JPH0463080B2 - - Google Patents

Description

Detailed Description of the Invention The present invention provides α-L-aspartyl-L-phenylalanine methyl ester (hereinafter referred to as “α-APM”).
It is sometimes abbreviated as. ) or its hydrochloride production method, more specifically, α-L-aspartyl-L-phenylalanine methyl ester in which the amino group is protected with a formyl group (hereinafter sometimes abbreviated as “For”); That is, N-formyl-α
Formyl group is efficiently and preferentially removed from -L-aspartyl-L-phenylalanine methyl ester (hereinafter sometimes abbreviated as "For-α-APM") in methanol-hydrochloric acid, resulting in α −
APM is isolated as its hydrochloride, that is, α-L-aspartyl-L-phenylalanine methyl ester hydrochloride (hereinafter sometimes abbreviated as “α-APM・HCl”), and if necessary, the hydrochloric acid α-APM or α- converts salt into free α-APM
Concerning a method for producing APM hydrochloride. Here, α−
APM.HCl may be changed into α-L-aspartyl-L-phenylalanine methyl ester according to a known method, if necessary. The present invention provides an effective means for synthesizing sweet peptides, and exhibits a superior effect in eliminating the For group from For-α-APM. Incidentally, For-α-APM can be easily obtained as a synthetic intermediate of α-APM by condensation of N-formyl-L-aspartic acid anhydride and L-phenylalanine methyl ester (Japanese Patent Application Laid-Open No. 52−23001
Such). Conventional methods for eliminating N-formyl groups include a method using dilute hydrochloric acid, a method using hydrogen peroxide (G. Losse et al., Ann. Chem., 636 , 140 (1960)), and a method using hydrazine (P. Lefrancier et al., Bull.
Soc. Chim. France, 1965, 3668), method using aniline (R. Geiger et al., Chem. Ber., 102 ,
2487 (1969), method using hydroxylamine (R.Geiger, Chem.Ber., 101 , 3386 (1968)), method by catalytic reduction (C.Losse et al., J.Prakt.Chem.,
24 118 (1964)) are known. Among these, the method using hydrochloric acid is easy to operate and inexpensive, and is excellent as an industrial method for eliminating N-formyl groups. A commonly used method for deformylation using dilute hydrochloric acid is N-
For simple compounds such as formyl amino acids, the acidic hydrolysis method involves heating and hydrolyzing with dilute hydrochloric acid (V.du Vigneaud et al., J.Biol.Chem., 98 , 577).
(1932) applies, but for peptides,
Usually 0.5 to avoid hydrolysis of peptide bonds
Method of leaving at room temperature for 48 hours in methanolic hydrochloric acid below the specification (JC Sheehan et al., J.Am.Chem.Soc.,
80, 1158 (1958)) shall apply. However, For−
In the case of a peptide such as α-APM, which has an ester group and a carboxyl group in addition to an amino group protected by a formyl group, the ester group and the peptide bond undergo hydrolysis in the acidic hydrolysis method; In the acidic alcohol decomposition method, free carboxyl groups are esterified, and in addition, it takes a long time (48 hours) or hydrolysis (Japanese Patent Publication No. 54-17727).
It is industrially disadvantageous to do so. The present inventors have previously developed a method using a strong acid salt of hydroxylamine (Japanese Patent Application Laid-Open No. 51-68520) and a method using a strong acid in a water-containing organic solvent (Japanese Patent Application Laid-open No. 52-23001) as methods that overcome the above drawbacks. However,
It is industrially disadvantageous to use hydroxylamine or an organic solvent. Therefore, as a deformylation method that does not use an organic solvent, we invented a method of short-term heat treatment at 70 to 150°C in 0.5 to 3N hydrochloric acid (Japanese Patent Application 18588-1981), but even with this method, the ester group It was found that this method was slightly hydrolyzed, so it cannot be said that this method is completely satisfactory. Therefore, the present inventors investigated an industrially advantageous N-formyl group elimination reaction that overcomes the above drawbacks and found that For-α-APM was mixed with methanol at a temperature of 0 to 40°C at a high concentration. The formyl group is removed by contacting with a mixed solvent of hydrochloric acid, and the generated α-APM is sequentially precipitated as a poorly soluble hydrochloride salt. By proceeding with the deformylation reaction while excluding it from the reaction system, side reactions such as ester groups can be performed. They discovered that it is possible to suppress hydrolysis of peptide bonds, esterification of free carboxyl groups, etc., and finally obtain α-APM in high yield, leading to the completion of the present invention (first invention). Ivy. Using formyl group as a protecting group, α-
When producing APM, it is very advantageous to apply the method of the invention. APM using formyl group
In the manufacturing process, N-formyl-aspartic acid anhydride and phenylalanine methyl ester are usually condensed, but in this case, the desired α-
In addition to For-α-APM, the precursor of APM, its isomer N-formyl-β-L-aspartyl-
L-phenylalanine methyl ester (hereinafter referred to as
) is produced as a by-product. To obtain the desired α-APM, conventionally, for example,
After treating a mixture of For-α-APM and For-β-APM with methanol and dilute hydrochloric acid, this solution was neutralized to obtain α-APM. However, in this method, α-APM hydrochloride does not precipitate during the reaction, so the side reactions mentioned above occur, and α-APM after neutralization
−When discussing APM, α−APM and β−
APM (herein, β-APM is an abbreviation for β-L-aspartyl-L-phenylalanine methyl ester) has approximately the same solubility, so β-
Almost the same amount of α-APM as APM remains in the mother liquor. However, if the method of the present invention is applied to such a case, that is, For-α-APM and For-β-
When a mixture of APM is brought into contact with methanol to highly concentrated hydrochloric acid, the formyl group is removed over time, producing α-APM and β-APM, respectively, but α-APM is a poorly soluble hydrochloride salt. Then,
Since it comes out of the system, side reactions of α-APM are suppressed, and β-APM is concentrated in the mother liquor, adding a purification effect and efficiently converting α-APM/HCl to α-APM. can be obtained. In addition,
Regarding the extremely low solubility of α-APM hydrochloride in hydrochloric acid, see US Pat. No. 3798207 (Japanese Patent Publication No. 49-41425). Regarding the reaction conditions for the formyl group elimination reaction in the present invention, as a result of examining the reaction under various conditions, it was found that the reaction is significantly affected by the concentrations of hydrochloric acid and ethanol used, and the reaction temperature. Ivy. According to the findings of the present inventors, when the concentration of hydrochloric acid is too low, precipitation of APM hydrochloride is small and side reactions such as hydrolysis of ester groups are involved. On the other hand, when the concentration of hydrochloric acid is too high, peptide bonds and ester groups are rapidly hydrolyzed without being differentiated from formyl groups. Regarding the reaction temperature, if the reaction temperature was too high, hydrolysis of ester groups and peptide bonds occurred without being distinguished from formyl groups, and the amount of precipitated hydrochloride was small. When the concentration of methanol was too low, the ester groups underwent hydrolysis, and when the concentration was too high, the free carboxyl groups were esterified and the amount of hydrochloride precipitated was also small. If the reaction time was too short, the formyl group was not removed sufficiently. On the other hand, a reaction time that is too long is not advantageous in terms of industrialization. Therefore, the concentration of hydrochloric acid is 2 to 12N, preferably 5 to 8N, the methanol concentration is 5 to 60% by volume relative to hydrochloric acid, preferably 10 to 30% by volume, and the reaction temperature is 0 to 40°C. A reaction time of about 1 to 5 days was selected as the reaction condition for the deformylation reaction. As a result of further research, the present inventors discovered that even better effects could be achieved by slightly modifying the first invention, and completed the second and third inventions. One is to maintain a mixture of For-α-APM, methanol, and highly concentrated hydrochloric acid at 0 to 40°C to generate For-α-
This is a heat treatment in which this mixture is once held at a high temperature for a short period of time as a pretreatment to deformylate APM. This heat treatment further promotes the precipitation of α-APM hydrochloride and reduces the time required for deformylation.
The above-mentioned 1-5 days can be shortened to 1-3 days. The invention according to claim 2. Here, from a practical point of view, high temperatures of 50-100℃ and 30℃
A short time of less than a minute is desirable. In addition, the amount of hydrochloric acid
For-α-APM, 0.5 to 3 times the mole, preferably 1
It is desirable to set it to about 1.3 times the mole. The other modification is to perform heat treatment at the same temperature and time for a short time at high temperature, but to perform this heat treatment at a slightly lower concentration of hydrochloric acid, and to add high-concentration hydrochloric acid after the heat treatment. According to such modification, in addition to the effects of promoting α-APM hydrochloride precipitation and shortening the time required for deformylation, side reactions are further suppressed, and therefore α-APM hydrochloride or free α-APM hydrochloride or free α- The effect of improving the yield of APM is also achieved.
The invention according to claim 3. Here, the slightly lowered hydrochloric acid concentration is about 2 to 6 normal,
Therefore, the concentration of methanol versus hydrochloric acid increases slightly,
The amount is 10 to 70% by volume, preferably 30 to 55% by volume (based on hydrochloric acid). Also, the amount of hydrochloric acid (vs. For−α−APM)
It is also desirable to adjust as above. The additional amount of high concentration hydrochloric acid is such that the hydrochloric acid concentration of the reaction mixture becomes the hydrochloric acid concentration of the first invention, that is, 2 to 12N, preferably 5 to 8N. The addition period is usually after the reaction mixture has been cooled after the heat treatment, but it may be before or during the cooling. The α-APM hydrochloride obtained in this way can be used as a sweetener as it is (Japanese Patent Application Laid-Open No. 13371/1983), but it is usually neutralized with an alkali such as sodium carbonate in an aqueous solvent. The peptide is isolated as a free peptide. As described above, according to the present invention, For-α-
After removing the formyl group from APM, α-APM
By precipitating and separating α-APM as a poorly soluble hydrochloride, the target peptide can be obtained with a higher isolation yield than ever before. Therefore, the present invention provides an industrially extremely useful means for producing α-APM or its hydrochloride. This is what we provide. The present invention will be explained in more detail with reference to Examples below. Example 1 A mixed solvent of 15 ml of methanol and 40 ml of 4N hydrochloric acid (hydrochloric acid concentration 2.9N) was heated to 60°C. For this
After adding 48.3 g of -α-APM and heating at the same temperature for 15 minutes, the reaction solution was quickly cooled to 25°C and concentrated hydrochloric acid was added.
28 ml was added (hydrochloric acid concentration in the reaction mixture: 5.2N) and stirred at the same temperature for 2 days. After further stirring at 5° C. for 3 hours, the precipitated crystals of α-APM hydrochloride dihydrate were collected by filtration. According to the amino 3 analyzer (model 835 manufactured by Hitachi), the α-APM content in this crystal was 36.1 g.
It was 81.8% compared to For-α-APM. Example 2 A mixed solvent of 10 ml of methanol and 17 ml of water (concentration of hydrochloric acid
2.6N), For−α−APM32.2g and For−β−
A mixture with 81.g of APM was added (hydrochloric acid concentration in the reaction mixture was 5.1N) and heated to 85°C. After adding 10 ml of concentrated hydrochloric acid and heating at the same temperature for 5 minutes, the reaction solution was quickly cooled to 20°C. Add 20ml of concentrated hydrochloric acid and heat at 20℃ for 2 hours.
Stir for days. After further stirring at 5° C. for 3 hours, the precipitated crystals of α-APM hydrochloride dihydrate were collected by filtration. The α-APM content in this crystal is 23.4g, and the For-APM content is 23.4g.
It was 79.5% relative to α-APM, and β-APM was not detected. Example 3 A mixed solvent of 15 ml of methanol and 65 ml of 7N hydrochloric acid (hydrochloric acid concentration 5.7N) was heated to 65°C. For−α−
After adding 48.3g of APM and processing at the same temperature for 10 minutes,
The reaction solution was quickly cooled to 20°C. After stirring at 20°C for 1 day and further stirring at 5°C for 3 hours, the precipitated crystals were collected by filtration. The α-APM content in the obtained crystals was 34.0 g, which was 77.0% based on For-α-APM. Example 4 A mixed solvent of 15 ml of methanol, 25 ml of water, and 14 ml of concentrated hydrochloric acid (hydrochloric acid concentration: 2.5N) was heated to 60°C. For−α
- After adding 48.3 g of APM and treating at the same temperature for 15 minutes, the reaction solution was quickly cooled to 20°C. concentrated hydrochloric acid 28
ml (hydrochloric acid concentration in the reaction mixture: 4.9N) and heated at 20°C.
The mixture was stirred for 2 days. After further stirring at 5° C. for 3 hours, the precipitated crystals were collected by filtration. The α-APM content in the obtained crystals was 35.0 g, which was 79.3% based on For-α-APM. Example 5 A mixed solvent of 15 ml of methanol, 25 ml of water, and 14 ml of concentrated hydrochloric acid (hydrochloric acid concentration: 2.5N) was heated to 70°C. For−α
- After adding 48.3 g of APM and treating at the same temperature for 15 minutes, the reaction solution was quickly cooled to 20°C. concentrated hydrochloric acid 14
ml (hydrochloric acid concentration in the reaction mixture: 4N), stirred at 20°C for 2 days, further stirred at 5°C for 3 hours, and precipitated crystals were collected by filtration. The α-APM content in the obtained crystals was 34.3 g, which was 77.7% based on For-α-APM. Example 6 An experiment similar to Example 4 was conducted using 10 ml of methanol (initial hydrochloric acid concentration in the mixed solvent: 2.7N; after adding 28 ml of concentrated hydrochloric acid, the hydrochloric acid concentration in the anti-concentrated mixture was 5.2N).
The isolated yield of α-APM hydrochloride dihydrate is For-α
-It was 82.3% against APM. Example 7 An experiment similar to Example 4 was conducted using 10 ml of methanol (initial hydrochloric acid concentration in the mixed solvent: 2.3N; after adding 28 ml of concentrated hydrochloric acid, the hydrochloric acid concentration in the anti-concentrated mixture was 5N). α
-The isolated yield of APM hydrochloride dihydrate is For-α
-It was 73.6% against APM. Example 8 A mixed solvent of 18 ml of methanol, 30 ml of water, and 17 ml of concentrated hydrochloric acid (hydrochloric acid concentration: 2.5N) was heated to 70°C. to this
After adding 48.3 g of For-α-APM and treating at the same temperature for 10 minutes, the reaction solution was quickly cooled to 20°C, and 24 ml of concentrated hydrochloric acid was added (hydrochloric acid concentration in the reaction mixture was 4.4N).
The mixture was stirred at 20°C for 2 days. After further stirring at 5° C. for 3 hours, the precipitated crystals of α-APM hydrochloride dihydrate were collected by filtration. The α-APM content in this crystal is 33.1g,
It was 75.0% for For-α-APM.

Claims (1)

[Claims] 1 N-formyl-α-L-aspartyl-L-
After holding phenylalanine methyl ester at 50 to 100°C for within 30 minutes in a mixture containing methanol and hydrochloric acid and having a hydrochloric acid concentration of 2 to 12N,
0-40 with or without addition of 2-12N hydrochloric acid
℃ and kept at this temperature to remove the formyl, and the resulting α-L-aspartyl-L-phenylalanine methyl ester is precipitated as a sparingly soluble hydrochloride, which is isolated and, if necessary, freed. A method for producing α-L-aspartyl-L-phenylalanine methyl ester or its hydrochloride, which comprises converting it into an ester.