JPH10259192A - Production of phosphorothioate diester - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simply obtain the subject compound for phosphorothioate oligonucleotide intermediate by allowing a nucleoside derivative to react with a thiophosphoryl compound and an alcohol in order, then removing the phosphoric acid-protecting group. SOLUTION: A nucleoside derivative of formula I [B is (protected) nucleic acid base; R1 is a protecting group for hydroxyl group; R2 and R3 are each H, (protected) hydroxyl group where at least one is hydroxyl group] is allowed to react with a thiophosphoryl compound as thiophospho-tris(triazolide), further with an alcohol represented by the formula: HO-R4 (R4 is phosphoric acid- protecting group) as ethylenecyanohydrin. Then, a deprotecting agent (M) as triethylamine is used to remove a part of the phosphoric acid-protecting group (R4 ) to prepare the objective phosphorothioate diester of formula II (M is deprotecting agent) that is useful as a starting substance for synthesizing phosphorothioate oligonucleotide in the one-pot manner according to the phosphoric triester process.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a simple synthesis of a phosphorothioate diester compound which is an intermediate for synthesizing a phosphorothioate triester compound used as a raw material for synthesizing a phosphorothioate oligonucleotide by a phosphoric acid triester method (liquid phase method). It is about the law.

[0002]

BACKGROUND OF THE INVENTION Generally,
When synthesizing a phosphorothioate oligonucleotide by the phosphate triester method (liquid phase method), a phosphorothioate triester compound is used as a raw material. As a method for synthesizing the phosphorothioate triester compound, various methods have been reported in the past, but any method may result in a low yield of the intended triester compound or a complicated reaction operation. Was not always satisfactory (J. Am. Chem. Soc., 113, 3972-3980).
(1991), Antisense Research and Development, 5, 39-
47 (1995), US Pat. No. 5,210,264).

[0003]

The present inventors have conducted intensive studies to establish a simple method for synthesizing phosphorothioate triester compounds. As a result, the present inventors have found that phosphorothioate diesters are intermediates or precursors of phosphorothioate triester compounds. The present inventors have found a method of synthesizing a compound in one pot, and have confirmed that a desired phosphorothioate triester compound can be easily derived from the obtained phosphorothioate diester compound by a known method, thereby completing the present invention.

That is, according to the present invention, after reacting a nucleoside derivative represented by the formula (I) with a thiophosphoryl compound, further reacting an alcohol (HO-R ₄ ), the deprotecting agent (M) A phosphorothioate diester compound represented by the formula (II) in one pot without isolating its intermediate by removing a part of the phosphate protecting group (R ₄ ) The method relates to a method for producing the same.

[0005]

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(Wherein B represents a nucleobase which may have a protecting group if necessary, R ₁ represents a protecting group for a hydroxyl group, and R ₂ and R ₃ represent a hydrogen atom, a hydroxyl group or a hydroxyl group having a protecting group. Wherein at least one is a hydroxyl group, R ₄ represents a phosphate protecting group, and M represents a deprotecting agent.)

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The flow chart of the method of the present invention is as follows. It is a simple method without isolating an intermediate synthesized in the course of the reaction. The greatest feature is that the phosphorothioate diester compound represented can be synthesized.

[0008]

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(Wherein B, R ₁ , R ₂ , R ₃ , R ₄ , and M have the same meanings as described above. X represents a heterocyclic residue such as imidazole, triazole, and tetrazole.)

The nucleoside derivative represented by the formula (I) is a general compound used for the synthesis of oligonucleotides, and is not particularly limited.
That is, the nucleobase represented by B includes cytosine,
Examples thereof include ordinary nucleobases such as thymine, uracil, adenine, and guanine or derivatives and analogs thereof, and the above-described nucleobases having ordinary protective groups such as benzoyl and isobutyryl. The protecting group for the hydroxyl group represented by R ₁ may be any one which is commonly used as a protecting group for the hydroxyl group at the 5′-position of a nucleoside, and specifically, dimethoxytrityl, methoxytrityl, trityl, t-butyldimethyl. Examples thereof include silyl and t-butoxydiphenylsilyl. The protecting group for the hydroxyl group represented by R ₂ and R ₃ may be any one which is usually used as a protecting group for a hydroxyl group of a nucleoside, and specifically, t-butyldimethylsilyl, tetrahydropyranyl, Acetyl, benzoyl and the like can be exemplified.

The thiophosphoryl compound (S) used in the reaction
= P (X) ₃ ) is a thiophosphorylated heterocycle,
Specific examples include thiophosphotrisimidazolide, thiophosphotristriazolide, thiophosphotristrizolidide, and the like, with thiophosphotristriazolidide being particularly preferred. Such thiophosphoryl compounds are known compounds and can be prepared by a conventional method (Bioact. Mol. 1987 (3) (Biophosphates their Analogu
es) 395-399). That is, in an organic solvent such as tetrahydrofuran, acetonitrile, dichloromethane, and dichloroethane, a halogenated thiophosphoryl such as thiophosphoryl trichloride and thiophosphoryl tribromide and a five-membered heterocyclic ring such as imidazole, triazole, and tetrazole are halogenated with thiophosphoryl halide. By using 3 moles of the 5-membered heterocycle per 1 mole, and stirring and reacting at 20 to 50 ° C for about 10 minutes to 5 hours in the presence of 3 to 20 moles of an acid trapping agent such as 1-methylimidazole, Thiophosphoryl compound can be obtained. Further, the reaction solution thus obtained can be used as it is in the method of the present invention.

The reaction between a nucleoside derivative and a thiophosphoryl compound is carried out in tetrahydrofuran, acetonitrile,
In a single or mixed solvent such as dichloromethane and dichloroethane, 1 to 2 moles of a thiophosphoryl compound is used per 1 mole of a nucleoside derivative, and is used at 20 to 50 ° C for 10 minutes to
The reaction can be carried out by stirring and reacting for about 5 hours.

After the above reaction, the alcohols (HO-
R ₄ ). The alcohol used in the reaction is not particularly limited as long as it has an protecting group for a phosphate residue represented by R ₄ which is generally used as a protecting group during oligonucleotide synthesis. In particular,
Examples thereof include alcohols having a cyanoethyl group, a cyanobutenyl group, a 4-nitrophenethyl group, and a 2-pyridylethyl group. The reaction is performed by using 2 to 5 moles of alcohol per 1 mole of the nucleoside derivative, and using 20 to 50 alcohols.
The reaction can be carried out by stirring the mixture at a temperature of about 10 minutes to about 5 hours.

Finally, a part of the phosphate protecting group (R ₄ ) is removed using a deprotecting agent (M) to obtain a phosphorothioate diester compound represented by the formula (II) in the above flowchart. As a deprotecting agent used in the reaction, a base usually used for removing a phosphate protecting group (R ₄ ) may be used. For example, when R ₄ is a cyanoethyl group, triethylamine, diisopropylamine, diisopropylethylamine, tributylamine, N-methylmorpholine, ammonia and the like can be used.
The reaction is carried out by reacting 1 mole of the nucleoside derivative with 1 mole of the deprotecting agent.
The reaction can be carried out by stirring and reacting at 20 to 50 ° C. for about 10 minutes to 5 hours.

The phosphorothioate diester compound represented by the formula (II) thus obtained can be isolated and purified by a conventional method. That is, the target compound can be isolated and purified by partitioning the reaction solution with an organic solvent such as chloroform and then subjecting the reaction solution to silica gel column chromatography. After isolation and purification, a phosphorothioate triester compound is synthesized by a conventional method and used as a raw material for synthesizing a phosphorothioate oligonucleotide.

[0016]

The method of the present invention is a very practical method which is extremely simple in operation, has a high yield and can synthesize a phosphorothioate diester compound in one pot as compared with the conventional method.

[0017]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to examples and reference examples, but the present invention is not limited to these examples. Example 1 Compound of Formula (II) wherein B = thymine, R
₁ = DMTr, R ₃ = H, R ₄ = cyanoethyl, M = triethylamine) Synthesis of 1,2,4-triazole (420 mg) in THF (3
Thiophosphorochloride (0.2 ml) was added to a 1-methylimidazole (0.5 ml) solution, and the mixture was stirred at room temperature for 30 minutes, and then 5′-O-DMTr-thymidine (436).
mg), and the mixture was further stirred for 90 minutes. To this reaction solution was added cyanoethylhydrin (0.28 ml), and the mixture was stirred for 2 hours. Triethylamine (1.4 ml) and water (5.0 ml) were added.
l) and pyridine (10 ml) were added, and the mixture was further stirred for 1 hour. This reaction solution was partitioned between water (50 ml) and chloroform (50 ml), and the aqueous layer was separated into chloroform (50 ml).
l), the combined chloroform layer was
lTEAB buffer (50ml), then water (50m
After washing with 1) and drying over sodium sulfate, the solvent was concentrated under reduced pressure. The residue was purified by a silica gel column [chloroform (containing 1% triethylamine) / methanol] to obtain 630 mg (99.2%) of the desired compound.

MS (FAB <0) 692 ³¹ P-NMR (CD ₃ CN) ¹ H-NMR (CDCl ₃ ) d 7.61 (s, 1H,
_{H 6), 7.42-6.83 (m,} 13H, aroma
tic H and CHCl ₃ ), 6.43 (m, 1
H, H1 _' ), 5.31 (m, 1H, H3 _' ), 4.37.
(M, 1H, H4 _' ), 4.28-3.95 (m, 2H,
_{P-OCH 3), 3.79 (} s, 6H, OCH 3), 3.
55-3.38 (m, 2H, H5 _' and H5 _'' ),
_{2.80 (q, Et 3 N)} , 2.75-2.36 (m,
4H, H _{2 ′} , H _{2 ″} and CH ₂ CN), 1.37 ｛2
s, 3H, CH ₃ (thymin)｝, 1.19 (t,
Et ₃ N)

Example 2: Compound of formula (II) wherein B
= N ² - isobutyryl _{guanine, R 1 = DMTr, R 3} =
H, R ₄ = cyanoethyl, M = triethylamine) As in Example 1, N ² -isobutyryl-5′-DMT
570 mg of the target compound was obtained from r-deoxyguanosine (512 mg) in a yield (73.3%). MS (FA
B <0) 787

Example 3: Compound of formula (II) wherein B
= N ⁶ -benzoyl adenine, R ₁ = DMTr, R ₃ =
H, R ₄ = cyanoethyl, M = triethylamine) Synthesis of N ⁶ -benzoyl-5′-DMTr in the same manner as in Example 1.
-The desired compound was obtained from deoxyadenosine (526 mg) in a yield of 670 mg (92.2%). MS (FAB
<0) 805

Example 4: Compound of formula (II) wherein B
= N ⁴ - benzoyl _{cytosine, R 1 = DMTr, R 3} =
Synthesis of H, R ₄ = cyanoethyl, M = triethylamine) As in Example 1, N ⁴ -benzoyl-5′-DMTr
-Deoxycytidine (507 mg) to give the desired compound 5
Obtained in 90 mg, yield (83.6%). MS (FAB <
0) 781

Reference Example 1: Synthesis of phosphorothioate triester compound

[0023]

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(Wherein B, R ₁ , R ₃ , R ₄ and M have the same meanings as described above.) The phosphorothioate diester compound obtained in Example 1 (where B = thymine, R ₁ = DMTr, R ₃ = H, R ₄
= Cyanoethyl, M = triethylamine) (6.35)
g) in acetonitrile (160 ml) solution
Lutidine (1.9 ml) and 4-nitrobenzyl bromide (6.9 g) were added, and after stirring at room temperature for 4 hours, the solvent was concentrated under reduced pressure. The residue was partitioned between ethyl acetate (100 ml) and water (100 ml). The ethyl acetate layer was washed with saturated aqueous sodium hydrogen carbonate, dried over sodium sulfate, and concentrated under reduced pressure.
The obtained residue was purified by a silica gel column (chloroform / methanol) to obtain 4.09 g (61.7%) of a phosphorothioate triester compound.

NMR (CDCl ₃ ) 8.18 [m, 2H, H meta (p-nitrob)
Enzyl)], 7.51 [m, 2H, Hortho
(P-nitrobenzyl)], 7.36-6.8.
_{2 (m, 14H, H 6} and DMTr), 6.36
(M, 1H, H1 _' ), 5.20 (m, 1H, H3 _' ),
4.25-4.09 (m, 5H, P- OCH 2, P-S
CH ₂ and H _{4 ′} ), 3.79 (s, 6H, OC
H ₃ ), 3.48 and 3.33 (2 m, 2H, H _{5 ′)}
and H5 _'' ), 2.73-2.39 (m, 4H, C
H ₂ CN, H _{2 ′} and H _{2 ″} ), 1.44 [s, 3
H, CH ₃ (thymine)].

Claims (10)

[Claims] 1. A method comprising reacting a nucleoside derivative represented by the formula (I) with a thiophosphoryl compound, further reacting an alcohol (HO-R ₄ ), and phosphoric acid using a deprotecting agent (M). A part of the protecting group (R ₄ ) is removed, and the compound represented by the formula (I)
A process for producing a phosphorothioate diester compound, which comprises synthesizing the phosphorothioate diester compound represented by I) in one pot without isolating an intermediate thereof. Embedded image (Wherein, B represents a nucleobase that may have a protecting group as necessary, R ₁ represents a protecting group for a hydroxyl group, R ₂ and R ₃ represent a hydrogen atom, a hydroxyl group or a hydroxyl group having a protecting group, However, at least one is a hydroxyl group.) (Wherein, R ₄ represents a phosphate protecting group, M represents a deprotecting agent, and B, R ₁ and R ₃ have the same meanings as described above.) 2. The method according to claim 1, wherein the thiophosphoryl compound is a thiophosphorylated heterocycle. 3. The thiophosphoryl compound is selected from thiophosphotrisimidazolide, thiophosphotristriazolide and thiophosphotristetrazolide.
The method of claim 1. 4. The method according to claim 1, wherein the thiophosphoryl compound is thiophosphotristriazolide. 5. The method according to claim 1, wherein the alcohol (HO-R ₄ ) is an alcohol having a cyanoethyl group, a cyanobutenyl group, a 4-nitrophenethyl group, or a 2-pyridylethyl group as R ₄ . 6. The method according to claim 1, wherein the alcohol (HO-R ₄ ) is cyanoethylhydrin. 7. The method according to claim 1, wherein the deprotecting agent (M) is a base.
The described method. 8. The method according to claim 1, wherein the deprotecting agent (M) is triethylamine. 9. The method according to claim 1, wherein the deprotecting agent (M) is ammonia. 10. The method according to claim 1, wherein the thiophosphoryl compound is thiophosphotristriazolide, the alcohols (HO-R ₄ ) are cyanoethylhydrin, and the deprotecting agent (M) is triethylamine.