CN109852660B - Process for preparing cefadroxil - Google Patents
Process for preparing cefadroxil Download PDFInfo
- Publication number
- CN109852660B CN109852660B CN201910012708.1A CN201910012708A CN109852660B CN 109852660 B CN109852660 B CN 109852660B CN 201910012708 A CN201910012708 A CN 201910012708A CN 109852660 B CN109852660 B CN 109852660B
- Authority
- CN
- China
- Prior art keywords
- cefadroxil
- acylase
- adca
- reaction
- magnetic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
Abstract
A preparation method of cefadroxil comprises the following steps: adding a cosolvent consisting of ionic liquid with a preset volume and phosphate buffer solution into a reactor with constant temperature in a water bath; adding D-HPGM and 7-ADCA in a preset molar ratio into the cosolvent and stirring, wherein the molar ratio of the D-HPGM to the 7-ADCA is 1: 1-1: 2.0; the concentration of 7-ADCA is 0.1-1.0 mol/L; adding magnetic immobilized penicillin G acylase into a cosolvent added with D-HPGM and 7-ADCA, and continuously stirring to ensure that the magnetic immobilized penicillin G acylase is fully contacted with the D-HPGM and the 7-ADCA until the reaction is finished; and (3) layering and standing the reaction system liquid after the reaction is finished, layering the upper-layer phosphate buffer solution in which the cefadroxil is dissolved and the lower-layer ionic liquid, adjusting the pH value of the separated phosphate buffer solution to 4.0-5.5, and crystallizing and separating out the cefadroxil and drying the cefadroxil.
Description
Technical Field
The invention relates to the technical field of medicines, and particularly relates to a preparation method of cefadroxil.
Background
Cefadroxil (cefadroxil) is an important semisynthetic beta-lactam antibiotic widely used in clinic, and has the advantages of strong activity, good oral absorption, low toxic and side effects and wide antibacterial spectrum. At present, the domestic production of cefadroxil generally adopts a mixed anhydride method (such as CN201310227621.9), the steps are complicated, the group protection process in the process needs to be carried out at a low temperature of-30 ℃, a large amount of toxic reagents are used, a large amount of wastes are generated, and serious harm is caused to the ecological environment. The enzyme catalysis method is to catalyze 7-amino-3-desacetoxycephalosporanic acid (7-ADCA) to react with D-p-hydroxyphenylglycine methyl ester (D-HPGM) by utilizing penicillin G acylase (EC 3.5.1.11, abbreviated as PGA) to synthesize cefadroxil, the reaction condition is mild, the use of toxic reagents is not involved, and the process is clean and environment-friendly. However, the existing process for enzymatically synthesizing cefadroxil has two defects, which severely restrict the application of large-scale production. On one hand, as the solubility of the substrates 7-amino-3-desacetoxycephalosporanic acid and D-p-hydroxyphenylglycine methyl ester in a neutral aqueous solution is low, the substrates are dissolved in a strong alkaline aqueous solution, the substrates are dissolved in a strong acid solution, and both the substrates can not be completely dissolved in an enzyme catalysis aqueous solution system close to the neutral, so that the reaction speed is low, and the synthesis yield is not high; on the other hand, the product cefadroxil is easily dissolved in water, penicillin G acylase can catalyze D-HPGM and the product cefadroxil to generate D-p-hydroxyphenylglycine (D-HPG) through hydrolysis side reaction, and the reaction process is as follows:
resulting in poor selectivity of the enzymatic synthesis of cefadroxil in aqueous solution and a low synthesis/hydrolysis ratio (synthesis/hydrolysis ratio refers to the ratio of moles of p-hydroxyphenylglycine produced by hydrolysis to moles of cefadroxil produced by synthesis). In order to improve the conversion rate of parent nucleus 7-ADCA, the dosage of side chain D-p-hydroxyphenylglycine methyl ester is increased in synthesis, the molar ratio of D-p-hydroxyphenylglycine methyl ester to 7-ADCA even reaches 4:1, which is far higher than the reaction dosage (1:1), most D-p-hydroxyphenylglycine methyl ester in a reaction system is not subjected to acylation reaction and is converted into a target product, and the consumption of D-p-hydroxyphenylglycine methyl ester is excessively high. In order to increase the solubility of the substrate in the reaction medium and to inhibit hydrolysis side reactions, researchers have attempted to perform an enzymatic synthesis of cefadroxil in organic solvents.
Although the hydrolysis reaction can be inhibited to a certain extent by using an organic reagent solvent, the catalytic activity of the penicillin acylase in the organic solvent capable of dissolving the substrate is greatly reduced, so that the yield of the target product is low; the volatility of organic solvents is harmful to the ecological environment. Therefore, the key problem to be solved urgently in the industrial production of cefadroxil by an enzyme method is to improve the synthesis efficiency and reduce the side chain consumption so as to reduce the production cost.
Disclosure of Invention
In view of the above, there is a need for a process for preparing cefadroxil with improved synthesis efficiency and reduced side chain consumption.
A preparation method of cefadroxil comprises the following steps:
adding a cosolvent consisting of ionic liquid with a preset volume and phosphate buffer solution into a reactor with constant temperature in a water bath;
adding D-HPGM and 7-ADCA in a preset molar ratio into the cosolvent and stirring, wherein the molar ratio of the D-HPGM to the 7-ADCA is 1: 1-1: 2.0; the concentration of 7-ADCA is 0.1-1.0 mol/L;
adding magnetic immobilized penicillin G acylase into a cosolvent added with D-HPGM and 7-ADCA, wherein the mass ratio of the magnetic immobilized penicillin G acylase to the 7-ADCA is 1: 10-1: 50, continuously stirring to ensure that the magnetic immobilized penicillin G acylase, the D-HPGM and the 7-ADCA are fully contacted until the reaction is finished, wherein the stirring speed is controlled to be 150-250 r/min;
and (3) layering and standing the reaction system liquid after the reaction is finished so as to layer the upper phosphate buffer solution in which the cefadroxil is dissolved and the lower ionic liquid, adjusting the pH value of the separated phosphate buffer solution to 4.0-5.5 by using 6mol/L HCl to separate out cefadroxil crystals, and separating out the crystals by conventional operation at 45 ℃ for drying.
Preferably, in the ionic liquid-phosphate buffer solution cosolvent, the mass of the ionic liquid is 40% -60% of the total solvent mass, the pH value of the phosphate buffer solution is 6.0-8.0, and the concentration is 0.10-0.50 mol/L.
Preferably, the ionic liquid may be any one of 1-butyl-3-methylimidazole bistrifluoromethylsulfonyl imide salt, 1-butyl-3-methylimidazole hexafluorophosphate, 1-butyl-3-methylimidazole tetrafluoroborate, 1-octyl-3-methylimidazole hexafluoroborate, 1-octyl-3-methylimidazole tetrafluoroborate ionic liquid, and 1-ethyl-3-methylimidazole tetrafluoroborate.
Preferably, the reaction temperature for synthesizing cefadroxil is 20-50 ℃; the reaction time is 5-15 h.
Preferably, the reaction temperature for synthesizing cefadroxil is 30-40 ℃, and the reaction time is 8-12 hours.
Preferably, the magnetic immobilized penicillin G acylase is obtained by immobilizing free penicillin G acylase by using a magnetic composite microsphere with a core-shell structure as a carrier.
Preferably, the magnetically immobilized penicillin G acylase is obtained by the following method:
preparing the magnetic composite microsphere with the core-shell structure: 2 parts of polyvinylpyrrolidone, 3.2 parts of NaCl, 8 parts of soluble starch and 180 parts of deionized water are filled into a reaction kettle, and the mixture is uniformly dispersed under stirring;
the flask was charged with a predetermined amount of FeCl2·4H2O and FeCl3·6H2O, wherein Fe2+With Fe3+The molar ratio of (1: 2) is added, 100 parts of deionized water is added and dissolved under mechanical stirring, strong ammonia water is dropped when the temperature is heated to 40 ℃, the pH value is adjusted to 9-10, then 0.2-0.5 part of oleic acid is added, the temperature is raised to 90 ℃, and then the temperature is kept for 2 hours;
naturally cooling to room temperature, and adding 10 parts of styrene, 3 parts of ethylene glycol dimethacrylate and 0.5 part of initiator azobisisobutyronitrile;
transferring the mixed liquid in the flask to the reaction kettle,heating the reaction kettle in water bath to 60 ℃, stirring and keeping the temperature constant for 2-5 hours to prepare Fe3O4-a polymer core body;
adding 0.5 part of glycidyl methacrylate and 0.2 part of N, N '-methylene-bisacrylamide into the same reaction kettle at the temperature of 30 ℃, uniformly stirring, heating to the temperature of 60 ℃, keeping the temperature for 3-6 hours, and reacting the glycidyl methacrylate and the N, N' -methylene-bisacrylamide in Fe3O4Surface cross-linking polymerization of polymer core to prepare epoxy-rich core-shell composite magnetic beads;
the preparation of the magnetic immobilized enzyme comprises the following steps: weighing 0.1 part of the core-shell composite magnetic beads, and adding PGA solution diluted by 0.1mol/L phosphoric acid buffer solution with pH of 7.0, wherein VPhosphoric acid buffer solution：VPGAUniformly mixing the components 2:1, and carrying out shaking reaction on an air shaking table at the temperature of 30 ℃ for 12-24 hours at the rotating speed of the shaking table of 100-;
preferably, the method further comprises the following steps: separating the magnetic immobilized penicillin G acylase under the action of magnetic force, and washing for 3 times by using phosphate buffer solution to obtain the magnetic immobilized penicillin G acylase.
In the method, a cosolvent consisting of ionic liquid and phosphate buffer solution is used as a reaction medium, and in the process of synthesizing cefadroxil by catalyzing 7-amino-3-desacetoxycephalosporanic acid and D-p-hydroxyphenylglycine methyl ester (7-ADCA and D-HPGM) through reaction by using immobilized penicillin G acylase, the cosolvent consisting of the ionic liquid and the phosphate buffer solution inhibits D-HPGM hydrolysis side reaction, so that the synthesis/hydrolysis ratio is improved, the yield of cefadroxil is not lower than 80%, and the synthesis/hydrolysis ratio is higher than 1.5; meanwhile, the cosolvent consisting of the ionic liquid and the phosphate buffer can completely dissolve the substrates 7-ADCA and D-HPGM in a near-neutral medium (the optimal pH value of the enzyme catalytic reaction) at the same time, so that the enzyme catalytic synthesis efficiency is higher than that of other solvents.
Drawings
FIG. 1 is a scanning electron micrograph of core-shell magnetic microspheres.
FIG. 2 is an infrared spectrum of magnetic microspheres.
Detailed Description
The invention provides a preparation method of cefadroxil, which comprises the following steps: adding a cosolvent consisting of ionic liquid with a preset volume and phosphate buffer solution into a reactor with constant temperature in a water bath; adding D-HPGM and 7-ADCA in a preset molar ratio into the cosolvent and stirring, wherein the molar ratio of the D-HPGM to the 7-ADCA is 1: 1-1: 2.0; the concentration of 7-ADCA is 0.1-1.0 mol/L; adding magnetic immobilized penicillin G acylase into a cosolvent added with D-HPGM and 7-ADCA, wherein the mass ratio of the magnetic immobilized penicillin G acylase to the 7-ADCA is 1: 10-1: 50, continuously stirring to ensure that the magnetic immobilized penicillin G acylase, the D-HPGM and the 7-ADCA are fully contacted until the reaction is finished, and controlling the stirring speed to be 150-250 r/min; and (3) layering and standing the reaction system liquid after the reaction is finished so as to layer the upper phosphate buffer solution in which the cefadroxil is dissolved and the lower ionic liquid, adjusting the pH value of the separated phosphate buffer solution to 4.0-5.5 by using 6mol/L HCl to separate out cefadroxil crystals, and separating out the crystals by conventional operation at 45 ℃ for drying. Further, the method also comprises the following steps: the magnetic immobilized penicillin G acylase is obtained by separating the magnetic immobilized penicillin G acylase under the action of magnetic force and washing with phosphate buffer solution, the step can be carried out before the liquid of the reaction system after the reaction is finished is layered and kept still, more phosphate buffer solution is used for dissolving cefadroxil, and of course, in other embodiments, the step can be set as a step after cefadroxil is crystallized and precipitated according to actual production requirements. For example, after washing the magnetically immobilized penicillin G acylase 2 times with a phosphate buffer solution having a pH of 7.0, the magnetically immobilized penicillin G acylase obtained is reused for the next batch of reactions.
In the ionic liquid-phosphate buffer solution cosolvent, the mass of the ionic liquid is 40% -60% of the total solvent mass, the pH value of the phosphoric acid buffer solution is 6.0-8.0, and the concentration is 0.10-0.50 mol/L; the ionic liquid can be any one of 1-butyl-3-methylimidazole bistrifluoromethylsulfonyl imide salt, 1-butyl-3-methylimidazole hexafluorophosphate, 1-butyl-3-methylimidazole tetrafluoroborate, 1-octyl-3-methylimidazole hexafluoroborate, 1-octyl-3-methylimidazole tetrafluoroborate ionic liquid and 1-ethyl-3-methylimidazole tetrafluoroborate; wherein the reaction temperature for synthesizing cefadroxil is 20-50 ℃; the reaction time is 5-15 h; the magnetic immobilized penicillin G acylase is obtained by immobilizing free penicillin G acylase by using a magnetic composite microsphere with a core-shell structure as a carrier.
Wherein the magnetically immobilized penicillin G acylase is obtained by a method wherein the "parts" appearing in the following steps are in different units of measurement depending on the nature of the material, e.g. 1 part may be 1G or 1 kg for a raw material that can be quantified by mass, 1 part may be 1 l or 1 ml for a raw material that can be quantified by volume:
preparing the magnetic composite microsphere with the core-shell structure: 2 parts of polyvinylpyrrolidone, 3.2 parts of NaCl, 8 parts of soluble starch and 180 parts of deionized water are filled into a reaction kettle, and the mixture is uniformly dispersed under stirring; a flask was charged with a predetermined amount of FeCl2·4H2O and FeCl3·6H2O, wherein Fe2+With Fe3+The molar ratio of (1: 2) is added, 100 parts of deionized water is added and dissolved under mechanical stirring, strong ammonia water is dropped when the temperature is heated to 40 ℃, the pH value is adjusted to 9-10, then 0.2-0.5 part of oleic acid is added, the temperature is raised to 90 ℃, and then the temperature is kept for 2 hours; naturally cooling to room temperature, and adding 10 parts of styrene, 3 parts of ethylene glycol dimethacrylate and 0.5 part of initiator azobisisobutyronitrile; transferring the mixed solution in the flask into the reaction kettle, heating the reaction kettle in water bath to 60 ℃, stirring, keeping the temperature constant for 2-5 hours, and preparing Fe3O4-a polymer core body; adding 0.5 part of glycidyl methacrylate and 0.2 part of N, N '-methylene bisacrylamide into a same reaction kettle at the temperature of 30 ℃, uniformly stirring, heating to the temperature of 60 ℃, keeping the temperature for 3-6 hours, and reacting the glycidyl methacrylate and the N, N' -methylene bisacrylamide in Fe3O4Surface cross-linking polymerization of polymer core to prepare epoxy-rich core-shell composite magnetic beads; referring to fig. 1 and fig. 2, it can be seen from fig. 1 that the prepared core-shell magnetic microspheres are regular spheres, and no adhesion occurs between the microspheres. Specific saturation magnetization (sigma) of5-15 emu/g, the diameter of the microsphere is about 180-250 μm; at 906cm in FIG. 2-1And 849cm-1The infrared absorption peak of (1) is a characteristic absorption peak of an epoxy group, and is 1546cm-1And 1727cm-1The absorption peaks are the absorption peak of an amide group and the characteristic absorption peak of a carbonyl group, which shows that a large amount of epoxy groups capable of being covalently bonded with enzyme and hydrophilic amide groups providing an enzyme immobilization microenvironment exist on the surface of the shell layer of the prepared magnetic microsphere, and the content of the functional epoxy groups of the core-shell type magnetic polymer microsphere is 300-345 mu mol/g.
The preparation of the magnetic immobilized enzyme comprises the following steps: weighing 0.1 part of the core-shell composite magnetic beads, and adding PGA solution diluted by 0.1mol/L phosphoric acid buffer solution with pH of 7.0, wherein VPhosphoric acid buffer solution：VPGAMixing uniformly 2:1, oscillating and reacting for 12-24 hours at 30 ℃ by using an air shaking table, wherein the rotating speed of the shaking table is 100-.
After the reaction is finished, the immobilized penicillin G acylase can be rapidly precipitated under the action of magnetic force, the separation is thorough, the recovery and the use are convenient, and the loss problem of the biocatalyst in the operation and use process is avoided.
In the method, a cosolvent consisting of ionic liquid and phosphate buffer solution is used as a reaction medium, and in the process of synthesizing cefadroxil by catalyzing 7-amino-3-desacetoxycephalosporanic acid and D-p-hydroxyphenylglycine methyl ester (7-ADCA and D-HPGM) through reaction by using immobilized penicillin G acylase, the cosolvent consisting of the ionic liquid and the phosphate buffer solution inhibits D-HPGM hydrolysis side reaction, so that the synthesis/hydrolysis ratio is improved, the yield of cefadroxil is not lower than 80%, and the synthesis/hydrolysis ratio is higher than 1.5; meanwhile, the cosolvent consisting of the ionic liquid and the phosphate buffer can completely dissolve the substrates 7-ADCA and D-HPGM in a near-neutral medium (the optimal pH value of the enzyme catalytic reaction) at the same time, so that the enzyme catalytic synthesis efficiency is higher than that of other solvents. After the reaction is finished, the immobilized penicillin G acylase can be rapidly precipitated under the action of magnetic force, the separation is thorough, the recovery and the use are convenient, and the loss problem of the biocatalyst in the operation and use process is avoided.
To facilitate understanding of the technical solution of the present invention, the following examples are given:
example 1, 60mL of an ionic liquid 1-butyl-3-methylimidazolium bistrifluoromethylsulfonyl imide salt and 40mL of a sodium phosphate buffer solution (0.10mol/L, pH 7.0) were added to a reactor, and after mixing well, 0.1mol of 7-ADCA and 0.15mol of D-HPGM were added, at which time the concentration of 7-ADCA was 214 g/L. Then 0.42G of magnetic immobilized penicillin G acylase is added, the mass ratio of the immobilized enzyme to 7-ADCA is 1:50, the temperature of the water bath is controlled to be constant at 30 ℃, the stirring speed is controlled to be 150r/min, the reaction is carried out for 10 hours, and the immobilized enzyme is separated out under the action of magnetic force. Adjusting the pH value of the upper solution of the separated reaction system to 5.2, and obtaining cefadroxil crystals by a crystallization method. The reactor may be a batch reactor which is commonly used in the industry.
Example 2, 60mL of ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate and 40mL of sodium phosphate buffer solution (0.10mol/L, pH 7.0) were added to the reactor, and after mixing well, 0.1mol of 7-ADCA and 0.15mol of D-HPGM were added, at which time the concentration of 7-ADCA was 214 g/L. Then 0.42G of magnetic immobilized penicillin G acylase is added, the mass ratio of the immobilized enzyme to 7-ADCA is 1:50, the temperature of the water bath is controlled to be 30 ℃, the stirring speed is controlled to be 150r/min, the reaction is carried out for 10 hours, and the immobilized enzyme is separated out under the action of magnetic force. Adjusting the pH value of the upper solution of the separated reaction system to 5.0, and obtaining cefadroxil crystal by a crystallization method.
Example 3, 60mL of ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate and 40mL of sodium phosphate buffer (0.5mol/L, pH 7.0) were added to the reactor, and after mixing, 0.1mol of 7-ADCA and 0.15mol of D-HPGM were added, at which time the concentration of 7-ADCA was 214 g/L. Then 0.85G of magnetic immobilized penicillin G acylase is added, the mass ratio of the immobilized enzyme to 7-ADCA is 1:25, the temperature of the water bath is controlled to be 30 ℃, the stirring speed is controlled to be 150r/min, the reaction is carried out for 8 hours, and the immobilized enzyme is separated out under the action of magnetic force. Adjusting the pH value of the upper solution of the separated reaction system to 5.0, and obtaining cefadroxil crystal by a crystallization method.
Example 4, 50mL of ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate and 50mL of sodium phosphate buffer (0.10mol/L, pH 7.0) were added to the reactor, and after mixing well, 0.1mol of 7-ADCA and 0.15mol of D-HPGM were added, at which time the concentration of 7-ADCA was 214 g/L. Then 0.42G of magnetic immobilized penicillin G acylase is added, the mass ratio of the immobilized enzyme to 7-ADCA is 1:50, the temperature of the water bath is controlled to be 30 ℃, the stirring speed is controlled to be 150r/min, the reaction is carried out for 8 hours, and the immobilized enzyme is separated out under the action of magnetic force. And adjusting the pH value of the upper layer aqueous solution of the separated reaction system to 4.5, and obtaining cefadroxil crystals by a crystallization method.
Example 5, 60mL of ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate and 40mL of sodium phosphate buffer solution (0.5mol/L, pH 8.0) were added to the reactor, and after mixing well, 0.1mol of 7-ADCA and 0.15mol of D-HPGM were added, at which time the concentration of 7-ADCA was 214 g/L. Then 0.63G of magnetic immobilized penicillin G acylase is added, the mass ratio of the immobilized enzyme to 7-ADCA is 1:34, the temperature of the water bath is controlled to be 35 ℃, the stirring speed is controlled to be 150r/min, the reaction is carried out for 8 hours, and the immobilized enzyme is separated out under the action of magnetic force. And adjusting the pH value of the upper layer aqueous solution of the separated reaction system to 4.5, and obtaining cefadroxil crystals by a crystallization method.
Example 6, 60mL of ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate and 40mL of sodium phosphate buffer solution (0.10mol/L, pH 7.0) were added to the reactor, and after mixing well, 0.1mol of 7-ADCA and 0.15mol of D-HPGM were added, at which time the concentration of 7-ADCA was 214 g/L. Then 0.42G of magnetic immobilized penicillin G acylase is added, the mass ratio of the immobilized enzyme to 7-ADCA is 1:50, the temperature of the water bath is controlled to be 30 ℃, the stirring speed is controlled to be 250r/min, the reaction is carried out for 8 hours, and the immobilized enzyme is separated out under the action of magnetic force. And adjusting the pH value of the upper layer aqueous solution of the separated reaction system to 5.0, and obtaining cefadroxil crystals by a crystallization method.
Example 7, 60mL of an ionic liquid 1-butyl-3-methylimidazolium bistrifluoromethylsulfonyl imide salt and 40mL of a sodium phosphate buffer solution (0.10mol/L, pH 7.0) were added to a reactor, and after mixing well, 0.1mol of 7-ADCA and 0.2mol of D-HPGM were added, at which time the concentration of 7-ADCA was 214 g/L. Then 0.42G of magnetic immobilized penicillin G acylase is added, the mass ratio of the immobilized enzyme to 7-ADCA is 1:50, the temperature of the water bath is controlled to be 30 ℃, the stirring speed is controlled to be 150r/min, the reaction is carried out for 10 hours, and the immobilized enzyme is separated out under the action of magnetic force. Adjusting the pH value of the upper layer aqueous solution of the separated reaction system to 5.0, and obtaining cefadroxil crystals by a crystallization method.
The concentrations of the respective substances in the reactions of the above examples were quantitatively analyzed by high performance liquid chromatography. The yield (Y) and synthesis/hydrolysis ratio (S/H) of cefadroxil were calculated as follows:
TABLE 1 Cefadroxil yield and S/H values
| Group of | Y(％) | S/H value |
| Example 1 | 80.6 | 1.56 |
| Example 2 | 81.4 | 1.59 |
| Example 3 | 81.8 | 1.61 |
| Example 4 | 80.3 | 1.55 |
| Example 5 | 81.5 | 1.63 |
| Example 6 | 81.0 | 1.60 |
| Example 7 | 83.9 | 1.58 |
As can be seen from Table 1, in the process of synthesizing cefadroxil by catalyzing (D-HPGM and 7-ADCA) 7-amino-3-desacetoxycephalosporanic acid and D-p-hydroxyphenylglycine methyl ester through magnetic immobilized penicillin G acylase by using the cosolvent composed of the ionic liquid and the phosphate buffer solution as a reaction medium, the D-HPGM hydrolysis side reaction is inhibited by the cosolvent composed of the ionic liquid and the phosphate buffer solution, and the synthesis/hydrolysis ratio is higher than 1.5, so that the yield of cefadroxil is higher than 80%.
Claims (6)
1. The preparation method of cefadroxil is characterized by comprising the following steps:
adding a cosolvent consisting of ionic liquid with a preset volume and phosphate buffer solution into a reactor with constant temperature in a water bath;
adding D-HPGM and 7-ADCA in a preset molar ratio into the cosolvent and stirring, wherein the molar ratio of the D-HPGM to the 7-ADCA is 1: 1-1: 2.0; the concentration of 7-ADCA is 0.1-1.0 mol/L;
adding magnetic immobilized penicillin G acylase into a cosolvent added with D-HPGM and 7-ADCA, wherein the mass ratio of the magnetic immobilized penicillin G acylase to the 7-ADCA is 1: 10-1: 50, continuously stirring to ensure that the magnetic immobilized penicillin G acylase, the D-HPGM and the 7-ADCA are fully contacted until the reaction is finished, wherein the stirring speed is controlled to be 150-250 r/min;
layering and standing reaction system liquid after the reaction is finished so as to layer an upper layer phosphate buffer solution in which cefadroxil is dissolved and a lower layer ionic liquid, adjusting the pH value of the separated phosphate buffer solution to 4.0-5.5 by using 6mol/L HCl to separate out cefadroxil crystals, and separating out the crystals by conventional operation at 45 ℃ for drying;
the magnetic immobilized penicillin G acylase is obtained by immobilizing free penicillin G acylase by using a core-shell structure magnetic composite microsphere as a carrier; the magnetic immobilized penicillin G acylase is obtained by adopting the following method:
preparing the magnetic composite microsphere with the core-shell structure: 2 parts of polyvinylpyrrolidone, 3.2 parts of NaCl, 8 parts of soluble starch and 180 parts of deionized water are filled into a reaction kettle, and the mixture is uniformly dispersed under stirring;
a flask was charged with a predetermined amount of FeCl2·4H2O and FeCl3·6H2O, wherein Fe2+With Fe3+The molar ratio of (1: 2) is added, 100 parts of deionized water is added and dissolved under mechanical stirring, strong ammonia water is dropped when the temperature is heated to 40 ℃, the pH value is adjusted to 9-10, then 0.2-0.5 part of oleic acid is added, the temperature is raised to 90 ℃, and then the temperature is kept for 2 hours;
naturally cooling to room temperature, and adding 10 parts of styrene, 3 parts of ethylene glycol dimethacrylate and 0.5 part of initiator azobisisobutyronitrile;
transferring the mixed solution in the flask into the reaction kettle, heating the reaction kettle in water bath to 60 ℃, stirring and keeping the temperature for 2-5 hours to prepare Fe3O4-a polymer core body;
adding 0.5 part of glycidyl methacrylate and 0.2 part of N, N '-methylene bisacrylamide into a same reaction kettle at the temperature of 30 ℃, uniformly stirring, heating to the temperature of 60 ℃, keeping the temperature for 3-6 hours, and reacting the glycidyl methacrylate and the N, N' -methylene bisacrylamide in Fe3O4Surface cross-linking polymerization of polymer core to prepare epoxy-rich core-shell composite magnetic beads;
the preparation of the magnetic immobilized penicillin G acylase comprises the following steps: weighing 0.1 part of the core-shell compound magnetic beads, adding into the core-shell compound magnetic beads0.1mol/L penicillin G acylase solution diluted with phosphate buffered solution having pH 7.0, wherein VPhosphoric acid buffer solution：VPenicillin G acylase solution1, uniformly mixing, carrying out oscillation reaction on an air shaking table at the temperature of 30 ℃ for 12-24 hours, wherein the rotating speed of the shaking table is 100-;
separating the immobilized enzyme under the action of magnetic force, and washing for 3 times by using phosphate buffer solution to obtain the magnetic immobilized penicillin G acylase.
2. Process for the preparation of cefadroxil according to claim 1, characterised in that: in the ionic liquid-phosphate buffer solution cosolvent, the mass of the ionic liquid is 40-60% of the total solvent mass, the pH value of the phosphoric acid buffer solution is 6.0-8.0, and the concentration is 0.10-0.50 mol/L.
3. Process for the preparation of cefadroxil according to claim 1, characterised in that: the ionic liquid is any one of 1-butyl-3-methylimidazole bistrifluoromethylsulfonyl imide salt, 1-butyl-3-methylimidazole hexafluorophosphate, 1-butyl-3-methylimidazole tetrafluoroborate, 1-octyl-3-methylimidazole hexafluoroborate, 1-octyl-3-methylimidazole tetrafluoroborate ionic liquid and 1-ethyl-3-methylimidazole tetrafluoroborate.
4. Process for the preparation of cefadroxil according to claim 1, characterised in that: the reaction temperature for synthesizing the cefadroxil is 20-50 ℃; the reaction time is 5-15 h.
5. Process for the preparation of cefadroxil according to claim 4, characterized in that: the reaction temperature for synthesizing the cefadroxil is 30-40 ℃, and the reaction time is 8-12 h.
6. Process for the preparation of cefadroxil according to claim 1, further comprising the steps of: separating the magnetic immobilized penicillin G acylase under the action of magnetic force, and washing the magnetic immobilized penicillin G acylase by using a phosphate buffer solution to obtain the magnetic immobilized penicillin G acylase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910012708.1A CN109852660B (en) | 2019-01-07 | 2019-01-07 | Process for preparing cefadroxil |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910012708.1A CN109852660B (en) | 2019-01-07 | 2019-01-07 | Process for preparing cefadroxil |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109852660A CN109852660A (en) | 2019-06-07 |
| CN109852660B true CN109852660B (en) | 2022-05-24 |
Family
ID=66894094
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910012708.1A Active CN109852660B (en) | 2019-01-07 | 2019-01-07 | Process for preparing cefadroxil |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109852660B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101502722A (en) * | 2008-12-29 | 2009-08-12 | 嘉兴学院 | Ionic liquid double-aqueous phase system for extracting residual antibiotic in foodstuffs and use thereof |
| CN102559833A (en) * | 2011-12-19 | 2012-07-11 | 浙江工业大学 | Method for preparing (S)-(+)-2,2-dimethyl cyclopropane methanoic acid by biological resolution in ionic liquid cosolvent |
| CN103451259A (en) * | 2013-08-07 | 2013-12-18 | 南通康鑫药业有限公司 | Method for enzymatic synthesis of cefprozil in recyclable aqueous two-phase system by using immobilized penicillin acylase |
| CN107058447A (en) * | 2016-12-23 | 2017-08-18 | 苏州中联化学制药有限公司 | A kind of method of enzymatic clarification cefadroxil |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110229580A1 (en) * | 2010-03-22 | 2011-09-22 | Indian Institute of Technology Bombay, School of Biosciences and Bioengineering | Compositions and methods for nano-in-micro particles |
| US9509009B2 (en) * | 2011-06-08 | 2016-11-29 | Cfd Research Corporation | Enzyme catalyzed oxidation of hydrocarbons |
-
2019
- 2019-01-07 CN CN201910012708.1A patent/CN109852660B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101502722A (en) * | 2008-12-29 | 2009-08-12 | 嘉兴学院 | Ionic liquid double-aqueous phase system for extracting residual antibiotic in foodstuffs and use thereof |
| CN102559833A (en) * | 2011-12-19 | 2012-07-11 | 浙江工业大学 | Method for preparing (S)-(+)-2,2-dimethyl cyclopropane methanoic acid by biological resolution in ionic liquid cosolvent |
| CN103451259A (en) * | 2013-08-07 | 2013-12-18 | 南通康鑫药业有限公司 | Method for enzymatic synthesis of cefprozil in recyclable aqueous two-phase system by using immobilized penicillin acylase |
| CN107058447A (en) * | 2016-12-23 | 2017-08-18 | 苏州中联化学制药有限公司 | A kind of method of enzymatic clarification cefadroxil |
Non-Patent Citations (5)
| Title |
|---|
| "Efficient synthesis of cefadroxil in [Bmim][NTf2]-phosphate cosolvent by magnetic immobilized penicillin G acylase";Zheng Zhaoyu et al.;《JOURNAL OF THE CHINESE CHEMICAL SOCIETY》;20191231;第1-9页 * |
| "Hofmeister effects: an explanation for the impact of ionic liquids on biocatalysis";Zhen Yang;《Journal of Biotechnology》;20091231;第144卷;第12-22页 * |
| "磁性聚合物微球的制备及其应用研究进展";王晔晨 等;《化工进展》;20171231;第36卷(第8期);第2971-2977页 * |
| "酶法合成头孢羟氨苄工艺研究";吴耀辉 等;《中国抗生素杂志》;20160430;第41卷(第4期);第255-260页 * |
| "青霉素Ｇ酰化酶在磁性复合载体上的固定化及其酶学性质";屈冠群 等;《宁夏大学学报（自然科学版）》;20110331;第32卷(第1期);第57页左栏第1段，第57-58页第1.2-1.4节，第60页结论部分 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109852660A (en) | 2019-06-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107312768B (en) | Immobilized tannase and preparation method and application thereof | |
| CN104928340A (en) | Process for enzymatic synthesis of cefprozil | |
| CN109576256B (en) | Method for encapsulating double enzymes by magnetic DNA hydrogel | |
| CN104404023A (en) | Preparation method of magnetic carrier immobilized lipase, and method for preparing biodiesel under catalysis of magnetic carrier immobilized lipase | |
| CN102337256A (en) | Method for entrapping and cross-linking phosphatidase A1 aggregates | |
| CN109852660B (en) | Process for preparing cefadroxil | |
| WO2024002326A1 (en) | Preparation method for and use of double-enzyme-inorganic hybrid nanoflower microspheres | |
| CN111041014B (en) | Magnetic immobilized lipase and application thereof in resolution of 1-methyl-3-amphetamine | |
| CN104830940A (en) | An enzymatic synthesis process of Amoxicillin | |
| CN107012137A (en) | A kind of method that sodium alginate-chitosan fixes zytase | |
| CN106148319B (en) | Method for preparing immobilized enzyme based on reaction adsorption method | |
| CN112626060B (en) | Immobilized multienzyme system for producing inositol and method for producing inositol | |
| CN1149284C (en) | Immobilized pencillin amidase using multi-element copolymerized porous microparticles as carrier and its preparing process | |
| CN104711248A (en) | Substrate toxicity eliminating method | |
| CN110760496B (en) | Co-crosslinking immobilization method of penicillin G acylase | |
| CN107326021B (en) | Preparation method of magnetic cellulose microsphere immobilized lipase catalyst | |
| JPS5838152B2 (en) | Water-insoluble enzyme complex | |
| CN101575595B (en) | Method for preparing calcium-alginate-immobilized marine bacterium MP-2 esterase | |
| CN111607584A (en) | Method for immobilizing marine cyclodextrin glucosyltransferase by resin | |
| CN103145257A (en) | Water quality stabilizer | |
| CN111979282A (en) | Method for producing alpha-arbutin by substrate and cell double-immobilization fermentation | |
| CN102875731A (en) | Preparation method and application of macroporous penicillin acylase immobilized magnetic bead polymer carrier | |
| CN107641623A (en) | Primary amine base immobilized enzyme vector and preparation method thereof | |
| CN114990101B (en) | Magnetic nanoparticle composite carrier immobilized lipase and preparation method thereof | |
| CN1732256A (en) | The guard method of insoluble enzymatic biological catalyst, the biological catalyst of its acquisition and the bio-reactor that has immobilized biocatalyst |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |