JPH0630597B2 - Method for producing (R) -γ-butyrolactone-γ-propionic acid ester - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing (R) -γ-butyrolactone-γ-propionic acid ester using a microorganism.

[Problems to be Solved by Conventional Techniques and Inventions] In recent years, the synthesis of physiologically active substances has been actively studied.
Compounds having optical activity have been attracting attention as important intermediate raw materials in the synthesis of these physiologically active substances. However, the compound usually obtained is a racemate containing 1-form of levorotatory and d-form of dextrorotatory in a ratio of 1: 1. Therefore, optical resolution is performed to obtain an optically active compound. Therefore, it is extremely difficult to synthesize a compound having optical activity. Conventionally known methods for such optical resolution include a crystallization method, a diastereomer method, and an optically active column chromatography method, but all of them are used as intermediate raw materials because they require expensive reagents. Has cost problems.

The optically active (R) -γ-butyrolactone-γ-propionic acid ester (I), which is the object compound of the present invention, is important as an intermediate raw material for the useful substances shown below.

For example, in the case of perfume, γ-jasmolactone, which is the main scent component of natural jasmine oil (Oil Chemistry, Vol. 29, No. 3
(0), pages 196-198) and Tuberose (Fragrance Journal, NO
77 (1986), pp. 124-129) and is useful as an important intermediate raw material.

In addition, erdanolide (Agric Cellular Biological Chemistry) 49, 2775 to 2776, 198, which is a male pheromone of African sugar cane gliding insect, is used.
6) or 4-hexanolide, which is a pheromone of the cuticle beetle (Tetrahedron 41, 919, 1980)
It is important as an important intermediate raw material when synthesizing insect pheromones such as.

Furthermore, Steganodon (Tetrahedron Letters) 21, 2709, which is a drug for leukemia,
It is also useful as an intermediate raw material when synthesizing (1980)).

Conventionally, as a method for producing (R) -γ-butyrolactone-γ-propionic acid ester or its analog, a chemical method and a biochemical method are known.

As a chemical method, for example, a method of chemically reducing 4-oxopimelic acid diester to form a lactone ring is known (Journal of the American Chemical Society (j. Am.Che.
m.Soc.) 1985, 107, 6404-6406).

However, since the lactone obtained by this method is a racemate, optical resolution is required to obtain an optically active lactone, which is practically disadvantageous. Piriro as an alternative
(Pirillo) reports that the optical purity is not sufficient (I, farmaco Ed. Sci. 40, 623-629 (1985)).

Γ-butyrolactone-γ-propionic acid (Journal of the American Chemical Society,
1985, 107, 6404-6406) are known, but in this case, a severe reaction condition of -78 ° C is required and, in addition, the optical purity decreases due to scale-up, which is not practical. ing. Other examples of similar compounds include lactone carboxylic acids derived from L-glutamic acid (Organic Syntheses 63, 12
1; Synthesis 1981, 265) is known, but in this case, the chemical reaction conditions are severe due to the use of nitrous acid.

On the other hand, the biochemical method has an advantage that the reaction condition is milder than that of the chemical method. Further, in the asymmetric reduction of ketone, there is a feature that it has enantioselectivity and a compound with high optical purity can be obtained.

An example of the biochemical method is a method of reducing the ketone of 4-oxopimelic acid diester with baker's yeast. However, in this method, since the bulkiness of the substituents on both sides of the plane of symmetry centering on the ketone at the 4-position is the same, asymmetric reduction is not carried out, and the obtained compound is a racemate. Therefore, in order to obtain an optically active lactone from a racemate, an operation of optical resolution is required, which is not advantageous.

In addition, asymmetric reduction of keto acids by utilizing the asymmetric reduction function of microorganisms has been proposed (Appl. Microbiol. Chem. 11, 38).
9, 1963). According to this method, an optically active γ-butyrolactone can be obtained, but it is not practical because the side chain is an alkyl group and has no functional group.

As described above, none of the conventional methods can be said to be a method for producing (R) -γ-butyrolactone-γ-propionic acid ester under mild reaction conditions, high yield and high optical purity.

As described above, there has been a long-felt demand for a production method capable of easily synthesizing (R) -γ-butyrolactone-γ-propionic acid ester under mild reaction conditions with high yield and high optical purity.

[Means for Solving Problems] The present inventors have found problems that cannot be solved by conventional techniques, that is, (R) -γ-, which has a high yield and high optical purity under mild reaction conditions. As a result of repeated studies to develop a method for producing butyrolactone-γ-propionic acid ester, focusing on the advantages of the biochemical method, 4-oxopimelic acid diester as a half ester, and then using a specific microorganism By performing reduction (R)
The inventors have found that -γ-butyrolactone-γ-propionic acid ester can be obtained, and have completed the present invention.

That is, the present invention has the general formula (II): (In the formula, R represents a C ₁ -C ₄ lower alkyl group)
The 4-oxopimelic acid half ester represented by the formula is neutralized with an aqueous alkaline solution, and then the obtained compound is Saccharomyces cerevisiae.
e), in contact with Hansenula anomala or Candida albicans to give the general formula (I): (Wherein R is the same as described above) leads to (R) -γ-butyrolactone-γ-propionic acid ester (R) -γ-butyrolactone-γ represented by the general formula (I). -A method for producing a propionate ester.

[Operations and Examples] The general formula (II) as a starting material of the production method of the present invention: (In the formula, R represents a C ₁ -C ₄ lower alkyl group)
Can be obtained, for example, by the following steps.

In the first-step reaction, furfural is used as a starting material, and malonic acid is dehydrated and condensed in the presence of ammonia or a base such as a primary or secondary amine, preferably pyridine to obtain furfuryl acrylic acid. In the second-stage reaction, 4-oxopimelic acid diester is obtained by dissolving the _first- stage reaction product in a C ₁ -C ₄ alcohol and then blowing in hydrogen chloride gas. By appropriately selecting and using the alcohols used here, ester chemicals corresponding to each are obtained (Journal of the American Chemical Society, 78, 3425, 1958). The method for synthesizing 4-oxopimelic acid diester is not limited to the above method, and any method can be used as long as the target chemical can be obtained.

Considering the method for converting the 4-oxopimelic acid diester thus obtained into a half ester, it is difficult to selectively synthesize the half ester by hydrolysis as a chemical method, and a separation operation is required later. To do. However, pig liver esterase or microorganisms having a strong esterase activity, such as Pseudomonas diminuta IFO 13181 and 13182, Achromobacter Iyticus IFO 1
2725 and 12726, Chromobacterium cocoratum
(Chromobacterium chocolatum) IFO 3578, Flavobacter ium lutescents I
The half ester (II) of 4-oxopimelic acid can be synthesized by contacting FO 3084 and 3085.

The compound represented by the general formula (II) thus obtained is neutralized with an alkaline aqueous solution such as an aqueous solution of sodium hydroxide, sodium hydroxide or ammonia.
By bringing the following microorganisms into contact with the obtained neutralized product, (R) -γ-butyrolactone-γ-propionic acid ester, which is the object compound of the present invention, can be obtained.

The microorganism used in the present invention is (R) -γ-butyrolactone-γ-propionic acid ester of the present invention, which is Saccharomyces cerevisiae, Hansenula anomala or Candida albicans. For production, first, the above-mentioned microorganism is inoculated into an appropriate medium and cultured. Culturing may be carried out by a usual yeast culturing method, for example, potato, sucrose,
The cells are grown by shaking or stirring culture for a certain period of time in a medium containing glucose, casein degradation product and the like. Alternatively, after the cells are grown, the cells may be separated by an operation such as centrifugation, and the starting material may be added to the cells together with the carbon source, water, etc., and the cells may be further cultured. The method of contacting the cells with the starting material may be any method as long as it does not significantly damage the cells,
For example, well-known immobilized carriers such as carrageenan, collagen, alginic acid and agar, urethane type, PVA
It can also be used after being immobilized on a synthetic polymer such as a resin, a superabsorbent resin, a photocurable resin or the like by an appropriate method.

The culturing temperature is usually about 28 to 37 ° C, preferably about 30 ° C, and the culturing time is usually about 1 to 120 hours, preferably 2 to 9 hours.
About 6 hours is appropriate.

The obtained culture is passed through, for example, Celite, the solution is adjusted to pH 7 to 10, and the unreacted starting material is extracted with an organic solvent in a usual manner. Then, the aqueous layer is adjusted to pH 2 to 4, extracted again with an ordinary organic solvent, dried and the solvent is distilled off to obtain crude (R) -γ-butyrolactone-γ-propionic acid ester. The obtained crude (R) -γ-butyrolactone-γ-propionic acid ester can be directly purified by silica gel chromatography or reverse phase chromatography, or by distillation.

Next, 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 Pseudomonas diminuta IFO 13181 cells cultivated in a peptone meat extract agar slant medium at 30 ° C for 48 hours were inoculated into a 2 x 18 cm test tube into which 10 m of medium having the composition shown in Table 1 was dispensed. Then, shaking culture was carried out at 30 ° C. for 24 hours to prepare an inoculum solution.

A 500 m Sakaguchi flask into which 100 m of the same liquid medium as the above composition was dispensed was inoculated with 1 m of the above inoculum solution and cultured at 30 ° C for 24 hours. The cells obtained by shaking culture for a total of 700 m were collected by centrifugation, suspended in 350 m of 0.2 M phosphate buffer (pH 8.2), and 17.5 g of 4-oxopimelic acid diethyl ester was added, and the temperature was adjusted to 30 ° C. And reacted for 72 hours. After the reaction was completed, the pH was adjusted to 9.0 with KOH, and unreacted 4-oxopimelic acid diethyl ester was extracted and removed.
The pH was adjusted to 2.0 with 1 and extracted with ethyl ether. The ether layer was dried over sodium sulfate and evaporated under reduced pressure to give 12.5 g of 4-oxopimelic acid monoethyl ester. This ester was almost pure from NMR and TLC and was used as such in the next reaction without purification.

Reference Example 2 4-oxopimelic acid diethyl ester 17.5 g was replaced with 4-oxopimelic acid dimethyl ester 10.0 g, and the same operation as in Reference Example 1 was carried out to obtain 4-oxopimelic acid monomethyl ester 5.6 g. This ester has NMR and
Almost pure than TLC.

Reference Example 3 The same operation as in Reference Example 1 was performed except that 17.5 g of 4-oxopimelic acid diethyl ester was replaced with 20.0 g of 4-oxopimelic acid dipropyl ester to obtain 13.2 g of 4-oxopimelic acid monopropyl ester. The ester was almost pure by NMR and TLC.

Reference Example 4 4-oxopimelic acid diethyl ester 17.5 g was replaced with 4-oxopimelic acid dibutyl ester 25.0 g, and the same operation as in Reference Example 1 was carried out to obtain 4-oxopimelic acid monobutyl ester 15.5 g. This ester has NMR and T
Almost purer than LC.

Example 1 5.0 g of the substrate obtained in Reference Example 1 was neutralized in a flask of Example 1 with a dilute aqueous potassium hydroxide solution, and sucrose 80 was added.
g, potassium dihydrogen phosphate 1.0 g, magnesium sulfate 0.5
g, calcium carbonate 2.5 g, ammonium sulfate 1.0 g and water 500 m, and after adjusting the pH to 6.8 with dilute potassium hydroxide aqueous solution, Saccharomyces cerevisiae-IFO 2044 (manufactured by Oriental Yeast Co., Ltd.) 15 g was added at 30 ° C. The reaction was carried out for 2 days. After the reaction is complete, the cells are removed by centrifugation,
The supernatant was adjusted to pH 2 under ice cooling and extracted 3 times with 100 m of ethyl acetate. The ethyl acetate layer was dried over sodium sulfate and concentrated under reduced pressure to obtain crude (R) -γ-butyrolactone-
An oily product of γ-propionic acid ethyl ester was obtained. This was purified by silica gel chromatography (chloroform / methanol = 20/1) and TLC, reverse phase chromatography (C ₁₈ column, 0.01% phosphoric acid aqueous solution / methanol = 7 /).
In (3), 2.5 g of (R) -γ-butyrolactone-γ-propionic acid ethyl ester having a purity of 100% was obtained. This product has a specific luminous intensity ▲ [α] ²³ _D ▼ ＝ ＋ 59.6 (C = 1.01, chloroform)
(R) -γ-butyrolactone-γ-propionic acid ethyl ester of D. PIRILLO et al.
40 , 623-629, 1985, the literature value is ▲ [α] ²⁰ _D ▼ ＝ ＋ 5
3.8 ° (C = 1, chloroform)).

Example 2 5.0 g of the substrate obtained in Reference Example 1 and 5.0 g of the substrate obtained in Reference Example 2 were used, and Saccharomyces cerevisiae I was used.
FO 2044 (manufactured by Oriental Yeast Co., Ltd.) was replaced with 20 g by the same procedure as in Example 1 except that the purity was 100%.
1.1 g of (R) -γ-butyrolactone-γ-propionic acid methyl ester was obtained. The specific rotation of this product is ▲ [α]
²⁵ _D ▼ = + 61.79 ° (C = 0.81, chloroform).

Example 3 5.0 g of the substrate obtained in Reference Example 1 was used as 5.0 g of the substrate obtained in Reference Example 3, and Saccharomyces cerevisiae IFO 2
044 (manufactured by Oriental Yeast Co., Ltd.) was replaced with 20 g,
During the reaction, the same operation as in Example 1 was performed except that 5 g of sucrose was added every 15 hours to obtain 1.2 g of 100% pure (R) -γ-butyrolactone-γ-propionic acid propyl ester. I got it. The specific rotation of this product is ▲ [α] ²⁵ _D ▼ ＝ ＋ 53.31 ° (C = 1.24, chloroform)
Met.

Example 4 The same operation as in Example 3 was carried out except that 5.0 g of the substrate obtained in Reference Example 3 was changed to 5.0 g of the substrate obtained in Reference Example 4, and (R) -γ-butyrolactone-γ-propione. 1.5 g of acid butyl ester was obtained. The specific rotation of this thing is ▲
[Α] ²⁵ _D ▼ = + 49.76 ° (C = 1.04, chloroform).

Example 5 Yeast extract 0.3%, Malt extract 0.3%, Polypeptone 0.5
%, Glucose 1%, agar 2% on a slant medium at 25 ° C,
One platinum loop of Hansenula anomala IFO 0136, which had been cultured for 48 hours, was dispensed with 10 m of the medium having the composition shown in Table 2 2 x 18 cm
Was inoculated into a test tube of No. 1 and cultured with shaking at 27 ° C. for 2 days to prepare an inoculum solution.

A 2 liter Sakaguchi flask into which 700 m of the same liquid medium as the above composition was dispensed was inoculated with 3 m of the above inoculum solution and cultured at 27 ° C for 48 hours. A total of 2.1 bacterial cells obtained by shaking culture were collected by centrifugation, and 80 g of sucrose, 1.0 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, 2.5 g of calcium carbonate, 1.0 g of ammonium sulfate and 500 of water were collected.
m was suspended in a reaction solution having a pH of 6.8. 5.0 g of the substrate obtained in Reference Example 1 was dispersed in this suspension in 50 m of water, neutralized with a dilute aqueous potassium hydroxide solution, added to the above reaction solution, and reacted at 30 ° C. for 48 hours. Furthermore, during the reaction
Every 15 hours 10 g of sucrose was added. After completion of the reaction, the same operation as in Example 1 was performed to obtain a purity of 100%.
(R) -γ-butyrolactone-γ-propionic acid ethyl ester (2.8 g) was obtained. The specific rotation of this product is ▲ [α]
²⁵ _D ▼ = + 55.86 ° (C = 1.51, chloroform).

Example 6 The same operation as in Example 5 was carried out except that Hansenula anomala IFO 0136 was changed to Candida albicans IFO 1061, and 0.8 g of ethyl ester of (R) -γ-butyrolactone-γ-propionic acid having a purity of 100% was obtained. I got it. The specific rotation of this product was ▲ [α] ²⁵ _D ▼ = + 53.25 ° (C = 1.25, chloroform).

Example 7 The same operation as in Example 1 was carried out except that 5.0 g of the substrate obtained in Reference Example 1 was neutralized with sodium hydroxide instead of neutralizing with dilute potassium hydroxide. 2.5 g of γ-butyrolactone-γ-propionic acid ethyl ester was obtained. The specific rotation of this product is ▲ [α] ²⁵ _D ▼ ＝ ＋ 52.98 ° (C
= 1.25, chloroform).

EFFECTS OF THE INVENTION According to the method of the present invention, (R) -γ-butyrolactone-γ-
Propionate ester can be easily synthesized under mild reaction conditions with high yield and high optical purity.

Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location (C12P 17/04 C12R 1: 725)

Claims (2)

[Claims] 1. General formula (II): (In the formula, R represents a C ₁ -C ₄ lower alkyl group)
The 4-oxopimelic acid half ester represented by the formula is neutralized with an aqueous alkaline solution, and then the obtained compound is Saccharomyces cerevisiae.
e), in contact with Hansenula anomala or Candida albicans general formula (I): (Wherein R is the same as described above) leads to (R) -γ-butyrolactone-γ-propionic acid ester (R) -γ-butyrolactone-γ represented by the general formula (I). -A method for producing a propionic ester. 2. The alkaline aqueous solution used for neutralizing 4-oxopimelic acid half ester represented by the general formula (II) is an aqueous solution of sodium hydroxide, potassium hydroxide or ammonia. Manufacturing method.