JPH09206089A - Process for producing optically active amino acid derivative - Google Patents

Abstract

(57) Abstract: An optically active N-substituted-L-amino acid and / or N-substituted-D-amino acid ester, especially a substituent on N is a lower alkanoyl group, and an amino acid is alanine or methionine. Efficiently produce a compound. SOLUTION: A racemic N-substituted-DL-amino acid ester has a weak base as a pH control reagent for a culture of a microorganism having a capability of asymmetrically hydrolyzing an L-type optically active substance in a racemate or a treated product thereof. After the reaction, the optically active N-substituted-L-amino acid and / or N-substituted-D-amino acid ester is separated and collected.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an optically active N-substituted-L-amino acid and an optically active N-substituted-D-using an enzyme.
The present invention relates to a novel method for producing an amino acid ester and an optically active D-amino acid.

[0002]

2. Description of the Related Art Optically active N-substituted-L-amino acids, particularly compounds in which the substituent on N is an acetyl group and the amino acid is alanine or methionine, have ideal characteristics as therapeutic agents for renal hypertension and the like. It is a compound useful as a synthetic intermediate for phenethylamine derivatives (dopamine precursors) and other pharmaceuticals (Japanese Patent Publication No. 57-4810).
No. 3).

Conventionally, various methods for producing these optically active N-substituted-L-amino acids have been known. Particularly, as a method for producing optically active N-acetyl-L-methionine, N-acetyl-DL is a chemical method. -Method of resolving methionine using a resolving agent such as α-phenethylamine (Jour
nal of American ChemicalS
ociety, Vol. 73, 4604 (1951)
Etc.), a method of acetylating L-methionine with acetic anhydride, acetyl chloride, etc. (Journal of Biol).
Official Chemistry, Vol. 98,3
00 (1932) and the like, and a biochemical method for dividing N-acetyl-DL-methionine by utilizing the action of D-aminoacylase (US Patent No. 52).
No. 06162) and N-acetyl-DL-methionine alkyl ester in the presence of a hydrolase such as protease, lipase, esterase, etc. to selectively asymmetrically hydrolyze the L-form and divide it (Japanese Patent Publication No. S58-58). -491
No. 60).

[0004]

[Problems to be Solved by the Invention] Racemic N-substituted-D
Among L-amino acid esters, for example, a method of asymmetrically hydrolyzing a racemic N-acetyl-DL-methionine alkyl ester to obtain optically active N-acetyl-L-methionine, DL-methionine is very inexpensive and available. It is a more useful method than easy. However, in the present hydrolysis reaction, the pH does not decrease due to the action of the buffer solution when the substrate concentration is as low as several millimolar to several tens of millimolar, but when the substrate concentration is several molar that can withstand the production, it is generated. Due to the effect of acid, the pH is lowered with the reaction. Due to this decrease in pH, the reaction rate remarkably decreases and the reaction efficiency decreases. Normal pH for efficient reaction
The control is carried out, for example, Japanese Patent Publication No. 58-4.
In the method described in 9160, sodium hydroxide is used as a pH control reagent. However, under this reaction condition, the N-acetyl-D-methionine alkyl ester is chemically hydrolyzed by sodium hydroxide, and N-acetyl-D-methionine is produced as a by-product in the reaction-terminated solution, so that the obtained optical activity is obtained. There is a problem that the yield of N-acetyl-L-methionine is low and the optical purity is low.

[0005]

Means for Solving the Problems As a result of intensive studies, the present inventors have efficiently obtained a target optically active N-substituted-L-amino acid from a racemic N-substituted-DL-amino acid ester. To find a method of
The inventors have found that -substituted-D-amino acid esters can be efficiently obtained, and completed the present invention.

That is, the present invention is a racemic N-substituted-D
The L-amino acid ester is preferentially hydrolyzed with the L-type optically active substance in the selected racemate to give an optically active N-amino acid ester.
An unreacted optically active N-substituted-D that remains after the reaction of an enzyme having the ability to convert to a substituted-L-amino acid, a culture of a microorganism, or a treated product thereof as a pH control reagent in the presence of a weak base. -Amino acid ester is separated and collected, or the produced optically active N-substituted-L-
An optically active N-substituted-L-amino acid and / or an optically active N characterized by separating and collecting an amino acid from a reaction solution
A method for producing a -substituted-D-amino acid ester.

In the racemic N-substituted-DL-amino acid ester of the present invention, the substituent on N may be any as long as the substituent can be a substituent of an amino group which is not affected in the reaction system. , For example, an acyl group. As the amino acid, any compound having an amino group and a carboxyl group in the same molecule may be used, and α-amino acid may be mentioned.

Examples of the acyl group include an aliphatic acyl group and an aromatic acyl group, and examples of the aliphatic acyl group include a lower alkanoyl group. Examples of such lower alkanoyl group include linear or branched alkanoyl groups having 2 to 6 carbon atoms such as acetyl group, propionyl group, isopropionyl group, butanoyl group and isobutanoyl group.

Examples of α-amino acids include alanine, cysteine, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, valine, and the like. Is mentioned.

Examples of the alkyl ester include lower alkyl ester, and examples of such lower alkyl group include those having 1 to 6 carbon atoms such as methyl group, ethyl group, propyl group, isopropyl group, butyl group and isobutyl group. A straight-chain or branched-chain alkyl group is mentioned, and examples of the active ester include active esters such as N-hydroxysuccinimide, N-hydroxyphthalimide, 1-hydroxybenzotriazole, or ester with p-nitrophenol.

Of these, preferred are those in which the substituent on N is a lower alkanoyl group, the amino acid is alanine or methionine, and the ester is a lower alkyl ester.
It is preferred that the above substituent is an acetyl group and the amino acid is methionine.

The weak base as a pH control reagent may be a weak base which does not chemically hydrolyze the racemic N-substituted-DL-amino acid ester and cannot produce an N-substituted-D-amino acid as a by-product. Good.

Examples of preferable weak bases include alkali metal carbonates (eg, sodium carbonate, potassium carbonate, etc.),
Alkaline earth metal carbonates (eg magnesium carbonate, calcium carbonate etc.), alkali metal bicarbonates (eg sodium bicarbonate, potassium bicarbonate etc.), alkali metal acetates (eg sodium acetate, potassium acetate etc.), alkali citrate Metals (eg, sodium citrate), alkaline earth citrates (eg, magnesium citrate, calcium citrate, etc.), alkaline earth metal phosphates (eg, magnesium phosphate, calcium phosphate, etc.), alkali metal hydrogen phosphates ( For example, disodium hydrogen phosphate,
Dipotassium hydrogen phosphate, etc.), alkali metal phosphates (eg,
Monopotassium phosphate, monosodium phosphate, etc.) or ammonia. Of these, alkali metal carbonates, alkali metal bicarbonates, alkali metal hydrogen phosphates, alkali metal citrates or ammonia are preferred.

As the enzyme in the present invention, for example, a group of enzymes called protease, lipase or esterase can be used. These enzymes may be of microbial origin, animal cell origin, or
It may be derived from plant cells. Further, it may be extracted from a microbial cell, animal cell or plant cell containing these hydrolytic enzymes by a known method, or may be a commercially available one. Specifically, for example, lipase-A [Aspergillus niger (Aspergill
us niger) origin, manufactured by Amano Pharmaceutical Co., Ltd.], Lipase-N
[Rhizopus niveu
s), manufactured by Amano Pharmaceutical Co., Ltd., professional leather [derived from Bacillus sp., manufactured by Amano Pharmaceutical Co., Ltd.], lipase-CE [Humicola Lanuginosa (Hum)
derived from icola lanuginosa), manufactured by Amano Pharmaceutical Co., Ltd., Neurase [Rhizopus nibius (Rhizo
derived from pus niveus, manufactured by Amano Pharmaceutical Co., Ltd.], papain W-40 [Carica papaya (Carica papaya)
a) origin, manufactured by Amano Pharmaceuticals], pancreatin [derived from hog pancreas, manufactured by Amano Pharmaceuticals], Protease-A (Aspergillus oryzae (A
spergillus oryzae), Amano Pharmaceuticals], Protease-N [Bacillus subtilis (Ba)
derived from Cillus subtilis, manufactured by Amano Pharmaceuticals, Inc.],
Protease-P [Aspergillus meleus (Aspe
derived from r. gillus melleus), manufactured by Amano Pharmaceutical Co., Ltd., Protease-M [Aspergillus oryzae (A
spergillus oryzae) -derived, manufactured by Amano Pharmaceutical Co., Ltd.], lipase A-5 [Rhizopus japonicas (Rhi
derived from zopus japonicus, manufactured by Nagase Seikagaku], XP-415 [Rhizopus delemmer (Rhi
derived from zopus delemer, manufactured by Nagase Seikagaku], Denazyme-AP [Aspergillus oryzae (A
spergillus oryzae) -derived, Nagase Seikagaku Corporation], Esperase-8.0SL [Bacillus
Derived from species (Bacillus sp.), Manufactured by Novo Nordix Co., Ltd., Neutrase-0.5 L [Bacillus subtilis (Bacillus subtil).
is), manufactured by Novo Nordix, Inc.], Savinase-
8.0L [Bacillus species
sp. ) Origin, Novo Nordix, Inc.], Alcalase-2.5L [Bacillus licheniformis (Bac
derived from illus licheniformis, manufactured by Novo Nordix, Inc., lipase type II [Porcine pancreas]
s), manufactured by Sigma], lipase [derived from Porcine pancreas, manufactured by Wako Pure Chemical Industries, Ltd.], lipase [porcine pancreas (P
orine pancreas), Katayama Chemical Co., Ltd.], lipase [Rhizopus genus (Rhizopus genus), Katayama Kagaku Co.] or Neurase [Rhizopus niveus (Rhizopus niveus, Amano Pharmaceuticals)], among others, professional leather. , Protease-P, Protease-M, Bioprase AL-1
5, Savinase-8.0L or Alcamyl-1.5 / 5
0L type B is preferred.

As the microorganism in the present invention, the racemic N-substituted-DL-amino acid ester as described above is used to preferentially hydrolyze the L-type optically active substance in the selected racemate to give optically active N-. As long as it has the ability to convert to a substituted-L-amino acid, Aspergillus (Asp
ergillus), Rhizopus
Genus, genus Bacillus or Humicola (H
The microorganisms belonging to the genus Umicola) are mentioned.

Specific examples of such microorganisms include Aspergillus niger.
r), Rhizopus nivus
eus), Bacillus species (Bacillus)
sp. ), Humicola Ranuginosa (Humicola)
lanuginosa), Carica papaya (Cari)
ca papaya), Aspergillus oryzae (As
pergillus oryzae, Bacillus subtilis, Aspergillus mereus
lleus, Rhizopus japonicas (Rhizopu)
s japonicus), Rhizopus delemar (Rh
izopus delemer or Bacillus licheniformis
mis).

These microorganisms may be any strains as long as they have the ability required for the present invention, and may be mutant strains obtained by artificial treatment such as ultraviolet irradiation or treatment with a mutagen. , Recombinant DNA from these microorganisms
It may be induced by a genetic engineering technique such as a cell fusion method or a cell fusion method, or a bioengineering method. For example,
According to genetic engineering techniques, from the chromosome fragment of the microorganism that produces the hydrolase, isolate the gene of the enzyme of interest,
After preparing a recombinant plasmid in which this is inserted into an appropriate plasmid vector, and transforming an appropriate host microorganism with this recombinant plasmid, a microorganism having enzyme-producing ability or a microorganism having further improved enzyme-producing ability is obtained. Obtainable. In addition, a host microorganism having other excellent properties (for example, easy culturing) may be transformed with this recombinant plasmid, if necessary.

The culture in the present invention includes, for example, a culture solution or viable cells of the above-mentioned microorganism, and a processed product of the culture is, for example, a processed product of the culture solution (culture supernatant or the like) or Examples include processed bacterial cells.

Further, the culture of the microorganism or its treated product may be any one containing a group of enzymes called protease, lipase or esterase.

The above-mentioned culture (culture solution, bacterial cells, etc.) is prepared, for example, in a medium usually used in this field of the above-mentioned microorganisms, for example, a medium containing a conventional carbon source, nitrogen source and inorganic salt, at room temperature or under heating. (Preferably about 20-40
C.) and under aerobic conditions at pH 4-9, and if necessary, cells can be isolated and collected from the thus obtained culture solution by a conventional method.

As a treated product of microorganisms, the culture is treated by various physicochemical methods such as ultrasonic wave, French press, osmotic pressure, freeze-thawing, freeze-drying, alumina destruction, lytic enzyme, surfactant or In addition to cells treated by means such as an organic solvent, partial purification prepared by a known method from the aforementioned culture (culture solution, cells, etc.) or its treated product (culture supernatant, treated cells, etc.) An enzyme or a purified enzyme having the ability to preferentially hydrolyze an L-type optically active substance among racemic N-substituted-DL-amino acid esters to convert it to an optically active N-substituted-L-amino acid Can be mentioned.

As such partially purified enzyme or purified enzyme, commercially available products similar to those described above can be used.

That is, in the reaction of the present invention, the ability to preferentially hydrolyze the L-form optically active form of the racemic N-substituted-DL-amino acid ester to convert it into the optically active N-substituted-L-amino acid. Any enzyme can be used as long as it has an enzyme.

Further, the enzyme, microbial cell or treated product of the present invention of the present invention is immobilized by, for example, a polyacrylamide method, a sulfur-containing polysaccharide gel method (carrageenan gel method or the like), an alginic acid gel method or an agar gel method. It can also be used.

[0025]

BEST MODE FOR CARRYING OUT THE INVENTION The asymmetric hydrolysis reaction according to the present invention is carried out by contacting a racemic N-substituted-DL-amino acid ester with an enzyme, a culture of a microorganism or a treated product thereof in a suitable solvent. can do.

The reaction temperature is not particularly limited and may be appropriately selected depending on the type of ester and the elimination method, but it is preferably room temperature or under heating, preferably about 0 to 70 ° C., particularly preferably about 0 to 50 ° C. And proceed appropriately. In addition, the reaction is such that addition of a weak base as a pH control reagent does not adversely affect the optically active N-substituted-D-amino acid ester, the enzyme used in the present hydrolysis reaction, the culture of the microorganism, or the processed product thereof. It is preferably carried out under neutral conditions, but preferably at a pH of about 6 to about 8, especially at a pH of about 6.5 to about 7.5. The above pH control reagent may be used as a solid or prepared as an aqueous solution, and as an aqueous solution, 1 to 80%, particularly 10 to 50%
% Is preferable.

Racemic N-substituted-DL- which serves as a reaction substrate
The charged concentration (w / v) of the amino acid ester is 10 to 4
0% is preferred. Further, when the raw material compound is poorly soluble, it can be used by suspending it in a solvent, and as a solubilizing agent, a small amount of dimethylformamide, dimethylsulfoxide, acetonitrile, tetrahydrofuran, acetone,
It is also possible to use polar organic solvents such as dioxane, methanol or ethanol. At that time, the reaction substrate may be added all at once, or may be added in several portions during the reaction. Further, the reaction can be carried out in an aqueous solution, a two-phase system of an aqueous solution and an organic solvent, or the like. As such an organic solvent, any organic solvent can be used as long as it can dissolve the racemic N-substituted-DL-amino acid ester serving as a reaction substrate and is immiscible with water. Examples of such an organic solvent include toluene, xylene, carbon tetrachloride, benzene, trichloroethane, chloroform and n-.
Hexane, n-heptane, n-octane, cyclohexane, isooctane, ethyl acetate, butyl acetate, diethyl ether, t-butyl methyl ether, isopropyl ether and the like can be mentioned, among which toluene, carbon tetrachloride,
Benzene, trichloroethane, n-hexane, t-butyl methyl ether and the like are preferable.

Further, a racemic N-substitution- which is a raw material compound
As the DL-amino acid ester, not only those containing equal amounts of the D-type optically active substance and L-type optically active substance, but also any substance containing both of these optically active substances can be used.

When using viable cells in the present invention, the reaction time can be shortened by adding a surfactant to the reaction solution. Examples of the surfactant used for this purpose include cetylpyridinium bromide, cetyltrimethylammonium bromide, or p-isooctylphenyl ether (trade name Triton X-100, manufactured by Rohm and Haas Company, USA). About 0.
It is preferable to use about 0001 to about 0.1%.

Separation and collection of the optically active N-substituted-D-amino acid ester and / or the optically active N-substituted-L-amino acid from the reaction solution can be carried out by a conventional method. That is, for example, when the hydrolysis reaction is carried out in a water-organic solvent two-phase system, the optically active N-substituted-D-amino acid ester can be obtained by remaining in an organic solvent and concentrating under reduced pressure. In addition, the optically active N- transferred to the water layer
The substituted -L-amino acid is acidified with hydrochloric acid or the like, and then salted. The salt to be added may be any salt as long as it is soluble in water, but ammonium sulfate, sodium chloride or the like is preferably used. The salt concentration to be added is 30 to 6 with respect to the aqueous solution.
0% is preferred.

An organic solvent is added to the salted-out aqueous solution for extraction, and then concentrated and crystallized and centrifuged to recrystallize the optically active N-substituted-L-amino acid in high yield and high optical purity. Can be separated. The solvent used for extraction may be a solvent that separates into two phases with water and dissolves the N-substituted-L-amino acid, and preferably ethyl acetate, chloroform and the like.

The assay of whether or not the enzyme, the culture of the microorganism or the processed product thereof has the ability to asymmetrically decompose the L-type optically active substance of the racemic N-substituted-DL-amino acid ester is carried out as described above. It can be easily carried out according to the reaction method, for example, as follows. That is, racemic N-acetyl-D
To the aqueous solution containing L-methionine methyl ester, an enzyme to be assayed, a culture of a microorganism or a treated product thereof is added, and 3
After leaving still for 4 hours at 0 ° C., the reaction-terminated liquid is used as an optically active column (eg, SUMICHIRAL manufactured by Sumika Chemical Analysis Center).
OA-5000) is used for analysis and quantification by high performance liquid chromatography, and when the produced N-acetyl-L-methionine has a high optical purity, it has the ability to selectively hydrolyze the ester of the L-type optically active substance. Is determined.

The optically active N-substituted-L- thus obtained
The amino acid and / or the optically active N-substituted-D-amino acid ester can be prepared according to a known method, for example, the method disclosed in JP-B-57-48103 or JP-B-7-8853 and represented by the general formula [I].

[0034]

Embedded image

(Wherein R represents a group obtained by removing a hydroxyl group from a carboxyl group of an N-substituted-amino acid), and can be converted into an optically active phenethylamine derivative,
Furthermore, if desired, the product can be made into its pharmaceutically acceptable salt.

In the above method, there are two kinds of optically active substances in the general formula [I], but the present invention includes both optically active substances and a mixture thereof. However, when the general formula [I] is used as a medicine, a compound obtained by using an N-substituted-L-amino acid is particularly preferable.

On the other hand, the optically active N-substituted-D-amino acid ester can be prepared by a known method, for example, Journal of of
Organic Chemistry, Vol. Four
3, 4593 (1978), hydrolyzing in the presence of a strong base aqueous solution such as sodium hydroxide to once give an optically active N-substituted-D-amino acid, and then hydrolyzing in a strong acid aqueous solution such as hydrochloric acid. It can be converted to a D-amino acid.

By the way, it has been found that when the optically active N-substituted-D-amino acid ester is heated in a strong base environment such as an aqueous solution of sodium hydroxide to carry out a hydrolysis reaction, further racemization occurs. That is, since this method is inefficient because the reaction solution is once made alkaline and then acidified, a method for easily and efficiently producing an optically active D-amino acid has long been desired.

However, it is also possible to obtain an optically active D-amino acid by hydrolyzing an optically active N-substituted-D-amino acid ester with only an acid without using a base.

As the acid used in the present hydrolysis, an ester of an optically active N-substituted-D-amino acid ester is hydrolyzed to give an optically active N-substituted-D-amino acid, and then N
Any acid can be used as long as it hydrolyzes the above-mentioned substituent to synthesize an optically active D-amino acid and can not racemize the optically active N-substituted-D-amino acid ester as a substrate. Examples of such an acid include 1 to 4N hydrochloric acid, hydrobromic acid, sulfuric acid and the like. Of these, hydrochloric acid is preferable, and 2N hydrochloric acid is particularly preferable.

The reaction may be carried out in the presence of an organic solvent,
The reaction temperature is preferably about 80 to 140 ° C. The reaction time may be monitored by an optically active column at any time, and the reaction is completed in about 2 to 12 hours. The charged concentration (w / v) of the optically active N-substituted-D-amino acid ester serving as a reaction substrate is preferably 1 to 50%. When the raw material compound is poorly soluble, it can be used by suspending it in a solvent.

Separation / collection of the optically active D-amino acid from the reaction solution can be carried out according to a conventional method. That is, for example, after setting the reaction residue to about 0 to 40 ° C., an aqueous alkali metal solution such as sodium hydroxide or potassium hydroxide is added dropwise to adjust the pH of the aqueous solution to neutral, and then crystallized at 0 to 5 ° C. To do. The optically active D-amino acid can be obtained by filtering the precipitate.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these.

In the present specification, "%" means "weight / volume (g / dl)". In addition, in this example, the quantitative determination of the optically active substance was carried out by using SUMICHIR
It was performed by high performance liquid chromatography using ALOA-5000 (manufactured by Sumika Chemical Analysis Service).

[0045]

【Example】

Example 1 0.1 M phosphate buffer (KH ₂ PO ₄ -NaOH, pH
7.5) 200 mg of Proleather (derived from Bacillus subtilis, manufactured by Amano Pharmaceutical Co., Ltd.) was dissolved in 100 ml and stirred at 40 ° C. N-acetyl-DL-methionine methyl ester 4
0 g was charged with stirring, and the stirring reaction was performed as it was. Gradually add sodium hydrogencarbonate during the enzymatic reaction to reach pH 6.
Adjusted to 5-7.0. The progress of the reaction is 100μ sample
l was collected, diluted with 1.9 ml of a 2 mM copper sulfate solution, and extracted with 2.0 ml of chloroform. Then, the residual amount of N-acetyl-L-methionine methyl ester in the chloroform layer was determined by the HPLC method (CHIRALCEL OD, 4. 6
mm × 250 mm, manufactured by Daicel Chemical Industries).
After 3 hours, the reaction was terminated after confirming the disappearance of the optically active N-acetyl-L-methionine methyl ester. After 2 g of activated carbon was added to the reaction solution to remove the enzyme, the mixture was stirred for 30 minutes and filtered with filter paper. The filtrate is chloroform 50
After washing 3 times with ml, unreacted optically active N-acetyl-D
-Methionine methyl ester was extracted and removed. On the other hand, acidify the aqueous layer with hydrochloric acid, add 60 g of ammonium sulfate,
After extraction with 200 ml of ethyl acetate, the ethyl acetate layer was dehydrated with magnesium sulfate and then filtered. The filtrate is concentrated under reduced pressure,
After crystallization for 1 hour under ice cooling, the crystals were collected by filtration. The obtained crystals were blow-dried at 40 ° C. for 16 hours. as a result,
100% optical purity by HPLC e. e. The crystal of optically active N-acetyl-L-methionine showing
%. The specific rotation and optical purity of the obtained crystal were as follows. Enzyme used, N-acetyl-
Table 1 below shows the reaction time at which L-methionine methyl ester disappeared, the crystal yield, and the amount of N-acetyl-D-methionine produced in the reaction-terminated liquid.

Crystal yield: 16.9 g, chemical purity: 10
0%, optical purity: 100% e. e. Optical rotation: [α] ^D ₂₀ = -21.4 ° (C = 4, H ₂ O) Incidentally, the chloroform extract of unreacted optically active N-acetyl-D-methionine methyl ester was concentrated to obtain the optically active substance. N-acetyl-D-methionine methyl ester 19.
0 g was collected.

Examples 2 to 3 Enzyme amount 100 mg, N
-Acetyl-DL-methionine methyl ester was treated with 20 g in the same manner as in Example 1, and N-acetyl-after the reaction was completed.
The amount of D-methionine produced was examined. Enzyme used, N-
Table 1 below shows the reaction time at which acetyl-L-methionine methyl ester disappeared, the crystal yield, and the amount of N-acetyl-D-methionine produced in the reaction-terminated liquid.

Comparative Examples 2 to 3 (Control Experiments of Examples 2 to 3) Examples 2 to 3 using 5N sodium hydroxide aqueous solution as a pH control reagent in place of sodium hydrogen carbonate.
The same treatment was performed, and the amount of N-acetyl-D-methionine produced after the reaction was examined. Reaction time when the enzyme used, N-acetyl-L-methionine methyl ester disappeared,
The yield of crystals and the amount of N-acetyl-D-methionine produced in the liquid after the reaction are shown in Table 1 below.

[0049]

[Table 1]

Examples 4 to 6 Using a pH control reagent (weak base) instead of sodium hydrogen carbonate, the amount of enzyme was 100 mg, and N-acetyl-D was used.
It was treated with 20 g of L-methionine methyl ester in the same manner as in Example 1, and the amount of N-acetyl-D-methionine produced after the reaction was examined. PH control reagent used, N
The following Table 2 shows the reaction time at which -acetyl-L-methionine methyl ester disappeared, the crystal yield, and the amount of N-acetyl-D-methionine produced in the reaction-terminated liquid.

Comparative Examples 4 to 6 Treatment was carried out in the same manner as in Examples 4 to 6 using an aqueous sodium hydroxide solution as a pH control reagent in place of a weak base, and the amount of N-acetyl-D-methionine produced after the reaction was examined. It was PH control reagent used, N-acetyl-
Table 2 below shows the reaction time at which L-methionine methyl ester disappeared, the crystal yield, and the amount of N-acetyl-D-methionine produced in the reaction-terminated liquid.

Comparative Example 7 The same treatment as in Examples 4 to 6 was carried out in the absence of a pH control reagent, and the amount of N-acetyl-D-methionine produced after the reaction was examined. Table 2 below shows the reaction time at which the enzyme used, N-acetyl-L-methionine methyl ester disappeared, the crystal yield, and the amount of N-acetyl-D-methionine produced in the reaction-terminated liquid.

[0053]

[Table 2]

Example 7 19 g of N-acetyl-D-methionine methyl ester was dissolved in 200 ml of a 2N aqueous hydrochloric acid solution, and the mixture was heated under reflux for 4 hours. After the reaction was completed, the reaction solution was concentrated to about 50 ml, and 10
A normal sodium hydroxide aqueous solution was added to adjust the pH to 6, and the precipitate was collected by filtration to obtain 10.5 g of D-methionine.

[0055]

INDUSTRIAL APPLICABILITY According to the present invention, by utilizing an enzyme capable of preferentially hydrolyzing an L-type optically active substance in the presence of a weak base as a pH control reagent, the presence of a conventionally known strong base is improved. As compared with the reaction in (1), the by-product of N-substituted-D-amino acid due to chemical hydrolysis can be suppressed. Therefore, an optically active N-substituted-L-amino acid and / or an optically active N-amino acid useful as a synthetic intermediate for a phenethylamine derivative (dopamine precursor) and other various drugs having ideal properties as a therapeutic agent for renal hypertension and the like.
A substituted -D-amino acid ester, especially a compound in which the substituent on N is a lower alkanoyl group, and the amino acid is alanine, methionine, or the like, is treated with racemic N-substituted-DL-
It can be industrially advantageously produced from an amino acid ester in high yield.

Further according to the present invention, an optically active N-substituted-
The D-amino acid ester can be hydrolyzed only with an acid to give an optically active D-amino acid. That is, in the present invention, since the hydrolysis reaction can be carried out without using a base, racemization can be prevented, an optically active D-amino acid can be obtained in high purity and high yield, and operability can be improved. Is an improved industrially advantageous method for producing D-amino acids.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C12R 1:07) (C12P 13/12 C12R 1:66) (C12P 13/12 C12R 1: 125)

Claims (19)

[Claims] 1. A racemic N-substituted-DL-amino acid ester is preferentially hydrolyzed to the optically active L-form in the selected racemate to convert it to an optically active N-substituted-L-amino acid. After reacting an enzyme having the ability to react, a culture of a microorganism or a treated product thereof in the presence of a weak base as a pH control reagent, the remaining unreacted optically active N-substituted-D-amino acid ester is separated and collected. Or
A method for producing an optically active N-substituted-L-amino acid and / or an optically active N-substituted-D-amino acid ester, characterized in that the produced optically active N-substituted-L-amino acid is separated and collected from a reaction solution. 2. A racemic N-substituted-DL-amino acid ester is preferentially hydrolyzed with an L-type optically active substance in the selected racemate, and then produced.
The method according to claim 1, wherein the L-amino acid is separated and collected from the reaction solution. 3. The racemic N-substituted-DL-amino acid ester is preferentially hydrolyzed with the L-type optically active substance in the selected racemate, and then remains unreacted optically active N.
The method according to claim 1, wherein the -substituted-D-amino acid ester is separated and collected. 4. The method according to claim 1, wherein the enzyme is a protease, a lipase or an esterase, or the culture of a microorganism or a treated product thereof contains a protease, a lipase or an esterase. 5. Among racemic N-substituted-DL-amino acid esters, the L-type optically active substance in the selected racemate is preferentially hydrolyzed to be converted into an optically active N-substituted-L-amino acid. The method according to claim 1 or 4, wherein the microorganism capable of performing is a microorganism belonging to the genus Aspergillus, the genus Rhizopus, the genus Bacillus, or the genus Humicola. 6. Among racemic N-substituted-DL-amino acid esters, the L-type optically active substance in the selected racemate is preferentially hydrolyzed to be converted into an optically active N-substituted-L-amino acid. Microorganisms that have the ability to function include Aspergius niger, Rhizopus nibius, Bacillus species, Humicola lanuginosa, Aspergillus oryzae,
Bacillus subtilis, Aspergillus mereus, Rhizopus japonicas, Rhizopus derema or Bacillus
The method according to claim 1, 4 or 5, which is Licheniformis. 7. The racemic N-substituted-DL-amino acid ester is preferentially hydrolyzed to the optically active L-form in the selected racemate to convert it to an optically active N-substituted-L-amino acid. The method according to claim 1 or 4, wherein an enzyme having the ability to do so is used. 8. A racemic N-substituted-DL-amino acid ester is preferentially hydrolyzed with the L-type optically active substance in the selected racemate to be converted into an optically active N-substituted-L-amino acid. 2. The method according to claim 1, wherein the reaction of reacting the enzyme having the ability to act, the culture of the microorganism, or the treated product thereof is performed under neutral conditions. 9. The method according to claim 1, wherein the pH control reagent is an alkali metal carbonate, an alkali metal bicarbonate, an alkali metal phosphate, an alkali metal citrate or ammonia. 10. The method according to claim 1, 8 or 9, which is carried out at pH 6-8. 11. A racemic N-acetyl-DL-methionine alkyl ester is treated with a protease in the presence of a weak base, and then N-acetyl-L-methionine is separated and collected from the reaction solution. A method for producing N-acetyl-L-methionine. 12. The method of claim 1, 2, 3, 4, 5, 6, 7,
Optically active N-substituted-D-amino acid ester or optically active N- which is obtained by the production method described in 8, 9, 10 or 11.
The substituted -L-amino acid can be prepared according to a known method by the general formula [I] (In the formula, R represents a group obtained by removing a hydroxyl group from a carboxyl group of an N-substituted-amino acid.), And an optically active phenethylamine derivative, and if desired, a product thereof is a pharmacologically acceptable salt thereof. A process for producing the optically active phenethylamine derivative or a pharmacologically acceptable salt thereof. 13. The method according to claim 1, 3, 4, 5, 6, 7, 8,
A method for producing a D-amino acid, which comprises converting the optically active N-substituted-D-amino acid ester obtained by the method according to 9 or 10 into an optically active D-amino acid according to a known method. 14. The substituent on N is an acyl group, and the amino acid is an α-amino acid, 1, 2, 3, 4, 5,
The production method according to 6, 7, 8, 9, 10, 12 or 13. 15. The substituent on N is a lower alkanoyl group, the amino acid is alanine or methionine, and the ester is a lower alkyl ester.
The method according to 4, 5, 6, 7, 8, 9, 10, 12, 13 or 14. 16. A method for producing an optically active D-amino acid, which comprises treating the optically active N-substituted-D-amino acid ester with an acid. 17. The method according to claim 16, wherein the substituent on N is an acyl group, and the amino acid is an α-amino acid. 18. The substituent on N is a lower alkanoyl group, the amino acid is alanine or methionine, and the ester is a lower alkyl ester.
The manufacturing method described. 19. The method according to claim 16, 17 or 18, wherein the substituent on N is an acetyl group, the amino acid is methionine, and the ester is a methyl ester.