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Clean Enzymatic Oxidation of 12α‐Hydroxysteroids to 12‐Oxo‐Derivatives Catalyzed by Hydroxysteroid Dehydrogenase
Author(s) -
Tonin Fabio,
Alvarenga Natália,
Ye Jia Zheng,
Arends Isabel W. C. E.,
Hanefeld Ulf
Publication year - 2019
Publication title -
advanced synthesis and catalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.541
H-Index - 155
eISSN - 1615-4169
pISSN - 1615-4150
DOI - 10.1002/adsc.201900144
Subject(s) - chemistry , cholic acid , nad+ kinase , cofactor , nicotinamide adenine dinucleotide , chenodeoxycholic acid , hydroxysteroid dehydrogenases , dehydrogenase , enzyme , formate dehydrogenase , nicotinamide adenine dinucleotide phosphate , biochemistry , hydroxysteroid dehydrogenase , oxidase test , combinatorial chemistry , bile acid
The C12 specific oxidation of hydroxysteroids is an essential reaction required for the preparation of pharmaceutical ingredients like ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA), which can be synthesized by Wolff‐Kishner reduction of the obtained 12‐oxo‐hydroxysteroids. 12α‐hydroxysteroid dehydrogenases (12α‐HSDHs) have been shown to perform this reaction with high yields, under mild conditions and without the need of protection and deprotection steps, required in chemical synthesis. Here, the recombinant expression and biochemical characterization of the nicotinamide adenine dinucleotide (NAD + )‐dependent HSDH from Eggerthella lenta ( El 12α‐HSDH) are reported. This enzyme shows comparable properties with the well‐known nicotinamide adenine dinucleotide phosphate (NADP + )‐dependent enzyme from Clostridium sp . 48–50. In order to perform a viable and atom efficient enzymatic hydroxysteroid oxidation, NAD(P)H oxidase (NOX) was employed as cofactor regeneration system: NOX uses oxygen (O 2 ) as sacrificial substrate and produces only water as side product. 10 mM of cholic acid was fully and selectively converted to 12‐oxo‐CDCA in 24 h. The possibility to employ this system on UCA and 7‐oxo‐deoxycholic acid (7‐oxo‐DCA) as substrates was additionally investigated. The performance of the El 12α‐HSDH was evaluated also in combination with a “classical” regeneration system (oxaloacetate/malate dehydrogenase) showing full conversion in 4 h. Finally, the feasibility of a catalytic aerobic‐NAD + ‐dependent enzymatic oxidation was shown on a preparative scale (oxidation of CA to 12‐oxo‐CDCA) employing the El 12α‐HSDH‐NOX system in a segmented‐flow‐reactor.