(R)-Citronellal is one of the key chiral intermediates in the synthesis of the isomer (−)-menthol, one of the most commercialised terpenoid flavours worldwide. Enzymatic approaches could represent a less energy-demanding alternative for its synthesis, such as a previously reported bienzymatic cascade starting from inexpensive, commercially available geraniol. A copper radical oxidase (CgrAlcOx) followed by a flavin-dependent ene reductase (OYE2) were used to obtain (R)-citronellal. Here, we used a metal-affinity immobilisation strategy on the His-tagged enzymes for the cascade and studied enzyme recovery and reusability as well as increased solvent tolerance. After screening a panel of resins for enzyme immobilisation and water-immiscible co-solvents, we successfully obtained 95% conversion to (R)-citronellal with 96.9% enantiomeric excess (ee) in a concurrent cascade after 7 h of reaction time, starting from 10 mM of geraniol.

The unmatched chemo-, regio-, and stereoselectivity of enzymes renders them powerful catalysts in the synthesis of chiral active pharmaceutical ingredients (APIs). Inspired by the discovery route toward the LPA₁-antagonist BMS-986278, access to the API building block (1S,3R)-3-hydroxycyclohexanecarbonitrile was envisaged using an ene reductase (ER) and alcohol dehydrogenase (ADH) to set both stereocenters. Starting from the commercially available cyclohexene-1-nitrile, a C-H oxyfunctionalization step was required to introduce the ketone functional group, yet several chemical allylic oxidation strategies proved unsuccessful. Enzymatic strategies for allylic oxidation are underdeveloped, with few examples on selected substrates with cytochrome P450s and unspecific peroxygenases (UPOs). In this case, UPOs were found to catalyze the desired allylic oxidation with high chemo- and regioselectivity, at substrate loadings of up to 200 mM, without the addition of organic cosolvents, thus enabling the subsequent ER and ADH steps in a three-step one-pot cascade. UPOs even displayed unreported enantioselective oxyfunctionalization and overoxidation of the substituted cyclohexene. After screening of enzyme panels, the final product was obtained at titers of 85% with 97% ee and 99% de, with a substrate loading of 50 mM, the ER being the limiting step. This synthetic approach provides the first example of a three-step, one-pot UPO-ER-ADH cascade and highlights the potential for UPOs to catalyze diverse enantioselective allylic hydroxylations and oxidations that are otherwise difficult to achieve.

Chiral N-heterocycles are a common motif in many active pharmaceutical ingredients; however, their synthesis often relies on the use of heavy metals. In recent years, several biocatalytic approaches have emerged to reach enantiopurity. Here, we describe the asymmetric synthesis of 2-substituted pyrrolidines and piperidines, starting from commercially available ω-chloroketones by using transaminases, which has not yet been comprehensively studied. Analytical yields of up to 90% and enantiomeric excesses of up to >99.5% for each enantiomer were achieved, which has not previously been shown for bulky substituents. This biocatalytic approach was applied to synthesize (R)-2-(p-chlorophenyl)pyrrolidine on a 300 mg scale, affording 84% isolated yield, with >99.5% ee.

Noncanonical redox cofactors are attractive low-cost alternatives to nicotinamide adenine dinucleotide (phosphate) (NAD(P)⁺) in biotransformation. However, engineering enzymes to utilize them is challenging. Here, we present a high-throughput directed evolution platform which couples cell growth to the in vivo cycling of a noncanonical cofactor, nicotinamide mononucleotide (NMN⁺). We achieve this by engineering the life-essential glutathione reductase in Escherichia coli to exclusively rely on the reduced NMN⁺ (NMNH). Using this system, we develop a phosphite dehydrogenase (PTDH) to cycle NMN⁺ with ~147-fold improved catalytic efficiency, which translates to an industrially viable total turnover number of ~45,000 in cell-free biotransformation without requiring high cofactor concentrations. Moreover, the PTDH variants also exhibit improved activity with another structurally deviant noncanonical cofactor, 1-benzylnicotinamide (BNA⁺), showcasing their broad applications. Structural modeling prediction reveals a general design principle where the mutations and the smaller, noncanonical cofactors together mimic the steric interactions of the larger, natural cofactors NAD(P)⁺.