JPH0751072B2 - Method for producing (R)-(+)-γ-butyrolactone-γ-3-propionic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention utilizes a culture of a microorganism, its bacterial cells or pig river esterase and has a high optical purity (R)-(+).
-Γ-butyrolactone-γ-3-propionic acid

[Prior Art] In recent years, the synthesis of physiologically active substances, fragrances, and the like has been actively studied, but substances having optical activity have been attracting attention as important intermediate raw materials when they are synthesized. It is generally known that the activity of physiologically active substances, fragrances and the like is greatly reduced when optical isomers are present, and therefore the optical purity of the optically active substance that can be used as a raw material must be high. That is, in the case of the same compound, it is desired that the compound has a high specific light rotation.

By the way, (R)-(+)-γ which is the target substance of the present invention
-Butyrolactone-γ-3-propionic acid can be a raw material for various organic compounds, but is particularly useful as a raw material for physiologically active substances, fragrances, or the precursor thereof, and examples thereof include the following. .

(1) Intermediate raw material of chasmolactone (main constituent of jasmine oil, which is a natural fragrance) (see Fragrance Journal, Vol. 77, pp. 124-129 (1986)).

(2) Ingredients for erdanolide, an insect pheromone (Agricultural Biological Chemistry (Ag
ric.Biol.Chem.), 49: 2775-2776 (1986)).

(3) Raw material of steganodon, a drug for leukemia (Tetrahedoron Letters
s), 21: 2709 (1980)).

(4) A beetle pheromone (see JP-A-59-157055).

Conventionally, (R)-(+)-γ-butyrolactone-γ-
As a method for producing 3-propionic acid, a method by D. Pirillo et al. (I1.Farmaco.Ed.Sc.
i.), 40, pp. 623-629 (1985)), but the specific illumination of the obtained target is ▲ [α] ²² _D ▼ = +35.
Low as 5 ° (c = 1.00, chloroform)
Japanese Patent Publication No. 205270-A also discloses a method for producing (R)-(+)-γ-butyrolactone-γ-3-propionic acid, but the specific irradiance is ▲ [α] ²⁵ _D ▼ = + 21.6 °. (C = 0.66, H
_Since it is as low as ₂ O), it has a drawback that it is difficult to use as a raw material for physiologically active substances and fragrances.

[Problems to be Solved by the Invention] In the present invention, (R)-(+)-γ-butyrolactone-γ-3
-An object of the present invention is to provide a method for producing propionic acid easily, in high yield and with high optical purity.

[Means for Solving Problems] However, as a result of intensive studies in view of the problems of the prior art, the present inventors have found that (R)-(+)-γ using a specific microorganism.
It was found that (R)-(+)-γ-butyrolactone-γ-3-propionic acid can be obtained in high yield and high optical purity by hydrolyzing -butyrolactone-γ-3-propionic acid ester. The invention was completed.

That is, the present invention has the general formula (I): (In the formula, R represents a lower alkyl group having 1 to 4 carbon atoms excluding t-butyl group) (R)-(+)-γ-
Butyrolactone-γ-3-propionic acid ester was used in the genus Chromobacterium, Flavobacterium, Pseudomonas.
monas) and a culture of a microorganism belonging to the genus selected from the group consisting of the genus Bacillus (Bacillus), by contacting the bacterial cells or pig river esterase,
General formula (II): (R)-(+)-γ-butyrolactone-γ-
(R) -characterized by leading to 3-propionic acid
It relates to a method for producing (+)-γ-butyrolactone-γ-3-propionic acid.

[Action and Example] The compound represented by the general formula (I) (hereinafter, referred to as compound (I)) used as a starting material of the present invention is, for example,
Although it can be obtained by the following series of steps already described in the specification filed by the present inventors (see Japanese Patent Application No. 61-206435 and Japanese Patent Application No. 61-229260). Of course, the starting material may be obtained by any method.

(In the formula, R is the same as above) The compound represented by the general formula (II) (hereinafter referred to as compound (II)), which is the target compound of the present invention, is obtained by converting the starting compound (I) into a microorganism culture, It can be obtained by contacting the cells or pig river esterase by an appropriate method. Here, when a compound in which R of compound (I) is a t-butyl group is used instead of compound (I) as a starting material, the compound cannot be hydrolyzed by microorganisms.
Further, it is not practical because it is difficult to synthesize a compound in which R is an alkyl group having 5 or more carbon atoms. In addition, compound (II)
When is hydrolyzed with acid, the lactone ring is hydrolyzed at the same time as the ester group to give a racemate.

Examples of the above-mentioned microorganism include microorganisms having a strong esterase activity, such as Pseudomonas diminu
ta, IFO13181, 13182), Chromobacterium chocolatum (IFO3758), Flavobacterium lutes (Flavobacterium lutes
cens, IFO3084), Pseudomonas dinitrificans (IFO13302), Bacillus pumilus (IFO3813), and the like.

In the present invention, the culturing of the microorganism is carried out by the usual culturing method of bacteria. For example, cells are grown by shaking or stirring culture for a certain period of time in a medium containing meat extract, glucose, casein degradation product and the like. The culturing temperature is usually 20 to 50 ° C, preferably 25 to 40 ° C, and the culturing time is usually 1 to 120 hours, preferably 2 to 72 hours.
The cells are separated from the obtained culture by an operation such as centrifugation.

The object of the present invention is achieved by contacting a starting material, compound (I), with a culture of a microorganism, its cells or pig river esterase by a suitable method. Examples of the method of contacting here include the methods described below. That is, when the culture is used as it is, the pH is 5 to 7, preferably 6 to 7 with an acid or alkaline solution.
After adjusting to pH, and when using bacterial cells or pig river esterase, the pH is adjusted to 5 to 7, preferably 6
After suspending in a buffer solution of ~ 7, compound (I) as a starting material is added and stirred. As a method other than the above, commonly used immobilization carriers such as color ginnan, collagen, alginic acid, and agar, or urethane-based, PVA-based, superabsorbent resin, synthetic polymers such as photocurable resin, the above-mentioned microorganism The same purpose can be achieved by immobilizing the culture, its cells or pig river esterase by a suitable method. The reaction time is usually 1 to 200 hours, preferably 1 to 120 hours. If it is less than 1 hour, a considerable amount of the raw material remains unreacted, and if it exceeds 200 hours, the enzyme is deactivated and the reaction rate is remarkably high. Tends to decline. The reaction temperature is usually 20 to 50 ° C., preferably 25 to 35 ° C. When the temperature is lower than 20 ° C., the reaction rate is slow and not practical, and when it exceeds 50 ° C., the enzyme is inactivated significantly and the yield is high. Tends to decline.

The end point of the reaction is confirmed and determined by TLC (thin-layer chromatography). After removing the cells by a method such as centrifugation, the supernatant liquid is adjusted to pH 7 to 7.5, and the unreacted compound is diluted with an organic solvent such as ethyl acetate. Extract (I). Then, the aqueous solution
The compound (II) can be obtained by adjusting the pH to 3 to 4, extracting again with an organic solvent, drying and distilling off the solvent.

Hereinafter, the present invention will be described in more detail with reference to Examples and Examples, but the present invention is not limited thereto.

Reference example 1 After preparing 500 ml of the reaction solution having the composition shown in Table 1 in a 1-volume flask and adjusting the pH to 6.8 with a dilute aqueous sodium hydroxide solution, Saccharomyces cervidiae (Saccharomyces
cerevisiae, IFO2044) (Oriental Yeast Co., Ltd.)
20 g, 4-oxopimelic acid monoethyl ester 5.0
g was added and reacted at 30 ° C. for 72 hours. After completion of the reaction, cells were removed by centrifugation and the supernatant was cooled at 0-5 ° C.
The pH was adjusted to 2.0 and the mixture was extracted 3 times with 100 ml of ethyl acetate. The ethyl acetate layer was dried over sodium sulfate and concentrated under reduced pressure to give an oily crude (R)-(+)-γ-butyrolactone-γ-3.
-3.0 g of ethyl propionate were obtained. This was purified by silica gel column chromatography (eluent: chloroform / ethyl acetate = 8/2 (volume ratio)) and further purified by T
Purified by LC, reverse phase chromatography (C ₁₈ column, 0.01% phosphoric acid aqueous solution (pH 3) / methanol = 7/3 (volume ratio)), and 100% pure (R)-(+)-γ-butyrolactone- 2.5 g of γ-3-propionic acid ethyl ester was obtained (yield 54.0%). The relative light intensity is ▲ [α] ²³ _D ▼ ＝ ＋ 59.6 ° (c
= 1.01, chloroform). ^The analysis results by ¹ H-NMR and IR are shown below. ¹ H-NMR (300 MHz, CDCl ₃ ) δ (ppm): 1.27 (s, 3H), 1.92 (m, 3H), 2.34 (m, 1H), 2.47 (m, 4H), 4.11 (q, 2H), 4.48 (m, 1H), IR (neat) (cm ^-1 ): 3000, 2950, 1780, 1740, 1380, 1350, 1260, 1180, 10
50 Reference Example 2 The same operation as in Reference Example 1 was carried out except that 5.0 g of 4-oxopimelic acid monomethyl ester was used instead of 5.0 g of 4-oxopimelic acid monoethyl ester, and 100% pure (R)-(+) was used. 1.1 g of -γ-butyrolactone-γ-3-propionic acid methyl ester was obtained (yield 24.0%). The specific optical rotation was ▲ [α] ²⁵ _D ▼ = + 61.79 ° (c = 0.81, chloroform). ^The analysis results by ¹ H-NMR and IR are shown below. ¹ H-NMR (300 MHz, CDCl ₃ ) δ (ppm): 1.65 to 1.92 (m, 3H) 2.15 to 2.40 (m, 5H), 3.52
(S, 3H), 4.35 (m, 1H) IR (neat) (cm ^-1 ): 2950, 1780, 1760, 1440, 1350, 1260, 1180, 1050 Reference Example 3 4-oxopimelic acid monoethyl ester 5.0 g The same operation as in Reference Example 1 was carried out except that 5.0 g of 4-oxopimelic acid monopropyl ester was used instead, and the purity was 100%.
(R)-(+)-γ-butyrolactone-γ-3-propionic acid propyl ester (1.2 g) was obtained (yield 26.0%).
The specific optical rotation was ▲ [α] ²⁵ _D ▼ = + 53.31 ° (c = 1.24, chloroform). ^The analysis results by ¹ H-NMR and IR are shown below. ¹ H-NMR (300 MHz, CDCl ₃ ) δ (ppm): 0.85 (t, 3H), 1.52 (m, 2H), 1.70 to 1.93 (m, 3)
H), 2.20 ~ 2.42 (m, 5H), 3.92 (t, 2H), 4.40
(M, 1H) IR (neat) (cm ^-1 ): 3500, 2950, 1780, 1740, 1460, 1420, 1350, 1270, 11
80, 1050, 960 Reference Example 4 The same operation as in Reference Example 1 was performed except that 5.0 g of 4-oxopimelic acid monobutyl ester was used in place of 5.0 g of 4-oxopimelic acid monoethyl ester, and the purity of 100% (R )-(+)-Γ-butyrolactone-γ-3-propionic acid butyl ester (1.5 g) was obtained (yield 32.0%). The specific optical rotation was ▲ [α] ²⁵ _D ▼ = + 49.76 ° (c = 1.04, chloroform). ^The analysis results by ¹ H-NMR and IR are shown below. ¹ H-NMR (300 MHz, CDCl ₃ ) δ (ppm): 0.85 (t, 3H), 1.28 (m, 2H), 1.50 (m, 2H), 1.
70 ~ 1.95 (m, 3H), 2.20 ~ 2.42 (m, 5H), 3.95
(M, 2H) 4.39 (m, 1H) IR (neat) (cm ^-1 ): 2960, 1780, 1740, 1460, 1420, 1360, 1260, 1180, 10
50, 960 Example 1 0.2M phosphate buffer (pH 7.0) 100m in a 200ml flask
and 270 units of pig river esterase (manufactured by Sigma) were added, and then (R)-(+)-γ-obtained in Reference Example 1 was added.
Butyrolactone-γ-3-propionic acid ethyl ester
5 g was added, and the mixture was stirred at 30 ° C. for 2 hours while adjusting the pH to 7.0 with potassium hydroxide solution.

After confirming the completion of the reaction by TLC, the pH was adjusted to 3.0 with hydrochloric acid under ice cooling, and the mixture was extracted 4 times with 30 ml of ethyl acetate. The ethyl acetate layer was dried over sodium sulfate and then evaporated under reduced pressure,
White crystals were obtained by cooling. Further, the crystal was recrystallized from benzene to obtain 3.2 g of 100% pure γ-butyrolactone-γ-3-propionic acid (yield 75%). The obtained compound has a specific irradiance of ▲ [α] ²⁵ _D ▼ ＝ ＋ 60.96 ° (c
= 0.976, H ₂ O) (R)-(+)-γ-butyrolactone-γ-3-propionic acid. The melting point is 54.5-55.
It was ℃. ^The analysis results by ¹ H-NMR and IR are shown below. ¹ H-NMR (300 MHz, CDCl ₃ ) δ (ppm): 1.80 to 2.05 (m, 3H), 2.30 to 2.6 (m, 5H), 4.53
(M, 1H), 9.25 (broad s, 1H) IR (KBr) (cm ^-1 ): 3190, 1740, 1720, 1460, 1440, 1420, 1380, 1360, 13
10, 1260, 1230, 1200, 1160, 1080, 1040, 960, 920 (R)-(+)-γ-butyrolactone-obtained here
¹ H-NMR and IR spectra of γ-3-propionic acid and (±) -γ-butyrolactone-γ-3-propionic acid, which can be obtained only by an organic chemical method, were in agreement.

Example 2 Instead of 5 g of (R)-(+)-γ-butyrolactone-γ-3-propionic acid ethyl ester obtained in Reference Example 1, (R)-(+)-γ obtained in Reference Example 2 -Butyrolactone-γ-3-propionic acid methyl ester 1 g was used, and the same operation as in Example 1 was performed except that the reaction time was changed to 30 minutes, and (R)-(+)-γ-butyrolactone having a purity of 100% was used. 0.45 g of -γ-3-propionic acid was obtained (yield 53%).
The specific optical rotation of this compound was ▲ [α] ²⁵ _D ▼ = + 59.76 ° (c = 0.900, H ₂ O), and the other analytical results were in agreement with those of Example 1.

Example 3 Instead of 5 g of (R)-(+)-γ-butyrolactone-γ-3-propionic acid ethyl ester obtained in Reference Example 1, (R)-(+)-obtained in Reference Example 3 Using the same procedure as in Example 1 except that 1 g of γ-butyrolactone-γ-3-propionic acid propyl ester was used and the reaction time was changed to 1 hour, 100% pure (R)-(+)-γ -Butyrolactone-γ-3-propionic acid 0.59 g was obtained (yield 69%). The specific irradiance of this compound is ▲ [α] ²⁵ _D ▼ =
At + 60.02 ° (c = 0.985, H ₂ O), the other analytical results were in agreement with those of Example 1.

Example 4 Instead of 5 g of (R)-(+)-γ-butyrolactone-γ-3-propionic acid ethyl ester obtained in Reference Example 1, (R)-(+)-γ obtained in Reference Example 4 -Butyrolactone-γ-3-propionic acid butyl ester was used in the same manner as in Example 1 except that 1 g of (R)-(+)-γ-butyrolactone-γ-3-propionic acid having a purity of 100% was used. g was obtained (yield 66%). The specific irradiance of this compound is ▲ [α] ²⁵ _D ▼ = + 60.56 ° (c = 1.01, H ₂ O)
The other analysis results were in agreement with those of Example 1.

Example 5 Pseudomonas diminuta (IFO13181) cell-platinum loops cultured in a peptone meat extract agar slant medium at 30 ° C. for 48 hours were dispensed with 10 ml of a liquid medium having the composition shown in Table 2 2 × 18.
After inoculating a cm test tube, shaking culture was carried out at 30 ° C. for 24 hours to prepare a seed culture solution.

Next, 1 ml of this inoculum solution was inoculated into a 500 ml Sakaguchi flask into which 100 ml of the liquid medium having the composition shown in Table 2 was dispensed,
The cells were cultured at 30 ° C for 24 hours. The obtained bacterial cells were collected by centrifugation and suspended in 50 ml of 0.2 M phosphate buffer (pH 7.0), and then (R)-(+)-γ-butyrolactone-obtained in Reference Example 1 was collected. 1 g of γ-3-propionic acid ethyl ester was added, and while adjusting the pH to 7.0 with potassium hydroxide solution, 3
The reaction was carried out at 0 ° C for 5 hours. After confirming the completion of the reaction by TLC, the cells were removed by centrifugation, and the unreacted (R)-(+)-γ-butyrolactone-γ-3-propionic acid ethyl ester was extracted with ethyl acetate from the supernatant. Then, the aqueous layer was adjusted to pH 3.0 with dilute hydrochloric acid under ice cooling and extracted with ethyl acetate. The ethyl acetate layer was dried over sodium sulfate, evaporated under reduced pressure, and cooled to give white crystals. Further, the crystal was recrystallized from benzene to obtain a purity of 10
0.57 g of 0% γ-butyrolactone-γ-3-propionic acid
Was obtained (yield 67%). This compound has a specific irradiance of ▲
[Α] ²⁵ _D ▼ = + 60.75 ° (c = 0.980, H ₂ O) (R)-
(+)-Γ-butyrolactone-γ-3-propionic acid, and the other analysis results were in agreement with those of Example 1.

Example 6 Chromobacterium chocolatum (IFO3758) was used in place of Pseudomonas diminuta (IFO13181), and the culture time was changed to 36 hours. The same operation as in Example 5 was carried out to obtain 0.22 g of (R). -(+)-Γ-butyrolactone-γ-3-propionic acid was obtained (yield 26%). The specific rotation of this compound is ▲ [α] ²⁵ _D ▼ = + 59.86 ° (c = 1.0
5, H ₂ O) and other analytical results were in agreement with those of Example 1.

Example 7 Pseudomonas diminuta (IFO13181) was replaced with Flavobacterium lutesense (IFO3084) and the culture time was changed to 36 hours. The same operation as in Example 5 was carried out to obtain 0.29 g of (R). -(+)-Γ-butyrolactone-γ-3-propionic acid was obtained (yield 34%). The specific irradiance of this compound is ▲ [α] ²⁵ _D ▼ = + 59.97 ° (c = 0.9
8, H ₂ O) and other analytical results were in agreement with those of Example 1.

Example 8 The same procedure as in Example 5 was repeated except that Bacillus pumilus (IFO3813) was used instead of Pseudomonas diminuta (IFO13181) and the reaction time was changed to 12 hours.
g (R)-(+)-γ-butyrolactone-γ-3-propionic acid was obtained (yield 22%). The specific optical rotation of this compound was ▲ [α] ²⁵ _D ▼ = + 59.76 ° (c = 0.975, H ₂ O), and the other analytical results were in agreement with those of Example 1.

Example 9 Pseudomonas diminuta (IFO131 cultured in Example 5
The bacterial cell-platinum loop of 81) was dispensed with 10 ml of 3.7% Brain Heart Infusion (manufactured by Difco) liquid medium 2
It was inoculated into a × 18 cm test tube and cultured at 30 ° C. for 24 hours. 1 ml of this inoculum solution was inoculated into a 500 ml Sakaguchi flask into which 100 ml of the Brain Heart Infusion medium was dispensed, and cultured at 30 ° C for 24 hours. The obtained cells were collected by centrifugation, reacted in the same manner as in Example 5, and extracted and purified to give 0.50 g of (R)-(+)-γ-butyrolactone-γ-3-propion. Acid was obtained (yield 59
%). The specific irradiance of this compound is ▲ [α] ²⁵ _D ▼ ＝ ＋ 60.16
(C = 1.10, H ₂ O), and other analytical results were in agreement with those of Example 1.

[Effect of the Invention] According to the present invention, there is an effect that (R)-(+)-γ-butyrolactone-γ-3-propionic acid can be easily synthesized with high yield and high optical purity.

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Claims (1)

[Claims] 1. General formula (I): (In the formula, R represents a lower alkyl group having 1 to 4 carbon atoms excluding t-butyl group) (R)-(+)-γ-butyrolactone-γ-
A culture of a microorganism belonging to the genus selected from the group consisting of the genus Chromobacterium, the genus Flavobacterium, the genus Pseudomonas and the genus Bacillus, wherein 3-propionate is
By bringing them into contact with the cells or pig river esterase, the compound of the general formula (II): (R)-(+)-γ-butyrolactone-γ-
(R) -characterized by leading to 3-propionic acid
A method for producing (+)-γ-butyrolactone-γ-3-propionic acid.