JPH03285689A - Production of optically active 3-hydroxyfatty ester derivative - Google Patents

Abstract

PURPOSE:To enable production of the subject compounds of high optical purity using a simple operation at a low cost by reducing a specified 3-oxofatty ester derivative using a specified enzyme component separated from baker's yeast and a specified coenzyue. CONSTITUTION:A 3-oxofatty ester derivative represented by formula I (In formula, X is 1-3C alkyl group which may be substituted with hydrogen atom or halogen atom. Y is hydrogen atom or 1-3C hydrocarbon group and R shows 1-10C hydrocarbon group) is suspended or dissolved in a solution of a component containing an enzyme component of about 32000 molecular weight separated from baker's yeast with a simple operation and non-adsorbable to a weakly basic ion-exchange resin and a coenzyme, nicotinamide adenine dinucleotide phosphate for reduction thereof. After completion of the reduction, extraction is carried out using a solvent to obtain the objective product expressed by formula II (In formula, X, Y and R are same as in the above-mentioned definition). The cost goes down thereby and a product of high optical purity can be obtained. Industrial significance is high.

Description

Detailed Description of the Invention [Industrial Field of Application] The present invention uses one component of an enzyme contained in baker's yeast to produce optically active 3-hydroxy fatty acid ester derivatives.
The present invention relates to a method for producing a corresponding 3-oxo fatty acid ester derivative by reduction reaction.

[Conventional technology]

Optically active 3-hydroxy fatty acid ester derivatives are important as synthetic intermediates for natural products, physiologically active substances, and the like. In particular, L-form 4-chloro3-hydroxybutyric acid ester is used as a raw material for the synthesis of L-lunithine.

When using a method of reducing a 3-oxo fatty acid ester derivative to obtain an optically active 3-hydroxy fatty acid ester derivative, the chemical synthesis method requires optical resolution of the 0-form and the L-form from the racemic body. However, the yield never exceeds 50%, and the optical resolution method is not simple. Furthermore, when a halogen atom is present at the 4-position, dehalogenation is likely to occur, resulting in a disadvantage that the yield cannot be improved.

Biological methods have been used to overcome this drawback, including methods using microorganisms and methods using enzymes.

Regarding the reduction reaction of 3-oxobutyric acid ester derivatives that do not have an alkyl group at the 2-position, a method using a microorganism includes a method using baker's yeast (J, Am.

Chem, Soc, 105.5925 (1983
)) In other words, a method using other bacterial cells (Unexamined Japanese Patent Publication No. 59-11
No. 8093), etc. are shown. Furthermore, as a method using an enzyme, a method using horse liver alcohol dehydrogenase (J, Am, Chem.

Soc, + H, 4028 (1985)), a method using an enzyme extracted from baker's yeast (J, Am, Chem.
, Soc, 107.2993 (1985) and Japanese Patent Application Laid-Open No. 64-60391).

In addition, as a method for obtaining an optically active form of a 3-hydroxy fatty acid ester derivative having an alkyl group at the 2-position by reduction of a 2-alkyl-3-oxo fatty acid ester derivative, there is a biological method using baker's yeast (Bull ,
Chem, Soc, Jpn, +61+2089
(1988) and Bull, Chem, Soc.
Jpn,, fll,, 1179 (1989)).

[Problem to be solved by the invention]

Methods using baker's yeast or microorganisms have problems in that a product with the desired steric configuration is not necessarily obtained and the optical yield is not high. Furthermore, in order to improve the optical yield, conversion to octyl ester or other esters is required, resulting in increased costs. In addition, although methods using enzymes reduce the disadvantages mentioned above, the enzymes themselves are expensive, the methods for isolating and purifying the enzymes are complicated, and the disadvantages are that they are difficult to use as boat fishing methods. be.

An object of the present invention is to provide a method for producing optically active 3-hydroxy fatty acid ester derivatives with high optical purity at low cost using simple operations.

[Means to solve the problem]

The method for producing an optically active 3-hydroxy fatty acid ester derivative of the present invention has the following general formula (I): (wherein, , Y represents a hydrogen atom or a hydrocarbon group having 1 to 3 carbon atoms, and R represents a hydrogen atom having 1 to 3 carbon atoms.
~1o represents a hydrocarbon group. ), a weakly basic ion exchange resin containing an enzyme component with a molecular weight of 32,000 separated from baker's yeast, and a non-adsorbable component of nicotinamide adenine dinucleotide phosphate (hereinafter referred to as rNADPH). The present invention is characterized by producing an optically active 3-hydroxy fatty acid ester derivative represented by the general formula (■):

In the formulas (I) and (rl), the alkyl group having 1 to 3 carbon atoms which may be substituted with a halogen atom represented by X includes a methyl group, an ethyl group, a propyl group, an isopropyl group, and Examples include those in which an alkyl group is substituted with a chlorine atom, a bromine atom, a fluorine atom, or an iodine atom, preferably a methyl group or a chloromethyl group. Also, Y
Examples of the hydrocarbon group having 1 to 3 carbon atoms represented by include, in addition to the alkyl group described above, an allyl group and a propargyl group. Further, examples of the hydrocarbon group having 1 to 1 o of carbon atoms represented by R include linear, branched, or cyclic saturated or unsaturated aliphatic hydrocarbon groups having 1 to 1 o of carbon atoms.

The molecular weight isolated from baker's yeast used in the present invention is approximately 30
The weakly basic ion exchange resin non-adsorbed component containing the enzyme component of No. 00 can be obtained, for example, as follows. That is, the cell walls of baker's yeast are destroyed by a known method, such as a French press, and the yeast is passed through a layer of diethylaminoethyl to obtain oral fluid.

As the weakly basic ion exchange resin used here, for example, diethylaminoethyltoyovar (manufactured by Tosoh Corporation, DE
DEAE cellulose can be used. Further, in the separation of the main fraction, it is sufficient to use a buffer solution to which an enzyme stabilizer is added as a washing solution. The enzyme contained in the obtained aliquot is fractionated as a mixture with other proteins, but for use in the present invention there is no need for any more complicated purification (no side reactions may occur even as a mixture). There are no drawbacks such as deterioration or decrease in optical yield.

In this way, you can use inexpensive baker's yeast with extremely simple operations.
To obtain a solution containing the desired enzyme,
It can be used as a low-cost and hassle-free method.

The activity of this enzyme component is 0.00000000000 at a temperature of 50μ℃.
In 3 ml of 1M phosphate buffer solution (pH 7.0),
0.1 mM NADPH and 1 μl of 3-oxo fatty acid ester derivative (I) (e.g., ethyl 4-chloroacetoacetate) were added, and the absorbance of NADPH was measured using a spectrophotometer at an absorption wavelength of 340 nm, and the amount of decrease was measured. .

The properties of the enzyme contained in the fraction used in the present invention are shown below.

0 action: Asymmetric reduction of 3-oxo fatty acid ester derivative (1) in the presence of NADPH.

■Substrate specificity: α-ketoester, β-ketoester,
It acts on aldehydes and α-diketones, but not on monoketones and β-diketones.

■Molecular weight: Approximately 32,000 The reaction between the 3-oxo fatty acid ester derivative and this enzyme component is carried out by adding appropriate amounts of the enzyme component and coenzyme NADPH to a 0.1M phosphate buffer solution (pH 7.0), and then stirring at room temperature for 30 minutes. - The oxo fatty acid ester derivative is suspended or dissolved at a concentration of 0.1 to 10% by weight, and the reaction is carried out with stirring for 1 to 2 days. At this time, glucose dehydrogenase and D-glucose, or glucose-6-phosphate dehydrogenase and D-glucose-6-
An appropriate amount of phosphoric acid may be added. The reaction temperature is usually 10~
40°C, preferably 20-35°C.

After the reaction is completed, extraction is performed using a solvent such as ethyl acetate, and after the extract is dehydrated, the solvent is distilled off to obtain the optically active 3-hydroxy fatty acid ester derivative that is the target product.

The method of the present invention will be explained in more detail with reference to Examples below. However, the following examples are specific examples of the present invention, and do not limit the present invention.

In the following examples, baker's yeast obtained from Oriental Yeast Industry (2) was used.

Example 1 (Separation of Enzyme Components)] A solution of 1mM dithiothretol, 1mM phenylmethylsulfonyl fluoride and 0.05% 2-mercaptoethanol in OmM phosphate buffer (pH 7,0). Suspend 1 kg of baker's yeast in 500 d of (basic buffer), and use a French press to
Cell membranes were disrupted at a pressure of 10 psi and a supernatant solution of 670 cells was obtained after centrifugation. After thoroughly dialyzing this solution, DE
After passing through the AEI-Yopal layer, the base buffer was washed to obtain an active fraction. The total activity of this solution is 60 units, 4 units based on 2-methyl acetate,
-590 units based on ethyl chloroacetoacetate
(One unit indicates the amount of enzyme required to reduce 1 μmol of substrate per minute.)

(Reduction of ethyl 4-chloroacetoacetate) 0.1M phosphate buffer solution (pH 7,0) NAD PH in 20mQ
After dissolving 10,111 g of glucose-6-phosphate, 300 mg of glucose-6-phosphate dehydrogenase, 50 units of glucose-6-phosphate dehydrogenase, and 1.54 units of an enzyme component derived from baker's yeast, 82,111 g of ethyl 4-chloroacetoacetate was added at room temperature.

After stirring at 30°C overnight, extraction with ethyl acetate and isolation by gas chromatography were carried out, yield 82 mg, yield 10
Ethyl 4-chloro-3-hydroxybutyrate was obtained at 0%.

When the optical rotation of the product thus obtained was measured, it was [α], +31.5 (1,0, benzene),
It turned out to be a 3g body. In addition, 10 mg of the product was dissolved in anhydrous benzene 1 relative in a nitrogen atmosphere, and pyridine 250
μl and 50 μl of R(+)-methoxytrifluoromethylphenylacetic acid chloride! Add the following rMT and stir at night.
(referred to as PA). The reaction mixture was extracted with diethyl ether, and optical isomers were determined by HPLC.

As a result, optical 1 [too% of 3 g of 4-chloro-3
- It was confirmed that ethyl hydroxybutyrate was obtained.

Comparative example Quoted from Japanese Patent Application Laid-open No. 64-60391.

Add 0.2M acetate buffer (pH
5,5) 32.5 mR of distilled water containing 50 mR and 0.1 g of polyoxyethylene sorbitan monooleate
After adding 100 mg of ethyl 4-chloroacetoacetate
(0.61 mmol) was added.

Next, 5 mfi of an aqueous solution (containing 100 units) of 4-chloroacetoacetate ethyl reductase extracted from baker's yeast and N
12.5 mQ (containing 0.61 mmol) of ADP H* solution was added and reacted with reciprocating shaking at 27°C for 1 hour. After the reaction was completed, hexane was added to extract the product, and the hexane layer was dried over anhydrous magnesium sulfate, and the hexane was evaporated under vacuum to obtain 94 ml of ethyl 4-chloro-3(R)-hydroxybutyrate. The specific rotation of this product is [α10
+23.0 (5,0, CHCl3).

Example 2 0.1 phosphate buffer (pH 7,0) 30 mg,
Contains 8+ng of NADPH, 30g of glucose-6-phosphate
0 mg, 9.2 units of glucose-6-phosphate dehydrogenase, and 0.44 units of the enzyme component derived from baker's yeast were dissolved, and 6 units of ethyl 2-methyl-3-oxopropanoate were dissolved at room temperature.
5 mg was added. After stirring overnight at 30°C, the mixture was extracted with ethyl acetate and isolated by silica gel column chromatography. Next, MTPA conversion was performed in the same manner as in Example], and the 2R form of ethyl 2-methyl-3-hydroxypropanoate was obtained by HPLC in an optical yield of 100%.

Example 3 0 mg of NADPH1, 300 mg of glucose in 30 mR of 0.1M phosphate buffered Na (pH 7,0),
9.2 units of glucose dehydrogenase and 0.88 units of an enzyme component derived from baker's yeast were dissolved, and 85 mg of ethyl 2-allylacetoacetate was added at room temperature. After stirring at 30°C for 48 hours, it was isolated in the same manner as in Example 2, and then converted to MTPA in the same manner as in Example 1, and the 2R,33-2-allyl was purified by gas chromatography in an optical yield of 100%. It was found that -3-hydroxyethyl acetate was produced.

Example 4-12 N in 3 ml of 0.1M phosphate buffer (pH 7.0)
AD P H8K, an enzyme component derived from baker's yeast, 0.16 units, was dissolved, and 5+ng of 2-alkyl acetoacetate was added at room temperature. After stirring at 30°C for 48 hours, it was isolated and quantified in the same manner as in Example 2. All products are 2R
There were 933 bodies.

(Margins below this page) 3 4 Because it can be obtained with scientific purity, it is useful as a method for synthesizing intermediates in the synthesis of pharmaceuticals, etc. Furthermore, glucose dehydrogenase or the like can be used to regenerate the coenzyme N A D P H used in the reduction reaction, and the amount of coenzyme used can be reduced. Therefore, the method can greatly reduce costs and provide high-quality products with high optical purity.

〔Effect of the invention〕

In the present invention, there is no need to perform complicated purification from inexpensive baker's yeast as in the above embodiment, and optically active 3-hydroxy fatty acids with high optical purity can be obtained by using an enzyme component that acts as a reaction catalyst with a very simple operation. Ester derivatives can be produced.

Claims (1)

[Claims] 1. General formula: ▲ Numerical formula, chemical formula, table, etc. ▼ (In the formula, X represents an alkyl group having 1 to 3 carbon atoms which may be substituted with a hydrogen atom or a halogen atom, Y represents a hydrogen atom or a hydrocarbon group having 1 to 3 carbon atoms, and R represents a hydrogen atom having 1 to 3 carbon atoms.
~10 hydrocarbon groups. ) is reduced using a weakly basic ion exchange resin non-adsorbent component containing an enzyme component with a molecular weight of about 32,000 separated from baker's yeast and nicotinamide adenine dinucleotide phosphate. Featured general formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ A method for producing an optically active 3-hydroxy fatty acid ester derivative represented by (wherein, X, Y, and R have the same meanings as above). 2. Optically active 3-hydroxy fatty acid ester derivative (
The method according to claim 1, wherein R) is 4-halo-3-hydroxybutyric acid ester. 3. Optically active 3-hydroxy fatty acid ester derivative (
The method according to claim 1, wherein R) is a 2-alkyl-3-hydroxypropanoic acid ester. 4. Optically active 3-hydroxy fatty acid ester derivative (
2. The method according to claim 1, wherein the ester is 2R,3S)2-substituted-3-hydroxybutyric acid ester. 5. The production method according to any one of claims 1 to 4, wherein glucose dehydrogenase or glucose-6-phosphate dehydrogenase is used to regenerate nicotinamide adenine dinucleotide phosphate consumed in the reaction.