DE69018815T2 - Process for the preparation of a mixture of inosic acid and guanosic acid by direct phosphorylation. - Google Patents

Description

The present invention relates to a new process for the phosphorylation of inosine and guanosine, more precisely to a direct process for the phosphorylation of the hydroxy group in the 5' position in a simple and advantageous manner without having to protect the hydroxy groups in the 2' and 3' positions of the ribose unit in the nucleoside.

The object is to provide an industrially applicable and economical process for producing a mixture of 5'-nucleotides, in particular a mixture of inosinic acid (inosine-5'-phosphate) and guanylic acid (guanosine-5'-phosphate), which can be used as a seasoning, medicine, etc.

Various methods are currently known for the chemical phosphorylation of unprotected nucleosides.

These procedures can be classified as follows:

1) Process using a trialkyl phosphate as solvent for the reaction:

Tetrahedron Lett., 50, 5065 (1967);

Bull. Chem. Soc. Jpn., 42, 3505(1969);

Japanese Patent Publication No. 42-01107

2) Process using a polar organic solvent and an organic amine or its salt of an inorganic acid:

Bull. Chem. Soc. Jpn., 48, 2084 (1975)

3) Process using a tri(alkoxyalkyl)phosphate as solvent for the reaction:

Japanese Patent Application Laid-Open No. 51- 86482

4) Process using an alkali metal salt of a nucleoside:

Soviet Patent No. 487 887

5) Method using mixed crystals of guanosine and inosine:

Japanese Patent Application Laid-Open No. 59-167599.

In the past, the processes for chemically phosphorylating a nucleoside mixture, especially a mixture of inosine and guanosine, have had some problems. In general, byproducts are often produced during the process for producing inosinic acid and guanylic acid by phosphorylating a mixture of inosinic acid and free guanosine. In particular, unless free inosine is present in excess relative to free guanosine, free inosine is partially converted to byproducts such as inosinic acid diphosphate, inosinic acid triphosphate, etc. via inosinic acid during the period of time in which the conversion of free guanosine, which has a lower phosphorylation rate than inosine, is completed. Specifically, free inosine is converted to inosine 5'-monophosphate approximately three times faster than free guanosine. At the time when the free guanosine is converted to more than 99% guanosine 5'-monophosphate, free inosine is phosphorylated at positions other than the 5'-position, forming many by-products such as inosine 3',5'-diphosphate, inosine 2',5'-diphosphate, inosine 5'-diphosphate, etc. For this reason, the phosphorylation of a mixture of free inosine and free guanosine, especially in a molar ratio of 1:1, is extremely difficult.

On the other hand, known methods for chemical phosphorylation of guanosine alone include the addition of picoline, pyridine base or their salts of inorganic Acids (Process 2 as described above) and a process using alkali metal salts of guanosine (Process 4 as described above) to improve the reactivity of guanosine. However, in Process 2 described above, the yield of guanylic acid is not improved as expected. In addition, the product is contaminated with amines, which are considered toxic. Therefore, this process is not suitable for application to food additives, etc.

Furthermore, in the above process 4, the phosphorylation of the alkali metal salt of guanosine proceeds at a reaction rate exceeding that of the phosphorylation of guanosine by 2 times or more. Furthermore, it is known that the reactive polar solvent (a trialkyl phosphate, etc.) and the phosphorylating agent (phosphorus oxychloride, etc.) are each reduced to less than 1/2.

Furthermore, as an example of phosphorylation of a mixture of inosine and guanosine, a method of treating the mixture in the form of solid solutions (method 5 described above) is known. It is said that according to this method, the formation of by-products is reduced and the desired mixture can be obtained in high yield. However, this method requires a step for preparing solid solutions, so that (1) the number of steps increases, (2) it is not easy to use inosine and free guanosine in an arbitrary mixing ratio, (3) the reaction time is considerably prolonged, and considerable amounts of a reaction solvent and a phosphorylating agent are required.

In view of this state of the art, the chemical process for phosphorylation of a mixture containing inosine and guanosine in freely selectable proportions always has still presents a number of problems. Against this background, the development of a process that would enable phosphorylation in high yield and with a shortened reaction time using reduced amounts of reactants and with reduced formation of by-products was desired.

The object of the present invention was to provide a process for phosphorylating a mixture of inosine and guanosine containing at least one alkali metal salt of inosine and guanosine, which leads to a high purity and yield of the product, whereby the amount of the solvent for the reaction and the phosphorylating agent can be reduced, and which can be carried out in a shortened period of time.

The inventors have conducted studies on chemical methods for the mixed phosphorylation of inosine and guanosine, using alkali metal salts at the site closest to the reaction site of these nucleosides to impart basicity to them and to accelerate the phosphorylation by the neighboring group effect. By these measures, the reactivity of guanosine and inosine is increased, thus providing a method for producing a mixture of inosinic acid and guanylic acid in such high purity and high yield as has not been possible heretofore.

That is, the present invention relates to:

(A) a chemical process for the preparation of a mixture of inosinic acid and guanosic acid by mixed phosphorylation of inosine and guanosine, characterized in that inosine and guanosine, which contain at least one of the respective alkali metal salts of inosine and guanosine, are reacted with a phosphorus oxyhalide or its hydrate in a polar organic solvent, wherein Inosine and guanosine are preferably present as alkali metal salt of guanosine and free inosine, and the molar ratio (free inosine)/(alkali metal salt of guanosine) is chosen in a range of 1/2 to 5/3;

(B) a process for the mixed phosphorylation of inosine and guanosine, which comprises subjecting an alkali metal salt of guanosine or an alkali metal salt of inosine to a phosphorylation step, and then subjecting the alkali metal salt of the nucleoside which has not been subjected to the phosphorylation step to a phosphorylation step in the presence of the alkali metal salt of the nucleoside, wherein the permeability of the reaction solution is in the range of 20 to 95%; and

(C) a process for the mixed phosphorylation of inosine and guanosine, which comprises subjecting an alkali metal salt of guanosine (or free inosine) to a phosphorylation step and then subjecting the free inosine (or an alkali metal salt of guanosine) to a phosphorylation step in the presence of the free inosine (or an alkali metal salt of guanosine), the permeability of the reaction solution being in the range of 20 to 95%.

In the following, the present invention will be described in details.

The mixed chemical phosphorylation of inosine and guanosine is carried out in a reaction system in which at least one of the alkali metal salts of inosine and guanosine is present. Preferably, for example, a combination of an alkali metal salt of guanosine and free inosine, a combination of an alkali metal salt of guanosine and an alkali metal salt of inosine, free guanosine and an alkali metal salt of inosine, an alkali metal salt of Guanosine and free inosine and an alkali metal salt of inosine, etc. It is sufficient that the reaction system contains at least one of the alkali metal salts of guanosine and inosine. A combination with an alkali metal salt of guanosine is preferably used

By reacting the nucleosides in such a combination with phosphorus oxyhalide or its hydrate, the mixed phosphorylation of inosine and guanosine can be carried out in an advantageous manner.

Furthermore, the phosphorylation of the nucleosides in the mixed phosphorylation of inosine and guanosine, which contain at least one of the alkali metal salts of inosine and guanosine, can be carried out in different orders. The order of phosphorylation is determined by the time at which the respective phosphorylations of inosine and guanosine begin.

As a typical example, the case in which the nucleosides are subjected to phosphorylation in the presence of the respective nucleosides from the beginning of the reaction, and the case in which first one nucleoside is subjected to phosphorylation and after the first reaction period another nucleoside is added and subjected to phosphorylation.

When carrying out the mixed phosphorylation in the presence of the respective nucleoside, namely the mixed phosphorylation of guanosine or an alkali metal salt of guanosine and inosine or an alkali metal salt of inosine, an alkali metal salt of guanosine and free inosine are preferably present together and the molar ratio of free inosine to the alkali metal salt of guanosine is 1/2 to 5/3. In this case, the recovery rate of inosinic acid and guanylic acid is more than 90%. On the other hand, in the phosphorylation in which the nucleosides are added simultaneously together with nucleosides other than those described above, the molar ratio of the alkali metal salt of inosine to guanosine (or its alkali metal salt) is considerably restricted to more than 9 or less than 1/9 in order to obtain inosinic acid and guanylic acid in a recovery rate of more than 90%.

Furthermore, during the phosphorylation of a nucleoside and the subsequent addition of another nucleoside and its phosphorylation, the alkali metal salt of guanosine is preferably reacted in combination with the alkali metal salt of inosine (or free inosine).

More specifically, after the phosphorylation of the alkali metal salt of guanosine or the alkali metal salt of inosine, the alkali metal salt of the nucleoside which has not been subjected to phosphorylation is added and phosphorylated at a transmittance (wavelength 600 nm) of 20 to 95% in the reaction solution.

It is also possible to add free inosine after phosphorylation of the alkali metal salt of guanosine and to phosphorylate it in the reaction solution at a transmittance (wavelength 600 nm) of 20 to 95%.

In addition, it is possible to add the alkali metal salt of guanosine after phosphorylation of the free inosine and to phosphorylate it in the reaction solution at a transmittance (wavelength 600 nm) of 20 to 95%.

In these cases, the molar ratio of free inosine (or its alkali metal salt) to the alkali metal salt of guanosine can be freely selected. Furthermore, it is possible to achieve a recovery rate of inosinic acid and guanylic acid of more than 90%. When phosphorylating the combination of free guanosine and an alkali metal salt of inosine as described above, it is not possible to freely choose the molar ratio of the alkali metal salt of inosine to free guanosine in order to obtain more than 90% recovery rate of inosinic acid and guanylic acid, but the molar ratio should be more than 9.

According to the present invention, the metal for the alkali metal salts of the nucleosides may be any one of lithium, sodium, potassium and the like.

Also significant is the fact that the alkali metal salts of the nucleosides used in the reaction are not limited in terms of their grain size and crystal form. The alkali metal salts of the nucleosides can be fed to the reaction in a simple manner as they are by dispersing them in the solvent for the reaction.

The water content of the alkali metal salts of nucleosides should be 2% or less. It is desirable to adjust it to less than 1%. The reason for this is that as the water content increases, the amount of phosphorus oxychloride must be increased, so that the amount of phosphates or sodium chloride produced during neutralization after phosphorylation increases and these compounds greatly affect the purification of sodium inosinate and sodium guanylate.

Any polar organic solvent can be used as the solvent for the reaction, and there can be used phosphates substituted three times with lower alkyl groups, for example triethyl phosphate, trimethyl phosphate, etc., tri(alkoxyalkyl)phosphates, such as tri(ethoxyethyl)phosphate, tri(methoxyethyl)phosphate, etc., ethylene glycol dialkyl ethers, such as ethylene glycol dimethyl ether, ethylene glycol diethyl ether, etc., cyclic ethers such as tetrahydrofuran, 1,4-dioxane, etc., halogenated hydrocarbons such as dichloromethane, chloroform, 1,2-dichloroethane, etc., nitro compounds such as nitromethane, nitroethane, nitrobenzene, etc. These solvents are used in 5 to 50 times, preferably 10 to 20 times, the molar amount based on the number of moles of nucleosides.

As the phosphorylating agent, phosphorus oxyhalides or their hydrates can be used. Examples include phosphorus oxychloride, phosphorus oxybromide, etc. The amount of the phosphorylating agent is 1 to 5 moles based on 1 mole of the nucleoside.

The reaction temperature is preferably in the range of -30ºC to 50ºC because the addition of the phosphorylating agent generates heat and the temperature rises rapidly to a certain extent at the start of the reaction. By avoiding the temperature rise, the formation of diphosphates and similar by-products can be prevented.

In the mixed phosphorylation described above, the reaction time is about 30 minutes to 10 hours. Compared with using the nucleoside alone, the reaction time can be shortened to about 1/2 to 1/4. In addition, the amounts of the reaction solvent and the phosphorylating agent can be reduced to about 1/2 compared with using the nucleoside alone.

After completion of the phosphorylation, the reaction solution is introduced into more than 20 times the molar amount of ice-cooled water to hydrolyze the phosphorylating agent. Furthermore, the pH is adjusted to 6 to 10 by adding alkali (sodium hydroxide, etc.), whereby a solution of the alkali metal salts of inosine and guanylic acid The resulting mixture of these nucleotides can be purified by various methods. For example, the reaction solution can be extracted with an organic solvent or, if a solvent having a lower boiling point is used, the mixture can be adjusted to a pH at which the nucleotides are stable, for example, in the case of inosinic acid, to a pH of about 10 and then distilled under reduced pressure. By removing the organic solvent from the nucleotide solution obtained by these methods, using activated carbon, etc., crystals can be easily obtained.

According to the present invention, the amounts of by-products such as inosine diphosphates, etc. can be reduced and the mixture of inosinic acid and guanylic acid can be obtained in high yield. In addition, the reaction time can be shortened and the amounts of the starting materials can be reduced.

In the accompanying drawings, Fig. 1 shows the permeability during phosphorylation of sodium guanosine as a function of time.

Fig. 2 shows the yield of the conversion of inosinic acid in the phosphorylation of sodium guanosine with addition of sodium inosine at each permeability.

Hereinafter, the present invention will be described in detail with reference to Examples and Comparative Examples.

example 1

(Relationship between the addition time of sodium inosine and the reaction yield)

10 g (33 mmol) of sodium guanosine was dispersed in 140 g (770 mmol) of triethyl phosphate. The dispersion was kept at -5°C, and 18 g (118 mmol) of phosphorus oxychloride was added to the dispersion to start the reaction. The intensity of transmitted light (wavelength 600 nm) of the reaction solution was determined by a densometer model K395 (manufactured by Anritsu Electric Co., Ltd.) using optical fibers. At each transmittance value as shown in Fig. 1, 9.5 g (33 mmol) of sodium inosine was added, the reaction was carried out for six hours, and then the reaction yield of sodium inosine was determined. The results are shown in Fig. 2. The reaction yield of sodium guanosine was 93 to 96% in all cases. At this point, sodium inosine showed a reaction yield of 90%, provided that the permeability was between 20 and 95% at the time of addition.

Example 2

10 g (33 mmol) of sodium guanosine was dispersed in 140 g (770 mmol) of triethyl phosphate. The dispersion was cooled to -10°C, 18 g (118 mmol) of phosphorus oxychloride was added to the dispersion, and the mixture was reacted for 3 hours. 9.5 g (33 mmol) of sodium inosine was added to the reaction mixture, and then reacted for 2 hours. After that, the reaction mixture was poured into cooled water and the phosphorus oxychloride was hydrolyzed. The reaction yields of guanylic acid and inosinic acid were 96.6% and 97.2%, respectively.

Example 3

10 g (37 mmol) of inosine was dispersed in 490 g (2.7 mmol) of triethyl phosphate. The dispersion was cooled to -10°C, 61 g (400 mmol) of phosphorus oxychloride was added to the dispersion, and the mixture was reacted. After 1 hour, 57 g (187 mmol) of sodium guanosine was added to the reaction mixture and then reacted for 5 hours. The reaction mixture was then hydrolyzed with water cooled to 5°C. The reaction yields of inosinic acid and guanylic acid were 97.5% and 95.8%, respectively.

Example 4

10 g (37 mmol) of inosine and 100 g (36 mmol) of sodium guanosine were dispersed in 156 g (864 mmol) of triethyl phosphate. The dispersion was cooled to -10ºC, 20 g (130 mmol) of phosphorus oxychloride was added to the dispersion and the mixture was reacted for 5 hours. Then the reaction solution was poured into water cooled to 5ºC and the phosphorus oxychloride was hydrolyzed. At this time, the reaction yields of inosinic acid and guanylic acid were 95.2% and 96.8%, respectively.

Example 5

50 g (164 mmol) of sodium guanosine and 24 g (83 mmol) of inosine were dispersed in 446 g (2.5 mol) of triethyl phosphate. The dispersion was cooled to -10ºC, 75 g (494 mmol) of phosphorus oxychloride was added to the dispersion, and the mixture was reacted for five hours. Then, the reaction solution was poured into water cooled to 5ºC and the phosphorus oxychloride was hydrolyzed. At this time, the reaction yields of guanylic acid and inosinic acid were 96.5% and 97.0%, respectively.

Comparison example 1

50 g (164 mmol) of guanosine was mixed with 48 g (164 mmol) of inosine, and the mixture was dispersed in 550 g of triethyl phosphate. The solution was cooled to -10°C, 115 g (754 mmol) of phosphorus oxychloride was added to the dispersion, and the mixture was reacted for six hours. The reaction solution was hydrolyzed with water at 5°C, and the yields of the guanylic acid and inosinic acid thus obtained were 85% and 94%, respectively.

Comparison example 2

50 g (164 mmol) of guanosine was mixed with 48 g (164 mmol) of inosine, and the mixture was dispersed in 550 g of acetonitrile. To the solution was added 137 g of pyridine, and the mixture was cooled to -10°C. After adding 120 g (787 mmol) of phosphorus oxychloride, the mixture was reacted for six hours. The phosphorus oxychloride in the reaction solution was hydrolyzed with water at 5°C to obtain guanylic acid and inosinic acid. Here, the yields of the thus obtained guanylic acid and inosinic acid were 85.1 0.6 and 93.2%, respectively.

Comparison example 3

50 g (164 mmol) of guanosine was mixed with 9.6 g (32 mmol) of inosine, and the mixture was dispersed in 780 g (4.3 mmol) of triethyl phosphate. The dispersion was cooled to -10°C, and 69 g (450 mmol) of phosphorus oxychloride was added to the dispersion. The mixture was reacted for 10 hours. The reaction solution was hydrolyzed with water at 5°C, and the yields of the guanylic acid and inosinic acid thus obtained were 85% and 88%, respectively.

Claims (5)

1. Process for the preparation of a mixture of inosinic acid and guanosinic acid by direct phosphorylation of the 5'-hydroxy group of inosine and guanosine, characterized in that a mixture of inosine and guanosine, which contains at least one alkali metal salt selected from alkali metal salts of inosine and/or guanosine, is reacted with a phosphorus oxyhalide or its hydrate in a polar organic solvent. 2. A process according to claim 1, wherein the inosine and guanosine are present in the reaction mixture in the form of an alkali metal salt of guanosine and free inosine, the molar ratio of free inosine/alkali metal salt of guanosine being in the range of 1/2 to 5/3. 3. Process for producing a mixture of inosinic acid and guanosic acid by direct phosphorylation of the 5'-hydroxy group of inosine and guanosine, characterized in that a first nucleoside alkali metal salt selected from an alkali metal salt of guanosine or an alkali metal salt of inosine is subjected to a phosphorylation step, and then a second, previously unphosphorylated nucleoside alkali metal salt is subjected to a phosphorylation step in the presence of the first nucleoside alkali metal salt, the permeability of the reaction solution being in a range from 20 to 95%. 4. A process for the preparation of a mixture of inosinic acid and guanosic acid by direct phosphorylation of the 5'-hydroxy group of inosine and guanosine, which comprises subjecting an alkali metal salt of guanosine to a phosphorylation step and then subjecting free Inosine undergoes a phosphorylation step in the presence of the alkali metal salt of guanosine, the permeability of the reaction solution being in the range of 20 to 95% 5. A process for preparing a mixture of inosinic acid and guanosinic acid by direct phosphorylation of the 5'-hydroxy group of inosine and guanosine, which comprises subjecting free inosine to a phosphorylation step and subsequently subjecting an alkali metal salt of guanosine to a phosphorylation step in the presence of the free inosine, the permeability of the reaction solution being in the range of 20 to 95%.