JPH10127299A - Optically active glycidic ester and production of optically active glycidic ester - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an optically active glycidic ester used for a raw material, etc., for medicines, agrochemicals, etc., in high yield by treating a racemic glycidic ester in the presence of microorganisms such as the one belonging to genus corynebacterium having stereoselective hydrolytic open ring ability of the compound. SOLUTION: This optically active glycidic ester of formula II (a carbon atom with (*) represents the asymmetric carbon atom) and/or optically active glyceric ester of formula III is obtained by performing stereoselective hydrolytic ring opening of racemic glycidic ester of formula I (R is an alkyl, a cycloalkyl or an aryl) in the presence of a cultured material, microbial cell or a treated material of the microbial cell, of microorganisms such as the one belonging to genus corynebacterium, agrobacterium or aurebacterium and having stereoselective hydrolytic open ring ability of the compound in high yield and optical purity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing optically active glycidic acid esters and optically active glyceric acid esters which are useful as raw materials or intermediates for pharmaceuticals, agricultural chemicals and the like.

[0002]

2. Description of the Related Art As a method for synthesizing an optically active glycidic ester, there is a method for synthesizing via an optically active 3-halolactic acid. For example, there is a method in which optically active 3-chlorolactic acid obtained by stereoselectively reducing chloropyruvic acid with an enzyme is synthesized by ring closure with an alkali (J. Am. Chem. Soc., 104 , 4).
458-4460 (1982)).

Further, as a method for producing optically active 3-halolactic acid,
As described in JP-A-61-268197, a method of metabolizing and synthesizing only one enantiomer of racemic 3-halolactic acid using a microorganism, or as disclosed in JP-A-2-113890. In addition, there is known a method of synthesizing α, β-dihalopropionic acid by reacting 2-haloacid dehalogenase. However, the reaction using an enzyme as described above has a disadvantage that productivity is extremely low.

As a method for synthesizing an optically active glycidic acid ester, a method is also known in which α-halogenation is performed using serine as a raw material, followed by ring closure to synthesize (Japanese Patent Application Laid-Open No. H6-6539,
FR2609032) has a disadvantage that a special device is required because a strong acid is used in this method. In addition, this method also has a problem that measures against harmful nitric oxide gas are required.

Further, the racemic glycidic acid ester is
There is known a method of synthesizing an optically active glycidic ester by asymmetric resolution using an ester asymmetric hydrolase (Japanese Patent Application Laid-Open No. 5-276966). However, with this method, satisfactory values have not been obtained for both the yield and the optical purity.

On the other hand, as a method of synthesizing optically active glyceric acid ester, a method of synthesizing serine by reacting with nitrous acid is known (Chem. Phys. Lipids, 16 , 115-122 (1.
976)), but this method also has the same problem as in the above-mentioned synthesis of optically active glycidic acid ester. Also, a method of synthesizing optically active glycerate ester from mannitol in multiple steps (Tetrahedron Lett., 37 (3) , 355-356 (1996))
Are known, but the yield is extremely low.

[0007]

Accordingly, an object of the present invention is to provide an effective method for producing optically active glycidic acid ester and optically active glyceric acid ester, which are effective intermediates for producing pharmaceuticals and agricultural chemicals. is there.

[0008]

Means for Solving the Problems In order to solve the above problems, the present inventors have conducted intensive studies on a method for producing an optically active glycidic acid ester and an optically active glyceric acid ester. The present inventors have found a method of hydrating and opening only one enantiomer by the action of a microorganism cell or a processed product thereof, thereby completing the present invention. The present invention provides a compound represented by the general formula (1):

[0009]

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(Wherein, R represents an alkyl group, a cycloalkyl group or an aryl group), a racemic glycidate ester represented by the following formula:
m) a culture, cell or treatment of a microorganism belonging to the genus Agrobacterium or Aureobacterium and having the ability to stereoselectively hydrate and open glycidate esters; General formula (2), characterized by stereoselective hydration ring opening in the presence of a compound
:

[0011]

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(Wherein R is as defined above, and the carbon atom marked with * is an asymmetric carbon atom) and / or a general formula (3):

[0013]

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(Wherein R is as defined above, and the carbon atom marked with * is an asymmetric carbon atom). Hereinafter, the present invention will be described in detail.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION In the above general formulas (1) to (3),
The alkyl group represented by R is usually an alkyl group having 1 to 18 carbon atoms, and may have a linear or branched structure. Specific examples include methyl, ethyl, n-butyl, iso-butyl, tert-butyl, n-hexyl, n-octyl, stearyl and the like.

In the above general formulas (1) to (3), examples of the cycloalkyl group represented by R include a cyclohexyl group. In the above general formulas (1) to (3), examples of the aryl group represented by R include phenyl and benzyl.

The glycidic acid ester used as a raw material in the present invention may be synthesized by any method. For example, it can be easily synthesized by epoxidizing an acrylic acid ester which is a general-purpose industrial raw material.

The microorganism used in the present invention may be of the genus Corynebacterium, the genus Agrobacterium, or the ureobacterium.
Any microorganism can be used as long as it belongs to the genus (Aureobacterium) and has the ability to stereoselectively hydrate and open glycidate esters. Specifically, Corynebacterium sp. Strain N-1074 (FERM BP-2643), Agrobacterium sp. Strain DH079 (FERMP-11651), Aureobacterium sp. Strain DH095 (FERM P-12360) Is exemplified. The above-mentioned Corynebacterium sp. Strain N-1074, Agrobacterium sp. Strain DH079, and Aureobacterium sp.
The bacteriological properties of the DH095 strain are described in JP-A-2-291280, JP-A-4-94689, and JP-A-5-199665, respectively.

As the medium composition for culturing the above microorganisms, any medium can be used, so long as these microorganisms can normally grow. Examples of the carbon source include sugars such as glucose, sucrose and maltose; organic acids such as acetic acid and citric acid and salts thereof; and alcohols such as ethanol and glycerol. As the nitrogen source, for example, various inorganic and organic acid ammonium salts can be used in addition to general natural nitrogen sources such as peptone, meat extract, yeast extract and amino acids. In addition, inorganic salts, trace metal salts, vitamins and the like are appropriately used as needed.
The cultivation of the microorganism may be performed by a conventional method.
10. Incubate aerobically for 10 to 96 hours at a temperature of 20 to 40 ° C.

In the present invention, a culture obtained by culturing the above microorganisms in a culture medium may be used as it is, or a cell obtained by a cell collection operation such as centrifugation from the culture or a processed product thereof may be used. Can be used. Examples of the processed bacterial cells include crushed bacterial cells, crude enzyme, and purified cell enzymes such as purified enzymes. In addition, cells immobilized on a carrier by a conventional method or processed cells can also be used.

In order to stereoselectively hydrate and open the racemic glycidic acid ester, a racemic glycidic acid ester serving as a substrate is added to the above-mentioned culture or suspension of the cells or the treated cells. Performed by The method of adding the substrate is not particularly limited, but it can be added all at once or in portions during the reaction. Further, the substrate can be added after being dissolved in an appropriate organic solvent. Examples of the organic solvent include methanol, ethanol, acetone and the like. The reaction is usually carried out in a state in which the substrate is dissolved or dispersed in an aqueous solvent such as a culture solution or a buffer, and a suitable organic solvent such as methanol, ethanol, tert-butyl alcohol, acetone or the like which is compatible with water is used. It can be added to increase the solubility of the substrate, or the reaction can be performed in a two-phase system using an organic solvent such as toluene or ether.

The concentration of the substrate in the reaction solution is not particularly limited, and is usually 0.01 to 30% by weight. The reaction temperature is
Usually, it is 1 to 60 ° C, preferably 10 to 40 ° C. Further, the pH of the reaction solution is usually 4 to 10, preferably 6 to 8. An optically active glyceric acid ester is produced by the stereoselective hydration ring-opening reaction of the racemic glycidic acid ester. The remaining substrate is an optically active glycidic ester.

The optically active glyceric acid ester formed and the optically active glycidic acid ester as a residual substrate can be isolated from the reaction solution according to a conventional method. For example, known methods such as extraction, distillation and column chromatography can be used. It can be done by a method.

The optically active glycidic acid ester and the optically active glyceric acid ester can be easily hydrolyzed to the corresponding carboxylic acids by a conventional method, and glycidic acid and glyceric acid can also be converted to the corresponding esters. Since they can be converted, they can be applied according to the purpose of use of these compounds.

[0025]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. [Example 1] Glycerol 1%, peptone 0.5%, meat extract 0.3%, yeast extract 0.3%, KH ₂ PO ₄ 0.15% and
500 ml of 100 ml medium containing 0.3% K ₂ HPO ₄ (pH 7.2)
Dispensed into a conical flask. These were sterilized in an autoclave at 121 ° C. for 15 minutes, inoculated with the microorganisms shown in Table 1, and cultured with shaking at 30 ° C. for 2 days. The cells were collected from this culture by centrifugation, and the same amount of 50 mM KH ₂ PO ₄
After washing the cells twice with -Na ₂ HPO ₄ buffer (pH 7.7), 10 ml
In the same buffer. To 2 ml of this bacterial suspension, 2 ml of the above buffer and 20 μl of racemic glycidic acid ethyl ester were added, and the mixture was stirred at 20 ° C. to carry out a reaction. As the racemic glycidic acid ethyl ester, an acrylic acid ester synthesized by a conventional method in which an aqueous sodium hypochlorite solution was allowed to act in a two-phase system medium of an organic phase / aqueous phase was used. After the reaction, the cells were removed by centrifugation, and the reaction solution was analyzed by gas chromatography. Table 1 shows the residual ratio and optical purity of glycidic acid ethyl ester in the reaction solution.

[0026]

[Table 1]

Example 2 Agrobacterium sp. DH079 strain was cultured in the same manner as in Example 1 to prepare a bacterial suspension. To 15 ml of this bacterial suspension was added 15 ml of a 10% (w / v) glycidic acid ethyl ester / toluene solution, and the mixture was stirred at 20 ° C for 24 hours.
A time reaction was performed. After the reaction, the toluene phase was separated and dried over anhydrous sodium sulfate, and then toluene was distilled off using an evaporator. Thus, 0.41 g of glycidic acid ethyl ester was obtained. Further, glyceric acid ethyl ester was extracted from the aqueous phase with ethyl acetate (15 ml × 7 times), then dried over anhydrous sodium sulfate, and then ethyl acetate was distilled off with an evaporator and purified by silica gel column chromatography. Thus, 0.56 g of glyceric acid ethyl ester was obtained.

The optical rotation of the obtained glycidic acid ethyl ester and glyceric acid ethyl ester was measured. The results are shown below. Glycidic acid ethyl ester [α] _D ²⁴ : + 12.4 ° (neat) (R-form; literature value [α] _D ²⁰ : + 12.3 ° (c = 5.0, MeOH) Bu
ll. Soc. Chim. Fr. , 1 , 130-139 (1989)) glyceric acid ethyl ester [α] _D ²⁴ : -10.05 ° (neat)

The results of other instrumental analysis of glycidic acid ethyl ester and glyceric acid ethyl ester are as follows. Glycidic acid ethyl ester IR (cm ^-1 ): 2987, 1750, 1412, 1294, 1253, 1205,
1032 ¹ H-NMR (270 MHz, DMSO-d6) (δ ppm): 1.23 (t, 3H,
CH ₃ -CH ₂ -O), 2.90 (dd, 2H, CH ₂ -O), 3.50 (dd, 1H, CH-
O), 4.17 (q, 2H , O-CH 2 -CH 3) 13 C-NMR (270MHz, DMSO-d6) (δ ppm): 14.0 (CH 3),
45.8 (CH ₂ -O), 46.9 (CH-O), 61.1 (O-CH ₂ -CH ₃ ), 169.0 (C
= O) MS (EI) m / z 101 [M-CH ₃ ] ⁺

Glyceric acid ethyl ester IR (cm ^-1 ): 3384 (br), 2983, 1736, 1216, 1120 ¹ H-NMR (270 MHz, DMSO-d6) (δ ppm): 1.21 (t, 3H,
CH ₃ -CH ₂ -O), 3.57 (m, 2H, CH ₂ -O), 4.07 (m, 1H, CH-O),
4.10 (q, 2H, O-CH ₂ -CH ₃ ), 4.78 (t, 1H, CH ₂ -O), 5.28
(d, 1H, CH-O) ¹³ C-NMR (270 MHz, DMSO-d6) (δ ppm): 14.1 (CH ₃ ),
_{60.1 (O-CH 2 -CH 3} ), 63.7 (CH 2 -OH), 72.0 (CH-OH), 172.7
(C = O) MS (EI) m / z 116 [MH ₂ O] ⁺

[Example 3] A 50 mM solution was added to 1 ml of the bacterial suspension of Agrobacterium sp. DH079 prepared in the same manner as in Example 1.
1 ml of KH ₂ PO ₄ —Na ₂ HPO ₄ buffer (pH 7.7) and 10 μl of glycidic acid methyl ester were added and reacted at 20 ° C. for 2.5 hours. The residual ratio of glycidic acid methyl ester was 47.6%, and the optical purity was 93.0% ee (R form).

Example 4 50 ml of Agrobacterium sp. DH079 strain suspension prepared in the same manner as in Example 1 was added to 1 ml of the bacterial suspension.
3 ml of KH ₂ PO ₄ —Na ₂ HPO ₄ buffer (pH 7.7) and 20 μl of normal butyl glycidate were added, and the reaction was carried out at 20 ° C. for 4.5 hours. The residual ratio of normal butyl glycidate was 49.1%, and its optical purity was 97.8% ee (R
Body).

Example 5 A 2 mM suspension of Agrobacterium sp. DH079 prepared in the same manner as in Example 1
₆ ml of KH ₂ PO ₄ -Na ₂ HPO ₄ buffer (pH 7.7) and 10 μl of glycidic acid cyclohexyl ester were added, and the reaction was carried out at 20 ° C. for 2.5 hours. The residual ratio of glycidic acid cyclohexyl ester is 50.4%, and its optical purity is 93.4% ee (R
Body).

Example 6 100 μl of toluene containing 10% by weight of benzyl glycylate was added to 1 ml of the bacterial suspension of Agrobacterium sp. DH079 prepared in the same manner as in Example 1.
Was added and shaken at 30 ° C. for 1 hour. As a result of analysis by gas chromatography, the residual ratio of glycidic acid benzyl ester was 64.3%, and the optical purity of the produced glyceric acid benzyl ester was 100% ee (S form).

[0035]

According to the present invention, an optically active glycidic acid ester and an optically active glyceric acid ester, which are effective optically intermediates for the production of medicines, agricultural chemicals, etc., can be produced with high yield and high optical purity. Can be.

 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Ryuichi Endo 10-1 Ogurocho, Tsurumi-ku, Yokohama-shi, Kanagawa Nippon Kagaku Kogyo Co., Ltd. 1-15 No. 203

Claims (1)

[Claims] [Claim 1] General formula (1): (Wherein, R represents an alkyl group, a cycloalkyl group or an aryl group) by adding a racemic glycidate ester represented by the genus Corynebacterium, the genus Agrobacterium or the ureobacterium ( (Aureobacterium), which is capable of stereoselectively hydrating and opening in the presence of cultures, microbial cells, or processed cells of microorganisms capable of stereoselectively hydrating and opening glycidic acid esters General formula (2): (Wherein, R is as described above. The carbon atom marked with * is an asymmetric carbon atom.) And / or an optically active glycidic acid ester represented by the general formula (3): (In the formula, R is as described above. The carbon atom marked with * is an asymmetric carbon atom.) A method for producing an optically active glycerate ester represented by the formula: