JPH0680071B2 - Phosphoramidite compound and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel nucleoside phosphoramidite compound and a method for producing the same, and more specifically, as a protecting group for a hydroxyl group of a phosphoric acid moiety and an amino group for a basic moiety, respectively. The present invention relates to a novel nucleoside phosphoramidite compound having a protecting group capable of leaving by β-cleavage and an allyloxycarbonyl type residue, and a method for producing the same.

(Prior art) With the recent development of genetic engineering, it is an important material.
BACKGROUND ART Research on methods for chemically synthesizing polynucleotides such as DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) has been actively conducted.

Conventionally, methods such as the phosphoric acid diester method, the phosphoric acid triester method, and the phosphite method are known as chemical synthesis methods for polynucleotides, but in any case, in order to avoid side reactions, the hydroxyl group of the phosphate moiety is avoided. And, after protecting the amino group of the nucleoside base, it is subjected to a condensation reaction.

Among these, regarding the protective group for the hydroxyl group, β-cyanoethyl group, β-cyanoethyl group, β-
Β such as halogenoethyl group and β-nitroethyl group
-Methods have been developed which use protective groups which are cleaved off (eg WO85 / 00816, Tetrahedron Letters 24, 52, 5843-5846).

According to this method, deprotection is easier than with a methyl group, and thus the treatment of thiophenol, which was a problem of the conventional method, can be avoided. When applied to the solid phase synthesis method, the carrier is treated with ammonia. It has an advantage that the hydroxyl group of the phosphoric acid moiety can be deprotected at the same time when it is removed by.

However, in the case of this method, a benzoyl group, an isobutyryl group, etc. are used as a protecting group for an amino group, and in order to remove these protecting groups, it is necessary to treat with hot ammonia water for a long time. was there.

(Problems to be Solved by the Invention) Then, as a result of intensive investigations by the present inventors to solve the drawbacks of the prior art, in addition to the use of a protecting group capable of leaving by β-cleavage as a protecting group for a hydroxyl group, It was found that the use of an allyloxycarbonyl type residue as a protecting group for an amino group enables rapid deprotection under mild conditions and is extremely stable in the course of a polynucleotide synthesis reaction, thus completing the present invention. Came to.

(Means for Solving Problems) Thus, according to the present invention, a phosphoramidite compound represented by the following general formula [I] is provided as a first invention, and a second general formula [II] is provided as a second invention. There is provided a process for producing a phosphoramidite compound represented by the above general formula [I], which comprises reacting a nucleoside represented by the following formula with a phosphorus amide compound represented by the following general formula [III].

(In the formula, R ₁ and R ₂ are a hydroxyl group having a protective group or —OR ₄
The stands, R ₃ represents a hydroxyl group or -OR ₄ has a hydrogen atom, a protecting group, R ₄ is Represents a secondary amino group having 10 or less carbon atoms or a cyclic amino group having 10 or less carbon atoms which may have an unsaturated bond,
R ₅ is Represents a protecting group which is eliminated by β-cleavage,
R ₆ , R ₇ and R ₈ represent a hydrogen atom or a lower alkyl group, Z represents an electron-withdrawing group, only one of R ₁ , R ₂ and R ₃ is —OR ₄ , and B ^AOC Represents a residue of a nucleoside base in which an amino group or an imino group is protected with an allyloxycarbonyl type. ) (In the formula, R ₁ ′ and R ₂ ′ represent a hydroxyl group optionally having a protecting group, R ₃ ′ represents a hydrogen atom or a hydroxyl group optionally having a protecting group, R ₁ ′, R only one of the ₂ 'and R _3' is a hydroxyl group, B ^AOC, X and R ₅ are as defined above, cyclic Y have a have the same secondary amino group, an unsaturated bond between said X Amino group or halogen atom is represented.) The nucleoside phosphoramidite compound of the present invention has a hydroxyl group of a phosphoric acid moiety protected by a protecting group leaving by β-cleavage, and an amino group or an imino group is an allyl group in a nucleoside base. It is protected by an oxycarbonyl type residue.

In the above formula, R ₁ and R ₂ are a hydroxyl group having a protective group or —OR
₄ and the protecting group may be any of those commonly used in nucleoside chemistry. Specific examples thereof include, for example, trityl group, monomethoxytrityl group, dimethoxytrityl group, trimethylsilyl group, triethylsilyl group, triphenylsilyl group, t-butyldimethylsilyl group, tetrahydropyranyl group, 4-methoxytetrahydrofuranyl group, benzoyl. Examples thereof include a group, a benzyl group, a tetrahydrofuranyl group, a methoxymethyl group, a methoxyethoxymethyl group, a phenoxymethyl group, a methylthiomethyl group and a phenylthiomethyl group.

R ₄ is Is the residue of phosphine. Here, R ₅ is a protecting group that is eliminated by β-cleavage, that is, the following general formula [IV]
Represents a protecting group represented by.

In the formula, R ₆ , R ₇ and R ₈ represent a hydrogen atom or a lower alkyl group, and specific examples of the lower alkyl group include a methyl group and an ethyl group. Z represents an electron-withdrawing residue, and specific examples thereof include a cyano group, a nitro group, a thiocyano group, fluorine, a base, a halogen atom such as bromine, a phenylsulfonyl group, a methylsulfonyl group, and a phenyl group. In this case, the ortho-position and / or the para-position of the phenyl group portion may have a substituent such as halogen, cyano group and nitro group.

Among these protecting groups, those in which Z is a cyano group are preferable, and a β-cyanoethyl group is particularly preferred.

Specific examples of the secondary amino group having 10 or less carbon atoms represented by X include dimethylamino group, diethylamino group, diisopropylamino group, di-t-butylamino group, methylpropylamino group, methylhexylamino group, Examples include methylcyclohexylamino group, ethylbenzylamino group, and the like, and specific examples of the cyclic amino group having 10 or less carbon atoms which may have an unsaturated bond include a morpholino group, a thiomorpholino group, a pyrrolidino group, a piperidino group, and 2, Examples thereof include a 6-dimethylpiperidino group, a piperazino group, an imidazolino group and a pyrrino group.

Further, Y is a secondary amino group similar to X or a cyclic amino group which may have an unsaturated bond, or a halogen atom such as chlorine, bromine or iodine, and among them, a secondary amino group is preferable.

R ₃ is a hydrogen atom, a hydroxyl group having a protective group or —OR ₄
And the contents of the protecting group and R ₄ are the same as described above.

The nucleoside phosphoramidite of the present invention is used as a monomer for synthesizing a polynucleotide, and therefore only one of R ₁ , R _{2 and} R ₃ has -OR ₄
Has been introduced.

Further, B ^AOC is a residue of a nucleoside base in which an amino group or an imino group is protected with an allyloxycarbonyl type residue. Specific examples of nucleosides having a nucleoside base include, for example, deoxyadenosine, deoxyguanosine, deoxycytidine, thymidine, adenosine,
Examples include guanosine, cytidine, uridine, inosine and the like.

The allyloxycarbonyl type residue used as a protecting group for the amino group of the base moiety may be any as long as it does not substantially impair the deprotection reaction, and specific examples thereof include an allyloxycarbonyl group and methallyloxy group. Examples thereof include a carbonyl group, a crotyloxycarbonyl group, a prenyloxycarbonyl group, a cinnamyloxycarbonyl group, a p-chlorocinnamyloxycarbonyl group and a chloroallyloxycarbonyl group.

The carbon number of the allyloxycarbonyl type residue may be appropriately selected in consideration of separation of by-products generated after the reaction, availability of raw materials, and the like, but a carbon number of 12 or less is usually used.

The phosphoramidite compound of the present invention has the above general formula [II].
It can be obtained by reacting the nucleoside represented by the formula with the phosphorus amide compound represented by the general formula [III].

In formula (II), any _one of R ₁ ′, R ₂ ′ and R ₃ ′ is a hydroxyl group, and when they are hydroxyl groups having a protecting group, the same protecting groups as those described above are used. Things are used.

X in the general formula [III] is the same as above, and Y
Is a secondary amino group similar to X or a cyclic amino group which may have an unsaturated bond, as well as a halogen atom such as chlorine, bromine or iodine, and among them, a secondary amino group is preferable.

The nucleosides used in the present invention include unprotected nucleosides, and an allylating agent such as allyl chlorocarbonate, allyl bromcarbonate, and allyl (1-benzotriazoyl) carbonate in the presence of a base such as triethylamine and n-butyllithium. It can be easily obtained by reacting in a solvent such as tetrahydrofuran or hexamethylphosphortriamide.

Further, the phosphorus amide compound is easily prepared by reacting a phosphorus halide represented by phosphorus trichloride with an alcohol represented by the following general formula [V] and then reacting it with a secondary amine in a suitable solvent such as ether. Can be obtained.

The method for producing a phosphoramidite compound in the present invention is, except that the nucleosides represented by the general formulas [II] and [III] and a phosphorus amide compound are used as raw materials,
It is performed according to the usual method.

For example, the latter 1 to 3 mol per 1 mol of the former is charged, and the target phosphoryl is obtained by reacting at 0 to 40 ° C. for 1 to 5 hours in the presence of a solvent such as tetrahydrofuran, acetonitrile, dimethylformamide, dichloromethane and dimethylsulfoxide. An amidite compound can be efficiently obtained.

Isolation / purification of the product from the reaction solution is carried out by appropriately selecting a known means such as adsorption chromatography or ion exchange chromatography, which is a means of ordinary organic synthesis reaction, or distribution or crystallization with an organic solvent, or It is possible to carry out in combination.

(Effects of the Invention) The phosphoramidite compound of the present invention thus obtained can rapidly remove both the hydroxyl group of the phosphoric acid moiety and the protective groups of both the amino group and imino group of the nucleoside base under mild conditions. For example, after subjecting such a phosphoramidite compound to a predetermined reaction, the phosphite moiety is oxidized to phosphate, and then an O-valent palladium compound and a nucleophilic reagent typified by an amine or a formate are used under neutral conditions. When treated below, the protecting group of the nucleoside base moiety can be removed in a short time at room temperature.

In addition, deprotection of the hydroxyl group part is performed at room temperature using ammonia water.
It can be easily performed by treating for 0.1 to 5 hours. Particularly in the case of the solid phase synthesis method using a carrier, the cleavage from the carrier can be simultaneously performed during the above treatment.

Further, the protecting group and allyloxycarbonyl type residue which are eliminated by β-cleavage are generally used in the deprotection reaction of the sugar group hydroxyl group (for example, deprotection of 5′-hydroxyl group with trichloroacetic acid or tetra-n-butyl). It exists extremely stably under deprotection of 3'-hydroxyl group with ammonium fluoride).

(Example) Hereinafter, the present invention will be described more specifically with reference to Examples.

Reference Example 1 156 mmol of diisopropylamine was dissolved in 80 ml of ether, and then 1 equivalent of β-cyanoethanol was reacted with 1 equivalent of phosphorus trichloride in an organic solvent at −5 ° C. for 3 hours.
At the room temperature, add 34.7 mmol of β-cyanoethoxydichlorophosphine obtained by collecting a fraction of 110-112 ° C / 9 mmHg.
Stir for hours. After the reaction, the produced diisopropylammonium chloride was separated, and the ether solution was distilled to obtain oily β-cyanoethoxymonochloro (N, N-diisopropylamino) phosphine. The yield was 73 mol%. Boiling point was 102-104 ℃ (0.08mmHg).

Example 1 1.5 'of 5'-o-dimethoxytrityl-N-allyloxydeoxycytidine obtained by reacting 5'-o-dimethoxytrityldeoxycytidine with allyl (1-benzotriazoyl) carbonate and 1-H-tetrazole 1.56 mmol was dissolved in 12 ml of a mixed solvent of tetrahydrofuran: acetonitrile = 1: 1, and then β
2.25 mmol of -cyanoethoxy monochloro (N, N-diisopropylamino) phosphine was added at 0 ° C, then 25
Stirred at 1.5 ° C for 1.5 hours.

The mixture was diluted with ethyl acetate washed with saturated aqueous sodium bicarbonate, washed with saturated brine, the organic layer was dried over magnesium sulfate, and concentrated. Then, the residue was dissolved in a mixed solvent of toluene: hexane (3: 1) and added to hexane with vigorous stirring at -78 ° C to cause precipitation, and the precipitate was separated by filtration. The yield was 65%. When this precipitate was analyzed, the physical properties were as follows, and it was found to be the following phosphoramidite (Compound 1 ).

Elemental analysis value (C ₄₃ H ₅₂ O ₉ N ₅ P) Calcd. C63.5 H6.40 N8.61 Found. C63.3 H6.46 N8.57 ¹ H-NMR (CDCl ₃ ) Example 2 5'-o-dimethoxytrityl-N-allyloxycarbonyl-2'-deoxyadenosine 0.2 mmol and 1
After 0.21 mmol of --H-tetrazole was dissolved in 2 ml of acetonitrile, 0.3 mmol of allyloxymonochloro (diisopropylamino) phosphine was added, and the mixture was stirred at 25 ° C. for 1.5 hours. The subsequent processing is the first embodiment.
The following phosphoramidite (compound 2 )
Was obtained in a yield of 62%. The physical properties are as follows.

Elemental analysis value (C ₄₄ H ₅₅ O ₈ N ₇ P) Calcd. C63.1 H6.21 N11.7 Found. C63.0 H6.29 N11.0 ¹ H-NMR (CDCl ₃ ) Reference Example 2 (two Synthesis of dimer) Compound 1 3.12 mmol, obtained in Example 1 tetrahydrofuran: acetonitrile = 1: After dissolving the 2 mixed solvent 15 ml of, 3'-o-t- butyldimethylsilyl thymidine 2.86 Mmol and 1-H-tetrazole (3.42 mmol) were added, and the mixture was stirred at 20 ° C. for 2 hours.

Then, the mixture was cooled to −78 ° C., a dichloromethane solution of nitrogen dioxide 4.86 mmol was added, and after oxidation for 30 minutes, 30 ml of 0.5 mol sodium sulfite aqueous solution was added. After returning to room temperature, chloroform and saturated saline were added, and the separated aqueous layer was extracted with chloroform. After drying, it was separated by chromatography (silica gel 120 g, methanol: chloroform = 1: 30-1: 20), and β-cyanoethyl-
5'-o-dimethoxytrityl cytidylyl- (3 '→
5 ')-3'-o-t-butyldimethylsilylthymidine (Compound 3 ) was obtained in a yield of 80%.

The physical properties of this substance are as follows.

・ Elemental analysis value (C ₅₃ H ₆₅ O ₁₅ N ₆ SiP) Calcd. C58.67 H6.00 N7.75 Found. C58.49 H6.02 N7.88 Reference Example 3 (Deprotection of dimer) Compound 3 0.084 mmol and triphenylphosphine 0.0
252 mmol was dissolved in 1 ml of tetrahydrofuran under an argon atmosphere, 0.0042 mmol of tetrakis (triphenylphosphine) palladium (O) was added, and the mixture was stirred at room temperature for 5 minutes. Next, 36 microliters of n-butylamine was added, and after stirring for 20 minutes, the solvent was distilled off, ammonia water was reacted at room temperature for 20 minutes, then ammonia was distilled off, and the residue was chromatographed (ODS, water:
Separation with methanol = 1: 4) gave the corresponding dinucleoside phosphate (compound 8 ) from which the allyloxycarbonyl group and the cyanoethyl group were removed in a yield of 77 mol%.

・ Elemental analysis value (C ₄₆ H ₅₈ O ₁₃ N ₅ SiP) Calcd. C58.29 H6.12 N7.39 Found. C58.33 H6.06 N7.51 Reference example 4 Ester bond to CPG (Controlled Pore Grass) resin 30 mg of 5'-o-dimethoxytritylthymidine (Compound 5 ) bound via the above was placed in a glass reactor, to which a dichloromethane solution of trichloroacetic acid was added to give 5 '.
-After deprotecting the hydroxyl group, it was washed with acetonitrile. Next, a phosphoramidite in which the amino group of the cytidine base is protected by an allyloxycarbonyl group (Compound 1 )
0.03 mmol and 1-H-tetrazole 0.04 mmol were dissolved in an acetonitrile-tetrahydrofuran mixed solvent and added, and the mixture was reacted at room temperature for 2 minutes.

Then, after washing with acetonitrile, the iodine solution 1.2
C-supported on the carrier was added by adding milliliters (iodine 11.6 g, water 18 ml, lutidine 180 ml, tetrahydrofuran 720 ml) for 25 seconds.
A T dimer (compound 6 ) was obtained. The yield was 80% (measured by color development due to elimination of 5'-dimethoxytrityl group described later).

The compound was confirmed according to the following procedure. First, the carrier containing the compound 6 was washed with acetonitrile, acetic anhydride was added to cap the unreacted 5'-hydroxyl group, and acetonitrile was added to wash. Then triphenylphosphine 0.095 mmol, n-butylamine
0.6 mmol, formic acid 0.6 mmol, and tetrakis (triphenylphosphine) palladium (O) 0.015 mmol were dissolved in tetrahydrofuran and added, and the mixture was reacted at room temperature for 10 minutes to deprotect the allyloxycarbonyl group in the base moiety.

After washing with tetrahydrofuran and dichloromethane, the dimethoxytrityl group of the 5'-hydroxyl group was eliminated with trichloroacetic acid to obtain compound 7 . Then, a 30% aqueous ammonia solution was added, and the mixture was allowed to stand at room temperature for 30 minutes to remove the β-cyanoethyl group in the phosphoric acid moiety and the carrier to obtain a CT dimer (Compound 8 ).

After distilling off the ammonia, the solution was dissolved in 200 microliters of water, 5 microliters of the aqueous solution was collected, and 1 microliter of [γ- ³² P] ATP (PB-170, Amersham) was added to this to dryness. did. To this, 2 μ of a cation buffer (× 2.5), 1 μ of T ₄ nucleotide kinase (Takara Shuzo, 2.5 u / μ), and 2 μ of water were added, and cation was performed at 37 ° C. TLC (Polygram, CELL300 DEAE / HR
-2/15, manufactured by Mackerey-Nagel) was developed by RNA homomixture to obtain a one-dimensional autoradiograph, and it was confirmed that there was one spot. The position of 1 spot was extracted from TLC, digested with venom phosphodiesterase and nuclease P1, and the digests were electrophoresed using DEAE-cellulose paper, transferred to TLC, and then homomixed in a homomixture. It was developed in two dimensions. After two-dimensional development, TLC autoradiograph was taken, and it was confirmed from the position of the spot that a CT dimer with the protecting group deprotected was produced.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) References Published in West Germany 3329892 (DE, A1) Tetrahedron Letters, Vol. 25, No. 19. PP. 1975-78 (1984) Nucleic Aid Research, Vol. 12, No. 11. PP. 4539-57 (1984)

Claims (2)

[Claims] 1. A phosphoramidide compound represented by the following general formula [I]. (In the formula, R ₁ and R ₂ are a hydroxyl group having a protective group or —OR ₄
The stands, R ₃ is a hydroxyl group having a hydrogen atom, a protecting group or -
Represents OR ₄ , where R ₄ is Wherein X is a secondary amino group having 10 or less carbon atoms or a cyclic amino group having 10 or less carbon atoms which may have an unsaturated bond, and R ₅ is Represents a protecting group which is eliminated by β-cleavage,
R ₆ , R ₇ and R ₈ represent a hydrogen atom or a lower alkyl group, Z represents an electron-withdrawing group, and only one of R ₁ , R ₂ and R ₃ is —OR ₄ , and B ^AOC represents a residue of a nucleoside base in which an amino group or an imino group is protected with an allyloxycarbonyl type. ) 2. A phosphonate represented by the above general formula [I], which comprises reacting a nucleoside represented by the following general formula [II] with a phosphorus amide compound represented by the following general formula [III]. Method for producing ruamidide compound. (In the formula, R ₁ ′ and R ₂ ′ represent a hydroxyl group optionally having a protecting group, R ₃ ′ represents a hydrogen atom or a hydroxyl group optionally having a protecting group, R ₁ ′, R Only one of ₂ ′ and R ₃ ′ is a hydroxyl group, B ^AOC , X and R ₅ are the same as the above, Y is the same secondary amino group as the above X, and a cyclic amino which may have an unsaturated bond. Represents a group or halogen atom.)