阅读说明：本技术 一种酶催化合成琥珀酸氯霉素的工艺方法 (Process method for synthesizing chloramphenicol succinate through enzyme catalysis ) 是由 胡惜朝 李自永 邓萌 范艳利 周双双 于 2020-12-28 设计创作，主要内容包括：本发明涉及一种酶催化合成琥珀酸氯霉素的工艺方法,属于生物医药技术领域,所述工艺方法以氯霉素为原料,使氯霉素与琥珀酸酐在水解酶的作用下,在溶剂中适宜条件下发生反应,完全转化后经简单过滤、脱色、重结晶即得高纯度琥珀酸氯霉素。所述的水解酶为蛋白酶、脂肪酶、脂肪酶、酰基转移酶中的任一种或多种组合,其中酶可以是天然来源或通过基因工程技术重组获得。本发明采用的酶催化工艺与已有的工艺相比,更方便、安全,污染更小,质量更高,成本更低,更适合琥珀酸氯霉素的大规模工业化生产。(The invention relates to a process method for synthesizing chloramphenicol succinate through enzyme catalysis, which belongs to the technical field of biological medicine. The hydrolase is any one or combination of protease, lipase and acyltransferase, wherein the enzyme can be natural source or obtained by recombination through genetic engineering technology. Compared with the existing process, the enzyme catalysis process adopted by the invention is more convenient and safer, has less pollution, higher quality and lower cost, and is more suitable for large-scale industrial production of chloramphenicol succinate.)

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

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

dissolving chloramphenicol and succinic anhydride in a solvent, adding hydrolase, controlling the temperature in a water bath at 0-60 ℃ for mixing reaction, and stopping the reaction when chloramphenicol conversion is completed; recovering enzyme catalyst, decolorizing the reaction solution with activated carbon, vacuum filtering, and distilling the filtrate under reduced pressure to remove solvent to obtain white solid product;

the molar ratio of the succinic anhydride to the chloramphenicol is 1-1.2: 1; 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 the 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 formed by mixing 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, separating out white needle crystals, filtering, and drying the obtained crystals for 2 hours in vacuum at 50 ℃ to obtain a refined product;

the good solvent is any one or more 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 adding amount of the good solvent and the poor solvent is 8-12 times of the mass of the white solid product.

2. The process according to claim 1, characterized in that: in the first step, the solvent is any one or a mixture of more of acetone, acetonitrile, tetrahydrofuran, methyl tert-butyl ether, dioxane, toluene and isopropyl ether.

3. The process according to claim 1, characterized in that: in the first step, the lipase is one or more of thermophilic fungi, rhizopus oryzae, candida antarctica, candida rugosa, pseudomonas cepacia, pseudomonas fluorescens, aspergillus niger, bacillus subtilis and rhizopus oryzae.

4. A process according to claim 3, characterized in that: the lipase is candida antarctica lipase CALB or rhizomucor miehei lipase RML.

5. A process according to any one of claims 1, 3 or 4, characterized in that: the hydrolase is immobilized enzyme.

6. The process of claim 5, wherein: in the first step, the adding amount of the hydrolase is 10-20% of the mass of the chloramphenicol.

7. The process according to claim 1, characterized in that: in the first step, the temperature of the water bath is controlled to be 40 ℃ to carry out mixing reaction.

8. 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 filled fixed bed reactor or a shaking table.

9. The process according to claim 1, characterized in that: step one, adding a water absorbent into a reaction system before the mixing reaction; the water absorbent is molecular sieve, silica gel, diatomite or anhydrous sodium sulfate, and the addition amount of the water absorbent is 5% of the mass of the solvent.

10. The process according to claim 1, characterized in that: in the second step, the refining process comprises the following specific steps: under the condition of 20-40 ℃, firstly adding methanol, stirring and dissolving a white solid product crude product of chloramphenicol succinate, then slowly adding purified water, slowly cooling to 0-5 ℃, and finally standing for 2-6 hours for crystallization.

Technical Field

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

Background

Chloramphenicol (Chloramphenicol) is an amidoalcohol wide spectrum antibiotic found in Streptomyces venezuelae. It has inhibitory effect on gram-positive and gram-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 in clinic, and is also effective for pertussis, trachoma, bacillary dysentery, urinary tract infection and the like. The chloramphenicol antibiotic is still a first-line clinical medicine because of its large use, low price and easy large-scale synthesis and preparation.

However, chloramphenicol has serious adverse effects of inhibiting bone marrow hemopoiesis, and can reduce various blood cells of human body, even produce irreversible aplastic anemia, thereby limiting its expanded use. Therefore, people modify the structure of chloramphenicol, so as to reduce toxic and side effects while maintaining drug effects, wherein chloramphenicol succinate is a more successful chloramphenicol derivative.

Chloramphenicol Succinate (Chloramphenicol Succinate) is named as D-threo- (-) -N-alpha- (hydroxymethyl) -beta-hydroxy-p-nitrophenylethyl-2, 2-dichloroacetamide-alpha-Succinate (DSA), can be rapidly hydrolyzed in vivo to free Chloramphenicol for generating effect, has the same or better drug effect as Chloramphenicol, and has greatly reduced side effect compared with Chloramphenicol. The classical preparation method is prepared by reacting chloramphenicol and succinic anhydride under the catalysis of organic base, and related chemical synthesis methods in patents, papers and other documents are reported more, but the traditional chemical synthesis methods have the disadvantages of more raw material consumption, lower yield, high energy consumption in the reaction process, high toxicity of reaction reagents and environmental pollution, and the activity of the used traditional chemical catalyst is lower, so that the reaction selectivity is poor, more byproducts are generated, the yield is not high, the product quality is lower, and the post-treatment refining is difficult. These disadvantages limit the industrial application of the process.

In summary, there is a need to develop a method for preparing chloramphenicol succinate with lower cost, more convenient operation, more safety and higher efficiency.

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 chloramphenicol and succinic anhydride to carry out esterification reaction to generate chloramphenicol succinate, has higher production efficiency and product quality, and has the advantages of convenient operation, low cost, environmental friendliness, safety, high efficiency and the like.

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

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

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

dissolving chloramphenicol and succinic anhydride in a solvent, adding hydrolase, controlling the temperature in a water bath at 0-60 ℃ for mixing reaction, and stopping the reaction when chloramphenicol conversion is completed; recovering enzyme catalyst, decolorizing the reaction solution with activated carbon, vacuum filtering, and distilling the filtrate under reduced pressure to remove solvent to obtain white solid product;

the molar ratio of the succinic anhydride to the chloramphenicol is 1-1.2: 1; 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 the 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, separating out white needle crystals, filtering, and drying the obtained crystals for 2 hours in vacuum at 50 ℃ to obtain a refined product;

the good solvent is any one or more 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 adding 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 tert-butyl ether, dioxane, toluene and isopropyl ether, and acetone is preferred.

In the first step, the lipase is one or more of thermophilic fungi, rhizopus oryzae, 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 adding amount of the hydrolase is 10-20% of the mass of the chloramphenicol.

In the first step, the temperature of the water bath is controlled to be 40 ℃ to carry out mixing reaction.

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

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

In the second step, the refining process comprises the following specific steps: under the condition of 20-40 ℃, firstly adding methanol, stirring and dissolving a white solid product crude product of chloramphenicol succinate, then slowly adding purified water, slowly cooling to 0-5 ℃, and finally standing for 2-6 hours for crystallization.

Has the advantages that:

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

Drawings

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

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

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

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

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

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

Detailed Description

A process method for synthesizing chloramphenicol succinate through enzyme catalysis comprises the following steps:

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

dissolving chloramphenicol and succinic anhydride in a certain proportion in a solvent, adding hydrolase, performing water bath temperature control mixing reaction, and stopping the reaction when chloramphenicol conversion is completed; recovering enzyme catalyst, decolorizing the reaction solution with activated carbon, vacuum filtering, and distilling the filtrate under reduced pressure to remove solvent to obtain white solid product;

step two, refining:

and (3) adding a good solvent into the white solid product obtained in the step one, stirring at room temperature until the good solvent is dissolved, then slowly adding a poor solvent until the product is saturated, slowly cooling to 4 ℃, standing at constant temperature, separating out white needle crystals, filtering, and drying the obtained crystals for 2 hours in vacuum at 50 ℃ 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), Rhizomucor miehei (Rhizomucor miehei), Candida Antarctica (Candida Antarctica), Candida rugosa (Candida rugosa), Pseudomonas cepacia (Pseudomonas cepacia), Pseudomonas fluorescens (Pseudomonas fluorescens), Aspergillus niger (Aspergillus niger), Bacillus subtilis (Bacillus subtilis), and 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.

The hydrolase used according to the invention is not limited to natural sources, but also includes enzymes which are recombined by molecular microbiology. The form of the enzyme to be used is not limited, and may be either dry powder or immobilized, and since the immobilized enzyme is used, the subsequent treatment is easy and the enzyme can be recovered and reused, the hydrolase is particularly preferably an immobilized enzyme. Among the above numerous lipases, we found that lipases derived from Candida Antarctica and Rhizomucor miehei have better activity for the selective esterification of chloramphenicol and succinic anhydride. Immobilized enzymes are generally used to make the enzymes more stable, convenient and reusable for post-treatment, and for example, Candida antarctica lipase B (Novozym 435) immobilized with macroporous adsorption resin and Mucor miehei lipase (Lipozyme RM IM) immobilized with ion exchange resin may be selected.

In the first step, the solvent is any one of acetone, acetonitrile, tetrahydrofuran, methyl tert-butyl ether, dioxane, toluene and isopropyl ether or a mixture thereof. In many cases, acetone is used as the solvent, so that the catalytic activity is higher, the conversion rate is more complete, the boiling point is lower, and the distillation recovery is easy, so that acetone is selected as the solvent, but the acetone is not limited.

In the first step, the molar ratio of the added raw material succinic anhydride to chloramphenicol can be 1-1.2, the enzymatic reaction has high activity and high selectivity and few side reactions, the succinic anhydride and chloramphenicol can completely react according to the molar ratio of 1:1 in a theoretical amount, and the raw material succinic anhydride with lower price is slightly excessive in the actual process, wherein the preferred molar ratio is 1.1.

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

The invention relates to an esterification reaction process between chloramphenicol and succinic anhydride catalyzed by lipase, the lipase is one of hydrolytic enzymes and can catalyze ester hydrolysis, so the system is required to be ensured to be anhydrous. In the process step (1), molecular sieves, silica gel, diatomite, anhydrous sodium sulfate and other water absorbents can be used for removing a small amount of water brought by a solvent, so that 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) according to the invention, the decolorization process by activated carbon is known to the person skilled in the art. Specifically, the activated carbon can be powder, 5% of the mass of the chloramphenicol raw material is added, and the mixture is heated to boil and stirred for 10 minutes.

According to the present invention, in the process step (1), the enzyme-catalyzed reaction process generally employs a tank-type stirred reactor, and the reaction may be carried out by shaking in a shaker, considering that the shearing force during the stirring may inactivate the enzyme. Especially 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 the enzyme can be reduced to the maximum extent, and the recycling rate can be improved.

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

According to the crystallization process in the step (2), methanol is firstly added and stirred to dissolve the chloramphenicol succinate solid crude product at the temperature of 20-40 ℃, then purified water is slowly added, the temperature is slowly reduced to 0-5 ℃, and finally standing is carried out 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 with reference to 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: and (4) room temperature. Conversion was 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 types and their sources.

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

Table 2: the enzymes used and their conversion correspond to the 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 (1mmol) of chloramphenicol, 0.11g (1.1mmol) of succinic anhydride, 0.065g of lipase E03, 5mL of acetone, and the reaction was stirred at room temperature for 4 hours, the reaction equation is shown in FIG. 2, and the solvents used and the corresponding conversions are shown in Table 3 below.

Table 3: the solvents used and the corresponding transformation tables.

Solvent

CH3CN

toluene

acetone

THF

dioxane

MeOtBu

iPr2O

Conv.％

74％

15％

80％

51％

32％

60％％

67％％

Example 3

Anhydrous test: 0.32g (1mmol) of chloramphenicol, 0.11g (1.1mmol) of succinic anhydride, 0.065g of lipase E03, a drying agent (5% by mass of the solvent) and 5mL of acetone were stirred at room temperature for 4 hours, 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: the desiccant used and the corresponding conversion table.

desiccant

Silica gel

4A M.S.

Na2SO4

MgSO4

CuSO4

Na2CO3

Conv.％

85％

88％

80％

76％

70％

82％％

Example 4

Temperature test: 0.32g (1mmol) of chloramphenicol, 0.11g (1.1mmol) of succinic anhydride, 0.065g of lipase E03, 4A molecular sieve (5% of the mass of the solvent) and 5mL of acetone were reacted for 4 hours with temperature-controlled stirring, the reaction equation is shown in FIG. 4, and the temperatures used and the corresponding conversions 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 test: 0.32g (1mmol) of chloramphenicol and 0.11g (1.1mmol) of succinic anhydride were added to the mixture, and the mixture was reacted at 40 ℃ for 8 hours by adding various amounts of lipase E03, 4A molecular sieve (5% by mass of the solvent) and 5mL of acetone, the reaction equation is shown in FIG. 5, and the amounts of the enzymes used and the corresponding conversions are shown in Table 6 below.

Table 6: the amount of enzyme used and the corresponding conversion table.

Example 6

Small test experiment: 3.23g (10mmol) of chloramphenicol, 1.1g (11mmol) of succinic anhydride, 0.48g of lipase E03, 15 particles of 4A molecular sieve and 50mL of acetone, stirring and reacting at 40 ℃ for 8h, filtering, adding 0.2g of activated carbon into the filtrate, heating and stirring for 10min, filtering, and removing the solvent from the filtrate under reduced pressure to obtain a white solid product. And (3) adding 5mL of methanol into the crude product at room temperature for dissolving, slowly adding 5mL of purified water while stirring, slowly cooling to 4 ℃, standing at a constant temperature for 6h, separating out white needle-shaped crystals, filtering, and drying the obtained crystals in vacuum at 50 ℃ for 2h to obtain 3.1g of chloramphenicol succinate pure product, wherein the yield is 73%, and the purity is more than 99%.

Example 7

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

(1) the reaction process is as follows: 32.3g (100mmol) of chloramphenicol, 11g (110mmol) of succinic anhydride, 4.8g of lipase, 150 particles of 4A molecular sieve and 500mL of acetone are stirred and reacted for 12h at 40 ℃, the mixture is filtered, 2g of activated carbon is added into the filtrate, the mixture is heated and stirred for 10min for decolorization, the filtrate is filtered, and the solvent is removed from the filtrate under reduced pressure to obtain a white solid product.

(2) And (3) refining: and (3) adding 50mL of methanol into the crude product at room temperature for dissolving, slowly adding 50mL of purified water while stirring, slowly cooling to 4 ℃, standing at constant temperature for 8h, separating out white needle-shaped crystals, filtering, and drying the obtained crystals at 50 ℃ in vacuum for 2h to obtain 34.2g of the chloramphenicol succinate pure product, wherein the yield is 79%, and the purity is more than 99%.

The chromatographic analysis of chloramphenicol succinate prepared by the method of the present invention showed that the purity of the chloramphenicol succinate was high as shown in fig. 6, as can be seen from fig. 6.

It should be noted that the above-mentioned embodiments illustrate rather than limit the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that certain insubstantial modifications and adaptations of the present invention can be made without departing from the spirit and scope of the invention.