JPH04346795A - Production of l-3,4-dihydroxyphenylserine derivative - Google Patents

Abstract

PURPOSE:To provide a method for simply and efficiently obtaining a L-3-(3,4- dihydroxyphenyl)serine derivative from racemic 3-(3,4-dihydroxyphenyl)serine derivatives. CONSTITUTION:A method for producing a L-3-(3,4-dihydroxyphenyl)serine derivative is characterized by treating racemic 3-(3,4-dihydroxyphenylserine derivatives with an enzyme having an ability to specifically decompose a D-3-(3,4- dihydroxyphenyl)serine derivative into a protocatechuic aldehyde derivative corresponding to glycine, with the cell of a microorganism producing the enzyme, or with the treated product of the cell.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] Racemic-3-(3,4-dihydroxyphenyl)serine derivatives, D-3-(3,4-dihydroxyphenyl)serine derivatives, and glycine-corresponding protocatechualdehyde derivatives specifically. L, which is known to be useful as a therapeutic agent for Parkinson's disease and familial amyloid polyneuropathy, is treated with an enzyme capable of decomposing the enzyme, a microorganism that produces the same enzyme, or a processed product of the bacterial cell. -Threo-3
The present invention relates to a method for producing L-3-(3,4-dihydroxyphenyl)serine derivatives (hereinafter abbreviated as L-DOPS derivatives) including -(3,4-dihydroxyphenyl)serine.

0002

BACKGROUND OF THE INVENTION Conventionally, L-threo-DOPS derivatives have been synthesized by the following method. That is, a protocatechualdehyde derivative with a protected catechol moiety and glycine are condensed in a strong alkali, and then racemic
A threo/erythro-DOPS derivative is obtained, the product thus obtained is subjected to mutual separation treatment of threo/erythro forms, and then a protecting group on the catechol moiety is removed in order to optically resolve the racemic-threo-DOPS derivative. After removal, a substituent is introduced into the amino group, optical resolution is performed using an optical resolving agent such as quinine or brucine, and finally the substituent at the amino group is removed (Unexamined Japanese Patent Publication No. Showa 5
No. 8-216146).

0003

[Problem to be solved by the invention] L-Threo-DOP
In the synthesis of S derivatives, it is necessary to introduce or remove various specific protecting groups, and in addition, multiple stereoisomers are produced during the synthesis, and separation of these stereoisomers from each other is difficult. , has become a major issue. Since two or more stereoisomers are generated in the synthesis process, at least multiple steps are essential to separate these isomers, and steps for introducing and removing various protecting groups are also necessary. For this reason, it has become a major problem in industrial manufacturing.

Among these methods, the optical resolution method in particular has problems as an industrial production method because not only the process is complicated and the yield is poor, but also the optical resolution agent used therein is expensive. Furthermore, conventional optical resolution methods always require separation treatment between threo/erythro forms before racemic separation, and furthermore, unnecessary products obtained as a result of these methods, namely DL-erythro derivatives and D- In order to use the threo derivative again, it is necessary to separately racemize or decompose it, making the manufacturing equipment increasingly complex.

[0005]

[Means for Solving the Problems] The present inventors have conducted intensive studies on a method for obtaining L-DOPS derivatives, which are important intermediates in the synthesis of L-threo-DOPS and L-erythro-DOPS. The inventors completed the present invention by discovering that only L-DOPS derivatives can be specifically obtained by treating racemic DOPS derivatives with enzymes having specific substrate specificity. The present invention
general formula

[0006]

[Formula 2] (In the formula, R1 and R2 are the same or different and represent hydrogen, lower alkyl or aralkyl group, and both R1 and R2 may be substituted with an alkylene group to form a ring)
Ability to specifically decompose D-3-(3,4-dihydroxyphenyl)serine derivative into glycine and the corresponding protocatechualdehyde derivative into racemic-3-(3,4-dihydroxyphenyl)serine derivative represented by L-3-(3,4-
The present invention relates to a method for producing dihydroxyphenyl)serine derivatives.

Raw material racemic DOPS used in the present invention
Examples of derivatives include compounds having the above formula (I), and these compounds have R1 and R2 of the protecting groups.
It may be a mixture of compounds in which are the same or different. Representative examples of the compound represented by the above formula (I) include racemic-threo/erythro-3-(3,4-methylenedioxyphenyl)serine, racemic-threo/erythro-3-(3,4- dibenzyloxyphenyl)serine, racemic threo/erythro-3-(3,4-dihydroxyphenyl)serine, racemic threo/erythro-
3-(3,4-dimethyloxyphenyl)serine or a mixture thereof.

[0008] The raw material racemic DOPS derivative may be a mixture of its erythro/threo isomers, or
It may be a threo or erythro body alone. Erythro and threo bodies can be separated from each other by methods such as crystallization, although it is complicated, or products consisting of only one of them can be obtained as a by-product.
It is also preferable to use a raw material containing each of these isomers alone. In addition, conventionally, it has been difficult to easily and efficiently optically resolve racemic erythro forms, but according to the method of the present invention, not only L-erythro forms can be easily obtained, but also the obtained glycine and proto-erythro forms can be easily obtained. The catechualdehyde derivative can be used again in the synthesis.

[0009] In the present invention, not only isomers other than L-DOPS derivatives, that is, D-threo-DOPS derivatives and D-erythro-DOPS derivatives, but also these isomers are decomposed in correspondence with glycine. The glycine thus obtained and the corresponding protocatechualdehyde derivative can be used again for synthesis.

[0010] Enzymes used in the present invention that have the ability to specifically decompose D-threo and erythro-DOPS derivatives into glycine and the corresponding protocatechualdehyde derivatives include D-threonine aldolase and other enzymes. Examples include substances having substantially the same activity as enzymes, such as microorganisms themselves that produce these enzymes, or processed products of microbial cells.

In the present invention, the above-mentioned enzyme or its equivalent may be reacted before separating the erythro form and the threo form, or may be reacted after the separation. In the present invention, as described above, D-DO
An example of this is the use of an enzyme that decomposes PS derivatives, but any enzyme can be used as long as it specifically decomposes D-threo and/or erythro-DOPS derivatives. Particularly preferred such enzymes include those having the ability to specifically decompose D-threo/erythro-DOPS derivatives into glycine and the corresponding protocatechualdehyde derivatives. Particularly preferred are those that specifically recognize the difference in steric structure between the D-form and the L-form, but are not affected by the structural difference between the threo-form and the erythro-form. Furthermore, there are compounds that have specific activity regardless of the presence of various protective groups introduced into (dihydroxyphenyl)serine.

A particularly preferred example of such an enzyme is D-threonine aldolase. As such D-threonine aldolase,
In addition to those described in No. 1279, examples include those obtained by manipulating the gene (for example, Japanese Patent Application No. 1-276126).

Although these D-threonine aldolases unexpectedly have very high specificity for the D-configuration, they are tolerable for the erythro/threo configuration. Not only is it tolerant, it can act not only on (dihydroxyphenyl)serine but also its derivatives, and has the excellent property of producing glycine and the corresponding protocatechualdehyde.

D-threonine aldolase used in the present invention can be obtained from a D-threonine aldolase-producing bacterium or its mutant or variant strain. Examples of D-threonine aldolase-producing bacteria include:
Alcaligenes faecalis
s faecalis) (IFO 12669),
Pseudomonas DK-2
(Feikoken Bacillus No. 6200), Aslobacter (Arth
robacter) DK-19 (Feikoken Bacteria 6201
No.), Xanthomonas oryzae
nasoryzae) (IAM1657), etc.
3586) can also be used without being limited to these.

[0015] Furthermore, a recombinant prepared by introducing DNA carrying a gene involved in the synthesis of D-threonine aldolase using techniques such as genetic recombination can also be suitably used. Examples of microorganisms into which such genes can be introduced and utilized include Escherichia
Bacillus (richia), Bacillus
Bacteria of the genus Xanthomonas, bacteria of the genus Acetobacter, bacteria of the genus Pseudomonas, bacteria of the genus Gluconobacter, bacteria of the genus Azotobacter, bacteria of the genus Rhizobacter um) bacteria, Alcaligenes ( Bacteria of the genus Alcaligenes, bacteria of the genus Klebsiella, Salmonella (S
Bacteria of the genus almonella, Serratia
Prokaryotic microorganisms such as bacteria of the genus ia) and lower eukaryotes such as yeast can be used.

Among such recombinants, examples of recombinants having the ability to produce D-threonine aldolase to a high degree include those belonging to bacteria of the genus Xanthomonas. Xanthomonas citri (Xanthomonas citri), which has been deposited as Microtechnical Research Institute No. 10979.
nas citri) IFO3311 (pDT64
8) has the ability to highly produce D-threonine aldolase (Japanese Patent Application No. 1-276126) and is particularly suitable.

In particular, this Xanthomonas citri
Since IFO3311 (pDT648) specifically produces only D-threonine aldolase in large quantities,
D-threonine aldolase with high activity can be obtained by simple processing, and when used, unexpected results such as shortening unit reaction time, increasing yield, and simplifying equipment are possible. Excellent effects can be obtained.

The microorganisms used in the present invention can be cultured according to conventional methods. The medium used for culture is a normal medium containing carbon sources, nitrogen sources, inorganic substances, etc. necessary for the growth of microorganisms. Carbon sources used here include glucose, ribose, galactose, soluble starch, etc., and nitrogen sources include ammonium chloride, ammonium sulfate, ammonium nitrate, peptone, meat extract, yeast extract, corn steep liquor, soy flour, and cotton. Examples include fruit scraps. In addition to sodium chloride, potassium salts, calcium salts, ammonium salts, phosphates, and the like, inorganic substances such as zinc, magnesium, iron, and manganese can also be used. Additionally, the addition of organic micronutrients such as vitamins, amino acids, etc. often provides desirable results. Cultivation is carried out under aerobic conditions at pH 4 to 10 and temperature 20 to 60°C.
The culture may be carried out for 1 to 10 days while controlling the amount within an arbitrary range. When using a recombinant, antibiotics such as ampicillin and chloramphenicol may be added to the culture solution depending on the strain used.

In the enzymatic reaction, bacterial culture solution, bacterial cells isolated from the culture solution, dried bacterial cells by freeze-drying, etc.
A bacterial cell disruption solution, a crude enzyme extract, a purified enzyme, the above-mentioned processed products and immobilized products, and any of the others can be used.
When producing L-threo/erythro form from racemic threo/erythro form, or when producing L-erythro form from racemic erythro form, the L-allothreonine aldolase activity contained in the enzyme solution or bacterial cells used is It is necessary to remove it beforehand before using it in the reaction. L-
Allothreonine aldolase is an enzyme that specifically decomposes L-allothreonine into glycine and acetaldehyde, and also specifically acts on L-erythro-DOPS derivatives, decomposing them into protocatechualdehyde derivatives and glycine. 6 is a method for removing only L-allothreonine aldolase activity from an enzyme solution having D-threonine aldolase activity and L-allothreonine aldolase activity.
A method using heat treatment at 0°C for about 30 minutes or a method using acetone fractionation of an enzyme solution is preferred, but any method can be used as long as it can selectively remove L-allothreonine aldolase activity. I can do it. The method for carrying out the enzymatic reaction is to mix the racemic threo and/or erythro DOPS derivative as a substrate in an aqueous medium with the above-mentioned microorganism culture solution, microorganisms, microorganism-treated products, enzymes, or these usually. It is sufficient if it is brought into contact with something that has been immobilized using the method described above. As the aqueous medium for such a reaction, for example, water, a buffer solution, a water-containing organic solvent, etc. can be used.

[0020] The concentration of the enzyme, microbial cells, or processed bacterial cells in the reaction is determined by D-Threo/Erythro DOPS.
There is no particular restriction as long as the derivative can be completely decomposed, but D
-Threonine aldolase activity (1 U represents the amount of enzyme required to produce 1 μmole of glycine per minute from D-threonine) is preferably 0.01 U/ml or more. In particular, if the concentration is 0.1 U/ml or more, the reaction can be terminated quickly. The concentration of the racemic threo and/or erythro DOPS derivative as a substrate may be 1 mm or less as long as it does not significantly inhibit the reaction.
The amount is preferably from le/liter to 100 mmole/liter, and the substrate can be supplied in bulk, continuously, or in portions.

[0021] The reaction temperature is preferably 10 to 55°C, and 20 to 55°C.
to 40°C is more suitable. pH during reaction is 5 to 1
0, preferably 6 to 8. D-threonine aldolase requires pyridoxal-5'-phosphate as a coenzyme, so the reaction system contains 10 nmole/liter ~
10 mmole/liter, preferably 1 μmole/liter
The reaction is accelerated by addition at a concentration of liter to 100 μmole/liter.

[0022] Furthermore, since D-threonine aldolase requires divalent metal ions as complementary factors, Mg2+,
100 μm of Mn2+, Co2+, Fe2+, etc. in salt form
The reaction is accelerated by adding at a concentration of ole/liter to 100 mmole/liter, preferably 1 mmole/liter to 10 mmole/liter.

[0023] The reaction type may be either a batch type or a continuous type, but the reaction is thus completed in about 0.5 to 50 hours. After the reaction is completed, the reaction solution is sterilized and protein removed by centrifugation, ultrafiltration, etc., and the produced protocatechualdehyde derivative is first separated by organic solvent extraction. For extraction, any solvent can be used as long as the protocatechualdehyde derivative is highly soluble and the L-DOPS derivative and glycine are almost insoluble, but good results are obtained when ether is used.

The protocatechualdehyde derivative is recovered from the solvent phase by concentration under reduced pressure. L from aqueous phase
The -DOPS derivative and glycine can be isolated and recovered by chromatographic separation using a conventional ion exchange resin. That is, the aqueous phase is passed through a column packed with a cation exchange resin, washed with water, and then eluted with dilute ammonia water or the like.

If the purity is low, the degree of purification can be increased by further performing crystallization and water recrystallization. The method for isolating and purifying the reaction product described above is one representative method and is a preferred method, but the present invention is not limited thereto and various separation and purification methods can be applied. Examples of such methods include alcohol solvents such as methanol and ethanol, aromatic hydrocarbon solvents such as benzene and toluene, ester solvents such as ethyl acetate, ketone solvents such as acetone and methyl ethyl ketone, and dimethyl sulfoxide. Sulfoxide solvent, N,N-
Amide solvents such as dimethylformamide, nitrile solvents such as acetonitrile, ethyl ether, dioxane,
Separation methods such as solvent extraction using ether solvents such as tetrahydrofuran, halogenated hydrocarbon solvents such as methylene chloride, mixed solvents thereof, or aqueous solvents thereof, column chromatography, reversed phase chromatography, high performance liquid chromatography , recrystallization method, and the like.

According to the method of the present invention, (1) There is no need to introduce or remove substituents for optical resolution, there is no need to use an expensive optical resolution agent, and the yield is high. (2) Since the process can be greatly simplified, equipment costs for the device can be greatly reduced. (3) Both D-threo and/or erythro-DOPS derivatives are decomposed into glycine and protocatechualdehyde derivatives, which are raw materials for synthesis, so they are recovered from the reaction solution again to synthesize racemic-threo/erythro-DOPS derivatives. It can be recycled and used, greatly reducing production costs. In particular, even when the treatment of the present invention is performed using a derivative with a protected catechol moiety as a substrate, the treatment of the present invention can be carried out satisfactorily, and the recovered protocatechualdehyde derivative has a protective group. remains as it is, so it can be used as a raw material for synthesis without performing the step of introducing a protecting group again. (4) The yield of the target product from raw materials can be greatly increased, and the target product of high purity can be easily obtained.

[0027]

EXAMPLES Next, the method of the present invention will be explained in more detail with reference to Examples, but the present invention is not limited by the Examples. In the examples, % indicates weight %.

Oxygen Production Example 1 A medium with a pH of 7.5 consisting of 0.5% polypeptone, 0.5% yeast extract, and 0.5% sodium chloride was prepared.
Add 3 liters of it to a liter culture tank and heat it to 120°C.
It was heat sterilized for 15 minutes. Arthroba in the medium
cter DK-19 (Feikoken Bokuyori No. 6201) was inoculated and cultured at 30° C. for 20 hours with aeration and stirring while maintaining the pH at 7.5. After culturing, the cells were collected from the culture solution by centrifugation, washed with water, and added to 0.2M HEPES (N-2), pH 7.5, containing 0.01mM pyridoxal-5'-phosphate and 1.0mM magnesium chloride. -H
ydroxyethylpiperazine-N'-
2-ethanesulfonic acid) buffer solution, and this bacterial cell suspension was heated at 20kHz,
Ultrasonic disruption treatment for 3 minutes was performed four times, and suspended matter in the disruption solution was removed by centrifugation to prepare a crude enzyme solution. Next, cold acetone was gradually added to this crude enzyme solution while stirring, and the precipitate in the acetone concentration fraction of 45 to 65% was collected and dissolved in 10 ml of the above buffer solution to prepare an acetone fractionated enzyme solution. . The D-threonine aldolase activity (1 U represents the amount of enzyme required to produce 1 μmole of glycine per minute from D-threonine) of the obtained bacterial cell suspension and acetone fractionated enzyme solution was measured. Bacterial cell suspension: 0.42 U/ml Acetone fractionated enzyme solution: 3.5 U/ml

Oxygen Production Example 2 In the same manner as in Enzyme Production Example 1, Pseudomonas
DK-2 (Feikoken Bokuyori No. 6200), Alcali
genes faecalis (IFO12669)
, Xanthomonas oryzae (IAM1
657), and Xanthomonas citri
A cell suspension of IFO3311 (pDT648) and an acetone fractionated enzyme solution were prepared. The D-threonine aldolase activity of the obtained bacterial cell suspension and acetone fractionated enzyme solution was measured.

[0030]

[Table 1]

Synthesis Example 1 Synthesis of racemic threo-3-(3,4-methylenedioxyphenyl)serine 48.8 g of potassium hydroxide and 26.4 g of glycine were dissolved in 844 ml of methanol and heated at 30°C.
After adding 116 g of piperonal, the mixture was stirred at 65° C. for 30 minutes, and then concentrated under reduced pressure. Pour the residue into 108ml of methanol.
Add 64.8g of acetic acid at 30°C or below, and heat at 45°C.
The mixture was stirred for 1 hour. 1000ml of toluene and 108ml of water
After stirring at 45°C for 30 minutes and further stirring at 5°C for 2 hours, the precipitated crystals were collected by filtration and dried.
．． 9 g of crystals were obtained. Add 1000 ml of water to the crystals, stir and dissolve at 80°C under reflux, filter, and remove the mother liquor at 5°C.
Recrystallization from water was performed by stirring for 2 hours. The filtered crystals were dried to obtain 42.3 g of racemic threo-3-(3,4-methylenedioxyphenyl)serine. Also, o-phthalaldehyde and N-acetyl-L-
After derivatization with cysteine, H
The ratio of each isomer was determined by isomer analysis using PLC.

Synthesis Example 2 Synthesis of Racemic-Threo-DOPS 2 g of racemic-threo-3-(3,4-methylenedioxyphenyl)serine obtained in Synthesis Example 1, 20 ml of dichloromethane, and 2 ml of ethyl mercaptan were mixed, and anhydrous 10 g of aluminum bromide was added while stirring under ice cooling, and the mixture was stirred for 3 hours under ice cooling and further at room temperature for 15 hours. 200 ml of 1N hydrochloric acid was added to this reaction solution, mixed and separated, the pH of the aqueous layer was adjusted to about 5 with sodium hydroxide, and the precipitated crystals were collected by filtration. The crystals were recrystallized from 40 ml of water containing 0.05% L-ascorbic acid, and the obtained crystals were dried to obtain 1.5 g of racemic-threo-DOPS.

Example 1 100 mg of D,L-threo-DOPS obtained in Synthesis Example 2 was added to 100 ml of the acetone fractionated enzyme solution obtained in Enzyme Production Example 1, and the mixture was reacted at 30° C. for 10 hours. After the reaction was completed, 10 ml of 1N hydrochloric acid was added, and insoluble matter was removed by centrifugation. When the remaining substrate and produced glycine were quantified by HPLC (Shimadzu amino acid analysis system), the substrate was 50.8% at the time of addition.
% remaining, glycine was produced at a molar concentration equal to the reduction in substrate. In addition, protocatechualdehyde is 2,4
- Reaction with dinitrophenylhydrazine and HPLC analysis using an ODS column revealed that the product was produced at a molar concentration equivalent to the decrease in the substrate. Add 200% to the above centrifuged supernatant.
ml of ethyl ether was added, stirred and allowed to stand, and the ether phase was separated and dried under reduced pressure to obtain 31 mg of protocatechualdehyde. The aqueous phase was replaced with strongly acidic cation exchange resin D.
Column packed with OWEX 50W x 8 (10mmφ
After thorough washing with deionized water, the adsorbed substances were eluted with 0.5N ammonia water, and the DO
The PS fraction and glycine fraction were collected separately. When both fractions were dried under reduced pressure, the amounts were 49 mg and 16 mg, respectively.
mg of crystals were obtained. The crystals obtained from the DOPS fraction are
The following analysis confirmed that it was L-threo-DOPS. mp: 208℃ Elemental analysis: Calculated value for C9 H11O5 N: C50
．． 71, H5.20, N6.57% Actual value: C50.9
7, H5.15, N6.46% Isomer analysis (after derivatization with o-phthalaldehyde and N-acetyl-L-cysteine, HPLC analysis using an ODS column): The peak is L-
It was seen only in the threo position. Specific optical rotation: [α] 20D = -39.9° (C = 0
．． 5,0.1N HCl)

Example 2 Using the acetone fractionated enzyme solution obtained in Enzyme Production Example 2, a reaction was carried out in the same manner as in Example 1, and the remaining substrate in the reaction solution was quantified. The results are shown below.

[0035]

[Table 2]

After the reaction was completed, the reaction solution was treated in the same manner as in Example 1, and the specific optical rotation was measured, and it was confirmed that all of the obtained DOPS were L-threo forms.

Example 3 The reaction was carried out in the same manner as in Example 1, except that the bacterial cell suspension obtained in Enzyme Production Examples 1 and 2 was used and the reaction time was 30 hours. The results of mass determination are shown.

[0038]

[Table 3]

After the reaction was completed, the reaction solution was treated in the same manner as in Example 1, and the specific optical rotation was measured, and it was confirmed that all of the obtained DOPS were L-threo forms.

Example 4 A derivative in which R1 and R2 in general formula (I) are both a methyl group or a benzyl group, and a derivative in which R1 and R2 are substituted with a methylene group to form a five-membered ring are used as substrates, Example 1 An acetone fractionated enzyme solution of the bacterial strains shown in the table below was used.
The results of the reaction were carried out in the same manner as above, and the remaining substrate in the reaction solution was quantified.

[0041]

[Table 4]

After the reaction was completed, the reaction solution was treated in the same manner as in Example 1, and the specific optical rotation was measured, and it was confirmed that all of the obtained DOPS derivatives were L-threo derivatives.

Synthesis Example 3 Synthesis of racemic erythro-3-(3,4-methylenedioxyphenyl)serine 48.8 g of potassium hydroxide and 26.4 g of glycine were dissolved in 844 ml of methanol.
116 g of piperonal was added below .degree. C., stirred at 65.degree. C. for 30 minutes, and then concentrated under reduced pressure. 108m of methanol from the residue
Add 64.8 g of acetic acid at 30°C or below,
The mixture was stirred at ℃ for 1 hour. 1000ml of toluene and 108ml of water
After stirring at 45°C for 30 minutes and further stirring at 5°C for 2 hours, the mother liquor excluding the precipitated crystals was further stirred at room temperature for 20 hours, and the precipitated crystals were filtered and dried. .28 g of crystals were obtained. 50m to this crystal
1 of water was added, stirred and dissolved at 80°C under reflux, filtered, and the mother liquor was stirred at 5°C for 2 hours to perform recrystallization from water. The filtered crystals were dried to obtain 2.9 g of racemic erythro-3-(3,4-methylenedioxyphenyl)serine. Also, o-phthalaldehyde and N-acetyl-L-
After derivatization with cysteine, H
The ratio of each isomer was determined by isomer analysis using PLC.

Synthesis Example 4 Synthesis of racemic-erythro-DOPS Racemic-erythro-3-(3,4-
Mix 2 g of (methylenedioxyphenyl)serine, 20 ml of dichloromethane, and 2 ml of ethyl mercaptan, and add 10 g of anhydrous aluminum bromide while stirring under ice cooling.
The mixture was stirred for 3 hours under ice cooling and further stirred for 15 hours at room temperature. 200 ml of 1N hydrochloric acid was added to this reaction solution, mixed and separated, the pH of the aqueous layer was adjusted to about 5 with sodium hydroxide, and the precipitated crystals were collected by filtration. The crystals were recrystallized from 40 ml of water containing 0.05% L-ascorbic acid, and the obtained crystals were dried to obtain 1.3 g of racemic erythro-DOPS.

Example 5 100 mg of D,L-erythro-DOPS obtained in Synthesis Example 4 was added to 100 ml of the acetone fractionated enzyme solution obtained in Enzyme Production Example 1, and the mixture was reacted at 30° C. for 10 hours. After the reaction is completed, 1N
After adding 10 ml of hydrochloric acid, insoluble matter was removed by centrifugation. When the remaining substrate and produced glycine were quantified using HPLC (Shimadzu Amino Acid Analysis System), the substrate was 48.
With 2% remaining, glycine was produced at a molar concentration equal to the reduction in substrate. In addition, protocatechualdehyde is 2,
When reacted with 4-dinitrophenylhydrazine and analyzed by HPLC using an ODS column, it was found that the product was produced at a molar concentration equivalent to the decrease in the substrate. Add 20% to the above centrifuged supernatant.
0 ml of ethyl ether was added, stirred and allowed to stand, and the ether phase was separated and dried under reduced pressure to obtain 30 mg of protocatechualdehyde. The aqueous phase was transferred to a column (10 mm
After thorough washing with deionized water, the adsorbed substance was eluted with 0.5N ammonia water, and D
The OPS fraction and glycine fraction were collected separately. When both fractions were dried under reduced pressure, 47 mg and 1
7 mg of crystals were obtained. The crystals obtained from the DOPS fraction were confirmed to be L-erythro-DOPS by the following analysis. Isomer analysis (after derivatization with o-phthalaldehyde and N-acetyl-L-cysteine, H
PLC analysis): A peak was observed only at the position of L-erythro form. Specific optical rotation: [α] 20D = +51.3° (C = 0
．． 5,0.1N HCl)

Example 6 Using the acetone fractionated enzyme solution obtained in Enzyme Production Example 2, a reaction was carried out in the same manner as in Example 5, and the remaining substrate in the reaction solution was quantified. The results are shown below.

[0047]

[Table 5]

After the reaction was completed, the reaction solution was treated in the same manner as in Example 5, and the specific optical rotation was measured, and it was confirmed that all of the DOPS obtained were L-erythro forms.

Example 7 A derivative in which R1 and R2 in general formula (I) are both a methyl group or a benzyl group, and a derivative in which R1 and R2 are substituted with a methylene group to form a five-membered ring are used as substrates, The acetone-fractionated enzyme solutions of the strains shown in the table below were reacted in the same manner as in Example 5, and the residual substrate in the reaction solution was quantified. The results are shown below.

[0050]

[Table 6]

After the reaction was completed, the reaction solution was treated in the same manner as in Example 5, and the specific optical rotation was measured, and it was confirmed that all of the obtained DOPS derivatives were L-erythro derivatives.

Enzyme Production Example 3 The crude enzyme solutions obtained in Enzyme Production Examples 1 and 2 were each treated at 60°C for 30 minutes, and then centrifuged, and the resulting supernatant was used as a heat-treated enzyme solution.

D-threonine aldolase activity and L-allothreonine aldolase activity of crude enzyme solution and heat-treated enzyme solution (1U is 1 μm from L-allothreonine per minute)
ole (representing the amount of enzyme required to produce glycine) was measured.

[0054]

[Table 7]

[0055]

[Table 8]

Example 8 Arthrobacter DK obtained in Enzyme Production Example 3
-100 mg of D,L-threo/erythro-DOPS (mixture of equal amounts of each isomer) in 100 ml of heat-treated enzyme solution of No. 19
was added and reacted at 30°C for 30 hours. After the reaction is complete, 1
After adding 10 ml of N-hydrochloric acid, insoluble matter was removed by centrifugation. When the remaining substrate and produced glycine were quantified by HPLC (Shimadzu amino acid analysis system), the substrate was 48% at the time of addition.
．． With 3% remaining, glycine was produced at a molar concentration equal to the reduction in substrate. In addition, protocatechualdehyde is 2
,4-dinitrophenylhydrazine, ODS
HPLC analysis using a column revealed that the product was produced at a molar concentration equivalent to the decrease in substrate. Add 2 to the above centrifuged supernatant.
00 ml of ethyl ether was added, stirred and allowed to stand, and the ether phase was separated and dried under reduced pressure to obtain 28 mg of protocatechualdehyde. The aqueous phase was transferred to a column (10 m
After thoroughly washing with deionized water, the adsorbed substance was eluted with 0.5N ammonia water.
The DOPS fraction and glycine fraction were collected separately. When both fractions were dried under reduced pressure, each yielded 48 mg,
18 mg of crystals were obtained. The crystals obtained from the DOPS fraction were confirmed to be a mixture of L-threo-DOPS and L-erythro-DOPS by the following analysis. Elemental analysis: C9 H11O5 Calculated value for N: C50
．． 71, H5.20, N6.57% Actual value: C50.8
3, H5.18, N6.49% Ratio of each isomer by isomer analysis (after derivatization with o-phthalaldehyde and N-acetyl-L-cysteine, HPLC analysis with ODS column): L-threo form: D -Threo body: L-Erythro body: D-Erythro body = 49:0:51:0

Example 9 Using the heat-treated enzyme solution (A) obtained in Enzyme Production Example 3 or the acetone fractionated enzyme solution (B) obtained in Enzyme Production Examples 1 and 2, a reaction was carried out in the same manner as in Example 8. The results of quantifying the remaining substrate in the reaction solution are shown.

[0058]

[Table 9]

After the reaction was completed, the reaction solution was treated in the same manner as in Example 8, and the isomer analysis was performed.
It was confirmed that both were mixtures of equal amounts of L-threo and L-erythro.

Example 10 Equivalent mixture of D,L-threo/erythro derivatives of derivatives in which R1 and R2 in general formula (I) are both methyl and benzyl groups, and a five-membered derivative in which R1 and R2 are substituted with methylene groups. The acetone fractionated enzyme solution obtained in Enzyme Production Examples 1 and 2 was reacted in the same manner as in Example 8, using a mixture of equivalent amounts of D,L-threo/erythro bodies of ring-forming derivatives as a substrate. , shows the results of quantifying the remaining substrate in the reaction solution.

[0061]

[Table 10]

After the reaction was completed, the reaction solution was treated in the same manner as in Example 8, and the isomer analysis was performed.
It was confirmed that all the derivatives were mixtures of equal amounts of L-threo and L-erythro forms.

[0063]

Effects of the Invention: Conventional optical resolution methods for obtaining L-DOPS require introduction and removal of substituents, resulting in complicated steps and low yields, expensive resolving agents, and There were problems as an industrial production method, such as the need to racemize the isomers. According to the present invention, there is no need to introduce or remove substituents for optical resolution, there is no need to use an expensive optical resolution agent, and the generated glycine and protocatechualdehyde derivatives can be recycled and used in raw material synthesis. This method is extremely advantageous compared to conventional methods because it does not require complicated racemization.

Claims (5)

[Claims] Claim 1: General formula [Formula 1] (wherein R1 and R2 are the same or different and represent hydrogen, lower alkyl or aralkyl group,
D-3-(3,4-dihydroxyphenyl)serine derivative is added to racemic-3-(3,4-dihydroxyphenyl)serine derivative represented by (both may be substituted with an alkylene group to form a ring). L-3-(3,4
-Production method of dihydroxyphenyl) serine derivative. 2. The enzyme according to claim 1, wherein the enzyme that specifically decomposes D-3-(3,4-dihydroxyphenyl)serine derivatives into protocatechualdehyde derivatives corresponding to glycine is D-threonine aldolase. the method of. 3. A microorganism that produces an enzyme that specifically decomposes D-3-(3,4-dihydroxyphenyl)serine derivatives into glycine and the corresponding protocatechualdehyde derivatives is a member of the genus Alcaligenes, Pseudom.
onas genus, Arthrobacter genus, Xanth
The method according to claim 1, comprising at least one type of microorganism selected from the group consisting of microorganisms belonging to the genus Omonas. 4. The racemic-3-(3,4-dihydroxyphenyl)serine derivative is racemic-threo-3-(
3,4-dihydroxyphenyl)serine derivative,
The method according to claim 1, wherein the L-3-(3,4-dihydroxyphenyl)serine derivative is an L-threo-3-(3,4-dihydroxyphenyl)serine derivative. 5. The racemic-3-(3,4-dihydroxyphenyl)serine derivative is racemic-erythro-3-
(3,4-dihydroxyphenyl)serine derivative, and the L-3-(3,4-dihydroxyphenyl)serine derivative is an L-erythro-3-(3,4-dihydroxyphenyl)serine derivative. The method described in 1.