KR100345994B1 - Process for enzymatically preparing 7-aminocephalosporanic acid - Google Patents

Abstract

PURPOSE: A process for enzymatically preparing 7-aminocephalosporanic acid is provided, thereby preparing high purity 7-aminocephalosporanic acid in higher yield. CONSTITUTION: A process for enzymatically preparing 7-aminocephalosporanic acid comprises the steps of: reacting cephalosporin C with immobilized D-amino acid oxidase to prepare 7-aminocephalosporanic acid (GL-7-ACA) and ketoadipyl 7-aminocephalosporanic acid (Keto-7-ACA); and reacting GL-7-ACA with GL-7-ACA acylase, wherein the initial pH is maintained during the reaction of cephalosporin C and D-amino acid oxidase and hydrogen peroxide is added into the reaction solution of cephalosporin C and D-amino acid oxidase when pH of the reaction solution is increased to 0.1 or more to convert remaining Keto-7-ACA to GL-7-ACA; pH of the reaction solution is maintained 7.1 to 8.0; and the concentration of hydrogen peroxide is 5 to 10%.

Description

Enzymatic Preparation of 7-Aminocephalosporanic Acid

The present invention improves the yield and purity of glutaryl 7-aminocephalosporranic acid (hereinafter referred to as GL-7-ACA), an intermediate in the preparation of 7-aminocephalosporonic acid (hereinafter referred to as 7-ACA) by the enzymatic method. It is about a method.

7-ACA, a precursor of cephatic antibiotics, can be produced by chemical or enzymatic synthesis. Currently, the chemical synthesis method, which is widely used worldwide, is a problem of environmental pollution caused by organic solvents used as raw materials and residual organic solvents in products. Increasingly regulated, the trend is increasingly being replaced by enzymatic synthesis.

Enzymatic 7-ACA preparation was carried out by the action of the enzyme D-amino acid oxidase immobilized on cephalosporin C (hereinafter referred to as Cepha C) to undergo an oxidative deamination reaction followed by ketoadifil 7-aminocephalosporonic acid (hereinafter referred to as Keto-). 7-ACA) is then converted to GL-7-ACA by adding a stoichiometric equivalent of hydrogen peroxide and then reacted with GL-7-ACA acylase to prepare 7-ACA. Therefore, the purity of the intermediate GL-7-ACA has a significant impact on the quality of the final product 7-ACA.

An object of the present invention is to develop a manufacturing process for improving the yield and purity of GL-7-ACA used as a substrate of the GL-7-ACA acylase reaction.

In Japanese Patent Laid-Open No. Hei 5-211890, which is a conventional method for preparing GL-7-ACA, an air flow rate of 0.5-1.0 is immobilized at 25 ° C. and pH 7.5 by immobilizing enzyme D-amino acid oxidase at Cepha C at a concentration of 20-100 g / l. Keto-7-ACA was prepared under the reaction conditions of vvm, and then the stoichiometric equivalent of hydrogen peroxide was added, followed by reaction at 20-25 ° C. for 10-15 minutes to produce GL-7-ACA, and excess hydrogen peroxide was added to reduce the reaction time. The remaining hydrogen peroxide after the reaction was removed using a reducing agent such as pyruvic acid or its salt or alkaline sulfite. This process requires the process of separating D-amino acid oxidase from the reactant and removing excess hydrogen peroxide for the oxidative carboxyl removal process, which does not simplify the process and decomposes the unstable Keto-7-ACA in the alkaline region, resulting in GL In addition to -7-ACA, there are disadvantages that other impurities are generated, and unreacted Keto-7-ACA has the disadvantage of remaining as impurities in the final product.

In the manufacturing process of PCT / US90 / 01696, the immobilized enzyme D-amino acid oxidase was recycled to Cepha C solution at 6 ml / min for 80 minutes at 50 psig, and the yield of GL-7-ACA reaction was 90.6%. In this process, however, unreacted Keto-7-ACA was detected from 20 minutes after the reaction and maintained at about the same amount until the end of the reaction, resulting in low purity and yield of GL-7-ACA. The conversion process to GL-7-ACA also takes a long time, and impurities are likely to be generated by natural decomposition.

Therefore, the present inventors have solved the problems caused by the existing manufacturing method to simplify the whole process and to study a method for increasing the reaction yield and purity of GL-7-ACA.

In this process, the immobilized D-amino acid oxidase was enzymatically reacted with 10-30 g / l Cepha C solution at a pressure of 45-55 psig using oxygen at a temperature of 20-25 ° C., pH 7.1-8.0, and a flow rate of 1 vvm. GL-7-ACA was prepared. The pH during the reaction was maintained at the initial pH in 2M-KOH solution. The higher the initial pH, the shorter the reaction time, but the intermediate pH Keto-7-ACA is unstable in the alkaline region, so the operating pH was set to 7.2-7.6.

However, the problem found in this manufacturing process is the lack of hydrogen peroxide at the end of the reaction, the rate of conversion of Keto-7-ACA to GL-7-ACA is slow and the pH value tended to increase from the beginning. Therefore, even if the reaction time is increased, unreacted Keto-7-ACA remains in the reaction solution, which causes a drop in purity and yield of GL-7-ACA.

In order to solve this problem, the inventors kept the reaction pH at the initial pH by adding 5-10% hydrogen peroxide solution to the reaction solution. After the reaction, the reaction solution was collected and analyzed. Keto-7-ACA was not detected, and the purity and reaction yield of GL-7-ACA were improved more than in the conventional process.

Advantages of the present invention compared to the existing technology are as follows.

First, the process can be simplified by omitting the addition of hydrogen peroxide and the reduction of the excess hydrogen peroxide in the conventional process. In other words, by converting Keto-7-ACA to GL-7-ACA in a continuous process in one reactor, the overall process time can be shortened, minimizing decomposition of unstable Keto-7-ACA to impurities in the alkaline region. I could make it.

Second, when the rate of conversion of Keto-7-ACA to GL-7-ACA is slowed due to the lack of hydrogen peroxide, it can be recognized simply by changing the pH, such as Keto-7-ACA, GL-7-ACA, hydrogen peroxide, etc. Eliminate the hassle of having to analyze the concentration every time.

Third, when the enzyme activity decreases and the reactivity decreases, the time and rate of hydrogen peroxide addition can be easily recognized by adjusting only the pH during the reaction.

Fourth, in this manufacturing process, all unreacted Keto-7-ACA is converted to GL-7-ACA, resulting in a 96-98% reaction that is improved from 90-95% of the conventional Japanese Patent Publication No. Hei 5-211890 in terms of yield. Yield can be obtained. In addition, in terms of the purity of GL-7-ACA, a small amount of Keto-7-ACA was present in the conventional process, but since there is no trace amount of Keto-7-ACA when using the manufacturing method of the present invention, the purity of GL-7-ACA is increased. Can be improved to more than 95%.

Analysis of the reactants in the present invention using HPLC and the RT values of Cepha C, Keto-7-ACA, GL-7-ACA were 6.2-6.3, 9.3-9.4, 27.1-27, 4 minutes, respectively.

The present invention is explained in more detail in the following examples.

Example 1

300 ml of a solution of 10 g of purified Cepha C was adjusted to pH 7.1 with 2M-KOH, and the immobilized D-amino acid oxidase (600 units) was reacted under a reaction condition of oxygen 1.0vvm flow rate at a temperature of 20 ° C. and a pressure of 45-55 psig. As a result of the reaction for 110 minutes, the pH started to increase above 7.1 at 70 minutes and the concentration of Keto-7-ACA was maintained at the same level until the end of the reaction. The reaction rate of GL-7-ACA was 91.9%, and the results are shown in Table 1.

Example 2

600 ml of a solution of 20 g of purified Cepha C was adjusted to pH 7.4 with 2-KOH and reacted with immobilized enzyme D-amino acid oxidase (1300 units) for 80 minutes under the same reaction conditions as in Example 1. After minutes, the amount of Keto-7-ACA remained constant and there was little conversion of Keto-7-ACA to GL-7-ACA when hydrogen peroxide was not added.

Example 3

After maintaining the pH of Cepha C at 7.3 under the same reaction conditions as in Example 1, 5% hydrogen peroxide solution was added to the reactor at a flow rate of 0.08 ml / min after 51 minutes of increasing the pH to 7.4. The reaction proceeded faster than when the pH was maintained at 7.1, but when the pH was increased again after 25 minutes, the reaction was terminated and the reaction solution was analyzed. As a result, a small amount of remaining Keto-7-ACA was present. Therefore, in order to shorten the reaction time, it is necessary to increase the flow rate of hydrogen peroxide.

Example 4

Under the same reaction conditions as in Example 2, the pH was maintained at 2M-KOH at 7.2, followed by 55 minutes of increasing the pH to 7.3, followed by the addition of 5% hydrogen peroxide at a flow rate of 0.24 ml / min for 25 minutes. Keto-7-ACA was not detected and the GL-7-ACA purity increased to 95.5%.

Example 5

Under the same reaction conditions as Example 2, the pH was maintained at 7.4 and when the pH was increased to 7.5, 10% hydrogen peroxide solution was added at a flow rate of 0.2 ml / min for 15 minutes. It was converted to GL-7-ACA and the yield of GL-7-ACA reaction was 96.7%.

Example 6

Comparing the purity and reaction completion time of the final reaction solution by pH of the initial Cepha C solution in the same manner as in Example 5, the reaction is possible from pH 7.1 to pH 8.0 as shown in Table 6, the higher the pH, the shorter the reaction completion time However, above pH 7.6, the purity was lowered as Keto-7-ACA was unstable and converted to impurities. Therefore, considering the reaction rate and GL-7-ACA purity, the reaction pH is less than 7.6.

1: Chromatogram by High Performance Liquid Chromatography (HPLC) of the reaction solution before hydrogen peroxide addition in Example 4

Figure 2: HPLC chromatogram 15 min after addition of 5% hydrogen peroxide in Example 4

Figure 3: HPLC chromatogram 25 min after addition of 5% hydrogen peroxide in Example 4

Claims (5)

Cephalosporin C is reacted with the immobilized enzyme D-amino acid oxidase to give glutaryl 7-aminocephalosporonic acid (GL-7-ACA) and ketoadifil 7-aminocephalosporonic acid (Keto-7 -ACA), and then reacting GL-7-ACA with CL-7-ACA acylase to produce 7-aminocephalosporanic acid, Maintain the initial pH during the reaction between the cephalosporin C and D-amino acid oxidase, and add hydrogen peroxide at the time when the pH of the reaction solution increases by 0.1 or more to convert the remaining Keto-7-ACA into GL-7-ACA. Enzymatic continuous preparation of 7-aminocephalosporranic acid characterized by conversion to The method of claim 1, wherein the pH is maintained between 7.1 and 8.0 under enzymatic reaction conditions. The method of claim 2, wherein the pH is maintained between 7.2 and 7.6 under enzymatic conditions. The method of claim 1 wherein the concentration of hydrogen peroxide solution is 5-10%. The method of claim 1 wherein the hydrogen peroxide is added at a flow rate of 0.17 to 0.67 ml / min per liter of reaction liquid.