CN111394415A - Method for synthesizing cefaclor by enzyme method - Google Patents
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Abstract
The invention provides a method for synthesizing cefaclor by an enzyme method, which comprises the following steps of adding 7-amino-3-chloro-cefaclor acid into a mixed solvent, adjusting the pH value to 8.1-8.3, adding immobilized cefaclor synthetase, adding D-p-phenylglycine methyl ester hydrochloride and D-phenylglycine methyl ester, carrying out enzyme catalytic synthesis reaction, separating reaction liquid and the immobilized cefaclor synthetase after the reaction is finished, obtaining a cefaclor crude product, and recrystallizing to obtain a cefaclor product, wherein the mixed solvent comprises methanol, glutaraldehyde and soluble phosphate.
Description
Technical Field
The invention relates to the technical field of pharmaceutical chemical production, in particular to a method for synthesizing cefaclor by an enzymatic method.
Background
Cefaclor is a second generation oral cephalosporin antibiotic medicament developed by American etiquette, has strong killing effect on various gram-positive bacteria and gram-negative bacteria, has the advantages of high efficiency, broad spectrum, good chemical stability and good clinical safety, is widely applied to the field of treatment of bacterial infection, and stably occupies the world sales of oral cephalosporin antibiotics for many years.
At present, the reported methods for synthesizing cefaclor mainly adopt a chemical synthesis method, and basically use 7-amino-3-chloro-3-cephem-4-acid (7-ACCA) as a key intermediate for synthesis, and the problems of complex process, high cost, large pollution, high solvent residue, low yield and the like mainly exist. With the continuous progress of biotechnology, the technology for synthesizing cefaclor by using biological enzyme catalysis has also been developed rapidly. Compared with the chemical synthesis method, the biological enzyme catalysis method has the advantages of high synthesis conversion rate, environmental friendliness, low production cost and the like due to the specificity and specificity of the enzyme. However, in the process of synthesizing cefaclor by a bio-enzyme catalysis method, cefaclor is generated by continuous crystallization in the presence of bio-enzyme, so that the obtained cefaclor is often mixed with synthetase and side chain substances in synthesis, so that the purity of the synthesized cefaclor is not high enough, the activity of the enzyme is reduced quickly after reaction, the reuse frequency of the enzyme is small, and the production cost is increased. Therefore, a new method for synthesizing cefaclor by an enzyme method is developed to improve the purity of the prepared cefaclor and improve the repeated use times of the enzyme, and has very important significance for the expanded production and wide application of cefaclor.
Disclosure of Invention
Aiming at the problems that the purity of cefaclor is not high enough and the recycling frequency of enzyme is low in the existing cefaclor synthesis method by adopting a bio-enzyme catalysis method, the invention provides a method for synthesizing cefaclor by an enzyme method.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
a method for synthesizing cefaclor by an enzymatic method comprises the following steps:
adding 7-amino-3-chloro-cefaclor into a mixed solvent, adjusting the pH value to 8.1-8.3, adding immobilized cefaclor synthetase, uniformly mixing, adding a side chain mixture of D-p-phenylglycine methyl ester hydrochloride and D-phenylglycine methyl ester, carrying out enzyme catalytic synthesis reaction, and separating reaction liquid and the immobilized cefaclor synthetase after the reaction is finished to obtain a cefaclor crude product;
step two, recrystallizing the cefaclor crude product to obtain a cefaclor product;
wherein the mixed solvent comprises methanol, glutaraldehyde and soluble phosphate.
Compared with the prior art, the method for synthesizing cefaclor by the enzyme method provided by the invention is characterized in that 7-amino-3-chloro-cefaclor is used as a mother nucleus substance, a mixture of D-p-phenylglycine methyl ester hydrochloride and D-phenylglycine methyl ester is used as a side chain substance, a mixed solvent consisting of methanol, glutaraldehyde and soluble phosphate is selected as a reaction solvent, and cefaclor is prepared by an enzyme catalytic synthesis reaction. The mixed solvent selected by the invention can not only increase the uniformity of a reaction system, effectively eliminate factors hindering the contact between reactants, increase the collision probability of the reactants and improve the conversion rate; the method can also improve the effective enzyme activity of the immobilized cefaclor synthetase, accelerate the reaction rate of a parent substance and a side chain substance, inhibit the cracking reaction of the side chain substance and cefaclor, reduce the solubility of cefaclor and prevent cefaclor from being hydrolyzed by enzyme, so that the reaction time is shorter and the purity of the prepared cefaclor is higher; in addition, the invention selects the mixture of D-p-phenylglycine methyl ester hydrochloride and D-phenylglycine methyl ester as a side chain substance, and cooperates with the soluble phosphate in the mixed solution, so that the reduction of the pH value of the system in the reaction process can be effectively inhibited, and the pH value of the reaction system is always maintained in a range with higher enzyme activity, therefore, the pH value of the reaction system does not need to be controlled in the reaction process, and the production process is simplified.
Preferably, the mixed solvent comprises the following components in percentage by mass: 0.5-10% of methanol, 0.5-1% of glutaraldehyde, 0.1-1% of soluble phosphate and the balance of water.
More preferably, the mixed solvent comprises the following components in percentage by mass: 5% of methanol, 0.5% of glutaraldehyde, 0.3% of soluble phosphate and the balance of water.
The substances in the preferable mixed solvent are cooperated, so that the bonding free energy between the immobilized cefaclor synthetase and the substrate can be reduced, the synthesis reaction rate is accelerated, the cracking reaction of a side chain substance and cefaclor can be effectively inhibited, the solubility of cefaclor is reduced, and the cefaclor is prevented from being hydrolyzed by enzyme. Meanwhile, glutaraldehyde also has a bactericidal effect, so that the problem of enzyme activity reduction caused by bacteria generated by the enzyme in the long-term use process can be effectively prevented, but glutaraldehyde is easy to polymerize in an aqueous solution and loses the bactericidal effect, and methanol in a mixed solvent can inhibit the polymerization problem of glutaraldehyde in the aqueous solution, so that the activity reduction of the enzyme activity in the long-term use process is reduced, and the repeated use times of the enzyme are increased.
Preferably, the soluble phosphate is at least one of sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, or dipotassium hydrogen phosphate.
More preferably, the soluble phosphate is disodium hydrogen phosphate.
The preferable soluble phosphate not only can cooperate with the side chain mixture to maintain the pH of the reaction system at a value with higher enzyme activity, thereby realizing the aim of not controlling the pH during the reaction; meanwhile, sodium ions and potassium ions can also improve the activity of the enzyme, accelerate the rate of enzyme catalytic reaction, shorten the reaction time and improve the reuse rate of the enzyme.
Preferably, the immobilized cefaclor synthase is immobilized penicillin acylase.
Preferably, the mass-volume ratio of the 7-amino-3-chloro-cephem acid to the mixed solvent is 6:45-55, wherein the unit of mass is gram, and the unit of volume is milliliter.
Preferably, the molar ratio of D-phenylglycine methyl ester to D-phenylglycine methyl ester hydrochloride in the side chain mixture is 1.6-2.1: 1.
More preferably, the molar ratio of D-phenylglycine methyl ester to D-phenylglycine methyl ester hydrochloride in the side chain mixture is 2.02: 1.
Adding 7-amino-3-chloro-cephem acid into the mixed solvent, adjusting the pH to 8.1-8.3 to completely dissolve the 7-amino-3-chloro-cephem acid in the mixed solvent, wherein when the pH is adjusted to 8.1-8.3, the reaction between the mother nucleus and the side chain substance is quicker, but the cracking reaction of the side chain and the generated cefaclor is easier to occur under the pH condition. The D-p-phenylglycine methyl ester hydrochloride and the D-phenylglycine methyl ester are selected to be mixed according to a specific proportion to serve as side chain substances, the side chain and the generated cracking reaction of cefaclor can be effectively inhibited, and meanwhile, the pH value of a reaction system is maintained in a range with higher enzyme activity by cooperating with phosphate in a mixed solvent, so that the pH value of the reaction system does not need to be controlled, and the production process is greatly simplified.
Preferably, the addition amount of the immobilized spore clorox synthetase is 0.25-0.5 time of the mass of the 7-amino-3-chloro-cephem acid.
The addition amount of the enzyme is small, and the reaction time is long; the addition of a large amount of enzyme leads to an increase in the amount of enzyme inclusion in the synthesized cefaclor, which leads to a decrease in product purity. The preferable addition amount of the immobilized cefaclor synthase can improve the reaction rate, shorten the reaction time and improve the purity of the product.
Preferably, in the step one, the reaction temperature of the enzymatic synthesis reaction is 15-25 ℃, and the reaction time is 80-150 min.
The preferable reaction temperature and reaction time can increase the reaction speed and reduce the occurrence of side reactions.
Preferably, in step one, the side chain mixture of D-p-phenylglycine methyl ester hydrochloride and D-phenylglycine methyl ester is divided into two equal parts, and added in two times: after addition of the immobilized cefaclor synthase, one aliquot of the side chain mixture is added, and after reaction for 15-30min, another aliquot of the side chain mixture is added.
The preferable method for adding the side chain substance can control the conversion reaction speed, prevent the reaction from being too fast, so that the product is exploded out, and the immobilized enzyme is wrapped, so that the conversion reaction can not be continued, and finally the problem of too low conversion rate is caused.
Optionally, in the first step, ammonia water with a mass concentration of 15-20 wt% is used for adjusting the pH value to 8.1-8.3.
Preferably, the specific process of recrystallization comprises the following steps: adjusting the pH value of the cefaclor crude product to 0.5-1.5 by using an acid solution, and filtering to obtain a liquid to be crystallized; rapidly mixing the solution to be crystallized with an alkali solution to obtain a mixed solution with the pH of 3.5-5.0, and growing the crystals at 10-15 ℃ for 30-60 min; adjusting pH to 3.5-4.0, and growing crystal at 0-5 deg.C for 30-60 min.
The preferable crystallization temperature, time and pH value can make the supersaturation degree in the system easier to control in a range which is favorable for the good growth of the crystal, and is not easy to generate impurities and enzyme occlusion, thereby being favorable for improving the purity of the product.
Preferably, the mixing time of the liquid to be crystallized and the alkali solution is 1 to 2 seconds.
The mixing time of the liquid to be crystallized and the alkali solution is 1-2 seconds, so that the system is prevented from being over-alkali, cefaclor is cracked under the alkali condition, and the optimal mixing time is favorable for improving the purity of cefaclor.
Optionally, a hydrochloric acid solution or a sulfuric acid solution with a mass concentration of 20-30 wt% is adopted to adjust the pH to 0.5-1.5 in the recrystallization process.
Optionally, the alkali solution in the recrystallization process is ammonia water or sodium hydroxide solution with the mass concentration of 10-15 wt%.
Preferably, in the step one, the immobilized cefaclor synthase separated after the reaction is finished can be soaked in the above mixed solvent with the pH of 4.5-6.0 for the next enzymatic synthesis reaction.
Optionally, the mass of the soaked mixed solvent is 1.5-2 times of the mass of the immobilized cefaclor synthetase.
The molar ratio of the 7-amino-3-chloro-cephem acid to the side chain mixture in the present invention can be selected according to conventional techniques.
Drawings
Fig. 1 is a high performance liquid chromatogram for content detection of cefaclor prepared in example 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
A method for synthesizing cefaclor by an enzymatic method comprises the following steps:
step one, adding 60.0g of 7-amino-3-chloro-cefaclor acid and 450m L serving as a mixed solvent into a reaction bottle at the temperature of 10 ℃, adjusting the pH to 8.1-8.3 by using refined ammonia water with the mass concentration of 18 wt%, adding 30g of immobilized penicillin acylase, adding a side chain mixture of 19g of D-p-phenylglycine methyl ester hydrochloride and 31.5g D-phenylglycine methyl ester, reacting for 100min at the temperature of 20 ℃, separating a reaction solution and immobilized cefaclor synthetase after the reaction is finished to obtain a cefaclor crude product, washing the immobilized penicillin acylase for 3 times by using the mixed solution of 90m L, and mixing a washing solution with the separated cefaclor crude product to obtain a cefaclor crude product suspension;
the mixed solvent comprises the following components in percentage by mass: 3% of methanol, 0.5% of glutaraldehyde, 0.3% of disodium hydrogen phosphate and 96.2% of water;
the side chain mixture was added in the following manner: dividing the side chain mixture of the D-p-phenylglycine methyl ester hydrochloride and the D-phenylglycine methyl ester into two equal parts, and adding the mixture in two times: adding immobilized cefaclor synthetase, adding one equal part of side chain mixture, reacting for 30min, and adding the other equal part of side chain mixture;
step two, regulating the pH of the cefaclor crude product suspension to 1.0 by using a hydrochloric acid solution with the mass concentration of 25 wt%, and filtering to obtain a to-be-crystallized liquid;
step three, reducing the temperature of the liquid to be crystallized and ammonia water with the mass concentration of 13 wt% to 0 ℃, quickly and uniformly mixing in 1-2s to obtain a mixed solution with the pH of 4.0, and growing the crystals for 50min at the temperature of 13 ℃; and regulating the pH value to 3.5, and continuously growing the crystals at the temperature of 3 ℃ for 50min to obtain the cefaclor product.
7-ACCA conversion (weight of 7-ACCA charged at reaction-weight of 7-ACCA remaining in solution after reaction)/weight of 7-ACCA charged at reaction
Weight yield of cefaclor (7-ACCA weight input during the reaction) is equal to the weight of cefaclor after purification
The conversion rate of 7-ACCA was 99.3%, the weight yield of cefaclor was 130%, the liquid phase purity of cefaclor was 99.8%, and the bulk density of cefaclor was 6.7g/m L.
The content of the prepared cefaclor is detected by adopting a conventional liquid phase detection method of cefaclor, and the result is shown in fig. 1.
Example 2
A method for synthesizing cefaclor by an enzymatic method comprises the following steps:
step one, adding 60.0g of 7-amino-3-chloro-cefaclor acid and 550m L serving as a mixed solvent into a reaction bottle at the temperature of 10 ℃, adjusting the pH to 8.1-8.3 by using 20 wt% of refined ammonia water, adding 15g of immobilized penicillin acylase, adding a side chain mixture of 20.2g D-p-phenylglycine methyl ester hydrochloride and 30.6g D-phenylglycine methyl ester at one time, reacting for 150min at the temperature of 15 ℃, separating a reaction solution and immobilized cefaclor synthetase after the reaction is finished to obtain a cefaclor crude product, washing the immobilized penicillin acylase by using 90m L mixed solution for 3 times, and mixing a washing solution with the separated cefaclor crude product to obtain a cefaclor crude product suspension;
the mixed solvent comprises the following components in percentage by mass: 0.5% of methanol, 0.8% of glutaraldehyde, 1% of disodium hydrogen phosphate and 97.7% of water;
step two, regulating the pH of the cefaclor crude product suspension to 0.5 by using a hydrochloric acid solution with the mass concentration of 30 wt%, and filtering to obtain a to-be-crystallized liquid;
step three, reducing the temperature of the liquid to be crystallized and a sodium hydroxide solution with the mass concentration of 10 wt% to 2 ℃, then quickly and uniformly mixing within 1-2s to obtain a mixed solution with the pH value of 3.5, and growing the crystals for 60min at the temperature of 10 ℃; and regulating the pH value to 3.8, and continuously growing the crystals at the temperature of 5 ℃ for 30min to obtain the cefaclor product.
7-ACCA conversion (weight of 7-ACCA charged at reaction-weight of 7-ACCA remaining in solution after reaction)/weight of 7-ACCA charged at reaction
Weight yield of cefaclor (7-ACCA weight input during the reaction) is equal to the weight of cefaclor after purification
The conversion rate of 7-ACCA is 96.5%, the weight yield of cefaclor is 125%, the purity of cefaclor is 99.5%, and the bulk density of cefaclor is 6.2g/m L.
Example 3
A method for synthesizing cefaclor by an enzymatic method comprises the following steps:
step one, adding 60.0g of 7-amino-3-chloro-cefaclor acid and 500m L of mixed solvent into a reaction bottle at the temperature of 10 ℃, adjusting the pH to 8.1-8.3 by using 15 wt% of refined ammonia water, adding 25g of immobilized penicillin acylase, adding 22g of a side chain mixture of D-p-phenylglycine methyl ester hydrochloride and 29g D-phenylglycine methyl ester, reacting for 80min at the temperature of 25 ℃, separating reaction liquid and immobilized cefaclor synthetase after the reaction is finished to obtain cefaclor crude products, washing the immobilized penicillin acylase for 3 times by using 90m L of the mixed solution, and mixing washing liquid with the separated cefaclor crude products to obtain cefaclor crude product suspension;
the mixed solvent comprises the following components in percentage by mass: 10% of methanol, 1% of glutaraldehyde, 0.1% of disodium hydrogen phosphate and 88.9% of water;
the side chain mixture was added in the following manner: dividing the side chain mixture of the D-p-phenylglycine methyl ester hydrochloride and the D-phenylglycine methyl ester into two equal parts, and adding the mixture in two times: adding immobilized cefaclor synthetase, adding one equal part of side chain mixture, reacting for 15min, and adding the other equal part of side chain mixture;
step two, regulating the pH of the cefaclor crude product suspension to 1.5 by using a sulfuric acid solution with the mass concentration of 20 wt%, and filtering to obtain a to-be-crystallized solution;
step three, reducing the temperature of the liquid to be crystallized and ammonia water with the mass concentration of 15 wt% to 1 ℃, then quickly and uniformly mixing within 1-2s to obtain a mixed solution with the pH of 5.0, and growing the crystals for 30min at 15 ℃; and regulating the pH value to 4.0, and continuously growing the crystals at the temperature of 0 ℃ for 60min to obtain the cefaclor product.
7-ACCA conversion (weight of 7-ACCA charged at reaction-weight of 7-ACCA remaining in solution after reaction)/weight of 7-ACCA charged at reaction
Weight yield of cefaclor (7-ACCA weight input during the reaction) is equal to the weight of cefaclor after purification
The conversion rate of 7-ACCA is 97%, the weight yield of cefaclor is 129%, the purity of cefaclor is 99.7%, and the bulk density of cefaclor is 6.5g/m L.
Comparative example 1
This comparative example provides an enzymatic synthesis of cefaclor, which is prepared in exactly the same manner as in example 1, except that the mixed solvent of example 1 is replaced with an equal amount of water.
Comparative example 2
This comparative example provides an enzymatic synthesis of cefaclor, which is prepared in exactly the same manner as in example 1, except that the mixed solvent of example 1 is replaced with glutaraldehyde and water in equal amounts, wherein the content of glutaraldehyde is 0.5 wt%.
Comparative example 3
This comparative example provides an enzymatic synthesis of cefaclor, which is prepared in exactly the same manner as in example 1, except that the mixed solvent of example 1 is replaced with equal amounts of methanol and water, wherein the methanol content is 3% by weight.
The immobilized penicillin acylase separated after the reaction in the embodiment 1 and the comparative examples 1-3 of the invention is soaked by the mixed solvent with the pH value of 4.5-6.0 which is 1.5-2 times of the mass of the immobilized penicillin acylase for the next enzyme-catalyzed synthesis reaction, the soaked recovered immobilized penicillin acylase is adopted to carry out a plurality of times of enzyme-catalyzed synthesis reactions according to the corresponding reaction conditions, the test is stopped until the reaction time exceeds 150min when the residual quantity of 7-ACCA in the system is less than 3.0mg/m L, the recycling times of the respectively recovered immobilized penicillin acylase are counted, and the results are shown in Table 1.
TABLE 1
| Number of times of recycling | Reaction time (min) | 7-ACCA residue | |
| Example 1 | 200 | <150 | Less than 3mg/ml |
| Comparative example 1 | 45 | <150 | Less than 3mg/ml |
| Comparative example 2 | 60 | <150 | Less than 3mg/ml |
| Comparative example 3 | 45 | <150 | Less than 3mg/ml |
The enzyme activities of the immobilized penicillin acylases used in the above examples and comparative examples were all 80-90U/g in the mixed solvent.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A method for synthesizing cefaclor by an enzymatic method is characterized by comprising the following steps:
adding 7-amino-3-chloro-cefaclor into a mixed solvent, adjusting the pH value to 8.1-8.3, adding immobilized cefaclor synthetase, uniformly mixing, adding a side chain mixture of D-p-phenylglycine methyl ester hydrochloride and D-phenylglycine methyl ester, carrying out enzyme catalytic synthesis reaction, and separating reaction liquid and the immobilized cefaclor synthetase after the reaction is finished to obtain a cefaclor crude product;
step two, recrystallizing the cefaclor crude product to obtain a cefaclor product;
wherein the mixed solvent comprises methanol, glutaraldehyde and soluble phosphate.
2. The enzymatic synthesis method of cefaclor as claimed in claim 1, wherein the mixed solvent comprises the following components in percentage by mass: 0.5-10% of methanol, 0.5-1% of glutaraldehyde, 0.1-1% of soluble phosphate and the balance of water.
3. The process for the enzymatic synthesis of cefaclor as claimed in claim 1 or 2, wherein the soluble phosphate is at least one of sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate or dipotassium hydrogen phosphate; and/or
The immobilized cefaclor synthetase is immobilized penicillin acylase.
4. The process for the enzymatic synthesis of cefaclor as claimed in claim 1 or 2, wherein the mass to volume ratio of 7-amino-3-chloro-cefaclor to the mixed solvent is 6:45-55, wherein the unit of mass is g and the unit of volume is ml.
5. The process for the enzymatic synthesis of cefaclor as claimed in claim 1, wherein the molar ratio of D-phenylglycine methyl ester to D-p-phenylglycine methyl ester hydrochloride in the side chain mixture is 1.6-2.1: 1.
6. The enzymatic synthesis of cefaclor as claimed in claim 1 or 3, wherein the immobilized cefaclor synthase is added in an amount of 0.25-0.5 times the mass of 7-amino-3-chloro-cefaclor acid.
7. The enzymatic synthesis of cefaclor as claimed in claim 1, wherein in step one, the reaction temperature of the enzymatic synthesis reaction is 15-25 ℃ and the reaction time is 80-150 min.
8. The process for the enzymatic synthesis of cefaclor according to claim 1 or 5, wherein in step one, the mixture of the side chains of D-p-phenylglycine methyl ester hydrochloride and D-phenylglycine methyl ester is divided into two equal portions, and the two portions are added: after addition of the immobilized cefaclor synthase, one aliquot of the side chain mixture is added, and after reaction for 15-30min, another aliquot of the side chain mixture is added.
9. The enzymatic synthesis method of cefaclor as claimed in claim 1, wherein in the second step, the recrystallization comprises the following steps: adjusting the pH value of the cefaclor crude product to 0.5-1.5 by using an acid solution, and filtering to obtain a liquid to be crystallized; rapidly mixing the solution to be crystallized with an alkali solution to obtain a mixed solution with the pH of 3.5-5.0, and growing the crystals at 10-15 ℃ for 30-60 min; adjusting pH to 3.5-4.0, and growing crystal at 0-5 deg.C for 30-60 min.
10. The process for the enzymatic synthesis of cefaclor as claimed in claim 9, wherein the mixing time of the liquid to be crystallized and the alkaline solution is 1-2 seconds.
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| CN111909046A (en) * | 2020-08-27 | 2020-11-10 | 天津大学 | D-phenylglycine methyl ester phosphate crystal, preparation method and solution |
| CN115058479A (en) * | 2022-08-08 | 2022-09-16 | 河北工业大学 | Method for preparing high-purity cefaclor through enzymatic reaction crystallization |
| CN115372498A (en) * | 2022-07-14 | 2022-11-22 | 石家庄四药有限公司 | Method for detecting residual impurities in cefaclor |
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