JPS5852252A - Method for decomposition of addition compound of dipeptide ester and amino acid ester - Google Patents

Abstract

PURPOSE:To decompose the titled compound in high efficiency, by adding an organic solvent and an acid to an aqueous mixture containing the addition compound of dipeptide ester and amino acid ester to effect the decomposition of the addition compound, and separating both components to the organic solvent phase and the aqueous phase. CONSTITUTION:The amino acid ester of formulaI(R1 is lower alkyl; R2 is side chain of amino acid) is made to react with the N-protected carboxylic acid of formula II (X is benzyloxycarbonyl; n is 1 or 2) in an aqueous medium in the presence of proteinase, and the resultant aqueous mixture containing the addition compound of formula III (R4 is R1; R3 is R2) as solid phase is mixed with an organic solvent capable of forming two phases with water (e.g. toluene) and an acid (e.g. inorganic Broensted acid). The amount of the acid is 1-10 equivalent per 1mol of the addition compound. The mixture is separated into the organic solvent phase containing the dipeptide ester of formula IV as a solid and an aqueous phase containing the amino acid ester of formulaI, to complete the decomposition of the addition compound to each component.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for decomposing an addition compound of a dipeptide ester and an amino acid ester.

More specifically, the present invention relates to a method of decomposing an addition compound of a dipeptide ester and an amino acid ester with an acid and separating both components using an organic solvent.

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 such as α-L-
It is a compound useful as an intermediate to aspartyl-L-7enylalanine lower alkyl ester or as an intermediate for optical resolution of racemic amino acid ester.

Such an addition compound can be produced by reacting N-carboxylic amino dicarboxylic acid and an amino acid ester in an aqueous medium in the presence of a protease (JP-A-53-92729, JP-A-54-9226). Alternatively, a dipeptide ester and an amino acid ester may be reacted in a solvent such as water (% 19234/1986; 73644/1984).
) etc. can be obtained.

The adduct compound thus obtained is sparingly soluble in water, but decomposes when treated with acid in an aqueous medium, giving a Jll-soluble dipeptide ester and an easily soluble amino acid ester salt in an acidic aqueous solution. The system then becomes an aqueous mixture containing the amino acid ester salt as the solute and the dipeptide ester in the solid phase. By removing the covalent group, the dipeptide ester separated from this aqueous mixture immediately becomes a useful compound 1 in itself, such as the above-mentioned α-L-aspartyl-L.
- It can be converted into phenylalanine lower alkyl ester, etc., and the amino acid ester can be obtained in a state of high optical purity even if the racemic form is used in preparing the above-mentioned addition compound.

How to efficiently perform this decomposition and separation is an extremely important issue.

In the above-mentioned publications, a solid addition compound is mixed and reacted with an acidic aqueous solution:・□・The reaction mixture is solid-liquid separated, the dipeptide ester is recovered as a solid phase, and one is added to the other component, aminosulfate. A method for separating and recovering pernicious ester as an aqueous solution (% 9226/1986) and a method in which a dipeptide ester such as ethyl acetate is well dissolved in a mixture of an addition compound and an acidic aqueous solution and form two phases with water. A method is disclosed in which an organic solvent is added and the dipeptide ester is separated into an organic phase and the other component is separated into an acidic aqueous phase as a homogeneous solution and recovered once (Japanese Patent Laid-Open No. 53-92729).

However, in this case, 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 dipeptide esters, but such organic solvents such as ethyl acetate Esters, chloroform, dichloroethane, etc., are relatively limited. However, esters have problems with hydrolysis, and f7't-rodanated alkyls have recently become a problem in the treatment process of dipeptide esters, which are raw materials for cracked and damaged products, etc. It is desirable to avoid its use as much as possible. On the other hand, in the case of organic solvents with low dissolving power for dipeptide esters, it is necessary to use large amounts! is necessary,
There are also economic problems.

In order to solve these problems, the present inventors have intensively investigated an industrially advantageous method for decomposing adduct compounds. When organic solvents capable of forming amino acid esters are mixed and contacted, surprisingly, a substantial portion of the dipeptide ester produced by the decomposition of the adduct is incorporated in the organic solvent phase in the form of a slurry, leaving the homogeneous aqueous phase containing the amino acid ester. They discovered that the separation can be carried out effectively and completed the present invention.

That is, the present invention provides an addition compound of a dipeptide ester and an amino acid ester represented by the general formula (wherein R8 and R1 are lower alkyl groups, R
2 and R3 are side chain groups of amino acids, x is a benzyloxycarbonyl group that may have a substituent on the nucleus, n is 1 or Fi2). The adduct is decomposed by mixing and contacting an organic solvent and an acid that can be formed to decompose the dipeptide ester of the general formula (wherein R3, R4).

an organic solvent phase containing a substantial portion of X and nti (as above) as a solid phase, and an aqueous phase containing an amino acid ester represented by the general formula (%) (wherein R□ and □ are as above) The present invention provides a method for decomposing an adduct compound of a dipeptide ester and an amino acid ester, which is characterized by forming two liquid phases with the organic solvent and separating the aqueous phase from the organic solvent phase.

The addition compound represented by the general formula (1) contained in the aqueous mixture used as a starting material in the present invention contains an aspartic acid skeleton when U is 1, and a glutamic acid skeleton when n is 2.

R1 and R4 are each a common or different lower alkyl group such as a methyl group, an ethyl group, or a propyl group.

R3 and Rs Fi are each a methyl group, an isopropyl group, an isopdel group, an isoamyl group, and a benzyl group.

69 in side chain groups of amino acids such as p-hydroxybenzyl group
．． Including times when they are common and times when they are different. A benzyl group is particularly preferred as Rs.

)I benzyloxycarbonyl group, which may have a nuclear Kt substituent such as p-methoxybenzyloxycarbonyl group.

An aqueous mixture #LFi containing the addition compound represented by the general formula (1) in the same phase, an amino acid ester represented by the general formula, and an N-polyaminodicarboxylic acid represented by the general formula (in the formula, X and uD are the same as above). It can be prepared by precipitating the addition compound represented by the general formula (1) in the aqueous medium by reacting the compound (representing the meaning) in the presence of a protease in an aqueous medium. In this case, R, FiRl, and R4 are common to R□. In this case.

Amino acid ester represented by general formula value) and general formula■
As the N-protected aminodicarboxylic acid represented by, the dipeptide moiety of the addition compound represented by the general formula (1), which can be obtained either by using each L monomer or by using the racemate, is always K
It is LL-type. The amino acid ester moiety follows the general formula (represented by V) when the amino acid ester is L-unit, but when it is racemic, it is mostly D-type.

In addition, the addition compound represented by general formula (1), the dipenotedoester represented by the general formula (phantom), the amino acid ester represented by the same (1), and the N-protected aminodicarboxylic acid represented by the same (2) are adduct compounds, respectively. They are called dipeptide esters, amino acid esters, and N-protected aminodicarboxylic acids.

The preparation of the addition compound by the above-mentioned method is described in JP-A-53-92.
Known conditions described in Publication No. 729, etc. and No. 55
It can also be prepared by reacting a dipeptide ester with an amino acid ester in an aqueous solvent as disclosed in Japanese Patent No. 73644 and the like. In this case, racemic amino@[Yes]・@
When reacted with acid ester, D = body amino...@0
Forms addition compounds with acid esters. The addition compound thus obtained can be turbid in an aqueous medium and used as a raw material for the present invention. In this case, the optically resolved amine acid ester can be separated and recovered from the aqueous phase, and the dipeptide ester can be separated and recovered from the organic phase.

The aqueous mixture used in the method of the present invention is not limited to the aqueous mixture prepared by the method exemplified above,
It may be a mixed solution in which the addition compound obtained by the above method or another method is suspended in water.

The content of the addition compound in the aqueous mixture used as a raw material in the present invention is within a content range that allows the amount of the aqueous phase after addition of the acid to be the amount described below.

The organic solvent used in the method of the present invention includes aromatic hydrocarbons such as toluene and benzene.

Suitable examples include aliphatic hydrocarbons such as n-hexane, n-hebutane, and cyclohexane, ethers such as genal ether and diinopropyl ether, 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 case where X of the dipeptide ester separated as a slurry in the organic phase in the method of the present invention is a benzyloxycarbonyl group, toluene is produced when debenzyloxycarbonyl is removed by reduction. Therefore, among the above solvents, toluene is particularly advantageous because it allows the reduction reaction to be carried out without replacing the solvent.

Yes! ! The amount of the solvent used is generally about 1 to about 20 parts by weight, preferably about 2 to about 15 parts by weight, and most preferably about 3 to about 10 parts by weight, per 1 part by weight of the addition compound in the aqueous mixture. Parts by weight.

In the method of the present invention, an inorganic or organic Bronste acid is used as the acid to be reacted with the compound (1). An example of an inorganic Bronsted acid is hydrochloric acid.

Hydrobromic acid, sulfuric acid, phosphoric acid, etc. can be avoided. An example of an organic Bronsted acid is formic acid.

Examples include acetic acid, citric acid, toluenesulfonic acid, and the like. These acids can be used in the form of aqueous solutions. Since there is no particular limitation on its concentration, it may be determined heteronomously from the amount of the aqueous phase described below.

Ma7'4. 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.

Use i1 of the acid of the present invention is that the present invention essentially ionizes the amino≠=≠; therapeutic ester part of the addition compound with an acid to form a solution of its salt in water (however, when a solid acid is used, Since the amino acid ester is ion-exchanged with hydrogen ions of the solid acid and exits the liquid phase system, the amount is stoichiometric or more, that is, about 1 to about 100 equivalents per mole of the addition compound.

Preferably from about 1 to about 20 equivalents, most preferably not required in some cases, and this does not preclude the use of substoichiometric amounts in such cases.

The method of the present invention is characterized in that the amount of water phase derived from the aqueous medium and acid salt of the aqueous mixture is typically 0.3 m/m of addition compound.
from about 20 parts by weight, preferably from about 0.5 to about 15 parts by weight
Parts by weight, most preferably within the range of about 1 to about 101 parts by weight.

In the method of the present invention, the temperature at which the adduct is decomposed and the phase separation is carried out is generally from 0 to about 100°C, preferably from about 5 to about 80°C. The decomposition reaction of the addition compound is usually completed within about 10 minutes if stirring is sufficient.

However, the cystic group X is a relatively easily hydrolyzed group 1, such as p-
In the case of a methoxybenzyloxycarbonyl group, this is not the case unless these groups are actively removed at the same time or there is no problem in allowing the removal. It is necessary to pay attention to the reaction time and reaction temperature so as not to cause separation of these groups.

Although the addition compound is a solid, it is decomposed by contact with an acid (acidic aqueous solution) to become a dipeptide ester and a salt of an amino acid ester with an acid. dipeptide ester IIi,
Because of its low solubility in acidic water baths, a substantial portion of it exists as a slurry in the organic solvent phase. On the other hand, salts of amino acid esters dissolve well in acidic aqueous solutions, so they dissolve into the aqueous phase unless a solid acid is used. When a solid acid is used, it precipitates as its salt. Thus, the reaction system can include an organic phase containing a slurry of dipeptide esters and amino acids. Recovery from amino acid ester solid acid salts can also be easily performed by conventional means.

The cydipegtide ester can be recovered from the organic slurry phase by a conventional method such as t'' filtration, centrifugal sedimentation, or distillation of the Fi solvent.

The organic slurry phase can be directly subjected to a debinding reaction in the next step. In particular, in the general formula (Otori) of dipeptide ester, X is benzyloxycal, f? When the organic solvent is toluene for a nyl group, the N-protecting group can be effectively removed because the organic solvent is transferred to this, and at the same time, the removed protecting group is converted to toluene, which is the same as the solvent toluene. Because it is.

This elimination reaction does not contaminate the solvent. The solvent toluene thus used can therefore be collected in a container and reused.

This method is particularly effective as an industrial method for producing aspartame as a low-calorie sweetener when applied to the case where 8□ and he is a Medel group in the general formula (1) of the addition compound, and n is 1.

In the method of the present invention, the acidic aqueous phase separated from the organic slurry phase is prepared by a conventional method 1, for example, by condensing 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 moat ester can be recovered by a method such as extraction with a suitable organic solvent.

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 portion thereof is of the LL-type, but the amino acid ester portion is of the L-type. in the case of.

There may be a D-type case or a mixed type of both.

The method of the present invention is applicable to any of these cases.

It is also applicable when the dipeptide ester moiety is DD-type, DL-type or LD-type.

Note that the method of the present invention can also be achieved by starting from a suspension containing the addition compound in an organic solvent and adding water and an acid thereto, so the present invention also includes such a method. . This is obvious from the fact that by mixing the raw materials in this manner, the same conditions as those obtained by the generally described technique of the present invention can be achieved.

Although the dipeptide esters obtained in the present invention are themselves useful as intermediates in pegtide synthesis.

A 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, from the addition compound formed by mixing a dipeptide ester and a racemic aminosansha foronic acid ester, an optically resolved amino #sac&=acid ester can be obtained, so it is not necessary to dissolve the optically resolved tide ester. Therefore, it is extremely advantageous industrially because it is possible to effectively decompose the adduct and separate the produced dipeptide ester and amino acid ester using a small amount of organic solvent.

Example 1 N-benzyloxycarbonyl-L-asuno (lagic acid 53.45f and DI, -7 enylalanine mether ester hydrochloride 107.84F were added to 21 flasks, distilled water 400d, 5N sodium hydroxide aqueous solution 100wL1
．． and crude thermolysin (Thermoase Pg-16
0, Trademark, manufactured by Daiwa Kasei ■) 7.21. 1.3 t of acetic acid hydrate was added, and the mixture was reacted at 40°C with stirring. After 8 hours, some of the solids were sampled from the suspended aqueous mixture, and since it matched the charger with cold water, N
-Benzyloxycarbonyl-α-L--T Spartel-L-phenylalanine methyl ester (hereinafter referred to as Z-A
It was confirmed that it is a 1=1 addition adduct of 7-enylalanine methyl ester (referred to as PM) and 7-enylalanine methyl ester, which is mainly composed of D.

60 rliu of toluene and 1001 liters of concentrated hydrochloric acid were added to the aqueous reaction mixture, and the mixture was stirred and mixed 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 is 200
After washing twice at 60°C with 11 J of distilled water, it was cooled to room temperature, the toluene phase crystals were collected under reduced pressure through a glass filter, and after drying, 2-APM74.452 (total yield from raw materials) was obtained. A yield of 86.3% and a purity of 99.31 were obtained.

Example 2 In Example 1, L-phenylalanine methyl ester hydrochloride was used as a substitute for DL-7 enylalanine methyl ester hydrochloride, 7.2 tons of crude thermolysin was substituted with 3.6 tons of crude thermolysin, 1. The reaction was carried out in the same manner as in Example 1 except for using 0.6F instead of 3t. After 8 hours, a portion of the solid matter was sampled from the suspended aqueous mixture.

It was confirmed that it was an x:i addition compound of d-oxycarbonyl-ζ-aspartyl-L-7znylalanine methyl ester (Z-APM) and L-phenylalanine methyl ester.

The aqueous reaction mixture was treated in the same manner as in Example 1, and after drying Z-APM by filtration from the toluene phase, the yield was 7.
4. s6y (total yield from raw materials 86.4% purity 9
9.3 Ch) Obtained in 1.

Example 3 N-benzyloxycarbonyl-a-L-7 spartyl-L-phenylalanine methyl ester (2-APM)
and D-phenyl methyl ester (hereinafter D-
A 1:1 addition compound (referred to as PM) with O2 in distilled water
Toluene 35- and IN-hydrochloric acid aqueous solution IOY were added to the suspension in 1111, and the mixture was stirred at 60°C for 30 minutes. Approximately 20
After standing for a minute, the toluene phase containing solids was separated from the homogeneous transparent aqueous phase. The toluene phase was washed twice with 2ONl of distilled water, and the solvent was distilled off using a rotary evaporator.
M3,49r (purity 99.5%) was obtained.

Example 4 The operation was carried out in the same manner as in Example 3, except that diisopropyl ether was used instead of toluene. Z-APM
3.36tC7iIJA degree 97.4%)ヲ411゜Example 5 Same as Example 3 except that the temperature and time of stirring and mixing of the suspended aqueous solution of the addition compound and the toluene and hydrochloric acid aqueous solution were changed to 25°C and 60 minutes. The operation was performed as follows. Z-
APM3.4! V (purity 99.7%) was obtained.

Example 6 The operation was carried out in the same manner as in Example 3, except that 951 sulfuric acid 10111j was used instead of hydrochloric acid, and 30-951 was used instead of 201 Lt of distilled water. Z-APM3, 42f (
A purity of 99.5% was obtained.

Example 7 The operation was carried out in the same manner as in Example 6 except that acetic acid x, 0+j was used instead of 95-thiosulfuric acid. Z-APM 3.2
1f (purity 91.896) as % fb Example 8 The same procedure as in Example 3 was carried out except that n-heptane was used instead of toluene. Z-APM 3.21r
(purity 91.8%) was obtained.

Example 9 N instead of 1:1 addition compound of Z-APM and D-PM
-benzyloxycarbonyl-, -L-aspartyl-
A 1:1 addition compound of L-phenylalanine ethyl ester (Z-APR) and D-PM at 5.00? The same operation as in Example 3 was performed except that fc was used. Z-APE i
3,389 (purity 99.1%).

Example 10 N-p-methoxybenzylox7carbonyl-α instead of the suspension of the l21 addition compound of Z-APM and D-PM
- Using a suspension of 5,001 of a 1:1 addition compound of -L-aspartyl-L-phenylalanine methyl ester (PMZ-APM) and D-PM, the contact temperature of this aqueous suspension with toluene and an aqueous hydrochloric acid solution was set to 20 The operation was carried out in the same manner as in Example 3 except that the temperature was changed to ℃. PMZ-APM 3
．． 41 f (N degree 98.4 inches) was obtained.

Example 11 1) 6.66 g of L-feralanine methyl ester 8
The addition formed after mixing a 0 mt hot ethyl acetate solution and a 200 d hot ethyl acetate solution of N-be/zyloxycarbonyl-α-L-7 spartyl-L-phenylalanine methyl ester '1.729 and leaving it at room temperature overnight. The compound was filtered, separated and dried to obtain an additional compound. The thus obtained addition compound 5,001 was suspended in distilled water 201 to obtain an aqueous mixture. To this aqueous mixture, add 35m of toluray and IN.
- Aqueous hydrochloric acid solution 1011 was added, stirred at 60°C for 30 minutes, and kept in a quiet device for 20 minutes.

The toluene phase containing solids was separated from the homogeneous clear aqueous phase. After washing the toluene phase twice with 20 d of distilled water, the solvent was distilled off using a rotary evaporator, and Z-APM3.
47F (purity 99.6%) was obtained.

On the other hand, the toluene phase, the separated aqueous phase, and the washing liquid of the toluene phase were combined, sodium carbonate was added to adjust the liquid pH to 8, and the mixture was extracted with dichloromethane. The dichloromethane phase was dried over anhydrous magnesium sulfate, hydrogen chloride gas was blown into it, and then concentrated. Furthermore, diethyl ether was added to this to precipitate crystals, which were then treated with tP. Crystals of D-7 enylalanine methyl ester hydrochloride 1.58 f (optical purity 97s) were obtained.

Examples 12-15 Z-APM and D-PM (7) 1:1 addition compound glue,
The operation was carried out in the same manner as in Example 3 using 5.0Of a 1=1 addition compound of DK,Z-APM and an amino acid ester other than phenylalanine methyl ester.

The results are shown in the table below.

Example Addition compound Recovered Z-APM (purity) 1
2 L-valine ether ester 3.58f (9
9,2U13 D-7 U=nlf xx chill 3.
92f (99,3%) 14 L-leucine ethyl ester 3.41f (99,1%) 15 D-tyrosine methyl ester 3.13f (98,8 thi) Patent applicant Toyo Soda Kogyo Co., Ltd.

Claims (1)

[Scope of Claims] An addition compound of a dipeptide ester and an amino acid ester represented by the general formula (in the formula, B and R4 are lower alkyl groups, B
, and R1 is a side chain group of an amino acid, X is a benzyloxycarbonyl group that may have a substituent on the nucleus, and Ton, m
is 1 or 2) as a solid phase, an organic solvent capable of forming two phases with water and an acid are added and brought into contact with each other to decompose the adduct, thereby producing a dipeptide represented by the general formula An organic solvent phase containing a substantial portion of an ester (in the formula R, R4, 1. A method for decomposing an addition compound of a dipeptide ester and an amino acid ester, which comprises completely forming a two-liquid phase with an aqueous phase containing a dipeptide ester and an aqueous phase, and separating the organic solvent phase and the aqueous phase. (2) The decomposition method according to claim 1, wherein the organic solvent capable of forming two phases with water is an aliphatic or aromatic hydrocarbon or an ether. (3) The decomposition method according to claim 1, in which toluene is used as the organic solvent capable of forming two phases with water. (4) The organic solvent capable of forming two phases with water is used as an addition compound. The decomposition method according to any one of claims 1 to 3, which is used in an amount less than the amount that can be completely dissolved. (5) Any one of claims 1 to 3, in which about 1 to about 20 parts by weight of an organic solvent is used based on 1 part by weight of the addition compound because it forms two phases with water. Decomposition method described. (6) The decomposition method according to any one of claims 1 to 5, in which an inorganic pronste acid is used as the acid. (7) The decomposition method according to any one of claims 1 to 6, in which an organic pronste acid is used as the acid. (8) The decomposition method according to any one of claims 1 to 7, wherein the amount of acid used is about 1 to about 10 equivalents per mole of the addition compound. (9) A decomposition method according to any one of claims 6 to 8, in which the acid is used in the form of an aqueous solution. (6) Any one of claims 1 to 9 in which the aqueous mixture and acid are used in an amount such that the amount of the aqueous phase is about 1 to about 10 parts by weight per 1 part by weight of the addition compound. Decomposition method described. The decomposition method according to any one of claims 1 to 10, wherein the dipeptide ester moiety of the I addition compound is LL-type. Q3 R□ and R4 are methyl or ethyl groups, town is benzyl group & R, is methyl group or isobutyl group. Isobutyl group, benzyl group or #'ip-hydrogen group:
/')IL'-#X is a benzyloxycarbonyl group or #'ip-methoxybenzyloxycarbonyl group, L
The decomposition method according to any one of claims 1 to 11, wherein m is 1. 0 An aqueous mixture contains an amino acid ester represented by the general formula % (in which R1 is a lower alkyl group and R2 is a side chain group of amic acid) and an N-protected amino dicarbonate represented by the general formula %. In the acid formula, X is a benzyloxycarbonyl group that may have a substituent, and n is 1 or 2) in an aqueous medium in the presence of a protease to produce a dipeptide ester represented by the general formula and an amino acid. An addition compound with an ester (in the formula, R1, R2, X and m are the same as above, and R3 and R4 are the same groups as R1 and R8 respectively) A decomposition method described in any one of the ranges 1 to 11. I In the general formula of addition compounds, dipeptide esters, amino acid esters and N-protected aminodicarboxylic acids, R1 and R4 are methyl or ethyl groups, R2 and R3 are benzyl groups, and X is benzyloxycarbonyl group or p-methoxybenzyloxy 14. The decomposition method according to claim 13, wherein the decomposition method is a carbonyl group and n is 1. aθ The decomposition method according to claim 11 or claim 12, wherein the amino acid ester and the N-protected amino dicarboxylic acid used are each independently of the L-type or a mixture of the L-type and the D-type. α0 The decomposition method according to any one of claims 1 to 13, in which the separated organic solvent phase is used for the elimination reaction of the dipeptide ester, The decomposition method described in any one of claims 1 to 14 to be recovered