KR100688770B1 - Method for preparing an optically active R-2-amino-1-butanol by enzymatic method - Google Patents

Abstract

The present invention relates to a method for preparing optically active (R) -2-amino-1-butanol ((R) -2-amino-1-butanol) by enzymatic method, more specifically racemic in aqueous solution The present invention relates to a method for producing optically active (R) -2-amino-1-butanol by an enzymatic method of selectively hydrolyzing an ester compound of 2-amino-1-butanol present in a sieve using a hydrolase. The production method of the present invention is very useful in the industry because it is possible to produce an optically active compound having a high optical purity by a simple method using a hydrolase.

2-amino-1-butanol, racemic body, hydrolysis, hydrolase, protease, esterase, lipase, L-(+)-tartaric acid

Description

Method for preparing an optically active (R) -2-amino-1-butanol by enzymatic method}

The present invention relates to a method for preparing optically active (R) -2-amino-1-butanol by enzymatic method, and more particularly, to buffer an ester compound of racemic 2-amino-1-butanol. After dissolving in a solution, it relates to a method for producing optically active (R) -2-amino-1-butanol by an enzymatic method of selectively hydrolyzing one ester by reacting a hydrolytic enzyme derived from a microorganism.

In racemic 2-amino-1-butanol, (S) -2-amino-1-butanol and (R) -2-amino-1-butanol are present in half and half. Of these, (S) -2-amino-1-butanol is used as an intermediate of Ethambutol, an anti-tuberculosis drug, and (R) -2-amino-1-butanol may also be useful as a pharmaceutical intermediate.

There are many known methods for producing (S) -2-amino-1-butanol from racemic 2-amino-1-butanol. For example, U.S. Patent No. 3,944,619, published in 1976 by American Cyanamid, describes the use of L-(+)-tartaric acid from (S) -2-amino from racemic 2-amino-1-butanol. A method for preparing -1-butanol is described. However, this method is not only difficult to recrystallize, but also has a low yield, and it is difficult to separate the two materials due to the low solubility difference between L-(+)-tartaric acid and (S) -2-amino-1-butanol tartarate. have. In addition, tartarate has a disadvantage in that it is very hygroscopic and difficult to handle.

Nippon Soda's U.S. Patent Publication No. 1471838 discloses dN-benzoyl-trans-2-aminocyclohexane carboxylic acid and lN-benzoyl-trans-2- (S) -2-amino-1-butanol, which is an optically active isomer from racemic 2-amino-1-butanol, with aminocyclohexane carboxylic acid (lN-benzoyl-trans-2-aminocyclohexane carboxylic acid); A method for preparing (R) -2-amino-1-butanol is described. However, this method also requires less than about 10 complex recrystallization steps, resulting in inefficiency, resulting in optical (S) -2-amino-1-butanol and (R) -2-amino-1-butanol. There is a disadvantage that the optical purity is not high as 92-94% ee.

On the other hand, unlike the above processes, a process for separating racemic compounds using lipase enzymes has been developed. Francalanci and Cesti et al. Describe racemic 2-amino-N-ethoxycarbonyl-1-butanol from racemic 2-amino-1-butanol (2-amino-N-ethoxycarbonyl-1). -butanol) and then reacted with ethyl acetate using Porcine pancreatic lipase (S) -2-amino-N-ethoxycarbonyl-1-butanol and ( R) -2-amino-N-ethoxycarbonyl-1-butanol acetate was synthesized and then (S) -2-amino-1-butanol and (R) -2-amino-1-butanol were prepared therefrom It was. The optical purity of (S) -2-amino-1-butanol was 98.4% ee, and the optical purity of (R) -2-amino-1-butanol was 60% ee. At this time, the optical purity of the (R) -2-amino-1-butanol produced as a result of the reaction of the enzyme substrate to racemic 2-amino-N-benzyloxycarbonyl-1-butanol was 90.4% ee. (Ep. 0222561, J. Org. Chem., 52 (23), pp. 5079-82,1987).

In addition, Francalanci and Cesti et al. Selectively hydrolyze racemic 2-amino-N-ethoxycarbonyl-1-butanol acetate using a lipase derived from porcine pancreas (S A method of preparing 2-amino-1-butanol and (R) -2-amino-1-butanol was also disclosed, wherein the optical purity of (S) -2-amino-1-butanol was 99.2% ee, ( The optical purity of R) -2-amino-1-butanol showed 75% ee (European Patent No. 0239122, J. Org. Chem., 52 (23), pp. 5079-82,1987).

In addition, Chisso of Japan has developed a method for producing (S) -2-amino-1-butanol using aminoacylase-containing microorganisms (Japanese Patent Laid-Open No. 58-198296), Bevinakatti et al. Transesterified racemic N, O-diacetyl-2-amino-1-butanol using phosphase pancreatic lipase. The reaction method was developed to produce optically active 2-amino-1-butanol (Tetrahedron: Asymmetry, 1 (9), 583-6, 1990), and Gotor was prepared using lipase (Porcine pancreatic lipase). A method of preparing optically active 2-amino-1-butanol by acylating racemic 2-amino-1-butanol (J. Chem. Soc., Perkin Trans. 1, 21, 2885-9, 1992 ).                         

The above-mentioned methods are all methods for preparing (S) -2-amino-1-butanol, which is an intermediate raw material of etabutol, a tuberculosis treatment agent, and for preparing (R) -2-amino-1-butanol with high purity. No announcement was made. Among the above-mentioned technologies, (R) -2-amino-1-butanol is more than 90% in Nippon Soda and the technology published by Francalanci (European Patent No. 0222561). Although it can be obtained with optical purity, there is a disadvantage that the optical purity is too low for use as an actual pharmaceutical intermediate.

Therefore, there is a need for the development of a process that is economically superior using a simple process and can produce optically active (R) -2-amino-1-butanol having a high optical purity of 98% or more.

Accordingly, the present inventors pay attention to the fact that by treating the ester compound of racemic 2-amino-1-butanol with a suitable enzyme or microorganism, it is possible to prepare (R) -2-amino-1-butanol of high optical purity. The invention has been completed.

It is therefore an object of the present invention to provide a method for producing optically active (R) -2-amino-1-butanol from ester compounds of racemic 2-amino-1-butanol using a hydrolase.

The production method of the present invention for achieving the above object is to selectively hydrolyze the ester compound of racemic 2-amino-1-butanol in the presence of a buffer solution of pH 6-9 using a microbial hydrolase It is composed.

Looking at the present invention in more detail as follows.

As described above, in the present invention, racemic N, O-diacetyl-2-amino-1-butanol (N, O-diacetyl-2-amino-1-butanol) as a substrate is added to a buffer solution of pH 6-9. After appropriate dissolution, the ratio of enzyme and substrate is 1: 10 to 10: 1, and hydrolysis enzymes derived from Rhizopus genus or Candida genus microorganisms are added without a separate treatment step. The mixture is stirred at 100 to 300 rpm at a temperature of 20 to 40 ° C. to selectively hydrolyze only the (S) -isomer to produce (R) -2-amino-1-butanol having optical activity.

The reaction process of the present invention is shown in Scheme 1 below.

The hydrolase used in the present invention is obtained from microorganisms of genus Rhizopus or Candida, which enzymes are immobilized in powder, liquid or carrier, or biological cells containing enzymes and immobilized biological cells. to be.

The microorganism of the genus Rizopus (Rhizopus) is the Rhizopus oryzae , Candida (Candida) is a Candida rugosa (Candida rugosa ) is preferred.

The hydrolase may be used commercially available or manufactured. Commercially available enzymes include, but are not limited to, Ppeptidase R (protease) from Amano and Europa Esterase 2 (Cr / F5) from Europa.

In the present invention, the enzyme reaction temperature is not particularly limited because it is changed depending on the activity temperature of the selected enzyme, the preferred reaction temperature is 20 to 40 ℃ suitable, if the temperature is less than 20 ℃ reaction rate is too slow There exists a problem that optical purity falls when it exceeds 40 degreeC. In addition, the pH of the enzyme reaction is in the range of pH 6 ~ 9 is appropriate, if there is a problem that the reaction rate and optical purity is lowered, the ratio of enzyme and substrate during the hydrolysis reaction is 1: 10 ~ 10: 1 is appropriate, and the ratio of the enzyme and the substrate is out of the range, there is a problem that the reaction rate is slow or the amount of the enzyme used is too large and economic efficiency is low.

Analysis conditions and methods of optically active N, O-diacetyl-2-amino-1-butanol prepared according to the present invention can be analyzed using gas chromatography as follows.

The CP-Chirasil-Dex CB column (length 25m, inner diameter 0.25mm), a Sufelco company chiral column, was heated at 100 ° C for 5 minutes, increased to 200 ° C at a rate of 5 ° C / min, and then stopped at 200 ° C for 15 minutes Let's do it. When helium gas was flowed at a rate of 1.4 ml / min as a carrier and detected using a Flame Ionization Detector (FID) at 250 ° C., (S) -N, O-diacetyl-2-amino-1-butanol Silver is detected at 19.3 minutes and (R) -N, O-diacetyl-2-amino-1-butanol at 19.5 minutes. N-acetyl-2-amino-1-butanol produced as a result of the hydrolysis reaction is detected at 20.1 minutes, in which case the (S) -isomer and the (R) -isomer are not distinguished.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.

Example 1

To a mixture of 50 g of 2-amino-1-butanol dissolved in 5 L of dichloromethane were added 114 g of trimethylamine and 115 g of acetic anhydride in order, followed by stirring at room temperature. After the reaction, water was added and extracted, MgSO ₄ was added to the organic layer to remove the water and filtered. After distilling the filtrate, 87.2 g of a white solid, racemic N, O-diacetyl-2-amino-1-butanol (yield 92%) was recovered.

Example 2

5 ml of 100 mM phosphoric acid solution (pH 7.0) was added 0.1 g of racemic N, O-diacetyl-2-amino-1-butanol obtained in Example 1, and the pH was adjusted to 7.0. 0.5 g of enzyme was added and the reaction was carried out at 30 ° C. 0.5 ml of the reaction solution was extracted with 1 ml of ethyl acetate and analyzed by gas chromatography using the aforementioned analytical method.

enzyme microbe Optical purity (% ee) Absolute configuration of N, O-diacetyl-2-amino-1-butanol Remarks Protease Rhizopus oryzae 100 S Aspergillus niger 0 100% hydrolysis Aspergillus oryzae 0 100% hydrolysis Lipase Pseudomonas cepacia 0 100% hydrolysis Candida a ntarctica 0 100% hydrolysis Alcaligenes sp. 0 100% hydrolysis Esterase Candida rugosa 91.2 S

As shown in Table 1, protease from Rhizopus oryzae and esterase from Candida rugosa selectively hydrolyzed only the (S) -isomer, while other hydrolysis was performed. The enzyme was not selective. In addition, experiments were carried out using about 60 hydrolytic enzymes in addition to the enzymes shown in Table 1, but most of them were found to have no selectivity.

Example 3

Among the enzymes selected in Example 2, the following reaction was performed using a protease derived from Rhizopus oryzae . 400 ml (10% w / v) of racemic N, O-diacetyl-2-amino-1-butanol was added to 400 ml of 100 mM phosphoric acid solution (pH 7.0), pH was adjusted to 7.0, and then 40 g of the protease was added thereto. The reaction was carried out at 30 ° C. The pH during the reaction was maintained at 6.5-7.5 using 20% sodium hydroxide solution. Reaction samples were taken at regular intervals to analyze the reaction progress. After 47 hours of reaction, when the optical purity of (R) -N, O-diacetyl-2-amino-1-butanol reached 98.0% ee or more, the reaction was stopped and dichloromethane (CH ₂ Cl ₂ ) was added to the reaction solution. Was added to extract N, O-diacetyl-2-amino-1-butanol which remained unhydrolyzed. The extraction process was repeated four times to separate the organic solvent layer and dichloromethane was removed by distillation under reduced pressure. As a result, 12.5 g of (R) -N, O-diacetyl-2-amino-1-butanol (yield 31.3%) Was recovered. A part of this was purified by a column, and the optical purity of (R) -N, O-diacetyl-2-amino-1-butanol was measured and found to be 99.1%.

Example 4

10 g of (R) -N, O-diacetyl-2-amino-1-butanol obtained in Example 3 was placed in a 1N hydrochloric acid solution and refluxed. After completion of the reaction, dichloromethane was added to the reaction mixture, followed by extraction. The organic solvent layer was distilled under reduced pressure to obtain (R) -O-acetyl-2-amino-1-butanol, which was dissolved in methanol and then 13 g of K ₂ CO _3. Was added and stirred. The reaction mixture was filtered to remove K ₂ CO ₃ to obtain 4.2 g of (R) -2-amino-1-butanol (yield: 82%). The optical rotation of the (R) -2-amino-1-butanol using a polarimeter was [α] _D ²⁵ = 12.38 (c = 2 in ethanol). This value corresponds to 99.1% ee of optical purity.

Example 5

The reaction was carried out using an esterase derived from Candida rugosa instead of the protease under Example 3 conditions. 13 g (10% w / v) of racemic N, O-diacetyl-2-amino-1-butanol was added to 130 ml of 100 mM phosphate solution (pH 7.0), pH was adjusted to 7.0, and 13 g of esterase was added thereto. The reaction was carried out at 30 ℃. The pH during the reaction was maintained at 6.5-7.5 using 20% sodium hydroxide solution. Reaction samples were taken at regular intervals to analyze the reaction progress. When the optical purity of (R) -N, O-diacetyl-2-amino-1-butanol reached 90.5% ee after 72 hours, the reaction was stopped and dichloromethane (CH ₂ Cl ₂ ) was added to the reaction solution. Was added to extract N, O-diacetyl-2-amino-1-butanol which remained unhydrolyzed. The extraction process was repeated four times to separate the organic solvent layer separately, and dichloromethane was removed by distillation under reduced pressure. As a result, 1.6 g of (R) -N, O-diacetyl-2-amino-1-butanol (yield 12.3%) was obtained. Was recovered. 1.6R of (R) -N, O-diacetyl-2-amino-1-butanol having an optical purity of 90.5% ee was recrystallized with diisopropyl ether to give (R)-of an optical purity of 99.3% ee. 1.24 g of N, O-diacetyl-2-amino-1-butanol (77.3% recrystallization yield) were obtained. (R) -N, O-diacetyl-2-amino-1-butanol obtained here was treated in the same manner as in Example 4 to give 0.5 g of (R) -2-amino-1-butanol (yield: 80%) Got. The optical rotation of this (R) -2-amino-1-butanol was measured using a polarimeter. The result was [α] _D ²⁵ = 12.4 (c = 2 in ethanol). This corresponds to an optical purity of 99.2% ee.

As described above, the method of the present invention can produce a high optical purity optically active (R) -2-amino-1-butanol by a simple method using a hydrolase, in particular a complex recrystallization process using an enzyme It is very useful industrially because it can achieve high optical purity of more than 98% at a time.

Claims (7)

In the presence of a buffer solution of pH 6-9, ester compounds of racemic 2-amino-1-butanol are selectively selected using a hydrolytic enzyme derived from Rhizopus sp. or Candida sp. A method for producing optically active (R) -2-amino-1-butanol by enzymatic method, characterized in that hydrolysis is carried out. The method according to claim 1, wherein the hydrolase is immobilized in powder, liquid or carrier and characterized in that it is a protease or esterase derived from a microorganism. delete The method of claim 1, wherein the genus of the genus of the genus is Rezopus duck (Rhizopus oryzae ). The method of claim 1, wherein the Candida genus microorganism is Candida rugosa . The method of claim 1, wherein the temperature of the hydrolysis reaction is 20 to 40 ℃. The method of claim 1, wherein the ratio of the enzyme and the substrate in the hydrolysis reaction is characterized in that 1: 10 to 10: 1.