JPH08322588A - Production of optically active aliphatic unsaturated alcohol - Google Patents

Abstract

PURPOSE: To efficiently obtain the subject compound useful as an intermediate for medicines, agricultural chemicals, etc., by subjecting the mixture of the racemate of an asymmetrical carbon-having aliphatic unsaturated alcohol with an organic acid ester to a steric ester interchange reaction in the presence of a lipase originating from an microorganism and subsequently hydrolyzing the obtained ester. CONSTITUTION: A method for producing the optically active aliphatic unsaturated alcohol comprises subjecting the mixture of an organic acid ester (e.g. vinyl butanoate) with the racemate of an aliphatic unsaturated alcohol [e.g. (R,S)- buten-3-ol] having an asymmetric carbon atom in the molecule and represented by formula I (R1 is a 1-20C aliphatic hydrocarbon; R2 is H, a 1-20C aliphatic hydrocarbon) in the presence of a lipase separated from a microorganism (Candida antatica SP) in a water-immiscible organic solvent (e.g. diisopropyl ether) in the absence of water to esterify only one of the optical isomers in the racemate of the alcohol, and subsequently hydrolyzing the ester into the aliphatic unsaturated alcohol (R) isomer of formula II.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for selectively and efficiently optically resolving a racemate of an unsaturated aliphatic alcohol by an oxygen reaction in an organic solvent. Further, the alcohol produced is 3,5-dinitrobenzoic acid. Acid ester,
The present invention relates to a purification method for improving the optical purity by recrystallizing it. The alcohol obtained by the present invention is an extremely versatile compound that can be used as an intermediate for the synthesis of optically active compounds such as pharmaceuticals, agricultural chemicals and physiologically active substances. For example, optically active (R) -buten-3-ol is used as a useful drug raw material. However, no conventional optically active (R) -buten-3-ol has been found to satisfy all of optical purity, economical efficiency and production efficiency.

[0002]

2. Description of the Related Art Generally, a method for separating an optical isomer from a racemic alcohol by a chemical resolution method (Bull, Soc, Chim. Fr., 2974 (1971)) requires expensive reagents and complicated steps. In addition, the optical purity of the obtained product is not so good. Therefore, in recent years, biochemical optical resolution methods utilizing stereoselectivity using enzymes derived from microorganisms have been actively studied, and many reports have been made (Review article: Zh
uo Feng Xie, Tetrahedron Asymmetry, 2 , 733 (1991)).
Those methods utilize asymmetric hydrolysis of racemic ester by lipase or esterase or asymmetric transesterification reaction between racemic alcohol and fatty acid ester. Manufactures fatty acid esters. In order to improve the chemical yield and optical purity, and to efficiently recover the enzyme after the reaction is completed, an irreversible reaction between a racemic alcohol and a vinyl or isopropenyl ester of a fatty acid using a non-water-miscible organic solvent. A method utilizing asymmetric asymmetric transesterification reaction is known (Japanese Patent Laid-Open No. 1-20
2296 and JP-A-1-171497).
However, in general, the types of enzymes that can be applied to the optical resolution reaction using these enzymes differ depending on the difference in the structure of the substrate, and the chemical yields and optical purities of the obtained products are also uneven. In order to solve this problem, it is necessary to screen many enzymes that satisfy these requirements and to develop enzymes suitable for the purpose.

[0003]

SUMMARY OF THE INVENTION Accordingly, the present inventors
In order to carry out the production of an optically active aliphatic unsaturated alcohol on an industrial scale, various enzymes and various enzyme-containing substances were examined, and the results were compared.
antatica sp.)-derived new lipase (NOVO
It was found that SP-525) is most suitable for asymmetric esterification of racemate of aliphatic unsaturated alcohol, and the present invention has been completed. That is, the present invention can solve the above problems in the conventional technology,
It is an object of the present invention to provide a method for producing an optically unsaturated alcohol, which is capable of optically resolving an aliphatic unsaturated alcohol, for example, a racemate of buten-3-ol, selectively and efficiently by using a specific enzyme.

The present invention is a microorganism (Candida antatica sp.)
Using lipase (NOVOSP-525) isolated from the general formula (R, S) -1:

Embedded image (In the formula, R ₁ represents an aliphatic hydrocarbon group having ₁ to 20 carbon atoms, and R ₂ represents hydrogen or an aliphatic hydrocarbon group having 1 to 20 carbon atoms). A racemic mixture of an aliphatic unsaturated alcohol having an atom and an organic acid ester in a non-water-miscible organic solvent, wherein the mixture undergoes a steric transesterification reaction substantially without adding water. One of the optical isomers in the body is esterified and then hydrolyzed to give the compound of the general formula (R)
-2

[Chemical 4] (In the formula, R ₁ and R ₂ have the same meanings as described above.) A method for producing an optically active alcohol, characterized in that an (R) unit of an aliphatic unsaturated alcohol represented by the formula is produced and collected. .

The above reaction is represented by the following chemical formula in the case of producing (R) -buten-3-ol.

Embedded image

The organic acid ester used in the present invention is not particularly limited, and specifically, vinyl acetate, vinyl butyrate,
Examples thereof include n-vinyl hexanoate, n-vinyl octanoate, and vinyl laurate. The carbon number of the organic acid ester is 2 to 4 (vinyl acetate ee, 56%),
(Vinyl butyrate ee, 82%), 6 (n-vinyl hexanoate ee, 84%), and 8 (n-vinyl octanoate ee, 89%), the optical purity tends to improve as the length increases. 12 carbons (vinyl laurate
ee, 78%). In the present invention, the non-water-miscible organic solvent for mixing the racemic alcohol and the organic acid ester is not particularly limited, for example, diethyl ether, diisopropyl ether, hexane, cyclohexane, benzene, toluene, chloroform, carbon tetrachloride, dichloromethane. , Dichloroethane and the like. As the enzyme used in the present invention, a new lipase derived from the microorganism Candida antatica sp. Can be mentioned (the details are shown in Japanese Patent Application Publication No. 1-501120). The Candida strain is cultivated under aerobic conditions in a medium containing components known in the art, an assimilable carbon source and a nitrogen source, and a medium containing a nitrogen source and other micronutrients to produce lipase. Furthermore, NOVO SP-525 can be obtained in high yield by the following three steps of recombining DNA. That is, (a) a step of supplying a suitable DNA cloning vector comprising a DNA sequence encoding a function of facilitating the discovery of a gene and a DNA sequence encoding candida lipase; (b) a cloning vector derived from the step (a) Using a suitable host organism, such as Aspe
rgillus oryzae, and (c) culturing the transformed host in a suitable medium, and optionally recovering lipase from the medium.

In the present invention, the racemic alcohol and the organic acid ester are put in an organic solvent, and the enzyme is allowed to act without substantially adding water. The ratio of the alcohol to the organic acid ester is the former: the latter = 1: 0.4-2 (molar ratio), preferably 1: 0.4-1 (molar ratio), and the concentration of the alcohol in the organic solvent is 0. 0.5-5 molar concentration,
The concentration is preferably 1 to 2 molar, and 1 to 100 g, preferably 5 to 50 g, of this enzyme racemic alcohol is added to this solution and stirred. Reaction is 0-5
It is carried out at 0 ° C., usually 20 to 30 ° C., with shaking or stirring for 1 to 24 so that the enzyme is appropriately dispersed in the reaction system
It is desirable to carry out the time, preferably 5 to 15 hours. After the reaction was completed, the enzyme was Toyo Filter Paper No. It can be separated from the reaction solution and reused by filtering with a filter paper such as 2.

The (R) monoalcohol ester thus obtained can be subjected to alkali hydrolysis and then subjected to solvent extraction with dichloromethane or the like to obtain an optically active alcohol. Further, the produced alcohol was changed to 3,5-dinitrobenzoic acid ester (DNB) and recrystallized in an appropriate organic solvent to improve the optical purity.

[0009]

The present invention will be described below in detail with reference to examples. [Example 1] Enzymatic optical resolution reaction racemic butene-3-ol (72.11 g, 1 mol) and vinyl butanoate (46 g, 0.4 mol) were dissolved in diisopropyl ether (500 ml) to prepare a solution. NOVO SP-525 (5g) (Novo Nordisk
Bio-Industry Co., Ltd.) is added and the temperature is 0 ℃, 300
The reaction was carried out for about 12 hours while stirring at rpm until vinyl butanoate disappeared. The (R) -buten-3-ol butanoic acid ester produced by the reaction and the other butene-3-ol were quantified by gas chromatography (hereinafter referred to as GC) to calculate the conversion rate. Table 1 below shows the GC conditions.

[0010]

[Table 1]

After completion of the reaction, NOVO SP-
To separate and reuse 525, the reaction solution was treated with Toyo Filter Paper N.
o. Filtered by 2. The filtrate was concentrated by an evaporator to give an oily substance. In order to remove a trace amount of residual alcohol, benzene was added and the concentration under reduced pressure was repeated three times to obtain (R) -buten-3-olbutanoic acid ester with a theoretical yield of 64%. When this was converted into the 3,5-dinitrobenzoic acid ester derivative by the method described in Example 2 and measured by HPLC using an optically active column, the optical purity (ee) of the target product was 82%. Further, when the enzyme recovered by filtration was used to repeat the reaction (under the same conditions), the same favorable result as the reaction result of the first time was obtained.

Example 2 Purification of Target Product The (R) -butene-3-olbutanoic acid ester (26.4 g, 0.2 mol) obtained as described above was added to THF-H.
Optically active (R) -alcohol was hydrolyzed by adding sodium hydroxide (20 g, 0.5 mol) dissolved in a mixed solvent of ₂ O (1: 1 200 ml) and heating under reflux for 4 hours. Got The reaction solution was cooled to room temperature, sodium chloride (50 g) was added, and the mixture was sufficiently stirred, and the produced alcohol was extracted with dichloromethane. Then, the organic layer was dried over anhydrous sodium sulfate. While cooling it to 0 ° C., 3,5-dinitrobenzoic acid (DNB, 42.4 g, 0.2 mol), N, N′-
Dicyclohexylcarbodiimide (DCC, 41 g,
0.2 mol) and 4-dimethylaminopyridine (0.2 mol)
Each of g) was added and reacted for 3 hours with stirring.
The reaction solution was returned to room temperature, hexane (500 ml) was added, and the mixture was left for 2 hours. The resulting solid was filtered off, the filtrate was concentrated under reduced pressure and measured by HPLC using an optically active column. As a result, (R) -buten-3-ol-3,5-dinitrobenzoic acid ester having an optical purity of 82% ee was obtained. Yield 84%
And was obtained as crude yellow crystals. Furthermore, this crude crystal was mixed with a hexane-ethyl acetate mixed solvent (10: 1,800 ml).
The optical purity of the ester is 92% by recrystallizing with
improved to ee. Similarly, when recrystallized again, (R) -buten-3-ol 3,5-dinitrobenzoic acid ester having an optical purity of 98% ee was obtained in a substantially pure form. The total recrystallization yield is 49%
Then, the specific rotation [α] _D = -32.1 ° (C = 1.1,
CHCl ₃ ). HPL using an optically active column
The optical purity measurement conditions by C are as described below.

[0013]

[Table 2] [Comparative Example 1] The same treatment as in Examples 1 and 2 was carried out except that Lipase AmanoPS (trade name of Amano Pharmaceutical Co., Ltd.) was used in place of NOVO SP-525, and the theoretical yield was 90%, optical purity (ee). 56% of 3,5-dinitrobenzoic acid ester was obtained. [Example 3] The same treatment as in Examples 1 and 2 except that n-vinyl hexanoate was used, and the yield was 3,5-dinitrobenzoic acid ester with a theoretical yield of 66% and an optical purity (ee) of 84%. Got [Example 4] The same treatment as in Examples 1 and 2 except that n-vinyl octanoate was used, and the 3,5-dinitrobenzoic acid ester had a theoretical yield of 74% and an optical purity (ee) of 89%. Got Example 5 The same procedure as in Examples 1 and 2 except that vinyl laurate was used, the 3,5-dinitrobenzoic acid ester was obtained with a theoretical yield of 75% and an optical purity (ee) of 78%. Obtained.

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Claims (2)

[Claims] 1. Using a lipase isolated from a microorganism (Candida antatica SP), a compound represented by the general formula (R, S) -1: (In the formula, R ₁ represents an aliphatic hydrocarbon group having ₁ to 20 carbon atoms, and R ₂ represents hydrogen or an aliphatic hydrocarbon group having 1 to 20 carbon atoms). A racemic form of an aliphatic unsaturated alcohol having an atom and an organic acid ester in a non-water-miscible organic solvent, the mixture is subjected to a steric transesterification reaction without substantially adding water. By esterifying only one of the optical isomers in the racemate and hydrolyzing it, the compound of the general formula (R)-
2 [Chemical 2] (In the formula, R ₁ and R ₂ have the same meanings as described above) A method for producing an optically active alcohol, which comprises producing and collecting (R) monolith of an aliphatic unsaturated alcohol. 2. The (R) unit of the resulting aliphatic unsaturated alcohol is 3,5-dinitrobenzoic acid ester,
Next, the method for producing an optically active alcohol according to claim 1, wherein purification is performed by recrystallization.