Method for producing cytidine-5'-diphosphate compounds

Abstract

The objective is to provide a method for producing cytidine-5'-diphosphate compounds that efficiently removes 5'-CMP from crude cytidine-5'-diphosphate compounds and concentrates the cytidine-5'-diphosphate compounds to a higher concentration than before purification. [Solution] The present invention relates to a method for producing a cytidine-5'-diphosphate compound, comprising the steps of (1) contacting a solution containing a cytidine-5'-diphosphate compound with a basic ion exchange resin to adsorb the cytidine-5'-diphosphate compound onto the basic ion exchange resin, and (2) using an aqueous solution containing an organic acid having 2 or more carbon atoms to elute the cytidine-5'-diphosphate compound adsorbed onto the basic ion exchange resin to obtain an eluate, wherein the cytidine-5'-diphosphate compound is a cytidine-5'-diphosphate compound having a cationic substituent.

Description

[Technical Field]

[0001] This invention relates to a method for producing cytidine-5'-diphosphate compounds. [Background technology]

[0002] Industrial production methods for cytidine-5'-diphosphate compounds, including choline cytidine-5'-diphosphate (hereinafter also abbreviated as CDP-choline), include chemical synthesis, fermentation using microorganisms, and enzymatic methods. In all of these methods, the reaction involves the condensation of cytidine-5'-phosphate (hereinafter also abbreviated as 5'-CMP) or its highly reactive derivatives with phosphocholine or phosphoethanolamine, etc., so 5'-CMP originating from the raw materials or produced as a by-product is present in the system as an impurity. Furthermore, cytidine-5'-diphosphate compounds are hydrolyzed by heating, producing 5'-CMP as a by-product.

[0003] Conventionally, a common method for purifying cytidine-5'-diphosphate compounds from a solution containing them is, for example, column chromatography using an ion exchange resin. For example, Patent Documents 1 to 3 disclose a method in which crude CDP-choline is adsorbed onto an anion exchange resin and then eluted using extremely dilute formic acid or hydrochloric acid of about 0.005 to 0.10 mol / L.

[0004] Furthermore, Patent Document 4 discloses a method using an aqueous sodium chloride solution as an eluent. Another method, as disclosed in Patent Document 5, involves passing crude CDP-choline through a weakly basic anion exchange resin to selectively adsorb and remove only the impurity 5'-CMP. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 48-86869 [Patent Document 2] Japanese Patent Publication No. 47-18881 [Patent Document 3] Indian Patent Application Publication No. 2012MUM02534 Specification [Patent Document 4] Chinese Patent Application Publication No. 101130797 Specification [Patent Document 5] Japan Special Publication No. 6-31306 [Overview of the project] [Problems that the invention aims to solve]

[0006] In the process of purifying cytidine-5'-diphosphate compounds from a solution containing them, it is difficult to remove impurities, particularly 5'-CMP, which has similar physicochemical properties, from the final product. Conventional column chromatography methods for removing 5'-CMP have the problem of diluting the cytidine-5'-diphosphate compound in the solution, requiring time and cost for subsequent concentration. Furthermore, there is a risk of generating impurities such as 5'-CMP due to the decomposition of the cytidine-5'-diphosphate compound by heating during the concentration process.

[0007] Therefore, efficiently separating cytidine-5'-diphosphate compounds from impurities, particularly 5'-CMP, from a crude solution containing cytidine-5'-diphosphate compounds, and obtaining a solution containing a higher concentration of cytidine-5'-diphosphate compounds than before purification, is an important challenge in producing high-purity cytidine-5'-diphosphate compounds.

[0008] However, the methods described in Patent Documents 1 to 3 require the recovery of cytidine-5'-diphosphate compounds using a large amount of eluate under mild elution conditions, particularly in order to separate 5'-CMP from cytidine-5'-diphosphate compounds. This reduces the concentration of cytidine-5'-diphosphate compounds in the eluate, thus necessitating a concentration step for crystallization and other processes. In particular, when eluting with volatile acids such as formic acid or hydrochloric acid, these eluents are mainly removed by evaporation, which is done by concentration under reduced pressure by heating. However, heating causes the cytidine-5'-diphosphate compounds to decompose, leading to an increase in 5'-CMP again, thus presenting problems as an industrial production method.

[0009] The method described in Reference 4 is not suitable as an industrial production method because it is difficult to ultimately remove the eluent that has been mixed into the fraction containing the cytidine-5'-diphosphate compound. Furthermore, the method described in Patent Document 5 requires washing the resin column with water after the liquid has passed through to recover the cytidine-5'-diphosphate compound, and, similar to the methods described in Patent Documents 1 to 3, the concentration of the cytidine-5'-diphosphate compound decreases compared to before the liquid has passed through the column. Therefore, its application as an industrial production method is limited to liquids with a certain concentration or higher, and its versatility is low.

[0010] As described above, no method has been reported for separating 5'-CMP from crude cytidine-5'-diphosphate compounds and concentrating the cytidine-5'-diphosphate compounds to a higher concentration than before purification. Therefore, the present invention aims to provide a method for producing cytidine-5'-diphosphate compounds that can efficiently remove 5'-CMP from crude cytidine-5'-diphosphate compounds and concentrate the cytidine-5'-diphosphate compounds to a higher concentration than before purification. [Means for solving the problem]

[0011] The inventors of the present invention have discovered that cytidine-5'-diphosphate compounds can be efficiently purified and concentrated by elution using a specific organic acid solution. In particular, they have found that cytidine-5'-diphosphate compounds and 5'-CMP can be efficiently separated, and that an eluate containing a higher concentration of cytidine-5'-diphosphate compounds than that before purification can be obtained, thus completing the present invention.

[0012] In other words, the present invention is as follows: 1. A method for producing a cytidine-5'-diphosphate compound, comprising the following steps (1) and (2), wherein the cytidine-5'-diphosphate compound is a cytidine-5'-diphosphate compound having a cationic substituent. (1) A step of contacting a solution containing a cytidine-5'-diphosphate compound with a basic ion exchange resin to adsorb the cytidine-5'-diphosphate compound onto the basic ion exchange resin. (2) A step of obtaining an eluate by eluting the cytidine-5'-diphosphate compound adsorbed on the basic ion exchange resin using an aqueous solution containing an organic acid having 2 or more carbon atoms. 2. The manufacturing method according to paragraph 1, wherein the concentration of the cytidine-5'-diphosphate compound contained in the eluate in step (2) is equal to or greater than the concentration of the cytidine-5'-diphosphate compound in the solution containing the cytidine-5'-diphosphate compound in step (1). 3. The manufacturing method according to 1 or 2, wherein in step (2), the temperature at which the cytidine-5'-diphosphate compound is eluted from the basic ion exchange resin is 20°C or higher. 4. The manufacturing method according to any one of 1 to 3 above, wherein the organic acid having 2 or more carbon atoms is at least one organic acid selected from acetic acid, propionic acid, and butyric acid. 5. The manufacturing method according to any one of 1 to 4, wherein in step (2), the concentration of the organic acid contained in the aqueous solution is 0.1 mol / L or more. 6. The production method according to any one of 1 to 5, wherein the cytidine-5'-diphosphate compound having a cationic substituent is a compound in which cytidine-5'-diphosphate and a cationic substituent are bonded together. 7. The production method according to any one of 1 to 6 above, wherein the cytidine-5'-diphosphate compound having a cationic substituent is a compound in which a cationic substituent is bonded to the β-phosphate group of cytidine-5'-diphosphate. 8. The production method according to any one of 1 to 7 above, wherein the organic acid having 2 or more carbon atoms is a carboxylic acid having 2 or more carbon atoms. 9. The production method according to any one of 1 to 8 above, wherein the cytidine-5'-diphosphate compound having a cationic substituent is at least one selected from cytidine-5'-diphosphate choline and cytidine-5'-diphosphate ethanolamine.

Advantages of the Invention

[0013] According to the production method of the present invention, by including an elution step using an organic acid solution having 2 or more carbon atoms such as acetic acid or propionic acid instead of conventional formic acid, it is possible to achieve both efficient removal of 5'-CMP and obtain an eluate containing a cytidine-5'-diphosphate compound at a higher concentration than before purification, and a high-purity cytidine-5'-diphosphate compound can be produced with high efficiency.

Brief Description of the Drawings

[0014] [Figure 1] Figure 1 is a diagram showing the CDP-choline concentration and 5'-CMP concentration for each elution fraction in Comparative Example 2. RV on the horizontal axis is an abbreviation for resin volume, and represents the volume ratio of the eluate when the resin volume is set to 1. [Figure 2] Figure 2 is a diagram showing the CDP-choline concentration and 5'-CMP concentration for each elution fraction in Example 2. RV on the horizontal axis is an abbreviation for resin volume, and represents the volume ratio of the eluate when the resin volume is set to 1. [Figure 3] Figure 3 is a diagram showing the CDP-choline concentration and 5'-CMP concentration for each elution fraction in Example 4. RV on the horizontal axis is an abbreviation for resin volume, and represents the volume ratio of the eluate when the resin volume is set to 1.

Modes for Carrying Out the Invention

[0015] <Manufacturing method> The manufacturing method of the present invention is characterized by comprising the following steps (1) and (2). (1) A step of contacting a solution containing a cytidine-5'-diphosphate compound with a basic ion exchange resin to adsorb the cytidine-5'-diphosphate compound onto the basic ion exchange resin. (2) A step of obtaining an eluate by eluting the cytidine-5'-diphosphate compound adsorbed on a basic ion exchange resin using an aqueous solution containing an organic acid having 2 or more carbon atoms.

[0016] (1) A step of contacting a solution containing a cytidine-5'-diphosphate compound with a basic ion exchange resin to adsorb the cytidine-5'-diphosphate compound onto the basic ion exchange resin.

[0017] In the present invention, the cytidine-5'-diphosphate compound is a cytidine-5'-diphosphate compound having a cationic substituent. Examples of cytidine-5'-diphosphate compounds having a cationic substituent include compounds in which cytidine-5'-diphosphate is bonded to a cationic substituent. More specifically, a cytidine-5'-diphosphate compound having a cationic substituent is a compound in which a cationic substituent is bonded to the β-phosphate group of cytidine-5'-diphosphate. Here, the positions of the phosphate groups in cytidine-5'-diphosphate are, in order from the cytidine side, the α-phosphate group and the β-phosphate group (Chemistry and Education, Vol. 46, No. 6, 1998, pp. 334-337). Examples of cationic substituents include substituents having an onium ion such as ammonium, oxonium, phosphonium, sulfonium, iminium, nitrilium, nitrosonium, diazonium, nitronium, or diazenium, and among these, those having an ammonium ion are preferred. Examples of cytidine-5'-diphosphate compounds having cationic substituents include cytidine-5'-diphosphate choline and cytidine-5'-diphosphate ethanolamine. Among these, cytidine-5'-diphosphate choline or cytidine-5'-diphosphate ethanolamine are preferred from the viewpoint of purification efficiency, and cytidine-5'-diphosphate choline is more preferred. These may be included in the solution individually or in combination of two or more. Modification of the cytidine-5'-diphosphate compound with functional groups is also permitted, as long as separation from 5'-CMP is maintained.

[0018] In step (1) of the manufacturing method of the present invention, the solution containing the cytidine-5'-diphosphate compound that is contacted with the basic ion exchange resin may be any solution containing the cytidine-5'-diphosphate compound prepared by any method, for example, a solution produced by chemical synthesis, fermentation, or a biocatalyst.

[0019] Examples of chemical synthesis methods for CDP-choline include the method described in K. Kikugawa, M. Ichino, Chem. Pharm. Bull., 19, 1011, 1971. Examples of chemical synthesis methods for CDP-ethanolamine include the methods described in H. Ngo, MF Dunn et al. Biochemistry, 46, 7713, 2007.; T. Tanaka yakugakuzasshi, 80, 439, 1959.; K. Oertell, CE McKenna, MF Goodman et al. Biochemistry, 53, 1842, 2014.; T. Tanaka et al. yakugakuzasshi, 85, 863, 1965.; T. Tanaka et al. yakugakuzasshi, 85, 863, 1965.

[0020] Examples of methods using fermentation or biocatalysis include those described in Japanese Patent No. 3369236; Y. Liu et al. Appl. Microbiol. Biotechnol., 101, 1409, 2017. The solution containing the cytidine-5'-diphosphate compound used in step (1) of the production method of the present invention is preferably a solution obtained by removing solid matter from a solution obtained by fermentation or biocatalysis.

[0021] Furthermore, as the solution containing the cytidine-5'-diphosphate compound in step (1) of the manufacturing method of the present invention, a solution produced by chemical synthesis, fermentation, or a method using a biocatalyst may be used, which has been treated with a strongly acidic cation exchange resin to reduce or remove impurities in advance. In addition, if necessary, a solution that has been decolorized using activated carbon treatment or a non-polar porous synthetic adsorbent, such as Mitsubishi Chemical's Diaion HP series (e.g., HP20, HP21, etc.), Mitsubishi Chemical's Diaion SP800 series (e.g., SP825, SP850, etc.), Mitsubishi Chemical's Diaion SP200 series (e.g., SP207, etc.), or Rohm & Haas's Amberlite XAD series (e.g., XAD4, XAD7HP, XAD16HP, XAD1180, XAD2000, etc.), may be used.

[0022] Cytidine-5'-diphosphate compounds and 5'-CMP can be analyzed using conventional methods with high-performance liquid chromatography (UV detection).

[0023] The concentration of the cytidine-5'-diphosphate compound in the solution containing the cytidine-5'-diphosphate compound in step (1) is not particularly limited, but from the viewpoint of purification efficiency, it is preferably 100 g / L or less, more preferably 85 g / L or less, even more preferably 40 g / L or less, even more preferably 15 g / L or less, and most preferably 13 g / L or less. The lower limit of the concentration of the cytidine-5'-diphosphate compound in the solution containing the cytidine-5'-diphosphate compound in step (1) is not particularly limited, but it is preferably 0.1 g / L or more, more preferably 0.2 g / L or more, even more preferably 0.3 g / L or more, and most preferably 0.4 g / L or more.

[0024] Furthermore, the ratio of the concentration of 5'-CMP to the concentration of the cytidine-5'-diphosphate compound in the solution containing the cytidine-5'-diphosphate compound in step (1) ("concentration of 5'-CMP (g / L) / concentration of cytidine-5'-diphosphate compound (g / L) × 100 (%)", hereinafter referred to as "5'-CMP ratio (ratio to cytidine-5'-diphosphate compound %)") is not particularly limited, but from the viewpoint of purification efficiency, it is preferably 150% or less, more preferably 100% or less, even more preferably 50% or less, and most preferably 15% or less.

[0025] The basic ion exchange resin of the present invention is not particularly limited, and examples include ion exchange resins having primary to tertiary amines or polyamines as exchange groups in weakly basic anion exchange resins, and ion exchange resins having quaternary ammonium as exchange groups in strongly basic anion exchange resins. As ion exchange resins having quaternary ammonium, both type I, which has trimethylammonium groups or triethylammonium groups, and type II, which has dimethylethanolammonium groups, can be suitably used. Furthermore, examples of the base material for the basic ion exchange resin include porous type, macroporous type, gel type, styrene type, and acrylic type.

[0026] Examples of basic ion exchange resins include, specifically, Dow Chemical's Marathon series (e.g., Marathon A, Marathon MSA, Marathon A2, etc.), Dow Chemical's Monosphere series (e.g., Monosphere 77, Monosphere 550A, etc.), Dow Chemical's 1x2, 1x4, 1x8, 22, 66, MSA-2, and Purolite's A400, A600, SGA550, A200, A300, A500, A501P, A502PS, A503, A510, A850, A860, A870, SSTA63. SSTA64, PFA520E, A100, A103S, A110, A111S, A133S, A830W, A845, A847, Organo's IRA400J, IRA402BL, IRA900J, IRA4580, SCAV4, HPR4002, IRA410J, IRA910CT, HPR4010, HPR4780, IRA67, IRA96SB, IRA98, Mitsubishi Chemical's Diaion PA series (e.g., PA306S, PA308, PA308L, PA312, PA312L, PA312LOH, P A312LTU, PA312LTUMB, PA316, PA316L, PA318L, PA318LOH, PA408, PA412, PA418, PA418L, PA418LL, PAF308L, HPA25L, HPA25M, HPA512L, HPA716, etc. (PA is a trademark), Mitsubishi Chemical's Diaion SA series (e.g., SA10A, SA10AL, SA10ALLP, SA10AOH, SA10AP, SA10DL, SA11A, SA11AL, SA12A, SA12AL, SA12ALL, SA2 Examples include 0A, SA20ALL, SA20ALLP, SA20AP, SA20AP2, SAF11AL, SANUPB, SAT10L, SAT20L, etc.), Mitsubishi Chemical's Diaion UBA series (e.g., UBA100, UBA100OH, UBA100OHUP, UBA120, UBA120OH, UBA120OHUP, UBA150, UBA200, etc.), and Mitsubishi Chemical's Diaion WA series (e.g., WA10, WA20, WA21J, WA30, WA30C, WA30LL, WA55, etc.).

[0027] As the basic ion exchange resin of the present invention, an ion exchange resin having a quaternary ammonium group in the exchange group of a strongly basic anion exchange resin is more preferred. Specifically, examples of ion exchange resins having a quaternary ammonium group in the exchange group of a strongly basic anion exchange resin include 1×2, 1×4, 1×8, 22, MSA-2, Marathon A, Marathon A2, Marathon MSA, Monosphere 550A from Dow Chemical, and A400, A600, SGA550, A200, A300, A500, A501P, A502PS, A503, A510, A850, A860, A from Purolite. 870, SSTA63, SSTA64, PFA520E, Organo's IRA400J, IRA402BL, IRA900J, IRA4580, SCAV4, HPR4002, IRA410J, IRA910CT, HPR4010, Mitsubishi Chemical's Diaion PA series (e.g., PA306S, PA308, PA308L, PA312, PA312L, PA312LOH, PA312LTU, PA312LTUMB, PA316, PA316L, PA318L, PA318LOH, PA408, PA412, PA418, PA418L, PA418LL, PAF308L, HPA25L, HPA25M, HPA512L, HPA716, etc. (PA is a trademark), Mitsubishi Chemical's Diaion SA series (e.g., SA10A, SA10AL, SA10ALLP, SA10AOH, SA10AP, SA10DL, SA11A, SA11AL, S Examples include A12A, SA12AL, SA12ALL, SA20A, SA20ALL, SA20ALLP, SA20AP, SA20AP2, SAF11AL, SANUPB, SAT10L, SAT20L, etc., and Mitsubishi Chemical's Diaion UBA series (for example, UBA100, UBA100OH, UBA100OHUP, UBA120, UBA120OH, UBA120OHUP, UBA150, UBA200, etc.).

[0028] The amount of basic ion exchange resin used should be such that the amount of cations in the solution containing the cytidine-5'-diphosphate compound, i.e., the total molar equivalent of impurities such as amino acids, organic acids, and chloride ions in addition to the cytidine-5'-diphosphate compound, is within the total exchange capacity of the basic ion exchange resin. Furthermore, the ionic form of the basic ion exchange resin can be either hydroxide ions, or a state to which organic acids with 2 or more carbon atoms used for elution are bound, or a combination of these.

[0029] The degree of crosslinking of the basic ion exchange resin is not particularly limited as long as it is sufficient to separate the cytidine-5'-diphosphate compound and 5'-CMP, but it is preferably 1 to 16%, more preferably 2 to 12%, and even more preferably 4 to 10%. The basic ion exchange resin can be used in a manner in which a solution containing the cytidine-5'-diphosphate compound is brought into contact with the basic ion exchange resin, but it is preferable to use it in the present invention in a form packed in a column, and any column may be used in the present invention.

[0030] When a solution containing a cytidine-5'-diphosphate compound is passed through a column packed with a basic ion exchange resin to bring the resin into contact with the resin, thereby adsorbing the cytidine-5'-diphosphate compound onto the resin, for example, when passing the solution through a column packed with a basic ion exchange resin with a degree of crosslinking of 1 to 16%, the passing rate is preferably such that the space velocity (the ratio of the volume of solution passed through the column per hour when the volume of the ion exchange resin is set to 1 ( / hour), hereinafter referred to as "SV") is SV = 0.1 to 5, more preferably SV = 0.2 to 4, and even more preferably SV = 0.4 to 3.

[0031] (2) A step of obtaining an eluate by eluting the cytidine-5'-diphosphate compound adsorbed on a basic ion exchange resin using an aqueous solution containing an organic acid having 2 or more carbon atoms. Step (2) is a step in which the cytidine-5'-diphosphate compound is separated and purified by passing an aqueous solution containing an organic acid having 2 or more carbon atoms through a basic ion exchange resin on which the cytidine-5'-diphosphate compound has been adsorbed in step (1), thereby obtaining an eluate and eluting the cytidine-5'-diphosphate compound from the resin.

[0032] The organic acid having two or more carbon atoms is preferably a carboxylic acid having two or more carbon atoms. Examples of organic acids having two or more carbon atoms include acetic acid, propionic acid, butyric acid, valeric acid, lactic acid, glycolic acid, pyruvic acid, gluconic acid, tartaric acid, oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, malic acid, fumaric acid, maleic acid, citric acid, isocitric acid, and ascorbic acid. Among these, acetic acid, propionic acid, and butyric acid are preferred, and acetic acid and propionic acid are more preferred. The organic acid having two or more carbon atoms may be used individually or in combination. There is no particular upper limit to the number of carbon atoms in the organic acid, but from the viewpoint of solubility in water, it is preferable to have eight or fewer carbon atoms, more preferably six or fewer carbon atoms, and even more preferably three or fewer carbon atoms. The organic acid having two or more carbon atoms may also be a salt of an organic acid having two or more carbon atoms.

[0033] In an aqueous solution containing an organic acid having two or more carbon atoms, the concentration of the organic acid having two or more carbon atoms is preferably 0.1 mol / L or higher, and more preferably 0.35 mol / L or higher, from the viewpoint of improving the purification efficiency of the cytidine-5'-diphosphate compound. There is no particular upper limit to the concentration of the organic acid having two or more carbon atoms in the aqueous solution, but from the viewpoint of balancing the amount of organic acid used and its effect, it is preferably 2.0 mol / L or lower, more preferably 1.0 mol / L or lower, even more preferably 0.6 mol / L or lower, and most preferably 0.45 mol / L or lower.

[0034] The eluate in step (2) (eluate containing cytidine-5'-diphosphate compound, hereinafter also abbreviated as cytidine-5'-diphosphate compound-containing eluate) is obtained by passing an aqueous solution containing an organic acid having 2 or more carbon atoms through a basic ion exchange resin, and then recovering the resulting liquid by dividing it into two or more fractions, in order to separate the cytidine-5'-diphosphate compound from impurities and, if necessary, to concentrate the cytidine-5'-diphosphate compound. Alternatively, the two or more fractions containing the obtained cytidine-5'-diphosphate compound may be mixed to obtain the cytidine-5'-diphosphate compound-containing eluate. The fractions can more preferably be divided into three or more, and even more preferably into four or more, for recovery. Before the step of mixing the fractions containing the cytidine-5'-diphosphate compound, a step of analyzing impurities such as the cytidine-5'-diphosphate compound and 5'-CMP contained in each fraction by HPLC may be included. Impurities such as cytidine-5'-diphosphate compounds and 5'-CMP can be analyzed by conventional methods, for example, high-performance liquid chromatography (UV detection).

[0035] The concentration of the cytidine-5'-diphosphate compound contained in the eluate in step (2) is preferably equal to or greater than the concentration of the cytidine-5'-diphosphate compound in the solution containing the cytidine-5'-diphosphate compound in step (1). Specifically, for example, the concentration ratio, i.e., the concentration of the cytidine-5'-diphosphate compound contained in the eluate in step (2) (g / L) / the concentration of the cytidine-5'-diphosphate compound in the solution contacted with the basic ion exchange resin in step (1) (g / L), is preferably 1.0 times or more, and more preferably 1.1 times or more. There is no particular upper limit to the concentration ratio, but for example, it is 160 times or less.

[0036] In step (2), the 5'-CMP ratio (ratio %) of the eluate to the cytidine-5'-diphosphate compound is preferably 1.00% or less, more preferably 0.20% or less, and even more preferably 0.10% or less. The lower limit of the ratio to the cytidine-5'-diphosphate compound is not particularly limited, but for example, it is 0% or more.

[0037] The recovery rate (%) of the cytidine-5'-diphosphate compound in step (2), i.e., the mass (g) of the cytidine-5'-diphosphate compound contained in the eluate in step (2) / the mass (g) of the cytidine-5'-diphosphate compound contained in the solution containing the cytidine-5'-diphosphate compound in step (1) × 100 (%), is preferably 60% or more, more preferably 80% or more, and even more preferably 89% or more. There is no particular upper limit to the recovery rate, but for example, it is less than 100%.

[0038] In step (2), the temperature at which the cytidine-5'-diphosphate compound is eluted from the basic ion exchange resin is preferably 20°C or higher, more preferably 30°C or higher, and even more preferably 40°C or higher, from the viewpoint of improving the separation of the cytidine-5'-diphosphate compound from 5'-CMP during elution. There is no particular upper limit to the elution temperature, but it is preferably 60°C or lower from the viewpoint of suppressing the generation of 5'-CMP by thermal decomposition of the cytidine-5'-diphosphate compound. In this invention, the elution temperature refers to the temperature of the column and / or the temperature of the aqueous solution containing an organic acid having 2 or more carbon atoms.

[0039] From the viewpoint of improving the separation of cytidine-5'-diphosphate compounds and 5'-CMP, when the organic acid used for elution is a C2 organic acid (e.g., acetic acid), the elution temperature is preferably 30 to 60°C, more preferably 40 to 60°C. On the other hand, when the organic acid used for elution is a C3 or more organic acid (e.g., propionic acid), the elution temperature is preferably 20 to 60°C, more preferably 30 to 60°C, and even more preferably 40 to 60°C.

[0040] It is known that changing the temperature in an ion exchange reaction alters the retention time of a compound due to changes in separation equilibrium, diffusion rate, degree of dissociation, and viscosity of the eluent. The effect of temperature in an ion exchange reaction varies depending on the compound, and is also said to change depending on the support and eluent. However, the inventors have found that increasing the elution temperature tends to improve the separation of cytidine-5'-diphosphate compounds and 5'-CMP. Furthermore, the inventors confirmed that hydrolysis of cytidine-5'-diphosphate compounds by heating proceeds even at 30°C, and that under conditions of 40°C or higher, more than 10 times the amount of 5'-CMP is produced as a by-product compared to conditions of 20°C, indicating that decomposition progresses significantly. However, they found that the effect of increasing the elution temperature on improving the separation of cytidine-5'-diphosphate compounds and 5'-CMP outweighs the disadvantages of decomposition by heating.

[0041] In step (2), the flow rate at which the aqueous solution containing an organic acid having 2 or more carbon atoms is passed through the basic ion exchange resin is preferably SV = 0.1 to 2, more preferably SV = 0.2 to 1.5, and even more preferably SV = 0.4 to 1.

[0042] By steps (1) and (2) described above, the cytidine-5'-diphosphate compound can be efficiently separated from impurities contained in the solution, such as 5'-CMP, UMP, UDP, UTP, UDP-choline, UDP-ethanolamine, CDP, CTP, inorganic phosphoric acid, and polyphosphate, and a high-purity cytidine-5'-diphosphate compound can be produced. In particular, by steps (1) and (2) described above, the cytidine-5'-diphosphate compound and 5'-CMP can be efficiently separated.

[0043] Furthermore, the eluate containing the cytidine-5'-diphosphate compound obtained in the elution process may be decolorized as needed using activated carbon treatment or a non-polar porous synthetic adsorbent, such as Mitsubishi Chemical's Diaion HP series (e.g., HP20, HP21, etc.), Mitsubishi Chemical's Diaion SP800 series (e.g., SP825, SP850, etc.), Mitsubishi Chemical's Diaion SP200 series (e.g., SP207, etc.), or Rohm & Haas's Amberlite XAD series (e.g., XAD4, XAD7HP, XAD16HP, XAD1180, XAD2000, etc.).

[0044] Furthermore, to obtain free crystals of the cytidine-5'-diphosphate compound, the above-mentioned eluate containing the cytidine-5'-diphosphate compound or the solution after decolorization treatment is adjusted to a pH of preferably 2.0 to 4.0 with an acid or alkali as needed, and after concentration as needed, the concentration of the cytidine-5'-diphosphate compound is adjusted to preferably 50 to 800 g / L, more preferably 100 to 700 g / L, and the cytidine-5'-diphosphate compound crystals can be obtained using an organic solvent, preferably a hydrophilic organic solvent such as acetone, ethanol, methanol, or propanol.

[0045] Furthermore, to obtain salt crystals of the cytidine-5'-diphosphate compound, for example, the above-mentioned eluate containing the cytidine-5'-diphosphate compound can be adjusted to a pH of preferably 5.0 to 9.5 with sodium hydroxide, and after decolorization and concentration treatment as necessary, the concentration of the cytidine-5'-diphosphate compound can be adjusted to preferably 50 to 800 g / L, more preferably 100 to 700 g / L, and sodium salt crystals of the cytidine-5'-diphosphate compound can be obtained using an organic solvent, preferably a hydrophilic organic solvent such as acetone, ethanol, methanol, or propanol.

[0046] Methods for obtaining cytidine-5'-diphosphate compound crystals using organic solvents include, for example, adding an organic solvent to a cytidine-5'-diphosphate compound solution to precipitate crystals, or dropping a cytidine-5'-diphosphate compound solution into a large amount of organic solvent to precipitate crystals.

[0047] The present invention will be further described by the following examples, but the present invention is not limited to these examples. [Examples]

[0048] [Analysis Example 1] Quantitative analysis of CDP-choline, 5'-CMP, and CDP-ethanolamine by HPLC was performed under the following conditions. Equipment used: System controller (CBM-20A), detector (SPD-20A), pump (LC-20AT), autosampler (SIL-20ACXR), column oven (CTO-10ASvp), data analysis software (LabSolutions Version 5.85) (all manufactured by Shimadzu Corporation) Detector: UV absorbance spectrophotometer (measurement wavelength 254 nm) Column: Shodex Asahipak NH2P-50 4E 4.6×250mm (manufactured by Showa Denko Corporation) Mobile phase: 0.03 mol / L potassium dihydrogen phosphate aqueous solution adjusted to pH 3.5 with phosphoric acid (20.41 g of potassium dihydrogen phosphate was dissolved in 5 L of distilled water, and then phosphoric acid was added to adjust the pH to 3.5). Column temperature: 40℃ Flow rate: 0.50mL / min Sample injection volume: 10 μL Retention time: CDP-choline 7.3 min, 5'-CMP 16.0 min, CDP-ethanolamine 9.1 min

[0049] Each eluted fraction in step (2) was diluted 100-fold or adjusted to approximate the concentration of the standard and used for analysis. The concentrations of CDP-choline, 5'-CMP, and CDP-ethanolamine in each eluted fraction were quantified using a single-check measure method from the peak area values ​​of the standard, with 0.02 g / L of CDP-choline, 0.01 g / L of 5'-CMP, and 0.018 g / L of CDP-ethanolamine used as the standard.

[0050] For example, regarding CDP-choline, if the peak area value of the CDP-choline standard is A and the peak area value of the eluted fraction after x-fold dilution is B, then the (CDP-choline concentration of the eluted fraction (g / L)) is calculated as (0.02 × B / A × x).

[0051] [Analysis Example 2] Quantitative analysis of CDP trisodium salt by HPLC was performed under the following conditions. Equipment used: System controller (CBM-20A), detector (SPD-20A), pump (LC-20AD), autosampler (SIL-20AC), column oven (CTO-20AC), data analysis software (LabSolutions Version 5.71 SP1) (all manufactured by Shimadzu Corporation) Detector: UV absorbance spectrophotometer (measurement wavelength 254 nm) Column: Two Partisil 10 SAX 4.0×250mm columns connected in series (Hichrom) Mobile phase: 0.06 mol / L potassium dihydrogen phosphate aqueous solution adjusted to pH 3.5 with phosphoric acid (40.83 g of potassium dihydrogen phosphate was dissolved in 5 L of distilled water, and then phosphoric acid was added to adjust the pH to 3.5). Column temperature: 30℃ Flow rate: 0.45mL / min Sample injection volume: 10 μL Retention time: CDP trisodium salt 82.5min

[0052] [Comparative Example 1] Based on the specifications of Indian Patent Application Publication No. 2012MUM02534, experiments were conducted using a 0.10 mol / L formic acid aqueous solution. As a solution containing cytidine-5'-diphosphate compounds, 210 mL of an aqueous solution was prepared containing 7.02 g / L of CDP-choline (manufactured by Kyowa Hakko Bio Co., Ltd.) and 0.37 g / L of 5'-CMP (manufactured by Tokyo Chemical Industry Co., Ltd.) [the ratio of the concentration of 5'-CMP to the concentration of CDP-choline in the solution (hereinafter referred to as the "CDP-choline ratio") is 5.3%]. - Adsorption was performed by passing the sample through 150 mL of Malathion A (manufactured by Dow Chemical) at an SV of 1.25.

[0053] Next, the outside of the column was adjusted to 20°C, and 225 mL of water was passed through at SV=1.10. Finally, while maintaining the column at 20°C, 3 L of 0.10 mol / L formic acid aqueous solution (diluted reagent grade from Fujifilm Wako Pure Chemical Industries, Ltd.) was passed through at SV=0.85, and 29.9 mL of eluate was obtained. After mixing the fractions containing CDP-choline, 239 mL of a solution with a CDP-choline concentration of 6.08 g / L was obtained. The 5'-CMP concentration in this solution was 0.40 g / L (6.6% of CDP-choline). The recovery rate of CDP-choline was 98.6%.

[0054] [Comparative Example 2] As a solution containing cytidine-5'-diphosphate compounds, a 3.20 L aqueous solution was prepared containing CDP-choline at a concentration of 7.11 g / L and 5'-CMP at a concentration of 0.36 g / L (5.1% relative to CDP-choline), and OH - Adsorption was performed by passing the eluate through 150 mL of Marathon MSA (Dow Chemical) at SV=1.25. Next, the outside of the column was adjusted to 20°C, and 225 mL of water was passed through at SV=1.10. Finally, while maintaining the column at 20°C, 1350 mL of 0.35 mol / L formic acid aqueous solution was passed through at SV=0.85, and 14.1 mL of eluate was collected. The CDP-choline and 5'-CMP concentrations for each eluted fraction were quantified by HPLC, and the results are shown in Figure 1.

[0055] After mixing the fraction containing CDP-choline, 381 mL of a solution with a CDP-choline concentration of 59.7 g / L was obtained. The 5'-CMP concentration in this solution was 2.7 g / L (4.5% of CDP-choline). Furthermore, the recovery rate of CDP-choline was 100%.

[0056] [Example 1] As a solution containing cytidine-5'-diphosphate compounds, a 3.20 L aqueous solution was prepared containing CDP-choline at a concentration of 7.11 g / L and 5'-CMP at a concentration of 0.36 g / L (5.1% relative to CDP-choline), and OH -Adsorption was performed by passing the CDP-choline solution through 150 mL of Marathon MSA (Dow Chemical) at an SV of 1.25. Next, the outside of the column was adjusted to 20°C, and 225 mL of water was passed through at an SV of 1.10. Finally, while maintaining the column at 20°C, 1350 mL of 0.35 mol / L aqueous acetic acid solution (diluted reagent grade from Fujifilm Wako Pure Chemical Industries, Ltd.) was passed through at an SV of 0.85, and 14.6 mL of eluate was obtained in two portions. After mixing the fractions containing CDP-choline, 351 mL of a solution with a CDP-choline concentration of 65.3 g / L was obtained. The 5'-CMP concentration in this solution was 0.10 g / L (0.15% of CDP-choline). The recovery rate of CDP-choline was 100%.

[0057] [Example 2] As a solution containing cytidine-5'-diphosphate compounds, 3.21 L of aqueous solution was prepared containing CDP-choline at a concentration of 7.13 g / L and 5'-CMP at a concentration of 0.38 g / L (5.3% relative to CDP-choline), and OH - Adsorption was performed by passing the compound through 150 mL of Marathon MSA (Dow Chemical) at an SV of 1.25. Next, the outside of the column was adjusted to 20°C, and 225 mL of water was passed through at an SV of 1.10. Finally, while maintaining the column at 20°C, 1350 mL of 0.35 mol / L propionic acid aqueous solution (diluted Wako Special Grade, Fujifilm Wako Pure Chemical Industries, Ltd.) was passed through at an SV of 0.85, and eluates were collected in 15.6 mL portions. The CDP-choline and 5'-CMP concentrations for each eluted fraction were quantified by HPLC, and the results are shown in Figure 2.

[0058] After mixing the fraction containing CDP-choline, 281 mL of a solution with a CDP-choline concentration of 78.2 g / L was obtained. The 5'-CMP concentration in this solution was 0.03 g / L (0.04% of CDP-choline). The recovery rate of CDP-choline was 96.0%.

[0059] [Example 3] As a solution containing cytidine-5'-diphosphate compounds, 3.64 L of an aqueous solution was prepared containing CDP-choline at a concentration of 7.12 g / L and 5'-CMP at a concentration of 0.41 g / L (CDP-choline ratio of 5.8%), and OH -Adsorption was performed by passing the eluate through a 150 mL column of Malathion A (Dow Chemical) at an SV of 1.25. Next, the outside of the column was adjusted to 20°C, and 225 mL of water was passed through at an SV of 1.10. Finally, while maintaining the column at 20°C, 1350 mL of 0.45 mol / L aqueous acetic acid solution was passed through at an SV of 0.85, and 14.8 mL of eluate was collected. After mixing the fractions containing CDP-choline, 415 mL of a solution with a CDP-choline concentration of 63.2 g / L was obtained. The 5'-CMP concentration in this solution was 0.32 g / L (0.51% of CDP-choline). The recovery rate of CDP-choline was 100%.

[0060] [Example 4] As a solution containing cytidine-5'-diphosphate compounds, 3.64 L of an aqueous solution was prepared containing CDP-choline at a concentration of 7.12 g / L and 5'-CMP at a concentration of 0.41 g / L (5.8% relative to CDP-choline), and OH - Adsorption was performed by passing the CDP-choline solution through a 150 mL column of Malathion A (Dow Chemical) at an SV of 1.25. Next, the outside of the column was adjusted to 30°C, and 225 mL of water was passed through at an SV of 1.10. Finally, while maintaining the column at 30°C, 1350 mL of 0.45 mol / L aqueous acetic acid solution was passed through at an SV of 0.85, and 14.6 mL of eluate was collected. The CDP-choline and 5'-CMP concentrations of each eluted fraction were quantified by HPLC, and the results are shown in Figure 3. After mixing the fractions containing CDP-choline, 349 mL of a solution with a CDP-choline concentration of 71.4 g / L was obtained. The 5'-CMP concentration in the same solution was 0.03 g / L (0.04% of CDP-choline). The recovery rate of CDP-choline was 96.1%.

[0061] [Example 5] As a solution containing cytidine-5'-diphosphate compounds, 3.64 L of aqueous solution was prepared containing CDP-choline at a concentration of 7.13 g / L and 5'-CMP at a concentration of 0.38 g / L (5.3% relative to CDP-choline), and OH -150 mL of Type 0 Marathon A (manufactured by Dow Chemical) was passed through at an SV of 1.25 for adsorption. Next, the outside of the column was heated to 60 °C, and 225 mL of water was passed through at an SV of 1.10. Finally, while maintaining the column at 60 °C, 1350 mL of 0.45 mol / L acetic acid aqueous solution was passed through at an SV of 0.85, and eluate was obtained in 14.8 mL portions. As a result of mixing the fractions containing CDP-choline, 354 mL of a solution with a CDP-choline concentration of 71.2 g / L was obtained. The 5'-CMP concentration of the same solution was 0.01 g / L (ratio to CDP-choline 0.01%). Also, the recovery rate of CDP-choline was 97.1%.

[0062] [Comparative Example 3] As a solution containing a cytidine-5'-diphosphate compound, 3.20 L of an aqueous solution containing CDP-choline at a concentration of 7.10 g / L and 5'-CMP at a concentration of 0.38 g / L (ratio to CDP-choline 5.4%) was prepared, and OH - 150 mL of Type 0 Marathon MSA (manufactured by Dow Chemical) was passed through at an SV of 1.25 for adsorption. Next, the outside of the column was heated to 40 °C, and 225 mL of water was passed through at an SV of 1.10. Finally, while maintaining the column at 40 °C, 1350 mL of 0.45 mol / L formic acid aqueous solution was passed through at an SV of 0.85, and eluate was obtained in 14.7 mL portions. As a result of mixing the fractions containing CDP-choline, 323 mL of a solution with a CDP-choline concentration of 67.4 g / L was obtained. The 5'-CMP concentration of the same solution was 3.5 g / L (ratio to CDP-choline 5.2%). Also, the recovery rate of CDP-choline was 95.8%.

[0063] [Example 6] As a solution containing a cytidine-5'-diphosphate compound, 3.20 L of an aqueous solution containing CDP-choline at a concentration of 7.10 g / L and 5'-CMP at a concentration of 0.38 g / L (ratio to CDP-choline 5.4%) was prepared, and OH -Adsorption was performed by passing the eluate through a 150 mL column of Malathion MSA (Dow Chemical) at an SV of 1.25. Next, the outside of the column was heated to 40°C, and 225 mL of water was passed through at an SV of 1.10. Finally, while maintaining the column at 40°C, 1350 mL of 0.45 mol / L aqueous acetic acid solution was passed through at an SV of 0.85, and 15.2 mL of eluate was collected. After mixing the fractions containing CDP-choline, 305 mL of a solution with a CDP-choline concentration of 70.8 g / L was obtained. The 5'-CMP concentration in this solution was 0.05 g / L (0.07% of CDP-choline). The recovery rate of CDP-choline was 95.0%.

[0064] [Example 7] As a solution containing cytidine-5'-diphosphate compounds, a 2.90 L aqueous solution was prepared containing CDP-choline at a concentration of 7.05 g / L and 5'-CMP at a concentration of 0.37 g / L (5.2% relative to CDP-choline), and OH - Adsorption was performed by passing the eluate through a 150 mL column of Malathion A (Dow Chemical) at an SV of 1.25. Next, the outside of the column was heated to 40°C, and 225 mL of water was passed through at an SV of 1.10. Finally, while maintaining the column at 40°C, 3 L of 0.10 mol / L aqueous acetic acid solution was passed through at an SV of 0.85, and 26.8 mL of eluate was collected. After mixing the fractions containing CDP-choline, 885 mL of a solution with a CDP-choline concentration of 22.6 g / L was obtained. The 5'-CMP concentration in this solution was 0.003 g / L (0.01% of CDP-choline). The recovery rate of CDP-choline was 97.8%.

[0065] [Example 8] As a solution containing cytidine-5'-diphosphate compounds, prepare 2.15 L of an aqueous solution containing CDP-choline at a concentration of 7.24 g / L and 5'-CMP at a concentration of 0.38 g / L (5.2% relative to CDP-choline), and OH -Adsorption was performed by passing the eluate through a 139 mL column of type PFA520E (Purolite) at an SV of 1.25. Next, the outside of the column was heated to 40°C, and 209 mL of water was passed through at an SV of 1.10. Finally, while maintaining the column at 40°C, 1500 mL of 0.10 mol / L aqueous acetic acid solution was passed through at an SV of 0.85, and 13.9 mL of eluate was collected. After mixing the fractions containing CDP-choline, 487 mL of a solution with a CDP-choline concentration of 31.0 g / L was obtained. The 5'-CMP concentration in this solution was 0.005 g / L (0.02% of CDP-choline). The recovery rate of CDP-choline was 97.0%.

[0066] [Example 9] A 1.00 L aqueous solution containing cytidine-5'-diphosphate compounds was prepared, containing CDP-choline at a concentration of 7.04 g / L and 5'-CMP at a concentration of 0.37 g / L (5.3% relative to CDP-choline). - Adsorption was performed by passing the CDP-choline solution through a 100 mL monosphere 77 column (Dow Chemical) at an SV of 1.25. Next, the outside of the column was heated to 40°C, and 150 mL of water was passed through at an SV of 1.10. Finally, while maintaining the column at 40°C, 1500 mL of 0.40 mol / L aqueous acetic acid solution was passed through at an SV of 0.85, and eluates were collected in 15.0 mL increments. After mixing the fractions containing CDP-choline, 285 mL of a solution with a CDP-choline concentration of 24.4 g / L was obtained. The 5'-CMP concentration in this solution was 0.005 g / L (0.02% of CDP-choline). The recovery rate of CDP-choline was 98.8%.

[0067] The results described above are summarized in Table 1.

[0068] [Table 1]

[0069] As shown in Table 1, in Examples 1 to 9, a solution containing a cytidine-5'-diphosphate compound was brought into contact with a basic ion exchange resin to adsorb the cytidine-5'-diphosphate compound onto the basic ion exchange resin, and then the eluate was obtained by eluting the cytidine-5'-diphosphate compound adsorbed on the basic ion exchange resin using an aqueous solution containing an organic acid having 2 or more carbon atoms. Compared to Comparative Examples 1 to 3, in which the cytidine-5'-diphosphate compound was eluted using an aqueous solution containing an organic acid having 1 carbon atom, it was found that high-purity cytidine-5'-diphosphate compound could be obtained with high efficiency.

[0070] [Example 10] The organic chemical synthesis of CDP-choline was carried out according to a known method (K. Kikugawa, M. Ichino, Chem. Pharm. Bull., 19, 1011, 1971). 6.6 g of Phosphocholine Chloride Calcium Salt Tetrahydrate (Tokyo Chemical Industries, Ltd.) was added to 20 mL of water and stirred at 80°C until dissolved. 2.52 g of oxalic acid dihydrate (reagent grade, Fujifilm Wako Pure Chemical Industries, Ltd.) was added to this solution and stirred for 10 minutes.

[0071] The resulting white precipitate was filtered off, and the filtrate was concentrated under reduced pressure at 50°C. Then, 16 mL of DMF (organic synthesis grade, Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the mixture was concentrated under reduced pressure at 70°C. After cooling to 0°C, a solution of 4.4 g of p-toluenesulfonyl chloride (Nacalai Tesque) dissolved in 6 mL of DMF was added dropwise, and the mixture was stirred until homogeneous. 2.0 g of cytidine 5'-monophosphate was added, and the mixture was stirred overnight at room temperature. After cooling again to 0°C, 250 mL of water was added, and the pH was adjusted to 3.5 with 28% by mass aqueous ammonia (reagent grade, Fujifilm Wako Pure Chemical Industries, Ltd.). Two of these operations were performed simultaneously, and after mixing the two solutions, water was added to obtain 5050 mL of a solution containing the cytidine-5'-diphosphate compound. At this time, the solution containing the cytidine-5'-diphosphate compound contained CDP-choline at a concentration of 0.486 g / L and 5'-CMP at a concentration of 0.0717 g / L (14.8% of CDP-choline).

[0072] 2450 mL of the solution containing the cytidine-5'-diphosphate compound obtained in this way was OH - The compound was passed through a 150 mL column of PA412 (manufactured by Mitsubishi Chemical Corporation) at an SV of 1.25 for adsorption. Then, the outside of the column was heated to 60°C, and 225 mL of water was passed through at an SV of 1.10. Finally, while maintaining the column at 60°C, 1350 mL of 0.50 mol / L aqueous propionic acid solution was passed through at an SV of 0.85, and 15.2 mL of eluate was obtained. After mixing the fractions containing CDP-choline, 106 mL of a solution with a CDP-choline concentration of 10.7 g / L was obtained. The 5'-CMP concentration in this solution was 0.003 g / L (0.03% of CDP-choline). The recovery rate of CDP-choline was 95.3%. From these results, it was found that the production method of the present invention can also be applied to solutions containing cytidine-5'-diphosphate compounds obtained by organic synthesis.

[0073] [Example 11] A culture medium with a CDP-choline concentration of 30.8 g / L was obtained using the method described in Japanese Patent Publication No. 3369236, and the pH was adjusted to 3.0 with sulfuric acid. Next, the bacterial cells were separated by centrifugation (8000 rpm, 10 minutes), and water was added to the obtained supernatant to obtain 10.9 L of an aqueous solution containing CDP-choline at a concentration of 12.2 g / L as a solution containing cytidine-5'-diphosphate compound.

[0074] A solution containing this cytidine-5'-diphosphate compound is H + Adsorption was carried out by passing the ion-exchanged cation exchange resin (8.4 L) with an SV of 0.26 and maintaining the column temperature at 10°C. Subsequently, cold water at 10°C was passed through the column, and once the elution of CDP-choline began, OH - The sample was continuously passed through a 900 mL column of PA412 to allow adsorption. Then, the outside of the PA412 column was heated to 40°C, and 2.7 L of water was passed through at an SV of 1.10. Finally, while maintaining the column at 40°C, 5.4 L of 0.47 mol / L aqueous acetic acid solution was passed through at an SV of 0.85, and 61 mL of eluate was collected.

[0075] As a result of mixing the fraction containing CDP-choline, 2.85 L of a solution with a CDP-choline concentration of 42.8 g / L was obtained. The 5'-CMP concentration in this solution was 0.01 g / L or less (CDP-choline ratio of 0.02% or less). Furthermore, the recovery rate of CDP-choline was 91.7%. From these results, it was found that the production method of the present invention can also be applied to solutions containing cytidine-5'-diphosphate compounds obtained by the culture method.

[0076] [Example 12] The reaction equation for the organic synthesis of CDP-ethanolamine is shown below. In the reaction equation below, "rt" represents room temperature.

[0077] [ka]

[0078] The synthesis of compound 2 was carried out based on known literature (H. Ngo, MF Dunn et al. Biochemistry, 46, 7713, 2007.; T. Tanaka yakugakuzasshi, 80, 439, 1959.). 6.80 g of sodium hydroxide (reagent grade, Fujifilm Wako Pure Chemical Industries, Ltd.) was added to 18.9 mL of water and stirred until completely dissolved. After cooling this solution to 0°C, 8.00 g of phosphoethanolamine (Tokyo Chemical Industries, Ltd.) was added and stirred until dissolved. 7.99 mL of Z-chloride (Tokyo Chemical Industries, Ltd.) was added dropwise and stirred at room temperature for 2 hours. Further stirring was carried out at 50°C for 1 hour, followed by stirring overnight at room temperature. 1 L of water was added to prepare the solution, which was then mixed with H + The eluate was passed through 850 mL of a strong acid cation exchange resin. After passing through the column, it was washed with water to obtain a total of 5 L of eluate. This eluate was concentrated under reduced pressure to obtain 500 mL of crude product.

[0079] The solution was neutralized with cyclohexylamine (Wako Special Grade, Fujifilm Wako Pure Chemical Industries, Ltd.) until the pH reached 4.5, and then the solvent was completely removed under reduced pressure. 98 mL of water and 28 mL of ethanol (Reagent Grade, Fujifilm Wako Pure Chemical Industries, Ltd.) were added to the precipitated white solid, and the mixture was completely dissolved at 66°C. This solution was slowly cooled to room temperature and allowed to stand overnight. The precipitated white crystals were obtained by suction filtration and dried overnight under reduced pressure. The yield was 12.2 g.

[0080] The synthesis of compound 3 was carried out based on known literature (K. Oertell, CE McKenna, MF Goodman et al. Biochemistry, 53, 1842, 2014). To a mixed solvent of 16.8 mL of t-BuOH (Wako Special Grade, Fujifilm Wako Pure Chemical Industries, Ltd.) and 16.8 mL of water, 953 mg of cytidine 5'-Monophosphate and 0.77 mL of morpholine (Wako Special Grade, Fujifilm Wako Pure Chemical Industries, Ltd.) were added and completely dissolved by stirring under reflux. In addition, 1.83 g of N,N'-Dicyclohexylcarbodiimide (Wako First Grade, Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 19.3 mL of t-BuOH.

[0081] One-eighth of this solution was added to the previously prepared cytidine 5'-monophosphate solution at a rate of once every 15 minutes. After the entire volume was added, reflux was continued for another 2.5 hours. Approximately 50 mL of water was added to the crude product, which had been cooled to room temperature, and the resulting white solid was removed by suction filtration. Further filtration was performed using a 0.45 μm pore size membrane filter until the filtrate was clear. After removing the solvent under reduced pressure, DMF was added and removed again by distillation, and the solution was used in the next reaction without further purification.

[0082] The synthesis of compound 4 was carried out based on known literature (T. Tanaka et al. yakugakuzasshi, 85, 863, 1965). 2.00 g of compound 2, which was synthesized earlier, was added to 178 mL of water and dissolved. This was then H +Cyclohexylamine was removed by passing the mixture through a strongly acidic cation exchange resin column, followed by washing with water to obtain a total of 300 mL of eluate. Next, the water was removed under reduced pressure, and an appropriate amount of DMF was added and removed again. More DMF was added to dissolve the compound. Compound 3, which had been synthesized separately, was then dissolved in DMF, and the two mixtures were mixed and stirred overnight at room temperature. At this time, 16.2 mL of DMF was used, including the amount used for washing. The next day, the temperature was raised to 60°C and stirred for 5 hours to complete the reaction. After removing the solvent under reduced pressure, water was added and removed again, and the mixture was used in the next reaction without purification.

[0083] The synthesis of CDP-ethanolamine was carried out based on known literature (T. Tanaka et al. yakugakuzasshi, 85, 863, 1965). Compound 4, synthesized earlier, was dissolved in 11.2 mL of water. After replacing the atmosphere with a nitrogen atmosphere, 636 mg of 5% by mass Pd / C (NECHEMCAT, standard type for organic synthesis) was added and the mixture was stirred. Next, the atmosphere was replaced with a hydrogen atmosphere and the mixture was vigorously stirred at room temperature for 4 hours. After again replacing the atmosphere with a nitrogen atmosphere, Pd / C was removed from the reaction mixture by suction filtration. Further removal of the remaining Pd / C was performed using a 0.45 μm pore size membrane filter. Water was added to the resulting filtrate to obtain a solution containing 1020 mL of cytidine-5'-diphosphate compound. At this time, the solution containing the cytidine-5'-diphosphate compound contained CDP-ethanolamine at a concentration of 1.17 g / L and 5'-CMP at a concentration of 0.0334 g / L (2.9% relative to CDP-ethanolamine).

[0084] 1000 mL of the solution containing the cytidine-5'-diphosphate compound obtained above, OH -Adsorption was carried out by passing the solution through 150 mL of PA412 column at SV=1.25. Then, the outside of the column was heated to 60°C, and 225 mL of water was passed through at SV=1.10. Finally, while maintaining the column at 60°C, 1500 mL of 0.5 mol / L aqueous propionic acid solution was passed through at SV=0.85, and 15.1 mL of eluate was obtained. After mixing the fractions containing CDP-ethanolamine, 151 mL of a solution with a CDP-ethanolamine concentration of 7.50 g / L was obtained. The 5'-CMP concentration in this solution was 0.008 g / L (0.11% of CDP-ethanolamine). The recovery rate of CDP-ethanolamine was 96.8%. From these results, it was found that the production method of the present invention can also be applied to CDP-ethanolamine solutions obtained by synthesis.

[0085] [Comparative Example 4] Prepare 140 mL of an aqueous solution containing 6.67 g / L of CDP trisodium salt (Combi-Blocks, Lot: B12560) and 0.43 g / L of 5'-CMP, and then OH - Adsorption was performed by passing 100 mL of Malathion A (Dow Chemical) column at SV=1.25. Next, the outside of the column was heated to 40°C, and 150 mL of water was passed through at SV=1.10. Finally, while maintaining the column at 40°C, 1500 mL of 0.50 mol / L aqueous acetic acid solution was passed through at SV=0.85, and 15.0 mL of eluate was obtained. CDP trisodium salt was not present in any of the fractions. From these results, it was found that the production method of the present invention is not applicable to CDP trisodium salt.

[0086] [Example 13] Prepare 140 mL of an aqueous solution containing cytidine-5'-diphosphate compounds, with CDP-choline at a concentration of 7.09 g / L and 5'-CMP at a concentration of 0.45 g / L (6.3% relative to CDP-choline), and OH -Adsorption was performed by passing the CDP-choline through 100 mL of Malathion A (Dow Chemical) column at SV=1.25. Next, the outside of the column was heated to 40°C, and 150 mL of water was passed through at SV=1.10. Finally, while maintaining the column at 40°C, 1000 mL of 0.50 mol / L aqueous acetic acid solution was passed through at SV=0.85, and 10.0 mL of eluate was collected. After mixing the fractions containing CDP-choline, 110 mL of a solution with a CDP-choline concentration of 8.06 g / L was obtained. This solution did not contain 5'-CMP, and the recovery rate of CDP-choline was 89.3%.

[0087] Although the present invention has been described in detail with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications are possible without departing from the spirit and scope of the invention. This application is based on the international application (PCT / JP2021 / 031429) filed on 26 August 2021, which is incorporated by reference in its entirety. All references incorporated herein are incorporated as a whole.

Claims

1. A method for producing a cytidine-5'-diphosphate compound, comprising the following steps (1) and (2), wherein the cytidine-5'-diphosphate compound is a cytidine-5'-diphosphate compound having a cationic substituent. (1) A step of bringing a solution containing a cytidine-5'-diphosphate compound into contact with a basic ion exchange resin to adsorb the cytidine-5'-diphosphate compound onto the basic ion exchange resin. (2) A step of obtaining an eluate by eluting the cytidine-5'-diphosphate compound adsorbed on the basic ion exchange resin using an aqueous solution containing an organic acid having 2 or more carbon atoms.

2. The manufacturing method according to claim 1, wherein the concentration of the cytidine-5'-diphosphate compound contained in the eluate in step (2) is equal to or greater than the concentration of the cytidine-5'-diphosphate compound in the solution containing the cytidine-5'-diphosphate compound in step (1).

3. The manufacturing method according to claim 1 or 2, wherein in step (2), the temperature at which the cytidine-5'-diphosphate compound is eluted from the basic ion exchange resin is 20°C or higher.

4. The method for producing an organic acid having 2 or more carbon atoms, wherein the organic acid is at least one organic acid selected from acetic acid, propionic acid, and butyric acid.

5. The manufacturing method according to any one of claims 1 to 4, wherein in step (2), the concentration of the organic acid contained in the aqueous solution is 0.1 mol / L or more.

6. The method for producing cytidine-5'-diphosphate compound having a cationic substituent is a compound in which cytidine-5'-diphosphate is bonded to a cationic substituent, according to any one of claims 1 to 5.

7. The method for producing cytidine-5'-diphosphate compound having a cationic substituent is a compound in which a cationic substituent is bonded to the β-phosphate group of cytidine-5'-diphosphate, according to any one of claims 1 to 6.

8. The manufacturing method according to any one of claims 1 to 7, wherein the organic acid having two or more carbon atoms is a carboxylic acid having two or more carbon atoms.

9. The production method according to any one of claims 1 to 8, wherein the cytidine-5'-diphosphate compound having a cationic substituent is at least one selected from choline cytidine-5'-diphosphate and ethanolamine cytidine-5'-diphosphate.

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