JPH0680070B2 - Phosphoramidide compound and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel nucleoside phosphoramidide compound and a method for producing the same, more specifically, as a protecting group for a hydroxyl group of a phosphoric acid moiety and an amino group of a base moiety. The present invention relates to a novel nucleoside phosphoramidide compound having an allyl type residue and an allyloxycarbonyl type residue, respectively, 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.

Thus, a methyl group or a cyanoethyl group has been conventionally known as a protective group for a hydroxyl group, and a benzoyl group, an isobutyryl group, an anisoyl group or the like is known as a protective group for an amino group (for example, JP-A-57- 176998, Tetrahedron Letters 24, 52, 5843-5846).

However, these methods have a problem that thiophenol must be used for deprotection after a predetermined reaction or treatment with hot ammonia water must be carried out for a long time, and the hydroxyl group and the amino group must be deprotected by separate operations. There is a problem that the operation becomes complicated because it has to be done.

(Problems to be Solved by the Invention) Then, as a result of intensive investigations by the present inventors to solve the above-mentioned drawbacks of the prior art, an allyl-type residue was used as a hydroxyl-protecting group, and as an amino-group protecting group It was found that the use of an allyloxycarbonyl type residue enables simultaneous and rapid deprotection under mild conditions, and is extremely stable in the process of polynucleotide synthesis reaction, leading to the completion of the present invention.

(Means for Solving Problems) Thus, according to the present invention, a phosphoramidide compound represented by the following general formula [I] is provided as a first invention, and a second general formula [II ] A method for producing a phosphoramidide compound represented by the above general formula [I] is provided by reacting a nucleoside represented by the above formula with a phosphoramide 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,
A represents an allyl group residue, only one of R ₁ , R ₂ and R ₃ is —OR ₄ , and B ^AOC is a nucleoside base having no amino group or an allyloxy group having an amino group or imino group. Represents a carbonyl-protected residue of a nucleoside base. ) (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 A are the same as above, Y is the same secondary amino group as X above, and a cyclic amino group which may have an unsaturated bond. Or a halogen atom.) The nucleoside phosphoramidide compound of the present invention has a hydroxyl group of a phosphoric acid moiety protected by an allyl-type residue, and when an amino group is present in the nucleoside base, the amino group is an allyl group. It is protected by an oxycarbonyl type residue. When the nucleoside base contains only an imino group, the imino group may or may not be protected by an allyloxycarbonyl 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. The allyl type residue represented by A here may be any as long as it does not substantially impair the deprotection reaction. Specific examples thereof include an allyl group,
Examples thereof include a methallyl group, a crotyl group, a prenyl group, a geranyl group, a cinnamyl group, and a p-chlorocinnamyl group. The number of carbon atoms of these protecting groups is not particularly limited,
Usually, 10 or less is used.

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 nucleoside base having no amino group or a nucleoside base residue 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, guanosine, cytidine, uridine, inosine and the like are exemplified, and among these, thymidine, uridine and inosine belong to those having no amino group.

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 phosphoramidide 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.

The phosphorus amide compound is prepared by reacting hydroxydihalogenophosphine such as hydroxydichlorophosphine with an allylating agent typified by allyl bromide to give allyloxydihalogenophosphine, and then using a secondary amine in an appropriate solvent such as ether. It can be easily obtained by reacting with.

The method for producing the phosphoramidide compound in the present invention is, except that the nucleosides represented by the general formulas [II] and [III] and the 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 amidide 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.

(Effect of the Invention) The phosphoramidide compound of the present invention thus obtained is capable of simultaneously and rapidly removing both the hydroxyl group of the phosphate moiety and the protective groups of both the amino group and the imino group of the nucleoside base under mild conditions. You can For example, after subjecting such a phosphoramidide compound to a predetermined reaction,
When the phosphite moiety is oxidized to phosphate and treated with neutral O-containing palladium compounds and nucleophiles represented by amines and formates under neutral conditions, both are protected in a short time at room temperature. The group can be removed.

Further, the allyl type residue and the allyloxycarbonyl type residue are generally used in the deprotection reaction of the sugar group hydroxyl group (for example, deprotection of 5'-hydroxyl group with trichloroacetic acid or 3 with tetra-n-butylammonium fluoride). It exists extremely stably under the conditions of ammonia treatment, which is used for the removal of carriers such as the deprotection of'-hydroxyl group).

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

Reference Example 1 Allyloxydichlorophosphine, 34.7, was prepared by dissolving 156 mmol of dimethylamine in 80 ml of ether.
After adding mmol, the mixture was stirred at room temperature for 12 hours. After the reaction, the produced dimethylammonium chloride was separated, and the ether solution was distilled to obtain oily allyloxybis (N, N-dimethylamino) phosphine. The yield was 76 mol%. The physical properties are as follows. Boiling point 93-94 ℃ (48mm
Hg) ¹ H-NMR (C ₆ D ₆ ) 2.55 (d, J = 12.8Hz, 12H, 4NC
H ₃ ), 4.15 (ddt, J = 11.0,4.8,1.5Hz, 2H, C = CC
H ₂ ), 5.08 (ddd, J = 10.1,2.0,15Hz, 1H, cis CH ＝ CHC
H ₂ ), 5.34 (ddd, J = 15.1, 2.0, 1.5Hz, 1H, trans CH
= CHCH ₂ ), 5.90 (ddt, J = 15.1, 10.1, 4.8Hz, 1H, CH ₂
= CHCH ₂ ) ³¹ P NMR (C ₆ D ₆ -C ₆ H ₆ 1: 4) 138.36 Reference Example 2 Reaction was performed in the same manner as in Example 1 except that diisopropylamine was used instead of dimethylamine, and allyloxy was performed. Bis (N, N-diisopropylamino) phosphine was obtained. The yield was 55 mol%.

Boiling point 130-133 ° C / 6mmHg ¹ H NMR (C ₆ D ₆ ) 1.17 (dd, J = 7.8,1.8Hz, 24H, 4NCH
(CH ₃ ) ₂ ), 3.53 (d · sept, J = 10.8,7.8Hz, 4H, 4
NCH), 4.10 (ddt, J = 10,5,2Hz, 2H, C = CCH ₂ ), 5.
07 (m, 1H, cis CH = CHCH ₂ ), 5.33 (m, 1H, trans
CH = CHCH ₂ ), 5.93 (ddt, J = 18,10,5Hz, 1H, CH ₂ = CH
CH ₂ ) ³¹ P NMR (C ₆ D ₆ —C ₆ H ₆ , 1: 4) 123.58 ppm Example 1 5 mmol of 5′-o-monomethoxytritylthymidine and 1.56 mmol of 1-H-tetrazole were added to tetrahydrofuran: acetonitrile = After dissolving in 12 ml of a 1: 1 mixed solvent, 2.25 mmol of allyloxybis (N, N-dimethylamino) phosphine was added at 0 ° C., then
The mixture was stirred at 25 ° 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 a white precipitate was separated by filtration. The yield was 71%. When this precipitate was analyzed, the physical properties were as follows:
It was found to be the phosphoramidide (Compound 1) shown in the table. ¹ H NMR (C ₆ D ₆ ) 1.55 (brs, 3H, C (5) CH ₃ ), 2.1−
2.6 (m, 8H, 2NCH ₃ , 2H ₂ '), 3.3-3.6 (m, 5H, 2
_{H 5 ', OCH 3),} 4.1-4.4 (m, 3H, H 4', C = CCH 2),
_{4.95-5.4 (m, 3H, CH 2} = CCH 3 '), 5.6-6.0 (m, 1
H, C = CHC), 6.53 (t-like, J = 6Hz, 1H, H ₁ '),
6.7-6.9, 7.0-7.7 (m, 15, Aromatic H, C (5)
H), 10.5 (twos, 1H, N (3) H) IR (KBr) 3440,3180,1720,1700,1610,1470,1260cm ^-1 UV (CH ₃ CN) λ _max = 265.3nm (ε = 1.11 × 10 ⁴ ) 233.9 (ε = 1.56 × 10 ⁴ ) Anal Calcd for C ₃₅ N ₄₀ N ₃ O ₇ P: C, 65.10; H, 6.26; N, 6.5
0Found: C, 65.42H, 6.20, N, 6.45 Example 2 3 mmol of 5'-o-monomethoxytritylthymidine and 3.1 mmol of 1-H-tetrazole were dissolved in 20 ml of a mixed solvent of tetrahydrofuran: acetonitrile = 1: 1. Then, 4.5 mmol of allyloxybis (N, N-diisopropylamino) phosphine was added at 0 ° C, and then the mixture was stirred at 25 ° C for 1.5 hours. Then, after mixing with an equal mixture of ethyl acetate and saturated saline, the separated organic layer was washed with saturated saline, and the organic layer was dried over magnesium sulfate and then concentrated. The residue is then dissolved in toluene,
The mixture was added to hexane while stirring at -78 ° C to cause precipitation, and the precipitate was separated by filtration. The yield was 76%. The physical properties of this precipitate were as follows, and were found to be the phosphoramidite (Compound 2) shown in Table 1. ¹ H NMR (C ₆ D ₆ ) 1.10 (dd, J = 8.2 Hz, 12 H, 2 NCH (C
H ₃ ) ₂ 1.57, 1.60 (brs, 3H, C (5) CH ₃ ), 2.1-2.7
(M, 2H, 2H ₂ '), 3.15 (m, 2H, 2NCH), 3.4 (m,
5H, 2H ₅ ', OCH ₃ ), 4.1 (m, 1H, H ₄ '), 4.38 (m,
2H, C = CCH ₂ ), 5.03 (m, 1H, cis CH = CHCH ₂ ), 5.1
0 (m, 1H, H ₃ '), 5.30 (m, 1H, trans CH = CHC
H ₂ ), 5.77 (m, 1H, C = CHCH ₂ ), 6.50 (t-like, J
= 6Hz, 1H, H ₁ '), 6.73-6.9, 7.0-7.7 (m, 15H, Ar
omatic H, C (5) H), 10.4 (brs, 1H, N (3)
H) IR (KBr) 3420,3180,1720,1690,1600,1450,1250cm ^-1 UV (CH ₃ CN) λ _max = 264.7nm (ε = 1.20 × 10 ⁴ ) 233.3nm (ε = 1.64 × 10 ⁴ ) Anal.Calcd for C ₃₉ H ₄₈ N ₃ O ₇ P: C, 66.74; H, 6.91; N, 5.98
Found: C, 66.44; H, 6.87; N, 5.96 Example 3 5'-o-monomethoxytrityl-N-allyloxycarbonyl-2'-deoxyadenosine 0.2 mmol and 1-H-tetrazole 0.21 mmol in acetonitrile 2
After dissolving in milliliter, 0.3 mmol of allyloxybis (morpholino) phosphine was added, and the mixture was stirred at 25 ° C. for 1.5 hours.

Then, dilute the reaction solution with 30 ml of ethyl acetate,
It was then mixed with 15 ml of 0.1 molar potassium dihydrogen phosphate-sodium hydroxide buffer (pH 7.0). The separated organic layer was washed with the same buffer solution, dried over magnesium sulfate, and then concentrated. Then, the residue was subjected to silica gel column chromatography (methanol: chloroform = 1: 5).
Separation in 0) gave phosphoramidide (Compound 3 ) shown in Table 1 in a yield of 83%.

The physical properties are as follows. ¹ H NMR (CDCl ₃ ) δ2.5-3.2 (m, 6H, 2H ₂ ′, 2PNC
_{H 2), 6.25-3.85 (m,} 9H, 2H 5 ', 2PNCCHO, OCH 3),
4.2-4.7 (m, 3H, allic CH ₂ , H ₄ ′), 4.74 (d, 2
H, J = 6.7Hz, ACO- CH 2), 5.1-5.7 (m, 5H, 2C = C
_{H 2, H 3 '),} 5.85-6.2 (m, 2H, 2CCH = C), 6.47 (d
d, J = 6,7 Hz, 1H, H ₁ ′) 6.7−6.93, 7.15−7.55 (m,
14H, aromatic H), 8.07 (s, 1H, H ₂ ), 8.60 (brs,
1H, NHCO), 8.64 (s , 1H, H 8) IR (CHCl 3) 3425,3050,1770,1620,1610,1520,1480,1260
cm ^-1 UV (CH ₃ ON) λ _max = 266.5,236.9,223.5nm Anal.Calcd for C ₄₁ H ₄₅ N ₆ O ₈ P: C, 63.06; H, 5.82; N, 10.76
Found C, 62.91; H, 6.03; N, 10.95 Reference Example 3 (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 4.86 mmol of nitrogen dioxide was added, and after oxidizing for 30 minutes, 30 ml of a 0.5 mol aqueous sodium malsulfate 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 allyl-5′-o-monomethoxytritylthymidylyl- (3 ′ → 5 ′)-3 ′.
-Ot-Butyldimethylsilylthymidine (Compound 4 )
Was obtained in a yield of 86%.

The yield value of this substance is as follows.

R _f (SiO ₂ , methanol-ethyl acetate-hexane = 1: 5
0:25) 0.38 ¹ H NMR (CDCl ₃ ) 0.09 (s, 6H, 2SiCH ₃ ), 0.89 (s, 9H,
_{SiC (CH 3) 3),} 1.41,1.90 (each s, 6H, 25-CH 3),
2.1-2.8 (m, 4H, 4H ₂ '), 3.44 (m, 2H, 2H ₅ '),
_{3.79 (s, 3H, OCH 3} ), 3.95-4.7 (m, 7H, 2H 4 ',
_{H 3 ', 2H 5',} CH 2 CH =), 5.1-5.5 (m, 3H, H 3 ', =
_{CH 2), 5.65-6.08 (m,} 1H, CH = CH 2), 6.1-6.4
(M, 2H, 2H ₁ '), 6.8, 7.15-7.5 (m, 14H, Ar-
H), 7.67 (brs, 2H, 6-H), 9.48 (brs, 2H, 2N)
H) IR (CHCl ₃ ) 3350,2900,1700,1680,1595,1250cm ^-1 UV (CH ₃ OH) λ _max = 266nm Anal.Calcd for C ₄₉ H ₆₁ N ₄ O ₁₃ PSi; C, 60.47; H, 6.33; N, 5.7
5 Found; C, 60.65; H, 6.08; N, 5.71 ³¹ P NMR (CDCl ₃ : C ₆ H ₆ , 1: 1) 10.30,9.42 Reference Example 4 (Deprotection of dimer) Compound 4 0.084 mmol and tri Phenylphosphine
0.0252 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. Then, n-butylamine 36
After adding microliters and stirring for 20 minutes, the solvent was distilled off and the residue was separated by chromatography (ODS, water: methanol = 1: 4) to give the corresponding dinucleoside phosphate (compound 5 ) Was obtained in a yield of 80 mol%.

Reference Example 5 (Synthesis of Dimer) Allyl-5'- was tested according to Reference Example 1 except that Compound 3 was used instead of Compound 1 used in Reference Example 3.
o-monomethoxytrityldeoxyadenosilyl-
(3 ′ → 5 ′)-3′-o-t-butyldimethylsilylthymidine (Compound 6 ) was obtained. The yield was 85%.

The physical properties of this substance are as follows.

Elemental analysis value Calculated value C, 59.70; H, 6.06; N, 9.20 Actual value C, 59.85; H, 6.01; N, 9.14 Reference example 6 (Deprotection of dimer) 0.103 mmol of compound 6 was dissolved in 2 ml of tetrahydrofuran. After that, 0.406 mmol of n-butylamine and 0.434 mmol of formic acid were sequentially added dropwise. Then tetrakis (triphenylphosphine) palladium (O)
0.0104 mmol was dissolved in 1.5 ml of tetrahydrofuran and added, and the mixture was stirred at room temperature for 20 minutes.

After the reaction, the solvent was distilled off, ethyl acetate and water were added to separate the organic layer and the aqueous layer, and the organic layer was washed with saturated saline. Then, the organic layer is dried over magnesium sulfate and then concentrated,
The residue was separated by chromatography to obtain a dinucleoside phosphate (compound 7 ) from which the allyl group of the phosphorus part of compound 6 and the allyloxycarbonyl group of the nucleoside base part were removed in a yield of 90 mol%.

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 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,
A represents an allyl-type residue, only one of R ₁ , R ₂ and R ₃ is —OR ₄ , and B ^AOC is a nucleoside base having no amino group or an allyloxy group having an amino group or imino group. Represents a carbonyl-protected nucleoside residue. ) 2. A phosphoramid 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 daid 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 A are the same as above, Y is the same secondary amino group as X above, and a cyclic amino group which may have an unsaturated bond. Or represents a halogen atom.)