CN113388646A - Method for synthesizing (R) -1- [3, 5-bis (trifluoromethyl) ] phenethyl alcohol under catalysis of optical enzyme system - Google Patents

Abstract

The invention relates to a method for synthesizing (R) -1- [3, 5-bis (trifluoromethyl) under the catalysis of a light enzyme system]Method of phenethyl alcohol, with TiO₂Nanotube bonding [ Cp Rh (bpy) ((H))₂O)]²⁺Catalytic regeneration of NADPH, the NADPH produced being used for the catalytic synthesis of (R) -1- [3, 5-bis (trifluoromethyl)]And (3) phenethyl alcohol. The method of the invention has low cost and uses TiO₂The nano tube photolyzes water to supply hydrogen, the used enzyme is a cross-linked enzyme aggregate, is insoluble in an organic solvent, can be recycled for many times, has high yield, is safe and reliable, has less environmental pollution, and is suitable for industrial production.

Description

Method for synthesizing (R) -1- [3, 5-bis (trifluoromethyl) ] phenethyl alcohol under catalysis of optical enzyme system

Technical Field

The invention belongs to the technical field of NADPH regeneration and the technical field of synthesis of anti-emetic drugs in tumors, and particularly relates to a method for synthesizing (R) -1- [3, 5-bis (trifluoromethyl) ] phenethyl alcohol under the catalysis of a photo-enzyme system.

Background

(R) -1- [3, 5-bis (trifluoromethyl) ] phenethyl alcohol (structural formula is shown as B) is an important chiral intermediate of a key precursor for synthesizing aprepitant (structural formula is shown as A). Obtained by direct reduction of 3,5-bis (trifluoromethyl) acetophenone (3,5-bis (trifluoromethyl) acetophenone). Among these, optically active secondary alcohols are an important component of many biologically active compounds in the pharmaceutical industry. In industrial production, however, Asymmetric Hydrogenation (AH) of prochiral ketones is a practical and simple method for obtaining enantiomerically pure secondary alcohols.

Li, Y.H. et al developed a method of using RuCl based on previous studies₂[(R)-Diop][(R)-BIMAH]Preparation of (R) -1- [3, 5-bis (trifluoromethyl)]A novel method for preparing phenethyl alcohol. Although the method for producing optical alcohol by AH has attracted extensive interest in academic and industrial research, most of the research is still in the case of small-scale production due to the complexity of the catalyst or the harsh reaction conditions. Thereafter, (R) -1- [3, 5-bis (trifluoromethyl) was prepared synthetically by Jianfei et al using iridium as a catalyst for the aminophosphonic acid ligand of ferrocene]The process of the phenethyl alcohol also has good effect.

In the production of drug intermediates, biocatalysis is more matched with chemical synthesis. The use of enzymatic catalysis is considered as a key step in the synthesis of industrial drugs through ongoing research and technological innovation. In recent years, the number of biocatalysts for the production of chiral compounds has increased rapidly. Chiral alcohols are the most valuable key intermediates for the manufacture of pharmaceuticals. Carbonyl reductases have previously only been used for the reduction of ketones and are now frequently used for their reliable chemo-and stereoselectivity. However, it is possible to catalyze the formation of (R) -1- [3, 5-bis (trifluoromethyl)]The carbonyl reductase of phenethyl alcohol requires expensive NAD (P)⁺As a cofactor, can be performed.

In biology, however, NAD (P) H-dependent oxidoreductases mayTo catalyze the reduction of specific substrates by withdrawing hydrogen from a reduced nicotinamide adenine dinucleotide (NAD (P) H) cofactor, resulting in the oxidation of NAD (P) H to NAD (P)⁺。NAD(P)H/NAD(P)⁺The nicotinamide ring moiety of (A) is directly involved in the electron transfer during the reaction catalyzed by NAD (P) H-dependent oxidoreductase, while the C4 carbon atom of the nicotinamide ring acts as acceptor/donor proton in NAD (P) H/NAD (P)⁺The addition of phosphate to the 2' -OH group of the adenine ribose ring does not alter the electron transport ability. Enzymatic regeneration of NADH, such as alcohol dehydrogenase and Formate Dehydrogenase (FDH), has been established in industrial biocatalysis, however, this process usually requires regeneration of the enzyme and the corresponding substrate, which can be limited by limited stability of the enzyme and high separation costs.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a method for synthesizing (R) -1- [3, 5-bis (trifluoromethyl) under the catalysis of a light enzyme system]Method for the production of phenethyl alcohol, i.e. the use of regenerated NADPH for the photocatalytic synthesis of (R) -1- [3, 5-bis (trifluoromethyl)]The method of the phenethyl alcohol has the advantages of reducing steps, reducing environmental pollution, lowering production cost, improving production safety and providing a competitive route for industrialization; the use of TiO in the invention₂Method for supplying hydrogen and electrons by nanotube splitting water with [ Cp Rh (bpy) (H)₂O)]²⁺As hydrogen transfer agent and electron transfer agent, assisted by high-efficiency mild enzyme catalysis reaction; the reaction process has mild conditions, simple operation and separation, easy recovery and recycling, less environmental pollution and greatly reduced production cost.

The technical scheme adopted by the invention is as follows:

optical enzyme system catalytic synthesis of (R) -1- [3, 5-bis (trifluoromethyl)]Method of phenethyl alcohol, with TiO₂Nanotube bonding [ Cp Rh (bpy) ((H))₂O)]²⁺Catalytic regeneration of NADPH, the NADPH produced being used for the catalytic synthesis of (R) -1- [3, 5-bis (trifluoromethyl)]And (3) phenethyl alcohol.

Preferably, the method for synthesizing (R) -1- [3, 5-bis (trifluoromethyl) ] phenethyl alcohol by the catalysis of the photo-enzyme system comprises the following steps:

(1) in the reactionAdding reaction solvent and NADP into the reactor⁺、[Cp*Rh(bpy)(H₂O)]²⁺Aqueous solution, TiO₂Reacting the nanotube, the zymoprotein and the 3,5-bis (trifluoromethyl) acetophenone for 24 hours under illumination by using a xenon lamp with the wavelength of 420 nm;

(2) and (2) taking the supernatant of the reaction liquid in the step (1), extracting the supernatant by using anhydrous n-hexane, and then heating, decompressing and spin-drying to obtain pure (R) -1- [3, 5-bis (trifluoromethyl) ] phenethyl alcohol.

Preparation of (R) -1- [3, 5-bis (trifluoromethyl)]The reaction process of phenethyl alcohol is shown in figure 1, and is carried out by using TiO₂Nanotube bonding [ Cp Rh (bpy) ((H))₂O)]²⁺Catalyzing and regenerating NADPH, using the generated NADPH for enzyme catalysis, and preparing (R) -1- [3, 5-bis (trifluoromethyl) by simulating illumination reaction for 24h at room temperature]And (3) phenethyl alcohol.

Preferably, TiO is used₂The nanotube is prepared by the following method:

1g of TiO₂Dispersing the nano powder in 100ml of 10M NaOH, and stirring in a beaker for 30 min; then transferring the mixture into a stainless steel autoclave lined with Teflon, wherein the temperature is 140 ℃, and the duration is 18 hours; after the autoclave was naturally cooled to room temperature, the resulting precipitate was recovered by filtration and washed with distilled water and 0.1MHCl solution for 6 hours; subsequently, the solution was washed several times until the pH was 7; drying the product at 80 ℃ for 24h, and then calcining the product in air at 400 ℃ for 2h to obtain TiO₂A nanotube.

TiO of the invention₂The nanotubes being made of TiO₂The nanometer powder is white solid prepared by a calcining method.

The [ Cp Rh (bpy) (H)₂O)]²⁺The hydrogen transfer agent and the electron transfer agent are synthesized by a chemical method; preferably, [ Cp + Rh (bpy) ((H))₂O)]²⁺The aqueous solution is prepared by the following method:

refluxing rhodium trichloride and 1,2,3,4, 5-pentamethylcyclopentadiene in methanol for 24 hours; the resulting red precipitate was filtered and suspended in methanol; adding 2, 2-bipyridine to form a light yellow solution; adding diethyl ether, during the addition, [ Cp Rh (bpy) Cl]Cl with diethyl etherGradually precipitating by adding; [ Cp + Rh (bpy) Cl]Hydrolysis of Cl to [ Cp Rh (bpy)) (H₂O)]²⁺(ii) a Water was added to bring the solution to a final concentration of 100mM and stored at room temperature.

Preferably, the enzyme protein is prepared by the following method:

cells containing 34. mu.g/mL of alcohol dehydrogenase mutant were inoculated^-1Chloramphenicol, 100. mu.g.mL^-1Kanamycin and 50. mu.g/mL^-1Ampicillin in TB medium and cultured in a shaking incubator at 34 ℃; when the OD600 is detected to be 0.6-0.8 by ultraviolet light, an inducer L- (+) -arabinose of tRNA aminoacyl synthetase is directly added until the final mass/volume concentration is 0.2%, and an inducer pZE21-ADH is added to reach 60 ng/mL^-1aTc and para-azido-L-phenylalanine are used to induce protein expression; unnatural amino acid to a final concentration of 1 mmol. L^-1；

Inducing expression in a shaking incubator at 23 deg.C for 16h, centrifuging at 8000rpm for 5min to obtain bacteria of unnatural amino acid modified alcohol dehydrogenase, resuspending the obtained bacteria precipitate with PBS buffer solution, lysing the bacteria with ultrasonic treatment, centrifuging at 10000rpm for 15min to obtain soluble fraction, separating the soluble fraction with phosphate buffer solution KPB and 2 mol. L^-1Washing twice with NaCl solution; under the assistance of microwave, the washed soluble part, the same amount of the bis-azide cross-linking agent and 0.4 equivalent of CuI participate in azide-alkyne cycloaddition click reaction; the immobilized enzyme was separated by centrifugation and eluted continuously with phosphate buffer KPB until no protein was detected in the supernatant by coomassie brilliant blue staining.

Preferably, the reaction solvent is one of methanol, n-hexane, PBS buffer solution, sodium hydroxide solution and hydrochloric acid solution.

Preferably, the method for synthesizing (R) -1- [3, 5-bis (trifluoromethyl) ] phenethyl alcohol by the catalysis of the photo-enzyme system comprises the following steps:

(1) the reaction vessel was charged with 100mM PBS buffer (pH 7.0) and 1mM NADP⁺1mM of [ Cp Rh (bpy) (H)₂O)]²⁺Aqueous solution, 250mu.M TiO₂Nanotubes with a final concentration of 32 mg.L^-1The zymoprotein and 50mM substrate 3,5-bis (trifluoromethyl) acetophenone have a total volume of 5ml, and react for 24 hours under illumination by using a xenon lamp with a wavelength of 420nm and a distance of 15 cm from the reactor;

(2) and (2) taking the supernatant of the reaction liquid in the step (1), extracting the supernatant by using anhydrous n-hexane, and then heating, decompressing and spin-drying to obtain pure (R) -1- [3, 5-bis (trifluoromethyl) ] phenethyl alcohol.

The method has the following beneficial effects:

(1) the operation is simple and can be realized in one step;

(2) TiO used₂The nano tube is insoluble in water, the zymoprotein is immobilized into soluble macromolecular substances through crosslinking, and the soluble macromolecular substances and the zymoprotein can be recovered through centrifugation after use and can be recycled;

(3) the reaction condition is mild, and the environmental pollution is less;

(4) the hydrogen is supplied by water, other sacrificial reagents are not required to be added, the cost is reduced, and the method is green and environment-friendly.

Drawings

FIG. 1 is a diagram showing a process for producing (R) -1- [3, 5-bis (trifluoromethyl) ] phenethyl alcohol;

FIG. 2 shows the TiO prepared₂Characterization spectra of the nanotubes, including XPS spectra, SEM spectra, TEM spectra, EDS spectra;

FIG. 3 shows LC-MS spectra of prepared [ Cp. multidot.Rh (bpy) Cl ] Cl;

FIG. 4 is a characterization diagram of the prepared enzyme protein (enzyme aggregate), including SDS-PAGE diagram, Maldi-tof high resolution mass spectrum diagram, SEM diagram, CLSM diagram and LC-MS diagram of acidolysis of the enzyme aggregate;

FIG. 5 is a high performance liquid chromatography chromatogram analysis chart of (R) -1- [3, 5-bis (trifluoromethyl) ] phenethyl alcohol in the reaction system after 3/6/9/12/24h (A/B/C/D/E) reaction respectively.

Having the embodiments

The present invention is further analyzed with reference to the following specific examples. It is to be understood that the following examples are illustrative of the present invention only and are not intended to limit the scope of the present invention.

Raw material TiO used in the invention₂The nanometer powder is purchased from Shanghai Merlin Biotechnology, Inc.; the raw material rhodium trichloride trihydrate is purchased from chemical engineering limited company of Waverrucke, Beijing; other raw materials of the present invention are commercially available unless otherwise specified.

Example 1

Preparation of TiO₂Nanotube and method of manufacturing the same

1g of TiO₂Dispersing the nano powder in 100ml of 10M NaOH, and stirring in a beaker for 30 min; then transferring the mixture into a stainless steel autoclave lined with Teflon, wherein the temperature is 140 ℃, and the duration is 18 hours; after the autoclave was naturally cooled to room temperature, the resulting precipitate was recovered by filtration and washed with distilled water and 0.1MHCl solution for 6 hours; subsequently, the solution was washed several times until the pH was 7; drying the product at 80 ℃ for 24h, and then calcining the product in air at 400 ℃ for 2h to obtain TiO₂A nanotube.

Preparing the obtained TiO₂The XPS spectrum, SEM spectrum, TEM spectrum and EDS spectrum of the nanotube are shown in figure 2. As can be seen from FIG. 2, the rhodium metal ligands were successfully adsorbed to the titanium dioxide nanotubes, and they also successfully attached to the surface of the enzyme aggregate to form the composite catalytic material.

Preparation of [ Cp Rh (bpy) (H)₂O)]²⁺Aqueous solution

80mg of rhodium trichloride and one equivalent of 1,2,3,4, 5-pentamethylcyclopentadiene are refluxed in methanol for 24h, and the resulting red precipitate is filtered and suspended in methanol; two equivalents of 2, 2-bipyridine were added and the suspension was almost immediately cleared and formed a pale yellow solution; adding diethyl ether, during the addition, [ Cp Rh (bpy) Cl]Cl gradually precipitated with the addition of diethyl ether; [ Cp + Rh (bpy) Cl]Cl is readily hydrolyzed to [ Cp Rh (bpy)) (H₂O)]²⁺. Appropriate amount of water was added to make the solution a final concentration of 100mM and stored at room temperature.

The LC-MS spectrum of the prepared [ Cp Rh (bpy) Cl ] Cl is shown in FIG. 3, m/z, 465.03. As can be seen from FIG. 3, the rhodium ligand was synthesized smoothly.

Preparation of enzyme proteins

Inoculating alcohol dehydrogenase mutant cellsTo contain 34. mu.g.mL^-1Chloramphenicol, 100. mu.g.mL^-1Kanamycin and 50. mu.g/mL^-1Ampicillin in TB medium and cultured in a shaking incubator at 34 ℃; when the OD600 is detected to be 0.6-0.8 by ultraviolet light, an inducer L- (+) -arabinose of tRNA aminoacyl synthetase is directly added to the final concentration of 0.2% (w/v), and an inducer pZE21-ADH is added to the mixture to obtain 60 ng/mL^-1The aTc and a proper amount of p-azido-L-phenylalanine are used for inducing protein expression; unnatural amino acid to a final concentration of 1 mmol. L^-1。

Inducing expression in a shaking incubator at 23 deg.C for 16h, centrifuging at 8000rpm for 5min to obtain bacterial precipitate of unnatural amino acid-modified alcohol dehydrogenase, resuspending the bacterial precipitate with appropriate amount of PBS buffer (20 mmol. multidot.L-1, pH 7.0), lysing the bacteria by ultrasonic treatment, centrifuging at 10000rpm for 15min to obtain soluble fraction, and subjecting the soluble fraction to KPB (0.01 mol. multidot.L)^-1pH 7.0) and 2mol · L^-1Washing twice with NaCl solution; the washed soluble fraction, equivalent amount of bis-azido crosslinker and 0.4 equivalent amount of CuI were subjected to azide-alkyne cycloaddition click reaction with the aid of microwave. The immobilized enzyme was separated by centrifugation and washed with phosphate buffer KPB (0.01 mol. L)^-1pH 7.0) until no protein was detected in the supernatant by coomassie brilliant blue staining.

The SDS-PAGE pattern, Maldi-tof high resolution mass spectrum pattern, SEM pattern, CLSM pattern and LC-MS pattern of acidolysis of the prepared enzyme protein (enzyme aggregate) are shown in figure 4. As can be seen from FIG. 4, propargyloxyphenylalanine was inserted at a predetermined site, and the enzyme aggregate was successfully prepared in the cell disruption solution, and a part of purification and immobilization of the target enzyme was achieved from the cell disruption solution.

The specific process for preparing (R) -1- [3, 5-bis (trifluoromethyl) ] phenethyl alcohol is shown in figure 1:

100mM PBS buffer (pH 7.0), 1mM NADP⁺1mM Cp-Rh (bpy) in water and 250. mu.M TiO₂Nanotubes at a final concentration of 32 mg.L^-1Enzyme protein, 50mM substrate 3,5-bis (trifluoro)Methyl) acetophenone, the total volume is 5ml, and the mixture is placed in a xenon lamp with the wavelength of 420nm and the distance of 15 cm from the reactor and is reacted for 24 hours under illumination; HPLC detection to obtain (R) -1- [3, 5-bis (trifluoromethyl)]The conversion of phenethyl alcohol was 41%, the conversion being determined by the standard ((R) -1- [3, 5-bis (trifluoromethyl)]Peak area of phenethyl alcohol) was calculated, and the standard for the standard was y 3444.6x +342.34, where x represents the concentration of the product in the reaction solution in mg/mL and y represents the peak area of the product.

After the reaction system is respectively reacted for 3/6/9/12/24h (A/B/C/D/E), the high performance liquid chromatography analysis spectrum result of (R) -1- [3, 5-bis (trifluoromethyl) ] phenethyl alcohol in the system is shown in figure 5; high performance liquid phase conditions: c18 column, column temperature 25 ℃, 210nm, 0.5mL/min, mobile phase: methanol: water 75:25, (R) -1- [3, 5-bis (trifluoromethyl) ] phenethyl alcohol retention time 15.837 min. As can be seen from FIG. 5, the photocatalytic regeneration of NADPH promoted chiral alcohol synthesis has the same effect as the artificial addition of NADPH, i.e., the method can be completely used for regenerating NADPH by taking water as a hydrogen donor, and the enzyme protein keeps stable catalytic performance.

Example 2

100mM PBS buffer (pH 7.0), 1mM NADP⁺1mM Cp-Rh (bpy) in water and 250. mu.M TiO₂Nanotubes at a final concentration of 32 mg.L^-1Enzyme protein, 50mM substrate 3,5-bis (trifluoromethyl) acetophenone, the total volume is 5ml, the reaction is carried out for 24h under the illumination by a xenon lamp with the wavelength of 420nm and the distance of 15 cm from the reactor, and TiO is added₂The nanotube and the enzyme protein are recycled, the recycling is carried out for 12h once, and after the recycling is carried out for 5 times, the HPLC (high performance liquid chromatography) detection is carried out on the (R) -1- [3, 5-bis (trifluoromethyl)]The conversion of the phenethyl alcohol was substantially unchanged.

(R) -1- [3, 5-bis (trifluoromethyl) ] phenethyl alcohol provided by TCI (Tokyo chemical industry Co., Ltd.) and the product prepared by the method of the invention were subjected to HPLC analysis, and the retention times of the two were consistent to determine the product structure.

The method of the invention has low cost and uses TiO₂The nano tube photolyzes water to supply hydrogen, the used enzyme is a cross-linked enzyme aggregate, is insoluble in an organic solvent, can be recycled for many times, has high yield, is safe and reliable, has less environmental pollution, and is suitable for industrial production.

The examples described below are merely illustrative of preferred embodiments of the invention and are not intended to limit the invention. Modifications and variations of the above-described embodiments can be made by those skilled in the art without departing from the scope of the invention as defined by the appended claims. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims (7)

1. Optical enzyme system catalytic synthesis of (R) -1- [3, 5-bis (trifluoromethyl)]A process for the production of phenethyl alcohol, characterized by: with TiO₂Nanotube bonding [ Cp Rh (bpy) ((H))₂O)]²⁺Catalytic regeneration of NADPH, the NADPH produced being used for the catalytic synthesis of (R) -1- [3, 5-bis (trifluoromethyl)]And (3) phenethyl alcohol.

2. The method for the photocatalytic synthesis of (R) -1- [3, 5-bis (trifluoromethyl) ] phenethyl alcohol by the photocatalyst system according to claim 1, characterized by comprising the following steps:

(1) adding reaction solvent and NADP into a reactor⁺、[Cp*Rh(bpy)(H₂O)]²⁺Aqueous solution, TiO₂Reacting the nanotube, the zymoprotein and the 3,5-bis (trifluoromethyl) acetophenone for 24 hours under illumination by using a xenon lamp with the wavelength of 420 nm;

(2) and (2) taking the supernatant of the reaction liquid in the step (1), extracting the supernatant by using anhydrous n-hexane, and then heating, decompressing and spin-drying to obtain pure (R) -1- [3, 5-bis (trifluoromethyl) ] phenethyl alcohol.

3. The method of claim 2, wherein the photocatalyst is used for the systematic catalytic synthesis of (R) -1- [3, 5-bis (trifluoromethyl)]Process for the preparation of phenethyl alcohol, characterised in that TiO₂The nanotube is prepared by the following method:

1g of TiO₂Dispersing the nano powder in 100ml of 10M NaOH, and stirring in a beaker for 30 min; then transferring the mixture into a stainless steel autoclave lined with Teflon, wherein the temperature is 140 ℃, and the duration is 18 hours; after the autoclave was naturally cooled to room temperature, the resulting precipitate was recovered by filtration and dissolved in distilled water and 0.1MHClWashing the solution for 6 hours; subsequently, the solution was washed several times until the pH was 7; drying the product at 80 ℃ for 24h, and then calcining the product in air at 400 ℃ for 2h to obtain TiO₂A nanotube.

4. The method of claim 2, wherein the photocatalyst is used for the systematic catalytic synthesis of (R) -1- [3, 5-bis (trifluoromethyl)]A process for the preparation of phenethyl alcohol, characterised in that [ Cp Rh (bpy) (H)₂O)]²⁺The aqueous solution is prepared by the following method:

refluxing rhodium trichloride and 1,2,3,4, 5-pentamethylcyclopentadiene in methanol for 24 hours; the resulting red precipitate was filtered and suspended in methanol; adding 2, 2-bipyridine to form a light yellow solution; adding diethyl ether, during the addition, [ Cp Rh (bpy) Cl]Cl gradually precipitated with the addition of diethyl ether; [ Cp + Rh (bpy) Cl]Hydrolysis of Cl to [ Cp Rh (bpy)) (H₂O)]²⁺(ii) a Water was added to bring the solution to a final concentration of 100mM and stored at room temperature.

5. The method for synthesizing (R) -1- [3, 5-bis (trifluoromethyl) ] phenethyl alcohol catalyzed by the photocatalyst system according to claim 2, wherein the enzyme protein is prepared by the following method:

cells containing 34. mu.g/mL of alcohol dehydrogenase mutant were inoculated^-1Chloramphenicol, 100. mu.g.mL^-1Kanamycin and 50. mu.g/mL^-1Ampicillin in TB medium and cultured in a shaking incubator at 34 ℃; when the OD600 is detected to be 0.6-0.8 by ultraviolet light, an inducer L- (+) -arabinose of tRNA aminoacyl synthetase is directly added until the final mass/volume concentration is 0.2%, and an inducer pZE21-ADH is added to reach 60 ng/mL^-1aTc and para-azido-L-phenylalanine are used to induce protein expression; unnatural amino acid to a final concentration of 1 mmol. L^-1；

Inducing expression in a shaking incubator at 23 deg.C for 16h, centrifuging at 8000rpm for 5min to obtain bacteria of unnatural amino acid modified alcohol dehydrogenase, resuspending the obtained bacteria precipitate with PBS buffer solution, treating with ultrasonic wave to lyse the bacteria, centrifuging at 10000rpm for 15min to obtain soluble fraction, and separating the soluble fraction with phosphate buffer solutionKPB solution and 2 mol.L^-1Washing twice with NaCl solution; under the assistance of microwave, the washed soluble part, the same amount of the bis-azide cross-linking agent and 0.4 equivalent of CuI participate in azide-alkyne cycloaddition click reaction; the immobilized enzyme was separated by centrifugation and eluted with phosphate buffer KPB until no protein was detected in the supernatant.

6. The method for the photocatalytic synthesis of (R) -1- [3, 5-bis (trifluoromethyl) ] phenethyl alcohol by the photocatalyst system as claimed in claim 2, wherein: the reaction solvent is one of methanol, n-hexane, PBS buffer solution, sodium hydroxide solution and hydrochloric acid solution.

7. The method for synthesizing (R) -1- [3, 5-bis (trifluoromethyl) ] phenethyl alcohol catalyzed by the photocatalyst system according to claim 2, which is characterized by comprising the following steps:

(1) the reaction vessel was charged with 100mM PBS buffer (pH 7.0) and 1mM NADP⁺1mM of [ Cp Rh (bpy) (H)₂O)]²⁺Aqueous solution, 250. mu.M TiO₂Nanotubes with a final concentration of 32 mg.L^-1The zymoprotein and 50mM substrate 3,5-bis (trifluoromethyl) acetophenone have a total volume of 5ml, and react for 24 hours under illumination by using a xenon lamp with a wavelength of 420nm and a distance of 15 cm from the reactor;

(2) and (2) taking the supernatant of the reaction liquid in the step (1), extracting the supernatant by using anhydrous n-hexane, and then heating, decompressing and spin-drying to obtain pure (R) -1- [3, 5-bis (trifluoromethyl) ] phenethyl alcohol.

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