JPH0191789A - Production of optically active beta-substituted glutaric acid monoester - Google Patents

Abstract

PURPOSE:To efficiently produce an optically active beta-substituted glutaric acid monoester, by alcoholizing a beta-substituted glutaric acid anhydride in the presence of an aliphatic alcohol in an organic solvent containing an enzyme suspended therein,. CONSTITUTION:A beta-substituted glutaric acid anhydride expressed by formula I (R is hydrogen, halogen or substituted or unsubstituted aliphatic hydrocarbon group or substituted or unsubstituted aromatic hydrocarbon group or at least one kind of substituent group containing a hetero-atom., such as oxygen, nitrogen or sulfur) is alcoholized in the presence of an aliphatic alcohol expressed by the formula Z-OH (Z is 1-10C straight-chain or branched chain alkyl) in an organic solvent, such as n-hexane, toluene, diethyl ether or n-dibutyl ether, containing an enzyme, such as lipase or esterase, suspended therein to afford the aimed optically active glutaric acid monoester expressed by formula II or III.

Description

Detailed Description of the Invention [Industrial Field of Application] The present invention provides an optically active β-substituted compound useful as an optically active synthetic intermediate that shortens the synthetic route when synthesizing an optically active substance having physiological activity. This invention relates to a method for producing glutaric acid monoester.

[Prior art]

Conventionally, as an example of the synthesis of optically active β-substituted glutaric acid monoester, Cohen and Ketooli (S.

G. Cohan, E. Nedohri, Nat.
ure, 186.75 (1960) and the proposal by Ohno (Japanese Patent Publication No. 57-713!34) are known. In the former, an optically active monoester is obtained from β-acedo≦toglutarate diethyl ester using α-chymotrypsin, a type of watermelon protease. On the other hand, in the latter, an optically active monoester is obtained from β-benzyloxycarbonylaminoglutarate dimethyl ester using an esterase.

However, these studies have not gone beyond the research on the reaction of enzymes in water, and in recent years, synthetic β-lactam antibiotics such as chenamycin have been attracting attention as fourth-generation antibiotics. It was far from the purpose of production. Namely: ■ Since expensive enzymes are dissolved in water, they cannot be recovered unless they are immobilized. ■When immobilization treatment is performed, enzyme activity decreases during the treatment process. ■Enzymes in aqueous systems are more unstable than in organic solvents under anhydrous conditions, regardless of whether they are immobilized or not. ■Furthermore, the materials used are generally lipophilic and poorly soluble in water, so acetone and DM
A solubilizing agent such as F must be used, which has many disadvantages such as making the enzyme even more unstable.

[Objectives] An object of the present invention is to overcome the drawbacks of the prior art and provide an industrially advantageous method for producing a β-substituted glutaric acid monoester with high optical purity.

〔composition〕

According to the present invention, General formula (1) % Formula % [In the formula, R is hydrogen, halogen, a substituted or M-substituted aliphatic hydrocarbon group, a substituted or unsubstituted aromatic hydrocarbon group, or oxygen, nitrogen , at least one substituent containing a heteroatom such as sulfur. ] In the presence of Z-011 (wherein 2 represents a straight or branched alkyl group having 1 to 10 carbon atoms), the β-substituted glutaric anhydride represented by is suspended in an enzyme-suspending organic solvent. Provided is a method for producing an optically active β-substituted glutaric acid monoester represented by the general formula (U) in which R and 2 are as described above, which is characterized by alcoholysis.

That is, the present invention has five improvements in enzyme stability and recoverability.

In order to improve reusability, a non-aqueous organic solvent in which the enzyme does not dissolve was selected as the reaction solvent, and an alcoholysis reaction was selected as a reaction that allows asymmetric synthesis to be carried out more efficiently.

The reaction formula of the present invention is illustrated as follows.

[Reaction formula of the present invention]

β-substituted gluta/La'1llt! Water optically active β-substituted gluta/14m monoester in the present invention. The mechanism of action regarding the asymmetric synthesis of optically active β-substituted glutaric acid monoesters by enzymatic alcoholysis of β-substituted glutaric acid anhydrides is thought to be as follows.

First, when no enzyme is added to the reaction system, β-substituted glutaric anhydride does not undergo an esterification reaction with alcohol;
As long as the reaction is confirmed by gas chromatography or high performance liquid chromatography, the reaction does not proceed. From this,
It is clear that enzymes are catalysts that drive reactions. Furthermore, when using lipase whose enzymatic activity has been completely lost with P-nitrophenyl diethyl phosphate, the esterification reaction does not proceed at all, indicating that the reaction is not proceeding due to the nucleophilicity of the enzyme protein. things can be estimated. From this, it can be inferred that the reaction is proceeding at the activation site of the enzyme.

In the present invention, the β-substituted glutaric anhydride used is represented by the following general formula (R) as described above.

General formula [wherein R is hydrogen, halogen, a substituted or unsubstituted aliphatic hydrocarbon group, a substituted or unsubstituted aromatic hydrocarbon group, or at least one group containing a heteroatom such as oxygen, nitrogen, or sulfur] It is a substituent of a species. ] In the formula, R is particularly preferably a straight or branched alkyl group having 1 to 6 carbon atoms such as methyl, ethyl or isopropyl, or a halogen such as chloro.

The alcohol (Z-011) used in the present invention represents an aliphatic alcohol.

More specifically, alcohols having no asymmetric carbon atoms include straight-chain or branched aliphatic alcohols having 1 to 10 carbon atoms, and especially straight-chain or branched aliphatic alcohols having 3 to 6 carbon atoms. Branched chain aliphatic alcohols are preferred.

In addition to the alcohols mentioned above, the organic solvents used in the present invention include the following non-aqueous organic solvents. Specific examples of non-aqueous n-solvents include n-pentane, n-
- straight chain aliphatic hydrocarbons such as hexane, n-hebutane,
Isobutane, isopentane.

Branched aliphatic hydrocarbons such as 2-methylpentane, alicyclic hydrocarbons such as cyclopentane and cyclohexane, halogenated hydrocarbons such as methylene dichloride, chloroform, ethylene tetrachloride, dichloroethane and trichloroethane,
Aromatic hydrocarbons such as benzene, toluene, and xylene; aliphatic ethers such as diethyl ether, diisopropyl ether, and n-dibutyl ether; Examples include alicyclic ethers such as herahydrofuran and tetrahydropyran, among which n-hexane, toluene, diethyl ether, and n-dibutyl ether are more suitable.

There is no particular restriction on the amount of organic solvent used, but it is usually 5 to 20 parts by weight per 1 part by weight of the raw material β-substituted glutaric anhydride.
It is desirable to use 0 parts by weight, preferably 20 to 100 parts by weight.

The enzyme in the present invention refers to a hydrolytic enzyme, and more specific examples include lipases such as swine pancreatic lipase, yeast lipase derived from the genus Candida, bacterial lipase derived from the genus Aspergillus, Mucor, and Pseudomonas, or pigs. Examples include esterase derived from the liver, and proteolytic enzymes such as 1-lipsin, chymotrypsin, and subtilisin. The amount of the enzyme may be a catalytic amount, but in consideration of the reaction rate, it is 0.1 to 30 parts by weight, preferably 0.5 to 17 parts by weight, based on 1 part by weight of the raw material β-substituted glutaric anhydride. It is better to do so.

In the present invention, the water content of the reaction system is kept extremely low and the reaction is carried out in a substantially non-aqueous system. Specifically, the total water content contained in the β-substituted glutaric anhydride, alcohol, and organic solvent is 2% (V/V) or less, and further, 0.
5% (W/V) or less is desirable.

〔effect〕

According to the method of the present invention, an optically active β-substituted glutaric acid monoester with high optical purity can be obtained in a very high yield from a relatively inexpensive β-substituted glutaric acid anhydride.

Furthermore, by replacing the conventional water-based enzyme reaction with an organic solvent system, it is possible to react an expensive enzyme in an insoluble state, resulting in excellent recoverability and reusability of the enzyme. Furthermore, problems related to the solubility of raw materials are also eliminated.
This method is suitable as an industrial method for producing optically active β-substituted glutaric acid monoester.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 3-Chloroglutaric anhydride (1,49a) was dissolved in 100ml of toluene, and enzyme powder (Amano P, Amano Pharmaceutical) 1
Add 7g and n-butyl alcohol (4.5g),
Stir for 160 minutes at 30°C. After the reaction was completed, the enzyme powder was filtered off, and the solvent was distilled off from the filtrate under reduced pressure, followed by purification using silica gel column chromatography to obtain (-)-3-chloroglutaric acid monobutyl ester with a yield of 80%. Next, this monoester was condensed with optically active 2-naphthylethylamine, and when liquid chromatography was measured, the optical purity was 89% ae.

Example 2 In order to examine the possibility of enzyme reuse, the enzyme powder used in Example 1 was used repeatedly. The content of the repeated experiment was carried out under exactly the same conditions as in Example 1, and the enzyme powder was used after the reaction was completed.
It was washed with toluene and reused. In the second reaction,
As a result of tracking 1 reaction by thin layer chromatography, 2
It was completed in 00 minutes, and the purification was performed in the same manner as in Example 1.
-Chloroglutaric acid monobutyl ester with optical purity of 89
Obtained in %ee. Furthermore, when a third reaction was carried out using the same enzyme powder, the reaction time was 200 minutes, and 3-chloroglutaric acid monobutyl ester was obtained with an optical purity of 89% ee.

Example 3 3-Methylglutaric anhydride (1.28 g) was dissolved in 100 ml of benzene, 1 g, base powder (Amano CES, Ohno Pharmaceutical) 17 g, and n-butyl alcohol (4.5 g).
Add and incubate at 30℃ for 24 hours. After the reaction is complete, r
After filtering the LII powder and distilling off the solvent from the filtrate under reduced pressure, it was purified by silica gel column chromatography (-)-
3-Methylglutaric acid monobutyl ester was obtained with a yield of 75%. This monoester was converted into a 591-2 compound with a known optical rotation through two steps, and the optical purity was 76%ee and the configuration was R.

Example 4 1.42 g of 3-ethylglutaric anhydride was dissolved in 100 ml of ethyl ether, and enzyme powder (Amano P, Ohno Pharmaceutical) was added.
17g and n-propyl alcohol (3.7g) were added and stirred at 30°C for 5 hours. After the reaction was completed, the enzyme powder was filtered off, and the solvent was distilled off from the filtrate under reduced pressure, followed by purification by silica gel column chromatography to obtain (-)-3-ditylglutarate monopropyl ester in a yield of 95%.

When this monoester was converted into a lactone with a known optical rotation, the optical purity was 70%ee and the configuration was R.

Example 5 3-chloroglutaric anhydride (1.49 g) was dissolved in 100 ml of ethyl ether, and 17 g of enzyme powder (Amano P, Ohno Pharmaceutical) and n-propyl alcohol (3.7 g) were added.
Add and stir at 30°C for 200 minutes. After the reaction is complete,
After separating the enzyme powder by filtration and distilling off the solvent from the filtrate under reduced pressure, purification was performed using silica gel column chromatography to obtain (-)-3-
Chloroglutarate monopropyl ester was obtained with a yield of 80%. Next, this monoester was condensed with optically active 2-naphthylamine, and 8
The optical purity was 80%ee.

Example 6 1.42 g of 3-ethylglutaric anhydride was added to 10 g of toluene.
Dissolved in 0ml, 17g of enzyme powder (PPL, manufactured by Sigma)
Then, add n-butyl alcohol (4.5 g) and add 30
Stir at ℃ for 5 hours. After the reaction is completed, the enzyme powder is filtered and
After distilling off the solvent from the filtrate under reduced pressure, the residue was purified by silica gel column chromatography to obtain (-)-3-ethylglutaric acid monobutyl ester. The yield and optical purity were the same as in Example 4.

Example 7 When 3-ace1-amidoglutaric anhydride was reacted under the same conditions as in Example 1, good results were obtained.

Example 8 When 3-benzylthioglutaric anhydride was reacted under the same conditions as in Example 1, good results were obtained.

Example 9 Purification of enzyme Amano P (50 g) was dissolved in 200 ml of 101011I phosphate buffer (pH 7). After stirring at 0°C for 1 hour and 30 minutes, the precipitate was separated using a centrifuge (1500G,
After 15 minutes), the supernatant was collected and fractionated with ammonium sulfate (ammonium sulfate a
35%). After stirring at 0°C for 1 hour, the precipitate was collected by centrifugation and washed with 35% ammonium sulfate solution. After centrifugation, the precipitate is dissolved in 20 ml of 18 mM phosphate buffer (pH 7) and dialyzed overnight.

Add 16 g of Hyfloth-Purcel to this protein solution, stir, and add acetone to 75% (W/V) at 0°C. Thereafter, the powder is filtered and dried under reduced pressure for 3 hours.

3-Methylglutaric anhydride (t, zgg) is dissolved in 100 ml of toluene, 3 g of the above Hyfloth-Purcel adsorbed enzyme and methanol (0.3 g) are added, and the mixture is stirred at 30°C for 23 hours. After the reaction was completed, the enzyme powder was filtered off, and the solvent was distilled off from the filtrate under reduced pressure, followed by purification by silica gel column chromatography to quantitatively obtain 3-methylglutaric acid monomethyl ester. Next, this monoester was condensed with optically active 2-naphthylethylamine to form a diastereomer, and then subjected to NMR analysis at 200 M 11 z.
When the optical purity was measured, the absolute configuration of the monomethyl form was R and the optical purity was 92% ea.

Example 10 3-Methylglutaric anhydride (1.28 g) was dissolved in 100 mQ of diisopropyl ether, and enzyme powder (Amano P) was dissolved.

2 g of Hitoko ffi) and n-butyl alcohol (1,
5g) and stirred at 25°C for 6 hours.

After the reaction was completed, the enzyme powder was filtered off, and the solvent was distilled off from the filtrate under reduced pressure, followed by purification by silica gel column chromatography to quantitatively obtain (-)-3-methylglutaric acid monobutyl ester. This monoester is processed through two steps to produce jM
When the lactone was introduced into a lactone with a known luminous intensity, its optical purity was found to be 90.8%.

Comparative Example 200 mg of Amano P (Jinko Pharmaceutical) was added to sodium phosphate.
1 buffer (20+nM, pl+7) after dissolving in LomQ.
3-Methylglutarate dimethyl ester (174B, )
, Ommol) and shaken at 37°C for 65 hours.

After the reaction is completed, the reaction solution is acidified with 3N hydrochloric acid and extracted three times with ethyl acetate (10 ml). After washing the extract three times with saturated aqueous sodium bicarbonate solution (10 ml), the washings are acidified with concentrated hydrochloric acid and extracted three times with ethyl acetate (10 ml). The extract was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain 80 mg (50% yield) of (+)-3-methylglutaric acid monomethyl ester. The optical purity of this monoester was 74%ee. As shown in this comparative example, the yield is low in the reaction in an aqueous solution, and the enzyme is dissolved in water.

It has the disadvantage that recovery and reuse of the enzyme is difficult.

Claims (2)

[Claims] (1) General formula (I) ▲Mathematical formulas, chemical formulas, tables, etc.▼(I) [In the formula, R is hydrogen, halogen, substituted or unsubstituted aliphatic hydrocarbon group, substituted or unsubstituted aromatic carbon At least one substituent containing a hydrogen group or a heteroatom such as oxygen, nitrogen, or sulfur. ] The β-substituted glutaric anhydride represented by is suspended in an enzyme-suspending organic solvent in the presence of Z-OH [wherein Z represents a straight or branched alkyl group having 1 to 10 carbon atoms]. General formula (II), which is characterized by alcoholysis ▲There are mathematical formulas, chemical formulas, tables, etc.▼or ▲There are mathematical formulas, chemical formulas, tables, etc.▼(II) [In the formula, R and Z are as above. ] A method for producing an optically active β-substituted glutaric acid monoester. (2) Claim No. 1 in which the enzyme is a hydrolase
Manufacturing method described in )