An amine transaminase was engineered for the efficient production of a chiral precursor to sacubitril, (2R,4S)-5-([1,1′-biphenyl]-4-yl)-4-amino-2-methylpentanoic acid, a key component in the blockbuster heart failure drug Entresto. Starting from an enzyme with trace activity and preference for the undesired diastereoisomer, 11 rounds of enzyme evolution were performed. The resultant variant, CDX-043, showed high productivity giving 90% conversion at 75 g/L substrate concentration with 1% enzyme loading with respect to the substrate in 24 h and without the use of an organic cosolvent. The product diastereomeric purity toward the desired (2R,4S)-stereoisomer was >99.9:0.1 d.r. This variant also exhibited high process robustness and could tolerate reaction temperatures up to 65 °C, isopropylamine concentrations of at least 2 M, and reaction times of at least 5 days. A structural analysis of the enzyme variants gave insight into how the mutations affected activity and selectivity. This enzyme variant allows for the efficient and cost-effective production of sacubitril at large scale.

(R)-2-Methylpentanol is an important chiral intermediate for the synthesis of certain medicinally important compounds, natural products, and liquid crystals. Here we describe the development of a practical kinetic resolution utilizing an enantiospecific biocatalytic reduction of racemic 2-methylvaleraldehyde. The process utilizes an evolved ketoreductase enzyme to selectively reduce the (R)-enantiomer of racemic 2-methylvaleraldehyde to the desired product with high volumetric productivity. A scaleable method for separating the desired product from the off-enantiomer of the starting material is also described. The process is cost-effective, green, and amenable to manufacturing scale.