JP2583862B2 - Process for producing optically active β-monoalkyl malate or optically active isoserine - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to an enzyme, a microbial cell or an enzyme capable of selectively hydrolyzing an α-position ester bond and asymmetrically hydrolyzing a dialkyl malate ester. Using those processed materials,
The present invention relates to a method for producing optically active β-monoalkyl malate, and a method for producing optically active isoserine using this method.

[Prior Art] The following equation [1], Isoserine represented by the antibiotics butyrosine A and B
(Ambutyrosine A, B), [Tetrahedron Letters, Vol. 28, 2
617-2630, 1971], an optically active isoserine, which is a biochemically interesting compound, is useful as a raw material for the synthesis of various pharmaceuticals.

As a method for producing optically active isoserine, several methods have been known so far. One of the methods is a method of subjecting optically active β-malamidic acid to a Hoffman reaction [Organic Reactions (Organic Reacti).
ons), Volume 3, p. 267, 1949]. The optically active β-malamide acid is, for example, esterified optically active malic acid to form an optically active dialkyl malate, which is chemically mono-deesterified to obtain optically active malic acid β-malamic acid. A monoalkyl ester, which was prepared by converting this ester group to an amide [Beilstein Organich Chemie, 3rd Edition.
Vol. 429, 435].

[Problems to be solved by the invention]

However, in the above-mentioned known method, since it is difficult to selectively deesterify the ester at the α-position of the dialkyl malate, the process for obtaining the β-monoalkyl malate from the dialkyl malate is difficult. The rate was bad, and this was an overflow of the above method. Therefore, development of a method for advantageously producing malic acid β-monoalkyl ester has been desired.

Under such circumstances, the present inventors have found that if an esterase is first allowed to act on a dialkyl malate, the ester at the α-position is selectively de-esterified to obtain a β-monoalkyl malate in high yield. (Japanese Patent Application No. 61-32583).

However, in order to obtain optically active isoserine that is more useful as a raw material for synthesizing various pharmaceuticals, optically active malic acid must be used as a starting material. For example, D-form isoserine or D-form malate β- In order to obtain a monoalkyl ester, it is difficult to obtain a starting material because a D-form which does not exist as natural malic acid is required.

[Means for solving the problem]

Under such circumstances, the present inventors have conducted further research and found that enzymes such as lipase and esterase or Bacillus, Protaminobacterium, Flavobacterium, Rhodoshudomonas, Sporoboromyces By using an esterase- or lipase-producing microorganism of the genus Rhodotorula, Saccharomycopsis, Cryptococcus, Trichosporone, Pichia, or the like, dialkyl malate can be converted into optically active β-malate in high yield.
It has been found that monoalkyl esters can be selectively obtained.

That is, the present invention relates to a dialkyl malate, a genus Bacillus, a genus Protaminobacterium, a genus Flavobacterium, a genus Rhodoshudomonas, a genus Sporobomyces, a genus Rhodotorula, a genus Saccharomycopsis, a genus Cryptococcus, and a genus Trichosporon. Optically active malic acid obtained by the action of an enzyme, a cell or a treated product thereof having the ability to selectively hydrolyze and asymmetrically hydrolyze the ester bond at the α-position selected from the group consisting of microorganisms belonging to the genus Pichia An object of the present invention is to provide a method for producing an optically active β-monoalkyl malate characterized by obtaining a β-mol alkyl ester.

Furthermore, the present invention relates to a dialkyl malate, wherein the genus Bacillus, the genus Protaminobacterium, the genus Flavobacterium, the genus Rhodoshudomonas, the genus Sporobomyces, the genus Rhodotorula, the genus Saccharomycopsis, the genus Cryptococcus, the genus Trichosporon An optically active apple which has an ability to selectively hydrolyze and asymmetrically hydrolyze an ester bond at the α-position selected from the group consisting of microorganisms belonging to the genus Pichia and Pichia; Production of an optically active isoserine characterized by forming an acid β-monoalkyl ester, then amidating the ester group thereof to obtain an optically active β-malamic acid, and further reacting the resulting product with an alkali hypohalite. It provides the law.

The dialkyl malate which is a starting material of the present invention is produced by reacting malic acid or a reactive derivative thereof with an alcohol or a reactive derivative thereof according to a conventional method.

Examples of an enzyme capable of selectively hydrolyzing an α-ester bond and asymmetrically hydrolyzing a dialkyl malate ester include, for example, Bacillus laterosporus (Ba
cillus laterosporus), Bacillus thiaminoricus (B)
acillus thiaminolyticus, Bacillus sphaericus, Protaminobacter alboflavus, Flavobacterium ferrugi
nem), Rhodosudomonas sphaeroides (Rhodops)
eudomonas spheroides, Sparobolomyces albo-rubescens, Rhodotorula glutinus, Rhodotorula minuta, Saccharomycopsis hybridis (Saccharomycopsis fibulig)
era), Cryptococcus lawrenty (Cryptococ)
cus laurentii), Cryptococcus albidas (Cry
ptococcus albidus), Trichosporone ctaneus (T
richosporon cutaneus), Pichia psudopolymorpha and the like, and lipases, esterases and the like obtained by microorganisms induced by mutants or gene manipulation thereof, cell fusion and the like.
Optically active malic acid β from dialkyl malate
-In order to produce a monoalkyl ester, the lipase or esterase is allowed to act on the dialkyl malate, but instead of the lipase or esterase, cells of the microorganism or a treated product thereof may be applied. The processed products include immobilized enzymes obtained by immobilizing the lipase or esterase, immobilized microorganisms obtained by immobilizing the cells of the microorganism, and the like.

The reaction is conducted at a dialkyl malate concentration of 5 to 40%.
(V / V), preferably 10-20%, enzyme concentration 0.1-10U / ml,
Preferably 1-5 U / ml, temperature 15-70 ° C, preferably 20-4
0 ° C., reaction time 1 to 24 hours, preferably 2 to 9 hours, pH
It is carried out at 4.0 to 10.0, preferably 6.5 to 9.0. In this case, the ester bond at the α-position of the dialkyl malate is selectively and asymmetrically hydrolyzed to obtain an optically active β-monoalkyl malate in high yield.

The thus obtained optically active β-monoalkyl malate is converted into an optically active β-malamic acid according to the method described in Beilstein Organisch Chemie, Vol. Optically active isoserine can be derived according to the method described in Reactions (Organic Reactions), vol. 3, p. 267, 1949.

That is, first, the optically active β-monoalkyl malate is reacted with concentrated aqueous ammonia to obtain optically active β-malamic acid. In order to obtain optically active isoserine from the optically active β-malamic acid thus obtained, a Hoffman rearrangement reaction may be used.For example, barium hydroxide and bromine, which are generally used, are dissolved in an aqueous solution. A reaction for reacting with optically active β-malamic acid [Organic Reactions, Vol. 3, p. 284, 1949] can also be used, but in the present invention, optically active β-malamic acid is used. The reaction is carried out with a dilute aqueous sodium hypochlorite solution. After the reaction, the reaction solution is diluted with water or neutralized with an acid and then diluted with water.The target substance is adsorbed on the resin by passing through a basic ion exchange resin, followed by washing with water and dilute acetic acid. The fraction containing the target substance is eluted and concentrated to dryness to obtain optically active isoserine. If necessary, an amino protecting group such as t-butoxycarbonyl may be introduced without isolating the crude optically active isoserine obtained by the Hoffman rearrangement reaction.

〔Example〕

Hereinafter, the present invention will be described with reference examples and examples, but the present invention is not limited to the examples.

Example 1 (1) Dissolve 50 g of malic acid in 500 ml of methanol,
Under stirring, 35 ml of thionyl chloride was added dropwise over 10 minutes, and the mixture was stirred and reacted at room temperature overnight. Methanol was distilled off from the reaction solution under reduced pressure to obtain 60 g of malic acid dimethyl ester.

(2) Glucose 2%, peptone 0.5%, malt extract
1000 ml of a medium (pH 6.0) containing 0.3%, yeast extract 0.3% and malic acid dimethyl ester 0.4% is sterilized, and Cryptococcus laurentii
ATCC 18803 strain was inoculated and cultured at 28 ° C. for 70 hours. The culture was centrifugally filtered to collect the cells, and 50 ml of physiological saline was added thereto to obtain a cell suspension.

(3) 60 g of dimethyl malate was added to 250 ml of water, adjusted to pH 7.0 with a 50% NaOH solution, and reacted at 30 ° C. for 20 hours. After the reaction, the reaction solution was centrifugally filtered to remove cells,
The remaining malic acid dimethyl ester was extracted and removed using 200 ml of ethyl acetate. This operation is performed with an additional 200 m
l was added and performed. The aqueous layer was adjusted to pH 2.0 by adding 2N HCl, and extracted twice with 200 ml of ethyl acetate, and the solution of the ethyl acetate layer was separated. Ethyl acetate was distilled off from the solution of the ethyl acetate layer under reduced pressure to obtain 19 g of L-malic acid β-monomethyl ester.

(4) 100 ml of concentrated aqueous ammonia was added to 19 g of L-malic acid β-monomethyl ester and stirred to form a homogeneous solution,
Let stand overnight. To this, 200 ml of water was added, and 90 ml of a 10% hypochlorous acid solution containing 4.4 g of NaOH was added dropwise at 0 ° C. with stirring over 30 minutes, followed by stirring at room temperature for 1 hour. Add Na to this reaction solution.
35 ml of an aqueous solution containing 20 g of OH was added, and the mixture was stirred at 80 to 90 ° C for 1 hour. After completion of the reaction, was neutralized with hydrochloric acid, ion exchange resin [DOWEX 1X2 (OH ^-)] was filled in 3, washed with 15 of water, then eluted with 5% acetic acid solution, identical to L- isoserine in chromatography Value [Rf = 0.19 by paper chromatography (butanol: acetic acid: water = 4: 1: 1)]
The fractions represented by were concentrated and dried to give 4.1 g of L-isoserine [(s) -α-hydroxy-β-amino-propionic acid]. If necessary, the product was recrystallized with 80% ethanol to obtain 3.5 g of colorless needles of L-isoserine.
The melting point of L-isoserine obtained here is 188-189 ° C., ▲
[Α] ²⁰ _D ▼ = -31.7 (C = 1.0, H ₂ O), which was the same as the standard product.

Example 2 100 ml of a medium (pH 7.0) containing 2% of peptone, 0.4% of yeast extract, and 0.4% of malic acid dimethyl ester was sterilized, and Flavobacterium ferguinem (Flavobacteri) was sterilized.
um ferruginem) ATCC13524 strain was inoculated and cultured at 28 ° C for 30 hours. The culture was centrifugally filtered to collect the cells, and 21 ml of physiological saline was added thereto to obtain a cell suspension. 1 g of malic acid dimethyl ester obtained in the same manner as in Example 1 (1) was 0.1 M
The cells were dissolved in 10 ml of a phosphate buffer (pH 7.0), and the whole amount of the cell suspension was added thereto, followed by a reaction at 30 ° C. for 3 hours. The reaction solution was subjected to extraction using ethyl acetate in the same manner as in Example 1 (2), and then the ethyl acetate solution layer was subjected to high performance liquid chromatography analysis (HPLC) under the following conditions. Disappeared, and only a peak of malic acid β-monomethyl ester was observed.

After the ethyl acetate in the ethyl acetate solution layer was distilled off under reduced pressure, the residue was distilled to obtain β-monomethyl malate.
When the optical rotation of the obtained malic acid β-monomethyl ester was measured, ▲ [α] ²⁰ _D ▼ = -7.70 (C = 1.0, MeO
H), and together with the measurement results of IR and H-NMR, L-
It was the same as malic acid β-monomethyl ester.

HPLC conditions Column: Shodex KC 810 (manufactured by Showa Denko KK) Elution solvent: 0.005% perchloric acid solution Flow rate: 1.0 ml / min Detection: RI: retention time of malic acid dimethyl ester 11.8
Example 3 to 15 100 ml of the medium described in Table 1 was sterilized, inoculated with each of the microorganisms described in Table 1, and cultured at 28 ° C. Thereafter, the cells were collected in the same manner as in Example 1 (2). Using the obtained microbial cells, dimethyl malate was used in the same manner as in Example 1 (3).
A reaction using 1.0 g as a substrate was performed under the reaction conditions described in Table 1, treated in the same manner as in Example 1 (3), and the solution of the ethyl acetate layer was examined under the HPLC conditions described in Example 2. Malic acid β
A single peak of monomethyl ester was observed, and the ethyl acetate layer solution was evaporated under reduced pressure to remove ethyl acetate to obtain optically active β-monomethyl malate. The optical rotation of the obtained optically active β-monomethyl malate was measured, and the results are shown in Table 1.

[Effects of the Invention] The present invention provides an optically active isoserine useful as a raw material for medicines and agricultural chemicals at a low cost. In particular, D-isoserine, which has not yet been found in nature, can be obtained more easily. Also, in the case of obtaining L-isoserine, it is useful without using an optically active substance as a raw material, and without having to go through a complicated optical resolution step in the production process.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location C12R 1:07) (C12P 41/00 C12R 1:01) (C12P 41/00 C12R 1:20) (C12P 41/00 C12R 1:84) (C12P 41/00 C12R 1: 645)

Claims (2)

(57) [Claims] (1) The dialkyl malate includes a genus Bacillus, a genus Protaminobacterium, a genus Flavobacterium, a genus Rhodoshudomonas, a genus Sporobomyces, a genus Rhodotorula, a genus Saccharomycopsis, a genus Cryptococcus, a genus Trichosporon, and Pichia Enzymes having the ability to selectively hydrolyze and asymmetrically hydrolyze ester bonds at the α-position selected from the group consisting of microorganisms belonging to the genus, fungi, or optically active malic acid β by treating them. -A process for producing an optically active β-monoalkyl malate, characterized by obtaining a monoalkyl ester. 2. A dialkyl malate, wherein the genus Bacillus, the genus Protaminobacterium, the genus Flavobacterium, the genus Rhodoshudomonas, the genus Sporobomyces, the genus Rhodotorula, the genus Saccharomycopsis, the genus Cryptococcus, the genus Trichosporon, and Pichia Enzymes having the ability to selectively hydrolyze and asymmetrically hydrolyze ester bonds at the α-position selected from the group consisting of microorganisms belonging to the genus, fungi, or optically active malic acid β by treating them. -A process for producing optically active isoserine, which comprises converting the product to a monoalkyl ester, then amidating the ester group thereof to obtain an optically active β-malamic acid, and further reacting the resulting product with an alkali hypohalite.