Chiral Cyanohydrins Research Articles

Hydroxynitrile Lyase Discovery, Engineering, and Promiscuity towards Asymmetric Synthesis: Recent Progress

AbstractHydroxynitrile 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.

Asymmetric Cyanosilylation of Aldehydes by a Lewis Acid/Base Synergistic Catalyst of Chiral Metal Clusters.

Metal clusters with well-defined crystal structures are extremely useful for studying the synergistic catalytic effects and associated catalytic mechanisms. In this study, two pairs of chiral lanthanide-transition metal clusters (R)/(S)-Co3Ln2 (Ln = Tb or Dy) were synthesized using Schiff-base ligands [(R)- or (S)-H3L] with multiple Lewis base sites (O sites). The as-prepared (R)/(S)-Co3Ln2 chiral metal clusters exhibited good catalytic functionality in the asymmetric synthesis of chiral cyanohydrins, with high conversions of up to 99% and medium-to-high enantiomeric excess values of up to 78%. The catalysis process followed a mechanism in which the bifunctional metal clusters of (R)/(S)-Co3Ln2, containing Lewis acid sites and Lewis base sites, simultaneously activated the aldehydes and trimethylsilyl cyanide, respectively. Consequently, synergistic catalysis was realized. The enantioselectivity of the different aldehydes and stereochemical configuration of the resulting products are attributed to the formation of a steric chiral pocket via the external chiral ligands on the clusters. In addition, heterogeneous asymmetric cyanosilylation using (R)/(S)-Co3Ln2 chiral metal clusters achieved high chemoselectivity and regioselectivity under mild conditions.

Pickering emulsion droplet-based biomimetic microreactors for continuous flow cascade reactions

A continuous flow cascade of multi-step catalytic reactions is a cutting-edge concept to revolutionize stepwise catalytic synthesis yet is still challenging in practical applications. Herein, a method for practical one-pot cascade catalysis is developed by combining Pickering emulsions with continuous flow. Our method involves co-localization of different catalytically active sub-compartments within droplets of a Pickering emulsion yielding cell-like microreactors, which can be packed in a column reactor for continuous flow cascade catalysis. As exemplified by two chemo-enzymatic cascade reactions for the synthesis of chiral cyanohydrins and chiral ester, 5 − 420 fold enhancement in the catalysis efficiency and as high as 99% enantioselectivity were obtained even over a period of 80 − 240 h. The compartmentalization effect and enriching-reactant properties arising from the biomimetic microreactor are theoretically and experimentally identified as the key factors for boosting the catalysis efficiency and for regulating the kinetics of cascade catalysis.

A High-Throughput Screening Method for the Directed Evolution of Hydroxynitrile Lyase towards Cyanohydrin Synthesis.

Chiral cyanohydrins are useful intermediates in the pharmaceutical and agricultural industries. In nature, hydroxynitrile lyases (HNLs) are a kind of elegant tool for enantioselective hydrocyanation of carbonyl compounds. However, currently available methods for demonstrating hydrocyanation are still stalled at precise, but low-throughput, GC or HPLC analyses. Herein, we report a chromogenic high-throughput screening (HTS) method that is feasible for the cyanohydrin synthesis reaction. This method was highly anti-interference and sensitive, and could be used to directly profile the substrate scope of HNLs either in cell-free extract or fermentation clear broth. This HTS method was also validated by generating new variants of PcHNL5 that presented higher catalytic efficiency and stronger acidic tolerance in variant libraries.

Pouteria sapota hydroxynitrile lyase catalyzed synthesis of optically active cyanohydrins from ortho-substituted benzaldehydes

Chiral cyanohydrins are important building blocks in the preparation of pharmaceuticals and agrochemicals. The synthesis of optically active o-substituted mandelonitriles was carried out in a biphasic system, using the defatted meal of Pouteria sapota seeds as (R)-hydroxynitrile lyase (PsHNL) source to biocatalyze the enantioselective addition of HCN to the sterically hindered o-chloro, o-bromo, o-fluoro, o-nitro, o-methyl, and 2,4-dichlobenzaldehydes, that are poor substrates for other HNLs. The enantiomeric excess (ee) of the cyanohydrins obtained was from 98 to 100 %, except in the case of o-nitromandelonitrile (82 % ee), the conversion was from 56 % to 100 %, at the optimum conditions of 2 mL diisopropyl ether (i-pr2O), 37.5 %, of aqueous phase and ratio 60 mg/0.5 mmol of PsHNL meal/aldehyde. The relationship between molecular descriptors and bioconversion of the o-substituted benzaldehydes was analyzed using Person´s correlation (r ≤ 0.8) and path analysis, the molecular descriptors that had a highest influence over the conversion were Total size indexes (T) weighted by atomic van der Waals volume (Tv), atomic polarizability (Tp), atomic Sanderson electronegativity (Te) and unweighted (Tu). The P. sapota seed is an inexpensive source of HNL, that was able to biocatalyze the conversion of sterically hindered benzaldehydes to the corresponding enantiopure cyanohydrins.

Enantio- and Diastereoselective Construction of Contiguous Tetrasubstituted Chiral Carbons in Organocatalytic Oxadecalin Synthesis.

The organocatalytic enantio- and diastereoselective cycloetherification of 1,3-cyclohexanedione-bearing enones involving the in situ generation of chiral cyanohydrins was developed. This transformation offers the first catalytic asymmetric approach to oxadecalin derivatives containing contiguous tetrasubstituted chiral carbons at the bridge heads of the fused ring systems. Depending on substituents, both cis- and trans-decalin-type scaffolds were synthesized with good to excellent stereoselectivities, and a range of functional groups accumulated on the chiral quaternary carbon moieties of the trans-oxadecalin derivatives.

Organocatalytic Enantio- and Diastereoselective Construction of syn-1,3-Diol Motifs via Dynamic Kinetic Resolution of In Situ Generated Chiral Cyanohydrins.

An organocatalytic method for the asymmetric synthesis of syn-1,3-dioxanes as protected 1,3-diols via dynamic kinetic resolution of in situ generated chiral cyanohydrins has been developed. This method involves a reversible cyanohydrin formation/hemiacetalization/intramolecular oxy-Michael addition reaction cascade, affording a chiral syn-1,3-diol structure with simultaneous construction of two stereogenic centers. The use of trifluoromethyl ketones is crucial for the efficient three-component cascade reaction, and a chiral bifunctional organocatalyst imparts high enantio- and diastereoselectivities in the formation of the chiral syn-1,3-diol motifs.

Synthesis and Stereostructure-Activity Relationship of Novel Pyrethroids Possessing two Asymmetric Centers on a Cyclopropane Ring.

2-Methylcyclopropane pyrethroid insecticides bearing chiral cyanohydrin esters or chiral ethers and two asymmetric centers on the cyclopropane ring, were synthesized. These compounds were designed using a “reverse connection approach” between the isopropyl group in Fenvalerate, and between two dimethyl groups in an Etofenprox analogue (the methyl, ethyl form), respectively. These syntheses were achieved by accessible ring opening reactions of commercially available (±)-, (R)-, and (S)-propylene oxides using 4-chlorobenzyl cyanide anion as the crucial step, giving good overall yield of the product with >98% ee. The insecticidal activity against the common mosquito (Culex pipiens pallens) was assessed for pairs of achiral diastereomeric (1R*,2S*)-, (1R*,2R*)-cyanohydrin esters, and (1R*,2S*)-, (1R*,2R*)-ethers; only the (1R*,2R*)-ether was significantly effective. For the enantiomeric (1S,2S)-ether and (1R,2R)-ether, the activity was clearly centered on the (1R,2R)-ether. The present stereostructure‒activity relationship revealed that (i) cyanohydrin esters derived from fenvalerate were unexpectedly inactive, whereas ethers derived from etofenprox were active, and (ii) apparent chiral discrimination between the (1S,2S)-ether and the (1R,2R)-ether was observed. During the present synthetic study, we performed alternative convergent syntheses of Etofenprox and novel 4-EtO-type (1S,2S)- and (1R,2R)-pyrethroids from the corresponding parent 4-Cl-type pyrethroids, by utilizing a recently-developed hydroxylation cross-coupling reaction.

Supported Ionic Liquid‐Like Phases (SILLPs) as Immobilised Catalysts for the Multistep and Multicatalytic Continuous Flow Synthesis of Chiral Cyanohydrins

AbstractSupported Ionic Liquid‐Like Phases have been found to be efficient organocatalysts for the synthesis of cyanohydrin esters under solvent‐free conditions by an “electrophile‐nucleophile dual activation” based on hydrogen bond formation. The combination of multiple and consecutive multicatalytic steps in a single and integrated cascade process of organocatalytic SILLPs with commercially available supported Candida antarctica lipase type B (CAL‐B) has allowed developing an efficient process for the multicatalytic synthesis of enantiopure cyanohydrins under flow conditions.

Immobilized Baliospermum montanum hydroxynitrile lyase catalyzed synthesis of chiral cyanohydrins

Hydroxynitrile lyase (HNL) catalyzed enantioselective CC 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 (BmHNL) 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-BmHNL. The optimized reaction parameters were: 20 min of reaction time, 7 U of CLEA-BmHNL, 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-BmHNL biocatalysis did not improve in % ee or conversion of product unlike other CLEA-HNLs. CLEA-BmHNL 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-BmHNL has improved the enantioselectivity of (S)-mandelonitrile synthesis compared to the use of purified BmHNL. Nine aldehydes not tested earlier with BmHNL were converted into their corresponding (S)-cyanohydrins for the first time using CLEA-BmHNL. 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-BmHNL and its biocatalytic application in the synthesis of different (S)-aromatic cyanohydrins.

Enantioselective Cyanosilylation of Alkynyl Ketones Catalyzed by Combined Systems Consisting of Chiral Ruthenium(II) Complex and Lithium Phenoxide

AbstractAsymmetric cyanosilylation of alkynyl ketones with the catalyst systems consisting of amino acid/2,2′‐bis(diphenylphosphino)‐1,1′‐binaphthyl (BINAP)/ruthenium(II) complex and lithium phenoxide (Ru⋅Li cat.) was studied. The reaction was conducted in tert‐butyl methyl ether (TBME) at −78 °C with a substrate‐to‐catalyst molar ratio (S/C) as high as 2000. A series of simple and functionalized ketones was converted into the alkynyl tertiary cyanohydrin derivatives in up to 99% ee. Appropriate selection of an amino‐acid ligand of the catalyst according to the substrate structure was crucially important to achieve high enantioselectivity and a wide scope of substrates. Transformation of the chiral cyanohydrin product into a functionalized lactone was also examined.magnified image

Organocatalytic enantio- and diastereoselective cycloetherification via dynamic kinetic resolution of chiral cyanohydrins

Enantioselective approaches to synthesize six-membered oxacycles with multiple stereogenic centres are in high demand to enable the discovery of new therapeutic agents. Here we present a concise organocatalytic cycloetherification for the highly enantio- and diastereoselective synthesis of tetrahydropyrans involving simultaneous construction of two chiral centres, one of which is fully substituted. This method involves dynamic kinetic resolution of reversibly generated chiral cyanohydrins. A chiral bifunctional organocatalyst selectively recognizes a specific chair-like conformation of the intermediate, in which the small steric effect of the linear cyano group as well as its anomeric effect play important roles in controlling stereoselectivity. The products offer additional utility as synthetic intermediates because the cyano group can be further transformed into a variety of important functional groups. This strategy provides a platform to design efficient approaches to obtain a wide range of optically active tetrahydropyrans, which are otherwise synthetically challenging materials.

Enzyme discovery beyond homology: a unique hydroxynitrile lyase in the Bet v1 superfamily

Homology and similarity based approaches are most widely used for the identification of new enzymes for biocatalysis. However, they are not suitable to find truly novel scaffolds with a desired function and this averts options and diversity. Hydroxynitrile lyases (HNLs) are an example of non-homologous isofunctional enzymes for the synthesis of chiral cyanohydrins. Due to their convergent evolution, finding new representatives is challenging. Here we show the discovery of unique HNL enzymes from the fern Davallia tyermannii by coalescence of transcriptomics, proteomics and enzymatic screening. It is the first protein with a Bet v1-like protein fold exhibiting HNL activity, and has a new catalytic center, as shown by protein crystallography. Biochemical properties of D. tyermannii HNLs open perspectives for the development of a complementary class of biocatalysts for the stereoselective synthesis of cyanohydrins. This work shows that systematic integration of -omics data facilitates discovery of enzymes with unpredictable sequences and helps to extend our knowledge about enzyme diversity.

Hydroxynitrile Lyase Isozymes from Prunus communis: Identification, Characterization and Synthetic Applications

AbstractBiocatalysts originating from Badamu (Prunus communis) have been applied to catalyze the asymmetric synthesis of (R)‐4‐methylsulfanylmandelonitrile, a key building block of thiamphenicol and florfenicol. Here, four hydroxynitrile lyase (HNL) isozymes from Badamu were cloned and heterologously expressed in Pichia pastoris. The biochemical properties and catalytic performances of these isozymes were comprehensively explored to evaluate their efficiency and selectivity in asymmetric synthesis. Among then, PcHNL5 was identified with outstanding activity and enantioselectivity in asymmetric hydrocyanation. Under the optimized mild biphasic reaction conditions, seventeen prochiral aromatic aldehydes were converted to valuable chiral cyanohydrins with good yields (up to 94%) and excellent optical purities (up to >99.9% ee), which provide a facile access to numerous chiral amino alcohols, hypoglycemic agents, angiotension converting enzyme (ACE) inhibitors and β‐blockers. This work therefore underlines the importance of discovering the most potent biocatalyst among a group of isozymes for converting unnatural substrates into value‐added products.magnified image

Asymmetric counteranion-directed Lewis acid organocatalysis for the scalable cyanosilylation of aldehydes.

Due to the high versatility of chiral cyanohydrins, the catalytic asymmetric cyanation reaction of carbonyl compounds has attracted widespread interest. However, efficient protocols that function at a preparative scale with low catalyst loading are still rare. Here, asymmetric counteranion-directed Lewis acid organocatalysis proves to be remarkably successful in addressing this problem and enabled a molar-scale cyanosilylation in quantitative yield and with excellent enantioselectivity. Also, the catalyst loading could be lowered to a part-per-million level (50 ppm: 0.005 mol%). A readily accessible chiral disulfonimide was used, which in combination with trimethylsilyl cyanide, turned into the active silylium Lewis acid organocatalyst. The nature of a peculiar phenomenon referred to as a “dormant period”, which is mainly induced by water, was systematically investigated by means of in situ Fourier transform infrared analysis.

Host-Guest Assembly of a Molecular Reporter with Chiral Cyanohydrins for Assignment of Absolute Stereochemistry.

The absolute stereochemistry of cyanohydrins, derived from ketones and aldehydes, is obtained routinely, in a microscale and derivatization-free manner, upon their complexation with Zn-MAPOL, a zincated porphyrin host with a binding pocket comprised of a biphenol core. The host-guest complex leads to observable exciton-coupled circular dichroism (ECCD), the sign of which is easily correlated to the absolute stereochemistry of the bound cyanohydrin. A working model, based on the ECCD signal of cyanohydrins with known configuration, is proposed.

Improving the Properties of Bacterial R‐Selective Hydroxynitrile Lyases for Industrial Applications

AbstractHydroxynitrile lyases (HNLs) catalyse the reversible cleavage of cyanohydrins to carbonyl compounds and HCN. The recent discovery of bacterial HNLs with a cupin fold gave rise to a new promising class of these enzymes. They are interesting candidates for the synthesis of cyanohydrins on an industrial scale owing to their high expression levels in Escherichia coli. The activity and enantioselectivity of the manganese‐dependent HNL from Granulicella tundricola (GtHNL) were significantly improved by site‐saturation mutagenesis of active site amino acids. The combination of beneficial mutations resulted in a variant with 490‐fold higher specific activity in comparison to the wild‐type enzyme. More importantly, GtHNL‐A40H/V42T/Q110H is a highly competitive alternative for the synthesis of chiral cyanohydrins, such as 2‐chlorobenzaldehyde cyanohydrin, (R)‐2‐hydroxy‐4‐phenylbutyronitrile, and (R)‐2‐hydroxy‐4‐phenyl‐3‐butene nitrile, which serve as intermediates for the synthesis of pharmaceuticals.

A QM/MM study of the reaction mechanism of (R)-hydroxynitrile lyases from Arabidopsis thaliana (AtHNL).

Hydroxynitrile lyases (HNLs) catalyze the conversion of chiral cyanohydrins to hydrocyanic acid (HCN) and aldehyde or ketone. Hydroxynitrile lyase from Arabidopsis thaliana (AtHNL) is the first R-selective HNL enzyme containing an α/β-hydrolases fold. In this article, the catalytic mechanism of AtHNL was theoretically studied by using QM/MM approach based on the recently obtained crystal structure in 2012. Two computational models were constructed, and two possible reaction pathways were considered. In Path A, the calculation results indicate that the proton transfer from the hydroxyl group of cyanohydrin occurs firstly, and then the cleavage of C1-C2 bond and the rotation of the generated cyanide ion (CN(-)) follow, afterwards, CN(-) abstracts a proton from His236 via Ser81. The C1-C2 bond cleavage and the protonation of CN(-) correspond to comparable free energy barriers (12.1 vs. 12.2 kcal mol(-1)), suggesting that both of the two processes contribute a lot to rate-limiting. In Path B, the deprotonation of the hydroxyl group of cyanohydrin and the cleavage of C1-C2 bond take place in a concerted manner, which corresponds to the highest free energy barrier of 13.2 kcal mol(-1). The free energy barriers of Path A and B are very similar and basically agree well with the experimental value of HbHNL, a similar enzyme of AtHNL. Therefore, both of the two pathways are possible. In the reaction, the catalytic triad (His236, Ser81, and Asp208) acts as the general acid/base, and the generated CN(-) is stabilized by the hydroxyl group of Ser81 and the main-chain NH-groups of Ala13 and Phe82.

Discovery of a novel (R)-selective bacterial hydroxynitrile lyase from Acidobacterium capsulatum

Hydroxynitrile lyases (HNLs) are powerful carbon–carbon bond forming enzymes. The reverse of their natural reaction – the stereoselective addition of hydrogen cyanide (HCN) to carbonyls – yields chiral cyanohydrins, versatile building blocks for the pharmaceutical and chemical industry. Recently, bacterial HNLs have been discovered, which represent a completely new type: HNLs with a cupin fold. Due to various benefits of cupins (e.g. high yield recombinant expression in Escherichia coli), the class of cupin HNLs provides a new source for interesting, powerful hydroxynitrile lyases in the ongoing search for HNLs with improved activity, enantioselectivity, stability and substrate scope. In this study, database mining revealed a novel cupin HNL from Acidobacterium capsulatum ATCC 51196 (AcHNL), which was able to catalyse the (R)-selective synthesis of mandelonitrile with significantly better conversion (97%) and enantioselectivity (96.7%) than other cupin HNLs.

Expression control of nitrile hydratase and amidase genes in Rhodococcus erythropolis and substrate specificities of the enzymes.

Bacterial amidases and nitrile hydratases can be used for the synthesis of various intermediates and products in the chemical and pharmaceutical industries and for the bioremediation of toxic pollutants. The aim of this study was to analyze the expression of the amidase and nitrile hydratase genes of Rhodococcus erythropolis and test the stereospecific nitrile hydratase and amidase activities on chiral cyanohydrins. The nucleotide sequences of the gene clusters containing the oxd (aldoxime dehydratase), ami (amidase), nha1, nha2 (subunits of the nitrile hydratase), nhr1, nhr2, nhr3 and nhr4 (putative regulatory proteins) genes of two R. erythropolis strains, A4 and CCM2595, were determined. All genes of both of the clusters are transcribed in the same direction. RT-PCR analysis, primer extension and promoter fusions with the gfp reporter gene showed that the ami, nha1 and nha2 genes of R. erythropolis A4 form an operon transcribed from the Pami promoter and an internal Pnha promoter. The activity of Pami was found to be weakly induced when the cells grew in the presence of acetonitrile, whereas the Pnha promoter was moderately induced by both the acetonitrile or acetamide used instead of the inorganic nitrogen source. However, R. erythropolis A4 cells showed no increase in amidase and nitrile hydratase activities in the presence of acetamide or acetonitrile in the medium. R. erythropolis A4 nitrile hydratase and amidase were found to be effective at hydrolysing cyanohydrins and 2-hydroxyamides, respectively.