A method for synthesizing rasagiline in a microfluidic field

Abstract

The present invention discloses a method for synthesizing rasagiline in a microfluidic field. The method is based on a double enzyme hydrogenation cascade reaction, which efficiently converts racemic alcohol into enantiomeric amines, realizes the direct synthesis of chiral amines, and has high atom economy and reaction efficiency. Racemic 1-indanol is completely oxidized to generate 1-indanone by alcohol dehydrogenase catalysis, and the cofactor NADP+ is reduced to NADPH to provide the required reducing power for subsequent reactions; using ( R )-selective reductive amino enzyme catalyzes the reductive amination reaction of 1-indanone to generate the target product rasagiline. The present invention adopts a dual enzyme synergistic catalytic strategy to successfully achieve the efficient conversion from racemic alcohol to chiral amine, has the advantages of mild reaction conditions, green sustainability, high product yield and optical purity, and provides a new efficient, economical and environmentally friendly way for the synthesis of rasagiline.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical synthesis, and particularly relates to a method for synthesizing rasagiline, a drug for treating Parkinson's disease, by using 1-indanol as a starting material and utilizing alcohol dehydrogenase and reductive amino enzyme in a microfluidic field. Background Art

[0002] Rasagiline ((R)-N-propargyl-1-aminoindan, (R)-N-2-Propynyl-1-indanamine, Rasagiline) is a second-generation monoamine oxidase inhibitor that can block the decomposition of the neurotransmitter dopamine. Compared with selegiline (a first-generation monoamine oxidase inhibitor), its inhibitory effect is 5-10 times stronger. It is used alone as a first-line drug for the early treatment of Parkinson's disease (PD). It also has an improvement effect on patients who have experienced a decline in the efficacy of long-term use of dopa preparations. It can be used in combination with levodopa to treat moderate to severe Parkinson's disease. The metabolite of rasagiline is an inactive non-amphetamine substance with few side effects.

[0003] However, most of the current methods for synthesizing rasagiline require expensive reagents as starting materials, have multiple steps including protection and deprotection, and the (R)-1-aminoindan prepared at the same time has the fatal disadvantages of low overall yield and low purity. In addition, the multi-step and long process makes it impossible to efficiently synthesize rasagiline. Therefore, more and more scientists are committed to studying methods for the efficient and green synthesis of rasagiline. Summary of the invention

[0004] Purpose of the invention: In view of the shortcomings of the prior art, the present invention provides a method for synthesizing rasagiline, a drug for treating Parkinson's disease, using 1-indanol as a starting material, alcohol dehydrogenase and reductive amino enzyme in a microfluidic field.

[0005] In order to solve the above technical problems, the present invention provides a method for synthesizing rasagiline in a microfluidic field, comprising the following steps:

[0006] Step S1: immobilizing alcohol dehydrogenase and reductive amino enzyme on resins to obtain immobilized alcohol dehydrogenase and immobilized reductive amino enzyme;

[0007] Step S2: mixing the immobilized alcohol dehydrogenase and the immobilized reductive amino enzyme evenly and then filling the mixture into a microchannel reactor;

[0008] Step S3: 1-indanol, NADP + The mixed solution of propargylamine and 1,2-dihydro-1,4 ...

[0009] The amino acid sequence of the alcohol dehydrogenase is shown in SEQ ID NO.1, and the amino acid sequence of the reductive aminoase is shown in SEQ ID NO.2.

[0010] The alcohol dehydrogenase is prepared by the following preparation method: constructing the alcohol dehydrogenase LsADH gene with a gene sequence as shown in SEQ ID NO.3 into a pET-28a (+) vector, and transferring it into Escherichia coli BL 21 (DE 3), culturing and purifying to obtain the corresponding alcohol dehydrogenase LsADH.

[0011] The reductive amino enzyme is prepared by the following method: constructing the reductive amino enzyme AspRedAm gene with a gene sequence as shown in SEQ ID NO.4 into a pET-28a (+) vector, and transferring it into Escherichia coli BL 21 (DE 3), culturing and purifying to obtain the corresponding reductive amino enzyme AspRedAm.

[0012] In step S1, the usage ratio of the resin to the alcohol dehydrogenase or reductive amino enzyme is 400-1400 mg: 20-70 mg.

[0013] The method for fixing the alcohol dehydrogenase or reductive amino enzyme on the resin is as follows: the alcohol dehydrogenase or reductive amino enzyme obtained by culture and purification is dispersed in a phosphate buffer soaked with pretreated epoxy resin, stirred continuously at 160-200 rpm for 4-8 h at 18-25°C, rinsed three times with 10-50 mM phosphate buffer to remove unfixed proteins, and the resin is removed by draining the filter funnel. The LsADH and AspRedAm are purified and dispersed in PBS buffer, fixed in the microchannel by resin adsorption to participate in the reaction, and the enzyme amount before immobilization and the enzyme amount in the supernatant after immobilization can be measured by an enzyme marker. The calculation formula of the protein adsorption rate is (total enzyme amount before immobilization-enzyme amount in the supernatant) / total enzyme amount before immobilization.

[0014] Preferably, in step S1, the resin needs to be activated before being put into use. The activation step is as follows: First, 1-2 g of epoxy resin is suspended in 10-20 mL of a modification solution (containing 0.1 M sodium borate and 2 M iminodiacetic acid). The mixture is then placed in a constant temperature shaker and gently incubated at 180 r / min for 2-6 h at 20-30°C. Subsequently, the mixture is filtered using a suction funnel to separate the epoxy resin and the modification solution. The resin is then rinsed thoroughly with deionized water, repeated three times, to ensure that the residual modification solution is removed. In the next step, the filtered epoxy resin is incubated with 10-20 mL of 0.5 M sulfuric acid solution at the same temperature and stirring conditions for 2-6 h. This step is intended to eliminate residual epoxy groups, thereby preventing unexpected reactions in future steps. After the incubation is completed, the resin is collected by filtration and rinsed three times with deionized water to remove residual acidic substances. After the acid treatment, the epoxy resin was further treated with 10-20 mL of 0.1 M sodium periodate solution at 20-30°C and 180 r / min for 2-6 h. This step introduced the key functional group aldehyde by promoting the oxidation of the epoxy resin. After incubation, the resin was filtered and rinsed three times with deionized water to remove the unreacted sodium periodate. Subsequently, the treated epoxy resin was mixed with 10-20 mL of metal modification solution and gently incubated for 2-6 h at 20-30°C and 180 r / min. This key step aims to introduce metal ions into the resin structure and enhance its affinity for binding to proteins. After the incubation, the resin was filtered and rinsed three times with deionized water to remove unbound metal ions. In the final step, the epoxy resin was incubated with 10-20 mL of desorption solution at 20-30°C and 180 r / min for 2-6 h to remove loosely bound metal ions and ensure that only tightly attached metal ions remain. The resin was then filtered, rinsed three times with deionized water, and dried using a pump to remove any residual moisture. The treated epoxy resin was then transferred to an appropriate container and stored at 4°C until further use.

[0015] Wherein, the 1-indanol, NADP + The reaction solvent of the mixed solution of propargylamine is a mixture of PBS buffer and an organic solvent, wherein the organic solvent is any one or more of n-heptane, isooctane, methanol, ethanol, isopropanol, acetonitrile, and dimethyl sulfoxide; wherein the volume of the organic solvent is less than or equal to 1% of the total volume of the reaction solvent.

[0016] Step S1, the resin is any one of the hydrophobic epoxy resins ES-1, ES-103B, ES-108, LX-1000EP, NKA and ESD-1.

[0017] Specifically, the concentration of 1-indanol was 5~20 mM, NADP + The concentration is 2~5 mM, and the concentration of propargylamine is 25~100mM.

[0018] The pipeline in the microchannel reaction device is a polytetrafluoroethylene tube with a length of 30-50 cm and a diameter of 2-3 mm.

[0019] Preferably, the microchannel reaction conditions are: 25-30° C. and a residence time of 15-60 min.

[0020] In summary, the present invention discloses a method for generating rasagiline by alcohol dehydrogenase and reductive amino enzyme synergistic catalysis in a microflow field. The method is based on a dual enzyme hydrogen cascade reaction, which efficiently converts racemic alcohol into enantiomeric amines, realizes the direct synthesis of chiral amines, and has high atom economy and reaction efficiency. The present invention adopts a dual enzyme synergistic catalysis strategy of immobilized enzymes, and successfully prepares high-purity rasagiline by using a microchannel reaction device, 1-indanol as a raw material, and an oxidation reaction catalyzed by alcohol dehydrogenase and a reductive amination reaction catalyzed by reductive amino enzyme. The method has the advantages of mild reaction conditions, green sustainability, high product yield, fast reaction and high optical purity, and provides a new efficient, economical and environmentally friendly way for the synthesis of rasagiline.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0022] (1) The present invention, for the first time, uses immobilized alcohol dehydrogenase and reductive amino enzyme in a microfluidic field to deracemize 1-indanol by two-step enzyme combination catalysis to obtain rasagiline, which is difficult to synthesize using chemical methods. This method avoids expensive raw materials, high temperatures, and serious pollution in the preparation method. It is simple to operate, has high optical purity, good selectivity, mild reaction conditions, strong sustainability, high product yield, high safety, and rapid reaction, and is more in line with the requirements of green chemistry.

[0023] (2) The present invention is based on a dual-enzyme hydrogen-borrowing cascade reaction and adopts a dual-enzyme synergistic catalytic strategy of immobilized enzymes to efficiently convert racemic alcohols into enantiomeric amines, thereby achieving direct synthesis of chiral amines with high atom economy and reaction efficiency;

[0024] (3) The present invention adopts a method of immobilizing pure enzymes to successfully immobilize reductive amino enzymes and alcohol dehydrogenases, which are finally filled and fixed in a microchannel reactor and can be stored at 4°C for easy reuse. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0026] Figure 1 is the reaction scheme of the present invention;

[0027] Figure 2 It is a schematic flow diagram of the reaction device of the present invention. DETAILED DESCRIPTION

[0028] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0029] The present invention provides a method for synthesizing rasagiline, a drug for treating Parkinson's disease, using 1-indanol as a starting material and alcohol dehydrogenase and reductive amino enzyme in a microfluidic field. The racemic 1-indanol is completely oxidized to generate 1-indanone by alcohol dehydrogenase (LsADH) catalysis, and the cofactor NADP+ is reduced to NADPH to provide the required reducing power for subsequent reactions. R )-selective reductive amino enzyme (AspRedAm) catalyzes the reductive amination reaction of 1-indanone to produce the target product rasagiline, such as Figure 1 shown.

[0030] The microchannel reaction device described in the following embodiments includes a syringe, a syringe pump, a microchannel reactor and a product collector. The pipeline in the microchannel reactor is a polytetrafluoroethylene tube, on which LsADH and AspRedAm (Immobilized LsADH / AspRedAm) are fixed, with a length of 30-50 cm and a diameter of 2-3 mm. Figure 2 shown.

[0031] The steps for constructing the alcohol dehydrogenase (LsADH) described in the following examples are as follows:

[0032] (1) The source Leifsonia sp. strain S749 The alcohol dehydrogenase LsADH gene (the gene sequence is shown in SEQ ID NO. 4) was constructed into the pET-28a (+) vector (the restriction endonuclease sites are NdeI and Xhol), and then transformed into Escherichia coli BL 21 (DE 3);

[0033] (2) Take 50 μL of the LsADH glycerol strain constructed in step (1) and inoculate it into 5 mL of LB medium, and add kanamycin with a final concentration of 100 μg / mL, and shake culture at 37°C and 200 rpm for 12 h to obtain a pre-cultured bacterial solution. Transfer 5 mL of the pre-cultured bacterial solution to 500 mL of TB (containing 100 mL of PBS) medium, and add kanamycin with a final concentration of 100 μg / mL, and shake culture at 37°C and 200 rpm for about 4 h until the OD reaches 0. 600 When the concentration reaches 0.6-0.8 and the culture medium temperature is cooled to 4°C, IPTG is added to a final concentration of 50 mM to induce expression, and the shaking culture is continued at 18°C ​​for 22 h. The culture solution is concentrated by centrifugation, the bacteria are collected, and the bacteria are suspended in the buffer with PBS buffer on a mixer to prepare a 0.2 g / mL whole cell solution.

[0034] (3) Use an ultrasonic cell disruptor to disrupt the whole cell solution at a power of 200 W, with ultrasound on for 2 s, ultrasound off for 2 s, and ice water bath for 25 min to obtain the whole cell lysate.

[0035] (4) After the cell lysate is fully centrifuged and filtered, the supernatant is taken and pumped into the pre-equilibrated Ni-NTA resin column by a peristaltic pump. After the supernatant is loaded, the resin is gradient eluted with 60 mL of lysis buffer with imidazole concentrations of 10 mM, 20 mM, 30 mM, 40 mM, and 50 mM, respectively. After the impurities are eluted by UV detection analysis, the Ni-NTA is rinsed with lysis buffer containing 250 mM imidazole to collect the pure enzyme. The collected pure enzyme contains a high concentration of imidazole. The imidazole is eluted by ultrafiltration centrifugation using an ultrafiltration tube until the imidazole concentration is less than 10 mM, and then the pure enzyme (20 mg / mL) is collected.

[0036] The steps for constructing the reductive amino enzyme (AspRedAm) described in the following examples are as follows:

[0037] (1) The source Aspergillus oryzae The reductive amino enzyme AspRedAm gene (the gene sequence is shown in SEQ ID NO.4) was constructed into the pET-28a (+) vector (the restriction endonuclease sites are NdeI and Xhol), and then transformed into Escherichia coli BL 21 (DE 3);

[0038] (2) Take 50 μL of the AspRedAm glycerol strain constructed in step (1) and inoculate it into 5 mL of LB medium, and add kanamycin with a final concentration of 100 μg / mL, and shake culture at 37°C and 200 rpm for 12 h to obtain a pre-cultured bacterial solution. Transfer 5 mL of the pre-cultured bacterial solution to 500 mL of TB (containing 100 mL of PBS) medium, and add kanamycin with a final concentration of 100 μg / mL, and shake culture at 37°C and 200 rpm for about 4 h until the OD reaches 0. 600 When the concentration reaches 0.6-0.8 and the culture medium temperature is cooled to 4°C, IPTG is added to a final concentration of 50 mM to induce expression, and the shaking culture is continued at 18°C ​​for 22 h. The culture solution is concentrated by centrifugation, the bacteria are collected, and the bacteria are suspended in the buffer with PBS buffer on a mixer to prepare a 0.2 g / mL whole cell solution.

[0039] (3) Use an ultrasonic cell disruptor to disrupt the whole cell solution at a power of 200 W, with ultrasound on for 2 s, ultrasound off for 2 s, and ice water bath for 25 min to obtain the whole cell lysate.

[0040] (4) After the cell lysate is fully centrifuged and filtered, the supernatant is taken and pumped into the pre-equilibrated Ni-NTA resin column by a peristaltic pump. After the supernatant is loaded, the resin is gradient eluted with 60 mL of lysis buffer with imidazole concentrations of 10 mM, 20 mM, 30 mM, 40 mM, and 50 mM, respectively. After the impurities are eluted by UV detection analysis, the Ni-NTA is rinsed with lysis buffer containing 250 mM imidazole to collect the pure enzyme. The collected pure enzyme contains a high concentration of imidazole. The imidazole is eluted by ultrafiltration centrifugation using an ultrafiltration tube until the imidazole concentration is less than 10 mM, and then the pure enzyme (15 mg / mL) is collected.

[0041] In the following examples, the resin needs to be activated before it is put into use. The activation steps are as follows: First, 1 g of epoxy resin is suspended in 10 mL of a modification solution (containing 0.1 M sodium borate and 2 M iminodiacetic acid). The mixture is then placed in a thermostatic shaker and gently incubated at 180 r / min for 4 h at 20°C. Subsequently, the mixture is filtered using a suction funnel to separate the epoxy resin and the modification solution. The resin is then rinsed thoroughly with deionized water, repeated three times, to ensure that the residual modification solution is removed. In the next step, the filtered epoxy resin is incubated with 10 mL of a 0.5 M sulfuric acid solution for 4 h at the same temperature and stirring conditions. This step is intended to eliminate residual epoxy groups, thereby preventing unexpected reactions in future steps. After the incubation is complete, the resin is collected by filtration and rinsed three times with deionized water to remove residual acidic substances. After the acid treatment, the epoxy resin was further treated with 10 mL of 0.1 M sodium periodate solution at 20°C and 180 r / min for 2-6 h. This step introduces the key functional group aldehyde by promoting the oxidation of the epoxy resin. After incubation, the resin was filtered and rinsed three times with deionized water to remove the unreacted sodium periodate. Subsequently, the treated epoxy resin was mixed with 10-20 mL of metal modification solution and gently incubated for 4 h at 20°C and 180 r / min. This key step aims to introduce metal ions into the resin structure and enhance its affinity for binding to proteins. After the incubation, the resin was filtered and rinsed three times with deionized water to remove unbound metal ions. In the final step, the epoxy resin was incubated with 10 mL of desorption solution at 20°C and 180 r / min for 4 h to remove loosely bound metal ions and ensure that only tightly attached metal ions remain. The resin was then filtered, rinsed three times with deionized water, and dried using a pump to remove any residual moisture. The treated epoxy resin was then transferred to an appropriate container and stored at 4°C until further use.

[0042] The present invention is described in detail below through specific embodiments.

[0043] Example 1

[0044] 800 mg of aqueous resin ES-1 was added to 2 mL of pure LsADH enzyme solution (20 mg / mL, PBS buffer, 50 mM, pH 7.4), and the mixture was slowly stirred at 20 °C and 180 rpm for 8 h, followed by centrifugation to obtain the immobilized enzyme and supernatant. The absorbance of the diluted supernatant was measured at a wavelength of 562 nm using an ELISA reader, and the amount of enzyme in the supernatant under this condition was calculated based on the protein standard curve. The protein adsorption rate of the hydrophobic resin ES-1 for pure LsADH enzyme was 29%. The protein adsorption rate was calculated as follows: protein adsorption rate = (total enzyme amount before immobilization - enzyme amount in supernatant) / total enzyme amount before immobilization.

[0045] Example 2

[0046] 800 mg of aqueous resin ES-103B was added to 2 mL of pure LsADH enzyme solution (20 mg / mL, PBS buffer, 50 mM, pH 7.4), and the mixture was slowly stirred at 20°C and 180 rpm for 8 h, followed by centrifugation to obtain the immobilized enzyme and supernatant. The absorbance of the diluted supernatant was measured at a wavelength of 562 nm using an ELISA reader, and the amount of enzyme in the supernatant under this condition was calculated based on the protein standard curve. The protein adsorption rate of the hydrophobic resin ES-103B for pure LsADH enzyme was 59%. The protein adsorption rate was calculated as follows: protein adsorption rate = (total enzyme amount before immobilization - enzyme amount in supernatant) / total enzyme amount before immobilization.

[0047] Example 3

[0048] 800 mg of resin LX-1000EP was added to 2 mL of LsADH pure enzyme solution (20 mg / mL, PBS buffer, 50 mM, pH 7.4), and the mixture was slowly stirred at 20°C and 180 rpm for 8 h, followed by centrifugation to obtain the immobilized enzyme and supernatant. The absorbance of the diluted supernatant was measured at a wavelength of 562 nm using an ELISA reader, and the amount of enzyme in the supernatant under this condition was calculated based on the protein standard curve. The protein adsorption rate of the hydrophobic resin LX-1000EP for LsADH pure enzyme was 98%. The protein adsorption rate was calculated as follows: protein adsorption rate = (total enzyme amount before immobilization - enzyme amount in supernatant) / total enzyme amount before immobilization.

[0049] Example 4

[0050] 600 mg of resin ES-1 was added to 2 mL of pure AspRedAm enzyme solution (15 mg / mL, PBS buffer, 50 mM, pH 7.4), and the mixture was slowly stirred at 20°C and 180 rpm for 8 h, followed by centrifugation to obtain the immobilized enzyme and supernatant. The absorbance of the diluted supernatant was measured at a wavelength of 562 nm using an ELISA reader, and the amount of enzyme in the supernatant under these conditions was calculated based on the protein standard curve. The protein adsorption rate of the hydrophobic resin ES-1 for pure AspRedAm enzyme was 78%. The protein adsorption rate was calculated as follows: protein adsorption rate = (total enzyme amount before immobilization - enzyme amount in supernatant) / total enzyme amount before immobilization.

[0051] Example 5

[0052] 600 mg of water-based resin ES-103B was added to 2 mL of pure AspRedAm enzyme solution (15 mg / mL, PBS buffer, 50 mM, pH 7.4). After slow stirring at 20°C and 180 rpm for 8 h, the immobilized enzyme and supernatant were separated by centrifugation. The absorbance of the diluted supernatant was measured at a wavelength of 562 nm using an ELISA reader, and the amount of enzyme in the supernatant under this condition was calculated based on the protein standard curve. The protein adsorption rate of the hydrophobic resin ES-103B for pure AspRedAm enzyme was 90%. The protein adsorption rate calculation formula is: protein adsorption rate = (total enzyme amount before immobilization - enzyme amount in supernatant) / total enzyme amount before immobilization.

[0053] Example 6

[0054] 600 mg of aqueous resin LX-1000EP was added to 2 mL of pure AspRedAm enzyme solution (15 mg / mL, PBS buffer, 50 mM, pH 7.4), and the mixture was slowly stirred at 20°C and 180 rpm for 8 h, followed by centrifugation to obtain the immobilized enzyme and supernatant. The absorbance of the diluted supernatant was measured at a wavelength of 562 nm using an ELISA reader, and the amount of enzyme in the supernatant under this condition was calculated based on the protein standard curve. The protein adsorption rate of the hydrophobic resin LX-1000EP for pure AspRedAm enzyme was 46%. The protein adsorption rate was calculated as follows: protein adsorption rate = (total enzyme amount before immobilization - enzyme amount in supernatant) / total enzyme amount before immobilization.

[0055] Example 7

[0056] 1 g of resin LX-1000EP was added to 4 mL of LsADH pure enzyme solution (concentration of 20 mg / mL) and slowly stirred at 20°C and 180 rpm for 8 hours. Then, the resin was removed, centrifuged and the water was drained with a suction funnel;

[0057] 1 g of resin ES-103B was added to 4 mL of pure AspRedAm enzyme solution (concentration 20 mg / mL), and stirred slowly at 20°C and 180 rpm for 8 hours. After removing the resin, centrifuge and drain the water.

[0058] Take 600 mg of each of the two immobilized enzyme resins and add 10 mM phosphate buffer to mix evenly, and use a suction funnel to drain the water in the mixed resin again. Finally, fill the mixed immobilized resin into a microchannel reactor with a diameter of 2.5 mm and a length of 40 cm for later use.

[0059] 50 mL of 10 mM 1-indanol solution (the reaction solvent is a mixture of phosphate buffer, 50 mM, pH 7.4 and an organic solvent, including NADP + : 2 mM, propargylamine: 50 mM, 10% DMSO) were placed in a syringe and injected into the microchannel reactor via the syringe. The reaction temperature was 25°C and the flow rate was controlled at 10 mL / min. After reacting for about 1 hour, sampling was performed and the formation of rasagiline was monitored by HPLC. The reaction conversion rate of 1-indanol was greater than or equal to 99%, and the ee value was greater than or equal to 99%.

[0060] Example 8

[0061] 1 g of resin LX-1000EP was added to 4 mL of LsADH pure enzyme solution (concentration of 20 mg / mL) and slowly stirred at 20°C and 180 rpm for 8 hours. Then, the resin was removed, centrifuged and the water was drained with a suction funnel;

[0062] 1 g of resin ES-103B was added to 4 mL of pure AspRedAm enzyme solution (concentration 20 mg / mL), and stirred slowly at 20°C and 180 rpm for 8 hours. After removing the resin, centrifuge and drain the water.

[0063] Take 600 mg of each of the two immobilized enzyme resins and mix them evenly, and use a suction funnel to drain the water in the mixed resin again. Finally, fill the mixed immobilized resin into a microchannel reactor with an inner diameter of 2.5 mm and a length of 40 cm for standby use.

[0064] 50 mL of 10 mM 1-indanol solution (the reaction solvent is a mixture of phosphate buffer, 50 mM, pH 7.4 and an organic solvent, including NADP +: 2 mM, propargylamine: 50 mM, 10% DMSO) were placed in a syringe and injected into the microchannel reactor via the syringe. The reaction temperature was 25°C and the flow rate was controlled at 15 mL / min. After reacting for about 40 min, samples were taken and the formation of rasagiline was monitored by HPLC. The reaction conversion rate of 1-indanol was greater than or equal to 99%, and the ee value was greater than or equal to 99%.

[0065] Example 9

[0066] 1 g of resin LX-1000EP was added to 4 mL of LsADH pure enzyme solution (concentration of 20 mg / mL) and slowly stirred at 20°C and 180 rpm for 8 hours. Then, the resin was removed, centrifuged and the water was drained with a suction funnel;

[0067] 1 g of resin ES-103B was added to 4 mL of pure AspRedAm enzyme solution (concentration 20 mg / mL), and the mixture was slowly stirred at 20°C and 180 rpm for 8 hours. After the resin was removed, it was centrifuged and the water was drained.

[0068] Take 600 mg of each of the two immobilized enzyme resins and mix them evenly, and use a suction funnel to drain the water in the mixed resin again. Finally, fill the mixed immobilized resin into a microchannel reactor with a diameter of 2.5 mm and a length of 40 cm for use.

[0069] 50 mL of 10 mM 1-indanol solution (containing NADP + : 2 mM, propargylamine: 50 mM) were placed in a syringe and injected into the microchannel reactor via the syringe. The reaction temperature was 25°C and the flow rate was controlled at 20 mL / min. After reacting for about 30 min, samples were taken and the formation of rasagiline was monitored by HPLC. The reaction conversion rate of 1-indanol was greater than or equal to 99%, and the ee value was greater than or equal to 99%.

[0070] Comparative Example 1

[0071] 1 g of resin LX-1000EP was added to 4 mL of LsADH pure enzyme solution (concentration of 20 mg / mL) and slowly stirred at 20°C and 180 rpm for 8 hours. Then, the resin was removed, centrifuged and the water was drained with a suction funnel;

[0072] 1 g of resin ES-103B was added to 4 mL of pure AspRedAm enzyme solution (concentration 20 mg / mL), and stirred slowly at 20°C and 180 rpm for 8 hours. After removing the resin, centrifuge and drain the water.

[0073] The two immobilized enzyme resins were mixed evenly, and the water in the mixed resin was drained again with a suction funnel. Finally, the mixed immobilized resin was filled into a microchannel reactor with a diameter of 2.5 mm and a length of 40 cm for standby use.

[0074] 50 mL of 10 mM 1-indanol solution (containing NADP + : 2 mM, propargylamine: 50 mM) were placed in a syringe and injected into the microchannel reactor via the syringe. The reaction temperature was 25°C and the flow rate was controlled at 40 mL / min. After reacting for about 15 minutes, sampling was performed and the formation of rasagiline was monitored by HPLC. The reaction conversion rate of 1-indanol was about 69%, and the ee value was greater than or equal to 99%.

[0075] Comparative Example 2

[0076] 1 g of resin LX-1000EP was added to 4 mL of LsADH pure enzyme solution (concentration of 20 mg / mL) and slowly stirred at 20°C and 180 rpm for 8 hours. Then, the resin was removed, centrifuged and the water was drained with a suction funnel;

[0077] 1 g of resin ES-103B was added to 4 mL of pure AspRedAm enzyme solution (concentration 20 mg / mL), and stirred slowly at 20°C and 180 rpm for 8 hours. After removing the resin, centrifuge and drain the water.

[0078] The two immobilized enzyme resins were mixed evenly, and the water in the mixed resin was drained again with a suction funnel. Finally, the mixed immobilized resin was filled into a microchannel reactor with a diameter of 2 mm and a length of 50 cm for standby use.

[0079] 50 mL of 10 mM 1-indanol solution (containing NADP +: 2 mM, propargylamine: 50 mM) were placed in a syringe and injected into the microchannel reactor through the syringe. The reaction temperature was 25°C and the flow rate was controlled at 10 mL / min. After reacting for about 1 h, sampling was performed and the generation of rasagiline was monitored by HPLC. The reaction conversion rate of 1-indanol was about 89%, and the ee value was greater than or equal to 99%; the flow rate was controlled at 15 mL / min, and after balancing for 30 min, the reaction was continued for about 40 min, sampling was performed and the generation of rasagiline was monitored by HPLC. The reaction conversion rate of 1-indanol was about 75%, and the ee value was greater than or equal to 99%; the flow rate was controlled at 20 mL / min, and after balancing for 30 min, the reaction was continued for about 30 min, sampling was performed and the generation of rasagiline was monitored by HPLC. The reaction conversion rate of 1-indanol was about 59%, and the ee value was greater than or equal to 99%; the flow rate was controlled at 40 mL / min, and after balancing for 30 min, the reaction was continued for about 15 min, sampling was performed, and the formation of rasagiline was monitored by HPLC. The reaction conversion rate of 1-indanol was about 32%, and the ee value was greater than or equal to 99%.

[0080] Comparative Example 3

[0081] 1 g of resin LX-1000EP was added to 4 mL of LsADH pure enzyme solution (concentration of 20 mg / mL) and slowly stirred at 20°C and 180 rpm for 8 hours. Then, the resin was removed, centrifuged and the water was drained with a suction funnel;

[0082] 1 g of resin ES-103B was added to 4 mL of pure AspRedAm enzyme solution (concentration 20 mg / mL), and stirred slowly at 20°C and 180 rpm for 8 hours. After removing the resin, centrifuge and drain the water.

[0083] The two immobilized enzyme resins were mixed evenly, and the water in the mixed resin was drained again with a suction funnel. Finally, the mixed immobilized resin was filled into a microchannel reactor with a diameter of 3 mm and a length of 30 cm for standby use.

[0084] 50 mL of 10 mM 1-indanol solution (containing NADP +: 2 mM, propargylamine: 50 mM) were placed in a syringe and injected into the microchannel reactor through the syringe. The reaction temperature was 25°C and the flow rate was controlled at 10 mL / min. After reacting for about 1 h, sampling was performed and the generation of rasagiline was monitored by HPLC. The reaction conversion rate of 1-indanol was about 85%, and the ee value was greater than or equal to 99%; the flow rate was controlled at 15 mL / min, and after balancing for 30 min, the reaction was continued for about 40 min, sampling was performed and the generation of rasagiline was monitored by HPLC. The reaction conversion rate of 1-indanol was about 79%, and the ee value was greater than or equal to 99%; the flow rate was controlled at 20 mL / min, and after balancing for 30 min, the reaction was continued for about 30 min, sampling was performed and the generation of rasagiline was monitored by HPLC. The reaction conversion rate of 1-indanol was about 56%, and the ee value was greater than or equal to 99%; the flow rate was controlled at 40 mL / min, and after balancing for 30 min, the reaction was continued for about 15 min, sampling was performed, and the formation of rasagiline was monitored by HPLC. The reaction conversion rate of 1-indanol was about 36%, and the ee value was greater than or equal to 99%.

[0085] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for synthesizing rasagiline in a microfluidic field, characterized in that: The steps include: Step S1: immobilizing alcohol dehydrogenase and reductive amino enzyme on resins to obtain immobilized alcohol dehydrogenase and immobilized reductive amino enzyme; Step S2: mixing the immobilized alcohol dehydrogenase and the immobilized reductive amino enzyme evenly and then filling the mixture into a microchannel reactor; Step S3: 1-indanol, NADP + The mixed solution of propargylamine and 1,2-dihydro-1,4 ... Wherein, the amino acid sequence of the alcohol dehydrogenase is shown in SEQ ID NO.1, and the amino acid sequence of the reductive amino enzyme is shown in SEQ ID NO.2; The alcohol dehydrogenase is prepared by the following preparation method: constructing the alcohol dehydrogenase LsADH gene with a gene sequence as shown in SEQ ID NO.3 into a pET-28a (+) vector, and transferring it into Escherichia coli BL 21 (DE 3), culturing and purifying to obtain the alcohol dehydrogenase LsADH; The reductive amino enzyme is prepared by the following method: constructing the reductive amino enzyme AspRedAm gene with a gene sequence as shown in SEQ ID NO.4 into a pET-28a (+) vector, and transferring it into Escherichia coli BL 21 (DE 3), culturing and purifying to obtain the reductive amino enzyme AspRedAm; In step S1, the ratio of the amount of the resin to the alcohol dehydrogenase or the reductive aminase is 400-1400 mg: 20-70 mg; the method for immobilizing the alcohol dehydrogenase or the reductive aminase on the resin is: dispersing the alcohol dehydrogenase or the reductive aminase obtained by culture and purification in a phosphate buffer soaked with the pretreated epoxy resin, stirring continuously at 160-200 rpm for 4-8 h at 18-25° C., washing several times with 10-50 mM phosphate buffer, and draining the immobilized resin with a suction funnel; The 1-indanol, NADP + The reaction solvent of the mixed solution of propargylamine is a mixture of PBS buffer and an organic solvent, wherein the organic solvent is any one or more of n-heptane, isooctane, methanol, ethanol, isopropanol, acetonitrile, and dimethyl sulfoxide; wherein the volume of the organic solvent is less than or equal to 1% of the total volume of the reaction solvent; In step S3, the concentration of 1-indanol is 5-20 mM, and NADP + The concentration is 2~5 mM, and the concentration of propargylamine is 25~100 mM; The microchannel reaction conditions are: reaction temperature is 25~30℃ and residence time is 15~60 min.

2. The method according to claim 1, characterized in that Step S1, the resin is any one of the hydrophobic epoxy resins ES-1, ES-103B, ES-108, LX-1000EP, NKA and ESD-1.

3. The method according to claim 1, characterized in that The pipe in the microchannel reactor is a polytetrafluoroethylene tube with a length of 30-50 cm and a diameter of 2-3 mm.

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