JPH09322786A - Production of (s)-4-halo-3-hydroxy-butyrate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently produce the subject compound useful as a raw material for synthesizing a medicinal or agrochemical agent, etc., by acting a specific microorganism belonging to the genus Agrobacterium or the genus Rhodococcus on the mixture of enantiomers of 4-halo-3-hydroxy-butyrate. SOLUTION: The objective compound is readily and efficiently produced by acting a microorganism belonging to the genus Agrobacterium or the genus Rhodococcus (e.g. Agrobacterium radiobacter IFO12664) having a capability of acting on the mixture of enantiomers of 4-halo-3-hydroxy-butyrate expressed by the formula (X is chlorine, bromine or iodine; R is a 1-4C alkyl) to remain (S)-4-halo-3-hydroxy-butyrate, or acting a substance obtained by treating the microorganism on the mixture of the enantiomers of 4-halo-3-hydroxy-butyrate expressed by the formula and collecting the remained (S)-4-halo-3-hydroxy- butyrate.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing (S) -4-halo-3-hydroxy-butyric acid ester. More specifically, Equation 1

Embedded image A specific microorganism is allowed to act on the enantiomer mixture of 4-halo-3-hydroxy-butyric acid ester represented by
The present invention relates to a method for producing (S) -4-halo-3-hydroxy-butyric acid ester, which comprises collecting (S) -4-halo-3-hydroxy-butyric acid ester. (S) -4-halo-3-hydroxy-butyric acid ester is useful as an important raw material for the synthesis of various medicines and agricultural chemicals such as antibiotics.

[0002]

2. Description of the Related Art The conventionally known method for producing (S) -4-halo-3-hydroxy-butyric acid ester is (1) 3α-obtained from a microorganism belonging to the genus Cellulomonas by a biochemical asymmetric reduction method. Method using hydroxysteroid dehydrogenase (JP-A-1-
277494), (2) a method using microorganisms of the genus Candida, genus Geotricum, genus Debaryomyces, genus Endomyces, etc. (JP-A-61-146191), a method using microorganisms of the genus Lactobacillus (biocatalysis, 5, 325- 332 (1
992)), Brevibacterium genus, Kluyveromyces genus, Stefanoascus genus, and Saccharomycopsis genus (Japanese Patent Laid-Open No. 6-209782) and the like, and there are chemical asymmetric reduction methods. (3) A method using a ruthenium-optically active phosphine complex as a catalyst.
1-211551) and (4) a method of reducing with an optically active amino acid derivative and an alcohol (JP-A-61-155354).

As a method using lipase, (5-a) 6- (3
-Chloro-2-hydroxypropyl) -1,3-dioxin-4-one was subjected to asymmetric transfer of the acetyl group from vinyl acetate, and then the acetylated product was separated and subjected to lipase hydrolysis, ozonolysis and esterification to give 4- Method for obtaining chloro-3-hydroxy-butyric acid ethyl ester (tetrahedron asymmetry, 2, 3
43-346 (1991)), (5-b) 4-chloro-3-acetoxy-butyric acid ethyl ester enantiomeric mixture method of reacting commercial pig liver lipase (Biocatalysis, 5, 325
-332 (1992)) is known.

[0004]

[Problems to be Solved by the Invention] Conventional (S) -4-halo-3-
The method for producing hydroxybutyric acid ester has the following problems. That is, the method of (1) is NADH, NADPH.
A coenzyme regeneration system is required, the reaction system is complicated, and the purified enzyme and coenzyme itself are expensive. In the asymmetric reduction method using microbial cells of (2), it is difficult to obtain a product with high optical purity with high reproducibility because the concentration of products generally obtained is low, and applied and environmental microbiology, 56 , 2374-2377 (1990), the substrate 4-halo-acetoacetic acid ester has drawbacks such as being extremely unstable in an aqueous solvent. Japanese Unexamined Patent Publication (Kokai) No. 6-209782 describes a method for producing an optically active 4-halo-3-hydroxybutyric acid ester by an asymmetric reduction method using a microorganism, but the same drawbacks still exist.

In the case of (3), it is not easy to synthesize an optically active phosphine complex and it is expensive. The method (4) also has a drawback that the optical purity of the product is low, and the reaction must be performed at a low temperature such as -78 ° C. Method using lipase
(5-a) and (5-b) are not economical methods because a large amount of enzyme is required for the substrate and the enzyme itself is expensive.

[0006]

[Means for Solving the Problems] As described above, the methods known up to now do not enable the production of (S) -4-halo-3-hydroxy-butyric acid ester at an industrial level at low cost. It has been desired to establish a method for producing (S) -4-halo-3-hydroxy-butyric acid ester by an economical and simple means. Therefore, in order to solve the above-mentioned drawbacks, the present inventors have focused on the stereoselective resolution of microorganisms, and as a result of the examination, bacterial cells of the genus Agrobacterium or Rhodococcus or their processed products have the formula Represented by 1 4-
It was found to act on a mixture of halo-3-hydroxy-butyric acid esters, leaving only one enantiomer.

No method has been known so far in which a mixture of 4-chloro-3-hydroxy-butyric acid enantiomers is allowed to act directly on a microbial cell or a treated product thereof to leave only one enantiomer. The present invention allows a microorganism selected from a group of microorganisms belonging to the genus Agrobacterium or the genus Rhodococcus or a treated product thereof to act on an enantiomeric mixture of 4-halo-3-hydroxy-butyric acid ester to remain.
The present invention provides a method for producing (S) -4-halo-3-hydroxy-butyric acid ester, which comprises collecting (S) -4-halo-3-hydroxy-butyric acid ester. According to this method, reproducibility of optical purity is good and it is possible to obtain almost pure (S) -4-halo-3-hydroxy-butyric acid ester. In addition, 4-halo-3-
Hydroxy-butyric acid esters also have the advantage of being much more stable in aqueous solvents than 4-halo-acetoacetic acid esters.

The microorganism used in the present invention is a microorganism selected from the group of microorganisms belonging to the genus Agrobacterium or the genus Rhodococcus, which acts on the enantiomer mixture of 4-halo-3-hydroxy-butyric acid ester, Any microorganism can be used as long as it has the ability of remaining (S) -4-halo-3-hydroxy-butyric acid ester.

Specifically, Agrobacterium radiobacter IFO 12664 or Rhodococcus equi JCM 68
Preference is given to using 17.

The microorganisms with IFO are described in the Microorganism Catalog issued by Fermentation Research Institute, 9th edition (1992) and can be obtained from the IFO. Microorganisms with JCM are listed in the Microorganism Catalog issued by RIKEN Microorganism Preservation Facility, 5th edition (1992).
Can be obtained from As these microorganisms, any strain such as a wild strain, a mutant strain, or a recombinant strain derived by a genetic technique such as cell fusion or gene manipulation can be preferably used.

The medium for culturing the microorganism used in the present invention is not particularly limited as long as the microorganism can grow. For example, as the carbon source, any of the above microorganisms can be used as long as it is available, specifically, glucose,
Sugars such as fructose, sucrose and dextrin, alcohols such as sorbitol and glycerol,
Organic acids and salts thereof such as fumaric acid, citric acid, acetic acid and propionic acid, hydrocarbons such as paraffin and the like or a mixture thereof can be used. Examples of the nitrogen source include ammonium salts of inorganic acids such as ammonium chloride, ammonium sulfate and ammonium phosphate, ammonium salts of organic acids such as ammonium fumarate and ammonium citrate, meat extract, yeast extract, corn steep liquor and casein hydrolysis. Inorganic organic nitrogen-containing compounds such as compounds, urea, etc. can be used.

In addition, inorganic salts such as MgSO4 · 7H2O, NaCl, KCl, trace metal salts such as FeSO4 · 7H2O, MnSO4 · nH2O, ZnSO4 · 7H2O, vitamins such as biotin, thiamine, p-aminobenzoic acid, etc. The nutrient sources used for culturing can be appropriately mixed and used. If necessary, a factor such as oleic acid or mevalonic acid that promotes the growth of microorganisms, or a substance such as CaCO3 that is effective in maintaining the pH of the medium can be added.

As a culture method, the medium pH is 3.0 to 9.0,
Preferably 4.0 to 8.0, the culture temperature is 20 to 45 ℃, preferably 25 to 40 ℃, anaerobically or aerobically, under conditions suitable for the growth of the microorganism 5 to 120 hours, preferably 12 to 72 hours Incubate to a degree.

As a method for selectively leaving the (S) -4-halo-3-hydroxy-butyric acid ester from the enantiomer mixture of 4-halo-3-hydroxy-butyric acid ester, for example,
(1) using the culture solution as it is, a method of adding an enantiomer mixture of 4-halo-3-hydroxy-butyric acid ester to the culture solution, (2) centrifuging from the culture solution, etc. to separate cells, There is a method in which a mixture of 4-halo-3-hydroxy-butyric acid enantiomers is added to a reaction product, which is resuspended in a buffer solution, water or the like, as it is or after being washed, and then reacted. In addition, the microbial cells may be intact microbial cells, or may be those that have been subjected to a treatment such as a crushed bacterial cell product, an acetone treatment, a toluene treatment, a freeze-drying treatment.

The enantiomeric mixture of the starting 4-halo-3-hydroxy-butyric acid ester used in the present invention can be easily obtained by reducing the corresponding 4-halo-aceto-acetic acid ester with NaBH 4. Mixtures of 4-halo-3-hydroxy-butyric acid ester enantiomers as they are, or by dissolving them in an organic solvent that dissolves in water or does not affect the reaction, or disperse them in a surfactant, etc. Or may be added in portions.

The reaction has a pH in the range of 3.0 to 10.0, preferably pH 5.0 to 9.0, and a temperature of 5 to 60 ° C., preferably 20 to 40.
It is carried out in the temperature range of 1 to 120 hours, preferably with stirring or standing. The concentration of the enantiomeric mixture of 4-halo-3-hydroxy-butyric acid ester which is the substrate is not particularly limited, but is preferably about 0.2 to 10%. Also as needed
If the pH of the reaction solution is maintained with NaOH, CaCO3, aqueous ammonia, HCl, H2SO4, etc., good results may be obtained.

The (S) -4-halo-3-hydroxy-butyric acid ester remaining after the reaction is collected directly from the reaction solution or after separation of the bacterial cells by purification using a combination of ordinary methods such as extraction with an organic solvent and distillation. Can be collected.

[0018]

EXAMPLES The present invention will now be specifically described with reference to examples, but the present invention is not limited to these examples. Incidentally, the quantification of ethyl 4-chloro-3-hydroxybutyrate in the reaction solution in the example was carried out by gas chromatography using the supernatant solution as an analysis sample after sterilizing the reaction solution by centrifugation. : Thermon 3000, φ3mm
X 2.1 m, column temperature: 150 ° C, detection: FID). Further, the analysis of optical purity was carried out by extracting the supernatant with ethyl acetate,
Desolvation was performed and high performance liquid chromatography was performed using Chiralpak AS (manufactured by Daicel Chemical Industries) (column: Ch
iralpak AS, mobile phase: hexane / ethanol / isopropyl alcohol / cyclohexanol [92: 2.5: 1.25:
0.25], flow rate: 1.0 ml / min, temperature: ice cooling, detection: UV 220n
m). Retention time for S body is 14.3 minutes, that for R body is 1.
It's 6.1 minutes.

(Example 1): A culture medium for preparing bacterial cells having the following composition was prepared.

<Medium for cell preparation> Dry broth "Nissui" 3% (manufactured by Nissui Pharmaceutical Co., Ltd.), pH
7.2 5 ml of the above medium was placed in a φ21 mm test tube and sterilized at 121 ° C for 15 minutes. After cooling, Agrobacterium radiobacter IFO 12664 was aseptically inoculated and shake-cultured at 30 ° C. Then, the cells were separated by centrifugation to obtain viable cells. Next, 0.5% (W / V) 4
It was suspended in 5 ml of 100 mM potassium phosphate buffer (pH 7.0) containing a racemate of -chloro-3-hydroxy-butyric acid ethyl ester. The bacterial cell suspension was put into a φ21 mm test tube, and reacted by shaking at 30 ° C. for 24 hours.

After the reaction, the reaction solution was sterilized by centrifugation, and 4-chloro-3-hydroxy-butyric acid ethyl ester remaining in the obtained supernatant was quantified and the optical purity was analyzed. The results are shown in Table 1.

(Example 2) 4-chloro-3- remained in the supernatant in the same manner as in Example 1 except that Rhodococcus equi JCM 6817 was used as the bacterial cell.
Quantitation of hydroxy-butyric acid ethyl ester and analysis of optical purity were performed. The results are shown in Table 1.

Table-1 -------------------------------------------- ---------- Absolute configuration Optical purity (% ee) Remaining amount (mg / ml) ------------------------- ----------------------------- Example 1 S 97.3 0.95 Example 2 S 43.0 2.64 --------- --------------------------------------------- (Example 3) 600 ml of the culture medium for preparing bacterial cells was placed in a 1.2-liter small culture tank manufactured by Maruhishi Bioengine, and sterilized at 121 ° C for 15 minutes. After cooling, shake culture of Agrobacterium radiobacter IFO 12664 in cell culture medium at 30 ° C for 24 hours (5ml / φ21mm test tube)
Aseptically inoculate the obtained culture solution at 30 ℃, 900rpm, 1.0v
It was cultured for 25.5 hours under the condition of vm.

After culturing, cells were separated by centrifugation while cooling to obtain viable cells. Next, viable cells were added to 1.0% (W / V) 4-chloro
-3-Hydroxy-butyric acid ethyl ester containing racemate 10
It was suspended in 600 ml of 0 mM potassium phosphate buffer (pH 7.0).
The cell suspension was placed in the 1.2-liter small culture tank at 30 ° C., 6
The reaction was carried out for 27 hours under the conditions of 00 rpm and 1.0 vvm.

The reaction-completed liquid was centrifuged to remove bacteria, and the resulting supernatant liquid was filtered through an ultrafiltration membrane (cut off molecular weight 10,000 cut) manufactured by Millipore to remove polymer substances. Next, this filtrate was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and then desolvated under reduced pressure. The residue was distilled at 2 mmHg and a temperature of 83 ° C. to obtain 0.65 g of purified (S) -4-chloro-3-hydroxy-butyric acid ethyl ester. The purity was 98.4% and the optical purity was 99.0% ee.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical indication C12R 1:01)

Claims (3)

[Claims] 1. Agrobacterium
m) or Rhodococcus genus,
Formula 1 (In the formula 1, X represents a chlorine, bromine or iodine atom, and R represents an alkyl group having 1 to 4 carbon atoms) 4-
A microorganism having a capability of acting on an enantiomer mixture of halo-3-hydroxy-butyric acid ester and leaving the (S) -4-halo-3-hydroxy-butyric acid ester or a treated product thereof is represented by the above formula 1 -Halo-3-hydroxy-butyric acid ester is allowed to act on the enantiomeric mixture, and the remaining (S)-
A method for producing (S) -4-halo-3-hydroxy-butyric acid ester, which comprises collecting 4-halo-3-hydroxy-butyric acid ester. 2. A 4-halo-3-hydroxy-butyric acid ester
The method for producing (S) -4-halo-3-hydroxy-butyric acid ester according to claim 1, which is 4-chloro-3-hydroxy-butyric acid ethyl ester. 3. The (S) -4-halo-3-hydroxy-butyric acid ester according to claim 1 or 2, wherein the microorganism is Agrobacterium radiobacter or Rhodococcus equi. Manufacturing method.