JPH027597B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a method for synthesizing nucleoside-5'-diphosphoric acid amide in which a highly active amino group is introduced into the phosphate group of a nucleotide. (Prior art) Nucleotides perform a wide range of physiological functions in living organisms, such as energy supply, transmission, and transmission of genetic information, and non-natural derivatives exhibit interesting activities such as anticancer properties and interferon-inducing properties. Since it is a compound, many chemical and biological methods have been attempted for its synthesis. The present inventors previously proposed the general formula (However, R ₁ and R ₂ are an alkyl group having 8 to 22 carbon atoms or an alkenyl group having 1 to 3 double bonds,
X ₁ ^- and X ₂ ^- represent anions. ) A highly hydrophobic diammonium salt, i.e., 1,4-diazabicyclo-(2,2,2)-
N,N'-dilong chain alkyl or alkenyl diammonium salt of octane or N-long chain alkyl or alkenyl ammonium salt of 1,4-diazabicyclo-(2,2,2)-octane (hereinafter referred to as
This compound is described as a highly hydrophobic diammonium salt) by extracting a compound with a pyrophosphate group.
We found that it is a highly specific reagent for interphase transport, and we developed a method to utilize this property to synthesize phosphorylated nucleoside derivatives, namely:
For example, we developed a method for synthesizing adenosine phosphorylate by reacting adenosine (a type of nucleic acid) with a phosphorylating agent in an organic solvent in the presence of the highly hydrophobic ammonium salt (Japanese Patent Application Laid-Open No. 124797/1983). (see official bulletin). (Problems to be Solved by the Invention) The present inventors have conducted further research and found that some nucleotides, for example, adenosine phosphorylated
5'-monophosphate (hereinafter referred to as AMP), adenosine-5'-diphosphate (hereinafter referred to as ADP), and adenosine-5'-triphosphate (hereinafter referred to as ATP) with different numbers of bound phosphates. There are various chemical compounds, and their reactivity and properties (physical properties) are very different. In particular, the differences in interactions with enzymes in living organisms are remarkable, and although each is important, ATP is especially required in biotechnology. Therefore, using the highly hydrophobic diammonium salt, AMP to ADP,
We attempted to synthesize ATP and its derivatives. In this synthesis, a nonpolar solvent such as chloroform or methylene chloride was used as a solvent, and a highly hydrophobic diammonium salt was dissolved therein.
This is because the phosphorylating agent is decomposed in polar solvents. For example, 20ml of AMP aqueous solution adjusted to pH8 (1.1×
10 ^-4 M) and 200 ml of a chloroform solution of a highly hydrophobic diammonium salt were gently stirred, and the organic phase was separated. Add a large excess to 10 ml of this organic phase solution.
POCl ₃ and bases such as K ₂ CO ₃ , CsCO ₃ and urea were added with cooling. After a suitable reaction time, the PH
Add 10 ml of 5-6 NaClO ₄ aqueous solution ( ^1.0
When AMP, ADP, and ATP were analyzed, the following results were obtained. Note that pyrophosphoric acids such as ADP and ATP are very strongly bound in nonpolar solvents, and it is necessary to use an electrolyte such as NaClO ₄ for their dissociation and elution.

[Table] Under conditions where this reaction is hidden, the main component of the reaction product is ADP, but a large amount of unreacted AMP remains. However, under vigorous reaction conditions, phosphorylation progresses further, and a considerable amount of ATP is generated simultaneously with ADP. The results in the table above also show that it is necessary to select and use an appropriate base. In this reaction, AMP is extracted from an aqueous solution into a chloroform solution by a highly hydrophobic diammonium salt, activated by the desolvation action that occurs, and reacts with POCl ₃ to form anhydrous chloride. The reaction formula is as follows. This chloride is unstable and easily converts into ADP (and even AMP) in the presence of a small amount of water. In this way, ATP and ADP compete with the reaction that decomposes them into ADP and AMP to give ATP and ADP.
There were problems in that ADP could not be synthesized in high yield and the purity of the product was poor. The present invention aims to solve this problem. (Means for Solving the Problems) The present inventors found that when ammonia or ammonium carbonate, which acts as an aminating reagent and a buffer base, acts on the anhydrous chloride produced in the reaction formula (2), it becomes extremely mild. In a very short time under the conditions, The ammonium salt shown can be produced at a good yield of 65% of the amount of AMP used and 80% of the amount of AMP consumed, and the yield is 95%.
% or more, and based on this finding, the present invention was completed. That is, the present invention provides 1,4-diazabicyclo-
N,N'-di-long-chain alkyl or alkenyldiammonium salt of (2,2,2)-octane or N-long-chain alkyl or alkenyl ammonium salt of 1,4-diazabicyclo-(2,2,2)-octane Nucleoside −5′− trapped in
Nucleoside-5'-diphosphoric acid amide, which is characterized in that monophosphoric acid is treated with a phosphorylating agent in a nonpolar solvent, and then ammonium carbonate or ammonia is treated to introduce an amino group into the phosphoric acid group. This is a synthesis method. As the highly hydrophobic diammonium salt used in the method of the present invention, R ₁ and R ₂ in general formula (1) include, for example, a palmityl group, a stearyl group, an oleyl group, a linolenyl group, and the like. X ₁ ^- , X ₂ ^-
For example, halogen ions such as Cl ^- and I ^- ,
Examples include mineral acid ions such as sulfate ions, nitrate ions, and phosphate ions, organic acid ions such as acetate ions, and hydroxide ions. Among them, Cl ^- , I ^- ,
Monovalent strong acid ions such as NO ₃ ^- and HSO ₄ ^- , especially
Cl ^- is preferred because it is very stable. 1,4-
diazabicyclo-(2,2,2)-octane N,
N'-di long chain alkyl or alkenyldiammonium salt or N-long chain alkyl or alkenyl ammonium salt is the iodide or bromide of 1,4-diazabicyclo-(2,2,2)-octane and long chain alkyl or alkenyl. can be synthesized by heating in an organic solvent. In order to obtain a product in which R ₁ and R ₂ are different, iodides or bromides corresponding to R ₁ and R ₂ may be mixed and added and reacted. Examples of organic solvents include dimethylformamide and methanol.
The reaction is carried out by heating to 50-100°C for several days. Separation and purification can be carried out by appropriately combining methods such as dissolving in a water-insoluble organic solvent and extracting impurities, dissolving in a solvent and cooling or concentrating and crystallizing. This compound captures nucleotides. Examples of nucleotides that can be captured include monophosphates, diphosphates, triphosphates of nucleosides such as inosine, guanosine, adenosine, xanthosine, cytidine, and uridine.
Includes tetraphosphate, pentaphosphate, etc. In order to make the nucleotide trapped in the highly hydrophobic diammonium salt, the pH of the nucleotide solution or suspension should be 3 or higher, preferably 7.
As described above, it is obtained by contacting the ammonium salt with the above-mentioned ammonium salt in the presence of a water-insoluble organic solvent (preferably a non-polar solvent such as chloroform, methylene chloride, etc.). In the present invention, the nucleoside 5'-monophosphate thus captured is treated with a phosphorylating agent such as phosphorus oxychloride or tetrachloropyrophosphoric acid in an organic solvent, and then ammonium carbonate or ammonia is treated. This yields nucleoside-5'-diphosphoric acid amide in which an amino group is introduced into the phosphoric acid group. As the organic solvent used at this time, it is preferable to use a nonpolar solvent. Nucleoside-5'-diphosphoric acid amide is produced in a short time (for example, within 2 minutes), with a high yield of 80%, and with high purity. Their separation etc. will be shown in Examples. Example () Synthesis of diammonium salt of diazabicyclooctane (hereinafter referred to as DABCO) DABCO (1.0 g, 8.9 mmol) was mixed with stearyl iodine (20 g, 53 mmol) in 50 ml of dimethylformamide (hereinafter referred to as DMF) at 70°C. 2
Heated for days. This DMF solution was dropped into 200 ml of benzene and filtered to remove excess stearyl iodine. Solid in 100ml water at 60℃
After washing for 1 hour, the monoammonium salt was removed by filtration. This solid was recrystallized in DMF,
Colorless columns were obtained. Its properties are: mp.234-237℃, IR (KBr), 2900, 2850,
1460, 1350, 1110, 1050, 870, 840, 710cm
^-1 , NMR (Me ₂ SO−d ₆ ), δ0.80 (br.6H, CH ₃ ),
δ1.18 (br.60HCCH ₂ ), δ1.64 (br.4H.
NCCH ₂ ), δ3.48 (br.4H.NCH ₂ ), δ3.82
(br.12H.NCH ₂ CH ₂ N) This iodide salt exhibits UV absorption at λmax = 218 nm, which overlaps with the UV absorption of nucleotide bases, making it difficult to determine the total amount of nucleotides in extraction experiments and The sex is also low. Therefore, the counterion was converted to chlorine via the hydroxyl group. That is, dissolve iodized salt (5.0 g, 3.0 mmol) in 1 methanol containing 10 ml of water,
Silver oxide was added to this and kept at 40°C for 40 minutes. Complete precipitation of iodide ions was confirmed by complete disappearance of UV absorption of iodide ions. After removing the silver salt from the solution by filtration, 2.3 ml of concentrated hydrochloric acid was added to make it acidic. The methanol was distilled off using a vacuum pump to obtain 3.81 g (96%) of a colorless solid. This was recrystallized from DMF and then from methanol. 2.1g
The purified salt was obtained at mp.92-93°C. () Synthesis and isolation of adenosine-5'-diphosphoric acid amide A chloroform solution of the diammonium salt of DABCO (2.4×10 ^-4 mol/, 2) and AMP-
Aqueous solution of 2Na (3.0×10 ^-4 mol/, 2, PH
8.0) and shaken at 10°C for several minutes. AMP was extracted from the aqueous layer into the chloroform layer. During this time, the pH of the aqueous solution is always 8.0 using 0.1N NaOH.
I adjusted it so that remains in the water layer
The concentration of AMP is λmax according to the UV spectrum
1.0× by measuring absorption at (260 nm)
It was confirmed that the amount was 10 ^-4 mol/. The AMP concentration in the chloroform layer was measured as follows. A 5 ml sample was taken from the lower chloroform layer, taking care not to contaminate it with the boundary layer between chloroform and water. This chloroform solution was mixed with an aqueous solution of perchloric acid (1.0×10 ^-2 mol/
, 5 ml, pH 5.0) and shaken. The concentration of AMP extracted from the chloroform layer to the aqueous layer is determined by UV
The spectrum showed 2.0 x 10 ^-4 mol/(67% of the AMP used). Chloroform solution of AMP obtained by the above method (2.0×10 ^-4 mol/, 1.995, 3.99×10 ^-4 mol)
The mixture was transferred to a three-neck flask, ammonium carbonate (5.75 g, 5.99×10 ⁻² mol) was added, and the mixture was vigorously stirred at 35° C. for 30 minutes. Then phosphorus oxychloride (3.06 g, 1.8 ml, 2.00 x 10 ^-2 mol) was added. 2
Minutes later, sodium perchlorate aqueous solution (1.0×
The reaction was stopped by adding 10 ^-2 mol/, 1, PH5.0), and stirring was continued for 30 minutes. During this time, the pH of the aqueous solution was maintained at approximately 8.0 with a 0.1N-NaOH solution. The chloroform layer was further extracted with an aqueous sodium perchlorate solution (1.0×10 ^-2 mol/, 1, PH5.0), and the aqueous perchlorate solutions were analyzed together. Quantitative analysis using high-performance liquid chromatography revealed that 51% of AMP2Na was converted to the corresponding 5'-diphosphoramidate. Further, this aqueous solution 2 was concentrated to 30 ml at a temperature below 40°C, 1 g of activated carbon was added, and the mixture was stirred for over 1 hour. Thereafter, the activated carbon was filtered with suction, and 0.1 g of activated carbon was added to the filtrate, which was then filtered with suction. UV spectroscopy showed that 98% of the nucleic acid species were adsorbed on the activated carbon. After washing the activated carbon with water, the nucleic acids from the activated carbon were released with a 50% aqueous ethanol solution containing concentrated aqueous ammonia (28-30%). The obtained aqueous nucleic acid solution was concentrated and adjusted to pH 8.0 with NaOH solution. This solution was subjected to chromatography on an anion exchange resin column. After washing the column with water, AMP was eluted with HCl solution. Other nucleic acids were then eluted with a mixture of HCl and LiCl. The eluate was neutralized with lithium hydroxide and concentrated to a syrupy state at 30-40°C. 10 ml of ethanol was added to this concentrated solution, and the solvent was distilled off at room temperature using a vacuum pump. Acetone was then added to precipitate the lithium salt of ADP amidite. This precipitate was centrifuged, washed with acetone containing ethanol, further washed with ether, and then dried. The total amount of lithium salt of ADP amidite obtained is
It was 31 mg. (Effects of the Invention) According to the present invention, highly pure nucleoside
5'-diphosphoric acid amide can be synthesized in high yield and in a short time.

Claims (1)

[Claims] 1 N,N'-di long chain alkyl or alkenyldiammonium salt of 1,4-diazabicyclo-(2,2,2)-octane or N- of 1,4-diazabicyclo-(2,2,2)-octane Nucleoside-5'-monophosphoric acid trapped in a long-chain alkyl or alkenylammonium salt is treated with a phosphorylating agent in a nonpolar solvent, and then ammonium carbonate or ammonia is treated to convert the phosphoric acid group into an amino acid. A method for synthesizing nucleoside-5'-diphosphoric acid amide, which comprises introducing a group.