Chemistry

Metallurgy

Materials science

Enantiomeric excess

Imine

Reductive amination

Biochemistry

Reductase

Enzyme

Active site

Organic chemistry

Amination

Catalysis

Streptomyces clavuligerus

Antibiotics

Enantioselective synthesis

Yield (engineering)

Combinatorial chemistry

Amoxicillin

Stereochemistry

Protein engineering

Clavulanic acid

ACS Catalysis

Imine reductases (IREDs) are increasingly identified and characterized for the reduction of imines and reductive amination of ketones. However, their practical application is still limited due to the low activity and poor stability of native enzymes. Herein, we developed an engineered IRED through three rounds of evolution from wild-type Streptomyces clavuligerus. The specific activity of the engineered enzyme, ScIRED-R3-V4, was increased >100-fold, and stability was increased >270-fold. Using the more active and stable ScIRED-R3-V4, 80 g L–1 cyclic imine 2-(2,5-difluorophenyl)-pyrroline was completely reduced, producing kilogram quantities of a key chiral intermediate of larotrectinib in 82.5% yield, with >99.5% enantiomeric excess and a space–time yield of 352 g L–1 day–1. In addition, crystal structures of enzyme–substrate complexes and molecular dynamics simulations were conducted to gain insight into the molecular mechanism for improved enzyme performance. The significantly improved process demonstrated the potential of the engineered IRED in industrial applications.

Engineered Imine Reductase for Larotrectinib Intermediate Manufacture