JPH0427992B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing nucleotides by phosphorylating various nucleosides. Deoxyribonucleotides and ribonucleotides are important as raw materials for the synthesis of various DNAs or RNAs. These are manufactured by reacting deoxyribonucleosides or ribonucleosides with a phosphorylating agent. Examples of the phosphorylating agent include p-chlorophenyl-β-cyanoethyl phosphorochloridate (Helv.Chim,
Acha, 64 2142 (1981)), cyclohexylammonium p-chlorophenyl-β-cyanoethyl phosphate (Tetrahedron Lett., 22 851)
(1981)), p-chlorophenyl phosphoroditriazolide (J.Biol.Chem. 250 4592 (1975))
etc. are used. However, and are inconvenient because they are prepared immediately before use due to stability problems, and although is a stable compound, it takes some time to remove the protective group after phosphorylation, so it is not always satisfactory. It wasn't. The present inventor completed the present invention as a result of research on the production of nucleotides using a more excellent phosphorylating agent. That is, the present invention provides nucleosides including cyclohexylammonium o-chlorophenyl-β
- A method for producing nucleotides by reacting cyanoethyl phosphate. The reaction formula for this method is as follows. In the formula, B means a base with or without a protecting group such as N-benzoylcytosine, N-benzoyladenine, N-isobutyrylguanine or thymine, and R ¹ is protected with H or a protecting group for the hydroxyl group of a sugar. and R ² means a protecting group for the hydroxyl group of sugars such as dimethoxytrityl. Phosphorizing agent cyclohexylammonium o-
Although chlorophenyl-β-cyanoethyl phosphate () is a new substance, it is produced by the method shown in the reference example. In order to produce a nucleotide (), the compound () may be reacted with equimolar to several times the mole of the nucleoside () at a temperature of 10° to 50°C, usually at room temperature. This reaction is advantageously carried out in the presence of a suitable condensing agent, such as arylsulfonyl chlorides such as 2,4,6-triisopropylphenylsulfonyl chloride and mesitylenesulfonyl chloride, and their combinations such as 1-methylimidazole. In combination with bases, nucleosides () usually have a
It is appropriate to use 10 times the molar amount. The reaction time varies depending on the type of condensing agent, but is usually about 30 minutes to 24 hours. The generated nucleotides () can be derived into various oligonucleotides by removing protective groups at appropriate positions and condensing them with other nucleotides. As methods for removing protective groups, for example, o-chlorophenyl groups can be removed using tetramethylguanidinium pyridine aldoxime, 4-nitrobenzaldoxime, etc., and cyanoethyl groups can be removed using a base such as triethylamine. can. Furthermore, the protective group for the sugar hydroxyl group can be removed by a known general method. For example, dimethoxytrityl can be removed using acids such as benzenesulfonic acid, trichloroacetic acid, or zinc bromide. The acyl group of the base moiety can be removed by a general method using concentrated aqueous ammonia or the like. Example 1 5'-O-dimethoxytrityl-2'-deoxyribonucleoside (: R ¹ = H, R ² = 4,4'-dimethoxytrityl) and cyclohexylammonium o-chlorophenyl-β-cyanoethyl phosphate (1.44 g, 4.00 mmol), add an appropriate amount of pyridine, and dry under reduced pressure. Do this operation three times. Add 8 ml of pyridine to the residue and add 2.
Add 4,6-triisopropylphenylsulfonyl chloride (2.42 g, 8 mmol) and N-methylimidazole (1.31 g, 16 mmol) and leave at room temperature for 30 or 60 minutes. Then, add 1 ml of ice-cooled water and concentrate. Add 50ml of chloroform to this
and 50 ml of 5% aqueous sodium bicarbonate solution. Extract with chloroform (50 ml x 2), wash the chloroform layer with 100 ml of water, concentrate, and purify by silica gel column chromatography. Concentrate the fraction eluted with chloroform-methanol (50/1, v/v), add n-pentane, and collect the powdered target product (: R ¹ = H, R ² = 4.4'-dimethoxytrityl) by filtration. . The base moieties of the raw materials and the target product, the number of moles of the raw materials used, the reaction time, the yield of the target product, and the Rf value are shown in the table.

[Table] Example 2 Using 1.09 g (2 mmol) of 5'-0-dimethoxytrityl-2'-deoxythymidine, mesitylenesulfonyl chloride was substituted for the 2,4,6-triisopropylphenyl sulfonyl chloride of Example 1. Using 1.75 g (8 mmol), the reaction was carried out in the same manner as in Example 1, and post-treatment was performed to obtain 5'-0-dimethoxytrityl-2'-deoxythymidine 3'-(o
-chlorophenyl-β-cyanoethyl phosphate) 1.0 g (64%) are obtained. Example 3 Using 1.09 g (2 mmol) of 5'-0-dimethoxytrityl-2'-deoxythymidine, the reaction was carried out in the same manner as in Example 1 except for the 1-methylimidazole in Example 1, and after 24 hours, the same reaction was carried out. After post-processing, 5′−
0-dimethoxytrityl-2'-deoxythymidine
1.06 g (67%) of 3'-(o-chlorophenyl-β-cyanoethyl phosphate) is obtained. Reference example o-chlorophenyl phosphorodichloridate
Dissolve 11.4g in 100ml of dioxane and add β-
Add 10.7 g of cyanoethanol and stir at room temperature for 30 minutes. Next, 8.7 g of pyridine was slowly added dropwise to this. After 4 hours, the mixture was checked by thin layer chromatography to confirm the formation of biscyanoethyl compound (Rf 0.50, chloroform-methanol = 10:1), and concentrated. The precipitate was removed by filtration, and the filtrate was concentrated. To the obtained oil, 200 ml of a 5% aqueous sodium bicarbonate solution and 200 ml of methylene chloride were added, and the mixture was further extracted with methylene chloride (200 ml x 2). The methylene chloride layer was washed with 300 ml of water and concentrated to obtain 15.42 g (98%) of o-chlorophenyl-bis(β-cyanoethyl) phosphate. Dissolve 3.09 g of this in 15 ml of acetonitrile, add 1.95 g of cyclohexylamine, and bring to room temperature.
If left for 30 minutes, crystals will precipitate. After leaving it in the refrigerator, filter the crystals. Cyclohexylammonium o-chlorophenyl-β-
3.10 g (87%) of cyanoethyl phosphate are obtained. Melting point 114-116℃. ¹ H-NMR (CDCl ₃ ) δ: 2.66 (t, 2H, J _HH = 7HzCH ₂ CH ₂ CN) 4.16 (dt, 2H, J _HH = J _HP = 7Hz POCH ₂ ) 6.93-7.58 (m, 4Harom) 8.21 (bs, 3H, _H3N +-) IR (KBr) ν (cm ^-1 ): 2930 (N+ _H3 ) 2250 (CN) 1240 (P=O).

Claims (1)

[Claims] 1. A method for producing nucleotides, which comprises reacting nucleosides with cyclohexylammonium o-chlorophenyl-β-cyanoethyl phosphate.