JP2002335989A - Method for purifying isobutyl d-lactate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an industrial method for purifying isobutyl D-lactate having an optical purity of >=99.60%ee and useful as a raw material for pharmaceuticals. SOLUTION: Isobutyl D-lactate having an optical purity of 90.00-99.60%ee is purified by preferentially hydrolyzing the existing isobutyl L-lactate with a lipase (abbreviated to C lipase) originated from a microbial strain of the genus Candida (especially Candida rugosa) in the presence of an organic solvent to obtain isobutyl D-lactate having an optical purity of >=99.60%ee. Isobutyl D-lactate having the optical purity of >=99.60%ee can be produced on an industrial scale with a simple procedure in high yield and high pot efficiency by using an industrially available isobutyl D-lactate as a starting raw material. The organic solvent used in the process can be recovered and reused to reduce the load on waste water treatment. The water phase containing C lipase is resistant to putrefaction and is repeatedly usable.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for purifying isobutyl D-lactate useful as a raw material for agrochemicals.

[0002]

2. Description of the Related Art In some cases, isobutyl D-lactate used as a raw material for medicines is required to have extremely high optical purity. However, a method for purifying isobutyl D-lactate to a high optical purity of 99.60 ee% or more on an industrial scale is not known. Therefore, there has been a demand for a method capable of purifying D-isobutyl lactate having an extremely high optical purity required for a pharmaceutical raw material on an industrial scale.

[0003]

DISCLOSURE OF THE INVENTION The present invention relates to a process which can be carried out industrially to purify, on an industrial scale, isobutyl D-lactate having a high optical purity of at least 99.60 ee%, which is useful as a pharmaceutical raw material. The task was to establish a way to do what could be done.

[0004]

In view of the above situation, the present inventors have made intensive studies on a method for industrially obtaining isobutyl D-lactate having extremely high optical purity. As a result, unexpectedly, an optical purity of about 95 ee% (90.00 ee% or more and 99.60 e) which can be industrially obtained as a raw material.
(less than e%) D-isobutyl lactate (hereinafter, "raw material D-isobutyl lactate" is synonymous) and hydrolyzed using a lipase derived from Candida genus in the presence of an organic solvent. However, isobutyl L-lactate is hydrolyzed and decomposed into L-lactic acid and isobutanol, and the raw material D-isobutyl lactate has an optical purity of 99.60 e.
It has been found that it is possible to purify into D-isobutyl lactate having a high optical purity of e% or more. Furthermore, this method has found that the yield of the target product is high, the operation is simple, the pot efficiency is good, the method can be carried out industrially as a whole, and the above-mentioned problems can be solved. It was completed.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.

The present invention has solved the above-mentioned problems by providing the inventions described in the following [1] to [3]. [1] Optical purity of 90.00ee% or more and 99.60ee
% Of isobutyl D-lactate in the presence of an organic solvent using L-lipase derived from the genus Candida.
A method for purifying D-isobutyl lactate, comprising purifying isobutyl lactate by hydrolysis to obtain D-isobutyl lactate having a high optical purity of 99.60 ee% or more. [2] The method for purifying isobutyl D-lactate according to [1], wherein the organic solvent is an aromatic hydrocarbon, an aliphatic hydrocarbon, an ether solvent or an aromatic halogenated hydrocarbon. [3] Lipase is Candida rugosa ( Candida)
(Rugosa ) -derived lipase according to [1] or [2].

The optical purity of the starting material D-isobutyl lactate and the target D-isobutyl lactate used in the present specification is as follows:
Enantiomeric excess commonly used in the field of optically active compounds (% enantiomeric excess, "ee
% ". ).

Hereinafter, the present invention will be described in detail.

In the purification method of the present invention, the starting material D-isobutyl lactate is hydrolyzed in the presence of an organic solvent using a lipase derived from Candida sp. The hydrolysis is performed by recovering the remaining isobutyl D-lactate that has not been hydrolyzed from the organic solvent layer used. This hydrolysis reaction is carried out in an organic solvent.
-A two-layer system consisting of a layer of an organic solvent in which isobutyl lactate is dissolved, and an aqueous layer in which a lipase derived from the genus Candida is dissolved. In the two-layer reaction, the amount of D-isobutyl lactate dissolved in the aqueous layer is reduced by the use of a solvent, and post-reaction processing such as removal of a target substance is easy.
It is suitable in terms of operation, and in the purification method of the present invention, the target substance can be easily recovered from the organic solvent layer separated from the aqueous layer. It is possible to use the enzyme repeatedly with or without supplementing the enzyme, and from these points, it is suitable for industrial practice from the viewpoint that the load on wastewater can be reduced.

First, the raw material D-isobutyl lactate used in the present invention will be described.

The purification method of the present invention can be applied in principle even when the optical purity of isobutyl D-lactate used as a raw material is less than 90.00 ee%. However, in consideration of the overall efficiency and load of the actual operation, the pot efficiency, the reaction time, the yield of the target product, the availability and cost of the raw material, etc., in the purification method of the present invention, as the raw material D-isobutyl lactate, , 90.00ee
% Or more and less than 99.60 ee%, preferably 95.00 e
e% or more and less than 99.60 ee% D-isobutyl lactate is used.

The lipase derived from the genus Candida used in the purification method of the present invention (hereinafter simply referred to as “C
Lipase ". ) Is, for example, lipase
AY [manufactured by Amano Enzyme Co., Ltd., Candida rugosa ( C
Andida rugosa) from,] can be used, specifically, Lipase AY "Amano" 30 [Amano Enzyme Co., Ltd., (30000U / g)], Lipase AY "Amano" 30G [Amano Enzyme Inc. Made,
(30000 U / g)], Lipase AYLS [enzyme aqueous solution manufactured by Amano Enzyme Co., Ltd. (20,000 U / m)
l)] etc., and tirazyme (C
HIRAZYME) L3 Lyo [manufactured by Rossie Co., Ltd.
Product No. 1600885] In addition, industrially available lipases derived from the genus Candida can be used.

U / g and U / ml described as values indicating the activity of the C lipase are expressed in terms of fat digestion [LAMP method using olive oil (Amano Enzyme), 37 ° C., pH
7.0, 30 minutes]. The amount of the enzyme that releases 1.0 microequivalent fatty acid from olive oil under the measurement conditions is defined as 1 U (unit), and multiplied by the dilution ratio of the enzyme. It is calculated and displayed (in the present specification, unless otherwise specified, the enzyme activity is indicated by this definition).

In the purification method of the present invention, C lipase may be used as a mixture of two or more kinds, and may be in the form of a powder, a liquid such as an aqueous solution, or immobilized on clay, diatomaceous earth, ceramic or the like. It may be used in the supplied form as it is.

The amount (weight and volume) of C lipase used in the purification method of the present invention depends on the degree of purification of the C lipase used, the form to be used (whether or not excipients and stabilizers are used, and whether it is solid or not). Liquid or the like) or active (U / g or U
/ Ml), etc., so it cannot be said unconditionally. For example, the lipase AY “Amano” 30 [Amano Enzyme Co., Ltd.]
When (30000 U / g) is used, the range of 0.01 to 100 g per 1 mol of isobutyl D-lactate can be exemplified as an actual usage amount, but such weight basis is used. It may be appropriately determined in consideration of the scale of the reaction system, the pot efficiency, and the like, and it is preferable to determine the amount to be used in consideration of the activity (U / g or U / ml) of C lipase. The amount of C lipase used is D
The activity as defined above is 100 to 5,000,000 U, preferably 300, based on 1 mol of isobutyl lactate
0-300000U, more preferably 10,000-1
The amount (weight / volume) in the range of 000000U can be exemplified.

The organic solvent used in the purification method of the present invention may be any solvent which dissolves raw material D-isobutyl lactate and does not inhibit the hydrolysis reaction by C lipase and separates from water. , Ether solvents, aliphatic hydrocarbons and aromatic halogenated hydrocarbons can be used. Examples of the aromatic hydrocarbons include benzene, toluene, xylene and the like. Examples of the ether solvents include diethyl ether and diisopropyl ether, and the like. Include, for example, pentane, n-hexane, heptane, cyclohexane and the like, and as the aromatic halogenated hydrocarbons, for example, chlorobenzene and the like can be mentioned. Preferably, they are aromatic hydrocarbons, ether solvents, or aliphatic hydrocarbons. These are given as examples, and the organic solvents that can be used in the present invention are not limited to these examples.

The organic solvent may be used alone or in any mixture. The amount of the organic solvent to be used should be at least an amount capable of sufficiently stirring, but in consideration of the pot efficiency and the like, 10 ml or more, preferably 30 to 2000 m, per 1 mol of the raw material D-isobutyl lactate.
1, more preferably in the range of 50 to 1000 ml.

In the purification method of the present invention, the amount of water in the aqueous layer is:
It suffices that the amount of C lipase dissolve or exceed the amount necessary for mixing. However, considering the kettle efficiency and the like, 5 ml or more, preferably 10 ml, per mol of raw material D-isobutyl lactate.
The range may be, for example, 2,000 ml, more preferably 50-1000 ml.

The hydrolysis reaction in the purification method of the present invention comprises:
The pH of the aqueous layer is preferably maintained in the range of 3 to 10, preferably 4 to 9, and more preferably 5 to 8. In the hydrolysis reaction, lactic acid is released into the system as the reaction progresses, so that the pH of the aqueous layer gradually decreases from the pH at the start of the reaction. Is dissolved in a known buffer (for example, 0.01
The pH during the hydrolysis reaction can be maintained in the above-mentioned pH range by setting the concentration to a concentration of ２2 M (mol / l) in a monopotassium phosphate / dipotassium phosphate buffer or the like.
In addition, even if the aqueous layer is not used as a buffer, the pH is measured and the pH is reduced, and phosphate phosphates such as dipotassium phosphate, monopotassium phosphate, disodium phosphate, monosodium phosphate, etc .; Alkali metal carbonates and alkali metal bicarbonates such as sodium carbonate, potassium bicarbonate and sodium bicarbonate; alkaline earth metal carbonates such as calcium carbonate; alkali metal hydroxides such as sodium hydroxide and potassium hydroxide; One or more pH adjusters such as organic amines such as triethylamine and pyridine; or ammonia or the like, as they are, or, for example, aqueous solutions having an appropriate concentration of about 0.1 to 50%, preferably about 1 to 25%. It is also possible to adjust to the above-mentioned pH range by adding as above.

The reaction temperature in the hydrolysis reaction in the purification method of the present invention may be not less than the freezing point of the solvent or 0 ° C. or higher.
Preferably 10 to 50 ° C, more preferably 20 to 40 ° C
The reaction time depends on the used amount and concentration of the raw material D-isobutyl lactate, the used amount and concentration of C lipase, the activity value, the reaction temperature, and the like.
There is no particular limitation as long as the optical purity of isobutyl lactate is at least 99.60 ee% or more. For example, from 1 hour to 14 days, preferably from the viewpoint of the yield of the objective D-isobutyl lactate and industrial viewpoint. Is preferably 5 to 48 hours.

After completion of the hydrolysis reaction, the aqueous layer that can be recovered by liquid separation can be used repeatedly with or without supplementation of enzymes as necessary.

[0022]

According to the present invention, a novel method for purifying D-isobutyl lactate having extremely high optical purity is provided. According to the purification method of the present invention, 90.
Using D-isobutyl lactate of 00% to 99.60 ee% as a raw material, D-isobutyl l-lactate having an extremely high optical purity of 99.60 ee% or more, which can be used as a raw material for medicine, can be obtained with good pot efficiency and good yield. It can be obtained on an industrial scale with a simple operation. In the purification method of the present invention, the target substance may be recovered from the layer of the organic solvent, and the used organic solvent can be reused after the recovery of the target substance, so that the load of wastewater treatment can be reduced. Further, in the aqueous layer containing C lipase separated after the completion of the reaction, the organic solvent used for the reaction may have been dissolved to almost its saturation limit, or the C lipase separated without handling in a special sterile environment. The aqueous layer containing the substance hardly rots, so the used aqueous layer can be used repeatedly without discarding.
As a whole, it is environmentally friendly and advantageous in terms of industrial economy, and the method of the present invention has high industrial utility value.

[0023]

Next, the purification method of the present invention will be described specifically with reference to examples, but the present invention is not limited to these examples.

Example 1 Isobutyl D-lactate 2.0 having an optical purity of 97.6 ee%
g (0.0146 mol) was dissolved in 2 ml of toluene.
Separately, 0.16 g of lipase AY “Amano” 30G
[Amano Enzyme Co., Ltd., (30000U / g)]
With a 0.67 M (mol / l) -phosphate buffer (pH =
6.7) The resulting solution was dissolved in 10 ml to form an aqueous layer. This aqueous layer was mixed with the toluene layer and mixed and stirred at 25 ° C. for 24 hours. After completion of the reaction, the reaction solution was separated, and the obtained toluene layer was analyzed by gas chromatography (column: Lipodex-E).
(MACHEREY & NAGEL (Germany) product), injection temperature: 200 ° C, column temperature: 40 ° C to 100 ° C (after holding at 40 ° C for 5 minutes, 3
℃ / min to 100 ℃) ~ 160 ℃ (100 ℃
After reaching the temperature, the temperature was raised to 160 ° C. at a rate of 10 ° C./min.
(Holding at 14 ° C. for 14 minutes), the injection amount: 1 μl, absolute calibration curve method), and 1.6 g of isobutyl D-lactate having an optical purity of 99.68 ee% was obtained. The yield was 80% (based on raw material D-isobutyl lactate).

Examples 2 to 4 The same procedure as in Example 1 was carried out except that the types of the solvent and the lipase in Example 1 were changed, and the analysis was carried out in the same manner. The results are shown in (Table 1).

[0026]

[Table 1]

Example 5 The starting material D-isobutyl lactate was changed to D-isobutyl lactate having an optical purity of 99.2 ee%, and lipase was changed.
The analysis was performed in the same manner as in Example 1. The results are shown in (Table 2).

[0028]

[Table 2]

Example 6 (Repeated Use of Aqueous Layer) (First time) 146.2 g (1 mol) of isobutyl D-lactate having an optical purity of 97.6 ee% was dissolved in 150 ml of toluene. 13. Separately, lipase AYLS [manufactured by Amano Enzyme Co., Ltd. (20,000 U / ml), aqueous enzyme solution]
6 ml was added to 50 ml of water, and the obtained aqueous layer was mixed with the toluene layer, and the mixture was reacted by mixing and stirring at 25 to 30 ° C. During the reaction, 5% -ammonia water is appropriately added to p of the reaction system.
It stirred for 48 hours, adding so that H might be in the range of 6-7. The used 5% -ammonia water was 139 g. After completion of the reaction, the reaction solution was separated, and the obtained toluene layer was subjected to gas chromatography analysis in the same manner as in Example 1. As a result, 102.3 g of isobutyl D-lactate having an optical purity of 99.72 ee% was obtained. found. 70% yield
Met. The results are shown in (Table 3).

(Second time) In the second time, an aqueous layer (182 g including dissolved lactate and the like) obtained by separating and collecting the reaction solution after the first reaction was used as the first aqueous layer. 7.3 m of lipase AYLS [manufactured by Amano Enzyme Co., Ltd., 20000 U / ml, aqueous enzyme solution]
A hydrolysis reaction was carried out in the same manner as the first time except that 1 was replenished, to obtain D-isobutyl lactate. The results are shown in (Table 3).

(Third time) In the third time, an aqueous layer (257 g including dissolved lactate and the like) obtained by separating and collecting the reaction solution after the completion of the second reaction is used as the first aqueous layer. 6. Repeatedly use and add lipase AYLS [manufactured by Amano Enzyme Co., Ltd., 20000 U / ml, aqueous enzyme solution] to this aqueous layer.
A hydrolysis reaction was carried out in the same manner as the first time except that 3 ml was replenished to obtain D-isobutyl lactate. (Table 3)
Shown in

[0032]

[Table 3]

Comparative Example 1: Example of use of Lipase L-3001 (lipase derived from wheat germ) D-isobutyl lactate 2.0 having an optical purity of 97.6 ee%
g (0.0146 mol) was dissolved in 2 ml of toluene.
Also, lipase L3001 [a lipase derived from wheat germ (Wheat germ) manufactured by Sigma, 8500 U / g (substrate triacetin, pH7.
4)] 200 mg of 0.67 M-phosphate buffer (pH =
6.7) Dissolved in 10 ml, mixed and stirred at 25 ° C. for 6 days, taken a part of the reaction solution, and analyzed the toluene layer obtained in the same manner as in Example 1. The optical purity of isobutyl D-lactate was determined. Was not improved to 98.60 ee% or more.
Add 80 mg of Lipase L3001 and add 8 more.
The reaction was carried out for 14 days (a total of 14 days), but no remarkable improvement in optical purity was observed, and only 0.78 g of isobutyl D-lactate having an optical purity of 98.82 ee% was obtained.

Comparative Example 2: Example of use of lipase L-3126 (lipase derived from porcine pancreas) D-isobutyl lactate 2.0 having an optical purity of 97.6 ee%
g (0.0146 mol) are dissolved in 2 ml of toluene.
In addition, L3126 [manufactured by Sigma, lipase derived from pig pancreas, 46000 U / g (substrate olive oil, pH 7.
7)] 0.21 g of a 0.67 M phosphate buffer (pH =
6.7) It was dissolved in 10 ml, mixed with the toluene layer obtained previously, and mixed and stirred at 25 ° C for 4 days. When a part of the reaction solution was analyzed in the same manner as in Example 1, the optical purity of D-isobutyl lactate was not improved to 98.80 ee% or more. 0.1 g of lipase L3126 was newly added, and the reaction was continued for another 6 days (total of 10 days). However, no improvement in optical purity was observed, and D with optical purity of 98.80 ee% was observed.
-Only 0.77 g of isobutyl lactate was obtained.

Comparative Example 3: Example of use of C lipase for methyl D-lactate D-methyl lactate having an optical purity of 96.6 ee% used in the present comparative example is methyl D-lactate and DL-methyl lactate (racemate). Was appropriately mixed and prepared. This optical purity is 96.
2.0 g (0.0194 mol) of 6ee% methyl D-lactate is dissolved in 2 ml of toluene. In addition, 0.10 ml
AYLS [manufactured by Amano Enzyme Co., Ltd., 20
000 U / ml, aqueous enzyme solution] in 10 ml of a 0.67 M phosphate buffer (pH = 6.7), mixed with the previously obtained toluene layer, and mixed and stirred at 25 ° C. for 2 hours. Did not rise by 96.7 ee% or more. In addition, 2
The reaction was continued for one day, and 0.4 g of D-methyl lactate having an optical purity of 97.0 ee% was obtained (gas chromatography analysis, column; Lipodex-E, absolute calibration curve method).

The results of Comparative Examples 1, 2, and 3 show that the purification method of the present invention exerts a remarkable effect particularly when a combination of C lipase as lipase and isobutyl D-lactate as substrate is used. It can be seen that it is.

Claims (3)

[Claims] An optical purity of 99.6 ee% or more and 99.6.
Isobutyl L-lactate of less than 0 ee% is hydrolyzed with lipase derived from the genus Candida in the presence of an organic solvent to give D with a high optical purity of 99.60 ee% or more. -A method for purifying D-isobutyl lactate, characterized by obtaining isobutyl lactate. 2. The method for purifying isobutyl D-lactate according to claim 1, wherein the organic solvent is an aromatic hydrocarbon, an aliphatic hydrocarbon, an ether solvent or an aromatic halogenated hydrocarbon. Wherein the lipase is Candida rugosa (Candi
da rugosa ).
Or the method for purifying isobutyl D-lactate according to claim 2.