Four-step continuous-flow biosynthesis of a chiral precursor for angiotensin-converting enzyme inhibitors

Optically active ethyl ( R )-2-hydroxy-4-phenylbutanoate (( R )-HPBE), is a key component in the production of angiotensin-converting enzyme (ACE) inhibitors, which are essential for treating hypertension. However, the high cost of starting materials has been a barrier to the industrial synthesis of ( R )-HPBE. Herein, we have established for the first time a four-step enzymatic reaction in continuous flow for the efficient synthesis of ( R )-HPBE from inexpensive benzaldehyde and pyruvic acid. This route also overcomes the issues of chemical disequilibrium and incompatibility of reaction conditions that are common in multi-enzyme cascades. The immobilization methods for aldolase/ene-reductase/keto-reductase were developed, and the immobilized enzymes were initially used in three individual flow modules to produce ( R )-2-hydroxy-4-phenylbutanoic acid (( R )-HPB), achieving high space–time yields of 3103 g (OPBEA)  L −1  d −1 , 1685 g (OPBE)  L −1  d −1 and 1709 g ( R )-HPB  L −1  d −1 respectively. Moreover, integrating three modules in continuous flow achieved a space–time yield of about 350 g ( R )-HPB  L −1  d −1 after 108 h, and maintained high operational stability with a conversion of >99 % and a product ee of >99.9 % after 192 h. Subsequently, the ethyl esterification of ( R )-HPB as the fourth module for producing ( R )-HPBE obtained a high space–time yield of 2117 g ( R )-HPBE  L −1  d −1 , marking a substantial 74.5-fold increase over the batch system.