JPH0959211A - Novel optically active dicarboxylic acid derivative and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new (2S, 5S)-dimethylhexanediacid dialkyl ester expected to find use as an intermediate for various optically active compounds. SOLUTION: This new compound is expressed by formula I (R is H or a 1-6C alkyl; * denotes an asymmetric carbon atom), e.g. (2S, 5S)- dimethlhexanediacid dimethyl ester. The compound of formula I is obtained by making microorganisms capable of asymmetrically hydrolyzing ester linkage (e.g. those belonging to Pseudomonas or Escherichia, or genetically engineered microorganisms transformed by a gene encoding an enzyme capable of asymmetrically hydrolyzing ester linkage), a cultured product thereof or treated product thereof, act on a racemic dicarboxylic diester of formula II.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel optically active carboxylic acid derivative and a method for producing the same.

[0002]

[Prior art]

 General formula (I):

[0003]

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The novel optically active dicarboxylic acid derivative represented by and the method for producing them have not been known so far.

[0005]

The object of the present invention is to provide novel optically active unsaturated dicarboxylic acid derivatives represented by the above general formula (I), which are expected to be used as synthetic intermediates for various optically active compounds, and their derivatives. It is to provide a manufacturing method of.

[0006]

That is, the present invention provides the following general formula (I):

[0007]

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A novel optically active dicarboxylic acid derivative represented by

Further, the present invention provides the following general formula (II):

[0010]

[Chemical 6]

The racemic dicarboxylic acid diester represented by the formula (I) is represented by the action of a culture of a microorganism having the ability to asymmetrically hydrolyze an ester bond, a microbial cell or a treated product of the microbial cell. Another method is to produce a novel optically active dicarboxylic acid derivative.

The racemic dicarboxylic acid diester represented by the general formula (II) used in the present invention has two asymmetric points in the molecule, and has (2S, 5S), (2S, 5)
There are four types of diastereomers, R), (2R, 5S) and (2R, 5R). This diastereomer mixture acts on a culture, a microbial cell or a treated product of a microorganism having an ability to recognize an ester site whose absolute configuration of α-methyl side chain corresponds to the (R) -form and asymmetrically hydrolyze. Let (2S,
5R), (2R, 5S) and (2R, 5R) isomers are asymmetrically hydrolyzed to carboxylic acids, and the unreacted (2S, 5S) isomer diester can be isolated optically pure. The present invention has been discovered and the present invention has been completed.

In the above formula, R may be any as long as it serves as a substrate for an enzymatic reaction, and examples thereof include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group and an isobutyl group. it can.

The microorganism used in the present invention has the general formula (I
Any compound having the ability to asymmetrically hydrolyze the ester bond of the racemic dicarboxylic acid diester represented by I) can be used. Typical examples include the genus Pseudomonas or Esericia.
Examples include microorganisms belonging to the genus cherichia. Specifically, Pseudomonas putida MR-2068
(FERM BP-3846), Escherichia coli
MR-2103 (FERM BP-3835) is mentioned. Escherichia coli MR-2103 (FERM BP-3835) is Pseudomonas putida MR-2068 (FERM
It is a strain transformed with the esterase gene derived from BP-3846).

The culture of the microorganism used in the present invention can be carried out in a liquid medium or a solid medium. As the medium, a medium appropriately mixed with components such as a carbon source, a nitrogen source, vitamins, and minerals that can normally be used by microorganisms is used. It is also possible to add a small amount of ester to the medium in order to improve the ability of the microorganism to hydrolyze. The culture is carried out at a temperature and pH at which the microorganism can grow, but it is preferably carried out under the optimum culture conditions of the strain to be used. Aeration and agitation may be performed to promote the growth of microorganisms.

When carrying out the hydrolysis reaction, the ester may be added to the medium at the start or during the culture, or the ester may be added to the culture solution 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 the ester. As the bacterial cells, bacterial cells treated with acetone, toluene or the like may be used.

Further, instead of the cells, a culture such as a culture solution, a disrupted cell, a cell extract, a crude enzyme, a purified enzyme or the like treated cell may be used. It is also possible to immobilize on a suitable carrier, carry out the reaction, and then collect and reuse. Here, as the enzyme, various microbial-derived lipases, proteases and esterases can be used.

As the reaction medium, for example, ion-exchanged water,
A buffer is used. The ester concentration in the reaction medium or culture medium is preferably 0.1 to 70% by weight, more preferably 5 to 40%. Methanol, acetone,
It is also possible to add a surfactant or the like to the reaction system. The pH of the reaction solution is in the range of 2 to 11, preferably 5 to 8. As the reaction progresses, the pH of the reaction solution decreases due to the carboxylic acid formed. In this case, it is preferable to maintain the pH at an optimum level with a suitable neutralizing agent. The reaction temperature is 5
70 degreeC is preferable and 10-60 degreeC is more preferable.

For separation and purification of the product from the reaction-terminated liquid, extraction with an organic solvent such as ethyl acetate, chloroform, diethyl ether, etc. is carried out, and then a conventional purification method such as distillation or column chromatography is applied.
An optically active dicarboxylic acid derivative can be obtained.

[0020]

EXAMPLES The present invention will be described in more detail with reference to examples below, but the invention is not limited thereto.

Example 1 Production of (2S, 5S) -dimethylhexanedioic acid dimethyl ester Escherichia coli MR-2103 (FERM BP-
3835) in LB medium containing 50 μg / ml of ampicillin (1% polypeptone, 0.5% yeast extract, 0.5% N).
(aCl) was inoculated into 500 ml and cultured with shaking at 37 ° C. for 20 hours. After the completion of the culture, the culture solution was centrifuged, the whole amount of the obtained bacterial cells was washed with ion-exchanged water, and then suspended in 500 ml of 50 mM phosphate buffer (pH 7.0). To this cell suspension, 50 g of racemic 2,5-dimethylhexanedioic acid dimethyl ester was added and reacted at 30 ° C. for 20 hours. During this period, the pH of the reaction solution was adjusted to 7.0 using a 1N NaOH aqueous solution. After completion of the reaction, cells were removed by centrifugation and unreacted 2,5-dimethylhexanedioic acid dimethyl ester was extracted with ethyl acetate. Anhydrous sodium sulfate was added to the organic layer for dehydration, the solvent was removed by evaporation, and the residue was further purified by distillation to obtain a diester fraction of 9.6 g.

Physical property data of the obtained diester fraction are shown below.

(2S, 5S) -Dimethylhexanedioic acid dimethyl ester < ¹ H-NMR spectrum> CDCl ₃ internal standard TMS (FIG. 1) δ _H 1.14 to 1.16 (6H, d, -CH ₃ ). δ _H 1.43 to 1.45 (2H, m, -CH _2- ) δ _H 1.63 to 1.66 (2H, m, -CH _2- ) δ _H 2.43 to 2.45 (2H, _{q, -CH-) δ H 3.68 (} 6H, s, -COOCH 3) <13 C-NMR spectrum> CDCl ₃ internal standard TMS (Figure _{2) δ c 16.98 (-CH 3)} δ c 31 _{.18 (-CH-) δ c 39.31 (} -CH 2 -) δ c 51.77 (-COOCH 3) δ c 176.83 (C = O) < specific _{^{rotation> [α] 25 D = +}} 28 .8 ° (neat)

[0024]

INDUSTRIAL APPLICABILITY According to the present invention, a novel optically active dicarboxylic acid derivative represented by the above general formula (I), which is expected to be used as a synthetic intermediate for various optically active compounds, can be obtained.

[Brief description of drawings]

FIG. 1 is a ¹ H-NMR spectrum diagram of (2S, 5S) -dimethylhexanedioic acid dimethyl ester obtained in Example 1.

FIG. 2 is a ¹³ C-NMR spectrum diagram of (2S, 5S) -dimethylhexanedioic acid dimethyl ester obtained in Example 1.

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Claims (4)

[Claims] 1. The following general formula (I): A novel optically active dicarboxylic acid derivative represented by: 2. The following general formula (II): The racemic dicarboxylic acid diester represented by the formula (I) below is reacted with a culture of a microorganism having the ability to asymmetrically hydrolyze an ester bond, a microbial cell or a treated product of the microbial cell. A method for producing a novel optically active dicarboxylic acid derivative represented by: 3. The novel optically active dicarboxylic acid derivative according to claim 2, wherein the microorganism having the ability to asymmetrically hydrolyze an ester bond is a microorganism belonging to the genus Pseudomonas or the genus Escherichia. Method of manufacturing. 4. The microorganism having the ability to asymmetrically hydrolyze an ester bond is a genetically engineered microorganism transformed with a gene encoding an enzyme that asymmetrically hydrolyzes an ester bond. A method for producing a novel optically active dicarboxylic acid derivative.