JPH0285294A - Preparation alpha-l-aspatyl-l-phenylalanine methyl ester having improved solubility - Google Patents

Abstract

PURPOSE:To prepare the subject substance having improved solubility in water without requiring any special apparatus or means by purifying alpha-L-aspartyl-L- phenylalanine methyl ester(salt) is the presence of a specific additive such as sodium alginate. CONSTITUTION:When alpha-L-aspartyl-L-phenylalanine methyl ester(salt) is purified from an aqueous medium, the purification is carried out in the presence of one or more of additives selected from sodium alginate, carboxymethylcellulose sodium salt, starch glycolic acid sodium salt and polyacrylic acid sodium salt preferably in an amount of 0.01-3wt.%.

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention provides α-L-aspartyl-
L-phenylalanine mediyl ester (hereinafter α-APM)
(abbreviated as)).

More specifically, when purifying α-APM from an aqueous medium, purification is performed in the presence of one or more additives selected from the group consisting of sodium alginate, sodium carboxymethyl cellulose, sodium starch glycolate, and sodium polyacrylate. The present invention relates to a method for producing α-APM with improved solubility.

α-APM is widely known as a dipeptide sweetener. It has good sweetness characteristics and a sweetness level nearly 200 times that of sucrose, and its demand as a dietary sweetener is growing rapidly.

(Prior art and problems) α-APM is a dipeptide compound consisting of L-aspartic acid and L-phenylalanine methyl ester, and its production methods are broadly divided into chemical methods and biochemical methods using microorganisms. Various methods have been disclosed.

For example, a chemical production method is to protect the amino group, condense monoaspartic acid anhydride and L-phenylalanine methyl ester in a suitable medium, and then remove the protecting group by a conventional method. (For example, U.S. Patent 3
．． 786□039) is one of the typical manufacturing methods. In addition, as a biochemical method, N-benzyloxycarbonyl-cycloaspartic acid and L-phenylalanine methyl ester are condensed in the presence of metalloprotease.
Benzyloxycarbonyl-alpha-aspartyl L-
A method of producing phenylalanine methyl ester and then removing the benzyloxycarbonyl group by catalytic reduction can be mentioned. When producing α-APM industrially, no matter which production method is used, when isolating α-APM from the reaction mass to produce the final product,
A step to purify crude α-APM is essential. This purification step is generally performed by recrystallization from water or a water-containing lower alcohol (hereinafter, water and a water-containing solvent will be referred to as an aqueous medium).

Depending on the quality of crude ct-APM, in an aqueous medium,
There is also a method of removing impurities by stirring in a suspended state. However, α-APM obtained by such a purification method forms a hard block when dried, and therefore, it is necessary to grind it into a product, which tends to cause difficulties in handling.

In addition, it takes a long time to dry, and as a result, diketopiperazine compounds, which are intramolecular cyclization products of α-APM, tend to increase, making this method problematic in terms of obtaining products with stable quality. I have no choice but to.

In addition, α-APM obtained by this conventional recrystallization purification method
has the disadvantage that the solubility (dissolution rate) of the product in water is poor. For example, 50% methanol aqueous solution (
In the case of α-APM obtained by recrystallization from 250! @! When the solubility of α-APM was investigated by adding it to water under stirring at room temperature and repeating stirring and standing still every 30 seconds, it was found that α-APM remained undissolved even after 5 minutes.
There is a considerable amount left behind, and it will take 1 hour to completely dissolve.
It takes more than 5 minutes. Furthermore, the solubility of α-APM obtained by recrystallization and purification from water is approximately the same.

The solubility of α-AP~1 in water is an important factor in determining product specifications, as most of the demand for α-APM as a sweetener is for use in soft drinks. It is clear that this will happen. There is little prior art regarding improving the solubility of α-APM drug substances. JP-A-58-1
No. 77952 discloses that when α-APM is cooled and crystallized from an aqueous solution, an aqueous solution with an initial M concentration of 2 to 10% by weight is controlled by conductive heat transfer without applying forced flow such as mechanical stirring. Cool to the bottom, and the overall appearance will be that of upper frozen confectionery (
After forming a sherbento-like pseudo-solid phase, the crystals of α-APM are improved by further cooling as necessary, which in turn improves filterability and powder properties such as bulk specific gravity. . And this one? ! It is stated that α-APM obtained by = is also superior in solubility compared to conventional products. However, although this method does significantly improve various powder properties including solubility, it does not require a method of cooling and crystallizing without applying forced flow such as mechanical stirring during crystallization from an aqueous solution. Since the scale of the crystallizer is large, it takes a very long time to cool down, which is a limit for industrial use.Therefore, this prior art In this paper, the maximum distance from the cooling surface to the object to be cooled is specified, and a special crystallization device corresponding to the maximum distance is specified.In this way, the method of JP-A-58-177952 uses a special crystallization device. Unless it is done, it cannot be an industrial method.

(Means for Solving the Problems) The present invention provides a method for purifying α-APM from an aqueous medium using one member selected from the group consisting of sodium alginate, sodium carboxymethyl cellulose, sodium starch glycolate, and sodium polyacrylate. This is a method for producing αAPM with improved solubility, which comprises performing a purification operation in the presence of the above-mentioned additives, followed by surface liquid separation to recover α-APM.

The α-APM used in the method of the present invention may be free or may be a mineral acid or sulfonic acid salt. Furthermore, the manufacturing method is not limited, and α-APM manufactured by the method of Wlh is used. When a salt of α-APM is used, an inorganic base is added to the solution of the salt of α-APM in an aqueous medium or to the same solution to adjust the pH of the α-APM salt.
After adjusting the isoelectric point of M, as in the case of free α-APM, 1 selected from the group consisting of sodium alginate, sodium carboxymethyl cellulose, sodium starch glycolate, and sodium polyacrylate.
This can be done by adding more than one type of additive.

Salts of α-APM include, for example, mineral acid salts such as hydrochloride, sulfate, phosphate, nitrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, trifluoromethane salt, etc. Examples include aliphatic sulfonates or aromatic sulfonates such as sulfonates and naphthalene sulfonates, and preferably hydrochlorides, sulfates, and methanesulfonates are used.

In the method of the present invention, an aqueous medium containing usually water or a lower alcohol such as methanol, ethanol, isopropanol or tertiary butanol is used as a solvent. Of course, it is also possible to use other water-miscible organic solvents within a range that does not impair the purpose of the present invention. When an aqueous medium containing a lower alcohol is used as a solvent, it can be used without any particular problem as long as the concentration of the lower alcohol is up to about 60% by weight. There are no particular restrictions on the amount of these aqueous media used, but from the standpoint of volumetric efficiency and workability,
Usually, it is used in a range of 3 to 50 times the weight of α-APM.

The additive used in the method of the invention is a water-soluble additive selected from the group consisting of sodium alginate, sodium carboxymethyl cellulose, sodium starch glycolate and sodium polyacrylate, comprising:
These additives are usually used alone, but there is no problem in using a mixture of two or more. If the amount of additive used is too small, the solubility (dissolution rate) of the purified α-APM will not be improved, and if it is too large, the solid-liquid separation property will deteriorate. .01 to 3% by weight, preferably 0.02 to 2% by weight.

These additives may be added in solid form, or may be added after being dissolved or suspended in water to an appropriate concentration.

The method of the present invention is characterized in that the purification treatment is carried out in the presence of the above-mentioned additives, and the purification method is not particularly limited. Examples include a method of crystallizing and purifying α-APM from an aqueous medium, or a method of treating α-APM in an aqueous medium in a substantially suspended state. α
- When crystallizing and purifying APM, first add α-AP to an aqueous medium.
Dissolve M. If there are insoluble matters, they are removed by filtration. Thereafter, a crystallization operation is performed under normal stirring conditions. The above-mentioned additives may be added beforehand, or may be added to the solution after α-APM is dissolved. Alternatively, the object of the present invention can also be achieved by adding it to the liquid during or after crystallization.

In this crystallization purification method, the temperature at which α-APM is dissolved in the aqueous medium is usually preferably 70° C. or lower in consideration of the thermal stability of the α-APM solution. If it is dissolved at a temperature higher than this, a diketopiperazine compound will be produced as a by-product, which is not preferable.

The dissolved concentration of α-APM is not particularly limited, and is usually 2% by weight.
It can be arbitrarily selected within the range from to the saturation solubility at the melting temperature.

In the case of a method in which α-APM is stirred in an aqueous medium in a substantially 2.4 state in the presence of the additive, the amount of the aqueous medium is not particularly limited as long as it is the amount necessary for purifying the crude α-APM. Furthermore, the treatment temperature can be arbitrarily selected within the range of 0 to 60°C.

On the other hand, when using a salt of α-APM as a raw material, the salt of α-APM is dissolved in an aqueous medium. If there are insoluble substances, they are removed by filtration. Thereafter, an inorganic base is added under normal stirring conditions to adjust the pH to the isoelectric point of α-APM. Thereafter, it can be purified by the same treatment as in the case of free α-APM.

Although the isoelectric point of α-APM is 5.6, the object of the present invention can be achieved even if the value is adjusted to a value near this value.

Examples of the inorganic base used in the method of the present invention include sodium hydrogen carbonate, potassium hydrogen carbonate, sodium carbonate, potassium carbonate, aqueous ammonia, and the like.

In this method, the temperature at which the mineral acid and organic sulfonate of α-APM are dissolved in the aqueous medium is determined by considering the thermal stability of the mineral acid and organic sulfonate of α-APM. Preferably 50″C or less, usually 25-30°C
It is. If it is dissolved at a temperature higher than this, a diketopiperazine compound will be produced as a by-product, which is not preferable.

The dissolved concentration of the mineral acid and organic sulfonate of α-APM is not particularly limited, and can be arbitrarily selected within the range of usually 2% by weight to the saturation solubility at the dissolution temperature.

(Function) α-APM obtained by the method of the present invention exhibits good solubility in water. Furthermore, during drying, there is relatively little formation of hard lumps as seen in ordinary crystallization purification, and handling after drying is easy.

(Example) Hereinafter, the present invention will be explained in detail with reference to Examples.

In addition, the solubility test in the examples was carried out by the following method.

[Solubility test method] 250 mg of a sample of pulverized α-APM was mixed with 26 ± 2
Pour the sample into 25°C pure water with stirring (stir at 350-360 rpm using a Mag-Futisoku cooler), repeat stirring and standing still every 30 seconds, and wait until the sample is completely dissolved. Measure.

Example 1 22 g of crude α-APM (purity 95%) was added to 40% (volume %)
) Methanol water was put into 420 g of the liquid, heated to 60°C to dissolve, and hot filtered to remove an amount of insoluble matter. 10. An aqueous solution in which sodium carboxymethyl cellulose was previously dissolved at a concentration of 1% by weight in the obtained solution.
After adding 0 g, the mixture was cooled with stirring and crystallized.

After cooling to 5°C, it was filtered and washed with cold water.

The obtained wet cake was dried at 50-60°C to obtain 17.8 g of purified α-APM. Purity analysis result by high performance liquid chromatography: purity 97.2
%Met. Also, the drying loss (105°C/4 hours) is 3
．． It was 2%.

A solubility test was conducted on the α-APM crystals obtained here, and as a result, they were completely dissolved within 5 minutes.

Example 2 22g of crude α-APM (purity 95%) at 10% (vol%)
Methanol water i6? 480g of the mixture was heated to 62°C to dissolve, and hot filtration was performed to remove trace amounts of insoluble matter.

The resulting solution was cooled to 30°C with stirring to obtain α-AP.
M was crystallized. Thereafter, 10.0% of an aqueous solution in which sodium carboxymethyl cellulose was dissolved at a concentration of 1% by weight.
After adding g and stirring at the same temperature for 1 hour,
The precipitated crystals were filtered, washed with a small amount of cold water, and dried at 50 to 60°C.

Yield Wk 17.6 g Purity 96.8% Loss on drying 3.6% A solubility test of the α-APM obtained here revealed that it was completely dissolved within 5 minutes.

Comparative Example 1 Crystallization purification was carried out in the same manner as in Example 1, except that sodium carboxymethyl cellulose was not added. The obtained α
-The purity of APM was almost the same, but as a result of a solubility test, it took 12.5 minutes to completely dissolve.

Comparative Example 2 Crystallization and purification was carried out in the same manner as in Example 2, except that sodium carboxymethyl cellulose was not added. The obtained α
-The purity of APM was almost the same, but as a result of a solubility test, it took 11.5 minutes to completely dissolve.

Example 3 36.6 g of α-APM hydrochloride dihydrate was added to 367.5 g of water.
g, dissolved at 25-30°C, and filtered to remove trace amounts of insoluble matter. 25-30 °C in the resulting solution
While stirring, add 6.4 g of 28% aqueous ammonia to pH 5.
, 6. Next, an aqueous solution 1 in which sodium carboxymethyl cellulose was dissolved in advance to a concentration of 1 M%
4.7g was added. The mixture was then cooled to 5°C with stirring, filtered and washed with cold water. 50% of the resulting wet cake
By drying at ~55 °C, 27.9 g of α-A
Got PM. As a result of analysis by high performance liquid chromatography, the purity was 97.2%.

The loss on drying (105°C/4 hours) was 3.2%.

A solubility test was conducted on the α-APM crystals obtained here, and as a result, they were completely dissolved within 5 minutes.

Example 4 39.2 g of α-APM sulfate was added to 420 g of water, dissolved at 25 to 30°C, and filtered to remove trace amounts of insoluble matter. Add 28% ammonia water 12 to the obtained solution at the same temperature.
．． 4 g was added and the pH was adjusted to 5.6. Next, 14.7 g of an aqueous solution in which sodium carboxymethyl cellulose was dissolved at a concentration of 1% by weight was added and stirred at the same temperature for 1 hour, then cooled to 5°C and precipitated crystals were filtered.
After washing with a small amount of cold water, it was dried at 50-55°C.

Yield 26.5 g, purity 97.0%, loss on drying (10
5°C/4 hours) was 3.6%.

A solubility test was conducted on the α-APM crystals obtained here, and as a result, they were completely dissolved within 5 minutes.

Comparative Example 3 Crystallization purification was carried out in the same manner as in Example 3, except that sodium carboxymethyl cellulose was not added.

The purity of the obtained α-APM crystals was almost the same as in Example 3, but a solubility test showed that it took 12.5 minutes to completely dissolve.

Comparative Example 4 Crystallization purification was carried out in the same manner as in Example 4, except that sodium carboxymethyl cellulose was not added.

The purity of the obtained α-APM crystals was almost the same as in Example 4, but as a result of a solubility test, it took 13.5 minutes to completely dissolve.

Examples 5 to 10, Comparative Example 5.6 Conditions such as medium composition, α-APM fi degree, amount and timing of addition of sodium carboxymethyl cellulose during crystallization and purification of crude α-APM (purity 95%) are shown in Table-1. Crystallization purification was performed in the same manner as in Example 1 with various changes as shown in Figure 1, and the results are summarized in Table 1.

(Left below) Example 11 22g of crude a-APM (purity 95%) was added to 40% (volume %)
) The mixture was poured into 420 g of an aqueous methanol solution, heated to 60°C to dissolve, and trace amounts of insoluble matter were removed. The obtained solution was cooled while stirring to perform crystallization. To this crystallization mixture, 4.0 g of a 2% aqueous solution of sodium alginate was added and stirred at 25°C for 1 hour, then cooled to 5°C and filtered with suction. After washing with a small amount of cold water, it was dried at 50-60''C.

Yield 17.7 g Purity 96.8% Dry area N3.8
As a result of the % solubility test, it was completely dissolved in 6.5 minutes.

Example 12 22 g of crude α-APM (purity 95%) was added to 10% (volume %)
) The mixture was poured into 460 g of an aqueous methanol solution, heated to 60"C to dissolve, and trace amounts of insoluble matter were removed. The resulting solution was cooled with stirring to perform crystallization.

5.0 g of a 1% aqueous solution of sodium starch glycolate was added to this crystallization mixture, and the mixture was stirred at 25°C for 1 hour. Thereafter, it was cooled to 5°C and filtered with suction.

After washing with a small amount of cold water, it was dried at 50-60°C.

Yield 17.8 g Purity 97.2% Loss on drying 3.4
% α-APM obtained here has a solubility test result of 5.5
It completely dissolved in minutes.

Example 13 α-AP containing 22 g (95% purity) of α-APM
Pour the wet cake of M into 180g of water and add 1% by weight.
An aqueous solution of sodium carboxymethyl cellulose of 10
g and stirred at room temperature for 2 hours.

Thereafter, the precipitated crystals were filtered, washed with a small amount of water, and dried at 50 to 60°C.

Yield 18.9 g Purity 97.0% Loss on drying 3.6
% α-APM obtained here has a solubility test result of 5.5
It completely dissolved in minutes.

Comparative Example 7 The same procedure as in Example 13 was conducted except that sodium carboxymethyl cellulose was not added. The purity of the obtained α-APM was almost the same as in Example 13, but as a result of a solubility test, it took 13.5 minutes to completely dissolve.

Example 14 αAPM containing 22g (95% purity) of α-APM
The wet cake was poured into 180 g of water and stirred at room temperature for 1 hour to sufficiently loosen the crystals. Thereafter, 1.0 g of a 1% by weight aqueous solution of carboxymethyl cellulose sodium salt was added and stirred at room temperature for an additional 2 hours. Thereafter, the precipitated crystals were filtered, washed with a small amount of water, and dried at 50 to 60°C. Yield 1 18.7g Purity 96.8% Loss on drying 3.8% As a result of a solubility test, the obtained crystals were completely dissolved in 6.5 minutes.

Example 15 1 wt% sodium polyacrylate in place of sodium carboxymethylcellulose in 1 volume of water, night 4, Og
The same procedure as in Example 14 was carried out except that .

The obtained α-APM purity was almost the same as in Example 13, but as a result of the cold piece test, it was completely dissolved in 5.5 minutes.

Example 16 36.6 g of α-APM/hydrochloride dihydrate was put into 367.5 g of 10% (volume %) methanol water 78 liquid,
It was dissolved at 25-30°C and filtered to remove traces of insoluble matter. 28% ammonia water under stirring at 25-30”CtW6
．． After adding 4 g of the solution and adjusting the pH to 5.6, 10.0 g of an aqueous solution in which sodium carboxymethyl cellulose was dissolved in advance at a concentration of 1% by weight was added, and the mixture was heated with stirring for 5.5 g.
Cooled to °C, then filtered and washed with cold water.

The resulting wet cake was dried at 50-55°C to obtain 27.5 g of α-APM. As a result of analysis by high performance liquid chromatography, the purity was 97.2%. The loss on drying (105°C/4 hours) was 3.2%.

Finally, a solubility test was conducted on the α-APM crystals obtained, and the crystals were completely dissolved within 5 minutes.

Example 17 36.6 g of α-APM hydrochloride dihydrate was added to 367 g of water.
5 g, melted at 25-30°C, and filtered to remove trace amounts of insoluble matter.

6.4 g of 28% ammonia water was added to the resulting solution under stirring at 25 to 30°C, and the pH was adjusted to 5.6.
The temperature was raised to ~60°C and stirred at the same temperature for 1 hour. 25-3
After stirring and cooling to 0°C, 2.9 g of an aqueous solution in which sodium carboxymethyl cellulose was dissolved at a concentration of 1% by weight.
After stirring at the same temperature for 1 hour, precipitated crystals were filtered and washed with a small amount of cold water.

The obtained wet cake was dried at 50 to 60°C to obtain 26.0 g of α-APM.

As a result of analysis by high performance liquid chromatography, the purity was 9.
It was 7.2%. The loss on drying (105'C/4 hours) was 3.2%.

A solubility test was conducted on the α-APM crystals obtained here, and as a result, they were completely dissolved within 5 minutes.

Example 18 Example 3 except using 8.8 g of sodium bicarbonate
I did the same thing. Yield 5 tons 27.0 g, purity 97.0%
, drying range it (105°C/4 hours) was 3.6%. As a result of a dissolution test, complete dissolution occurred within 5 minutes.

Examples 19 to 22 The same procedure as Example 17 was conducted except that the amount of water and additives shown in Table 2 were used. The results are shown in Table-2.

Example 23 Crystallization and purification was carried out in the same manner as in Example 3, except that 39.0 g of the methanesulfonate of α-APM was used.

Yield: 27.3 g.

Purity 97 as analyzed by high performance liquid chromatography
．． It was 1%. Also, drying loss (105°C/4 hours)
was 3.2%.

A solubility test was conducted on the α-APM crystals obtained here, and as a result, they were completely dissolved within 5 minutes.

The method of the present invention does not require special equipment or means, and crystallization is carried out under stirring conditions using a crystallizer equipped with ordinary stirring means, or the stirring treatment is carried out in a substantially suspended state. This method has great industrial advantages because α-L-aspartyl-L-phenylalanine methyl ester with significantly improved water solubility (dissolution rate) can be obtained.

Claims (1)

[Claims] 1) When purifying α-L-aspartyl-L-phenylalanine methyl ester or its salt from an aqueous medium, a group consisting of sodium alginate, sodium carboxymethyl cellulose, sodium starch glycolate, and sodium polyacrylate is used. A method for producing α-L-aspartyl-L-phenylalanine methyl ester with improved solubility, the method comprising purifying it in the presence of one or more additives selected from the following. 2) A patent claim in which a mineral acid and an organic sulfonate of α-L-aspartyl-L-phenylalanine methyl ester are adjusted to the isoelectric point of α-L-aspartyl-L-phenylalanine methyl ester with an inorganic base in an aqueous medium. Manufacturing method according to scope 1 3) The amount of the additive added is 0.01 to 3% by weight based on the α-L-aspartyl-L-phenylalanine methyl ester or salt thereof to be treated. The manufacturing method described in paragraph 1. 4) The manufacturing method according to claim 1 or 2, wherein the purification method is crystallization purification. 5) The manufacturing method according to claim 1 or 2, wherein the purification method is carried out by stirring in a substantially suspended state in an aqueous medium.