TY - JOUR
T1 - Clean Enzymatic Oxidation of 12α-Hydroxysteroids to 12-Oxo-Derivatives Catalyzed by Hydroxysteroid Dehydrogenase
AU - Tonin, Fabio
AU - Alvarenga, Natália
AU - Ye, Jia Zheng
AU - Arends, Isabel W.C.E.
AU - Hanefeld, Ulf
PY - 2019
Y1 - 2019
N2 -
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 (El12α-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 El12α-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 El12α-HSDH-NOX system in a segmented-flow-reactor. (Figure presented.).
AB -
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 (El12α-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 El12α-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 El12α-HSDH-NOX system in a segmented-flow-reactor. (Figure presented.).
KW - 12α-hydroxysteroid dehydrogenases
KW - Bile acids
KW - Flow-reactor
KW - NAD(P)H oxidase
KW - NAD -dependent
UR - http://www.scopus.com/inward/record.url?scp=85065785841&partnerID=8YFLogxK
U2 - 10.1002/adsc.201900144
DO - 10.1002/adsc.201900144
M3 - Article
AN - SCOPUS:85065785841
SN - 1615-4150
VL - 361
SP - 2448
EP - 2455
JO - Advanced Synthesis and Catalysis
JF - Advanced Synthesis and Catalysis
IS - 11
ER -