Enantiopure Cyanohydrins Research Articles

Immobilization of Arabidopsis thaliana Hydroxynitrile Lyase (AtHNL) on EziG Opal

Arabidopsis thaliana hydroxynitrile lyase (AtHNL) catalyzes the selective synthesis of (R)-cyanohydrins. This enzyme is unstable under acidic conditions, therefore its immobilization is necessary for the synthesis of enantiopure cyanohydrins. EziG Opal is a controlled porosity glass material for the immobilization of His-tagged enzymes. The immobilization of His6-tagged AtHNL on EziG Opal was optimized for higher enzyme stability and tested for the synthesis of (R)-mandelonitrile in batch and continuous flow systems. AtHNL-EziG Opal achieved 95% of conversion after 30 min of reaction time in batch and it was recycled up to eight times with a final conversion of 80% and excellent enantioselectivity. The EziG Opal carrier catalyzed the racemic background reaction; however, the high enantioselectivity observed in the recycling study demonstrated that this was efficiently suppressed by using citrate/phosphate buffer saturated methyl-tert-butylether (MTBE) pH 5 as reaction medium. The continuous flow system achieved 96% of conversion and excellent enantioselectivity at 0.1 mL min−1. Lower conversion and enantioselectivity were observed at higher flow rates. The specific rate of AtHNL-EziG Opal in flow was 0.26 mol h−1 genzyme−1 at 0.1 mL min−1 and 96% of conversion whereas in batch, the immobilized enzyme displayed a specific rate of 0.51 mol h−1 genzyme−1 after 30 min of reaction time at a similar level of conversion. However, in terms of productivity the continuous flow system proved to be almost four times more productive than the batch approach, displaying a space-time-yield (STY) of 690 molproduct h−1 L−1 genzyme−1 compared to 187 molproduct h−1 L−1 genzyme−1 achieved with the batch system.

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.

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.

Purification and characterization of a hydroxynitrile lyase from Amygdalus pedunculata Pall

Hydroxynitrile lyases (HNLs) are widely used in the asymmetric synthesis of cyanohydrins which are organic compounds used in the production of fine chemicals and pharmaceuticals, because these enzymes exhibit high catalytic efficiency and are very economical. In the present study, seeds of A. pedunculata Pall were identified as new potential source of HNLs. The HNL from A. pedunculata Pall (APHNL) was purified 138 fold and 4.20% yield with a specific activity of 661 U/mg. SDS-PAGE result showed the enzyme to be present as a monomer and the relative molecular mass determined by MALDI-TOF MS was 61 kDa. APHNL owned highest activity at pH 6.0 and at 60 °C temperature, showing activity up to 80 °C and stable up to 60 °C. APHNL has a Km of 0.5 mM, Vmax of 665.9 μmol mg−1 min−1, Kcat of 676.5 s−1 and Kcat/Km of 1353 s−1 mM−1 using mandelonitrile as substrate. Syntheses of (R)-mandelonitrile and (R)-2-Hydroxy-2-(3-phenoxy-phenyl)-acetonitrile were carried out using APHNL and molar conversion of (R)-mandelonitrile and (R)-2-Hydroxy-2-(3-phenoxy-phenyl)-acetonitrile were 90% and 98% with 94% and 93% ee, respectively. These results indicated that APHNL was an excellent biocatalyst and has very high potential for synthesis of enantiopure cyanohydrins.

Hydroxynitrile Lyase of Wild Apricot (Prunus armeniaca L.): Purification, Characterization and Application in Synthesis of Enantiopure Mandelonitrile

Hydroxynitrile lyases (HNLs) are increasingly finding application in synthesis of enantiomerically pure cyanohydrins. Cyanohydrins are important intermediates in the production of pharmaceuticals and agrochemicals. In the present studies seeds of wild apricot (Prunus armeniaca L.) have emerged as potential source of hydroxynitrile lyase. The HNL of wild apricot (ParsHNL) was purified 8.1 fold and 18.2 % yield with a specific activity of 141 units mg−1 protein. The SDS-PAGE of the enzyme revealed that it consists of subunits of 40 and 37 kDa. However, the molecular weight of holoenzyme was assessed to be 360 kDa. The enzyme showed maximum activity in 0.1 M sodium-citrate buffer having pH 4.75 at 25 °C. Thermostability studies revealed that this HNL showed activity up to 70 °C temperature and quite stable up to 50 °C. Activation energy of ParsHNL was calculated to be 37.83 kJ mol−1. This enzyme has K m of 3.76 mM, V max of 188.4 µmol mg−1 min−1 and k cat of 1130.4 s−1 using mandelonitrile as substrate while for reverse reaction using benzaldehyde as substrate it showed K m of 16.1 mM, V max of 7.21 µmol mg−1 min−1 and k cat of 43.3 s−1. Synthesis of mandelonitrile was carried out using ParsHNL and finally 8.88 mmole (1.184 g) of mandelonitrile was recovered which corresponded to 89 % molar conversion with 96 % ee for R-mandelonitrile. The yield of mandelonitrile was 411 µmol mg−1h−1. These results indicated that ParsHNL has very high potential for synthesis of cyanohydrins and can be used for the production of enantiopure cyanohydrins.

Enantioselective Titanium‐Catalyzed Cyanation Reactions of Carbonyl Compounds

AbstractThis review surveys the recent developments in enantioselective titanium‐catalyzed cyanation reactions of carbonyl compounds, covering the literature since the beginning of 2008. It well demonstrates that this reaction constitutes an important tool in organic synthesis, still attracting a considerable interest due to the potential use of enantiopure cyanohydrins as natural products and useful synthetic intermediates in the synthesis of biologically active molecules. Abbreviations: Ac: acetyl; Ar: aryl; BINOL: 1,1′‐bi‐2‐naphthol; BINOLAM: bis(diethylaminomethyl)‐1,1‘‐binaphthol; Bn: benzyl; Cy: cyclohexyl; DFT: density functional theory; DMAP: 4‐(dimethylamino)pyridine; ee: enantiomeric excess; Naph: naphthyl; r.t.: room temperature; salen: 1,2‐bis(salicylidenamino)ethane; TADDOL: α,α,α′,α′‐tetraphenyl‐2,2‐dimethyl‐1,3‐dioxolane‐4,5‐dimethanol; TEA: trimethylamine; THF: tetrahydrofuran; TMS: trimethylsilyl; Tol: tolyl.magnified image

Crosslinked enzyme aggregates of hydroxynitrile lyase partially purified from Prunus dulcis seeds and its application for the synthesis of enantiopure cyanohydrins

Hydroxynitrile lyases are powerful catalysts in the synthesis of enantiopure cyanohydrins which are key synthons in the preparations of a variety of important chemicals. The response surface methodology including three-factor and three-level Box-Behnken design was applied to optimize immobilization of hydroxynitrile lyase purified partially from Prunus dulcis seeds as crosslinked enzyme aggregates (PdHNL-CLEAs). The quadratic model was developed for predicting the response and its adequacy was validated with the analysis of variance test. The optimized immobilization parameters were initial glutaraldehyde concentration, ammonium sulfate saturation concentration, and crosslinking time, and the response was relative activity of PdHNL-CLEA. The optimal conditions were determined as initial glutaraldehyde concentration of 25% w/v, ammonium sulfate saturation concentration of 43% w/v, and crosslinking time of 18 h. The preparations of PdHNL-CLEA were examined for the synthesis of (R)-mandelonitrile, (R)-2-chloromandelonitrile, (R)-3,4-dihydroxymandelonitrile, (R)-2-hydroxy-4-phenyl butyronitrile, (R)-4-bromomandelonitrile, (R)-4-fluoromandelonitrile, and (R)-4-nitromandelonitrile from their corresponding aldehydes and hydrocyanic acid. After 96-h reaction time, the yield-enantiomeric excess values (%) were 100-99, 100-21, 100-99, 83-91, 100-99, 100-72, and 100-14%, respectively, for (R)-mandelonitrile, (R)-2-chloromandelonitrile, (R)-3,4-dihydroxymandelonitrile, (R)-2-hydroxy-4-phenyl butyronitrile, (R)-4-bromomandelonitrile, (R)-4-fluoromandelonitrile, and (R)-4-nitromandelonitrile. The results show that PdHNL-CLEA offers a promising potential for the preparation of enantiopure (R)-mandelonitrile, (R)-3,4-dihydroxymandelonitrile, (R)-2-hydroxy-4-phenyl butyronitrile, and (R)-4-bromomandelonitrile with a high yield and enantiopurity.

Hydroxynitrile Lyase‐Catalyzed Synthesis of Enantiopure Cyanohydrins in Biocatalytic Active Static Emulsions (BASE) with Suppression of the Non‐Enzymatic Side Reaction

AbstractThe use of biocatalytic active static emulsions (BASE) was investigated for the hydroxynitrile lyase‐catalyzed synthesis of enantiopure cyanohydrins. With this technique a full suppression of the undesired racemic non‐enzymatic side reaction was facilitated, even at unusually high pH within the aqueous phase. Thus the full catalytic potential at the optimum pH was achieved with the hydroxynitrile lyase from Manihot esculenta. As proof of principle, a series of carbonyl compounds was successfully converted to the corresponding cyanohydrins with high yields and enantioselectivities, including several benzaldehyde derivatives and aliphatic ketones. Furthermore, several mandelic acid derivatives were obtained on a multi‐gram scale after hydrolysis, which proves the synthetic applicability of BASE‐immobilized hydroxynitrile lyases.magnified image

Purification, immobilization and characterization of (R)-hydroxynitrile lyase from Prunus amygdalus turcomanica seeds and their applicability for synthesis of enantiopure cyanohydrins

Abstract A hydroxynitrile lyase (HNL) was purified from wild almond seeds (Prunus amygdalus turcomanica Lincz.) for the first time. Native and subunit molecular masses of the HNL were determined as 100 and 25 kDa, respectively indicating that the enzyme is a homotetramer. The purified enzyme was immobilized onto Eupergit CM and Eupergit C 250 L supports and their lyase and carboligation (synthetic) activities were characterized in terms of optimal pH, temperature and kinetic parameters. While the optimal pH of the free HNL for the lyase activity was 6.0, it was 5.5 for both of the immobilized HNLs. Optimal temperature was determined as 25 °C for all HNL preparations. For mandelonitrile cleavage, the apparent Km – Vmax values were 0.38 mM – 197.0 U mg protein−1 for the free HNL, 1.30 mM – 26.0 U mg protein−1 for HNL immobilized onto Eupergit CM (HNL-Eup CM) and 0.95 mM – 17.5 U mg protein−1 for HNL immobilized onto Eupergit C 250 L (HNL-Eup C 250 L), respectively. For the carboligation activity, the optimal pH was measured as 4.0 and optimal temperature was determined as 5 °C for all of the HNL preparations. For mandelonitrile synthesis, the apparent Km – Vmax values were 14.0 mM – 2.70 U mg protein−1 for the free HNL, 41.0 mM – 0.49 U mg protein−1 for HNL-Eup CM and 38.0 mM – 0.54 U mg protein−1 for HNL-Eup C 250 L, respectively. All of the HNL preparations were employed for the synthesis of mandelonitrile, 2-chloromandelonitrile, 3,4-dihydroxymandelonitrile and 2-hydroxy-4-phenyl butyronitrile in a biphasic tert-butyl methyl ether–citrate buffer (pH 4.0) medium. The results showed that the immobilized HNL preparations were better than the free HNL in the synthesis of abovementioned cyanohydrins except 2-chloromandelonitrile with higher yields and enantiopurities.

Biochemical and structural characterization of a novel bacterial manganese‐dependent hydroxynitrile lyase

Hydroxynitrile lyases (HNLs), which catalyse the decomposition of cyanohydrins, are found mainly in plants. In vitro, they are able to catalyse the synthesis of enantiopure cyanohydrins, which are versatile building blocks in the chemical industry. Recently, HNLs have also been discovered in bacteria. Here, we report on the detailed biochemical and structural characterization of a hydroxynitrile lyase from Granulicella tundricola (GtHNL), which was successfully heterologously expressed in Escherichia coli. The crystal structure was solved at a crystallographic resolution of 2.5 Å and exhibits a cupin fold. As GtHNL does not show any sequence or structural similarity to any other HNL and does not contain conserved motifs typical of HNLs, cupins represent a new class of HNLs. GtHNL is metal-dependent, as confirmed by inductively coupled plasma/optical emission spectroscopy, and in the crystal structure, manganese is bound to three histidine and one glutamine residue. GtHNL displayed a specific activity of 1.74 U·mg(-1) at pH 6 with (R)-mandelonitrile, and synthesized (R)-mandelonitrile with 90% enantiomeric excess at 80% conversion using 0.5 m benzaldehyde in a biphasic reaction system with methyl tertiary butyl ether.

Chemoenzymatic asymmetric synthesis of fluoxetine, atomoxetine, nisoxetine, and duloxetine

The asymmetric synthesis of two well-known anti-depressant drugs, fluoxetine and duloxetine has been accomplished in a chemoenzymatic manner. The main highlight of the synthesis is the enantioselective cyanohydrin formation by a plant (R)-HNL (hydroxynitrile lyase). The enantiopure cyanohydrins are then synthetically manipulated into the above two drug molecules and two of their structural analogues, atomoxetine and nisoxetine.

Synthesis of Methoxyisopropyl (MIP)-Protected (R)-Mandelonitrile and Derivatives in a Flow Reactor

Cyanohydrins are synthetically versatile chiral building blocks in organic synthesis. They can be conveniently synthesized in enantiomerically pure form via chemoenzymatic hydrogen cyanide addition onto the corresponding aldehyde using hydroxynitrile lyase. Recently, we reported that such transformations can be efficiently carried out in a continuous flow manner using microreactors. Since racemization of enantiopure cyanohydrins occurs readily under slightly basic conditions, they should be protected before the follow-up reactions, preferably under acidic conditions. In this contribution, we demonstrate that the methoxyisopropyl protection of mandelonitrile can be conveniently optimized in an automated microscale continuous flow system and subsequently scaled up under the same conditions by applying a larger flow reactor.

Hydroxynitrile lyases with α/β-hydrolase fold: two enzymes with almost identical 3D structures but opposite enantioselectivities and different reaction mechanisms.

Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins to yield hydrocyanic acid (HCN) and the respective carbonyl compound and are key enzymes in the process of cyanogenesis in plants. In organic syntheses, HNLs are used as biocatalysts for the formation of enantiopure cyanohydrins. We determined the structure of the recently identified, R-selective HNL from Arabidopsis thaliana (AtHNL) at a crystallographic resolution of 2.5 Å. The structure exhibits an α/β-hydrolase fold, very similar to the homologous, but S-selective, HNL from Hevea brasiliensis (HbHNL). The similarities also extend to the active sites of these enzymes, with a Ser-His-Asp catalytic triad present in all three cases. In order to elucidate the mode of substrate binding and to understand the unexpected opposite enantioselectivity of AtHNL, complexes of the enzyme with both (R)- and (S)-mandelonitrile were modeled using molecular docking simulations. Compared to the complex of HbHNL with (S)-mandelonitrile, the calculations produced an approximate mirror image binding mode of the substrate with the phenyl rings located at very similar positions, but with the cyano groups pointing in opposite directions. A catalytic mechanism for AtHNL is proposed, in which His236 from the catalytic triad acts as a general base and the emerging negative charge on the cyano group is stabilized by main-chain amide groups and an α-helix dipole very similar to α/β-hydrolases. This mechanistic proposal is additionally supported by mutagenesis studies.

Synthesis of enantiomerically enriched (R)- and (S)-benzofuranyl- and benzo[b]thiophenyl-1,2-ethanediols via enantiopure cyanohydrins as intermediates

The chemoenzymatic synthesis of highly enantiopure (R)- and (S)-benzofuranyl- and benzo[b]thiophenyl-cyanohydrins and their chemical transformation into the corresponding heteroaryl-1,2-ethanediols is described.

How to overcome limitations in biotechnological processes - examples from hydroxynitrile lyase applications

During the last decades, enzymes became very versatile catalysts for a variety of reactions including natural and unnatural compounds. However, many enzyme-catalysed reactions suffer from diverse restrictions because of limitations related to process parameters or the enzyme. The understanding and overcoming of those undesired side effects is therefore mandatory for the implementation of optimal process parameters. To achieve this aim, various methods from molecular biology and reaction engineering can be employed. By focusing on the hydroxynitrile lyase-catalysed synthesis of enantiopure cyanohydrins, we give an overview of strategies to improve commercially utilized enzymes and to suppress non-enzymatic reactions. Particular emphasis is placed on the necessity to combine approaches from different fields, such as enzyme engineering and reaction engineering.

Potential and capabilities of hydroxynitrile lyases as biocatalysts in the chemical industry

The application of hydroxynitrile lyases (HNLs) as catalysts for the stereoselective condensation of HCN with carbonyl compounds has been reported as early as 1908. This enzymatic C-C bond coupling reaction furnishes enantiopure cyanohydrins which serve as versatile bifunctional building blocks for chemical synthesis. Screening of natural sources led to the discovery of both (R)- and (S)-selective HNLs, and several distinctly different classes of these enzymes with substantial differences concerning sequence, structure, and mechanism have been found. Especially during the last two centuries, HNLs have been developed into valuable biocatalysts, which can be produced in recombinant form by overexpression in microbial hosts, resulting in the implementation of industrial processes utilizing these enzymes. Recently, protein engineering in combination with in silico methods gave rise to the development of a tailor-made HNL for large-scale manufacturing of a specific target cyanohydrin.

Study of an Enzyme Activity in Extracts of Ginkgo biloba Leaves

Biocatalyst is a strong tool for preparation of enantiopure compounds through kinetic resolution or direct synthesis. However, enzymes do not always satisfy the needs of researchers for this purpose because they often do not have enough activity or selectivity toward unnatural substrates. To solve the problems, it is important to find new enzymes from nature, although researchers have tried to change enzyme activity or selectivity by mutagenesis such as random mutagenesis and rational design. Researchers have isolated a variety of enzymes from various sources such as bacteria, fungi, mammals, and plants. Diabetic enzymes such as lipases and proteases from bacteria and mammals are well characterized and utilized successfully in academic and industrial areas. The enzymes involved in mechanisms for protecting plants from insect or fungal attack are also useful in organic synthesis. Such enzymes generally catalyze reactions to produce toxic chemicals in the last step of secondary metabolism to kill predators. As an example, hydroxynitrilases from cyanogenic plants catalyze the biodegradation of cyanogenic glycosides to release hydrogen cyanide. The reverse reaction is interesting in organic synthesis because hydroxynitrilases catalyze formation of enantiopure cyanohydrins from aldehydes or ketones and hydrogen cyanide. Searching enzymes involved in the secondary metabolism of plants may provide useful biocatalysts in organic synthesis because the enzymes have a key role for synthesis of many biologically active compounds. Ginkgo biloba leaves have high anti-insect activity and biological activity. Thus, they might contain an interesting enzyme involved in the secondary metabolism and the enzyme can be useful for organic synthesis. We have chosen Ginkgo biloba leaves for finding a new enzyme because Ginkgo biloba leaves potentially contain enzymes involved in the secondary metabolism and also the study of the enzymatic activity of extracts from Ginkgo leaves has not been focused while the chemicals in them have been well characterized. Herein, we represent an enzyme activity in Ginkgo biloba leaves. Most enzymes are classified into six groups, such as oxidoreductase, transferases, hydrolases, lyases, isomerases, and ligases. Activities for oxidoreductase, hydrolase, and lyase were chosen for screening enzyme activities in Ginkgo biloba leaves because these enzymes are important in organic synthesis. We assayed the activities of peroxidase for oxidoreductase, β-amylase, nitrilase, and esterase for hydrolase, and hydroxynitrilase for lyase. Ginkgo biloba leaves were ground by a homogenizer and washed with ethyl acetate to remove organic components. Ginkgo powders were extracted with the reaction buffers for activity screening. Under the assay condition tested, Ginkgo extracts showed only the peroxidase activity (Table 1). In the assay condition, peroxidases oxidize pyrogallol to purpurogallin using hydrogenperoxide (eq. 1). The absorbance change can be monitored at 420 nm. The reaction coordinate showed clear difference between the reaction by Ginkgo extracts and the blank reaction (Figure 1).

Biocatalytic hydrolysis of cyanohydrins: an efficient approach to enantiopure α-hydroxy carboxylic acids

Rhodococcus erythropolis NCIMB 11540 was found to have a highly active nitrile hydratase/amidase enzyme system present which accepts the nitrile function of α-hydroxynitriles (cyanohydrins) as substrates. This biocatalytic hydrolysis using whole bacterial cells leads to α-hydroxy carboxylic acids which are much valued chiral building blocks in organic synthesis. Employing enantiopure cyanohydrins, which are easy available using ( R)- or ( S)-hydroxynitrile lyases, the products were obtained in high yield without racemization, decomposition or side reactions. Herein, the application of this biotransformation for preparative scale applications is described. To clarify the substrate acceptance of the nitrile hydrolyzing enzymes of R. erythropolis NCIMB 11540, several selected model compounds were subjected to biocatalytic hydrolysis. Reaction conditions were optimized to enable preparative scale conversions. In this manner, ( R)-2-chloromandelic acid and ( R)-2-hydroxy-4-phenylbutyric acid, two important pharmaceutical intermediates, were prepared in a gram scale. The substrate concentrations used were 9.3 and 13 g/l, respectively. The process yielded both acids in high optical (ee>99 and 98%) and chemical (98%) yield after short reaction times (3 and 1.5 h).