JPH0636756B2 - 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 further conducted extensive research on a method for asymmetrically hydrolyzing DL-carboxylic acid ester by the action of microorganisms, and as a result, Brevibacterium sp.
-6224 [FERM P-9820] or Brevibacterium acetylicum ATCC 953 strain was used to find that the optically active carboxylic acid represented by Formula I and its enantiomer ester can be efficiently produced. The present invention has been completed.

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 Brevibacterium (Brevibacterium) having the ability to asymmetrically hydrolyze an ester bond into an ester
bacterim) sp.N-6224 (FERM P-9820) or Brevib-acterium acetylicum AT
A general formula, characterized in that CC 953 is allowed to act on a culture solution, bacterial cells or a treated product of bacterial cells. (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 above-mentioned two strains used in the present invention have extremely high ability to asymmetrically hydrolyze the ester bond of the compound, that is, esterase activity and optical selectivity, as compared with the conventional strains having the esterase activity of Brevibacterium. .

Brevibacterium sp.N-6224 was newly obtained by the present inventors from the natural world, and has been deposited with the Institute of Microbial Science and Technology as Microindustry Research Institute No. 9820 (FERM P-9820). The mycological properties are as shown below. In addition, Brevibacterium acetylicum (Breviba
The cterium acetylicum) ATCC 953 strain is publicly known and can be easily obtained through the preservation organization of the American Type Culture Collection (ATCC).

Burgey's Man
ual of Determinative Bacteriology, 8th Ed. (1974) and Bergey's Manual of Systematic Bacteriology, Vo
L.2 (1986), etc., spores are not formed, and it is a bacillus showing Gram-positive polymorphism, catalase,
+, OF test is oxidative acid production, but gradually fermentatively produces acid, cell wall amino acid type is meso-
Being diaminopimelic acid, cell fatty acids have 15 to 15 carbon atoms
It was identified as a bacterium of the genus Brevibacterium based on the fact that 17 branched (iso, antiiso) fatty acids occupy the majority.

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 A liquid medium (pH 7.2) 100 containing Brevibacterium sp. N-6224 and 1.0% meat extract, 1.0% peptone and 0.5% NaCl.
The cells were inoculated into m. After culturing, collect the whole amount of the cultured cells and use 1 / 10M phosphate buffer (pH
7) Suspended in 100 m. To this bacterial cell suspension was added 2 m of (±) -S-acetyl-β-mercaptoisobutyrate,
The reaction was carried out by shaking at 30 ° C for 24 hours. 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- during reaction at 30 ° C for 24 hours
The hydrolysis rate of methyl mercaptoisobutyrate was 48.0%.

The reaction solution was adjusted to pH 7.0 with NaOH, and methyl S-acetyl-β-mercaptoisobutyrate was extracted and separated with ethyl acetate. Next, the aqueous layer was adjusted to pH 2.0 with sulfuric acid, and then the aqueous layer Nakano S-acetyl-β-mercaptoisobutyric acid 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).

The results are shown in Table 1. From this table, it is understood that optically active carboxylic acid and its antipodal ester are produced.

Example 2 The same operation as in Example 1 was carried out except that Brevibacterium acetylicum ATCC 953 was used in place of Brevibacterium sp. N-6224, and the results shown in Table 2 were obtained.

From this, it can be seen that the optically active carboxylic acid and its antipodal ester are produced as in Example 1. The hydrolysis rate was 41.0%.

Comparative Examples 1 to 3 The same operation as in Example 1 was carried out except that the strain shown in Table 3 was used instead of Brevibacterium sp. N-6224, and the results shown in Table 3 were obtained.

As described above, from Tables 1, 2 and 3, the strain of the present invention has a higher ester hydrolysis rate than the strains of the genus Brevibacterium in the comparative example, and the optically active carboxylic acid and its enantiomer ester are efficiently produced. You can see that it is generating.

 ─────────────────────────────────────────────────── ─── 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, and n is 1 or 2), and the ester represented by Brevibacterium sp. N-622 has the ability to asymmetrically hydrolyze the ester bond.
4 [FERM P-9820] or Brevibacterium acetylicum ATCC 953 culture solution, bacterial cells or a treated product of bacterial cells, the general formula 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.