阅读说明：本技术 (E)-α-氰基-β-芳基丙烯酰胺类化合物的绿色制备方法 ((E) Green preparation method of (E) -alpha-cyano-beta-aryl acrylamide compound ) 是由 贾娴 游松 左伟国 邢亚洁 杨顺彬 肖茜雯 范多纳 于 2021-01-22 设计创作，主要内容包括：本发明属于化学合成技术领域,涉及(E)-α-氰基-β-芳基丙烯酰胺类化合物的绿色制备方法,具体涉及基于金属卟啉-酶连续催化法的(E)-α-氰基-β-芳基丙烯酰胺类化合物的绿色制备方法。本发明所述的方法以芳香醛和丙二腈为底物,以卟啉或金属卟啉为催化剂,在无溶剂条件下发生Knoevenagel反应,生成的芳基亚甲基丙二腈类化合物用于腈水合酶催化下的水合反应,可选择性地生成(E)-α-氰基-β-芳基丙烯酰胺类化合物。本发明利用腈水合酶对芳基亚甲基丙二腈类化合物进行生物转化,可以合成高顺反选择性的(E)-α-氰基-β-芳基丙烯酰胺类化合物,其转化率>99％,顺反选择性>99％。(The invention belongs to the technical field of chemical synthesis, and relates to a green preparation method of (E) -alpha-cyano-beta-aryl acrylamide compounds, in particular to a green preparation method of (E) -alpha-cyano-beta-aryl acrylamide compounds based on a metalloporphyrin-enzyme continuous catalysis method. According to the method, aromatic aldehyde and malononitrile are used as substrates, porphyrin or metalloporphyrin is used as a catalyst, Knoevenagel reaction is carried out under the solvent-free condition, the generated aryl methylene malononitrile compound is used for hydration reaction under the catalysis of nitrile hydratase, and the (E) -alpha-cyano-beta-aryl acrylamide compound can be selectively generated. The invention relates to aryl methylene malononitrile by using nitrile hydrataseThe compound is subjected to biotransformation to synthesize the (E) -alpha-cyano-beta-aryl acrylamide compound with high cis-trans selectivity, and the transformation rate of the compound>99% cis-trans selectivity>99％。)

A green process for producing (E) -alpha-cyano-beta-arylacrylamide compounds,

according to the method, aromatic aldehyde and malononitrile are used as substrates, porphyrin or metalloporphyrin is used as a catalyst, Knoevenagel reaction is carried out under the solvent-free condition, and the generated aryl methylene malononitrile compound is used for hydration reaction under the catalysis of nitrile hydratase to generate the (E) -alpha-cyano-beta-aryl acrylamide compound.

2. The method of claim 1, comprising the steps of:

(1) fully mixing aromatic aldehyde and malononitrile according to a certain proportion, taking porphyrin or metalloporphyrin as a catalyst, and carrying out Knoevenagel reaction at 30-80 ℃ under the condition of no solvent for 0.5-6 hours to obtain an aryl methylene malononitrile compound;

(2) dissolving the compound obtained in the step (1) in a proper amount of solvent, and filtering out porphyrin or metalloporphyrin to obtain a solution of an arylmethylenemalononitrile compound; adding a proper amount of the solution into a phosphate buffer solution system of a biocatalyst nitrile hydratase, carrying out hydration reaction, and extracting with ethyl acetate to obtain the (E) -alpha-cyano-beta-aryl acrylamide compound.

3. The method according to claim 1 or 2, wherein the aromatic aldehyde is a substituted or unsubstituted 5-10 membered aromatic aldehyde or a 5-10 membered heteroaromatic aldehyde, the substitution may be mono-or poly-substituted, and the substituent is halogen, nitro, cyano, hydroxy, C1-C10 alkyl, C1-C10 alkoxy.

4. The method according to claim 1 or 2, wherein the aromatic aldehyde is substituted or unsubstituted benzaldehyde, furaldehyde, thiophenecarboxaldehyde, imidazolecarboxaldehyde, pyrazolecarboxaldehyde, pyridinecarboxaldehyde, pyrazinecarboxaldehyde, cinnamaldehyde, naphthaldehyde, biphenylcarboxaldehyde, and the substituents are halogen, nitro, cyano, hydroxy, C1-C10 alkyl, C1-C10 alkoxy.

5. The method according to claim 2, wherein the aromatic aldehyde and the malononitrile in the step (1) are present in a molar ratio of 1:1 to 1: 3; the dosage of the catalyst is 0.1-5% of the dosage of the aromatic aldehyde, and preferably 1-2%; the reaction temperature is 60-80 ℃.

6. The method of claim 1 or 2, wherein the metalloporphyrin catalyst has a structure represented by formula (I):

in the formula (I), R₁、R₂、R₃、R₄Each independently is: substituted or unsubstituted phenyl, imidazolyl, pyridyl and biphenyl, wherein the substituent is amino, nitro or carboxyl; r₁、R₂、R₃、R₄Preferably 4-aminophenyl, 4-nitrophenyl, 4-carboxyphenyl, 3, 5-dicarboxyphenyl, 4- (4' -carboxybiphenyl) yl, 4-imidazolyl or 4-pyridyl, and M is any one of cobalt (II), iron (II), cadmium (II), manganese (II), nickel (II), zinc (II) or copper (II).

7. The method of claim 1 or 2, wherein the porphyrin or metalloporphyrin catalyst is: 5,10,15, 20-tetrakis (4-carboxyphenyl) porphyrin, 5, 15-bis (4-imidazolyl) -10, 20-bis (4-carboxyphenyl) ferriporphyrin, 5, 15-bis (4-imidazolyl) -10, 20-bis (4-carboxyphenyl) porphyrin, 5, 15-bis (4-imidazolyl) -10, 20-bis (4-carboxyphenyl) zinc porphyrin.

8. The process according to claim 1 or 2, wherein the biocatalyst nitrile hydratase is derived from Rhodococcus rhodochrous J1(Rhodococcus rhodochrous J1); the form of the biocatalyst is cell, crude enzyme powder, enzyme solution or immobilized enzyme.

9. The method according to claim 2, wherein the solvent in the step (2) is methanol, ethanol, dimethylsulfoxide, dioxane or tetrahydrofuran; the concentration of the aryl methylene malononitrile compound solution is 0.05-1 mol/mL; the concentration of the phosphate buffer solution is 50-100 mM, and the pH value is 5-9.

10. Application of metalloporphyrin-enzyme catalyst in preparation of (E) -alpha-cyano-beta-aryl acrylamide compounds.

Technical Field

The invention belongs to the technical field of chemical synthesis, and relates to a green preparation method of (E) -alpha-cyano-beta-aryl acrylamide compounds, in particular to a green preparation method of (E) -alpha-cyano-beta-aryl acrylamide compounds based on a metalloporphyrin-enzyme continuous catalysis method.

Background

The amide compounds can be used for constructing proteins, polypeptides, enzymes and the like in bioscience, and also have wide application in organic, medical and material science. Wherein the acrylamide compound is averagely produced into 20 ten thousand tons per year (Angewandte Chemie-International Edition 2004,43: 1576-1580; Organometallics 2003,22:1203-1211), and has important industrial application value. The alpha-cyano-beta-aryl acrylamide compounds and derivatives thereof play important roles in life sciences, such as inhibiting dengue fever, West Nile virus serine protease (NS2B-NS3) (Bioorganic & Medicinal Chemistry 2011,19:7318-7337), and also have potential biological activities of inhibiting reversible cysteine receptors of RSK2 and MSK1 kinases (Nature Chemical Biology 2014,10: 1066-1072; Journal of the American Chemical Society 2014,136: 12624-12630; Nature Chemical Biology 2012,8: 471-476). Therefore, it is very valuable to develop a new strategy for synthesizing α -cyano- β -aryl acrylamides simply, efficiently, economically and practically.

The current synthetic strategies for α -cyano- β -aryl acrylamides are classified into the following ones:

(1) the aromatic aldehyde is condensed with cyanoacetamide under strong acid or strong base conditions to directly produce alpha-cyano-beta-aryl acrylamide compounds (tetrahedron, 1987,43: 537-542; Catalysis Communications 2008,9: 403-405).

(2) Benzylamine, dibenzylamine or tribenzylamine is oxidized with cyanoacetamide under the mediation of high-valent iodine to give α -cyano- β -arylacrylamide compounds (European Journal of Organic Chemistry 2019,36: 62326239).

(3) Aromatic aldehyde and malononitrile are subjected to Knoevenagel condensation to generate aryl methylene malononitrile compounds, and then alpha-cyano-beta-aryl acrylamide compounds are generated through the single hydration reaction of cyano groups (ChemCatchem 2016,8: 1-11; ChemstrySelect 2018,3: 3534-3538).

(4) Aromatic aldehyde and acrylonitrile are subjected to Baylis-Hillman reaction, and the obtained product is converted into alpha-cyano-beta-aryl acrylic aldehyde compounds under the action of ionic liquid and then converted into alpha-cyano-beta-aryl acrylamide compounds in hydroxylamine methanol solution (New Journal of Chemistry 2017,41: 9203-.

The above synthetic strategy still suffers from the following problems: the reaction conditions are harsh and the reaction time is long; both cyano groups of the arylmethylenemalononitrile compound are likely to become reactive functional groups for hydration reaction; under some conditions the amide hydrolysis rate is faster than the conversion of cyano groups to amides, and the amide formed may continue to hydrolyze to carboxylic acids. Therefore, the development of a non-toxic, non-corrosive, carboxylic acid by-product free process to selectively produce the monohydrate product, i.e., (E) - α -cyano- β -arylacrylamide compounds, remains a challenging task.

The porphyrin compound has better thermal stability and chemical stability due to the special conjugated macrocyclic structure. Metalloporphyrin can simulate the biological functions of proteins such as peroxidase, cytochrome P450 and the like, and is one of important biomimetic catalysts (Chemical Reviews 2017,117,4: 2910-3043). The advantages of porphyrins and metalloporphyrins as catalysts include: stable structure, no toxicity, no smell, easy separation from the reaction system, high repeated utilization, easy synthesis, etc.

Nitrile hydratase (NHase, EC 4.2.1.84) is a key catabolic enzyme in the metabolism of nitrile compounds (Biotechnology Advances 2010,28: 725-containing 741), and is capable of efficiently hydrolyzing nitriles to the corresponding amides under mild conditions.

Disclosure of Invention

In order to overcome the defects of the traditional chemical synthesis of the (E) -alpha-cyano-beta-aryl acrylamide compound, the invention provides a method for continuously synthesizing the (E) -alpha-cyano-beta-aryl acrylamide compound by a chemical-enzymatic method, which is green, efficient and high in cis-trans selectivity.

The invention is realized by the following technical scheme:

a green preparation method of (E) -alpha-cyano-beta-aryl acrylamide compounds based on a metalloporphyrin chemical-enzyme continuous catalysis method is characterized in that aromatic aldehyde and malononitrile are used as substrates, porphyrin or metalloporphyrin is used as a catalyst, Knoevenagel reaction is carried out under the solvent-free condition, the generated aryl methylene malononitrile compounds are used for hydration reaction under catalysis of nitrile hydratase, and the (E) -alpha-cyano-beta-aryl acrylamide compounds can be selectively generated.

Wherein Ar is a 5-10 membered aryl or 5-10 membered heteroaryl.

The method specifically comprises the following steps:

(1) aromatic aldehyde and malononitrile are fully mixed according to a certain proportion, porphyrin or metalloporphyrin is used as a catalyst, Knoevenagel reaction is carried out at 30-80 ℃ under the condition of no solvent, and the reaction time is 0.5-6 hours, so that the aryl methylene malononitrile compound is obtained.

(2) And (2) adding a proper amount of solvent into the compound obtained in the step (1) for dissolving, and filtering out the metalloporphyrin to obtain a solution of the arylmethylenemalononitrile compound. Adding a proper amount of the solution into a phosphate buffer solution system of a biocatalyst nitrile hydratase, carrying out hydration reaction, and extracting with ethyl acetate to obtain the (E) -alpha-cyano-beta-aryl acrylamide compound.

Wherein the content of the first and second substances,

the aromatic aldehyde in the step (1) is substituted or unsubstituted 5-10-membered aromatic aldehyde and 5-10-membered heteroaromatic aldehyde, the substitution can be single substitution or polysubstitution, and the substituent is halogen, nitro, cyano, hydroxyl, C1-C10 alkyl and C1-C10 alkoxy;

further, the aromatic aldehyde includes, but is not limited to, substituted or unsubstituted benzaldehyde, furaldehyde, thiophenecarboxaldehyde, imidazolecarboxaldehyde, pyrazolecarboxaldehyde, pyridinecarboxaldehyde, pyrazinecarboxaldehyde, cinnamaldehyde, naphthaldehyde, biphenylcarboxaldehyde, and the substituents are halogen, nitro, cyano, hydroxyl, C1-C10 alkyl, and C1-C10 alkoxy.

The dosage of the catalyst in the step (1) is 0.1-5% of the dosage of the aromatic aldehyde;

the preferable reaction temperature is 60-80 ℃, the reaction time is 0.5-1 hour, and the dosage of the catalyst is 1-2%.

The molar ratio of the aromatic aldehyde to the malononitrile in the step (1) is 1: 1-1: 3.

The metalloporphyrin catalyst in the step (1) has a structure shown as a formula (I):

in the formula (I), R₁、R₂、R₃、R₄Each independently is: substituted or unsubstituted phenyl, imidazolyl, pyridyl and biphenyl, wherein the substituent is amino, nitro or carboxyl, and specifically can be 4-aminophenyl, 4-nitrophenyl or 4-carboxyphenylA group, a 3, 5-dicarboxyphenyl group, a 4- (4' -carboxybiphenyl) group, a 4-imidazolyl group or a 4-pyridyl group, wherein M is any one of cobalt (II), iron (II), cadmium (II), manganese (II), nickel (II), zinc (II) or copper (II).

Further, the porphyrin or metalloporphyrin catalyst in the step (1) is: 5,10,15, 20-tetrakis (4-carboxyphenyl) porphyrin, 5, 15-bis (4-imidazolyl) -10, 20-bis (4-carboxyphenyl) ferriporphyrin, 5, 15-bis (4-imidazolyl) -10, 20-bis (4-carboxyphenyl) porphyrin, 5, 15-bis (4-imidazolyl) -10, 20-bis (4-carboxyphenyl) zinc porphyrin.

The metalloporphyrin can be synthesized by a conventional method.

The biocatalyst nitrile hydratase in the step (2) is derived from Rhodococcus rhodochrous J1(Rhodococcus rhodochrous J1); the form of the biocatalyst can be cells, crude enzyme powder, enzyme solution or immobilized enzyme.

The solvent in the step (2) is methanol, ethanol, dimethyl sulfoxide, dioxane or tetrahydrofuran; the concentration of the aryl methylene malononitrile compound solution is 0.05-1 mol/mL; the concentration of the phosphate buffer solution is 50-100 mM, and the pH value is 5-9.

The metalloporphyrin in the step (2) can be recycled.

According to the invention, aromatic aldehyde and malononitrile are used as substrates, metalloporphyrin is used as a catalyst, Knoevenagel reaction is carried out under the solvent-free condition, the generated aryl methylene malononitrile compound is simply treated and then directly used for hydration reaction under the catalysis of nitrile hydratase, and the (E) -alpha-cyano-beta-aryl acrylamide compound can be selectively generated, so that an efficient, mild and green synthetic route is constructed for the preparation of the (E) -alpha-cyano-beta-aryl acrylamide compound.

Compared with the prior art, the invention has the following advantages:

(1) experiments prove that the metalloporphyrin can efficiently catalyze the Knoevenagel condensation reaction, the conversion rate is more than 99 percent, the properties are stable, and the metalloporphyrin can be repeatedly used.

(2) The invention firstly utilizes nitrile hydratase to carry out biotransformation on aryl methylene malononitrile compounds, and can synthesize (E) -alpha-cyano-beta-aryl acrylamide compounds with high cis-trans selectivity, wherein the conversion rate is more than 99 percent, and the cis-trans selectivity is more than 99 percent.

(3) The method adopts a solvent-free metalloporphyrin catalysis method in the first step and an enzyme catalysis method in the second step, compared with the traditional chemical synthesis method, the method is more efficient, green and environment-friendly, and both metalloporphyrin and nitrile hydratase can be obtained commercially, so that a new idea is provided for preparing the (E) -alpha-cyano-beta-aryl acrylamide compound.

Drawings

FIG. 1 is an HPLC chromatogram of benzaldehyde and phenylmethylenedinitrile for detecting conversion,

wherein: a is substrate benzaldehyde, B is product phenyl methylene malononitrile;

FIG. 2 is an HPLC chromatogram of phenylmethylenedinitrile and (E) -alpha-cyano-beta-phenylacrylamide, which is used for detecting conversion rate and cis-trans selectivity,

wherein: a is a substrate, phenyl methylene malononitrile, and B is a product (E) -alpha-cyano-beta-phenyl acrylamide.

FIG. 3 is a scheme showing that (E) -alpha-cyano-beta-phenylacrylamide¹H-¹H COSY spectrum.

Detailed Description

The objects, features, advantages, technical means of the present invention will be understood by the following examples, but are not limited thereto. The implementation conditions adopted in the examples can be further adjusted according to different requirements of specific applications, and the implementation conditions not indicated are those in routine experiments or those suggested by manufacturers.

The examples relate to the formulation of the medium:

(1) rhodococcus rhodochrous seed medium: KH (Perkin Elmer)₂PO₄ 2.4g，K₂HPO₄2.4g, 10.0g of glucose, 8.0g of yeast extract, 2.0g of urea, 1.6g of monosodium glutamate and MgSO₄2.4g, adding distilled water to a constant volume of 1000ml (if a solid plate culture medium is required to be prepared, 15g of agar can be added before sterilization), and sterilizing at 115 ℃ for 30 min.

(2) Rhodococcus rhodochrous fermentationCulture medium: KH (Perkin Elmer)₂PO₄ 6.0g，K₂HPO₄6.0g, 30.0g of glucose, 8.0g of yeast extract, 9.2g of urea and CoCl₂.6H₂O24 mg, monosodium glutamate 0.96g, MgSO₄0.6g, adding distilled water to 1000ml, sterilizing at 115 deg.C for 30 min.

Example 15, 10,15, 20-preparation of tetrakis (4-carboxyphenyl) porphyrin

Adding 25mL of propionic acid into a 50mL round-bottom flask, adding 1.5g (10mmol) of 4-formylbenzoic acid, heating to 80 ℃, dropwise adding 0.68mL (10mmol) of freshly distilled pyrrole, raising the temperature to 140 ℃, refluxing for 2h, detecting the completion of the reaction of raw materials by TLC, stopping the reaction, cooling to room temperature, and standing in a 4 ℃ refrigerator overnight. And (4) carrying out suction filtration, and washing a filter cake by using dichloromethane to obtain a target product. The crude product was subjected to column chromatography to give 0.39g of a black-purple powdery solid with a conversion of 20%.

EXAMPLE 25, 15-bis (4-imidazolyl) -10, 20-bis (4-carboxyphenyl) porphyrin

Adding 25mL of propionic acid into a 50mL round-bottom flask, heating to 80 ℃, adding 0.85g (3.2mmol) of 2,2' - ((4-carboxyphenyl) methylene) bis (1H-pyrrole), stirring until the solution is completely dissolved, adding 0.307g (3.2mmol) of 4-imidazole formaldehyde, raising the temperature to 140 ℃, carrying out reflux reaction for 2 hours, detecting that the raw materials are completely reacted by TLC, stopping the reaction, cooling to room temperature, and placing in a refrigerator at 4 ℃ for standing overnight. And (4) carrying out suction filtration, and washing a filter cake by using dichloromethane to obtain a target product. The crude product was subjected to column chromatography to give 0.37g of a dark purple powdery solid with a conversion of 17%.

EXAMPLE 35 preparation of 15, 15-bis (4-imidazolyl) -10, 20-bis (4-carboxyphenyl) ferriporphyrin

0.1g (0.15mmol) of 5, 15-bis (4-imidazolyl) -10, 20-bis (4-carboxyphenyl) porphyrin was dissolved in 40mL of DMF, and 0.15g (1.2mmol) of ferrous chloride was dissolved in 10mL of DMF, and the above reaction systems were mixed and reacted at 120 ℃ for 12 hours. After the reaction is finished, cooling to room temperature, adding 50mL of distilled water, standing, performing suction filtration, washing with DMF for 3 times, washing with distilled water and ethanol respectively, and drying a filter cake in vacuum to obtain 0.11g of a target product with the conversion rate of 93%.

EXAMPLE 45 preparation of 15-bis (4-imidazolyl) -10, 20-bis (4-carboxyphenyl) zinc porphyrin

0.1g (0.15mmol) of 5, 15-bis (4-imidazolyl) -10, 20-bis (4-carboxyphenyl) porphyrin was dissolved in 40mL of DMF, and 0.14g (1.2mmol) of zinc chloride was dissolved in 10mL of DMF, and the above reaction systems were mixed and reacted at 120 ℃ for 12 hours. After the reaction is finished, cooling to room temperature, adding 50mL of distilled water, standing, performing suction filtration, washing with DMF for 3 times, washing with distilled water and ethanol respectively, and drying a filter cake in vacuum to obtain 0.11g of a target product with the conversion rate of 93%.

Example 5 preparation of nitrile hydratase (EC 4.2.1.84) -Induction culture of Rhodococcus rhodochrous J1

Rhodococcus rhodochrous J1(Rhodococcus rhodochrous J1) was inoculated into 3mL of liquid seed medium and cultured at 28 ℃ and 200rpm for 48 hours. Inoculating 1mL of cultured seed solution into 100mL of fermentation medium, placing the mixture in a shaking table with the temperature of 28 ℃ and the rpm of 200 for shaking culture, when the OD600 of the culture solution reaches 1.0, centrifuging the mixture for 15min at the rpm of 4000, enriching thalli, discarding supernatant, washing the thalli twice by using 100mM phosphate buffer solution with the pH of 7.5, centrifuging the thalli again, and subpackaging the obtained thalli by using 100mM phosphate buffer solution with the pH of 7.5 in a refrigerator with the temperature of 4 ℃.

Example 6 Effect of metalloporphyrin catalyst amount and reaction temperature on Knoevenagel reaction

TABLE 1

Reaction conditions are as follows: benzaldehyde (1mmol), malononitrile (2mmol), 5, 15-bis (4-imidazolyl) -10, 20-bis (4-carboxyphenyl) ferriporphyrin were reacted with stirring under heating for 30 minutes.^aThe conversion is determined by high performance liquid chromatography.

EXAMPLE 7 preparation of Phenylmethylenemalononitrile

To a 10mL round-bottomed flask were added 1mmol of benzaldehyde, 100mg (1.5mmol) of malononitrile, and 10mg (1.4% mmol) of 5, 15-bis (4-imidazolyl) -10, 20-bis (4-carboxyphenyl) ferriporphyrin. And (3) stirring the mixture at 60 ℃, detecting by TLC until benzaldehyde completely reacts, adding 5mL of dimethyl sulfoxide, filtering out metalloporphyrin, and repeatedly using the metalloporphyrin to obtain a phenylmethyleneamalononitrile solution for the next enzyme catalysis. Reaction conversion was calculated to be > 99% by HPLC detection.

HPLC detection conditions of a substrate benzaldehyde and a product, namely phenyl methylene malononitrile, are as follows: a chromatographic column: ODS-C18 column; a detector: a UV detector; wavelength: 254 nm; mobile phase: acetonitrile: 50 parts of water: 50 (v/v); flow rate of mobile phase: 1 mL/min; column temperature: at 25 ℃.

EXAMPLE 8 preparation of (2-fluorophenyl) methylenemalononitrile

To a 10mL round-bottomed flask were added 1mmol of 2-fluorobenzaldehyde, 132mg (2mmol) of malononitrile, and 10mg (1.4% mmol) of 5, 15-bis (4-imidazolyl) -10, 20-bis (4-carboxyphenyl) zinc porphyrin. The mixture is stirred at 60 ℃, TLC detection is carried out until 2-fluorobenzaldehyde completely reacts, 5mL of ethanol is added, and metalloporphyrin is filtered out and can be recycled, so that (2-fluorophenyl) methylene malononitrile solution is obtained for the next enzyme catalysis. Reaction conversion was calculated to be > 99% by HPLC detection.

EXAMPLE 9 preparation of (E) - α -cyano- β -phenylacrylamide

50mg of nitrile hydratase (NHase) wet cells were suspended in 950. mu.L of 50mM phosphate buffer pH 7.5 and added to a 1.5mL Eppendorf tube, followed by 50. mu.L of a 0.1mmol/mL solution of phenylmethylenedinitrile in DMSO. The reaction was placed on a shaker at 200rpm at room temperature and checked by TLC until the substrate reaction was complete. After completion of the reaction, 10. mu.L of 6N HCl was added to terminate the reaction, and the reaction solution was centrifuged at 12000rpm, and then the supernatant was separated from the precipitate. The supernatant was then extracted with 3X 1mL of ethyl acetate, the organic layers were combined, and the solvent was removed under reduced pressure to give (E) - α -cyano- β -phenylacrylamide 1.7 mg. Conversion rate is more than 99% and cis-trans selectivity is more than 99% by HPLC detection.

HPLC detection conditions of a substrate phenyl methylene malononitrile and a product (E) -alpha-cyano-beta-phenyl acrylamide: a chromatographic column: ODS-C18 column; a detector: a UV detector; wavelength: 254 nm; mobile phase: acetonitrile: water: phosphoric acid 33: 66: 1 (v/v); flow rate of mobile phase: 1 mL/min; column temperature: at 25 ℃.

EXAMPLE 10 preparation of (E) - α -cyano- β - (2-fluorophenyl) acrylamide

50mg of the NHase wet cells were suspended in 950. mu.L of 50mM phosphate buffer pH 7.5 and added to a 1.5mL Eppendorf tube, followed by 50. mu.L of a 0.1mmol/mL ethanol solution of (2-fluorophenyl) methylenemalononitrile. The reaction was placed on a shaker at 200rpm at room temperature and checked by TLC until the substrate reaction was complete. After completion of the reaction, 10. mu.L of 6N HCl was added to terminate the reaction, and the reaction solution was centrifuged at 12000rpm, and then the supernatant was separated from the precipitate. The supernatant was then extracted with 3X 1mL of ethyl acetate, the organic layers were combined, and the solvent was removed under reduced pressure to give 1.9mg of (E) - α -cyano- β - (2-fluorophenyl) acrylamide. Conversion rate is more than 99% and cis-trans selectivity is more than 99% by HPLC detection.