CA1331255C - Method of producing –-l-aspartyl-l-phenylalanine methyl ester hydrochloride - Google Patents

Abstract

ABSTRACT
Crystals of .alpha.-L-aspartyl-L-phenylalanine methyl ester hydrochloride is produce in high yield while using lesser amounts of hydrogen chloride than are required by prior methods by carrying out the reaction with the decreased content of hydrogen.
chloride in the presence of an inorganic chloride salt, in particular an ammonium or metal halide.

Description

- 1 - 13312~
The present invention relates to a method of producing d -L-aspartyl-L-phenylalanine methyl ester hydrochloride.
~ -L-aspartyl-L-phenylalanine methyl ester (hereinafter referred to as ~-APM) is a substance which has attracted a wide attention for its useful-ness as a low calorie sweetening agent having a sweet-ness of excellent quality.
In known methods for producing ~ -APM, an N-protected-L-aspartic anhydride is reacted with L-phenylalanine methyl ester in an organic solvent, and then the protecting group is eliminated by a con ventional method ~U.S. Patent No. 3,786jO39); an addition salt of L-aspartic anhydride with a strong acid is directly reacted with L-phenylalanine methyl _ ester (Japanese Patent Publication No. 14,217/74); or L phenylalanine is reacted with N-formyl-L-aspartic `anhydride in glacial acetic acid, and the formyl :
group is eliminated from the product to give d -L-20 aspartyl-L-phenylalanine (hereinafter referred to as '~

~-AP), which is then esterified with methanol (U.S.

Patent No. 3,933,781).

In the known methods, however, ~-isomers of the desired ~-derivatives, such as ~-APM and ~ . ~
~ ~ -AP, are formed as a by-product in large quantities.

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r.. ',.. -." ,: . `. ,. - ~'. .. ': : .. ,, .; ~ ,: , . - 2 - 13312~a In addition, unreacted starting materials (for example, derivatives of L-aspartic acid and L-phenylalanine) also re~ain in the reaction mixture. It is, therefore, of vital importance in the commercial production of ~ -APM employing such prior methods, to isolate and purify o~-APM from a reaction mixture containing such impurities in an efficient manner.
In a known purification method, ~-APM con-taminated with ~ -L-aspartyl-L-phenylalanine methyl ester (hereinafter referred to as ~ -APM) and other impurities is brought into contact with a hydrogen halide (for example, hydrogen chloride) in water or : ln an aqueous medium comprising water and methanol in : order to precipitate crystals of the salt of the hydrogen halide and ~-APM, and the crystals pre-cipitated are then separated therefrom (U.S. Patent No. 3,798,207).
~ In an improvement of the method described ~ in U.S. Patent.No. 3,933,781, c~-AP is contacted with a reaction mixture containing methanol, a hydrogen : ` halide and water, so as to convert the ~-AP into c~ -APM, which precipitates immediately in the form : :, of a salt with the hydrogen halide (U.S. Patent No. : :
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The above-described known m~thods, which comprise precipitating crystals of a hydrogen halide salt of ~-APM, can be useful in enhancing the efficiency in its commercial production since they make it possible to convert ~-AP into ~-APM
and/or to separate and purify J~-~PM quite eficiently.
However, in order to precipitate the salt of c~-APM
in a high yield, such precipitation must be carried out in the presence of a large excess of hydrogen halides. For example, hydrogen halides must be used in an excess of not less than ca. 2 mol., preferably not less than ca. 3 mol., per liter of the aqueous medium.
The methods, therefore, suffer from the dis-advantages that ~ -APM can hardly be produced in a ._ :
high yield since the ester and peptide bonds con-tained in its molecule are subjected to hydrolysis, and that a large quantity of alkali is required to neutralize the excess hydrogen halides. In addition, hydrogen halides are highly corrosive to ordinary metals and, therefore, reaction equipment made of high-quality materials must be used in a commercial scale production of ~-APM. This requlres an undesir-ably high cost for equipment.

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The inventors have conducted intensive investigations for the purpose of overcoming the above disadvantages, thereby using hydrochloric acid as a hydrohalogenic acid for a commercial scale production. As a result, it has been found that ~ -APM hydrochloride can be efficiently precipitated out of an aqueous medium by carrying out its precipitation in the presence of more than a certain amount of inorganic chloride salt, even when hydrogen chloride is used in a considerably decreased quantity. The present invention has been accomplished based on the above finding.
The invention is concerned with a method of producing ~ -APM hydrochloride which comprises, upon ~;.
precipitation of ~-APM out o~ an aqueous medium, :
bringing it into contact with a considerably decreased quantity of hydrogen chloride in an aqueous medium ~, , ~; in the presence of an inorganic chloride salt.

According to the invention, there is pro-vided a method of producing ~-APM hydrochloride ~: which is free from the above-described disadvantages ~: ` with regard to its commercial production, wherein ~-APM hydrochloride is precipitated by bringing ; c~-APM into contact with a low concentration of : : hydrogen chloride in an aqueous medium in the pre-~:~` sence of an inorganic chloride salt, or (ii) ~-AP
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is brought into contact with methanol and a low con-centration of hydrogen chloride in an aqueous medium in the presence of an inorganic chloride salt, thus allowing ~-APM formed in accordance with the follow-ing equilibrium:

: d-AP ~ MeOH~ -APM + H2O

to precipitate immediately in the form of a hydro-chloride salt.
The concentration of hydrogen chloride to 10 be used in the invention can be decided by experi- 1.
ments, taking into consideration its corrosiveness to the materials, in particular, metals used for the reaction equipment to be employed, as well as the hydrolysis of the ester and peptide bonds contained : in ~-APM.
The inventors have conducted extensive investigations on the influence of the concentration of hydrogen chloride in the method according to , ~ , the invention. In particular such investig~tions, have been conducted at temperatures not higher than 30C. in view of the poor thermal stability of ~-APM, : and in particular, under highly acidic conditions : : ~
: as is the case of the method according to the invention : ~
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- 6 - 13 312 ~ 3 As a result, it has been established, as shown in Tables 1 and 2, that the adverse effects can be decreased to a fairly low level when hydrogen chloride is used in an amount not greater than 2 mol., per liter of the aqueous medium, and almost no adverse effects are caused when it is used in an amount not greater than 1 mol., per liter of the aqueous medium. The method of the present invention, there-fore, is practised in the presence of not more than 2 mol., preferably not more than 1 mol., of hydrogen chloride, per liter of the aqueous medium. The method is also practised with the amount of hydrogen chloride being not less than 1 mole, per mole of APM or ~-AP so as to provide an at`least equi-molar amount of HCl.

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9 13312~a In the method according to the invention, there can be used any inorganic chloride salt which is soluble in an amount not less than ca. 50 g., per liter of the aqueous medium, such that it is dissolved , in the medium, and does not hinder the precipitation of ~-APM hydrochloride, in particular ammonium and metal chlorides, as exemplified by NaCl, KCl, NH4Cl, CaC12, ZnC12 and the like. In order to precipitate ~-APM hydrochloride efficiently, the concentration of the inorganic salt must be above a certain level, which depends on the concentration of hydrogen ; chlorlde. In general, a concentration higher than such level is preferred.
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The required concentration of the inorganic ch-loride salt varies to some extent, depending on , the kind of inorganic chloride salt used. However, , the inorganic chloride salts can be used with advantage in an amount not lower than ca. 50 g., per liter of the aqueous medium with concentrations of hydrogen chloride of from 1 mol. to 2 mol., per .
liter of the aqueous solutions containing hydrogen chloride in relatively low concentrations, i.e., in concentrations up to 1 mol., per liter of the aqueous medium, the inorganic chloride salts can : :::: ~ ~ .:
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ca. 100 g. It is a matter of course that the ` ' `

133~2~a inorganic chloride salts are to be used in concent-rations at which no precipitates of the chlorides are generatea.
An inorganic chloride salt-containing aqueous medium can be prepared by adding a crystal-line inorganic chloride salt or an aqueous solution of an inorganic chloride salt to an aqueous medium, or by neutralizing hydrogen chloride contained in an aqueous medium with a solution of a metal hydroxide, for example, NaOH, KOH, Ca(OH)2 and Zn(OH)2, or aqueous ammonia, thereby forming an inorganic chloride salt in situ. It is also possible to use an inorganic chloride salt formed as a by-product in a .
commercial production process.

In the method according to the invention, ~; there can be used a solvent consisting of water alone, : as well as a mixture of methanol and water. The solvent used in the invention can additionally be . admixed with other solvents which do not hinder the addition reaction between ~-APM and hydrogen ~ , .
; : :chloride, for example, ethanol, ethylene glycol and acetone, in an amount not reducing the solubility of the inorganic chloride salts to an extremely low level . ;~ .

33125~ ~

In the method according to the invention, "~
-AP is brought into contact with methanol in the presence of hydrogen chloride and an inorganic chloride salt, in order to immediately precipitate the addition salt formed in situ from ~-APM and hydrogen chloride. The present inventors have con-ducted further investigations on the amount of methanol to be contained in the aqueous medium and, as a result, found that ~-AP can be efficiently converted into ~-APM hydrochloride even when methanol is contained in the aqueous medium in a relatively low concentration.
When methanol is used in an unnecessarily large amount, it was found that ~-APM formed by the , conversion tended to be further esterified to give a derivative in which the ~-carboxyl group contained in : . ~
~ the aspartic residue is esterified with methanol ;~ ~:: (hereinafter referred to as ~'-A(M)PM), in a markedly ; increased amount, thus resulting in a significant :~ 20 reduction in the conversion from C~-AP into ~(-APM.
: On the other hand, if the amount of methanol used is reduced too much, a decreased rate of conversion from ~-AP into ~-APM results, although it could be advantageous in preventing the undesirable side reaction from ~-APM into ~-A(M)PM.

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- 12 - ~33~2~

In view of the above, the inventoxs have conducted investigations on the amount of methanol to be used for the conversion from ~-AP into ~-APM
according to the invention. As a result, it has been established, as shown in Example 6, ~hat the most effective amount of methanol is in the range of from ca. 0.5 mol. to ca. 2 mol., per liter of the reaction medium, or the aqueous solvent.
Accordingly, it can be advantageous, in the case where the methanol contained in a reaction medium is consumed through the esterification of ~-AP and its concentration is reduced to a level . :: ;: :
below the lower limit of the above range, to add an ` - additional methanol to the reaction mixture in the course of the reaction. When all methanol used for the reaction is allowed to be present in the reaction mixture from the beginning of the reaction, an undesirable increase in the formation of ~-A(M)PM may result. Needless to say, at least 1 mol., in total, 20 ~ of methanol is required to convert 1 mol of ~-AP into APM.

, The present invention will be further illustrated by way~of the following Examples.

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Into 1,000 ml. of 2N hydrochloric acid was dissolved 25 g. of c~-APM at 40C., and the resulting solution was divided into three equal portions, each of which was then placed separately in 500 ml. flasks equipped with stirrers. One flask was set on a water bath maintained at 5C. and stirred overnight to effect precipitation (Case A). To the solutions placed in the other flasks was added 3.3 g. ~Case B) or 16.7 g. (Case C) of sodium chloride, and the sodium salt was dissolved. The flasks were then treated under the same conditions as in Case A.
The crystals precipitated were filtered off, washed with small portions of cold 2N hydrochloric - :, acid, and then vacuum dried at 50c. The crystals obtained in either case gave infrared spectra identical with that of an authentic ~-APM and had a purity of 98~i or ~above (determined by chromatography).
Results obtained are shown in Table 3.

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An aqueous solution (495 ml.) containing 15 g. of ~-APM and 30 g. of calcium chloride was placed in a 1 liter flask equipped with a stirrer.
To the solution was gradually added 105 ml. of 35%
hydrochloric acid, and the resulting mixture was stirred overnight, while maintaining its temperature at 5C
The crystals precipitated were treated in - 10 the same manner as in Example 1 to obtain 17.2 g. of crystals having a purity of 98.7%. Yield: 90.2%.

; EXAMPLE 3 In a mixture of 500 ml. of 1.1 N hydro-chloric acid and 50 ml. of ~-APM, and the resulting solution was divided into two equal portions. One :
of the portions was stored overnight in a refrigerator (Càse A). On the other hand, 60 g.~of ammonium chloride was added to the other portion and dissolved, and then the resulting mixture was stored overnight : : ~
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a purity of 97.6~ wer~ obtained ~the purities were determined by chromatography). Yields: 64.1% ~Case A); 90.8% ~Case B).

In 500 ml. of water was suspended 28 g~ of ~-APM and 12 g. of ~ APM, and the pH of the result-ing suspension was adjusted to 2.2 with 35~ hydro-chloric acid in order to dissolve the suspended sub-stances. The thus obtained solution was then divided ~-1~ into two equal portions. To one of them was added 150 ml. of 35~ hydrochloric acid, and the resulting mixture was stirred for 15 hours at 15C. to effect precipitation (Case A). To the other portion was , added 150 ml. of 35% hydrochloric acid. Thereafter, 61 g. of powdered anhydrous sodium carbonate wa - gradually added to the mixture in order to neutralize part of the excess hydrochloric acid. The resulting ` ~; -~ mixture was stirred for 15 hours at 15C. to effect .'~; : :
precipitation.
The crystals obtained were treated in the same manner as in Example 1. Results obtained are shown in Table 4. In this Example, Case A is a control.

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3 ~ a In a mixture of 38 ml. of methanol and 900 ml. of 2N hydrochloric acid was dissolved 143 g. of -AP having a purity of 98.2%, and the resulting solution was divided into two equal portions. One portion was stored with stirring at 25C. for 3 days and then at 10C. for 24 hours to precipitate the ;l cxystals of c~-APM hydrochloride (Case A). To the other portion was added 51 g. of crystals of ammonium chloride. The ammonium chloride was dissolved with stirring and the resulting mixtllre was treated in the same manner as above (Case B).

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as shown in Table 5 (the compositions were determined by liquid chromatography). In this Example, Case A
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In this Example, the initial concentration of methanol was 0.92 mol./l. and the concentration of NH4Cl in the mother liquid in Case B was 100 g./l.

In 1,500 ml. of lN hydrochloric acid were dissolved 215.6 g. of ~-APM having a purity of 97.5%
and 200 g. of sodium chloride. The resulting soiution was divided into 5 equal portions, and each portion was placed in a four necked flask equipped with a stirrer.
To them was added 5 ml. (Case A), 7 ml.
(Case B), 14 ml. (Case C), 30 ml. (Case D) or 50 ml.
(Case E) of methanol, and the resulting mixtures were treated in the same manner as in Example 5~ Crystals of ~-APM hydrochloride were obtained in an amount of ~ . .
17.5 g. (Case A), 28.6 g. (Case B), 33.3 g. (Case C), 33.0 g. (Case D) or 21.6 g. (Case E).
The compositions of the crystals were analyzed by liquid chromatography. Results obtained are shown in Table 6, together with the yields of ~-APM. In this example, Cases A and E are controls.
It will be understood that a large quantity of d~AP
is formed in the case where the concentration of methanol is low and a large quantity of ~-A(M)PM is formed when the concentration of methanol is high.

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The invention is further illustrated by reference to the accompanying drawings in which:
FIG. 1 is a graph showing the relationship between the density of hyrdogen chloride and the solubility of ~-APM at different temperatures; and FIG. 2 is a graph showing the relationship between the density of chloride salts and the solubility of ~-APM in the presence of different concentrations of hydrogen chloride.
With further reference to the drawings f Fig. 1 demonstrates that the hydrogen halides are used alone, in accordance with the prior art methods, they must be used in an excess of not less than ca.
2 mol., preferably not less than ca. 3 mol., per liter of the aqueous medium in order to precipitate ~; the salt of ~-APM in a high yield.
Fig. 2 shows that inorganic chloride salts can be used with advantage in an amount not lower than ca. 50 g. per liter of the aqueous medium with concentrations of-hydrogen chloride of from 1 molO to 2 mol. per liter of aqueous medium.

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Claims (5)

1. A method of producing .alpha.-L-aspartyl-L-phenylalanine methyl ester hydrochloride which comprises:
A) contacting .alpha.-aspartyl-L-phenylalanine methyl ester with an aqueous medium containing:
a) hydrogen chloride in an amount not more than ca. 2 mol., per liter of said aqueous medium, but not less than 1 mol., per mole of said .alpha.-L-aspartyl-L-phenylalanine methyl ester, and b) an inorganic chloride salt which does not hinder precipitation of .alpha.-APM hydrochloride, dissolved in said medium in an amount of at least ca.
50 g., per liter of said aqueous medium, or B) contacting .alpha.-L-aspartyl-L-phenylalanine with an aqueous medium containing:
i) hydrogen chloride in an amount not more than ca. 2 mol., per liter of said aqueous medium, but not less than 1 mol., per mole of said .alpha.-L-aspartyl-L-phenylalanine, ii) an inorganic chloride salt which does not hinder precipitation of .alpha.-APM hydrochloride, dissolved in said medium in an amount not less than ca. 50 g., per liter of said aqueous medium, and iii) methanol in an amount not less than 1 mol., per mole of .alpha.-L-aspartyl-L-phenylalanine.

2. A method of producing .alpha.-L-aspartyl-L-phenylalanine methyl ester hydrochloride, which comprises contacting .alpha.-aspartyl-L-phenylalanine methyl ester with an aqueous medium containing:

(a) hydrogen chloride in an amount not more than ca. 2 mol., per liter of said aqueous medium, but not less than 1 mol., per mole of said .alpha.-L-aspartyl-L-phenylalanine methyl ester, and (b) an inorganic chloride salt which does not hinder precipitation of .alpha.-APM hydrochloride, in an amount of at least ca. 50 g., per liter of said aqueous medium, to form crystals of .alpha.-L-aspartyl-L-phenylalanine methyl ester hydrochloride, and then recovering the crystals therefrom.

3. A method as set forth in claim 2, wherein said aqueous medium contains:
(a) hydrogen chloride in an amount not more than ca. 1 mol., per liter of said aqueous medium, but not less than 1 mol., per mole of said .alpha.-L-aspartyl-L-phenylalanine methyl ester, and (b) an inorganic chloride salt which does not hinder precipitation of .alpha.-APM hydrochloride, in an amount not less than ca. 100 g., per liter of said aqueous medium.

4. A method of producing .alpha.-L-aspartyl-L-phenylalanine methyl ester hydrochloride, which comprises:
contacting .alpha.-L-aspartyl-L-phenylalanine with an aqueous medium containing:
(i) hydrogen chloride in an amount not more than ca. 2 mol., per liter of said aqueous medium, but not less than 1 mol., per mole of said .alpha.-L-aspartyl-L-phenylalanine, (ii) an inorganic chloride salt which does not hinder precipitation of .alpha.-APM hydrochloride, in an amount not less than ca. 50 g., per liter of said aqueous medium, and (iii) methanol in an amount not less than 1 mol., per mole of .alpha.-L-aspartyl-L-phenylalanine, to form crystals of .alpha.-L-aspartyl-L-phenylalanine methyl ester hydrochloride, and then recovering the crystals therefrom.

5. A method as set forth in claim 4, wherein said aqueous medium contains:
(i) hydrogen chloride in an amount not more than ca. 1 mol., per liter of said aqueous medium, but not less than 1 mol., per mole of said .alpha.-L-aspartyl-L-phenylalanine, (ii) an inorganic chloride salt which does not hinder precipitation of .alpha.-APM hydrochloride, in an amount not less than ca. 100 g., per liter of said aqueous medium, and (iii) methanol in an amount of from ca. 0.5 mol. to ca. 2 mol., per liter of said aqueous medium, but not less than 1 mol., per mole of said .alpha.-L
aspartyl-L-phenylalanine.