Hydroxynitrile lyase engineering for promiscuous asymmetric Henry reaction with enhanced conversion, enantioselectivity and catalytic efficiency.

Arabidopsis thaliana hydroxynitrile lyase (AtHNL) engineering has uncovered variants that showed up to 12-fold improved catalytic efficiency than the wild-type towards asymmetric Henry reaction. The AtHNL variants have displayed excellent enantioselectivity, up to >99%, and higher conversion in the synthesis of 13 different (R)-ß-nitroalcohols from their corresponding aldehydes. Using cell lysates of Y14M/F179W, we demonstrated a preparative scale synthesis of (R)-1-(4-methoxyphenyl)-2-nitroethanol, a tembamide chiral intermediate, in >99% ee and 52% yield.

Biocatalytic approaches for enantio and diastereoselective synthesis of chiral ß-nitroalcohols.

Chiral ß-nitroalcohols find significant application in organic synthesis due to the versatile reactivity of hydroxyl and nitro functionalities attached to one or two vicinal asymmetric centers. They are key building blocks of several important pharmaceuticals, bioactive molecules, and fine chemicals. With the growing demand to develop clean and green methods for their synthesis, biocatalytic methods have gained tremendous importance among the existing asymmetric synthesis routes. Over the years, different biocatalytic strategies for the asymmetric synthesis of ß-nitroalcohol stereoisomers have been developed. They can be majorly classified as (a) kinetic resolution, (b) dynamic kinetic resolution, (c) Henry reaction, (d) retro-Henry reaction, (e) asymmetric reduction, and (f) enantioselective epoxide ring-opening. This review aims to provide an overview of the above biocatalytic strategies, and their comparison along with future prospects. Essentially, it presents an enzyme-toolbox for the asymmetric synthesis of ß-nitroalcohol enantiomers and diastereomers.

Immobilized Arabidopsis thaliana Hydroxynitrile Lyase-Catalyzed Retro-Henry Reaction in the Synthesis of (S)-ß-Nitroalcohols.

Enantiopure ß-nitroalcohols are versatile intermediates used in the synthesis of important pharmaceuticals and chiral synthons. In this article, immobilized Arabidopsis thaliana HNL (AtHNL)-catalyzed preparation of (S)-ß-nitroalcohols from their racemic mixtures via retro-Henry reaction was studied. AtHNL used in biocatalysis was immobilized by physical adsorption in inexpensive celite®545. Under optimized biocatalytic conditions, the total turnover number of the catalyst has improved 2.3-fold for (S)-2-nitro-1-phenylethanol (NPE) synthesis, than free enzyme catalysis. This study reported for the first time celite-AtHNL-catalyzed retro-Henry reaction at low pH. At pH 4.5 and 5.0, 62% ee and 41% conversion, and 97% ee and 42% conversion of (S)-NPE were obtained respectively, while the free enzyme inactivates at pH < 5.0. The increased catalytic efficiency and pH stability of the catalyst could be possibly due to increased stability of AtHNL by immobilization. A dozen of racemic ß-nitroalcohols were converted into their corresponding (S)-ß-nitroalcohols using this reaction; among them, eight were not tested earlier. The immobilized enzyme has showed broad substrate selectivity in the retro-Henry reaction, and products were obtained up to 98.5% ee.

Production of (S)-ß-Nitro Alcohols by Enantioselective C-C Bond Cleavage with an R-Selective Hydroxynitrile Lyase.

Hydroxynitrile lyase (HNL)-catalysed stereoselective synthesis of ß-nitro alcohols from aldehydes and nitroalkanes is considered an efficient biocatalytic approach. However, only one S-selective HNL-Hevea brasiliensis (HbHNL)-exists that is appropriate for the synthesis of (S)-ß-nitro alcohols from the corresponding aldehydes. Further, synthesis catalysed by HbHNL is limited by low specific activity and moderate yields. We have prepared a number of (S)-ß-nitro alcohols, by kinetic resolution with the aid of an R-selective HNL from Arabidopsis thaliana (AtHNL). Optimization of the reaction conditions for AtHNL-catalysed stereoselective C-C bond cleavage of racemic 2-nitro-1-phenylethanol (NPE) produced (S)-NPE (together with benzaldehyde and nitromethane, largely from the R enantiomer) in up to 99 % ee and with 47 % conversion. This is the fastest HNL-catalysed route known so far for the synthesis of a series of (S)-ß-nitro alcohols. This approach widens the application of AtHNL for the synthesis not only of (R)- but also of (S)-ß-nitro alcohols from the appropriate substrates. Without the need for the discovery of a new enzyme, but rather by use of a retro-Henry approach, it was used to generate a number of (S)-ß-nitro alcohols by taking advantage of the substrate selectivity of AtHNL.