JPS60120897A - Production of novel nucleoside 3'-phosphate - Google Patents

Abstract

PURPOSE:To produce the titled compound useful as an important raw material in genetic engineering, without causing side reactions, in high yield, by using a specific dialkyl phosphite as an agent for the phosphorylation of the nucleoside used as the raw material. CONSTITUTION:The objective compound can be produced by reacting (A) the nucleoside of formula I (R is H or OH; A is monomethyoxytrityl or dimethoxytrityl; B is purine nucleus of formula II which may have substituent groups at 2, 6 or 8 positions, or pyrimidine nucleus of formula III which may have substituent groups at 2, 4 or 5 positions) (e.g. 5'-o-monomethoxytritylthymidine) with (B) the dialkyl phosphite of formula IV (n is 1-3) [e.g. bis(2,2,2-trifluoroethyl)phosphite], and oxidizing the resultant nucleoside 3'-phosphite to phosphate with an oxidizing agent such as perbenzoic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a novel nucleoside 3'-phosphate, and more particularly, it relates to a method for producing a novel nucleoside 3'-phosphate, and more specifically, it is represented by the general formula [1] (wherein R represents a hydrogen atom or a hydroxyl group, and A represents monomethoxy nucleosides of the general formula []][CFs(
CH2)no)2POH(II) (wherein n is an integer of 1 to 3) A dialkyl phosphite represented by (in which R, A, B have the above-mentioned meanings) is oxidized to obtain the nucleoside 3'-phosphite represented by --P-OH 0 (CHz) nCF3 This invention relates to a novel method for synthesizing nucleoside 3'-phosphates. Nucleoside 3'-phosphate has become an important starting material in genetic engineering, and various synthetic methods have been developed to date, but none have yet been found to be satisfactory.

For example, 4-70 lophenyl phosphoroditriazole [R
, W., Adamiak et al., Nucleic A.
c1d Re5s, 3.

3397 (1976)], phosphoromonotetrazole [
H, Takakn etal Chem Lett 1
92 (1982) ] are known, but in these methods, phosphorylation of the base moiety of guanosine or uridine occurs as a side reaction.

Furthermore, in the synthesis method using a condensing agent, a side reaction occurs in which arylsulfonyl as a condensing agent is introduced into the base moiety or hydroxyl group of guanosine or chrysine.

[P, RBr1dson etal J, C, S, Ch
em, Gown, 447.

791, (1977)) As described above, the currently used methods are not very desirable because some side reactions occur, resulting in poor purity of the desired product and low yield.

Therefore, we have conducted extensive research to develop a synthetic method that does not cause side reactions during the production of nucleoside 3'-phosphates, and as a result, we have found that using a certain type of dialkyl phosphite as a phosphorylation agent for the raw 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, or condensing agents, the method for producing itanucleoside 3'-phosphate 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 a high yield. In other words, the present invention is a novel method that meets the above-mentioned demands in the field of cough technology and overcomes the drawbacks of conventional methods. A method for producing nucleoside 3'-phosphates is provided.

Next, the reaction route of the present invention is shown as follows: [1]
(1) [1) (IV) (In the formula, R, A, and B have the above-mentioned meanings.) The present invention preferably contains 1 to 3 equivalents of dialkyl phosphite [Il] to nucleoside M (1). In addition, the nucleoside 3′ is reacted in a solvent under heating or preferably under a nitrogen gas atmosphere.
- Obtain phosphite. Thereafter, when an oxidizing agent is added and treated, the compound [Otori] is oxidized and the corresponding nucleoside 3'-phosphene (IVI) is obtained in high yield.

Examples of solvents for the reaction with [1] and 0th include basic solvents such as pyridine, aromatic hydrocarbon solvents such as alcohol and toluene, ketone solvents such as acetone, ester solvents such as acetic acid ethyl ester, diethyl ether, etc. The reaction temperature is 25-80℃.
, preferably 40 to 60°C. Compound [from 13 (
The oxidizing agent in the reaction to IVI is hydrogen peroxide,
Perbenzoic acid, iodine, etc. are used. The amount of oxidizing agent used is preferably 3 to 6 equivalents relative to the nucleoside. Used in the compounds of general formula (IXI) CIVI above, each shows a pyrimidine nucleus that may have 1, but the substituent at position 2.6 on the purine nucleus may be substituted with a hydrogen atom, an alkyl group, or an acyl group. It represents an amino group or a keto group, and the 8th position is a hydrogen atom or substituted with a halogen atom. The 2.4-position substituent on the pyrimidine nucleus represents an amino group or a keto group which may be substituted with an alkyl group or an acyl group, and the 5-position substituent is a hydrogen atom or an alkyl group, a halogen atom, or a cyano group. Show. Here, the alkyl group represents a saturated hydrocarbon bond chain having 1 to 3 carbon atoms.

As stated above, the present invention provides a method for synthesizing nucleoside 3I-phosphate using a phosphorylating reagent without using an active group, and is selective and does not cause side reactions. The ability to obtain target products in high yields represents an important technological advance in the fields of nucleic acid chemistry and agrochemicals.

Next, typical synthetic methods of the present invention will be explained in detail with reference to Examples.

Example 1 Synthesis of 5'-o-monomethoxytritylthymidine 3'-()lifluoroethyl)phosphate 5'-o-monomethoxytritylthymidine (MMTr
T) (0,515g, 1.Ommol) in pyridine (7i+/)KI! and bis(2,2,2-)lifluoroethyl)phosphite (0.46 ml, 3 mm
ol) was added and reacted for 3 hours at 50°C under a room floor gas atmosphere, the reaction solution was cooled to room temperature, further cooled to 0°C, and m-chloroperbenzoin #it (1.04g,
After adding 6 mmol) and treating for 10 minutes, the product is oxidized to phosphate. The reaction solution was poured into ice water (5 m/
) K, and after extraction with methylene chloride (10 + + + J), the organic layer is washed with 5% NaHCO3 (5111jX 3 ) and then with water (5mJX2). Remove methylene chloride under reduced pressure, add a small amount of toluene, repeat under reduced pressure until the pyridine odor disappears, dissolve the residue in a small amount of methylene chloride, and add hexane-ether with vigorous stirring.
10:1), a white precipitate was formed, and when this was collected in F, 5'-〇-monomethoxytritylthymidine 3'-
()lifluoroethyl)phosphate) (m, p = 1
48-150°C) was obtained.

The physical properties of the obtained product are as follows.

Rr-o,ot (CH2Cgz:CHaOH-9:1
);Rr-0,97((OHs)zoo:H2O-7:
3) seOH Uv: λ -265,230nm; ax MeOH λ -247,226nm.

in Example 2 Synthesis of 5'-〇-dimethoxytritylinzoyldeoxymitidine 3'-()lifluoroethyl)phosphate 5'-〇-dimethoxytritylbenzoyldeoxythymidiney (d-DMTrC") (0,664 g .

The corresponding 5'-〇-dimethoxytritylbenzoyldeoxythymidine 3'-()U fluoroethyl)phosphate (mp=112-114°C) was prepared in the same manner as in Example 1 except that 1.0 mmol) was used. is 0.78
1i90%) was obtained. The physical properties of the obtained product are as follows.

Rf-0,11 (CH2CJ2:CH30H=9:1)
.

Rf-0,97 ((OH3)200:H2O-7:3)
; Uy: 2 MeOH, 3o 4.257.235 nm
;ax was enzymatically degraded and its chemical structure was confirmed. Specific examples similar to Examples 1 and 2 above are summarized in Table IK: Table I Nusileoside 3'-phosphate synthesis (2,0) 2 MMTrT 3.0 6.0 92 (1,0) Z 3 d-MMTrA 3.0 6.0 92(0,5) 4 d-MM'l1rC"' 1.5 3.0 85(
0,5) (O,S) (3 others)

Claims (1)

[Claims] General formula (1) (In the formula, R represents a hydrogen atom or a hydroxyl group, and A represents a monomethoxytrityl group or a phosphoryl nucleus which may have a substituent at the 2nd or 6th position.) nucleosides with the general formula (n) (CFa(GH2)no)2POH(If), where n is an integer from 1 to 3; A method for producing nucleoside 3'-phosphate, which comprises reacting phosphite and oxidizing the obtained nucleoside 3'-phosphite to phosphate.