JPS6270390A - Production of nucleotide compound - Google Patents

Abstract

PURPOSE:To obtain the titled compound containing a phosphate bond without dehydrating a system, by oxidizing a raw material nucleotide compound containing a phosphite bond with an organic oxidizing agent in the presence of an acid in the nonaqueous system. CONSTITUTION:A raw material nucleotide compound (e.g., deoxyadenosine, etc.,) containing a phosphite bond is oxidized with an organic oxidizing agent [e.g., bis(trimethylsilyl) peroxide, etc.,] in the presence of an acid (e.g., Lewis acid,formic acid, etc.,) in an nonaqueous system and the phosphite bond is converted into phosphate bond, to give the aimed compound.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for efficiently oxidizing the phosphite moiety of a raw nucleotide compound having a phosphite bond to phosphate.

(Conventional technology) DNA (deoxynucleotide nucleic acid) and RNA (riga nucleic acid)
As a method for chemically synthesizing polyscleotides such as No. 57-176998, etc.)
.

In this method, a phosphite-forming agent, which is a monomer component, is condensed with the hydroxyl group of a nucleoside or nucleotide to form an intermediate having a phosphite bond, and then oxidation to the corresponding phosphate is performed.

As a method for such an oxidation reaction, a method of oxidizing with iodine in the presence of water and a method of oxidizing with a peroxide such as t-butyl peroxide are conventionally known (for example, see the above-mentioned publication). .

However, the former method, in which the reaction is carried out in an aqueous system, has the problem of requiring a great deal of effort to return the system to an anhydrous state after the reaction, and the latter method, although the reaction can be carried out in a non-aqueous system, has the problem of inferior reactivity. was there.

(Problems to be Solved by the Invention) Therefore, the present inventors have made extensive efforts to solve the drawbacks of the prior art, and have found that when oxidized with an organic oxidizing agent in the presence of an acid, even non-aqueous They have discovered that the oxidation reaction proceeds extremely efficiently and have completed the present invention.

(Means for Solving the Problems) Thus, according to the present invention, a raw nucleotide compound having a phosphite bond is oxidized using an organic oxidizing agent in a non-aqueous system in the presence of an acid to convert the phosphite bond into a phosphate bond. A method for producing a nucleotide compound is provided.

The raw nucleotide compounds used in the present invention are known to have at least one phosphite bond. Here, the phosphite bond is represented by +o-p-o+, and R is an alkyl group, an anoethyl group, an O-chlorophenyl group, or a P-chlorophenyl group.
- means a protecting group such as a nitrophenylethyl group, a 2-methylsulfoethyl group, an allyl group, or a P-chlorocinnamyl group.

Such a raw nucleotide compound may be one in which two or more nucleosides form a phosphite triester (i.e., a compound in which all phosphorus moieties are phosphites, such as dinucleoside phosphites and trinucleoside diphosphites), Further, only one phosphorus may be a phosphite and the other phosphorus may be oxidized to a phosphate. The latter raw material nucleotide compound can be obtained by sequentially carrying out the condensation of the phosphite-forming agent, which is a monomer component, and the subsequent oxidation reaction by warping.

The nucleoside that is a component of the raw nucleotide compound is not particularly limited as long as it is a commonly used nucleoside, and specific examples include deoxyadenosine, deoxyadesine, deoxycytidine, thymidine, adenosine, and guanosine.

Examples include cytidine, uridine, and inosine.

In addition, the hydroxyl group of the sugar moiety and the amine group of the nucleoside base moiety of the starting nucleotide compound may be appropriately protected with a protecting group generally used in the field of nucleoside chemistry, and if the degree of polymerization is higher than that of a dimer, There are no particular restrictions.

The oxidation reaction in the present invention is carried out using an organic oxidizing agent in the presence of an acid. Any organic oxidizing agent may be used as long as it is substantially anhydrous, and specific examples thereof include bis(trimethylsilyl) peroxide, di-t-butyl peroxide, benzoyl peroxide, di-t-butyl hydroperoxide, cumene hydroperoxide,
Diisoprobyl peroxydical? peroxides such as N-methylmorpholine oxide, non-peroxide type oxidizing agents such as N-methylmorpholine oxide, 2-phenylsulfonyl-3-phenyl-1-4xa-2-aza-cyclopropane, triphenylphosphine oscinide, etc. is exemplified.

The acid that is incorporated together with the organic oxidizing agent may be either solvent-soluble or solvent-insoluble, and specific examples include zinc chloride, aluminum chloride, tin tetrachloride, titanium tetrachloride, boron trifluoride etherate, zinc bromide, and dichloride. Lewis acids such as iron, dialkyl aluminum chloride, formic acid,
Acetic acid, monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, trifluoroacetic acid, propionic acid, benzoic acid, hydrochloric acid,
Protonic acids such as sulfuric acid, phosphoric acid, methanesulfonic acid, trifluoromethanesulfonic acid, P-toluenesulfonic acid, strongly acidic ion exchange resins, perfluorinated sulfonic acid resins, trimethylsilyl triflate, perfluorine containing trinotylsilyl groups, etc. Examples include other acidic materials such as sulfonic acid resins (see Tetrahedron Letters, 21°767, 1980).

Use of an organic oxidizing agent in the present invention: t is selected as appropriate, but is usually 1 to 5 equivalents, preferably 2 to 4 equivalents relative to the phosphite moiety, and the acid is an organic oxidizing agent, usually O,
OO is incorporated in a proportion of 5 to 1 mole, preferably 0.01 to 0.1 mole.

The reaction is usually carried out in the presence of a solvent at -30 to 50°C, preferably -20 to 40°C for 1 minute to 2 hours, preferably 2 minutes to
It will last for one hour. The solvent used may be any commonly used solvent, and specific examples include pyridines such as pyridine, picoline, and lutidine, quinoline, inquinoline, and oxazole. Ethers such as tetrahydrofuran and dioxane may be used in combination as appropriate.

... in the system may be selected appropriately, but when a trityl group type protecting group is used, if -I in the system becomes excessively low, the trityl group may be eliminated.

The reaction may be a liquid phase reaction or a solid phase reaction, and in the case of a solid phase reaction, it is carried out by flowing an oxidizing agent solution and an acid solution through a layer of the raw material nucleotide compound bound to a carrier. I can do it.

Furthermore, when a solid acid is used, it is also possible to adopt a method in which the oxidizing agent is passed through a layer consisting of a mixture of the raw nucleotide compound and the solid acid.

When a Lewis acid or a protonic acid is used as the acid in the present invention, decore protection of the trityl group-based protecting group may occur, but this may occur if a deprotection reaction is planned after the oxidation reaction. This is an advantage.

After the oxidation reaction is completed, the generated nucleotide compound can be subjected to the next condensation reaction, and in order to isolate and purify the product from the reaction solution, adsorption chromatography, which is a means of ordinary organic synthesis reaction, or It is possible to appropriately select known means such as ion exchange chromatography, distribution using an organic solvent, or crystallization, and to carry out certain b in combination.

In addition, if the nucleotide compound is obtained by a solid phase synthesis method using a carrier, the carrier is removed according to a conventional method, and if necessary, the disosaccharide hydroxyl group and/or the protecting group of the nucleotide base are removed, and then the above-mentioned It can be isolated using a similar method.

(Effects of the Invention) According to the present invention, oxidation of phosphite to phosphate can be carried out non-aqueously and efficiently. Therefore, there is no need to dehydrate the system when performing the next condensation reaction after the oxidation reaction, which is an extremely large advantage in terms of polynucleotide synthesis operations.

(Example) The present invention will be described in more detail with reference to Examples below.

Example 1 The following dinucleoside fostumite (compound 1) obtained by reacting 5'-〇-monomethoxytritylthymidine, bis(dimethylamino)methoxyphosphine and 3'-〇-ditertiarybutyldimethylsilylthymidine (compound 1)0 ．．
After dissolving 25 mmol in 5.5 ml of dichloromethane, 0.014 mmol of triethylamine was added and then 10.0 mmol of dichloromethane containing 2 parts of bis(trimethylsilyl)peroxide (0.5 mmol) per compound 1 was added. 39mi IJ! 0.11 mmol of dichloromethane containing 0.012 mmol of various acids shown in Table 1 was added thereto, and the reaction was carried out at -20° C. for a predetermined period of time. After the reaction, after removing the solid content as necessary, the solvent was distilled off, and the residue was subjected to chromatography (methanol: ethyl acetate: hexane = 1:60).
:60) and the following dinucleoside phosphate (
Compound 2) was obtained. The results are shown in Table 1.

For comparison, a similar experiment was conducted on a system in which no acid was added. The experimental conditions were as shown in Table 1.

Usuhiki 〆＼ Brain - Example 2 The reaction was carried out according to Example 1 except that another organic oxidizing agent was used in place of bis(trimethylsilyl)peroxide. The results are shown in Table 2.

Example 3 Compound 4 was obtained by carrying out a reaction according to Example 1 except for using Compound 3 below as the dinucleoside phosphite. The results are shown in Table 3.

Compound 3 Compound 4 Table 3 Example 4 (Synthesis of dimer) CPG (Contro
114 m9 of 5'-〇-dimethoxytrityl thymidine (compound 5) bonded via an ester bond was added to the resin (Pore Glass), and a dichloromethane solution of 0.5 mol dichloroacetic acid was added. After shaking at room temperature, the liquid phase was filtered. did. This reaction removed the dimethoxytrityl group of the 57-hydroxyl group. Then phosphite of 5'-〇-dimethoxytritylthymidine (compound 6) 0
．． 087 mmot and 1-H-tetrazole 0.21
A 1M solution of mmot in acetonitrile was added and the mixture was allowed to lose color at room temperature for 30 minutes, and then the liquid phase was filtered. After washing with acetonitrile, the 5'-〇-dimethoxytrityl group was removed by the same procedure as above, and the amount of the removed dimethoxytrityl group was measured by absorption at 499 nm. As a result, the condensation rate was 87. After shaking at room temperature for 5 minutes, the liquid phase was filtered and then washed with dichloromethane.

This operation yielded Compound 8 in which the diphosphite moiety was phosphate-oxidized.

(Deprotection reaction) A mixture of thiophenol, trimethylamine and dioxane (1:2:2, v/v) was added to the above resin.
After adding 4 ml and stirring for 30 minutes, the liquid phase was filtered.
Next, after washing with methanol, it was treated with concentrated aqueous ammonia for 2 hours. Thereafter, tear fluid and lavage water were collected and concentrated. When the obtained concentrate was analyzed by HPLC using 5'-ATP as an internal standard, it was found that thymidyl (3'→5') thymidine (
The yield of compound 9) was 91%.

/IQ'X Compound 5 0CH3 Compound 8

Claims (1)

[Claims] 1. A method for producing a nucleotide compound, which comprises oxidizing a raw material nucleotide compound having a phosphite bond using an organic oxidizing agent in a non-aqueous system in the presence of an acid to convert the phosphite bond into a phosphate bond.