JP3007461B2 - Method for producing optically active 2-cyclohexenylacetic acid and its ester - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a process for producing optically active 2-cyclohexenylacetic acid and its ester by optical resolution, which is useful as a synthetic intermediate for pharmaceuticals.

[0002]

BACKGROUND OF THE INVENTION As a method for obtaining optically active 2-cyclohexenylacetic acid and its ester, there is known a method of synthesizing optically active 2-cyclohexen-1-ol and ethyl orthoacetate by Claisen rearrangement. [Tetrahedr Letters
on Letters), vol. 26, p. 2031 (1985
Year)]. However, this method is not suitable for optically active 2-
Since it is difficult to obtain cyclohexen-1-ol and the process is complicated, the compound having high optical purity cannot be obtained easily and efficiently, and it cannot be said to be an industrial production method.

Accordingly, an object of the present invention is to provide a method for industrially producing optically active 2-cyclohexenylacetic acid and its ester having high optical purity simply and efficiently.

[0004]

The present inventors have made intensive studies in order to achieve the above object, and as a result, by allowing a specific enzyme to act on a mixture of enantiomers of 2-cyclohexenyl acetate, a selective and efficient method has been developed. The inventors have found that optically active 2-cyclohexenyl acetic acid and optically active 2-cyclohexenyl acetic acid ester which can be subjected to asymmetric hydrolysis and have high optical purity can be easily obtained, and the present invention has been completed.

[0005] That is, the present invention relates to an optically active enzyme which is capable of acting on an enantiomeric mixture of 2-cyclohexenylacetic acid ester with an enzyme capable of asymmetrically hydrolyzing the compound, or a microorganism producing the enzyme or a processed product thereof. A method for producing cyclohexenyl acetic acid and optically active 2-cyclohexenyl acetic acid ester.

The 2-cyclohexenyl acetate of the present invention is represented by the general formula [I] .

[0007]

Embedded image In the formula, R ¹ is C _1-6 which may be substituted with a halogen atom.
It represents an alkyl group or a C _1-6 alkenyl group.

The alkyl group includes, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t
- butyl, pentyl, isopentyl, neopentyl, e.g., a straight chain or branched chain alkyl groups such as F <br/> alkoxy Le groups include.

The alkenyl group includes, for example, vinyl, 1-
Propenyl, allyl, isopropenyl, 2-butenyl,
A linear or branched alkenyl group such as a 3-butenyl, 4-pentenyl and 1,3-pentadienyl group is included. Alkynyl groups include, for example, ethynyl, propynyl groups and the like.

Furthermore the alkyl group, alkenyl group,
It may have one or more substituents which are inactive under the reaction conditions of the present invention. Examples of such a substituent include a halogen atom .

[0011] Good preferable substituent R ¹ is an alkyl group having 1 to 6 carbon atoms, an alkenyl group, halogenated alkyl group, and the like halogenated alkenyl group. Particularly preferred substituents are alkyl groups having 1 to 4 carbon atoms.

[0012] The 2-enantiomeric mixture of cyclohexenyl acetate is advantageous to use the racemate in terms of economic efficiency, the mixing ratio is not particularly limited, it can be applied to of any mixing ratio .

[0013] As enzymes for use in the present invention include enzymes having a 2-cyclohexenyl acetate asymmetric ability to hydrolyze, ie include lipases. like this
Specific examples of lipase, for example, key Yandida (Can
dida), Chromobacterium,
Lipases derived from microorganisms such as the genus Mucor, the genus Rhizopus, the genus Pseudomonas, and pig organs are included. Suitable examples include Candida cylindracea,
Candida lipolytica, Chromobacterium viscosum
cosum), Mucor javanicus
, Rhizopus japonicus, Pseudomonas fluores
cens) and lipase derived from pig pancreas.
These enzymes are obtained by culturing microorganisms that produce them or by extracting them from organs.

The product derived from Candida cylindracea is designated as Lipase OF (manufactured by Meito Sangyo Co., Ltd.), and the product derived from Candida lipolytica is designated as Lipase L "Amano" (manufactured by Amano Pharmaceutical Co., Ltd.). Bacterium viscosum-derived lipase is lipase (manufactured by Toyo Brewing Co., Ltd.), Mucor Javanicus-derived lipase M is "Amano" (manufactured by Amano Pharmaceutical Co., Ltd.), and lysops-derived lipase F (Amano) Pharmaceutical Co., Ltd.
) And lipase (manufactured by Osaka Hoken)
The lipase F-AP “Amano” (manufactured by Amano Pharmaceutical Co., Ltd.) is derived from Javanese, and the lipase PS “Amano” is derived from Pseudomonas fluorescens.
Pig pancreas-derived products are commercially available as lipase (Type II) (manufactured by Sigma) and lipase (manufactured by Wako Pure Chemical Industries) as Amano Pharmaceutical Co., Ltd.

In the present invention, in addition to using a purified enzyme or a crude enzyme, a cell culture solution or cells of a microorganism producing the enzyme may be used, or the cells of the microorganism may be crushed, treated with acetone,
A processed product subjected to a process such as freeze-drying may be used.

The above enzymes and the like can be used alone or as a mixture as needed. In addition, these enzymes and the like can be used after being immobilized by a conventional method such as a polyacrylamide gel method, a sulfur-containing polysaccharide gel method (such as a carrageenan gel method), an alginate gel method, and an agar gel method.

When performing the hydrolysis reaction, it is preferable to adjust the pH during the reaction to the optimum pH of the enzyme to be used. A preferred pH range is 4-9, particularly preferably 6-8. For pH adjustment, an appropriate buffer solution, an aqueous alkali solution such as sodium hydroxide or potassium hydroxide, p
H-stats can be used. Water is usually used as the reaction solvent, but an organic solvent can be used in a range not adversely affecting the reaction.

The reaction temperature varies depending on the enzyme used and the like, but is usually 0 ° C to 60 ° C, preferably 4 ° C to 50 ° C, more preferably about 15 ° C to 45 ° C.

The substrate concentration in the reaction mixture may suitably be selected within the range hydrolysis reaction is not impaired, usually 0.1
It is about 50% by weight, preferably about 1 to 30% by weight.
The enzyme concentration in the reaction mixture can be appropriately selected in consideration of the enzyme activity and economy. When a commercially available product is used, it can be determined according to the enzymatic activity of each enzyme preparation. For example, about 0.05 to 10% by weight, preferably
It is about 0.1 to 5% by weight.

The reaction can be carried out by any method with stirring or standing, but is preferably carried out with stirring.

In the present invention, when the above-mentioned enzyme and the like are allowed to act on 2-cyclohexenyl acetate, one of the enantiomers, S-form or R-form, is asymmetrically hydrolyzed to form an optically active S-form or R-form. 2-Cyclohexenylacetic acid is selectively and efficiently produced, and the other enantiomer, R-form or S-form 2-cyclohexenylacetic acid ester, remains as it is. Thus, according to the present invention,
The optically active 2-cyclohexenyl acetic acid and 2-cyclohexenyl acetic acid having different absolute configurations from the enantiomer mixture of 2-cyclohexenyl acetic acid ester can be obtained by simply separating the resulting optically active form from the remaining optically active form. Esters can be efficiently obtained with high optical purity.

The optically active 2-cyclohexenylacetic acid produced by the reaction and the remaining optically active 2-cyclohexenylacetic acid ester can be isolated by conventional separation and purification means. For example, an appropriate organic solvent, for example, ethyl acetate or the like is added to the reaction solution, and the mixture is extracted.The compound is easily subjected to ordinary purification methods such as column chromatography, concentration under reduced pressure, distillation, and resolution with an optical resolving agent, whereby the compound is easily obtained. Can be obtained.

[0023]

According to the production method of the present invention, optically active 2-cyclohexenylacetic acid and 2-cyclohexenylacetic acid ester having high optical purity can be produced simply and efficiently by the action of an enzyme.

[0024]

EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples.

The screw cap test tube of Examples 1 to 7 in diameter 21 mm, taking the lipase 0.05g shown below, 1M phosphate buffer (pH 7.3)
0.5 ml, 0.1 ml of 2-cyclohexenylacetic acid ethyl ester and 2.0 ml of water were added, and the mixture was shaken at 30 ° C. for 72 hours.

[0026] Example 1: Lipase M "Amano" (Amano Pharmaceutical Co., Ltd.) Example 2: Lipase F (Amano Pharmaceutical Co., Ltd.) Example 3: Lipase F-AP "Amano" (Amano Pharmaceutical (Co. )) Example 4: Lipase L “Amano” (manufactured by Amano Pharmaceutical Co., Ltd.) Example 5: Lipase (manufactured by Osaka Seiken) Example 6: Lipase (Type II) (manufactured by Sigma) Example 7: lipase ( Then, after acidifying the reaction mixture with hydrochloric acid, sodium chloride was added thereto, and a mixed solution (n-hexane / 2-propanol = 9 /
1) Extracted with 1.0 ml. The extract was subjected to high performance liquid chromatography using an optical isomer separation column (column:
(Chiral Cell OB, trade name, manufactured by Daicel Chemical Industries, Ltd., solvent: n-hexane / 2-propanol / trifluoroacetic acid = 1000/10/1), and the produced 2-cyclohexenylacetic acid was analyzed and its absolute configuration was analyzed. , Optical purity and reaction yield were determined. Table 1 shows the results.

[0027]

[Table 1] Examples 8 to 10 The reactions were carried out in the same manner as in Examples 1 to 7, except that the reaction time was changed to 6 hours using the lipase shown below.

[0028] Example 8: lipase (Toyo Jozo Co., Ltd.) Example 9: Lipase OF (Meito made Co., Ltd.) Example 10: Lipase PS "Amano" (Amano Pharmaceutical Co., Ltd.
The produced 2-cyclohexenylacetic acid was analyzed in the same manner as described above, and the absolute configuration, optical purity, and reaction yield were determined.
Table 2 shows the results.

[0029]

[Table 2] Examples 11 to 12 0.2 g of the following lipase was placed in a 100-ml baffled Erlenmeyer flask, and 1 M phosphate buffer (pH 7.3) was used.
4.0 ml, 2.0 ml of 2-cyclohexenyl acetic acid ethyl ester and 16.0 ml of water were added,
Shake for hours.

[0030] Example 11: Lipase (Toyo Jozo Co.) Example 12: Lipase OF (manufactured by Meito Sangyo Co.) The resulting 2-cyclohexenyl acetate was analyzed in the same manner as described above, the absolute configuration, Optical purity and reaction yield were determined.
Table 3 shows the results.

[0031]

[Table 3] Example 13 0.2 g of lipase OF (manufactured by Meito Sangyo Co., Ltd.) was placed in a 300 ml separable flask, 150.0 ml of a 0.01 M phosphate buffer (pH 7.3) and n-propyl 2-cyclohexenyl acetate were used. 10.0 g of ester was added and p
The mixture was stirred at 30 ° C. for 75 hours while adjusting the pH of the reaction solution to 7.3 using an H-stat. The reaction solution was subjected to high performance liquid chromatography using a reversed phase column (column: ODS of trade name DC-PAKS, manufactured by Daicel Chemical Industries, Ltd.).
20-5-100, solvent: 40 mM phosphate buffer (pH
(3.0) / acetonitrile = 1/1) to determine the reaction yield.

Further, after adjusting the pH addition of 6M aqueous potassium hydroxide to the reaction solution to 9, 2-cyclohexenyl acetate n- propyl ester remaining separated and extracted with n- hexane. Then, concentrated hydrochloric acid was added to the remaining aqueous layer to adjust the pH to 1 or less, and the generated 2-cyclohexenylacetic acid was extracted with ether. The absolute configuration and the optical purity were determined in the same manner as in Examples 1 to 7.

[0033] As a result, the absolute configuration of the resulting 2-cyclohexenyl acetate is R, optical purity 70.1% e
e, the reaction yield was 34.8%.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI (C12P 41/00 C12R 1: 845) (C12P 41/00 C12R 1:38)

Claims (1)

(57) [Claims] 1. An enantiomer mixture of 2-cyclohexenyl acetate represented by the following general formula [I] is added to a Candida genus, Chromobacterium
bacterium), Mucor, Rhizopu
s) a microorganism belonging to the genus Pseudomonas
Or a porcine organ, lipase having asymmetrically ability to hydrolyze the mixture, wherein the lipase is allowed to act a microorganism or a processed product thereof to produce, and the resulting optically active substance remaining optically active isomer separation For producing optically active 2-cyclohexenyl acetic acid and optically active 2-cyclohexenyl acetic acid ester. Embedded image (Wherein, R ¹ is C ¹ , which may be substituted with a halogen atom)
_1-6 alkyl group or C _1-6 alkenyl group)