Engineered amine dehydrogenase offers a cleaner route to a key mirogabalin building block

Mirogabalin belongs to the gabapentinoid class, together with gabapentin and pregabalin, and is used for peripheral neuropathic pain, including diabetic peripheral neuropathic pain, postherpetic neuralgia and fibromyalgia. It binds preferentially and selectively to the α2δ-1 subunit of voltage-gated calcium channels and dissociates from α2δ-1 more slowly than from α2δ-2; this binding profile is associated with stronger analgesia and fewer central-nervous-system side effects than pregabalin and gabapentin. The bicyclic ketone itself cannot be built asymmetrically. It is available only by resolving the racemate, and the two established routes both pay for that in downstream processing. In the biocatalytic route, a carbonyl reductase reduces the unwanted (1S,5R)-ketone to the corresponding alcohol, which must then be esterified with a diacid anhydride before the target ketone can be isolated: 41% yield and 97.7% ee, at the price of an extra derivatization step.

A research team at the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, working with colleagues at Tianjin University of Science and Technology and the National Technology Innovation Center of Synthetic Biology, has engineered an amine dehydrogenase that resolves racemic (1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one, the chiral ketone from which the analgesic mirogabalin is synthesized. In a 50 mL preparation at a substrate loading of 150 mmol/L, the best variant returned the target ketone with 97.1% enantiomeric excess (ee) and 45.4% isolated yield, after a single solvent extraction and an acid wash.

The team published their work in Chinese Journal of Biotechnology on Sep. 25, 2026.

Amine dehydrogenases use NAD(P)H as cofactor and inexpensive inorganic ammonia as the nitrogen donor to convert a ketone into a chiral amine, with water as the only by-product. Applied to the racemic ketone, the enzyme aminates the unwanted (1S,5R)-ketone and leaves the target (1R,5S)-ketone untouched. Because the resulting amine forms a water-soluble ammonium salt under acidic conditions while the ketone stays in the organic phase, the two compounds are separated by extraction and a wash with dilute hydrochloric acid — no derivatization or recrystallization is needed.

The template enzyme came from a screen of the laboratory's amine dehydrogenase collection. Structure-guided evolution of the enzyme was performed. The final variant was tested at preparative scale Escherichia coli whole cells co-expressing L40T/L125R/V291A and a formate dehydrogenase for cofactor recycling were used for the reaction at 150 mmol/L substrate concentration at 30 °C for 16 h. After extraction with dichloromethane and washing three times with 0.1 mol/L hydrochloric acid, which moved the amine into the aqueous phase as its salt, the target ketone was recovered at 97.1% ee and 45.4% isolated yield.

The engineered enzymes also were tested on a broader set of ketones. Across ten additional substrates covering cyclic, aliphatic, arylalkyl and aromatic ketones, the variants retain high stereoselectivity, exceeding 99% ee for most of them, although conversion varies on the substrates: the straight-chain 2-pentanone, 2-hexanone and 2-octanone react efficiently, 2-heptanone less active, while the bulky cyclic ketones show little activity..

According to the authors, this is the first report of an amine dehydrogenase being used for the kinetic resolution of a ketone; the enzyme family has previously employed for asymmetric reductive amination for chiral amine synthesis, not the resolution of a racemic ketone. Because the separation depends on a phase difference created by the reaction itself, the route avoids the derivatization and recrystallization steps in the product isolation process and cuts usage of the organic solvents — offering greener, lower-cost method for the manufacture of mirogabalin intermediate.