JPH0636755B2 - Process for producing optically active carboxylic acid and its enantiomer ester - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (In the formula, R ₁ is an alkyl group, an aralkyl group or an aryl group,
R ₂ is an alkyl group, and n is 1 or 2, and relates to a process for producing an optically active carboxylic acid and its enantiomer ester.

The carboxylic acid of formula I and its antipodal ester are used as raw materials for synthesizing various physiologically active substances having optical activity.

Conventionally, as a method for producing an optically active carboxylic acid of formula I,
A method is known in which a racemic carboxylic acid is first synthesized organically and then resolved using an optical resolving agent, that is, a method of physically and physically separating one optically active substance and its enantiomer. (JP-A-55-118455, JP-A-56-81557,
57-188563, European Patent Publication No. 79200477. On the other hand, for the optically active carboxylic acid ester, a method in which an optically active carboxylic acid is optically resolved and then an esterification reaction is performed to obtain an optically active ester is adopted. But,
In these methods, there are disadvantages that a large amount of expensive resolving agent is required, that this resolving agent easily mixes into the product as an impurity, and the resolving process is complicated. Is not always satisfactory.

As a method for improving these drawbacks, recently, a method of producing an optically active carboxylic acid represented by the formula I or its enantiomer ester by the action of a microorganism has been proposed (JP-A-60-12993). 60-30692 and 60-141297).

The present inventors, as a result of earnest research on a method for asymmetric hydrolysis of DL-carboxylic acid ester by the action of microorganisms, by using a new cellulomonas (Cellulomonas) genus or Rhodococcus (Rhodococcus) genus microorganisms. The inventors have found that the optically active carboxylic acid represented by the formula I and its antipodal ester can be efficiently produced, and completed the present invention.

That is, the present invention has the general formula (Wherein R ₃ represents an alkyl group, R ₁ , R ₂ and n have the above-mentioned meanings), and Cellulomona having the ability to asymmetrically hydrolyze an ester bond into an ester
s) genus or Rhodococcus (Rhodococcus) genus of the culture solution of microorganisms, characterized by the action of bacterial cells or treated cells, the general formula (Wherein R ₁ , R ₂ and n have the above-mentioned meanings), and a process for producing an optically active carboxylic acid and its enantiomer ester.

Examples of the alkyl group of the substituent R ₁ of the compounds of the formulas I and II include a methyl group and an ethyl group, an aralkyl group such as a benzyl group, and an aryl group such as a phenyl group.

Examples of the ester of formula II used in the present invention include S-
Acetyl-β-mercaptoisobutyrate methyl, S-acetyl-γ-mercapto-α-methyl-n-butyrate methyl, S-benzoyl-β-mercaptoisobutyrate methyl, S-phenylacetyl-β-mercaptoisobutyrate methyl etc. Can be mentioned. The mixing ratio of the D-form and the L-form of these esters is not particularly limited.

The microorganism used in the present invention belongs to the genus Cellulomonas or Rhodococcus and is a microorganism having the ability to asymmetrically hydrolyze the ester bond of the compound, for example, Cellulomonas flavigena ATCC.
484, Rhodococcus eryt
hropolis) IFO 12538 and the like.

All of these strains are known, and American T
ype Culture Collection (ATCC), Fermentation Research Institute (I
It can be easily obtained through preservation organizations such as FO).

Cultivation of the microorganism in the present invention is usually performed by liquid culture.
As the medium, a carbon source, a nitrogen source, vitamins, inorganic salts and the like that can be assimilated by the microorganism are appropriately used, but it is also possible to add a small amount of ester or the like to the medium in order to improve the hydrolytic ability of the microorganism. The culture is carried out at a temperature and pH at which the microorganism can grow, but usually, the temperature is 5 to 50 ° C and the pH is 2 to
The range is 11, preferably 5 to 8. Aeration and agitation may be performed to promote the growth of microorganisms.

When carrying out the hydrolysis reaction, the ester (formula II) may be added to the medium at the start or during the culture, or the ester (formula II) may be added to the culture medium after culturing the microorganism in advance. Alternatively, the cells of the grown microorganism may be collected by centrifugation or the like and added to the reaction medium containing ester. In this case, the cells are dried cells for convenience of handling,
For example, freeze-dried bacterial cells, spray-dried bacterial cells or bacterial cells treated with an organic solvent such as acetone, toluene, etc., or disrupted bacterial cells or bacterial cell extracts such as bacterial cell extracts can also be used. As the reaction medium, for example, ion-exchanged water or a buffer solution is used. The concentration of the ester in the reaction medium or the culture solution is preferably 0.01 to 50% by weight. The ester can also be added in suspension in water. Further, an organic solvent such as methanol or acetone may be added to the reaction solution to improve the solubility of the ester. The pH of the reaction solution is in the range of 2 to 11, preferably 5 to 8. As the reaction proceeds, the pH of the reaction solution decreases due to the generated carboxylic acid. In this case, it is preferable to maintain the pH at an optimum level with a suitable neutralizing agent. The reaction temperature is preferably 5 to 50 ° C.

Separation and purification of the product from the reaction solution or the culture solution can be carried out by an ordinary method such as extraction, recrystallization, column chromatography and the like.

Hereinafter, the present invention will be described in detail according to examples.

In the following examples,% means% by weight unless otherwise specified.

Example 1 Cellulomonas flavigena ATCC 484, meat extract 1.0
%, Peptone 1.0% and NaCl 0.5%
H7.2) was inoculated into 100 m and shake culture was performed at 30 ° C. for 2 days.
After the completion of the culture, the whole amount of the cultured bacterial cells was collected and suspended in 1/10 M phosphate buffer (pH 7) 100 m. Methyl (±) -S-acetyl-β-mercaptoisobutyrate 2 was added to this cell suspension.
m was added and shaken at 30 ° C. for 48 hours for reaction. After completion of the reaction, 5 m of the reaction solution was sterilized, and it was confirmed by high performance liquid chromatography that the reaction product was S-acetyl-β-mercaptoisobutyric acid. S-acetyl-β- at this time
The hydrolysis rate of methyl mercaptoisobutyrate was 43%.

The reaction solution was adjusted to pH 7.0 with NaOH, and methyl S-acetyl-β-mercaptoisobutyrate was extracted and separated with ethyl acetate. Then, the aqueous layer was adjusted to pH 2.0 with sulfuric acid, and then S-acetyl-β-mercaptoisobutyric acid in the aqueous layer was extracted with ethyl acetate. Anhydrous sodium sulfate was added to the ethyl acetate extract for dehydration, and then the solvent was removed by evaporation. Separated and extracted S-
Acetyl-β-mercaptoisobutyric acid and S-acetyl-β
-The specific optical rotation of methyl mercaptoisobutyrate was measured with a JASCO polarimeter (DIP-360 type).

From Table 1 showing the results, it can be seen that an optically active carboxylic acid and its antipodal ester are produced.

Example 2 In Example 1, Cellulomonas flavigena ATCC 484
Was replaced with Rhodococcus erythropolis IFO 12538 and the same operation as in Example 1 was carried out except that the reaction time was changed to 24 hours, and the results shown in Table 2 were obtained. The hydrolysis rate was 42.5%.

From this, it can be seen that the optically active carboxylic acid and its antipodal ester are produced as in Example 1.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Akihiro Sakimae 20-1 Miyuki-cho, Otake-shi, Hiroshima Prefecture Mitsubishi Rayon Co., Ltd. (72) Hisao Onishi 20-1 Miyuki-cho, Otake-shi, Hiroshima Mitsubishi Rayo Mayumi Saito, Corporate Auditor

Claims (1)

[Claims] 1. A general formula (In the formula, R ₁ is an alkyl group, an aralkyl group or an aryl group,
R ₂ and R ₃ are alkyl groups, n is 1 or 2), and a culture of a microorganism belonging to the genus Cellulomonas or Rhodococcus having the ability to asymmetrically hydrolyze the ester bond A general formula, characterized in that a liquid, a microbial cell, or a treated product of the microbial cell is allowed to act on it. A process for producing an optically active carboxylic acid represented by the formula (wherein R ₁ , R ₂ and n have the above-mentioned meanings) and an antipodal ester thereof.