JPH04341195A - Production of optically active mandelic acid - Google Patents

Abstract

PURPOSE:To obtain the subject compound useful as a raw material for pharmaceuticals, etc., on an industrial scale at a low cost by treating racemic mandelic acid with a cultured liquid or cells of microorganism capable of asymmetrically decomposing the acid to effect the decomposition of one of the optically active isomers and separating the remaining antipode. CONSTITUTION:A microbial strain capable of asymmetrically decomposing racemic mandelic acid of formula (e.g. Pseudomonas polycolor IFO 3918) is inoculated in a medium containing DL-mandelic acid and cultured at 30 deg.C for 72hr under shaking. An aqueous solution containing DL-mandelic acid is made to react with the cultured liquid, the bacterial cell recovered from the liquid by centrifugal separation or the treated product of the cell to effect the asymmetric decomposition of L-mandelic acid which is one of the optically active isomers. The reaction liquid is extracted with ethyl acetate, the extracted liquid is concentrated under reduced pressure and the produced crystal is recrystallized with 1:1 mixture of hexane and ethyl acetate to obtain the optically active mandelic acid consisting of the antipode D-mandelic acid.

Description

[Detailed description of the invention]

0001

TECHNICAL FIELD The present invention relates to a new method for producing optically active mandelic acid.

0002

[Prior art] Optically active mandelic acid is a penicillin,
It is a compound useful as a raw material or synthetic intermediate for various pharmaceuticals, including cephalosporin antibiotics and sympathomimetic agents such as ephedrine. Conventionally, methods for producing optically active mandelic acid include physicochemical methods such as optical resolution by fractional crystallization of a racemate, optical resolution by chromatography, organic chemical asymmetric synthesis, and microbial production of benzoylformic acid. A method of asymmetric reduction using an enzyme
-198096, 57-198097, 63-32
Biochemical methods such as 492) and a method for asymmetric hydrolysis of racemic mandelic acid ester (Japanese Unexamined Patent Publication No. 2-53497) are known.

0003

[Problems to be Solved by the Invention] However, the above-mentioned physicochemical method has drawbacks such as complicated operations and low product yield and optical purity, and the biochemical method This method has disadvantages in that it requires expensive coenzymes such as adenine dinucleotide, it requires esterification of mandelic acid, and the optical purity of the product is low.

0004

[Means for Solving the Problem] As a result of intensive research to solve these difficulties, the present inventors discovered the existence of microorganisms that have the ability to asymmetrically decompose racemic mandelic acid, and completed the present invention. I ended up doing it.

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

[0006]

[Chemical formula 1]

Racemic mandelic acid represented by the following formula is treated with a culture solution of a microorganism having the ability to asymmetrically decompose racemic mandelic acid, microorganisms obtained from the culture solution, or processed products of the microorganisms. This is a method for producing optically active mandelic acid, which is characterized by separating and collecting the enantiomer after decomposing the optically active form. In the present invention, racemic mandelic acid (I), which is a raw material compound, includes not only one containing equal amounts of D-form and L-form, but also any one containing both of these optically active forms in an arbitrary mixing ratio. be able to.

[0008] The microorganisms used in the present invention may be any microorganisms as long as they have the ability to asymmetrically decompose racemic mandelic acid, and for example, microorganisms such as molds, bacteria, and yeasts having such abilities are preferably used. can do. Specifically, molds include microorganisms belonging to the genus Gibberella, Fusarium, and Aspergillus, bacteria include microorganisms belonging to the genus Pseudomonas or Brevibacterium, and yeast include microorganisms belonging to the genus Rhodotorula and Rhodosporidium. Examples include microorganisms belonging to the genus. A specific example of such a microorganism is Gibberella fujikuroi (Gibberella fujikuroi).
ella fujikuroi) IFO 5268
, Aspergillus sidoui (Aspergillus
sydowi) IFO 4284, Fusarium
Fusarium oxysporum
um) IFO 5942, Pseudomonas aerugino
sa) ATCC 7700, same OUT 8252, Pseudomonas fluorescens
Pseudomonas fluorescens IAM 1219, Pseudomonas polycolor
polycolor) IFO 3918, Pseudomonas puti
da) ATCC12633, Brevibacterium ammonium
moniagenes) IAM 1641, Rhodotorula glu
tinis) IFO 0389, IFO 075
8, IFO 0898, OUT 6152, Rhodotorula rubra
ra) IFO 0001, IFO0918, IF
O 1100, 0UT 6158, Rhodosporidium toruroides
(toruloides) IFO 0559, etc. These may be wild strains or mutant strains, or may be derived from these microorganisms by biotechnological techniques such as genetic recombination and cell fusion.

Among these microorganisms, if a microorganism that selectively decomposes L-mandelic acid is used, D-mandelic acid can be obtained as the target product, and if a microorganism that selectively decomposes D-mandelic acid is used, L-mandelic acid can be obtained. - Mandelic acid can be obtained. Among the microorganisms exemplified above, microorganisms that selectively decompose L-mandelic acid include microorganisms belonging to the genus Gibberella, Pseudomonas, Brevibacterium, Rhodotorula, and Rhodosporidium; Examples of microorganisms that selectively decompose acids include microorganisms belonging to the genus Fusarium and Aspergillus.

[0010] The culture solution and bacterial cells of the above microorganism can be cultured in a medium normally used in this field, such as a medium containing a conventional carbon source, nitrogen source, and inorganic salts, at room temperature or under heating (preferably at 20°C). ~40°C) and under aerobic conditions at a pH of approximately 5 to 8, and if necessary, the bacterial cells can be isolated and collected from the culture solution by a conventional method. In addition, when culturing, racemic mandelic acid (1) is added to the medium.
It is also possible to increase the enzyme activity by adding about 0.001% or more, especially about 0.1 to 1%.

[0011] Examples of the processed products of microorganisms include freeze-dried cells, acetone-dried cells, cell autolysates, cell extracts, crushed cells, and supernatants of the above-mentioned microorganisms. Examples include sonicated products, and enzymes purified from bacterial cell extracts using a combination of known methods can also be used. Furthermore, the microbial cells or treated microbial cells of the present invention may be used after being immobilized by known methods such as the polyacrylamide method, sulfur-containing polysaccharide gel method (carrageenan gel method, etc.), alginate gel method, agar gel method, etc. You can also do it.

[0012] The asymmetric decomposition reaction according to the present invention includes, for example,
(1) It may be carried out by culturing the above microorganism in a medium containing racemic mandelic acid (I), or (2) racemic mandelic acid (I) is added to the culture solution of the above microorganism in an appropriate solvent. It may be carried out by contacting with bacterial cells collected from a culture solution or a treated product of the bacterial cells.

[0013] When the asymmetric decomposition reaction is carried out by method (2), under the above culture conditions, racemic mandelic acid (I)
This can be carried out by culturing microorganisms using a medium to which 0.5 to 10%, particularly 1 to 5%, of . In addition, when the asymmetric decomposition reaction is carried out by the method (2), the concentration of the substrate in the reaction solution is preferably approximately 0.05 to 10%, particularly 0.5 to 5%, and the reaction is carried out at room temperature or heated. below, preferably 10 to 50°C, particularly preferably 2
It proceeds suitably at 5-40°C. Examples of solvents include:
Water, ethanol, isopropyl alcohol, ethyl acetate, or a mixture of these organic solvents and water can be used. Also, during the reaction, the pH of the reaction solution should be 5 to 1.
It is preferable to adjust the value to 0, especially 6 to 9.

[0014] Furthermore, when using live microorganisms in this method, it is preferable to add a surfactant to the reaction solution in order to shorten the reaction time. Examples of surfactants used for this purpose include cetylpyridinium bromide, cetyltrimethylammonium bromide, and P-isooctylphenyl ether (manufactured by Rohm and Haas, USA, trade name: Triton X-100). , 0 for the reaction solution
．． It is preferable to use about 0001 to 0.1%.

The optically active mandelic acid (I
) can be easily isolated according to conventional methods. For example, after removing insoluble substances such as bacterial cells from the reaction solution by centrifugation, the pH of the reaction solution is adjusted to 1.0,
After extraction with ethyl acetate, optically active mandelic acid can be collected as crystals by concentrating it under reduced pressure.

[0016] The present invention will now be explained in detail by way of examples. In addition, in this specification, "%" refers to "weight/volume".
(g/dl)". In addition, in this example, the optically active form of mandelic acid was quantified using SUMI
The analysis was performed by high performance liquid chromatography using CHIRAL 0A-5000 (manufactured by Sumika Analysis Center).

[0017]

【Example】

Example 1 DL-mandelic acid 2%, ammonium sulfate 0.2%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.0
100 ml of medium consisting of 5% yeast extract and 0.02% yeast extract (
(pH 7.0) in a 500 ml shake flask, and
Sterilized at 0°C for 10 minutes. In this medium, Pseudomonas
Polycolor (Pseudomonas polycolo)
r) One platinum loop of IFO 3918 was inoculated and incubated at 30°C for 7 days.
The culture was incubated with shaking for 2 hours. 1000 ml of the above culture solution was centrifuged to remove bacterial cells and obtain a supernatant. After adjusting the pH to 1.0 by adding hydrochloric acid to the supernatant, 2000 ml of ethyl acetate was added for extraction, and the ethyl acetate layer was separated and concentrated under reduced pressure.
- 8.78 g of mandelic acid was obtained as crude crystals. A solution of hexane:ethyl acetate = 1:1 was added to the crude crystals, and the mixture was heated and dissolved, then cooled and recrystallized to obtain 7.02 g of D-mandelic acid crystals. Optical rotation: -155.4° (C=2, H2O) Optical purity: 100%

Example 2 100 ml of the culture medium shown in Example 1 was inoculated with the microorganisms shown in Table 1 below, and after culturing with shaking at 30°C for 72 hours, the amount of D-mandelic acid remaining in the culture solution was quantified. The content of D-mandelic acid is as shown in Table 1 below, and its enantiomer L-mandelic acid was hardly detected in the culture solution.

[0019]

[Table 1]

Example 3 100 ml of a medium (pH 7.0) consisting of 0.5% DL-mandelic acid, 1.0% polypeptone, 1.0% yeast extract, and 0.5% sodium chloride was placed in a 500 ml shaking flask. , sterilized at 120°C for 10 minutes. Pseudomonas polycolor (Pseudomonas polycolor) was added to this medium.
One platinum loop of IFO 3918 (polycolor) was inoculated and cultured at 30°C for 20 hours. The above culture solution 100
Bacterial cells collected from 0 ml by centrifugation were suspended in physiological saline and further collected by centrifugation. DL to the bacterial cells
- 100ml of 50mM phosphate buffer containing 2g of mandelic acid
L-mandelic acid was completely decomposed by adding L-mandelic acid (pH 7.0) and carrying out an asymmetric decomposition reaction at 30° C. for 60 hours. After the reaction, bacteria were removed by centrifugation and treated in the same manner as in Example 1 to obtain 820 mg of D-mandelic acid. Optical rotation: -155.2° (C=2, H2O) Optical purity: 100%

Example 4 The bacteria shown in Table 2 below were inoculated into the culture medium shown in Example 3, and cultured at 30°C for 20 hours. The bacterial cells collected from 100 ml of the above culture solution by centrifugation were suspended in physiological saline and further collected by centrifugation. 20 ml of 50 mM phosphate buffer containing 400 mg of DL-mandelic acid (p
H7.0) was added, and the asymmetric decomposition reaction was carried out at 30°C for 60 hours. The content of D-mandelic acid in this reaction solution is shown in Table 2 below, and its enantiomer, L-mandelic acid, was hardly detected in the reaction solution.

[0022]

[Table 2]

Example 5 DL-mandelic acid 2%, ammonium sulfate 0.2%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.0
100 ml of medium consisting of 5% yeast extract and 0.02% yeast extract (
(pH 7.0) in a 500 ml shake flask, and
Sterilized at 0°C for 10 minutes. In this medium, Aspergillus
Aspergillus sydowi
One platinum loop of IFO 4284 was inoculated and incubated at 30°C for 9
The culture was incubated with shaking for 6 hours. 1000 ml of the above culture solution was centrifuged to remove bacterial cells and obtain a supernatant. After adjusting the pH to 1.0 by adding hydrochloric acid to the supernatant, 2000 ml of ethyl acetate was added for extraction, and the ethyl acetate layer was separated and concentrated under reduced pressure.
- 8.96 g of mandelic acid was obtained as crude crystals. A solution of hexane:ethyl acetate = 1:1 was added to the crude crystals, and the mixture was heated and dissolved, then cooled and recrystallized to obtain 7.17 g of L-mandelic acid crystals. Optical rotation: +155.2° (C=2, H2O) Optical purity: 100%

Example 6 Fusarium oxysporum was added to 100 ml of the medium shown in Example 1.
As a result of inoculating IFO 5942 and culturing with shaking at 30° C. for 96 hours, 244 mg of L-mandelic acid remained, and almost no D-mandelic acid, which is its enantiomer, was detected in the culture solution.

[0025]

[Effects of the Invention] The method of the present invention allows optically active mandelic acid to be obtained from inexpensive racemic mandelic acid very efficiently, and a product with 100% optical purity can be obtained.
This is an industrially advantageous manufacturing method.

Claims (8)

[Claims] Claim 1: A culture solution of a microorganism having the ability to asymmetrically decompose racemic mandelic acid, bacterial cells collected from the culture solution, or a treated product of the microorganism is applied to racemic mandelic acid to obtain one optically active compound. A method for producing optically active mandelic acid, which is characterized by separating and collecting the enantiomer after decomposing the body. Claim 2: Cultivating a microorganism capable of asymmetrically decomposing racemic mandelic acid in a medium containing racemic mandelic acid to decompose one optically active form, and then separating the enantiomer from the culture solution. A method for producing optically active mandelic acid, which comprises collecting . 3. Racemic mandelic acid is brought into contact with a culture solution of a microorganism having the ability to asymmetrically decompose racemic mandelic acid, bacterial cells collected from the culture solution, or a treated product of the bacterial cells in a solvent. A method for producing optically active mandelic acid, which comprises decomposing the optically active form of , and then separating and collecting the enantiomer from the reaction solution. 4. The method according to claim 1, 2 or 3, wherein the enantiomer to be separated and collected is D-mandelic acid. 5. The method according to claim 1, 2 or 3, wherein the enantiomer to be separated and collected is L-mandelic acid. 6. The method according to claim 1, 2 or 3, wherein the microorganism belongs to the genus Aspergillus, Fusarium, Gibberella, Brevibacterium, Pseudomonas, Rhodosporidium, or Rhodotorula. 7. The method according to claim 4, wherein the microorganism belongs to the genus Gibberella, Previbacterium, Pseudomonas, Rhodosporidium, or Rhodotorula. 8. The method according to claim 5, wherein the microorganism belongs to the genus Aspergillus or Fusarium.