JP7264874B2 - Method for producing reduced glutathione - Google Patents

Description

The present invention relates to an industrially practicable method for producing reduced glutathione.

Reduced glutathione is a tripeptide consisting of glutamic acid, cysteine, and glycine, and is treated as a pharmaceutical in Japan as an antioxidant that has an auxiliary role in protecting cells from reactive oxygen species such as free radicals and peroxides. .
It is known that reduced glutathione can be produced by reductively cleaving the disulfide bond of oxidized glutathione. For example, Patent Document 1 discloses a method for producing reduced glutathione by reducing oxidized glutathione with an alkali borohydride. Further, Patent Document 2 discloses a method for producing reduced glutathione by reducing oxidized glutathione with an iron compound, sulfur and hydrogen sulfide. Patent Document 3 discloses a method for producing reduced glutathione by electrolytically reducing oxidized glutathione using a cathode chamber and an anode chamber separated by a diaphragm.
In addition, although it does not produce reduced glutathione, Non-Patent Document 1 discloses that after modifying the amino group of cystine in which two cysteines are bonded via a disulfide bond with a p-nitrocarbobenzoxy group, Pd/ A method of obtaining cysteine by catalytic reduction at normal temperature and pressure in the presence of C is disclosed.

Japanese Patent Publication No. 46-33939 JP 2007-254325 A International Publication No. 2014/133129 Pamphlet

CASIMIR BERSE et al., J. Am. Org. Chem. , 22(7), pp. 805-808 (1957)

As described above, various methods have been developed for reducing oxidized glutathione to produce reduced glutathione. However, in order to implement the conventional method industrially, there are problems that cannot be ignored.
For example, the method described in Patent Document 1 has a problem that the reagent must be used in a large excess of 10-fold molar equivalents. In addition, the method described in Patent Document 2 has a low conversion rate from oxidized glutathione to reduced glutathione and has a problem in yield, and the method of Patent Document 3 requires an electrolytic cell. In addition, the reduction method described in Non-Patent Document 1 requires an extra step of modifying the amino group with a p-nitrocarbobenzoxy group, and in reduced glutathione, which is a tripeptide, cysteine exists second. Therefore, it is doubtful whether the technique of Non-Patent Document 1 can be applied to the production of reduced glutathione.
Accordingly, an object of the present invention is to provide an industrially feasible method for producing reduced glutathione by reducing oxidized glutathione.

The present inventors have made intensive studies to solve the above problems. As a result, they found that S-sulfoglutathione produced by the cleavage can be easily reduced by cleaving the disulfide bond of oxidized glutathione with sulfite ions or hydrogen sulfite ions, and completed the present invention.
The present invention is shown below.

[1] A method for producing reduced glutathione, comprising:
cleaving the disulfide bonds of oxidized glutathione with sulfite ions or hydrogen sulfite ions to obtain S-sulfoglutathione and reduced glutathione; and
A method comprising the step of reducing the S-sulfoglutathione.
[2] The method according to [1] above, wherein the S-sulfoglutathione is reduced using a reducing agent.
[3] The method according to [2] above, wherein an alkali metal borohydride is used as the reducing agent.
[4] The method according to [3] above, wherein sodium borohydride is used as the alkali metal borohydride.
[5] The method according to any one of [1] to [4] above, wherein the sulfite ion or hydrogen sulfite ion is used in an amount of 0.1 or more molar equivalents relative to the oxidized glutathione.
[6] The method according to any one of [1] to [5] above, wherein a sulfite or hydrogen sulfite is used as the compound containing sulfite ions or hydrogen sulfite ions.
[7] The method according to [6] above, wherein an alkali metal salt or an ammonium salt is used as the sulfite or hydrogen sulfite.

The reagents essentially used in the method of the present invention are oxidized glutathione, which is a raw material, and inexpensive sulfite ions or hydrogen sulfite ions. Since these are water-soluble, water can be used as a solvent in the method of the present invention. In addition, in the method of the present invention, the disulfide bond of oxidized glutathione is first cleaved by sulfite ions or hydrogen sulfite ions, and the resulting S-sulfoglutathione is more easily reduced than oxidized glutathione. A reduction in the amount is possible. Moreover, according to the method of the present invention, reduced glutathione can be produced in high yield. Therefore, the method of the present invention is suitable for industrial large-scale synthesis of reduced glutathione, and is industrially excellent.

Each step of the method of the present invention will be described in detail below.
Step A: Step of Cleavage of Disulfide Bonds In this step, the disulfide bonds of oxidized glutathione are cleaved with sulfite ions or hydrogen sulfite ions to obtain S-sulfoglutathione and reduced glutathione.

The oxidized glutathione used as a raw material in the present invention is not particularly limited. For example, its origin is not limited, and it may be an extract extracted from a microbial culture medium containing oxidized glutathione, or a purified product. In addition, oxidized glutathione may be mixed with reduced glutathione at any ratio.

Sulfite ions (SO ₃ ²⁻ ) and hydrogen sulfite ions (HSO ₃ ⁻ ) are also not particularly limited, and can be obtained by dissolving sulfites or hydrogen sulfites in water or by dissolving sulfur dioxide in water. . Reagents containing sulfite or bisulfite ions are inexpensive. It is known that sulfite ions and hydrogen sulfite ions exist in an equilibrium state in an aqueous solution and exist as sulfur dioxide at low pH. Sulfites and hydrogen sulfites include, for example, alkali metal salts such as lithium salts, sodium salts and potassium salts; group 2 metal salts such as magnesium salts, calcium salts and barium salts; ammonium salts; transition metal salts such as iron salts. Examples include salt.

The amounts of sulfite ions and hydrogen sulfite ions to be used are not particularly limited and may be determined as appropriate. More preferably 5-fold molar equivalents or more, or 1-fold molar equivalents or more, and even more preferably 2-fold molar equivalents or more. Although the upper limit of the above ratio is not particularly limited, it can be, for example, 10-fold molar equivalent or less. It is believed that sulfite ions cleave oxidized glutathione into S-sulfoglutathione and reduced glutathione. However, as shown in the following reaction formula, the sulfite group of S-sulfoglutathione may be -S-SO3-, and the thiol group of reduced glutathione may be -S-.

This step is preferably carried out in a solvent. The solvent is not particularly limited as long as it does not inhibit the reaction, but water can be preferably used. As water, for example, ultrapure water, pure water, distilled water, purified water, reverse osmosis water, deionized water, industrial water, tap water, well water, etc. can be used without particular limitation.

The concentration of oxidized glutathione during the reaction may be adjusted as appropriate, and is not particularly limited. .

The reaction temperature is not particularly limited as long as the reaction proceeds, but can be, for example, 0° C. or higher and 80° C. or lower. The temperature is preferably 5° C. or higher, preferably 60° C. or lower, more preferably 40° C. or lower, and even more preferably 30° C. or lower. Moreover, you may react at normal temperature, without temperature control.

The cleavage reaction of the disulfide bond in this step is a pH-dependent equilibrium reaction. Under excessively low pH conditions, sulfite ions or hydrogen sulfite ions become sulfur dioxide, and the reaction does not proceed. equilibrium tilts to Therefore, the reaction pH is preferably 2 or more and 10 or less. The pH is more preferably 4 or higher, more preferably 5 or higher, more preferably 9 or lower, and even more preferably 8.5 or 8 or lower. The pH of the reaction solution can be adjusted with sulfuric acid, hydrogen chloride, sodium hydroxide, an aqueous solution thereof, or the like.

Since this reaction proceeds very rapidly, it can be carried out simultaneously with the next reduction reaction.

Step B: Reduction Step In this step, at least S-sulfoglutathione is reduced to obtain reduced glutathione. In the above step A, the disulfide bond of oxidized glutathione is cleaved by sulfite ions or hydrogen sulfite ions into S-sulfoglutathione and reduced glutathione, and S-sulfoglutathione is more easily reduced than oxidized glutathione. can be reduced under milder conditions than the conventional method, and the amount of reducing agent used can be reduced.

The reduction method is not particularly limited as long as it can reduce S-sulfoglutathione produced by the disulfide cleavage reaction of oxidized glutathione, and examples thereof include a method using a reducing agent and an electrolytic reduction method. In addition, even when the thiol group of reduced glutathione is present as -S- in the reaction solution of step A above, -S- is easily converted to a thiol group by protons, and S-sulfoglutathione is It easily becomes a thiol group under reducing conditions. Furthermore, when the disulfide bond is not cleaved in step A above and oxidized glutathione remains, there is a possibility that the oxidized glutathione can be directly reduced to reduced glutathione in this step.

The reducing agent that can be used in this step is not particularly limited as long as it can reduce S-sulfoglutathione produced by the disulfide cleavage reaction of oxidized glutathione. Combinations of catalyst and hydrogen, phosphines, iodide salts, cuprous oxide, and the like can be mentioned.

Examples of alkali borohydride salts include sodium borohydride, potassium borohydride, and lithium borohydride. Examples of metal catalysts include catalysts containing nickel, ruthenium, palladium, platinum, rhodium, copper, and the like. The metal catalyst may be oxides or salts of these elements as long as they contain these elements. Examples of phosphines include tributylphosphine and triphenylphosphine. Phosphonium iodide etc. can be mentioned as an iodide salt.

The amount of the reducing agent to be used may be adjusted as appropriate and is not particularly limited. A double molar equivalent or more is more preferable. The upper limit of the amount of the reducing agent used is not particularly limited.

The reduction reaction of S-sulfoglutathione is carried out using the disulfide cleavage reaction solution of oxidized glutathione with sulfite ions or hydrogen sulfite ions as it is. That is, for example, by adding a reducing agent to the solution after the reaction in the above step A, the steps A and B can be carried out in the same system (one pot). Moreover, since the disulfide cleavage reaction is a very fast reaction, the reaction proceeds without any problem even if the disulfide cleavage reaction and the reduction reaction are carried out simultaneously.

The reduction reaction of S-sulfoglutathione with a reducing agent is carried out, for example, by adding a reducing agent to the disulfide cleavage reaction solution of oxidized glutathione with sulfite ions or hydrogen sulfite ions and mixing. The reducing agent may be added to the reaction solution as it is, or may be dispersed or dissolved in a solvent such as water and added as a dispersion or solution.

The reducing agent is preferably added to the reaction solution containing S-sulfoglutathione in the form of a solution or dispersion. By adding a reducing agent solution or dispersion, local side reactions can be suppressed, and at least S-sulfoglutathione can be efficiently reduced with a smaller amount of reducing agent. The concentration of the reducing agent in the solution or dispersion may be appropriately adjusted within a range in which reduction proceeds efficiently, and may be, for example, 1% by mass or more and 20% by mass or less. In the case of using an alkali borohydride or the like, a basic aqueous solution other than water may be used as the solvent for the above solution or dispersion. The base of the basic aqueous solution is not particularly limited, but for example, alkali metal hydroxides such as lithium hydroxide, sodium hydroxide and potassium hydroxide; alkali metal hydrogen carbonates such as sodium hydrogen carbonate and potassium hydrogen carbonate; Alkali metal carbonates such as sodium carbonate and potassium carbonate; Group 2 metal hydroxides such as calcium hydroxide; and organic bases such as triethylamine and pyridine. The concentration of the base in the basic aqueous solution may be adjusted as appropriate, and may be, for example, 0.01% by mass or more and 50% by mass or less.

The reaction temperature is not particularly limited as long as it is a temperature at which the reduction reaction proceeds. The temperature is preferably 5° C. or higher, preferably 60° C. or lower, more preferably 40° C. or lower, and even more preferably 30° C. or lower. Moreover, you may react at normal temperature, without temperature control.

The pH of the reaction solution is preferably 4 or more and 10 or less because the reducing agent may react with water and be consumed under excessively low pH conditions. The pH is more preferably 6 or higher, still more preferably 6.5 or higher, more preferably 9 or lower, even more preferably 8.5 or lower, and particularly preferably 8 or lower.

Although the reaction time varies depending on the reaction conditions, about 1 hour or more and 5 hours or less is sufficient.

After completion of the reaction, reduced glutathione may be purified according to a known method. For example, cuprous oxide is added to form a water-insoluble reduced glutathione copper salt and the precipitate is filtered off. The resulting precipitate is suspended in water and hydrogen sulfide liberates reduced glutathione. After concentrating and cooling the resulting aqueous solution of reduced glutathione, seed crystals and ethanol can be added to precipitate crystals of reduced glutathione.

This application claims the benefit of priority based on Japanese Patent Application No. 2018-57970 filed on March 26, 2018. The entire contents of the specification of Japanese Patent Application No. 2018-57970 filed on March 26, 2018 are incorporated herein by reference.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited by the following examples, and can be modified appropriately within the scope that can conform to the gist of the above and later descriptions. It is of course possible to implement them, and all of them are included in the technical scope of the present invention.

Example 1: Reduction with sodium sulfite and sodium borohydride Oxidized glutathione (4.5 g, 7.35 mmol) was dissolved in water (295.5 g) to prepare a 15 g/L oxidized glutathione aqueous solution. A 4-fold molar equivalent of sodium sulfite (3.7 g, 29.4 mmol) was added to the oxidized glutathione, and the mixture was stirred at 25°C for 5 minutes. The pH of the resulting reaction solution was 7.0. Sodium borohydride (41.6 mg, 11.0 mmol) in an amount of 1.5 molar equivalents relative to oxidized glutathione was added to the reaction solution, and the mixture was stirred at 25° C. for 10 minutes. Then, the pH of the reaction solution was adjusted to 6.0 using 25% sulfuric acid, and the solution was further stirred for 2 hours. As a result of analyzing the obtained reaction solution by high performance liquid chromatography, it was confirmed that 4.48 g (conversion rate: 99.3%) of reduced glutathione was produced.

Comparative Example 1: Reduction with sodium borohydride alone Oxidized glutathione (4.5 g, 7.35 mmol) was dissolved in water (295.5 g) to prepare a 15 g/L oxidized glutathione aqueous solution. After adjusting the pH of the aqueous solution to 7.0 with a 10% sodium hydroxide solution, sodium borohydride (41.6 mg, 11.0 mmol) was added in an amount 1.5 times the molar equivalent of oxidized glutathione, and the mixture was heated at 25°C. and stirred for 10 minutes. Then, the pH of the reaction solution was adjusted to 6.0 with 25% sulfuric acid, and the mixture was further stirred for 2 hours. As a result of analyzing the resulting reaction solution by high performance liquid chromatography, the amount of reduced glutathione produced was 0.33 g (conversion rate: 7.3%).

Example 2: Reduction reaction of S-sulfoglutathione Oxidized glutathione (16.54 mg, 0.0270 mmol) was dissolved in water (983.5 mg) to prepare a 16.5 g/L oxidized glutathione aqueous solution. A 6-fold molar equivalent of sodium sulfite (20.4 mg, 0.162 mmol) was added to the oxidized glutathione, and the mixture was stirred at 25°C for 5 minutes. Next, sodium borohydride (17.4 mg, 0.459 mmol) was added in an amount 17 times molar equivalent to oxidized glutathione, and the mixture was stirred at 30°C for 60 minutes. The resulting reaction solution was analyzed by high-performance liquid chromatography to determine the amount of residual oxidized glutathione and the amount of produced reduced glutathione, and the yield of reduced glutathione was calculated. Table 1 shows the results. In the table, "GSH" indicates reduced glutathione, and "GSSG" indicates oxidized glutathione.

Comparative Example 2: Acid hydrolysis of S-sulfoglutathione Oxidized glutathione (16.54 mg, 0.0270 mmol) was dissolved in water (983.5 mg) to prepare a 16.5 g/L oxidized glutathione aqueous solution. A 6-fold molar equivalent of sodium sulfite (20.4 mg, 0.162 mmol) was added to the oxidized glutathione, and the mixture was stirred at 25°C for 5 minutes. Then, hydrochloric acid was added to adjust the pH of the reaction solution to 1.0, and the mixture was stirred at 30°C for 60 minutes. The resulting reaction solution was analyzed in the same manner as in Example 2 above. Table 1 shows the results.

Comparative Example 3: Acid hydrolysis of S-sulfoglutathione Acid hydrolysis of S-sulfoglutathione was carried out in the same manner as in Comparative Example 2 except that the reaction temperature after adding hydrochloric acid was 60° C., and the reaction solution was analyzed. Table 1 shows the results.

As the results shown in Table 1, after cleaving the disulfide bond of oxidized glutathione with sodium sulfite, when sodium borohydride is used as a reducing agent, compared to hydrolysis using hydrochloric acid, the reduced form The yield of glutathione was overwhelmingly high.

Reference Example 1: Examination of the amount of sodium sulfite to be used 0.5 to 4 times molar equivalent to the oxidized glutathione contained in the crude aqueous solution of oxidized glutathione obtained by hot water extraction of the culture solution of oxidized glutathione-producing bacteria of sodium sulfite was added and stirred at 25° C. for 5 minutes. The pH of the reaction solution was 6. The reaction solution was analyzed by high-performance liquid chromatography to determine the cleavage rate of disulfide bonds of oxidized glutathione. In this experiment, since 1 mol of oxidized glutathione produces 1 mol of reduced glutathione and 1 mol of S-sulfoglutathione, the molar conversion rate from oxidized glutathione to reduced glutathione is the GSSG cleavage rate. can be regarded Table 2 shows the results.

As shown in Table 2, the disulfide bond of oxidized glutathione is cleaved even when sodium sulfite is used in an amount of 0.5 times the molar equivalent of oxidized glutathione, which is sufficient for the reduction reaction as shown in Example 3. It was revealed.

Example 3: Effect of pH on reduction reaction of S-sulfoglutathione Oxidized glutathione (145 mg, 0.237 mmol) was dissolved in water (9.855 g) to prepare a 14.5 g/L oxidized glutathione aqueous solution. Add 4-fold molar equivalents of sodium sulfite (119.5 mg, 0.948 mmol) or 0.5-fold molar equivalents of sodium sulfite (14.9 mg, 0.119 mmol) to oxidized glutathione, and add 25% sulfuric acid or 10% After adjusting the pH of the reaction solution to 6.0, 7.0 or 8.0 using an aqueous sodium hydroxide solution, the solution was stirred at 25°C for 5 minutes. Next, sodium borohydride (13.4 mg, 0.356 mmol) was added in a molar equivalent of 1.5 times the oxidized glutathione, and the mixture was stirred at 25°C for 10 minutes. Furthermore, 10 minutes after the addition of sodium borohydride, the pH of the reaction solution was adjusted to 6.0 with 25% sulfuric acid, and the mixture was stirred at 25° C. for 60 minutes. The resulting reaction solution was analyzed by high-performance liquid chromatography to determine the amount of residual oxidized glutathione and the amount of produced reduced glutathione, and the yield of reduced glutathione was calculated. Table 3 shows the results.

Comparative Example 4 Effect of pH on Reduction Reaction of Oxidized Glutathione For comparison, direct reduction of oxidized glutathione was performed without performing a cleavage reaction with sulfite ions. Oxidized glutathione (145 mg, 0.237 mmol) was dissolved in water (9.855 g) to prepare a 14.5 g/L oxidized glutathione aqueous solution. After adjusting the pH of the oxidized glutathione aqueous solution to 6.0, 7.0 or 8.0 with 25% sulfuric acid or 10% sodium hydroxide, sodium borohydride in an amount 1.5 times the molar amount of oxidized glutathione (13.4 mg, 0.356 mmol) was added and stirred at 25° C. for 10 minutes. Furthermore, 10 minutes after the addition of sodium borohydride, the pH of the reaction solution was adjusted to 6.0 with 25% sulfuric acid, and the mixture was stirred at 25° C. for 60 minutes. The resulting reaction solution was analyzed by high-performance liquid chromatography to determine the amount of residual oxidized glutathione and the amount of produced reduced glutathione, and the yield of reduced glutathione was calculated. Table 3 shows the results.

As the results shown in Table 3 show, the reduction reaction of oxidized glutathione hardly progressed when only sodium borohydride was used without using sodium sulfite.
In contrast, when sodium sulfite was followed by sodium borohydride, oxidized glutathione was successfully reduced to reduced glutathione.

Example 4: Confirmation of the influence of the amount of sodium borohydride used in the reduction reaction of S-sulfoglutathione Oxidized glutathione (145 mg, 0.237 mmol) was dissolved in water (9.855 g) and oxidized to 14.5 g/L. type glutathione aqueous solution was prepared. Sodium sulfite (59.7 mg, 0.474 mmol) was added in an amount two times the molar equivalent of oxidized glutathione, the pH was adjusted to 7.0, and the mixture was stirred at 25°C for 5 minutes. Next, after dissolving the amount of sodium borohydride shown in Table 4 in 9 times by mass of a 0.5% sodium hydroxide aqueous solution, the resulting solution was added to the reaction solution and stirred at 25° C. for 10 minutes. After that, the pH of the reaction solution was adjusted to 7.0 with 25% sulfuric acid and stirred at 25° C. for 60 minutes. The reaction solution was analyzed by high-performance liquid chromatography, the amount of reduced glutathione produced was determined, and the yield of reduced glutathione was calculated. Table 4 shows the results.

Comparative Example 5: Confirmation of influence of amount of sodium borohydride used on reduction reaction of oxidized glutathione For comparison, direct reduction of oxidized glutathione was carried out without carrying out a cleavage reaction with sulfite ions. Oxidized glutathione (145 mg, 0.237 mmol) was dissolved in water (9.855 g) to prepare a 14.5 g/L oxidized glutathione aqueous solution. After adjusting the pH of the reaction solution to 7.0 with 10% sodium hydroxide, the amount of sodium borohydride shown in Table 4 was dissolved in 9 times the mass of a 0.5% sodium hydroxide aqueous solution, and the resulting The solution was added to the above solution and stirred for 10 minutes at 25°C. Next, the pH of the reaction solution was adjusted to 7.0 with 25% sulfuric acid and stirred at 25°C for 60 minutes. Thereafter, the reaction solution was analyzed by high-performance liquid chromatography to determine the production amount of reduced glutathione, and the yield of reduced glutathione was calculated. Table 4 shows the results.

As the results shown in Table 4, it is preferable to use sodium borohydride in an amount of 0.5 or more molar equivalents relative to oxidized glutathione, and it is particularly preferable to use sodium borohydride in an amount of 0.7 or more molar equivalents. It became clear.

Claims (7)

A method for producing reduced glutathione, comprising:
cleaving the disulfide bonds of oxidized glutathione with sulfite ions or hydrogen sulfite ions to obtain S-sulfoglutathione and reduced glutathione; and
A method comprising the step of reducing the S-sulfoglutathione. 2. The method of claim 1, wherein the S-sulfoglutathione is reduced using a reducing agent. 3. The method of claim 2, wherein an alkali metal borohydride is used as said reducing agent. 4. The method of claim 3, wherein sodium borohydride is used as said alkali metal borohydride salt. 5. The method according to any one of claims 1 to 4, wherein the sulfite ion or hydrogen sulfite ion is used in an amount of 0.1 or more molar equivalents relative to the oxidized glutathione. The method according to any one of claims 1 to 5, wherein a sulfite or hydrogen sulfite is used as the compound containing sulfite or hydrogen sulfite ions. 7. The method according to claim 6, wherein alkali metal salts or ammonium salts are used as sulfites or hydrogen sulfites.