CN104293875B - The method that biological enzyme prepares (S) 2 chlorobenzene glycine methyl ester single enantiomer - Google Patents

Abstract

The invention discloses a method for preparing a single enantiomer of (S)-2-chlorophenylglycine methyl ester catalyzed by biological enzymes, comprising the following steps: adding racemic 2-chlorobenzene to a reactor with a constant temperature of 30°C in a water bath Glycine methyl ester and sodium phosphate buffer solution, after stirring, add penicillin G acylase, control the stirring speed to 100-500r/min, control the reaction time for 20-60 hours, keep stirring until the end of the reaction, and finally recover penicillin G acylase. The present invention is beneficial in that: using PGA as a catalyst, utilizing its activity, high selectivity and specificity to the substrate, reacting at normal temperature and normal pressure, one-step preparation of (S)-2-chlorophenylglycine methyl ester, which not only saves the protection and deprotection process of the reactive group in chlorophenylglycine, but also obtains a single enantiomer of (S)‑2‑chlorophenylglycine methyl ester with higher optical purity, and the enantiomeric ratio The value reaches more than 95%; the method of the invention is simple and easy to control, has low energy consumption and less emission in the preparation process, and is an environmentally friendly and green preparation method.

Description

Method for preparing single enantiomer of (S)-2-chlorophenylglycine methyl ester catalyzed by biological enzyme

technical field

The invention relates to a method for preparing a single enantiomer of (S)-2-chlorophenylglycine methyl ester, in particular to a method for preparing a single enantiomer of (S)-2-chlorophenylglycine methyl ester catalyzed by biological enzymes, which belongs to medical technology field.

Background technique

2-Chlorophenylglycine is a biologically active unnatural amino acid, and (S)-2-chlorophenylglycine methyl ester is the key chiral structural unit of the new drug clopidogrel for the treatment of thrombotic diseases. Clopidogrel is a safe and effective platelet aggregation inhibitor with remarkable efficacy, safety and strong tolerance, and has been widely used in clinical treatment.

Clopidogrel is a chiral drug, and its S-configuration molecule has strong platelet aggregation inhibitory activity, and its tolerance is 40 times that of the R-configuration, and the R-configuration will produce convulsions after administration, so clopidogrel Gray is marketed as a drug in the S-configuration. The direct synthesis of (S)-clopidogrel with the single enantiomer of (S)-2-chlorophenylglycine methyl ester not only saves the resolution process after the chiral reagent, but also improves the utilization rate of raw materials and the quality of the product. It is the most ideal way to produce clopidogrel chiral drug. The preparation of (S)-2-chlorophenylglycine methyl ester by chemical method is usually resolved with chiral reagents such as D-camphorsulfonic acid and L-tartaric acid (US6812363B2, 2002-10-15), but the required chiral The resolving agent is expensive, the product purity is low, the operation process is complicated, and the production efficiency is low.

Therefore, industrial production urgently needs to develop a high-efficiency, low-cost (S)-2-chlorophenylglycine methyl ester single enantiomer preparation technology.

Contents of the invention

In order to solve the deficiencies of the prior art, the object of the present invention is to provide a method for preparing (S)-2-chlorophenylglycine methyl ester single enantiomer with high efficiency and low cost by utilizing biological enzyme catalysis.

In order to achieve the above object, the present invention adopts the following technical solutions:

The method for preparing the single enantiomer of (S)-2-chlorophenylglycine methyl ester catalyzed by biological enzymes is characterized in that it comprises the following steps:

(1) Add racemic 2-chlorophenylglycine methyl ester and sodium phosphate buffer solution in a reactor with a constant temperature of 30°C in a water bath, the concentration of the aforementioned racemic 2-chlorophenylglycine methyl ester is 20-200g/L, the aforementioned The pH value of the sodium phosphate buffer solution is 6.0-9.0, and the concentration is 0.01-0.50mol/L;

(2) After stirring, add penicillin G acylase, the mass ratio of penicillin G acylase to racemic 2-chlorophenylglycine methyl ester is 1:1-100;

(3) Continue stirring to make the penicillin G acylase fully contact with racemic 2-chlorophenylglycine methyl ester, control the stirring speed to be 100-500r/min, control the reaction time for 20-60 hours, and continue stirring until the reaction ends;

(4) After the reaction is finished, the penicillin G acylase is recovered.

The aforementioned preparation method is characterized in that the concentration of the aforementioned racemic 2-chlorophenylglycine methyl ester is 50-150 g/L.

The aforementioned preparation method is characterized in that the pH value of the aforementioned sodium phosphate buffer solution is 7.0-8.0, and the concentration is 0.05-0.20 mol/L.

The aforementioned preparation method is characterized in that the aforementioned penicillin G acylase is commercial immobilized penicillin G acylase IPA750 or Eupergit C.

The aforementioned preparation method is characterized in that the mass ratio of the aforementioned penicillin G acylase to 2-chlorophenylglycine methyl ester is 1:10-50.

The aforementioned preparation method is characterized in that the stirring speed is controlled to be 200-400r/min.

The benefits of the present invention are:

1. The present invention adopts penicillin G acylase (Penicillin G acylase, EC 3.5.1.11, referred to as PGA) as a catalyst, utilizes the high activity, high selectivity and specificity to substrate of PGA, under normal temperature and normal pressure Carrying out the reaction to prepare (S)-2-chlorophenylglycine methyl ester in one step, which not only saves the protection and deprotection process of reactive groups in chlorophenylglycine, but also obtains (S)-2- Chlorphenylglycine methyl ester is a single enantiomer, and the enantiomer pair value is over 95%;

2. At present, penicillin G acylase has been able to be efficiently expressed and mass-produced with low cost. It catalyzes the asymmetric hydrolysis reaction of racemic 2-chlorophenylglycine methyl ester. The operation process is simple and easy to control, and the energy consumption of the preparation process is low. , less emission, and is an environmentally friendly and green preparation method.

detailed description

The method for the preparation of (S)-2-chlorophenylglycine methyl ester single enantiomer catalyzed by the biological enzyme of the present invention, its key lies in: the asymmetric hydrolysis of racemic 2-chlorophenylglycine methyl ester catalyzed by penicillin G acylase, namely :

Utilizing the high activity, high selectivity and specificity of PGA to the substrate, the hydrolysis reaction is carried out at normal temperature and normal pressure to prepare (S)-2-chlorophenylglycine methyl ester in one step, which saves the chlorophenylglycine. During the protection and deprotection process of the reactive group, a single enantiomer of (S)-2-chlorophenylglycine methyl ester with higher optical purity was obtained.

The present invention will be specifically introduced below in conjunction with specific embodiments.

Example 1

In reactor, add 5.0g racemic 2-chlorophenylglycine methyl ester and 50mL pH 7.0, 0.2mol/L sodium phosphate buffer solution, now the concentration of racemic 2-chlorophenylglycine methyl ester is 100g/L, Then add 0.1 g of immobilized penicillin G acylase IPA750. At this time, the mass ratio of immobilized penicillin G acylase to 2-chlorophenylglycine methyl ester is 1:50. The temperature of the water bath is controlled at 30°C and the speed is controlled at 300r. /min, reacted for 30 hours, and finally separated the immobilized enzyme.

The reactor can adopt the batch reactor commonly used in industry.

Example 2

In reactor, add 5.0g racemic 2-chlorophenylglycine methyl ester and 50mL pH 7.0, 0.2mol/L sodium phosphate buffer solution, now the concentration of racemic 2-chlorophenylglycine methyl ester is 100g/L, Then add 0.2 g of immobilized penicillin G acylase IPA750, at this time the mass ratio of immobilized penicillin G acylase to 2-chlorophenylglycine methyl ester is 2:50, the temperature of the water bath is controlled at 30°C, and the control speed is 300r /min, reacted for 20 hours, and finally separated the immobilized enzyme.

Example 3

Add 5.0g racemic 2-chlorophenylglycine methyl ester and 50mL pH 7.0, 0.01mol/L sodium phosphate buffer solution in reactor, the concentration of racemic 2-chlorophenylglycine methyl ester is 100g/L now, Then add 0.1 g of immobilized penicillin G acylase IPA750. At this time, the mass ratio of immobilized penicillin G acylase to 2-chlorophenylglycine methyl ester is 1:50. The temperature of the water bath is controlled at 30°C and the speed is controlled at 300r. /min, reacted for 30 hours, and finally separated the immobilized enzyme.

Example 4

In reactor, add 5.0g racemic 2-chlorophenylglycine methyl ester and 50mL pH 8.0, 0.05mol/L sodium phosphate buffer solution, now the concentration of racemic 2-chlorophenylglycine methyl ester is 100g/L, Then add 0.1 g of immobilized penicillin G acylase IPA750. At this time, the mass ratio of immobilized penicillin G acylase to 2-chlorophenylglycine methyl ester is 1:50. The temperature of the water bath is controlled at 30°C and the speed is controlled at 300r. /min, reacted for 30 hours, and finally separated the immobilized enzyme.

Example 5

In reactor, add 2.5g racemic 2-chlorophenylglycine methyl ester and 50mL pH 7.0, 0.2mol/L sodium phosphate buffer solution, now the concentration of racemic 2-chlorophenylglycine methyl ester is 50g/L, Then add 0.1 g of immobilized penicillin G acylase Eupergit C. At this time, the mass ratio of immobilized penicillin G acylase to 2-chlorophenylglycine methyl ester is 1:25. 200r/min, react for 30 hours, and finally separate the immobilized enzyme.

Example 6

In reactor, add 2.5g racemic 2-chlorophenylglycine methyl ester and 50mL pH 7.0, 0.1mol/L sodium phosphate buffer solution, now the concentration of racemic 2-chlorophenylglycine methyl ester is 50g/L, Then add 0.1 g of immobilized penicillin G acylase Eupergit C. At this time, the mass ratio of immobilized penicillin G acylase to 2-chlorophenylglycine methyl ester is 1:25. 300r/min, react for 30 hours, and finally separate the immobilized enzyme.

Example 7

Add 5.0g racemic 2-chlorophenylglycine methyl ester and 50mL pH 7.0, 0.1mol/L sodium phosphate buffer solution in reactor, the concentration of racemic 2-chlorophenylglycine methyl ester is 100g/L now, Then add 0.1 g of immobilized penicillin G acylase Eupergit C. At this time, the mass ratio of immobilized penicillin G acylase to 2-chlorophenylglycine methyl ester is 1:50. 400r/min, react for 40 hours, and finally separate the immobilized enzyme.

Example 8

Add 1.0g racemic 2-chlorophenylglycine methyl ester and 50mL pH 8.0, 0.01mol/L sodium phosphate buffer solution in reactor, the concentration of racemic 2-chlorophenylglycine methyl ester is 20g/L now, Then add 0.01g of immobilized penicillin G acylase IPA750. At this time, the mass ratio of immobilized penicillin G acylase to 2-chlorophenylglycine methyl ester is 1:100. The temperature of the water bath is controlled at 30°C, and the control speed is 300r. /min, reacted for 30 hours, and finally separated the immobilized enzyme.

Example 9

In reactor, add 10.0g racemic 2-chlorophenylglycine methyl ester and 50mL pH 8.0, 0.05mol/L sodium phosphate buffer solution, now the concentration of racemic 2-chlorophenylglycine methyl ester is 200g/L, Then add 10.0 g of immobilized penicillin G acylase Eupergit C. At this time, the mass ratio of immobilized penicillin G acylase to 2-chlorophenylglycine methyl ester is 1:1. 300r/min, react for 60 hours, and finally separate the immobilized enzyme.

Example 10

Add 7.5g racemic 2-chlorophenylglycine methyl ester and 50mL pH 8.0, 0.01mol/L sodium phosphate buffer solution in reactor, the concentration of racemic 2-chlorophenylglycine methyl ester is 150g/L now, Then add 0.75g of immobilized penicillin G acylase IPA750. At this time, the mass ratio of immobilized penicillin G acylase to 2-chlorophenylglycine methyl ester is 1:10. The temperature of the water bath is controlled at 30°C, and the control speed is 300r. /min, reacted for 50 hours, and finally separated the immobilized enzyme.

It should be noted that, in the preparation method of the present invention:

(1) The pH value of sodium phosphate buffer solution can be adjusted in the scope of 6.0-9.0, and preferred pH value is 7.0-8.0, and the concentration of sodium phosphate buffer solution can be adjusted in the scope of 0.01-0.50mol/L, and preferred concentration is 0.05-0.20mol/L, penicillin G acylase has higher catalytic activity within the above range;

(2) According to the needs of the actual production process, the stirring speed can be adjusted within the range of 100-500r/min.

(S)-2-chlorophenylglycine methyl ester and (R)-2-chlorophenylglycine in the reaction solution are quantitatively analyzed by high-performance liquid chromatography equipped with imported chiral columns, and (S)-2-chlorophenylglycine The enantiomeric excess values of methyl ester and (R)-2-chlorophenylglycine are represented by ee _s (%) and ee _p (%) respectively, and the total conversion rate of the reaction is represented by C (%), which is calculated as follows:

ee _s = (c _SS –c _RS )/(c _SS +c _RS )

ee _p = (c _RP -c _SP )/(c _RP +c _SP )

C(%)＝ee _s /(ee _s +ee _p )

In the formula, c _SS is the concentration of (S)-2-chlorophenylglycine methyl ester, mol/L;

c _RS is the concentration of (R)-2-chlorophenylglycine methyl ester, mol/L;

c _RP is the concentration of (R)-2-chlorophenylglycine, mol/L;

c _SP is the concentration of (S)-2-chlorophenylglycine, mol/L.

The single enantiomer of (S)-2-chlorophenylglycine methyl ester prepared by the existing method generally has an optical purity of 70-90%.

The optical purity of the single enantiomer of (S)-2-chlorophenylglycine methyl ester prepared by the method of the present invention is shown in Table 1.

Table 1 (S) - Optical purity of the single enantiomer of 2-chlorophenylglycine methyl ester

group enantiomeric excess value ee _s Conversion rate C Example 1 96.5% 69.2% Example 2 95.3% 69.9% Example 3 97.1% 70.9% Example 4 95.1% 70.2% Example 5 95.8% 72.0% Example 6 95.5% 69.4% Example 7 95.1% 68.4% Example 8 96.1% 70.5% Example 9 95.0% 66.1% Example 10 95.3% 68.0%

It can be seen that the present invention uses penicillin G acylase to catalyze racemic 2-chlorophenylglycine methyl ester to prepare (S)-2-chlorophenylglycine methyl ester, and the outstanding advantage is that the (S)-2-chlorophenylglycine methyl ester obtained is The single enantiomer of glycine methyl ester has a higher purity, and the ee _s can reach more than 95%.

In addition, it can also be seen from the above examples that the method of the present invention has the characteristics of simple and convenient preparation process, mild reaction conditions and low cost, and is very suitable for industrial production.

It should be noted that the above embodiments do not limit the present invention in any form, and all technical solutions obtained by means of equivalent replacement or equivalent transformation fall within the protection scope of the present invention.

Claims (5)

1. The method for preparing (S)-2-chlorophenylglycine methyl ester single enantiomer by biological enzyme catalysis, is characterized in that, comprises the following steps: (1) Add racemic 2-chlorophenylglycine methyl ester and sodium phosphate buffer solution in a reactor with a constant temperature of 30°C in a water bath, the concentration of said racemic 2-chlorophenylglycine methyl ester is 20-200g/L, The pH value of the sodium phosphate buffer solution is 6.0-9.0, and the concentration is 0.01-0.50mol/L; (2) add penicillin G acylase after stirring, and described penicillin G acylase is commercial immobilized penicillin G acylase IPA750 or Eupergit C, the mixture of penicillin G acylase and racemic 2-chlorophenylglycine methyl ester The mass ratio is 1:1~100; (3) Continue stirring to make the penicillin G acylase fully contact with racemic 2-chlorophenylglycine methyl ester, control the stirring speed to be 100-500r/min, control the reaction time for 20-60 hours, and continue stirring until the reaction ends; (4) After the reaction is finished, the penicillin G acylase is recovered. 2. The preparation method according to claim 1, characterized in that, the concentration of said racemic 2-chlorophenylglycine methyl ester is 50-150g/L. 3. The preparation method according to claim 1, characterized in that, the pH value of the sodium phosphate buffer solution is 7.0-8.0, and the concentration is 0.05-0.20mol/L. 4. The preparation method according to claim 1, characterized in that the mass ratio of the penicillin G acylase to 2-chlorophenylglycine methyl ester is 1:10-50. 5. The preparation method according to claim 1, characterized in that the controlled stirring speed is 200-400r/min.