JPH06113885A - Production of 3-hydroxybutyric acid and its ester - Google Patents

Abstract

PURPOSE:To efficiently produce a 3-hydroxybutyric acid and its ester, useful as medicines such as antibiotic substances or biodegradable polymers and having optical activity. CONSTITUTION:The objective 3-hydroxybutyric acid at a ratio [(R)/(S)] different from that of a raw material mixture is obtained by using a mixture of an (R)-3-hydroxybutyric acid ester with an (S)-3-hydroxybutyric acid ester and reacting a hydrolase therewith. Furthermore, the objective 3-hydroxybutyric acid ester at a ratio [(R)/(S)] different from that of the raw material mixture is obtained.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to optically active 3-hydroxybutyric acid and 3-hydroxybutyric acid which are useful as raw materials for the production of pharmaceuticals such as antibiotics and monomer components of biodegradable polymers.
The present invention relates to a method for efficiently producing hydroxybutyric acid ester using a hydrolase.

[0002]

2. Description of the Related Art 3-Hydroxybutyric acid and its ester (hereinafter sometimes referred to as 3-hydroxybutyric acid) are raw materials for synthesizing biodegradable polymers (bioplastics),
It is useful as a starting material for optically active 4-acetoxy-β-lactam, or as a raw material for various active natural products, for example, fine chemical syntheses such as kamikirimushino defense substance, citronetol, various pheromones, choletodiol, and esperin. It is expected to increase more and more.

These 3-hydroxybutyric acids are abundant compounds in the living body, and the history of synthesizing optically active substances utilizing organisms, particularly microorganisms, is quite old. However, it was only recently that the demand for 3-hydroxybutyric acids increased, that full-scale synthetic research came to be carried out.

By the way, the 3-hydroxybutyric acids present in the living body have optical activity, and most of the 3-hydroxybutyric acids used in the above synthetic reaction are the (R) form. On the other hand, the 3-hydroxy acids produced by the synthetic reaction are racemic bodies consisting of (R / S) isomers. Therefore, in order to effectively utilize them as optically active compounds, the racemic isomers are separated after the synthetic reaction. Or, it is necessary to devise a synthetic method.

Under these circumstances, as methods for synthesizing optically active 3-hydroxybutyric acids, stereoselective reduction of 3-oxyacid, stereoselective β-hydroxylation of fatty acid, and bacterial cells are available. Hydrolysis of the poly-3-hydroxy acid produced is classified into three categories. However, the above method cannot be said to be suitable for practical use on an industrial scale because the resolution of the racemate is very complicated or the yield of the target substance is very low.

[0006]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and its object is to provide optically active 3-hydroxybutyric acid and its ester,
It is intended to provide a method which can be efficiently produced by utilizing an enzymatic method.

[0007]

The process for producing optically active 3-hydroxybutyric acid and its ester according to the present invention, which has been able to achieve the above object, includes (R) -3-hydroxybutyric acid ester and (S) By using a mixture with -3-hydroxybutyrate as a raw material and reacting it with a hydrolase, a (R) / (S) ratio different from the (R) / (S) ratio of the raw material mixture of 3- The gist is to obtain hydroxybutyric acid or to obtain 3-hydroxybutyric acid ester having a different (R) / (S) ratio as an unhydrolyzed product.

In carrying out this production method, the enzymatic activity and the optical selectivity of the hydrolase vary depending on the type of solvent used. Therefore, a reaction containing a limited amount of water depending on the type of the enzyme. A system or a non-aqueous organic solvent system is appropriately selected and used. As a preferable hydrolase, Candida genus, Aspergillus
Genus, Rhizopus, Rhizomucor, M
ucor, Arthrobacter, Bacil
Examples thereof include one kind of bacterium selected from the group of bacteria belonging to the genus lus and the genus Pseudomonus, or a hydrolase derived from porcine pancreas.

[0009]

In the present invention, a mixture of (R) -3-hydroxybutyric acid ester and (S) -3-hydroxybutyric acid ester is used as a raw material as described above, and a hydrolase is allowed to act on it to carry out an enzymatic reaction. By selectively hydrolyzing one of the (R) form and the (S) form, the ratio (R) / (S) of the raw material is different from the ratio (R) /
A (S) ratio of 3-hydroxybutyric acid or an unhydrolyzed product of a (R) / (S) ratio of 3-hydroxybutyric acid ester different from the (R) / (S) ratio of the raw material is obtained. By appropriately selecting a solvent according to the type of hydrolase used, optically active 3-hydroxybutyric acid or its ester can be obtained in good yield. This enzymatic reaction is shown as a reaction formula as follows.

[0010]

[Chemical 1]

As the hydrolase used in this reaction, lipase, esterase, protease and the like can be mentioned. More specifically, Candida genus, A
spergillus, Rhizopus, Rhi
genus zomucor, genus Mucor, Arthrobac
ter, Bacillus, Pseudomonu
One kind selected from the group consisting of s genus, or a hydrolase derived from porcine pancreas is exemplified as a preferable one.

The substrate concentration during the enzymatic reaction using these hydrolases cannot be uniformly determined because it depends on the type of solvent used, but usually 0.005 to 7.
It is selected from the range of 7 mol, and when the reaction is carried out in a water solvent, it is preferably about 0.1 mol, in a water-limited system, about 7 mol, and in an organic solvent system, about 0.2 mol is particularly preferable.

The pH of the reaction solution is also the optimum pH for each hydrolase.
However, it is preferable that p
It is in the range of H3 to 8, more preferably in the range of pH 5 to 7.5. In this case, it is preferable to use a buffer solution to keep the pH of the reaction solution stable, and usually 0.01 to 0.15 mol of phosphate buffer solution is used. When the buffer solution is not used, the pH of the reaction solution may be adjusted to an optimum pH using a mineral acid such as hydrochloric acid or sulfuric acid and an alkali such as sodium hydroxide or potassium hydroxide. It is not appropriate to uniformly determine the reaction temperature because the optimum temperature of each hydrolase is different, but it is usually selected from the range of 5 to 65 ° C, and more generally 25 to 45 ° C.

As described above, when the present invention is carried out, the type of optimum solvent varies depending on the type of hydrolase.
0.1-1000 mol%, more preferably 5 mol% relative to the 3-hydroxybutyric acid ester form of the (R) form or the (S) form
It is preferable to use 0 to 300 mol% of water, and when the reaction is carried out in an organic solvent, 10 to the ester is used.
~ 1000 mol%, more preferably 50-300 mol%
The range of is recommended. Needless to say, even when the enzymatic reaction is carried out in an organic solvent, an appropriate amount of water must be present in the reaction system for hydrolysis.

However, when 3-hydroxybutyric acid and its ester are separated, if the amount of water present in the product is small, the reaction product can be easily dehydrated with an appropriate desiccant. If a large amount of water is present, for example, when a reduced pressure drying method or the like is adopted, azeotropy between the target substance and water occurs, and the separation efficiency of them decreases. Therefore, the water present in the reaction system should be kept to a necessary minimum, and in carrying out the present invention, a system having a limited amount of water or a system in which a necessary amount of water is present using an organic solvent is used. It is preferable to carry out the enzymatic reaction with.

The organic solvent used here is n-
Hexane, cyclohexane, benzene, toluene, diethyl ether, dimethyl ether, dibutyl ether,
It is possible to use poorly water-soluble organic solvents such as chloroform, carbon tetrachloride, and methyl ethyl ketone; water-soluble organic solvents such as dioxane, dimethyl sulfoxide, and acetone, which may be used alone or, if necessary, two or more kinds. Can be used as a mixed solvent of.

By the way, depending on the type of reaction solvent, the activity and optical selectivity of the hydrolase are different, and in some cases the enzyme activity is expressed and in the other case it is not expressed. The ratio of the (S) body may be reversed. Therefore, it is desired to select an optimum solvent according to the type of hydrolase to be used and to carry out the enzyme reaction at the optimum temperature and pH range. It should be noted that those who carry out the present invention may make such selections each time with reference to the examples described later.

After completion of the reaction, a known method, for example, the reaction product may be separated into 3-hydroxybutyric acid and its ester by distillation under reduced pressure or the like, whereby 3-hydroxy having a higher optical activity with respect to the raw material mixture. Butyric acid or its ester can be obtained in good yield.

The thus obtained optically enhanced 3
-Hydroxybutyric acid or its ester can be widely used, for example, as a synthetic raw material for biodegradable polymers (bioplastics), a starting raw material for optically active 4-acetoxy-β-lactam, or a pharmaceutical raw material such as antibiotics. .

Hereinafter, the constitution and effects of the present invention will be described in more detail with reference to examples, but the present invention is not limited by the following examples and can be adapted to the gist of the preceding and the following. Any appropriate modification within the scope is included in the technical scope of the present invention.

[0021]

【Example】

Example 1 (Reaction in aqueous medium) 10 mg of the hydrolase shown in Table 1 was dissolved in 5 ml of 0.1 mol of phosphate buffer solution (pH 7.0, otherwise, described in Table 1). Then, 25.1 mg (24 μl) of methyl ester of (R, S) -3-hydroxybutyric acid was added, and the reaction was carried out for 24 hours while shaking at 30 ° C., followed by cooling to stop the reaction and centrifugation. Then, (R) -3-hydroxybutyric acid and (S) -3-hydroxybutyric acid in the supernatant were quantified. The quantitative determination of optically active 3-hydroxybutyric acid was performed by adding L-alanylnaphthylamide and DCC to a predetermined amount of the supernatant and allowing the mixture to react at room temperature for 1 hour, followed by high performance liquid chromatography loaded with a silica normal phase column. It was

Example 2 (Reaction of Water Restriction System) 1.02 mg of the hydrolase described in Table 1 was added to distilled water 76
It was dissolved in .mu.l, 1040 mg (996 .mu.l) of methyl ester of (R, S) -3-hydroxybutyric acid was added, and the reaction was carried out for 24 hours while shaking at 30.degree. C., followed by cooling to stop the reaction. After centrifugation, (R) -3-hydroxybutyric acid and (S) -3-hydroxybutyric acid in the supernatant were quantified in the same manner as in Example 1 above.

Example 3 (Reaction in organic solvent) 3.0 mg of the hydrolase described in Table 1 was added to distilled water 15.
Dissolve in 2 μl, and mix organic solvent (n-hexane /
5.0 ml of dioxane = 7/4) was added. 104 mg (10.3 μl) of methyl ester of (R, S) -3-hydroxybutyric acid was added thereto, and the reaction was carried out for 24 hours while shaking at 30 ° C., followed by cooling to stop the reaction and centrifugation. Then, (R) -3-hydroxybutyric acid and (S) -3-hydroxybutyric acid in the supernatant were quantified in the same manner as in Example 1 above.

The results of Examples 1 to 3 are shown in Tables 1 to 3. In addition, in the reaction in the aqueous medium shown in Table 1, the pH of the reaction solution was adjusted to As
pergillus oryzae, Aspergill
lus niger and Rhizopus delemo
r is pH 3, Bacillus subtilis (1)
And Pseudomonus sp. PH8, Baci
The llus subtilis (2) had a pH of 10, and the others had a pH of 7.

[0025]

[Table 1]

[0026]

[Table 2]

[0027]

[Table 3]

As is clear from Tables 1 to 3, the type of optimum solvent varies depending on the type of hydrolase, and the optical activity or optical selectivity also varies depending on the type of solvent. By selecting the optimum solvent according to the type, it is possible to selectively hydrolyze the (R) body or the (S) body of the 3-hydroxybutyric acid ester mixture consisting of the (R) body and the (S) body. This makes it possible to obtain 3-hydroxybutyric acid having an (R) / (S) ratio different from the (R) / (S) ratio of the raw material mixture.

In addition, the fact that one of the (R) form and the (S) form in the raw material mixture is selectively hydrolyzed in this way means that the (R) of the 3-hydroxybutyric acid ester remaining as an unhydrolyzed product is (R). ) / (S) ratio is (R) / of the raw material mixture
It means that the ratio is different from the (S) ratio, which makes it possible to obtain a 3-hydroxybutyric acid ester having higher optical activity.

[0030]

EFFECTS OF THE INVENTION The present invention is constituted as described above, and provides optically active 3-hydroxybutyric acid and its ester, which are useful as raw materials for biodegradable polymers and pharmaceuticals.
It has become possible to efficiently produce by an enzymatic reaction using (R, S) -3-hydroxybutyrate as a starting material.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location (C12P 41/00 C12R 1:06) (C12P 41/00 C12R 1:72) (C12P 41/00 C12R 1: 845) (C12P 41/00 C12R 1: 125) (C12P 41/00 C12R 1:38) (C12P 41/00 C12R 1: 785) (C12P 41/00 C12R 1:91)

Claims (5)

[Claims] 1. A mixture of (R) -3-hydroxybutyric acid ester and (S) -3-hydroxybutyric acid ester is used as a raw material, and a hydrolase is allowed to act on the mixture to give (R) / (of the raw material mixture. S) different from (R) /
A method for producing 3-hydroxybutyric acid, characterized in that (S) ratio of 3-hydroxybutyric acid is obtained. 2. A mixture of (R) -3-hydroxybutyric acid ester and (S) -3-hydroxybutyric acid ester is used as a raw material, and a hydrolase is allowed to act on the mixture to give (R) / (of the raw material mixture. S) different from (R) /
A method for producing 3-hydroxybutyric acid, characterized in that a (S) ratio of 3-hydroxybutyric acid ester is obtained. 3. The process according to claim 1, wherein the enzymatic reaction is carried out in the presence of a limited amount of water. 4. The method according to claim 3, wherein the enzymatic reaction is carried out in an organic solvent. 5. The hydrolase is Candida genus, A
spergillus, Rhizopus, Rhi
genus zomucor, genus Mucor, Arthrobac
ter, Bacillus, Pseudomonu
The method according to any one of claims 1 to 4, which is a hydrolase derived from a bacterium belonging to the genus s or a porcine pancreas.