JP7623802B2 - Biotin and its manufacturing method, and biotin-amine salt manufacturing method - Google Patents

Description

The present invention relates to biotin and a method for producing the same, as well as a method for producing biotin-amine salts.

Biotin is a water-soluble vitamin that belongs to the vitamin B group (Non-Patent Document 1). Biotin is expected to have diabetes prevention effects, skin disease improvement effects, and biotin deficiency improvement effects, and demand for it as a pharmaceutical and feed additive is increasing. Biotin is represented by the following formula (1) and is also called d-biotin.

As shown below, biotin (BIOF) is synthesized, for example, from a ureido derivative (URD) through seven steps (Patent Document 1).

Here, biotin has seven analogs other than d-biotin represented by the above formula (1) (Non-Patent Document 2). In the process of synthesizing d-biotin, for example, analogs such as l-biotin represented by the following formula (2) may be contained in the product. These analogs other than d-biotin do not have biological activity, so it is desirable to remove them from the product.

A method has been disclosed for purifying a mixture of d-biotin and l-biotin and increasing the purity of d-biotin (Non-Patent Document 3).

International Publication No. 2018/025722

Pierre J. de Clercq, “Biotin: A Timeless Challenge for Total Synthesis” Chemical Reviews, 1997, Vol. 97, No. 6. Toru YAMANO, Isao AOKI, and Kunio TAKANOHASHI “Direct Optical Resolution of (±)－Biotin and (±)－Epibiotin by a "Reserved-Phase High-Performance Liquid Chromatography" J. Nutr. Sci. Vitaminol. , 39, 419-423, 1993. DONALD E. WOLF, RALPH MOZINGO, SANTON A. HARRIS, R. CHRISTIAN ANDERSON AND KARL FOLKERS “Biotin. VI. Resolution of dl-Biotin” CONTRIBUTION FROM THE RESEARCH LABORATORIES, MERCK AND Co. , INC. RECEIVED AUGUST 17, 1945.

The object of the present invention is to provide highly pure biotin and a method for producing the same, as well as a method for producing biotin-amine salts.

According to one embodiment, a method for producing biotin-amine salts is provided. The method includes contacting a first mixture containing biotin or a salt thereof and epibiotin or a salt thereof with an amine to obtain a second mixture containing biotin and a salt of the amine.

According to another embodiment, a method for producing biotin is provided. The method includes contacting the salt of biotin and amines obtained by the method according to the embodiment with an acid to obtain a third mixture containing biotin.

In another embodiment, biotin is provided that has a purity of 99.93% or more as measured by area percentage in high performance liquid chromatography.

According to the embodiment, highly pure biotin and a method for producing the same, as well as a method for producing biotin-amine salts, are provided.

The manufacturing method according to the embodiment includes contacting a first mixture containing biotin or a salt thereof and epibiotin or a salt thereof with amines to obtain a second mixture containing biotin and salts of amines.

In addition, a manufacturing method according to another embodiment includes contacting the salt of biotin and amines obtained by the method according to the embodiment with an acid to obtain a third mixture containing biotin.

Hereinafter, salts of biotin and amines will also be referred to as biotin-amine salts. Also, salts of epibiotin and amines will also be referred to as epibiotin-amine salts.

As mentioned above, when biotin is synthesized via an intermediate, the product containing biotin may contain analogues such as diastereomers as impurities. Among diastereomers, epibiotin, represented by the following formula (3), is an impurity that is particularly difficult to remove from the product.

The area percentage of epibiotin in the product by high performance liquid chromatography (HPLC) is approximately 5%, which can be a factor in reducing the purity of biotin. If purification processes such as neutralization crystallization and activated carbon treatment are repeatedly performed to remove this epibiotin, the yield of biotin can decrease.

Therefore, a method for efficiently removing epibiotin from the product and increasing the purity of biotin is desired. As a result of intensive research, the present inventors have found that highly pure biotin can be obtained by contacting a mixture containing biotin and epibiotin with amines to form a biotin-amine salt, and then contacting this biotin-amine salt with an acid to perform a desalting process for biotin.

That is, since biotin and epibiotin are weak acids, biotin-amine salts and epibiotin-amine salts can be produced by contacting them with highly basic amines. Biotin-amine salts and epibiotin or epibiotin-amine salts have different solubilities in a specific solvent. The magnitude of this difference in solubility is greater than the difference in solubility in a specific solvent between other biotin-base salts and epibiotin-base salts. Therefore, by contacting biotin-amine salts and epibiotin or epibiotin-amine salts with a solvent in which one salt has a high solubility and the other salt has a low solubility, a solution of the highly soluble salt and a solid of the less soluble salt can be obtained. This allows the biotin-amine salts and epibiotin or epibiotin-amine salts to be separated with higher accuracy. Biotin can be isolated by contacting the separated biotin-amine salts with an acid.

This method allows epibiotin to be removed from the product with high precision, increasing the purity of biotin. Therefore, highly pure biotin can be obtained in high yield without multiple purification treatments of the product. Since the biotin obtained in this way has a low epibiotin content, it is possible to achieve ultra-high purity, for example, purity of 99.93% or more based on HPLC area percentage.

The details of the present invention are explained below.

(First mixture)
The first mixture contains biotin or a salt thereof and epibiotin or a salt thereof. The first mixture is typically a solid. In one example, the purity of biotin in the first mixture by HPLC area percentage is 90% or more and less than 99.93%. In one example, the purity of epibiotin in the first mixture by HPLC area percentage is 0.01% or more and 5% or less. Note that the biotin salt and the epibiotin salt may dissociate into biotin and a base, and epibiotin and a base in HPLC measurement. Therefore, when the first mixture contains a biotin salt and an epibiotin salt, the biotin contained in the biotin salt and the epibiotin contained in the epibiotin salt are counted as biotin and epibiotin, respectively, in HPLC measurement.

The first mixture may be a product obtained in the seventh step (Stage 7) of the seven steps of the biotin synthesis described above. That is, in the sixth step of the seven steps of the biotin synthesis described above, when the vinyl sulfide (DVE) is reduced to N,N'-dibenzylbiotin (HVC), an epimer of N,N'-dibenzylbiotin is considered to be a by-product. In the seventh step, a deprotection reaction is performed on this epimer of N,N'-dibenzylbiotin, and epibiotin is considered to be produced. Note that the first mixture may be a product obtained after subjecting the product obtained in the seventh step to a purification treatment such as a neutralization crystallization treatment or an activated carbon treatment. The first mixture may also be commercially available biotin.

An example of a method for producing the first mixture is as follows.
First, a vinyl sulfide compound represented by the following formula (4) (hereinafter also referred to as a vinyl sulfide compound) is prepared. As the vinyl sulfide compound, for example, a compound synthesized by a known method or a commercially available compound may be used.

By reducing this vinyl sulfide by a known method, a product containing N,N'-dibenzylbiotin shown in the following formula (5) is obtained. This product contains the epimer of N,N'-dibenzylbiotin as a by-product.

By mixing this product with an acid, a debenzylation reaction of N,N'-dibenzylbiotin and its epimer occurs to obtain a product containing biotin and epibiotin. As the first mixture, the product thus obtained may be used, or the product after purification may be used. The product may be purified multiple times.

Purification treatments include neutralization crystallization treatments, activated carbon treatments, and the like. Neutralization crystallization treatments are a method of removing impurities by repeatedly contacting biotin and epibiotin with a base other than amines to produce biotin salts and epibiotin salts, and then acidifying the aqueous solution of these salts and filtering the resulting solid. Examples of bases other than amines include sodium hydroxide and potassium hydroxide. This mixture of biotin salts and epibiotin salts may be used as the first mixture. Activated carbon treatments are a method of removing impurities from the first mixture by mixing the first mixture with activated carbon.

(Biotin or its salts)
Biotin is represented by the following formula (1) and is also called d-biotin.

Biotin salts can be produced by reacting biotin, which is a weak acid, with a base. Specific examples of bases include sodium hydroxide and potassium hydroxide. Specific examples of biotin salts include biotin sodium salt and biotin potassium salt. The base is preferably an inorganic compound.

Whether biotin or a biotin salt is used, a biotin-amine salt can be obtained by contacting it with an amine. Note that biotin or its salt may be a mixture of biotin and a biotin salt.

(Epibiotin or its salt)
Epibiotin is represented by the following formula (3) and is also called d-epibiotin. Epibiotin may include l-epibiotin.

Epibiotin can be produced by reacting epibiotin, which is a weak acid, with a base. Specific examples of bases include sodium hydroxide and potassium hydroxide. Specific examples of epibiotin salts include epibiotin sodium salt and epibiotin potassium salt. The base is preferably an inorganic compound.

Whether epibiotin or an epibiotin salt is used, a salt of epibiotin/amines can be produced by contacting it with an amine. Note that epibiotin or its salt may be a mixture of epibiotin and an epibiotin salt.

(Amines)
The amines include amines, amine derivatives, and alkaloids. The amines can form salts when in contact with biotin. The amines are, for example, represented by the following formula (6).

In the above formula (6), R ¹ , R ² , and R ³ are each a hydrogen atom, an alkyl group, an aralkyl group, or a heterocycle. The number of carbon atoms in the alkyl group, aralkyl group, or heterocycle is preferably 1 to 30. When at least two of R ¹ , R ² , and R ³ are an alkyl group, an aralkyl group, or a heterocycle, they may be bonded to each other to form a ring.

As the amine, it is preferable to use at least one selected from the group consisting of arginine, quinidine, and dicyclohexylamine. Since there is a large difference in solubility in a solvent between the biotin amine salts and the epibiotin amine salts obtained from these amine derivatives, it is easier to increase the purity of biotin. As the amine, it is more preferable to use arginine. Arginine may be in the L-form or the D-form, but it is preferable to use L-arginine.

When a low-toxicity substance such as L-arginine is used as the amine, a low-toxicity and highly safe biotin amine salt can be obtained. Like biotin, such biotin amine salt can be used as a pharmaceutical or feed additive.

The amount of amines per mole of biotin or its salt is preferably 0.5 moles or more and 2 moles or less, and more preferably 1.0 moles or more and 1.5 moles or less.

(Method for producing biotin-amine salts)
The biotin-amine salt is represented, for example, by the following formula (7): In the following formula (7), R ¹ , R ² and R ³ are the same as those in the above formula (6).

By contacting the first mixture with amines, a second mixture containing biotin-amine salts is obtained. The synthesis of biotin-amine salts may be performed by a solid phase method that does not use a reaction solvent, or by a liquid phase method that uses a reaction solvent. From the viewpoint of increasing efficiency, it is preferable to synthesize biotin-amine salts in the liquid phase.

The first mixture is contacted with the amines, for example, in an environment of -40°C or higher and 100°C or lower. The contact temperature is preferably -30°C or higher and 80°C or lower, and more preferably -20°C or higher and 60°C or lower. In another example, the first mixture is contacted with the amines in an environment of 50°C or higher and 80°C or lower. When the first mixture is contacted with the amines at a high temperature, the fluidity of the second mixture increases, and the yield of biotin-amine salts tends to increase.

The first mixture and the amines are preferably stirred after contacting each other. The stirring time is, for example, from 30 seconds to 72 hours, preferably from 1 minute to 48 hours, and more preferably from 3 minutes to 24 hours. In addition, the first mixture and the amines may be heated to 30°C to 80°C after contacting each other. By heating, unnecessary solvent in the crystal lattice can be removed.

The contact of the first mixture with the amines is preferably carried out in the presence of a first reaction solvent. That is, it is preferable to further include mixing the first mixture with the first reaction solvent to obtain a first mixed solution, and then contacting the first mixed solution with the amines to obtain a second mixture. As the first reaction solvent, it is preferable to use one capable of dissolving biotin or a salt thereof, epibiotin or a salt thereof, and amines. As the first reaction solvent, for example, at least one selected from the group consisting of water, N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, methanol, ethanol, and 2-propanol is used. As the first reaction solvent, it is preferable to use water. Water includes distilled water, purified water, pure water, ultrapure water, tap water, or a mixture thereof.

The amount of the first reaction solvent per 1 g of the first mixture is, for example, 1 mL to 30 mL, and preferably 10 mL to 20 mL. In another example, the amount of the first reaction solvent per 1 g of the first mixture is preferably 2 mL to 30 mL, and more preferably 3 mL to 10 mL.

When the first mixture is contacted with amines in the presence of a first reaction solvent, the resulting biotin-amine salt and epibiotin-amine salt may be in a dissolved or solvated state in the reaction solvent.

The formation of salts of biotin and amines in the second mixture can be confirmed, for example, by nuclear magnetic resonance (NMR) spectroscopic analysis.

(Method for separating biotin-amine salts)
The second mixture may contain epibiotin and its amine salts in addition to the biotin-amine salt. Therefore, it is preferable to separate only the biotin-amine salt from the second mixture. As a method for separating the biotin-amine salt from the second mixture, a solvent that selectively dissolves or selectively precipitates the biotin-amine salt may be mixed with the second mixture. Because of ease of operation, it is preferable to contact the second mixture with a solvent that selectively precipitates the biotin-amine salt (hereinafter also referred to as a precipitation solvent) to obtain a precipitate of the biotin-amine salt.

As the precipitation solvent, for example, at least one selected from the group consisting of water, methanol, ethanol, 2-propanol, acetone, methyl ethyl ketone, acetic acid, ethyl acetate, and methyl acetate is used. As the precipitation solvent, it is preferable to use at least one selected from the group consisting of 2-propanol and acetone because of its high selectivity. From the viewpoint of facilitating an increase in the purity of biotin, it is preferable to use 2-propanol, and from the viewpoint of increasing the yield of biotin-amine salts, it is preferable to use a mixed solvent of 2-propanol and acetone. In the mixed solvent, the volume ratio V2/V1 of the volume V1 of 2-propanol to the volume V2 of acetone is preferably 1 or more and 20 or less, more preferably 2 or more and 10 or less. In addition, when the first reaction solvent is used, it is preferable that the first reaction solvent and the precipitation solvent are different solvents.

The amount of the precipitation solvent per 1 g of the first mixture is, for example, 1 mL to 200 mL, preferably 1 mL to 150 mL, and more preferably 20 mL to 100 mL. In another example, the amount of the precipitation solvent per 1 g of the first mixture is 5 mL to 50 mL, and preferably 10 mL to 30 mL.

When the first reaction solvent is used, the ratio V4/V3 of the volume V4 of the precipitation solvent to the volume V3 of the first reaction solvent is, for example, 0.1 or more and 20 or less. The ratio V4/V3 is preferably 1 or more and 10 or less, and more preferably 2 or more and 7 or less. When the ratio V4/V3 is large, the yield of the biotin-amine salt tends to increase. On the other hand, when the ratio V4/V3 is small, the amount of epibiotin or its salt contained in the biotin-amine salt tends to decrease. Note that when multiple types of solvents are used as the first reaction solvent and the precipitation solvent, the volume V3 of the first reaction solvent and the volume V4 of the precipitation solvent each mean the total volume of the multiple types of solvents.

The mixing temperature of the second mixture and the precipitation solvent is, for example, -40°C to 100°C, preferably -30°C to 80°C, more preferably -20°C to 50°C, and even more preferably 0°C to 30°C. In another example, the mixing temperature of the second mixture and the precipitation solvent is 40°C to 100°C, preferably 60°C to 90°C. If the second mixture and the precipitation solvent are mixed at a high temperature, the fluidity of the second mixture tends to increase, and the yield of the biotin-amine salt tends to increase. In addition, the precipitation solvent may be mixed with the second mixture that has been heated or cooled to the above mixing temperature, and the resulting mixture may be further heated.

After mixing the second mixture with the precipitation solvent, the mixture may be stirred for, for example, 1 minute to 72 hours. The stirring time is preferably 5 minutes to 48 hours, and more preferably 10 minutes to 24 hours.

The precipitation solvent may be added to the second mixture in multiple batches. For example, the first precipitation solvent may be added to the second mixture and stirred for a certain period of time, and then the second precipitation solvent may be added and stirred for a certain period of time. The first and second precipitation solvents may be different from each other, or may be the same. From the viewpoint of increasing the yield of biotin-amine salts, it is preferable to add 2-propanol as the first precipitation solvent and then add acetone as the second precipitation solvent. This can increase the fluidity of the second mixture from which crystals are precipitated. The amount of the first precipitation solvent and the amount of the second precipitation solvent may be the same or different. The temperature of the second mixture to which the first precipitation solvent is added and the temperature of the second mixture to which the second precipitation solvent is added may be the same or different.

The precipitate of biotin-amine salt is preferably removed by filtration or the like from the second mixture to which the precipitation solvent has been added, and then washed with a washing solvent. The washing solvent may be the same type of solvent as the precipitation solvent. The precipitate after washing is dried to obtain a solid product of biotin-amine salt. Note that this neutralization crystallization process of biotin with amines may be carried out multiple times.

(Method of Producing Biotin)
The biotin-amine salt obtained by the above method can be decomposed by contacting the biotin-amine salt with an acid to obtain a third mixture containing biotin. Typically, solid biotin is precipitated by contacting the biotin-amine salt with an acid.

The third mixture is obtained by decomposing biotin-amine salts that contain almost no epibiotin, so the HPLC purity of epibiotin in the third mixture can be made very low. In one example, the HPLC area percentage purity of epibiotin in the third mixture is 0.1% or less, and in another example, 0.01% or less. The lower limit of this purity is 0% or the detection limit.

As the acid, for example, at least one selected from the group consisting of hydrochloric acid, sulfuric acid, methanesulfonic acid, phosphoric acid, sodium hydrogen phosphate, potassium hydrogen phosphate, and citric acid is used. As the acid, it is preferable to use hydrochloric acid. When hydrochloric acid is used, the amount of impurities such as epibiotin that may be contained in the biotin-amine salt tends to decrease. The concentration of hydrochloric acid is, for example, 1% by mass or more and 10% by mass or less. Also, instead of the acid, an acid salt capable of generating the above-mentioned acid may be used. As the acid salt, it is preferable to use at least one selected from the group consisting of potassium hydrogen sulfate, sodium hydrogen sulfate, sodium hydrogen phosphate, and potassium hydrogen phosphate. An acid and an acid salt may be used in combination.

The amount of acid per mole of biotin amine salt is, for example, 0.1 moles or more and 100 moles or less, and preferably 0.5 moles or more and 50 moles or less.

The contact between the biotin/amine salt and the acid is carried out, for example, in an environment of -40°C or higher and 100°C or lower. The contact temperature is preferably -30°C or higher and 90°C or lower, and more preferably -20°C or higher and 50°C or lower. In another example, the contact temperature between the biotin/amine salt and the acid is 50°C or higher and 100°C or lower, and preferably 80°C or higher and 100°C or lower. Increasing the contact temperature between the biotin/amine salt and the acid tends to increase the particle size of the crystals, making it easier to remove the crystals by filtration.

After contacting the biotin amine salt with the acid, the mixture may be stirred for, for example, 1 second to 24 hours. The stirring time is preferably 1 minute to 12 hours, and more preferably 3 minutes to 4 hours.

The biotin/amine salt and the acid are preferably contacted at a temperature of, for example, 60°C to 100°C, and then stirred for 1 minute to 1 hour while maintaining this temperature. The mixture of biotin/amine salt and acid after this stirring is preferably stirred for 1 minute to 1 hour at a temperature of, for example, -40°C to 10°C. In other words, it is preferable to contact the biotin/amine salt and the acid at a high temperature, stir at this temperature for a certain period of time, and then cool the resulting mixture and stir it further. This reduces the amount of impurities such as epibiotin that may be contained in the biotin/amine salt, and tends to increase the yield of biotin.

The biotin/amine salt may be contacted with the acid after dissolving the biotin/amine salt in the second reaction solvent. As the second reaction solvent, for example, at least one selected from the group consisting of water, methanol, ethanol, 2-propanol, acetone, and acetic acid is used. As the second reaction solvent, water is preferably used.

The amount of solvent per 1 g of biotin amine salt is, for example, from 1 mL to 100 mL, preferably from 1 mL to 30 mL, and more preferably from 5 mL to 15 mL.

The solid biotin separated from the biotin-amine salt may be washed with a washing solvent. The washing solvent may be the same as the solvent capable of dissolving the biotin-amine salt. High-purity biotin can be obtained by drying the washed solid, for example, at room temperature.

The biotin obtained in this manner does not contain impurities such as epibiotin, and therefore achieves extremely high purity. In one example, the purity of high-purity biotin measured by HPLC area percentage is 99.93% or more, in another example, 99.95% or more, and in yet another example, 99.98% or more. The upper limit of the purity measured by HPLC area percentage is 100%. The HPLC measurement conditions are described later in the Examples.

In order to further increase the purity of the biotin obtained by the above method, it may be further purified by the above-mentioned neutralization crystallization treatment, activated carbon treatment, etc., and the neutralization crystallization treatment with amines may be repeated multiple times.

The present invention will be explained in detail below with examples.

<Production Example 1>
Biotin was purified by the following method to obtain purified biotin.
First, commercially available biotin was prepared. 900 mg (3.68 mmol) of biotin was dissolved in an aqueous sodium hydroxide solution to prepare a solution. As the aqueous sodium hydroxide solution, 152 mg (3.79 mmol) of sodium hydroxide was dissolved in 8.6 mL of water. After the above solution was heated to a temperature of 90 ° C., 1.6 mL of 10% by mass hydrochloric acid was dropped therein over 30 minutes to obtain a reaction solution. The reaction solution was cooled to 5 ° C. and stirred for 30 minutes while maintaining this temperature to precipitate biotin crystals. Next, the biotin crystals were dissolved again in sodium hydroxide, and then hydrochloric acid was dropped in the same manner as above to reprecipitate biotin crystals. This operation was repeated two more times, and a total of four neutralization crystallization treatments using sodium hydroxide were performed. The obtained precipitate was dried to obtain a first mixture.

Example 1
(Production of Biotin-Arginine Salt BAS1)
The first mixture obtained in Production Example 1 was mixed with 1.13 g (6.48 mmol) of L-arginine and 20 mL of distilled water, and the mixture was stirred for 5 minutes to obtain a second mixture containing biotin-arginine salt. The amount of the first mixture was 1.44 g (5.89 mmol in terms of biotin).

In an environment of 25°C, 20 mL of 2-propanol (IPA) was added dropwise to the second mixture, which was then cooled to 5°C. While maintaining this temperature, the cooled second mixture was stirred for 20 minutes to precipitate the biotin-arginine salt. The precipitated biotin-arginine salt was removed by filtration and washed with 7 mL of acetone. The washed biotin-arginine salt was dried under reduced pressure at 25°C to obtain a dried biotin-arginine salt. The amount of the dried biotin-arginine salt was 1.22 g, and the yield was 49.4%. Hereinafter, this biotin-arginine salt is also referred to as biotin-arginine salt BAS1.

(Production of Biotin)
1.0 g (2.39 mmol) of the dried biotin-arginine salt BAS1 was dissolved in 11 mL of distilled water to obtain a solution. In an environment of 25° C., 1.74 g (2.39 mmol) of 5% by mass hydrochloric acid (HCl aqueous solution) was dropped into this solution to precipitate biotin to obtain a third mixture. The precipitated biotin was removed from the third mixture by filtration and washed with 5 mL of distilled water. The washed biotin was dried under reduced pressure at 25° C. to obtain purified biotin. The amount of biotin was 507 mg, and the yield was 86.8%.

Example 2
(Preparation of Biotin-Arginine Salt BAS2)
The first mixture obtained in Production Example 1 was mixed with 1.13 g (6.48 mmol) of L-arginine and 10 mL of distilled water to obtain a mixture. This mixture was heated to 60° C. and stirred for 5 minutes to obtain a second mixture containing biotin-arginine salt. The amount of the first mixture was 1.44 g (5.89 mmol in terms of biotin).

10 mL of 2-propanol was added dropwise to the second mixture at 60°C, and then cooled to 25°C. 58 mL of acetone was further added to the cooled second mixture, and the mixture was stirred for 5 minutes to precipitate the biotin-arginine salt. The precipitated biotin-arginine salt was removed by filtration and washed with 7 mL of acetone. The washed biotin-arginine salt was dried under reduced pressure at 25°C to obtain the dried biotin-arginine salt. The amount of the dried biotin-arginine salt was 2.45 g, and the yield was 99.2%. Hereinafter, this biotin-arginine salt is also referred to as biotin-arginine salt BAS2.

(Production of Biotin)
1.0 g (2.39 mmol) of dried biotin-arginine salt BAS2 was dissolved in 7 mL of distilled water to obtain a solution. This solution was heated to 90° C., and then 3.48 g (4.78 mmol) of 5% by mass hydrochloric acid was added dropwise. The solution after the addition of hydrochloric acid was cooled to 5° C. and stirred for 30 minutes to precipitate biotin to obtain a third mixture. The precipitated biotin was removed from the third mixture by filtration and washed with 5 mL of distilled water. The washed biotin was dried under reduced pressure at 25° C. to obtain purified biotin. The amount of biotin was 478 mg, and the yield was 81.9%.

Example 3
(Preparation of Biotin-Arginine Salt BAS3)
The first mixture obtained in Production Example 1 was mixed with 7.2 mL of distilled water to obtain a first mixed solution. This first mixed solution was heated to 75° C. After heating, 1.13 g (6.48 mmol) of L-arginine was added to the first mixed solution to obtain a second mixture containing biotin-arginine salt. The amount of the first mixture was 1.44 g (5.89 mmol in terms of biotin).

22 mL of 2-propanol was added dropwise to the second mixture at 75°C, and the mixture was stirred for 30 minutes while maintaining the temperature. The stirred second mixture was cooled to 25°C and stirred for another 30 minutes while maintaining the temperature, to precipitate the biotin-arginine salt. The precipitated biotin-arginine salt was removed by filtration and washed with 14 mL of acetone. The washed biotin-arginine salt was dried under reduced pressure at 25°C to obtain the dried biotin-arginine salt. The amount of the dried biotin-arginine salt was 2.34 g, and the yield was 94.7%. Hereinafter, this biotin-arginine salt is also referred to as biotin-arginine salt BAS3.

(Production of Biotin)
1.0 g (2.39 mmol) of dried biotin-arginine salt BAS3 was dissolved in 7 mL of distilled water to obtain a solution. This solution was heated to 90° C., and then 3.48 g (4.78 mmol) of 5% by mass hydrochloric acid was dropped into it. The solution after the drop of hydrochloric acid was stirred for 30 minutes while maintaining the temperature at 90° C. The stirred mixture was cooled to 5° C., and then further stirred for 30 minutes to precipitate biotin to obtain a third mixture. The precipitated biotin was removed from the third mixture by filtration and washed with 5 mL of distilled water. The washed biotin was dried under reduced pressure at 25° C. to obtain purified biotin. The amount of biotin was 538 mg, and the yield was 92.2%.

Example 4
(Preparation of Biotin-Arginine Salt BAS4)
The first mixture obtained in Production Example 1 was mixed with 50 mL of distilled water to obtain a first mixed solution. This first mixed solution was heated to 75° C. After heating, 7.84 g (5.0 mmol) of L-arginine was added to the first mixed solution to obtain a second mixture containing biotin-arginine salt. The amount of the first mixture was 10 g (40.9 mmol in terms of biotin).

150 mL of 2-propanol was added dropwise to the second mixture at 75°C, and the mixture was heated to 85°C and stirred for 30 minutes while maintaining this temperature. The stirred second mixture was cooled to 25°C and stirred for another 30 minutes while maintaining this temperature to precipitate biotin-arginine salt. The precipitated biotin-arginine salt was removed by filtration and washed with 100 mL of acetone. The washed biotin-arginine salt was dried under reduced pressure at 25°C to obtain a dried biotin-arginine salt. The amount of the dried biotin-arginine salt was 16.6 g, and the yield was 97.1%. Hereinafter, this biotin-arginine salt is also referred to as biotin-arginine salt BAS4.

Example 5
(Production of Biotin)
1.0 g (2.39 mmol) of dried biotin-arginine salt BAS2 was dissolved in 7 mL of distilled water to obtain a solution. This solution was heated to 90° C., and then 1.74 g (2.39 mmol) of 5% by mass hydrochloric acid was added dropwise. The solution after the addition of hydrochloric acid was cooled to 5° C., and then stirred for 30 minutes to precipitate biotin to obtain a third mixture. The precipitated biotin was removed from the third mixture by filtration and washed with 5 mL of distilled water. The washed biotin was dried under reduced pressure at 25° C. to obtain purified biotin. The amount of biotin was 469 mg, and the yield was 80.3%.

Example 6
(Production of Biotin)
1.0 g (2.39 mmol) of dried biotin-arginine salt BAS2 was dissolved in 7 mL of distilled water to obtain a solution. After heating this solution to 90° C., an aqueous solution of potassium hydrogen sulfate (KHSO ₄ ) was added dropwise. As the aqueous solution of potassium hydrogen sulfate, 651 mg (4.78 mmol) of potassium hydrogen sulfate was dissolved in 3.5 mL of distilled water. The solution after the aqueous solution of potassium hydrogen sulfate was cooled to 5° C. and stirred for 30 minutes to precipitate biotin to obtain a third mixture. The precipitated biotin was removed from the third mixture by filtration and washed with 5 mL of distilled water. The washed biotin was dried under reduced pressure at 25° C. to obtain purified biotin. The amount of biotin was 536 mg, and the yield was 91.8%.

Example 7
(Production of Biotin)
1.0 g (2.39 mmol) of dried biotin-arginine salt BAS3 was dissolved in 3.5 mL of distilled water to obtain a solution. After heating this solution to 90° C., 3.48 g (4.78 mmol) of 5% by mass hydrochloric acid was dropped. The solution after the drop of hydrochloric acid was stirred for 30 minutes while maintaining the temperature at 90° C. The stirred mixture was cooled to 5° C. and further stirred for 30 minutes to precipitate biotin to obtain a third mixture. The precipitated biotin was removed from the third mixture by filtration and washed with 5 mL of distilled water. The washed biotin was dried under reduced pressure at 25° C. to obtain purified biotin. The amount of biotin was 553 mg, and the yield was 94.7%.

NMR Spectroscopic Analysis
The biotin-arginine salts obtained in Examples 1 to 4 were subjected to ¹ H-NMR spectroscopic analysis at 400 MHz. Heavy water (D ₂ O) was used as the solvent. The results are shown below.
δ4.62 (m, 1H)
4.47 (m, 1H)
3.77 (t, 1H)
3.38 (m, 1H)
3.28 (t, 2H)
3.01 (dd, 1H)
2.79 (d, 1H)
2.21 (t, 2H)
1.91 (m, 1H)
1.56-1.78 (m, 6H)
1.43 (m, 2H).

<Purity measurement>
The purities of biotin, arginine, and epibiotin in the first mixture obtained in Production Example 1, the dried biotin-arginine salts obtained in Examples 1 to 4, and the purified biotin obtained in Examples 1 to 3 and 5 to 7 were measured by high performance liquid chromatography (HPLC) under the following conditions.

Apparatus: Waters Alliance (registered trademark) e2695 manufactured by Waters Corporation
Detector: UV spectrophotometer Waters 2489
Measurement wavelength: 210nm
Flow rate: 1.0mL/min
Mobile phase A: 50mM KH ₂ PO ₄ aqueous solution (pH 3)
Mobile phase B: Acetonitrile Mobile phase delivery:
0-30 minutes: Mobile phase A 95%, mobile phase B 5%
30-40 minutes: Mobile phase A 15%, mobile phase B 85%
40-50 minutes: Mobile phase A 95%, mobile phase B 5%
Flow rate: 1.0mL/min
Column temperature: 40°C
Column, packing material: XBridge C18, 5 μm (4.6 x 150 mm)
Retention times: arginine 1.4 min; biotin 17.297 min; epibiotin 18.444 min.

The manufacturing conditions and measurement results for the biotin-arginine salts of Examples 1 to 4 are summarized in Table 1 below.

In the above Table 1, the column labeled "First reaction solvent" lists the amount of the first reaction solvent per 1 g of the first mixture. In addition, in the column below the column labeled "Precipitation solvent", the column labeled "Amount (mL)" lists the amount of the precipitating solvent per 1 g of the first mixture. In addition, the column labeled "Contact temperature (°C)" lists the temperature when the precipitating solvent is contacted with the second mixture, or the temperature when the first precipitating solvent and the second precipitating solvent are contacted with the second mixture, respectively. In addition, the column labeled "Stirring temperature (°C)" lists the temperature when the precipitating solvent or the second precipitating solvent is contacted with the second mixture and then stirred.

The manufacturing conditions for Examples 1 to 3 and 5 to 7, as well as the measurement results for Manufacturing Example 1, Examples 1 to 3 and 5 to 7, are summarized in Table 2 below.

As shown in Table 2, epibiotin could not be completely removed even in the first mixture of Production Example 1, which was subjected to neutralization crystallization treatment with sodium hydroxide four times. In contrast, the purified biotin in Examples 1 to 3 and 5 to 7, which were subjected to neutralization crystallization treatment with arginine, contained almost no epibiotin, and the purity of biotin could be increased.

Claims (5)

contacting a first mixture containing biotin or a salt thereof and epibiotin or a salt thereof with an amine to obtain a second mixture containing the biotin and a salt of the amine;
mixing the second mixture with a precipitating solvent comprising 2-propanol and acetone to precipitate the salts of the biotin and the amines ;
The method for producing biotin-amine salts, wherein the amines include at least one selected from the group consisting of arginine, quinidine, and dicyclohexylamine. The method for producing biotin-amine salts according to claim 1, wherein the amount of the amines per mole of biotin or its salt is 0.5 moles or more and 2 moles or less. mixing the first mixture with a first reaction solvent to obtain a first mixture;
contacting the first mixture with the amines to obtain the second mixture,
a ratio of the volume of the deposition solvent to the volume of the first reaction solvent is 0.1 or more and 20 or less;
the first reaction solvent is water;
The method for producing the biotin-amine salt according to claim 1 . 4. A method for producing biotin, comprising obtaining a salt of biotin and an amine by the method according to any one of claims 1 to 3 , and contacting the obtained salt of biotin and an amine with an acid to obtain a third mixture containing biotin. 5. The method for producing biotin according to claim 4 , wherein the acid comprises at least one acid selected from the group consisting of hydrochloric acid, sulfuric acid, methanesulfonic acid, phosphoric acid, and citric acid.