JP2000264897A - Production of 5'-o-tritylnucleosides - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject compound by selectively tritylating the 5'-position of an unprotected nucleoside by reacting a nucleoside with a tritylating agent using an azole salt as a reaction promoter. SOLUTION: An unprotected nucleoside (preferably ribonucleoside, deoxyribonucleoside or a 2'-O-alkylribonucleoside) in an amount of 1 mol is reacted with 1-2 mols of a tritylating agent (preferably a trityl halide) by using preferably 1-3 mols of an azole salt (preferably imidazole, triazole, tetrazole or its derivative or its homolog hydrochloride, nitrate, perchlorate or sulfonate) and preferably further 1-3 mols of an amine (e.g. diisopropylethylamine, etc.), preferably at 20-40 deg.C for 1-50 hours to give the objective compound. The reaction is carried out in dimethylformamide alone or in its mixed solvent.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for selectively tritylating the 5'-position of an unprotected nucleoside.

[0002]

2. Description of the Related Art In the current chemical synthesis of oligonucleotides such as DNA, a method called a phosphoramidite method is most widely used in both solid phase and liquid phase methods. The phosphoramidite method is a method of synthesizing an oligonucleotide having a target sequence by sequentially condensing amidite blocks of nucleosides.However, in order to suppress a side reaction during synthesis, the amino acid of the nucleobase portion of the amidite block is synthesized. A group protected with a protecting group was usually used.

However, the use of such a protecting group requires two extra steps of introduction and deprotection of the protecting group, which is one of the causes of a decrease in the yield of the target oligonucleotide. This is not necessarily the preferred method. Recently, as a method of overcoming such a problem, studies have been actively conducted to chemically synthesize an oligonucleotide using an unprotected amidite block that does not protect the amino group of the nucleobase portion of the amidite block with a protecting group. It is getting big results.

[0004]

In order to make a method for chemically synthesizing an oligonucleotide using such an unprotected amidite block more practical, it is necessary to prepare an amidite block of the corresponding nucleoside and an amidite block for preparing the amidite block. It is necessary to efficiently synthesize a trityl form of a nucleoside such as a dimethoxytrityl (DMT) form by a simple method. Like the oligonucleotides described above,
For example, if a method for selectively tritylating the 5'-position of an unprotected nucleoside is developed, it is expected that an unprotected amidite block can be efficiently synthesized, and that an oligonucleotide can be prepared with high yield.

Heretofore, some methods have been reported for selectively tritylating the 5'-position of unprotected nucleosides (J. Am. Chem. Soc., 104, 1316-1319 (1982);
Am. Chem. Soc., 85, 3821-3827 (1963), JP-A-1-30
8294), all of which have problems such as a complicated synthesis method, a low synthesis yield, and application only to specific nucleosides, and are applicable to all nucleosides, and are efficient. No specific method has been reported. In particular, guanosine is a nucleoside which is difficult to selectively trityl at the 5'-position in an unprotected state, and once protecting the amino group at the 2-position and tritylating the 5'-position, the protecting group for the amino group is removed. Deprotection is currently considered the most practical and efficient method.

[0006]

Means for Solving the Problems The present inventors have made intensive studies to overcome the above problems, and as a result, by using a reaction accelerator such as imidazolium methanesulfonate,
The present inventors have found that the 5'-position of the unprotected nucleoside can be selectively tritylated, and that this method is not limited to a particular nucleoside, but is applicable to all nucleosides, thus completing the present invention. Therefore, the present invention
The unprotected nucleoside is reacted with a trityling agent to form 5 ′
The present invention relates to a method for producing -O-trityl nucleosides, which uses azole salts as a reaction accelerator. The present invention also relates to a method for producing 5'-O-trityl nucleosides by reacting an unprotected nucleoside with a tritylating agent, wherein the sulfonate of imidazole as a salt of azole and the trialkyl as an amine. It relates to a method using an amine. Further, the present invention relates to a method for producing 5'-O-trityl nucleosides by reacting an unprotected nucleoside with a tritylating agent, comprising imidazolium methanesulfonateol as an azole salt and diisopropyl as an amine. The present invention relates to a method using ethylamine.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The nucleosides to be used in the present invention can be used as long as they are used for the synthesis of oligonucleotides, for example, ribonucleosides such as guanosine, adenosine, cytidine and uridine. 2'-deoxyribonucleoside such as' -deoxyguanosine, 2'-deoxyadenosine, 2'-deoxycytidine, thymidine, 2'-O-methylguanosine, 2'-O-methyladenosine, 2'-O-methylcytidine, 2'-O-methyluridine, 2'-O-
Methyl-5-methyluridine, 2′-O-methoxyethyl guanosine, 2′-O-methoxyethyl adenosine,
2'-O-methoxyethyl cytidine, 2'-O-methoxyethyl uridine, 2'-O-alkyl ribonucleosides such as 2'-O-methoxyethyl-5-methyluridine and the like can be exemplified. It is not limited.

As the trityling agent, a trityl halide may be used. Specific examples include dimethoxytrityl chloride, dimethoxytrityl bromide, monomethoxytrityl chloride, monomethoxytrityl bromide, trityl chloride, trityl bromide and the like. can do.

The present invention is characterized in that one or more azole salts are used as a reaction accelerator.
Examples of such azole salts include hydrochloride, nitrate, perchlorate and sulfonate of imidazole, triazole, tetrazole and derivatives or homologs thereof. Specifically, hydrochloride, nitrate, perchlorate or sulfonate of imidazole, benzimidazole, 1-methylimidazole, triazole or tetrazole is exemplified.

[0010] Among such azole salts, a sulfonate of imidazole or a derivative thereof is preferable.
Specifically, imidazolium methanesulfonate, benzimidazolium methanesulfonate, 1-methylimidazolium methanesulfonate, imidazolium toluenesulfonate, benzimidazolium toluenesulfonate, 1-methylimidazolium toluenesulfonate, imidazolium trifluoromethanesulfonate,
Examples include benzimidazolium trifluoromethanesulfonate, 1-methylimidazolium trifluoromethanesulfonate, imidazoliumbenzenesulfonate, benzimidazoliumbenzenesulfonate, 1-methylimidazoliumbenzenesulfonate and the like. Of these, methanesulfonate, particularly imidazolium methanesulfonate, is preferred.

Further, amines are not essential for the reaction,
In many cases, the reaction is accelerated and the synthesis yield may be improved. Therefore, it is desirable to use one or more amines in combination. Examples of the amines used in combination include trialkylamines such as diisopropylethylamine and triethylamine, guanidine, imidazole, 1,8-diazabicyclo [5,4,0] undec-7-ene (DBU),
Examples thereof include 1,5-diazabicyclo [4,3,0] non-5-ene (DBN) and 1,4-diazabicyclo [2,2,2] octane (DABCO), and in particular, trialkylamines And more preferably diisopropylethylamine.

The reaction for selectively tritylating the 5'-position of the unprotected nucleoside is carried out by using dimethylformamide (DM
F), in a single or mixed solvent such as formamide, dimethylamine, acetonitrile, dimethyl carbonate, and pyridine, with respect to 1 mol of unprotected nucleoside, 1 to 2 mol of a tritylating agent, 0.5 to 5 mol of a reaction accelerator, preferably Is used in an amount of 1 to 3 mol, and if necessary, 0.5 to 5 mol, preferably 1 to 3 mol of amines.
The desired 5′-O-trityl nucleosides can be obtained by reacting at 1 ° C., preferably 20 to 40 ° C. for about 1 to 50 hours. The above reaction is preferably performed under non-aqueous conditions, and it is preferable to use a dried reaction solvent and the like. The thus obtained 5'-O-trityl nucleosides may be isolated and purified by a usual method of nucleoside, and specifically, may be purified by adsorption chromatography such as silica gel.

[0013]

The method of the present invention is not a method specific to a specific nucleoside, but is applicable to all nucleosides.
It is a very practical and highly versatile method that can selectively trityl the 5'-position of a nucleoside with a yield of 5% or more.

[0014]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited thereto. Example 1 (1) Preparation of imidazolium methanesulfonate Imidazole (34.0 g, 0.499 mol) was dissolved in methylene chloride (500 ml), and methanesulfonic acid (48.0 g, 32.4 ml) was added to this solution at 0 ° C. , 0.4
99 mol) was added dropwise. The precipitated crystals were collected by filtration and washed with methylene chloride (100 ml) to give imidazolium methanesulfonate (IMS) as colorless crystals (8).
1.1 g, 0.494 mol, yield 99%).

Melting point: 184-186 ° C IR (KBr, cm ^-1 ): 3140, 1589, 146
0, 1437, 1339, 1194 ¹ H-NMR (DMSO-d ₆ , ppm): 2.49
(S, 3H), 7.62 (s, 2H), 9.03
^{(S, 1H) 13 C-} NMR (DMSO-d 6, ppm): 40.1,
119.7, 134.8

(2) Method for selective dimethoxytritylation at 5'-position Deoxyribonucleoside (40.0 mmol), IM
S (13.1 g, 80.0 mmol), diisopropylethylamine (10.3 g, 13.9 ml, 80.0 m)
mol) is suspended in DMF (dry) (100 ml),
Further, p, p'-dimethoxytrityl chloride (13.
6g, 40.0 mmol) was added in several portions, and the mixture was stirred at room temperature for 2 hours. The obtained homogeneous reaction solution was mixed with water (3.
00 liters) and the precipitate was collected by filtration. This precipitate was purified by silica gel column chromatography, and 5 ′
-O- (p, p'-dimethoxytrityl) -2'-deoxyribonucleoside was obtained. Yield of the obtained compound,
The spectrum data is as follows.

5'-O- (p, p'-dimethoxytrityl) -2'-deoxyadenosine Property: colorless amorphous solid Yield: 79% ¹ H-NMR (CDCl ₃ , ppm): 2.22 (br)
s, 1H), 2.53 (ddd, 1H, J = 13.4,
4.0, 6.3 Hz), 2.78 (ddd, 1H, J =
13.4, 6.0, 6.3 Hz, H-2 "), 3.40
(Dd, 2H, J = 1.6, 4.4 Hz), 3.76
(S, 6H), 4.16 (dt, 1H, J = 3.9,
4.4 Hz), 4.67 (ddd, 1H, J = 6.0,
4.0, 3.9 Hz), 5.98 (brs, 2H),
6.44 (t, 1H, J = 6.3 Hz), 6.78
(D, 4H, J = 9.3 Hz), 7.16-7.39
(M, 9H), 7.96 (s, 1H), 8.26 (s,
1H)

5'-O- (p, p'-dimethoxytrityl) -2'-deoxycytidine Properties: colorless powder Yield: 76% ¹ H-NMR (CDCl ₃ , ppm): 2.01-2. 1
0 (m, 1H), 2.39-2.48 (m, 1H),
3.22-3.34 (m, 2H), 3.63 (s, 6
H), 3.86-3.91 (m, 1H), 4.32-
4.38 (m, 1H), 5.41 (d, 1H, J = 6.
8Hz), 6.10 (t, 1H, J = 5.9Hz),
6.64-6.69 (m, 5H), 7.05-7.24
(M, 11H), 7.75 (d, 1H, J = 7.3H
z)

[0019] 5'-O- (p, p'- dimethoxytrityl) -2'-deoxyguanosine Property: colorless needles Yield: 78% ^{mp: 190-191 ℃ 1 H-NMR (} DMSO-d 6, ppm ): 2.22-
2.31 (m, 1H), 2.57-2.67 (m, 1
H), 3.09-3.15 (m, 2H), 3.71.
(S, 6H), 3.87-3.91 (m, 1H), 4.
32-4.35 (m, 1H), 5.28 (d, 1H, J
= 4.4 Hz), 6.12 (dd, 1H, J = 6.3,
6.8 Hz), 6.42 (brs, 2H), 6.81
(Dd, 4H, J = 6.8, 8.8 Hz), 7.17−
7.34 (m, 9H), 7.76 (s, 1H), 10.
58 (brs, 1H)

5'-O- (p, p'-dimethoxytrityl) -2'-deoxythymidine Properties: colorless amorphous solid Yield: 82% ¹ H-NMR (CDCl ₃ , ppm): 2.12-2 .1
9 (m, 1H), 2.24-2.29 (m, 1H),
2.57 (brs, 1H), 3.20-3.33 (m,
2H), 3.63 (s, 6H), 3.90-3.99.
(M, 1H), 4.32-4.38 (m, 1H), 6.
27 (t, 1H, J = 7.3 Hz), 6.68 (d, 4
H, J = 8.8 Hz), 7.08-7.26 (m, 10
H), 7.43 (s, 1H), 8.79-8.98 (b
rs, 1H)

(3) Method for selective monomethoxytritylation at the 5′-position In the dimethoxytritylation method of the above (2), p,
Using p-methoxytrityl chloride (40.0 mmol) instead of p'-dimethoxytrityl chloride, the same reaction was performed to obtain 5'-O- (p-methoxytrityl) -2'-deoxyribonucleoside. The yield and spectrum data of the obtained compound are as follows.

5'-O- (p-methoxytrityl)-
2′-Deoxyadenosine Properties: colorless amorphous solid Yield: 76% ¹ H-NMR (CDCl ₃ , ppm): 2.21 (br
s, 1H), 2.48-2.54 (m, 1H), 2.6
3-2.81 (m, 1H), 3.40 (dd, 2H, J
= 1.5, 4.4 Hz), 3.77 (s, 3H), 4.
14 (dt, 1H, J = 3.9, 4.5 Hz), 4.5
5-4.78 (m, 1H), 5.91 (brs, 2
H), 6.46 (t, 1H, J = 6.3 Hz), 6.7.
8 (d, 2H, J = 9.1 Hz), 7.16-7.39
(M, 12H), 7.96 (s, 1H), 8.26
(S, 1H)

5'-O- (p-methoxytrityl)-
2'-Deoxycytidine Properties: colorless amorphous solid Yield: 75% ¹ H-NMR (CDCl ₃ , ppm): 2.05-2.1
3 (m, 1H), 2.33-2.49 (m, 1H),
3.21-3.36 (m, 2H), 3.61 (s, 3
H), 3.85-3.90 (m, 1H), 4.38-
4.45 (m, 1H), 5.44 (d, 1H, J = 6.
7 Hz), 6.10 (t, 1H, J = 5.9 Hz),
6.64-6.69 (m, 3H), 7.05-7.24
(M, 14H), 7.75 (d, 1H, J = 6.7H
z)

5'-O- (p-methoxytrityl)-
2'-Deoxyguanosine Property: colorless amorphous solid Yield: 76% ¹ H-NMR (DMSO-d ₆ , ppm): 2.20-
2.30 (m, 1H), 2.54-2.63 (m, 1H)
H), 3.12-3.18 (m, 2H), 3.75
(S, 3H), 3.89-4.01 (m, 1H), 4.
33-4.42 (m, 1H), 5.24-5.35
(M, 1H), 6.06-6.18 (m, 1H), 6.
42 (brs, 2H), 6.81 (d, 2H, J = 8.
8 Hz), 7.17-7.34 (m, 12H), 7.7
8 (s, 1H), 10.73 (brs, 1H)

5'-O- (p-methoxytrityl)-
2'-Deoxythymidine Property: colorless amorphous solid Yield: 81% ¹ H-NMR (DMSO-d ₆ , ppm): 2.13
2.20 (m, 1H), 2.21-2.28 (m, 1
H), 2.56 (brs, 1H), 3.15-3.31
(M, 2H), 3.61 (s, 3H), 3.85-3.
98 (m, 1H), 4.35-4.44 (m, 1H),
6.25 (t, 1H, J = 7.2 Hz), 6.65
(D, 2H, J = 8.8 Hz), 7.08-7.26
(M, 13H), 7.46 (s, 1H), 8.69-
8.91 (brs, 1H)

(4) Method for selective tritylation at the 5'-position In the dimethoxytritylation method of the above (2), p,
The same reaction was carried out using trityl chloride (40.0 mmol) instead of p'-dimethoxytrityl chloride to obtain 5'-O-trityl-2'-deoxyribonucleoside. The yield and spectrum data of the obtained compound are as follows.

5'-O-trityl-2'-deoxyadenosine Property: colorless amorphous solid Yield: 78% ¹ H-NMR (CDCl ₃ , ppm): 2.23 (br)
s, 1H), 2.31-2.48 (m, 1H), 2.6.
1-2.80 (m, 1H), 3.44 (dd, 2H, J
= 1.5, 4.4 Hz), 4.14 (dt, 1H, J =
3.8, 4.4 Hz), 4.51-4.76 (m, 1
H), 5.90 (brs, 2H), 6.46 (t, 1
H, J = 6.2 Hz), 6.78 (d, 2H, J = 8.
9 Hz), 7.14-7.28 (m, 15H), 7.9
1 (s, 1H), 8.24 (s, 1H)

5'-O-trityl-2'-deoxycytidine Properties: colorless amorphous solid Yield: 79% ¹ H-NMR (CDCl ₃ , ppm): 2.08-2.1
4 (m, 1H), 2.37-2.45 (m, 1H),
3.24-3.33 (m, 2H), 3.87-3.93
(M, 1H), 4.33-4.41 (m, 1H), 5.
44 (d, 1H, J = 6.7 Hz), 6.10 (t, 1
H, J = 5.8 Hz), 6.59-6.67 (m, 1
H), 7.04-7.18 (m, 16H), 7.74
(D, 1H, J = 7.3 Hz)

5'-O-trityl-2'-deoxyguanosine Property: colorless amorphous solid Yield: 76% ¹ H-NMR (DMSO-d ₆ , ppm): 2.18-
2.28 (m, 1H), 2.55-2.64 (m, 1
H), 3.11-3.18 (m, 2H), 3.88-
3.98 (m, 1H), 4.35-4.42 (m, 1
H), 5.23 (d, 1H, J = 4.5 Hz), 6.1.
2 (t, 1H, J = 6.4 Hz), 6.55 (brs,
2H), 7.12-7.26 (m, 15H), 7.81
(S, 1H), 10.31 (brs, 1H) Elemental analysis:
Calcd _{C 29 H 27 N 5 O 4} : C68.36, H5.34, N13.74 Found: C68.50, H5.28, N13.77

5'-O-trityl-2'-deoxythymidine Properties: colorless amorphous solid Yield: 80% ¹ H-NMR (DMSO-d ₆ , ppm): 2.11
2.19 (m, 1H), 2.26-2.32 (m, 1
H), 2.55 (brs, 1H), 3.21-3.37.
(M, 2H), 3.87-3.93 (m, 1H), 4.
35-4.43 (m, 1H), 6.34 (t, 1H, J
= 6.7 Hz), 7.08-7.23 (m, 16H),
7.42 (s, 1H), 8.75-8.92 (brs,
1H)

Claims (7)

[Claims] 1. A method for producing 5′-O-trityl nucleosides by reacting an unprotected nucleoside with a trityling agent, wherein azole salts are used as a reaction accelerator. 2. The method according to claim 1, wherein the salt of the azole is imidazole, triazole, tetrazole or a derivative thereof or a homolog thereof, such as hydrochloride, nitrate, perchlorate or sulfonate. 3. An amine is used in combination with the reaction accelerator.
The method of claim 1. 4. The method according to claim 1, wherein the nucleoside is a ribonucleoside, a deoxyribonucleoside or a 2′-O-alkyl ribonucleoside. 5. The method of claim 1, wherein the trityling agent is a trityl halide. 6. A method for producing 5′-O-trityl nucleosides by reacting an unprotected nucleoside with a tritylating agent, comprising a sulfonate of imidazole as an azole salt and a trialkylamine as an amine. How to use. 7. A method for producing 5′-O-trityl nucleosides by reacting an unprotected nucleoside with a trityling agent, comprising imidazolium methanesulfonateol as an azole salt and diisopropylethylamine as an amine How to use.