An Effective Chemo-Enzymatic method with An Evolved L-Threonine Aldolase for Preparing L-threo-4-Methylsulfonylphenylserine Ethyl Ester of High Optical Purity

Abstract

L-threo-4-methylsulfonylphenylserine ethyl ester [(2S, 3R)-1d], a key building block of florfenicol which synthesized by esterification using ethanol and L-threo-4-methylsulfonylphenylserine [(2S, 3R)-1b] as substrates. L-threonine aldolase (LTA) is a promising biocatalyst for producing (2S, 3R)-1b through a one-step process taking 4-Methylsulphonyl benzaldehyde (1a) and glycine as substrates under the mild condition. However, the moderate Cβ-stereoselective blocked the industrial application of LTA. To address this issue, rational design by combination with prereaction state molecular dynamics (MD) simulation and per-residue energy decomposition algorithm are employed to engineer LTA for enhancing Cβ-stereoselective. As a result, a triple mutant N16A/E98S/Y314R (Mu3) is screened out to produce (2S, 3R)-1b with 93.7% de and 90.2% conversion under the 300 mM 1a substrate loading. Furthermore, esterification is applied to synthesize (2S, 3R)-1d with over 99% chiral purity and 99% product purity. The success of this study provides new insights for the rational design of LTA with improved Cβ-stereoselectivity and proves a chemo-enzymatic route for green synthesis of (2S, 3R)-1d.