Preparation method of epinacillin
The synthesis of ipicillin in one step by enzymatic method simplifies the process flow, solves the problems of complex preparation processes and high energy consumption in the existing technology, and realizes high purity, low energy consumption and environmentally friendly ipicillin preparation, which is suitable for industrial production.
Patent Information
- Application Number
- CN202411646218.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-18
- Publication Date
- 2024-12-31
AI Technical Summary
The existing preparation process of ipicillin is complex and carried out under high temperature and high pressure conditions, resulting in high energy consumption, poor environmental pollution and product stability, and lack of simple and efficient synthesis methods.
Epicillin was synthesized by enzymatic method, and 6-APA was dissolved in dilute ammonia and reacted with immobilized penicillin acylase, and 2,5-dihydrophenylglycine methyl ester hydrochloride was added to control pH and temperature to achieve one-step synthesis and crystallization. , avoid high temperature and high pressure conditions and simplify the process flow.
It realizes the high purity preparation of ipicillin, with mild process conditions, low energy consumption, environmentally friendly, suitable for industrial scale production, and improves the stability and safety of the product.
Smart Images
Figure SMS_1 
Figure HDA0005139762010000011
Abstract
Description
Technical Field
[0001] The invention relates to a preparation process of epicillin, in particular to a method for preparing epicillin by using an enzyme method. Background Art
[0002] The chemical structure of epicillin is shown below. The molecular formula is C 16 H 21 N3O4S, with a molecular weight of 351.42, is a broad-spectrum penicillin. Clinical studies have shown that epicillin exhibits high antibacterial activity against Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, and Proteus. In addition, when tested against Pseudomonas aeruginosa, epicillin has a higher intrinsic activity than similar antibiotics. Epicillin is an amphoteric substance that is sensitive to penicillinase, acid-stable, and minimally binds to human serum proteins, and the process is reversible.
[0003]
[0004] Patent application DE2256330A1 uses recrystallization process conditions to prepare epicillin. The crystal morphology of epicillin is regulated by adding surfactants, such as tetradecyltrimethylammonium bromide, benzalkonium chloride, hexadecylpyridinium chloride, etc. Under different surfactant conditions, epicillin can present needle-shaped and cubic states. However, no relevant synthesis process has been reported. Summary of the invention
[0005] The purpose of the present invention is to provide a method for preparing epicillin. The method of the present invention has fewer reaction steps and can realize the preparation of epicillin under relatively mild conditions. The prepared product has high purity and good stability.
[0006] The present invention is achieved in that:
[0007] The invention adopts an enzymatic method to synthesize epicillin. First, 6-APA is dissolved in dilute ammonia water, and immobilized penicillin acylase is added. Then, a 2,5-dihydrophenylglycine methyl ester hydrochloride solution is prepared. The prepared solution is added dropwise into a 6-APA solution, and the solution is fully stirred. After the reaction is completed, an enzyme separation operation is performed. After acidification, the pH is adjusted, and the solution is cooled and allowed to stand for crystallization.
[0008] The preparation process of epicillin of the present invention is described in more detail below, specifically as follows:
[0009] (a) Add penicillin acyltransferase and 6-APA to water, and add alkali to dissolve 6-APA to obtain a mixed solution.
[0010] (b) 2,5-dihydrophenylglycine methyl ester hydrochloride is added to water and dissolved to obtain a 2,5-dihydrophenylglycine methyl ester hydrochloride solution.
[0011] (c) adding the 2,5-dihydrophenylglycine methyl ester hydrochloride solution dropwise to the mixed solution for reaction, and adding alkali to adjust the pH of the reaction solution and control the reaction temperature. After the addition is complete, the pH and temperature are continuously controlled and the reaction is continued for a period of time.
[0012] (d) cooling the reaction solution to 0-5° C., adding alkali to adjust the pH of the reaction solution, filtering the enzyme and the reaction solution to separate them, washing the surface of the enzyme with water, combining the filtrate and the washing solution to obtain a reaction solution containing the crude epicillin.
[0013] (e) The reaction solution containing the crude epicillin product is heated to 40-50°C, acid is added to adjust the pH, and the broken enzyme is removed by filtering with a 1 μm filter membrane. Alkali is added to adjust the pH, and the solution is cooled to 0-5°C and allowed to stand for crystallization to obtain epicillin crystal solution.
[0014] (f) The epicillin crystal solution was filtered, and the filter cake was washed with cold water and acetone in sequence, and dried in vacuum at 45° C. for 2 h.
[0015] Preferably, the type of penicillin acylase in step (a) is immobilized penicillin acylase, and its synthetic activity is 100 U / g.
[0016] Preferably, in step (a), the ratio of 6-APA to water is 1 g:20 mL to 1 g:30 mL.
[0017] Preferably, the alkali in step (a), step (c), step (d) and step (e) is aqueous ammonia or sodium hydroxide solution.
[0018] Preferably, in step (b), the ratio of 2,5-dihydrophenylglycine methyl ester hydrochloride to water is 1 g:1.9 mL to 1 g:2.5 mL.
[0019] Preferably, in step (c), the molar ratio of 6-APA to 2,5-dihydrophenylglycine methyl ester hydrochloride is 1:1 to 1.2.
[0020] Preferably, the dropping time of 2,5-dihydrophenylglycine methyl ester hydrochloride in step (c) is 60-90 min.
[0021] Preferably, in step (c), alkali is added to adjust the pH of the reaction solution to 6.5-7.5, and the stirring time is 3-5 hours.
[0022] Preferably, the pH in step (d) is adjusted to a range of pH=8.0-9.0.
[0023] Preferably, in step (e), the temperature of the crude reaction solution is raised to 40-50° C., the pH of the acidified solution is adjusted to 1.0-1.5, and the pH of the crystallized solution is adjusted to 4.5-5.5.
[0024] The beneficial effects of the present invention are as follows:
[0025] The present invention uses 6-APA and 2,5-dihydrophenylglycine methyl ester hydrochloride as raw materials, uses water as a reaction medium, and utilizes biological enzyme catalysis to synthesize and prepare epicillin in a one-step process. The present invention has the outstanding characteristics of simple preparation process, mild reaction conditions, low energy consumption, green environmental protection, high safety, etc., and is easy to realize large-scale industrial production. In addition, the present invention can also avoid the use of organic solvents, generate less waste, and is environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 This is a crystal picture of the product prepared in Example 1 of the present invention; wherein, the size of the left picture corresponds to 10×10 times, and the size of the right picture corresponds to 10×40 times. DETAILED DESCRIPTION
[0027] The invention discloses a method for preparing epicillin, the process of which includes dissolving 6-APA, enzyme catalytic reaction, crystallization, filtering, washing and drying. The preparation process has the outstanding characteristics of mild reaction conditions, low energy consumption and green environmental protection, and is of great significance for the industrial large-scale production of epicillin.
[0028] The process of the present invention is described in detail below with reference to specific embodiments.
[0029] Example 1
[0030] The preparation method of epicillin provided in this embodiment is as follows:
[0031] (1) 10 g of immobilized penicillin acylase (synthetic activity: 100 U / g) was added to a 1 L four-necked bottle, 15 g of 6-APA (0.07 mol) was added at 20°C, 300 mL of purified water was added, and the pH was adjusted to 7.00 with dilute ammonia water.
[0032] (2) Add 15.7 g of 2,5-dihydrophenylglycine methyl ester hydrochloride (0.077 mol) and 30 mL of purified water into a 100 mL conical flask and stir until dissolved.
[0033] (3) At 20°C, add the solution obtained in step (2) dropwise to the solution in the four-necked flask in step (1) within 60 minutes, during which the pH value is maintained at 6.5 with dilute ammonia water. After the addition is complete, stir at the same temperature and pH value for 3 hours.
[0034] (4) The solution obtained in step (3) is cooled to 0-5°C, adjusted to pH=8.0 with dilute ammonia water, the enzyme and the reaction solution are separated by filtration, the reactants on the surface of the enzyme are rinsed with 30 mL of purified water, and the rinse solution obtained is combined with the reaction solution obtained by filtration to obtain a reaction solution containing the crude epicillin.
[0035] (5) The reaction solution containing the crude epicillin was heated to 40°C, the pH was adjusted to 1.0 with dilute hydrochloric acid, the enzyme was filtered through a 1 μm filter membrane to remove the enzyme, the pH was adjusted to 4.5, and the solution was cooled to 3-5°C and allowed to stand for crystallization to obtain epicillin crystal solution.
[0036] (6) The epicillin crystal solution was filtered, and the filter cake was washed with cold water and acetone in sequence, and vacuum dried at 45°C for 2 h.
[0037] Figure 1 This is a crystal picture of the product obtained in Example 1 of the present invention. Figure 1 It can be seen that the epicillin prepared in Example 1 of the present invention is in the form of short rods with uniform size and good dispersibility.
[0038] Example 2
[0039] The preparation method of epicillin provided in this embodiment is as follows:
[0040] (1) 10 g of immobilized penicillin acylase (synthetic activity: 100 U / g) was added to a 1 L four-necked bottle, 15 g of 6-APA (0.07 mol) was added at 20°C, 375 mL of purified water was added, and the pH was adjusted to 7.00 with dilute ammonia water.
[0041] (2) Add 16.4 g of 2,5-dihydrophenylglycine methyl ester hydrochloride (0.08 mol) and 35 mL of purified water into a 100 mL conical flask and stir until dissolved.
[0042] (3) At 20°C, add the solution obtained in step (2) dropwise to the solution in the four-necked flask in step (1) over 75 minutes, during which the pH is maintained at 7.00 with dilute ammonia water. After the addition is complete, stir at the same temperature and pH for 4 hours.
[0043] (4) The solution obtained in step (3) is cooled to 0-5°C, adjusted to pH = 8.5 with dilute ammonia water, the enzyme and the reaction solution are separated by filtration, the reactants on the surface of the enzyme are rinsed with 30 mL of purified water, and the rinse solution obtained is combined with the reaction solution obtained by filtration to obtain a reaction solution containing the crude epicillin.
[0044] (5) The reaction solution containing the crude epicillin was heated to 45°C, the pH was adjusted to 1.25 with dilute hydrochloric acid, the enzyme was filtered through a 1 μm filter membrane to remove the enzyme, the pH was adjusted to 5.0, and the solution was cooled to 3-5°C and allowed to stand for crystallization to obtain epicillin crystal solution.
[0045] (6) The epicillin crystal solution was filtered, and the filter cake was washed with cold water and acetone in sequence, and vacuum dried at 45°C for 2 h.
[0046] Example 3
[0047] The preparation method of epicillin provided in this embodiment is as follows:
[0048] (1) 10 g of immobilized penicillin acylase (synthetic activity: 100 U / g) was added to a 1 L four-necked bottle, 15 g of 6-APA (0.07 mol) was added at 20°C, 450 mL of purified water was added, and the pH was adjusted to 7.00 with dilute ammonia water.
[0049] (2) Add 17.1 g of 2,5-dihydrophenylglycine methyl ester hydrochloride (0.084 mol) and 40 mL of purified water into a 100 mL conical flask and stir until dissolved.
[0050] (3) At 20°C, the solution obtained in step (2) was added dropwise to the solution in the four-necked flask in step (1) within 90 minutes, during which the pH value was maintained at 7.5 with dilute ammonia water. After the addition was completed, the mixture was stirred at the same temperature and pH value for 5 hours.
[0051] (4) The solution obtained in step (3) is cooled to 0-5°C, adjusted to pH = 9.0 with dilute ammonia water, the enzyme and the reaction solution are separated by filtration, the reactants on the surface of the enzyme are rinsed with 30 mL of purified water, and the rinse solution obtained is combined with the reaction solution obtained by filtration to obtain a reaction solution containing the crude epicillin.
[0052] (5) The reaction solution containing the crude epicillin product was heated to 50° C., the pH was adjusted to 1.5 with dilute hydrochloric acid, the enzyme was filtered through a 1 μm filter membrane to remove the enzyme, the pH was adjusted to 5.5, and the solution was cooled to 3-5° C. and allowed to stand for crystallization to obtain epicillin crystal solution.
[0053] (6) The epicillin crystal solution was filtered, and the filter cake was washed with cold water and acetone in sequence, and vacuum dried at 45°C for 2 h.
[0054] After testing, the yields of the products obtained in Examples 1 to 3 were 65.4%, 87.6% and 70.2%, respectively. Liquid phase tracking revealed that the conversion rates of 6-APA in step (4) of Examples 1 to 3 were all above 90%, but the yields of the products finally crystallized were quite different. The main reason was that there was an isoelectric point. Under the pH conditions corresponding to the isoelectric point, the solubility of epicillin in the crystallization system was low and the yield was high. Under the pH conditions far from the isoelectric point, the solubility was high and the yield was low.
[0055] The products prepared in Examples 1 to 3 were tested by liquid phase determination method. The results showed that the purity of epicillin in Examples 1 to 3 was 96.74%, 98.83% and 97.45%, respectively. It can be seen that the epicillin prepared by the method of the present invention has a higher purity, and the safety and stability of the drug can be greatly improved.
[0056] In addition, since phenylglycine methyl ester hydrochloride exists in the raw material 2,5-dihydrophenylglycine methyl ester hydrochloride, if the content of phenylglycine methyl ester hydrochloride in the raw material can be controlled, the purity of epicillin will be higher than the current level.
[0057] Comparative Example 1
[0058] The method for preparing epicillin in this comparative example is as follows:
[0059] (1) 10 g of immobilized penicillin acylase (synthetic activity: 100 U / g) was added to a 1 L four-necked bottle, 15 g of 6-APA (0.07 mol) was added at 20°C, 300 mL of purified water was added, and the pH was adjusted to 6.0 with dilute ammonia water.
[0060] (2) Add 15.7 g of 2,5-dihydrophenylglycine methyl ester hydrochloride (0.077 mol) and 30 mL of purified water into a 100 mL conical flask and stir until dissolved.
[0061] (3) At 20°C, add the solution obtained in step (2) dropwise to the solution in the four-necked flask in step (1) within 60 min, during which the pH value is maintained at 5.0 with dilute ammonia water. After the addition is complete, stir at the same temperature and pH value for 6 h.
[0062] (4) The solution obtained in step (3) is cooled to 0-5°C, adjusted to pH = 9.5 with dilute ammonia water, the enzyme and the reaction solution are separated by filtration, the reactants on the surface of the enzyme are rinsed with 30 mL of purified water, and the rinse solution obtained is combined with the reaction solution obtained by filtration to obtain a reaction solution containing the crude epicillin.
[0063] (5) The reaction solution containing the crude epicillin product was heated to 55° C., the pH was adjusted to 1.0 with dilute hydrochloric acid, the enzyme was filtered out with a 1 μm filter membrane, the pH was adjusted to 6.0, and the solution was cooled to 3-5° C. and allowed to stand for crystallization to obtain epicillin crystal solution.
[0064] (6) The epicillin crystal solution was filtered, and the filter cake was washed with cold water and acetone in sequence, and vacuum dried at 45°C for 2 h.
[0065] The test results show that the purity of the product obtained in this comparative example is 92.54%, which is lower than that of Examples 1 to 3. In terms of the molar yield test, the molar yield of this comparative example is only 40.2%.
Claims
1. A method for preparing epicillin, characterized in that: The steps include: (a) adding penicillin acyltransferase and 6-APA into water, and adding alkali to dissolve 6-APA to obtain a mixed solution; (b) adding 2,5-dihydrophenylglycine methyl ester hydrochloride to water and dissolving it to obtain a 2,5-dihydrophenylglycine methyl ester hydrochloride solution; (c) adding the 2,5-dihydrophenylglycine methyl ester hydrochloride solution dropwise to the mixed solution obtained in step (a) to carry out a reaction, and adding a base to adjust the pH of the reaction solution and control the reaction temperature; after the addition is completed, controlling the pH and temperature to continue the reaction; (d) cooling the reaction solution obtained in step (c) to 0-5° C., adding alkali to adjust the pH of the reaction solution, filtering and separating the enzyme and the reaction solution, washing the surface of the enzyme with water, combining the filtrate and the washing solution to obtain a reaction solution containing a crude product of epicillin; (e) heating the reaction solution containing the crude epicillin to 40-50° C., adding acid to adjust the pH, filtering with a 1 μm filter membrane to remove the enzyme fragments, adding alkali to adjust the pH, cooling to 0-5° C. and standing for crystallization to obtain epicillin crystal solution; (f) Filter the epicillin crystal solution, wash the filter cake with cold water and acetone in sequence, and dry it in vacuum at 45±5°C for 2 h.
2. The method for preparing epicillin according to claim 1, characterized in that: The type of penicillin acylase in step (a) is immobilized penicillin acylase, and its synthetic activity is 100 U / g.
3. The method for preparing epicillin according to claim 1, characterized in that: The ratio of 6-APA to water in step (a) is 1 g:20 mL to 1 g:30 mL.
4. The method for preparing epicillin according to claim 1, characterized in that: In step (a), step (c), step (d) and step (e), the alkali is aqueous ammonia or sodium hydroxide solution.
5. The method for preparing epicillin according to claim 1, characterized in that: In step (b), the ratio of 2,5-dihydrophenylglycine methyl ester hydrochloride to water is 1 g:1.9 mL to 1 g:2.5 mL.
6. The method for preparing epicillin according to claim 1, characterized in that: In step (c), the molar ratio of 6-APA to 2,5-dihydrophenylglycine methyl ester hydrochloride is 1:1 to 1.
2.
7. The method for preparing epicillin according to claim 1, characterized in that: The dropwise addition time of 2,5-dihydrophenylglycine methyl ester hydrochloride in step (c) is 60-90 min.
8. The method for preparing epicillin according to claim 1, characterized in that: In step (c), alkali is added to adjust the pH of the reaction solution to 6.5-7.5, and the stirring time is 3-5 hours.
9. The method for preparing epicillin according to claim 1, characterized in that: The pH in step (d) is adjusted to a range of pH=8.0-9.
0.
10. The method for preparing epicillin according to claim 1, characterized in that: In step (e), the temperature of the crude product reaction solution is raised to 40-50° C., the pH of the acidified solution is adjusted to 1.0-1.5, and the pH of the crystallized solution is adjusted to 4.5-5.5.