Method for preparing L-aminopropanol by splitting DL-aminopropanol

Abstract

The invention belongs to the technical field of fine chemical engineering, and particularly relates to a method for preparing L-aminopropanol by splitting DL-aminopropanol. The resolution method comprises the following steps: (1) preparing immobilized CALB lipase; (2) preparing a bimetallic catalyst; (3) performing transesterification with vinyl acetate under the action of a bimetallic catalyst and the immobilized CALB lipase; and (4) carrying out alkaline hydrolysis reaction with sodium hydroxide, and then carrying out post-treatment to obtain the L-aminopropanol. According to the method for preparing the L-aminopropanol by splitting the DL-aminopropanol, all the steps have a synergistic effect, and it is ensured that the prepared L-aminopropanol has the high yield and purity.

Description

Technical Field

[0001] This invention belongs to the field of fine chemical technology, specifically relating to a method for resolving DL-aminopropanol to prepare L-aminopropanol. Background Technology

[0002] Levofloxacin is the levorotatory isomer of ofloxacin, and its in vitro antibacterial activity is approximately twice that of ofloxacin. Its mechanism of action involves inhibiting the activity of bacterial DNA gyrase, thereby preventing bacterial DNA synthesis and replication, leading to bacterial death. It is primarily used to treat acute and chronic lower respiratory tract infections. Levofloxacin has two optical configurations: the S configuration and the R configuration. The R configuration has no pharmacological activity.

[0003] The chiral group in levofloxacin comes from L-aminopropanol, and the ability to effectively control the content of the enantiomer D-aminopropanol in L-aminopropanol plays a crucial role in the quality of levofloxacin.

[0004] Chiral L-aminopropanol is generally prepared by reducing chiral 2-aminopropionic acid or its derivatives, but the chiral raw materials and reducing agents are relatively expensive. Furthermore, most 2-aminopropanols obtained by conventional synthetic methods are racemic mixtures; therefore, there is currently no economically feasible method for resolving racemic DL-aminopropanol to prepare L-aminopropanol. Summary of the Invention

[0005] The purpose of this invention is to explore a novel method for resolving DL-aminopropanol to prepare L-aminopropanol, which has the advantages of high yield and high purity of the prepared L-aminopropanol.

[0006] The method for preparing L-aminopropanol by resolving DL-aminopropanol according to the present invention comprises the following steps: (1) Dissolve CALB lipase in 0.1 mol / L phosphate buffer with pH=7 for 1-1.2 h to neutralize the surface charge and prepare enzyme solution. Use SBA-15 mesoporous molecular sieve as carrier, mix the carrier with enzyme solution, shake and adsorb at 33-35℃ for 7 hours, then filter, rinse with buffer, dry and store to prepare immobilized CALB lipase. (2) Preparation of bimetallic catalysts ① Mix silane coupling agent KH550, anhydrous ethanol and silica powder, add acetic acid to adjust the pH of the system to 5 and mechanically stir. After drying and pretreatment, the resulting mixture is transferred to a high-temperature reactor for surface modification. The modified product is filtered, washed and dried to prepare an amino-grafted silica carrier. ② The amino-grafted silica carrier was dispersed in anhydrous ethanol and subjected to ultrasonic treatment to prepare an amino-grafted silica carrier dispersion. ③ Under a nitrogen atmosphere, Ru3(CO) 12 Ru nanoparticles were prepared by placing (ruthenium dodecylcarbonyltriruthenium) into a tube furnace and heating it to 500°C at a rate of 5°C / min and holding it for 2 hours. ④ Disperse Ru nanoparticles in anhydrous ethanol and perform ultrasonic dispersion to prepare a nano Ru dispersion. Disperse WC (tungsten carbide) nanoparticles in anhydrous ethanol and perform ultrasonic dispersion to prepare a nano WC dispersion. Mix the nano Ru dispersion and the nano WC dispersion evenly and then dropwise add them to the amino-grafted silica carrier dispersion for stirring reaction. ⑤ The mixture obtained in ④ was rotary evaporated and then dried under vacuum to remove the solvent. Then it was kept at a temperature under a nitrogen atmosphere to prepare the Ru-WC / NH2-SiO2 catalyst. (3) Dissolve the Ru-WC / NH2-SiO2 catalyst and the immobilized CALB lipase in tert-butanol-buffer, add DL-aminopropanol, and then add vinyl acetate dropwise to the above reaction system. React in a constant temperature water bath at 48-50℃ for 3.8-4h. After the reaction is complete, filter to remove the Ru-WC / NH2-SiO2 catalyst particles, then add hydrochloric acid to acidify to pH=3, and finally filter to remove the immobilized CALB lipase. Extract the filtrate three times with ethyl acetate and combine the organic phases. (4) The combined ethyl acetate extract was transferred to a reaction vessel and an equal volume of sodium hydroxide solution was added for hydrolysis. After the reaction was completed, the organic phase was removed by separation. The pH of the aqueous phase was adjusted to 7.0 with hydrochloric acid. The aqueous phase was then rotary evaporated to obtain crude L-aminopropanol. Finally, high-purity L-aminopropanol was prepared by recrystallization.

[0007] in: In step (1), the CALB lipase is manufactured by Guangdong Maike Baisheng Biotechnology Co., Ltd., and the SBA-15 mesoporous molecular sieve is manufactured by Jiangsu Xianfeng Nanomaterials Technology Co., Ltd.

[0008] In step (1), the mass ratio of CALB lipase to phosphate buffer is 1:1.5, and the unit is g / mL.

[0009] In step (1), the mass ratio of SBA-15 mesoporous molecular sieve to enzyme solution is 1:2, with units of g / mL.

[0010] The buffer rinsing in step (1) is to rinse three times with a phosphate buffer solution with a concentration of 0.1 mol / L and pH=7. The drying is to dry at 40°C for 2.5 h. The storage is to keep at a temperature of 4°C.

[0011] In step (2)①, the mass of silane coupling agent KH550 accounts for 10 wt% of the mass of silica powder.

[0012] In step (2)①, the mass ratio of silica powder to anhydrous ethanol is 1:30.

[0013] In step (2)①, the mechanical stirring speed is 500 r / min, the stirring time is 30 min, the drying pretreatment is carried out under vacuum, the drying pretreatment temperature is 80℃, and the drying pretreatment time is 2 h.

[0014] In step (2)①, the surface modification temperature is 180℃, the surface modification time is 2h, the surface modification atmosphere is nitrogen, and the pressure is 0.3MPa.

[0015] The washing process described in step (2) ① involves first washing with anhydrous ethanol three times, and then washing with deionized water twice.

[0016] The drying described in step (2) ① is carried out under vacuum at 70°C for 9 hours.

[0017] The ultrasonic treatment time in step (2) ② is 18-20 min.

[0018] In step (2) ②, the mass-to-volume ratio of amino-grafted silica carrier to anhydrous ethanol is 1:30, with units of g / mL.

[0019] In step (2)④, the mass ratio of Ru nanoparticles to anhydrous ethanol in the nano Ru dispersion is 0.07:1, with units of g / mL. The mass ratio of WC nanoparticles to anhydrous ethanol in the nano WC dispersion is 0.5:1, with units of 0.5 g / mL.

[0020] The mass of Ru nanoparticles in step (2) ④ accounts for 3.5-4 wt% of the mass of amino-grafted silica carrier in step (2) ②, and the mass of nano WC in step (2) ④ accounts for 1 wt% of the mass of amino-grafted silica carrier in step (2) ②.

[0021] In step (2) ④, the stirring reaction time is 2 hours, the stirring reaction temperature is 55-57℃, and the stirring reaction speed is 500 r / min.

[0022] In step (2) ④, the ultrasonic time for preparing nano Ru dispersion and nano WC dispersion is 15 min.

[0023] In step (2) ⑤, the rotary evaporation temperature is 55℃, the drying temperature is 75℃, and the drying time is 5h. The heat preservation treatment is to raise the temperature to 200℃ at a heating rate of 5°C / min and keep it warm for 2 hours.

[0024] In step (3), the concentration of DL-aminopropanol in tert-butanol-buffered solution is 1.0-1.2 mol / L; the concentration of vinyl acetate in tert-butanol-buffered solution is 1.8-2.0 mol / L.

[0025] In step (3), the volume ratio of tert-butanol to phosphate buffer with a concentration of 0.1 mol / L and pH=7 in the tert-butanol-buffered solution is 3:2.

[0026] In step (3), the concentration of Ru-WC / NH2-SiO2 catalyst in tert-butanol-buffered solution is 18-20 g / L.

[0027] In step (3), the mass of the immobilized CALB lipase is 8-10% of the mass of DL-aminopropanol.

[0028] The concentration of hydrochloric acid in step (3) is 2 mol / L.

[0029] In step (3), the addition of vinyl acetate must be completed within 1 hour.

[0030] In step (3), the immobilized CALB lipase and Ru-WC / NH2-SiO2 catalyst are used in combination to enable the in-situ racemization and recycling of the D-isomer, and the Ru-WC / NH2-SiO2 catalyst is used as a metal racemization catalyst; the metal catalyst is removed first and then acidified to prevent the acid from damaging the structure of the metal catalyst.

[0031] In step (3), the first filtration uses a 0.22 μm polytetrafluoroethylene filter membrane, the filter residue is a Ru-WC / NH2-SiO2 catalyst, and the filtrate is L-aminopropanol acetate and residual substrate, etc.; the second filtration can be done with ordinary filter paper. After adding hydrochloric acid to the filtrate, CALB lipase detaches from the carrier due to the change in surface charge. The filter residue is an SBA-15 carrier, and the filtrate contains L-aminopropanol acetate and free enzyme.

[0032] In step (3), the Ru-WC / NH2-SiO2 catalyst racemizes the unreacted D-aminopropanol into a mixture of DL-aminopropanols, while the immobilized enzyme selectively esterifies L-aminopropanol into L-aminopropanol acetate. The D-type remains hidden until it is racemized, and the racemization rate is greater than the enzyme catalysis rate, ensuring that the D-type does not accumulate.

[0033] In step (4), the volume ratio of ethyl acetate extract to sodium hydroxide solution is 1:1, and the concentration of sodium hydroxide solution is 0.5 mol / L.

[0034] In step (4), the hydrolysis reaction temperature is 53-55℃ and the hydrolysis reaction time is 2h.

[0035] In step (4), add 2.0 mol / L hydrochloric acid to adjust the pH value to 7.0.

[0036] In step (4), the rotary evaporation temperature is 60℃ and the rotary evaporation pressure is 0.1MPa.

[0037] In step (4), recrystallization is performed using a mixture of ethanol and water, wherein the volume ratio of ethanol to water is 4:1.

[0038] Compared with the prior art, the present invention has the following advantages: (1) The method for preparing L-aminopropanol by resolving DL-aminopropanol according to the present invention uses SBA-15 mesoporous molecular sieve as a support in the preparation of immobilized CALB lipase. The enzyme is anchored in the pores by physical adsorption. When preparing the bimetallic catalyst Ru-WC / NH2-SiO2 catalyst, KH550 forms -NH2 groups on the surface of SiO2, providing anchoring sites for metal loading. Highly active Ru is obtained by carbonyl thermal decomposition. Ru and WC are used in combination. WC promotes hydrogen overflow, and Ru accelerates substrate dehydrogenation / addition, which together enhance the D-type racemization rate. Nitrogen treatment at 200°C enhances the interaction between the metal and the support and prevents it from falling off during the reaction. In step (3), the immobilized CALB lipase and Ru-WC / NH2-SiO2 catalyst are used in combination. The immobilized CALB lipase specifically catalyzes the esterification reaction of L-aminopropanol and vinyl acetate, while the Ru-WC / NH2-SiO2 catalyst races D-aminopropanol into a DL- mixture through dehydrogenation / rehydrogenation. The amount of Ru-WC / NH2-SiO2 catalyst and immobilized CALB lipase is strictly controlled to ensure that the racemization rate is greater than the esterification rate. In step (4), high-purity L-aminopropanol is prepared through hydrolysis and purification reactions. Thus, each step works synergistically and is indispensable to achieve the resolution of DL-aminopropanol.

[0039] (2) The method for preparing L-aminopropanol by resolving DL-aminopropanol according to the present invention utilizes the synergistic effect of metal Ru and WC in the bimetallic catalyst Ru-WC / NH2-SiO2. Ru activates the α-CH bond of D-aminopropanol to generate an imine intermediate. The imine intermediate undergoes non-stereoselective hydrogenation on the Ru surface to generate a racemic mixture. WC acts as a hydrogen overflow channel, rapidly transferring the H atoms dissociated from Ru to the imine while enhancing the stability of the prepared catalyst. Using NH2-SiO2 as a support, the -NH2 group is protonated at pH=7, and negatively charged Ru / WC nanoparticles are electrostatically adsorbed. The pore size of SiO2 constrains the bimetallic particle size, preventing agglomeration during the reaction. Thus, the bimetallic catalyst Ru-WC / NH2-SiO2 plays a good catalytic role in the racemic reaction. Detailed Implementation

[0040] The present invention will be further described below with reference to embodiments.

[0041] Example 1 The method for preparing L-aminopropanol by resolving DL-aminopropanol as described in Example 1 consists of the following steps: (1) The lipase CALB was dissolved in phosphate buffer with a concentration of 0.1 mol / L and pH=7 and soaked for 1.1 h to neutralize the surface charge and prepare an enzyme solution. Using SBA-15 mesoporous molecular sieve as a carrier, the carrier and enzyme solution were mixed and adsorbed at 34℃ for 7 hours. Then, the mixture was separated by vacuum filtration, rinsed with buffer, dried and stored to prepare immobilized CALB lipase. (2) Preparation of bimetallic catalysts ① Mix silane coupling agent KH550, anhydrous ethanol and silica powder, add acetic acid to adjust the pH of the system to 5 and mechanically stir. After drying and pretreatment, the resulting mixture is transferred to a high-temperature reactor for surface modification. The modified product is filtered, washed and dried to prepare an amino-grafted silica carrier. ② The amino-grafted silica carrier was dispersed in anhydrous ethanol and subjected to ultrasonic treatment to prepare an amino-grafted silica carrier dispersion. ③ Under a nitrogen atmosphere, Ru3(CO) 12 Ru nanoparticles were prepared by placing (ruthenium dodecylcarbonyltriruthenium) into a tube furnace and heating it to 500°C at a rate of 5°C / min and holding it for 2 hours. ④ Disperse Ru nanoparticles in anhydrous ethanol and perform ultrasonic dispersion to prepare a nano Ru dispersion. Disperse WC (tungsten carbide) nanoparticles in anhydrous ethanol and perform ultrasonic dispersion to prepare a nano WC dispersion. Mix the nano Ru dispersion and the nano WC dispersion evenly and then dropwise add them to the amino-grafted silica carrier dispersion for stirring reaction. ⑤ The mixture obtained in ④ was rotary evaporated and then dried under vacuum to remove the solvent. Then it was kept at a temperature under a nitrogen atmosphere to prepare the Ru-WC / NH2-SiO2 catalyst. (3) The Ru-WC / NH2-SiO2 catalyst and the immobilized CALB lipase were dissolved in tert-butanol-buffered solution, DL-aminopropanol was added, and vinyl acetate was added dropwise to the above reaction system. The reaction was carried out in a constant temperature water bath at 49°C for 3.9 h. After the reaction was completed, the Ru-WC / NH2-SiO2 catalyst particles were removed by vacuum filtration. Then, hydrochloric acid was added to acidify to pH=3. Finally, the immobilized CALB lipase was removed by vacuum filtration. The filtrate was extracted three times with ethyl acetate and the organic phases were combined. (4) The combined ethyl acetate extract was transferred to a reaction vessel and an equal volume of sodium hydroxide solution was added for hydrolysis. After the reaction was completed, the organic phase was removed by separation. The pH of the aqueous phase was adjusted to 7.0 with hydrochloric acid. The aqueous phase was then rotary evaporated to obtain crude L-aminopropanol. Finally, high-purity L-aminopropanol was prepared by recrystallization.

[0042] in: In step (1), the CALB lipase is manufactured by Guangdong Maike Baisheng Biotechnology Co., Ltd., and the SBA-15 mesoporous molecular sieve is manufactured by Jiangsu Xianfeng Nanomaterials Technology Co., Ltd.

[0043] In step (1), the mass ratio of CALB lipase to phosphate buffer is 1:1.5, and the unit is g / mL.

[0044] In step (1), the mass ratio of SBA-15 mesoporous molecular sieve to enzyme solution is 1:2, with units of g / mL.

[0045] The buffer rinsing in step (1) is to rinse three times with a phosphate buffer solution with a concentration of 0.1 mol / L and pH=7. The drying is to dry at 40°C for 2.5 h. The storage is to keep at a temperature of 4°C.

[0046] In step (2)①, the mass of silane coupling agent KH550 accounts for 10 wt% of the mass of silica powder.

[0047] In step (2)①, the mass ratio of silica powder to anhydrous ethanol is 1:30.

[0048] In step (2)①, the mechanical stirring speed is 500 r / min, the stirring time is 30 min, the drying pretreatment is carried out under vacuum, the drying pretreatment temperature is 80℃, and the drying pretreatment time is 2 h.

[0049] In step (2)①, the surface modification temperature is 180℃, the surface modification time is 2h, the surface modification atmosphere is nitrogen, and the pressure is 0.3MPa.

[0050] The washing process described in step (2) ① involves first washing with anhydrous ethanol three times, and then washing with deionized water twice.

[0051] The drying described in step (2) ① is carried out under vacuum at 70°C for 9 hours.

[0052] The ultrasonic treatment time in step (2) ② is 19 min.

[0053] In step (2) ②, the mass-to-volume ratio of amino-grafted silica carrier to anhydrous ethanol is 1:30, with units of g / mL.

[0054] In step (2)④, the mass ratio of Ru nanoparticles to anhydrous ethanol in the nano Ru dispersion is 0.07:1, with units of g / mL. The mass ratio of WC nanoparticles to anhydrous ethanol in the nano WC dispersion is 0.5:1, with units of 0.5 g / mL.

[0055] The mass of Ru nanoparticles in step (2) ④ accounts for 3.7 wt% of the mass of amino-grafted silica carrier in step (2) ②, and the mass of nano WC in step (2) ④ accounts for 1 wt% of the mass of amino-grafted silica carrier in step (2) ②.

[0056] In step (2) ④, the stirring reaction time is 2 hours, the stirring reaction temperature is 56℃, and the stirring reaction speed is 500r / min.

[0057] In step (2) ④, the ultrasonic time for preparing nano Ru dispersion and nano WC dispersion is 15 min.

[0058] In step (2) ⑤, the rotary evaporation temperature is 55℃, the drying temperature is 75℃, and the drying time is 5h. The heat preservation treatment is to raise the temperature to 200℃ at a heating rate of 5°C / min and keep it warm for 2 hours.

[0059] In step (3), the concentration of DL-aminopropanol in tert-butanol-buffered solution is 1.1 mol / L; the concentration of vinyl acetate in tert-butanol-buffered solution is 1.9 mol / L.

[0060] In step (3), the volume ratio of tert-butanol to phosphate buffer with a concentration of 0.1 mol / L and pH=7 in the tert-butanol-buffered solution is 3:2.

[0061] In step (3), the concentration of Ru-WC / NH2-SiO2 catalyst in tert-butanol-buffered solution is 19 g / L.

[0062] In step (3), the mass of the immobilized CALB lipase is 9% of the mass of DL-aminopropanol.

[0063] The concentration of hydrochloric acid in step (3) is 2 mol / L.

[0064] In step (3), the addition of vinyl acetate must be completed within 1 hour.

[0065] In step (3), the immobilized CALB lipase and Ru-WC / NH2-SiO2 catalyst are used in combination to enable the in-situ racemization and recycling of the D-isomer, and the Ru-WC / NH2-SiO2 catalyst is used as a metal racemization catalyst; the metal catalyst is removed first and then acidified to prevent the acid from damaging the structure of the metal catalyst.

[0066] In step (3), the first filtration uses a 0.22 μm polytetrafluoroethylene filter membrane, the filter residue is a Ru-WC / NH2-SiO2 catalyst, and the filtrate is L-aminopropanol acetate and residual substrate, etc.; the second filtration can be done with ordinary filter paper. After adding hydrochloric acid to the filtrate, CALB lipase detaches from the carrier due to the change in surface charge. The filter residue is an SBA-15 carrier, and the filtrate contains L-aminopropanol acetate and free enzyme.

[0067] In step (3), the Ru-WC / NH2-SiO2 catalyst racemizes the unreacted D-aminopropanol into a mixture of DL-aminopropanols, while the immobilized enzyme selectively esterifies L-aminopropanol into L-aminopropanol acetate. The D-type remains hidden until it is racemized, and the racemization rate is greater than the enzyme catalysis rate, ensuring that the D-type does not accumulate.

[0068] In step (4), the volume ratio of ethyl acetate extract to sodium hydroxide solution is 1:1, and the concentration of sodium hydroxide solution is 0.5 mol / L.

[0069] In step (4), the hydrolysis reaction temperature is 54℃ and the hydrolysis reaction time is 2h.

[0070] In step (4), add 2.0 mol / L hydrochloric acid to adjust the pH value to 7.0.

[0071] In step (4), the rotary evaporation temperature is 60℃ and the rotary evaporation pressure is 0.1MPa.

[0072] In step (4), recrystallization is performed using a mixture of ethanol and water, wherein the volume ratio of ethanol to water is 4:1.

[0073] Chiral high-performance liquid chromatography analysis showed that the conversion rate of D-aminopropanol was 98.5%, the yield of L-aminopropanol was 88.2% (yield = actual molar amount of L-aminopropanol obtained / initial total molar amount of DL-aminopropanol × 100%), and the optical purity of L-aminopropanol was 99.6%.

[0074] Example 2 The method for preparing L-aminopropanol by resolving DL-aminopropanol as described in Example 2 consists of the following steps: (1) The lipase CALB was dissolved in phosphate buffer with a concentration of 0.1 mol / L and pH=7 for 1 h to neutralize the surface charge and prepare an enzyme solution. Using SBA-15 mesoporous molecular sieve as a carrier, the carrier and enzyme solution were mixed and adsorbed at 33℃ for 7 h. Then, the mixture was separated by vacuum filtration, rinsed with buffer, dried and stored to prepare immobilized CALB lipase. (2) Preparation of bimetallic catalysts ① Mix silane coupling agent KH550, anhydrous ethanol and silica powder, add acetic acid to adjust the pH of the system to 5 and mechanically stir. After drying and pretreatment, the resulting mixture is transferred to a high-temperature reactor for surface modification. The modified product is filtered, washed and dried to prepare an amino-grafted silica carrier. ② The amino-grafted silica carrier was dispersed in anhydrous ethanol and subjected to ultrasonic treatment to prepare an amino-grafted silica carrier dispersion. ③ Under a nitrogen atmosphere, Ru3(CO) 12 Ru nanoparticles were prepared by placing (ruthenium dodecylcarbonyltriruthenium) into a tube furnace and heating it to 500°C at a rate of 5°C / min and holding it for 2 hours. ④ Disperse Ru nanoparticles in anhydrous ethanol and perform ultrasonic dispersion to prepare a nano Ru dispersion. Disperse WC (tungsten carbide) nanoparticles in anhydrous ethanol and perform ultrasonic dispersion to prepare a nano WC dispersion. Mix the nano Ru dispersion and the nano WC dispersion evenly and then dropwise add them to the amino-grafted silica carrier dispersion for stirring reaction. ⑤ The mixture obtained in ④ was rotary evaporated and then dried under vacuum to remove the solvent. Then it was kept at a temperature under a nitrogen atmosphere to prepare the Ru-WC / NH2-SiO2 catalyst. (3) The Ru-WC / NH2-SiO2 catalyst and the immobilized CALB lipase were dissolved in tert-butanol-buffered solution, DL-aminopropanol was added, and vinyl acetate was added dropwise to the above reaction system. The reaction was carried out in a constant temperature water bath at 48°C for 3.8 h. After the reaction was completed, the Ru-WC / NH2-SiO2 catalyst particles were removed by vacuum filtration. Then, hydrochloric acid was added to acidify to pH=3. Finally, the immobilized CALB lipase was removed by vacuum filtration. The filtrate was extracted three times with ethyl acetate and the organic phases were combined. (4) The combined ethyl acetate extract was transferred to a reaction vessel and an equal volume of sodium hydroxide solution was added for hydrolysis. After the reaction was completed, the organic phase was removed by separation. The pH of the aqueous phase was adjusted to 7.0 with hydrochloric acid. The aqueous phase was then rotary evaporated to obtain crude L-aminopropanol. Finally, high-purity L-aminopropanol was prepared by recrystallization.

[0075] in: In step (1), the CALB lipase is manufactured by Guangdong Maike Baisheng Biotechnology Co., Ltd., and the SBA-15 mesoporous molecular sieve is manufactured by Jiangsu Xianfeng Nanomaterials Technology Co., Ltd.

[0076] In step (1), the mass ratio of CALB lipase to phosphate buffer is 1:1.5, and the unit is g / mL.

[0077] In step (1), the mass ratio of SBA-15 mesoporous molecular sieve to enzyme solution is 1:2, with units of g / mL.

[0078] The buffer rinsing in step (1) is to rinse three times with a phosphate buffer solution with a concentration of 0.1 mol / L and pH=7. The drying is to dry at 40°C for 2.5 h. The storage is to keep at a temperature of 4°C.

[0079] In step (2)①, the mass of silane coupling agent KH550 accounts for 10 wt% of the mass of silica powder.

[0080] In step (2)①, the mass ratio of silica powder to anhydrous ethanol is 1:30.

[0081] In step (2)①, the mechanical stirring speed is 500 r / min, the stirring time is 30 min, the drying pretreatment is carried out under vacuum, the drying pretreatment temperature is 80℃, and the drying pretreatment time is 2 h.

[0082] In step (2)①, the surface modification temperature is 180℃, the surface modification time is 2h, the surface modification atmosphere is nitrogen, and the pressure is 0.3MPa.

[0083] The washing process described in step (2) ① involves first washing with anhydrous ethanol three times, and then washing with deionized water twice.

[0084] The drying described in step (2) ① is carried out under vacuum at 70°C for 9 hours.

[0085] The ultrasonic treatment time in step (2) ② is 20 min.

[0086] In step (2) ②, the mass-to-volume ratio of amino-grafted silica carrier to anhydrous ethanol is 1:30, with units of g / mL.

[0087] In step (2)④, the mass ratio of Ru nanoparticles to anhydrous ethanol in the nano Ru dispersion is 0.07:1, with units of g / mL. The mass ratio of WC nanoparticles to anhydrous ethanol in the nano WC dispersion is 0.5:1, with units of 0.5 g / mL.

[0088] The mass of Ru nanoparticles in step (2) ④ accounts for 3.5 wt% of the mass of amino-grafted silica carrier in step (2) ②, and the mass of nano WC in step (2) ④ accounts for 1 wt% of the mass of amino-grafted silica carrier in step (2) ②.

[0089] In step (2) ④, the stirring reaction time is 2 hours, the stirring reaction temperature is 55℃, and the stirring reaction speed is 500r / min.

[0090] In step (2) ④, the ultrasonic time for preparing nano Ru dispersion and nano WC dispersion is 15 min.

[0091] In step (2) ⑤, the rotary evaporation temperature is 55℃, the drying temperature is 75℃, and the drying time is 5h. The heat preservation treatment is to raise the temperature to 200℃ at a heating rate of 5°C / min and keep it warm for 2 hours.

[0092] In step (3), the concentration of DL-aminopropanol in tert-butanol-buffered solution is 1.0 mol / L; the concentration of vinyl acetate in tert-butanol-buffered solution is 1.8 mol / L.

[0093] In step (3), the volume ratio of tert-butanol to phosphate buffer with a concentration of 0.1 mol / L and pH=7 in the tert-butanol-buffered solution is 3:2.

[0094] In step (3), the concentration of Ru-WC / NH2-SiO2 catalyst in tert-butanol-buffered solution is 18 g / L.

[0095] In step (3), the mass of the immobilized CALB lipase is 10% of the mass of DL-aminopropanol.

[0096] The concentration of hydrochloric acid in step (3) is 2 mol / L.

[0097] In step (3), the addition of vinyl acetate must be completed within 1 hour.

[0098] In step (3), the immobilized CALB lipase and Ru-WC / NH2-SiO2 catalyst are used in combination to enable the in-situ racemization and recycling of the D-isomer, and the Ru-WC / NH2-SiO2 catalyst is used as a metal racemization catalyst; the metal catalyst is removed first and then acidified to prevent the acid from damaging the structure of the metal catalyst.

[0099] In step (3), the first filtration uses a 0.22 μm polytetrafluoroethylene filter membrane, the filter residue is a Ru-WC / NH2-SiO2 catalyst, and the filtrate is L-aminopropanol acetate and residual substrate, etc.; the second filtration can be done with ordinary filter paper. After adding hydrochloric acid to the filtrate, CALB lipase detaches from the carrier due to the change in surface charge. The filter residue is an SBA-15 carrier, and the filtrate contains L-aminopropanol acetate and free enzyme.

[0100] In step (3), the Ru-WC / NH2-SiO2 catalyst racemizes the unreacted D-aminopropanol into a mixture of DL-aminopropanols, while the immobilized enzyme selectively esterifies L-aminopropanol into L-aminopropanol acetate. The D-type remains hidden until it is racemized, and the racemization rate is greater than the enzyme catalysis rate, ensuring that the D-type does not accumulate.

[0101] In step (4), the volume ratio of ethyl acetate extract to sodium hydroxide solution is 1:1, and the concentration of sodium hydroxide solution is 0.5 mol / L.

[0102] In step (4), the hydrolysis reaction temperature is 53℃ and the hydrolysis reaction time is 2h.

[0103] In step (4), add 2.0 mol / L hydrochloric acid to adjust the pH value to 7.0.

[0104] In step (4), the rotary evaporation temperature is 60℃ and the rotary evaporation pressure is 0.1MPa.

[0105] In step (4), recrystallization is performed using a mixture of ethanol and water, wherein the volume ratio of ethanol to water is 4:1.

[0106] Chiral high-performance liquid chromatography analysis showed that the conversion rate of D-aminopropanol was 98.0%, the yield of L-aminopropanol was 87.5%, and the optical purity of L-aminopropanol was 99.5%.

[0107] Example 3 The method for preparing L-aminopropanol by resolving DL-aminopropanol as described in Example 3 consists of the following steps: (1) The lipase CALB was dissolved in phosphate buffer with a concentration of 0.1 mol / L and pH=7 and soaked for 1.2 h to neutralize the surface charge and prepare an enzyme solution. Using SBA-15 mesoporous molecular sieve as a carrier, the carrier and enzyme solution were mixed and adsorbed at 35℃ for 7 hours. Then, the mixture was separated by vacuum filtration, rinsed with buffer, dried and stored to prepare immobilized CALB lipase. (2) Preparation of bimetallic catalysts ① Mix silane coupling agent KH550, anhydrous ethanol and silica powder, add acetic acid to adjust the pH of the system to 5 and mechanically stir. After drying and pretreatment, the resulting mixture is transferred to a high-temperature reactor for surface modification. The modified product is filtered, washed and dried to prepare an amino-grafted silica carrier. ② The amino-grafted silica carrier was dispersed in anhydrous ethanol and subjected to ultrasonic treatment to prepare an amino-grafted silica carrier dispersion. ③ Under a nitrogen atmosphere, Ru3(CO) 12 Ru nanoparticles were prepared by placing (ruthenium dodecylcarbonyltriruthenium) into a tube furnace and heating it to 500°C at a rate of 5°C / min and holding it for 2 hours. ④ Disperse Ru nanoparticles in anhydrous ethanol and perform ultrasonic dispersion to prepare a nano Ru dispersion. Disperse WC (tungsten carbide) nanoparticles in anhydrous ethanol and perform ultrasonic dispersion to prepare a nano WC dispersion. Mix the nano Ru dispersion and the nano WC dispersion evenly and then dropwise add them to the amino-grafted silica carrier dispersion for stirring reaction. ⑤ The mixture obtained in ④ was rotary evaporated and then dried under vacuum to remove the solvent. Then it was kept at a temperature under a nitrogen atmosphere to prepare the Ru-WC / NH2-SiO2 catalyst. (3) The Ru-WC / NH2-SiO2 catalyst and the immobilized CALB lipase were dissolved in tert-butanol-buffered solution, DL-aminopropanol was added, and then vinyl acetate was added dropwise to the above reaction system. The reaction was carried out in a constant temperature water bath at 50°C for 4 hours. After the reaction was completed, the Ru-WC / NH2-SiO2 catalyst particles were removed by vacuum filtration. Then hydrochloric acid was added to acidify to pH=3. Finally, the immobilized CALB lipase was removed by vacuum filtration. The filtrate was extracted three times with ethyl acetate and the organic phases were combined. (4) The combined ethyl acetate extract was transferred to a reaction vessel and an equal volume of sodium hydroxide solution was added for hydrolysis. After the reaction was completed, the organic phase was removed by separation. The pH of the aqueous phase was adjusted to 7.0 with hydrochloric acid. The aqueous phase was then rotary evaporated to obtain crude L-aminopropanol. Finally, high-purity L-aminopropanol was prepared by recrystallization.

[0108] in: In step (1), the CALB lipase is manufactured by Guangdong Maike Baisheng Biotechnology Co., Ltd., and the SBA-15 mesoporous molecular sieve is manufactured by Jiangsu Xianfeng Nanomaterials Technology Co., Ltd.

[0109] In step (1), the mass ratio of CALB lipase to phosphate buffer is 1:1.5, and the unit is g / mL.

[0110] In step (1), the mass ratio of SBA-15 mesoporous molecular sieve to enzyme solution is 1:2, with units of g / mL.

[0111] The buffer rinsing in step (1) is to rinse three times with a phosphate buffer solution with a concentration of 0.1 mol / L and pH=7. The drying is to dry at 40°C for 2.5 h. The storage is to keep at a temperature of 4°C.

[0112] In step (2)①, the mass of silane coupling agent KH550 accounts for 10 wt% of the mass of silica powder.

[0113] In step (2)①, the mass ratio of silica powder to anhydrous ethanol is 1:30.

[0114] In step (2)①, the mechanical stirring speed is 500 r / min, the stirring time is 30 min, the drying pretreatment is carried out under vacuum, the drying pretreatment temperature is 80℃, and the drying pretreatment time is 2 h.

[0115] In step (2)①, the surface modification temperature is 180℃, the surface modification time is 2h, the surface modification atmosphere is nitrogen, and the pressure is 0.3MPa.

[0116] The washing process described in step (2) ① involves first washing with anhydrous ethanol three times, and then washing with deionized water twice.

[0117] The drying described in step (2) ① is carried out under vacuum at 70°C for 9 hours.

[0118] The ultrasonic treatment time in step (2) ② is 18 min.

[0119] In step (2) ②, the mass-to-volume ratio of amino-grafted silica carrier to anhydrous ethanol is 1:30, with units of g / mL.

[0120] In step (2)④, the mass ratio of Ru nanoparticles to anhydrous ethanol in the nano Ru dispersion is 0.07:1, with units of g / mL. The mass ratio of WC nanoparticles to anhydrous ethanol in the nano WC dispersion is 0.5:1, with units of 0.5 g / mL.

[0121] The mass of Ru nanoparticles in step (2) ④ accounts for 4 wt% of the mass of amino-grafted silica carrier in step (2) ②, and the mass of nano WC in step (2) ④ accounts for 1 wt% of the mass of amino-grafted silica carrier in step (2) ②.

[0122] In step (2) ④, the stirring reaction time is 2 hours, the stirring reaction temperature is 57℃, and the stirring reaction speed is 500r / min.

[0123] In step (2) ④, the ultrasonic time for preparing nano Ru dispersion and nano WC dispersion is 15 min.

[0124] In step (2) ⑤, the rotary evaporation temperature is 55℃, the drying temperature is 75℃, and the drying time is 5h. The heat preservation treatment is to raise the temperature to 200℃ at a heating rate of 5°C / min and keep it warm for 2 hours.

[0125] In step (3), the concentration of DL-aminopropanol in tert-butanol-buffered solution is 1.2 mol / L; the concentration of vinyl acetate in tert-butanol-buffered solution is 2.0 mol / L.

[0126] In step (3), the volume ratio of tert-butanol to phosphate buffer with a concentration of 0.1 mol / L and pH=7 in the tert-butanol-buffered solution is 3:2.

[0127] In step (3), the concentration of Ru-WC / NH2-SiO2 catalyst in tert-butanol-buffered solution is 20 g / L.

[0128] In step (3), the mass of the immobilized CALB lipase is 8% of the mass of DL-aminopropanol.

[0129] The concentration of hydrochloric acid in step (3) is 2 mol / L.

[0130] In step (3), the addition of vinyl acetate must be completed within 1 hour.

[0131] In step (3), the immobilized CALB lipase and Ru-WC / NH2-SiO2 catalyst are used in combination to enable the in-situ racemization and recycling of the D-isomer, and the Ru-WC / NH2-SiO2 catalyst is used as a metal racemization catalyst; the metal catalyst is removed first and then acidified to prevent the acid from damaging the structure of the metal catalyst.

[0132] In step (3), the first filtration uses a 0.22 μm polytetrafluoroethylene filter membrane, the filter residue is a Ru-WC / NH2-SiO2 catalyst, and the filtrate is L-aminopropanol acetate and residual substrate, etc.; the second filtration can be done with ordinary filter paper. After adding hydrochloric acid to the filtrate, CALB lipase detaches from the carrier due to the change in surface charge. The filter residue is an SBA-15 carrier, and the filtrate contains L-aminopropanol acetate and free enzyme.

[0133] In step (3), the Ru-WC / NH2-SiO2 catalyst racemizes the unreacted D-aminopropanol into a mixture of DL-aminopropanols, while the immobilized enzyme selectively esterifies L-aminopropanol into L-aminopropanol acetate. The D-type remains hidden until it is racemized, and the racemization rate is greater than the enzyme catalysis rate, ensuring that the D-type does not accumulate.

[0134] In step (4), the volume ratio of ethyl acetate extract to sodium hydroxide solution is 1:1, and the concentration of sodium hydroxide solution is 0.5 mol / L.

[0135] In step (4), the hydrolysis reaction temperature is 55℃ and the hydrolysis reaction time is 2h.

[0136] In step (4), add 2.0 mol / L hydrochloric acid to adjust the pH value to 7.0.

[0137] In step (4), the rotary evaporation temperature is 60℃ and the rotary evaporation pressure is 0.1MPa.

[0138] In step (4), recrystallization is performed using a mixture of ethanol and water, wherein the volume ratio of ethanol to water is 4:1.

[0139] Chiral high-performance liquid chromatography analysis showed that the conversion rate of D-aminopropanol was 99.1%, the yield of L-aminopropanol was 89.0%, and the optical purity of L-aminopropanol was 99.7%.

[0140] Comparative Example 1 The method for preparing L-aminopropanol by resolving DL-aminopropanol in Comparative Example 1 differs from that in Example 1 in that the catalyst prepared in step (2) is a Ru / NH2-SiO2 catalyst, while the remaining steps and process parameters are the same as in Example 1.

[0141] Chiral high-performance liquid chromatography analysis showed that the conversion rate of D-aminopropanol was 88.5%, the yield of L-aminopropanol was 77.8%, and the optical purity of L-aminopropanol was 99.2%.

[0142] Comparative Example 2 The method for preparing L-aminopropanol by resolving DL-aminopropanol in Comparative Example 2 differs from that in Example 1 in that the catalyst prepared in step (2) is a WC / NH2-SiO2 catalyst, while the remaining steps and process parameters are the same as in Example 1.

[0143] Chiral high-performance liquid chromatography analysis showed that the conversion rate of D-aminopropanol was 50.2%, the yield of L-aminopropanol was 48.5%, and the optical purity of L-aminopropanol was 99.8%.

Claims

1. A method for resolving DL-aminopropanol to prepare L-aminopropanol, characterized in that: It consists of the following steps: (1) Dissolve CALB lipase in 0.1 mol / L phosphate buffer with pH=7 for 1-1.2 h to neutralize the surface charge and prepare enzyme solution. Use SBA-15 mesoporous molecular sieve as carrier, mix the carrier with enzyme solution, shake and adsorb at 33-35℃ for 7 hours, then filter, rinse with buffer, dry and store to prepare immobilized CALB lipase. (2) Preparation of bimetallic catalysts ① Mix silane coupling agent KH550, anhydrous ethanol and silica powder, add acetic acid to adjust the pH of the system to 5 and mechanically stir. After drying and pretreatment, the resulting mixture is transferred to a high-temperature reactor for surface modification. The modified product is filtered, washed and dried to prepare an amino-grafted silica carrier. ② The amino-grafted silica carrier was dispersed in anhydrous ethanol and subjected to ultrasonic treatment to prepare an amino-grafted silica carrier dispersion. ③ Under a nitrogen atmosphere, Ru3(CO) 12 The material was placed in a tube furnace and heated to 500℃ at a rate of 5℃ / min and held for 2 hours to prepare Ru nanoparticles. ④ Disperse Ru nanoparticles in anhydrous ethanol and perform ultrasonic dispersion to prepare a Ru nanoparticle dispersion. Disperse WC nanoparticles in anhydrous ethanol and perform ultrasonic dispersion to prepare a WC nanoparticle dispersion. Mix the Ru nanoparticle dispersion and the WC nanoparticle dispersion evenly and then add them dropwise to the amino-grafted silica carrier dispersion for stirring and reaction. ⑤ The mixture obtained in ④ was rotary evaporated and then dried under vacuum to remove the solvent. Then it was kept at a temperature under a nitrogen atmosphere to prepare the Ru-WC / NH2-SiO2 catalyst. (3) Dissolve the Ru-WC / NH2-SiO2 catalyst and the immobilized CALB lipase in tert-butanol-buffer, add DL-aminopropanol, and then add vinyl acetate dropwise to the above reaction system. React in a constant temperature water bath at 48-50℃ for 3.8-4h. After the reaction is complete, filter to remove the Ru-WC / NH2-SiO2 catalyst particles, then add hydrochloric acid to acidify to pH=3, and finally filter to remove the immobilized CALB lipase. Extract the filtrate three times with ethyl acetate and combine the organic phases. (4) The combined ethyl acetate extract was transferred to a reaction vessel and an equal volume of sodium hydroxide solution was added for hydrolysis. After the reaction was completed, the organic phase was removed by separation. The pH of the aqueous phase was adjusted to 7.0 with hydrochloric acid. The aqueous phase was then rotary evaporated to obtain crude L-aminopropanol. Finally, high-purity L-aminopropanol was prepared by recrystallization.

2. The method for preparing L-aminopropanol by resolving DL-aminopropanol according to claim 1, characterized in that: In step (1), the mass ratio of CALB lipase to phosphate buffer is 1:1.5, with units of g / mL. In step (1), the mass ratio of SBA-15 mesoporous molecular sieve to enzyme solution is 1:2, with units of g / mL; The buffer rinsing in step (1) is to rinse three times with a phosphate buffer solution with a concentration of 0.1 mol / L and pH=7. The drying is to dry at 40°C for 2.5 h. The storage is to keep at a temperature of 4°C.

3. The method for preparing L-aminopropanol by resolving DL-aminopropanol according to claim 1, characterized in that: In step (2) ①, the mass of silane coupling agent KH550 accounts for 10 wt% of the mass of silica powder. In step (2)①, the mass ratio of silica powder to anhydrous ethanol is 1:30; In step (2)①, the mechanical stirring speed is 500 r / min, the stirring time is 30 min, the drying pretreatment is carried out under vacuum, the drying pretreatment temperature is 80℃, and the drying pretreatment time is 2 h. In step (2)①, the surface modification temperature is 180℃, the surface modification time is 2h, the surface modification atmosphere is nitrogen, and the pressure is 0.3MPa. The washing process described in step (2) ① involves first washing with anhydrous ethanol three times, and then washing with deionized water twice. The drying described in step (2) ① is carried out under vacuum at 70°C for 9 hours.

4. The method for preparing L-aminopropanol by resolving DL-aminopropanol according to claim 1, characterized in that: The ultrasonic treatment time in step (2) ② is 18-20 min; In step (2) ②, the mass-to-volume ratio of amino-grafted silica carrier to anhydrous ethanol is 1:30, with units of g / mL.

5. The method for preparing L-aminopropanol by resolving DL-aminopropanol according to claim 1, characterized in that: In step (2)④, the mass ratio of Ru nanoparticles to anhydrous ethanol in the nano Ru dispersion is 0.07:1, with units of g / mL; and the mass ratio of WC nanoparticles to anhydrous ethanol in the nano WC dispersion is 0.5:1, with units of 0.5 g / mL. The mass of Ru nanoparticles in step (2) ④ accounts for 3.5-4 wt% of the mass of amino-grafted silica carrier in step (2) ②, and the mass of nano WC in step (2) ④ accounts for 1 wt% of the mass of amino-grafted silica carrier in step (2) ②.

6. The method for preparing L-aminopropanol by resolving DL-aminopropanol according to claim 1, characterized in that: In step (2) ④, the stirring reaction time is 2 hours, the stirring reaction temperature is 55-57℃, and the stirring reaction speed is 500 r / min; In step (2) ④, the ultrasonic time for preparing nano-Ru dispersion and nano-WC dispersion is 15 min; In step (2) ⑤, the rotary evaporation temperature is 55℃, the drying temperature is 75℃, and the drying time is 5h. The heat preservation treatment is to raise the temperature to 200℃ at a heating rate of 5°C / min and keep it warm for 2 hours.

7. The method for preparing L-aminopropanol by resolving DL-aminopropanol according to claim 1, characterized in that: In step (3), the concentration of DL-aminopropanol in tert-butanol-buffered solution is 1.0-1.2 mol / L; the concentration of vinyl acetate in tert-butanol-buffered solution is 1.8-2.0 mol / L. In step (3), the volume ratio of tert-butanol to phosphate buffer with a concentration of 0.1 mol / L and pH=7 in the tert-butanol-buffered solution is 3:

2. In step (3), the concentration of Ru-WC / NH2-SiO2 catalyst in tert-butanol-buffered solution is 18-20 g / L.

8. The method for preparing L-aminopropanol by resolving DL-aminopropanol according to claim 1, characterized in that: In step (3), the mass of the immobilized CALB lipase is 8-10% of the mass of DL-aminopropanol; The concentration of hydrochloric acid mentioned in step (3) is 2 mol / L; In step (3), the addition of vinyl acetate must be completed within 1 hour.

9. The method for preparing L-aminopropanol by resolving DL-aminopropanol according to claim 1, characterized in that: In step (4), the volume ratio of ethyl acetate extract to sodium hydroxide solution is 1:1, and the concentration of sodium hydroxide solution is 0.5 mol / L. In step (4), the hydrolysis reaction temperature is 53-55℃ and the hydrolysis reaction time is 2h; In step (4), add 2.0 mol / L hydrochloric acid to adjust the pH value to 7.

0.

10. The method for preparing L-aminopropanol by resolving DL-aminopropanol according to claim 1, characterized in that: In step (4), the rotary evaporation temperature is 60℃ and the rotary evaporation pressure is 0.1MPa; In step (4), recrystallization is performed using a mixture of ethanol and water, wherein the volume ratio of ethanol to water is 4:1.