JPH0430398B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel method for producing nucleoside 3'-phosphates, more specifically, the general formula [] (In the formula, R represents a hydrogen atom or a hydroxyl group, A represents a monomethoxytrityl group or a dimethoxytrityl group, and B represents a

A purine nucleus that may have substituents at the 2, 6, and 8 positions represented by [Formula] or

The nucleoside represented by the general formula [] [CF ₃ (CH ₂ ) _o O] ₂ POH [] ( (wherein n is an integer of 1 to 3) A dialkyl phosphite represented by the following is reacted in a solvent under heating, resulting in the general formula [] (In the formula, R, A, and B have the above-mentioned meanings) A general formula characterized in that a phosphate is obtained by oxidizing a nucleoside 3'-phosphite represented by [] This paper relates to a new method for synthesizing nucleoside 3'-phosphate represented by nucleosides
3'-phosphate has become an important starting material for genetic engineering, and various synthetic methods have been developed to date, but none have yet been found to be satisfactory. For example, 4-fluorophenylphosphoroditriazole [RWAdamiak etal. Nucleic Acid Ress.3,
3397 (1976)], phosphoromonotetrazole [H.
Synthesis methods using phosphorylating agents such as Takakn etal Chem Lett 192 (1982) are known;
In these methods, phosphorylation of guanosine or uridine to the base moiety occurs as a side reaction. Furthermore, in the synthesis method using a condensing agent, a side reaction occurs in which the arylsulfonyl condensing agent is introduced into the base moiety or hydroxyl group of guanosine or uridine. [PR Bridson etal JCSChem.447, 791,
(1977)] As described above, in the methods currently used, some side reactions occur, resulting in poor purity of the desired product and low yield, so it cannot be said to be very preferable. Therefore, we have conducted extensive research to develop a synthetic method that does not cause side reactions during the production of nucleoside 3'-phosphate, and as a result, we have found that using a certain type of dialkyl phosphite as a phosphorylation agent for the raw material nucleoside can be used for this purpose. I found that it fits. Since this phosphorylating agent does not use active groups such as triazole, tetrazole, and condensing agents, the method for producing nucleoside 3'-phosphates using the phosphorylating agent according to the present invention does not need to worry about side reactions. Moreover, it has the advantage of producing nucleoside 3'-phosphate in high yield. That is, the present invention provides a novel method for producing nucleoside 3'-phosphates that meets the needs of the technical field as described above and overcomes the drawbacks of conventional methods. Next, the reaction route of the present invention is shown as follows: (In the formula, R, A, and B have the above-mentioned meanings.) In the present invention, preferably 1 to 3 equivalents of dialkyl phosphite [] are added to the nucleoside [], and the mixture is heated in a solvent or preferably in a nitrogen gas atmosphere. The reaction is performed below to obtain nucleoside 3'-phosphite. Then, when an oxidizing agent is added and treated, the compound [] is oxidized and the corresponding nucleoside
3′-phosphate [ ] is obtained in high yield. Solvents for the reaction between [] and [] include basic solvents such as pyridine, aromatic hydrocarbon solvents such as benzene and toluene, ketone solvents such as acetone, ester solvents such as acetic acid ethyl ester, and ethers such as diethyl ether. Examples include solvents. The reaction temperature is 25 to 80℃, preferably 40℃.
~60℃. Hydrogen peroxide, perbenzoic acid, iodine, etc. are used as the oxidizing agent in the reaction from compound [] to []. The amount of oxidizing agent used is preferably 3 to 6 equivalents relative to the nucleoside. The substituent B used in the compound of the above general formula [] [] [] is of the formula

Purine that may have substituents at the 2, 6, and 8 positions represented by [Formula] or the formula

[Formula] These are pyrimidines that may have substituents at the 2, 4, and 5 positions, but the substituents at the 2, 6 positions of purine are substituted with hydrogen atoms, alkyl groups, or acyl groups. sometimes an amino group or a keto group, the 8th position being a hydrogen atom or substituted by a halogen atom. The substituents at the 2 and 4 positions of pyrimidine are an amino group or a keto group which may be substituted with an alkyl group or an acyl group, and the substituent at the 5 position is a hydrogen atom or an alkyl group, a halogen atom, or a cyano group. shows. Here, the alkyl group represents a saturated hydrocarbon bond chain having 1 to 3 carbon atoms. As described above, according to the present invention, screoside production using a reagent that phosphorylates without using an active group.
The method for synthesizing 3'-phosphate is unique, and the ability to selectively obtain the desired product in high yield without side reactions is an important technology in the fields of acid chemistry and agrochemicals. It is something that gives you a lot of progress. Next, typical synthetic methods of the present invention will be explained in detail with reference to Examples. Example 1 5'-o-monomethoxytritylthymidine 3'-
Synthesis of (trifluoroethyl) phosphate 5'-o-monomethoxytrityl thymidine (MMTrT) (0.515 g, 1.0 mmol) was dissolved in pyridine (7 ml) and bis(2,2,2-trifluoroethyl) phosphite ( 0.46ml, 3mmol)
was added and reacted for 3 hours at 50°C under a nitrogen gas atmosphere, then the reaction solution was cooled to room temperature and further heated to 0°C.
m-chloroperbenzoic acid (1.04 g, 6
mmol) and treated for 10 minutes, the product is oxidized to phosphate. Pour the reaction solution into ice water (5 ml)
After extraction with methylene chloride (10 ml), the organic layer was mixed with 5% NaHCO ₃ (5 ml x 3) and water (5 ml).
Wash with ×2). Remove methylene chloride under reduced pressure,
Add a small amount of toluene and repeat the vacuum until the pyridine odor disappears. The residue was dissolved in a small amount of methylene chloride and dissolved in hexane with vigorous stirring.
When added to ether (10:1), a white precipitate formed, which was removed to yield 0.69 g of 5'-o-monomethoxytritylthymidine 3'-(trifluoroethyl) phosphate (mp = 148-150°C). Obtained. The physical properties of the obtained product are as follows. Rf = 0.01 (CH ₂ C ₂ : CH ₃ OH = 9:1); Rf = 0.97 ((CH ₃ ) ₂ CO: H ₂ O = 7:3); UV; λ ^MeOH _nax = 265, 260 nm; λ ^MeOH _nio = 247, 226nm. Example 2 Synthesis of 5'-o-dimethoxytritylbenzoyldeoxymitidine 3'-(trifluoroethyl)phosphate 5'-o-dimethoxytritylbenzoyldeoxymitidine (d-DMTrC ^bz ) (0.664 g, 1.0 mmol)
The reaction was carried out in the same manner as in Example 1 except that 0.781 g (90
%) obtained. The physical properties of the obtained product are as follows. Rf=0.11 (CH ₂ C ₂ :CH ₃ OH=9:0); Rf=0.97 ((CH ₃ ) ₂ CO:H ₂ O=7:3); UV: λ ^MeOH _nax = 304, 275, 235 nm; λ ^MeOH _nio = 289, 249 nm. The chemical structures of these compounds were confirmed by completely deprotecting the protecting groups and then enzymatically decomposing them. Note that specific examples similar to Examples 1 and 2 above are summarized in Table 1.

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【table】

Claims (1)

[Claims] 1. General formula [] (In the formula, R represents a hydrogen atom or a hydroxyl group, A represents a monomethoxytrityl group or a dimethoxytrityl group, and B represents a purine nucleus that may have substituents at the 2, 6, and 8 positions represented by the formula) or a pyrimidine nucleus that may have a substituent at the 2, 4, or 5 positions represented by the formula] and a nucleoside represented by the general formula [] [CF ₃ (CH ₂ ) _o O] ₂ POH [] (In the formula, n is an integer of 1 to 3) The production of a nucleoside 3'-phosphate characterized by reacting a dialkyl phosphite represented by the formula and oxidizing the obtained nucleoside 3'-phosphite to a phosphate. Method.