Efficient Asymmetric Synthesis of Ethyl (S)-4-Chloro-3-hydroxybutyrate Using Alcohol Dehydrogenase SmADH31 with High Tolerance of Substrate and Product in a Monophasic Aqueous System

Abstract

Bioreductions catalyzed by alcohol dehydrogenases (ADHs) play an important role in the synthesis of chiral alcohols. However, the synthesis of ethyl (S)-4-chloro-3-hydroxybutyrate [(S)-CHBE], an important drug intermediate, has significant challenges concerning high substrate or product inhibition toward ADHs, which complicates its production. Herein, we evaluated a novel ADH, SmADH31, obtained from the Stenotrophomonas maltophilia genome, which can tolerate extremely high concentrations (6 M) of both substrate and product. The coexpression of SmADH31 and glucose dehydrogenase from Bacillus subtilis in Escherichia coli meant that as much as 660 g L–1 (4.0 M) ethyl 4-chloroacetoacetate was completely converted into (S)-CHBE in a monophasic aqueous system with a >99.9% ee value and a high space-time yield (2664 g L–1 d–1). Molecular dynamics simulation shed light on the high activity and stereoselectivity of SmADH31. Moreover, five other optically pure chiral alcohols were synthesized at high concentrations (100–462 g L–1) as a result of the broad substrate spectrum of SmADH31. All these compounds act as important drug intermediates, demonstrating the industrial potential of SmADH31-mediated bioreductions.

Single‐Point Mutant Inverts the Stereoselectivity of a Carbonyl Reductase toward β‐Ketoesters with Enhanced Activity

“…For instance, methyl ( R )‐3‐hydroxybutanoate is a precursor of antibiotic candidate (−)‐albocycline; [17] whereas methyl ( S )‐3‐hydroxybutanoate is an important synthon for the preparation of chaetoviridin A [18] . Ethyl ( R )‐4‐chloro‐3‐hydroxybutyrate serves as a versatile precursor for compounds with pharmacological activity, such as l ‐carnitine, ( R )‐4‐amino‐3‐hydroxybutyric acid, and ( R )‐4‐hydroxy‐pyrrolidone; [19] whereas its ( S )‐stereoisomer is a key intermediate to synthesize the cholesterol‐lowering blockbuster drug atorvastatin [20] . The demand for each stereoisomer of chiral β‐hydroxy esters inevitably leads to the demand for the specific enzymes.…”

“…[18] Ethyl (R)-4-chloro-3-hydroxybutyrate serves as av ersatile precursor forc ompounds with pharmacological activity,s uch as l-carnitine, (R)-4-amino-3-hydroxybutyric acid, and (R)-4-hydroxy-pyrrolidone; [19] whereas its (S)-stereoisomer is ak ey intermediate to synthesize the cholesterol-lowering blockbuster drug atorvastatin. [20] The demandf or each stereoisomer of chiral b-hydroxy esters inevitably leads to the demandf or the specific enzymes. Although enzyme-catalyzed asymmetricr eduction of b-ketoesters to chiral b-hydroxy esters has been described previously, [21] studies specifically involvingt he nature of enzyme stereo-recognition toward b-ketoesters are seldom reported.…”

Alcohol Dehydrogenases as Catalysts in Organic Synthesis

“…The large repertoire of halohydrins derived from ADHcatalysed reduction of the corresponding ketones (Figure 12) also includes propargylic chlorohydrins (Schubert et al, 2002) and compounds bearing functional groups such as esters (Yang et al, 2020), amino-protected groups (Patel, 2001;de Miranda et al, 2015) and heteroaryl moieties (Borzecka et al, 2013). Importantly, some relevant chlorohydrins have been produced by ADH in practical scale, such as the enantiopure ethyl (S)-chloroacetoacetate (Yang et al, 2020), a chiral synthetic intermediate of the "blockbuster" drug atorvastatin, and (S)-2chloro-1-(2,4-dichlorophenyl) ethanol (Zheng et al, 2021), an intermediate of the antifungal agent luliconazole.…”

Application of Ketoreductase in Asymmetric Synthesis of Pharmaceuticals and Bioactive Molecules: An Update (2018–2020)

“…Wang and co‐workers recently discovered a novel KRED named Sm ADH31 from Stenotrophomonase maltophilia and successfully expressed it heterologously in E. coli BL21 (DE3) [12] . Sm ADH31 was identified to possess a broad substrate spectrum with anti‐Prelog selectivity.…”