JP3160879B2 - Preparation of optically active amino acid derivatives - Google Patents

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-amino acid 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 properties as a therapeutic agent for renal hypertension and the like. Compounds useful as synthetic intermediates for phenethylamine derivatives (dopamine precursors) and other various pharmaceuticals (JP-B-57-4810)
No. 3).

Hitherto, 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 used in 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)
Acetylation of L-methionine with acetic anhydride, acetyl chloride, etc. (Journal of Biol)
Official Chemistry, Vol. 98,3
00 (1932)), and a method of splitting N-acetyl-DL-methionine using the action of D-aminoacylase in a biochemical method (U.S. Pat.
No. 06162) and a method of separating an N-acetyl-DL-methionine alkyl ester by selectively asymmetrically hydrolyzing the L-isomer in the presence of a hydrolase such as protease, lipase, esterase or the like (Japanese Patent Publication No. Sho 58). -491
No. 60).

[0004]

SUMMARY OF THE INVENTION Racemic N-substituted-D
Among the L-amino acid esters, for example, a method of obtaining an optically active N-acetyl-L-methionine by asymmetrically hydrolyzing a racemic N-acetyl-DL-methionine alkyl ester requires that DL-methionine is very inexpensive and available. It is a more useful method than being easy. However, in this hydrolysis reaction, when the substrate concentration is as low as several millimoles to several tens of millimoles, the pH does not decrease due to the action of the buffer solution, but when the substrate concentration is several mols that can withstand the production, the generated carboxylic acid is not used. Under the influence of the acid, the pH decreases with the reaction. Due to this decrease in pH, the reaction rate is significantly reduced, and the reaction efficiency is reduced. Normal pH for efficient reaction
Control is performed. For example, Japanese Patent Publication No. 58-4
In the method described in No. 9160, sodium hydroxide is used as a pH control reagent. However, under these reaction conditions, the N-acetyl-D-methionine alkyl ester is chemically hydrolyzed by sodium hydroxide and N-acetyl-D-methionine is by-produced in the reaction-finished solution. There was a problem that the yield of N-acetyl-L-methionine was low and the optical purity was 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 for
The present inventors have found that -substituted-D-amino acid esters can be obtained efficiently, and have completed the present invention.

That is, the present invention relates to a racemic N-substituted-D
The L-amino acid ester is preferentially hydrolyzed to the optically active L-form in the selected racemic form to give an optically active N-
An enzyme having the ability to convert to a substituted-L-amino acid, a culture of a microorganism or a treated product thereof is allowed to act as a pH control reagent in the presence of a weak base, and the remaining unreacted optically active N-substituted-D -Separating and collecting the amino acid ester, or the optically active N-substituted-L-
An optically active N-substituted-L-amino acid and / or an optically active N
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 present reaction system. , For example, an acyl group. The amino acid may be any compound having an amino group and a carboxyl group in the same molecule, and includes α-amino acids.

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

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

Of these, 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 are preferred.
Preferably, the above substituent is an acetyl group and the amino acid is methionine.

The weak base used as the pH control reagent is a weak base which does not chemically hydrolyze the racemic N-substituted-DL-amino acid ester and cannot produce N-substituted-D-amino acid as a by-product. I just need.

Examples of preferred weak bases include alkali metal carbonates (eg, sodium carbonate, potassium carbonate, etc.),
Alkaline earth metal carbonate (eg, magnesium carbonate, calcium carbonate, etc.), alkali metal bicarbonate (eg, sodium bicarbonate, potassium bicarbonate, etc.), alkali metal acetate (eg, sodium acetate, potassium acetate, etc.), alkali citrate Metal (eg, sodium citrate, etc.), alkaline earth metal citrate (eg, magnesium citrate, calcium citrate, etc.), alkaline earth metal phosphate (eg, magnesium phosphate, calcium phosphate, etc.), alkali metal hydrogen phosphate (eg, For example, disodium hydrogen phosphate,
Dipotassium hydrogen phosphate, etc.), alkali metal phosphate (for example,
Monopotassium phosphate, monosodium phosphate) or ammonia. Of these, alkali metal carbonate, alkali metal bicarbonate, alkali metal hydrogen phosphate, alkali metal citrate 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. Even if these enzymes are derived from microorganisms, even from animal cells,
It may be derived from plant cells. In addition, it may be extracted by a known method from microbial cells, animal cells or plant cells containing these hydrolases, or may be commercially available. Specifically, for example, lipase-A [Aspergillus niger (Aspergill)
us niger), manufactured by Amano Pharmaceutical Co., Ltd.], Lipase-N
[Rhizopus niveu
s), Prono Leather [from Bacillus sp., Amano Pharmaceutical], Lipase-CE [Fumicola Ranuginosa (Hum)
icola lanuginosa), Amano Pharmaceutical], Newase [Rhizops Nibius (Rhizo)
pus niveus, manufactured by Amano Pharmaceutical Co., Ltd.], Papain W-40 [Carica papaya
a), Amano Pharmaceutical], Pancreatin [hog pancreas, Amano Pharmaceutical], Protease-A (Aspergillus oryzae (A)
spergillus oryzae), manufactured by Amano Pharmaceutical], Protease-N [Bacillus subtilis (Ba)
C. subtilis), manufactured by Amano Pharmaceuticals],
Protease-P [Aspergillus mereus (Aspe
rgillus melleus, manufactured by Amano Pharmaceutical], Protease-M [Aspergillus oryzae (A
spergillus oryzae), manufactured by Amano Pharmaceutical Co., Ltd.], Lipase A-5 [Rhizopus japonicas (Rhi
zopus japonicus, Nagase Seikagaku Kogyo], XP-415 [Rhizopus delemar (Rhi
zopus delemer), manufactured by Nagase Seikagaku Kogyo], Denazyme-AP [Aspergillus oryzae (A
spergillus oryzae), manufactured by Nagase Seikagaku Corporation], Esperase-8.0SL [Bacillus
Species (Bacillus sp.), Manufactured by Novo Nordics Co., Ltd.), Neutrase-0.5 L [Bacillus subtilis (Bacillus subtilis)
is), from Novo Nordics, Inc.], Savinase-
8.0L [Bacillus species
sp. ), From Novo Nordics, Inc.), Alcalase-2.5 L [Bacillus licheniformis (Bac
illus licheniformis), lipase type II [porcine pancreas (Porcine pancreas)
s), lipase [from Porcine pancreas, from Wako Pure Chemical Industries], lipase [from Porcine pancreas (P)
orcine pancreas, Katayama Chemical Co., Ltd.], lipase [Rhizopus sp., Katayama Chemical Co., Ltd.] or Newase [Rhizopus niveus, Amano Pharmaceutical Co.] , Protease-P, Protease-M, Bioprase AL-1
5, Savinase-8.0 L or alcamyl-1.5 / 5
0L type B is preferred.

The microorganism in the present invention is preferably a racemic N-substituted-DL-amino acid ester as described above, which is obtained by preferentially hydrolyzing the optically active L-form in the selected racemic form. As long as it has the ability to convert to a substituted-L-amino acid, Aspergillus (Asp.
ergillus, Rhizopus
Genus, Bacillus genus or Humicola (H
microorganisms belonging to the genus Umicola).

As a specific example of such a microorganism, Aspergillus niger (Aspergillus niger)
r), Rhizopus niv
eus), Bacillus species
sp. ), Humicola Ranuginosa (Humicola)
lanuginosa), Carica Papaya (Cari)
ca papaya), Aspergillus oryzae (As
pergillus oryzae, Bacillus subtilis, Aspergillus meleus (Aspergillus mele)
lleus), Rhizopus Yaponicas (Rhizopu)
japonicus), Rhizopus delemar (Rh)
izopus delemer or Bacillus lichenifor
mis)).

These microorganisms may be any strains as long as they have the necessary ability for the present invention, and may be mutants obtained by artificial treatment such as ultraviolet irradiation or treatment with a mutagen. , Recombinant DNA from these microorganisms
It may be induced by genetic engineering or biotechnological techniques such as cell fusion and cell fusion. For example,
According to genetic engineering techniques, from the chromosome fragment of the microorganism producing the hydrolase, the gene of the target enzyme is isolated,
After preparing a recombinant plasmid into which this has been inserted into an appropriate plasmid vector, by transforming an appropriate host microorganism with this recombinant plasmid, a microorganism having an enzyme-producing ability or a microorganism having further improved enzyme-producing ability can be obtained. Obtainable. The recombinant plasmid may be used to transform a host microorganism having other excellent properties (for example, easy cultivation), if necessary.

The culture in the present invention includes, for example, a culture solution or viable cells of the microorganism, and the processed product of the culture includes, for example, a processed product of the culture solution (culture supernatant, etc.) or A treated bacterial cell can be used.

The culture of the microorganism or the processed product thereof may be any as long as it contains a group of enzymes called protease, lipase or esterase.

The above culture (culture solution, cells, etc.) can be prepared, for example, by subjecting the microorganism to a medium usually used in this field, such as a conventional medium containing a carbon source, a nitrogen source and inorganic salts, at normal temperature or under heating. (Preferably about 20-40
C) under aerobic conditions at pH 4 to 9, and if necessary, the cells can be obtained by separating and collecting cells from the thus obtained culture solution by a conventional method.

As a treated product of microorganisms, the above-mentioned culture is subjected to various physicochemical methods, for example, ultrasonic wave, French press, osmotic pressure, freeze-thawing, freeze-drying, alumina destruction, lytic enzyme, surfactant or In addition to the cells treated with an organic solvent or the like, a partial purification prepared by a known method from the 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 and convert it into an optically active N-substituted-L-amino acid among racemic N-substituted-DL-amino acid esters. No.

As such a partially purified enzyme or a purified enzyme, those commercially available as described above can be used.

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

Further, the enzyme, microbial cells or treated cells of the present invention may be immobilized by, for example, a polyacrylamide method, a sulfur-containing polysaccharide gel method (such as a carrageenan gel method), an alginic acid gel method or an agar gel method. Can also be used.

[0025]

BEST MODE FOR CARRYING OUT THE INVENTION The asymmetric hydrolysis according to the present invention is carried out by contacting a racemic N-substituted-DL-amino acid ester with a culture of an enzyme or 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 kind of the ester and the elimination method, but is preferably from about 0 to 70 ° C, more preferably from about 0 to 50 ° C, at room temperature or under heating. The process proceeds favorably. The reaction is carried out by adding a weak base as a pH control reagent so as not to adversely affect the optically active N-substituted-D-amino acid ester and the enzyme used in the hydrolysis reaction, the culture of microorganisms 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 pH control reagent may be used as a solid or as an aqueous solution. The aqueous solution may be 1 to 80%, particularly 10 to 50%.
% Is preferable.

Racemic N-substituted-DL- serving as a reaction substrate
The charged concentration (w / v) of the amino acid ester is 10 to 4
0% is preferred. When the raw material compound is hardly soluble, it can be used by suspending it in a solvent. Further, 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. The reaction can be carried out in an aqueous solution, or in a two-phase system of an aqueous solution and an organic solvent. As such an organic solvent, any organic solvent that dissolves a racemic N-substituted-DL-amino acid ester as a reaction substrate and is immiscible with water can be used. Such organic solvents include, for example, toluene, xylene, carbon tetrachloride, benzene, trichloroethane, chloroform, n-
Hexane, n-heptane, n-octane, cyclohexane, isooctane, ethyl acetate, butyl acetate, diethyl ether, t-butyl methyl ether, isopropyl ether and the like, among which toluene, carbon tetrachloride,
Benzene, trichloroethane, n-hexane, t-butyl methyl ether and the like are preferred.

The racemic N-substituted starting compound
As the DL-amino acid ester, not only those containing equal amounts of D-type and L-type optically active substances, but also those containing both of these optically active substances can be used.

In the case where viable cells are used in the present invention, the reaction time can be reduced by adding a surfactant to the reaction solution. Examples of the surfactant used for this purpose include cetylpyridinium bromide, cetyltrimethylammonium bromide, and 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%.

The 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 according to a conventional method. That is, for example, when the hydrolysis reaction is performed 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-
The substituted-L-amino acid is acidified with hydrochloric acid or the like, and salting is further performed. The salt to be added may be any salt as long as it is dissolved in water, but ammonium sulfate, sodium chloride and the like are 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 aqueous solution subjected to the salting-out, and extraction is performed. Then, the optically active N-substituted-L-amino acid having a high yield and a high optical purity can be obtained without recrystallization by concentration crystallization and centrifugation. Can be released. The solvent used for the extraction may be any solvent that separates water and two phases and dissolves the N-substituted-L-amino acid, and preferably includes ethyl acetate, chloroform and the like.

The test for determining whether an enzyme, a culture of a microorganism or a processed product thereof has the ability to asymmetrically degrade the L-type optically active form of a racemic N-substituted-DL-amino acid ester is carried out by the above-mentioned test. According to the reaction method, for example, it can be easily carried out as follows. That is, racemic N-acetyl-D
To an aqueous solution containing L-methionine methyl ester, a culture of an enzyme or a microorganism to be assayed or a processed product thereof is added.
The mixture was allowed to stand at 0 ° C. for 4 hours, and the reaction-terminated liquid was applied to an optically active column (for example, SUMICHIRAL manufactured by Sumika Chemical Analysis Center)
OA-5000) is analyzed and quantified by high performance liquid chromatography using N-acetyl-L-methionine and has the ability to selectively hydrolyze the ester of L-type optically active substance when the optical purity of the produced N-acetyl-L-methionine is high. Is determined.

The thus obtained optically active N-substituted-L-
Amino acids and / or optically active N-substituted-D-amino acid esters can be prepared by a known method, for example, a method disclosed in JP-B-57-48103 or JP-B-7-88553 according to 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 to an optically active phenethylamine derivative represented by the formula:
Further, if desired, the product can be converted into its pharmaceutically acceptable salt.

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

On the other hand, optically active N-substituted-D-amino acid esters can be prepared by known methods, for example, Journal of
Organic Chemistry, Vol. 4
3,4593 (1978), hydrolyze in the presence of an aqueous solution of a strong base such as sodium hydroxide to produce an optically active N-substituted-D-amino acid, and then hydrolyze with an aqueous solution of a strong acid such as hydrochloric acid to obtain an optically active solution. It can be converted to D-amino acids.

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

However, the optically active N-substituted-D-amino acid ester can be hydrolyzed with an acid alone without using a base to obtain an optically active D-amino acid.

The acid used in the present hydrolysis includes hydrolyzing an ester of an optically active N-substituted-D-amino acid ester to generate an optically active N-substituted-D-amino acid.
An acid which synthesizes an optically active D-amino acid by hydrolyzing the above substituents and which cannot racemize an optically active N-substituted-D-amino acid ester as a substrate may be used. Examples of such an acid include 1 to 4 N hydrochloric acid, hydrobromic acid, sulfuric acid and the like. Among these, hydrochloric acid is preferable, and 2 N 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 140C. The reaction time may be monitored at any time with an optically active column, 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 starting compound is hardly soluble, it can be used by suspending it in a solvent.

Separation and 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 the reaction residue is set at about 0 to 40 ° C., an aqueous solution of an alkali metal 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. I do. By filtering the precipitate, an optically active D-amino acid can be obtained.

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

In this specification, "%" means "weight / volume (g / dl)". Further, in this example, the quantitative determination of the optically active substance was carried out by using SUMICHIR.
This 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 a stirring reaction was performed as it was. Sodium bicarbonate was gradually added during the enzymatic reaction to achieve a pH of 6.
Adjusted to 5-7.0. The progress of the reaction is 100μ
After extracting 1 l, diluting with 1.9 ml of 2 mM copper sulfate solution and extracting with 2.0 ml of chloroform, the remaining amount of N-acetyl-L-methionine methyl ester in the chloroform layer was determined by HPLC method (CHIRALCEL OD, 4. 6
mm x 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 a filter paper. The filtrate is chloroform 50
Washed 3 times with unreacted optically active N-acetyl-D
-The methionine methyl ester was extracted and removed. On the other hand, the aqueous layer was acidified with hydrochloric acid, and 60 g of ammonium sulfate was added.
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. A crystal of optically active N-acetyl-L-methionine having a yield of 45.4 was obtained.
%. The specific rotation and the optical purity of the obtained crystal were as follows. The enzyme used, N-acetyl-
The reaction time at which L-methionine methyl ester disappeared, the yield of crystal acquisition, and the amount of N-acetyl-D-methionine produced in the reaction completed solution are shown in Table 1 below.

Crystal acquisition amount: 16.9 g, chemical purity: 10
0%, optical purity: 100% e. e. Optical rotation: [α] ^D ₂₀ = -21.4 ° (C = 4, H ₂ O) The chloroform extract of unreacted optically active N-acetyl-D-methionine methyl ester was concentrated to obtain optical activity. 18. N-acetyl-D-methionine methyl ester
0 g was recovered.

Examples 2-3 Using 100% of enzyme,
-Acetyl-DL-methionine methyl ester was treated in the same manner as in Example 1, and N-acetyl-
The amount of D-methionine produced was examined. The enzyme used, N-
The reaction time at which acetyl-L-methionine methyl ester disappeared, the yield of crystal acquisition, and the amount of N-acetyl-D-methionine produced in the reaction completed solution are shown in Table 1 below.

Comparative Examples 2-3 (Control Experiments of Examples 2-3) Examples 2-3 were conducted using a 5N aqueous sodium hydroxide solution as a pH control reagent instead of sodium hydrogen carbonate.
And the amount of N-acetyl-D-methionine produced after completion of the reaction was examined. Reaction time when the enzyme used, N-acetyl-L-methionine methyl ester disappeared,
The yield of crystal acquisition and the amount of N-acetyl-D-methionine produced in the reaction mixture are shown in Table 1 below.

[0049]

[Table 1]

Examples 4 to 6 Using a pH control reagent (weak base) instead of sodium bicarbonate, 100 mg of enzyme, N-acetyl-D
The mixture 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 completion of the reaction was examined. PH control reagent used, N
Table 2 below shows the reaction time at which -acetyl-L-methionine methyl ester disappeared, the yield of crystal acquisition, and the amount of N-acetyl-D-methionine produced in the reaction-terminated liquid.

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

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 completion of the reaction was examined. Table 2 below shows the reaction time in which the enzyme used, N-acetyl-L-methionine methyl ester disappeared, the yield of crystal acquisition, and the amount of N-acetyl-D-methionine produced in the reaction solution.

[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 heated under reflux for 4 hours. After completion of the reaction, the reaction solution was concentrated to about 50 ml,
The pH was adjusted to 6 by adding a normal aqueous sodium hydroxide solution, and the precipitate was collected by filtration to obtain 10.5 g of D-methionine.

[0055]

According to the present invention, by utilizing an enzyme having the ability to preferentially hydrolyze an L-type optically active substance in the presence of a weak base as a pH control reagent, As compared with the reaction described in (1), by-products of N-substituted-D-amino acids due to chemical hydrolysis can be suppressed. Therefore, optically active N-substituted-L-amino acids and / or optically active N-amino acids useful as synthetic intermediates of phenethylamine derivatives (dopamine precursors) and other various pharmaceuticals having ideal properties as therapeutic agents for renal hypertension and the like.
Substituted-D-amino acid esters, especially compounds in which the substituent on N is a lower alkanoyl group and the amino acid is alanine or methionine, are racemic N-substituted-DL-
It can be produced industrially and advantageously from amino acid esters in high yield.

Further, according to the present invention, an optically active N-substituted
The D-amino acid ester can be hydrolyzed with only an acid to lead to an optically active D-amino acid. That is, in the present invention, the hydrolysis reaction can be carried out without using a base, so that 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 also an improved industrially advantageous process for producing D-amino acids.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI C12R 1:66) (C12P 13/04 C12R 1: 125) (56) References JP-A-6-22789 (JP, A) JP-A-9-287 (JP, A) JP-A-6-46885 (JP, A) JP-B-57-48103 (JP, B2) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12P 13 / 00-13/24 BIOSIS (DIALOG) WPI (DIALOG)

Claims (12)

(57) [Claims] 1. An optically active L-form in a selected racemic form is preferentially hydrolyzed to a racemic N-substituted-DL-amino acid ester to be converted to an optically active N-substituted-L-amino acid. Bacillus subtilis with the ability to
An enzyme derived from Chils or Aspergillus meleus ,
After allowing a culture of a microorganism or a processed product thereof to act as a pH control reagent in the presence of a weak base, the remaining unreacted optically active N-substituted-D-amino acid ester is separated.
The optically active N-substituted
A method for producing an optically active N-substituted-L-amino acid and / or an optically active N-substituted-D-amino acid ester, comprising separating and collecting an L-amino acid from a reaction solution. 2. The optically active N-substituted compound formed after preferentially hydrolyzing the L-type optically active substance in the selected racemic form into a racemic N-substituted-DL-amino acid ester.
The method according to claim 1, wherein the L-amino acid is separated and collected from the reaction solution. 3. An unreacted optically active N-reacted N-substituted DL-amino acid ester which remains after preferentially hydrolyzing the L-type optically active substance in the selected racemic form.
The method according to claim 1, wherein the -substituted-D-amino acid ester is separated and collected. 4. The enzyme is a protease , or
A method according to claim 1, wherein the culture or a treated product of a microorganism is intended to include protease. 5. The racemic N-substituted-DL-amino acid ester is preferentially hydrolyzed to an optically active L-form in the selected racemic form to be converted to an optically active N-substituted-L-amino acid. The method according to claim 1 or 4, wherein an enzyme having an ability to perform the reaction is used. 6. The racemic N-substituted-DL-amino acid ester is preferentially hydrolyzed to the optically active L-form in the selected racemic form to be converted to an optically active N-substituted-L-amino acid. Bacillus subtilis with the ability to
An enzyme derived from Chils or Aspergillus meleus ,
2. The process according to claim 1, wherein the reaction for causing the culture of the microorganism or the treated product to act is carried out under neutral conditions. 7. A pH control reagents are alkali metal carbonates, alkali metal bicarbonate, phosphates alkali metal, method according to claim 1 or 6, wherein an alkali metal or ammonium citrate. 8. The method according to claim 1, which is carried out at a pH of 6 to 8.
7. The manufacturing method according to 7 . 9. A racemic N-substituted-DL-amino acid residue.
Ter is racemic N-acetyl-DL-methionine alkyl
The ester according to any one of claims 1 to 8, which is an ester.
Manufacturing method. 10. A racemic N-substituted-DL-amino acid
Stele is racemic N-acetyl-DL-methionine meth
The ester according to any one of claims 1 to 8, which is an ester.
Manufacturing method. 11. The method according to claim 1, wherein the optically active N
-Substituted-L-amino acid is obtained, and the obtained optically active N-substituted-L-amino acid is converted to a compound of the general formula [I] according to a known method. (Wherein R represents a group obtained by removing a hydroxyl group from a carboxyl group of an N-substituted-amino acid). An optically active phenethylamine derivative represented by the formula: A process for producing a characteristic optically active phenethylamine derivative or a pharmaceutically acceptable salt thereof. 12. The optically active N according to the method of claim 1.
-Substituted-D-amino acid ester is obtained, and the obtained optically active N-substituted-D-amino acid ester is converted into an optically active D-amino acid according to a known method.
-Preparation of amino acids.