One‐Pot Multienzymatic Synthesis of 12‐Ketoursodeoxycholic Acid: Subtle Cofactor Specificities Rule the Reaction Equilibria of Five Biocatalysts Working in a Row

Abstract

Abstract:The hydroxysteroid dehydrogenases (HSDHs)-catalyzed one-pot enzymatic synthesis of 12-ketoursodeoxycholic acid (3a,7b-dihydroxy-12-oxo-5b-cholanoic acid), a key intermediate for the synthesis of ursodeoxycholic acid, from cholic acid has been investigated. This goal has been achieved by alternating oxidative and reductive steps in a onepot system employing HSDHs with different cofactor specificity, namely NADH-dependent HSDHs in the oxidative step and an NADPH-dependent 7b-HSDH in the reductive one. Coupled in situ regeneration systems have been exploited not only to allow the use of catalytic amounts of the cofactors, but also to provide the necessary driving force to opposite reactions (i.e., oxidation and reduction) acting on different sites of the substrate molecule. Biocatalysts suitable for the set-up of this process have been selected and their kinetic behaviour in respect of the reactions of interest has been evaluated. Finally, the process has been studied employing the enzymes both in free and compartmentalized form.

Multi‐Enzymatic Cascade Reactions: Overview and Perspectives

“…[33] In this study, two alcohol oxidations and one ketone reduction could be realized in one pot using different hydroxysteroid dehydrogenases (HSDHs) and independent regeneration systems for NAD + and NADPH. Overall, one secondary alcohol moiety was oxidized while another one was stereoinverted.…”

Structural and biochemical insights into 7β‐hydroxysteroid dehydrogenase stereoselectivity

“…Years ago, the substrate specificity of the C. absonum 7β‐HSDH was investigated and the enzyme was applied to the synthesis of a cholic acid derivative . More recently, the synthetic exploitation of 7β‐HSDHs for the preparative production of ursodeoxycholic acid, a FDA‐approved drug for treatment of cholesterol gallstones, and current sole alternative to surgical intervention, has been widely investigated by different groups …”

A new concept for production of (3S,4R)-6-[(benzyloxycarbonyl)amino]-5,6-dideoxyhex-2-ulose, a precursor of d-fagomine

“…9 These reactions are simple to operate and analyze for pathway engineering and process optimization. 6 Up to now, a large number of biocatalytic cascade reactions has been developed; from simple oxido-reduction reaction coupled with cofactor regeneration, to highly complex multi-enzyme synthesis. 4,9 Furthermore, unfavorable equilibrium reactions can also benet from the cascade concept since continuous removal of product in a subsequent reaction step can drive the reaction towards product formation.…”