JP2566962B2 - Method for producing γ-substituted-β-hydroxybutyric acid ester - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] In recent years, the usefulness of various oxyacids as synthetic intermediates for medical and agricultural chemicals has been recognized. Among these oxyacids, the present invention provides a γ-substituted-which is an intermediate of carnitine synthesis
The present invention relates to an efficient method for producing β-hydroxybutyric acid ester.

[Conventional technology]

Conventionally, an aqueous medium alone system has been used to convert γ-substituted acetoacetic acid ester (hereinafter referred to as AAE) into γ-substituted-β-hydroxybutyric acid ester (hereinafter referred to as HBE) with a reductase. (JP-A-59-118093).

[Problems to be solved by the invention]

The present inventors have investigated the reaction conditions in an aqueous medium single reaction system, but there is a limit to the concentration of HBE accumulated, and a large amount of enzyme and coenzyme is required to increase the yield more than that. There was a problem such as a decrease in

[Means for Solving Problems] The present invention uses a reductase having an ability to reduce a γ-substituted acetoacetic acid ester to a γ-substituted-β-hydroxybutyric acid ester in an aqueous medium, and uses a γ-substituted acetoacetate. When producing a γ-substituted-β-hydroxybutyric acid ester from an acetic acid ester, a method for producing the γ-substituted-hydroxybutyric acid ester is characterized in that an organic solvent capable of forming two phases with water is present.

The minimum conditions for the organic solvent used in the present invention are (1) capable of dissolving the substrate AAE and product HBE (2) capable of forming two phases with an aqueous medium (3) inhibition of reductase reaction and inactivation A low effect is desirable. Specifically, organic acid esters such as formic acid ester, acetic acid ester, propionic acid ester, and butyric acid ester, and alcohols having 4 or more carbon atoms in the alkyl group such as n-butyl alcohol, n-amyl alcohol, and n-octyl alcohol. Aromatic solvents such as benzene, toluene and xylene, ethers such as diethyl ether, methyl ethyl ether and isopropyl ether, halogenated hydrocarbons such as chloroform, 1,2-dichloroethane and carbon tetrachloride. . The use ratio of these organic solvents to the aqueous medium in the reaction system is not particularly limited, but in practice, the organic solvent / aqueous medium = 95/5 to 10/90 is preferable on a weight basis.

Next, the enzyme source used for the reduction reaction in these two-phase media is an enzyme having the ability to convert AAE to HBE,
A microbial cell, a treated product of the cell, a crude enzyme extracted from the cell, or a purified enzyme can be used. Furthermore, an immobilized product obtained by immobilizing these by a known method can also be effectively used. Specifically, the genus Candida, the genus Saccharomyces, the genus Torulopsis,
Trichosporon, Pichia
a), Hansenula, Geotricom (Ge
otrichum, Endomyces, Debaryomyces, Sporobolomyces, and other yeasts, Aureobacterium, Alcallgenes, Agrobacterium, Agrobacterium Arthrobacter spp. Amorphosporangium spp. Ampullariella spp. Brevibacterium spp. Bacillus spp. Corynebacterium spp. Corulobacterium spp. Cellulomonas spp. Escherichia spp. Genus (Enterobacter) Genus Flavobacterium (genus Hafinia) Genus Kurthia (genus Kurthia) (genus Lactobacillus) (genus Micrococcus) (genus Micrococcus) (genus Methanomonas) (genus Methylobaci) genus Microbispora genus Micromonospora genus Nocardia genus Proteus genus Pseudomonas genus Pediococcus genus Planomonospora genus Protomonas genus Protomonas ) Genus Serratia genus Streptomyces genus Thermoactinomyces genus Xanthomonas genus Yersinia genus bacteria, aspergillus genus (Aspergillus) genus (Mucor) ), Genus Fusarium, genus Rhizopus, genus Penicillium, and genus Neurospor
a), Pullularia, Ustilago (Ust)
ilago), Verticillium and the like.

For these enzyme sources, AAE is asymmetrically reduced to (R) -H
Some produce BE or (S) -HBE, or have no optical selectivity and produce HBE in which the (R) form / (S) form is mixed. However, the method of the present invention is not limited to these enzyme sources, and can be applied to reactions other than those described above for the reaction of reducing AAE to HBE.

In the reduction reaction, in addition to reductase, reduced nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NA
DPH) is required, so add it to the reaction system or NADH or N
A reaction system that produces ADPH must coexist in the reduction reaction system. For example, NAD in the gluconic acid production reaction from glucose by glucose dehydrogenase
Or NADH using conversion of NADP to NADH or NADPH respectively
Alternatively, a NADPH regeneration system or the like can be preferably used.

With respect to the type of AAE used, the γ-position substituent is preferably a chloro, bromo, fluoro, azido group or the like, and the ester group is preferably an alkyl group having 1 to 10 carbon atoms.

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 effects of the present invention will be sufficiently exhibited. The reaction solution thus obtained is allowed to stand until the two phases are separated or separated by a centrifuge or the like, and the organic layer part containing most of HBE is collected. The aqueous layer residual HBE can be recovered with the same or another extraction solvent or the like, if necessary. After the organic layers containing HBE are combined and dehydrated with a dehydrating agent such as sodium sulfate, the organic solvent is removed under reduced pressure, and if necessary, further distillation under reduced pressure or chromatographic separation is performed to obtain high-purity HBE in high yield. Can be obtained at.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto.

〔Example〕

Example 1 Glucose 5% by weight, corn staple liquor 5
Put 5 ml of a medium (pH 6.0) containing 1% by weight into a test tube and use Sporobolomyces Salmonicolor.
monicolor) IFO1038 was inoculated and cultured at 28 ° C for 2 days.
Take 100 ml of the same composition medium into a 500 ml flask,
A seed culture (5 ml) was inoculated and shake culture was carried out at 28 ° C. for 4 days. The cells were collected by a centrifuge, and 0.01M phosphate buffer solution (pH
After washing with 7.0), the total volume was adjusted to 10 ml with the same buffer solution, and the cells were disrupted for 5 minutes at 20 KHz with an ultrasonic wave disruptor under ice cooling, and used as a crude enzyme solution in the reaction. Reaction is 10 ml of crude enzyme solution
As a trapping enzyme, 300 μmol of NADPH, 300 μmol of the substrate γ-chloroacetoacetate and 10 ml of ethyl acetate were added to the above, the pH was adjusted to 8, and the mixture was started at 30 ° C. with stirring. After 20 hours, the organic layer and the aqueous layer were separated, and the aqueous layer section was extracted twice with 5 ml of ethyl acetate. The ethyl acetate fractions were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give an oil. This product was distilled under reduced pressure to obtain IR (IR-435 made by Shimadzu Corporation) and NMR (made by JEOL Ltd.).
PMX 60SI), gas chromatograph (Shimadzu GC
-9 APF, PEG 20M × 1 m, 150 ° C., N ₂ 30 ml / min), it was confirmed to be ethyl γ-chloro-β-hydroxybutyrate.

NMR (CDCl ₃ ) δ (ppm): 1.25 (3H, tr) 4.20 (3H, q) 3.6 (2H, d) 3.2 (1H, br) 2.6 (2H, d) Add ethyl acetate for comparison. Table 1 also shows the results of the reaction under the same conditions in the aqueous medium alone.

Example 2 The procedure of Example 1 was repeated, except that various organic solvents shown in Table 2 were added and reacted. The yield was as shown in Table 2.

Example 3 The procedure of Example 1 was repeated except that the strains shown in Table 3 were used. The results are shown in Table 3.

The optical purity was measured as follows.

The resulting ester of ethyl γ-chloro-β-hydroxybutyrate and (+)-α-methoxy-α-trifluoromethylphenylacetic acid was synthesized to form a diastereomer compound, which was then analyzed by HPLC to calculate the optical purity. did.

HPLC analysis conditions Column: Partisil 5 (φ4.6 x 250 mm, manufactured by Wattmann) Mobile phase: Hexane: Tetrahydrofuran: Methanol = 600: 100: 1 (weight basis) Speed: 2.0 ml / min Detection: The absorbance at 217 nm was a (S) -form having a high optical purity.

Example 4 It carried out like Example 1 except having used the strain of Table 4. The results are shown in Table 4.

The optical activity analysis of the produced ethyl γ-chloro-β-hydroxybutyrate was carried out by the HPLC method described in Example 3. As a result, it was found to be a mixture of the (R) form and the (S) form.

Example 5 The procedure of Example 1 was repeated except that the substrates shown in Table 5 were used. The results are shown in Table 5.

Example 6 Glucose 5% by weight, corn staple liquor 5
Take 5 ml of a medium (pH 6.0) consisting of weight% in a test tube and inoculate it with Sporoboromyces salmonicolor IFO1038.
Seed culture was obtained by shaking culture at 2 ° C for 2 days. Seed culture is performed by taking 500 m of the same composition as above into 10 2 l flasks.
5 ml was added and shake culture was carried out at 28 ° C. for 4 days.

Next, centrifuge from culture solution of 5 (28000G, 20 minutes)
After washing the cultured cells recovered in step 1 above with 0.01 M phosphate buffer (pH 7.4), treat them with Dynomill (Shinmaru Enterprises Co., Ltd., beads 0.25-0.5 mmφ) for 20 minutes, and use 28,000 G for 2 minutes.
The crude enzyme solution was obtained by centrifuging for 0 minutes to remove suspended solids. Add ammonium sulphate to this to get 60-80% saturation
Was collected by centrifugation (28000G x 30 minutes),
It was dialyzed for 20 hours against M phosphate buffer (pH 7.0). Next is DEAE-
After being adsorbed on Sephacel (manufactured by Pharmacia) column chromatography (1.6φ × 30 cm) and washed with the above buffer solution,
A linear gradient elution was performed with the same buffer containing 0 to 0.6 M sodium chloride. Fractions showing activity were collected, concentrated with an ultrafilter (Amicon, YM10), supplied to a glu-filtration column chromatograph (Sephadex, G-100, 2.0 × 90 cm), and containing the above buffer containing 0.1 M NaCl. The liquid was chromatographed and the fractions showing activity were collected. Gel filtration chromatography was performed in the same manner as above to prepare a purified enzyme solution. This product electrophoretically showed a single band. The purified enzyme thus obtained was used and reacted under the conditions shown in Table 6. The results are shown together with comparative examples.

The ethyl acetate containing the product was dehydrated with anhydrous sodium sulfate and then removed under reduced pressure. The colorless liquid obtained was analyzed in the same manner as in Example 1 to find that it was ethyl γ-chloro-β-hydroxybutyrate having a purity of 98%. It was The optical purity of this product measured by the method of Example 3 was 97% ee (R) -form.

〔The invention's effect〕

According to the method of the present invention, it is possible to obtain a high-yield, high-purity and high-concentration HBE which cannot be obtained by a conventional aqueous medium alone system.

Claims (2)

(57) [Claims] 1. A reductase having the ability to reduce a γ-substituted acetoacetic ester to a γ-substituted-β-hydroxybutyric acid ester in an aqueous medium is used to convert the γ-substituted acetoacetic ester to γ-substituted-β-. A method for producing the γ-substituted-β-hydroxybutyric acid ester, characterized in that an organic solvent capable of forming two phases with water is present in the production of the hydroxybutyric acid ester. 2. The method according to claim 1, wherein the organic solvent capable of forming a two-phase with water is an ester, an alcohol, an aromatic, an ether or a halogenated hydrocarbon.