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We report the discovery of a new imine reductase (IRED), named AtIRED, by genome mining. Site-saturation mutagenesis on AtIRED generated two single mutants M118′L and P120′G and the double mutant M118′L/P120′G with improved specific activity toward sterically hindered 1-substituted dihydro-β-carbolines. The synthetic potential of these engineered IREDs was showcased by the preparative-scale synthesis of nine chiral 1-substituted tetrahydro-β-carbolines (THβCs), including (S)-1-t-butyl-THβC and (S)-1-t-pentyl-THβC, in 30–87% isolated yields with excellent optical purities (98–99% ee).

Development of an efficient, stereoselective, sustainable synthesis of chiral aryl β‐hydroxy α‐amino acids and their derivatives is of paramount importance, owing to the broad utility of these molecules in pharmaceutical application and asymmetric synthesis. We report a systematic study on ketoreductase (KRED)‐catalyzed dynamic reductive kinetic resolution (DYRKR) of aryl α‐amino β‐keto esters 6, furnishing 20 structurally diverse chiral syn‐aryl β‐hydroxy α‐amino esters (syn‐(2S,3R)‐7) in moderate‐to‐excellent isolated yield (up to 93%), along with moderate‐to‐excellent diastereoselectivity (up to >99 : 1 dr) and excellent enantioselectivity (mostly >99% ee). The practical synthesis potential of our developed method was showcased by the asymmetric, chemo‐enzymatic total synthesis of thiamphenicol (1). A generic, efficient, and highly stereoselective synthesis of valuable chiral syn‐aryl β‐hydroxy α‐amino esters (syn‐(2S,3R)‐7) was developed through ketoreductase‐catalyzed dynamic reductive kinetic resolution of aryl α‐amino β‐keto esters 6. The practical synthesis potential of this developed method was showcased by the asymmetric, chemo‐enzymatic total synthesis of thiamphenicol (1).

The HMG-CoA reductase inhibitors, statins, are one of the most effective and bestselling cholesterol-lowering drugs. The use of statins has greatly extended people’s lives and improved the quality of their life. Development of a more efficient, stereoselective, and sustainable synthesis of statins is continuingly of utmost importance. In the present study, through screening of ketoreductases (KREDs) and reaction optimization, we have successfully performed a highly stereoselective reduction of ketoester 1acatalyzed by KRED-06 at a pilot-plant scale without the addition of exogenous NADP+, generating 3.21 kg of enantiomerically pure tert-butyl (R)-3-hydroxyl-5-hexenoate ((R)-2a) (96.2% yield, >99.9% enantiomeric excess (ee)). This newly developed biocatalytic process alleviates the cryogenic conditions (−40 °C) employed in our first-generation synthesis of (R)-2ausing NaBH4and (l)-tartaric acid. Coupled with our previously established synthesis of bromocarbonate 3avia a one-pot diastereoselective carboxylation/bromocyclization of (R)-2a, we have developed an innovative, practical synthesis route to statin side chain, possessing great potential to be implemented into industrial production of statins.

(S)-1-(4-Methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline [(S)-1-(4-methoxybenzyl)-OHIQ, (S)-1a] is a key synthetic intermediate in the industrial production of dextromethorphan, one of the most widely used over-the-counter antitussives. We report here that a new cyclohexylamine oxidase discovered by genome mining, named CHAOCCH12-C2, was able to completely deracemize 100 mM 1aunder Turner’s deracemization conditions to afford (S)-1ain 80% isolated yield and 99% ee at a semipreparative scale (0.4 mmol). When this biocatalytic reaction was scaled up to a gram scale (5.8 mmol), without reaction optimization (S)-1awas still isolated in 67% yield and 96% ee. The relatively higher kcatdetermined for CHAOCCH12-C2was rationalized as one major factor rendering this enzyme capable of oxidizing 1aeffectively at elevated substrate concentrations. Protein sequence alignment, analysis of our co-crystal structure of CHAOCCH12-C2complexed with the product 1-(4-methoxybenzyl)-3,4,5,6,7,8-hexahydroisoquinoline [1-(4-methoxybenzyl)-HHIQ, 2a], and the structure-guided mutagenesis study together indicated L295 is one of the critical residues for this efficient enzymatic oxidation process and supported the presence of two cavities as well as a catalytically important “aromatic cage” formed by F342, Y433, and FAD. The synthetic applicability of CHAOCCH12-C2was further underscored by the stereoselective synthesis of various enantioenriched 1-benzyl-OHIQ derivatives of potential pharmaceutical importance at a semipreparative scale.

Given their ability to reduce activated alkenes thereby generating up to two stereogenic centers, ene reductases from the old yellow enzyme (OYE) family have received much attention as effective biocatalysts. Through genome mining, a “classical” OYE, KYE2, was identified from Kluyveromyces marxianusCBS4857 and fully characterized in vitro. This NADPH‐dependent enzyme displayed a broad substrate spectrum for the bioreduction of activated alkenes. The highly stereoselective synthesis (>95 % ee) of various (R)‐profen methyl esters, including the synthesis of (R)‐flurbiprofen methyl ester on a semi‐preparative scale further demonstrates the potential of KYE2 to be developed as a general biocatalyst for chemical synthesis. Minecrafting new tools for synthesis: KYE2, a new ene reductase, was identified from Kluyveromyces marxianusCBS4857 through genome mining. KYE2 displayed a broad substrate spectrum for the bioreduction of activated alkenes and was successfully applied in highly stereoselective syntheses of various (R)‐profen methyl esters.

We report here a generic, green synthesis of 17 valuable syn-aryl-(2S,3R)-2-chloro-3-hydroxy esters (syn-(2S,3R)-1) in 73%-99% isolated yields along with 6.1:1-83:1 drand 31%->99% ee, through dynamic reductive kinetic resolution of racemic aryl α-chloro β-keto esters (2) catalyzed by an engineered ketoreductase which was obtained viaepPCR-based directed evolution. The hectogram scale synthesis of syn-(2S,3R)-1bat a substrate concentration of 120 g/L showcased the application potential of the biocatalytic method developed presently.

KYE2, a new ene‐reductase, belonging to the Old Yellow Enzyme family was identified from Kluyveromyces marxianusCBS4857 through genome mining. KYE2 displayed a broad substrate spectrum for the bioreduction of activated alkenes and was further successfully applied in the highly enantioselective syntheses of various (R)‐profen methyl esters, including (R)‐flurbiprofen methyl ester as shown in the Front Cover. More information can be found in the Full Paper by Zedu Huang, Fener Chen et al. on page 763 in Issue 4, 2018(DOI: 10.1002/ajoc.201800059).

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