JPS585039B2 - Method for producing α-aspartyl-phenylalanine alkyl ester - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing α-aspartyl-phenylalanine alkyl ester.

α-L-Akupartyl-L-phenylalanine alkyl ester is known to have a sucrose-like sweetness,
In particular, methyl ester is about 200 times sweeter than sugar.
It is attracting attention as a new sweetener.

Conventionally known methods for producing α-L-aspartyl-L-phenylalanine alkyl ester include, for example, the following methods.

(1) Protected or unprotected L- of the amine group
A method involving the reaction of aspartic acid anhydride and L-phenylalanine alkyl ester (Japanese Patent Publication No. 49-14217
, JP 48-76835, JP 48-61451,
JP-A-50-58025 and JP-A-5O-7164
2).

(2) A method involving the reaction of L-aspartic acid-N-carboxylic anhydride in which the amino group and β-carboxyl group are protected with appropriate substituents and L-phenylalanine alkyl ester (Japanese Patent Laid-Open No. 48-96557).

(3) α[3-monosubstituted-5-oxooxazolidinyl-(
4)] - A method involving a reaction between acetic acid and L-phenylalanine alkyl ester (Japanese Patent Application Laid-open No. 49-35352).

These chemical synthesis methods tend to cause racemization and other undesirable side reactions during the reaction, and in many cases C
There was a drawback that β-aspartyl was produced as a by-product.

The present invention is represented by a general formula, at least a part of which is an L-type optical isomer, phenylalanine alkyl ester (wherein R is a lower alkoxyl group); is an L-type optical isomer, N-p-methoxybenzyloxycarbonyl aspartic acid, is reacted with N-p-methoxybenzyloxycarbonyl aspartic acid in the presence of a protease in an aqueous medium to obtain a compound represented by the general formula and containing water of crystallization. producing and precipitating a 1:1 addition compound of N-p-methoxybenzyloxycarbonyl-α-L-aspartyl-L-phenylalanine alkyl ester and phenylalanine alkyl ester (in the formula, R has the same meaning as above), α-L-aspartyl-L-phenylalanine alkyl ester ( In the formula, R has the same meaning as above).

The phenylalanine alkyl ester represented by the general formula (I) and the N-p-methoxybenzyloxycarbonyl aspartic acid represented by the formula (II) used as starting materials in the present invention are both at least partially L-
It must be an optical isomer of type.

Only the two Ls can form a peptide bond in the enzymatic reaction, which is the first step of the reaction of the present invention.

However, regarding the phenylalanine alkyl ester that participates in the reaction of forming an addition compound represented by the general formula (■) by combining with the generated N-p-methoxybenzyloxycarbonyl-α-L-aspartyl-phenylalanine alkyl ester, The optical activity does not matter, except that the D-unit participates more predominantly in this formation.

Therefore, when L-type phenylalanine alkyl ester is used, an addition compound of N-p-methoxybenzyloxycarbonyl-α-L-aspartyl-L-phenylalanine alkyl ester and L-phenylalanine alkyl ester is obtained as an intermediate, and D When using a phenylalanine alkyl ester containing monomers, such as racemic phenylalanine alkyl ester, N-p-methoxybenzyloxycarbonyl-α-L-aspartyl-L-phenylalanine alkyl ester and D-phenylalanine alkyl ester or D- and L-phenylalanine Addition compounds with alkyl esters are formed.

The addition compound represented by the general formula (■) obtained in this way is decomposed in the latter reaction of the present invention, and the addition compound represented by the general formula (I
α-L-aspartyl-L-phenylalanine alkyl ester represented by V) is obtained.

Therefore, in the present invention, as the starting material phenylalanine alkyl ester, not only the L monomer but also those containing the D monomer, such as a racemate, can be used as well.

Further, R in the general formula (I) represents a lower alkoxyl group obtained by esterifying the carboxyl group of phenylalanine, and specific examples thereof include a methoxyl group, an ethoxyl group, a propoxyl group, a butoxyl group, an amyloxyl group, etc. I can do it.

The other starting material, N-p represented by the general formula (■)
In -methoxybenzyloxycarbonyl aspartic acid, only its L-form optical isomer can participate in the reaction.

However, D does not interfere with the reaction at all, and remains in the reaction solution without being incorporated into the intermediate addition compound represented by the general formula (■).

Since this intermediate product is removed when it is separated from the reaction solution, it is harmless even if it coexists in the starting materials.

Therefore, in the present invention, as the starting material N-p-methoxybenzyloxycarbonyl aspartic acid, not only the L monomer but also those containing the D monomer, such as a racemic form, can be used.

The most preferred protease to be used is an enzyme having a metal ion in its active center, ie, metalloprotease.

Examples include those of microbial origin, such as neutral protease of actinomycete origin, prolysin, thermolysin, collagenase, and Crotalus atrotchus protease.

Crude enzymes such as samoase can also be used.

At that time, in order to avoid the effects of contaminating esterase, etc.
Inhibitors such as potato inhibitors may be used in combination.

Thiol proteases such as papain or serine proteases such as trypsin cannot be used, but since they also have esterase action, the reaction must be carried out with care to avoid hydrolysis of the ester.

The reaction is carried out in an aqueous medium, preferably an aqueous solution, under pH conditions such that the protease used exhibits enzymatic activity.

The range is pH about 4 to about 9, preferably about 5 to 8.

Therefore, the N-p-methoxybenzyloxycarbonyl-L-aspartic acid and phenylalanine alkyl ester used may be in free form or salt;
When these two components are dissolved in an aqueous medium, it is necessary to adjust the pH conditions.

As pH regulators, conventional inorganic or organic acids such as hydrochloric acid, sulfuric acid, acetic acid, alkali hydroxides such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium hydrogen carbonate, potassium carbonate, potassium hydrogen carbonate, etc. Conventional inorganic or organic bases can be used, such as alkali carbonates, ammonia, organic or inorganic amines such as trimethylamine, triethylamine, ethanolamine.

These regulators may be further added when the pH changes due to the progress of the reaction, and an appropriate buffer may be used to suppress pH fluctuations.

It is possible to add a water-soluble organic solvent to the aqueous medium to the extent that it does not significantly inhibit the precipitation of the reaction product.

The reaction temperature is 10°C to 90°C, preferably 20°C to 50°C from the viewpoint of maintaining enzyme activity.

The reaction usually completes in about 30 minutes to 24 hours, but
This reaction time is not limited in any way.

The concentrations of N-p-methoxybenzyloxycarbonyl-L-aspartic acid and phenylalanine alkyl ester during the reaction are about 0.001M to 7M, respectively.
Preferably it is about 0.1M to 4M.

Since this reaction utilizes the very low solubility of the adduct compound, which is an intermediate product of the present invention, in water, it is preferable that the concentration thereof be relatively high.

These two components react in a stoichiometric molar ratio of 1:2, but
Actually it is 100:1 to 1:100. Preferably 5:
They are used in a molar ratio of about 1 to 1:5, most preferably about 2:1 to 1:3.

The amount of enzyme used in this reaction is not necessarily limited, but is generally 2 to 400 per mmol of substrate.
mg (5 x 10-5 to 1 x 10-2 mmol), preferably about 5 to 100 mg (1 x 10-4 to 3 x 10-3 mmol).

As a liquid medium for dissolving the addition compound represented by the general formula (■), a solvent capable of dissolving this compound is naturally used.

Such solvents include organic solvents, especially ketones such as acetone; oxygenated organic solvents such as dioxane and tetrahydrofuran; chlorinated lower hydrocarbons such as chloroform, methylene dichloride, and ethylene dichloride; and aprotic solvents such as dimethylformamide and dimethyl sulfoxide. Polar organic solvents; examples include liquid carboxylic acids such as acetic acid and formic acid.

Of course, two or more of these solvents can be used in combination.

Esters such as ethyl acetate, alcohols such as methanol, ethanol, and propatool can also be used, but when such solvents are used, preferable reactions such as transesterification or esterification of carboxyl groups may occur. The yield of the target compound often decreases due to side reactions.

Also, N-p-methoxybenzyloxycarbonyl-α-L
Since the 1:1 addition compound of -aspartyl-L-phenylalanine alkyl ester and phenylalanine alkyl ester has a low solubility in water, water alone is not suitable as the liquid medium of the present invention.

However, there is no problem in adding it to an extent that does not significantly impair the solubility of the addition compound in the liquid medium used.

The amount of these liquid media to be used may vary depending on the type of liquid medium, as the solubility for the adduct differs, but it is usually 10 parts by weight or more, preferably 20 to 100 parts by weight, per 1 part by weight of the adduct. .

The acid used to decompose the adduct is an inorganic or organic Bronsted acid.

Examples of inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, perchloric acid, and the like.

Examples of organic acids include trifluoroacetic acid, p-toluenesulfonic acid, and the like.

The amount of acid used is at least stoichiometric, i.e., at least stoichiometric amount, since the present invention uses the acid to ionize the phenylalanine alkyl ester moiety of the adduct and further eliminate the p-methoxybenzyloxycarbonyl moiety. It is used in an amount of 2 equivalents or more based on mole.

The concentration of acid in the liquid medium is determined by taking these reaction conditions into consideration, since the time of the decomposition reaction also depends on the reaction temperature, the type of acid, etc.

Generally, it is about 0.1N to 10N, preferably about 1N to 5N.

If the acid concentration is too high, undesirable side reactions such as decomposition of the alkyl ester may occur, so it is desirable to avoid concentrations higher than this.

The acid used may be either aqueous or anhydrous.

However, when using a liquid medium that is immiscible with water, such as a halogenated hydrocarbon, it is preferable to use it in an anhydrous form, since if it is used in an aqueous form, two phases will be formed and the reaction will be extremely slow.

Although there are no particular limitations on the temperature and time of the decomposition reaction with an acid, the decomposition is usually carried out at a temperature of 20 to 100°C and a reaction time of about 10 minutes to 6 hours.

When the concentration of acid used is low, the reaction time is increased or the reaction temperature is increased.

On the other hand, when the acid concentration is high, it is appropriate to shorten the reaction time or lower the reaction temperature.

After acid decomposition of the addition compound in the present invention, the decomposition products α-L-aspartyl-L-phenylalanine alkyl ester, anis alcohol, and phenylalanine alkyl ester are separated and recovered by the following conventional means, respectively. be able to.

For example, after the reaction is completed, anis alcohol is extracted and separated into a solvent phase by adding an appropriate amount of water and a solvent capable of forming two phases with water, such as chloroform or diethyl ether, as necessary. The pH is adjusted to 5 to 6 with a basic substance such as sodium bicarbonate, sodium carbonate, ammonia, or triethylamine, and the precipitated α-aspartyl-phenylalanine alkyl ester is separated and collected by a method such as filtration.

The filtrate can be further adjusted to pH 8 to 10 with the above-mentioned basic substance, and the liberated phenylalanine alkyl ester can be extracted and recovered with a solvent such as chloroform, diethyl ether, or ethyl acetate.

α-L-Akupartyl-L-phenylalanine alkyl ester and phenylalanine alkyl ester can be easily recovered by a conventional method using a cation exchange resin.

The phenylalanine alkyl ester thus recovered may contain L monomers or D monomers depending on the optical activity of the phenylalanine alkyl ester represented by general formula (I) used as a starting material.

When a racemate is used as a raw material, it is possible to recover a phenylalanine alkyl ester that is substantially almost D-integrated.

According to the method of the present invention, from L-unit of N-p-methoxybenzyloxycarbonyl-aspartic acid and L-unit or L-unit of phenylalanine alkyl ester, Cβ-
α-L-Aspartyl-L- which does not contain aspartyl bodies
Phenylalanine alkyl ester can be efficiently produced.

Moreover, as a starting material, a racemate, which cannot be used when using general chemical synthesis methods, can be used as is.

Both the phenylalanine eliminated during the reaction and the anis alcohol produced from the p-methoxybenzyloxycarbonyl group also eliminated can be recovered, and the former can be racemized and reused as a starting material if necessary. The latter can be reacted with phosgene to form p
- Can be used cyclically as a methoxybenzyloxycarbonylating agent.

In addition, when racemic phenylalanine alkyl ester is used as a starting material, α-L-aspartyl-L
- Simultaneously with the production of phenylalanine alkyl ester,
Since it is possible to recover a phenylalanine alkyl ester that is substantially nearly D-integrated, optical resolution of racemic phenylalanine alkyl ester can also be achieved.

The present invention will be explained in more detail below with reference to Examples.

Example I 5.00 g (16,82 mmol) of N-p-methoxy-benzyloxycarbonyl-L-aspartic acid and L
- Phenylalanine methyl ester hydrochloride 7.26 g (
33.64 mmol) was placed in a flask with an internal volume of about 100 ml, and a sodium hydroxide solution (1N) was added to dissolve it, and the pH was adjusted to 6.0.

2.0 g of thermoase and 0.4 g of potato inhibitor were added to this solution, and the mixture was stirred at 38 to 40°C for 5 hours.

The deposited precipitate was collected by filtration, washed with 100 ml of water, and dried to obtain 8.11 g of crystals with a melting point of 88 to 92°C (
Yield (near 5.6%) as a 1:1 addition compound of N-p-methoxy-benzyloxycarbonyl-asbartyl-phenylalanine methyl ester and phenylalanine methyl ester.

This substance was confirmed to be a 1:1 addition compound of Np-methoxy-benzyloxycarbonyl-α-L-aspartyl-L-phenylalanine methyl ester and L-phenylalanine methyl ester from the following.

That is, the physical properties and elemental analysis results of the product obtained by recrystallizing this product from a mixed solvent of ethyl acetate and n-hexane are as follows: Melting point: 90 to 94°C [α] ■: +6.2 (C = 1. methanol ) The infrared absorption spectrum is shown in Figure 1, with N-H stretching at 3.280 cm-1 and C at 3,020 and 2.930 cm-1.
-H expansion and contraction, ester C=O at 1,735 cm-1, l, urethane C=O at 700 cm-1, 1,64
Amide first absorption at 9 cm-1, 1.500-1,54
Amide first absorption at 0 cm-1, C-H bending at 1.435 cm-1, carboxylate at 1,380 cm-1, C-0-C stretching and contraction from 1,210 to 1,240 cm-1 Absorptions originating from the amide No. 1 absorption, phenyl in-plane bending at 1.030 cm-1, and phenyl out-of-plane bending at 690, 740 and 810 cm-1 are observed, respectively.

Nuclear magnetic resonance spectra have δ values of (1) 2.7 ppm (2H); (2) 3.1 ppm (4
H); (3) 3.6 ppm (3H) and 3.7 ppm
(3H); (4) 3.8ppm (3H); (5) 4.0
ppm (1H); (6) 4.5ppm (1H); (7)
4.8ppm (1H); (8) 5.0ppm (2H);
(9) 5.65ppm (3H) (10) 5.65ppm
(1H) (11) 6.2ppm (1H) and (12)
It is characterized by 6.8 to 7.3 ppm (14H), and these can be attributed to the protons of the corresponding numbers in the following formula.

0.3 g of the thus obtained addition compound of N-p-methoxybenzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester and L-phenylalanine methyl ester was placed in a flask with an internal volume of 20 ml,
Add 3 ml of acetone to dissolve, then add hydrochloric acid (2,4N)
2 ml was added and reacted at 60°C for 1 hour.

A test solution was prepared by adding water, an aqueous sodium bicarbonate solution (1,2N), and cyclohexanone as an internal standard to the reaction solution, and the conversion to α-L-aspartyl-L-phenylalanine methyl ester was confirmed by high-performance liquid chromatography analysis. .

The yield was 72.7%.

The measuring device and measurement conditions are as follows.

Equipment: High performance liquid chromatograph (manufactured by Toyo Soda Kogyo Co., Ltd., TSK-HLC801) Column: inner diameter 7.5 mm x length 300 mm Packing material: starch gel particle size 5 μ (manufactured by Toyo Soda Kogyo Co., Ltd., TSK-GEL, LS-170, P5) Eluent: Sodium acetate aqueous solution (0.5% by weight) Flow rate:
0.8 ml/min Pressure loss: 20 Kg/cm2 Detector: Differential refractometer In the following examples, the same apparatus and conditions were used to measure the yield of the product unless otherwise specified.

Example 2 0.3 g of the addition compound of N-p-methoxybenzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester and L-phenylalanine methyl ester obtained in Example 1 was placed in a flask with an internal volume of 20 ml, Thereafter, the decomposition reaction and analysis of the reaction products were carried out in the same manner as in Example 1, except that dioxane was used instead of acetone.

The yield of α-L-aspartyl-L-phenylalanine methyl ester was 73.0%.

Example 3 Example 2 except that methanol was used instead of dioxane
The operation was performed in the same manner.

The yield of α-L-aspartyl-L-phenylalanine methyl ester was 63.3%.

Example 4 The same procedure as in Example 2 was carried out except that N,N-dimethylformamide was used instead of dioxane.

The yield of α-L-aspartyl-L-phenylalanine methyl ester was 28.1%.

Example 5 The amount of dioxane used was 4a, and the amount of hydrochloric acid (2,4N) was 2m.
1 ml of hydrogen chloride-dioxane solution (5,3N) instead of 1
The procedure was carried out in the same manner as in Example 2, except that triethanolamine was used in place of the aqueous sodium hydrogen carbonate solution (1,2N) used as the reaction wave neutralizing agent.

The yield of α-L-aspartyl-N-phenylalanine methyl ester was 98.6%.

Example 6 The operation was carried out in the same manner as in Example 5, except that the reaction temperature of the decomposition reaction was changed to 90° C. and the reaction time was changed to 20 minutes.

The yield of α-L-aspartyl-L-phenylalanine methyl ester was 88.5%.

Example 7 Dioxane and hydrogen chloride-dioxane solution (5,3N
) are used as 4.5ml and 0.5a, respectively.
Furthermore, the same operation as in Example 5 was carried out except that the reaction temperature was changed to 90°C.

The yield of α-L-aspartyl-L-phenylalanine methyl ester was 84.4%.

Example 8 Dioxane and hydrogen chloride-dioxane solution (5,3N
) was used in the same manner as in Example 5, except that the amounts used were 3 ml and 2 M, respectively, the reaction temperature was changed to 30° C., and the reaction time was changed to 120 minutes.

The yield of α-L-aspartyl-L-phenylalanine methyl ester was 98.6%.

Example 9 The amount of dioxane used was 4.5 ml, and 1 ml of hydrogen chloride-dioxane solution (5,3N) was mixed with perchloric acid (60%) 0.
．． The operation was carried out in the same manner as in Example 5 except that the volume was changed to 5 ml.

The yield of α-L-aspartyl-L-phenylalanine methyl ester was 65.5%.

Example 10 The operation was carried out in the same manner as in Example 5, except that the amount of dioxane used was 4.85 ml, and 1 ml of hydrogen chloride-dioxane solution (5,3N) was replaced with 0.15 a of concentrated sulfuric acid.

The yield of α-L-aspartyl-L-phenylalanine methyl ester was 89.9%.

Example 11 0.3 g of the 1:1 addition compound of N-p-methoxybenzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester and L-phenylalanine methyl ester obtained in Example 1 was dissolved in 2 ml of dioxane. , trifluoroacetic acid 3rd was added thereto, and the mixture was reacted at 60° C. for 1 hour.

After the reaction solution was evaporated under reduced pressure, a test solution was prepared by adding water, triethylamine, and cyclohexanone as an internal standard.

High performance liquid chromatography analysis was performed on this liquid.

The yield of α-L-aspartyl-L-phenylalanine methyl ester was 96.4%.

Example 12 1,000 kg of the 1:1 addition compound of N-p-methoxybenzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester and L-phenylalanine methyl ester obtained in Example 1, 14 ml of dioxane
Then, 4 ml of hydrogen chloride-dioxane solution (5,3N) was placed in a flask having an internal volume of about 50 ml, and the mixture was stirred at 60° C. for 1 hour to react.

Dioxane was distilled off from the reaction solution under reduced pressure, and 6 ml of water and 20 M diethyl ether were added to the remaining oil, mixed with stirring, and then separated into two layers.

The operation of adding 10 ml of diethyl ether to the aqueous layer and extracting in the same manner was repeated three times.

The diethyl ether layer was collected and dissolved in sodium bicarbonate solution (
After washing twice with 5 ml of (5% by weight), separating with anhydrous magnesium sulfate and drying, diethyl ether was distilled off under reduced pressure to obtain 0.176 g of crude anise alcohol (yield: 8
1.2%).

The aqueous layer was adjusted to pH 6 with sodium hydroxide solution (7% by weight) and left overnight at about 5°C.

The precipitated crystals were collected, washed with 2 ml of water, and dried to obtain 0.316 g (yield: 68.5%) of mono-aspartyl-L-phenylalanine methyl ester.

An aqueous sodium hydroxide solution (7% by weight) was added to the filtrate and the pH was adjusted to 9, followed by extraction three times with 15 ml of dichloromethane.

The dichloromethane layer was collected, washed with 5 ml of water, dried over anhydrous magnesium sulfate, and the dichloromethane layer was distilled off under reduced pressure to obtain 0.234 ml of coarse phenylalanine methyl ester.
．． 9 (yield 83.4%) was obtained.

Example 13 1.837 g (8 mmol) of L-phenylalanine ethyl ester hydrochloride was added to 1.189 g (4 mmol) of N-p-methoxybenzyloxycarbonyl-L-aspartic acid in a flask with an internal volume of about 30 ml, and hydroxylated. An aqueous sodium solution (IN) was added to dissolve and adjust the pH to 6.0.

After adding water to bring the total volume to 15 ml, 0.1 g of thermolysin was added to this solution and stirred at 38 to 40°C for 7 hours.

The precipitate was collected by filtration, washed with 30 ml of water, and dried to give 2.401 g of crystals having a melting point of 60 to 65°C (1:1 of N-p-methoxybenzyloxycarbonyl-asbartyl-phenylalanine ethyl ester and phenylalanine ethyl ester). Yield as addition compound: 90.2%
) was obtained.

This substance was confirmed to be a 1:1 addition compound of Np-methoxybenzyloxycarbonyl-L-aspartyl-L-phenylalanine ethyl ester and L-phenylalanine ethyl ester from the following.

That is, the physical properties and elemental analysis results of the product obtained by recrystallizing this product from a mixed solvent of methanol and diethyl ether are as follows: The infrared absorption spectrum is 3.300c as shown in Figure 2.
m-1 for N-H expansion and contraction, 2,900 to 3,050 cm-
1 to C-H stretch, 1,720, 1730 and 1.7
C=O2 of ester and urethane at 40 cm-1,
Amide first absorption at 1,650 cm-1, 1,510~
At 1.540cm-1, amide No. 1 absorption, 1,440c
C-H bending angle at m-1, carboxylate at 1,390 cm-1, C-0 at 1,220-1,280 cm-1
-C stretching and absorption, phenyl in-plane bending at 1,030 cm-1, and 690,760 and 810
Absorption derived from the phenyl out-of-plane bending angle is observed at cm-1.

Nuclear magnetic resonance spectrum has δ values: (1) 1.2 ppm (6H); (2) 2.7 ppm
(2H); (3) 3.1 ppm (4H); (4) 3.
8 ppm (3H); (5) 4.0 ppm (4H);
(6) 4.1 ppm (1H); (7) 4.5 ppm
(1H); (8) 4.7 ppm (1H): (9)5.
0 ppm (2H); (10) 5.5 ppm (4H)
(11) 6.1 ppm (1H); (12) 6.8~
It is characterized by 7.4 ppm (14H); these can be attributed to the protons of the corresponding numbers in the following formula.

1 of the thus obtained N-p-methoxybenzyloxycarbonyl-L-aspartyl-L-phenylalanine ethyl ester and L-phenylalanine ethyl ester:
1 addition compound with N-p-methoxycarbonyl-L-aspartyl-L-phenylalanine methyl ester and L-
The decomposition reaction of Example 5 was repeated using a 1=1 adduct with phenylalanine methyl ester instead.

α-L-aspartyl-L-phenylalanine ethyl ester was obtained, and the yield was 95.5%.

Example 14 N-p-methoxy-benzyloxycarbonyl-L-aspartic acid 1.18914 mmol) and DL-phenylalanine methyl ester hydrochloride 1.725 g (8
mmol) in a flask with an internal volume of about 30 ml, add and dissolve sodium hydroxide solution (1N), and adjust the pH to 6.0.
Adjusted to.

After adding water to bring the total volume to 15 ml, 0.1 g of thermolysin was added to this solution and stirred at 38 to 40°C for 50 minutes.

The deposited precipitate was collected by filtration, washed with 30 ml of water, and then dried to obtain 2.109 g of crystals having a melting point of 119 to 128°C (N-p-methoxybenzyloxycarbonyl-asbarthyl-phenylalanine methyl ester and phenylalanine methyl ester). (yield 86.8% as the 1/2 hydrate salt of the 1:1 addition compound).

This was recrystallized from a mixed solvent of ethyl acetate and n-hexane.

When the obtained crystals were dried at 80°C under reduced pressure for 7 hours,
A substance exhibiting the following physical properties and elemental analysis values was obtained.

In addition, the infrared absorption spectrum of this substance was the same as that of N obtained in Example 1.
It exhibits exactly the same characteristics as that of the 1:1 addition compound of -p-methoxybenzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester and L-phenylalanine methyl ester.

The nuclear magnetic resonance absorption spectrum is as described below.

Since the sample contains water of crystallization, the absorption of its protons and the absorption of protons of one NH- group and one NH3+ group, which were shifted due to the disturbance caused by the presence of crystallization water, appeared at 4,1 ppm, but the same as in Example 1 was obtained. It showed the same characteristics as the case of the above-mentioned addition compound.

1.5024 g of this compound was heated in a microwave heater (2,4
5GHz.

When dried by microwave irradiation for 12 minutes in a 1.2 Kw (output: 1.2 Kw), the weight was 1.48152 g (loss on drying: 0.
The elemental analysis result of the product after microwave drying is 02O2091°, and its infrared absorption spectrum and nuclear magnetic resonance absorption spectrum are N-p-methoxybendioxycarbonyl- obtained in Example 1.
It showed exactly the same characteristics as the 1=1 addition compound of L-aspartyl-L-phenylalanine methyl ester and L-phenylalanine methyl ester.

In addition, 1.0 g of the compound thus obtained was added to 4 ml of water and 2 d of hydrochloric acid (IN) and stirred at 60°C for 3 minutes. The resulting slurry was filtered, and the solid phase was washed with 10 ml of water and dried. -Methoxybenzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester was obtained, while D-phenylalanine methyl ester was obtained from the filtrate.

From these facts, the weight loss due to microwave drying is due to the scattering of crystal water, and the products precipitated by the enzyme reaction are N-p-methoxybenzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester and D
It was confirmed that this was a product in which 1/2 molecule of crystal water was added to 1 molecule of a 1:1 addition compound with -phenylalanine methyl ester.

Example 5 Using 0.3 g of the 1:1 addition compound of N-p-methoxybenzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester and D-phenylalanine methyl ester containing crystal water obtained in this manner,
The operation was performed according to.

The yield of α-L-aspartyl-L-phenylalanine methyl ester and D-phenylalanine methyl ester was 95.9%.

Example 15 0.3 g of the 1:1 addition compound of N-p-methoxybenzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester and L-phenylalanine methyl ester obtained in Example 1 was added to a hydrogen chloride-chloroform solution. (0,31N) and reacted at 60°C for 2 hours.

The reaction solution was evaporated under reduced pressure, and water, triethylamine, and cyclohexanone as an internal standard were added to the residue to prepare a test solution.

High performance liquid chromatography analysis was performed on this liquid.

The yield of α-L-aspartyl-L-phenylalanine methyl ester was 94.3%.

[Brief explanation of drawings]

FIGS. 1 and 2 show N that is passed through as an intermediate in the present invention.
FIG. 2 is a diagram showing an infrared absorption spectrum of a 1:1 addition compound of -p-methoxybenzyloxycarbonyl-α-L-aspartyl-L-phenylalanine alkyl ester and phenylalanine alkyl ester.

Claims (1)

[Scope of Claims] 1 Represented by the general formula, at least a part of which is represented by the formula phenylalanine alkyl ester (in the formula, R is a lower alkoxyl group) which is an L-type optical isomer, and at least A part of it is obtained by reacting N-p-methoxybenzyloxycarbonyl aspartic acid, which is an optical isomer of the L-type, in the presence of a protease in an aqueous medium to form a compound represented by the general formula and crystalline water. to produce a 1:1 addition compound of N-p-methoxybenzyloxycarbonyl-α-L-aspartyl-L-phenylalanine alkyl ester and phenylalanine alkyl ester (wherein R has the same meaning as above), which may include α-L-aspartyl-L-phenylalanine represented by the general formula is characterized by precipitating it, separating it from the reaction solution, dissolving the separated 1:1 addition compound in a liquid medium, and decomposing it with an acid. A method for producing an alkyl ester (in the formula, R has the same meaning as above). 2 Claim 1 in which R in the general formula is a methoxy group
Manufacturing method described in section. 3 Claim 1 in which R in the general formula is an ethoxy group
Manufacturing method described in section. 4. The production method according to any one of claims 1 to 3, wherein the aqueous medium is an aqueous solution and the reaction in the presence of an enzyme is carried out at about 20 to about 50°C. 5. The production method according to any one of claims 1 to 4, wherein the protease is a metalloprotease, and the enzyme reaction is carried out at a pH of about 5 to about 8. 6. The manufacturing method according to any one of claims 1 to 5, wherein the liquid medium is an organic solvent. 7. The manufacturing method according to claim 6, wherein the organic solvent is a ketone. 8. The manufacturing method according to claim 6, wherein the organic solvent is an oxygen-containing organic solvent. 9. The manufacturing method according to claim 6, wherein the organic solvent is a chlorinated lower hydrocarbon. 10. The manufacturing method according to claim 6, wherein the organic solvent is an apronto polar organic solvent. 11. The manufacturing method according to claim 6, wherein the organic solvent is a liquid carboxylic acid. 12 The amount of the liquid medium used is about 10 to 1 part by weight of the 1:1 addition compound of N-p-methoxybenzyloxycarbonyl-α-L-aspartyl-L-phenylalanine alkyl ester and phenylalanine alkyl ester represented by the general formula. 12. The manufacturing method according to any one of claims 1 to 11, wherein the amount is about 100 parts by weight. 13. The production method according to claim 12, wherein the acid is an inorganic or organic Bronsted acid. 14 The amount of acid used is 2 equivalents or more per mole of the 1:1 addition compound of N-p-methoxybenzyloxycarbonyl-α-L-aspartyl-L-phenylalanine alkyl ester and phenylalanine alkyl ester represented by the general formula. A manufacturing method according to claim 13. 15. The manufacturing method according to claim 12 or 14, wherein the concentration of acid in the liquid medium is about 0.1 to about 10 normal. 16. The manufacturing method according to any one of claims 1 to 15, wherein the decomposition with an acid is carried out at a temperature of about 20 to about 100°C. 17. The production method according to any one of claims 1 to 16, wherein the phenylalanine alkyl ester and the N-p-methoxybenzyloxycarbonyl aspartic acid used as starting materials are both L-monotypes. 18. The method according to any one of claims 1 to 16, wherein one or both of phenylalanine alkyl ester and N-p-methoxybenzyloxycarbonyl aspartic acid used as starting materials contain D monomers. Production method.