CN105907829B

CN105907829B - Method for preparing N-bromoacetyl-7-aminocephalosporanic acid through enzyme catalysis

Info

Publication number: CN105907829B
Application number: CN201610392858.6A
Authority: CN
Inventors: 李宁; 张晓利; 宗敏华; 罗春
Original assignee: White Cloud Mountain Chemical Pharmaceutical Factory Of Guangzhou Baiyunshan Pharmaceutical Group Co Ltd; South China University of Technology SCUT
Current assignee: White Cloud Mountain Chemical Pharmaceutical Factory Of Guangzhou Baiyunshan Pharmaceutical Group Co Ltd; South China University of Technology SCUT
Priority date: 2016-06-06
Filing date: 2016-06-06
Publication date: 2020-02-18
Anticipated expiration: 2036-06-06
Also published as: CN105907829A

Abstract

The invention belongs to the field of enzyme catalysis and biological pharmacy, and discloses a method for preparing N-bromoacetyl-7-aminocephalosporanic acid by enzyme catalysis, which comprises the following steps: adding 7-aminocephalosporanic acid and acyl donor into buffer solution, mixing uniformly, adding immobilized penicillin acylase for reaction, and separating after reaction to obtain N-bromoacetyl-7-aminocephalosporanic acid. The method has the advantages of mild reaction conditions, environmental friendliness, simple process, high yield of the target product and the like.

Description

Method for preparing N-bromoacetyl-7-aminocephalosporanic acid through enzyme catalysis

Technical Field

The invention belongs to the field of enzyme catalysis and biological pharmacy, and particularly relates to a method for preparing N-bromoacetyl-7-aminocephalosporanic acid by catalysis of penicillin acylase.

Background

Cefathiamidine is the first semisynthetic cephalosporin which is successfully researched and developed independently in China. Cefathiamidine is a broad-spectrum antibacterial agent, has strong killing effect on most gram-positive bacteria and part gram-negative bacteria, and especially has better killing effect on enterococcus than similar species used clinically. In addition, cefathiamidine also has certain curative effect on methicillin-resistant staphylococcus aureus and methicillin-resistant staphylococcus epidermidis infection (China J.antibiotics, 2005,30(2): 107). Clinical research shows that the cefathiamidine has the characteristics of high blood concentration, good clinical curative effect and the like. In recent years, the market for cefathiamidine is steadily increasing. However, cefathiamidine is still produced by chemical method in industry, which comprises two steps: (1) taking 7-aminocephalosporanic acid (7-ACA) as a raw material, adding high-activity bromoacetyl bromide at low temperature to prepare N-bromoacetyl-7-ACA; (2) N-bromoacetyl-7-ACA is condensed with N, N' -diisopropylthiourea to obtain cefathiamidine (Chinese journal of pharmaceutical industry, 1978,9(4): 1). Or chloroacetyl chloride is used for replacing bromoacetyl bromide to prepare cefathiamidine (China medicine industry journal, 2009,40(12): 888; CN 102285999B; CN 101486718B) through similar chemical acylation and condensation processes, but the activity of chlorine in chloroacetyl is low, so that the yield of the second-step condensation reaction is low, and therefore, in industry, bromoacetyl bromide is still used as an acyl donor in the first step of cefathiamidine synthesis. Another method for synthesizing cefathiamidine is that 1, 3-diisopropyl amidino-2-thio-acetate acid salt with side chain is synthesized, then activated by Vilsmeier reagent, finally condensed with 7-ACA with protective group, and deprotected to obtain cefathiamidine (Chinese journal of pharmaceutical chemistry, 2001,11(5): 293; CN 101704827B); although the yield of the target product of the synthetic route is relatively high (56%), the operation is complicated, the conditions are harsh, and the used silanization reagent is expensive and has higher cost; in addition, highly toxic active reagents such as triphosgene and triphenylphosphine oxide are needed in the activation process, and highly toxic reagents such as toluene, xylene, chlorobenzene, dichlorobenzene and the like are needed. Therefore, the chemical method for preparing cefathiamidine not only needs to use high-activity acyl donor (acyl chloride or acyl bromide), volatile organic solvent and alkali, but also needs low-temperature reaction, and has the disadvantages of harsh reaction conditions, complex process and serious environmental pollution, thus not meeting the requirements of the current sustainable society and environmental development. In addition, the chemical method for preparing the key intermediate of cefathiamidine, namely N-bromoacetyl-7-ACA, has undesirable yield which is only 73 percent at most (Chinese modern application pharmacy, 201027 (2): 126).

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a green, simple and efficient method for preparing N-bromoacetyl-7-ACA through enzyme catalysis.

The invention adopts the following technical scheme:

a method for preparing N-bromoacetyl-7-aminocephalosporanic acid (key intermediate of cefathiamidine) through enzyme catalysis comprises the following steps: adding 7-ACA and acyl donor into buffer solution, mixing, adding immobilized penicillin acylase, reacting, and separating to obtain N-bromoacetyl-7-ACA.

The acyl donor is bromoacetic acid alkyl ester or bromoacetamide.

The alkyl chain length of the bromoacetic acid alkyl ester is C1-C6.

The alkyl bromoacetate is methyl bromoacetate or ethyl bromoacetate.

The concentration of the 7-ACA is 20 mM-70 mM.

The molar ratio of the 7-ACA to the acyl donor is 1: 1-1: 6.

The reaction conditions are that the temperature is 10-40 ℃, the reaction time is 1-24 h, and the oscillation speed is 150-300 r/min.

The dosage of the immobilized penicillin acylase is 2U/mL-5U/mL.

The immobilized penicillin acylase is penicillin G acylase II, penicillin G acylase IV, penicillin acylase SIPA-III, penicillin acylase SIPA-IV, penicillin acylase SIPA-V or penicillin acylase PGA-750.

Preferably, the molar ratio of the 7-ACA to the acyl donor is 1: 2-1: 3; the immobilized penicillin acylase is penicillin acylase PGA-750; the dosage of the enzyme is 3U/mL-5U/mL; the reaction time is 1-5 h.

The buffer solution is phosphate buffer solution or Tris-HCl buffer solution, and the pH value is 5.0-9.0.

And the target product separation method comprises the steps of filtering to remove enzyme after the reaction is finished, adding hydrochloric acid into the filtrate to adjust the pH value to be 0.5-2.0, filtering, washing and drying to obtain the N-bromoacetyl-7-ACA.

Compared with the prior art, the invention has the following advantages:

1) the N-bromoacetyl-7-ACA is prepared by adopting a high-efficiency biocatalyst, namely penicillin acylase.

The enzyme is used as a natural biocatalyst and can effectively reduce the reaction activation energy, so that only bromoacetic acid alkyl ester or bromoacetamide with moderate activity is used as an acyl donor, and high-activity bromoacetyl bromide or chloroacetyl chloride is not used, thereby greatly improving the reaction safety.

2) The enzyme is a biological macromolecule easy to biodegrade, and overcomes the disadvantage that a chemical catalyst is not environment-friendly.

3) The reaction process is simple and easy to control, and the cheap and easily-obtained environment-friendly aqueous solution is used as a reaction medium without using an organic solvent.

4) The immobilized penicillin acylase is used as a catalyst, and the immobilized enzyme can be recovered by simple filtration after the reaction is finished, so that the repeated utilization of the biocatalyst can be realized, and the separation and purification of the product are easy.

5) The invention has mild reaction conditions, and the cephalosporin nucleus 7-ACA is not easy to decompose in the reaction process, so the product quality is high.

Drawings

FIG. 1 is a liquid chromatogram before reaction.

FIG. 2 is a liquid chromatogram after the reaction.

FIG. 3 is a liquid chromatogram of N-bromoacetyl-7-ACA standard.

Detailed Description

The invention is further illustrated by the examples.

Example 1

10mL of phosphate buffer (100mM, pH 7.5), 40mM7-ACA and 120mM methyl bromoacetate were added to a 50mL triangular flask with a cover, and mixed well, followed by addition of 30U of penicillin G acylase II (available from Shiyao corporation) and reaction at 20 ℃ and 200 r/min. After 11h, liquid chromatography analysis showed that the yield of N-bromoacetyl-7-ACA was 34%.

Example 2

10mL of phosphate buffer (100mM, pH 7.5), 40mM7-ACA and 120mM methyl bromoacetate were added to a 50mL triangular flask with a cover, and mixed well, followed by addition of 30U of penicillin G acylase IV (available from Shiyao corporation) and reaction at 20 ℃ and 200 r/min. After 7h, the liquid chromatography analysis showed that the yield of N-bromoacetyl-7-ACA was 32%.

Example 3

10mL of phosphate buffer (100mM, pH 7.5), 40mM7-ACA and 120mM methyl bromoacetate were added to a 50mL Erlenmeyer flask with a lid, and mixed well, followed by addition of 30U of penicillin acylase PGA-750 (available from Shunharder Co., Ltd., Zhejiang) and reaction at 20 ℃ and 200 r/min. After 3h, the liquid chromatography analysis showed that the yield of N-bromoacetyl-7-ACA was 91%. The liquid chromatogram before and after the reaction are shown in figure 1 and figure 2 respectively, and the retention time of 7-ACA and N-bromoacetyl-7-ACA is 2.58 min and 4.10min respectively. Then, filtering to remove enzyme, adding hydrochloric acid into the filtrate to adjust the pH value to 1.0, filtering, washing and drying to obtain the N-bromoacetyl-7-ACA with the yield of 88%.

Example 4

10mL of phosphate buffer (100mM, pH 7.5), 40mM7-ACA and 120mM methyl bromoacetate were added to a 50mL triangular flask with a cover, mixed well, followed by addition of 30U of penicillin acylase SIPA-III (available from Hunan Fulaige Biotech Co., Ltd.) and reaction at 20 ℃ and 200 r/min. After 11h, the liquid chromatography analysis showed that the yield of N-bromoacetyl-7-ACA was 32%.

Example 5

10mL of phosphate buffer (100mM, pH 7.5), 40mM7-ACA and 120mM methyl bromoacetate were added to a 50mL triangular flask with a cover, and mixed well, followed by addition of 30U of penicillin acylase SIPA-IV (available from Hunan Fulaige Biotech Co., Ltd.) and reaction at 20 ℃ and 200 r/min. After 11h, liquid chromatography analysis showed that the yield of N-bromoacetyl-7-ACA was 52%.

Example 6

10mL of phosphate buffer (100mM, pH 7.5), 40mM7-ACA and 120mM methyl bromoacetate were added to a 50mL triangular flask with a cover, and mixed well, followed by addition of 30U of penicillin acylase SIPA-V (available from Hunan Fulaige Biotech Co., Ltd.) and reaction at 20 ℃ and 200 r/min. After 24h, the liquid chromatography analysis showed that the yield of N-bromoacetyl-7-ACA was 16%.

Example 7

10mL of phosphate buffer (100mM, pH 7.5), 40mM7-ACA and 120mM ethyl bromoacetate were added to a 50mL triangular flask with a cap, mixed well, followed by addition of 30U of penicillin acylase PGA-750, and reaction was carried out at 20 ℃ and 200 r/min. After 5h, the liquid chromatography analysis showed that the yield of N-bromoacetyl-7-ACA was 87%. Then, filtering to remove enzyme, adding hydrochloric acid into the filtrate to adjust the pH value to 1.0, filtering, washing and drying to obtain the N-bromoacetyl-7-ACA with the yield of 85%.

Example 8

10mL of phosphate buffer (100mM, pH 7.5), 40mM7-ACA and 120mM bromoacetamide were added to a 50mL triangular flask with a cap, mixed well, followed by addition of 30U of penicillin acylase PGA-750, and reaction was carried out at 20 ℃ and 200 r/min. After 5h, the liquid chromatography analysis showed an N-bromoacetyl-7-ACA yield of 84%. Then, filtering to remove enzyme, adding hydrochloric acid into the filtrate to adjust the pH value to 1.0, filtering, washing and drying to obtain the N-bromoacetyl-7-ACA with the yield of 83%.

Example 9

10mL of phosphate buffer (100mM, pH 7.5), 40mM7-ACA and 40mM methyl bromoacetate were added to a 50mL triangular flask with a cap, mixed well, followed by addition of 30U of penicillin acylase PGA-750, and reaction was carried out at 20 ℃ and 200 r/min. After 1h, liquid chromatography analysis showed that the yield of N-bromoacetyl-7-ACA was 53%.

Example 10

10mL of Tris-HCl buffer (100mM, pH 9.0), 40mM7-ACA and 120mM methyl bromoacetate were added to a 50mL triangular flask with a cover, mixed well, followed by addition of 30U of penicillin acylase PGA-750, and reacted at 20 ℃ and 200 r/min. After 2h, the liquid chromatography analysis showed an N-bromoacetyl-7-ACA yield of 88%. Then, filtering to remove enzyme, adding hydrochloric acid into the filtrate to adjust the pH value to 1.0, filtering, washing and drying to obtain the N-bromoacetyl-7-ACA with the yield of 88%.

Example 11

10mL of phosphate buffer (100mM, pH 7.5), 40mM7-ACA and 120mM methyl bromoacetate were added to a 50mL triangular flask with a cap, mixed well, followed by addition of 30U of penicillin acylase PGA-750, and reaction was carried out at 40 ℃ and 200 r/min. After 1h, liquid chromatography analysis showed that the yield of N-bromoacetyl-7-ACA was 73%.

Example 12

10mL of phosphate buffer (100mM, pH 7.5), 40mM7-ACA and 100mM methyl bromoacetate were added to a 50mL triangular flask with a cap, mixed well, followed by addition of 30U of penicillin acylase PGA-750, and reaction was carried out at 20 ℃ and 200 r/min. After 3h, liquid chromatography analysis showed that the yield of N-bromoacetyl-7-ACA was 85%. Then, filtering to remove enzyme, adding hydrochloric acid into the filtrate to adjust the pH value to 1.0, filtering, washing and drying to obtain the N-bromoacetyl-7-ACA with the yield of 83%.

Example 13

10mL of phosphate buffer (100mM, pH 7.5), 60mM7-ACA and 180mM methyl bromoacetate were added to a 50mL triangular flask with a cap, mixed well, followed by addition of 30U of penicillin acylase PGA-750, and reaction was carried out at 20 ℃ and 200 r/min. After 5h, the liquid chromatography analysis showed that the yield of N-bromoacetyl-7-ACA was 79%.

Example 14

10mL of phosphate buffer (100mM, pH 7.5), 50mM7-ACA and 150mM methyl bromoacetate were added to a 50mL triangular flask with a cap, mixed well, followed by addition of 40U of penicillin acylase PGA-750, and reaction was carried out at 20 ℃ and 200 r/min. After 2h, the liquid chromatography analysis showed that the yield of N-bromoacetyl-7-ACA was 85%. Then filtering to remove enzyme, adding hydrochloric acid into the filtrate to adjust the pH value to 1.0, filtering, washing and drying to obtain the N-bromoacetyl-7-ACA with the yield of 82%.

Comparative example

10mL of phosphate buffer (100mM, pH 7.5), 40mM7-ACA and 100mM methyl bromoacetate were added to a 50mL triangular flask with a cap, mixed well, and reacted at 20 ℃ and 200 r/min. After 24 hours, the generation of the target product N-bromoacetyl-7-ACA is not found.

Claims

1. A method for preparing N-bromoacetyl-7-aminocephalosporanic acid through enzyme catalysis is characterized by comprising the following steps: adding 7-aminocephalosporanic acid and an acyl donor into a buffer solution, uniformly mixing, adding immobilized penicillin acylase for reaction, and separating after the reaction to obtain N-bromoacetyl-7-aminocephalosporanic acid; the acyl donor is bromoacetic acid alkyl ester or bromoacetamide;

the immobilized penicillin acylase is penicillin acylase PGA-750.

2. The method of claim 1, wherein the alkyl bromoacetate has an alkyl chain length of from C1 to C6.

3. The method of claim 2, wherein the alkyl bromoacetate is methyl bromoacetate or ethyl bromoacetate.

4. The method according to claim 1, wherein the 7-aminocephalosporanic acid concentration is 20mM to 70 mM.

5. The method according to any one of claims 1 to 4, wherein the molar ratio of the 7-aminocephalosporanic acid to the acyl donor is 1:1 to 1: 6.

6. The method according to claim 5, wherein the reaction conditions are a temperature of 10 ℃ to 40 ℃, a reaction time of 1h to 24h, and an oscillation speed of 150r/min to 300 r/min.

7. The method as claimed in claim 6, wherein the amount of immobilized penicillin acylase is 2 to 5U/mL.

8. The method according to claim 7, wherein the molar ratio of the 7-aminocephalosporanic acid to the acyl donor is 1:2 to 1: 3; the immobilized penicillin acylase is penicillin acylase PGA-750; the dosage of the enzyme is 3U/mL-5U/mL; the reaction time is 1-5 h.

9. The method according to claim 8, wherein the buffer is phosphate buffer or Tris-HCl buffer, and the pH is 5.0-9.0.