Isabel W. C. E. Arends

The specific oxidation of 12α-OH group of hydroxysteroids is required for the preparation of cheno- and ursodeoxycholic acid (CDCA and UDCA, respectively). The C₁₂ oxidation of hydroxysteroids into their 12-oxo derivatives can selectively be performed by employing 12α-hydroxysteroid dehydrogenases. These enzymes use NAD(P)⁺ as an electron acceptor, which has to be re-oxidized in a so-called “regeneration system”. Recently, the enzyme NAD(P)H oxidase (NOX) was applied for the regeneration of NAD⁺ in the enzymatic preparation of 12-oxo-CDCA from cholic acid (CA), which allows air to be used as an oxidant. However, the NOX system suffers from low activity and low stability. Moreover, the substrate loading is limited to 10 mM. In this study, the laccase/mediator system was investigated as a possible alternative to NOX, employing air as an oxidant. The laccase/mediator system shows higher productivity and scalability than the NOX system. This was proven with a preparative biotransformation of 20 g of CA into 12-oxo-CDCA (92% isolated yield) by employing a substrate loading of 120 mM (corresponding to 50 g/L). Additionally, the performance of the laccase/mediator system was compared with a classical ADH/acetone regeneration system and with other regeneration systems reported in literature.

The scale-up of the chemoenzymatic bromolactonization to 100 g-scale is presented together with an identification of current limitations. The preparative-scale reaction also allowed for meaningful mass balances identifying current bottlenecks of the chemoenzymatic reaction.

H₂O₂ can be accepted by several peroxygenases as a clean oxidant, able to supply both the necessary electrons and oxygen atom at the same time. The biocatalysts, in turn, are able to catalyse an array of interesting oxygen insertion reactions at enantio‐ and regio‐selectivities hard to attain with classical chemical methods. The sensitivity of most peroxygenases towards H₂O₂, however, requires this oxidant to be generated in situ. Here, we suggest the application of (modified) sulfite oxidases to couple the oxidation of sulfites to the reduction of oxygen. This enables us to use calcium sulfite, an industrial waste product from scrubbing flue gases, as an electron donor to reduce oxygen. This will supply the required peroxide in a controlled manner and enables us to perform these challenging reactions at the expense of simple salts.

Elemental metals are shown to be suitable sacrificial electron donors to drive the stereoselective reduction of conjugated C=C double bonds using Old Yellow Enzymes as catalysts. Both direct electron transfer from the metal to the enzyme as well as mediated electron transfer is feasible, although the latter excels by higher reaction rates. The general applicability of this new chemoenzymatic reduction method is demonstrated, and current limitations are outlined.

Biocatalytic oxyfunctionalisation reactions are traditionally conducted in aqueous media limiting their production yield. Here we report the application of a peroxygenase in neat reaction conditions reaching product concentrations of up to 360 mM.

Peroxygenases are very interesting catalysts for specific oxyfunctionalization chemistry. Instead of relying on complicated electron transport chains, they rely on simple hydrogen peroxide as stoichiometric oxidant. Their poor robustness against H2O2 can be addressed via in situ generation of H2O2. Here we report that simple graphitic carbon nitride (g-C3N4) is a promising photocatalyst to drive peroxygenase-catalyzed hydroxylation reactions. The system has been characterized outlining its scope but also its current limitations. In particular, spatial separation of the photocatalyst from the enzyme is shown as solution to circumvent the undesired inactivation of the biocatalyst. Overall, very promising turnover numbers of the biocatalyst of more than 60.000 have been achieved.

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 (O₂) 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. magnified image

The consecutive photooxidation and reductive amination of various alcohols in a cascade reaction were realized by the combination of a photocatalyst and several enzymes. Whereas the photocatalyst (sodium anthraquinone-2-sulfonate) mediated the light-driven, aerobic oxidation of primary and secondary alcohols, the enzymes (various ω-transaminases) catalyzed the enantio-specific reductive amination of the intermediate aldehydes and ketones. The system worked in a one-pot one-step fashion, whereas the productivity was significantly improved by switching to a one-pot two-step procedure. A wide range of aliphatic and aromatic compounds was transformed into the enantiomerically pure corresponding amines via the photo-enzymatic cascade.

Two-component-diffusible-flavomonooxygenases are versatile biocatalysts for selective epoxidation-, hydroxylation- or halogenation reactions. Their complicated molecular architecture can be simplified using photochemical regeneration of the catalytically active, reduced FADH₂ prosthetic group. In this contribution we provide the proof-of-concept and characterization for the direct regeneration of the styrene monooxygenase from Pseudomonas.