CN112538506B

CN112538506B - Process method for synthesizing chloramphenicol succinate by enzyme catalysis

Info

Publication number: CN112538506B
Application number: CN202011577379.4A
Authority: CN
Inventors: 胡惜朝; 李自永; 邓萌; 范艳利; 周双双
Original assignee: Luoyang Normal University
Current assignee: Luoyang Normal University
Priority date: 2020-12-28
Filing date: 2020-12-28
Publication date: 2023-04-25
Anticipated expiration: 2040-12-28
Also published as: CN112538506A

Abstract

The invention relates to a process method for synthesizing succinic acid chloramphenicol by enzyme catalysis, which belongs to the technical field of biological medicine. The hydrolase is any one or a combination of a plurality of protease, lipase and acyltransferase, wherein the enzyme can be of natural source or obtained by recombination of genetic engineering technology. Compared with the existing technology, the enzyme catalysis technology adopted by the invention is more convenient and safer, has smaller pollution, higher quality and lower cost, and is more suitable for large-scale industrial production of the succinic acid chloramphenicol.

Description

Process method for synthesizing chloramphenicol succinate by enzyme catalysis

Technical Field

The invention belongs to the technical field of biological medicine, and particularly relates to a synthesis method of chloramphenicol succinate.

Background

Chloramphenicol (chlorhenicol) is an amidic broad-spectrum antibiotic found in streptomyces venezuelae. Has inhibiting effects on gram positive and negative bacteria, anaerobe, spirochete, chlamydia and rickettsia. The traditional Chinese medicine composition is mainly used for treating typhoid fever, influenza, pneumonia, meningitis and the like clinically, and is also effective on pertussis, trachoma, bacillary dysentery, urinary tract infection and the like. Chloramphenicol antibiotics are easy to synthesize and prepare in large scale due to large application, low cost and are still first-line clinical medicines.

However, chloramphenicol has serious adverse reactions of inhibiting the hematopoietic function of bone marrow, can reduce various blood cells of human body and even produce irreversible aplastic anemia, thus limiting the expansion of the use of chloramphenicol. Therefore, the structure of chloramphenicol is modified to reduce toxic and side effects while maintaining the efficacy, wherein chloramphenicol succinate is a more successful chloramphenicol derivative.

The system name of the succinic acid chloramphenicol (Chloramphenicol Succinate) is D-threo- (-) -N-alpha- (hydroxymethyl) -beta-hydroxy-p-nitrophenyl ethyl-2, 2-dichloroacetamide-alpha-succinate, chloramphenicol can be rapidly hydrolyzed in vivo to generate action, the drug effect is the same as or even better than that of chloramphenicol, and the side effect is greatly reduced compared with that of chloramphenicol. The classical preparation method is prepared by reacting chloramphenicol and succinic anhydride under the catalysis of organic base, and more reports are reported on related chemical synthesis methods in patent, paper and other documents, but the traditional chemical synthesis method has the advantages of more raw material consumption, lower yield, high energy consumption in the reaction process, high toxicity of reaction reagents, environmental pollution, and lower activity of the traditional chemical catalyst, so that the reaction selectivity is poor, more byproducts are generated, the yield is low, the product quality is low, and the post-treatment refining is difficult. These drawbacks limit the industrial application of the method.

In summary, development of a method for preparing chloramphenicol succinate with lower cost, more convenient operation, safety and high efficiency is needed.

Disclosure of Invention

Aiming at the problems, the invention aims to provide a process method for synthesizing chloramphenicol succinate by enzyme catalysis, which adopts hydrolase to catalyze the esterification reaction of chloramphenicol and succinic anhydride to generate chloramphenicol succinate, has higher production efficiency and product quality, and has the advantages of convenient operation, low cost, environmental protection, safety, high efficiency and the like.

In order to achieve the above purpose, the invention adopts the following specific scheme:

a process method for synthesizing chloramphenicol succinate by enzyme catalysis comprises the following steps:

step one, chloramphenicol and succinic anhydride react under the catalysis of biological enzyme to generate chloramphenicol succinate:

mixing chloramphenicol and succinic anhydride into a solvent, adding hydrolase, performing mixed reaction in water bath at 0-60 ℃, and stopping the reaction when the chloramphenicol is completely converted; recovering the enzyme catalyst, adding active carbon into the reaction solution for decoloring, carrying out suction filtration, and carrying out reduced pressure distillation on the filtrate to remove the solvent to obtain a white solid product;

the molar ratio of the succinic anhydride to the chloramphenicol is 1-1.2:1, a step of; the hydrolase is one or more of protease, lipase and acyltransferase, and the addition amount of the hydrolase is 5-50% of the mass of chloramphenicol;

step two, refining:

adding a good solvent into the white solid product obtained in the step one, stirring at room temperature until the good solvent is dissolved, and then slowly adding a poor solvent; or, adding a crystallization solvent consisting of a good solvent and a poor solvent into the white solid product obtained in the step one; slowly cooling to 0-5 ℃, standing at constant temperature, precipitating white needle-like crystals, filtering, and vacuum drying the obtained crystals at 50 ℃ for 2 hours to obtain a refined product;

the good solvent is any one or more mixed solvents of methanol, ethanol or acetone; the poor solvent is water; the volume ratio of the good solvent to the poor solvent is 1:1, and the total addition amount of the good solvent and the poor solvent is 8-12 times of the mass of the white solid product.

In the first step, the solvent is any one or a mixture of more of acetone, acetonitrile, tetrahydrofuran, methyl tertiary butyl ether, dioxane, toluene and isopropyl ether, and is preferably acetone.

In the first step, the lipase is one or more of thermophilic fungi, rhizomucor miehei, candida antarctica, candida rugosa, pseudomonas cepacia, pseudomonas fluorescens, aspergillus niger, bacillus subtilis and rhizopus oryzae. More preferably, the lipase is candida antarctica lipase CALB or rhizomucor miehei lipase RML. Most preferably, the lipase is candida antarctica lipase CALB.

In the first step, the hydrolase is an immobilized enzyme.

In the first step, the addition amount of the hydrolase is 10-20% of the mass of chloramphenicol.

In the first step, the water bath is controlled to be at a temperature of 40 ℃ for mixing reaction.

In the first step, the mixing reaction is carried out in a kettle type stirring reactor, an enzyme filling fixed bed reactor or a shaking table.

In the first step, a water absorbing agent is added to the reaction system before the mixing reaction. Preferably, the water absorbing agent is molecular sieve, silica gel, bentonite or anhydrous sodium sulfate, and the addition amount of the water absorbing agent is 5% of the mass of the solvent.

In the second step, the specific refining process is as follows: under the condition of 20-40 ℃, methanol is firstly added to stir and dissolve crude product of white solid product of succinic chloramphenicol, then purified water is slowly added, and then the temperature is slowly reduced to 0-5 ℃, and finally the mixture is kept stand for 2-6 hours for crystallization.

The beneficial effects are that:

compared with the prior art, the method has the advantages of higher production efficiency and product quality, convenient operation, low cost, environment friendliness, safety, high efficiency and the like, and is more suitable for industrial production of the chloramphenicol succinate.

Drawings

FIG. 1 is a reaction equation for the reaction described in example 1;

FIG. 2 is a reaction equation for the reaction described in example 2;

FIG. 3 is a reaction equation for the reaction described in example 3;

FIG. 4 is a reaction equation for the reaction described in example 4;

FIG. 5 is a reaction equation for the reaction described in example 5;

FIG. 6 is a chromatogram of chloramphenicol succinate prepared using the present invention.

Detailed Description

mixing chloramphenicol and succinic anhydride in a certain proportion into a solvent, adding hydrolase, carrying out water bath temperature control mixing reaction, and stopping the reaction when the chloramphenicol conversion is monitored; recovering the enzyme catalyst, adding active carbon into the reaction solution for decoloring, carrying out suction filtration, and carrying out reduced pressure distillation on the filtrate to remove the solvent to obtain a white solid product;

step two, refining:

and (3) adding a good solvent into the white solid product obtained in the step (I), stirring at room temperature until the good solvent is dissolved, slowly adding the poor solvent until the product is saturated, slowly cooling to 4 ℃, standing at constant temperature, precipitating white needle-like crystals, filtering, and vacuum drying the obtained crystals at 50 ℃ for 2 hours to obtain a refined product.

In the first step, the hydrolase is one or more of protease, lipase and acyltransferase.

Further, the lipase is derived from any one or more of thermophilic fungi (Thermomyces lanuginosa), rhizopus oryzae (Rhizomucor miehei), candida antarctica (Candida Antarctica), candida rugosa (Candida rugosa), pseudomonas cepa (Pseudomonas cepacia), pseudomonas fluorescens (Pseudomonas fluorescens), aspergillus niger (Aspergillus niger), bacillus subtilis (Bacillus subtilis), rhizopus oryzae (Rhizopus oryzae). More preferably, the lipases used are candida antarctica lipase CALB and rhizomucor miehei lipase RML. Most preferably, the lipase used is candida antarctica lipase CALB.

According to the present invention, the hydrolytic enzymes used are not limited to natural sources but include enzymes that are recombinant by molecular biological micro-means. The form of the enzyme used is not limited, and may be either dry powder or immobilized, and since the immobilized enzyme is used, the subsequent treatment is convenient and the enzyme can be recovered for reuse, the immobilized enzyme is particularly preferable. Among the above numerous lipases, we found that lipases derived from candida antarctica (Candida Antarctica) and rhizomucor miehei (Rhizomucor miehei) have better activity for the selective esterification of chloramphenicol and succinic anhydride. Generally, immobilized enzymes are used to make the enzymes more stable, post-treatment convenient and reusable, for example, candida antarctica lipase B (Novozym 435) immobilized on macroporous adsorption resin and rhizomucor miehei lipase (Lipozyme RM IM) immobilized on ion exchange resin can be selected.

In the first step, the solvent is any one of acetone, acetonitrile, tetrahydrofuran, methyl tertiary butyl ether, dioxane, toluene and isopropyl ether or a mixture thereof. In various examples, acetone is used as solvent, so that the catalytic activity is higher, the conversion rate is more complete, the boiling point is lower, and the acetone is easy to distill and recycle, and therefore, the acetone is selected as the solvent, but the acetone is not limited to the acetone.

In the first step, the molar ratio of the added raw material succinic anhydride to the chloramphenicol can be 1-1.2, and the succinic anhydride and the chloramphenicol can completely react according to the theoretical amount of 1:1 molar ratio because of high activity and high selectivity of enzyme catalytic reaction, and the molar ratio of the succinic anhydride to the chloramphenicol is 1.1.

In the first step, the specific enzyme activity 10000U/g of the immobilized lipase is taken as a standard, the mass ratio of the added lipase to the chloramphenicol is 5-50%, and if recycling of the enzyme is not considered, the mass ratio is more preferably 10-20%, and most preferably 15%. The immobilized lipase catalytic reaction is carried out at the temperature of 0-60 ℃, and the enzyme is more stable and difficult to deactivate under the condition of lower temperature, but the catalytic activity is lower and the reaction is slower; at a higher temperature, the enzyme activity is higher, the reaction is quick, but the enzyme is unstable and easy to inactivate. Considering the stability, catalytic activity and energy consumption of lipase, the preferred reaction temperature is 40 ℃, and the esterification reaction between succinic anhydride and chloramphenicol can be completely converted within 8 hours under the condition of ensuring the addition amount of 15% of immobilized lipase.

The invention is the esterification reaction process between chloramphenicol and succinic anhydride catalyzed by lipase, which is one of hydrolytic enzymes and can catalyze the hydrolysis of ester, so that the system is required to be ensured to be anhydrous. In the process step (1), water absorbing agents such as molecular sieve, silica gel, bentonite, anhydrous sodium sulfate and the like can be used, a small amount of water brought by a solvent is removed, the hydrolysis reverse reaction is reduced to the maximum extent, the esterification reaction is promoted, and the reaction conversion rate is improved.

In process step (1), activated carbon decolorization methods are well known to those skilled in the art in accordance with the present invention. Specifically, the activated carbon can be in powder form, the mass of the activated carbon is 5% of the mass of the chloramphenicol raw material, and the activated carbon is heated, boiled and stirred for 10 minutes.

In the process step (1) according to the invention, the enzymatic reaction is carried out using a stirred tank reactor, or by shaking the reaction in a shaker, taking into account the possible deactivation of the enzyme by shear forces during stirring. In particular, for immobilized enzyme, the optimal method is to use an immobilized enzyme packed bed reactor, so that the production process of chloramphenicol succinate can be continuous, the inactivation of enzyme can be reduced to the maximum extent, and the recycling rate can be improved.

According to the present invention, in the step (2), a mixed solvent of a good solvent and a poor solvent in a certain ratio is used as a crystallization solvent in the purification of chloramphenicol succinate, and the good solvent may be any one of methanol, ethanol, acetone, or a mixed solvent thereof, and the poor solvent is water. In the examples, methanol/water is preferably mixed in a volume ratio of 1:1 to be used as a crystallization solvent, and the addition amount of the mixed solvent is 8 to 12 times, preferably 10 times, the mass of chloramphenicol succinate solid to be purified and crystallized.

According to the crystallization process of the step (2), methanol is firstly added under the condition of 20-40 ℃ to stir and dissolve a solid crude product of succinic chloramphenicol, then purified water is slowly added, the temperature is slowly reduced to 0-5 ℃, and finally the mixture is stood for 2-6 hours for crystallization.

In the examples, the dissolution temperature is preferably 30 ℃, the standing crystallization temperature is preferably 4 ℃, and the standing crystallization time is preferably 4 hours.

The technical solutions in the embodiments of the present invention will be clearly and completely described below in connection with the embodiments of the present invention.

The conversion in the examples was determined by high performance liquid chromatography: agilent 1200 liquid chromatograph, column: c182.1mm×250mm, mobile phase: water/acetonitrile=85/15, flow rate 0.5mL/min, detection wavelength: 280nm, column temperature: room temperature. Conversion = product peak area/(residual chloramphenicol peak area + product peak area) ×100%.

The lipases used in the examples are shown in Table 1.

Table 1: lipase species and their sources are listed.

(Code)	Enzymes	Source
			E01	Lipozyme RMIM	Rhizomucor miehei
E02	Lipozyme TLIM	Thermomyces lanuginosa
			E03	Novozyme 435	Candida Antarctica
E04	Lipase PS	Pseudomonas cepacia
			E05	Lipase AK	Pseudomonas fluorescens
E06	Lipase AYS	Candida rugosa
			E07	Lipase AS	Aspergillus niger
E08	IMMOZYME CALA	Candida Antarctica

Example 1

Enzyme screening assay: the reaction was stirred at room temperature for 4h with 0.32g (1 mmol) chloramphenicol, 0.11g (1.1 mmol) succinic anhydride, 0.065g lipase, 5mL acetonitrile, the reaction equation being shown in FIG. 1, the enzymes used and the corresponding conversions being shown in Table 2 below.

Table 2: enzyme used and conversion correspond to table.

Lipase

E01

E02

E03

E04

E05

E06

E07

E08

Conv.％

52％

38％

74％

25％

0

5％

12％

45％

Example 2

Solvent screening test: 32g (1 mmol) chloramphenicol, 0.11g (1.1 mmol) succinic anhydride, 0.065g lipase E03,5mL acetone, and the reaction was stirred at room temperature for 4h, the reaction equation is shown in FIG. 2, and the solvents used and the corresponding conversions are shown in Table 3 below.

Table 3: solvent used and corresponding conversion table.

Solvent

CH ₃ CN

toluene

acetone

THF

dioxane

MeOtBu

iPr ₂ O

Conv.％

74％

15％

80％

51％

32％

60％％

67％％

Example 3

Anhydrous test: 0.32g (1 mmol) chloramphenicol, 0.11g (1.1 mmol) succinic anhydride, 0.065g lipase E03, drying agent (5% of solvent mass) and 5mL acetone were stirred at room temperature for 4h, the reaction equation is shown in FIG. 3, and the drying agent used and the corresponding conversion are shown in Table 4 below.

Table 4: desiccant used and corresponding conversion table.

desiccant

Silica gel

4A M.S.

Na ₂ SO ₄

MgSO ₄

CuSO ₄

Na ₂ CO ₃

Conv.％

85％

88％

80％

76％

70％

82％％

Example 4

Temperature test: 0.32g (1 mmol) of chloramphenicol, 0.11g (1.1 mmol) of succinic anhydride, 0.065g of lipase E03,4A molecular sieve (5% of solvent mass) and 5mL of acetone, and stirring and reacting for 4h under a controlled temperature, wherein the reaction equation is shown in FIG. 4, and the used temperature and the corresponding conversion rate are shown in Table 5 below.

Table 5: the temperatures used and the corresponding conversion tables.

Temp.	30℃	35℃	40℃	45℃	50℃
						Conv.％	88％	90％	93％	94％	85％

Example 5

Enzyme addition assay: 0.32g (1 mmol) chloramphenicol, 0.11g (1.1 mmol) succinic anhydride, and various amounts of lipase E03,4A molecular sieves (5% of solvent mass) and 5mL acetone were added and reacted at 40℃for 8 hours, the reaction equation is shown in FIG. 5, and the amounts of enzyme used and the corresponding conversions are shown in Table 6 below.

Table 6: enzyme amounts used and corresponding conversion tables.

Example 6

Pilot test: 3.23g (10 mmol) of chloramphenicol, 1.1g (11 mmol) of succinic anhydride, 0.48g of lipase E03, 15 particles of 4A molecular sieve and 50mL of acetone are stirred and reacted for 8 hours at 40 ℃, the mixture is filtered, 0.2g of activated carbon is added into the filtrate, the mixture is heated and stirred for 10 minutes, the filtrate is filtered, and the solvent is removed from the filtrate under reduced pressure, so that a white solid product is obtained. Dissolving the crude product with 5mL of methanol at room temperature, slowly adding 5mL of purified water while stirring, slowly cooling to 4 ℃, standing at constant temperature for 6 hours, precipitating white needle-like crystals, filtering, and vacuum drying the obtained crystals at 50 ℃ for 2 hours to obtain 3.1g of chloramphenicol succinate with a yield of 73% and a purity of >99%.

Example 7

The embodiment provides a technology for synthesizing chloramphenicol succinate by enzyme catalysis, which comprises the following steps:

(1) The reaction process comprises the following steps: 32.3g (100 mmol) of chloramphenicol, 11g (110 mmol) of succinic anhydride, 4.8g of lipase, 150 grains of 4A molecular sieve and 500mL of acetone are stirred and reacted for 12 hours at 40 ℃, filtered, 2g of active carbon is added into the filtrate, heated and stirred for 10 minutes for decoloration, filtered, and the solvent is removed from the filtrate under reduced pressure, thus obtaining a white solid product.

(2) Refining: dissolving the crude product with 50mL of methanol at room temperature, slowly adding 50mL of purified water while stirring, slowly cooling to 4 ℃, standing at constant temperature for 8 hours, precipitating white needle-like crystals, filtering, and vacuum drying the obtained crystals at 50 ℃ for 2 hours to obtain 34.2g of pure chloramphenicol succinate with a yield of 79% and a purity of >99%.

The result of chromatographic analysis of chloramphenicol succinate prepared by the method of the present invention is shown in fig. 6, and as can be seen from fig. 6, the chloramphenicol succinate has higher purity.

It should be noted that the above-mentioned embodiments are to be understood as illustrative, and not limiting, the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made to the present invention without departing from its spirit or scope.

Claims

1. A process method for synthesizing chloramphenicol succinate by enzyme catalysis is characterized in that: the method comprises the following steps:

mixing chloramphenicol and succinic anhydride into a solvent, adding hydrolase, performing mixed reaction in water bath at 30-50deg.C, and stopping reaction when chloramphenicol conversion is detected; recovering the enzyme catalyst, adding active carbon into the reaction solution for decoloring, carrying out suction filtration, and carrying out reduced pressure distillation on the filtrate to remove the solvent to obtain a white solid product;

the molar ratio of the succinic anhydride to the chloramphenicol is 1-1.2:1, a step of; the hydrolase is candida antarctica lipase CALB, and the addition amount of the hydrolase is 10% -20% of the mass of chloramphenicol;

step two, refining:

adding a good solvent into the white solid product obtained in the step one, stirring at room temperature until the good solvent is dissolved, and then slowly adding a poor solvent; slowly cooling to 0-5 ℃, standing at constant temperature, precipitating white needle-like crystals, filtering, and vacuum drying the obtained crystals at 50 ℃ for 2 hours to obtain a refined product;

the good solvent is methanol; the poor solvent is water; the volume ratio of the good solvent to the poor solvent is 1:1, and the total addition amount of the good solvent and the poor solvent is 8-12 times of the mass of the white solid product;

in the first step, the solvent is acetone;

step one, adding a water absorbent into a reaction system before the mixing reaction; the water absorbing agent is a molecular sieve, and the addition amount of the water absorbing agent is 5% of the mass of the solvent.

2. The process according to claim 1, characterized in that: the hydrolase is an immobilized enzyme.

3. The process according to claim 1, characterized in that: in the first step, the water bath is controlled to be at a temperature of 40 ℃ for mixing reaction.

4. The process according to claim 1, characterized in that: in the first step, the mixing reaction is carried out in a kettle type stirring reactor, an enzyme filling fixed bed reactor or a shaking table.

5. The process according to claim 1, characterized in that: in the second step, the specific refining process is as follows: under the condition of 20-40 ℃, methanol is firstly added to stir and dissolve crude product of white solid product of succinic chloramphenicol, then purified water is slowly added, and then the temperature is slowly reduced to 0-5 ℃, and finally the mixture is kept stand for 2-6 hours for crystallization.