Method for producing biotin, and L-lysine salt of biotin and method for producing the same.

Abstract

The purpose of the present invention is to provide: a method which is for preparing biotin, and can effectively remove epibiotin from a product and increase the purity of biotin to obtain high-purity biotin; an L-lysine salt of the biotin; and a method for preparing same. The present invention provides: a method for preparing an L-lysine salt of biotin, the method comprising (a) a step for bringing crude biotin into contact with L-lysine in a solvent to obtain a solution or suspension of an L-lysine salt of the biotin, and (b) a step for precipitating the L-lysine salt of the biotin from the solution of the L-lysine salt of the biotin when the solution of the biotin is obtained in step (a); and a method for preparing biotin, the method comprising a step for preparing an L-lysine salt of the biotin using said preparation method, and then bringing the L-lysine salt of the biotin into contact with an acid to obtain biotin.

Description

Technical Field

[0001] The present invention relates to a method for producing biotin, and to the L-lysine salt of biotin and a method for producing the same.

Background Art

[0002] Biotin is a water-soluble vitamin belonging to the vitamin B group (Non-Patent Document 1). Biotin is expected to have effects such as preventing diabetes, improving skin diseases, and improving biotin deficiency, and the demand as pharmaceuticals, feed additives, etc. is increasing. Biotin is represented by the following formula (1) and is also referred to as D-biotin.

[0003]

Chemical Formula

[0004] As shown below, biotin (BIF) is synthesized from, for example, the ureido form (URD) through seven steps (Patent Document 1).

[0005]

Chemical Formula

[0006] Here, biotin has seven isomers in addition to D-biotin represented by the above formula (1) (Non-Patent Document 2). In the process of synthesizing D-biotin, isomers such as L-biotin represented by the following formula (2) may be included in the product. It is desirable to remove these isomers other than D-biotin from the product from the viewpoint of safety.

[0007]

Chemical Formula

[0008] [[ID=4i7]] A method has been disclosed for purifying D-biotin by reacting a racemic mixture of D-biotin and L-biotin with L(+)-arginine and crystallizing it as a sparingly soluble salt, thereby increasing the purity of D-biotin. However, there is no description of the separation and removal of epibiotin, a diastereomer of biotin (Non-Patent Literature 3). [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] International Publication No. 2018 / 025722 [Non-patent literature]

[0010] [Non-Patent Document 1] Pierre J. de Clercq, “Biotin: A Timeless Challenge for Total Synthesis” Chemical Reviews, 1997, Vol. 97, No. 6. [Non-Patent Document 2] 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. [Non-Patent Document 3] DONALD E.WOLF,RALPH MOZINGO,STANTON A.HARRIS,R.CHRISTIAN ANDERSON AND KARL FOLKERS “Biotin.VI.Resolution of dl-Biotin” CONTRIBUTION FROM THE RESEARCH LABORATORIES,JOURNAL of the American Chemical Society,1945,Vol.67,No.12,2100-2102 [Overview of the project] [Problems that the invention aims to solve]

[0011] When biotin is synthesized via the pathway described above, the biotin-containing product may contain diastereomers and other impurities. Among these diastereomers, epibiotin, represented by formula (3) below, is a particularly difficult impurity to remove from the product.

[0012] [ka]

[0013] The area percentage of epibiotin in the product, as determined by high-performance liquid chromatography (HPLC), can reach approximately 5%, potentially reducing the purity of biotin. While epibiotin can be removed through repeated purification processes such as neutralization crystallization or activated carbon treatment, this can lead to a decrease in biotin yield. Therefore, there is a need for a method to efficiently remove epibiotin from the product and increase the purity of biotin.

[0014] Therefore, an object of the present invention is to provide a method for producing biotin that enables the efficient removal of epibiotin from the product, thereby increasing the purity of biotin and obtaining highly pure biotin. Another object of the present invention is to provide an L-lysine salt of biotin and a method for producing the same that enables the efficient removal of epibiotin from the product, thereby increasing the purity of biotin and obtaining highly pure biotin. [Means for solving the problem]

[0015] As a result of diligent research by the present inventors, it was discovered that high-purity biotin can be obtained by contacting crude biotin containing biotin and epibiotin with L-lysine in a solvent to obtain an L-lysine salt of biotin, and then desalting the biotin by contacting this L-lysine salt of biotin with an acid.

[0016] That is, the present invention includes the following inventions. [1] The following steps: (a) A step of contacting crude biotin and L-lysine in a solvent to obtain a solution or suspension of the L-lysine salt of biotin, and (b) When a solution of the L-lysine salt of biotin is obtained in step (a), a step of precipitating the L-lysine salt of biotin from the solution of the L-lysine salt of biotin A method for producing the L-lysine salt of biotin, comprising: [2] The method for producing the L-lysine salt of biotin according to [1], wherein the solvent contains at least one selected from the group consisting of water, methanol, ethanol, 2-propanol, acetone and acetonitrile. [3] The method for producing the L-lysine salt of biotin according to [1] or [2], wherein step (a) is a step of contacting crude biotin and L-lysine in water to obtain a solution of the L-lysine salt of biotin. [4] The method for producing the L-lysine salt of biotin according to any one of [1] to [3], wherein step (b) is a step of precipitating the L-lysine salt of biotin by contacting the solution of the L-lysine salt of biotin with a precipitation solvent. [5] The method for producing the L-lysine salt of biotin according to [4], wherein the precipitation solvent contains at least one selected from the group consisting of methanol, ethanol, 2-propanol, acetone and acetonitrile. [6] The method for producing the L-lysine salt of biotin according to [4] or [5], wherein the volume ratio of the precipitation solvent to water (precipitation solvent / water) is 5 or more and 30 or less. [7] The method for producing the L-lysine salt of biotin according to any one of [4] to [6], wherein step (b) is a step of heating the solution of the L-lysine salt of biotin to 40°C or higher and 80°C or lower, then contacting it with a precipitation solvent, and then cooling it to -10°C or higher and 10°C or lower to precipitate the L-lysine salt of biotin. [8] The method for producing a biotin L-lysine salt according to [1] or [2], wherein step (a) is a step of contacting crude biotin with L-lysine in an organic solvent containing at least one selected from the group consisting of alcohol, acetone, and acetonitrile to obtain a suspension of the biotin L-lysine salt. [9] A method for producing L-lysine salt of biotin according to [8], wherein the organic solvent is an alcohol.

[10] A method for producing L-lysine salt of biotin according to [8] or [9], wherein the amount of organic solvent used is 8 mL or more per 1 g of crude biotin. A method for producing biotin, comprising the step of producing an L-lysine salt of biotin by any of the methods in

[11] [1] to

[10] , and then contacting the L-lysine salt of biotin with an acid to obtain biotin.

[12] L-lysine salt of biotin.

[13] The L-lysine salt of biotin as described in

[12] , wherein the epibiotin content by HPLC area percentage is 0.035% or less. [Effects of the Invention]

[0017] The present invention's method for producing biotin L-lysine salt efficiently removes epibiotin from crude biotin, yielding a highly pure biotin L-lysine salt. This obtained biotin L-lysine salt can then be used to produce highly pure biotin. Furthermore, since the biotin production method of the present invention uses the highly pure biotin L-lysine salt obtained by the present invention, highly pure biotin can be obtained. Therefore, highly pure biotin can be obtained in high yield without having to perform multiple purification processes on the product. The biotin thus obtained has a low epibiotin content, so, for example, an ultra-high purity of 99.96% or higher can be achieved in terms of biotin content (purity) as measured by HPLC area percentage. In the present invention, the "content as measured by HPLC area percentage" is measured according to the method described in the examples, and the HPLC is performed under the conditions described in the examples.

[0018] Since biotin and epibiotin are weak acids, contact with highly basic L-lysine can produce L-lysine salts of biotin and epibiotin. These L-lysine salts have different solubility in specific solvents. This difference in solubility is greater than the difference between the solubility of other biotin base salts and epibiotin base salts in specific solvents. Therefore, by contacting the L-lysine salts of biotin and epibiotin with a solvent in which one salt is highly soluble and the other is less soluble, a solution of the highly soluble salt and a solid of the less soluble salt can be obtained. This allows for more efficient separation of the L-lysine salts of biotin and epibiotin. Biotin can then be isolated by contacting the separated L-lysine salt with an acid. [Modes for carrying out the invention]

[0019] The details of the present invention will be described below.

[0020] The present invention provides a method for producing L-lysine salt of biotin, comprising the following steps: (a) A step of contacting crude biotin with L-lysine in a solvent to obtain a solution or suspension of the L-lysine salt of biotin, (b) If a solution of biotin L-lysine salt is obtained in step (a), a step of precipitating biotin L-lysine salt from the biotin L-lysine salt solution. This includes the following. Step (b) is performed if a solution of biotin L-lysine salt is obtained in step (a), but not if a suspension of biotin L-lysine salt is obtained in step (a).

[0021] (Crude biotin) Crude biotin contains biotin and epibiotin. Crude biotin is typically a solid. The biotin content (purity) in crude biotin, based on HPLC area percentage, is, in one example, between 90% and less than 99.92%. The epibiotin content in crude biotin, based on HPLC area percentage, is, in one example, approximately 5% or less.

[0022] Crude biotin may be the product obtained in the seventh step (stage 7) of the biotin synthesis described above. Specifically, in the sixth step of the seven steps of biotin synthesis described above, when the vinyl sulfide compound (DVE) is reduced to N,N'-dibenzylbiotin (HVC), it is thought that an epimer of N,N'-dibenzylbiotin is produced as a by-product. It is thought that epibiotin is produced by deprotecting this N,N'-dibenzylbiotin epimer in the seventh step. Crude biotin may also be the product obtained after purification treatment such as activated carbon treatment of the product obtained in the seventh step. Furthermore, crude biotin may be commercially available biotin.

[0023] An example of a method for producing crude biotin is as follows:

[0024] First, a vinyl sulfide compound (DVE) shown in formula (6) below is synthesized from a ureid compound (URD) ​​using a known method described in Patent Document 1, etc.

[0025] [ka]

[0026] Alternatively, the vinyl sulfide compound (DVE) may be synthesized by first synthesizing the lactone compound (LCT) shown in formula (4) below using a known method such as Patent Document 1 (for example, stages 1 to 3 of Patent Document 1), and then by the method described below.

[0027] [ka]

[0028] After contacting the lactone compound (4) with an alkali metal acetate salt, the mixture is reacted with a thiocarboxylic acid to obtain a mixture containing the thiolactone compound (DTL) shown in formula (5) below.

[0029] [ka]

[0030] Under a nitrogen atmosphere, zinc powder is activated by adding dihalogenoethane, and then contacted with a halogenated alkane derivative to prepare an alkyl zinc halide. A thiolactone compound (5) dissolved in an organic solvent is mixed with this mixture, and an acid is added to the reaction mixture to obtain a vinyl sulfide compound (DVE).

[0031] By reducing the synthesized vinyl sulfide compound using a known method, a product containing N,N'-dibenzylbiotin (HVC) as shown in formula (7) below is obtained. This product contains the by-product epimer of N,N'-dibenzylbiotin.

[0032] [ka]

[0033] 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 crude biotin, the product obtained in this way may be used, or the product after activated carbon treatment may be used.

[0034] Activated carbon treatment is a method of removing impurities from crude biotin by mixing it with activated carbon.

[0035] (Biotin) Biotin is represented by the following formula (1) and is also called D-biotin.

[0036] [ka]

[0037] By contacting biotin with L-lysine in a solvent, L-lysine salts of biotin can be produced.

[0038] (Epiviotin) Epiviotin is represented by the following formula (3) and is also called D-epibiotin. Epiviotin may include L-epibiotin.

[0039] [ka]

[0040] By contacting epibiotin with L-lysine in a solvent, an L-lysine salt of epibiotin can be produced.

[0041] (L-lysine) The L-lysine used in the present invention is not particularly limited.

[0042] The amount of L-lysine used per mole of biotin is preferably 0.50 moles or more and 2.0 moles or less, and more preferably 1.0 moles or more and 1.5 moles or less.

[0043] (solvent) The solvent used to contact crude biotin with L-lysine preferably contains at least one selected from the group consisting of water, alcohol, acetone, and acetonitrile. In particular, it is preferable to use water or an organic solvent consisting of at least one selected from the group consisting of alcohol, acetone, and acetonitrile, and more preferably to use water or alcohol. Water includes distilled water, purified water, pure water, ultrapure water, tap water, or mixtures thereof. Aqueous organic solvents may also be used. The volume-based water content of the aqueous organic solvent is preferably 50% or less, more preferably 20% or less.

[0044] The alcohol preferably contains at least one selected from the group consisting of methanol, ethanol, and 2-propanol, and among these, ethanol is preferred.

[0045] (Production of L-lysine salt) The present invention provides a method for producing L-lysine salt of biotin, (1) A method for producing biotin L-lysine salt (hereinafter also referred to as "production method (1)"), comprising the steps of: (1) contacting crude biotin with L-lysine in water to obtain a solution of biotin L-lysine salt; and (2) precipitating biotin L-lysine salt from the solution of biotin L-lysine salt; and (2) A method for producing biotin L-lysine salt, comprising the step of contacting crude biotin with L-lysine in an organic solvent containing at least one selected from the group consisting of alcohol, acetone, and acetonitrile to obtain a suspension of biotin L-lysine salt (hereinafter also referred to as "production method (2)"). One could list these:

[0046] According to manufacturing methods (1) and (2), a high-purity biotin L-lysine salt with low epibiotin or its salt content can be obtained. The epibiotin content in the obtained high-purity biotin L-lysine salt, as determined by HPLC area percentage, is, in one example, 0.035% or less, in another example, 0.020% or less, in yet another example, 0.010% or less, and in yet another example, 0.004% or less. The lower limit is 0% or the detection limit. The biotin content (purity) in the obtained high-purity biotin L-lysine salt, as determined by HPLC area percentage, is, in one example, 99.92% or more, in another example, 99.94% or more, in yet another example, 99.95% or more, and in yet another example, 99.96% or more. The upper limit is 100%.

[0047] (Manufacturing method (1)) (Step to obtain a solution of biotin L-lysine salt) The L-lysine salt of biotin is represented by the following formula (8).

[0048] [ka]

[0049] In manufacturing method (1), first, a solution of biotin's L-lysine salt is obtained by contacting crude biotin with L-lysine in water. This step is one embodiment of step (a).

[0050] The method for contacting crude biotin and L-lysine in water is not particularly limited. By stirring and mixing crude biotin and L-lysine in water, a solution of biotin's L-lysine salt can be obtained. The order in which water, crude biotin, and L-lysine are added is also not particularly limited.

[0051] For example, the amount of water used per 1 g of crude biotin is, for example, 1 mL to 10 mL, preferably 3 mL to 5 mL. For another example, the amount of water used per 1 g of crude biotin is, for example, 0.5 mL to 5 mL, preferably 0.5 mL to 2 mL.

[0052] The temperature at which crude biotin and L-lysine come into contact is not particularly limited. Contact may occur at room temperature, or at the temperature at which the biotin-L-lysine solution and the precipitation solvent come into contact in the next step (the step of precipitating the biotin-L-lysine salt).

[0053] Crude biotin and L-lysine may be brought into contact in water, and the resulting mixture may be stirred. If stirred, the stirring time may be, for example, 1 minute to 24 hours, preferably 10 minutes to 12 hours. The stirring may be carried out at the temperature of contact or higher, at room temperature, similar to the temperature of contact, or at the temperature at which the biotin L-lysine salt solution and the precipitation solvent come into contact in the next step.

[0054] The resulting biotin L-lysine salt and epibiotin L-lysine salt may be dissolved or solvated in water, and in this invention, these states are collectively referred to as a biotin L-lysine salt solution.

[0055] The formation of biotin L-lysine salt in a solution of biotin L-lysine salt can be confirmed, for example, by nuclear magnetic resonance (NMR) spectroscopy.

[0056] (Process for precipitating biotin L-lysine salt) In manufacturing method (1), the biotin L-lysine salt is then precipitated from the biotin L-lysine salt solution obtained in the above step (the step of obtaining a biotin L-lysine salt solution) to obtain a biotin L-lysine salt precipitate. This step corresponds to step (b).

[0057] A solution of biotin L-lysine salt may contain epibiotin L-lysine salt in addition to biotin L-lysine salt. Therefore, it is preferable to separate only biotin L-lysine salt from the biotin L-lysine salt solution. One method for separating biotin L-lysine salt from a biotin L-lysine salt solution is to mix the biotin L-lysine salt solution with a solvent that selectively dissolves or precipitates biotin L-lysine salt. Because the operation is easy, it is preferable to precipitate biotin L-lysine salt by contacting the biotin L-lysine salt solution with a solvent that selectively precipitates biotin L-lysine salt (hereinafter also referred to as the "precipitation solvent") to obtain a biotin L-lysine salt precipitate.

[0058] As the precipitation solvent, it is preferable to use an organic solvent containing at least one selected from the group consisting of methanol, ethanol, 2-propanol, acetone, and acetonitrile, and more preferably an organic solvent containing at least one selected from the group consisting of methanol, ethanol, 2-propanol, acetone, and acetonitrile. As the precipitation solvent, it is preferable to use an organic solvent containing at least one selected from the group consisting of ethanol and 2-propanol, due to its high selectivity for the L-lysine salt of biotin, and more preferably an organic solvent containing at least one selected from the group consisting of ethanol and 2-propanol. From the viewpoint of increasing the purity and yield of the L-lysine salt of biotin, it is more preferable to use 2-propanol.

[0059] In one example, the amount of precipitation solvent used per 1 g of crude biotin is, for example, 1 mL to 200 mL, preferably 10 mL to 100 mL, and more preferably 32 mL to 50 mL. In another example, the amount of precipitation solvent used per 1 g of crude biotin is, for example, 5 mL to 50 mL, preferably 8 mL to 20 mL.

[0060] The volume ratio of water to the precipitation solvent (precipitation solvent / water) is, for example, 1 to 50, preferably 5 to 30. A larger volume ratio tends to increase the yield of biotin L-lysine salt. On the other hand, a smaller volume ratio tends to decrease the amount of epibiotin or its salt contained in the biotin L-lysine salt. Since it is possible to obtain a high-purity biotin L-lysine salt with a small amount of epibiotin or its salt, it is particularly preferable to set the amount of precipitation solvent used per 1 g of crude biotin and the volume ratio of water to the precipitation solvent (precipitation solvent / water) within the above range. Note that when multiple types of precipitation solvents are used as the precipitation solvent, the volume of the precipitation solvent refers to the total volume of the multiple types of precipitation solvents.

[0061] The temperature at which the biotin L-lysine salt solution is brought into contact with the precipitation solvent is, for example, between 0°C and 100°C, preferably between 40°C and 80°C. When the biotin L-lysine salt solution is brought into contact with the precipitation solvent, the biotin L-lysine salt precipitates, forming a suspension of biotin L-lysine salt. If the contact between the biotin L-lysine salt solution and the precipitation solvent is carried out at a higher temperature, the fluidity of the biotin L-lysine salt solution increases, and the biotin L-lysine salt solution and the precipitation solvent come into sufficient contact, which tends to increase the amount of biotin L-lysine salt precipitated and thus the yield of biotin L-lysine salt. Alternatively, after bringing the precipitation solvent into contact with the biotin L-lysine salt solution heated or cooled to the above contact temperature, the resulting mixture may be further heated.

[0062] Contact between the biotin L-lysine salt solution and the precipitation solvent can be brought into contact by adding the precipitation solvent to the biotin L-lysine salt solution. The precipitation solvent may be added to the biotin L-lysine salt solution in multiple stages. For example, the precipitation solvent may be added dropwise while stirring, or the first precipitation solvent may be added to the biotin L-lysine salt solution 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 of different types or of the same type. The amounts of the first and second precipitation solvents may be the same or different.

[0063] When obtaining a suspension of biotin L-lysine salt, it is preferable to bring the biotin L-lysine salt solution into contact with the precipitation solvent and then stir-mix the resulting mixture. The stirring time is, for example, 1 minute to 24 hours, preferably 10 minutes to 12 hours. Stirring is preferably carried out at a temperature equal to or higher than the temperature at which contact occurred, for example, 0°C to 100°C, preferably 40°C to 80°C. Stirring the mixture of biotin L-lysine salt solution and precipitation solvent at a higher temperature increases the fluidity of the mixture, allowing sufficient contact between the biotin L-lysine salt solution and the precipitation solvent, which tends to increase the amount of biotin L-lysine salt precipitated and thus increase the yield of biotin L-lysine salt.

[0064] When obtaining a suspension of biotin L-lysine salt, it is preferable to stir-mix the biotin L-lysine salt solution and the precipitation solvent at the above temperature, then cool and stir-mix the mixture. The cooling temperature of the biotin L-lysine salt solution and precipitation solvent mixture can be, for example, -20°C to 10°C, and preferably -10°C to 10°C. Cooling the biotin L-lysine salt solution and precipitation solvent mixture tends to increase the amount of biotin L-lysine salt precipitated and raise the yield of biotin L-lysine salt. The stirring time can be, for example, 1 minute to 24 hours, and preferably 10 minutes to 12 hours.

[0065] The precipitate of biotin L-lysine salt can be separated from the biotin L-lysine salt suspension by filtration or the like. It is then preferably washed with a washing solvent. The same type of solvent used for precipitation should be used as the washing solvent. By drying the precipitate after washing, a solid of biotin L-lysine salt can be obtained. Biotin L-lysine salt is, for example, in powder form.

[0066] (Manufacturing method (2)) In manufacturing method (2), crude biotin and L-lysine are contacted in an organic solvent containing at least one selected from the group consisting of alcohol, acetone, and acetonitrile to obtain a suspension of the biotin L-lysine salt. This step is one embodiment of step (a).

[0067] The method of contacting crude biotin and L-lysine in an organic solvent is not particularly limited. It is sufficient to mix crude biotin and L-lysine in an organic solvent, and the order of addition of the organic solvent, crude biotin, and L-lysine is not particularly limited.

[0068] The amount of organic solvent used per 1 g of crude biotin is, for example, 1 mL or more, preferably 5 mL or more, and more preferably 8 mL or more. Since the L-lysine salt of biotin is practically insoluble in alcohol, it does not affect the yield. Therefore, there is no upper limit on the amount of organic solvent used, but considering ease of handling, it is preferably 100 mL or less, and more preferably 50 mL or less.

[0069] The organic solvent used to contact crude biotin with L-lysine is preferably an organic solvent consisting of at least one selected from the group consisting of alcohol, acetone, and acetonitrile, and more preferably an alcohol. As described above, since alcohol can be used to obtain a high-purity biotin L-lysine salt with a low amount of epibiotin or its salt, it is preferable that the alcohol contains at least one selected from the group consisting of methanol, ethanol, and 2-propanol, and among these, ethanol is preferred.

[0070] The temperature at which crude biotin and L-lysine come into contact is not particularly limited, but is, for example, between 0°C and 100°C, preferably between 20°C and 80°C.

[0071] When crude biotin and L-lysine are brought into contact in an organic solvent, the crude biotin and L-lysine gradually dissolve in the organic solvent, and the resulting biotin-L-lysine salt precipitates, ultimately forming a suspension of biotin-L-lysine salt.

[0072] It is preferable to contact crude biotin and L-lysine in an organic solvent and then stir-mix the resulting mixture. The stirring time is, for example, 1 minute to 24 hours, preferably 30 minutes to 12 hours. It is preferable to stir at a temperature equal to or higher than the temperature at which the mixture was contacted, for example, 0°C to 100°C, preferably 40°C to 80°C. Stirring the mixture at a higher temperature increases the fluidity of the mixture, allowing biotin and L-lysine to come into sufficient contact, which tends to increase the amount of biotin-L-lysine salt precipitated and thus increase the yield of biotin-L-lysine salt.

[0073] The obtained suspension of biotin L-lysine salt is preferably cooled and stirred after being stirred at the above temperature. The cooling temperature of the suspension of biotin L-lysine salt can be, for example, -20°C to 10°C, and preferably -10°C to 10°C. Cooling the suspension of biotin L-lysine salt tends to increase the amount of biotin L-lysine salt precipitated and increase the yield of biotin L-lysine salt. The stirring time is, for example, 1 minute to 24 hours, and preferably 30 minutes to 12 hours.

[0074] The precipitate of biotin L-lysine salt can be separated from the biotin L-lysine salt suspension by filtration or the like. It is then preferable to wash it with a washing solvent. The washing solvent should be the same type of alcohol used when contacting crude biotin and L-lysine. By drying the precipitate after washing, a solid of biotin L-lysine salt can be obtained. Biotin L-lysine salt is, for example, in powder form.

[0075] (Method of producing biotin) By contacting the biotin L-lysine salt obtained by the present invention with an acid, the biotin L-lysine salt can be decomposed to obtain biotin. Typically, contact between the biotin L-lysine salt and an acid results in the precipitation of solid biotin.

[0076] In the biotin production method of the present invention, biotin is obtained by decomposing a high-purity biotin L-lysine salt that contains almost no epibiotin or its salts. Therefore, the epibiotin content in the obtained biotin, as measured by HPLC area percentage, can be made very low. In one example, the epibiotin content in the obtained biotin, as measured by HPLC area percentage, is 0.1% or less; in another example, it is 0.01% or less; and in yet another example, it is 0.006% or less. The lower limit of this content is 0% or the detection limit.

[0077] As the acid, at least one acid selected from the group consisting of hydrochloric acid, sulfuric acid, methanesulfonic acid, phosphoric acid, sodium hydrogen phosphate, potassium hydrogen phosphate, and citric acid can be used. Hydrochloric acid is preferred as the acid. When hydrochloric acid is used, the amount of impurities such as epibiotin that may be contained in the L-lysine salt of biotin tends to decrease. The concentration of hydrochloric acid should be, for example, 1% by mass or more and 20% by mass or less. Alternatively, an acidic salt capable of generating the above acid may be used instead of the acid. As the acidic salt, at least one selected from the group consisting of potassium bisulfate, sodium bisulfate, sodium hydrogen phosphate, and potassium hydrogen phosphate is preferred. Acids and acidic salts may be used in combination.

[0078] The amount of acid used per mole of biotin L-lysine salt is, for example, 0.1 moles to 20 moles, preferably 0.5 moles to 10 moles, and more preferably 1.0 mole to 3.0 moles.

[0079] Contact between the biotin L-lysine salt and the acid may be carried out after the biotin L-lysine salt has been dissolved in the dissolving solvent. Biotin precipitates upon contact with the acid. The dissolving solvent for the biotin L-lysine salt preferably contains at least one selected from the group consisting of water, methanol, ethanol, 2-propanol, acetone, and acetonitrile. Water is more preferably used as the dissolving solvent.

[0080] The amount of solvent used to dissolve 1 g of biotin in L-lysine salt is, for example, 1 mL to 100 mL, preferably 1 mL to 30 mL, and more preferably 1 mL to 15 mL.

[0081] After dissolving the L-lysine salt of biotin in a solvent, contact with the acid is preferably carried out under heating. The temperature at contact is, for example, 50°C to 120°C, preferably 70°C to 100°C.

[0082] Contact with the acid is preferably carried out by adding the acid dropwise to a solution in which the L-lysine salt of biotin is dissolved in a solvent. The time for adding the acid dropwise is, for example, 10 minutes to 2 hours, preferably 15 minutes to 2 hours.

[0083] It is preferable to add an acid to a solution of biotin's L-lysine salt while stirring, and to continue stirring at the temperature at which contact occurred after the acid was added. The stirring time after the acid was added should be, for example, 1 minute to 5 hours, preferably 20 minutes to 2 hours.

[0084] It is preferable to stir the solution of biotin L-lysine salt and the acid mixture at the above temperature and then cool it. The cooling temperature of the biotin L-lysine salt solution and acid mixture should be, for example, between -20°C and 10°C, preferably between -10°C and 10°C. Cooling the biotin L-lysine salt solution and acid mixture tends to increase the amount of biotin precipitated and thus increase the biotin yield.

[0085] It is preferable to cool the mixture of the biotin L-lysine salt solution and the acid while stirring. The cooling time for the mixture of the biotin L-lysine salt solution and the acid should be, for example, 1 minute to 5 hours, preferably 20 minutes to 2 hours.

[0086] The biotin precipitate can be removed from the solution of biotin L-lysine salt to which acid has been added by filtration or other means. The biotin solid separated from the biotin L-lysine salt may then be washed with a washing solvent. The washing solvent can be the same as the solvent capable of dissolving biotin L-lysine salt. High-purity biotin can be obtained by drying the washed solid, for example, at room temperature.

[0087] The biotin obtained in this way does not contain impurities such as epibiotin, and therefore can achieve very high purity. The biotin content (purity) in the obtained high-purity biotin, as measured by HPLC area percentage, is, for example, 99.96% or higher. The upper limit for the biotin content (purity) as measured by HPLC area percentage is 100%.

[0088] Furthermore, the biotin obtained by the above method may be subjected to further purification treatments such as the neutralization crystallization treatment and activated carbon treatment described above in order to increase its purity, and the neutralization crystallization treatment with L-lysine may be repeated multiple times. [Examples]

[0089] The present invention will be further described with the following examples, but these examples are not intended to limit the scope of the invention.

[0090] <Purity measurement> The biotin and epibiotin content, expressed as HPLC area percentages, in the crude biotin obtained in the production example, the L-lysine salt of the dried biotin obtained in the examples, and the dried biotin obtained in the examples was measured using high-performance liquid chromatography (HPLC) under the following conditions.

[0091] The biotin content, measured by HPLC area percentage, was determined as the ratio of the biotin peak area value to the sum of the area values ​​of all peaks excluding the solvent peak and the L-lysine peak, measured under the following conditions. The epibiotin content, measured by HPLC area percentage, was determined in the same manner as above, except that the area value of the epibiotin peak was used instead of the area value of the biotin peak.

[0092] Equipment: Waters Corporation, Waters Alliance® e2695 Detector: Ultraviolet absorbance spectrophotometer Waters 2489 Measurement wavelength: 200nm Mobile phase A: 20mM KH2PO4 aqueous solution (pH3) Mobile phase B: Acetonitrile Mobile phase delivery: 0-30 minutes: Mobile phase A92%, mobile phase B8% 30-35 minutes: Mobile phase A20%, mobile phase B80% 35-40 minutes: 92% mobile phase A, 8% mobile phase B Flow rate: 1.0mL / min Column temperature: 40℃ Column and packing material: XBridge C18, 5μm (4.6x150mm) L-lysine RT: 1.493 Biotin RT: 11.912 Epiviotin RT: 15.729.

[0093] <Example 1 of crude biotin production> (Synthesis of lactone compounds (LCTs)) First, 15.00 g of lactone compound (LCT) was synthesized from the ureid (URD) ​​through three steps using the method described in Patent Document 1.

[0094] (Synthesis of thiolactone compounds (DTLs)) 15.00 g (0.047 mol) of lactone compound, 7.31 g (0.074 mol) of potassium acetate salt, and 22.5 mL of N,N-dimethylacetamide (DMA) were placed in a 200 mL four-necked flask and stirred at room temperature under a nitrogen atmosphere to obtain a mixture.

[0095] This mixture was heated to a temperature of 125°C, then 4.25 g (0.056 mol) of thioacetic acid was added in 1 / 10th portions at 3-minute intervals, and the mixture was stirred at 125°C for 2 hours to obtain a mixture containing a thiolactone compound.

[0096] Next, the mixture was cooled to 80°C, and then 15 mL of DMA was added. Then, 40 mL of water was added over 30 minutes, and the mixture was stirred at a temperature between 25°C and 30°C for 2 hours to obtain a mixture containing a precipitate of the thiolactone compound. This mixture was filtered to separate the precipitate of the thiolactone compound. The separated precipitate of the thiolactone compound was washed with a washing solution. A mixed solvent of 10 mL of acetone and 20 mL of water was used as the washing solution. The amount of precipitate of the thiolactone compound after washing was 16.34 g. Measurement using HPLC under the above conditions showed that the amount of thiolactone compound in the precipitate (wet) after washing was 13.39 g, and the yield from the lactone compound was 85% (stage 4).

[0097] (Synthesis of ethylzinc 5-iodovalerate) Under a nitrogen atmosphere, zinc powder (Hakusui Tech R powder, 4.29 g, 65.6 mmol) was suspended in a mixed solvent of tetrahydrofuran (THF, 7.0 mL) and toluene (4.9 mL). 1,2-dibromoethane (2.57 g, 13.7 mmol) was added dropwise over 30 minutes at 75°C, and the mixture was stirred at the same temperature for 30 minutes. Subsequently, ethyl 5-iodovalerate (I-TAI, 7.0 g, 27.3 mmol) was added dropwise over 30 minutes at 55°C, and the mixture was stirred at the same temperature for 3 hours. A portion of the reaction solution was taken and analyzed by gas chromatography using the above method, and the conversion rate to the zinc compound was found to be 97.96%.

[0098] (Synthesis of vinyl sulfide compounds (DVE)) The reaction mixture was cooled to 25°C, and thiolactone compound (6.48 g, 19.1 mmol) and toluene (14 mL) were added. Then, at the same temperature, a suspension of 10% Pd / C (179.4 mg, 0.169 mmol) N,N-dimethylformamide (DMF, 1.8 mL) was added, and the mixture was stirred at 40°C for 3 hours and at 25°C for 14 hours.

[0099] After the reaction was complete, 14 mL of 16% hydrochloric acid was added at 25°C, and the mixture was stirred at the same temperature for 1 hour. The reaction mixture was then filtered. The filtrate was stirred at 40°C for 1 hour. The organic layer was then washed with water (14 mL, 2 x 21 mL), 5% sodium sulfite aqueous solution (21 mL), 5% sodium bicarbonate aqueous solution (21 mL), and water (14 mL), washed with water, and concentrated to obtain the vinyl sulfide compound. Analysis of the obtained organic layer by high-performance liquid chromatography showed a conversion rate to vinyl sulfide of 98.37% (stage 5).

[0100] (Synthesis of N,N'-dibenzylbiotin (HVC)) This vinyl sulfide compound (maximum 19.1 mmol) was dissolved in a mixture of methanol (59 mL) and water (16 mL), and Pd(OH)2 / C (50% by mass wet, 0.59 g) was added. Catalytic reduction was carried out at 11°C and a hydrogen pressure of 0.9 MPa for 12 hours. After the reaction was complete, the reaction mixture was filtered, and 31% by mass NaOH aqueous solution (7.01 g) was added to the filtrate and stirred at 40°C for 2 hours. After hydrogenation was complete, 10% by mass hydrochloric acid was added to the reaction mixture to adjust the pH to 1. Methanol was removed by distillation under reduced pressure, and the product was extracted with ethyl acetate, washed with water, and concentrated to obtain a product containing N,N'-dibenzylbiotin. This product contains the by-product epimer of N,N'-dibenzylbiotin.

[0101] (Biotin (BIF) synthesis) N,N'-Dibenzylbiotin (2.04 g, 4.81 mmol) was mixed with mesitylene (6.6 mL) and sulfuric acid (1.7 mL) at room temperature and stirred at 100 °C for 2.5 hours. After the reaction was complete, the mesitylene was removed, and toluene (7.7 mL) was added and stirred for 10 minutes. After removing the toluene, water (25 mL) was added in eight equal parts at 10-minute intervals while the reaction mixture was kept at 80 °C, and stirred at the same temperature for 10 minutes. After the reaction mixture was allowed to cool, it was cooled to 5 °C and stirred for 3 hours. The obtained crystals were filtered and washed with water (8.2 mL) and acetone (10 mL). Then, biotin (1.02 g, yield 86.5%) was obtained by vacuum drying at 60 °C for 16 hours. Hereafter, this biotin will also be referred to as crude biotin. The biotin content (purity) in this crude biotin, as determined by HPLC area percentage, was 99.914%, and the epibiotin content, as determined by HPLC area percentage, was 0.037%.

[0102] <Example 1> (Production of biotin L-lysine salt BLS1) 1.0 g (4.09 mmol) of crude biotin obtained in Preparation Example 1, 758 mg (4.50 mmol) of L-lysine, and 5 mL of distilled water were added to a reaction vessel in this order, stirred, mixed, and dissolved to obtain a solution of biotin L-lysine salt. This solution was heated to 60°C with stirring, and then 30 mL of 2-isopropanol (IPA) was added dropwise, and the mixture was stirred at the same temperature for 30 minutes. After the dropwise addition, the solution was cooled to 5°C with stirring, and then kept at 5°C with stirring overnight to precipitate biotin L-lysine salt. The precipitated biotin L-lysine salt was removed by filtration and dried under reduced pressure at 25°C to obtain dried biotin L-lysine salt. The amount of dried biotin L-lysine salt was 1.32 g, and the yield was 82.67%. Hereafter, this biotin L-lysine salt will also be referred to as biotin L-lysine salt BLS1.

[0103] The results of 1H-NMR spectroscopy analysis performed at 400 MHz on the obtained biotin L-lysine salt BLS1 are shown below. Heavy water (D2O) was used as the solvent. δ4.60(m,1H) 4.46 (m, 1H) 3.72(t,1H) 3.36 (m, 1H) 3.02 (m, 3H) 2.80 (d, 1H) 2.20(t,2H) 1.88 (m, 2H) 1.75 (m, 3H) 1.62 (m, 3H) 1.40-1.55 (m, 4H).

[0104] <Example 2> (Production of biotin L-lysine salt BLS2) 1.0 g (4.09 mmol) of crude biotin obtained in Preparation Example 1, 758 mg (4.50 mmol) of L-lysine, and 5 mL of distilled water were added to a reaction vessel in this order, stirred, mixed, and dissolved to obtain a solution of biotin L-lysine salt. This solution was heated to 60°C under stirring, and then 40 mL of 2-isopropanol (IPA) was added dropwise, and the mixture was stirred at the same temperature for 30 minutes. After the dropwise addition, the solution was cooled to 5°C under stirring, and then kept at 5°C and stirred overnight to precipitate biotin L-lysine salt. The precipitated biotin L-lysine salt was removed by filtration and dried under reduced pressure at 25°C to obtain dried biotin L-lysine salt. The amount of dried biotin L-lysine salt was 1.58 g, and the yield was 98.67%. Hereafter, this biotin L-lysine salt will also be referred to as biotin L-lysine salt BLS2.

[0105] <Example 3> (Production of biotin L-lysine salt BLS3) 1.0 g (4.09 mmol) of crude biotin obtained in Preparation Example 1, 758 mg (4.50 mmol) of L-lysine, and 5 mL of distilled water were added to a reaction vessel in this order, stirred, mixed, and dissolved to obtain a solution of biotin L-lysine salt. This solution was heated to 60°C with stirring, and then 50 mL of 2-isopropanol (IPA) was added dropwise, and the mixture was stirred at the same temperature for 30 minutes. After the dropwise addition, the solution was cooled to 5°C with stirring, and then kept at 5°C with stirring for 1 hour to precipitate the biotin L-lysine salt. The precipitated biotin L-lysine salt was removed by filtration and washed with 25 mL of 2-isopropanol. It was dried under reduced pressure at 25°C to obtain the dried biotin L-lysine salt. The amount of dried biotin L-lysine salt was 1.61 g, and the yield was quant. Hereafter, this biotin L-lysine salt will also be referred to as biotin L-lysine salt BLS3.

[0106] <Example 4> (Production of biotin L-lysine salt BLS4) 1.0 g (4.09 mmol) of crude biotin obtained in Preparation Example 1, 758 mg (4.50 mmol) of L-lysine, and 4 mL of distilled water were added to a reaction vessel in this order, stirred, mixed, and dissolved to obtain a solution of biotin L-lysine salt. This solution was heated to 60°C with stirring, and then 32 mL of 2-isopropanol (IPA) was added dropwise, and the mixture was stirred at the same temperature for 30 minutes. After the dropwise addition, the solution was cooled to 5°C with stirring, and then kept at 5°C with stirring for 1 hour to precipitate the biotin L-lysine salt. The precipitated biotin L-lysine salt was removed by filtration and washed with 16 mL of 2-isopropanol. It was dried under reduced pressure at 25°C to obtain the dried biotin L-lysine salt. The amount of dried biotin L-lysine salt was 1.61 g, and the yield was quant. Hereafter, this biotin L-lysine salt will also be referred to as biotin L-lysine salt BLS4.

[0107] <Example 5> (Production of biotin L-lysine salt BLS5) 1.0 g (4.09 mmol) of crude biotin obtained in Preparation Example 1, 758 mg (4.50 mmol) of L-lysine, and 1 mL of distilled water were added to a reaction vessel in this order, stirred, mixed, and dissolved to obtain a solution of biotin L-lysine salt. This solution was heated to 60°C with stirring, then 20 mL of ethanol (EtOH) was added dropwise, and the mixture was stirred at the same temperature for 30 minutes. After the dropwise addition, the solution was cooled to 5°C with stirring, and then kept at 5°C with stirring for 1 hour to precipitate the biotin L-lysine salt. The precipitated biotin L-lysine salt was removed by filtration and washed with 10 mL of cold ethanol. It was dried under reduced pressure at 25°C to obtain the dried biotin L-lysine salt. The amount of dried biotin L-lysine salt was 1.58 g, and the yield was 98.60%. Hereafter, this biotin L-lysine salt will also be referred to as biotin L-lysine salt BLS5.

[0108] <Example 6> (Production of biotin L-lysine salt BLS6) 1.0 g (4.09 mmol) of crude biotin obtained in Preparation Example 1, 758 mg (4.50 mmol) of L-lysine, and 0.5 mL of distilled water were added to a reaction vessel in this order, stirred, mixed, and dissolved to obtain a solution of biotin L-lysine salt. This solution was heated to 60°C with stirring, then 10 mL of ethanol (EtOH) was added dropwise, and the mixture was stirred at the same temperature for 30 minutes. After the dropwise addition, the solution was cooled to 5°C with stirring, and then kept at 5°C with stirring for 1 hour to precipitate the biotin L-lysine salt. The precipitated biotin L-lysine salt was removed by filtration and washed with 25 mL of cold ethanol. It was dried under reduced pressure at 25°C to obtain the dried biotin L-lysine salt. The amount of dried biotin L-lysine salt was 1.61 g, and the yield was quant. Hereafter, this biotin L-lysine salt will also be referred to as biotin L-lysine salt BLS6.

[0109] <Example 7> (Production of biotin L-lysine salt BLS7) 5 g (20.47 mmol) of crude biotin obtained in Preparation Example 1, 3.29 g (20.51 mmol) of L-lysine, and 5 mL of distilled water were added to a reaction vessel in this order, stirred, mixed, and dissolved to obtain a solution of biotin L-lysine salt. This solution was heated to 60°C with stirring, then 100 mL of ethanol (EtOH) was added dropwise, and the mixture was stirred at the same temperature for 30 minutes. After the dropwise addition, the solution was cooled to 5°C with stirring, and then kept at 5°C with stirring for 1 hour to precipitate the biotin L-lysine salt. The precipitated biotin L-lysine salt was removed by filtration and washed with 25 mL of cold ethanol. It was dried under reduced pressure at 25°C to obtain the dried biotin L-lysine salt. The amount of dried biotin L-lysine salt was 7.93 g, and the yield was 99.24%. Hereafter, this biotin L-lysine salt will also be referred to as biotin L-lysine salt BLS7.

[0110] <Example 8> (Production of biotin L-lysine salt BLS8) 1.0 g (4.09 mmol) of crude biotin obtained in Preparation Example 1, 758 mg (4.50 mmol) of L-lysine, and 20 mL of ethanol (EtOH) were added to a reaction vessel in this order, stirred, and mixed to obtain a suspension of biotin L-lysine salt. This suspension was heated to 60°C under stirring, stirred at the same temperature for 30 minutes, then cooled to 5°C and stirred at the same temperature for 1 hour. The biotin L-lysine salt was extracted by filtration and washed with 25 mL of ethanol. It was dried under reduced pressure at 25°C to obtain the dried biotin L-lysine salt. The amount of dried biotin L-lysine salt was 1.61 g, and the yield was quant. Hereafter, this biotin L-lysine salt will also be referred to as biotin L-lysine salt BLS8.

[0111] <Example 9> (Production of biotin L-lysine salt BLS9) 1.0 g (4.09 mmol) of crude biotin obtained in Preparation Example 1, 758 mg (4.50 mmol) of L-lysine, and 10 mL of ethanol (EtOH) were added to a reaction vessel in this order, stirred, and mixed to obtain a suspension of biotin L-lysine salt. This suspension was heated to 60°C under stirring, stirred at the same temperature for 30 minutes, then cooled to 5°C and stirred at the same temperature for 1 hour. The biotin L-lysine salt was extracted by filtration and washed with 15 mL of cold ethanol. It was dried under reduced pressure at 25°C to obtain the dried biotin L-lysine salt. The amount of dried biotin L-lysine salt was 1.61 g, and the yield was quant. Hereafter, this biotin L-lysine salt will also be referred to as biotin L-lysine salt BLS9.

[0112] <Example 10> (Manufacturing of biotin L-lysine salt BLS10) 1.0 g (4.09 mmol) of crude biotin obtained in Preparation Example 1, 758 mg (4.50 mmol) of L-lysine, and 8 mL of ethanol (EtOH) were added to a reaction vessel in this order, stirred, and mixed to obtain a suspension of biotin L-lysine salt. This suspension was heated to 60°C under stirring, stirred at the same temperature for 30 minutes, then cooled to 5°C and stirred at the same temperature for 1 hour. The biotin L-lysine salt was extracted by filtration and washed with 20 mL of cold ethanol. It was dried under reduced pressure at 25°C to obtain the dried biotin L-lysine salt. The amount of dried biotin L-lysine salt was 1.62 g, and the yield was quant. Hereafter, this biotin L-lysine salt will also be referred to as biotin L-lysine salt BLS10.

[0113] <Example 11> (Biotin production) A solution was obtained by dissolving 6.0 g (15.37 mmol) of the L-lysine salt BLS7 of dried biotin in 21.0 mL of distilled water. This solution was heated to 90°C with stirring, and then 11.2 g (15.37 mmol) of 10% by mass hydrochloric acid was added dropwise over 30 minutes while maintaining the temperature at 90°C. The suspension after the addition of hydrochloric acid was stirred at the same temperature for 30 minutes, cooled to 5°C with stirring, and then stirred at the same temperature for 30 minutes. The precipitated biotin was removed by filtration and washed with 12 mL of distilled water. The washed biotin was dried under reduced pressure at 25°C to obtain dried biotin. The amount of dried biotin was 3.59 g, and the yield was 95.58%.

[0114] The conditions and measurement results for the production of biotin L-lysine salts in Examples 1 to 10 are summarized in Table 1 below. In Table 1, "Content" refers to the content (%) calculated as HPLC area percentage.

[0115] [Table 1]

[0116] The conditions for the production of biotin in Example 11, as well as the measurement results for Production Example 1 and Example 11, are summarized in Table 2 below. In Table 2, "Content" refers to the content (%) calculated as HPLC area percentage.

[0117] [Table 2]

[0118] As shown in Table 1, by contacting crude biotin with L-lysine in a solvent and separating the L-lysine salt of biotin from a solution or suspension of the biotin L-lysine salt, the biotin L-lysine salt was obtained, and the amount of epibiotin was reduced. Furthermore, as shown in Table 2, the biotin obtained after drying in Example 11, in which the biotin L-lysine salt was contacted with an acid in a solvent, contained almost no epibiotin, and the purity of biotin was increased.

Claims

1. The following steps: (a) A step of contacting crude biotin containing biotin and epibiotin with L-lysine in water to obtain a solution of the L-lysine salt of biotin, and (b) A step of precipitating the biotin L-lysine salt by heating a solution of biotin L-lysine salt to 40°C or higher and 80°C or lower, then contacting it with a precipitation solvent, and then cooling it to -10°C or higher and 10°C or lower. A method for producing L-lysine salt of biotin, which includes: The amount of water used in process (a) is 0.5 mL or more and 10 mL or less per 1 g of crude biotin. The precipitation solvent used in step (b) is at least one selected from the group consisting of methanol, ethanol, and 2-propanol. A method for producing L-lysine salt of biotin, wherein the volume ratio of water to precipitation solvent (precipitation solvent / water) in step (b) is 8 or more and 30 or less.

2. The method for producing L-lysine salt of biotin according to Claim 1, wherein the amount of water used in step (a) is 0.5 mL or more and 5 mL or less per 1 g of crude biotin.

3. A method for producing biotin, comprising the step of producing an L-lysine salt of biotin by the method of claim 1 or 2, and then contacting the L-lysine salt of biotin with an acid to obtain biotin.

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