Method for synthesizing pyrrole-2-ethyl formate under catalysis of lipase

Abstract

The invention discloses a method for synthesizing pyrrole-2-ethyl formate through lipase catalysis, and belongs to the field of enzyme catalysis chemical reaction. The method for synthesizing the pyrrole-2-ethyl formate specifically comprises the following steps: firstly, preparing PS-HMDP microspheres, then preparing a lipase catalyst by using the PS-HMDP microspheres, and finally preparing the pyrrole-2-ethyl formate by using lipase to catalyze pyrrole-2-formic acid. And the yield reaches 80-85%. The method adopts enzyme as a catalyst, has the advantages of mild reaction conditions, few toxic and side effects and the like, avoids the steps of protection and deprotection, and provides a novel green, simple and convenient method for preparing the ethyl pyrrole-2-formate.

Description

Technical Field

[0001] The invention relates to a method for synthesizing ethyl pyrrole-2-carboxylate catalyzed by lipase, and belongs to the field of enzyme-catalyzed chemical reactions. Background Art

[0002] Pyrrole-2-carboxylic acid ethyl ester is used as a standard substance for chromatography analysis, and is also used in organic synthesis and the pharmaceutical industry, mainly as a solvent for polyester fiber spinning. Pyrrole-Chemicalbook2-carboxylic acid ethyl ester is also commonly used as a metal preservative, epoxy resin curing agent, catalyst for olefin polymerization, and raw materials in the pharmaceutical industry, and is also used as a photosensitizer.

[0003] The existing preparation method of ethyl pyrrole-2-carboxylate: Denis M. Bailey et al. added 1.0 g of sodium to 300 ml of anhydrous ethanol. After the sodium was dissolved, 75 g of 2-pyrrolyl trichloromethyl ketone was added in batches within 10 minutes, stirred for reaction for 30 minutes, and then concentrated to dryness using a rotary evaporator. The oily residue was extracted, washed, dried, and distilled under reduced pressure to obtain 44.0-44.5 g of ethyl pyrrole-2-carboxylate (Org. Synth. 1971, 51, 100); Chinese patent CN108299270A discloses that 2-formyl pyrrole is dissolved in methanol and tetrachloromethane, cobalt chloride is added, refluxed at 150° C. for reaction for 2 hours, and ethyl acetate is recrystallized to obtain dimethyl 2,5-pyrrole dicarboxylate; Li Zifan et al. (Analytical Chemistry, 2008, 36 (7): 5) dissolved 2.0 g of sodium ethoxide in 15 mL of ethanol, and then dropped 4.0 g of 2-pyrrolyl trichloromethyl ketone 20mL ethanol solution, stir, reflux 12h, vacuum distillation, recrystallization to obtain 2,5-pyrrole dicarboxylic acid dimethyl ester 1.8g, yield 69%. Zhang Xuehui et al. (Journal of PLA Pharmaceutical Sciences 1 (2013): 3.) In 60ml anhydrous 1,2-dichloroethane, 40ml anhydrous nitromethane, 6.7g (0.1mmol) freshly distilled pyrrole and 0.1mmol ethyl chloroformate, under stirring conditions, 13.3g (0.1mmol) AlCl3 powder was added in batches, the temperature and intensity of the reaction system were controlled, and after the addition was completed, the reaction mixture was cooled, washed, dried, and vacuum distilled to obtain pyrrole-2-carboxylate, with a yield of 66.3%.

[0004] The existing chemical synthesis of ethyl pyrrole-2-carboxylate often has complex processes and green problems such as the need for protection and deprotection steps (such as using 2-pyrrolyltrichloromethyl ketone as a raw material) or severe reaction conditions (such as using AlCl3 powder as a catalyst), and the yield is also low. Summary of the invention

[0005] [Technical issues]

[0006] The preparation of pyrrole-2-carboxylate in the prior art has the problems of complex process, such as the need for protection and deprotection steps (such as using 2-pyrrolyltrichloromethyl ketone as a raw material), severe reaction conditions (such as using AlCl3 powder as a catalyst), unsafe, environmentally unfriendly, and low yield.

[0007] [Technical solution]

[0008] In view of the above technical problems, the purpose of the present invention is to provide a method for lipase-catalyzed synthesis of ethyl pyrrole-2-carboxylate, which utilizes PS-HMDP microspheres as carriers to load lipase as a catalyst to synthesize ethyl pyrrole-2-carboxylate, has mild conditions, is environmentally friendly, and effectively improves the synthesis yield of ethyl pyrrole-2-carboxylate.

[0009] In order to achieve the above purpose, the technical solutions adopted are as follows:

[0010] A method for synthesizing ethyl pyrrole-2-carboxylate catalyzed by lipase, the method comprising the following steps:

[0011] (1) Preparation of PS-HMDP microspheres

[0012] Chlorinated polystyrene microspheres are added to toluene to swell, and then K2CO3, tetrabutylammonium chloride and polyethylene glycol are added to react to obtain PS-PEG microspheres; PS-PEG microspheres are added to toluene, and phosphorus tribromide is slowly added dropwise to react to obtain PS-PEG-Br microspheres; then PS-PEG-Br microspheres are added to butan-2-one, and p-hydroxybenzaldehyde and K2CO3 are added to react to obtain PS-PHBA microspheres; then PS-PHBA microspheres are added to methanol, and urea and concentrated hydrochloric acid are added to react to obtain PS-HMD microspheres; finally, PS-HMD microspheres are added to toluene, and epichlorohydrin and NaOH are added to react to obtain PS-HMDP microspheres;

[0013] (2) Preparation of lipase catalyst

[0014] Add the PS-HMDP microspheres prepared in step (1) to the sodium hydrogen phosphate-citric acid buffer solution containing lipase, shake and react, wash and filter after the reaction is completed, and obtain a lipase catalyst;

[0015] (3) Preparation of ethyl pyrrole-2-carboxylate

[0016] The lipase catalyst prepared in step (2), pyrrole-2-carboxylic acid and ethanol are added to dimethyl sulfoxide, and the mixture is continuously shaken at 25-45° C. for 9-24 hours. After the reaction is completed, the mixture is centrifuged, and the supernatant is taken. The content of ethyl pyrrole-2-carboxylate is determined by gas chromatography; the supernatant is distilled to 100° C., and the residual liquid is poured into a separatory funnel, and washed with a saturated Na2CO3 solution until neutral, and the aqueous layer is separated and then washed with a saturated NaCl solution. The organic layer is placed in a drying bottle of anhydrous magnesium sulfate and dried. The dried product is subjected to reduced pressure distillation, and the fraction at 108-128° C. (2 mmHg) is collected to obtain ethyl pyrrole-2-carboxylate.

[0017] In one embodiment, the conditions for the determination by gas chromatography in step (3) are as follows: the content of the ester synthesis product is determined on a Nexis GC-2030 gas chromatograph; the equipment includes a flame ionization detector (FID) and an SLB-5ms capillary column (30m×0.25mm ID×0.25μm df, Merck Life Science), nitrogen is used as the carrier gas (20mL / min), the detector temperature is 300°C, and the injection volume is 1μL; the temperature control program for detecting ethyl pyrrole-2-carboxylate is: 100°C (5 minutes), then heated to 230°C at 5°C / min, maintained for 10 minutes, then heated to 290°C at 5°C / min, and maintained for 5 minutes.

[0018] In one embodiment, the preparation process of the PS-PEG microspheres in step (1) is specifically as follows: weigh 6.0-20 g of chlorinated polystyrene microspheres, add 50-200 mL of toluene to swell for 12-24 h, then add 5.0-20 g of K2CO3 and 0.5-2 g of tetrabutylammonium chloride, add polyethylene glycol according to the molar ratio of the chlorine content on the surface of the chlorinated polystyrene microspheres to the hydroxyl content of polyethylene glycol, heat to 60-130° C., stir to react for 12-24 h, filter and collect the microspheres, wash with deionized water and ethanol, respectively, to obtain PS-PEG microspheres.

[0019] In one embodiment, the chlorinated polystyrene microspheres are first washed with 10 to 15 times the volume of acetone and anhydrous ethanol in sequence for 30 minutes before use, and then vacuum dried at 40 to 80°C.

[0020] In one embodiment, the molar ratio of the chlorine group content on the surface of the chlorinated polystyrene microspheres to the hydroxyl group content of the polyethylene glycol is 1:6 to 1:12.

[0021] In one embodiment, the average molecular weight of the polyethylene glycol is 300-2000, preferably 550.

[0022] In one embodiment, the preparation process of the PS-PEG-Br microspheres in step (1) is specifically as follows: 4.0-20 g of PS-PEG microspheres are added to 60-200 mL of toluene, 0.8-2.0 mL of phosphorus tribromide is slowly added at 50-100° C., the temperature is raised to 80-100° C. after the addition is completed, and the reaction is performed for 5-10 hours. After the reaction is completed, the microspheres are filtered and collected, and washed with deionized water and ethanol, respectively, to obtain PS-PEG-Br microspheres.

[0023] In one embodiment, the preparation process of the PS-PHBA microspheres in step (1) is specifically as follows: 3.3 to 15 g of PS-PEG-Br microspheres are added with 30 to 150 mL of butan-2-one, and then 0.015 to 0.06 mol of p-hydroxybenzaldehyde and 0.0025 to 0.01 mol of K2CO3 are added, and magnetic stirring is performed at 60 to 100° C., and refluxed for 6 to 12 hours. After the reaction is completed, the microspheres are washed three times with anhydrous ethanol, deionized water and acetone, respectively, to obtain PS-PHBA microspheres.

[0024] In one embodiment, the preparation process of the PS-HMD microspheres in step (1) is specifically as follows: 3.8 to 15 g of PS-PHBA microspheres are added to 30 to 150 mL of methanol, and then 0.03 to 0.15 mol of urea and 10 to 40 μL of concentrated hydrochloric acid are added to react, and magnetic stirring is performed at room temperature for 12 to 16 hours. After the reaction is completed, the microspheres are washed three times with anhydrous ethanol, deionized water and acetone respectively to obtain PS-HMD microspheres.

[0025] In one embodiment, the preparation process of the PS-HMDP microspheres in step (1) is as follows: 3.7 to 15 g PS-HMD is added to 30 to 150 mL toluene, and then 0.03 to 0.3 mol epichlorohydrin is added. After magnetic stirring at room temperature for 24 to 48 hours, 0.05 to 0.2 mol NaOH is added and reacted for 3 to 8 hours. After the reaction is completed, the PS-HMDP microspheres are washed three times with anhydrous ethanol, deionized water and acetone respectively to obtain PS-HMDP microspheres.

[0026] In one embodiment, the concentration of the lipase in step (2) is 5-10 mg / mL.

[0027] In one embodiment, the concentration of the sodium hydrogen phosphate-citrate buffer in step (2) is 0.2-0.5 mol / L, and the pH is 6.5-9.0.

[0028] In one embodiment, the volume mass ratio of the sodium hydrogen phosphate-citrate buffer containing lipase to the PS-HMDP microspheres in step (2) is 30-100: 0.5-1.5; mL / g.

[0029] In one embodiment, the shaking reaction conditions of step (2) are: shaking reaction at 25-60° C. and 800-1500 rpm for 6-12 hours.

[0030] In one embodiment, the washing in step (2) is performed using a blank phosphate buffer.

[0031] In one embodiment, the lipase in step (2) is any one of Candida rugosa lipase (product model 013435157), Aspergillus niger lipase (product model 011480691), Thermomyces lanuginosus lipase (product model 013435159), and Candida antarctica lipase (product model 011480689).

[0032] In one embodiment, the ratio of the lipase catalyst to pyrrole-2-carboxylic acid and ethanol in step (3) is: 1.5-2: 0.3-4: 1.5-3; g: mmol: mmol.

[0033] In one embodiment, the ratio of dimethyl sulfoxide (DMSO) to lipase catalyst in step (3) is: 30-100: 1.5-2; mL / g.

[0034] In one embodiment, the yield of ethyl pyrrole-2-carboxylate is 80-85%.

[0035] The present invention also provides an application of the method for synthesizing ethyl pyrrole-2-carboxylate by catalysis of the above-mentioned lipase in production monitoring and quality inspection of ethyl pyrrole-2-carboxylate.

[0036] Beneficial effects:

[0037] The invention uses PS-HMDP microspheres as carriers to load lipase as a catalyst to synthesize ethyl pyrrole-2-carboxylate, has mild conditions, few toxic side effects, is environmentally friendly, avoids the use of complex protection and deprotection processes, and effectively improves the synthesis yield of ethyl pyrrole-2-carboxylate, with the yield reaching 80-85%. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The following specific implementation methods further describe the present invention.

[0039] The present invention relates to a method for determining the content of ethyl pyrrole-2-carboxylate:

[0040] The content of the ester synthesis product was determined on a Nexis GC-2030 gas chromatograph. The equipment included a flame ionization detector (FID) and an SLB-5ms capillary column (30m×0.25mm ID×0.25μm df, Merck Life Science). Nitrogen was used as the carrier gas (20mL / min), the detector temperature was 300°C, and the injection volume was 1μL; the temperature control program for detecting ethyl pyrrole-2-carboxylate was: 100°C (5 minutes), then heated to 230°C at 5°C / min, maintained for 10 minutes, then heated to 290°C at 5°C / min, maintained for 5 minutes.

[0041] The raw material sources involved in the present invention are:

[0042] Chlorinated polystyrene microspheres were purchased from Jiangsu Yonghe Technology Co., Ltd.; the crosslinking degree was 7, the chlorine content was 7.51%, and the particle size was 0.02-2 μm.

[0043] Candida rugosa lipase, enzyme activity ≥700U / mg, purchased from Shanghai Titan Technology Co., Ltd., product model 01148069.

[0044] Aspergillus niger lipase, enzyme activity ≥50000U / g, purchased from Shanghai Titan Technology Co., Ltd., product model 1013435157.

[0045] Candida antarctica lipase, enzyme activity ≥ 20000U / g, purchased from Shanghai Titan Technology Co., Ltd., product model 011480689.

[0046] Example 1

[0047] The preparation of PS-HMDP microspheres includes the following steps:

[0048] (1) Chlorinated polystyrene microspheres were washed with 10 volumes of acetone and anhydrous ethanol for 30 minutes, and then vacuum dried at 40°C; 6.0 g of the dried microspheres were weighed, 50 mL of toluene was added to swell for 12 hours, and then 5.0 g of K2CO3 and 0.5 g of tetrabutylammonium chloride were added, and polyethylene glycol (average molecular weight of 2000) was added according to the molar ratio of the chlorine content on the surface of the chlorinated polystyrene microspheres to the hydroxyl content of polyethylene glycol (1:6), and the temperature was raised to 130°C. After stirring for 24 hours, the microspheres were collected by filtration and washed with deionized water and ethanol, respectively, to obtain PS-PEG microspheres;

[0049] (2) Add 4.0 g of PS-PEG microspheres prepared in step (1) to 60 mL of toluene, slowly drop 1.8 mL of phosphorus tribromide at 50° C., heat to 80° C. after the addition is complete, and react for 5 h. After the reaction is complete, filter and collect the microspheres, wash with deionized water and ethanol, respectively, to obtain PS-PEG-Br microspheres;

[0050] (3) 3.3 g of the PS-PEG-Br microspheres obtained in step (2) were added with 60 mL of butanone, 0.03 mol of p-hydroxybenzaldehyde and 0.0025 mol of K2CO3, and the mixture was magnetically stirred at 60° C. and refluxed for 12 h. After the reaction was completed, the microspheres were washed three times with anhydrous ethanol, deionized water and acetone, respectively, to obtain PS-PHBA microspheres;

[0051] (4) 3.8 g of the PS-PHBA microspheres obtained in step (3) were added to 30 mL of methanol, and then 0.03 mol of urea and 10 μL of 12 mol / L concentrated hydrochloric acid were added to react, and stirred at room temperature for 12 h. After the reaction was completed, anhydrous ethanol, deionized water and acetone were used in sequence, and each was washed three times to obtain PS-HMD microspheres;

[0052] (5) 3.7 g of the PS-HMD microspheres obtained in step (4) were added to 30 mL of toluene, and then 0.03 mol of epichlorohydrin was added. After stirring at room temperature for 24 h, 0.05 mol of NaOH was added and reacted for 3 h. After the reaction was completed, anhydrous ethanol, deionized water and acetone were used in sequence, and each was washed three times to obtain PS-HMDP microspheres.

[0053] The preparation of lipase catalyst comprises the following steps:

[0054] 0.5 g of PS-HMDP microspheres were added to 30 mL of sodium hydrogen phosphate-citric acid buffer (0.2 mol / L, pH = 6.5) containing 5 mg / mL Candida rugosa lipase at a protein concentration of 5 mg / mL, and the mixture was shaken at 35° C. and 800 rpm for 6 h. After the reaction, the mixture was filtered and washed with blank sodium hydrogen phosphate-citric acid buffer (0.2 mol / L, pH = 6.5). After filtration, the lipase catalyst was obtained and stored in a refrigerator at 4° C. for use.

[0055] A method for preparing ethyl pyrrole-2-carboxylate by lipase-catalyzed pyrrole-2-carboxylic acid, the method comprising the following steps:

[0056] 1.5 g of the lipase catalyst prepared above, 0.3 mmol of pyrrole-2-carboxylic acid and 1.5 mmol of ethanol were added to 30 mL of DMSO, and the mixture was continuously shaken at 25° C. for 9 h. After the reaction was completed, the mixture was centrifuged at 10,000 rpm for 5 min, and the supernatant was taken. The content of ethyl pyrrole-2-carboxylate was determined by gas chromatography to be 8.2 mmol / L.

[0057] The supernatant after centrifugation was distilled on an atmospheric distillation apparatus and evaporated to 100°C. The residual liquid was poured into a separatory funnel and washed with 1 volume of saturated Na2CO3 solution until neutral. The aqueous layer was separated, and the organic layer was washed once with 1 volume of saturated NaCl solution and then washed twice with 1 volume of saturated CaCl2 solution. The organic layer was placed in a dry conical flask and dried with 10g of anhydrous magnesium sulfate. The dried ester was distilled under reduced pressure and the fraction at 108-128°C (2mmHg) was collected, i.e., ethyl pyrrole-2-carboxylate, with a yield of 82%.

[0058] Example 2

[0059] The preparation of PS-HMDP microspheres includes the following steps:

[0060] (1) The chlorinated polystyrene microspheres were washed with 15 times the volume of acetone and anhydrous ethanol for 30 minutes, and then vacuum dried at 80°C. Weigh 20g of the treated microspheres, add 200mL of toluene to swell for 24h, then add 20g of K2CO3 and 2g of tetrabutylammonium chloride, add polyethylene glycol (average molecular weight of 550) according to the molar ratio of the chlorine content on the surface of the chlorinated polystyrene microspheres to the hydroxyl content of polyethylene glycol (1:12), heat to 130°C, stir and react for 24h, filter and collect the microspheres PS-PEG, wash with deionized water and ethanol, respectively, to obtain PS-PEG microspheres;

[0061] (2) Add 20 g of PS-PEG microspheres to 200 mL of toluene, slowly drop 2.0 mL of phosphorus tribromide at 100° C., heat to 100° C. after the addition, and react for 10 h. After the reaction, filter and collect the microspheres, wash with deionized water and ethanol, respectively, to obtain PS-PEG-Br microspheres;

[0062] (3) Add 150 mL of butanone, 0.06 mol of p-hydroxybenzaldehyde and 0.01 mol of K2CO3 to 15 g of PS-PEG-Br microspheres, stir magnetically at 100 °C, and reflux for 12 h. After the reaction is completed, wash with anhydrous ethanol, deionized water and acetone three times each to obtain PS-PHBA microspheres;

[0063] (4) 15 g of PS-PHBA microspheres were added to 150 mL of methanol, and then 0.15 mol of urea and 40 μL of 12 mol / L concentrated hydrochloric acid were added to react, and stirred at room temperature for 16 h. After the reaction, anhydrous ethanol, deionized water and acetone were used to wash three times respectively to obtain PS-HMD microspheres;

[0064] (5) 15 g of PS-HMD was added to 150 mL of toluene, and then 0.3 mol of epichlorohydrin was added. After stirring at room temperature for 48 h, 0.2 mol of NaOH was added and the reaction was continued for 8 h. After the reaction was completed, anhydrous ethanol, deionized water and acetone were used to wash three times each to obtain PS-HMDP microspheres.

[0065] The preparation of lipase catalyst comprises the following steps:

[0066] 1.5 g of PS-HMDP microspheres were added to 100 mL of sodium hydrogen phosphate-citric acid buffer (0.5 mol / L, pH = 9.0) containing 8 mg / mL protein concentration of Aspergillus niger lipase, and the mixture was shaken at 60°C and 1500 rpm for 12 h. After the reaction, the mixture was filtered and washed with blank sodium hydrogen phosphate-citric acid buffer (0.5 mol / L, pH = 9). After filtration, the lipase catalyst was obtained and stored in a refrigerator at 4°C for use.

[0067] A method for preparing ethyl pyrrole-2-carboxylate by lipase-catalyzed pyrrole-2-carboxylic acid, the method comprising the following steps:

[0068] 2 g of lipase catalyst, 4 mmol of pyrrole-2-carboxylic acid and 3 mmol of ethanol were added to 100 mL of DMSO, and the mixture was shaken continuously at 45°C for 24 h. After the reaction was completed, the mixture was centrifuged at 10,000 rpm for 5 minutes, and the supernatant was taken. The content of ethyl pyrrole-2-carboxylate was determined by gas chromatography to be 34 mmol / L.

[0069] The supernatant after centrifugation is distilled on an atmospheric distillation apparatus and evaporated to 100°C. The residual liquid is poured into a separatory funnel and washed with 1 volume of saturated Na2CO3 solution until neutral. The aqueous layer is separated, and the organic layer is washed once with 1 volume of saturated NaCl solution and then washed twice with 1 volume of saturated CaCl2 solution. The organic layer is placed in a dry conical flask and dried with 10g of anhydrous magnesium sulfate. The dried ester is distilled under reduced pressure and the fraction at 108-128°C (2mmHg) is collected, i.e., ethyl pyrrole-2-carboxylate, with a yield of 85%.

[0070] Example 3

[0071] The preparation of PS-HMDP microspheres includes the following steps:

[0072] The chlorinated polystyrene microspheres were shaken and washed with 15 times the volume of acetone and anhydrous ethanol for 30 minutes, and then vacuum dried at 70°C; 12g of the treated microspheres were weighed, 100mL of toluene was added to swell for 14h, and then 15g of K2CO3 and 1.5g of tetrabutylammonium chloride were added, and polyethylene glycol (average molecular weight of 300) was added according to the molar ratio of the chlorine content on the surface of the chlorinated polystyrene microspheres to the hydroxyl content of polyethylene glycol (1:9), the temperature was raised to 90°C, and the reaction was stirred for 16h, and the microspheres were collected by filtration, and washed with deionized water and ethanol, respectively, to obtain PS-PEG microspheres;

[0073] (2) Add 10 g of PS-PEG microspheres to 150 mL of toluene, slowly drop 1.6 mL of phosphorus tribromide at 80 ° C, heat to 90 ° C after the addition is completed, react for 8 h, filter and collect the microspheres, wash with deionized water and ethanol, respectively, to obtain PS-PEG-Br microspheres;

[0074] (3) 10 g of PS-PEG-Br microspheres were added with 80 mL of butanone, 0.05 mol of p-hydroxybenzaldehyde and 0.01 mol of K2CO3, and the mixture was stirred magnetically at 100 °C and refluxed for 10 h. After the reaction was completed, the microspheres were washed three times with anhydrous ethanol, deionized water and acetone respectively to obtain PS-PHBA microspheres;

[0075] (4) 10 g of PS-PHBA was added to 50 mL of methanol, and then 0.10 mol of urea and 30 μL of 12 mol / L concentrated hydrochloric acid were added for reaction. The mixture was stirred at room temperature for 14 h. After the reaction, anhydrous ethanol, deionized water and acetone were used to wash the mixture three times respectively to obtain PS-HMD microspheres.

[0076] (5) 7 g of PS-HMD was added to 90 mL of toluene, and then 0.15 mol of epichlorohydrin was added. After stirring at room temperature for 36 h, 0.15 mol of NaOH was added and reacted for 7 h. After the reaction was completed, anhydrous ethanol, deionized water and acetone were used to wash three times each to obtain PS-HMDP microspheres.

[0077] The preparation method of the lipase catalyst comprises the following steps:

[0078] 1.2 g of PS-HMDP microspheres were added to 80 mL of sodium hydrogen phosphate-citric acid buffer (0.3 mol / L, pH = 8.0) containing 8 mg / mL protein concentration of Candida antarctica lipase, and the mixture was shaken at 40°C and 1000 rpm for 10 h. After the reaction, the mixture was filtered and washed with blank sodium hydrogen phosphate-citric acid buffer (0.3 mol / L, pH = 8.0). After filtration, the lipase catalyst was obtained and stored in a refrigerator at 4°C for use.

[0079] A method for preparing ethyl pyrrole-2-carboxylate by lipase-catalyzed pyrrole-2-carboxylic acid, comprising the following steps:

[0080] 2 g of lipase catalyst, 2.4 mmol of pyrrole-2-carboxylic acid and 2.5 mmol of ethanol were added to 80 mL of DMSO, and the mixture was continuously shaken at 45°C for 20 h. After the reaction was completed, the mixture was centrifuged at 10,000 rpm for 5 min, and the supernatant was taken. The content of ethyl pyrrole-2-carboxylate was determined by gas chromatography to be 24 mmol / L.

[0081] The supernatant after centrifugation is distilled on an atmospheric distillation apparatus and evaporated to 100°C. The residual liquid is poured into a separatory funnel and washed with 1 volume of saturated Na2CO3 solution until neutral. The aqueous layer is separated, and the organic layer is washed once with 1 volume of saturated NaCl solution and then washed twice with 1 volume of saturated CaCl2 solution. The organic layer is placed in a dry conical flask and dried with 10g of anhydrous magnesium sulfate. The dried ester is distilled under reduced pressure and the fraction at 108-128°C (2mmHg) is collected, i.e., ethyl pyrrole-2-carboxylate, with a yield of 80%.

[0082] Comparative Example 1

[0083] 0.08 g of Candida rugosa lipase was added to 150 mL of dimethyl sulfoxide (DMSO), pyrrole-2-carboxylic acid (1 mmol) and ethanol (3 mmol), and the mixture was shaken continuously at 45° C. for 24 h. After the reaction was completed, the mixture was centrifuged at 10,000 rpm for 5 minutes, and the supernatant was taken. The content of ethyl pyrrole-2-carboxylate was determined by gas chromatography to be 4.3 mmol / L.

[0084] The supernatant after centrifugation was distilled on an atmospheric distillation apparatus and evaporated to 100° C. The residual liquid was poured into a separatory funnel and washed with 1 volume of saturated Na2CO3 solution until neutral. The aqueous layer was separated, and the organic layer was washed once with 1 volume of saturated NaCl solution and then washed twice with 1 volume of saturated CaCl2 solution. The organic layer was placed in a dry conical flask and dried with 10 g of anhydrous magnesium sulfate. The dried ester was distilled under reduced pressure and the fraction at 108-128° C. (2 mmHg) was collected, i.e., ethyl pyrrole-2-carboxylate, with a yield of 64.6%.

[0085] Comparative Example 2

[0086] 0.15 g of Aspergillus niger lipase was added to 80 mL of dimethyl sulfoxide (DMSO), and pyrrole-2-carboxylic acid (0.5 mmol) and ethanol (2 mmol) were added. The mixture was shaken continuously at 35°C for 20 h. After the reaction was completed, the mixture was centrifuged at 10,000 rpm for 5 min, and the supernatant was taken. The content of ethyl pyrrole-2-carboxylate was determined by gas chromatography, and the result was 4.2 mmol / L.

[0087] The supernatant after centrifugation was distilled on an atmospheric distillation apparatus and evaporated to 100° C. The residual liquid was poured into a separatory funnel and washed with 1 volume of saturated Na2CO3 solution until neutral. The aqueous layer was separated, and the organic layer was washed once with 1 volume of saturated NaCl solution and then washed twice with 1 volume of saturated CaCl2 solution. The organic layer was placed in a dry conical flask and dried with 5 g of anhydrous magnesium sulfate. The dried ester was distilled under reduced pressure and the fraction at 108-128° C. (2 mmHg) was collected, i.e., ethyl pyrrole-2-carboxylate, with a yield of 67.3%.

[0088] Comparative Example 3

[0089] 0.2 g of Candida antarctica lipase was added to 50 mL of dimethyl sulfoxide (DMSO), pyrrole-2-carboxylic acid (0.6 mmol) and ethanol (3 mmol), and the mixture was shaken continuously at 45°C for 24 h. After the reaction, the mixture was centrifuged at 10,000 rpm for 5 minutes, and the supernatant was taken and the content of ethyl pyrrole-2-carboxylate was determined by gas chromatography to be 6.6 mmol / L.

[0090] The supernatant after centrifugation was distilled on an atmospheric distillation apparatus and evaporated to 100°C. The residual liquid was poured into a separatory funnel and washed with 1 volume of saturated Na2CO3 solution until neutral. The aqueous layer was separated, and the organic layer was washed once with 1 volume of saturated NaCl solution and then washed twice with 1 volume of saturated CaCl2 solution. The organic layer was placed in a dry conical flask and dried with 5g of anhydrous magnesium sulfate. The dried ester was distilled under reduced pressure and the fraction at 108-128°C (2mmHg) was collected, i.e., ethyl pyrrole-2-carboxylate, with a yield of 55.3%.

[0091] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for synthesizing ethyl pyrrole-2-carboxylate catalyzed by lipase, characterized in that: The method comprises the following steps: (1) Preparation of PS-HMDP microspheres Chlorinated polystyrene microspheres are added to toluene to swell, and then K2CO3, tetrabutylammonium chloride and polyethylene glycol are added to react to obtain PS-PEG microspheres; PS-PEG microspheres are added to toluene, and phosphorus tribromide is slowly added dropwise to react to obtain PS-PEG-Br microspheres; then PS-PEG-Br microspheres are added to butan-2-one, and p-hydroxybenzaldehyde and K2CO3 are added to react to obtain PS-PHBA microspheres; then PS-PHBA microspheres are added to methanol, and urea and concentrated hydrochloric acid are added to react to obtain PS-HMD microspheres; finally, PS-HMD microspheres are added to toluene, and epichlorohydrin and NaOH are added to react to obtain PS-HMDP microspheres; (2) Preparation of lipase catalyst Add the PS-HMDP microspheres prepared in step (1) to the sodium hydrogen phosphate-citric acid buffer solution containing lipase, shake and react, wash and filter after the reaction is completed, and obtain a lipase catalyst; (3) Preparation of ethyl pyrrole-2-carboxylate The lipase catalyst prepared in step (2), pyrrole-2-carboxylic acid and ethanol are added to dimethyl sulfoxide, and the mixture is continuously shaken at 25-45° C. for 9-24 hours. After the reaction is completed, the mixture is centrifuged, the supernatant is taken, and the supernatant is distilled to 100° C. The residual liquid is poured into a separatory funnel, and washed with a saturated Na2CO3 solution until neutral. The aqueous layer is separated and then washed with a saturated NaCl solution. The organic layer is placed in a drying bottle over anhydrous magnesium sulfate and dried. The dried product is subjected to reduced pressure distillation, and the fraction at 108-128° C. is collected to obtain ethyl pyrrole-2-carboxylate.

2. The method according to claim 1, characterized in that The preparation process of the PS-PEG microspheres in step (1) is as follows: 6.0-20 g of chlorinated polystyrene microspheres are weighed, 50-200 mL of toluene is added to swell for 12-24 h, 5.0-20 g of K2CO3 and 0.5-2 g of tetrabutylammonium chloride are added, polyethylene glycol is added according to the molar ratio of the chlorine content on the surface of the chlorinated polystyrene microspheres to the hydroxyl content of polyethylene glycol, the temperature is raised to 60-130° C., the reaction is stirred for 12-24 h, the microspheres are collected by filtration, and they are washed with deionized water and ethanol, respectively, to obtain PS-PEG microspheres.

3. The method according to claim 2, characterized in that The molar ratio of the chlorine group content on the surface of the chlorinated polystyrene microspheres to the hydroxyl group content of the polyethylene glycol is 1:6 to 1:

12.

4. The method according to claim 2, characterized in that: The average molecular weight of the polyethylene glycol is 300-2000.

5. The method according to claim 1, characterized in that The concentration of the lipase in step (2) is 5-10 mg / mL.

6. The method according to claim 1, characterized in that The volume mass ratio of the sodium hydrogen phosphate-citrate buffer solution containing lipase to the PS-HMDP microspheres in step (2) is 30-100: 0.5-1.5; mL / g.

7. The method according to claim 1, characterized in that The shaking reaction conditions of step (2) are as follows: shaking reaction at 25-60° C. and 800-1500 rpm for 6-12 hours.

8. The method according to claim 1, characterized in that The lipase in step (2) is any one of Candida lipase, Aspergillus niger lipase, Thermomyces lanuginosus lipase and Candida antarctica lipase.

9. The method according to claim 1, characterized in that: In step (3), the ratio of the lipase catalyst to pyrrole-2-carboxylic acid and ethanol is: 1.5-2: 0.3-4: 1.5-3; g: mmol: mmol.

10. Use of the method according to any one of claims 1 to 9 in production monitoring and quality inspection of ethyl pyrrole-2-carboxylate.