WO2002012264A1

WO2002012264A1 - Process for producing 2'-o-alkylguanosine

Info

Publication number: WO2002012264A1
Application number: PCT/JP2001/006737
Authority: WO
Inventors: Shinji Sakata; Toshio Yamada
Original assignee: Yamasa Corporation
Priority date: 2000-08-08
Filing date: 2001-08-06
Publication date: 2002-02-14
Also published as: JP3596669B2; JP2002053591A; AU2001276736A1

Abstract

A process for producing a 2'-O-alkylguanosine from 2,6-diaminopurine riboside, characterized by using an aqueous medium to treat 2,6-diaminopurine riboside with an alkyl phosphate or alkyl sulfate as an alkylating agent under basic conditions to yield a 2'-O-alkyl-2,6-diaminopurine riboside and then deaminating the diamino compound.

Description

Specification

2'-10-Alkylguanosine production technology

The present invention provides a method for producing 2'-0-alkylguanosine from 2,6-diaminopurine riboside which is safe, excellent in operability and practicality.

Background technology

As a method for producing 2′-dialkylguanosine represented by 2′-0-methylguanosine, there is a method using guanosine as a raw material. In this case, the alkylation reaction of the 2′-hydroxyl group proceeds in good yield. In general, a method using 2,6-diaminopurine riboside as a raw material has been practiced.

As a method for obtaining 2'-0-methylguanosine from this 2,6-diaminopurine liposide, conventionally, 2'-position of 2,6-diaminopurine riboside using diazomethane under the stannous chloride catalyst has been used. A method has been known in which a hydroxyl group is methylated and then deaminated with adenosine deaminase to obtain 2'-0-methylguanosine (Can. J. Chem, 59: 3360 (1981)).

However, this method uses explosive and highly toxic diazomethane, so it could not be an industrially viable method. For this reason, various methods that do not use diazomethane have been studied. As a result, as a modification of the above method, the 2′-formation of 2,6-diaminopurine riboside using an alkyl halide in the presence of a base was carried out. There has been reported a method of methylating a hydroxyl group, followed by deamination with adenosine kinase to obtain 2′-0-methylguanosine (Japanese Patent No. 3015404).

However, even with the above-mentioned improved method, sodium hydride, which is a base coexisting during the methylation reaction, is ignitable, and methyl iodide used as a methylating agent is less toxic than diazomethane. Although it was low, it was still toxic and had problems such as strict control of these chemicals when implementing the above improved method.

In addition, all of the above-mentioned conventional methods use methylazomethane or methyl iodide. This process consists of a discontinuous process in which the hydrolysis reaction is carried out in a non-aqueous system such as an organic solvent containing no water, and the deamination reaction using adenosine deaminase is carried out in an aqueous system such as water. When such a discontinuous process is performed as a series of processes, there is a problem that the handling of each process must be strictly distinguished and equipment for executing the process must be prepared, so that it can be performed easily by anyone. It wasn't a simple method.

Disclosure of the invention

The present inventors have conducted intensive studies to solve the above-mentioned problems of the conventional method. As a result, when producing 2′-0-methylguanosine from 2,6-diaminopurine riboside, basic Under the conditions, by using trimethyl phosphate as a methylating agent, the methylation reaction can be carried out in an aqueous solvent, and the 2′-hydroxyl group of 2,6-diaminopurine riboside can be selectively formed. It was found that 2′-10-methylguanosine could be efficiently obtained after deamination by adenosine deminase.

Conventionally, methylation of nucleosides using trimethyl phosphate has focused on methylation of nucleoside nucleobases, and no report has been made on methylation of sugar hydroxyl groups. However, in the case of nucleobase methylation experiments, in the case of inosine and adenosine, which were performed as comparative experiments, methylation of the sugar hydroxyl group slightly progressed with trimethyl phosphate.For example, inosine was methylated using trimethyl phosphate. If you, 6 5% of N ¹ - methyl inosine and ^{3 3%] ^, 0 2} '| 3' 1 - form dimethyl inosine raw, when it is methylated adenosine with trimethyl phosphate 3 of ^{8% 0 2... '} | 3' 1 - methyl adenosine and 2 4% ^{^{^{N 6, 0 2 ', 3}}} ' 1 - dimethyl adenosine produces, has been reported that (Bull Chem Soc Jpn , 54, 1569-1570 (1981)). Based on these reports, even if 2,6-diaminopurine riboside was methylated using trimethyl phosphate, it was never thought that the 2'-hydroxyl group would be selectively methylated. The above findings of the inventors were completely surprising.

The present inventors have further studied based on the above findings, and completed the present invention. That is, the present invention relates to a method for producing 2′-0-alkylguanosine from 2,6-diaminopurine riboside, wherein an aqueous solvent is used as a solvent, , 2,6-Diaminopurine liposide is treated with an alkylating agent of an alkyl phosphate or an alkyl sulfate to form 2'-0-alkyl-2,6-diaminopurine riboside, which is then formed. A process for producing 2'-0-alkylguanosine, which comprises deaminating the product to obtain 2'-0-alkylguanosine _c

BEST MODE FOR CARRYING OUT THE INVENTION

The starting compound, 2,6-diaminopurine riboside, is a known compound having the following formula (I). Such a compound can be synthesized chemically or enzymatically, or a commercially available product may be used. In particular, when 2,6-diaminopurine riboside is synthesized enzymatically, it is not necessary to isolate the synthesized product, and the enzyme reaction solution itself or a crude product from the enzyme reaction solution is used directly as a raw material. No problem.

As described above, the method of the present invention comprises the steps of: first, in a water-based solvent, under basic conditions,

2, 6-di § amino riboside is treated with an alkylating agent, 2 ⁷ -0- alkyl one 2 having the formula (II), 6 - to produce a di § amino riboside.

(Wherein, R represents lower alkyl having 5 or less carbon atoms.) As the alkylating agent used here, an alkyl phosphate or an alkyl sulfate can be used. Specifically, trialkyl phosphates such as trimethyl phosphate, triethyl phosphate, tripropyl phosphate, and ethyl dimethyl phosphate; Dialkyl phosphates such as dimethyl phosphate and getyl phosphate; and dialkyl sulfates such as dimethyl sulfate and getyl sulfate. In particular, in the case of methylation, trimethyl phosphate is preferred.

The alkylation reaction is carried out under basic conditions in an aqueous solvent such as water or a buffer solution, under basic conditions, with 1 to 100 mol of alkyl phosphate or alkyl sulfate per mol of 2,6-diaminopurine riboside. The reaction can be carried out preferably by using 10 to 30 moles and reacting at 10 to 90 ° C, preferably 30 to 70 ° C for about 1 to 20 hours.

It is important that this alkylation reaction is performed under basic conditions. Basic conditions include, for example, pH 11 or higher, preferably pH 11 to 13. When the pH is less than 11, the alkylation reaction becomes slow, and when the pH exceeds 13, the selectivity to the 2'-hydroxyl group in the methylation reaction decreases. Not preferred. It is desirable that the above basic conditions be maintained or adjusted from the start to the end of the reaction, but it is important to adjust at least the pH at the beginning of the reaction to the basic conditions described above. It is.

The 2'-0-alkyl-1,2,6-diaminopurine riboside thus produced can be purified by a known purification method (eg, solvent extraction method; ion exchange resin, activated carbon, etc.) if necessary. And subject it to the next deamination reaction.

The deamination reaction can be carried out by a known method using adenosine deaminase. As the adenosine deaminase, any enzyme derived from animals and microorganisms can be used as long as it can deaminate the amino group at the 6-position of 2'-0-alkyl-1,2,6-diaminopurine riboside. can do. In particular, E. coli-derived enzymes are advantageous in terms of reaction efficiency and price.

Although the deamination reaction varies depending on the enzyme used, an excess amount of adenosine deaminase is used in water or a buffer at a pH of 6 to 8 and at a temperature of 20 to 50 ° C. The reaction can be carried out for about 100 hours.

After the reaction, the resulting 2'-0-alkylguanosine (compound of the following formula (III)) is It can be isolated and purified by known methods (eg, crystallization method, solvent extraction method, chromatography method using an adsorption resin, etc.).

(In the formula, R means lower alkyl having 5 or less carbon atoms.)

Hereinafter, the present invention will be described more specifically with reference to Examples, but it is apparent that the present invention is not limited to these.

Example 1: Synthesis of 2'-0-methylguanosine (Part 1)

2,6-Diaminopurine riboside (5.65 _s 2 Ommo 1) is suspended in a mixture of trimethyl phosphate (40 ml) and deionized water (40 ml), heated to 50 ° C, The reaction was carried out with stirring while adjusting with an aqueous solution of sodium hydroxide so that the pH of the reaction solution became 12.0 to 12.5.

Seven hours after the start of the reaction, the reaction solution was analyzed by HPLC, and as a result, 2'-0-methyl-2,6-diaminopurine riboside and 3'-0-methyl-1,2,6-diaminopurine riboside However, it was confirmed that 41% and 8.5% (HPLC: analyzed by UV260 nm) were generated respectively.

Dilute the reaction solution to 40 Oml with deionized water, adjust the pH to 7.6 with hydrochloric acid, add adenosine deaminase (1500 units) from E. coli, and stir at 37 ° C. It was allowed to react. During the reaction, the deamination reaction was performed for 88 hours while adjusting the pH to 7.6 with an aqueous hydrochloric acid solution, and then the reaction solution was boiled for 10 minutes to stop the reaction.

After filtering the reaction solution and passing the filtrate through an adsorption resin column, the adsorbate is eluted with deionized water, and the eluted fraction of 2'-0-methylguanosine thus obtained is decompressed to 5 Oml. Concentrate and cool overnight. The resulting precipitate was collected by filtration and dried to obtain 1.72 g of a crystal of —0-methylguanosine.

Example 2: Synthesis of 2'-0-methylguanosine (Part 2)

Perisine (14.6 g, 60 mmo 1) and 2,6-diaminopurine (6 g, 40 mmo 1) are suspended in 25 mM phosphate buffer (pH 7.0, 1000 ml), and then suspended. Pyrimidine nucleoside phosphorylase (2100 units) and purine nucleoside phosphorylase (5400 units) were added to the suspension, followed by stirring at 50 ° C for 100 minutes. The reaction was boiled for 10 minutes to stop the reaction.

After completion of the reaction, analysis by HPLC revealed that the conversion to 2,6-diaminopurine liposide per 9,6-diaminopurine was 95.7%.

The reaction solution was concentrated to 20 Oml, trimethyl phosphate (15 Oml) was added, and the mixture was reacted at 50 ° C for 7 hours while adjusting the pH to 12 to 12.5 with an aqueous solution of sodium hydroxide. -Analysis of the reaction mixture obtained by HP LC shows that 2'-〇-methyl-2,6-diaminopurine riboside is produced from 2,6-diaminopurine riboside at a conversion of 47%. Was confirmed.

This reaction solution was deaminated in the same manner as in Example 1 to obtain 2′-0-methylguanosine.

Industrial applicability

According to the method of the present invention, 2′-0-alkylguanosine can be efficiently produced from 2,6-diaminopurine riboside using an aqueous solvent without using any flammable or toxic chemical. Since it is an extremely useful method and does not require special equipment or work management, it is an extremely practical method for mass synthesis of 2'-0-alkylguanosine.

Claims

The scope of the claims

This is a method for producing 2'-0-alkylguanosine from 1,2,6-diaminopurine riboside, using an aqueous solvent as a solvent under basic conditions. The minopurine riboside is treated with an alkyl phosphate or an alkyl sulfate to produce 2′—0—alkyl-1,2,6-diaminopurine riboside, which is then deaminated to 2 ′ — A method for producing a 2′-1 0-alkylguanosine, which comprises obtaining a 0-alkylguanosine.

2. The method according to claim 1, wherein the alkyl in the 2.2'-10-alkylguanosine is a lower alkyl having 5 or less carbon atoms.

3. The method according to claim 1, wherein the aqueous solvent is water or a buffer. .

4. The method according to claim 1, wherein the basic condition is a condition of pH 11 or more.

5. The method according to claim 1, wherein the basic condition is a condition of pH 12 to 13.

6. The method according to claim 1, wherein the alkyl phosphate is a trialkyl phosphate or a dialkyl phosphate.

7. The method of claim 1, wherein the alkyl sulfate is a dialkyl sulfate.

8. The method of claim 1, wherein the deamination is an enzymatic deamination using adenosine kinase.

9. The method of claim 1, wherein the 9.2'-0-alkylguanosine is 2'-10-methylguanosine and the alkyl phosphate is trimethyl phosphate or dimethyl phosphate.

The method of claim 1, wherein the 10.2'-0-alkylguanosine is 2'10-methylguanosine and the alkyl sulfate is dimethyl sulfate.