is incorporated into the product (1.11.2)
    • C12Y111/02001—Unspecific peroxygenase (1.11.2.1)

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  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
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  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Steroid Compounds (AREA)

Abstract

Gegenstand der Erfindung ist ein Verfahren zur enzymatischen selektiven Einführung von Sauerstoff in das Grundgerüst von vorzugsweise Testosteron und Testosteron-ähnlichen Steroiden in einem wasserhaltigen Lösungsmittel in Gegenwart einer Peroxygenase (EC 1.11.2.1), vorzugsweise aus einem Ascomyceten, unter Erhalten eines 16α-Hydroxids des 3-on 4-en Sterangrundgerüstes und eines Epoxids am 4-en des Sterangrundgerüstes. Die Erfindung ermöglicht vorteilhaft die Synthese schwerzugänglicher hydroxylierter Steroidstrukturen.The invention relates to a process for the enzymatic selective introduction of oxygen into the skeleton of preferably testosterone and testosterone-like steroids in an aqueous solvent in the presence of a peroxygenase (EC 1.11.2.1), preferably from an ascomycete, to obtain a 16α-hydroxide of the 3-on 4-star steroid scaffold and an epoxide on the 4-in of the steroid scaffold. The invention advantageously allows the synthesis of difficult-to-access hydroxylated steroid structures.

Description

Gegenstand der Erfindung ist ein Verfahren zur enzymatischen selektiven Einführung von Sauerstoff in das Grundgerüst von vorzugsweise Testosteron und Testosteron-ähnlichen Steroiden in einem wasserhaltigen Lösungsmittel in Gegenwart einer Peroxygenase (EC 1.11.2.1), vorzugsweise aus einem Ascomyceten, unter Erhalten eines 16α-Hydroxids des 3-on 4-en Sterangrundgerüstes und eines Epoxids am 4-en des Sterangrundgerüstes. Die Erfindung ermöglicht vorteilhaft die Synthese schwerzugänglicher hydroxylierter Steroidstrukturen.The invention relates to a process for the enzymatic selective introduction of oxygen into the skeleton of preferably testosterone and testosterone-like steroids in an aqueous solvent in the presence of a peroxygenase (EC 1.11.2.1), preferably from an ascomycete, to obtain a 16α-hydroxide of the 3-on 4-star steroid scaffold and an epoxide on the 4-in of the steroid scaffold. The invention advantageously allows the synthesis of difficult-to-access hydroxylated steroid structures.

Steroide bilden eine der wichtigsten Klassen von Naturstoffen, welche ubiquitär in allen Lebewesen vorkommen. Sie gehören zu den Terpenoiden und stellen Derivate des Kohlenwasserstoffs Steran dar. Unter ihnen finden sich zahlreiche physiologisch aktive Substanzgruppen wieder; von Vitaminen über Gallensäure, Sexual- und Nebennierenrindenhormone bis hin zu herzaktiven Verbindungen, Corticoiden u. v. a. ( Nuhn, Naturstoffchemie, S. Hirzel Verlag, Stuttgart, 2006 ).Steroids are one of the most important classes of natural substances that are ubiquitous in all living things. They belong to the terpenoids and represent derivatives of the hydrocarbon sterane. Among them, numerous physiologically active substance groups are found again; from vitamins to bile acids, sex and adrenocortical hormones, to heart-active compounds, corticoids and many others ( Nuhn, Natural Products Chemistry, S. Hirzel Verlag, Stuttgart, 2006 ).

Steroide, insbesondere Steroidhormone, gehören zu den wichtigsten Verbindungen in der pharmazeutischen Industrie zur Behandlung und Vorsorge diverser Krankheiten. Steroidale Pharmazeutika sind die am meisten vermarkteten medizinischen Produkte und werden weitverbreitet als antikanzerogene, antiinflammatorische, antimikrobielle, antivirale, antifungale, antiestrogene, anticonvulsive und antiallergene Mittel eingesetzt. Sie finden Verwendung zur Prophylaxe und Therapie verschiedener Krankheiten wie Hormon-abhängige Formen von Brust- und Prostatakrebs, bestimmte Formen von Darmkrebs, Adipositas, Diabetes, rheumatoide Arthritis, Bluthochdruck, Asthma, Ekzeme, Entzündungen, Stoffwechselstörungen, neurodegenerative Erkrankungen, Störung des zentralen Nervensystems, anaphylaktischer Schock; als Ersatzmittel bei der Behandlung von Niereninsuffizienz, zur Inhibierung der HIV Integrase, zur Prävention und Behandlung bei Infektionen mit HIV und zur Behandlung von AIDS. Steroide spielen auch eine entscheidende Rolle bei der Regulierung des Cholesterol-Spiegels sowie bei cardiovaskulären und neuroprotektiven Funktionen ( Backström, Haage et al., Paradoxical effects of GABA-A modulators may explain sex steroid induced negative mood symptoms in some persons, Neuroscience, 15, 46–54, 2011 ; Bäckstrom and Ragagnin, The use of pregnane and androstane steroids for the manufacture of a pharmaceutical composition for the treatment of CNS disorders, WO2008/063128 , 2008; Craigie, Mullins et al., Glucocorticoids and mineralocorticoids, Bader M (ed) Cardiovascular hormone systems: from molecular mechanism to novel therapeutics, 2009 ; Donova and Egorova, Microbial steroid transformations: current state and prospects, Appl. Microbiol. Biotechnol., 94, 1423–1447, 2012 ; Finocchi and Ferrari, Female reproductive steroids and neuronal excitability, Neurol. Sci., 32, 31–35, 2011 ; Garcia-Segura and Balthazart, Steroids and neuroprotection: new advances, Front Neuroendocrinol., 30, 5–9, 2009 ; Rugutt and Rugutt, Antimycobacterial activity of steroids, long-chain alcohols and lytic peptides, Nat. Prod. Res., 26, 1004–1011, 2012 ; Tong and Dong, Microbial transformations: recent developements an steroid drugs, Recent Patents an Biotechnol., 3, 141–153, 2009 ).Steroids, in particular steroid hormones, are among the most important compounds in the pharmaceutical industry for the treatment and prevention of various diseases. Steroidal pharmaceuticals are the most widely marketed medical products and are widely used as anticancer, anti-inflammatory, antimicrobial, antiviral, antifungal, antiestrogenic, anticonvulsant and antiallergenic agents. They are used for the prophylaxis and therapy of various diseases such as hormone-dependent forms of breast and prostate cancer, certain forms of colon cancer, obesity, diabetes, rheumatoid arthritis, hypertension, asthma, eczema, inflammation, metabolic disorders, neurodegenerative diseases, central nervous system disorder, anaphylactic shock; as a replacement in the treatment of renal failure, to inhibit HIV integrase, to prevent and treat infections with HIV and to treat AIDS. Steroids also play a crucial role in the regulation of cholesterol levels as well as cardiovascular and neuroprotective functions ( Backström, Haage et al., Paradoxical effects of GABA-A modulators may explain sexually induced negative mood symptoms in some persons, Neuroscience, 15, 46-54, 2011 ; Bäckstrom and Ragagnin, The use of pregnane and androstane steroids for the manufacture of CNS disorders, WO2008 / 063128 , 2008; Craigie, Mullins et al., Glucocorticoids and mineralocorticoids, Bader M (ed) Cardiovascular hormone systems: from molecular mechanics to novel therapeutics, 2009 ; Donova and Egorova, Microbial steroid transformations: current state and prospects, Appl. Microbiol. Biotechnol., 94, 1423-1447, 2012 ; Finocchi and Ferrari, Female reproductive steroid and neuronal excitability, Neurol. Sci., 32, 31-35, 2011 ; Garcia-Segura and Balthazart, Steroids and neuroprotection: new advances, Front Neuroendocrinol., 30, 5-9, 2009 ; Rugutt and Rugutt, Antimycobacterial activity of steroids, long-chain alcohols and lytic peptides, Nat. Prod. Res., 26, 1004-1011, 2012 ; Tong and Dong, Microbial transformations: recent developements of steroid drugs, Recent Patents to Biotechnol., 3, 141-153, 2009 ).

Die physiologische Aktivität von Steroiden hängt unmittelbar von ihrer Struktur ab: die Art, Anzahl und Regio- sowie Stereoposition ihrer funktionellen Gruppe und dem Oxidationszustand des Ringsystems. Beispielsweise ist eine Sauerstofffunktionalität in C-11β-Position notwendig für eine inflammatorische Wirkung; eine 17β-Hydroxylgruppe bestimmt die androgenen Eigenschaften, eine Aromatisierung des Ringes A resultiert in estrogenen Effekten ( Donova and Egorova, Microbial steroid transformations: current state and prospects, Appl. Microbiol. Biotechnol., 94, 1423–1447, 2012 ).The physiological activity of steroids depends directly on their structure: the nature, number and regio- and stereoposition of their functional group and the oxidation state of the ring system. For example, an oxygen functionality in C-11β position is necessary for an inflammatory effect; a 17β-hydroxyl group determines the androgenic properties, aromatization of the ring A results in estrogenic effects ( Donova and Egorova, Microbial steroid transformations: current state and prospects, Appl. Microbiol. Biotechnol., 94, 1423-1447, 2012 ).

Die Herstellung steroidaler Wirkstoffe und Hormone basiert im Stand der Technik auf einer Kombination chemischer und mikrobiologischer Verfahren. Die chemischen Methoden sind meist aufwändige Mehrstufen-Synthesen und mit Schutzgruppenchemie verbunden, wodurch diese Verfahren zu geringen Ausbeuten und hohem Zeitaufwand führen. Infolge der Regioselektivität und Spezifität von Steroidmolekülen ist die Nutzung von Biokatalysatoren für regio- und stereoselektive Synthesestufen von enorm hohem Interesse ( Bortolini, Medici et al., Biotransformations of the steroid nucleus of bile acids, Steroids, 62, 564–577, 1997 ). Zu den wichtigsten Reaktionen gehören die Steroid-Hydroxylierungen, Dehydrierungen und Sterol-Seitenkettenabspaltungen. Diese Modifikationen der natürlichen Steroide führen, wie oben erwähnt, zu veränderten therapeutischen Wirkungen, wie zum Beispiel erhöhte Potenz, längere Halbwertszeiten im Blutstrom, vereinfachter Transport und verringerte Nebenwirkungen.The preparation of steroidal drugs and hormones is based in the prior art on a combination of chemical and microbiological processes. The chemical methods are usually elaborate multi-step syntheses and associated with protective group chemistry, whereby these methods lead to low yields and high expenditure of time. Due to the regioselectivity and specificity of steroid molecules, the use of biocatalysts for regio- and stereoselective synthesis steps is of tremendous interest ( Bortolini, Medici et al., Biotransformations of the steroid nucleus of bile acids, Steroids, 62, 564-577, 1997 ). Key reactions include steroid hydroxylations, dehydrations, and sterol side-chain cleavages. These modifications of the natural steroids, as mentioned above, result in altered therapeutic effects, such as increased potency, longer half-lives in the bloodstream, easier transport, and reduced side effects.

Für gewöhnlich werden Ganzzell-Systeme gegenüber Enzymsystemen als Biokatalysatoren vorgezogen, um Kosten für Enzymisolierung, -reinigung und -stabilisierung zu vermeiden. Als Ausgangsmaterialien für die pharmazeutische Industrie dienen hauptsächlich Phytosterole (Stigmasterol, β-Sitosterol, Campesterol), Sapogenine (Diosgenine), Hecogenin, Solasodin und Cholesterol, welche aus natürlichen Quellen isoliert werden ( Fernandes, Cruz et al., Microbial conversion of steroid compounds: recent developments, Enzyme Microb. Technol., 32, 688–705, 2003 ).Usually, whole-cell systems are preferred over enzyme systems as biocatalysts to avoid costs for enzyme isolation, purification, and stabilization. The starting materials for the pharmaceutical industry are mainly phytosterols (stigmasterol, β-sitosterol, campesterol), sapogenins (diosgenins), hecogenin, solasodine and cholesterol, which are isolated from natural sources become ( Fernandes, Cruz et al., Microbial conversion of steroid compounds: recent developments, Enzyme Microb. Technol., 32, 688-705, 2003 ).

Die Biotransformation von Steroiden ist ein nützliches Werkzeug für die Synthese neuer steroidaler Wirkstoffe sowie für die effiziente Herstellung steroidaler aktiver pharmazeutischer Wirkstoffe (API's) und verschiedener synthetischer Zwischenstufen. Die bedeutendsten Vorstufen sind 4-Androsten-3,17-dion (AD) und 1,4-Androstadien-3,17-dion (ADD), welche für die industrielle Herstellung von diversen Steroiden benötigt werden. Beide Verbindungen können aus Phytosterolen in einem biotechnologischen Schritt mittels Actinobakterien der Gattungen Mycobacterium und Rhodococcus gewonnen werden ( Malavija and Gomes, Androstenedione production by biotransformation of phytosterols, Bioresour. Technol., 99, 6725–6737, 2008 ; Molchenova, Andryushina et al., Preparation of androsta-1,4-diene-3,17-dione from sterols using Mycobacterium neoaurum VKPM Ac-1656 strain, Russ. J. Bioorg. Chem., 33, 354–358, 2007 ; Sripalakit, Wichai et al., Biotransformation of various natural sterols to androstenones by Mycobacterium sp. and some steroid converting, microbial strains, J. Mol. Cat. B: Enzym, 41, 2006 ). Ebenso kann Testosteron durch Actinobakterien aus Sterolen produziert werden ( Egorova, Nikolayeva et al., Transformation of C19-steroids and testosterone production by sterol-transforming strains of Mycobacterium spp., J. Mol. Cat. B: Enzym, 5, 2009 ). Ebenfalls wird die Kombination verschiedener mikrobiologischen Umsetzungen häufig angewandt. Beispielsweise wird Boldenon durch 1-Dehydrogenierung von AD durch Fusarium sp. gewonnen (Kutney, Herrington et al., Process for fermentation of phytosterols to androstadienon, WO2003064674A2 , 2003). Neben den genannten Beispielen werden noch zahlreiche weitere Steroide durch Biotransformation hergestellt, welche Anwendung in zahlreichen Wirkstoffsynthesen finden ( Donova and Egorova, Microbial steroid transformations: current state and prospects, Appl. Microbiol. Biotechnol., 94, 1423–1447, 2012 ).Steroids biotransformation is a useful tool for the synthesis of novel steroidal drugs as well as for the efficient production of steroidal active pharmaceutical ingredients (APIs) and various synthetic intermediates. The most important precursors are 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD), which are needed for the industrial production of various steroids. Both compounds can be obtained from phytosterols in a biotechnological step by means of actinobacteria of the genera Mycobacterium and Rhodococcus ( Malavija and Gomes, Androstenedione production by biotransformation of phytosterols, Bioresour. Technol., 99, 6725-6737, 2008 ; Molchenova, Andryushina et al., Preparation of androsta-1,4-dienes-3,17-diones from sterols using Mycobacterium neoaurum VKPM Ac-1656 strain, Russ. J. Bioorg. Chem., 33, 354-358, 2007 ; Sripalakit, Wichai et al., Biotransformation of various natural sterols to androstenones by Mycobacterium sp. and some steroid converting, microbial strains, J. Mol. Cat. B: Enzyme, 41, 2006 ). Likewise, testosterone can be produced by actinobacteria from sterols ( Egorova, Nikolayeva et al., Transformation of C19-steroids and testosterone production by sterol-transforming strains of Mycobacterium spp., J. Mol. Cat. B: Enzyme, 5, 2009 ). Also, the combination of various microbiological reactions is often used. For example, boldenone is obtained by 1-dehydrogenation of AD by Fusarium sp. Kutney, Herrington et al., Process for the fermentation of phytosterols to androstadienone, WO2003064674A2 , 2003). In addition to the above examples, numerous other steroids are produced by biotransformation, which find application in numerous drug syntheses ( Donova and Egorova, Microbial steroid transformations: current state and prospects, Appl. Microbiol. Biotechnol., 94, 1423-1447, 2012 ).

Eine der wichtigsten Reaktionen zur Steroid-Funktionalisierung ist jedoch die Hydroxylierung am inaktiven, gesättigten Kohlenstoff des Sterangrundkörpers. Hydroxylierte Steroide weisen oft eine gesteigerte biologische Aktivität im Vergleich zu ihren weniger polaren Analoga auf. Beispielsweise besitzt das 7β-Hydroxyderivat von Dehydroepiandrosteron (DHEA) siebenfach höhere immunoprotektive und immunoregulative Eigenschaften im Vergleich zu DHEA ( Janeczko, Dmochowska-Gladysz et al., Biotransformation of steroid compounds by Chaetomium sp. KCH 6651, Steroids, 74, 2009 ). Hydroxylierungen führen zu einer Veränderung der Polarität von Steroiden, beeinflussen ihre Toxizität sowie ihre Ausscheidung aus der Zelle. Der aktuelle Trend in diversen Studien mikrobieller Hydroxylierungen geht in Richtung

  • a) der Suche nach neuen Biokatalysatoren, welche die wichtigsten Reaktionen an Steroiden katalysieren (7α, 9α, 11α, 11β, 16α, 17α),
  • b) der Herstellung von neuen Hydroxysteroiden mit therapeutischen Eigenschaften,
  • c) der Untersuchung der Effekte der Steroidstruktur in Abhängigkeit der Position der Hydroxylgruppe ( Donova and Egorova, Microbial steroid transformations: current state and prospects, Appl. Microbiol. Biotechnol., 94, 1423–1447, 2012 ).
However, one of the most important reactions for steroid functionalization is the hydroxylation on the inactive, saturated carbon of the steroidal matrix. Hydroxylated steroids often have increased biological activity compared to their less polar analogs. For example, the 7β-hydroxy derivative of dehydroepiandrosterone (DHEA) has seven times higher immunoprotective and immunoregulatory properties compared to DHEA ( Janeczko, Dmochowska-Gladysz et al., Biotransformation of steroid compounds by Chaetomium sp. KCH 6651, Steroids, 74, 2009 ). Hydroxylations alter the polarity of steroids, affecting their toxicity as well as their excretion from the cell. The current trend in various studies of microbial hydroxylation is heading in the direction
  • a) the search for new biocatalysts which catalyze the most important reactions to steroids (7α, 9α, 11α, 11β, 16α, 17α),
  • b) the production of novel hydroxysteroids with therapeutic properties,
  • c) investigating the effects of the steroid structure as a function of the position of the hydroxyl group ( Donova and Egorova, Microbial steroid transformations: current state and prospects, Appl. Microbiol. Biotechnol., 94, 1423-1447, 2012 ).

Testosteron gehört zu den pharmazeutisch wichtigsten Steroiden. Zahlreiche mikrobielle Transformationen sind bereits in der Literatur beschrieben, so die Bildung von 7α-OH-Testosteron durch Botrytis cinerea AM235 ( Huszcza and Dmochowska-Gladysz, Transformations of testosterone and related steroids by Botrytis cinerea, Phytochem., 62, 155–158, 2003 ), 9α-OH-Testosteron durch Rhodococcus equi ATCC 14887 ( Kim, Han et al., Steroid 9-hydroxylation during testosterone degradation by resting Rhodococcus equi cells, Arch. Pharm., 340, 209–214, 2007 ), 11α-OH-Testosteron durch Rhizopus stolonifer ATCC 10404 und Fusarium lini NRRL 68751 ( Al-Aboudi, Mohammad et al., Microbial transformation of testosterone by Rhizopus stolofiner and Fusarium lini, Natl. Product Res., 22, 1498–1509, 2008 ), 14α-OH-Testosteron durch Aspergillus wentii MRC 200316 ( Yildirim, Kupcu et al., Biotransformation of some steroids by Aspergillus wentii, Z. Naturforsch. C., 65, 688–692, 2010 ), 15α-OH-Testosteron durch Fusarium oxysporum var. cubense ( Reese, Biotransformation of terpenes and steroids by fungi, Natural products: essential resources for human survival, 2007 ), 15β-OH-Testosteron Pseudomonas putida S12 ( Ruijssenaars, Sperling et al., Testosterone 15b-hydroxylation by solvent tolerant Pseudomonas putida S12, J. Biotechnol., 131, 205–208, 2007 ) und 16β-OH-Testosteron durch Whetzelinia sclerotiorum ATCC 18687 ( Lamm, Chen et al., Steroid hydroxylation by Whetzelinia sclerotiorum, Phanerochaete chrysosporium and Mucor plumbeus, Steroids, 72, 713–722, 2007 ). Steroid-Hydroxylasen sind meist Multienzym-Komplexe mit Cytochrom P450 als Substrat-bindende Oxidase. Trotz der interessanten katalytischen Eigenschaften sind Anwendungen im größeren synthetischen Maßstab mit diesen Enzymen, welche NAD(P)H als Elektronendonor und auxilare Flavinreduktasen als Elektronenüberträger benötigen, bislang nicht umsetzbar ( Beilen and Funhoff, Expanding the alkane oxygenase toolbox: new enzymes and application, Curr. Opin. Biotechnol., 16, 308–314, 2005 ; U rlacher and Girhard, Cytochrom P450 monooxygenases: an update an perspectives for synthetic application Trends Biotechnol., 30, 26–36, 2012 ).Testosterone is one of the most important pharmaceutical steroids. Numerous microbial transformations are already described in the literature, such as the formation of 7α-OH-testosterone by Botrytis cinerea AM235 ( Huszcza and Dmochowska-Gladysz, Transformations of testosterone and related steroids by Botrytis cinerea, Phytochem., 62, 155-158, 2003 ), 9α-OH testosterone by Rhodococcus equi ATCC 14887 ( Kim, Han et al., Steroid 9-hydroxylation during testosterone degradation by resting Rhodococcus equi cells, Arch. Pharm., 340, 209-214, 2007 ), 11α-OH testosterone by Rhizopus stolonifer ATCC 10404 and Fusarium lini NRRL 68751 ( Al-Aboudi, Mohammad et al., Microbial transformation of testosterone by Rhizopus stolofiner and Fusarium lini, Natl. Product Res., 22, 1498-1509, 2008 ), 14α-OH testosterone by Aspergillus wentii MRC 200316 ( Yildirim, Kupcu et al., Biotransformation of some steroids by Aspergillus wentii, Z. Naturforsch. C., 65, 688-692, 2010 ), 15α-OH testosterone by Fusarium oxysporum var. Cubense ( Reese, Biotransformation of terpenes and steroids by fungi, Natural products: essential resources for human survival, 2007 ), 15β-OH testosterone Pseudomonas putida S12 ( Ruijssenaars, Sperling et al., Testosterone 15b-hydroxylation by solvent tolerant Pseudomonas putida S12, J. Biotechnol., 131, 205-208, 2007 ) and 16β-OH testosterone by Whetzelinia sclerotiorum ATCC 18687 ( Lamm, Chen et al., Steroid hydroxylation by Whetzelinia sclerotiorum, Phanerochaete chrysosporium and Mucor plumbeus, Steroids, 72, 713-722, 2007 ). Steroid hydroxylases are mostly multi-enzyme complexes with cytochrome P450 as a substrate-binding oxidase. Despite the interesting catalytic properties, applications on a larger synthetic scale with these enzymes, which require NAD (P) H as electron donor and auxiliaries flavin reductases as electron transfer agents, have not been feasible ( Beilen and Funhoff, Expanding the alkane oxygenase toolbox: new enzymes and application, Curr. Opin. Biotechnol., 16, 308-314, 2005 ; U rlacher and Girhard, Cytochrome P450 monooxygenases: an update to perspectives for synthetic application Trends Biotechnol., 30, 26-36, 2012 ).

16α-OH-Testosteron (16α-OHT) wurde als Metabolit von Testosteron in Hühner-, Maus- und Rattenleber sowie bei Umsetzungen mit humaner Plazenta identifiziert (Harada and Negishi, Mouse liver testosterone 16a-Hydroxylase ( Cytochrom P-45016a), J. Biol. Chem., 259, 12285–12290, 1984; Paolini, Pozzetti et al., Developement of basal and induced testosterone hydroxylase activity in the chicken embryo in ovo, Brit. J. Pharmacol., 122, 344–350, 1997 ; Sugiyama, Nagata et al., Theoratical kinetics of sequential metabolism in vitro. Study of the formation of 16 alpha-hydroxyandrostenedione from testosterone by purified rat P450 2C11, Drug Metab. Dispos., 22, 584–591, 1994 ; Waxman, Ko et al., Regioselectivity and stereoselectivity of androgen hydroxylations catalyzed by cytochrom P-450 isozymes purified from phenobarbital-induced rat liver, J. Biol. Chem., 258, 11937–11947, 1983 ). 16α-OHT wird als mögliche Zwischenstufe in der Biosynthese von Estriol vermutet ( Ryan, Metabolism of C-16-oxygenated steroids by human placenta: the formation of estriol, J. Biol. Chem., 234, 2006–2008, 1959 ; Stevenson, Wright et al., Synthesis of 19-functionalised derivatives of 16a-hydroxy-testosterone: mechanistic studies an oestriol biosynthesis, J. Chem. Soc., Chem. Commun., 1078–1080, 1985 ). 16α-OH-Testosterone (16α-OHT) has been identified as a metabolite of testosterone in chicken, mouse and rat liver as well as in human placental reactions (Harada and Negishi, Mouse liver testosterone 16α-hydroxylase ( Cytochrome P-45016a), J. Biol. Chem., 259, 12285-12290, 1984; Paolini, Pozzetti et al., Developement of basal and induced testosterone hydroxylase activity in the chicken embryo in ovo, Brit. J. Pharmacol., 122, 344-350, 1997 ; Sugiyama, Nagata et al., Theoratical kinetics of sequential metabolism in vitro. Study of the formation of 16 alpha-hydroxyandrostenedione from testosterone by P450 2C11, Drug Metab. Dispos., 22, 584-591, 1994 ; Waxman, Ko et al., Regioselectivity and stereoselectivity of androgen hydroxylations catalyzed by cytochrome P-450 isozymes purified from phenobarbital-induced rat liver, J. Biol. Chem., 258, 11937-11947, 1983 ). 16α-OHT is thought to be a possible intermediate in the biosynthesis of estriol ( Ryan, Metabolism of C-16 oxygenated steroids by human placenta: the formation of estriol, J. Biol. Chem., 234, 2006-2008, 1959 ; Stevenson, Wright et al., Synthesis of 19-functionalized derivatives of 16α-hydroxy-testosterone: mechanistic studies on oestriol biosynthesis, J. Chem. Soc., Chem. Commun., 1078-1080, 1985 ).

Eine chemische Synthese von 16α-OHT wurde von Numazama und Osawa ausgehend von Dehydroepiandrosteron (DHEA) mit einer Gesamtausbeute von 3–4% beschrieben ( Numazawa and Osawa, Improved synthesis of 16α-hydroxylated androgens: intermediates of estriol formation in pregnancy, Steroids, 32, 519–527, 1978 ). Der Metabolit tritt auch als Nebenprodukt in Umsetzungen mit Actinobakterien auf ( Donova, Transformation of steroids by actinobacteria: a review, Appl. Biochem. Microbiol., 43, 1–14, 2007 ).A chemical synthesis of 16α-OHT was reported by Numazama and Osawa starting from dehydroepiandrosterone (DHEA) with an overall yield of 3-4% ( Numazawa and Osawa, Improved synthesis of 16α-hydroxylated androgens: intermediates of estriol formation in pregnancy, Steroids, 32, 519-527, 1978 ). The metabolite also occurs as a by-product in reactions with actinobacteria ( Donova, Transformation of steroids by actinobacteria: a review, Appl. Biochem. Microbiol., 43, 1-14, 2007 ).

Ein mikrobielles Verfahren zur Herstellung von 16α-OHT ist im Stand der Technik nicht beschrieben.A microbial process for the preparation of 16α-OHT is not described in the prior art.

Daher betrifft die Erfindung ein Verfahren zur Herstellung von einem Hydroxid eines 3-on 4-en Steroids gemäß Formel I

Figure DE102015120587A1_0001
wobei jeweils unabhängig voneinander
R1 H oder CH3 ist,
R2 H oder CH3 ist,
R3 H, OH oder eine Ketogruppe (=O) ist,
R4 H, CO-CH3, CO-CH2OH,
R5 H, OH,
oder R4 und R5 bilden eine Ketogruppe (=O);
insbesondere solche wie 16α,17β-Dihydroxy-4-androsten-3-on, 16α-Hydroxy-4-androsten-3,17-dion, 16α-Hydroxy-4-pregnen-3,20-dion, 11β,16α,17,21-Tetrahydroxypregn-4-en-3,20-dion, 16α,17α,21-Trihydroxy-pregnen-3,11,20-trion, 11β,16α,21-Trihydroxy-pregn-4-en-3,20-dion,
mittels einer Peroxygenase aus einem 3-on 4-en Steroid gemäß Formel II
Figure DE102015120587A1_0002
wobei jeweils unabhängig voneinander
R1 H oder CH3 ist,
R2 H oder CH3 ist,
R3 H, OH oder eine Ketogruppe (=O) ist,
R4 H, CO-CH3, CO-CH2OH,
R5 H, OH,
oder R4 und R5 bilden eine Ketogruppe (=O);
insbesondere solche umfassend 4-Androsten-3,17-dion, 17β-Hydroxy-4-androsten-3-on (INN: Testosteron), 4-Pregnen-3,20-dion (INN: Progesteron), 11β,17,21-Trihydroxypregn-4-en-3,20-dion (INN: Hydrocortison), 17α,21-Dihydroxy-pregnen-3,11,20-trion (INN: Cortison), 11β,21-Dihydroxy-pregn-4-en-3,20-dion (INN: Corticosteron).Therefore, the invention relates to a process for the preparation of a hydroxide of a 3-one 4-ene steroid according to formula I.
Figure DE102015120587A1_0001
each being independent of each other
R1 is H or CH3,
R2 is H or CH3,
R 3 is H, OH or a keto group (= O),
R 4 is H, CO-CH 3, CO-CH 2 OH,
R5 H, OH,
or R4 and R5 form a keto group (= O);
especially those such as 16α, 17β-dihydroxy-4-androsten-3-one, 16α-hydroxy-4-androstene-3,17-dione, 16α-hydroxy-4-pregnene-3,20-dione, 11β, 16α, 17 , 21-Tetrahydroxypregn-4-ene-3,20-dione, 16α, 17α, 21-trihydroxy-pregnene-3,11,20-trione, 11β, 16α, 21-trihydroxy-pregn-4-ene-3,20 -dione
by means of a peroxygenase from a 3-one 4-ene steroid according to formula II
Figure DE102015120587A1_0002
each being independent of each other
R1 is H or CH3,
R2 is H or CH3,
R 3 is H, OH or a keto group (= O),
R 4 is H, CO-CH 3, CO-CH 2 OH,
R5 H, OH,
or R4 and R5 form a keto group (= O);
in particular those comprising 4-androstene-3,17-dione, 17β-hydroxy-4-androsten-3-one (INN: testosterone), 4-pregnene-3,20-dione (INN: progesterone), 11β, 17,21 -Trihydroxypregn-4-ene-3,20-dione (INN: hydrocortisone), 17α, 21-dihydroxy-pregnene-3,11,20-trione (INN: cortisone), 11β, 21-dihydroxy-pregn-4-ene -3,20-dione (INN: corticosterone).

Besonders bevorzugt ist jedoch für Formel I 16α-OH-Testosteron (16α,17β-Hydoxy-4-androsten-3-on) und für Formel II Testosteron (17β-Hydoxy-4-androsten-3-on).However, particularly preferred for formula I is 16α-OH-testosterone (16α, 17β-hydroxy-4-androsten-3-one) and for formula II testosterone (17β-hydroxy-4-androsten-3-one).

Bei dem erfindungsgemäßen Verfahren wird für die bevorzugte Ausführungsform Testosteron gemäß Formel II neben dem 16α-OH-Testosteron ein 4-Epoxi-Testosteron erhalten.

Figure DE102015120587A1_0003
Abbildung 1: Peroxygenase-katalysierte Oxygenierung von Testosteron gemäß Formel II zu einem 16α-Hydroxi-Testosteron gemäß Formel I (rechts unten) und 4-Epoxid-Testosteron gemäß Formel III (links unten). In the method according to the invention testosterone according to formula II is obtained in addition to the 16α-OH-testosterone a 4-epoxy testosterone for the preferred embodiment.
Figure DE102015120587A1_0003
Figure 1: Peroxygenase-catalyzed oxygenation of testosterone according to formula II to a 16α-hydroxy testosterone according to formula I (bottom right) and 4-epoxide testosterone according to formula III (bottom left).

Entsprechend werden aus Formel II die Epoxide der Formel III gemäß dem erfindunsggemäßen Verfahren erhalten:

Figure DE102015120587A1_0004
wobei jeweils unabhängig voneinander
R1 H oder CH3 ist,
R2 H oder CH3 ist,
R3 H, OH oder eine Ketogruppe (=O) ist,
R4 H, CO-CH3, CO-CH2OH,
R5 H, OH,
oder R4 und R5 bilden eine Ketogruppe (=O);
insbesondere solche wie 4,5-Epoxi-androstan-3,17-dion, 4,5-Epoxi-17β-Hydroxy-androstan-3-on, 4,5-Epoxi-pregnan-3,20-dion, 4,5-Epoxi-11β,17,21-Trihydroxypregnan-3,20-dion, 4,5-Epoxi-17α,21-Dihydroxy-pregnan-3,11,20-trion, 4,5-Epoxi-11β,21-Dihydroxy-pregnan-3,20-dion.Accordingly, the epoxides of the formula III are obtained from formula II according to the process according to the invention:
Figure DE102015120587A1_0004
each being independent of each other
R1 is H or CH3,
R2 is H or CH3,
R 3 is H, OH or a keto group (= O),
R 4 is H, CO-CH 3, CO-CH 2 OH,
R5 H, OH,
or R4 and R5 form a keto group (= O);
in particular, such as 4,5-epoxy-androstane-3,17-dione, 4,5-epoxy-17β-hydroxy-androstane-3-one, 4,5-epoxy-pregnane-3,20-dione, 4,5 Epoxi-11β, 17,21-trihydroxypregnan-3,20-dione, 4,5-epoxi-17α, 21-dihydroxy-pregnane-3,11,20-trione, 4,5-epoxy-11β, 21-dihydroxy pregnane-3,20-dione.

Die Herstellung der Verbindungen gemäß Formel I und Formel III gelingt vorteilhaft mit geringem verfahrenstechnischen und apparativem Aufwand sowie unter Einsatz kostengünstiger Cosubstrate. Die Umsetzung von Testosteron oder einer Formel II erfolgt bei Raumtemperatur, unter Normaldruck, im wässrigen Milieu sowie ohne erhöhte Anforderungen an sterile bzw. semisterile Reaktionsbedingungen in einem vorteilhaften einstufigen Verfahren, auch im Eintopfverfahren. Die Reaktionsprodukte sind mit geringem Aufwand zu isolieren und zu reinigen, vorzugsweise mittels Chromatographie.The preparation of the compounds of formula I and formula III is advantageously achieved with low process engineering and equipment expense and using cost cosubstrates. The reaction of testosterone or a formula II is carried out at room temperature, under atmospheric pressure, in an aqueous medium and without increased requirements for sterile or semisterile reaction conditions in an advantageous one-step process, also in a one-pot process. The reaction products can be isolated and purified with little effort, preferably by chromatography.

Zur biokatalytischen Oxygenierung gemäß der vorliegenden Erfindung wird eine Peroxygenase gemäß EC 1.11.2.1 eingesetzt, welche die Reaktion „RH + H2O2 (Peroxid) <=> ROH + H2O” katalysiert.For biocatalytic oxygenation according to the present invention, a peroxygenase according to EC 1.11.2.1 is used, which catalyzes the reaction "RH + H 2 O 2 (peroxide) <=> ROH + H 2 O".

Das erfindungsmäßige Verfahren kann nach dem Fachmann geläufigen Standardprozeduren für enzymkatalysierte Reaktionen durchgeführt werden, indem das Substrat (Formel II), das Oxidationsmittel (Wasserstoff)Peroxid, die Peroxygenase und gegebenenfalls Hilfsstoffe in geeigneter Weise in einem wasserhaltigen Lösungsmittel vereint werden und das Reaktionsgemisch nach Ablauf der Reaktion nach Standardmethoden aufgearbeitet wird.The process according to the invention can be carried out according to standard procedures familiar to the person skilled in the art for enzyme-catalyzed reactions by suitably combining the substrate (formula II), the oxidant (hydrogen) peroxide, the peroxygenase and optionally auxiliaries in an aqueous solvent and the reaction mixture after the end of the reaction Reaction is processed according to standard methods.

Erfindungsgemäß können unspezifische Peroxygenasen (EC 1.11.2.1) verwendet werden, hierbei handelt es sich beispielsweise um extrazelluläre Häm-Thiolat-Proteine, die Eigenschaften von klassischen Peroxidasen und Monooxygenasen in sich vereinen und daher als Hybridenzyme verstanden werden können ( Hofrichter and Ullrich, Heme-thiolate haloperoxidases: versatile biocatalysts with biotechnological and environmental significance, Appl. Microbiol. Biotechnol., 71, 276–288, 2006 ; Hofrichter and Ullrich, Oxidations catalyzed by fungal peroxygenases, Curr. Opin. Chem. biol., 19, 116–125, 2014 ; Hofrichter, Ullrich et al., New and classic families of secreted fungal heme peroxidases, Appl. Microbiol. Biotechnol., 87, 871–897, 2010 ). Eine unspezifische Peroxygenase, welche vormals als Haloperoxidase oder als aromatische Peroxygenase (APO) bezeichnet wurde, wurde erstmals aus dem Basidiomycet Agrocybe aegerita ( Südlicher Ackerling) isoliert (Ullrich, Nüske et al., Novel haloperoxidase from the agaric basidiomycete agrocybe aegerita oxidizes aryl alcohols and aldehydes, Appl. Environ. Microbiol., 70, 4575–4581, 2004 ). Ähnliche Enzymaktivitäten wurden unter anderem in den Kulturmedien der Basidiomyceten Coprinellus radians ( Anh, Ullrich et al., The coprphilous mushroom Coprinus radians secreted a haloperoxidase that catalyzes aromatic peroxgenation, Appl. Environ. Microbiol., 73, 5477–5485, 2007 ) und Marasmius rotula ( Gröbe, Ullrich et al., High-yield production of aromatic peroxygenase by the agaric fungus Marasmius rotula, AMB Express, 1, 31, 2011 ) nachgewiesen. Bei den unspezifischen Peroxygenasen handelt es sich um stark glycosylierte Proteine mit einem Molekulargewicht von 32 bis 46 kDa und isoelektrischen Punkten zwischen pI 3.5 und pI 6.0. In Abhängigkeit vom Enzym, Substrat und den Reaktionsbedingungen katalysieren die unspezifischen Peroxygenasen eine Vielzahl von Reaktionen wie Halogenierungen, Oxygenierungen und Oxidationen durch Ein-Elektronen-Abstraktion. Die Enzyme benötigen keine Cofaktoren wie NADP(H), sondern lediglich (Wasserstoff)Peroxid, wobei ein Sauerstoffatom des Peroxids auf das Substrat übertragen wird. Mechanistisch kann die Katalyse als Analogie zum H2O2-shunt pathway der Cytochrom P450-Monoxygenase angesehen werden.Unspecific peroxygenases (EC 1.11.2.1) can be used according to the invention; these are, for example, extracellular heme thiolate proteins which combine the properties of classical peroxidases and monooxygenases and can therefore be understood as hybrid enzymes ( Hofrichter and Ullrich, Heme-thiolate haloperoxidases: versatile biocatalysts with biotechnological and environmental significance, Appl. Microbiol. Biotechnol., 71, 276-288, 2006 ; Hofrichter and Ullrich, Oxidations catalyzed by fungal peroxygenases, Curr. Opin. Chem. Biol., 19, 116-125, 2014 ; Hofrichter, Ullrich et al., New and classic families of secreted fungal heme peroxidases, Appl. Microbiol. Biotechnol., 87, 871-897, 2010 ). A non-specific peroxygenase, formerly known as haloperoxidase or aromatic peroxygenase (APO), was first extracted from Basidiomycet Agrocybe aegerita ( Southern fieldling) (Ullrich, Nüske et al., Novel haloperoxidase from the agaric basidiomycete agrocyte aegerita oxidizes aryl alcohols and aldehydes, Appl. Environ. Microbiol., 70, 4575-4581, 2004 ). Similar enzyme activities have been reported, inter alia, in the culture media of the Basidiomycetes Coprinellus radians ( Anh, Ullrich et al., The coprphilic mushroom Coprinus radians secreted a haloperoxidase that catalyzes aromatic peroxgenation, Appl. Environ. Microbiol., 73, 5477-5485, 2007 ) and Marasmius rotula ( Gröbe, Ullrich et al., High-yield production of aromatic peroxygenase by the agaric fungus Marasmius rotula, AMB Express, 1, 31, 2011 ). The non-specific peroxygenases are highly glycosylated proteins with a molecular weight of 32 to 46 kDa and isoelectric points between pI 3.5 and pI 6.0. Depending on the enzyme, substrate, and reaction conditions, the nonspecific peroxygenases catalyze a variety of reactions such as halogenation, oxygenation, and oxidation by one-electron abstraction. The enzymes do not require cofactors such as NADP (H), but only (hydrogen) peroxide, whereby an oxygen atom of the peroxide is transferred to the substrate. Mechanistically, catalysis can be considered as analogous to the H 2 O 2 shunt pathway of cytochrome P450 monoxygenase.

In einer bevorzugten Ausführungsform der Erfindung ist die Peroxygenase aus dem Ascomyceten Chaetomium globosum Isolat ausgewählt. Die Peroxygenase kann in fester Form, zum Beispiel lyophilisiert, in gelöster Form zum Beispiel in wässriger Lösung, oder in immobilisierter Form, zum Beispiel auf einem Träger oder in einem Polyvinylalkohol-Polyethylenglykol-Gel verkapselt ( Poraj-Kobielska, Peter et al., Immobilization of unspecific peroxygenases (EC 1.11.2.1) in PVA/PEG gel and hollow fiber modules, Biochem. Eng. J., 98, 144–150, 2015 ) eingesetzt werden. Die Enzymmenge wird in Abhängigkeit der Geschwindigkeit der durchzuführenden Reaktion bzw. von der angestrebten Reaktionszeit, von der eingesetzten Form und von anderen Parametern wie der Temperatur gewählt und kann durch Vorversuche leicht ermittelt werden. Im Allgemeinen wird pro mmol Substrat das Enzym in einer Menge von ca. 100 U bis ca. 10.000 U eingesetzt. Gemäß Definition bedeutet im Fall der Peroxygenasen 1 U (unit) die Enzymmenge, die 1 μmol Veratrylalkohol (3,4-Dimethoxybenzylalkohol) in 1 Minute bei 23°C und pH 7 zu 3,4-Diemthoxybenzylaldehyd oxidiert ( Ullrich, Nüskeet al., Novel haloperoxidase from the agaric basidiomycete agrocybe aegerita oxidizes aryl alcohols and aldehydes, Appl. Environ. Microbiol., 70, 4575–4581, 2004 ).In a preferred embodiment of the invention, the peroxygenase is selected from the Ascomycete Chaetomium globosum isolate. The peroxygenase can be encapsulated in solid form, for example lyophilized, in dissolved form, for example in aqueous solution, or in immobilized form, for example on a support or in a polyvinyl alcohol-polyethylene glycol gel ( Poraj-Kobielska, Peter et al., Immobilization of unspecific peroxygenases (EC 1.11.2.1) in PVA / PEG gel and hollow fiber modules, Biochem. Closely. J., 98, 144-150, 2015 ) are used. The amount of enzyme is chosen as a function of the speed of the reaction to be carried out or of the desired reaction time, the shape used and other parameters such as the temperature and can be easily determined by preliminary experiments. In general, the enzyme is used in an amount of about 100 U to about 10,000 U per mmol of substrate. By definition, in the case of peroxygenases 1 U (unit), the amount of enzyme oxidizing 1 μmol of veratryl alcohol (3,4-dimethoxybenzyl alcohol) to 3,4-dimethoxybenzylaldehyde in 1 minute at 23 ° C and pH 7 ( Ullrich, Nüske et al., Novel haloperoxidase from the agaric basidiomycete agrocyte aegerita oxidizes aryl alcohols and aldehydes, Appl. Environ. Microbiol., 70, 4575-4581, 2004 ).

Als Oxidationsmittel werden erfindungsgemäß vorzugsweise Wasserstoffperoxid (H2O2), organische Hydroperoxide (R-OOH, z. B. tert-Butylhydroperoxid) oder Peroxycarbonsäuren (R-CO-OOH, z. B. meta-Chlorperbenzoesäure) eingesetzt. Das Wasserstoffperoxid kann in Form einer Lösung eingesetzt werden, zum Beispiel in Form einer wässrigen Lösung mit einem Gehalt von 0,1% (Prozentangaben sind Gewichtsprozent, wenn nicht anders angegeben) bis ca. 35% Wasserstoffperoxid, wobei die zweckmäßige Konzentration wie üblich unter anderem von der Ansatzgröße abhängt und bei größeren Ansätzen eher eine konzentriertere Lösung, bei kleineren Ansätzen eher eine verdünntere Lösung eingesetzt wird. Das Oxidationsmittel kann direkt dem Ansatz oder kontinuierlich mittels Spritzenpumpe zugegeben werden. Alternativ ist die in situ Erzeugung des Oxidationsmittels durch die Zusetzung von H2O2-generierenden Enzymen, insbesondere Oxidasen, wie Zucker-Oxidasen und/oder Alkohol-Oxidasen sowie deren Substrate (z. B. Glucose bzw. Benzylalkohol). Wasserstoffperoxid wird im Allgemeinen im Überschuss eingesetzt, zum Beispiel in der ca. 1-fachen bis ca. 100-fachen molaren Menge, bezogen auf das Substrat. Bei der Durchführung des erfindungsgemäßen Verfahrens kann die Wasserstoffperoxidkonzentration im Reaktionsgemisch zum Beispiel ca. 0,1 mM (mMol/l) bis ca. 20 mM liegen. Dies ist anhängig von der Verfahrensdurchführung und kann sich während der Durchführung der Reaktion verändern.As an oxidizing agent (for example meta-chloroperbenzoic acid R-CO-OOH, z.) According to the invention preferably hydrogen peroxide (H 2 O 2), organic hydroperoxides (R-OOH, z. B. tert-butyl hydroperoxide), or peroxycarboxylic acids used. The hydrogen peroxide may be used in the form of a solution, for example in the form of an aqueous solution containing 0.1% (percentages are by weight unless otherwise specified) to about 35% hydrogen peroxide, with the appropriate concentration as usual among others depends on the size of the batch and, in the case of larger batches, tends to be a more concentrated solution, with smaller batches a more dilute solution. The oxidizer may be added directly to the batch or continuously via a syringe pump. Alternatively, the in situ generation of the oxidizing agent is due to the addition of H2O2-generating enzymes, in particular oxidases, such as sugar oxidases and / or alcohol oxidases and their substrates (for example glucose or benzyl alcohol). Hydrogen peroxide is generally used in excess, for example in about 1 to about 100 times. times the molar amount, based on the substrate. When carrying out the process according to the invention, the hydrogen peroxide concentration in the reaction mixture can be, for example, about 0.1 mM (mmol / l) to about 20 mM. This is dependent on the procedure and may change during the performance of the reaction.

Das wasserhaltige oder wässrige Lösungsmittel, in dem das Verfahren durchgeführt wird, kann Wasser sein oder ein Gemisch aus Wasser und einem oder mehreren organischen Lösungsmitteln, durch die als Cosolventien die Löslichkeit des Substrats erhöht wird und die uneingeschränkt mit Wasser mischbar sein können oder nicht uneingeschränkt mit Wasser mischbar sein können. Die geeignete Menge bzw. Konzentration an zugesetztem organischen Lösungsmittel hängt zum Beispiel von der Löslichkeit des Substrats ab und kann sich während der Durchführung der Reaktion auch verändern, zum Beispiel durch Zudosieren von Wasserstoffperoxid und beträgt im Allgemeinen 0,5 Volumen-% bis ca. 50 Volumen-% bezogen auf das gesamte wasserhaltige Lösungsmittel. Geeignete organische Lösungsmittel sind zum Beispiel Alkohole, etwa (C1-C4)-Alkanole wie Methanol, Ethanol, tert-Butanol, Ketone, etwa (C3-C5)-Alkanone wie Aceton, Nitrile wie Acetonitril, chlorierte Kohlenwasserstoffe wie Dichlormethan, Amide wie Dimethylformamid (DMF), Sulfoxide wie Dimethylsulfoxid (DMSO), Ether wie 1,2-Dimethoxyethan (DME), Tetrahydrofuran (THF), Dioxan und Ester wie Essigsäureethylester. In der Ausführungsform der Erfindung wird das Substrat in einer geeigneten Menge Acetonitril oder Aceton oder einer Mischung der organischen Lösungsmittel und einem geringen Anteil Wasser gelöst und die Lösung mit Wasser, Peroxygenase, Wasserstoffperoxid und gegebenenfalls mit Hilfsstoffen versetzt.The hydrous or aqueous solvent in which the process is carried out may be water or a mixture of water and one or more organic solvents which, as cosolvents, increase the solubility of the substrate and which may or may not be fully miscible with water Water can be mixed. The appropriate amount of organic solvent added depends, for example, on the solubility of the substrate and may also change as the reaction is conducted, for example by metering in hydrogen peroxide, and is generally from 0.5% to about 50% by volume Volume% based on the total water-containing solvent. Suitable organic solvents are, for example, alcohols such as (C 1 -C 4) -alkanols such as methanol, ethanol, tert-butanol, ketones, such as (C 3 -C 5) -alkanones such as acetone, nitriles such as acetonitrile, chlorinated hydrocarbons such as dichloromethane, amides such as dimethylformamide (DMF), sulfoxides such as dimethyl sulfoxide (DMSO), ethers such as 1,2-dimethoxyethane (DME), tetrahydrofuran (THF), dioxane and esters such as ethyl acetate. In the embodiment of the invention, the substrate is dissolved in a suitable amount of acetonitrile or acetone or a mixture of the organic solvents and a small amount of water, and the solution is treated with water, peroxygenase, hydrogen peroxide and optionally with excipients.

Das erfindungsmäßige Verfahren wird bei einem pH-Wert von ca. 4 bis 9, insbesondere bei einem pH-Wert von ca. 7 durchgeführt. Der pH-Wert des Reaktionsgemisches, der durch saure oder basische Gruppen im Substrat und dem Produkt beeinflusst wird und sich im Laufe der Reaktion ändern kann, kann durch Zudosieren von Säuren, zum Beispiel Salzsäure, oder Basen, zum Beispiel Natronlauge, im gewünschten Bereich gehalten werden oder durch Zugabe von Puffersubstanzen bzw. Pufferlösungen eingestellt werden. Säuren, Basen, Puffersubstanzen und Pufferlösungen sind Beispiele für Hilfsstoffe, die bei der Durchführung des erfindungsgemäßen Verfahrens zugegeben werden können. In der Ausführungsform wird die Reaktion in Gegenwart eines Puffers durchgeführt, also unter Zugabe von Puffersubstanzen oder Pufferlösungen. Zur Pufferung des Reaktionsgemisches können übliche Puffer verwendet werden, zum Beispiel Phosphatpuffer, Tris-Puffer (Tris-(hydroxymethyl)-methylamin-Puffer) oder der Phosphat und Citronensäure enthaltende McIIvaine-Puffer. Die Konzentration an zugesetzter Puffersubstanz im Reaktionsgemisch kann zum Beispiel bei ca. 0,01 M bis ca. 1 M, in der Ausführungsform bei ca. 0,05 M liegen.The process according to the invention is carried out at a pH of about 4 to 9, in particular at a pH of about 7. The pH of the reaction mixture, which is influenced by acidic or basic groups in the substrate and the product and may change in the course of the reaction, can be kept in the desired range by metering in acids, for example hydrochloric acid, or bases, for example sodium hydroxide solution be adjusted or by addition of buffer substances or buffer solutions. Acids, bases, buffer substances and buffer solutions are examples of auxiliaries which can be added when carrying out the process according to the invention. In the embodiment, the reaction is carried out in the presence of a buffer, ie with the addition of buffer substances or buffer solutions. Conventional buffers can be used to buffer the reaction mixture, for example phosphate buffer, Tris buffer (tris- (hydroxymethyl) -methylamine buffer) or the phosphate and citric acid-containing McIIvaine buffer. The concentration of added buffer substance in the reaction mixture may be, for example, about 0.01 M to about 1 M, in the embodiment about 0.05 M.

Ein weiteres Beispiel für Hilfsstoffe, die bei der Durchführung des erfindungsmäßigen Verfahrens zugegeben werden können, sind Radikalfänger, zum Beispiel Ascorbinsäure, deren Gegenwart bei der Durchführung des Verfahrens einen stabilisierenden Effekt haben kann, insbesondere bei den Verbindungen, die aromatische Gruppen enthalten ( Karich, Kluge et al., Benzene oxygenation and oxidation by the peroxygenase Agrocybe aegerita, AMB Express, 3, 1–8, 2013 ). Die Konzentration an zugesetztem Radikalfänger, beispielsweise Ascorbinsäure, im Reaktionsgemisch kann zum Beispiel bei ca. 0,01 mM bis ca. 100 mM, in der Ausführungsform bei 4 mM liegen. Ein Radikalfänger, beispielsweise Ascorbinsäure, kann zu Beginn der Reaktion dem Reaktionsgemisch zugesetzt werden und kann auch während des Ablaufs der Reaktion sukzessive, zum Beispiel portionsweise oder kontinuierlich, zudosiert werden, zum Beispiel in Form einer wässrigen Lösung.Another example of auxiliaries which may be added in carrying out the process according to the invention are free-radical scavengers, for example ascorbic acid, the presence of which may have a stabilizing effect when carrying out the process, in particular in the compounds which contain aromatic groups ( Karich, Kluge et al., Benzene oxygenation and oxidation by the peroxygenase Agrocybe aegerita, AMB Express, 3, 1-8, 2013 ). The concentration of added radical scavenger, for example ascorbic acid, in the reaction mixture may be, for example, from about 0.01 mM to about 100 mM, in the embodiment at 4 mM. A radical scavenger, for example ascorbic acid, can be added to the reaction mixture at the beginning of the reaction and can also be metered in successively, for example in portions or continuously, during the course of the reaction, for example in the form of an aqueous solution.

Die Reaktionstemperatur bei der Durchführung des erfindungsmäßigen Verfahrens liegt im Allgemeinen bei ca. 10°C bis ca. 70°C, in der Ausführungsform bei ca. 20°C bis ca. 25°C, beispielsweise Raumtemperatur. Die Temperatur kann auch variiert werden und zum Beispiel im Laufe der Reaktion zur Vervollständigung der Umsetzung erhöht werden. Das erfindungsmäßige Verfahren kann auch unter der Behandlung des Reaktionsgemisches mit Ultraschall durchgeführt werden.The reaction temperature in carrying out the process according to the invention is generally from about 10 ° C. to about 70 ° C., in the embodiment at about 20 ° C. to about 25 ° C., for example room temperature. The temperature can also be varied and increased, for example, in the course of the reaction to complete the reaction. The process according to the invention can also be carried out under the treatment of the reaction mixture with ultrasound.

Nachfolgende Beispiele und Figuren dienen zur näheren Erläuterung der Erfindung, ohne jedoch die Erfindung auf diese Beipiele und Figuren zu begrenzen.The following examples and figures serve to illustrate the invention without, however, limiting the invention to these examples and figures.

Beispiele:Examples:

Beispiel 1:Example 1:

Es wurden 100 mg (0,35 mmol) Testosteron in einem 500 ml-Zweihalskolben in 10 ml Aceton, 78 ml Wasser, 2 ml Ascorbinsäure-Lösung (400 mM) und 100 ml 0,1 M Kaliumphosphatpuffer (pH 7) unter Rühren bei Raumtemperatur gelöst. Anschließend wurden 700 U CglUPO (10 ml, 70 U/ml) hinzugegeben. Die Reaktion wurde durch die Zugabe von Wasserstoffperoxid mittels Spritzenpumpe initiiert. Hierfür wurden 28 ml einer 100 mM H2O2-Lösung mit einer Geschwindigkeit von 4 ml/h zudosiert. In regelmäßigen Abständen wurden 50 μl Probe entnommen, mit 10 μl einer 10 mM Natriumazidlösung und 50 μl Acetonitril versetzt und mittel HPLC und DC untersucht. Für die DC-Analytik wurden fluoreszenzmarkierte Kieselgelplatten (TLC Silica gel 60 F254, Merck) verwendet. Als Laufmittel diente Essigester/n-Hexan 9:1 und als Detektionsmittel wurde MOPS-Reagenz eingesetzt. Nach der Reaktionszeit von 7 Stunden wurde die Zufuhr von Wasserstoffperoxid eingestellt und das Reaktionsgemisch mehrfach mit Essigsäureethylester extrahiert. Die vereinten organischen Phasen wurden mit Natriumsulfat getrocknet. Die Lösung wurde am Rotationsverdampfer eingeengt und die Substanz auf wenig Kieselgel aufgebracht. Anschließend erfolgte die Reinigung der Produkte mittels Chromatographie an Kieselgel mit Essigester/n-Hexan (9:1).100 mg (0.35 mmol) of testosterone in a 500 ml two-necked flask in 10 ml of acetone, 78 ml of water, 2 ml of ascorbic acid solution (400 mM) and 100 ml of 0.1 M potassium phosphate buffer (pH 7) were added with stirring Room temperature dissolved. Then, 700 U of CglUPO (10 ml, 70 U / ml) was added. The Reaction was initiated by the addition of hydrogen peroxide by means of a syringe pump. For this purpose, 28 ml of a 100 mM H2O2 solution were added at a rate of 4 ml / h. At regular intervals, 50 .mu.l of sample were taken, mixed with 10 .mu.l of a 10 mM sodium azide solution and 50 .mu.l of acetonitrile and analyzed by HPLC and TLC. Fluorescent-labeled silica gel plates (TLC Silica gel 60 F254, Merck) were used for the TLC analysis. The mobile phase used was ethyl acetate / n-hexane 9: 1 and the detection agent used was MOPS reagent. After the reaction time of 7 hours, the supply of hydrogen peroxide was adjusted and the reaction mixture extracted several times with ethyl acetate. The combined organic phases were dried with sodium sulfate. The solution was concentrated on a rotary evaporator and the substance was applied to a little silica gel. Subsequently, the products were purified by chromatography on silica gel with ethyl acetate / n-hexane (9: 1).

4,5-Epoxy-17β-hydroxy-5β-androstan-3-on4,5-epoxy-17β-hydroxy-5β-androstan-3-one

  • Ausbeute: 65 mg (61,1%)Yield: 65 mg (61.1%)
  • Reinheit: 96,3% (UV, 245 nm)Purity: 96.3% (UV, 245 nm)
  • DC (EE/n-Hexan): Rf = 0,67TLC (EA / n-hexane): Rf = 0.67
  • MS (ESI+): m/z (%) = 304,2 (100)MS (ESI + ): m / z (%) = 304.2 (100)
  • NMR: 1H-NMR (400 MHz, CD3OD): δ = 0.79 (s, 3H, H-18), 1.02–1.21 (m, 5H, H-9α, H-7α, H-7β, H-12α, H-14α,), 1.18 (s, 3H, H-19), 1.35 (qd, 2J = 12.1 Hz, 3J = 12.1 Hz (2x), 5.8 Hz, 1H, H-15β), 1.45–1.57 (m, 3H, H-11β, H-16β, H-11α), 1.60–1.70 (m, 3H, H-15α, H-8β, H-1α), 1.73–1.82 (m, 1H, H-1β), 1.85–1.92 (m, 2H, H-12β, H-6α), 1.96–2.06 (m, 1H, 1H, H-16a), 2.14–2.28 (m, 3H, H-6β, H-2β, H-2α), 2.96 (s, 1H, H-4α) 3.61 (t, 3J = 8.6 Hz, 1H, H-17α) 13C-NMR (100 MHz, CD3OD): δ = 10.23 (C-18), 18.01 (C-19), 20.84 (C-11), 22.89 (C-15), 25.85 (C-1), 29.18 (C-16), 29.36 (C-6), 29.62 (C-7), 32.00 (C-2), 35.04 (C-8), 36.12 (C-12), 36.90 (C-10), 42.77 (C-13), 46.29 (C-9), 50.31 (C-14), 62.13 (C-4), 69.71 (C-5), 80.85 (C-5), 206.83 (C-3)NMR: 1 H-NMR (400 MHz, CD 3 OD): δ = 0.79 (s, 3H, H-18), 1.02-1.21 (m, 5H, H-9α, H-7α, H-7β, H- 12α, H-14α,), 1.18 (s, 3H, H-19), 1.35 (qd, 2 J = 12.1 Hz, 3 J = 12.1 Hz (2x), 5.8 Hz, 1H, H-15β), 1.45- 1.57 (m, 3H, H-11β, H-16β, H-11α), 1.60-1.70 (m, 3H, H-15α, H-8β, H-1α), 1.73-1.82 (m, 1H, H) 1β), 1.85-1.92 (m, 2H, H-12β, H-6α), 1.96-2.06 (m, 1H, 1H, H-16a), 2.14-2.28 (m, 3H, H-6β, H-2β , H-2α), 2.96 (s, 1H, H-4α) 3.61 (t, 3 J = 8.6 Hz, 1H, H-17α) 13 C-NMR (100 MHz, CD 3 OD): δ = 10.23 (C -18), 18.01 (C-19), 20.84 (C-11), 22.89 (C-15), 25.85 (C-1), 29.18 (C-16), 29.36 (C-6), 29.62 (C-1) 7), 32.00 (C-2), 35.04 (C-8), 36.12 (C-12), 36.90 (C-10), 42.77 (C-13), 46.29 (C-9), 50.31 (C-14 ), 62.13 (C-4), 69.71 (C-5), 80.85 (C-5), 206.83 (C-3)

16α,17β-Dihydroxyandrost-4-en-3-on (16α-Hydroxytestosteron, 16α-OHT)16α, 17β-Dihydroxyandrost-4-en-3-one (16α-hydroxytestosterone, 16α-OHT)

  • Ausbeute: 7 mg (6,6%)Yield: 7 mg (6.6%)
  • Reinheit: 93,7% (UV, 245 nm)Purity: 93.7% (UV, 245 nm)
  • DC (EE/n-Hexan): Rf = 0,11TLC (EE / n-hexane): R f = 0.11
  • MS (ESI+): m/z (%) = 304,2 (100)MS (ESI + ): m / z (%) = 304.2 (100)
  • NMR: 1H-NMR (400 MHz, CD3OD): δ = 0.71 (s, 3H, H-18), 0.87–1.11 (m, 3H, H-9α, H-7α, H-12α), 1.14 (s, 3H, H-19), 1.19–1.26 (m, 2H, H-14α, H-11β), 1.32–1.44 (m, 3H, H-15β, H-11α, H-8β), 1.50–1.80 (m, 4H, H-1α, H-15α, H-12β, H-7β), 2.11 (ddd, 2J = 13.5 Hz, 3J = 5.3 Hz, 3.1 Hz, 1H, H-1β), 3.29 (d, 3J = 5.8 Hz, 1H, H17), 3.92 (ddd, 2J = 9.5 Hz, 3J = 5.9 Hz, 2.1 Hz, 1H, H-16β), 5.61 (s, 1H, H-4) 13C-NMR (100 MHz, CD3OD): δ = 11.32 (C-18), 16.29 (C-19), 19.93 (C-11), 31.43 (C-7), 32.43 (C-6), 32.29 (C-2), 33.69 (C-15), 35.00 (C-8), 35.30 (C-1), 36.33 (C-12), 38.64 (C-10), 43.16 (C-13), 48.12 (C-14), 54.03 (C-9), 77.22 (C-16), 88.99 (C-17), 122.79 (C-4), 173.64 (C-5), 200.93 (C-3)NMR: 1 H-NMR (400 MHz, CD 3 OD): δ = 0.71 (s, 3H, H-18), 0.87-1.11 (m, 3H, H-9α, H-7α, H-12α), 1.14 (s, 3H, H-19), 1.19-1.26 (m, 2H, H-14α, H-11β), 1.32-1.44 (m, 3H, H-15β, H-11α, H-8β), 1.50- 1.80 (m, 4H, H-1α, H-15α, H-12β, H-7β), 2.11 (ddd, 2 J = 13.5 Hz, 3 J = 5.3 Hz, 3.1 Hz, 1H, H-1β), 3.29 (d, 3 J = 5.8 Hz, 1H, H17), 3.92 (ddd, 2 J = 9.5 Hz, 3 J = 5.9 Hz, 2.1 Hz, 1H, H-16β), 5.61 (s, 1H, H-4) 13 C-NMR (100 MHz, CD 3 OD): δ = 11.32 (C-18), 16.29 (C-19), 19.93 (C-11), 31.43 (C-7), 32.43 (C-6), 32.29 (C-2), 33.69 (C-15), 35.00 (C-8), 35.30 (C-1), 36.33 (C-12), 38.64 (C-10), 43.16 (C-13), 48.12 (C-14), 54.03 (C-9), 77.22 (C-16), 88.99 (C-17), 122.79 (C-4), 173.64 (C-5), 200.93 (C-3)

ZITATE ENTHALTEN IN DER BESCHREIBUNG QUOTES INCLUDE IN THE DESCRIPTION

Diese Liste der vom Anmelder aufgeführten Dokumente wurde automatisiert erzeugt und ist ausschließlich zur besseren Information des Lesers aufgenommen. Die Liste ist nicht Bestandteil der deutschen Patent- bzw. Gebrauchsmusteranmeldung. Das DPMA übernimmt keinerlei Haftung für etwaige Fehler oder Auslassungen.This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

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  • Donova and Egorova, Microbial steroid transformations: current state and prospects, Appl. Microbiol. Biotechnol., 94, 1423–1447, 2012 [0007] Donova and Egorova, Microbial steroid transformations: current state and prospects, Appl. Microbiol. Biotechnol., 94, 1423-1447, 2012 [0007]
  • Janeczko, Dmochowska-Gladysz et al., Biotransformation of steroid compounds by Chaetomium sp. KCH 6651, Steroids, 74, 2009 [0008] Janeczko, Dmochowska-Gladysz et al., Biotransformation of steroid compounds by Chaetomium sp. KCH 6651, Steroids, 74, 2009 [0008]
  • Donova and Egorova, Microbial steroid transformations: current state and prospects, Appl. Microbiol. Biotechnol., 94, 1423–1447, 2012 [0008] Donova and Egorova, Microbial steroid transformations: current state and prospects, Appl. Microbiol. Biotechnol., 94, 1423-1447, 2012 [0008]
  • Huszcza and Dmochowska-Gladysz, Transformations of testosterone and related steroids by Botrytis cinerea, Phytochem., 62, 155–158, 2003 [0009] Huszcza and Dmochowska-Gladysz, Transformations of testosterone and related steroids by Botrytis cinerea, Phytochem., 62, 155-158, 2003 [0009]
  • Kim, Han et al., Steroid 9-hydroxylation during testosterone degradation by resting Rhodococcus equi cells, Arch. Pharm., 340, 209–214, 2007 [0009] Kim, Han et al., Steroid 9-hydroxylation during testosterone degradation by resting Rhodococcus equi cells, Arch. Pharm., 340, 209-214, 2007 [0009]
  • Al-Aboudi, Mohammad et al., Microbial transformation of testosterone by Rhizopus stolofiner and Fusarium lini, Natl. Product Res., 22, 1498–1509, 2008 [0009] Al-Aboudi, Mohammad et al., Microbial transformation of testosterone by Rhizopus stolofiner and Fusarium lini, Natl. Product Res., 22, 1498-1509, 2008 [0009]
  • Yildirim, Kupcu et al., Biotransformation of some steroids by Aspergillus wentii, Z. Naturforsch. C., 65, 688–692, 2010 [0009] Yildirim, Kupcu et al., Biotransformation of some steroids by Aspergillus wentii, Z. Naturforsch. C., 65, 688-692, 2010 [0009]
  • Reese, Biotransformation of terpenes and steroids by fungi, Natural products: essential resources for human survival, 2007 [0009] Reese, Biotransformation of terpenes and steroids by fungi, Natural products: essential resources for human survival, 2007 [0009]
  • Ruijssenaars, Sperling et al., Testosterone 15b-hydroxylation by solvent tolerant Pseudomonas putida S12, J. Biotechnol., 131, 205–208, 2007 [0009] Ruijssenaars, Sperling et al., Testosterone 15b-hydroxylation by solvent tolerant Pseudomonas putida S12, J. Biotechnol., 131, 205-208, 2007 [0009]
  • Lamm, Chen et al., Steroid hydroxylation by Whetzelinia sclerotiorum, Phanerochaete chrysosporium and Mucor plumbeus, Steroids, 72, 713–722, 2007 [0009] Lamm, Chen et al., Steroid hydroxylation by Whetzelinia sclerotiorum, Phanerochaete chrysosporium and Mucor plumbeus, Steroids, 72, 713-722, 2007 [0009]
  • Beilen and Funhoff, Expanding the alkane oxygenase toolbox: new enzymes and application, Curr. Opin. Biotechnol., 16, 308–314, 2005 [0009] Beilen and Funhoff, Expanding the alkane oxygenase toolbox: new enzymes and application, Curr. Opin. Biotechnol., 16, 308-314, 2005 [0009]
  • rlacher and Girhard, Cytochrom P450 monooxygenases: an update an perspectives for synthetic application Trends Biotechnol., 30, 26–36, 2012 [0009] rlacher and Girhard, Cytochrome P450 monooxygenases: an update to perspectives for synthetic application Trends Biotechnol., 30, 26-36, 2012 [0009]
  • Cytochrom P-45016a), J. Biol. Chem., 259, 12285–12290, 1984; Paolini, Pozzetti et al., Developement of basal and induced testosterone hydroxylase activity in the chicken embryo in ovo, Brit. J. Pharmacol., 122, 344–350, 1997 [0010] Cytochrome P-45016a), J. Biol. Chem., 259, 12285-12290, 1984; Paolini, Pozzetti et al., Developement of basal and induced testosterone hydroxylase activity in the chicken embryo in ovo, Brit. J. Pharmacol., 122, 344-350, 1997 [0010]
  • Sugiyama, Nagata et al., Theoratical kinetics of sequential metabolism in vitro. Study of the formation of 16 alpha-hydroxyandrostenedione from testosterone by purified rat P450 2C11, Drug Metab. Dispos., 22, 584–591, 1994 [0010] Sugiyama, Nagata et al., Theoratical kinetics of sequential metabolism in vitro. Study of the formation of 16 alpha-hydroxyandrostenedione from testosterone by P450 2C11, Drug Metab. Dispos., 22, 584-591, 1994 [0010]
  • Waxman, Ko et al., Regioselectivity and stereoselectivity of androgen hydroxylations catalyzed by cytochrom P-450 isozymes purified from phenobarbital-induced rat liver, J. Biol. Chem., 258, 11937–11947, 1983 [0010] Waxman, Ko et al., Regioselectivity and stereoselectivity of androgen hydroxylations catalyzed by cytochrome P-450 isozymes purified from phenobarbital-induced rat liver, J. Biol. Chem., 258, 11937-11947, 1983 [0010]
  • Ryan, Metabolism of C-16-oxygenated steroids by human placenta: the formation of estriol, J. Biol. Chem., 234, 2006–2008, 1959 [0010] Ryan, Metabolism of C-16 oxygenated steroids by human placenta: the formation of estriol, J. Biol. Chem., 234, 2006-2008, 1959 [0010]
  • Stevenson, Wright et al., Synthesis of 19-functionalised derivatives of 16a-hydroxy-testosterone: mechanistic studies an oestriol biosynthesis, J. Chem. Soc., Chem. Commun., 1078–1080, 1985 [0010] Stevenson, Wright et al., Synthesis of 19-functionalized derivatives of 16α-hydroxy-testosterone: mechanistic studies on oestriol biosynthesis, J. Chem. Soc., Chem. Commun., 1078-1080, 1985. [0010]
  • Numazawa and Osawa, Improved synthesis of 16α-hydroxylated androgens: intermediates of estriol formation in pregnancy, Steroids, 32, 519–527, 1978 [0011] Numazawa and Osawa, Improved synthesis of 16α-hydroxylated androgens: intermediates of estriol formation in pregnancy, Steroids, 32, 519-527, 1978 [0011]
  • Donova, Transformation of steroids by actinobacteria: a review, Appl. Biochem. Microbiol., 43, 1–14, 2007 [0011] Donova, Transformation of steroids by actinobacteria: a review, Appl. Biochem. Microbiol., 43, 1-14, 2007 [0011]
  • Hofrichter and Ullrich, Heme-thiolate haloperoxidases: versatile biocatalysts with biotechnological and environmental significance, Appl. Microbiol. Biotechnol., 71, 276–288, 2006 [0020] Hofrichter and Ullrich, Heme-thiolate haloperoxidases: versatile biocatalysts with biotechnological and environmental significance, Appl. Microbiol. Biotechnol., 71, 276-288, 2006 [0020]
  • Hofrichter and Ullrich, Oxidations catalyzed by fungal peroxygenases, Curr. Opin. Chem. biol., 19, 116–125, 2014 [0020] Hofrichter and Ullrich, Oxidations catalyzed by fungal peroxygenases, Curr. Opin. Chem. Biol., 19, 116-125, 2014 [0020]
  • Hofrichter, Ullrich et al., New and classic families of secreted fungal heme peroxidases, Appl. Microbiol. Biotechnol., 87, 871–897, 2010 [0020] Hofrichter, Ullrich et al., New and classic families of secreted fungal heme peroxidases, Appl. Microbiol. Biotechnol., 87, 871-897, 2010 [0020]
  • Südlicher Ackerling) isoliert (Ullrich, Nüske et al., Novel haloperoxidase from the agaric basidiomycete agrocybe aegerita oxidizes aryl alcohols and aldehydes, Appl. Environ. Microbiol., 70, 4575–4581, 2004 [0020] Southern fieldling) (Ullrich, Nüske et al., Novel haloperoxidase from the agaric basidiomycete agrocyte aegerita oxidizes aryl alcohols and aldehydes, Appl. Environ. Microbiol., 70, 4575-4581, 2004 [0020]
  • Anh, Ullrich et al., The coprphilous mushroom Coprinus radians secreted a haloperoxidase that catalyzes aromatic peroxgenation, Appl. Environ. Microbiol., 73, 5477–5485, 2007 [0020] Anh, Ullrich et al., The coprphilic mushroom Coprinus radians secreted a haloperoxidase that catalyzes aromatic peroxgenation, Appl. Environ. Microbiol., 73, 5477-5485, 2007 [0020]
  • Gröbe, Ullrich et al., High-yield production of aromatic peroxygenase by the agaric fungus Marasmius rotula, AMB Express, 1, 31, 2011 [0020] Gröbe, Ullrich et al., High-yield production of aromatic peroxygenase by the agaric fungus Marasmius rotula, AMB Express, 1, 31, 2011 [0020]
  • Poraj-Kobielska, Peter et al., Immobilization of unspecific peroxygenases (EC 1.11.2.1) in PVA/PEG gel and hollow fiber modules, Biochem. Eng. J., 98, 144–150, 2015 [0021] Poraj-Kobielska, Peter et al., Immobilization of unspecific peroxygenases (EC 1.11.2.1) in PVA / PEG gel and hollow fiber modules, Biochem. Closely. J., 98, 144-150, 2015 [0021]
  • Ullrich, Nüskeet al., Novel haloperoxidase from the agaric basidiomycete agrocybe aegerita oxidizes aryl alcohols and aldehydes, Appl. Environ. Microbiol., 70, 4575–4581, 2004 [0021] Ullrich, Nüske et al., Novel haloperoxidase from the agaric basidiomycete agrocyte aegerita oxidizes aryl alcohols and aldehydes, Appl. Environ. Microbiol., 70, 4575-4581, 2004 [0021]
  • Karich, Kluge et al., Benzene oxygenation and oxidation by the peroxygenase Agrocybe aegerita, AMB Express, 3, 1–8, 2013 [0025] Karich, Kluge et al., Benzene oxygenation and oxidation by the peroxygenase Agrocybe aegerita, AMB Express, 3, 1-8, 2013 [0025]

Claims (10)

Verfahren zur Herstellung von einem Hydroxid eines 3-on 4-en Steroids gemäß Formel I
Figure DE102015120587A1_0005
wobei jeweils unabhängig voneinander R1 H oder CH3 ist, R2 H oder CH3 ist, R3 H, OH oder eine Ketogruppe (=O) ist, R4 H, CO-CH3, CO-CH2OH, R5 H, OH, oder R4 und R5 bilden eine Ketogruppe (=O); mittels einer Peroxygenase aus einem 3-on 4-en Steroid gemäß Formel II
Figure DE102015120587A1_0006
wobei jeweils unabhängig voneinander R1 H oder CH3 ist, R2 H oder CH3 ist, R3 H, OH oder eine Ketogruppe (=O) ist, R4 H, CO-CH3, CO-CH2OH, R5 H, OH, oder R4 und R5 bilden eine Ketogruppe (=O);Process for the preparation of a hydroxide of a 3-one 4-ene steroid according to formula I.
Figure DE102015120587A1_0005
wherein each independently of one another R 1 is H or CH 3, R 2 is H or CH 3, R 3 is H, OH or a keto group (= O), R 4 is H, CO-CH 3, CO-CH 2 OH, R 5 is H, OH, or R 4 and R 5 form a keto group (= O); by means of a peroxygenase from a 3-one 4-ene steroid according to formula II
Figure DE102015120587A1_0006
wherein each independently of one another R 1 is H or CH 3, R 2 is H or CH 3, R 3 is H, OH or a keto group (= O), R 4 is H, CO-CH 3, CO-CH 2 OH, R 5 is H, OH, or R 4 and R 5 form a keto group (= O); Verfahren nach Anspruch 1, wobei Formel II ausgewählt ist aus 4-Androsten-3,17-dion, 17β-Hydroxy-4-androsten-3-on (INN: Testosteron), 4-Pregnen-3,20-dion (INN: Progesteron), 11β,17,21-Trihydroxypregn-4-en-3,20-dion (INN: Hydrocortison), 17α,21-Dihydroxy-pregnen-3,11,20-trion (INN: Cortison), 11β,21-Dihydroxy-pregn-4-en-3,20-dion (INN: Corticosteron).The method of claim 1, wherein formula II is selected from 4-androstene-3,17-dione, 17β-hydroxy-4-androsten-3-one (INN: testosterone), 4-pregnene-3,20-dione (INN: Progesterone), 11β, 17,21-trihydroxypregn-4-ene-3,20-dione (INN: hydrocortisone), 17α, 21-dihydroxy-pregnene-3,11,20-trione (INN: cortisone), 11β, 21 -Dihydroxy-pregn-4-ene-3,20-dione (INN: corticosterone). Verfahren nach Anspruch 1 oder 2, wobei Formel I ausgewählt ist aus 16α,17β-Dihydroxy-4-androsten-3-on, 16α-Hydroxy-4-androsten-3,17-dion, 16α-Hydroxy-4-pregnen-3,20-dion, 11β,16α,17,21-Tetrahydroxypregn-4-en-3,20-dion, 16α,17α,21-Trihydroxy-pregnen-3,11,20-trion, 11β,16α,21-Trihydroxy-pregn-4-en-3,20-dion.The method of claim 1 or 2, wherein formula I is selected from 16α, 17β-dihydroxy-4-androsten-3-one, 16α-hydroxy-4-androstene-3,17-dione, 16α-hydroxy-4-pregnene-3 , 20-dione, 11β, 16α, 17,21-tetrahydroxypregn-4-ene-3,20-dione, 16α, 17α, 21-trihydroxy-pregnene-3,11,20-trione, 11β, 16α, 21-trihydroxy pregn-4-ene-3,20-dione. Verfahren nach einem der Ansprüche 1 bis 3, dadurch gekennzeichnet, dass ein 4,5-Epoxid der Formel III erhalten wird:
Figure DE102015120587A1_0007
wobei jeweils unabhängig voneinander R1 H oder CH3 ist, R2 H oder CH3 ist, R3 H, OH oder eine Ketogruppe (=O) ist, R4 H, CO-CH3, CO-CH2OH, R5 H, OH, oder R4 und R5 bilden eine Ketogruppe (=O); insbesondere 4,5-Epoxi-17β-Hydroxy-androstan-3-on, 4,5-Epoxi-pregnan-3,20-dion, 4,5-Epoxi-11β,17,21-Trihydroxypregnan-3,20-dion, 4,5-Epoxi-17α,21-Dihydroxy-pregnan-3,11,20-trion, 4,5-Epoxi-11β,21-Dihydroxy-pregnan-3,20-dion.Method according to one of claims 1 to 3, characterized in that a 4,5-epoxide of the formula III is obtained:
Figure DE102015120587A1_0007
wherein each independently of one another R 1 is H or CH 3, R 2 is H or CH 3, R 3 is H, OH or a keto group (= O), R 4 is H, CO-CH 3, CO-CH 2 OH, R 5 is H, OH, or R 4 and R 5 form a keto group (= O); in particular 4,5-epoxi-17β-hydroxy-androstan-3-one, 4,5-epi-pregnane-3,20-dione, 4,5-epoxy-11β, 17,21-trihydroxypregnan-3,20-dione , 4,5-epoxy-17α, 21-dihydroxy-pregnane-3,11,20-trione, 4,5-epoxy-11β, 21-dihydroxy-pregnane-3,20-dione. Verfahren nach einem der Ansprüche 1 bis 4, dadurch gekennzeichnet, dass ein Oxidationsmittel, insbesondere Wasserstoffperoxid, organische Hydroperoxide oder Peroxycarbonsäuren zugesetzt wird.Method according to one of claims 1 to 4, characterized in that an oxidizing agent, in particular hydrogen peroxide, organic hydroperoxides or peroxycarboxylic acids is added. Verfahren nach einem der Ansprüche 1 bis 5, dadurch gekennzeichnet, dass eine Peroxygenase aus dem Ascomyceten Chaetomium globosum eingesetzt wird.Method according to one of claims 1 to 5, characterized in that a peroxygenase from the Ascomyceten Chaetomium globosum is used. Verfahren nach einem der Ansprüche 1 bis 6, dadurch gekennzeichnet, dass der pH-Wert von 4 bis 9 beträgt, inbesondere in Form eines Puffers.Method according to one of claims 1 to 6, characterized in that the pH of 4 to 9, in particular in the form of a buffer. Verfahren nach einem der Ansprüche 1 bis 7, dadurch gekennzeichnet, dass das Verfahren in einem wässrigen oder wässrig-organischem Medium bei 10°C bis 70°C durchgeführt wird.Method according to one of claims 1 to 7, characterized in that the method is carried out in an aqueous or aqueous-organic medium at 10 ° C to 70 ° C. 16α-Hydroxy-4-androsten-3,17-dion, 16α-Hydroxy-4-Pregnen-3,20-dion, 11β,16α,17,21-Tetrahydroxypregn-4-en-3,20-dion, 16α,17α,21-Trihydroxy-pregnen-3,11,20-trion, 11β,16α,21-Trihydroxy-pregn-4-en-3,20-dion.16α-hydroxy-4-androstene-3,17-dione, 16α-hydroxy-4-pregnene-3,20-dione, 11β, 16α, 17,21-tetrahydroxypregn-4-ene-3,20-dione, 16α, 17α, 21-trihydroxy-pregnene-3,11,20-trione, 11β, 16α, 21-trihydroxy-pregn-4-ene-3,20-dione. 4,5-Epoxi-androstan-3,17-dion, 4,5-Epoxi-17β-Hydroxy-androstan-3-on, 4,5-Epoxi-pregnan-3,20-dion, 4,5-Epoxi-11β,17,21-Trihydroxypregnan-3,20-dion, 4,5-Epoxi-17α,21-Dihydroxy-pregnan-3,11,20-trion, 4,5-Epoxi-11β,21-Dihydroxy-pregnan-3,20-dion.4,5-epoxy-androstane-3,17-dione, 4,5-epoxy-17β-hydroxy-androstane-3-one, 4,5-epoxy-pregnane-3,20-dione, 4,5-epoxide 11β, 17,21-trihydroxypregnane-3,20-dione, 4,5-epoxy-17α, 21-dihydroxy-pregnane-3,11,20-trione, 4,5-epoxy-11β, 21-dihydroxy-pregnane 3,20-dione.
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