JPH09248197A - Production of dipeptide ester - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently obtain a dipeptide ester useful as e.g. a sweetener without any loss of expensive raw materials by reaction a specific amount of an N-substituted aspartic acid with a phenylalanine alkyl ester (salt). SOLUTION: One mol of a L-phenylalanine alkyl ester or its hydrochloride (e.g. L-phenylalanine methyl ester hydrochloride) and two or more mol of an N-substituted aspartic acid (e.g. N-benzyloxycarbonyl-L-aspartic acid) are weighed and put into a beaker followed by addition of water to the compounds; an aqueous solution of sodium hydroxide is then added to the mixture under agitation to adjust of the pH of the resultant solution to 4-9, and the resultant mixture is allowed to react in the presence of a protease as catalyst (e.g. thermolysin) at 10-40 deg.C to reduce the amount of the phenylalanine alkyl ester to be recavered and recycled, thus obtaining the objective dipeptide ester useful as e.g. a sweetener in high yield.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a dipeptide ester, which is particularly useful as a sweetener, and more specifically, a specific amount or more of N-substituted aspartic acid per 1 mol of phenylalanine alkyl ester or its hydrochloride. The present invention relates to a method for producing a dipeptide ester, which comprises reacting

[0002]

2. Description of the Related Art The production of dipeptide esters using phenylalanine alkyl ester and N-substituted aspartic acid as raw materials is widely carried out as peptide synthesis utilizing the reverse reaction of proteolytic enzyme. Conventionally, in the production of dipeptide esters, the yield is improved by inducing and precipitating an addition compound of a product dipeptide ester and a raw material phenylalanine alkyl ester (JP-A-53-92729). Japanese Patent Laid-Open No. 54-9226).

That is, since the enzyme used for production is originally a degrading enzyme, the reaction equilibrium on the synthesis side is low. Therefore, a method of inducing an addition compound of a dipeptide ester and a phenylalanine alkyl ester, precipitating it as a crystal having low solubility, and taking out the product out of the system to shift the apparent equilibrium to the synthesis side is adopted.

[0004]

In this method, since the addition compound of the dipeptide ester and the phenylalanine alkyl ester is derived, the amount of the phenylalanine alkyl ester as the raw material is at least twice the amount of the other N-substituted aspartic acid as the raw material. It is necessary excessively. Although phenylalanine alkyl ester is expensive, it will be recovered and reused, but it is unstable in an aqueous solution, so about 10% of the amount input in the synthesis reaction step is lost due to decomposition, and it is also recovered in the subsequent recovery step. Approximately 10% of the amount thrown in was lost. Therefore, it cannot be denied that this is an uneconomical method since the recovery rate of expensive phenylalanine alkyl ester is poor.

[0005]

[Means for Solving the Problem] Therefore, contrary to the conventional method, we excessively add N-substituted aspartic acid stable in an aqueous solution to a reaction system to separate the product as a dipeptide ester itself. As a result, a phenylalanine alkyl ester to be recovered and reused was reduced, a decomposition amount thereof was reduced, and a method for producing a dipeptide ester capable of maintaining a high yield was found, and the present invention was completed. That is, the present invention is a method for producing a dipeptide ester, which comprises reacting 1 mol of phenylalanine alkyl ester or its hydrochloride with 2 mol or more of N-substituted aspartic acid.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION According to the production method of the present invention, 1 mol of a phenylalanine alkyl ester, for example, L-phenylalanine methyl ester (hydrochloride) is used with N-substituted aspartic acid, for example, N-benzyloxycarbonyl-L-. A dipeptide ester is produced without wasting L-phenylalanine methyl ester (hydrochloride) by reacting 2 parts or more of aspartic acid under known reaction conditions and separating it from the reaction system as the desired product dipeptide ester itself. can do.

That is, the solubility of the addition compound used in the conventional method in an aqueous solvent is as low as 5 mM, and the apparent reaction equilibrium was promoted to the synthesis side by utilizing the low solubility. The solubility of the dipeptide ester itself is 8 mM, which is slightly higher than that of the addition compound, but the absolute value is very low, and the dipeptide ester itself can be taken out of the system.

The effect of the present invention can be obtained by reacting 2 moles or more of N-substituted aspartic acid with 1 mole of phenylalanine alkyl ester or its hydrochloride, but a proteolytic enzyme that can be used for dipeptide ester synthesis is If the concentration of N-substituted aspartic acid is high, the reaction becomes unstable, so if the ratio is too high, the yield will decrease. Therefore, upon conducting a detailed study, the reaction molar ratio was preferably phenylalanine alkyl ester 1
It was revealed that a high yield can be maintained by setting the amount of N-substituted aspartic acid to 2 to 8 mol, more preferably 3.5 to 4.5 mol, based on mol.

Further, according to the production method of the present invention, since the amount of unreacted phenylalanine alkyl ester is very small, even if the cleaning liquid containing the phenylalanine alkyl ester is discarded without being recovered, substantially no phenylalanine alkyl ester is produced. There is no loss. In this case, one recovery process can be reduced, and labor and equipment can be significantly reduced.

In the production method of the present invention, a proteolytic enzyme is usually used as a catalyst. Examples of proteolytic enzymes that can be used include thermolysin, protease,
Prolysin, collagenase, crotalus astrotus protease, etc. are mentioned.

In the production method of the present invention, it is preferable to carry out the reaction in an aqueous solution under a pH condition at which the protease used is active. The pH may be any level as long as it can react with a proteolytic enzyme, but in order to suppress the decomposition of phenylalanine alkyl ester as a raw material,
Neutral to slightly acidic side, specifically 4 to 9 is preferable, 5
To 8 are more preferable. If necessary, a pH adjusting agent such as hydrochloric acid, acetic acid, sodium hydroxide, potassium hydroxide, sodium carbonate, ammonia or ethanolamine may be used.

The reaction temperature in the production method of the present invention is preferably a temperature at which the enzyme used as the catalyst shows activity. For example, the optimum temperature of thermolyticin, a proteolytic enzyme, is 65 ° C. Generally, a temperature of 40 ° C. or higher is desirable in order to proceed the enzyme reaction rapidly, but phenylalanine alkyl ester is easily decomposed at a high temperature, and therefore, a temperature of 40 ° C. or lower is desirable. The decomposition rate of the phenylalanine alkyl ester during the reaction decreases from about 10% at 40 ° C to about 2% at 10 ° C. Therefore, in order to suppress the decomposition rate of phenylalanine alkyl ester,
It can be said that the low temperature side is desirable. However, conversely, the time until the reaction is completed extends from about 6 hours at 40 ° C. to about 24 hours at 10 ° C. Therefore, the temperature of the reaction system is 10
In the range of -40 ° C, it is desirable to carry out the reaction on the low temperature side when the reduction of the decomposition rate of the phenylalanine alkyl ester is prioritized, and on the high temperature side when the reduction of the reaction end time is prioritized.

Specific steps are shown below. Phenylalanine alkyl ester as a raw material and N-substituted aspartic acid are dissolved in water, proteolytic enzyme is added, pH is adjusted and maintained at a constant temperature, and the precipitated dipeptide ester crystals are separated by a method such as filtration and washed with water. To do. Since the filtrate contains the protease and unreacted N-substituted aspartic acid, it is returned to the beginning of the process and reused as it is. The washing solution can also be reused by concentrating it.

The crystals thus obtained are mixed with an aqueous acid solution such as hydrochloric acid, filtered and washed again with water to remove a slight amount of the remaining phenylalanine alkyl ester, whereby a high-purity dipeptide ester is obtained. When the removed phenylalanine alkyl ester is reused, the filtrate is concentrated and used. When it is recovered as a solid, a conventional method such as solvent extraction, hydrochloride formation, and solvent precipitation is used.

When the washing liquid is concentrated, it may be concentrated under reduced pressure or concentrated by heating, but a concentration method using a membrane is preferable from the viewpoints of reducing energy cost and suppressing alteration and decomposition of the recovered material. As the membrane to be used, an ultrafiltration membrane, a nanofiltration membrane, a reverse osmosis membrane or the like is used as necessary.

[0016]

EXAMPLES The present invention will be specifically described below with reference to Examples and Comparative Examples. Example 1 Phenylalanine alkyl ester: N-substituted aspartic acid = 1: 4 (molar ratio, the same applies hereinafter) L-phenylalanine methyl ester hydrochloride 2.7 g,
N-benzyloxycarbonyl-L-aspartic acid 1
Weigh 3.4 g into a 200 mL beaker and water 68.
1 g was added, and while stirring, about 13.8 g of saturated aqueous sodium hydroxide was added to adjust the pH to 6.0, 0.3 g of the enzyme thermolysin was added, and the mixture was reacted at 40 ° C. with stirring. After 6 hours, a reaction product was precipitated in this solution to give a creamy form, so the precipitate was filtered (Toyo Roshi No.
2), the first washing with water (100 mL, 4 times).

This was mixed with 100 mL of 0.5N hydrochloric acid, stirred for 10 minutes, filtered (Toyo Filter Paper No. 2), second
It was washed with water (100 mL, 3 times) and dried to obtain 4.7 g of a white powder (85% yield based on L-phenylalanine methyl ester). When this white powder was compared with an N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester standard product by liquid chromatography, the purity was 99% or more.

The 0.5N hydrochloric acid filtrate and the second washing water were collected, neutralized, concentrated by a rotary evaporator, and mixed with ethyl acetate. The ethyl acetate layer was dried over sodium sulfate and then evaporated to dryness to recover L-phenylalanine methyl ester, and 0.11 g (recovery rate 4.8) was obtained.
%)Met. The amount of phenylalanine alkyl ester lost relative to the amount of dipeptide produced (mol%, the same applies below) was 6.3%.

Example 2 Phenylalanine alkyl ester: N-substituted aspartic acid = 1: 2 L-phenylalanine methyl ester hydrochloride 5.4 g,
N-benzyloxycarbonyl-L-aspartic acid 1
Weigh 3.4 g into a 200 mL beaker and water 63.
9 g was added, and about 15.3 g of saturated aqueous sodium hydroxide was added with stirring, and dipeptides were synthesized in the same manner as in Example 1 under all other conditions, and L-phenylalanine methyl ester was recovered. Finally dipeptide powder 6.
4 g was obtained (60% yield based on L-phenylalanine methyl ester), and 1.2 g (recovery rate 27%) of L-phenylalanine methyl ester was recovered. The amount of phenylalanine alkyl ester lost relative to the amount of dipeptide produced was 11%.

Example 3 Phenylalanine alkyl ester: N-substituted aspartic acid = 1: 8 L-phenylalanine methyl ester hydrochloride 1.4 g,
N-benzyloxycarbonyl-L-aspartic acid 1
Weigh 3.4 g into a 200 mL beaker and add 70.
2 g was added and about 13.0 g of saturated aqueous sodium hydroxide was added with stirring, and dipeptides were synthesized in the same manner as in Example 1 under all other conditions, and L-phenylalanine methyl ester was recovered. Finally dipeptide powder 1.
7 g was obtained (67% yield based on L-phenylalanine methyl ester), and 0.23 g (recovery rate 21%) of L-phenylalanine methyl ester was recovered. The amount of phenylalanine alkyl ester lost relative to the amount of dipeptide produced was 9.3%.

Comparative Example 1 Phenylalanine alkyl ester: N-substituted aspartic acid = 1: 1 L-phenylalanine methyl ester hydrochloride 10.8
g, 13.4 g of N-benzyloxycarbonyl-L-aspartic acid were weighed in a 200 mL beaker and water 5
5.4 g was added, and about 18.4 g of saturated sodium hydroxide aqueous solution was added with stirring. All other conditions were the same as in Example 1.
The dipeptide was synthesized in the same manner as in, and L-phenylalanine methyl ester was recovered. Finally, 10.3 g of dipeptide powder was obtained (48% yield based on L-phenylalanine methyl ester), and 3.4 g (recovery rate 38%) of L-phenylalanine methyl ester was recovered. The amount of phenylalanine alkyl ester lost relative to the amount of dipeptide produced was 15%.

Comparative Example 2 Phenylalanine alkyl ester: N-substituted aspartic acid = 2: 1 L-phenylalanine methyl ester hydrochloride 10.8
g, N-benzyloxycarbonyl-L-aspartic acid 6.7 g was weighed in a 200 mL beaker, and water 6 was added.
8.2 g was added, and about 12.3 g of saturated sodium hydroxide aqueous solution was added with stirring.
The dipeptide was synthesized in the same manner as in, and L-phenylalanine methyl ester was recovered. Finally, 9.4 g of dipeptide powder was obtained (44% yield based on L-phenylalanine methyl ester), and 3.7 g (recovery rate 41%) of L-phenylalanine methyl ester was recovered. The amount of phenylalanine alkyl ester lost relative to the amount of dipeptide produced was 16%.

The above results are shown in the following table. Table 1

As described above, according to the production method of the present invention, the yield of dipeptide based on phenylalanine alkyl ester can be improved. In addition, the dipeptide can be produced without losing the expensive raw material phenylalanine alkyl ester.

[0025]

The effect of the present invention is to provide a novel method for producing a dipeptide ester. According to the manufacturing method of the present invention, it is possible to efficiently manufacture a target product without loss of raw materials.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kenji Fukushima 2087-8 Negishi, Yasuyuki Ryogishi, Kawaguchi City, Saitama Prefecture Cotto Negishidai C (72) Inventor Yasuyuki Isono 2-176-1-B403 (72) Akayamacho, Koshigaya City, Saitama Prefecture ) Inventor Akira Hoshino 3-4 Koshigayahoncho, Koshigaya City, Saitama Prefecture

Claims (4)

[Claims] 1. A process for producing a dipeptide ester, which comprises reacting 1 mol of a phenylalanine alkyl ester or its hydrochloride with 2 mol or more of N-substituted aspartic acid. 2. The method for producing a dipeptide ester according to claim 1, wherein a proteolytic enzyme is used as a catalyst in an aqueous solution. 3. The pH in the reaction system is 4-9.
Or the method for producing a dipeptide ester according to item 2. 4. The method for producing a dipeptide ester according to claim 1, 2 or 3, wherein the temperature in the reaction system is 10 to 40 ° C.