JPH0212239B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for treating an addition compound of a dipeptide ester and an amino acid ester. More specifically, it relates to a method in which an aqueous suspension containing an addition compound of a dipeptide ester and an amino acid ester is separated using an organic solvent and then decomposed with an acid to separate both components. 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, can be used as sweeteners. It is a compound useful as an intermediate for α-L-aspartyl-L-phenylalanine lower alkyl ester, or as an intermediate for optical resolution of racemic amino acid ester. Such an addition compound can be prepared by reacting an N-protected aminodicarboxylic acid with an amino acid ester in an aqueous medium in the presence of a proteolytic enzyme (Japanese Patent Application Laid-Open No. 53-1991).
-92729, JP-A-54-9226), or reacting a dipeptide ester and an amino acid ester in a solvent such as water (JP-A-55-19234, JP-A-55)
−73644) etc. In these reactions, the adduct precipitates into the solvent in the form of a solid phase. Therefore, it is an important issue how to efficiently recover this adduct from such a solvent, especially an aqueous medium, and how to decompose it into its constituent components, dipeptide ester and amino acid ester, and separate the two components. In the above-mentioned known method, the adduct is separated by filtration, and the decomposition into the dipeptide ester and separation from the amino acid ester are carried out by decomposition with an aqueous acid solution and subsequent extraction with filtration or an organic solvent. Further, the adduct is separated by filtration and then dissolved in a mixed solution of water and an organic solvent.
It is also disclosed that decomposition separation is performed by treatment with an H-type ion exchange resin. On the other hand, it is also known to separate this adduct as a homogeneous solution in an organic solvent by solvent extraction (Japanese Patent Application Laid-open No. 11295-1983). However, in order to perform extraction efficiently, it is necessary to use an organic solvent that is phase-separable from water and has a high dissolving power for adducts, etc.; such organic solvents include esters such as ethyl acetate, chloroform, Alkyl halides such as ethane chloride are relatively limited. However, esters may be subject to hydrolysis. Furthermore, since halogenated alkyl compounds have the problem of carcinogenicity, their use in the process of processing addition compounds that can be used as food raw materials should be avoided as much as possible. On the other hand, if an organic solvent having a low dissolving power for adducts and the like is used, it will be necessary to use a large amount as long as extraction by dissolution is assumed, which is also economically problematic. In order to solve such problems, the present inventors
As a result of further studies on the separation, decomposition, and separation of decomposition products of adduct compounds, we found that in a two-phase system of an aqueous phase and an organic solvent phase, the crystals of the adduct compound are incorporated into the organic solvent phase in a solid phase, and that this organic solvent The present invention was completed based on the discovery that when the phase is separated from the aqueous phase and then treated with water and an acid, the decomposed dipeptide ester remains in the organic solvent phase, while the amino acid ester moves to the aqueous phase. That is, the present invention is based on the general formula An adduct compound _{of a} dipeptide _ester and an amino acid _ester represented by An organic solvent capable of forming two phases with water is added and mixed to an aqueous mixture containing a carbonyl group (n is 1 or 2) in a solid phase, and a substantial portion of this addition compound is transferred to a solid phase. A two-liquid phase of an organic solvent phase and an aqueous phase is formed, the organic solvent phase is separated from the aqueous phase, water and an acid are added to the organic solvent phase, and the mixture is brought into contact with the general formula. A dipeptide ester represented by (in the formula R ₃ , R ₄ ,
X and n are the same as above); and an organic solvent phase containing the general formula Amino acid ester represented by (in the formula R ₁ and R ₂
The present invention provides a method for treating an addition compound of a dipeptide ester and an amino acid ester, which comprises forming a two-liquid phase with an aqueous phase containing (same as above) and separating the organic solvent phase and the aqueous phase. be. In the present invention, the addition compound of the general formula () contained in the solid phase in the aqueous mixture used as a starting material is:
In the formula, when n is 1, it contains aspartic acid, and when n is 2, it contains a brutamic acid skeleton. R ₂ and R ₄ each represent a lower alkyl group such as a methyl group, an ethyl group, a propyl group, or a butyl group, which may be common or different from each other. R ₂ and R ₃ each represent a side chain group of an amino acid such as a methyl group, an isopropyl group, an isobutyl group, an isoamyl group, a benzyl group, or a p-hydroxybenzyl group, and may be common to each other or different from each other. A benzyl group is particularly preferred as R ₃ . X is a benzyloxycarbonyl group which may have a substituent on the nucleus, such as a benzyloxycarbonyl group or a p-methoxybenzyloxycarbonyl group. 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 An N-protected aminodicarboxylic acid represented by the formula (in the formula, X and n represent the same meanings as above) is reacted in an aqueous medium in the presence of a protease to obtain a compound represented by the general formula () in an aqueous medium. It can be prepared by precipitating an adduct. In this case, R ₃ is common to R ₂ and R ₄ is common to R ₁ . In this case, as the amino acid ester represented by the general formula () and the N-protected aminodicarboxylic acid represented by the general formula (), the general formula () can be obtained by using the L-form or racemic form of each. The dipeptide moiety of the adduct represented by is always in the LL-form. The amino acid ester moiety conforms to this when the amino acid ester represented by the general formula () used is in the L-form, but when it is in the racemic form, it is mostly in the D-form. In addition, the following addition compounds represented by the general formula (), dipeptide esters represented by the same (),
The amino acid ester represented by the same () and the N-protected amino dicarboxylic acid represented by the same () are respectively referred to as an addition compound, a dipeptide ester, an amino acid ester, and an N-protected amino dicarboxylic acid. The preparation of the addition compound by the above method is described in Japanese Patent Application Laid-open No. 53
The known conditions described in JP-A-92729 and the like may be followed. This aqueous mixture can also be prepared by reacting a dipeptide ester with an amino acid ester in an aqueous solvent as disclosed in JP-A-55-19234 and JP-A-55-73644. In this case, when the LL-form of the dipeptide ester is reacted with the racemic amino acid ester, an adduct is mainly formed with the D-form amino acid ester. If the aqueous mixture containing the addition compound thus obtained is treated according to the present invention, the optically resolved amino acid ester can be recovered from the aqueous phase, and the dipeptide ester can be recovered from the organic phase. The amount of water relative to the adduct compound in the aqueous mixture used in the present invention is approximately 0.3 parts by weight of the adduct compound.
from about 20 parts by weight, preferably from about 0.5 to about
15 parts by weight, most preferably about 1 to about 10 parts by weight. The organic solvents used in the method of the present invention include aromatic hydrocarbons such as toluene and benzene, aliphatic hydrocarbons such as n-hexane, n-heptane, and cyclohexane, and ethers such as diethyl ether and diisopropyl ether. Preferable examples include ketones that can form two phases with water, such as , methyl isobutyl ketone, and dibutyl ketone, and mixtures thereof. Organic solvents other than these may also be used in combination with these organic solvents as long as they do not interfere with the formation of two phases. In the present invention, since the addition compound is separated and treated as a slurry in an organic solvent, the organic solvent is not used in an amount so large as to dissolve a substantial part of the addition compound, and the amount added is limited to 1.
From about 1 to about 20 parts by weight, preferably from about 2 to about 15 parts by weight, and most preferably from about 3 to about 10 parts by weight. In the method of the present invention, when X of the dipeptide ester is a benzyloxycarbonyl group, it is particularly advantageous to use toluene as the organic solvent because it can be easily recovered and used repeatedly as described below. In the present invention, the amount of water added to the organic solvent phase separated as an organic solvent phase containing a substantial portion of the addition compound in solid phase is about 0.5 in total with the water derived from the acid described below per 1 part by weight of the addition compound. from about 15 parts by weight, preferably from about 1 to about 10 parts by weight. When the acid is used as an aqueous solution, the necessary water may be added in the form of an aqueous solution of the acid. In that case, it is not necessary to add water separately. In the method of the present invention, inorganic or organic Bronsted acid is used as the acid added together with water to react with the addition compound. Examples of inorganic Bronsted acids include hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid. Examples of organic Bronsted acids include formic acid, acetic acid, citric acid, and toluenesulfonic acid. These acids can be used in the form of aqueous solutions. Since there is no particular limitation on the concentration, the amount of water added may be determined heteronomously so that it falls within the above-mentioned range. Solid acids such as H-type cation exchange resins can also be used. In this case, such solid acids settle to the bottom of the system. The amount of acid to be used relative to the addition compound in the aqueous mixture that is the starting material of the present invention is that the present invention essentially ionizes the amino acid ester portion of the addition compound with an acid to form an aqueous solution of its salt. (However, when a solid acid is used, the amino acid ester is ion-exchanged with the hydrogen ions of the solid acid and exits the liquid phase system.) Therefore, the amount is stoichiometric or more, that is, about 1 to about 1% per mole of the addition compound. 100 equivalents, preferably about 1 to about 20 equivalents, most preferably about 1
or about 10 equivalents. However, depending on the purpose, the addition compound may not necessarily require such high purity, and in such cases, this does not preclude the use of a less than stoichiometric amount. In the present invention, separation of the dipeptide ester after decomposition of the adduct is usually carried out in the form of a slurry in an organic solvent. However, when ketones capable of forming two phases with water are used as the organic solvent, the reaction can be carried out in the form of a solution thereof. The temperature at which the adduct is treated in the method of the present invention is generally about 0 to about 100°C, preferably about 5 to about 80°C. If stirring is sufficient, the decomposition reaction of the addition compound is usually completed within about 10 minutes. However, the protecting group X is a group that is relatively easily hydrolyzed,
For example, in the case of a p-methoxybenzyloxycarbonyl group, care should be taken not to cause separation of these groups, unless the separation of these groups is actively carried out at the same time or there is no problem in allowing the separation. , it is necessary to pay attention to the reaction time and reaction temperature. Although the adduct is a solid, it decomposes on contact with an acid to form a dipeptide ester and a salt of an amino acid ester and an acid. Since the dipeptide ester has low solubility in acidic aqueous solutions, a substantial portion thereof is present in the organic phase as a slurry or solute. On the other hand, salts of amino acid esters dissolve well in acidic aqueous solutions, so they dissolve in the liquid phase. When a solid acid is used, it precipitates as its salt. The reaction system thus becomes a two-phase system consisting of an organic phase containing the addition compound as a slurry or solute and an aqueous solution of the salt of the amino acid ester. The resulting two-phase system can be separated by a conventional liquid-liquid separation method. Recovery from amino acid ester solid acid salts can also be easily performed by conventional means. The dipeptide ester can be recovered from the organic slurry phase by conventional methods such as filtration, centrifugal sedimentation, or distillation of the organic solvent. Further, the organic slurry phase can be directly subjected to a deprotection reaction, etc. in the next step. In particular, when X in the general formula () of the dipeptide ester is a benzyloxycarbonyl group and the organic solvent is toluene, water is added to the separated organic slurry phase and the benzyloxycarbonyl group is reductively removed. Since the dipeptide ester from which the protecting group has been removed is transferred to the aqueous phase, the N-protecting group can be effectively removed, and at the same time, the removed protecting group becomes toluene, which is the same as the solvent toluene. This elimination reaction does not contaminate the solvent. The solvent toluene thus used can therefore be easily recovered and reused. This method uses R ₁ in the general formula () of the addition compound.
When R ₄ is a methyl group and n is 1, it is particularly effective as an industrial method for producing aspartame, a low-calorie sweetener. The acidic aqueous phase separated from the organic phase in the method of the present invention can be prepared by a conventional method, for example, by concentrating it to crystallize the amino acid ester in the form of a salt, or by making the liquid alkaline and then using a suitable method. The amino acid ester can be recovered by a method such as extraction with an organic solvent. Amino acid esters can also be easily recovered from solid acid salts using conventional methods. Note that when the addition compound, which is a component of the raw material used in the present invention, is obtained by the method described in JP-A No. 53-92729, the dipeptide ester moiety is LL-
However, the amino acid ester moiety may be L-type, D-type, or a mixture of both. The method of the present invention is applicable to any of these cases. Also, the dipeptide ester part is DD
- type, DL- type, or LD- type. The dipeptide ester obtained in the present invention is itself useful as an intermediate in peptide synthesis, but the dipeptide ester obtained by removing the protecting group X from the amino group is also a useful compound.
For example, as mentioned above, α-L-aspartyl-L-phenylalanine methyl ester is useful as a sweetening agent. Furthermore, since an optically resolved amino acid ester can be obtained from an addition compound formed by mixing a dipeptide ester and a racemic amino acid ester, it is also useful as an optical resolution method. As is clear from the above explanation, according to the method of the present invention, there is no need to dissolve the adduct, and the adduct can be effectively treated using a small amount of organic solvent, which is industrially advantageous. That is clear. Example 1 53.45 g of N-benzyloxycarbonyl-L-aspartic acid and 107.84 g of DL-phenylalanine methyl ester hydrochloride were placed in a second flask, and 400 ml of distilled water, 100 ml of 5N aqueous sodium hydroxide solution, and crude thermolysin (Thermoase PS-
160, trademark, manufactured by Daiwa Kasei Co., Ltd.) and 1.3 g of calcium acetate monohydrate were added, and the mixture was reacted at 40° C. with stirring. After 8 hours, the reaction mixture became an aqueous mixture containing the reaction product in suspension. 700 ml of toluene was added to this aqueous mixture, and the mixture was stirred and mixed at 40°C for 20 minutes. When stirring was stopped, a toluene phase containing suspended solids and a homogeneous clear aqueous phase were separated. A portion of the solid matter was sampled and recrystallized from ethyl acetate-n-hexane, and then analyzed for elemental analysis.
Since the NMR, IR and optical rotation agree with the data disclosed in JP-A No. 53-92729, N-
1:1 addition of benzyloxycarbonyl-α-L-aspartyl-L-phenylalanine methyl ester (hereinafter referred to as Z-APM) and mainly D-form phenylalanine methyl ester (hereinafter referred to as D-PM) It was confirmed that it was a compound. After the toluene phase was separated from the aqueous phase, 500 ml of 1N hydrochloric acid aqueous solution was added and mixed with stirring at 60°C for 1 hour.
After standing for about 20 minutes, the toluene phase containing solids was separated from the homogeneous transparent aqueous phase. The toluene phase was washed twice with 200 ml of distilled water at 60°C, cooled to room temperature, the crystals of the toluene phase were collected under reduced pressure using a glass filter, and after drying, Z-
APM73.42g (total yield from raw materials 85.2%, purity
99.4%). Example 2 53.45 g of N-benzyloxycarbonyl-α-L-aspartic acid and 86.27 g of L-phenylalanine methyl ester hydrochloride were placed in a second flask.
Distilled water 350ml, 5N sodium hydroxide aqueous solution 90ml
and crude thermolysin (Thermoase PS
-160, trademark, manufactured by Daiwa Kasei Co., Ltd.) and 0.9 g of calcium acetate monohydrate were added and reacted at 40°C with stirring. After 5 hours, 600 ml of toluene was added to the reaction mixture and stirred and mixed at 40°C for 20 minutes. When stirring was stopped, a toluene phase containing suspended solids and a homogeneous clear aqueous phase were separated. A portion of the solid was sampled and analyzed after recrystallization from ethyl acetate-n-hexane, and the elemental analysis, NMR, IR, and optical rotation were found to match the data disclosed in JP-A-53-92729. , N-benzyloxycarbonyl-
It was confirmed that it was a 1:1 addition compound of α-L-aspartyl-L-phenylalanine methyl ester (Z-APM) and L-phenylalanine methyl ester (hereinafter referred to as L-PM). After the toluene phase was separated from the aqueous phase, 500 ml of 1N hydrochloric acid aqueous solution was added and mixed with stirring at 60°C for 1 hour.
After standing for about 20 minutes, a toluene phase containing solids and a homogeneous transparent aqueous phase were separated. After cooling to room temperature, the toluene phase crystals were collected through a glass filter under reduced pressure and dried to obtain 73.27 g of Z-APM (total yield from raw materials: 85.3%, purity: 99.7%). Example 3 1:1 addition compound of Z-APM and D-PM
To a suspension of 4.25 g in 35 ml of toluene were added 10 ml of a 1N hydrochloric acid solution and 20 ml of distilled water, and the mixture was stirred at 60°C for 30 minutes. After standing for about 20 minutes, the toluene phase containing solids was separated from the homogeneous transparent aqueous phase. After washing the toluene phase twice with 20 ml of distilled water, the solvent was distilled off using a rotary evaporator to obtain 2.92 g of Z-APM (purity
99.7%). Example 4 The same procedure as in Example 3 was carried out except that diisopropyl ether was used instead of toluene. The isopropyl ether phase containing solids is removed by distilling off the solvent using a rotary evaporator.
2.96 g of APM (purity 98.0%) was obtained. Example 5 The same operation as in Example 3 was carried out except that the temperature and time for stirring and mixing the toluene suspension of the addition compound and the aqueous hydrochloric acid solution were changed to 25°C and 60 minutes. Z
-2.96 g of APM (purity 98.1%) was obtained. Example 6 In Example 3, 95% sulfuric acid was used instead of hydrochloric acid.
Use 0.715ml and use 26.3ml instead of 20ml of distilled water.
The operation was carried out in the same manner as in Example 3, except that . 2.96 g of Z-APM (purity 99.6%) was obtained. Example 7 The same procedure as in Example 6 was carried out except that 0.733 ml of acetic acid was used instead of 95% sulfuric acid. Z-
3.06 g of APM (purity 91.7%) was obtained. Example 8 The operation was carried out in the same manner as in Example 3, except that n-heptane was used instead of toluene. 3.12 g of Z-APM (purity 89.6%) was obtained. Example 9 5.0 g of sodium salt of N-benzyloxycarbonyl-α-L-aspatyl-L-phenylalanine methyl ester was dissolved in 20 ml of water, and this solution was
Contains 5.0g of DL-valine methyl ester hydrochloride
When added dropwise to 20ml of aqueous solution while stirring,
The reaction product precipitated and the reaction mixture became a suspension. After this suspension was allowed to stand at room temperature for 2 hours, 50 ml of methyl isobutyl ketone was added to it, and when the suspension was stirred and allowed to settle, it was separated into a methyl isobutyl ketone suspended phase containing the reaction product and a homogeneous transparent aqueous phase. did. The methyl isobutyl ketone suspended phase is separated from the aqueous phase and added with 10 ml of 1N aqueous hydrochloric acid and 20 ml of water.
When the mixture was stirred at 60°C for 30 minutes and allowed to settle, it was separated into a homogeneous methyl isobutyl ketone phase and a homogeneous aqueous phase. Methyl isobutyl ketone is distilled off from the methyl isobutyl ketone phase using a rotary evaporator.
4.5 g of Z-APM (purity 99.5%) was obtained. On the other hand, the aqueous phase was adjusted to pH 8 by adding sodium carbonate, and then extracted with dichloromethane. The dichloromethane phase was dried by adding anhydrous magnesium sulfate,
After blowing in hydrogen chloride gas, it was concentrated. Furthermore, diethyl ether was added to this to precipitate crystals, which were collected. 1.6 g (optical purity: 69%) of crystals of D-valine methyl ester hydrochloride was obtained. Examples 10 to 13 Examples using 5.00 g of a 1:1 addition compound of Z-APM and another amino acid ester other than phenylalanine methyl ester instead of the 1:1 addition compound of Z-APM and D-PM. The same operation as in 3 was performed. The results are shown in the table below.

[Table] Ester Example 14 N-benzyloxycarbonyl-α-L- instead of 1:1 addition compound of Z-APM and D-PM
1:1 addition compound of aspartyl-L-phenylalanine ethyl ester (Z-APE) and D-PM
The operation was carried out in the same manner as in Example 3 except that 5.00 g was used. 3.42g (purity 98.8%) of Z-APE was obtained. Example 15 N-p-methoxybenzyloxycarbonyl-α-L-aspartel-L-phenylalanine methyl ester (PMZ-APM) and D-PM instead of the 1:1 addition compound of Z-APM and D-PM The operation was carried out in the same manner as in Example 3, except that 5.00 g of the 1:1 addition compound was used and the melting temperature of the toluene suspension and the aqueous hydrochloric acid solution was set at 20°C. 3.45g (purity 97.2%) of PMZ-APM was obtained.

Claims (1)

[Claims] 1. General formula An adduct compound _{of a} dipeptide _ester and an amino acid _ester represented by An organic solvent capable of forming two phases with water is added and mixed to an aqueous mixture containing a carbonyl group (n is 1 or 2) in a solid phase, and a substantial portion of this addition compound is transferred to a solid phase. A two-liquid phase of an organic solvent phase and an aqueous phase is formed, the organic solvent phase is separated from the aqueous phase, water and an acid are added to the organic solvent phase, and the mixture is brought into contact with the general formula. A dipeptide ester represented by (in the formula R ₃ , R ₄ ,
X and n are the same as above); and an organic solvent phase containing the general formula Amino acid ester represented by (in the formula R ₁ and R ₂
A method for treating an addition compound of a dipeptide ester and an amino acid ester, the method comprising forming a two-liquid phase with an aqueous phase containing (same as above) and separating the organic solvent phase and the aqueous phase. 2. A patent claim in which the organic solvent capable of forming two phases with water is a ketone, an aliphatic or aromatic hydrocarbon, an ether, or a mixture thereof that can form two phases with water. Range 1
Treatment method described in section. 3. The treatment method according to claim 1, in which toluene is used as the organic solvent capable of forming two phases with water. 4. The treatment method according to any one of claims 1 to 3, in which the organic solvent capable of forming two phases with water is used in an amount less than the amount that can completely dissolve the addition compound. 5. Addition of an organic solvent capable of forming two phases with water in an amount of about 1 to about 20 parts by weight per 1 part by weight of the addition compound. Processing method. 6. Process according to any one of claims 1 to 5, wherein the amount of water in the aqueous mixture is about 1 to 10 parts by weight per part by weight of the addition compound. 7. The treatment method according to any one of claims 1 to 6, wherein inorganic Bronsted acid is used as the acid. 8. The treatment method according to any one of claims 1 to 7, wherein organic Bronsted acid is used as the acid. 9. The treatment method according to any one of claims 1 to 8, wherein the amount of acid used is about 1 to about 10 equivalents per mole of addition compound. 10 The amount of water added with the acid is the addition compound 1
10. Process according to claims 1 to 9, in which the amount is from about 1 part by weight to about 10 parts by weight. 11. The treatment method according to any one of claims 1 to 10, wherein the dipeptide moiety of the addition compound is LL-type. 12 R ₁ and R ₄ are methyl groups, R ₂ is benzyl group,
R ₃ is a methyl group, isopropyl group, isobutyl group,
The treatment according to any one of claims 1 to 11, wherein X is a benzyl group or a p-hydroxybenzyl group, X is a benzyloxycarbonyl group or a p-methoxybenzyloxycarbonyl group, and n is 1. Method. 13 General formula for aqueous mixture 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 on the nucleus, and n is 1 or 2) is reacted in the presence of a protease in an aqueous medium. general formula _An adduct compound _of a dipeptide _ester and _an amino acid ester _{represented by}
12. The treatment method according to any one of claims 1 to 11, wherein the reaction product solution is a reaction product solution in which the same group as . 14 In the general formula of addition compounds, dipeptide esters, amino acid esters, and N-protected aminocarboxylic acids, R ₁ and R ₄ are methyl or ethyl groups, R ₂ and R ₃ are benzyl groups, and X is benzyloxycarbonyl group or p -methoxybenzyloxycarbonyl group, and n is 1. Claim 13
Treatment method described in section. 15 The amino acid ester and N-protected aminodicarboxylic acid used are each independently L-type or L-
Claim 13, which is a mixture of type and D-type.
or 14. 16 Claim 1 in which the separated organic solvent phase is used in the elimination reaction of the protecting group of the dipeptide ester
The processing method described in any one of Items 1 to 15. 17. The treatment method according to any one of claims 1 to 16, wherein the amino acid ester is recovered from the separated aqueous phase.