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Hydroxynitrile lyases (HNLs) primarily catalyze the stereoselective synthesis of cyanohydrins, but some of them also carry out asymmetric nitroaldol synthesis (Henry reaction). By virtue of the importance of asymmetric C−C bond formation, and the wide application of chiral cyanohydrins and β‐nitroalcohols, HNLs have gained significance in organic synthesis. Over the years new HNLs have been discovered and their synthetic scope are evaluated. Engineering of HNLs has contributed towards increasing not only the catalytic toolbox but also broad substrate selectivity, stereoselectivity, enzyme stability, and application of the enzymes for different transformations other than the conventional ones. This review describes the recent discoveries of diverse hydroxynitrile lyases (HNLs) and the developments to date since 2016, with respect to their biocatalytic applications in the asymmetric synthesis using both natural and promiscuous reactions. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Preparation and characterization of CLEA- Bm HNL reported first time. • Reusability of CLEA- Bm HNL in (S)-cyanohydrin synthesis up to eight cycles without loss of conversion. • CLEA has improved enantioselectivity of (S)-cyanohydrin synthesis compared to the use of purified enzyme. • Among the 11 (S)-cyanohydrins synthesized here, 8 were not synthesized by any CLEA-HNL and 9 were not tested by BmHNL earlier. Abstract Hydroxynitrile lyase (HNL) catalyzed enantioselective C C bond formation is an efficient approach to synthesize chiral cyanohydrins which are important building blocks in the synthesis of a number of fine chemicals, agrochemicals and pharmaceuticals. Immobilization of HNL is known to provide robustness, reusability and in some cases also enhances activity and selectivity. We optimized the preparation of immobilization of Baliospermium montanum HNL (Bm HNL) by cross linking enzyme aggregate (CLEA) method and characterized it by SEM. Optimization of biocatalytic parameters was performed to obtain highest % conversion and ee of (S)-mandelonitrile from benzaldehyde using CLEA- Bm HNL. The optimized reaction parameters were: 20 min of reaction time, 7 U of CLEA- Bm HNL, 1.2 mM substrate, and 300 mM citrate buffer pH 4.2, that synthesized (S)-mandelonitrile in ∼99% ee and ∼60% conversion. Addition of organic solvent in CLEA- Bm HNL biocatalysis did not improve in % ee or conversion of product unlike other CLEA-HNLs. CLEA- Bm HNL could be successfully reused for eight consecutive cycles without loss of conversion or product formation and five cycles with a little loss in enantioselectivity. Eleven different chiral cyanohydrins were synthesized under optimal biocatalytic conditions in up to 99% ee and 59% conversion, however the % conversion and ee varied for different products. CLEA- Bm HNL has improved the enantioselectivity of (S)-mandelonitrile synthesis compared to the use of purified Bm HNL. Nine aldehydes not tested earlier with Bm HNL were converted into their corresponding (S)-cyanohydrins for the first time using CLEA- Bm HNL. Among the eleven (S)-cyanohydrins syntheses reported here, eight of them have not been synthesized by any CLEA-HNL. Overall, this study showed preparation, characterization of a stable, robust and recyclable biocatalyst i.e. CLEA- Bm HNL and its biocatalytic application in the synthesis of different (S)-aromatic cyanohydrins. [ABSTRACT FROM AUTHOR]

Abstract This study presents asymmetric synthesis of cyanohydrins in aqueous–organic biphasic system using crude Baliospermum montanum hydroxynitrile lyase (Bm HNL). The effect of various biocatalytic parameters e.g. different organic solvents, ratio of organic solvents, substrate concentration (benzaldehyde and HCN), pH, and temperature were studied to achieve highest % conversion and enantiomeric excess of (S)-mandelonitrile. Among the organic solvents i.e. hexane, toluene, acetonitrile, tert -butyl methyl ether (TBME), di -isopropyl ether (DIPE) and n -butyl acetate studied, 10% n -butyl acetate showed best results. Optimal results were also found with 0.8 mM benzaldehyde, 10 min reaction time, pH 4.2 and substrate to KCN ratio of 1:125. Under optimal biocatalytic conditions more than a dozen of different aromatic aldehydes were converted into corresponding chiral (S)-cyanohydrins. This method also reports the synthesis of eight chiral (S)-cyanohydrins for the first time, not synthesized by any (S)-selective HNL earlier. Graphical abstract fx1 Highlights • Bm HNL catalyzed synthesis of (S)-cyanohydrins was studied for the first time in biphasic media. • Use of crude Bm HNL in biocatalysis have resulted in comparable % ee and conversion of products with that of the use of purified enzymes in case of a few substrates. • Sixteen (S)-cyanohydrins were synthesized using Bm HNL in biphasic media. • Thirteen (S)-cyanohydrins were synthesized by Bm HNL for the first time and eight among them have not been synthesized by any other (S)-selective HNL except this report. [ABSTRACT FROM AUTHOR]