JPS63309195A - Production of gamma-substituted-beta-hydroxybutyric acid ester - Google Patents

Abstract

PURPOSE:To produce gamma-substituted-beta-hydroxybutyric acid ester in high yield, by treating gamma-substituted acetoacetic acid ester with a reductase in the presence of an organic solvent which can form two phases together with water in an aqueous medium. CONSTITUTION:A reductase is used to convert a gamma-substituted acetoacetic acid ester into a gamma-substituted-beta-hydroxybutyric acid ester using an organic solvent which can form two phases in an aqueous medium, dissolve the substrate and the product and has low actions to inhibit or deactivate the reaction of the reductase. The organic solvent is, e.g., an organic acid ester or alcohol, preferably the weight ratio of the organic solvent to the aqueous medium is 95/5-10/90.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] In recent years, the usefulness of various oxyacids as pharmaceutical synthesis intermediates has been recognized. The present invention relates to an efficient method for producing γ-substituted monoβ-hydroxybutyric acid ester, which is an intermediate for cartin synthesis among these oxyacids.

[Conventional technology]

Conventionally, an aqueous medium-only system has been used to convert -substituted acetoacetate (hereinafter referred to as AAE) to i-substituted -β-hydroxybutyric acid ester (hereinafter referred to as HBE) using a reductase. (Japanese Unexamined Patent Publication No. 118093/1983).

[Problem that the invention seeks to solve]

The present inventors investigated the reaction conditions in an aqueous medium-only reaction system, but there was a limit to the accumulated concentration of HBlli, and in order to further increase the yield, a large amount of enzyme and coenzyme was required, and the yield was reduced. There was a layer that caused a decrease in

[Means for solving problems]

The present invention provides for -substituted acetoacetic acid x step h in an aqueous medium.
t Forming two phases with water when producing 1-substituted -β-hydroxybutyrate from L-substituted acetoacetate using a reducing # element that has the ability to reduce to 1-a-substituted -β-hydroxybutyrate. The process of manufacturing the t-substituted -β-no-idroxybutyric acid ester is characterized by the presence of an organic solvent that can be used.

The minimum conditions for the organic solvent used in the present invention are: (1) It must be able to dissolve the substrate AAE and the product HBE (
2) capable of forming two phases with an aqueous medium; and (3) desirably low reaction inhibition and inactivating effects on reductases. Specifically, organic acid esters such as icate ester, acetate ester, fropionate ester, and butyrate ester, and the carbon of an alkyl group such as n-butyl alcohol, n-7 methyl alcohol, and n-octyl 7A/cole, etc. Alcohols of number 4 or more, aromatic solvents such as benzene, toluene, xylene, ethers such as diethyl ether, methyl ethyl ether, inglovir ether, halogenated substances such as chloroform, 1,2-dichloroethane, carbon tetrachloride, etc. Examples include hydrocarbons. The ratio of these organic solvents to the aqueous medium in the reaction system is not particularly limited, but in practice, the range of organic solvent/aqueous medium = 9515 to 50150 on a weight basis is preferred.

Next, the enzyme source used for the reduction reaction in these two-phase media is an enzyme capable of converting AAE to HBE, including microbial cells, processed bacterial cells, crude enzymes extracted from bacterial cells, and purified enzymes. can be used. Furthermore, immobilized products obtained by immobilizing these by known methods can also be effectively used. Specifically, the genus Candida, the genus Saccharomyces, the genus Torulo-psis, the genus Trichosporon, and the genus Pichia M.
p i Ch i a ), Hansenula spp.
enu-1a), Geotrichum (C) eozric
hum ), Endomyces spp.
), Debaryomyces manuscript
), Sporopolomyces sp.
Yces) and other yeasts to Aureobacterium (Au
reobacierium ) 754 Alcallgenes genus Agrobacterium (
Agrobaczerium ) genus Arilobacter (
ArthrObaCter) Genus Amorphosporan-gium Genus Ampullariella Genus Brevibaczerium
Genus Bacillus Genus Corynebacterium
m) Genus Cellulomonas
14 Escherichia genus Enterobacter (Ent, erobaczer) genus Flavobacterium (Fl, avobacterium)
) genus Haftnia I/f4 Kurzhia 84 Lactobacillus genus Micrococcus uicrOcOccus genus MsZhanomonas lf4 Methylobacillus cillus ) Genus Miku 1
Genus Microbispora (Micromonospora) Genus Nocardia Genus Prozeus 14 Genus Pseudomonas Pe
diococcus ) genus Cyranomonospora (Plan
omonospora) Genus Protomonas (Prot.
, omonas ) genus Rhodococcus
s) Genus Serratia Genus Strepzomyces - Thermoaczinomyces
Bacteria belonging to the genus Xant, homonas, genus Yersinia, and fungi such as Aspergilla and Mucor, respectively.
ucor), Fusarium,
Rhizopus (Rh1zopus), Penicillium (Penicillium), Neurospora (Ne
urospo-ra), Plularia sp.
ria), Uszilago, and Verzicilium.

These enzyme sources include asymmetric reduction of AAE and (8)-
Some produce HBE or (s)-HBE, while others produce HBE with no optical selectivity (a mixture of P5 form/(S) form). However, according to the method of the present invention,
Not limited to these enzyme sources, AAE can be
Reactions other than those described above can also be applied to the reaction for reducing to E.

In the reduction reaction, reduced nicotinamide adenine dinucleotide (NADH) is usually used as a coenzyme in addition to the reductase.
or nicotinamide adenine dinucleotide phosphate (
NADPH) is required, so either add it to the reaction system or
It is necessary to coexist a reaction system that produces ADH or NADPH' in the reduction reaction system. For example, NADH or NADPH that utilizes the conversion of NAD or NADP to NADH or NADPH, respectively, in the gluconic acid production reaction from glucose by glucose dehydrodenase.
A playback system etc. can be suitably used.

Regarding the type of AAE used, as the r-position substituent, chloro, bromo, fluoro, acid group, etc. are preferable, and as the ester group, an alkyl group having 1 to 10 carbon atoms is preferable.

The reaction temperature is 5-70°C1, preferably 20-400°C1
If the reaction value is adjusted to 4 to 10, preferably 6 to 8, the effects of the present invention can be fully exhibited. The reaction solution thus obtained is either left to stand until the two phases are separated, or separated using a centrifuge, etc.
Collect the portion of the organic layer containing the majority of the HBE. The HBE remaining in the aqueous layer can be recovered using the same or other extraction solvent, if necessary.

After combining the organic layers containing HBE and dehydrating with a dehydrating agent such as sodium sulfate, the organic solvent is removed under reduced pressure, and if necessary, further treatment such as vacuum distillation or chromatographic separation can be performed to obtain high purity HBg. It can be obtained with

Hereinafter, the present invention will be explained in detail with reference to Examples.
It is not limited to these.

〔Example〕

Example 1 51 d of a medium (p) 16.0) consisting of 5 parts by weight of glucose and 5 parts by weight of corn steep liquor was placed in a test tube, and Sporozaromyces salmonicolar
bolomyces Salmonicolor)
IFO1038 was inoculated and cultured at 28°C for 2 days.

100 rL of a medium having the same composition as this was placed in a 5,001-volume flask, inoculated with 5 at of seed culture, and cultured with shaking at 28°C for 4 days. Bacteria were collected using a centrifuge and 0. O
After washing with IM phosphate buffer (CF) 17.0), the total volume was adjusted to 10 tnl with the same buffer, and the bacterial cells were disrupted for 5 minutes at 2Q KHz using an ultrasonic disrupter under water cooling, and then reacted as a crude solution. It was used for. The reaction was started by adding 300 μmol of NADPH as a coenzyme, 600 μmol of substrate ethyl t-chloroacetoacetate, and 10 μmol of ethyl acetate to 101 L of the crude enzyme solution, and after adjusting - to 8, the reaction was started at 300 C with stirring. 20
After a period of time, separate the organic layer and aqueous layer, and separate the aqueous layer with ethyl acetate.
Extracted twice with IrLt. The ethyl acetate fractions were combined, dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain an oil. This product was distilled under reduced pressure and IR (IR4 manufactured by Konsei Manufacturing Co., Ltd.)
55), N"' (JEOL HPMX 60SI)
, gas chromatography (Kinsei Seisakusho HGC-9AP
F, PEG 2Q M xll, 150℃, N260
ILt/min) analysis confirmed that it was ethyl 1-yro-β-hydroxybutyrate.

NMR (CDC!3) J (pI)m): 1.2
5 (3H, tr) 4.20 (3B, q) 3.6 (2H, d) 3.2 (IH, br) 2.6 (2H, d) For comparison, without adding ethyl acetate, Table 1 also shows the results of the reaction in an aqueous medium alone under the same conditions.

Table 1 Example 2 The same procedure as Example 1 was carried out except that the various organic solvents shown in Table 2 were added and reacted. The yield was as shown in Table 2.

Table 2 Example 6 The same procedure as Example 1 was carried out except that the strains shown in Table 6 were used. Show the results to the 6th group.

The optical purity was measured as follows.

Generate? -Chloro-β-hydroxybutyric acid ethyltoke)-
After synthesizing an ester with α-methoxy-α-trifluoromethylphenylacetic acid to form a diastereomer compound, HPLC analysis was performed to calculate the optical purity.

HPLC analysis conditions Column: Parzisil 5 (φ4
．． (6 x 250 fl, manufactured by Whatman) Mobile phase: Hexane: Tetrahydro 7 run: Methanol = 600:100:1 (J & amount basis) Speed: 2. Od/min detection: Both absorbances at 217 nm were (S) monolithic with high optical purity.

Example 4 The same procedure as Example 1 was carried out except that the strains shown in Table 4 were used. The results are shown in Table 4.

The optical activity of the produced ethyl r-chloro-β-hydroxybutyrate was analyzed by the HPLC method described in Example 6. As a result, it was found to be a mixture of (R) and (S) forms.

Example 5 The same procedure as Example 1 was carried out except that the substrates shown in Table 5 were used. The results are shown in Table 5.

Example 6 Glucose 5% by weight, corn sugar 7' liquor 5
A medium (p86.0) 5aZ consisting of 1i% was taken into a test tube, and Sporobolomyces salmonicolar-IF0103
8 was inoculated and cultured with shaking at 28°C for 2 days to obtain a seed culture. A medium 500 sake having the same composition as above was placed in 10 21-volume flasks, seed culture 51 was added thereto, and cultured with shaking at 28°C for 4 days.

Next, the culture solution of 51 was centrifuged (28000G, 20
After washing the cultured bacterial cells collected with 0.01 MIJ acid buffer (-7,4), they were washed with Dynomil (manufactured by Shinmaru Enterprises, beads 0.25-0.5 taφ) for 2 minutes.
The mixture was treated for 0 minutes and centrifuged at 28,000G for 20 minutes to remove suspended substances to obtain a crude enzyme solution. Ammonium sulfate was added to this, and the 60 to 80 saturated fraction was collected by centrifugation (28,000 GX 30 minutes).
Dialysis was performed for 20 hours against M phosphate buffer (p87.0).

Next, it was adsorbed on DEAE-Sephacel (manufactured by Pharmacia) column chromatography (1,6φ x 30cIrL), and after washing with the above buffer, sodium chloride D ~ 0.6
Linear gradient elution was performed using the same buffer containing M.

Collect the fractions that showed activity and use an ultrafilter (Amicon, Y
Concentrate with Mlo), feed to Delfiltration 2M chromatograph (Sephadex, G-100,2, OX 90GrrL), perform chromatography with the above buffer containing 0.1 M Na1Lf, and collect the fractions that showed activity. collected.

Purified enzyme solution was prepared by performing Delfiltration chromatography in the same manner as above. This product showed a single band electrophoretically. Using the thus obtained purified #6
The reaction was carried out under the conditions shown in the table. The results are shown together with comparative examples.

6th Person City Activity Measurement Method Two groups jj600nmol and NADPH192
nmol to 0.61j enzyme solution (0.1M phosphate buffer,
p) 17.0) 3 4 at 37°C with KIIS
It was determined by measuring the change in absorbance at Q nm.

Ethyl acetate containing the product was dehydrated with anhydrous sodium sulfate, then removed under reduced pressure, and the resulting colorless liquid was analyzed in the same manner as in Example 1. As a result, it was found to be ethyl t-chloro-β-hydroxybutyrate with a purity of 98%. Ta. Further, the optical purity of this product measured by the method of Example 3 was 97% ee of (R).

〔Effect of the invention〕

According to the method of the present invention, it is possible to obtain HBE with high yield, high purity, and high melting strength, which could not be achieved using conventional aqueous media alone.

Patent Applicant: Denki Kagaku Kogyo Co., Ltd. Procedural Amendment July 11, 1988 Director General of the Patent Office Kunio Ogawa 2. Title of Invention: Process for producing T-substituted-β-hydroxybutyric acid ester 3. Case made by the person making the amendment Relationship with Patent Applicant Address ■100 1-4-1 Yurakucho, Chiyoda-ku, Tokyo Column 5 of the detailed description of the invention in the specification, Contents of amendment l) r50150 J on page 4, line 4 rlo/9
Correct to 0 J.

Claims (2)

[Claims] (1) In an aqueous medium, convert γ-substituted acetoacetate to γ
- An organic solvent that can form two phases with water when producing γ-substituted β-hydroxybutyrate from γ-substituted acetoacetate using a reductase that has the ability to reduce to substituted β-hydroxybutyrate. A method for producing the γ-substituted β-hydroxybutyric acid ester, characterized in that the γ-substituted β-hydroxybutyric acid ester is present. (2) The production method according to claim (1), wherein the organic solvent capable of forming two phases with water is an ester, an alcohol, an aromatic, an ether, or a halogenated hydrocarbon.