KR100657204B1 - The method of making optically active 3-hydroxy-?-butyrolactone by enzymatic method - Google Patents

Abstract

The present invention is generalized by enzymatic method from racemic 4-chloro-3-hydroxybutyric acid alkyl ester represented by the general formula (1) in the following [Scheme 1]. It relates to a method for producing optically active 3-hydroxy-gamma-butyrolactone represented by formula (3). More specifically, the racemic 4-chloro-3-hydroxybutyl acid alkyl ester, which is a reactant, in an aqueous solution or a solvent containing the aqueous solution is subjected to stereoselective hydrolysis reaction using a microorganism having a lipase or lipase producing ability to optically react. A method for preparing active 3-hydroxy-gamma-butyrolactone.

Compared to the previously reported methods, the method of the present invention is easy to process, and easy to separate and recover.

Scheme 1

(R = C _n H _{2n + 1} (n = 1 ~ 8))

Methyl 4-chloro-3-hydroxybutyrate, ethyl 4-chloro-3-hydroxybutyrate, butyl 4-chloro-3-hydroxybutyrate, 3-hydroxy-gamma-butyrolactone, lipase, hydrolysis reaction, Optical activity

Description

The method of making optically active 3-hydroxy-γ-butyrolactone by enzymatic method}

( R = C_nH_2n+1(n=1~8) )The present invention relates to optically active 3-hydroxy-gamma-butyrolactone by enzymatic method from racemic 4-chloro-3-hydroxybutyric acid alkyl ester. hydroxy-γ-butyrolactone). In more detail, using a microorganism having a lipase or lipase-producing ability in an aqueous solution, stereoselective hydrolysis of the racemic 4-chloro-3-hydroxybutyl acid alkyl ester represented by the general formula (1) in [Scheme 1] Reaction relates to a method for producing optically active 3-hydroxy-gamma-butyrolactone represented by the general formula (3).

(R = C _n H _{2n + 1} (n = 1 ~ 8))

The above-mentioned optically active 3-hydroxy-gamma-butyrolactone is L-carnitine, gamma-amino-beta-hydroxybutyric acid, therapeutic agents for hyperlipidemia, Wide range of applications as pharmaceutical intermediates and pesticide intermediates such as AIDS treatments. In addition, the production method according to the present invention can be advantageously used in the actual process because it is simpler than the existing method, and easy to separate and recover after the reaction.

The technology for producing (S) -3-hydroxy-gamma-butyrolactone reported to date is as follows.

Yuasa et al. (Liebigs Ann. IRecueil. 1997, 1877-1879) used ethyl 4-chloro-3-oxobutanoate as a Ru- (R) -p-tolyl-BINAP catalyst. Was synthesized ethyl (S) -4-chloro-3-hydroxybutanoate having 94% ee optical purity using hydrochloric acid and treated with (S) -3- Hydroxy-gamma-butyrolactone was prepared. At this time, the yield was 83%, the optical purity was maintained. However, in the above process, the quality of (S) -3-hydroxy-gamma-butyrolactone is determined by the optical purity of (S) -ethyl 4-chloro-3-hydroxybutanoate and is applied to the actual process. It's hard to do.

Suzuki et al. (Enzyme Microbiology and Technology, 1999, 24. 13-20) have an optical purity of 99.8% ee from racemic ethyl 4-chloro-3-hydroxybutyrate using a microorganism with dechlorination (R). ) -Ethyl 4-chloro-3-hydroxybutyrate and (S) -3-hydroxy-gamma-butyrolactone having an optical purity of 92.4% ee were prepared.

On the other hand, Chun Jong Pil et al. (Japanese Patent No. 10-0310935, U.S. Patent No. 6,122,122) prepare oligosaccharides by reacting amylopectin with enzymes and reacting them with basic anion exchange resins and oxidizing agents (S) -3,4-dihydroxy-butyl. An acid was obtained, and a method of preparing (S) -beta-hydroxy-gamma-butyrolactone in which the optical purity was maintained through the esterification reaction and the cyclization reaction was developed. In addition to the above method, a method of producing a higher yield using starch or tetrigo (maltooligosaccharide) as a raw material (US Pat. No. 6,124,122) is known, but these methods are decomposed because they are prepared by decomposing polysaccharide molecules. Various undesired compounds are produced during the process, and most of the resulting compounds have similar physical properties due to structural similarities to the target compound, which is very difficult to purify or remove.

In addition, Kwak, Byung-Sung et al. (Sec. 2003-0004902) cyclized a di-hydroxy-butyl acid methyl ester produced by hydrogenating a substituted malic acid derivative in the presence of an acid catalyst (S) -beta-hydroxy-gamma. Butyrolactone was prepared. However, this method has a disadvantage in that the optical purity and yield of the product is lowered and the deactivation rate of the catalyst is increased when the pressure and the temperature range are out of the high temperature and high pressure reaction.

Lee Ho-Sung et al. (Sec. 2003-0065192) synthesized (S) -4-chloro-3-hydroxybutyronitrile from high purity (S) -epichlorohydrin. (S) -3-hydroxy-gamma-butyrolactone was prepared as a starting material. However, the manufacturing method has a disadvantage in that the manufacturing cost is high because the price of the first raw material (S)-epichlorohydrin is expensive.

Japanese Patent Publication No. 2002-204699 published by Daicel Co., Ltd. discloses optically active 3-hydroxy-gamma-butyro from optically active ethyl 4-chloro-3-hydroxy-butyrate using microorganisms. Lactones were prepared. At this time, the optical purity was more than 93.9% ee but the concentration of the reactants is low, so it is difficult to apply to the actual process. They also prepared optically active 3-hydroxy-gamma-butyrolactone from racemic ethyl 4-chloro-3-hydroxy-butyrate, but the concentration and optical purity of the reactants were very low. In addition, the reaction mechanism in preparing optically active 3-hydroxy-gamma-butyrolactone from ethyl 4-chloro-3-hydroxy-butyrate using microorganisms is not accurately described.

The production method according to the present invention hydrolyzes racemic 4-chloro-3-hydroxybutyl acid alkyl esters using microorganisms having lipase or lipase-producing ability to produce optically active 3-hydroxy-gamma-butyrolactone. It is a new process that has not been reported to date as a manufacturing method.

The inventors of the present invention have optically active 3-hydrides in a hydrolysis reaction using microorganisms having lipase or lipase production ability in reacting racemic 4-chloro-3-hydroxybutyl acid alkyl ester represented by the general formula (1). Roxy-gamma-butyrolactone was prepared.

Unlike the conventional method, the method according to the present invention is a method by hydrolysis reaction, and thus, 3-hydroxy-gamma-butyrolactone having a very simple process and high optical purity can be prepared. It is therefore an object of the present invention to provide a process for producing optically active 3-hydroxy-gamma-butyrolactone from an ester compound represented by the general formula (1) using an enzyme or a microorganism.

The production method of the present invention for achieving the above object consists of stereoselective hydrolysis reaction using a racemic ester compound in the aqueous solution phase or a solvent containing an aqueous solution using a microorganism having a lipase or lipase production capacity as a catalyst.

Hereinafter, the present invention will be described in more detail. As described above, the present invention is a method for producing an optically active 3-hydroxy-gamma-butyrolactone by a hydrolysis reaction by adding an enzyme or a microorganism as a biocatalyst to an ester compound represented by the general formula (1). It is about.

R of the racemic 4-chloro-3-hydroxybutyl acid alkyl ester represented by the general formula (1) used in the present invention includes C _n H _{2n + 1} (n = 1 to 8), but is not limited thereto. no. In Formula (1), R may be isopropyl, n-propyl, isobutyl, benzyl, or the like.

The lipases used in the present invention include Amano's PS, AK, Novozymes' CALB, and the like. Candida rugosa , Rhodococcus butanica, Cunninghamella echinylata, Candida magnoliae, Burkholderia cepacia, Geotrichum candidum, etc. Microorganisms are possible but are not limited thereto.

In the present invention, the reactants and products were analyzed using gas chromatography (Donam Instruments, Model DS6200), and after the reaction was analyzed by extraction with ethyl acetate (ethyl acetate).

The racemic 4-chloro-3-hydroxybutyl acid alkyl ester was heated in capillary column G-TA (Astec, 30m × 0.32mm) at 100 ° C. for 5 minutes and then raised to 170 ° C. by 20 ° C. per minute. 15 minutes was maintained at 170 degreeC. Helium gas was used as the carrier and was detected using FID at 170 ° C. while maintaining the column head pressure at 6 psi. In this case, methyl 4-chloro-3-hydroxybutyrate is 9.31 minutes, ethyl 4-chloro-3-hydroxybutyrate is 10.05 minutes, butyl 4-chloro-3-hydroxybutyrate is 12.92 minutes, 3-hydroxy-gamma- Butyrolactone was detected at 18.11 minutes respectively.

Optically active (R)-and (S) -3-hydroxy-gamma-butyrolactone was heated to 170 ° C. after heating a capillary column, G-TA (Astec, 30 m × 0.32 mm) for 10 minutes at 120 ° C. The temperature was raised by 10 ° C. per minute and maintained 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. At this time, (R) -3-hydroxy-gamma-butyrolactone was detected at 25.83 minutes and (S) -3-hydroxy-gamma-butyrolactone at 26.52 minutes, respectively.

In addition, methyl 4-chloro-3-hydroxybutyrate, ethyl 4-chloro-3-hydroxybutyrate, and butyl 4-chloro-3-hydroxybutyrate were confirmed by FT-NMR (Burker, model DPX300), respectively. The analysis results of are as follows.

Methyl 4-chloro-3-hydroxybutyrate:

¹ H-NMR (CDCl ₃ ) δ (ppm) = 2.64 (2H, d), 3.61 (2H, d), 3.72 (3H, s)

 4.15 (1H, br), 4.26-4.31 (1H, m)

Ethyl 4-chloro-3-hydroxybutyrate:

¹ H-NMR (CDCl ₃ ) δ (ppm) = 1.28 (3H, t), 2.62 (2H, d), 3.53 (1H, br),

 3.60 (2H, d), 4.20 (2H, q), 4.33 (1H, m)

n-butyl 4-chloro-3-hydroxybutyrate:

¹ H-NMR (CDCl ₃ ) δ (ppm) = 0.94 (3H, t), 1.36 to 1.43 (2H, m), 1.59 to 1.66

 (2H, m), 2.64 (2H, d), 3.45 (1H, br), 3.63 (2H, d), 4.12 (2H, t), 4.24-

 4.29 (1 H, m)

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

Example 1

To a vial containing 4.5 ml of 0.1 M potassium phosphate buffer (pH 8.0) add racemic methyl 4-chloro-3-hydroxybutyrate to 10% (v / v) and add lipase CAL B 2% (w / v) was added and the reaction was carried out at 30 ℃. After the reaction for 10 minutes, the reaction solution was extracted with ethyl acetate and analyzed according to the above analysis method. At this time, the conversion was 21.2% and the optical purity of (S) -3-hydroxy-gamma-butyrolactone was 94.6% e.e.

Example 2-3

 The reaction was carried out using ethyl 4-chloro-3-hydroxybutyrate and butyl 4-chloro-3-hydroxybutyrate instead of methyl 3-hydroxybutyrate used as the reactant in Example 1, and the conversion and optical Purity is shown in Table 1.

Example Reactant Response time (minutes) % Conversion (S) -3-hydroxy-gamma-butyrolactone optical purity (% ee) 2 Ethyl 4-chloro-3-hydroxybutyrate 10 39.9 90.4 3 Butyl 4-chloro-3-hydroxybutyrate 10 59.1 63.5
Example 4-5

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In Example 1, the reaction was performed using the lipase shown in Table 2 instead of the lipase CAL B, and the conversion and optical purity of each reaction are shown in Table 2.

Example Lipase Type Response time (hours) % Conversion (R) -3-hydroxy-gamma-butyrolactone optical purity (% e.e) 4 PS (Amano company) 9 39.1 51.8 5 AK (Amano) 24 34.5 52.6

Example 6-12

In Example 1, instead of the lipase CAL B, the reaction was performed using the microorganisms shown in Table 2, wherein the substrate was 5% and the microbial cell was 20%. The conversion and optical purity of each reaction are shown in Table 3.

Example microbe Response time (hours) % Conversion 3-hydroxy-gamma-butyrolactone optical purity (% e.e) 6 Candida rugosa KTCC 7292 6 13.4 63.4 (R) 7 Cunninghamella echinylata ATCC 26269 4.5 20.4 45.4 (S) 8 Rhodococcus butanica ATCC 21197 4.5 36.8 85.4 (S) 9 Burkholderia cepacia ATCC 21808 32 16.8 78.7 (S) 10 Candida magnoliae KCCM 50046 72 21.9 80.7 (S) 11 Candida magnoliae KCCM 50567 100 6.1 92.0 (S) 12 Geotrichum candidum IFO 4597 2 8.4 50.7 (S)

As can be seen through Examples 1-12, in the method for producing optically active 3-hydroxy-gamma-butyrolactone hydrolysis reaction using a microorganism having a lipase or lipase production capacity according to the present invention Is very simple. In addition, the use of suitable lipase enzymes or monarchs can be used to synthesize high optical purity of 3-hydroxy-gamma-butyrolactone. The method of the present invention is environmentally friendly in terms of the use of enzymes and microorganisms, the reaction concentration is high and can be used in actual mass production, and the use of immobilized enzymes or immobilized strains can be repeated and used to reduce costs in the manufacturing process. There is this.

Claims (3)

The racemic 4-chloro-3-hydroxybutyl acid alkyl ester represented by the general formula (1) in the following [Reaction Scheme 1] is Rhodococcus butanica and Candida magnoli in an aqueous solution phase or a solvent containing an aqueous solution. Hydrolysis using a lipase derived from a microorganism selected from Candida magnoliae or Candida antarctica , or a microorganism having a lipase producing ability selected from Rhodococcus butanica, Candida magnolia or Candida antarctica A method for producing an optically active 3-hydroxy-gamma-butyrolactone represented by the general formula (3), which is reacted. Scheme 1

(R = C _n H _{2n + 1} (n = 1 ~ 4)) The microorganism strain of claim 1, wherein the microorganism strain is Rhodococcus butanica ATCC 21197, Candida Magnolia. KCCM 50046 or Candida Magnolia Method characterized in that KCCM 50567. delete