KR19980052661A - Method for preparing riboflavin 5'-phosphate salt - Google Patents

Abstract

The present invention relates to a method for preparing riboflavin 5'-phosphate salt, and more particularly, to a method for preparing riboflavin 5'-phosphate salt represented by the following general formula (1), which is useful in medicine and foodstuffs.

[Formula 1]

Wherein M is a hydrogen atom or a sodium atom.

Description

Method for preparing riboflavin 5'-phosphate salt

The present invention relates to a method for preparing riboflavin 5'-phosphate salt, and more particularly, to a method for preparing riboflavin 5'-phosphate salt represented by the following general formula (1), which is useful in medicine and foodstuffs.

Wherein M is a hydrogen atom or a sodium atom.

Riboflavin 5'-phosphate (hereinafter referred to as 5'-FMN) plays an important role as a coenzyme of various enzymatic reactions of biological tissues. In particular, the monosodium salt of riboflavin 5'-phosphate (hereinafter referred to as monosodium 5'-FMN) is much more soluble in water than riboflavin itself, which is useful for preparing riboflavin solutions injectables or foodstuffs. It is also used as a starting material for flavin adenine dinucleotides used as a treatment for B ₂ deficiency.

In the industrial production method of sodium 5'-FMN, a phosphorylating agent such as POCl ₃ is directly reacted with riboflavin in an organic solvent and then partially hydrolyzed. However, selective phosphorylation of the 5'-position of riboflavin is not straightforward.

For example, US Pat. No. 2,610,177 uses 5'-FMN to synthesize about 20 equivalents of partially hydrolyzed POCl ₃ . In this process, excess POCl ₃ is used, and besides the problem of environmental pollution due to waste water generation, the product contains a large amount of unreacted riboflavin and side reaction products (isomers and monophosphate of monophosphate), so that the purity and preparation Yields are very low, and the technique of purifying 5'-FMN from the reaction product is also very complex. Purification method of 5'-FMN [Chemical Engineering, Nov. 1954, page 120, which is treated several times with ethanolamine or morpholine to separate the initial product dissolved in the form of a monoammonium salt from unreacted riboflavin, which is not efficient.

US Pat. No. 2,111,491 discloses a method of phosphorylating with POCl ₃ in excess pyridine. In addition to the excessive use of toxic pyridine in this method, there is a problem that the post-reaction treatment is complicated because of the large amount of unpleasant odors and impurities.

Further, when 5'-FMN is prepared by reacting with riboflavin using a little more than POCl ₃ equivalent, tetrahydrofuran (THF), diethylene glycol dimethyl ether, monoethylene glycol dimethyl ether (DME), triethyl phosphate , 1,2-dichloroethane, 1,2-dibromoethane and the like [CA 83 (1975), 79549f, 79550z, 79551a; Japanese Patent Application Laid-Open Nos. 75-25597, 75-25598, and 75-25599 are known. In practice, the production yield was very low due to an error due to poor compound analysis method at that time.

In addition, US Pat. Nos. 5,017,700 and 5,095,115 synthesize monosodium 5′-FMN via cyclized phosphate intermediates. In the case of US Pat. No. 5,017,700, riboflavin potassium salt in powder form obtained by reacting riboflavin with potassium metal and 3 equivalents of POCl ₃ in a diethylene glycol dimethyl ether solvent is used to react riboflavin 4 ', 5'-cyclophosphoric acid ester chloride. Was separated into an intermediate and treated with a base to obtain monosodium 5'-FMN, which was separated, purified and dried. However, the separation and purification process for each step until the production of monosodium 5'-FMN is complicated, the production of by-products still has not been solved, and has the disadvantage of using a relatively expensive solvent. In the case of US Pat. No. 5,095,115, lactone is used as a solvent, which also has disadvantages in that the solvent is not commercially useful and it is difficult to control the formation of by-products.

In the conventional manufacturing process as described above, since 4'-FMN having a very similar property to 5'-FMN is generated as a by-product, its purification process is very complicated. In order to solve the problem of the separation process, US Patent No. 4,476,304 is separated by column chromatography using a polymer adsorbent, while US Patent No. 4,987,229 is separated using a weakly basic anion exchange resin, but it is applied to industrial applications. Has many difficulties.

The present invention suppresses the wastewater by using halogen-containing phosphate compounds instead of POCl ₃ , which generates wastewater containing excess Cl as a phosphorylating agent, causing environmental pollution problems, and prevents γ-butyrolactone or diethylene. Compared to glycol dimethyl ether, N, N -dimethylformamide , which is relatively inexpensive , was used as a solvent to improve the economic process. In addition, the use of dialkyl metal oxides to prevent the formation of 4'-FMN as a by-product in the production of 5'-FMN and to play an important role to help the selective phosphorylation. In addition, while the conventional 5'-FMN manufacturing method requires a heating of 80 ~ 90 ℃ or more, the present invention, except for heating for about 2 hours at 60 ℃ after the addition of dibutyltin oxide, all processes are below room temperature It has the advantage of improving the experimental conditions to proceed.

The present invention is a method for producing a riboflavin phosphate salt by 5'-phosphorylated riboflavin, the dialkyl metal oxide is added to the riboflavin and stirred, followed by a phosphorylation reaction by adding a phosphorylation agent represented by the following formula (2), It is characterized by a method of producing a riboflavin phosphate salt obtained by treating with a base solution and a dilute acid solution and then adding alcohol to precipitate.

Formula 1

Formula 2

In the above formula,

M is hydrogen atom or sodium atom;

R is 2- ( p -toluenesulfonyl) ethyl, 2-methanesulfonylethyl, 2-phenylsulfonylethyl, 2- ( p -nitrophenyl) ethyl, 2-halophenylethyl or 2- (trimethylsilyl) ethyl group to be.

Referring to the present invention in more detail as follows.

The present invention uses a compound represented by the formula (2) as a phosphorylating agent, using a dialkyl metal oxide to increase the selectivity of the phosphorylating agent and inhibit the formation of by-products, N, N -dimethylformamide as a solvent The present invention relates to a method for preparing the target compound represented by Chemical Formula 1 in a high yield even under relatively low temperature conditions. Thus, the process of the present invention is useful for industrial mass production of riboflavin phosphate salts.

Referring to the manufacturing process according to the invention in more detail as follows.

First, dialkyl metal oxide is added and stirred to riboflavin, and then a phosphorylating agent is added and stirred.

Examples of the dialkyl metal oxides include straight-chain or pulverized alkyl groups having 4 to 8 carbon atoms, or metal oxides in which aryl groups are bonded to metals such as tin (Sn), germanium (Ge), titanium (Ti), and zirconium (Zr). do. For example, dibutyltin oxide, di-t-butyltin oxide, diphenyltin oxide or diphenylgermanium oxide may be used, most preferably dibutyltin oxide. The dialkyl metal oxide is preferably used in an amount of 1.05 to 1.10 equivalents based on riboflavin. If the amount is less than this, the amount of by-product 4'-FMN increases, and in excess, more unidentified side reactions are produced. there is a problem. Dialkyl metal oxide is charged at 50 ~ 80 ℃, most preferably 60 ℃ and stirred for 1 to 2 hours. If the agitation temperature of the dialkyl metal oxide is less than 50 ° C., the dialkyl metal oxide is present in a solid state without melting in the reaction solvent, thereby preventing the reaction with riboflavin. If the dialkyl metal oxide exceeds 80 ° C., the selectivity is lowered. In addition, the dialkyl metal oxide separated by filtration after the reaction may be reused.

Examples of the compound represented by Chemical Formula 2 as the phosphorylating agent include di (2- ( p -toluenesulfonyl) ethyloxy) phosphorous chloride, di (2-methanesulfonylethyloxy) phosphorus chloride, di ( 2- (phenylsulfonyl) ethyloxy) phosphorus chloride, di (2- ( p -nitrophenyl) ethyloxy) phosphorus chloride, di (2- (halophenyl) ethyloxy) phosphorus chloride or di (2- (Trimethylsilyl) ethyloxy) phosphorus chloride. Most preferably, di (2- ( p -toluenesulfonyl) ethyloxy) phosphorus chloride is used. It is preferable to use 1.05 to 1.10 equivalents of the phosphorylating agent with respect to riboflavin. If the amount is less than this, the amount of unreacted riboflavin increases, and if the amount is excessive, 3'-FMN, a byproduct, is generated. Phosphorylating agent is added at 0 ~ 5 ℃ and stirred at room temperature for 2 to 5 hours. If the input temperature of the phosphorylating agent is less than 0 ℃ and the reaction does not proceed if there is a problem that the secondary reactant is generated in the initial reaction.

N, N -dimethylformamide is used as the reaction solvent, which has better solubility in riboflavin compared to other organic solvents, and it is easy to separate unreacted riboflavin due to poor solubility in 5'-FMN. There is this.

When the phosphorylation reaction is completed, the reactant is diluted with a basic solution to adjust the pH of the reaction to 10.0 ~ 11.0 and stirred for 1 to 3 hours at room temperature, and then added a diluted acid solution to the reactant pH of 1.0 Adjust to ~ 2.0 and stir at room temperature for 0.5 ~ 1 hour. When the base solution is treated, the pH of the reactant is limited to 10.0 to 11.0, which is to remove the protecting group of the phosphorylating agent, and the pH of the reactant is limited to 1.0 to 2.0 when treating the acid solution, which is 5'-phosphate due to the base solution treatment. To convert to the desired 5'-diphosphoric acid in the form converted to disodium salt. The base solution and the acid solution are preferably used in a concentration of 0.1N. In this invention, it is preferable to use sodium hydroxide, potassium hydroxide, and ammonia aqueous solution as a base solution, and to use aqueous solution of hydrochloric acid, nitric acid, and sulfuric acid as an acid solution.

When the base and acid treatment is completed, alcohol is added to the reactant to precipitate the riboflavin 5'-phosphate or monosodium salt represented by Chemical Formula 1 as an object of the present invention.

Such a manufacturing method of the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1. Synthesis of Riboflavin 5'-Phosphate (5'FMN)

Riboflavin (0.75 g, 2.0 mmol) and dibutyltin oxide (0.52 g, 2.1 mmol) were added to N, N -dimethylformamide (15 mL) and reacted at 60 ° C for 1 hour, and then cooled to 0 ° C. Di [2- ( p -toluenesulfonyl) ethyloxy] phosphorous chloride (0.95 g, 2.1 mmol) was added at 0 ° C., and after 1 hour of stirring, the solution was further stirred at room temperature for 3 hours, whereby the yellow solution turned green. Butyl tin oxide falls into precipitation. After dibutyltin oxide was removed by filtration, the filtrate was treated with 0.1 N NaOH solution to adjust the pH to 10.0 to 11.0 and stirred at room temperature for 2 hours. 0.1 N hydrochloric acid was added thereto to adjust the pH to 1.0 to 2.0, and then methanol (20 mL) was added, and 5'-FMN fell to yellow precipitate. The precipitate was filtered off and the product was washed with methanol. After washing with distilled water and then again with methanol and drying under reduced pressure, 0.8 g (yield 90%) of 5'-FMN of yellow crystals was obtained. HPLC analysis showed that 4'-FMN and 3'-FMN were not formed at all, but only 4% of unreacted riboflavin was recovered.

Example 2 Synthesis of Monosodium Riboflavin 5'-Phosphate (Na 5'-FMN)

Riboflavin (3.76 g, 10.0 mmol) and dibutyltin oxide (2.61 g, 10.5 mmol) were added to N, N -dimethylformamide (60 mL), and reacted at 60 ° C for 1 hour, and then cooled to 0 ° C. Di [2- ( p -toluenesulfonyl) ethyloxy] phosphorus chloride (4.76 g, 10.5 mmol) was added at 0 ° C, and after stirring for 1 hour at room temperature for 3 hours, the yellow solution turned green. Butyl tin oxide falls into precipitation. After dibutyltin oxide was removed by filtration, the filtrate was treated with 0.1N NaOH solution to adjust the pH to 10.0 to 11.0 and stirred at room temperature for 2 hours. At this time, in order to convert the produced 5'-phosphate disodium salt into the desired monosodium salt 5'-FMN, 0.1N hydrochloric acid was added to adjust the pH to 5.0 to 6.0, and then methanol (100 ml) was added to monosodium 5'-FMN. This falls into a yellow precipitate. The precipitate was filtered off and the product was washed with methanol. After washing with distilled water and washing with methanol and drying under reduced pressure, 4.2 g (yield 88%) of yellow sodium monocrystal 5'-FMN was obtained.

Comparative example

Riboflavin (0.75 g, 2.0 mmol) was dissolved in N, N -dimethylformamide (15 mL) and then di [2- ( p -toluenesulfonyl) ethyloxy] phosphorous chloride (0.95 g, 2.1 mmol) at 0 ° C. ) Was added. The reaction mixture was stirred at 0 ° C. for 1 hour and then further stirred at room temperature for 3 hours. The reaction mixture was treated with 0.1N NaOH solution to adjust the pH to 10.0 to 11.0 and stirred at room temperature for 2 hours. 0.1N hydrochloric acid was added thereto to adjust the pH to 1.0-2.0, and then 20 ml of methanol dropped 5'-FMN to yellow precipitate. The precipitate was filtered off and the product was washed with methanol. After washing with distilled water and then again with methanol and drying under reduced pressure, 0.69 g (yield 78%) of 5-FMN of yellow crystals was obtained. HPLC analysis showed 78% of 5'-FMN, 12% of 4'-FNM, and 6% of unreacted riboflavin.

Table 1 is a table comparing the manufacturing method according to the present invention and the conventional manufacturing method.

division menstruum Phosphorylating agent Dialkyl Metal Oxide % Composition of the product 5'-FMN 4'-FMN 3'-FMN Riboflavin Etc Example 1 DMF ¹⁾ p-TOSCl ²⁾ Bu ₂ Sn = O 90 - - 4 6 Example 2 DMF p-TOSCl Bu ₂ Sn = O 88 ³⁾ - - - 12 Example 3 DMF MESCl ⁴⁾ Bu ₂ Sn = O 84 - - 5 11 Example 4 DMF PhSCl ⁵⁾ Bu ₂ Sn = O 85 - - 7 8 Example 5 DMF p-NPhCl ⁶⁾ Bu ₂ Sn = O 84 - - 8 8 Example 6 DMF p-TOSCl Ph ₂ Sn = O 83 4 - 6 7 Example 7 DMF p-TOSCl t-Bu ₂ Sn = O 78 10 - 7 5 Example 8 DMF PhSCl Bu ₂ Sn = O 84 - - 10 6 Comparative example DMF p-TOSCl - 78 12 - 6 4 Japanese Patent Publication No. 81-2559 Triethylphosphate POCl ₃ - 41.1 10.3 11.6 22.8 14.2 U.S. Patent No. 5095115 γ-butyrolactone POCl ₃ - 76-80 9 ~ 11 5 ~ 7 4 ~ 5.8 U.S. Patent No. 5017700 Diethylene glycol dimethyl ether POCl ₃ - 75-78 9 ~ 11 5 ~ 7 4 ~ 5.8 1) DMF: N, N -dimethylformamide 2) p-TOSCl: Di [2- (p-toluenesulfonyl) ethyloxy] phosphate chloride 3) Synthesis yield of monosodium 5'-FMN 4) MESCl : Di [2- (p-methanesulfonyl) ethyloxy] phosphorus chloride 5) PhSCl: di [2- (p-phenylsulfonyl) ethyloxy] phosphorus chloride 6) p-NPhCl: di [2- ( p-nitrophenyl) ethyloxy] phosphorus chloride

In the preparation method according to the present invention, riboflavin is phosphorylated with halogen-free phosphating agent and dialkyl metal oxide in order to phosphorylate with high selectivity for the 5'-position of riboflavin, thereby reducing the formation of by-products that are difficult to separate. It is possible to apply to industrial mass production because the recovery of riboflavin 5'- phosphate salt at a high recovery rate without a separate separation process.

Claims (12)

In the process for preparing riboflavin phosphate salt by 5'-phosphorylated riboflavin, The dialkyl metal oxide was added to the riboflavin and stirred, followed by the phosphorylation reaction by adding a phosphating agent represented by the following Chemical Formula 2, followed by treatment with a dilute base solution and a dilute acid solution, followed by precipitation by adding alcohol. Process for preparing riboflavin 5'-phosphate salt. Formula 1 Formula 2 In the above formula, M is hydrogen atom or sodium atom; R is 2- ( p -toluenesulfonyl) ethyl, 2-methanesulfonylethyl, 2-phenylsulfonylethyl, 2- ( p -nitrophenyl) ethyl, 2-halophenylethyl or 2- (trimethylsilyl) ethyl group to be. [Claim 2] The dialkyl metal oxide of claim 1, wherein a linear or pulverized alkyl group or aryl group having 4 to 8 carbon atoms is bonded to a tin (Sn), germanium (Ge), titanium (Ti) or zirconium (Zr) metal. A method for producing riboflavin 5'-phosphate salt, characterized by using a metal oxide. The method of claim 2, wherein the dialkyl metal oxide is dibutyltin oxide, di-t-butyltin oxide, diphenyltin oxide or diphenylgermanium oxide. . The method for producing riboflavin 5'-phosphate salt according to claim 1, wherein the dialkyl metal oxide is added and stirred at a temperature of 50 to 80 ° C. The method of claim 1, wherein the phosphorylating agent is di (2- ( p -toluenesulfonyl) ethyloxy) phosphorous chloride, di (2-methanesulfonylethyloxy) phosphorous chloride, di (2- (phenylsulfonyl ) Ethyloxy) phosphorus chloride, di (2- ( p -nitrophenyl) ethyloxy) phosphorus chloride, di (2- (halophenyl) ethyloxy) phosphor chloride or di (2- (trimethylsilyl) ethyloxy A method for producing riboflavin 5'-phosphate salt, characterized by using phosphorus chloride. The method for producing riboflavin 5'-phosphate salt according to claim 5, wherein di (2- ( p -toluenesulfonyl) ethyloxy) phosphorus chloride is used as the phosphorylating agent. The method for preparing riboflavin 5'-phosphate salt according to claim 1, wherein the phosphorylating agent is added at 0˜5 ° C. and stirred at room temperature. The method for producing riboflavin 5'-phosphate salt according to claim 1, wherein N, N -dimethylformamide is used as the reaction solvent. The method for preparing riboflavin 5'-phosphate salt according to claim 1, wherein the pH of the reaction solution is adjusted to 10.0 to 11.0 using the base solution. The method for preparing riboflavin 5'-phosphate salt according to claim 1, wherein the acid solution is used to adjust the pH of the reaction solution to 1.0 to 2.0. The method of claim 1, wherein the alcohol is a lower alcohol having 1 to 4 carbon atoms to prepare a riboflavin 5'- phosphate salt. 12. The method for producing riboflavin 5'-phosphate salt according to claim 11, wherein methanol is used as the alcohol.