Bienzymatic Cascade for the Synthesis of an Optically Active O-benzoyl Cyanohydrin

Abstract

A concurrent bienzymatic cascade for the synthesis of optically pure (S)-4-methoxymandelonitrile benzoate ((S)-3) starting from 4-anisaldehyde (1) has been developed. The cascade involves an enantioselective Manihot esculenta hydroxynitrile lyase-catalyzed hydrocyanation of 1, and the subsequent benzoylation of the resulting cyanohydrin (S)-2 catalyzed by Candida antarctica lipase A in organic solvent. To accomplish this new direct synthesis of the protected enantiopure cyanohydrin, both enzymes were immobilized and each biocatalytic step was studied separately in search for a window of compatibility. In addition, potential cross-interactions between the two reactions were identified. Optimization of the cascade resulted in 81% conversion of the aldehyde to the corresponding benzoyl cyanohydrin with 98% enantiomeric excess.

Multi-Catalytic Route for the Synthesis of (S)-Tembamide

“…Once all hydrogenation conditions had been optimized, we aimed for a one-pot, two-step catalytic synthesis of (S)-tembamide. Based on our previous studies [36], the enzymatic cascade leading to (S)-3 afforded a crude solution containing approximately 80-85 mM (S)-3, 10-15 mM 2, 5-10 mM 1, 180 mM phenyl benzoate, 40 mM benzoic acid and a maximum of 550 mM HCN. Given the high concentration of HCN and its susceptibility to hydrogenation [62,63], its effect on the hydrogenation of 3 was evaluated.…”

“…Hydroxynitrile lyase from Manihot esculenta was heterologously expressed in E. coli K12 Top 10F' harboring the pSE420-MeHNL plasmid, which was kindly provided by the Austrian Centre of Industrial Biotechnology (ACIB GmbH), Graz, Austria. The procedure is described in detail in our previous publication [36]. Candida antarctica lipase A (Novozym ® CALA L, LDN 00025) was purchased from Novozymes A/S, Bagsvaerd, Denmark.…”

“…The combination of MeHNL and CALA to convert aldehyde 1 into (S)-3 in organic solvent could be accomplished at low water activity by carefully selecting the acylating agent and further optimizing the reaction conditions [36]. The present work focuses on the identification of a proper catalytic system to reduce the chiral benzoylated cyanohydrin (S)-3 to form (S)-tembamide.…”

“…Considering this trend, a two-step route to (S)-tembamide ((S)-4) starting from 4-anisaldehyde (1) was envisioned (see Scheme 1). The first step consists in a concurrent bi-enzymatic synthesis of (S)-4-methoxymandelonitrile benzoate ((S)-3), using immobilized Manihot esculenta hydroxynitrile lyase (MeHNL) and Candida antarctica lipase A (CALA) as catalysts [36]. In a second step, the nitrile group is catalytically reduced to give (S)-tembamide.…”

“…While metal hydrides are generally effective, their use results in the co-production of stoichiometric amounts of metal salts, which may cause elaborate work-up procedures. The alternative catalytic hydrogenation typically yields secondary and tertiary amines as side products, due to the The first step consists in a concurrent bi-enzymatic synthesis of (S)-4-methoxymandelonitrile benzoate ((S)-3), using immobilized Manihot esculenta hydroxynitrile lyase (MeHNL) and Candida antarctica lipase A (CALA) as catalysts [36]. In a second step, the nitrile group is catalytically reduced to give (S)-tembamide.…”