KR100748897B1 - The method of making optically active 3-hydroxybutyric acid and their esters by enzymatic method - Google Patents

Abstract

The present invention relates to a method for preparing racemic 3-hydroxybutyric acid represented by the general formula (1) in [Scheme 1] with optically active 3-hydroxybutyric acid and esters thereof by enzymatic method. It is about. More specifically, the racemic 3-hydroxy butyric acid represented by the general formula (1) in the presence of an acyl donor in an organic solvent and a nonsolvent is esterified with a lipase enzyme to give general formulas (2) and ( It relates to a method for producing optically active 3-hydroxy butyric acid and esters thereof represented by 3).

Since the method of the present invention uses a lipase as a manufacturing method that has not been reported before, the process is not only simple and environmentally friendly, but also easy to separate the acid and the ester, thereby obtaining a high yield of optically active 3-hydroxybutyric acid after the reaction. have.

Scheme 1

(R = CH ₃ (CH ₂ ) _n CO, n = 0 ~ 6 or R = C ₆ H ₅ CO.)

3-hydroxy butyric acid, optical activity, acyl donor, lipase, ester reaction

Description

The method of making optically active 3-hydroxybutyric acid and their esters by enzymatic method

( R = CH₃(CH₂)_nCO, n=0~6 또는 R = C₆H₅CO 임.)The present invention relates to a process for preparing optically active 3-hydroxy butyric acid and esters thereof by enzymatic methods. More specifically, the racemic 3-hydroxy butyric acid represented by the general formula (1) in the following [Scheme 1] in the presence of an acyl donor is esterified with a lipase enzyme to be represented by the general formulas (2) and (3). A method of preparing optically active 3-hydroxy butyric acid and esters thereof.

(R = CH ₃ (CH ₂ ) _n CO, n = 0 ~ 6 or R = C ₆ H ₅ CO.)

The ester derivative of (R) -3-hydroxy butyl acid represented by the general formula (3) produced by the present invention can be easily converted to (R) -3-hydroxy butyl acid by deacylation reaction. In addition, (R)-and (S) -3-hydroxybutyric acid are precursors of a wide range of optically active ethyl 3-hydroxy butyrates as major chiral intermediates, and ethyl 3-hydride by general esterification under alcohol and acid catalyst conditions. It can be easily prepared with oxy butyrate.

Ethyl (R) -3-hydroxybutyrate is anti glaucoma

chirality in industry II. Chichester, UK: Wiley, 1997, 245-262, and ethyl (S) -3-hydroxybutyrate is a pheromone (Tetrahedron, 1989, 45: 3233-3298) and carbapenem (Journal of the Chemical Society.Perkin Transaction, 1999, 1: 2489-2494).

The preparation method of optically active ethyl 3-hydroxy butyrate reported to date is as follows.

Ethyl Acetoacetate (ethylacetoacetate) using the BINAP-coordinated Ru (II) complex to prepare ethyl (S) -3-hydroxybutyrate by asymmetric synthesis (Journal of the American Chemical Society, 1987, 109: 5856-5858) There is a method, but high pressure (100 atm) is not suitable for mass production.

Jayasinghe et al. (Tetrahedron Letters, 1993, 34: 3949-3950) converted ethylacetoacetate to ethyl (S) -3-hydroxybutyrate on petroleum ether using lyophilized yeast as a biocatalyst. At this time, the yield was 58%, optical purity was 94% ee. Medson et al. (Tetrahedron: Asymmetry, 1997, 8: 1049-1054) reduced ethylacetoacetate to organic solvents using yeast, yield 69%, optical purity 99% ee, ethyl (S) -3- Hydroxybutyrate was prepared. Chin-Joe et al. (Biotechnology and Bioengineering, 2000, 69: 370-376) also reduced ethylacetoacetate with Baker's yeast as a catalyst and ethyl (S) -3 with optical purity of 99% ee. Hydroxybutyrate was obtained, yield being 85%.

However, the production method by the above reduction reaction is difficult to apply to the actual process because the yield of the reduction reaction is lowered as well as the concentration of the reactants is low.

Sugai et al. (Agricultural and Biological Chemistry, 1989, 53: 2009-2010) reported that 99.4% ee using vinyl butanoate as the acyl donor in racemic ethyl 3-hydroxybutyrate and lipase in the pig pancreas as a catalyst. Ethyl (S) -3-hydroxybutyrate having an optical purity of was obtained. In addition, Fishman et al. (Biotechnoogy and Bioengineering, 2001, 74: 256-263) used vinyl acetate as an acyl donor and used CAL B ( Candida antartica lipase B) as a catalyst to provide an optical purity of 96% ee. S) -3-hydroxybutyrate was obtained in a yield of 40%, and the reaction of ethyl (R) -3-acetoxybutyrate (ethyl-3-acetoxybutyrate) produced by the reaction with lipase CAL B again (alcololysis) 96% ee of ethyl (R) -3-hydroxybutyrate was obtained in 33% yield.

As described above, the method of preparing the optically active ethyl 3-hydroxybutyrate using the reaction of the microorganism or the enzyme and the chiral catalyst is not high in the yield and the reactant concentration of the optically active ethyl 3-hydroxybutyrate, or after the reaction. There is a disadvantage that the separation and recovery of the product is difficult. However, the optically active 3-hydroxy butyric acid prepared according to the present invention can be easily prepared and the optically active ethyl 3-hydroxybutyrate having excellent quality.

Accordingly, the present inventors have described the optically active 3-hydroxy butyric acid and 3-hydride represented by the general formulas (2) and (3) by ester reaction from the racemic 3-hydroxybutyl acid represented by the general formula (1). The research on the preparation of the oxy-butyl acid ester derivative has not been reported to date, and the production of the present invention focusing on the fact that the acid and the ester can be extracted with an organic solvent and an aqueous solution after the ester reaction, so that the separation and recovery are easy. The method was developed.

We have developed a process for preparing racemic 3-hydroxy butyric acid ester derivatives of (S) -3-hydroxy butyric acid and (R) -3-hydroxy butyric acid using lipase enzymes, and also suitable lipase enzymes. The opposite is also possible with.

Unlike the conventional method, the method according to the present invention is economical because it is easy to separate and recover the acid and the ester, and the process is simple to prepare (R)-or (S) -3-hydroxybutyric acid having high optical purity. can do. Accordingly, it is an object of the present invention to use optically active 3-hydroxybutyric acid represented by the general formulas (2) and (3) from racemic 3-hydroxybutyric acid represented by the general formula (1) using a lipase and its It is to provide a method for preparing an ester derivative.

The production method of the present invention for achieving the above object is a racemic 3-hydroxy butyl acid represented by the general formula (1) in the presence of an acyl donor in an organic solvent, or in the presence of only an acyl donor without a separate organic solvent Stereoselective esterification using a lipase enzyme.

Looking at the present invention in more detail as follows. As described above, the present invention relates to a process for preparing high optical purity 3-hydroxy butyric acid and 3-hydroxy butyric acid ester derivatives from racemic 3-hydroxy butyric acid using a lipase enzyme.

Lipases used in the present invention include enzymes or immobilized lipases provided in the form of a powder. In particular, the lipase may be commercially available or may be prepared and used as necessary. Commercially available lipases include, for example, Novozyme 435 (CAL B) from Novo, Lipase PS from Amano, PS-C, AK or Candida rugosa lipase (CRL) from Sigma. However, it is not limited thereto.

Organic solvents usable in the present invention include isopropyl ether, t-butylmethylether, t-butylmethylether, tetrahydrofuran, methylenechloride, and the like. Vinyl acetate, vinyl propionate, isopropenyl acetate, acetic anhydride, and the like are also used.

On the other hand, racemic 3-hydroxybutyric acid and (R)-and (S) -3-hydroxybutyric acid, racemic 3- O -acyl butyric acid and optical activity (R)-and (S) -3- O- Acyl butyric acid was analyzed using gas chromatography (Donam Instruments, Model DS 6200), at which time the reaction solvent was collected and analyzed as it was without any pretreatment.

The racemic 3-hydroxybutyric acid was heated by capillary column G-TA (Astec, 30 m × 0.32 mm) at 100 ° C. for 5 minutes and then heated to 170 ° C. by 20 ° C. per minute, and then heated at 170 ° C. for 15 minutes. Maintained. Helium gas was used as the carrier and was detected using FID at 170 ° C. while maintaining the column head pressure at 6 psi. The 3-hydroxy-butyl acid was detected at 11.0 minutes, 3-O-acetyl-butyric acid (3- O -acetyl-3-butyric acid) was 12.5 minutes, 3-O-propionyl-butyric acid (3- O -propionyl-3-butyric acid) was detected at 13.8 min.

Optically active (R)-and (S) -3-hydroxybutyric acid was quantified using gas chromatography equipped with G-TA (30m × 0.32mm, Astec, Inc.), a capillary column. Analytical conditions for (R)-and (S) -3-hydroxybutyric acid heated the column at 120 ° C. for 10 minutes, and then raised 10 ° C. per minute to 170 ° C. and maintained the temperature at 170 ° C. for 15 minutes. Helium gas was used as the carrier and was detected using FID at 170 ° C. while maintaining the column head pressure at 10 psi. (R) -3-hydroxy butyric acid was detected in 8.3 minutes and (S) -3-hydroxy butyric acid in 7.9 minutes, respectively. (R) -3- O -acetyl-butyl acid is 12.0 minutes, (S) -3- O -acetyl-butyl acid is 12.5 minutes, (R) -3- O -propionyl-butyl acid is 13.4 minutes, (S) 3- O -propionyl-butyl acid was detected at 13.6 minutes respectively.

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

Example 1

1 ml (v / v) of liquid racemic 3-hydroxybutyric acid was added to a vial containing 2 ml of t-butyl methyl ether containing 1% of vinyl acetate (v / v), followed by lipase About 4% (w / v) of CAL B (Novozyme) was added, and the reaction was performed at 30 ° C.

After 2 hours of reaction time, when the reaction solution was analyzed according to the above conditions without pretreatment, the conversion rate of 3-hydroxybutyric acid was 38.1%. The optical purity of (S) -3-hydroxybutyric acid was 58.1 ee%. The optical purity of (R) -3- O -acetyl-butyl acid was 90.6 ee%. After the reaction for about 3 hours, the reaction solution was extracted with an aqueous solution, and (R) -3- O -acetyl-butyl acid was separated on an organic solvent, and (S) -3-hydroxy-butyl acid was separated on an aqueous solution. Was analyzed as it was, the aqueous solution was acidified, extracted with ethyl acetate and analyzed according to the above method. In this case, the conversion rate of 3-hydroxy butyric acid was 53.1%, the optical purity of (S) -3-hydroxy-butyl acid was 99 ee%, and the optical purity of (R) -3- O -acetyl-butyl acid was 77.4. It was ee%.

Example 2-4

The reaction was carried out using the acyl donors specified in Table 1 below instead of the vinyl acetate used in Example 1. The reaction conversion rate of 3-hydroxy butyric acid and the optical purity of (S) -3-hydroxy butyric acid and (R) -3-hydroxy butyric acid ester derivative are as follows.

Example Acyl donor Response time (hours) % Conversion (S) -acid optical purity (ee%) (R) -ester optical purity (ee%) 2 Vinyl propionate 3 53.1 99 50.1 3 Acetic acid isopropenyl 3 51.2 99 84.0 4 Acetic anhydride 3 55.7 99 77.4

Example 5-8

The reaction was carried out using the lipases shown in Table 2 below instead of lipase CAL B in Example 1. At this time, the reaction conversion rate of 3-hydroxy butyric acid and optical purity of (S) -3-hydroxy butyric acid and (R) -3- O -acetyl-butyl acid are as follows.

Example Lipase Type Response time (hours) % Conversion Acid Optical Purity (ee%) Ester Optical Purity (ee%) 5 PS 19 16.1 20.3 (S) 81.5 (R) 6 PS-C 19 37.1 56.1 (S) 66.8 (R) 7 AK 19 12.8 3.7 (S) 73.0 (R) 8 CRL 19 41.6 8.0 (R) 13.6 (S)

Example 9-11

The reaction was carried out using the organic solvent shown in the following [Table 3] instead of t-butyl methyl ether used as the organic solvent in Example 1. At this time, the reaction conversion rate of 3-hydroxy butyric acid and optical purity of (S) -3-hydroxy butyric acid and (R) -3- O -acetyl-butyl acid are as follows.

Example Organic solvent Response time (hours) % Conversion (S) -acid optical purity (ee%) (R) -ester optical purity (ee%) 9 Methylene chloride 5 42.8 43.9 85.2 10 Tetrahydrofuran 3 45.1 49.3 82.8 11 Isopropyl ether 3 55.0 99 77.5

Example 12

The reaction was carried out by adding only vinyl acetate used as an acyl donor without using t-butylmethylether used as the organic solvent in Example 1. After about 19 hours, the supernatant was collected and analyzed according to the above analysis method without pretreatment. In this case, the conversion rate of 3-hydroxy butyric acid was 64.4%, the optical purity of (S) -3-hydroxy-butyl acid was 96.0 ee%, and the optical purity of (R) -3- O -acetyl-butyl acid was 45.0. It was ee%.

As can be seen in Examples 1-12, in the synthesis of (S) -3-hydroxy-butyl acid or (R) -3- O -acyl-butyl acid, the appropriate lipase enzyme and acyl donor are selected. Compounds having high optical purity can be easily prepared, and the acid and ester can be easily separated, which is useful for the actual manufacturing process. In addition, the method of the present invention has the advantage of being environmentally friendly in that it uses enzymes, and can be used repeatedly, thereby reducing costs in the manufacturing process.

Claims (4)

Optical represented by the general formula (2) characterized by esterifying the racemic 3-hydroxy-butyl acid represented by the general formula (1) in the following [Scheme 1] in the presence of an acyl donor in an organic solvent with a lipase enzyme. Method for preparing an active 3-hydroxy-butyl acid and the ester represented by the general formula (3). Scheme 1

(R = CH ₃ (CH ₂ ) _n CO, n = 0 ~ 6 or R = C ₆ H ₅ CO.) Optical represented by the general formula (2), characterized in that in the presence of an acyl donor without an organic solvent, the racemic 3-hydroxy-butyl acid represented by the general formula (1) in [Scheme 1] is esterified with a lipase enzyme Method for preparing an active 3-hydroxy-butyl acid and the ester represented by the general formula (3). The method of claim 1 or 2, wherein the acyl donor is selected from a vinyl ester compound, isopropenyl acetate, and an anhydride compound. The method of claim 1, wherein the organic solvent is characterized in that isopropyl ether, t-butyl methyl ether, tetrahydrofuran, methylene chloride is used alone or in combination.