JPH01191697A - Production of optically active carboxylic acid and its antipode ester - Google Patents

Abstract

PURPOSE:To obtain the above compound useful as a raw material for the synthesis of physiologically active substance, in high efficiency, by treating a DL- carboxylic acid ester with a specific microorganism belonging to genus Brevibacterium, thereby asymmetrically hydrolyzing said ester. CONSTITUTION:The objective compound of formula II is produced by treating an ester of formula I (R1 is alkyl, aralkyl, etc.; R2 and R3 are alkyl; n is 1 or 2) (e.g., S-acetyl-beta-mercaptoisobutyric acid methyl ester) with cultured liquid or (treated) cell of Brevibacterium sp. N-6224 (FERM P-9820) or Brevibacterium acetilicum ATCC 953 capable of asymmetrically hydrolyzing an ester bond.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides an optical compound represented by the general formula % (1) (wherein R1 is an alkyl group, an aralkyl group, or an aryl group, R2 is an alkyl group, and n is l or 2). This invention relates to a method for producing active carboxylic acids and their enantiomers.

Carboxylic acid of formula (2) and its diagonal ester are used as raw materials for synthesizing various physiologically active substances having optical activity.

Conventionally, the method for producing optically active carboxylic acid of formula (■) is as follows:
A known method is to synthesize a racemic carboxylic acid in advance using organic synthesis and then resolve it using an optical resolving agent, that is, to physicochemically separate it into one optically active form and its enantiomer. (Unexamined Japanese Patent Publication No. 55-118455, No. 56-
No. 81557, No. 57-188563, and European Patent Publication No. 79200477).

On the other hand, optically active carboxylic acid esters are produced by optically resolving carboxylic acids and then subjecting them to an esterification reaction to produce optically active esters.

However, these methods have drawbacks such as the need for large quantities of expensive resolving agents, the ease with which these resolving agents are mixed into the product as impurities, and the complexity of the resolving process. The manufacturing method is not necessarily satisfactory.

Recently, as a way to improve these shortcomings, expression! A method has been proposed for producing carboxylic acids and their enantiomer esters having the optical activity expressed by the following by the action of microorganisms (JP-A-60-12993, JP-A-60-30692,
60-141297).

The present inventors further conducted intensive research on a method for asymmetrically hydrolyzing OL-carboxylic acid esters by the action of microorganisms, and as a result, they discovered that Brevibacterium
terium) sp, N-6224 (F[!R
M P-9820) or Brevibacterium acetylicum (Brevib-acterium acety
The present inventors have discovered that the optically active carboxylic acid represented by formula (2) and its enantiomer ester can be efficiently produced by using the strain ATCC953 (Licum), and have completed the present invention.

That is, the present invention has the ability to asymmetrically hydrolyze an ester bond into an ester represented by the general formula % (in which R1 represents an alkyl group, and R9, R2, and n have the above-mentioned meanings). Brevibacterium sp, N-622
4 (FIERMP-9820) or Brevibacterium acetylicum (Brevib-acterium a
cetylicum) 8TCC953, expressed by the general formula % formula % (1) (wherein R3, R2 and n have the above-mentioned meanings). This is a method for producing an optically active carboxylic acid and its enantiomer ester.

Examples of the alkyl group of the substituted convex RI of the compounds of formula I and formula (2) include methyl group and ethyl group, examples of the aralkyl group include benzyl group, and examples of the aryl group include phenyl group.

As the ester of formula (1) used in the present invention, for example, S
- methyl acetyl-β-mercaptoisobutyrate, S-acetyl-γ-mercapto α-methyl-n-methyl butyrate,
Methyl S-benzoyl-β-mercaptoisoacetate, S-
Examples include methyl phenylacetyl-β-mercaptoisobutyrate. The mixing ratio of D and L in these esters is not particularly limited.

The above 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, compared to conventional Brevibacterium strains having esterase activity. .

Brevibacterium 3p, N-6224 was newly isolated and obtained from the natural world by the present inventors, and was submitted to the Microbial Technology Research Institute as part of the Microbiological Research Institute No. 9820 (FER).
M P-9820), and its mycological properties are as shown below. Also, Brevibacterium acetylicum (Brevibacterium
ace-tylicum) ATCC953 strain is a known strain, Allerican Type C
It is readily available through the Ulture Collection (ATCC) repository.

The above mycological properties are summarized in Virginie's classification book: Ber-g.
ey's Manual of Determi
native Bacteriology+8Lh
Ed, (1974) and Bergey's Manu
al of Sys-tematic Bacteri
I searched based on ology + Vol. 2 (1986), etc., and found that it is a bacillus that does not form spores and exhibits Durham-positive polymorphism, catalase +, OF test indicates oxidative acid production, but gradual fermentation. also produce acid,
It was identified as a bacterium of the genus Brevibacterium based on the fact that the cell wall amino acid type was meso-diaminopimelic acid and that the bacterial cell fatty acids were mostly branched (iso, antiiso) fatty acids having 15 to 17 carbon atoms.

The cultivation of microorganisms in the present invention is usually carried out by liquid culture.
As the medium, carbon sources, nitrogen sources, vitamins, inorganic salts, etc. that can be assimilated by microorganisms are used as appropriate, but it is also possible to add small amounts of esters, etc. to the medium in order to improve the hydrolytic ability of microorganisms. Cultivation is carried out at a temperature and pl+ at which microorganisms can grow, but usually at a temperature of 5 to 50'C.
, pH is in the range of 2-11, preferably 5-8. Aeration and stirring may be performed to promote the growth of microorganisms.

When carrying out the hydrolysis reaction, the ester (Formula 2) may be added to the medium at the beginning or during the culture, or the ester (Formula 2) may be added to the culture solution after culturing the microorganisms in advance. Alternatively, the cells of the proliferated microorganisms may be collected by centrifugation or the like and added to the reaction medium containing the ester.In this case, for convenience of handling, the cells may be dried,
For example, freeze-dried bacterial cells, spray-dried bacterial cells, bacterial cells treated with an organic solvent such as acetone or toluene, or bacterial cell-treated products such as crushed bacterial cells and Japanese extracts can 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 culture solution is preferably 0.01 to 50% by weight; the ester can also be added in grams in water. In addition, the solubility of the ester can be improved by adding an organic solvent such as methanol or acetone to the reaction solution.The po of the reaction solution is 2 to 2.
11, preferably in the range of 5 to 8.0 As the reaction progresses, the PL+ of the reaction solution decreases due to the generated carboxylic acid, but in this case, it is possible to maintain the optimum pH with an appropriate neutralizing agent. The preferred reaction temperature is preferably 5 to 50°C.

Separation and purification of the product from the reaction solution or culture solution can be carried out by conventional methods such as extraction, recrystallization, column chromatography, etc.

Hereinafter, the present invention will be explained in detail according to Examples.

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

Example 1 Brevibacterium sp, N-6224 was inoculated into 100 tel of a liquid medium (pH 7.2) consisting of 1.0% meat extract, 1.0% peptone and 10.5% NaC, and incubated at 30°C for 2 days. Shaking culture was performed. After culturing, collect the entire amount of cultured Kokutai and add l/10M phosphate buffer solution (pH
7) Suspended in 100 trdl. Methyl (±)-5-acetyl-β-mercaptoisobutyrate was added to this hedron suspension and reacted by shaking at 30°C for 24 hours.

After the reaction was completed, 5 ran of the reaction solution was sterilized and the reaction product was confirmed to be S-acetyl-β-mercaptoisobutyric acid by high performance liquid chromatography. 30°C12
The hydrolysis rate of methyl S-acetyl-β-mercaptoisoacetate during the 4-hour reaction was 48.0%.

The reaction solution was adjusted to pl+7.0 with NaOIf, and S-
Methyl acetyl-β-mercaptoisoacetate was extracted and separated with ethyl acetate.The aqueous layer was then lowered to pl+2.0 with sulfuric acid, and 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. The specific optical rotation of the separated and extracted S-acetyl β-mercaptoisobutyric acid and methyl S-acetyl-β-mercaptoisobutyrate was measured using a JASCO film-forming photometer (DIP-3Ci).
o type).

The results are shown in Table 1. From this table, it can be seen that an optically active carboxylic acid and its enantiomer ester were produced.

Table 1 Example 2 In Example 1, Brevibacterium sp, N-62
Brevibacterium acetylicum AT instead of 24
The same operation as in Example 1 was performed except that CC953 was used, and the results shown in Table 2 were obtained.

This shows that, as in Example 1, an optically active carboxylic acid and its enantiomer ester were produced. Note that the hydrolysis rate was 41.0%.

Table 2 Comparative examples 1 to 3 In Example 1, Brevibacterium sp.

The same operation as in Example 1 was performed except that the strain shown in Table 3 was used instead of N-6224, and the results shown in Table 3 were obtained.

Table 3 From the above, Tables 1.2 and 3, the strain of the present invention has a higher ester hydrolysis rate than the Brevibacterium strain in the comparative example, and can efficiently remove optically active carboxylic acids and their counterpart esters. It can be seen that it is produced well.

Claims (1)

[Claims] Represented by the general formula ▲ Numerical formulas, chemical formulas, tables, etc. Brevibacterium has the ability to asymmetrically hydrolyze ester bonds into esters.
bacterium) sp. N-6224 [FERMP
-9820] or Brevibacterium acetylicum (
Brevibacterium acetylicum
) A general formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (in the formula, R_1, R_2 and n have the above meanings), which is characterized by the action of ATCC953 culture solution, bacterial cells or treated bacterial cells. A method for producing the optically active carboxylic acid and its enantiomer ester.