JPH0243756B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for recovering an adduct compound of a dipeptide ester and an amino acid ester, and more particularly to a method for extracting this adduct compound from an aqueous suspension containing this compound with tetrahydrofuran and recovering it. It is. Addition compounds of dipeptide esters, such as N-benzyloxycarbonyl-α-L-aspartyl-L-phenylalanine lower alkyl esters, and amino acid esters, such as phenylalanine lower alkyl esters and valine lower alkyl esters, are sweeteners. It is a compound useful as an intermediate to a certain α-L-aspartyl-L-phenylalanine lower alkyl ester or as an intermediate for optical resolution of racemic amino acid ester. Such addition compounds can be prepared by reacting an N-protected amino dicarboxylic acid with an amino acid ester in an aqueous medium in the presence of a proteolytic enzyme (as described in
53-92729, JP 54-9226), or reacting a dipeptide ester and an amino acid ester in a solvent such as water (JP 55-19234, JP 1987-1929).
55-73644) etc. In these reactions, the adduct precipitates into the solvent in the form of a solid phase.
Therefore, how to efficiently recover this adduct from such a solvent, especially an aqueous medium, is an extremely important issue. In the above-mentioned known methods, this recovery is carried out in vain. It is also known that an organic solvent capable of forming two phases with water is added to the reaction-completed solution to dissolve and extract the adduct and separate it as a homogeneous liquid in the organic solvent (Japanese Patent Application Laid-Open No. 11295-1982). . However, in order to perform extraction efficiently, it is necessary to use an organic solvent that can form two phases with water and has a high dissolving power for the addition compound. and chloroform, alkyl halides such as ethane dichloride, etc. are relatively limited. However, esters have problems with hydrolysis, and halogenated alkyls have recently become a carcinogenic problem, so they must be treated as much as possible in the process of processing addition compounds that are used as raw materials for foods, etc. It is advisable to avoid its use. On the other hand, in the case of an organic solvent having a small dissolving power for the adduct, it is necessary to use a large amount. In order to solve these problems, the inventors of the present invention have intensively investigated a more industrially advantageous method for separating the adduct, and found that tetrahydrofuran, which is naturally miscible with water, can separate the adduct. They discovered that when dissolved, they do not mix freely with water and form two phases with water, and have completed the present invention. That is, the present invention is based on the general formula Tetrahydrofuran is added to an aqueous mixture containing an adduct compound of a dipeptide ester and an amino acid ester represented as a solid phase and mixed to form a two-liquid phase of a tetrahydrofuran phase containing this adduct compound and an aqueous phase. The present invention provides a method for recovering an adduct, which comprises separating the adduct from the aqueous phase and recovering the adduct as a solution in tetrahydrofuran. In the general formula (), R ₁ and R ₄ are lower alkyl groups such as methyl group and ethyl group, R ₂ and R ₃ are isopropyl groups, side chain groups of amino acids such as benzyl group,
X is a benzyloxycarbonyl group, a benzyloxycarbonyl group which may have a substituent such as a p-methoxybenzyloxycarbonyl group, and n is 1 or 2. An aqueous mixture containing an adduct compound represented by the general formula () in the solid phase has the general formula Amino acid ester and general formula represented by It can be prepared by reacting the N-protected aminodicarboxylic acid represented by in an aqueous medium in the presence of a protease to precipitate an addition compound represented by the general formula () in the aqueous medium. In general formulas () and (), R ₁ , R ₂ , R ₃ ,
R ₄ , X and n have the same meanings as in the general formula (). However, in this case R ₃ is R ₂ and R ₄ is R ₁
It is common to In addition, the addition compound represented by the general formula (), the amino acid ester represented by the same (), and the N-protected aminodicarboxylic acid represented by the same () below refer to the addition compound, amino acid ester, and N-protected aminodicarboxylic acid, respectively. say. The preparation of adduct compounds by the above method is described in JP-A-Sho.
The known conditions described in JP 53-92729 and the like may be followed. Examples of these conditions are as follows.

[Table] In this method, the amino acid ester and the N-protected aminodicarboxylic acid are each used in the L-form or a mixture of the L-form and the D-form. When L-form is used as amino acid ester, LL
- type dipeptide ester and L-type amino acid ester, and when a mixture of L-type and D-type is used, LL-type dipeptide ester and D
An addition compound with an amino acid ester of the -form or a mixture of the D-form and the L-form is produced. The aqueous mixture of the present invention comprises an amino acid ester and a general formula It can be prepared by reacting a dipeptide ester represented by in an aqueous medium. The addition compound obtained in this way is
It can be used for the optical resolution of DL-amino acid esters, as disclosed in Japanese Patent Application Laid-open No. 19234 and Japanese Patent Application Laid-open No. 73644/1983. In the thus obtained aqueous mixture, which is originally freely miscible with water in the absence of the third component, when this addition compound is dissolved, that is, in the presence of the addition compound, it is not freely miscible with water and is mixed with water. When tetrahydrofuran is added, forming two phases, this organic solvent dissolves the adduct and forms a phase separate from the aqueous phase. Since this tetrahydrofuran phase contains most of the adduct, the adduct can be recovered in the form of a tetrahydrofuran solution by separating this tetrahydrofuran phase from the aqueous phase. Tetrahydrofuran used in the present invention may be mixed with other organic solvents as long as its action is not substantially hindered. The amount of tetrahydrofuran used in the present invention is generally about 2.5 to about 25 parts by weight, preferably about 5 parts by weight, per 1 part by weight of the addition compound.
or about 15 parts by weight. The amount of water in the aqueous mixture is about 0.5 to about 5 parts by weight per 1 part by weight of the water. Therefore, the content of the additional compound in the aqueous liquid mixture is adjusted as necessary so as to ensure these quantitative ratios. Tetrahydrofuran used in the present invention can dissolve an extremely large amount of the adduct compound compared to organic solvents that can form two phases with water in the absence of the adduct compound. Therefore, a small amount thereof can be used to extract the adduct. In the method of the present invention, the temperature at which the aqueous mixture containing the addition compound and tetrahydrofuran are brought into contact is usually about 0 to about 65°C. However, if the purpose is to separate the two phases formed and recover the remaining enzyme from the aqueous phase, it is desirable to carry out the mixing at a temperature of about 5 to about 50°C. The mixing time and the two-phase separation time are not particularly limited, and the mixing time can usually be about one hour or less. The tetrahydrofuran phase containing the adduct and the aqueous phase can be separated by conventional means such as those used in liquid-liquid extraction. Most of the unreacted amino acid esters, N-protected aminodicarboxylic acids, enzymes, etc. that remain in the aqueous phase in the reaction for producing the adduct as described above remain in the aqueous phase, so it is possible to separate the adduct from them. can. The adduct can be isolated from the separated tetrahydrofuran phase by conventional means, for example, by removing tetrahydrofuran etc. by evaporation. Alternatively, the tetrahydrofuran phase containing the addition compound can be immediately subjected to the step of removing the protecting group (X in the general formula ()) of the amino group of the addition compound. When an aqueous mixture is prepared by generating an adduct compound through the enzymatic reaction described above, most of the enzyme used in the reaction and still active is contained in the aqueous phase from which the tetrahydrofuran phase containing the adduct compound is separated. This aqueous phase can be used again by concentrating and recovering the enzyme using an ultrafiltration membrane or the like. Also,
The enzyme can also be used after being separated from the aqueous phase by other conventional means, such as salting out.
The recovered enzyme solution, together with the unreacted amino acid ester and N-protected aminodicarboxylic acid, can be used as a raw material for the next addition compound production reaction. As is clear from the above description, according to the present invention, it is possible to efficiently separate the adduct in an aqueous mixture from other components in the form of a highly concentrated solution in tetrahydrofuran. Furthermore, the amount used is significantly smaller than in the case of extraction with conventional organic solvents.
Industrially advantageous. Furthermore, the enzyme can be recovered and reused from the aqueous phase, which is economically advantageous. Hereinafter, the present invention will be explained in more detail with reference to Examples. Example 1 2.67 g of N-benzyloxycarbonyl-L-aspartic acid and 5.39 g of DL-phenylalanine methyl ester hydrochloride were placed in a 100 ml flask, and 20 ml of evaporated water, 5 ml of 5N aqueous sodium hydroxide solution and crude thermolysin (Thermoase PS −160,
1.2 g (trade name, manufactured by Daiwa Kasei Co., Ltd.) and 0.06 g of calcium acetate hydrate were added, and the mixture was reacted at 40° C. with stirring.
After 3 hours, a suspension-like reaction mixture was obtained. 50 ml of tetrahydrofuran was added to this liquid, and the mixture was stirred and mixed at 40°C for 10 minutes. The two phases formed 10 minutes after the end of stirring were separated, and the upper tetrahydrofuran phase was transferred to a rotary evaporator and evaporated to dryness. After drying the residue, it was recrystallized from a mixed solvent of ethyl acetate and n-hexane to obtain N-benzyloxycarbonyl-α-L-aspartyl-L-phenylalanine methyl ester (hereinafter referred to as Z-APM). 5.01 g (yield: 82.4%) of a 1:1 addition compound mainly with D-phenylalanine methyl ester (hereinafter referred to as D-PM) was obtained. This crystal is Z-APM and mainly D-PM 1:
It was confirmed that the compound was an addition compound of No. 1 because its NMR, IR, elemental analysis, and optical rotation were the same as those disclosed in JP-A-53-92729. On the other hand, when the enzyme activity in the aqueous phase after two-phase separation was measured by the casein digestion method, it was found that there was a residual activity of 86% of the charged enzyme. Example 2 L-phenylalanine methyl ester hydrochloride was used instead of DL-phenylalanine methyl ester hydrochloride, and the amount of crude thermolysin used was changed.
The reaction and post-treatment were carried out in the same manner as in Example 1, except that the amount was 0.36 g and the reaction time for the peptide production and adduct formation reactions was 8 hours. From the tetrahydrofuran phase, after evaporation and recrystallization,
An addition compound of Z-APM and L-phenylalanine methyl ester (hereinafter referred to as L-PM)
4.99g (yield 82.1%) was obtained. It was confirmed that this is a 1:1 addition compound of Z-APM and L-PM because the NMR, IR, elemental analysis, and optical rotation data are the same as those disclosed in JP-A-53-92729. did. On the other hand, when the enzyme activity in the aqueous phase was measured by the casein digestion method, it was found that there was a residual activity of 83% of the charged enzyme. Example 3 Thermolysin instead of crude thermolysin
The reaction and post-treatment were carried out in the same manner as in Example 1 except that 300 mg was used, and Z-APM and mainly D-
5.11g (yield 84.2%) of an adduct with PM was obtained.
On the other hand, when the enzyme activity in the aqueous phase was measured by the casein digestion method, it was found that there was a residual activity of 87% of the charged enzyme. Example 4 1 g of PS-protease instead of thermolysin
The reaction and post-treatment were carried out in the same manner as in Example 3, except that 4.94 g (yield: 81.3%) of an adduct of Z-APM and mainly D-PM was obtained. On the other hand, when the enzyme activity in the aqueous phase was measured by the casein digestion method, it was found that there was a residual activity of 84% of the charged enzyme.

Claims (1)

[Claims] 1. General formula An adduct compound _of a dipeptide _{ester and} an amino _acid ester represented by and n is 1 or 2) in the solid phase, tetrahydrofuran is added and mixed to form two liquid phases of tetrahydrofuran containing this addition compound and an aqueous phase, and the tetrahydrofuran phase is converted into an aqueous phase. A method for recovering an adduct compound of a dipeptide ester and an amino acid ester, the method comprising separating the adduct compound from the dipeptide ester and recovering the adduct compound as a solution in tetrahydrofuran. 2. The recovery method according to claim 1, wherein tetrahydrofuran is used in an amount of about 2.5 to about 25 parts by weight per part by weight of the adduct. 3. The recovery method according to claim 1 or 2, wherein tetrahydrofuran is added in an amount of about 0.5 to about 5 parts by weight per 1 part by weight of water in the aqueous mixture. 4. The recovery method according to any one of claims 1 to 3, wherein the dipeptide moiety of the adduct is LL-type. 5 Aqueous mixture is general formula An amino acid ester represented by (in the formula, R ₁ is a lower alkyl group, R ₂ is a side chain group of the amino acid) and a general formula An N-protected aminodicarboxylic acid represented by (in the formula, X is a benzyloxycarbonyl group that may have a substituent, and n is 1 or 2) is reacted in an aqueous medium in the presence of a protease. general formula _An addition compound of a dipeptide _{ester and an} amino acid ester _{represented by}
The recovery method according to any one of claims 1 to 4, wherein the reaction product solution is a reaction product solution in which the same group as . 6. The recovery method according to claim 5, wherein R1 and R4 in the general formula of the addition compound, amino acid ester, and N-protected aminodicarboxylic acid are methyl groups, _R2 and _R3 are benzyl groups, and n is 1. . 7. The recovery method according to claim 5 or 6, wherein the amino acid ester and N-protected aminodicarboxylic acid used are each independently of the L-type or a mixture of the L-type and the D-type. 8. The recovery method according to any one of claims 5 to 7, wherein the protease used is a metalloprotease. 9. The recovery method according to any one of claims 5 to 8, which recovers a protease from an aqueous phase.