JP2000063339A - Production of n-protective group-amino acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the subject industrial method which needs no step for removing by-product impurities or for purification. SOLUTION: This method introduces a protective group in the amino group of the amino acid in an alkaline aqueous solvent. The amino acid useful as the stock for the present invention may be natural or synthetic, and may be a racemate, optically active one or derivative thereof. The examples include alanine, arginine, isoleucine, glycine, glutamine, cystine, cysteine, serine, tyrosine, tryptophan, valine, phenylalanine, S-phenylcysteine, methionine, and leucine. Of these, S-phenylcysteine, phenylalanine, tryptophan or methionine are preferable.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a high-purity N-protecting group-amino acid, and more specifically to an N-protecting group-
The present invention relates to a method in which the production of by-product impurities can be suppressed by adding an organic solvent immiscible with water to an alkaline solution of an amino acid and the reaction can be performed, and the substance can be highly purified. That is, the present invention relates to a method for efficiently producing an N-protecting group-amino acid having a small amount of by-product impurities in a high yield.

[0002]

The benzyloxycarbonyl group, which is also known as Z group, is a typical amino group-protecting group for amino acids and amino acid derivatives.

As a method for introducing a Z group into an amino acid and an amino acid derivative, benzyloxycarbonyl chloride (abbreviated as Z-Cl) is used as a protecting group reagent, and Schott is used.
In most cases, the ten-Baumann method is used. This method is carried out by dissolving an amino acid in an aqueous solution of sodium hydroxide and dropping Z-Cl in an equimolar amount to the amino acid while maintaining alkalinity. (Peptide Synthesis, published by Maruzen Co., Ltd., author; Nobuo Izumiya et al., P24-28 (197).
5)).

However, in the above-mentioned reaction, by-product impurities are produced, and particularly in the reaction at a high concentration, the production of by-product impurities is increased. Even by the method of extracting the Z-compound into an organic solvent under acidic conditions after completion of the reaction, many by-product impurities are extracted into the organic solvent and separation is difficult. In any case, subsequent removal of by-product impurities and purification steps are required, which is not preferable as an industrial production method. Therefore, how to suppress the production of by-product impurities in the reaction process has been a problem.

[0005]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems, the present inventors have made an organic solvent immiscible with water coexist in the reaction of introducing a protecting group into an amino acid in an alkaline aqueous medium. It has been found that the production of by-product impurities can be suppressed by carrying out the reaction in a layer system, and the present invention has been completed.

That is, the present invention provides a method for introducing a protecting group into an amino group of an amino acid in an alkaline aqueous medium,
A method for producing an N-protecting group-amino acid, which is characterized in that an organic solvent immiscible with water is allowed to coexist.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The mode of the method for producing a high-purity N-protecting group-amino acid in the present invention is described in detail below.

First, the amino acid is dissolved in an alkaline aqueous medium, and then an organic solvent immiscible with water is added.

The amino acid used as a raw material in the present invention may be a natural product or a non-natural product, and may be a racemic body, an optically active body or a derivative thereof. Examples of amino acids include alanine, arginine, isoleucine, glycine, glutamine, cystine, cysteine, serine, tyrosine, tryptophan, valine, phenylalanine, S-phenylcysteine, methionine,
Examples thereof include leucine and the like, preferably S-phenylcysteine, phenylalanine, tryptophan or methionine, and particularly preferably S-phenylcysteine.

Regarding the concentration of the amino acid in the alkaline aqueous medium in the present invention, there is no problem as long as the amino acid used is soluble. The concentration is preferably 4% to 50%.

As the alkali used in the present invention, there are generally used alkali metal hydroxides, alkaline earth metal hydroxides, alkali metal carbonates, alkali metal bicarbonates and the like which are used when introducing a protective group into an amino group. There are specifically, sodium hydroxide, potassium hydroxide, sodium hydrogencarbonate and the like. There is no problem as long as the amount of alkali used is a sufficient amount for the reaction, preferably 1 to 5 times the molar equivalent relative to the amino acid, particularly preferably 2.0 times to
It is 2.5 times the molar equivalent.

The organic solvent used in the present invention may be any one that is immiscible with water, and examples thereof include aromatic compounds such as benzene, toluene, xylene and the like, with toluene being particularly preferred. Two or more of these may be mixed and used. Among the water immiscible solvents, chloroform,
Halides such as dichloromethane react with the raw materials in the reaction process of introducing a protecting group to generate a large amount of by-product impurities, and therefore are not preferably added to the reaction system. Similarly, acetic acid esters and the like are decomposed under alkali in the same reaction process, and a large amount of by-product impurities are generated, so that addition to the reaction system is not preferable. The amount of the organic solvent immiscible with water is not particularly limited, but it is 1/50 of the weight of the water layer.
To 3 times, preferably from 1/10 to an equivalent amount.

As described above, a reagent (for example, Z-Cl) which serves as a protective group for an amino group is added dropwise to a two-layer liquid of an amino acid alkaline aqueous medium in which an organic solvent coexists, and the amino group is added to the amino group. Introduce a protecting group.

The amino-protecting group used in the present invention may be, for example, a nucleus-substituted or unsubstituted benzyloxycarbonyl group, and the protecting-group reagent may be Z-Cl. There is no problem as long as the amount of the reagent used as a protective group is sufficient for the reaction, and it is preferably 0.9 to 1.1 times the molar equivalent relative to the amino acid. The reaction temperature is 50 ° C. or lower, preferably 10 ° C. to 25 ° C.

The N-protecting group-amino acid produced according to the present invention can be isolated and purified by a known method.
For example, hydrochloric acid is added to the aqueous layer from which the organic solvent has been separated and removed under alkali to crystallize and isolate it, or after the reaction, add hydrochloric acid to add N.
The protecting group amino acid may be extracted into the organic solvent layer, and after liquid separation, a poor solvent may be added to the organic solvent layer for crystallization or cooling crystallization. When the produced N-protecting group-amino acid is insoluble in the organic solvent used for the reaction, after removing the organic solvent by liquid separation, an organic solvent in which the N-protecting group-amino acid is soluble is newly added to the aqueous layer, Hydrochloric acid may be added to extract the N-protecting group amino acid in an organic solvent, and after liquid separation, a poor solvent may be added to the organic solvent layer for crystallization or may be isolated by cooling crystallization. In this case, the organic solvent to be newly added is not particularly specified, aromatic compounds such as benzene, toluene, xylene, chloroform, halides such as dichloromethane, methyl acetate, ethyl acetate, acetic acid esters such as butyl acetate, butanol, Any alcohol, such as benzyl alcohol, that is immiscible with water may be used. Of these, acetic acid esters such as methyl acetate, ethyl acetate and butyl acetate are preferable.

[0016]

EXAMPLES The method of the present invention will be described in detail below with reference to examples. Example 1 100.0 g (0.50) of S-phenyl-L-cysteine
7 mol) was suspended in 227.2 g of water, and 92.4 g (1.039 mol) of 45% sodium hydroxide solution was added and dissolved. After adding 339 g of toluene to this, 10
86.4 g (0.507 mo) of Z-Cl under stirring at ℃.
1) was slowly added dropwise. After completion of dropping, the mixture was aged for about 1 hour. After aging, the temperature was raised and the internal temperature reached 40 ° C.
% Hydrochloric acid 105.7 g (1.014 mol) was added to make the pH of the aqueous layer acidic. Then, the temperature is raised and the internal temperature is 60 ° C.
-Carbobenzoxy-S-phenyl-L-cysteine was extracted into the toluene layer. After extraction operation, N
513.1 g of a toluene solution of -carbobenzoxy-S-phenyl-L-cysteine was obtained. N- by concentration analysis
Carbobenzoxy-S-phenyl-L-cysteine is 3
At a 2.2% concentration, 165 g (0.498 mol) was obtained. The reaction result was 98.3% in terms of yield relative to S-phenyl-L-cysteine. HPLC of the toluene solution of N-carbobenzoxy-S-phenyl-L-cysteine
Analysis was carried out. N-carbobenzoxy-S-phenyl-
L-cysteine is 95.7% and other by-product impurities are 4.
3% (toluene excluded). It was confirmed that the amount of by-product impurities was smaller than that in the aqueous layer reaction (Comparative Example 1) and the purity was high. The comparison results are shown in Table 1.

Example 2 L-phenylalanine 100.0 g (0.605 mo)
l) was suspended in 292.4 g of water, and 107.6 g (1.211 mol) of 45% sodium hydroxide solution was added and dissolved. After adding 292 g of toluene to this, 10 ° C,
103.2 g (0.605 mol) of Z-Cl under stirring
Was slowly added dropwise. After completion of dropping, the mixture was aged for about 1 hour. After aging, the temperature was raised and when the internal temperature reached 40 ° C., 126.2 g (1.210 mol) of 35% hydrochloric acid was added, and the p
H was made acidic. Subsequently, the temperature was raised and the internal temperature was 60 ° C., and N-carbobenzoxy-L-phenylalanine was extracted into the toluene layer. After the extraction operation, N-carbobenzoxy-L-phenylalanine toluene solution 467 g by liquid separation operation
Got According to the concentration analysis, N-carbobenzoxy-L-phenylalanine was 37.4% and had a concentration of 167.4 g.
(0.559 mol) was obtained. The reaction result was 92.4% based on the yield of L-phenylalanine. When the toluene solution of the N-carbobenzoxy-L-phenylalanine was analyzed by HPLC, N-carbobenzoxy-L-
Phenylalanine was 96.1% and other by-product impurities were 3.9% (toluene was excluded). It was confirmed that the amount of by-product impurities produced was smaller and the purity was higher than in the aqueous layer reaction (Comparative Example 2). The comparison results are shown in Table 2.

Example 3 L-tryptophan 100.0 g (0.490 mo)
l) was suspended in 312.9 g of water, and 87.1 g (0.980 mol) of 45% sodium hydroxide solution was added and dissolved. After adding 292 g of toluene to this, 10 ° C,
With stirring, Z-Cl (87.9 g, 0.515 mol) was slowly added dropwise. After completion of dropping, the mixture was aged for about 1 hour. After aging, the toluene was separated and removed, and the aqueous layer was treated with 292 g of ethyl acetate and 102.2 g of 35% hydrochloric acid (0.980 mo).
1) was added to make the pH of the aqueous layer acidic, and N-carbobenzoxy-L-tryptophan was extracted into the ethyl acetate layer.
After the extraction operation, N-carbobenzoxy-L was separated by liquid separation operation.
450.4 g of a solution of tryptophan in ethyl acetate was obtained. According to the concentration analysis, N-carbobenzoxy-L-tryptophan was found to be 153.4 g (0.45%) at a concentration of 34.1%.
3 mol) was obtained. The reaction result was 92.5% with respect to the yield of L-tryptophan. The N-carbobenzoxy-L
HPLC analysis of a tryptophan-ethyl acetate solution revealed that N-carbobenzoxy-L-tryptophan was 97.9% and other by-product impurities were 2.1% (toluene was excluded). It was confirmed that the amount of by-product impurities produced was smaller and the purity was higher than in the aqueous layer reaction (Comparative Example 3). The comparison results are shown in Table 3.

Example 4 100.0 g (0.670 mol) of L-methionine was suspended in 280.9 g of water, and 119.1 g (1.34 mol) of 45% sodium hydroxide solution was added and dissolved. After adding 292 g of toluene thereto, 120.0 g (0.704 mol) of Z-Cl was slowly added dropwise at 10 ° C. under stirring. After completion of dropping, the mixture was aged for about 1 hour. After aging, the toluene was separated and removed, and the aqueous layer was 292 g of ethyl acetate.
And 359.7% hydrochloric acid 139.7 g (1.34 mol) were added,
The pH of the aqueous layer was made acidic and N-carbobenzoxy-L-methionine was extracted into the ethyl acetate layer. After the extraction operation, a liquid separation operation was performed to obtain 483.3 g of an ethyl acetate solution of N-carbobenzoxy-L-methionine. N- by concentration analysis
Carbobenzoxy-L-methionine was obtained at a concentration of 38.7% to obtain 187.0 g (0.660 mol). The reaction result was 98.5% based on the yield of L-methionine. The N
HPLC analysis of a solution of -carbobenzoxy-L-methionine in ethyl acetate revealed that N-carbobenzoxy-L-methionine was 93.0% and other by-product impurities were 7.0% (toluene: Excluded). It was confirmed that the amount of by-product impurities produced was smaller and the purity was higher than in the aqueous layer reaction (Comparative Example 4). The comparison results are shown in Table 4.

Comparative Example 1 The reaction of Example 1 was carried out without adding toluene, and after completion of dropping, aging was carried out for 1 hour. After aging, 339 g of toluene was added, the temperature was raised, and when the internal temperature reached 40 ° C, 105.7 g of 35% hydrochloric acid was added.
(1.014 mol) was added to make the pH of the aqueous layer acidic. Subsequently, the temperature was raised and the internal temperature was 60 ° C., and N-carbobenzoxy-S-phenyl-L-cysteine was extracted into the toluene layer. After the extraction operation, N-carbobenzoxy-S-phenyl-L-cysteine was added to a toluene solution 47 by a liquid separation operation.
6.7 g was obtained. According to the concentration analysis, N-carbobenzoxy-S-phenyl-L-cysteine was 28.5% concentration,
136.1 g (0.411 mol) was obtained. The reaction result was 81.0% in terms of yield relative to L-phenylalanine. HPLC analysis of the toluene solution of N-carbobenzoxy-S-phenyl-L-cysteine revealed that N-carbobenzoxy-S-phenyl-L-cysteine was 7%.
4.2% and other by-product impurities were 25.8% (excluding toluene).

Comparative Example 2 The reaction of Example 2 was carried out without adding toluene. After completion of dropping, the mixture was aged for 1 hour. After aging, add 292 g of toluene,
When the temperature rises and the internal temperature reaches 40 ° C, 35% hydrochloric acid 126.2
g (1.210 mol) was added to make the pH of the aqueous layer acidic. Subsequently, the temperature was raised and the internal temperature was 60 ° C., and N-carbobenzoxy-L-phenylalanine was extracted into the toluene layer. After the extraction operation, N-carbobenzoxy-L-
465.8 g of a toluene solution of phenylalanine was obtained.
According to the concentration analysis, N-carbobenzoxy-L-phenylalanine had a concentration of 35.6% and contained 165.9 g (0.554%).
mol) was obtained. The reaction result was 91.6% in terms of yield relative to L-phenylalanine. The N-carbobenzoxy-L
HPLC analysis of a toluene solution of -phenylalanine revealed that N-carbobenzoxy-L-phenylalanine was 87.2% and other by-product impurities were 12.8% (toluene was excluded).

Comparative Example 3 The reaction of Example 3 was carried out without the addition of toluene. After completion of dropping, the mixture was aged for 1 hour. After the aging, in order to meet the same conditions as in Example 3, 292 g of toluene was added, and after washing, the toluene layer was separated and removed. After that, 292 g of ethyl acetate and 3 were added to the water layer.
102.2 g (0.980 mol) of 5% hydrochloric acid was added to make the pH of the aqueous layer acidic, and N-carbobenzoxy-L-tryptophan was extracted into the ethyl acetate layer. After the extraction operation, a liquid separation operation was performed to obtain 448.0 g of an ethyl acetate solution of N-carbobenzoxy-L-tryptophan. Concentration analysis revealed that N-carbobenzoxy-L-tryptophan was 33.
151.0 g (0.446 mol) was obtained at a 7% concentration. The reaction result was 91.1% in terms of yield relative to L-tryptophan.
Met. HPLC analysis of the ethyl acetate solution of N-carbobenzoxy-L-tryptophan revealed that N
-Carbobenzoxy-L-tryptophan is 93.0
%, And other by-product impurities were 7.0% (toluene was excluded).

Comparative Example 4 The reaction of Example 4 was carried out without the addition of toluene. After completion of dropping, the mixture was aged for 1 hour. After the aging, in order to meet the same conditions as in Example 4, 292 g of toluene was added, and after washing, the toluene layer was separated and removed. After that, 292 g of ethyl acetate and 3 were added to the water layer.
139.7 g (1.34 mol) of 5% hydrochloric acid was added to make the pH of the aqueous layer acidic, and N-carbobenzoxy-L-methionine was extracted into the ethyl acetate layer. After the extraction operation, a liquid separation operation was performed to obtain 478.8 g of an ethyl acetate solution of N-carbobenzoxy-L-methionine. Concentration analysis revealed that N-carbobenzoxy-L-methionine had a concentration of 38.2% and 1
83.0 g (0.645 mol) was obtained. The reaction result was 96.4% in terms of yield relative to L-methionine. When HPLC analysis was performed on the ethyl acetate solution of N-carbobenzoxy-L-methionine, N-carbobenzoxy-L-
Methionine 89.4%, other by-product impurities 10.6
% (Toluene excluded).

[0024]

[Table 1]

[Table 2]

[Table 3]

[Table 4]

Impurity analysis of N-carbobenzoxy-amino acid was carried out by ODS column CAPCELL PAK C18.
(Manufactured by SHISEIDO) was used for HPLC analysis. The analysis conditions are shown below. [Analytical HPLC conditions] Column: CAPCELL PAK C18 (SHIS
Φ6.0 mm × 250 mm mobile phase; 10 mM phosphoric acid / acetonitrile = 53 /
47 temperature; 40 ° C. flow rate; 1.5 ml / min detection; UV (210 nm) retention time; N-carbobenzoxy-S-phenyl-L-
Cysteine = 6.2 min N-carbobenzoxy-L-phenylalanine = 4.9
Min N-carbobenzoxy-L-tryptophan = 4.3 min N-carbobenzoxy-L-methionine = 3.5 min

[0026]

INDUSTRIAL APPLICABILITY According to the present invention, particularly in a high-concentration reaction system, it is possible to suppress the production of by-product impurities in the reaction step, and thus a high-purity N-protecting group amino acid can be obtained in high yield and efficiency. It has become possible to manufacture well.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Seto Yoshino             30 Asamu-cho, Omuta-shi, Fukuoka Mitsui Chemicals             Within the corporation (72) Inventor Nobuhiro Fukuhara             30 Asamu-cho, Omuta-shi, Fukuoka Mitsui Chemicals             Within the corporation F-term (reference) 4H006 AA02 AC80 BB11 BB31 BC35                       BJ50 BS10 NB22

Claims (5)

[Claims] 1. A process for producing an N-protecting group-amino acid, which comprises allowing an organic solvent immiscible with water to coexist in a reaction of introducing a protecting group into an amino group of an amino acid in an alkaline aqueous medium. 2. The method according to claim 1, wherein the amino acid is selected from the group consisting of S-phenyl-L-cysteine, phenylalanine, tryptophan and methionine. 3. The method according to claim 1, wherein the amino-protecting group is a nucleus-substituted or unsubstituted benzyloxycarbonyl group. 4. The method according to claim 1, wherein the water-immiscible organic solvent is an aromatic hydrocarbon. 5. The method according to claim 1, wherein the charged concentration of amino acid is in the range of 4% to 50% with respect to water.