JPS60120890A - Novel phosphorylation agent - Google Patents

Abstract

NEW MATERIAL:The compound of formula I (n is integer of 1-3). EXAMPLE:Bis(2,2,2-trifluoroethyl)phosphite. USE:Phosphorylation agent for nucleoside, etc. PREPARATION:The compound of formula I can be produced e.g. dissolving t- butanol in a solvent such as methylene chloride, adding phosphorus trichloride dropwise to the solution at 0 deg.C, and reacting the reaction product with 2,2,2- trifluoroethanol of formula II. A dinucleoside useful as a raw material of nucleic acids, agricultural chemicals, etc. can be obtained in high yield, by reacting the compound of formula I with a nucleoside such as 5'-o-monomethoxytritylthymidine to phosphorylate the nucleoside, oxidizing the product to a phosphate compound with m-chloroperbenzoic acid, etc., and reacting the product with a nucleoside.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel phosphorylating agent for nucleoside 9, and more particularly to a compound represented by the general formula [1] and uses thereof.

[CF3(CH2)no]2POH[l] (in the formula, n is 1
Nucleoside phosphorylating agents are one of the most important starting materials for genetic engineering, and various phosphorylating agents have been developed up to now, but no satisfactory one has yet been obtained. Not yet. Examples of phosphorylating agents for nucleoside 9 include 4
-chlorophenylphosphoroditriazole [R, W, A
Damialc et al, NucleicAci
dRess, 3.3397 (1976)], phosphoromonotetrazole [HoTakaku at aL Ch
Lett 192 (1982) E and the like are known, but these phosphorylating agents cause phosphorylation of the base moiety of guanosine or uridine as a side reaction. Sarar
In the phosphorylation reaction using condensing agent c, a side reaction occurs in which arylsulfonyl as condensing agent 1 is introduced into the base moiety or hydroxyl group of guanosine or uridine (Pj (,
Br1dson et al., J.O.S.
, Ooram.

447.791 (1977)). As described above, in the currently used method, some side reactions occur, resulting in poor purity of the target product (low yield). After extensive research to develop a phosphorylating agent that does not cause phosphorylation, it was discovered that a novel dialkyl phosphite is suitable for this purpose. Since it does not use active groups, there is no need to worry about side reactions, and it is a new compound that can achieve phosphorylation of nucleosides in high yields.

The phosphorylating agent of the present invention is a dialkyl phosphite derivative represented by the general formula % (1). In this general formula, n is an integer from 1 to 3, preferably n=1.

The compounds of the present invention are all novel compounds that have not been described in literature and are produced according to the following formula: (OF3(CH2)nO]2POH[1] (wherein, n is an integer from 1 to 3) The compound [1] of the present invention can be obtained in high yield by reacting t-butanol and phosphorus trichloride in a solvent and reacting the obtained compound 10 with any alcohol.The dialkyl compound thus obtained Attempting to attach a phosphoryl group to a nucleoside using a phosphite gives favorable results according to the following reaction: VII where the Aft conjugate group, such as the monomethoxytrityl group,
represents a dimethoxytrityl group, B is a base, f/
i + Qi is benzoylguanine, acyladenine, isoditylguanine, indiyladenine, acyladenine,
Anisoylcytosine, (nsoylcytosine, thymine, uracil, etc.) When the 5'-hydroxyl protected nucleoside [B] and compound [1] are reacted in a solvent such as pyridine, the desired nucleoside 3'- Phosphite [V] is obtained. When treated with an oxidizing agent, the corresponding nucleonobi-3'-bosphae) [,
V1] is obtained at a commercial yield. When oligomers are synthesized using the nucleoside 13'-phosphate thus obtained, dinucleosides can be obtained in high yield.

Unlike conventional methods, the novel phosphorylation reagent of the present invention, which can perform phosphorylation without using an active group, is unique in the prior art and does not cause side reactions. The fact that nucleosides can be selectively phosphorylated without phosphorylation and that the desired product can be obtained in high yield represents an important technological advance in the fields of nucleic acid chemistry and laxative medicine.

Next, representative methods of synthesizing and using the novel compounds of the present invention will be explained in detail with reference to Examples.

Example 1 Synthesis of bis(2,2,2-trifluoroethyl)phosphite T-tutar (10,6me, 0.125M) was dissolved in CH20e2,
2 (25 ml) K-dissolved PGlh (10,9
rrLl, 0.125 M). After the reaction was completed for 30 minutes, 2.2.2-trifluoroethanol (2.5 g, 0.25 M) was added to CH2Ce2.
(25'ml) and gradually add the solution. The desired bis(2,2,2-
) fluoroethyl) phosphite (b, p, 66~
69℃/8mTlH,? ) is obtained as 2.8I.

Elemental analysis results; C4H5F603P Actual value Calculated value C; 1946%, 19.52% H; 1.97%, 2.05% Engineering R; νC1n''-', 2485 (P-H), 125
0 (p=o), 1150 (CF3), 1080 (CF3), 960 (P-H).

Example 2 Synthesis of 5'-o-monomethoxytritylthymidine 3'-()rifluoroethyl)phosphate 5'-o-monomethoxytritylthymidine (MMTr
T) (0,515,9,1,0mM) in pyridine (
7 ml) f was dissolved, bis(2,2,2-trifluoroether) phosphite (0.46ml 1.3 mM) was added, and the mixture was reacted for 3 hours at 50°C under a room floor gas atmosphere. was cooled to room temperature, further cooled to 0°C, m
- Add chloroperbenzoic acid (1.04g, 5mM) to 1
When treated for 0 minutes, the product is oxidized to phosphate. The reaction solution was added to ice water (5 mJ) and diluted with methylene chloride (10 mJ).
After extraction with A, the organic layer was extracted with 5% NaHCO
lx 3 ) and further washed with water (smJx2). Remove methylene chloride under reduced pressure, add a small amount of toluene, and repeat under reduced pressure until the pyridine odor disappears. Dissolve the residue in a small amount of methylene chloride and pour into hexane-ether (10:1) with vigorous stirring. When added, a white precipitate is formed, which is converted into 5'-o-monomethoxytritylthymidine (3'-()rifluoroether)phosphate).
(m-1), -r148-150°C) is obtained. The physical properties of the obtained product are as follows.

Rf = 0.01 (c) (2ce2:CH30H
= 9:1) pRf-0,97((CH3)2
00:H20=7:3);, MeOH UV, λ = 265.230 nm; 11aX MeOH λ -247,226 nm.

1n Example 3 Synthesis of dinucleoside 5'-o-monomethoxytritylthymidine 3'-(trifluoroethyl)phosphate) (0,6,9°0.8m
M) and 3'-〇-nonsilamidine 0.17
&, 0.53mM) in pyridine (2.7ml) KM
H, 8-quinolinesulfonyl-3-nitro 1.2
．． 4-) After adding Liashizu (0,83,9,2mM) and reacting at room temperature for 2 hours, it was processed according to the usual operation method, packed into a silica gel column chromatograph, and CH2Ce2-MeOH (98: 2) When released using (・#), the target compound is eluted.

The eluate is concentrated and the residue is dissolved in CH2Ce2 and added dropwise into vigorously stirring hexane-ether (io:i) resulting in a white precipitate. The number of precipitates corresponds to the dinucleoside [Peng TrTp-(OCH2GF3)
0.4'l of TOBz] is obtained. The physical properties of this product are as follows.

Rf=0.48 (CHzC6z:MeOH=9:
1) seOH-λ-246nm.

The chemical structures of these products were confirmed by complete deprotection of the protective groups and enzymatic decomposition. In addition, specific examples according to the above Example 2 are shown in Table 1: Table-1

Claims (1)

[Claims] 1. A dialkyl phosphite brocade conductor represented by the general formula (1) % formula % (1 (in the formula, n is an integer from 1 to 3). 2. General formula (1) % formula % (wherein n is an integer from 1 to 3).