JPH01309694A - Production of optically-active alcohol - Google Patents

Abstract

PURPOSE:To industrially advantageously the subject compound for raw material of drug, etc., by reacting stereoselective ester transition enzyme to racemic modification of aliphatic alcohols having asymmetric carbon in molecule and enol esters of fatty acid without addition of water. CONSTITUTION:Racemic modification of aliphatic alcohols having asymmetric carbon atom in molecule (e.g., 2-octanol) is mixed with enol esters of fatty acid (e.g., vinyl acetate) and reacted with enzyme having stereoselective ester transition action (e.g., lipase) without adding water to be subjected to asymmetric ester transition reaction esterifying one of the optical isomer in a racemic modified alcohol, then resultant alcohol and ester are separated by silica gel chromatography to afford the aimed optically active alcohol.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention utilizes the stereoselectivity of enzymatic reactions to produce racemic aliphatic alcohols (hereinafter referred to as racemic alcohols) having an asymmetric carbon atom in the molecule. The present invention relates to a method for producing an optically active alcohol by esterifying one optical isomer of the alcohol from enol esters of fatty acids and enol esters of fatty acids, and then separating the enol esters.

The optically active alcohol obtained by the present invention is an extremely versatile compound that can be used as a starting material for the synthesis of optically active compounds such as medicines, agricultural chemicals, physiologically active substances, and liquid crystalline compounds.

[Prior Art] Generally, the method of separating optical isomers from racemic alcohols by chemical resolution requires expensive reagents and complicated steps, and the optical purity of the obtained product is also not very good. Therefore, in recent years, biochemical optical resolution methods using enzymes derived from microorganisms and utilizing their stereoselectivity have been actively researched, and many reports have been made. These methods include: 1) A method in which racemic alcohol is esterified by a chemical method, and then the ester bond of one isomer is hydrolyzed and separated using lipase (asymmetric decomposition); 2) Racemic alcohol and fatty acid are separated in an organic solvent. Method 5: One of the optical isomers is esterified by the ester synthesis action of lipase from a mixture of the two, and then separated (asymmetric synthesis). 1 optical isomer of
It can be divided into three methods, ie, separation after stellation (asymmetric rearrangement), but all of them have the following drawbacks.

First, as a report on the asymmetric decomposition of
No. 9152, JP-A-62-29993, JP-A No. 6
No. 2-65688, No. 63-63398,
S, llamaguchi et al. (Agric, Bi
ol.

Chem, 48.2055 (1984)), D
, Breitgoff et al. (J, Chem, So
Examples include the method described in J. C., 1523 (1986). However, since both methods utilize the hydrolysis reaction of an enzyme in an aqueous solution, when using a water-insoluble substrate, the reaction becomes an emulsion type reaction and the reaction efficiency is poor. It is impossible to reuse it, and it is difficult to create a reactor, which increases the cost of the catalyst.Furthermore, both methods hydrolyze esters with lower fatty acids such as acetic acid, which slows down the progress of the reaction. As a result, these lower fatty acids may accumulate in the aqueous solution, lowering the 111 content of the reaction solution and inactivating the enzyme.Therefore, in the above method, a buffer solution with a fairly high concentration is used, but this method However, these methods also require an extraction step for the product.

Next, as a report on the asymmetric synthesis in an organic solvent of ①, S. Koshiro et al., J. Biotechn.
Examples include the method described in J. Ol., +2+47 (1985).

However, in general, the reaction rate of ester synthesis from a mixture of alcohol and fatty acid using lipase is extremely slow, and in the above report, the reaction takes more than 80 hours. Additionally, as the reaction progresses, water is generated in the reaction system, and the enzyme is dissolved in the generated water, reducing the efficiency of ester synthesis. moreover,
When an inexpensive lower fatty acid is used as a substrate to reduce costs, it may cause a decrease in pu and deactivate the enzyme. In addition, it is difficult to convert the reaction system into an active material, and the cost of the catalyst increases.

Additionally, a report on the asymmetric transition of (2) in an organic solvent is published in JP-A-62-166898, 8° Cambou et al., J. Am. Chem, Sac, + Dan 6.26.
87 (1984) and the like. In this reaction, no water is produced as the reaction progresses, so the efficiency of esterification is high, and since there is no free fatty acid, the pH does not decrease. Furthermore, since the enzyme remains insoluble until the end of its action, it can be easily recovered and reused. It is also possible to use it as a reactor. However, in the above report, 1 uses esters such as tributyrin and methylpropionate as substrates, which are extremely difficult to transfer acyl groups to alcohols, so it is necessary to add a large amount of enzyme or to take a long reaction time. It has to be long. Therefore, when performing mass production, it is necessary to remove a large amount of added enzyme from the reaction solution.

Recovering it would be a difficult task, and even if it were made into a reactor, it would be quite large-scale, so the reactor would lose its meaning.

Comparing the three methods for the biochemical asymmetric resolution of racemic alcohols shown above, it is clear that method (2) using asymmetric transesterification using lipase is preferable, but this method also provides a more suitable acyl. It is necessary to select a donor substrate to increase reaction efficiency, and furthermore, in consideration of safety, it is preferable not to add an organic solvent or the like.

It has been desired to develop a method for solving these problems and efficiently resolving and isolating racemic alcohols on an industrial scale.

:Problem to be solved by the invention] As mentioned above, the optical resolution method of racemic alcohol by chemical method has problems such as requiring expensive reagents and complicated steps. In the biochemical optical resolution of racemic alcohols using the enzymatic method, in the method using the asymmetric hydrolysis reaction of esters shown above or the asymmetric ester synthesis reaction in an organic solvent, there are However, the method using asymmetric transesterification reaction in an organic solvent using lipase does not have the above-mentioned problems. However, as previously reported, when tributyrin or methylpropionate are used as acyl donor substrates, the reaction is very slow and inefficient. Efficient production of optically active alcohols by biochemical optical resolution of racemic alcohols on an industrial scale is as difficult as conventional chemical methods.

Therefore, there has been a need for a method for producing large amounts of optically active alcohol efficiently, cheaply, easily, and safely, and the present invention attempts to achieve this.

[Means to solve the problem]

At present, it is difficult to optically resolve waxy semi-alcohols on an industrial scale using chemical and biochemical methods that utilize the three reactions of lipase for the reasons stated above. However, among these methods, if an acyl donor substrate with high reaction efficiency is found and a reaction system that increases the efficiency of the alcohol esterification reaction is developed in the method using the asymmetric transesterification reaction of lipase, racemic alcohol We thought that it would be extremely practical to produce optically active alcohols on an industrial scale by optical resolution. Therefore, the present inventors focused on the asymmetric transesterification reaction of lipase described in (1) above, searched for an acyl donor substrate that can more efficiently esterify alcohol, and used it to An attempt was made to optically resolve racemic alcohols by esterifying and separating the optical isomers. That is, industrial lipase powder is added to a mixture of a racemic alcohol and a highly reactive fatty acid ester without substantially adding water, and one optical isomer in the racemic alcohol is converted through the asymmetric transesterification reaction. After esterification, the ester and remaining alcohol are separated.

Note that, after separation, ester can be easily converted into alcohol by hydrolysis.

In these reaction systems, water is not present, and water is not produced during the reaction, so the esterification reaction proceeds efficiently without being inhibited by water.

Furthermore, since the enzyme remains insoluble until the end of its action, it can be easily recovered and reused. Furthermore, it can also be made into a reactor. Furthermore, when forming esters with lower fatty acids, the fatty acids do not remain free, so
There is no drop in the pH of the reaction system, and there is no fear of enzyme deactivation.

That is, the present invention provides a mixture of a racemic aliphatic alcohol having an asymmetric carbon atom in the molecule and a fatty acid enol ester which is a highly reactive acyl donor substrate without substantially adding water. The present invention provides a method for producing an optically active alcohol, which comprises esterifying and separating one optical isomer in the racemic alcohol by using an enzyme having a stereoselective transesterification action.

The highly reactive fatty acid esters that are acyl donor substrates for the enzymatic asymmetric transesterification reaction used in the present invention include:
fatty acid vinyl esters such as vinyl acetate and vinyl butyrate;
Examples include fatty acid enol esters such as fatty acid phenyl esters such as phenyl acetate and phenyl acetate, and fatty acid isopropenyl esters such as isopropenyl acetate and isopropenyl butyrate.

Next, there are no particular restrictions on the racemic alcohol (it may be saturated, unsaturated, straight, with a side chain, cyclic, or with a substituent). Specific examples include R3-(-+)-2-octatool and R3-(+-)-1-phenylethanol.

Further, as the enzyme used in the present invention, any enzyme may be used as long as it can selectively esterify one of the optical isomers in the racemic alcohol by a transesterification reaction with the above-mentioned enol esters. is a member of the genus Pseudomonas, Chromobacteria
i a) Genus or Candida
Examples include lipases derived from microorganisms belonging to the genus. For more information, see Pseudomonas fluorescens (Pse
udomonasf 1uorescens),
Pseudomonas flavo
+ Chromobacterium viscosum (Chromo
bacterium viscosum>, Candida ° Cylindracea (9μJida c 1indr,
cea) and the like. Commercial products of these lipases include Lipase P (Ohno'!A)
(manufactured by Yakusha).

Lipase B (manufactured by Sarapolo Beer Co., Ltd.), Lipase LP (manufactured by Toyo Jozo Co., Ltd.), Lipase OF, Lipase MY (manufactured by Awa-Nori Sangyo Co., Ltd.), and the like can be used.

In the present invention, the racemic alcohol and enol esters of fatty acids are mixed and the enzyme is allowed to act without substantially adding water. In the present invention, "substantially without addition of water" means a slightly aqueous state in which the reaction system is conducted with water necessary for enzyme stability secured.

In the present invention, the blending ratio of the racemic alcohol and fatty acid enol ester is the former: the latter = 1:1 to 5
(molar ratio), preferably 1:1 to 2 (molar ratio), where the above enzyme is added to 1 to 5 mol of racemic alcohol to 1 mol of racemic alcohol.
0 g, preferably 3 to 20 g, is added and reacted. The reaction is preferably carried out at 0 to 80°C, usually 15 to 65°C, for 1 to 24 hours, preferably 2 to 12 hours, with shaking or stirring so that the enzyme is properly dispersed in the reaction system. Furthermore, an organic solvent may be added to the reaction system as necessary. After the reaction is complete, the enzyme can be easily removed from the reaction solution and reused by filtering it through a filter paper such as Toyo Roshi No. 2. After the reaction, the optically active ester produced by the reaction and the remaining optically active alcohol can be easily separated and purified by, for example, silica gel column chromatography.

〔Example〕

Next, the present invention will be explained with reference to Examples, but the present invention is not limited to these Examples.

Experimental Example 1 Selection of Acyl Donor Substrate In a mixture of 10 mmol of 1-decanol and 10 mmol of acetic acid or various acetate esters shown in Table 1 as the acyl donor substrate, 1 portion of Lipase B manufactured by Sarapolo Beer was added.
0 mg was added, and the transesterification reaction was carried out for 3 hours while shaking at 25°C.

The n-decyl acetate produced by the reaction was quantified by liver LC, and its synthesis rate was calculated. IIPLC conditions are shown below.

Column; Lichrosorb RP-18, 5tl
(4X250mm.

(manufactured by Merck & Co., Ltd.) Mobile phase solvent; Acetonitrile: Water - 9=1 Detector; Differential refractometer (SE-51, manufactured by Showa Denko) Column temperature: 25
°C Table 1 shows the synthesis rate of n-decyl acetate for each acyl donor substrate after 3 hours of reaction.

Table 1 Acyl donor substrate synthesis rate, (%) Acetic acid 0 Vinyl acetate
96 Phenyl acetate 72 Isopropenyl acetate 14 Ethyl acetate 2 Propyl acetate 2 Butyl acetate 2 Benzyl acetate 15 Isopropyl acetate O Cyclohexyl acetate O Triacetin 14 From Table 1, enol esters of fatty acids such as vinyl acetate are excellent as acyl donor substrates. It was confirmed that there is.

Example I Optical resolution of R3-(-+)-2-octatool A mixture of 100 mmol of R3-(-+)-2-octatool and 150 mmol of vinyl acetate was mixed with 150 mg of Lipase B manufactured by Sarapolo Beer Co., Ltd. or Ohno 2.0 g of Lipase P manufactured by Pharmaceutical Co., Ltd. was added, and an asymmetric transesterification reaction was carried out at 25°C for G hours while shaking. The acetate ester of 2-octatool produced by the reaction is IIP
It was quantified by LC and the conversion rate was calculated. HPLC below
Indicates conditions.

Column; Asahipak 0DP-5 sail φ4; 6X
150 mm mobile phase solvent; Acetonitrile: Water = 7:3 Detector; Differential refractometer (S [-51, manufactured by Showa Denko) Column temperature: 25 °C After the reaction, the alcohol and ester produced were purified by silica gel chromatography. After concentration, the weight was measured and the yield was calculated. The conditions for silica gel chromatography are shown below.

Column size: 5 x 20 cm Gel: Silica gel 60 (manufactured by Merck & Co., Ltd.) Mobile phase solvent: Hexane:ethanol = 401 Next, the optical purity (χee) of the obtained alcohol and ester was determined by optical rotation measurement. In addition, the reference substance R-(
The optical rotations [α]85 of -)-2-octatool and S-(+)-2-octatool were -10 and +10, respectively. In addition, the optical rotation of R-(-)~2-octyl acetate and S-(+)-2-octyl acetate-(-
α] and 5 were -3, 1 and +3.1, respectively. The optical rotation needle used was a DIP-4 model manufactured by JASCO Corporation. The results obtained are shown in Table 2.

Example 2 Optical resolution of R3-(-1-)-1-phenylethanol To a mixture of 100 mmol of R3-(+-)-1-phenylethanol and 150 mmol of vinyl acetate was added 150 mg of lipase B manufactured by Sarapolo Beer Co., Ltd. or Ohno 2.0g of Lipase P manufactured by Pharmaceutical Company! The asymmetric transesterification reaction was carried out for δ hours with shaking at 25°C.

The acetate ester of l-phenylethanol produced by the reaction was quantified by HPLC, and the conversion rate was calculated. IIPLc conditions are described below.

Column; 85ahipak 0DP-50tφ4.6
X 150mm mobile phase solvent; acetonitrile: water-6;
4 Detector: Differential refractometer (SE-51, manufactured by Showa Denko) Column temperature: 25°C After the reaction, the alcohol and ester produced were purified by silica gel chromatography, and after concentration, the weight was measured to calculate the yield. I put it out. The conditions for silica gel chromatography are described below.

Column size: φ5X20c+n Gel: Silica gel 60 (manufactured by Merck & Co., Ltd.) Mobile phase solvent: Hexane:ethanol = 50:1 Next, optical purity (χee) of the obtained alcohol and ester
was determined by optical rotation measurement. In addition, the reference material R-
(+)-1-phenylethanol and S-(+)-1
-The optical rotation [α]t5 of phenylethanol is +
42 and -42.

Further, the optical rotations [α] of R-(+)-1-phenylethyl acetate and 5-(-)-1-phenylethyl acetate were +110 and -110, respectively. For the optical rotation, a DIP-4 model manufactured by JASCO Corporation was used. The results obtained are shown in Table 3.

(Effects of the Invention) According to the present invention, various racemic alcohols can be optically resolved by the asymmetric transesterification reaction by utilizing the stereoselectivity of the enzymatic reaction.

Furthermore, since inexpensive industrial raw materials such as vinyl acetate are used as the acyl donor substrate, the present invention is a practical method that can efficiently, easily, and inexpensively produce optically active alcohol 1 on an industrial scale. , which is unprecedented and revolutionary.

Patent applicant: Sapporo Beer Co., Ltd. Achiha - male Procedural master's letter (voluntary) June 28, 1988

Claims (1)

[Claims] (1) An enzyme having a stereoselective transesterification effect is added to a mixture of a racemic aliphatic alcohol having an asymmetric carbon atom in the molecule and an enol ester of a fatty acid without substantially adding water. A method for producing an optically active alcohol, which comprises esterifying and separating one of the optical isomers in the racemic alcohol.