JP2566960B2 - Method for producing (R) -r-substituted-β-hydroxybutyric acid ester - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] In recent years, the usefulness of optically active oxyacid derivatives has been increasing as a synthetic intermediate for medicines and agricultural chemicals.

Among these optically active oxyacid derivatives, (R) -γ-
As a result of various studies on a method for producing a substituted -β-hydroxybutyric acid ester, it was found that a novel reductase found by the present inventors can be used to effectively produce a γ-substituted acetoacetic acid ester, thereby completing the present invention. did.

[Conventional technology]

Conventionally, acting on γ-substituted acetoacetic ester,
To produce a (R) -γ-substituted-β-hydroxy enzyme ester, yeast-derived L-β-hydroxyacyl CoA dehydrogenase [EC1, 1, 1,
35] (JP-A-59-118093) and alcohol dehydrogenase derived from Thermoanaerobium brckii [EC1.1.1.2] (JACS, 198).
5, 107 , 4028) are known.

[Problems to be solved by the invention]

However, the former has room for improvement in terms of reactivity and temperature stability, and the latter is anaerobic culture. Therefore, a method for producing (R) -γ-substituted-β-hydroxybutyric acid ester using these is practical. It was hard to call it a target.

[Means for Solving Problems] The present invention relates to a reduced nicotinamide / adenine / dinucleotide / phosphate (hereinafter referred to as NADPH) -dependent reductase derived from Sporoboromyces sp. (R) -γ-substitution-β characterized in that
A method for producing hydroxybutyric acid ester.

The above-mentioned reductase used in the present invention is Sporobolomyces salmonicolor I
An enzyme having reducing ability was extracted and purified from FO1038. This enzyme was newly proposed because it is clear that it is a novel reductase having various physical properties that are practically superior to known enzymes.

Next, the preparation of the enzyme used in the present invention and its various physical properties will be specifically described.

Sporoboromomyces salmonicolor IFO1038 grown in a medium (pH 5.0) consisting of 5% glucose and 5% corn stay liquor was collected by centrifugation,
After washing with a phosphate buffer, the cells are crushed with Dynomill (manufactured by Shinmaru Enterprise Co., Ltd.) to extract the enzyme. After centrifugation, the resulting cell-free extract was ammonium sulfate fractionated and
%, Collect the saturated fraction. After dialysis overnight, it is adsorbed on a DEAE-Sephacel (manufactured by Pharmacia) column, and then linear gradient elution with 0 to 0.6 M of sodium chloride is performed to collect the active fraction. Furthermore, the active fractions are collected by gel filtration chromatography (Sephadex G-100 manufactured by Pharmacia), and gel filtration chromatography is performed again in the same manner as above to obtain a purified enzyme liquid preparation. It is electrophoretically uniform. A crystallized product of this enzyme has not been obtained.

 Next, the physicochemical properties of the enzyme used in the present invention will be described.

Action: In the presence of coenzyme NADPH, 1 mol of aldehyde or ketone is used as a substrate to purify 1 mol of alcohol.

Substrate specificity: The activity for each of the following substrates is expressed based on ethyl γ-chloroacetoacetate. The activity measuring method is as follows.

Substrate 600 nmol and NADPH 192 nmol in 0.6 ml of enzyme solution (0.1 M
Add to phosphate buffer, pH 7.0) and measure the change in absorbance at 340nm at 37 ℃ to obtain the value.

(I) Activity against ketones (Ii) Coenzyme ・ Active against NADPH.

・ Does not act on NADPH (nicotinamide adenine dinucleotide).

(Iii) Alcohols Substantially no activity is observed against the alcohols shown in Table 2.

Optimum pH and stable pH range The optimum pH is around 7, and 80% or more of the activity remains even when treated at pH 6 to 10 at 55 ° C for 10 minutes.

Optimal temperature and stable temperature range The optimal temperature is around 60 ° C. When treated at pH 7.0 for 10 minutes, 100% activity at 40 ° C and 70% activity at 60 ° C remain.

Effects of metal ions and various drugs Optical Selectivity The reduction reaction product of γ-substituted acetoacetic acid ester is also the (R) -form, and the optical purity is 97% ee or more.

The optical purity of the product was determined by high-performance liquid chromatography (HPLC) after synthesizing an ester with (+)-α-methoxy-α-trifluoromethylphenylacetic acid (MTPA) to obtain a diastereomeric compound. Separate and quantify.

HPLC conditions Column: Partisil5 (manufactured by Wattman) (4.6φ x 250 mm) Mobile phase: Hexane: Tetrachlorofuran: Methanol =
600: 100: 1 Velocity: 2.0 ml / min Detection (Absorbance wavelength): 217 nm Stability against organic solvents 28 in pH 7.0 phosphate buffer containing organic solvents in Table 5
It retains 80% or more of activity even when it is treated at ℃ for 24 hours.

Isoelectric point pH 4.7 Molecular weight 32,000 (Sephadex G-100 gel filtration method) 36,500 [SDS (sodium dodecyl sulphate) electrophoresis method] To produce the novel reductase used in the present invention, a conventional method is used. Then, the bacterium can be cultured. The medium used for culturing is an ordinary medium containing a carbon source, a nitrogen source, an inorganic substance and the like necessary for the growth of microorganisms. In addition, the addition of organic micronutrients such as vitamins and amino acids often produces desirable results.

The culture is carried out under aerobic conditions for 1 to 10 days while controlling the pH to be in the range of 3 to 8 and the temperature to be in the range of 10 to 40 ° C.

In order to obtain the electrophoretically uniform aldehyde reductase by culturing the bacterium, ordinary ammonium sulfate fractionation, affinity chromatography, ion exchange chromatography, gel filtration chromatography and the like are used.

The form of the enzyme used in the synthesis of (R) -γ-substituted-β-hydroxybutyric acid ester from γ-substituted acetoacetic acid ester is as follows: purified, semi-purified, enzyme-containing cells, treated cells and Any of the immobilized products can be used.
The strain used, Sporoboromyces salmonicolor IFO1
Since 038 also contains an enzyme that produces (S) -γ-substituted-β-hydroxybutyric acid ester, it must be removed in advance.

The concentration of γ-substituted acetoacetic acid ester used as a substrate can be up to about 20% by weight, but the reaction tends to be inhibited as the concentration increases, so 10% by weight or less or low concentration addition or continuous addition is possible. desirable.

As the γ-position substituent of γ-substituted acetoacetic acid ester,
Chlorine, bromine, fluoro, azido group and the like are preferable,
As the ester group, an alkyl group having 1 to 10 carbon atoms is preferable.

Since NADPH is essential as a coenzyme, NAD is added to the reaction system.
The required amount of PH is added in advance, or a system for producing NADPH is allowed to coexist. This system includes, for example, NAD that utilizes the conversion of NADP to NADPH in the gluconic acid production reaction from glucose by glucose dehydrogenase.
It is possible to use a PH playback system, etc.

If the reaction temperature is adjusted to 5 to 70 ° C., preferably 20 to 40 ° C., and the reaction pH is adjusted to 4 to 10, preferably 6 to 8, the object of the present invention is sufficiently exhibited.

〔Example〕

Hereinafter, the present invention will be specifically described with reference to Examples. In the examples,% means% by weight unless otherwise specified.

Oxygen isolation example 5 ml of glucose 6.0 wt% and corn stay liquor 5 wt% of pH 6.0 was placed in a test tube, inoculated with Sporoboromyces salmonicolor IFO1038, and shake culture was performed at 28 ° C for 2 days. Seed culture was obtained.

500 ml of the medium having the same composition as described above was placed in a 2-volume flask, 5 ml of the seed culture was added, and shaking culture was carried out at 28 ° C. for 4 days. Next, the above 10 flasks were combined, and the cultured bacterial cells recovered by centrifugation (2800 G, 20 minutes) from the culture solution of 5 were washed with 0.01 M phosphate buffer (pH 7.4), and then Dynomill (beads 0.25 ~ 0.5mmφ) for 20 minutes, 28000
The crude enzyme solution was obtained by centrifugation at G for 20 minutes to remove suspended matter. Ammonium sulphate was added to this, and the fraction of 60-80% saturation was collected by centrifugation (28,000 G x 30 minutes) and dialyzed against 0.01 M acid buffer (pH 7.0) for 20 hours. Next, it was adsorbed on DEAE-Sephacel column chromatography (1.6 ^φ × 30 cm),
After washing with the above buffer, linear gradient elution with the same buffer containing 0 to 0.6 M of sodium chloride was performed. Fractions showing activity were collected and concentrated by an ultrafiltration machine (Amicon, YM10), and then gel filtration column (Sephadex, G-100,
2.0 ^φ × 90 cm), and chromatograph with the above buffer containing 0.1 M NaCl to collect the fractions showing activity,
Gel filtration chromatography was performed in the same manner as above to prepare a purified enzyme solution. This product electrophoretically showed a single band.

Example 1 Using the purified enzyme obtained in the enzyme isolation example, under the conditions of Table 6,
A reduction reaction was performed. The reaction-terminated liquid was extracted twice with 200 ml of ethyl acetate, dehydrated with Na ₂ SO ₄ and the ethyl acetate was removed under reduced pressure. As a result of NMR analysis of the remaining colorless and transparent liquid, it was confirmed to be ethyl γ-chloro-β-hydroxybutyrate as shown below.

NMR (CDCl ₃ ) δ (ppm): 1.25 (3H, tr, CH ₃ CH ₂ −) 3.22 (1H, br, -OH) In addition, GC analysis showed a purity of 98.2% and a yield of 0.474 g (yield 92
%). This product was the (R) -form with an optical purity of 97% ee as determined by HPLC analysis of MTPA ester.

 The oxygen activity U in Table 6 is as follows.

1 per minute from γ-chloroacetoacetic acid ethyl ester
The capacity to produce μmol of γ-chloro-β-hydroxybutyric acid ethyl ester is defined as 1 unit (U).

Example 2 Under conditions including the NADPH regeneration system shown in Table 7, the reaction was started using the γ-substituted acetoacetic acid ester shown in Table 8 as a substrate, and the reaction was carried out for 20 hours while maintaining the pH at 6.5 with 0.5N-NaOH. did. After the completion of the reaction, the same operation as in Example 1 was conducted to analyze the product. As a result of NMR analysis, the product was confirmed. Table 8 shows yield (rate), optical purity and the like.

γ-Chloro-β-methyl hydroxybutyrate NMR (CDCl ₃ ) δ (ppm): 3.6 (3H, s, CH ₃ −) 3.22 (1H, br, -OH) γ-Chloro-β-ethyl hydroxybutyrate Same as in Example 1.

γ-Chloro-β-octyl hydroxybutyrate NMR (CDCl ₃ ) δ (ppm): 0.88 (3H, tr, CH _3- (CH ₂ ) _n- ) 1.28 (10, s,-( CH ₂ ) _5- ) 3.22 (1H, br, -OH) γ-Bromo-β-hydroxybutyric acid ethyl γ-fluoro-β-hydroxybutyric acid ethyl All are almost the same as the chloro ethyl ester.

γ-Azido-β-ethyl hydroxybutyrate NMR (CDCl ₃ ) δ (ppm): 1.25 (3H, tr, CH ₃ CH ₂ —) 3.22 (1H, br, -OH) EFFECTS OF THE INVENTION In the present invention, it is possible to obtain (R) -γ-substituted-β-hydroxybutyric acid ester excellent in high yield and optical purity by using reduced oxygen which does not require anaerobic culture and has good temperature stability. It can be manufactured and has great industrial significance.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Masamichi Hashimoto Inventor Masamichi Hashimoto 3-5-1, Asahimachi, Machida-shi Denki Kagaku Co., Ltd. Central Research Laboratory Examiner Hiroshi Taniguchi

Claims (1)

(57) [Claims] 1. A (R) -γ-substituted, characterized in that a reduced nicotinamide / adenine / dinucleotide / phosphate-dependent reductase derived from the genus Sporoboromyces is allowed to act on a γ-substituted acetoacetic acid ester. Process for producing -β-hydroxybutyric acid ester