Engineering an Amine Transaminase for the Efficient Production of a Chiral Sacubitril Precursor

Abstract

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.

Application of Transition-Metal Catalysis, Biocatalysis, and Flow Chemistry as State-of-the-Art Technologies in the Synthesis of LCZ696

“…Transformation of γ-ketoacid 11 to the corresponding (2 S ,4 R )-amino acid 12 was enabled by a biocatalytic enzymatic transamination using CDX-043, a highly evolved S -selective transaminase (E.C. 2.6.1) obtained by directed enzyme evolution over multiple evolution rounds, ,− with pyridoxal 5′-phosphate (PLP, vitamin B 6 ) as cofactor and isopropylamine as amine donor. Due to the low solubility of both substrate 11 and product 12 in aqueous conditions, the reaction was run as a slurry-to-slurry process, remaining heterogeneous throughout the whole reaction time.…”

The Development of Biocatalysis as a Tool for Drug Discovery

“…With increasing access to novel enzymes as a result of rich and vibrant discovery and engineering efforts and a collaborative research community, this technology can be applied routinely on commercial manufacturing scale. A second fundamental advantage of biocatalysis is the ability to support green and sustainable manufacturing of drugs at Novartis [69,70] and elsewhere. Advances in the development of retrosynthetic software and machine learning ensure the application of biocatalysis in the future.…”

The identification and application of a robust ω-transaminase with high tolerance towards substrates and isopropylamine from a directed soil metagenome

“…There are few successful implementations of ω-TAs with yields >50 g L −1 . 6,[16][17][18][19] Among them, the most representative applications are the biomanufacturing of sitagliptin, performed with >200 g L −1 ketone substrate by employing mutant Arthrobacter sp. ATA-117-Rd11, 6 and a sacubitril precursor with ketone substrate loading of 75 g L −1 catalyzed by mutant Vibrio fluvialis CDX-043, 19 both of which were conducted by Codexis.…”

“…6,[16][17][18][19] Among them, the most representative applications are the biomanufacturing of sitagliptin, performed with >200 g L −1 ketone substrate by employing mutant Arthrobacter sp. ATA-117-Rd11, 6 and a sacubitril precursor with ketone substrate loading of 75 g L −1 catalyzed by mutant Vibrio fluvialis CDX-043, 19 both of which were conducted by Codexis. However, both cases have been subjected to numerous rounds of evolution, and ultimately 8.2% of the ATA-117-Rd11 sequence (27 mutations in 330 amino acids) and 5.4% of CDX-043 (26 mutations in 478 amino acids) were modified, which requires large amounts of work and is difficult to implement easily in other ω-TAs.…”