Method for continuous flow biocatalytic synthesis of chiral butenafine

Abstract

The present application relates to a kind of continuous flow biocatalytic synthesis method of chiral butylphenylalcohol, using two different carbonyl reductase mutant catalysis 2-pentanoyl benzyl cyanide asymmetric reduction preparation optical active n-butyl phenylalcohol, glucose dehydrogenase provides coenzyme circulation, enzymatic reaction occurs in continuous flow microreactor.Compared with prior art, the present application provides a kind of continuous flow biocatalytic synthesis method of chiral butylphenylalcohol, can stereoselectively catalyze the asymmetric reduction of 2-pentanoyl benzyl cyanide in continuous flow microreactor, generate corresponding two different configuration optical active n-butyl phenylalcohol, reaction condition is mild, catalytic efficiency is high, conversion rate is high, product optical purity is good, ee value can be as high as 94% (S), 99% (R), with good industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to a method for synthesizing chiral butylphthalide, and more particularly to a method for synthesizing chiral butylphthalide by continuous flow biocatalysis. Background Technology

[0002] N-Butylphthalide (NBP), a novel drug independently developed in my country, has shown good therapeutic effects on acute ischemic stroke. In recent years, one configuration of this drug, (S)-NBP, has been discovered to have a series of new medicinal uses, including improving cognitive function, alleviating epilepsy, and treating Alzheimer's disease. Currently, the main synthesis method for this chiral drug is chemical.

[0003] In 2007, Chang et al. reported a method for the chiral synthesis of chiral lactones from phthalides derived from n-butyraldehyde and n-heptaldehyde, using cobalt complexes to promote the addition of iodobenzoates to aldehydes followed by lactone formation. The optically pure butylphthalides produced by this scheme exhibited unstable enantioselectivity, fluctuating between 70% and 98%.

[0004] In 2009, Zhang et al. reported the reaction of ethyl 2-pentanoylbenzoate with 2N-((1S,2S)-2-amino-1,2-bis(3,5-di-tert-butylphenyl)ethyl)-4-methylbenzenesulfonamide in the presence of sodium formate and hexadecyltrimethylammonium bromide to give (S)-NBP, with an overall yield of 61%.

[0005] In 2017, Lu et al. reported the enantioselective tandem hydrogenation / lactoneation of 2-p-phenylcarboxylate catalyzed by [RuCl(benzene)(S)-SunPhos]Cl. This method was applied to the synthesis of (S)-NBP using methyl 2-pentanoylbenzoate as a precursor, achieving yields as high as 94% and an ee value of 99.4% (S). However, the catalysts used in the reaction were expensive, and the added value of the product was not high.

[0006] In summary, chiral butylphthalide can currently be obtained through chemical catalysis, but the reaction conditions are very demanding, with very high requirements for gas pressure, temperature, and reaction environment. Moreover, it is often accompanied by the generation of a large number of byproducts, posing a high safety risk. Furthermore, some precious metal catalysts are expensive and not suitable for large-scale drug preparation. Summary of the Invention

[0007] The present invention addresses the issue of stringent reaction conditions in the preparation of optically active butylphthalide using various chemical methods. The aim of this invention is to overcome the shortcomings of existing technologies, such as demanding reaction conditions, low yields, and high costs, by providing a continuous-flow biocatalytic method for the synthesis of chiral butylphthalide.

[0008] This invention catalyzes the asymmetric reduction of 2-pentanoylbenzyl nitrile using a carbonyl reductase derived from Serratia marcescens and another carbonyl reductase derived from Scheffersomyces stipitis as catalysts. The reaction is carried out in a microreactor with two co-current pumps operating simultaneously to prepare chiral butylphthalide.

[0009] The objective of this invention can be achieved through the following technical solutions:

[0010] This invention provides a carbonyl reductase SmCR K6 Or carbonyl reductase SsCR K1 Application of the carbonyl reductase SmCR in catalyzing the reduction of 2-pentanoylbenzyl nitrile to the corresponding chiral butylphthalide K6 The amino acid sequence is shown in SEQ ID No. 2, and the carbonyl reductase SsCR K1 The amino acid sequence is shown in SEQ ID No. 4.

[0011] In one embodiment of the present invention, the carbonyl reductase SmCR K6 Derived from *Serratia marcescens*. The carbonyl reductase SmCR described in this invention... K6 It was obtained by mutation based on carbonyl reductase SmCR. Those skilled in the art can further combine it with carbonyl reductase SmCR. K6 The amino acid sequence is shown in SEQ ID No. 2, and the carbonyl reductase SmCR was prepared. K6 It is easy. The carbonyl reductase SmCR is derived from Serratia sp. CGMCC 1219, as disclosed in patent CN107142251B. This carbonyl reductase SmCR K6 It exhibits high catalytic activity and excellent stereoselectivity.

[0012] In one embodiment of the present invention, the carbonyl reductase SmCR K6 It is NADPH dependent.

[0013] Another carbonyl reductase, SsCR K1It was obtained by mutation of the carbonyl reductase SsCR. The carbonyl reductase SsCR is derived from *Scheffersomyces stipitis* CBS 6045, which has been reported in the article "Y.-P.Shang,Q.Chen,X.-D.Kong,Y.-J.Zhang,J.-H.Xu,H.-L.Yu*. Efficient synthesis of (R)-2-chloro-1-(2,4-dichlorophenyl)ethanol with a ketoreductase from *Scheffersomyces stipitis* CBS6045, Adv. Synth. Catal., 2017, 359, 426–431". Those skilled in the art have further developed this by combining it with the carbonyl reductase SsCR. K1 The amino acid sequence is shown in SEQ ID No. 4, and the carbonyl reductase SsCR was prepared accordingly. K1 It is easy. The carbonyl reductase SsCR K1 It has a broad substrate spectrum and excellent stereoselectivity.

[0014] In one embodiment of the present invention, the carbonyl reductase SsCR K1 It is NADPH dependent.

[0015] This invention also provides a carbonyl reductase gene, the nucleotide sequence of which is shown in SEQ ID No. 1. The protein encoded by this gene is the carbonyl reductase SmCR. K6 .

[0016] This invention also provides a carbonyl reductase gene, the nucleotide sequence of which is shown in SEQ ID No. 3. The protein encoded by this gene is the carbonyl reductase SsCR. K1 .

[0017] This invention provides a method for the continuous flow biocatalytic synthesis of chiral butylphthalide, using the carbonyl reductase SmCR K6 Or carbonyl reductase SsCR K1 Optically active butylphthalide was prepared by catalytic asymmetric reduction of 2-pentanoylbenzyl nitrile, with glucose dehydrogenase providing the coenzyme cycle. The enzymatic reaction took place in a continuous flow microreactor.

[0018] In one embodiment of the present invention, carbonyl reductase SmCR K6 Or carbonyl reductase SsCR K1The solution and substrate 2-pentanoylbenzyl nitrile solution were continuously fed into a continuous flow microreactor in a certain proportion to carry out the reaction. After biocatalytic reaction, chiral butylphthalide was obtained by cyano hydrolysis cyclization and purification.

[0019] In one embodiment of the present invention, carbonyl reductase SmCR K6 Or carbonyl reductase SsCR K1 glucose dehydrogenase, glucose and NADP + Solution 1 was prepared, and 2-pentanoylbenzyl nitrile was prepared into solution 2. Solutions 1 and 2 were continuously fed into a continuous flow microreactor in proportion to carry out the reaction. After biocatalytic reaction, chiral butylphthalide was obtained by cyano hydrolysis cyclization and purification.

[0020] In one embodiment of the invention, 2-pentanoylbenzylnitrile is dissolved using dimethyl sulfoxide (DMSO).

[0021] In one embodiment of the present invention, a sodium phosphate buffer solution with a concentration of 100 mM and a pH of 6.0 to 7.0 is used to prepare the first solution to participate in the reaction.

[0022] In one embodiment of the present invention, the carbonyl reductase SmCR K6 Or carbonyl reductase SsCR K1 The activity ratio with glucose dehydrogenase is 1:1.5.

[0023] In one embodiment of the present invention, the molar ratio of glucose to 2-pentanoylbenzylnitrile is 1.5:1.

[0024] The volume ratio of dimethyl sulfoxide to the reaction solution is 1:6, and the reaction solution refers to the total solution after mixing solution one and solution two.

[0025] The concentration of NADP+ in the reaction solution is 0.2 mmol / L. The reaction solution refers to the total solution after mixing solution one and solution two.

[0026] In one embodiment of the present invention, the continuous flow microreactor is a continuous flow microchannel reactor, which is composed of 1 to 20 microreactor chips connected together, and has two feed ports and one discharge port. The two feed ports are used to inject solution one and solution two respectively for continuous mixing reaction, and the discharge port is used for discharging the reaction product.

[0027] In one embodiment of the present invention, the continuous flow microreactor is a 3D three-dimensional continuous flow microchannel reactor, composed of three connected microreactor chips. The three microreactor chips are arranged in three layers. The first layer of microreactor chips has two inlets, the third layer of microreactor chips has one outlet, and the second layer of microreactor chips is connected to the first and third layers of microreactor chips. Each microreactor chip has several mixing reaction chambers for liquid mixing reactions. Solution 1 and Solution 2 enter from the two inlets respectively and mix, then pass through each mixing reaction chamber of the first layer of microreactor chips, each mixing reaction chamber of the second layer of microreactor chips, and each mixing reaction chamber of the third layer of microreactor chips in sequence, and then flow out from the outlet of the third layer of microreactor chips. The mixing reaction chambers on the first, second, and third layers of microreactor chips all serve as sites for continuous mixing reactions.

[0028] In one embodiment of the present invention, both feed inlets are connected to relatively long inlet channels, namely feed channel 1 and feed channel 2. Feed channel 1 is divided into two parts and forms a cross-shaped design with feed channel 2, which is beneficial to improving the mixing efficiency of the two fluids.

[0029] In this invention, each microreactor chip has several mixing reaction chambers for liquid mixing reactions. These chambers are connected by pipes. This structural design facilitates localized fluid reflux within the mixing reaction chambers, improving the backmixing degree of different reaction liquids and thus enhancing the mixing and mass transfer efficiency of reactants and catalysts. Furthermore, the cascaded mixing reaction chambers in this invention further enhance the microscopic mixing intensity of the reactants, contributing to a stronger reaction process.

[0030] In one embodiment of the present invention, the microreactor chip is arranged in a serpentine, square, or sawtooth pattern, and the mixing reaction chambers on the microreactor chip are teardrop-shaped, lantern-shaped, or heart-shaped structures. The mixing reaction chambers on the microreactor chip are connected by a microtubular or groove-shaped structure.

[0031] In one embodiment of the present invention, the microreactor chip is made of one of glass, ceramic, corrosion-resistant alloy or fluoropolymer.

[0032] In one embodiment of the present invention, the carbonyl reductase mutant SmCR K6Derived from *Serratia marcescens*, which was discovered in the UniProt biological resource database, strain number: WP_060438329.1 (SmCR); the carbonyl reductase mutant SsCR K1 It was derived from Scheffersomyces stipitis CBS 6045, which was discovered in the UniProt biological resource database, strain number: A3LWG4(SsCR).

[0033] carbonyl reductase SmCR K6 carbonyl reductase SsCR K1 It can catalyze the reduction of 2-pentanoylbenzyl nitrile to produce chiral butylphthalides with different configurations. Among them, the carbonyl reductase SmCR K6 The selectivity for 2-pentanoylbenzylnitrile is 94% (S), and the carbonyl reductase SsCR K1 The selectivity for 2-pentanoylbenzylnitrile is 99% (R).

[0034] In one embodiment of the present invention, the chemical structure of the 2-pentanoylbenzylnitrile is shown below:

[0035]

[0036] In one embodiment of the present invention, the asymmetric reduction of the 2-pentanoylbenzyl nitrile can be carried out according to the following exemplary method: in a phosphate buffer solution at pH 5.5-7.5, with glucose dehydrogenase, glucose, and NADP... + In the presence of the carbonyl reductase SmCR K6 or SsCR K1 Under the action of [unclear], the asymmetric reduction reaction of the 2-pentanoylbenzyl nitrile is catalyzed.

[0037] In one embodiment of the invention, the concentration of the substrate in the reaction solution can be 3–5 mmol / L. Depending on the reaction system used, the amount of carbonyl reductase can be 1–500 U / L.

[0038] During the enzymatic asymmetric reduction of 2-pentanoylbenzyl nitrile, the coenzyme NADPH is oxidized to NADP. + In order to carry out the cyclic regeneration of coenzyme NADPH, glucose and glucose dehydrogenase from Bacillus megaterium were added to the reaction system (J IndMicrob Biotechnol, 2011, 38: 633–641).

[0039] The reaction conversion rate and enantiomeric excess (ee) of the product can be analyzed by liquid chromatography. Preferably, a Chiralpak AD-H (25cm×4.6mm×5μm) microscope is used for conversion rate and ee value analysis, with a mobile phase of n-hexane and isopropanol in a ratio of 95:5. The detector wavelength is 254nm, and the column temperature is kept constant at 35℃.

[0040] In one embodiment of the present invention, after the enzymatic reaction is completed, the reaction solution is cooled to room temperature and extracted with an equal volume of a conventional water-insoluble organic solvent, such as ethyl acetate, butyl acetate, toluene, dichloromethane, chloroform, isopropyl ether, methyl tert-butyl ether, etc. The extraction is repeated twice, and the extracts are combined, washed with saturated brine, and dried overnight with anhydrous sodium sulfate. The solvent is removed by rotary evaporation to obtain the corresponding optically active intermediate. The cyano substituent is then reacted with NaOH under reflux and heated to a carboxyl group. The pH is then adjusted with HCl, and the intermediate is heated at 70-80°C to cyclize, yielding the pure product chiral butylphthalide, which is a yellow oil.

[0041] This invention provides a continuous-flow biocatalytic method for the synthesis of chiral butylphthalide, which exhibits higher catalytic efficiency compared to traditional mechanical stirring methods. The carbonyl reductases used are derived from *Serratia marcescens* and *Scheffersomyces stipitis* CBS 6045, demonstrating high catalytic activity and excellent stereoselectivity. This invention also discloses the use of two parallel-flow pumps to introduce different substances into a continuous-flow microreactor, enabling them to undergo enzymatic reactions within the reactor.

[0042] No previous research has been reported on the synthesis of this chiral drug using biocatalysts. Therefore, this invention utilizes asymmetric reduction of a pre-chiral aromatic ketone with a carbonyl reductase in a continuous flow reactor, followed by cyclization of the molecule by adjusting the pH through heating to obtain chiral butylphthalide. This is an atom-economical synthetic route with mild reaction conditions. The chemical synthetic route of chiral butylphthalide is as follows: Figure 1 As shown.

[0043] Compared with the prior art, the positive progress of the present invention is as follows: The present invention provides a method for the continuous flow biocatalytic synthesis of chiral butylphthalide, which can stereoselectively catalyze the asymmetric reduction of 2-pentanoylbenzyl nitrile in a continuous flow microreactor to generate the corresponding optically active butylphthalide. The reaction conditions are mild, the catalytic efficiency is high, the conversion rate is high, the product has good optical purity, and the ee value can be as high as 94% (S) and 99% (R), which has a very good prospect for industrial application. Attached Figure Description

[0044] Figure 1 This describes the synthetic route for chiral butylphthalide.

[0045] Figure 2 This is a schematic diagram of the structure of a continuous flow microreactor in one embodiment of the present invention.

[0046] Figure 3 This is an enlarged structural diagram illustrating the mixing reaction chamber and inlet channel structure in the first layer of the continuous flow microreactor chip in one embodiment of the present invention.

[0047] Figure 4 For SmCR K6 (A), SsCR K1 (B) Substrate conversion rates in different reaction systems. Detailed Implementation

[0048] This invention provides a method for the continuous flow biocatalytic synthesis of chiral butylphthalide, which uses carbonyl reductase to catalyze the asymmetric reduction of 2-pentanoylbenzyl nitrile to prepare optically active butylphthalide, with glucose dehydrogenase providing the coenzyme cycle, and the enzymatic reaction taking place in a continuous flow microreactor.

[0049] In one embodiment of the present invention, a carbonyl reductase solution and a substrate 2-pentanoylbenzyl nitrile solution are continuously fed into a continuous flow microreactor in a certain proportion to carry out the reaction. After the biocatalytic reaction, chiral butylphthalide is obtained by cyano hydrolysis cyclization and purification.

[0050] In one embodiment of the present invention, carbonyl reductase, glucose dehydrogenase, glucose and NADP are used. + Solution 1 was prepared, and 2-pentanoylbenzyl nitrile was prepared into solution 2. Solutions 1 and 2 were continuously fed into a continuous flow microreactor in proportion to carry out the reaction. After biocatalytic reaction, chiral butylphthalide was obtained by cyano hydrolysis cyclization and purification.

[0051] In one embodiment of the invention, 2-pentanoylbenzylnitrile is dissolved using dimethyl sulfoxide (DMSO).

[0052] In one embodiment of the present invention, a sodium phosphate buffer solution with a concentration of 100 mM and a pH of 6.0 to 7.0 is used to prepare the first solution to participate in the reaction.

[0053] In one embodiment of the present invention, the activity ratio of the carbonyl reductase to glucose dehydrogenase is 1:1.5.

[0054] In one embodiment of the present invention, the molar ratio of glucose to 2-pentanoylbenzylnitrile is 1.5:1.

[0055] The volume ratio of dimethyl sulfoxide to the reaction solution is 1:6, and the reaction solution refers to the total solution after mixing solution one and solution two.

[0056] The concentration of NADP+ in the reaction solution is 0.2 mmol / L. The reaction solution refers to the total solution after mixing solution one and solution two.

[0057] refer to Figure 2 , Figure 3 The microreactor device is a continuous flow microchannel reactor with a 3D three-dimensional structure, consisting of three interconnected microreactor chips. The three microreactor chips are arranged in three layers. The first layer of microreactor chips has two inlets, and the third layer of microreactor chips has one outlet. The second layer of microreactor chips is connected to the first and third layers of microreactor chips. Each microreactor chip has several mixing reaction chambers for liquid mixing reactions. Solution 1 and Solution 2 enter from the two inlets and mix, then pass through each mixing reaction chamber of the first, second, and third layers of microreactor chips in sequence, and finally flow out from the outlet of the third layer of microreactor chips. The mixing reaction chambers on the first, second, and third layers of microreactor chips all serve as sites for continuous mixing reactions.

[0058] Both feed inlets are connected to relatively long inlet channels, namely feed channel 1 and feed channel 2. Feed channel 1 is divided into two parts, forming a cross-shaped design with feed channel 2, which helps to improve the mixing efficiency of the two fluids.

[0059] In this invention, each microreactor chip has several mixing reaction chambers for liquid mixing reactions. These chambers are connected by pipes. This structural design facilitates localized fluid reflux within the mixing reaction chambers, improving the backmixing degree of different reaction liquids and thus enhancing the mixing and mass transfer efficiency of reactants and catalysts. Furthermore, the cascaded mixing reaction chambers in this invention further enhance the microscopic mixing intensity of the reactants, contributing to a stronger reaction process.

[0060] In one embodiment of the present invention, the microreactor chip is arranged in a serpentine, square, or sawtooth pattern, and the mixing reaction chambers on the microreactor chip are teardrop-shaped, lantern-shaped, or heart-shaped structures. The mixing reaction chambers on the microreactor chip are connected by a microtubular or groove-shaped structure.

[0061] In one embodiment of the present invention, the microreactor chip is made of one of glass, ceramic, corrosion-resistant alloy or fluoropolymer.

[0062] In one embodiment of the present invention, the carbonyl reductase mutant SmCR K6Derived from *Serratia marcescens*, which was discovered in the UniProt biological resource database, strain number: WP_060438329.1 (SmCR); the carbonyl reductase mutant SsCR K1 It was derived from Scheffersomyces stipitis CBS 6045, which was discovered in the UniProt biological resource database, strain number: A3LWG4(SsCR).

[0063] carbonyl reductase SmCR K6 and carbonyl reductase SsCR K1 It can catalyze the reduction of 2-pentanoylbenzyl nitrile to produce chiral butylphthalide with different configurations.

[0064] This invention also provides the carbonyl reductase SmCR K6 、SsCR K1 Application in the asymmetric reduction of 2-pentanoylbenzylnitrile. The chemical structure of said 2-pentanoylbenzylnitrile is shown below:

[0065]

[0066] In one embodiment of the present invention, the asymmetric reduction of the 2-pentanoylbenzyl nitrile can be carried out according to the following exemplary method: in a phosphate buffer solution at pH 5.5-7.5, with glucose dehydrogenase, glucose, and NADP... + In the presence of the carbonyl reductase SmCR K6 、SsCR K1 Under the action of [unclear], the asymmetric reduction reaction of the 2-pentanoylbenzyl nitrile is catalyzed.

[0067] In one embodiment of the invention, the concentration of the substrate in the reaction solution can be 3-5 mmol / L. Depending on the reaction system used, the amount of carbonyl reductase can be 1-500 U / L.

[0068] During the enzymatic asymmetric reduction of 2-pentanoylbenzyl nitrile, the coenzyme NADPH is oxidized to NADP. + In order to carry out the cyclic regeneration of coenzyme NADPH, glucose and glucose dehydrogenase from Bacillus megaterium were added to the reaction system (J IndMicrob Biotechnol, 2011, 38: 633–641).

[0069] The reaction conversion rate and enantiomeric excess (ee) of the product can be analyzed by liquid chromatography. Preferably, a Chiralpak AD-H (25cm×4.6mm×5μm) microscope is used for conversion rate and ee value analysis, with a mobile phase of n-hexane and isopropanol in a ratio of 95:5. The detector wavelength is 254nm, and the column temperature is kept constant at 35℃.

[0070] In one embodiment of the present invention, after the enzymatic reaction is completed, the reaction solution is cooled to room temperature and extracted with an equal volume of a conventional water-insoluble organic solvent, such as ethyl acetate, butyl acetate, toluene, dichloromethane, chloroform, isopropyl ether, methyl tert-butyl ether, etc. The extraction is repeated twice, and the extracts are combined, washed with saturated brine, and dried overnight with anhydrous sodium sulfate. The solvent is removed by rotary evaporation to obtain the corresponding optically active intermediate. The cyano substituent is then reacted with NaOH under reflux and heated to a carboxyl group. The pH is then adjusted with HCl, and the intermediate is heated at 70-80°C to cyclize, yielding the pure product chiral butylphthalide, which is a yellow oil.

[0071] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0072] The reaction or detection conditions described in the invention can be combined or modified based on common knowledge in the art, and can be verified experimentally. The invention is further illustrated below by way of examples. It should be understood that although the listed examples illustrate preferred embodiments of the invention, the specific examples are given only to better explain the invention and are not intended to limit the invention to the scope of the described examples.

[0073] The materials used in the following embodiments are sourced from:

[0074] Microreactor.

[0075] The horizontal flow pump was purchased from Beijing Xingda Technology Development Co., Ltd.

[0076] The expression plasmid pET28a was purchased from Novagen.

[0077] E. coli DH5α and E. coli BL21(DE3) competent cells, 2×Taq PCR MasterMix, and agarose gel DNA recovery kit were all purchased from Beijing Tiangen Biotech Co., Ltd.

[0078] Both restriction endonucleases EcoRI and HindIII are commercially available products from New England Biolabs (NEB).

[0079] Unless otherwise stated, the specific experiments in the following examples were performed according to conventional methods and conditions in the art, or in accordance with the product instructions for the kit.

[0080] refer to Figure 2 , Figure 3 The microreactor device is a continuous flow microchannel reactor with a 3D three-dimensional structure, consisting of three interconnected microreactor chips. The three microreactor chips are arranged in three layers. The first layer of microreactor chips has two inlets, and the third layer of microreactor chips has one outlet. The second layer of microreactor chips is connected to the first and third layers of microreactor chips. Each microreactor chip has several mixing reaction chambers for liquid mixing reactions. Solution 1 and Solution 2 enter from the two inlets and mix. Then, they pass through each mixing reaction chamber of the first, second, and third layers of microreactor chips in sequence, and finally flow out from the outlet of the third layer of microreactor chips. The mixing reaction chambers on the first, second, and third layers of microreactor chips all serve as sites for continuous mixing reactions.

[0081] Both feed inlets are connected to relatively long inlet channels, namely feed channel 1 and feed channel 2. Feed channel 1 is divided into two parts, forming a cross-shaped design with feed channel 2, which helps to improve the mixing efficiency of the two fluids.

[0082] Solution 1 (containing carbonyl reductase, glucose dehydrogenase, glucose, and NADP) was prepared using sodium phosphate buffer (100 mM, pH 7.0). + The enzyme solution and substrate solution (2-pentanoylbenzyl nitrile (DMSO co-solvent)) enter the mixing zone of the microreactor through feed channels 1 and 2, respectively, and are instantly and uniformly mixed, ensuring that the two different materials do not come into contact with each other before entering the mixing zone of the microreactor. After the enzyme solution and substrate solution enter the microchannel reactor, the temperature and concentration are homogenized and microscopically controlled in each unit reactor, which enables efficient and rapid catalytic reaction in the tube side of the microreactor, and finally produces the product chiral butylphthalide, which is discharged from the outlet into a 1 mol / L sulfuric acid solution to terminate the reaction.

[0083] The reaction solution was cooled to room temperature and extracted with an equal volume of a conventional water-insoluble organic solvent, such as ethyl acetate, butyl acetate, toluene, dichloromethane, chloroform, isopropyl ether, or methyl tert-butyl ether. The extraction was repeated twice, and the extracts were combined, washed with saturated brine, and dried overnight with anhydrous sodium sulfate. The solvent was removed by rotary evaporation to obtain the corresponding optically active intermediate. The cyano group was then refluxed with NaOH to react with a carboxyl group, and the pH was adjusted with HCl. Heating at 70-80°C cyclized the intermediate to obtain the pure product, chiral butylphthalide, which was a yellow oil.

[0084] Example 1

[0085] The continuous-flow biocatalytic synthesis of chiral butylphthalide from 2-pentanoylbenzyl nitrile was conducted entirely in a microreactor, requiring two parallel-flow pumps to flow through it. Pump 1 injected 2-pentanoylbenzyl nitrile (dissolved in DMSO) into the microreactor, while pump 2 injected carbonyl reductase, glucose dehydrogenase, glucose, and NADP. + The liquids from the two pumps converge in the microreactor, causing the enzyme-catalyzed reaction to occur.

[0086] SmCR, a carbonyl reductase in channel 1 K6 The volumetric activities of glucose dehydrogenase and NADP+ were 1 U / mL and 2 U / mL, respectively. The concentration of NADP+ was 0.2 mmol / L, and the concentration of the substrate 2-pentanoylbenzyl nitrile in pore two was 3 mmol / L.

[0087] In another different reactor, carbonyl reductase SsCR in channel one K1 The volumetric activities of glucose dehydrogenase and NADP+ were 5 U / mL and 10 U / mL, respectively. The concentration of NADP+ was 0.2 mmol / L, and the concentration of the substrate 2-pentanoylbenzyl nitrile in pore two was 3 mmol / L.

[0088] Continuous flow reactions control the residence time of the reaction system in the chip by controlling the injection pump flow rate. Simultaneously, batch reactions under identical reaction conditions are used to sample and test substrate conversion at the same residence time. SmCR K6 In a continuous flow reaction with a residence time of 90 min, the substrate conversion rate reached 98.2%, while in a batch reaction under the same conditions, the substrate conversion rate reached 97.6% after 90 min. (SmCR) K1 In both continuous flow and batch reactions with a residence time of 20 min, substrate conversion reached 100%. Detailed results can be found... Figure 4 .

[0089] The sequences involved in this invention are as follows:

[0090] SEQ ID No.1

[0091] SmCR K6 Nucleotide sequence:

[0092] ATGAGCTCGAAGGTAAAATCGTTCTGGTCACCGGCGCGAGCCGCGGTATTG

[0093] GCCGAGCTATTGCAGAAACGTTTTGTGGCCCGCGGCGCCAAAGTTATCGGCAC

[0094] CGCGACCAGCGGAGAGCGGCGCTGAAGCGATCAGCGGCTACCTGGGCGCAAA

[0095] CGGCAAAGGGTTTATGTTGAACGTTAAGGATGCGCAATCTATCGACAGCGTG

[0096] CTGGCATCGATTCGCGCCGAATTTGGCGAAATCGACATTTTAGTGAATAATGC

[0097] CGGCATTTCGCGTGATAACCTGCTGATGCGTATGAAGGATGATGAGTGGGAG

[0098] GATATCCTCGACACTAACCTGACTTCCGTATTCCGCCTGTCAAAAGCGGTAAT

[0099] GTGCGCTATGATGAAAAAGCGGTTTGGCCGTATCACCATCGGTTCCGTTG

[0100] TCGGCACCATGGGGTTGGCACGGCATGCGAACTACGCGGCGGCTAAAGCCG

[0101] GTCTGATTGGTTTTAGCAAATCTTTGGCACGTGAAGTTGCTTCTCGTGGCATT

[0102] ACGGTCAACGTCGTGGCACCTGGCTACATTGATACGGACATGACACGGGCGT

[0103] TGACAGATGATCAACGCGCAGGCATTTTGTCATCAGTTCCAGCCAACCGGCC

[0104] GGGCGATGCTAAAGAAATCGCCAGCGCTGTTGCATTTTTGGCCTCTGATGAG

[0105] GCCGGCTATATCACCGGTGAAACGTTACATGTCAATGGCGGCATGTACATGAT

[0106] TTAA

[0107] SEQ ID No.2

[0108] SmCR K6 Amino acid sequence:

[0109] MSFEGKIVLVTGASRGIGRAIAETFVARGAKVIGTATSESGAEAISGYLGANGKG

[0110] FMLNVKDAQSIDSVLASIRAEFGEIDILVNNAGISRDNLLMRMKDDEWEDILDT

[0111] NLTSVFRLSKAVMCAMMKKRFGRIITIGSVVGTMGLARHANYAAAKAGLIGFS

[0112] KSLAREVASRGITVNVVAPGYIDTDMTRALTDDQRAGILSSVPANRPGDAKEIASAVAFLASDEAGYITGETLHVNGGMYMI*

[0113] SEQ ID No. 3

[0114] SsCR K1 Nucleotide sequence:

[0115] ATGACTACCTCAGTTTTCGTTTCAGGTGCAACCGGTTACCTTGCCCAACAAAT

[0116] TATTGCACTTGTTCTCTCCAAGGGCTACAAGGTCGTTGGTTCGGTCAGATCTG

[0117] AAGAAAAGGGTGCAAACTTAAAAAAATTGTATGGTGACGATTTCTCCTATGA

[0118] AGTTGTCAAGGTCTTGGAACAGAAGGGTGCTTTCGATGAAGCCTTGAAGAA

[0119] GCACCCAGAAGTTACAATTTTCTTACACACTGCCTCTCCAGTTACCTTCGAAG

[0120] TTGAAGATACCGAAAAGGAAATCTTGATTCCTGCCATTAATGGAACAAAGTAC

[0121] GTCTTGCAATCTATCAAGGACGTTGCTCCTCAAATCACCAGAGTTGTTTACAC

[0122] CAGTTCTGTCGTTGCTATGAGCGTCCCAGAGGAATTAGGTAGCCCAGATGTGG

[0123] TCCTCTCTGAAGCTTCTTGGAGTAGTCTCTCTTACGAGCAATCCAAGACTCAT

[0124] GGAGTTTTGGCTTACTTCGGTTCGAAGCAATTTGCTGAAAGGGCTGCATGGG

[0125] AGTTTGTTGAACAGGAAAAGCCAAACTTTGCTCTCTCGACCGTAAACCCTGC

[0126] ATACATTTTTGGTCCTCAAGCTAAGGACGAGGAAGTTAAGGGTACCTTGAAC

[0127] CTTTCTGCCGAAATGGTTAATTCCGTATTGAAGTTGAATAAGGACGACGATGT

[0128] TCCAGCAACTACTGGTACTTTCATTGATGTTAGAGATGTGGCTAAAGCTCACC

[0129] TTGCAGCCTTCGAAAAGGACGAAGCAAAGGGTGAAAGACTTCTCCTCTCTA

[0130] ACACCAGATTCAATGGTCAAACTCTTTTGGACGTTGTTAGAAAGAACTTCCC

[0131] ACAACTTGCTGACAAGCTTCCAGTTGGAAAGCCACATTCTGACGATTTCTCT

[0132] GCTTTTAAGGAATGGAACGACAAGAAGACCAAGAAGATTCTTGGATTTGAAT

[0133] ACTTCGACTTTGAAACTTCTGTTGTTGACTCAATCAAGCAAGTTTTGAAGGTA

[0134] CAAGGTTAA

[0135] SEQ ID No. 4

[0136] SsCR K1 amino acid sequence:

[0137] MTTSVFVSGATGYLAQQIIALVLSKGYKVVGSVRSEEKGANLKKLYGDDFSYE

[0138] VVKVLEQKGAFDEALKKHPEVTIFLHTASPVTFEVEDTEKEILIPAINGTKYVLQ

[0139] SIKDVAPQITRVVYTSSVVAMSVPEELGSPDVVLSEASWSSLSYEQSKTHGVLA

[0140] YFGSKQFAERAAWEFVEQEKPNFALSTVNPAYIFGPQAKDEEVKGTLNLSAEM

[0141] VNSVLKLNKDDDVPATTGTFIDVRDVAKAHLAAFEKDEAKGERLLLSNTRFNG

[0142] QTLLDVVRKNFPQLADKLPVGKPHSDDFSAFKEWNDKKTKKILGFEYFDFETSVVDSIKQVLKVQG*

[0143] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for continuous flow biocatalytic synthesis of chiral butylphthalide, characterized in that, Optically active n-butylphthalide was prepared by the asymmetric reduction of 2-pentanoylbenzyl nitrile catalyzed by carbonyl reductase, with glucose dehydrogenase providing the coenzyme cycle. The enzymatic reaction occurred in a continuous flow microreactor, and the carbonyl reductase was selected as a carbonyl reductase mutant. Ss CR K1 The carbonyl reductase mutant Ss CR K1 The amino acid sequence is shown in SEQ ID No. 4; Carbonyl reductase, glucose dehydrogenase, glucose and NADP + Solution 1 was prepared, and 2-pentanoylbenzyl nitrile was prepared into solution 2. Solutions 1 and 2 were continuously fed into a continuous flow microreactor in proportion to carry out the reaction. After biocatalytic reaction, chiral butylphthalide was obtained by cyano hydrolysis cyclization and purification. In phosphate buffer at pH 5.5–7.5, glucose dehydrogenase, glucose, and NADP... + In the presence of the carbonyl reductase Ss CR K1 Under the action of [unclear], the asymmetric reduction reaction of the 2-pentanoylbenzyl nitrile is catalyzed; The continuous flow microreactor is a 3D three-dimensional continuous flow microchannel reactor, composed of three interconnected microreactor chips. The three microreactor chips are arranged in three layers. The first layer of microreactor chips has two inlets, and the third layer of microreactor chips has one outlet. The second layer of microreactor chips is connected to the first and third layers of microreactor chips. Each microreactor chip has several mixing reaction chambers for liquid mixing reactions. Solution 1 and Solution 2 enter from the two inlets and mix, then pass through each mixing reaction chamber of the first layer of microreactor chips, each mixing reaction chamber of the second layer of microreactor chips, and each mixing reaction chamber of the third layer of microreactor chips in sequence, and then flow out from the outlet of the third layer of microreactor chips. The mixing reaction chambers on the first, second, and third layers of microreactor chips all serve as sites for continuous mixing reactions. carbonyl reductase Ss CR K1 The volumetric activities of glucose dehydrogenase were 5 U / mL and 10 U / mL, respectively; the concentration of NADP+ was 0.2 mmol / L, and the concentration of the substrate 2-pentanoylbenzylnitrile was 3 mmol / L.

2. The method for continuous flow biocatalytic synthesis of chiral butylphthalide according to claim 1, characterized in that, After the reaction was completed, the reaction solution was cooled to room temperature and extracted with a water-insoluble organic solvent. The solution was washed with saturated brine, dried overnight with anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain the corresponding optically active intermediate. The cyano substituent was then converted to a carboxyl group by reflux with NaOH. The pH was then adjusted with HCl and heated at 70-80℃ to cyclize the intermediate to obtain the pure product, chiral butylphthalide.

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