Sequential resolution of (S) and (R)-6-fluoro-chroman-2-carboxylic acid by two esterases in turn

Abstract

Optically pure 6-fluoro-chroman-2-carboxylic acids (FCCAs), with (S) and (R)-configurations, are pivotal chiral building blocks in the pharmaceutical industry. To date, the production of (S) and (R)-FCCAs mainly depends on chemical resolution, which is a complex, low yield, and highly polluting process. Here, we present a practical enzymatic resolution method of FCCAs based on two esterases, EstS and EstR, isolated from Geobacillus thermocatenulatus. Using the racemic methyl 6-fluoro-chroman-2-carboxylate (MFCC) as the substrate in an aqueous–toluene biphasic system, (S) and (R)-FCCAs were produced by EstS and EstR catalysis with an enantiomeric excess (ee) value >99% and 95–96%, respectively, and the highly enantioselective mechanisms were revealed by molecular simulations. To simplify the resolution process and enhance the productivity, we further designed an innovative methodology for the “sequential biphasic batch resolution” of MFCC with immobilized cells. In each batch, only the aqueous phase needs to be replaced to sequentially change the immobilized cells of EstS or EstR and recover optically pure FCCAs in turn, while the organic phase was retained and MFCC was continually supplemented after every two batches. In this study, ten batches of sequential resolution were performed, and 229.3 mM (S)-FCCAs with 96.9% ee, and 224.1 mM (R)-FCCAs with 99.1% ee were obtained in 40 h, affording a 93.5% total mole yield. This is the first reported enzymatic resolution technique of FCCAs, and represents significant advantages over those chemical resolution methods.