JPH01222789A - Extraction of carboxylic acid derivative - Google Patents

Abstract

PURPOSE:To efficiently and readily extract a carboxylic acid derivative, by treating an aqueous medium containing both the carboxylic acid derivative and biological ingredients with an organic solvent of a specified concentration at a specified pH to flocculate and remove the biological ingredients. CONSTITUTION:Firstly, the pH value of an aqueous medium containing (A) a carboxylic acid derivative of formula I (R1 is alkyl, aralkyl or aryl group; R2 and R3 are each H or alkyl group; n is 1 or 2) produced by e.g., biological asymmetric hydrolysis of a racemic carboxylic ester and (B) such biological ingredients as bacteria decomposed product, protein, nucleic acid or the like is adjusted to <=5. Thence, said aqueous medium is incorporated with an organic solvent such as a halogenated hydrocarbon, alcohol or ester so that the concentration is at or below the saturated solubility for said aqueous medium to flocculate the biological ingredients. Said biological ingredients are then removed, and the resultant aqueous medium is spiked with an organic solvent, thus extracting the objective carboxylic acid derivative.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention is directed to the general formula % [1 (where R1 is an alkyl group, an aPA/k group or an aryl group, R , and R3 are alkyl groups or hydrogen atoms, n is 1
or 2)).

Carboxylic acid derivatives of formula (1) are used as raw materials for synthesizing various physiologically active substances.

[Prior Art] The present inventors have recently proposed a method for producing an optically active carboxylic acid and its enantiomer ester by biochemically asymmetrically decomposing a racemic carboxylic acid ester of formula I (particularly Kaisho 60-12992, 60-12993, 60-
No. 50692, No. 60-94091 and No. 60-1412
(Refer to each publication No. 97). If an attempt is made to extract and separate the carboxylic acid derivative of formula I from an aqueous medium such as a reaction solution obtained by such biochemical treatment using a water-immiscible solvent, an emulsion will be formed and extraction will be difficult. There were drawbacks.

In order to improve this drawback, the present inventors proposed a method for extracting the carboxylic acid derivative represented by the formula rl by adjusting the pH of the aqueous medium to below 5 and then extracting it, thereby eliminating the generation of an emulsion and increasing the extraction efficiency. proposed a method to improve this (Japanese Unexamined Patent Publication No. 60-248189).

[Problem that the invention seeks to solve]

Although this method has made it possible to prevent the formation of emulsions during extraction, even when extracted with this method, the ability to form an interface is low, it takes a long time to form an interface, and It became clear that precipitates still accumulated at the interface between the solvent layer and the water layer.

The purpose of the present invention is to provide a more efficient power that eliminates this drawback.
An object of the present invention is to provide a method for extracting a bonic acid derivative.

[Means for Solving the Problems] That is, the gist of the present invention is to obtain a carboxylic acid derivative represented by the general formula % [11 (wherein R1m ``le R3 and n have the above-mentioned meanings] using biochemical methods. The aqueous medium containing this compound and biological components obtained by the above treatment was made below pus, and the precipitate was removed by dissolving the organic solvent in the aqueous medium to a concentration below the saturation solubility of the aqueous medium. Thereafter, the method for extracting a carboxylic acid derivative is characterized in that the carboxylic acid derivative is extracted from an aqueous medium using an organic solvent.

In the present invention, a precipitate generated from a mixed acid of an aqueous medium and an organic solvent having a saturation solubility or less of pH 5 or less is
It is caused by biological components mixed in the aqueous medium, such as bacterial cells, bacterial decomposition products, proteins, and nucleic acids.

According to the method of the present invention, when extracting the carboxylic acid derivative of formula I, the pH of the aqueous medium is set to 5 or less, and an organic solvent is added so that the concentration is below the saturated solubility.
Biological components aggregate and are removed by centrifugation, filtration, etc., which facilitates the formation of an interface between the aqueous medium and organic solvent during the extraction of carboxylic acid derivatives from an aqueous medium, and prevents the accumulation of insoluble matter at the interface. There is a characteristic that there is no

The amount of organic solvent added must be below the saturation solubility; if it is added above the saturation solubility, the organic solvent and the aqueous medium will separate, forming an interface, and accumulation of precipitates at the interface will occur. A problem arises in that this precipitate cannot be completely removed, and the operation in the next extraction step becomes inconvenient due to the influence of this precipitate.

In the present invention, the alkyl group for the substituent R1 of the carboxylic acid derivative [13] includes, for example, a methyl group, an ethyl group, etc., the affix group includes, for example, a benzy group, and the aryl group includes, for example, a phenyl group and a Examples of the alkyl group include a methyl group and an ethyl group. Hydroxylic acid derivatives [13 include S-7 cetyl-β-merkadetoisobutyric acid and its methyl ester, S-acetyl-γ-mercapto α-methyl-n-butyric acid, and its methyl ester A/, S-benzoyl- β-mercaptoisobutyric acid and its methyl ester, S-phenylacetyl-β-mercaptoisobutyric acid and its methyl ester, S
-acetyl-β-mercap)-n-butyric acid and its methyl ester.

Examples of biological components include microbial cells, decomposition products thereof, enzyme proteins, and the like. Examples of the aqueous medium containing the carboxylic acid derivative of formula (2) and biological components include biochemically treated water such as microbial culture solution and enzyme reaction solution.

Organic solvents used to remove biological components from this aqueous medium include 03 ash alcohols such as propatool and butano-y, esters such as ethyl acetate, and halogenated hydrocarbons such as chloroform and carbon tetrachloride. etc. are used. In addition, in order to extract and separate carboxylic acid derivatives,
Halogenated hydrocarbons such as chloroform and carbon tetrachloride, esters such as ethyl acetate, ethers such as ethyl ether, and hydrogen subhydrides such as echahensen, toluene, nclohexane and n-hexane are used.

The organic solvent for precipitation and the extraction solvent may be of the same type or may be of different types.

When using different types of materials, the combination and quantitative ratio may be selected so that the ternary mixed system of these materials and the aqueous medium does not emulsify.

In addition, in order to adjust the pH to 5 or less, hydrochloric acid, sulfuric acid,
This is done by adding mineral acids such as phosphoric acid.

When carrying out the present invention, the pH of the aqueous medium containing the carboxylic acid derivative of formula I and the biological component is adjusted to 5 or less, and an organic solvent having a solubility or less is added. Note that after adding an organic solvent having a solubility or lower, the pH may be adjusted to 5 or lower. This causes biological components such as proteins in the aqueous medium to aggregate. This aggregate is removed by centrifugation, filtration, etc. before extraction.

Next, the carboxylic acid derivative of formula (1) is extracted using an organic solvent. The extraction method is carried out by adding an organic solvent to an aqueous medium, mixing well, leaving to form an interface, and then separating the liquids.

This causes the dicarboxylic acid derivative to migrate from the aqueous medium to the organic solvent. The amount of organic solvent used is preferably α01 to 100 parts by volume per 1 part by volume of the aqueous medium.

In addition, when extracting separately the optically active carboxylic acid and its enantiomer ester that are mixed in the aqueous medium, the p of the aqueous medium is
After reducing g to 5 or less, adding an organic solvent and removing the generated aggregates, adjusting the pH to around neutrality and extracting with an organic solvent, then making the pH of the aqueous medium acidic and extracting with an organic solvent. do it. The obtained extract can be further purified by distillation, column chromatography, etc.

[Effects of the Invention] According to the method of the present invention, carboxylic acid derivatives can be efficiently and easily extracted from a medium that conventionally takes time to form an interface during extraction and is difficult to extract due to the accumulation of insoluble matter at the interface. can. Therefore, the present invention is particularly suitable for extracting and separating optically active carboxylic acids and their enantiomers obtained by biochemically asymmetrically hydrolyzing racemic carboxylic esters.

〔Example〕

The present invention will be further explained below using Examples. In the following examples, the term "byte" means % by weight.

Example 1 Pseudomonas putida FG9677 was cultured in a liquid medium consisting of 05% meat extract, 175% peptone, 125% salt, 5% glucose a, 0.15% malt extract, and 0.15% yeast extract. I) H&8) 100-
The bacteria were cultured at 30°C for 1 day with shaking. After the culture is completed, the culture solution is centrifuged, and the total amount of the obtained bacterial cells is
After washing with /10 phosphate buffer (pH 7,0), it was suspended in the same phosphate buffer 200-. To this bacterial suspension (±
)-day-acetyl-β-mercagtoisobutyric acid methyl/L/
10 trt was added, and the mixture was shaken and reacted at 60° C. for 48 hours.

This reaction solution was divided into two parts, the pH of the reaction solution 100- was adjusted to 3, and ethyl acetate A15- was added. In this state, the reaction solution and ethyl acetate were not phase separated.

Next, when the supernatant liquid obtained by removing the precipitate generated by centrifugation was extracted by adding 45-ethyl acetate, an interface was formed within 5 minutes, and almost no precipitate was observed at the interface. Ta. It was extracted with Methyl B-acetyl-β-mercaptoisobutyric acid and d volume Jr. of H-acetyl-β-mercaptoisobutyric acid to obtain an extract of 7.69%.

Comparative Example 1 Reaction solution 10 obtained in the same manner as in Example 1 (LD was adjusted to pH 3, insoluble matter was removed by centrifugation, and extracted by adding 50% of ethyl acetate. It took 3 hours for the formation of , and insoluble matter accumulated at the interface.

Example 2 P of 500 d of reaction-completed liquid obtained in the same manner as Example 1
After adjusting H to 2 and adding ethyl acetate p25-, the precipitated insoluble biological components were removed by centrifugation, and then the pH was adjusted to
was adjusted to 7, and S-acetyl-β-mercaptoisobutyric acid methyl p was extracted three times with ethyl acetate 100-. oil layer
Separation of the liquid layers was good and an interface was formed within 5 minutes.

Next, the pH of the aqueous layer was lowered to pH 2 with sulfuric acid, and then 8-acetyl-β-mercaptoisobutyric acid in the aqueous layer was extracted three times with ethyl acetate/l/100-.

After dehydrating the extract by adding anhydrous sodium sulfate, the solvent was removed by evaporation.

Extracted methyl S-acetyl-β-mercaptoisobutyrate is 15.75f, and methyl B-acetyl p-β-mercaptoisobutyrate is 11.8? , 8-acetyl-β-mercaptoisobutyric acid gave 1α2f. According to the measurement results of specific optical power, υ, optical activity: boronic acid ester y and its enantiomer,
It was confirmed that carboxylic acid was produced.

Comparative Example 2 When extraction was attempted by adjusting the reaction solution to pu 2 and adjusting the centrifuged reaction solution to pH 7 without adding ethyl acetate, the formation of an interface was poor and the separation of the oil layer and water layer It took 4 hours to form the interface, and precipitates remained at the interface.

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Claims (1)

[Claims] General formula ▲ Numerical formula, chemical formula, table, etc. ▼ (In the formula, R_1 is an alkyl group, aralkyl group, or aryl group, R_2 and R_3 are an alkyl group or a hydrogen atom, and n represents 1 or 2) An aqueous medium containing this compound obtained by biochemically treating a carboxylic acid derivative represented by the formula and a biological component is adjusted to pH 5 or less, and an organic solvent is added to the aqueous medium at a saturation solubility of the organic solvent in the aqueous medium. A method for extracting a carboxylic acid derivative, which comprises dissolving the carboxylic acid derivative to the following concentration, removing precipitates, and then extracting the carboxylic acid derivative from the aqueous medium using an organic solvent.