JP3078597B2 - Method for producing L-threo-3- (3,4-dihydroxyphenyl) serine derivative - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to L-threo-3- which is known to be useful as a remedy for Parkinson's disease and a familial amyloid polyneuropathy.
The present invention relates to a method for producing (3,4-dihydroxyphenyl) serine and a derivative thereof (hereinafter, abbreviated as L-threo-DOPS derivative). L-threo-DOPS derivatives are important intermediates in the synthesis of L-threo-DOPS useful as pharmaceuticals.

[0002]

2. Description of the Related Art Conventionally, the synthesis of L-threo-DOPS derivatives has been carried out by the following method. That is, a protocatechualdehyde derivative having a protected catechol moiety and glycine are condensed under a strong alkali to obtain a racemic-threo / erythro-DOPS derivative, and the thus obtained product is converted into a threo / erythro form. After performing a separation treatment and then optically resolving the racemic-threo-DOPS derivative, after removing the protecting group of the catechol portion, introducing a substituent into the amino group portion, using an optical resolving agent such as quinine or brucine. After optical resolution is performed, the substituent at the amino group is finally removed (Japanese Patent Application Laid-Open No. 58-158).
-216146).

[0003]

Problems to be Solved by the Invention L-Threo-DOP
In the synthesis of the S derivative, it is necessary to introduce or remove various specific protecting groups, and in addition, a plurality of stereoisomers are produced in this synthesis, and the separation of the stereoisomers from each other is required. Has become a major challenge. As described above, since two or more stereoisomers are generated in the synthesis step, at least a plurality of steps are indispensable for the separation of the isomers, and a step of introducing and removing various protecting groups is also required accordingly. This has been a major problem in industrial production.

[0004] Among them, the optical resolution method has a problem as an industrial production method, because not only is the process complicated and the yield is poor, but also the optical resolution agent used is expensive. Furthermore, the conventional optical resolution method always requires a separation treatment between the threo / erythro isomer before the racemic separation, and furthermore, unnecessary products obtained as a result of these methods, that is, DL-erythro derivative and D-erythro derivative. The threo derivative had to be separately racemized or decomposed in order to use it again, and the production equipment and the like became more complicated.

[0005]

Means for Solving the Problems The present inventors have intensively studied a method for obtaining only an L-threo-DOPS derivative which is an important intermediate in the synthesis of L-threo-DOPS, and as a result, have found that racemic-threo-DOES / The present invention has been completed by finding that only an L-threo-DOPS derivative can be specifically obtained by allowing an enzyme having a specific substrate specificity to act on an erythro-DOPS derivative.
The present invention has the general formula

[0006]

Embedded image (Wherein R ₁ and R ₂ are the same or different and represent hydrogen, lower alkyl or aralkyl group, and both R ₁ and R ₂ may be substituted with an alkylene group to form a ring) -Threo / erythro-3- (3,4-dihydroxyphenyl) serine derivative, D-threo / erythro-3- (3,4-dihydroxyphenyl) serine derivative and L-erythro-3- (3,4- An enzyme having the ability to specifically decompose a dihydroxyphenyl) serine derivative into a glycine and a corresponding protocatechualdehyde derivative, a microbial cell producing the enzyme, or a treated product of an L-cell. Threo-3- (3,
The present invention relates to a method for producing a 4-dihydroxyphenyl) serine derivative. In particular, the present invention provides a compound of the general formula

[0007]

Embedded image (Wherein R ₁ and R ₂ are the same or different and represent hydrogen, lower alkyl or aralkyl group, and both R ₁ and R ₂ may be substituted with an alkylene group to form a ring) A protocatechualdehyde derivative specifically corresponding to glycine by adding a D-threo / erythro-3- (3,4-dihydroxyphenyl) serine derivative to a threo / erythro-3- (3,4-dihydroxyphenyl) serine derivative An enzyme having the ability to decompose into L-erythro-
Enzyme capable of specifically decomposing a 3- (3,4-dihydroxyphenyl) serine derivative into glycine and a corresponding protocatechualdehyde derivative, a microbial cell producing the enzyme, or a treated product of the microbial cell And a method for producing an L-threo-3- (3,4-dihydroxyphenyl) serine derivative.

[0008] The raw material racemic-threo /
Examples of the erythro-DOPS derivative include compounds having the above formula (I), and these compounds may be a mixture of compounds in which the protecting groups R ₁ and R ₂ are the same or different. Representative compounds of 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-dimethoxyphenyl) serine or a mixture thereof.

[0009] In the present invention, L-threo-DOPS
Isomers other than derivatives, ie, D-threo-DOPS
Not only specifically decomposes the derivative and the DL-erythro-DOPS derivative, but also decomposes their isomers into glycine and the corresponding protocatechualdehyde derivative. The aldehyde derivative can be used again in the synthesis.

The enzymes used in the present invention which have the ability to specifically degrade D-threo and erythro-DOPS derivatives and L-erythro-DOPS derivatives into glycine and the corresponding protocatechualdehyde derivative include D-threonine. Examples thereof include those obtained by combining aldolase and L-arothreonine aldolase, and those having substantially the same activity as the enzyme, for example, a microorganism itself that produces the enzyme, or a processed product of the cells. .

In the present invention, the above enzymes or their equivalents may be reacted simultaneously or sequentially. In the present invention, as described above, an enzyme that degrades a D-threo / erythro-DOPS derivative may be used. As such an enzyme, a D-threo / erythro-DOPS derivative is specifically degraded. Any of these can be used. Particularly preferred examples of such enzymes include those having the ability to specifically decompose a D-threo / erythro-DOPS derivative into glycine and a corresponding protocatechualdehyde derivative. Particularly preferred are D-form and L-form
Specifically recognizes the difference between the three-dimensional structure of, threo body and the d
Some are not affected by differences in the structure of the lithro body . Furthermore, those having a specific activity irrespective of the presence of various protecting groups and the like introduced into (dihydroxyphenyl) serine can be mentioned.

Particularly preferred as such an enzyme is D-threonine aldolase. Such D-threonine aldolase is disclosed in
Other than those described in JP-A-4-1279, those obtained by manipulating the gene (for example, Japanese Patent Application No. 1-276126).
Issue). D used in the present invention
-Threonine aldolase can be obtained from a D-threonine aldolase producing bacterium, or a mutant or variant thereof.

[0013] Examples of D-threonine aldolase producing bacteria include Alcaligenes faecalis (Alcaligene).
s faecalis) (IFO 12669), Pseudomo
nas) DK-2 (No. 6200)
throbacter) DK-19 (BikokenkinYadoriki No. 6201), Xanthomonas oryzae (Xa nt homonas oryzae) (IAM 1657) but the like are known, can be used without being limited to these.

[0014] A recombinant produced by introducing a DNA carrying a gene involved in the synthesis of D-threonine aldolase by a technique such as gene recombination can also be suitably used. The microorganisms can be utilized to introduce such genes, for example Esherihi A (Escherichia) genus <br/> bacteria, Bacillus (Baci11us) bacteria, Xanthomonas (Xanthomonas) bacteria, Acetobacter (Acetobacter)
Bacteria, Pseudomonas bacteria, Gluconobacter bacteria, Azotobacter bacteria, Rhizobium bacteria, Alcaligenes bacteria, K1ebsie11a bacteria, Salmonella Prokaryotic microorganisms such as genus bacteria and Serratia bacteria, and lower eukaryotes such as yeast can be used.

Among such recombinants, examples of the recombinants having the ability to produce D-threonine aldolase at a high level include those belonging to the genus Xanthomonas. Xanthomonas citri, deposited as Microtechnical Research Bacteria No. 10979
nas citri) IFO3311 (pDT648)
Has the ability to highly produce D-threonine aldolase (Japanese Patent Application No. 1-276126), and is particularly suitable.

In particular, this Xanthomonas citri IF
O3311 (pDT648) specifically produces D-threonine aldolase in a large amount, so that D-threonine aldolase having high activity can be obtained by a simple treatment. It is possible to obtain unexpectedly excellent effects such as shortening of the time, increase of the yield, and simplification of the apparatus.

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

In the present invention, an enzyme that degrades an L-erythro-DOPS derivative may be used as described above. Examples of such an enzyme include one that specifically degrades an L-erythro-DOPS derivative. Any can be used. Particularly preferred examples of such enzymes include those having the ability to specifically degrade L-erythro-DOPS derivatives into glycine and the corresponding protocatechualdehyde derivative . It is particularly preferable that not only specifically recognize the difference in the three-dimensional structure between the D-form and the L-form , but also specifically recognize the difference in the three-dimensional structure between the erythro-form and the threo-form. Those having specific activity irrespective of not only the skeleton but also the presence of various protecting groups introduced into the skeleton.

Particularly preferred as such an enzyme is L-arothreonine aldolase. Examples of such an L-arothreonine aldolase include not only those obtained from microorganisms described in JP-B-63-54359, but also those obtained by manipulating the gene. The L- allo-threonine aldolase (dihydroxyphenyl) not only serine, which can also act on the derivative, while having the property that allowed to produce a proto catheter queue aldehyde derivative corresponding to glycine, the DOPS derivative It has stereospecificity to act only on the L-erythro body.

The L-allothreonine aldolase used in the present invention can be obtained from an L-threonine aldolase-producing bacterium, or a mutant or variant thereof.
Examples of L-threonine aldolase-producing bacteria include, for example, Bacillus DK-39 (Microtechnical Laboratories No. 6202), Alcaligenes faecalis (IF
O 12669), Pseudomonas DK-2 (Microtechnical Laboratory No. 6200), Arthrobacter DK
-19 (BikokenkinYadoriki No. 6201), Xanthomonas oryzae (Xa nt homonas oryzae) (IAM 1657) are known, can also be used without limited to these.

In the same manner as in the case of D-threonine aldolase, L-allothreonine aldolase is introduced into a microorganism or the like by introducing a DNA carrying a gene involved in its synthesis into a microorganism or the like by a genetic manipulation or the like. The microorganism obtained by the above method can also be used. Examples of the microorganism used for such a purpose include those described above for D-threonine aldolase.

Also, D-threonine aldolase and L-
Alothreonine aldolase can be used in the same manner, whether it is derived from the same strain or from different strains. What was obtained from the thing which was made to produce | generate in a special way, or the thing which made both produce efficiently can be used.

The microorganism used in the present invention can be cultured according to a conventional method. The medium used for the culture is a usual medium containing a carbon source, a nitrogen source, an inorganic substance, and the like necessary for the growth of the microorganism. Examples of the carbon source used here include glucose, ribose, galactose, and soluble starch, and examples of the nitrogen source include ammonium chloride, ammonium sulfate, ammonium nitrate, peptone, meat extract, yeast extract, corn steep liquor, soy flour, and cotton. There is a real scum. In addition, sodium chloride, potassium salt, calcium salt, ammonium salt, phosphate and the like,
Inorganic substances such as zinc, magnesium, iron and manganese can also be used. Further, the addition of organic micronutrients such as vitamins and amino acids often provides desirable results. The cultivation is carried out under aerobic conditions at pH 4 to 10, at a temperature of
Culturing may be performed for 1 to 10 days while controlling the temperature to an arbitrary range of ° C. When a recombinant is used, an antibiotic such as ampicillin or chloramphenicol may be added to the culture solution depending on the strain used.

In the enzymatic reaction, a culture of the cells, cells separated from the culture, dried cells by freeze-drying,
A cell lysate, a crude enzyme extract, a purified enzyme, and the above-mentioned processed and immobilized substances, and others can be used. The method of carrying out the enzymatic reaction is carried out by using a racemic-threo / erythro-DOPS derivative as a substrate in an aqueous medium as described above in a culture solution of a microorganism, a cell, a treated cell, an enzyme, or a conventional method. What is necessary is just to make it contact with the thing fixed with. As the aqueous medium at the time of such a reaction, for example, water, a buffer, a water-containing organic solvent and the like can be used.

The concentration of the enzyme, the microbial cells or the processed microbial cells in the reaction is determined by D-threo / erythro-DOPS.
There is no particular limitation as long as the derivative and the L-erythro-DOPS derivative can be completely decomposed, but D-threonine aldolase activity (1 U is 1 μmol / min from D-threonine per minute)
(Denotes the amount of enzyme required to produce glycine in le)
And L-arothreonine aldolase activity (1 U represents the amount of enzyme required to produce 1 μmole of glycine per minute from L-allothreonine) are both 0.01
It is desirable that it be not less than U / ml. In particular, in both cases.
If it is 1 U / ml or more, the reaction can be terminated promptly.

The reaction can be carried out by allowing two kinds of enzymes to act simultaneously or sequentially, and when they act sequentially, the order does not matter. The concentration of the substrate, the racemic-threo / thrisulo-DOPS derivative, may be any value as long as it does not significantly inhibit the reaction, and is preferably from 1 mmole / liter to 100 mmole / liter. Can be implemented.

The reaction temperature is preferably from 10 to 55.degree.
To 40 ° C. is more preferred. PH during the reaction is 5 to 1
0, preferably 6 to 8. Since D-threonine aldolase and L-arothreonine aldolase require pyridoxal-5'-phosphate as a coenzyme, the reaction system is required to be 10 nmole / liter to 10 mmole / liter, preferably 1 μmol / liter to 100 μl.
The reaction is promoted by adding at a concentration of mole / liter.

Also, D-threonine aldolase requires a divalent metal ion as a prosthetic factor, so that Mg ²⁺ , Mn ²⁺ ,
Co ²⁺ , Fe ^2+, etc. in the form of salt 100 μmole / l to 100 mm
ole / liter, preferably 1 mmole / liter to 10 mmo
The reaction is accelerated by adding le / liter.

The reaction system may be either a batch system or a continuous system, but the reaction is completed in about 0.5 to 50 hours. After completion of the reaction, the reaction solution is subjected to sterilization and protein removal by centrifugation, ultrafiltration, or the like, and firstly, the produced protocatechualdehyde derivative is separated by extraction with an organic solvent. For the extraction, any solvent can be used as long as the solubility of the protocatechualdehyde derivative is high and the L-threo-DOPS derivative and glycine are almost insoluble, but good results can be obtained by using ether. Can be

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

When the purity is low, the degree of purification can be increased by further crystallization and recrystallization with water. The above-described method for isolating and purifying the reaction product exemplifies one typical method and is a preferable method. However, 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 and the like. Sulfoxides 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 and their mixed solvents or their aqueous solvents, column chromatography, reverse phase chromatography, high performance liquid chromatography And a recrystallization method.

According to the method of the present invention, (1) the step of separating the threo form from the erythro form can be omitted. (2) Introduction and removal of substituents for optical resolution are not required, and there is no need to use an expensive optical resolution agent, and the yield is high. (3) Since the equipment process can be greatly simplified, equipment costs for the equipment can be greatly reduced. (4) Since both the D-threo / erythro-DOPS derivative and the L-erythro-DOPS derivative are decomposed into glycine and protocatechualdehyde derivatives, which are synthetic raw materials,
They can be recovered from the reaction solution again and recycled for the synthesis of the racemic-threo / erythro-DOPS derivative, which can greatly reduce the production cost. In particular, even when the treatment of the present invention is carried out using a derivative in which the catechol moiety is protected as a substrate, the treatment of the present invention can be carried out well, and the recovered protocatechualdehyde derivative has a protecting group. Is left as it is, so that it can be used as it is as a raw material for synthesis without performing the operation of introducing a protecting group again. (5) The yield of the target substance from the raw material can be greatly increased, and the high-purity target substance can be easily obtained.

[0033]

Next, the method of the present invention will be described in more detail by way of examples, but the present invention is not limited to the examples. In the examples,% indicates% by weight.

Enzyme Production Example 1 A pH 7.5 medium containing 0.5% of polypeptone, 0.5% of yeast extract and 0.5% of sodium chloride was prepared.
Add 3 liters to a liter culture tank,
For 15 minutes. Arthroba in the medium
cter DK-19 (Microtechnical Laboratories No. 6201) was inoculated and cultured at 30 ° C. for 20 hours with aeration and stirring while maintaining the pH at 7.5. After completion of the culture, cells were collected from the culture by centrifugation, washed with water, and then washed with 0.2 mM HEPES (N-2) containing 0.01 mM pyridoxal-5'-phosphate and 1.0 mM magnesium chloride at pH 7.5. -Hyd
roxyethylpiperazine-N'-2-
ethanesulfonic acid) buffer 10
The suspension was suspended in 0 ml to obtain a cell suspension. This cell suspension was subjected to ultrasonic crushing at 20 kHz for 3 minutes four times, and suspended substances in the crushed liquid were removed by centrifugation to prepare a crude enzyme solution. Next, this crude enzyme solution was treated at 60 ° C. for 30 minutes, and the supernatant obtained by centrifugation was used as a heat-treated enzyme solution.
D- of the obtained cell suspension, crude enzyme solution and heat-treated enzyme solution
Threonine aldolase activity (1 U represents the amount of enzyme required to produce 1 μmole of glycine per minute from D-threonine) and L-arosthreonine aldolase activity (1 U is 1 μmole per minute from L-arothreonine)
(Denotes the amount of enzyme required to produce glycine in le)
Was measured.

[0035]

[Table 1]

Enzyme Production Example 2 In the same manner as in Enzyme Production Example 1, Pseu domonas DK-2 (Microtechnical Laboratories No. 6200), Alcaligenes faecalis (IFO 12669) ,
Xanthomonas oryzae (IAM 1657), Xanthomonas citri
(IFO 3311) ( pDT648) and Bacillus DK-39
No. 6202), a crude enzyme solution and a heat-treated enzyme solution were prepared. D-threonine aldolase activity and L-arothreonine aldolase activity of the obtained cell suspension, crude enzyme solution and heat-treated enzyme solution were measured.

[0037]

[Table 2]

[0038]

[Table 3]

[0039]

[Table 4]

Synthesis Example 1 Racemic-threo / erythro-3- (3,4-methylenedi
Synthesis of oxyphenyl) 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 at 30 ° C. or lower, and the mixture was stirred at 65 ° C. for 30 minutes and concentrated under reduced pressure. The residue was dissolved in methanol (108 ml), acetic acid (64.8 g) was added at 30 ° C. or lower, and the mixture was stirred at 45 ° C. for 1 hour.
1000 ml of toluene and 108 ml of water were added, and the mixture was stirred at 45 ° C. for 30 minutes and further at 5 ° C. for 2 hours. The precipitated crystals were collected by filtration and dried to obtain 86.9 g of crystal A. 85 g of this crystal A was recrystallized from 1000 ml of water, and the obtained crystal was dried to obtain 48.7 g of racemic
Threo / erythro-3- (3,4-methylenedioxyphenyl) serine was obtained. mp: 191 ° C. (decomposition) IR (Nujol) ν (cm ⁻¹ ): 3400, 320
0,1600,1300,1240,1020,910 After derivatization with o-phthalaldehyde and N-acetyl-L-cysteine, HPL was obtained using an ODS column.
The ratio of each isomer was determined by isomer analysis with C. L-threo body: D-threo body: L-erythro body: D-erythro body = 39: 38: 12: 11

Synthesis Example 2 Synthesis of racemic-threo / erythro-DOPS Racemic-threo / erythro-3- obtained in Synthesis Example 1
2 g of (3,4-methylenedioxyphenyl) serine, 20 ml of dichloromethane and 2 ml of ethyl mercaptan were mixed, and 10 g of anhydrous aluminum bromide was added with stirring under ice-cooling, and the mixture was cooled for 3 hours under ice-cooling and further for 15 hours at room temperature. Stirred. 200 ml of 1N hydrochloric acid was added to the 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 1.8 g of racemic-threo / erythro-DOPS. Elemental analysis: C ₉ H ₁₁ O ₅ N Calculated value: C 50.71, H 5.20, N 6.57% Actual value: C 50.99, H 5.15, N 6.44% IR (Nujol) ν (cm) ^-1 ): 3600, 350
0,3200,1620,1600,1500,140
0,1350,1280,1040 After derivatization with o-phthalaldehyde and N-acetyl-L-cysteine, HPL was obtained using an ODS column.
The ratio of each isomer was determined by isomer analysis with C. L-threo body: D-threo body: L-erythro body: D-erythro body = 41: 39: 10: 10

Example 1 100 mg of D, L-threo / erythro-DOPS obtained in Synthesis Example 2 was added to 100 ml of the crude enzyme solution obtained in Enzyme Production Example 1, and reacted at 30 ° C. for 30 hours. After the completion of the reaction, 10 ml of 1N hydrochloric acid was added, and insoluble materials were removed by centrifugation.
When the residual substrate and glycine produced were quantified by HPLC (Shimadzu amino acid analysis system), the substrate was 39.2 at the time of addition.
% Residual, glycine was produced at a molar concentration equal to the reduction of substrate. Also, protocatechualdehyde is 2,4
When reacted with -dinitrophenylhydrazine and subjected to HPLC analysis using an ODS column, it was found to be produced at a molar concentration equivalent to the reduction of the substrate. 200 centrifugation
Ethyl ether (ml) was added, the mixture was stirred and allowed to stand, the ether phase was separated, and dried under reduced pressure to obtain 36 mg of protocatechualdehyde. Convert the aqueous phase to a strongly acidic cation exchange resin D
Column packed with OWEX 50W × 8 (10mmφ ×
100mm), and after sufficient washing with deionized water,
The adsorbed substance is eluted with 0.5N ammonia water, and DOP
The S fraction and the glycine fraction were collected separately. Both fractions were dried under reduced pressure to give 33 mg and 25 m, respectively.
g of crystals were obtained. The crystals obtained from the DOPS fraction were confirmed to be L-threo-DOPS by the following analysis. mp: 208 ° C. Elemental analysis: for C ₃ H ₁₁ O ₅ N Calculated: C 50.71, H 5.20, N 6.57% Actual: C 50.83, H 5.18, N 6.49% Isomer analysis ( o-phthalaldehyde and N-acetyl-L
-After derivatization with cysteine, HPL
C analysis): The peak was observed only at the position of the L-threo compound. Specific rotation: [α] ²⁸ _D = −39.7 ° (C = 0.5,
0.1N HCl)

Example 2 As in Example 1, the crude enzyme solution (A) and the heat-treated enzyme solution (B) (50 ml each when using two types of enzyme solutions) obtained in the enzyme production examples 1 and 2 were used. The results obtained by carrying out the reaction and quantifying the remaining substrate in the reaction solution are shown.

[0044]

[Table 5]

After completion of the reaction, the reaction solution was treated in the same manner as in Example 1, and the specific rotation was measured. As a result, it was confirmed that all the DOPS obtained were L-threo compounds.

Example 3 A reaction was carried out in the same manner as in Example 1 by using the cell suspensions obtained in Enzyme Production Examples 1 and 2 (in the case of using two kinds of cell suspensions, 50 ml each). The result of having quantified the residual group mass in the liquid is shown.

[0047]

[Table 6]

After the completion of the reaction, the reaction solution was treated in the same manner as in Example 1, and the specific rotation was measured. As a result, it was confirmed that all the DOPS obtained were L-threo compounds.

Example 4 A derivative in which R ₁ and R ₂ in the formula (I) are both hydrogen / methyl and benzyl, and R ₁ and R ₂ are substituted with a methylene group to form a five-membered ring Using an equal mixture of the four isomers of the derivative as the substrate, the crude enzyme solutions obtained in Enzyme Production Examples 1 and 2 were reacted in the same manner as in Example 1, and the remaining substrate in the reaction solution was quantified. The results are shown.

[0050]

[Table 7]

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

[0052]

According to the conventional optical resolution method for obtaining L-threo-DOPS, the introduction and removal of substituents are required, the process is complicated, the yield is poor, the resolving agent is expensive, and the remaining There is a problem as an industrial production method, for example, it is necessary to racemize the optical isomer of According to the present invention, it is not necessary to introduce or remove a substituent for optical resolution, there is no need to use an expensive optical resolution agent, and the glycine and protocatechualdehyde derivative generated can be recycled for raw material synthesis. This is a very advantageous method as compared with the conventional method because it is not necessary to perform complicated racemization.

Continuation of the front page (56) References JP-A-50-63192 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12P 41/00 BIOSIS (DIALOG) WPI (DIALOG)

Claims (3)

(57) [Claims] 1. A compound of the general formula (Wherein R ₁ and R ₂ are the same or different and represent hydrogen, lower alkyl or aralkyl group, but R ₁ and R ₂ may be substituted with an alkylene group to form a ring) (A) D-threo / erythro-3- (3,4-dihydroxyphenyl) serine derivative specifically corresponds to glycine Proto
D-source with the ability to decompose into catechualdehyde derivatives
Leo Nin aldolase, (b) L- erythro-3- (3,4-dihydroxyphenyl) having an ability to resolve the proto catheter queue aldehyde derivative corresponding to the specific glycine serine <br/> induction body L- Arosureoni
L-threo characterized by acting on aldolase
A method for producing a -3- (3,4-dihydroxyphenyl) serine derivative. 2. The method according to claim 1, wherein the D-threonine aldolase is Alcalige.
Nes, Pseudomonas, Arthrobacter, Xanthomona
The method according to claim 1, which is obtained from at least one kind of microorganism selected from the group consisting of microorganisms belonging to the s genus. 3. The method according to claim 1, wherein the L- arothreonine aldolase is Alca
Ligenes, Pseudomonas, Arthrobacter, Xantho
obtained from at least one microorganism selected from the group consisting of microorganisms belonging to the genus monas, Bacillus, and Escherichia.
The method of the is claim 1, wherein those.