Engineered Amine Dehydrogenase Provides Cleaner Kinetic Resolution for Mirogabalin Precursor

A research team at the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, collaborating with colleagues from Tianjin University of Science and Technology and the National Technology Innovation Center of Synthetic Biology, has developed an engineered amine dehydrogenase that performs kinetic resolution on a racemic bicyclic ketone to produce a key chiral precursor for the analgesic medication mirogabalin. Reported in the Chinese Journal of Biotechnology, the biocatalytic method isolates the target ketone, (1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one, from a racemic mixture through an enzymatic reaction followed by standard phase separation. In a 50-milliliter preparative reaction running at a substrate concentration of 150 millimoles per liter, the optimized enzyme variant yielded the desired ketone with 97.1 percent enantiomeric excess and an isolated yield of 45.4 percent after a single solvent extraction and an acid wash.

Mirogabalin is an analgesic pharmaceutical belonging to the gabapentinoid therapeutic class, which also includes gabapentin and pregabalin. The therapeutic agent is utilized in the management of peripheral neuropathic pain disorders, including diabetic peripheral neuropathic pain, postherpetic neuralgia, and fibromyalgia. Mechanistically, mirogabalin binds preferentially and selectively to the α2δ-1 subunit of voltage-gated calcium channels while dissociating from the α2δ-1 subunit at a slower rate than it dissociates from the α2δ-2 subunit. According to pharmacological research, this selective binding profile is directly associated with stronger analgesia as well as fewer central nervous system side effects in comparison to older gabapentinoids such as pregabalin and gabapentin.

Synthesizing mirogabalin relies on the specific chiral building block (1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one, but this bicyclic ketone framework cannot be synthesized directly through asymmetric chemical construction. Because the building block is available only by resolving the racemate, established synthetic routes have historically incurred substantial penalties in downstream processing and product purification. In one established biocatalytic route, a carbonyl reductase enzyme reduces the unwanted (1S,5R)-ketone enantiomer into an alcohol intermediate, leaving the target ketone behind. However, because the resulting alcohol cannot be cleanly separated from the remaining ketone by simple extraction, it must first be chemically esterified using a diacid anhydride before the target ketone can be isolated. Although that carbonyl reductase method achieves an isolated yield of 41 percent and an enantiomeric excess of 97.7 percent, it requires an extra chemical derivatization step.

To eliminate these downstream processing complications, the research group investigated amine dehydrogenases, a class of biocatalysts that utilize nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate cofactors alongside inexpensive inorganic ammonia as a nitrogen donor. In this catalytic process, the enzyme converts a carbonyl group into a chiral amine while producing water as the sole chemical by-product. When introduced to the racemic ketone mixture, the newly engineered amine dehydrogenase selectively aminates the unwanted (1S,5R)-ketone into an amine while leaving the target (1R,5S)-ketone intact. Under acidic conditions, the newly formed amine readily protonates into a water-soluble ammonium salt, whereas the unreacted target ketone remains in the organic phase. Consequently, the two compounds separate cleanly through liquid-liquid extraction followed by a dilute hydrochloric acid wash, entirely circumventing the derivatization or recrystallization steps required by earlier methods.

The researchers selected their initial template enzyme from an in-house screen of the laboratory’s amine dehydrogenase collection and performed structure-guided evolution to optimize its catalytic properties. The resulting lead variant incorporated three amino acid substitutions: L40T, L125R, and V291A. To validate the catalytic performance at a preparative scale, the investigators utilized Escherichia coli whole cells co-expressing the triple-mutant amine dehydrogenase together with a formate dehydrogenase intended for cofactor recycling. The preparative reaction operated at a substrate loading of 150 millimoles per liter at 30 degrees Celsius for 16 hours. Following the incubation, the reaction mixture was extracted with dichloromethane and washed three times with 0.1 mole per liter hydrochloric acid to transfer the amine into the aqueous phase as its salt, recovering the purified ketone at 97.1 percent enantiomeric excess and 45.4 percent isolated yield.

In addition to resolving the mirogabalin intermediate, the research team evaluated their engineered enzyme variants against a broader panel of ten alternative ketones, encompassing cyclic, aliphatic, arylalkyl, and aromatic molecular structures. Across these ten substrates, the engineered biocatalysts retained high stereoselectivity, achieving enantiomeric excess values greater than 99 percent for most tested compounds. Nevertheless, catalytic conversion rates varied substantially across the different structural classes. Straight-chain aliphatic ketones such as 2-pentanone, 2-hexanone, and 2-octanone reacted efficiently in the presence of the enzyme, whereas the linear 2-heptanone exhibited noticeably lower activity.

According to the study authors, this investigation represents the first reported application of an amine dehydrogenase for the kinetic resolution of a racemic ketone, as the enzyme family had previously been employed only for asymmetric reductive amination to synthesize chiral amines. Because separation depends directly on an intrinsic phase difference generated during the enzymatic amination itself, the new method avoids both chemical derivatization and recrystallization steps while simultaneously reducing organic solvent usage during downstream isolation.

Subject of Research: Biocatalytic kinetic resolution of a chiral mirogabalin intermediate using engineered amine dehydrogenase

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

Article References: Engineered amine dehydrogenase offers a cleaner route to a key mirogabalin building block. Original publication (Bibliographic details unavailable.)

Image Credits: AI Generated

DOI: Not provided

Keywords: amine dehydrogenase, mirogabalin, kinetic resolution, biocatalysis, directed evolution, gabapentinoids, chiral synthesis, enzyme engineering, Engineered, amine, dehydrogenase, offers