CN113388646B - Method for synthesizing (R) -1- [3,5-bis (trifluoromethyl) ] phenethyl alcohol by catalysis of optical enzyme system - Google Patents
Method for synthesizing (R) -1- [3,5-bis (trifluoromethyl) ] phenethyl alcohol by catalysis of optical enzyme system Download PDFInfo
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Abstract
The invention relates to a method for synthesizing (R) -1- [3,5-bis (trifluoromethyl) by using a photocatalyst system as a catalyst]Method of phenethyl alcohol, with TiO 2 Nanotube bonding [ Cp Rh (bpy) (H) 2 O)] 2+ Catalytically regenerating NADPH, the generated NADPH is used for catalytically synthesizing (R) -1- [3,5-bis (trifluoromethyl)]And (3) phenethyl alcohol. The method of the invention has low cost and uses TiO 2 The 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
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 by a photocatalysis 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 (trifluoromethylphenyl) 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 studies 2 [(R)-Diop][(R)-BIMAH]Preparation of (R) -1- [3,5-bis (trifluoromethyl)]A novel method for preparing phenethyl alcohol. Although the method for preparing optical alcohol by AH has attracted extensive interest in academic and industrial research, it is complicated by the catalyst orThe conditions should be harsh and most studies are still in the case of small scale production. Thereafter, yu Jianfei et al prepared (R) -1- [3,5-bis (trifluoromethyl) synthetically by 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 the drug intermediate, biocatalysis and chemical synthesis are more matched. 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 can catalyze the formation of (R) -1- [3,5-bis (trifluoromethyl)]Carbonyl reductases of phenethyl alcohol require expensive NAD (P) + As a cofactor, can only be performed.
In biology, however, an NAD (P) H-dependent oxidoreductase can be used to catalyze the reduction of a specific substrate 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 the NAD (P) H-dependent oxidoreductase, while the C4 carbon atom of the nicotinamide ring acts as an 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 capability. 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) by using a photocatalyst system]Method for the preparation of phenethyl alcohol, a method for the regeneration of NADPH for the photocatalytic synthesis of (R) -1- [3,5-bis (trifluoromethyl)]The method of the phenethyl alcohol can reduce steps and environmental pollution,the production cost is reduced, the production safety is improved, and a competitive route is provided for industrialization; the use of TiO in the invention 2 Method for supplying hydrogen and electrons by nanotube cracking water by using [ Cp Rh (bpy) (H) 2 O)] 2+ As hydrogen transfer agent and electron transfer agent, assisted by high-efficiency mild enzyme catalysis reaction; the reaction process has mild condition, 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 TiO 2 Nanotube binding [ Cp Rh (bpy) (H) 2 O)] 2+ Catalytically regenerating NADPH, the generated NADPH is used for catalytically synthesizing (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 photocatalyst system comprises the following steps:
(1) Adding reaction solvent and NADP into a reactor + 、[Cp*Rh(bpy)(H 2 O)] 2+ Aqueous solution, tiO 2 Reacting the nanotube, the zymoprotein and the 3,5-bis (trifluoromethyl) acetophenone for 24 hours under illumination by using a xenon lamp at 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 TiO 2 Nanotube binding [ Cp Rh (bpy) (H) 2 O)] 2+ Catalytically regenerating NADPH, using the generated NADPH for enzyme catalysis, and preparing (R) -1- [3,5-bis (trifluoromethyl) by simulating an illumination reaction for 24h under the room temperature condition]And (3) phenethyl alcohol.
Preferably, tiO is used 2 The nanotube is prepared by the following method:
1g of TiO 2 Dispersing the nano powder in 100ml of 10M NaOH, and stirring in a beaker for 30min; the mixture was then transferred to a teflon lined stainless steel autoclave at room temperatureThe temperature is 140 ℃, and the duration is 18h; 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 TiO 2 A nanotube.
TiO of the invention 2 The nanotubes being made of TiO 2 The nanometer powder is white solid prepared by a calcining method.
The [ Cp Rh (bpy) (H) 2 O)] 2+ The hydrogen transfer agent and the electron transfer agent are synthesized by a chemical method; preferably, [ Cp × Rh (bpy) (H) 2 O)] 2+ The aqueous solution is prepared by the following method:
refluxing rhodium trichloride and 1,2,3,4,5-pentamethylcyclopentadiene in methanol for 24h; 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) (H) 2 O)] 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 -1 Chloramphenicol, 100. Mu.g.mL -1 Kanamycin and 50. Mu.g/mL -1 Ampicillin in TB medium and cultured in a shaking incubator at 34 ℃; when the OD600 is detected to be 0.6-0.8 by ultraviolet, the inducer L- (+) -arabinose of tRNA aminoacyl synthetase is directly added until the final concentration of the mass/volume is 0.2%, and the inducer pZE21-ADH is added to be 60 ng/mL -1 aTc and para-azido-L-phenylalanine are used to induce protein expression; unnatural amino acid to a final concentration of 1 mmol/L -1 ;
After inducing expression at 23 ℃ for 16 hours in a shaking incubator, the bacteria of unnatural amino acid-modified alcohol dehydrogenase were harvested by centrifuging at 8000rpm for 5min, the resulting bacterial pellet was resuspended in PBS buffer, and the bacteria were sonicatedLysing, centrifuging at 10000rpm for 15min to obtain soluble fraction, and separating the soluble fraction with phosphate buffer solution KPB and 2 mol. L -1 Washing 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 photocatalyst system comprises the following steps:
(1) 100mM PBS buffer, pH =7.0, 1mM NADP were added to the reactor + 1mM of [ Cp Rh (bpy) (H) 2 O)] 2+ Aqueous solution, 250. Mu.M TiO 2 Nanotubes with a final concentration of 32 mg.L -1 The 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 used 2 The 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 of a process for preparing (R) -1- [3,5-bis (trifluoromethyl) ] phenethyl alcohol;
FIG. 2 shows the TiO prepared 2 Characterization spectra of the nanotubes, including XPS spectra, SEM spectra, TEM spectra, EDS spectra;
FIG. 3 is an LC-MS spectrum of [ Cp. Multidot.Rh (bpy) Cl ] Cl prepared;
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 the HPLC analysis spectrum of (R) -1- [3,5-bis (trifluoromethyl) ] phenethyl alcohol after the reaction system reacts for 3/6/9/12/24h (A/B/C/D/E), respectively.
Having the embodiments
The 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 by the invention 2 The nanometer powder is purchased from Shanghai Merlin Biotechnology, inc.; the raw material rhodium trichloride trihydrate is purchased from Beijing Hua Weirui Ke chemical Co., ltd; other raw materials of the present invention are commercially available unless otherwise specified.
Example 1
Preparation of TiO 2 Nanotube and method of manufacturing the same
1g of TiO 2 Dispersing the nano powder in 100ml of 10M NaOH, and stirring in a beaker for 30min; then transferring the mixture into a stainless steel autoclave lined with Teflon, and keeping the temperature at 140 ℃ for 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 TiO 2 A nanotube.
Preparing the obtained TiO 2 The 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 ligand was successfully adsorbed toTitanium dioxide nanotubes, and they also smoothly adhere to the surface of the enzyme aggregate to form the composite catalytic material.
Preparation of [ Cp Rh (bpy) (H) 2 O)] 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; diethyl ether was added during which time [ Cp. About. Rh (bpy) Cl]Cl gradually precipitated with the addition of diethyl ether; [ Cp + Rh (bpy) Cl]Cl is easily hydrolyzed to [ Cp Rh (bpy) (H) 2 O)] 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
Cells containing 34. Mu.g/mL of alcohol dehydrogenase mutant were inoculated -1 Chloramphenicol, 100. Mu.g.mL -1 Kanamycin and 50. Mu.g/mL -1 Ampicillin in TB medium, and culturing at 34 ℃ in a shaking incubator; when the OD600 is detected to be 0.6-0.8 by ultraviolet, the inducer L- (+) -arabinose of tRNA aminoacyl synthetase is directly added to the final concentration of 0.2% (w/v), and the inducer pZE21-ADH is added to the final concentration of 60 ng/mL -1 The 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 bacteria of unnatural amino acid modified alcohol dehydrogenase, resuspending the obtained bacteria precipitate with appropriate amount of PBS buffer (20 mmol. 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. L) -1 pH = 7.0) solution and 2mol · L -1 Washing twice with NaCl solution; the washed soluble part, the same amount of the double-fold nitrogen cross-linking agent and 0.4 equivalent of CuI participate in the azide under the assistance of microwaveCycloaddition click reaction of hydride-alkyne. The immobilized enzyme was separated by centrifugation and washed with phosphate buffer KPB (0.01 mol. L) -1 pH = 7.0) until no protein is detected in the supernatant by means of a coomassie brilliant blue staining solution.
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 preparation process of (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 TiO 2 Nanotubes with a final concentration of 32 mg.L -1 Zymoprotein, 50mM substrate 3,5-bis (trifluoromethyl) acetophenone, the total volume is 5ml, and the reaction is carried out for 24h under illumination by using a xenon lamp with the wavelength of 420nm and the distance of 15 cm from the reactor; 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), the standard curve of the standard sample is y =3444.6x +342.34, wherein x represents the concentration of the product in the reaction solution and is in mg/mL, and y represents the peak area of the product.
After the reaction system reacts for 3/6/9/12/24h (A/B/C/D/E), the HPLC analysis spectrum result of (R) -1- [3,5-bis (trifluoromethyl) ] phenethyl alcohol in the system is shown in FIG. 5; high performance liquid phase conditions: c18 column, column temperature 25 ℃,210nm,0.5mL/min, mobile phase: methanol: water =75, (R) -1- [3,5-bis (trifluoromethyl) ] phenethyl alcohol retention time 15.837min. 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 of PBS buffer (pH = 7.0), 1mM NADP + 1mM Cp/Rh (bpy) in water and 250. Mu.M TiO 2 Nanotubes with a final concentration of 32 mg.L -1 Zymoprotein, 50mM substrate 3,5-bis (trifluoromethyl) acetophenone, total volume is 5ml, and reacting under illumination for 24h with xenon lamp 420nm wavelength 15 cm away from the reactor, and adding TiO 2 The nanotube and the zymoprotein are recycled, the recycling is carried out once for 12h, and after the recycling is carried out for 5 times, (R) -1- [3,5-bis (trifluoromethyl)]The conversion of the phenethyl alcohol was substantially unchanged.
HPLC analysis is carried out on (R) -1- [3,5-bis (trifluoromethyl) ] phenethyl alcohol provided by TCI (Tokyo chemical industry Co., ltd.) and a product prepared by the method of the invention, and the retention time of the two products is consistent, so as to determine the structure of the product.
The method of the invention has low cost and uses TiO 2 The 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 (3)
1. A method for synthesizing (R) -1- [3,5-bis (trifluoromethyl) ] phenethyl alcohol by using a photocatalyst system catalyst is characterized by comprising the following steps:
(1) Adding reaction solvent and NADP into a reactor + 、[Cp*Rh(bpy)(H 2 O)] 2+ Aqueous solution, tiO 2 Reacting the nanotube, the zymoprotein and the 3,5-bis (trifluoromethyl) acetophenone for 24 hours under illumination by using a xenon lamp at the wavelength of 420 nm;
(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;
wherein, tiO 2 The nanotube is prepared by the following method:
1g of TiO 2 Dispersing the nano powder in 100ml of 10M NaOH, and stirring in a beaker for 30min; 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 0.1M HCl 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 TiO 2 A nanotube;
[Cp*Rh(bpy)(H 2 O)] 2+ the aqueous solution is prepared by the following method:
refluxing rhodium trichloride and 1,2,3,4,5-pentamethylcyclopentadiene in methanol for 24h; 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) (H) 2 O)] 2+ (ii) a Adding water to make the solution have final concentration of 100mM and storing at room temperature;
the enzyme protein is prepared by the following method:
cells containing 34. Mu.g.mL of the alcohol dehydrogenase mutant were inoculated -1 Chloramphenicol, 100. Mu.g.mL -1 Kanamycin and 50. Mu.g.mL -1 Ampicillin in TB medium and cultured in a shaking incubator at 34 ℃; when the OD600 is detected to be 0.6-0.8 by ultraviolet, the inducer L- (+) -arabinose of tRNA aminoacyl synthetase is directly added until the final concentration of the mass/volume is 0.2%, and the inducer pZE21-ADH is added to be 60ng -1 aTc and para-azido-L-phenylalanine are used to induce protein expression; unnatural amino acid to a final concentration of 1 mmol.L -1 ;
After inducing expression of 16h in a shaking incubator at 23 ℃, the precipitate was centrifuged at 8000rpm for 5min to harvest the unnatural amino acid-modified ethanol dehydrogenase-containing bacteria, and the resulting bacterial precipitate was resuspended in PBS bufferAnd lysing the bacteria by ultrasonic treatment, centrifuging at 10000rpm for 15min to obtain soluble fraction, and separating the soluble fraction with phosphate buffer solution KPB and 2 mol.L -1 Washing 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.
2. The method for synthesizing (R) -1- [3,5-bis (trifluoromethyl) ] phenethyl alcohol by the catalysis of the photocatalyst system according to claim 1, which is characterized in that: the reaction solvent is one of methanol, normal hexane, PBS buffer solution, sodium hydroxide solution and hydrochloric acid solution.
3. The method for synthesizing (R) -1- [3,5-bis (trifluoromethyl) ] phenethyl alcohol by the catalysis of the photocatalyst system according to claim 1, which is characterized by comprising the following steps:
(1) The reactor was charged with 100mM, PBS buffer pH =7.0, 1mM NADP + 1mM [ Cp Rh (bpy) (H) 2 O)] 2+ Aqueous solution, 250. Mu.M TiO 2 Nanotube with final concentration of 32 mg. L -1 The 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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