DE2038262C2 - - Google Patents

Description

The invention relates to a new method for producing nucleotide anhydrides. The term “nucleotide anhydrides” here denotes anhydrides of a nucleotide 5′-monophosphate with an acid which is weakly acidic than diphenylphosphoric acid. The anhydrides have the general formula in which N _{s is} the 5′-residue of a nucleoside and X denotes the residue of an acid which is weaker than diphenylphosphoric acid. Important compounds are listed in Table I.

Table I

The nucleotide anhydrides play an important role in metabolism, furthermore they are called biochemical reagents and Drugs used. So z. B. cytidine-5'-diphosphocholine (CDP choline) used to treat head injuries. Trisodium inosine-5'-triphosphate (ITP-Na₃) and  Trisodium thymidine-5'-triphosphate (TTP-Na₃) are contained in the muscle and valuable reagents to research metabolism. Adenosine triphosphate (ATP) is used to treat peripheral vascular disease, edema, beriberi and muscle fatigue and for the treatment of myositis (myitis, sarcitis), Myasthenia, rheumatism, arthritis, neuralgia and similar diseases used.

Flavin adenine dinucleotide (FAD) is used to treat liver damage, Nutritional diseases in pregnant and lactating women Women, as well as poisoning, such as alcohol damage and nicotinism applied. The disodium salt of uridine-5'-diphosphoglucose (UDPG-Na₂) has valuable physiological activity, and CDP ethanolamine can be used as the nucleotide coenzyme. Uridyl sulfate, adenosine 5'-2,4-dinitrophenyl phosphate, Adenyl-5'-yl-carbobenzoxyglycine and other nucleotide anhydrides are also valuable drugs and reagents in the Biochemistry.

There are already various methods of making them Nucleotide anhydrides known. The anion exchange is of particular value Method that is carried out in two stages. In the The first stage is a nucleoside 5'-monophosphate with pyrophosphoric acid tetraphenyl ester or phosphoric acid di-phenyl ester chloride in the presence of a tertiary base in dioxane or a mixture Dioxane and dimethylformamide implemented. The reaction product is a P¹-nucleoside-5'-P²-diphenyl pyrophosphate, which is purified. In the second stage is the purified P¹-nucleoside-5'-P²-diphenylpyrophosphate reacted with an acid that is weakly acidic than diphenylphosphoric acid. A wide variety of nucleoside 5'-monophosphates and acids together to form the corresponding Nucleotide anhydrides are implemented.  

Purification of the intermediate obtained in the first stage is carried out as follows. First, the reaction mixture freed from solvent under reduced pressure. The residue is mixed with diethyl ether and 30 to 60 minutes left at 0 ° C. The ether is formed by the Precipitation decanted. This intermediate product is moisture and sensitive to heat. Therefore, evaporation must be done under anhydrous Conditions, at low temperature and under greatly reduced conditions Pressure. They are also very large Amounts of diethyl ether required to precipitate the product. This is dangerous when working on a large scale. thats why the process is disadvantageous in industrial terms. Furthermore there are large losses in the isolation of the intermediate product on. The water or diethyl ether obtained from the precipitation Product interferes in the second stage of implementation and reduces the yield. For this reason it is necessary in the second stage to take up the precipitation in dioxane and the distill solution obtained under reduced pressure to the accompanying solvents, d. H. Water or diethyl ether, separate as an azeotropic mixture.

This two-stage process, which was developed by AM Michelson (cf. "Biochem. Biophys. Acta" 91 (1964), pp. 1 to 13) already represents an improvement in the Khorana process, in which the expensive and only extremely Dicyclohexylcarboxamide to be handled with care is used as the condensing agent, but it is not yet suitable in practice because of the shortcomings indicated above.

The object of the invention was therefore to describe the above Avoid disadvantages and create a new process of nucleotide anhydrides to create that easy to do and in which intermediates do not have to be isolated. This object is achieved by the invention.

The invention therefore relates to a process for the preparation of nucleotide anhydrides of the general formula in which N _{s is} the 5′-residue of a nucleoside and X is the residue of an acid which is weaker than diphenylphosphoric acid, starting from a reaction mixture which is obtained by reacting an alkylammonium salt of a nucleoside-5′-monophosphate in 5 to 50 mol of dioxane, Dimethylformamide and / or dimethylacetamide and in the presence of an alkylamine with 1 to 2 moles of phosphoric acid-di-phenyl ester chloride or pyrophosphoric acid tetraphenyl ester per mole of nucleoside-5'-monophosphate, which is characterized in that the reaction mixture obtained with a solution of an alkylammonium salt with the phosphate residue of the corresponding nucleoside 5'-monophosphate acid to be condensed in pyridine or one of its alkyl derivatives and optionally formamide, which, if necessary, has previously been used for dissolution.

Nucleoside 5'-monophosphate is preferred in the form of salts of Trialkylamines or quaternary ammonium hydroxides, e.g. B. from Tri-n- butylamine, tri-n-octylamine, methyl-tri-n-butylammonium hydroxide or methyl-tri-n-octyl-ammonium hydroxide.  

Depending on the molar ratio of the nucleoside 5'-monophosphate and used alkyl amines, the mono-alkyl ammonium salt and the di-alkyl ammonium salt. Both salts can be used however, the mono-alkylammonium salt due to its reactivity prefers.

The solvent is used in a minimum amount of 5 mol per mol of the nucleoside-5'- phosphate used. The aforementioned solvents cannot only used alone but also in a mixture with each other. In addition, the reactants can increase the solubility still tetrahydrofuran, n-hexane, benzene, toluene, ethyl acetate, Acetone or chloroform can be used. These solvents are preferably in at most two to three times Volume amount, based on the reaction solvent used. In particular, these solvents are used in at most the same Volume used.

The molar ratio of phosphoric acid di-phenyl ester chloride or pyrophosphoric acid tetraphenyl ester -nucleoside-5'-phosphate is preferably 1 to 1.5: 1.

The alkylamine is added to the reaction mixture as a stabilizer. Examples of suitable alkylamines are tri-n-ethylamine, Tri-n-butylamine, tri-n-octylamine, N-methylpiperidine and diethylaniline. The molar ratio of stabilizer to nucleoside 5'-phosphate should preferably be at least 0.5: 1, in particular 1.2 to 2: 1.

Examples of suitable acids that are weaker than diphenylphosphoric acid are anhydrous sulfuric acid, phosphoric acids, such as Orthophosphoric acid, pyrophosphoric acid and triphosphoric acid, sugar phosphates, such as glucose-1-phosphate, mannose-1-phosphate, galactose-  1-phosphate and glucuronic acid 1-phosphate, carboxylic acids, such as Acetic acid and propionic acid, amino acids such as carbobenzoxyglycine, Peptides, phenols such as 2,4-dinitrophenol, nucleotides and the acidic components of coenzymes, such as panthetin 4 ′, 4′-bisphosphate, Flavin mononucleotide, phenyl phosphate, choline mononucleotide and nicotinamide phosphate.

The alkylammonium salt of this reactant can e.g. B. derived from tri-n-butylamine or tri-n-octylamine. The molar ratio the acid to the nucleoside 5'phosphate should preferably be at least 1: 1, in particular 1 to 2: 1.

Alkylammonium salts of acids that are found in pyridine, or its alkyl derivatives only difficult to solve, like that of flavin nucleotide, before adding them dissolved in another solvent such as formamide.

In the process of the invention it is essential that the pyridine or to add its alkyl derivative together with the alkylammonium salt at the same time. The reaction of the acid with the P¹-nucleoside-5'-P²-diphenylpyrophosphate is considered an anion exchange reaction, with the rest of the diphenyl phosphate (a strong acid) through the weak acid is replaced. In this anion exchange reaction pyridine or its alkyl derivatives play a crucial role Role. Examples of alkylpyridines that can be used are Picolin, Lutidin and Collidin. The molar ratio of pyridine or pyridine derivative to the nucleoside 5'-monophosphate is preferably 5 to 30: 1.  

The implementation is generally completed within a short time, however, the response time depends on the type and amount of used solvent and the temperature.

Forms under the reaction conditions described above are composed of the nucleoside 5′-phosphate and the phosphoric acid di phenyl ester chloride or pyrophosphoric acid tetraphenyl ester practically instantly the P¹-nucleoside-5'-P²-diphenylpyrophosphate. Therefore, the alkyl ammonium salt of the acid, dissolved in the pyridine compound, the reaction mixture practically immediately after mixing the Nucleoside 5'-phosphate with the phosphoric acid di-phenyl ester chloride or pyrophosphoric acid tetraphenyl ester are added. The anion exchange reaction proceeds within a few minutes up to a few hours in high yield and it just forms small amounts of by-products.

The nucleotide anhydrides produced in the process of the invention can be cleaned by conventional methods, e.g. B. by Chromatography on resin exchangers or by precipitation with organic Solvents. The cleaned products can be chromatographed, physically, chemically or by enzymatic Reactions are detected.

Example 1 Production of trisodium cytidine 5'-diphosphate (CDP-Na₃)

1 mol of mono- (methyl-tri-n-butylammonium) -5′-cytidylate in 1.5 mol of dimethylacetamide is mixed with 250 ml of phosphoric acid di phenyl ester chloride added. The mixture is 5 min. touched, until everything is in solution. Then 500 ml of Tri-n- butylamine and 2 l of dioxane added. After another 5 min. 2 l of pyridine are added, the 2 mol of mono- (tri-n- contain butylammonium) phosphate. The mixture is stirred and then left for 1 h. After completion of the implementation the reaction mixture is evaporated under reduced pressure, the residue was mixed with 7 l of water and 1 l of benzene extracted. The aqueous solution is diluted to 50 l and on a Duolite AlOlD anion resin exchange column (chloride form, 10 l). The cytidine 5'-diphosphate is made the column eluted with 0.1 mol / l hydrochloric acid. The eluate becomes that crystalline trisodium salt obtained. Yield 387 g (73.5% of the Theory). The product contains 11% water. The molar ratio of Ingredients are given in Table II.

Table II

Example 2 Production of trisodium inosine-5′-triphosphate (ITP-Na₃)

200 mmol of mono- (tri-n-butylammonium) -5'-inosinate are with 120 ml of dimethylacetamide and a mixture of 250 mmol of pyrophosphoric acid tetraphenyl ester, 100 ml tri-n-butylamine and 600 ml Dioxane added. After stirring, the mixture with 200 ml of pyridine are added, the 400 mmol di- (tri-n-butylammonium) - contains pyrophosphate.

After standing for 40 minutes, the inosine-5'-triphosphate formed from the reaction mixture using an anion resin exchanger Duolite® AlOlD (chloride form, 10 l) separated. The trisodium salt is obtained as an amorphous powder. yield 107.9 g, 84.3% of theory. The product contains 10.3% water. The molar ratio of the components is shown in Table III.

Table III

Example 3 Preparation of trisodium thymidine-5′-triphosphate (TTP-Na₃)

10 mmol mono- (tri-n-butylammonium) -5'-thymidylate are successively with 5 ml of dimethylformamide, a mixture of 2.3 ml Phosphoric acid-di-phenyl ester chloride, 5 ml of tri-n-butylamine and 20 ml of dioxane added. After stirring, 100 ml of pyridine  added the 20 mmol di- (tri-n-butylammonium) pyrophosphate contains. After 30 min. the resulting thymidine-5'-triphosphate isolated in the usual way from the reaction mixture. Yield 79.7% of theory.

Example 4 Production of disodium-uridine-5'-diphospho-glucose (UDPG-Na₂)

10 mmol of mono- (tri-n-butylammonium) -5'-uridylate are dissolved in 5 ml of dimethylformamide and mixed with 5 ml of dioxane, 6 ml of tri-n-butylamine and 2.3 ml of phosphoric acid di-phenyl ester chloride. After 10 min. the mixture is mixed with 15 ml of pyridine containing 12 mmol of mono- (tri-n-butylammonium) glucose-1-phosphate. After standing for 2 hours, the solvent is distilled off under reduced pressure, the residue is dissolved in water and added to a Dowex® 1 × 8 anion resin exchanger (chloride form, 100 ml). The uridine-5'-diphosphoglucose is eluted in the usual way. The disodium salt is obtained as an amorphous powder. Yield 4.12 g, 67.5% of theory. The molar ratio of the components is shown in Table IV.

By the action of UDPG dehydrogenase 98.6% of the compound dehydrated.

Example 5 Production of adenosine-5′-diphospho-glucose (ADPG)

1 mmol of mono- (methyl-tri-n-butylammonium) -5'-adenylate are with 1 ml dimethylformamide, 0.2 ml diphenyl ester of phosphoric acid, 1 ml of dioxane and 0.5 ml of tri-n-butylamine are added. To 30 min. the mixture is mixed with 3 ml of pyridine, the Contains 1.5 mmol mono- (tri-n-butylammonium) glucose-1-phosphate. After 1 hour of reaction, adenosine-5'-diphospho-glucose is in 81.5 percent yield.

Example 6 Production of flavin adenine dinucleotide (FAD)

1 mmol of mono- (tri-n-octylammonium) -5′-adenylate in 1 ml of dimethylacetamide are mixed with 0.25 ml of phosphoric acid di-phenyl ester chloride, 2 ml of dioxane and 0.5 ml of tri-n-butylamine are added. Then it will Mix with 2 ml of formamide, the 1.8 mmol mono- (methyl- tri-n-butylammonium) salt of flavin mononucleotide and 4 ml Contains pyridine. After 90 minutes of reaction, the yield is on flavin adenine dinucleotide 78.3% of theory.

Example 7 Manufacture of CDP ethanolamine

2 mmol mono- (methyl-tri-n-octylammonium) -5′-cytidylate in 2 ml Dimethylformamide with 0.5 ml of phosphoric acid di-phenyl ester chloride, 1 ml of tri-n-butylamine and 15 ml of dioxane were added. After this The mixture is mixed with 5 ml of pyridine, which is 3 mmol  Mono- (methyl-tri-n-butylammonium) salt of orthophosphoethanol contains amine. After 30 minutes of reaction, the yield is of CDP-ethanolamine 52.7% of theory.

Example 8 Manufacture of uridylyl sulfate

10 mmol mono- (tri-n-octylammonium) -5'-uridylate in 20 ml Dioxane dissolved. The solution is mixed with 2.5 ml of phosphoric acid di phenyl ester chloride and 2.1 ml of triethylamine. To Standing at room temperature for 5 minutes, the mixture is mixed with 20 mmol of tri-n-butylammonium sulfate and 5 ml of pyridine were added. After 1 hour of reaction, the yield of uridylyl sulfate is 40% of theory.

Example 9 Preparation of adenosine 5'-2,4-dinitrophenyl phosphate

1 mmol of mono- (methyl-tri-n-butylammonium) -5'-adenylate with 2 ml of dimethylacetamide and then with 0.25 ml of phosphoric acid di-phenyl ester chloride and 0.6 ml of tri-n-butylamine. The mixture is left for 1 h at room temperature and then mixed with 2 ml of pyridine, the 2 mmol of 2,4-dinitrophenol contains. After 2 hours of reaction at 0 ° C, the yield is on adenosine 5'-2,4-dinitrophenylphosphate 91% of theory.

Example 10 Production of adenyl-5'-yl-carbobenzyloxyglycine

1 mmol of mono- (tri-n-octylammonium) -5'-adenylate with 2 ml Dimethylacetamide added. After stirring, the mixture with 0.25 ml of phosphoric acid di-phenyl ester chloride, 4 ml of dioxane,  and 0.5 ml of tri-n-butylamine were added. The mixture becomes thorough stirred until everything went into solution. Then 2 ml Added pyridine, which contains 250 mg carbobenzyloxyglycine. After 3 hours of reaction, the yield of adenyl-5'- yl-carbobenzyloxyglycine 32% of theory.  

Example 11 Preparation of P¹, P³-di- (adenosine-5 ′) - triphosphate

53.3 g of mono- (tri-n-butylammonium) -5'-adenylate become 50 ml Given N, N-dimethylacetamide. This suspension comes with 25 ml Phosphoric acid diphenyl ester chloride was added to start the reaction bring, then 60 ml of tri-n-butylamine are added and 30 min. reacted calmly. The reaction mixture is with a Solution of the tri (tri-n-butylamine) salt of adenosine 5'-diphosphate in N, N-dimethylacetamide and 50 ml of pyridine and 60 min. implemented long. (The solution of tri- (tri-n-butylamine) - Salt of adenosine 5'-diphosphate in N, N-dimethylacetamide was prepared so that 50 ml of water to 51.5 g of adenosine-5'-di- phosphoric acid there, 86 ml of tri-n-butylamine salt and the Solution moved 1 h. Then 50 ml of N, N-dimethylacetamide added, the solution is concentrated under reduced pressure, mixed with a further 50 ml of dioxane and concentrated; finally the solution is dewatered azeotropically).

2 l of the solution obtained in water with 200 ml of xylene extracted. After removing the xylene phase, the aqueous Phase 25 g of sodium hydroxide added, the released tri-n-butylamine is separated. The remaining solution will be under reduced pressure to remove the organic solvent concentrated, the residual solution is made up to 2 l with water diluted.

This solution is on a column with 2 l of an anion exchange resin is filled, then on a column that is filled with 4 l of granulated activated carbon, chromatographed. A fraction is obtained, the P¹, P³-di- (adenosine-5 ′) - tri- contains phosphate, which is converted into the sodium salt and out Water / ethanol is recrystallized. Finally 29 g is obtained of the trisodium salt of P¹, P³-di- (adenosine-5 ′) - triphosphate.

Claims (2)

1. Process for the preparation of nucleotide anhydrides of the general formula in which N _{s is} the 5′-residue of a nucleoside and X is the residue of an acid which is weaker than diphenylphosphoric acid, starting from a reaction mixture which is obtained by reacting an alkylammonium salt of a nucleoside-5′-monophosphate in 5 to 50 mol of dioxane, Dimethylformamide and / or dimethylacetamide and in the presence of an alkylamine with 1 to 2 mol of phosphoric acid-di-phenyl ester chloride or pyrophosphoric acid tetraphenyl ester per mol of nucleoside-5'-monophosphate has been obtained, characterized in that the reaction mixture obtained with a solution of an alkylammonium salt with Phosphate residue of the corresponding nucleoside 5'-monophosphate acid to be condensed in pyridine or one of its alkyl derivatives and optionally formamide, which, if necessary, has been used previously for dissolving. 2. The method according to claim 1, characterized in that one 1 to 10 mol of the alkylammonium salt per mol of nucleoside 5′-monophosphate the acid and 2 to 50 moles of the pyridine compound  used and the process at temperatures from 0 to 30 ° C. carries out.