JPH0753587A - Method for synthesizing oligonucleotide - Google Patents

Abstract

PURPOSE:To simply obtain the subject compound having an anti-AIDS viral action at a low cost and in high purity by synthesizing a substituent-having oligonucleotide on the 3'-terminal hydroxyl group of an oligonucleotide having a basic sequence through phosphorous acid using a DNA synthesizing machine. CONSTITUTION:A compound of formula I [R<1>-R<3> are each H, a 1-4C alkoxy; X<1> is S', S'O, etc.; A is (CH2)h ((h) is 1-6), etc.; Y<1> is hydroxyl group, phenyloxy, etc.,] is reacted with a polymeric substance of formula II (X<2> is oxygen atom, sulfur atom, etc.,), and a DNA chain is elongated on the obtained polymeric substance of formula III using a DNA-synthesizing machine to obtain the objective compound of formula IV [R<4>-R<6> are each H, a 1-4C alkyl, etc.; Z is C, silicon, etc.,); R<7> is H, a 1-4C aralkyl; Y<2> is O2, sulfur atom, etc.; Y<3> is O2, NH group, etc.; X<3> is O2, etc., D is oligonucleotide having a chain length of 2-30].

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing an oligonucleotide having a substituent at the 3'-terminal hydroxyl group via a phosphoric acid.

The present invention also relates to a method for producing a substituted oligonucleotide having an excellent anti-AIDS virus activity.

[0003]

2. Description of the Related Art Conventionally, as a method for synthesizing an oligonucleotide having a substituent through a phosphoric acid at a 3'-terminal hydroxyl group, a method in which a phosphoric acid triester liquid phase synthesis method is used is known.

The phosphoric acid triester liquid phase synthesis method is carried out in the following order.

(I) A phosphoric acid group having a deprotectable substituent at the 3'position, a dimethoxytrityl group (DMT group) at the 5'position hydroxyl group, and a base moiety if necessary. A mononucleotide having an acyl-type protecting group used in ordinary DNA synthesis is synthesized.

(II) removing the DMT group, which is a protecting group for the hydroxyl group at the 5'end of the mononucleotide unit,
(III) The protecting group of the phosphate residue at the 3'end of the protected nucleotide monomer or dimer used in the triester method corresponding to the desired nucleotide sequence is removed to remove the 3 '
A phosphoric acid diester, wherein the free hydroxyl group at the 5 ′ end generated in (IV) (II) and the free phosphoric acid diester at the 3 ′ end obtained in (III) are condensed using a condensing agent, V) removing the DMT group, which is a protecting group for the hydroxyl group at the 5'end of the nucleotide obtained in (IV), (VI)
Hereinafter, the operations (III), (IV) and (V) are repeated until an oligonucleotide having a desired nucleotide sequence is obtained, and a desired substituent is provided at the 3'end via a phosphoric acid, and a base moiety and To obtain the desired oligonucleotide having a protecting group on the acid moiety (VII) under basic conditions,
By removing the protecting groups on the base moiety and the phosphate moiety, the desired oligonucleotide can be obtained.

(For (I), refer to page 1 of the Second Biochemical Experiment Course "Gene Research Method I").

As another method, as a carrier, polyacrylamide (E. Felder et. Al, Tetrahedron Letters
25 , 3967-3970 (1984)), polystyrene (VAEfimov
et.al, Nucleic Acids Res., 11 , 8369 (1983)) or cellulose acetate (K. Kamaike et. al, Nucleic
Acids, Symposium Series 17 , 89 (1986)) is known to synthesize the desired oligonucleotide by the phosphate triester solid-phase synthesis method.

[0009]

However, in the above liquid phase method, a highly pure target compound cannot be obtained unless it is sufficiently purified each time the condensation reaction is carried out. There has been a demand for a convenient method for synthesizing an oligonucleotide having a desired substituent at the ′ -terminal via a phosphoric acid.

Further, in the above solid phase method, an oligonucleotide in which limited substituents (eg, orthochlorophenyl group) are bound to the 3'end via phosphoric acid is synthesized, which is versatile. poor.

The present inventors have conducted extensive studies for many years, and as a result, 3'of an oligonucleotide having a desired nucleotide sequence has been obtained.
An oligonucleotide with a desired substituent on the terminal hydroxyl group via phosphoric acid is easily synthesized on a DNA synthesizer. Easy purification, high versatility, low cost, and high purity target product are obtained. In addition, the inventors have found a method suitable for mass synthesis and completed the present invention.

[0012]

The manufacturing method of the present invention has the general formula

[0013]

[Chemical 5]

[In the formula, R ¹ , R ² and R ³ independently represent the same or different and each represent a hydrogen atom or an alkoxy group having 1 to 4 carbon atoms, and X ¹ represents an -S- group or -SO- group. A group or a —SO ₂ — group, A is a — (CH ₂ ) h — group (h is 1
To 16 of an _{integer), - (CH 2) h} O- group (h is an integer of 1 to _{16), - (CH 2)} j -O-CO
- (CH ₂₎ k - group (j is a positive integer, k is 0 or a positive integer, and j + k is 2 to 16), -
_{(CH 2) j -NH-CO-} (CH 2) k - group (j is a positive integer, k is 0 or a positive integer, and j + k
It is 2 to _{16), - (CH 2)} j -S-CO-
(CH ₂ ) k-group (j is a positive integer, k is 0 or a positive integer, and j + k is 2 to 16),-
_{(CH 2) n-O-} CO-CH 2 (OCH 2 CH 2) p
-OCH ₂ - group (n is a positive integer, p is 0 or a positive integer, and n + p is 2 to 100) indicates, Y ¹ is hydroxyl group, which may have a substituent It represents a phenyloxy group or an ethyloxy group which may be substituted with halogen. ] To the compound represented by the general formula

[0015]

[Chemical 6]

[In the formula, X ² represents an oxygen atom, a sulfur atom or an NH group, and HX ² -P (circled) represents a polymer substance. ] The polymer substance represented by the formula (2) is reacted (however, when Y ¹ in the formula (2) is a hydroxyl group, it is reacted with the carboxylic acid activator and then with the polymer substance (3)). General formula

[0017]

[Chemical 7]

[Wherein R ¹ , R ² , R ³ , X ¹ , A, X
² and HX ² -P (circled) have the same meaning as described above. ] The high molecular substance (4) represented by

[0019]

[Chemical 8]

[In the formula, R ⁴ , R ⁵ , R ⁶ and Z groups (in the formula, R ⁴ , R ⁵ and R ⁶ are independently the same or different and each is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms) Represents an optionally substituted aryl group or an optionally substituted anthraquinonyl group, Z represents a carbon or silicon atom, or R ⁵ , R ⁶ and Z together form fluorenyl. A group or a xanthenyl group, or R ⁴ ,
R ⁵ , R ⁶ and Z together represent a hydrogen atom), and R ⁷ represents a hydrogen atom, an optionally substituted alkyl group having 1 to 4 carbon atoms, or a substituent. An optionally substituted aryl group or an optionally substituted aralkyl group, Y ² is the same or different and represents an oxygen atom, a sulfur atom or an NH group, and Y ³ is an oxygen atom, a sulfur atom or an NH group. Represents an alkylene group having 1 to 4 carbon atoms or a phenylene group, X ³ represents an oxygen atom or a sulfur atom, and D represents an oligonucleotide having a chain length of 2 to 30. However, D does not include hydroxyl groups at the 5'end and 3'end of each oligonucleotide. ] It is a manufacturing method of the compound represented by these.

The method of the present invention is shown in process charts 1 and 2.

[0022]

[Chemical 9]

[0023]

[Chemical 10]

[0024]

[Chemical 11]

[0025]

[Chemical 12]

In the table, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R
^{6, R 7, X 1,} X 2, X 3, HX 2 -P ( circled),
Y ¹ , Y ² , Y ³ , A and Z have the same meanings as described above (however, in compounds other than compound (1), R ⁴ , R 3
⁵ , R ⁶ and Z together do not represent a hydrogen atom). R ^8a represents a methyl group or a cyanoethyl group, and R ^8b
Represents a phenyl group which may be substituted with a chloro group,
R ⁸ represents a methyl group, a phenyl group which may be substituted with a cyanoethyl group or a chloro group, a hydrogen atom, X represents a halogen atom (preferably chlorine, bromine or iodine), and R ^9a
Represents a hydrogen atom or a protected hydroxyl group, A ¹ represents an alkylene group having 1 to 20 carbon atoms, m represents an integer of 1 to 14, n represents an integer of 1 to 28, B, B ′ and B ″ represents a base portion of an adenine nucleotide, a guanine nucleotide, a thymine nucleotide, a cytosine nucleotide, and a uracil nucleotide in which a base portion and a phosphate portion are protected (provided that a desired base sequence is selected), B'represents the base portion of the 3'terminal nucleotide of D having the desired base sequence, B "represents the base portion of the 5'terminal nucleotide of D having the desired base sequence, and D represents the desired oligonucleotide. (However,
5'-hydroxyl group at the 5'-terminal and 3'-hydroxyl group at the 3'-terminal are not included), Q represents a halogenoalkyl group, a halogenophenyl group or a nitrophenyl group, and preferably 2,2,2
-Trichloroethyl group, 2,2-dichloroethyl group, 2
-Chloroethyl group, 2,2,2-tribromoethyl group,
2,2-dibromoethyl group, 2-bromoethyl group, 2-
A nitrophenyl group, a 4-nitrophenyl group, a 2,4-dinitrophenyl group, a 2,4,5-trichlorophenyl group, a 2,3,4,5,6-pentachlorophenyl group (more preferably, trichlorophenyl group) (Ethyl group or orthonitrophenyl group), V is a lower alkyl group (preferably methyl, ethyl, isopropyl group, especially isopropyl group).

Next, each step will be described in detail.

(First Step) In this step, in an inert solvent,
In the presence of a deoxidizing agent, 2-mercaptoethanol (6),
In this step, ω-halogenocarboxylic acid (5) is reacted to obtain compound (7).

The solvent used is not particularly limited as long as it does not inhibit the reaction, but aromatic hydrocarbons such as benzene, toluene and xylene; halogenated hydrocarbons such as methylene chloride and chloroform. ;
Ethers such as ether, tetrahydrofuran, dioxane, dimethoxyethane; dimethylformamide,
Amides such as dimethylacetamide and hexamethylphosphorotriamide; sulfoxides such as dimethylsulfoxide; alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, isoamyl alcohol; sulfuric acid water Diluted acids such as sodium hydroxide; dilute bases such as aqueous sodium hydroxide; water; ketones such as acetone and methyl ethyl ketone; heterocyclic amines such as pyridine or nitrites such as acetonitrile. , Ketones such as acetone, and alcohols such as ethanol and propanol.

The deoxidizer used is potassium carbonate,
Examples thereof include alkali metal carbonates such as sodium carbonate, alkali metal hydrogen carbonates such as sodium hydrogen carbonate, alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, and organic amines such as triethylamine. However, it is preferably an alkali metal carbonate (particularly potassium carbonate).

The reaction temperature and reaction time vary depending on the solvent, deoxidizing agent, etc. used, but when potassium carbonate is used,
Heat at reflux for 8 hours.

After completion of the reaction, the desired compound is collected from the reaction mixture according to a conventional method. For example, the reaction mixture is appropriately neutralized, and if insolubles are present, they are removed by filtration, water and an immiscible organic solvent such as ethyl acetate are added, and the mixture is washed with water to form an organic layer containing the target compound. Separate and
It is obtained by drying over anhydrous magnesium sulfate or the like and then distilling off the solvent. The obtained target compound is, if necessary,
It can be further purified by a conventional method such as recrystallization, reprecipitation or chromatography.

(Second Step) In this step, a protecting reagent (preferably dimethoxytrityl chloride) which can be removed from compound (7) in an inert solvent in the presence of a deoxidizing agent under acidic conditions is used.
Is a step of obtaining a compound (8) in which the hydroxyl group of the compound (7) is protected.

The solvent used is not particularly limited as long as it does not inhibit the reaction and dissolves the starting material to some extent, but aromatic hydrocarbons such as benzene, toluene and xylene; methylene chloride and chloroform. Halogenated hydrocarbons such as; ethers such as ether, tetrahydrofuran, dioxane, dimethoxyethane; amides such as dimethylformamide, dimethylacetamide, hexamethylphosphorotriamide; sulfoxides such as dimethylsulfoxide; acetone,
Mention may be made of ketones such as methyl ethyl ketone; heterocyclic amines such as pyridine or nitriles such as acetonitrile, and preferably heterocyclic amines (particularly pyridine).

Examples of the protecting reagent used include trityl halides such as trityl chloride, monomethoxytrityl chloride, dimethoxytrityl chloride and trimethoxytrityl chloride, but dimethoxytrityl chloride is preferred.

The deoxidizing agent used is not particularly limited as long as it does not inhibit the reaction and does not decompose the product and the starting material, but aromatic amines such as pyridine and dimethylaminopyridine are preferred. Is.

The reaction temperature and reaction time will vary depending on the type of protecting reagent and deoxidizing agent used, but when dimethoxytrityl chloride is used as the protecting reagent and pyridine is used as both a solvent and a deoxidizing agent. Is for 2 hours at room temperature.

After completion of the reaction, the desired compound is collected from the reaction mixture according to a conventional method. For example, the reaction mixture is appropriately neutralized, and if insolubles are present, they are removed by filtration, water and an immiscible organic solvent such as ethyl acetate are added, and the mixture is washed with water to form an organic layer containing the target compound. Separate and
It is obtained by drying over anhydrous magnesium sulfate or the like and then distilling off the solvent. The obtained target compound is, if necessary,
It can be further purified by a conventional method such as recrystallization, reprecipitation or chromatography.

(Third step) In this step, compound (9) (obtained by tritylating an easily available diol in the same manner as in the second step) in an inert solvent. Is reacted with dicarboxylic acid anhydride (10),
In this step, compound (8) is obtained.

The solvent used is not particularly limited as long as it does not inhibit the reaction and dissolves the starting material to some extent, but aromatic hydrocarbons such as benzene, toluene and xylene; methylene chloride and chloroform. Halogenated hydrocarbons such as; ethers such as ether, tetrahydrofuran, dioxane, dimethoxyethane; amides such as dimethylformamide, dimethylacetamide, hexamethylphosphorotriamide; sulfoxides such as dimethylsulfoxide; acetone,
Mention may be made of ketones such as methyl ethyl ketone; heterocyclic amines such as pyridine or nitrites such as acetonitrile, with halogenated hydrocarbons such as methylene chloride being preferred.

Examples of the deoxidizing agent used include pyridines such as pyridine, dimethylaminopyridine and pyrrolidinopyridine, but dimethylaminopyridine is preferred.

The dicarboxylic acid anhydride used is
The anhydride of an α, ω-alkyldicarboxylic acid having 3 to 16 carbon atoms is not particularly limited, but succinic anhydride is preferred.

Although the reaction temperature and reaction time vary depending on the acid anhydride, deoxidizing agent, etc. used, succinic anhydride is used.
When dimethylaminopyridine is used as a deoxidizing agent, it is 30 minutes at room temperature.

After completion of the reaction, the desired compound is collected from the reaction mixture according to a conventional method. For example, the reaction mixture is appropriately neutralized, and if insolubles are present, they are removed by filtration, water and an immiscible organic solvent such as ethyl acetate are added, and the mixture is washed with water to form an organic layer containing the target compound. Separate and
It is obtained by drying over anhydrous magnesium sulfate or the like and then distilling off the solvent. The obtained target compound is, if necessary,
It can be further purified by a conventional method such as recrystallization, reprecipitation or chromatography.

(Step 4) In this step, the carboxyl group of the compound (8) having a free carboxyl group is reacted with an ester forming reagent in an inert solvent, and then a phenol optionally having a substituent is used. Is a step of forming an active ester (2) by reacting with.

The solvent used is not particularly limited as long as it does not inhibit the reaction, but aromatic hydrocarbons such as benzene, toluene and xylene; methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene. , Halogenated hydrocarbons such as dichlorobenzene; ethyl formate, ethyl acetate, propyl acetate, butyl acetate, esters such as diethyl carbonate, acetone, methyl ethyl ketone methyl isobutyl ketone, ketones such as isophorone, cyclohexanone; nitroethane, Nitro compounds such as nitrobenzene; Nitriles such as acetonitrile and isobutyronitrile; Amides such as formamide, dimethylformamide (DMF), dimethylacetamide, hexamethylphosphorotriamide; Dimethy Sulfoxides, sulfoxides such as sulfolane and the like, preferably a halogenated hydrocarbon (particularly methylene chloride), amides (particularly dimethylformamide).

The phenol used is not particularly limited as long as it can be used as an active ester.
4-nitrophenol, 2,4-dinitrophenol,
2,4,5-Trichlorophenol, 2,3,4,5
6-Pentachlorophenol and 2,3,5,6-tetrafluorophenol can be mentioned, but pentachlorophenol is preferred.

Examples of the ester-forming reagent used include N-hydroxy compounds such as N-hydroxysuccinimide, 1-hydroxybenzotriazole, N-hydroxy-5-norbornene-2,3-dicarboximide; 1,1'-oxalyldiimidazole, N,
Diimidazole compounds such as N'-carbonyldiimidazole; Disulfide compounds such as 2,2'-dipyridyldisulfide; Succinic acid compounds such as N, N'-disuccinimidyl carbonate; N,
Phosphinic chloride compounds such as N'-bis (2-oxo-3-oxazolidinyl) phosphinic chloride; N, N'-disuccinimidyl oxalate (DSO), N, N-diphtalimidylo Xizarate (DPO), N, N′-bis (norbornenylsuccinimidyl) oxalate (BNO), 1,1′-bis (benzotriazolyl) oxalate (BBTO),
1,1'-bis (6-chlorobenzotriazolyl) oxalate (BCTO), 1,1'-bis (6-trifluoromethylbenzotriazolyl) oxalate (BTB)
Examples thereof include oxalate compounds such as O) and carbodiimides such as dicyclohexylcarbodiimide (DCC), and preferably diimidazole compounds and carbodiimides (particularly DCC).

The reaction temperature and reaction time will vary depending on the type of ester-forming reagent and solvent used, but may range from 0 ° C. to 100 ° C. for 5 to 50 hours, especially when pentachlorophenol and DCC are used in DMF. 18 at room temperature
It's time.

After completion of the reaction, the desired compound is collected from the reaction mixture according to a conventional method. For example, the reaction mixture is appropriately neutralized, and if insolubles are present, they are removed by filtration, water and an immiscible organic solvent such as ethyl acetate are added, and the mixture is washed with water to form an organic layer containing the target compound. Separate and
It is obtained by drying over anhydrous magnesium sulfate or the like and then distilling off the solvent. The obtained target compound is, if necessary,
It can be further purified by a conventional method such as recrystallization, reprecipitation or chromatography.

(Step 5) In this step, compound (9) is reacted with compound (11) in the presence of a deoxidizing agent in an inert solvent to obtain compound (2).

The solvent used is not particularly limited as long as it does not inhibit the reaction, but benzene, toluene,
Aromatic hydrocarbons such as xylene; halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene and dichlorobenzene; such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, diethyl carbonate Esters; acetone,
Ketones such as methyl ethyl ketone, methyl isobutyl ketone, isophorone, and cyclohexanone; nitro compounds such as nitroethane and nitrobenzene; nitriles such as acetonitrile and isobutyronitrile; formamide, dimethylformamide (DMF), dimethylacetamide, hexamethyl Examples thereof include amides such as phosphorotriamide; sulfoxides such as dimethyl sulfoxide and sulfolane, preferably halogenated hydrocarbons, particularly methylene chloride.

Deoxidizing agents used include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, pyridine and 4- (N, N-
Dimethylamino) pyridine, N, N-dimethylaniline, N, N-diethylaniline, 1,5-diazabicyclo [4.3.0] non-5-ene, 1,4-diazabicyclo [2.2.2] octane (DABCO), 1,8-
Diazabicyclo [5.4.0] undec-7-ene (D
BU) organic bases, preferably organic bases, especially pyridine, N-methylmorpholine.

The reaction temperature and reaction time will differ depending on the deoxidizing agent used, but will usually be 10 to 40 ° C. and 1 to 5 hours.

After completion of the reaction, the desired compound is collected from the reaction mixture according to a conventional method. For example, the reaction mixture is appropriately neutralized, and if insolubles are present, they are removed by filtration, water and an immiscible organic solvent such as ethyl acetate are added, and the mixture is washed with water to form an organic layer containing the target compound. Separate and
It is obtained by drying over anhydrous magnesium sulfate or the like and then distilling off the solvent. The obtained target compound is, if necessary,
It can be further purified by a conventional method such as recrystallization, reprecipitation or chromatography.

(Sixth step) In this step, the compound (2) having an activated carboxyl group obtained in the fifth step is treated with an amino group, a hydroxyl group, and
In this step, a polymer derivative (4) that can be used as a carrier for oligonucleotide synthesis is obtained by reacting with a polymer substance (3) such as control pore glass (CPG) having a sulfhydryl group bonded thereto.

The polymer substance (3) used in this step is
There is no particular limitation as long as it is generally used as a carrier, but the carrier size, particle size, surface area due to three-dimensional network structure, ratio of hydrophilic groups, chemical composition, strength against pressure, etc. are examined. There is a need to.

Examples of the carrier to be used include polysaccharide derivatives such as cellulose, dextran and agarose, polyacrylamide gel, polystyrene jusi, synthetic polymers such as polyethylene glycol, silica gel, porous glass and metal oxides. Examples of such inorganic substances include, specifically, aminopropyl-CPG, long-chain aminoalkyl-CPG (above, manufactured by CPG Inc.), Cosmoseal NH ₂ , Cosmosir Diol (above, Nacalai Tesque, Inc.). Manufactured by CPC-Silica Carrier SilaneCoated,
Aminopropyl-CPG-550Å, Aminopropyl-
CPG-1400Å, polyethylene glycol 50
00 monomethyl ether (above, manufactured by Furuka), p-alkoxybenzyl alcohol resin,
Aminomethyl resin, hydroxymethyl resin (all manufactured by Kokusan Kagaku Co., Ltd.), polyethylene glycol 140
Examples thereof include commercially available carriers such as 00 monomethyl ether (above, manufactured by Union Carbide Co.).
It is not limited to these.

The functional group bonded to the carrier is preferably an amino group, a sulfhydryl group or a hydroxyl group.

The solvent used in this step is not particularly limited as long as it does not inhibit the reaction and dissolves the starting material to some extent, but preferably, an aromatic hydrocarbon such as benzene, toluene or xylene. Halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene and dichlorobenzene;
Ethyl formate, ethyl acetate, propyl acetate, butyl acetate,
Esters such as diethyl carbonate, ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone, cyclohexanone; nitro compounds such as nitroethane and nitrobenzene; acetonitrile,
Nitriles such as isobutyronitrile; amides such as formamide, dimethylformamide (DMF), dimethylacetamide, hexamethylphosphorotriamide; sulfoxides such as dimethyl sulfoxide and sulfolane, and preferably halogenated Hydrocarbons (especially methylene chloride) and amides (especially dimethylformamide). The reaction temperature is usually -20 to 150.
C., preferably 0 to 50.degree. While the reaction time varies depending on the starting materials used, solvent, reaction temperature, etc., it is generally 1 to 200 hours, and preferably 24 to 100.
It's time. After completion of the reaction, the target compound is collected from the reaction mixture according to a conventional method. For example, the polymer carrier is recovered from the reaction mixture by filtration, washed with an organic solvent such as methylene chloride, and then dried under reduced pressure. (Seventh Step) This step is a step of reacting the compound (4) with a deprotecting reagent in an inert solvent to selectively remove the hydroxyl-protecting group to produce a compound (12). . The steps from the 10th step to the 10th step are usually carried out on a DNA synthesizer.

The solvent used is preferably an aromatic hydrocarbon such as benzene, toluene or xylene;
Halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, dichlorobenzene; esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, diethyl carbonate; diethyl ether, diisopropyl ether, Ethers such as tetrahydrofuran, dioxane, dimethoxyethane, diethylene glycol dimethyl ether; methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, t-
Alcohols such as butanol, isoamyl alcohol, diethylene glycol, glycerin, octanol, cyclohexanol, methyl cellosolve; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone, cyclohexanone; nitro compounds such as nitroethane, nitrobenzene; Nitriles such as acetonitrile and isobutyronitrile;
Examples include amides such as formamide, dimethylformamide, dimethylacetamide, and hexamethylphosphorotriamide; sulfoxides such as dimethylsulfoxide and sulfolane, and more preferably alcohols (especially methanol and ethanol), methylene chloride, and demethylated. When acetic acid is used as the protecting reagent, a mixed solution of acetic acid and water can be used. The deprotecting reagent used is not particularly limited as long as it is usually used, but when the protecting group is a triarylmethyl group, for example, acetic acid, dichloroacetic acid, trifluoroacetic acid, hydrochloric acid and bromide. Examples of Lewis acids such as zinc are acetic acid, dichloroacetic acid and trifluoroacetic acid. The reaction temperature will differ depending on the reagents, raw materials, solvent, etc. used, but is usually −10 to 100 ° C.
And preferably 0 to 50 ° C. While the reaction time varies depending on the raw materials used, solvent, reaction temperature, etc., it is generally 1 minute to 50 hours, and preferably 1 minute to 24 hours.
It's time. After completion of the reaction, the target compound is collected from the reaction mixture according to a conventional method. For example, the polymer carrier is recovered from the reaction mixture by filtration, washed with an organic solvent such as methylene chloride, and then dried under reduced pressure. (Eighth step) In this step, each has a dimethoxytrityl group at the 5'-hydroxyl group, the base portion is the base at the 3'-terminal portion of the desired base sequence, and the 3'-hydroxyl group is bonded via phosphorus. Desired alkyloxy group to be bonded, phenyloxy group, aralkyloxy group, alkyl group, aralkyl group or phenyl group bonded (13), (1
3 ') or 3'-H-phosphonate (13 ")
Is reacted with a polymer substance (12) and, if necessary, subjected to thioation and alkylamination to bond the 3'-terminal nucleotide unit of a desired base sequence to the polymer substance such as CPG ( This is the step of obtaining 14).
Hereinafter, the compounds (13), (13 ') and (1
3 ″) will be described.

(A) When the compound (13) is reacted in this step, it is carried out using an acidic substance in an inert solvent, that is, in acetonitrile, tetrahydrofuran, methylene chloride (preferably acetonitrile) or the like. . To do.

Examples of the acidic substance used include acidic substances such as tetrazole, and preferably tetrazole.

After completion of the reaction, the desired product is washed with acetonitrile and used as it is in the next oxidation step.

The oxidizing agent used in the oxidation reaction in this step is not particularly limited as long as it is usually used in the oxidation reaction, but preferably potassium permanganate,
Manganese oxides such as manganese dioxide; Ruthenium oxides such as ruthenium tetroxide; Zelene compounds such as selene dioxide; Iron compounds such as iron chloride: Osmium compounds such as osmium tetroxide; Silver such as silver oxide Compounds: mercury compounds such as mercury acetate, lead oxide compounds such as lead oxide and lead tetroxide; potassium chromate, chromic acid compounds such as chromic acid-sulfuric acid complex, chromic acid-pyridine complex, cerium ammonium nitrate ( Inorganic metal oxidizers such as cerium compounds such as CAN); halogen molecules such as chlorine molecules, bromine molecules, iodine molecules; periodic acids such as sodium periodate; ozone, hydrogen peroxide water; nitrous acid, etc. Nitrite compounds; chlorite compounds such as potassium chlorite, sodium chlorite; potassium persulfate, sodium persulfate Inorganic oxidizers such as persulfate compounds such as; reagents used in DMSO oxidation (complexes of dimethylsulfoxide with dicyclohexylcarbodiimide, oxalyl chloride, acetic anhydride or phosphorus pentoxide or pyridine-sulfuric anhydride complexes); t -Peroxides such as butyl hydroperoxide; stable cations such as the triphenylmethyl cation; succinimides such as N-bromosuccinimide, hypochlorous acid such as t-butyl hypochlorite Compounds; azodicarboxylic acid compounds such as azodicarboxylic acid esters; disulfides such as dimethyl disulfide, diphenyl disulfide, dipyridyl disulfide and triphenylphosphine; nitrite esters such as methyl nitrite; methane tetrabromide Tetrahalogenated carbon, 2,3 Dichloro -5,
An organic oxidizing agent such as a quinone compound such as 6-dicyano-p-benzoquinone (DDQ) can be mentioned, and iodine is preferable.

The solvent used is not particularly limited as long as it does not inhibit the reaction and dissolves the starting material to some extent, but aromatic hydrocarbons such as benzene, toluene and xylene; methylene chloride and chloroform. Halogenated hydrocarbons such as; ethers such as ether, tetrahydrofuran, dioxane, dimethoxyethane; amides such as dimethylformamide, dimethylacetamide, hexamethylphosphorotriamide; sulfoxides such as dimethylsulfoxide; acetone,
Mention may be made of ketones such as methyl ethyl ketone; heterocyclic amines such as pyridine or nitriles such as acetonitrile, preferably heterocyclic amines (particularly pyridine), nitriles (particularly acetonitrile), ethers. (Especially tetrahydrofuran) and halogenated hydrocarbons (especially methylene chloride).

The reaction temperature is -50 to 100 ° C., and the reaction time is usually 5 minutes to 15 hours, varying mainly depending on the reaction temperature, the starting compound or the type of solvent used.

The solvent used when the compound (13 ') (b) is reacted is not particularly limited as long as it does not inhibit the reaction, but an aromatic amine such as pyridine is preferably used.

When the compound (13 ') is reacted,
Usually, a condensing agent is used.

Examples of the condensing agent used include dicyclocarbodiimide (DCC), mesitylenesulfonic acid chloride (Ms-Cl), triisopropylbenzenesulfonic acid chloride, mesitylenesulfonic acid triazolide (MS).
T), mesitylenesulfonic acid-3-nitrotriazolide (MSNT), triisopropylbenzenesulfonic acid tetrazolid (TPS-Te), triisopropylbenzenesulfonic acid nitroimidazolide (TPS-NI), and triisopropylbenzenesulfonic acid pyridyl Examples thereof include tetrazolide, but MSNT, TPS-Te and TPS-NI are preferably used.

The reaction temperature is not particularly limited to -10 to 100 ° C, but is usually room temperature. The reaction time varies depending on the solvent used and the reaction temperature, but it is 30 minutes when the reaction is carried out at room temperature using pyridine.

(C) The solvent used when reacting the compound (13 ″) is not particularly limited as long as it does not inhibit the reaction, but anhydrous acetonitrile is preferably used. As the reagent to be used, acid chloride of carboxylic acid or phosphoric acid is used, but pivaloyl chloride is preferably used Oxidation for oxidizing an oligonucleotide having an H-phosphonic acid bond into a phosphodiester type oligonucleotide The agent is not particularly limited as long as it is usually used in an oxidation reaction, and manganese oxides such as potassium permanganate and manganese dioxide; ruthenium oxides such as ruthenium tetroxide; Zelene compounds; Iron compounds such as iron chloride; Osmium compounds such as osmium tetroxide; Silver compounds such as silver oxide Mercury compounds such as mercury acetate, lead oxide compounds such as lead oxide and lead tetroxide; potassium chromate, chromic acid compounds such as chromic acid-sulfuric acid complex, chromic acid-pyridine complex, cerium ammonium nitrate (CAN) ) Inorganic metal oxidizers such as cerium compounds; halogen molecules such as chlorine molecules, bromine molecules, iodine molecules; periodic acids such as sodium periodate; ozone;
Hydrogen peroxide water; nitrite compounds such as nitrous acid; chlorite compounds such as potassium chlorite and sodium chlorite; inorganic oxidizers such as persulfate compounds such as potassium persulfate and sodium persulfate; Reagents used for DMSO oxidation (dimethylsulfoxide and dicyclohexylcarbodiimide, oxalyl chloride, acetic anhydride or phosphorus pentoxide complex or pyridine-acetic anhydride complex); peroxides such as t-butyl hydroperoxide; Stable cations such as phenylmethyl cation; N-
Succinimides such as bromosuccinimide, hypochlorite compounds such as t-butyl hypochlorite; azodicarboxylic acid compounds such as methyl azodicarboxylate; dimethyl disulfide, diphenyl disulfide, dipyridyl disulfide Disulfides and triphenylphosphine; nitrites such as methyl nitrite;
Tetrahalogenated carbon such as methane tetrabromide, 2,3-
Dichloro-5,6-dicyano-p-benzoquinone (DD
Examples thereof include organic oxidizers such as quinone compounds such as Q), and iodine molecules are preferable. Examples of the deoxidizing agent to be used include heterocyclic amines such as pyridine and dimethylaminopyridine, and aliphatic amines such as trimethylamine, triethylamine and diisopropylethylamine, preferably heterocyclic amines (particularly pyridine). ). The reaction temperature is not particularly limited, but usually-
The temperature is 50 to 50 ° C., preferably room temperature.

The reaction time varies depending on the starting materials used, reagents, temperature and the like, but is usually 5 minutes to 30 hours, and preferably 30 minutes when reacted at room temperature. The thioation reaction optionally carried out in this step can be carried out by binding the compound (13) according to the above-mentioned method and then reacting it with a thioation reagent instead of oxidation by an iodine molecule, which has a thioate bond. (14) which is a polymer carrier such as CPG can be obtained. The thioation reagent is not particularly limited as long as it is a reagent capable of reacting with trivalent phosphorus to form a thioate, but in addition to sulfur, tetraethylthiuram disulfide (TET) sold by Applied Biosystems, Inc.
D) and Be released by Mirigen / Biosearch
The aucage reagent and the like are preferable, and in the case of the former, the method shown in a known document (Tetrahedron Letters, 32,3005 (1991)) or a modification thereof, and in the case of the latter, a known document (JA
m.Chem.Soc. 112 1253 (1990)) or a modification thereof can be mentioned, but the invention is not limited thereto.

The alkylamination optionally carried out in this step can be carried out according to the above-mentioned method.
3 ″) and then reacting with a desired alkylamine at room temperature instead of reacting with an oxidizing agent such as iodine molecule. It is possible to obtain (14) in which the 3'terminal nucleotide of the desired base sequence is linked by a phosphoramidate bond.

(Ninth Step) In this step, the polymer substance (14) obtained in the eighth step is 1) treated with acid to remove the DMT group, and 2) reacted with an amidite reagent and an acid catalyst.
3) Oxidation reaction is performed with an oxidizing agent, and 4) The unreacted portion is capped with acetic anhydride.
This is a process of repeating 1) to 4) to obtain (15) in which only the nucleotide at the 5'end of the desired base sequence is not yet bound.

DN on the DNA synthesizer used here
The extension reaction of the A chain is carried out, for example, by a modified stick method of the phosphoramidite method using Applied Biosystems 380-B (J. Am. Chem. Soc., 106, 6077-6089 (198).
4)) and a cyclone from Milligen / Biosearch
Phosphoramidite method (Nuc
leic Acids Res., 12 4539 (1984) by H. Koester et al.), but is not limited to these methods.

(Step 10) In this step, the DMT group at the 5'end of the polymer substance (15) obtained in step 9 is removed according to the method of step 7, and then the compound ( 13), (13 ′) and (13 ″) instead of the following compounds (16), (16 ′) and (1
6 ″) is treated in the same manner as above, and a polymeric substance having a desired base sequence and a desired substituent and having a protecting group (1
This is the step of obtaining 7).

Compounds (16) and (1 used in this step
6 ') and (16 ") are 2'-deoxyribonucleosides each having a protected base portion of the nucleotide at the 5'end of the desired base sequence as a base portion, or are protected at the 2'position. A ribonucleoside having a hydroxyl group, which has a desired modifying group at the 5'-position, and a normal DN at the 3'-position in (16).
It has a phosphoramidite bond having a protecting group for the phosphoric acid moiety used in A synthesis.

Similarly, (16 ') has a phosphodiester bond and (16 ") has an H-phosphonic acid bond.

(Eleventh step) This step is carried on the polymer substance obtained in the tenth step and has a desired base sequence and substituents on the 5'end and 3'end sides, and ,
The oligonucleotide portion is cleaved from the polymer substance (17) which is still protected, and then the protecting group other than the desired substituents at the 5'end and / or the 3'end is removed to remove the desired oligonucleotide (1 ) Is a process of obtaining.

Removal of the protecting group is carried out by a known method (J. Am. Chem. So.
c., 103, 3185 (1981)).

The reaction mixture containing the compound of the general formula (1) thus obtained is subjected to nucleic acid chromatography such as reverse phase and / or various chromatography such as ion exchange chromatography (including high performance liquid chromatography). The compound having the above-mentioned general formula (1) can be obtained by purification by a conventional purification operation used for purification.

(Twelfth Step) This step is carried out in an inert solvent,
Free hydroxyl group of compound (19) or compound (20)
Is a step of obtaining a half ester of a dicarboxylic acid by reacting a free sulfhydryl group of the above with an anhydride of a dicarboxylic acid such as the compound (18) in the presence of a basic catalyst. In this step, a compound (5) is prepared.

The anhydride of the dicarboxylic acid used is not particularly limited, but preferably has 1 to 16 carbon atoms, and most preferably succinic anhydride or glutaric anhydride.

Examples of the basic catalyst used include aminopyridines such as dimethylaminopyridine and pyrrolidinopyridine, tertiary amines such as trimethylamine and triethylamine, alkali metal carbonates such as sodium hydrogencarbonate and potassium carbonate. Are preferred, but dimethylaminopyridine and pyrrolidinopyridine are most preferred.

The solvent used is not particularly limited as long as it does not inhibit the reaction and dissolves the starting material to some extent, but preferably, aromatic hydrocarbons such as benzene, toluene and xylene; Halogenated hydrocarbons such as methylene chloride, chloroform; ethers such as ether, tetrahydrofuran, dioxane, dimethoxyethane; amides such as dimethylformamide, dimethylacetamide, hexamethylphosphorotriamide; sulfoxides such as dimethylsulfoxide Alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, isoamyl alcohol; dilute acids such as sulfuric acid;
Dilute base such as sodium hydroxide water; water; acetone;
Mention may be made of ketones such as methyl ethyl ketone; heterocyclic amines such as pyridine or nitriles such as acetonitrile, preferably nitriles (especially acetonitrile), ethers (especially tetrahydrofuran), halogenated hydrocarbons. Class (especially methylene chloride)
Is.

The reaction temperature is -50 to 100 ° C,
The reaction time varies depending mainly on the reaction temperature, the starting compound or the type of solvent used, but it is usually 30 minutes to 15 hours.

After completion of the reaction, the desired compound is collected from the reaction mixture according to a conventional method. For example, the reaction mixture is appropriately neutralized, and if insolubles are present, they are removed by filtration, water and an immiscible organic solvent such as ethyl acetate are added, and the mixture is washed with water to form an organic layer containing the target compound. Separate and
It is obtained by drying over anhydrous magnesium sulfate or the like and then distilling off the solvent. The obtained target compound is, if necessary,
It can be further purified by a conventional method such as recrystallization, reprecipitation or chromatography.

(13th step) This step is carried out in an inert solvent,
In this step, compound (8) is obtained by reacting compound (9) with dicarboxylic acid (21) in the presence of an ester-forming reagent. This step can be performed in the same manner as the fourth step.

[0090]

INDUSTRIAL APPLICABILITY According to the present method, a modified oligonucleotide having a desired modification group at the 3'end of the oligonucleotide via a phosphate can be easily synthesized on a DNA synthesizer,
Purification is simple, cost is low, and a large amount of high-purity products can be obtained.

Useful oligonucleotides having a modifying group at the 3'end include those having an anti-AIDS effect described in Japanese Patent Application No. 5-301744.

[0092]

EXAMPLES The present invention will be described in more detail below with reference to examples.

In the examples, the base sequence (TGGGAG) represents the triethylamine salt of oligodeoxyribonucleotide having the corresponding base sequence from the 5'end, and does not include hydroxyl groups at the 5'end and 3'end.

Chemical names or structural formulas were used as appropriate to indicate the target compound.

[0095]

Example 1 2- [2-O- (4,4′-dimethoxytrityloxy)
Si) ethylsulfonyl] ethyl monosuccinate 2- [2-O- (4,4'-dimethoxytrityloxy) ethylsulfonyl] ethanol (Tetrahedron Let
t. 27, 4705 (1986)) 1.37 g (3.0 mmol)
Pyridine was added to the mixture, and the mixture was evaporated under reduced pressure and dried. This was dissolved in methylene chloride (12 ml) and succinic anhydride 315 m
g (3.15 mmol) and dimethylaminopyridine 38
4 mg (3.15 mmol) was added and stirred for 30 minutes. After confirming the completion of the reaction by TLC, 80 ml of methylene chloride was added to the reaction solution, and 0.5M KH ₂ PO ₄ (pH 5.
0) and water were washed in this order, and the organic layer was dried over anhydrous sodium sulfate. The solvent was distilled off to obtain 1.60 g (96%) of the target compound.

1H-NMR (270 MHz, CDCl ₃ , TMS) δppm: 7.40-6.
83 (13H, m, Ph), 4.58-4.53 (2H, t, SO ₂ CH ₂ C H ₂ OCO-, J = 5.94H
z), 3.79 (6H, s, CH _3- ), 3.68-3.64 (2H, t, DMT-O-CH ₂ C H ₂
-, J = 5.61Hz), 3.50-3.45 (2H, t, SO ₂ C H ₂ CH ₂ OCO-, J = 5.94H
z), 3.18-3.14 (2H, t, DMT-OC H ₂ CH _2- , J = 5.61Hz), 2.71-
2.61 (4H, m, -COCH ₂ CH ₂ CO-).

[0097]

Example 2

[0098]

[Chemical 13]

1.60 g (2.87 m) of the compound of Example 1
mol) was dissolved in DMF (18 ml), and pentachlorophenol 0.96 g (3.6 mmol) and 1,3-dicyclohexylcarbodiimide 0.96 g (4.5 m).
mol) was added and stirred for 18 hours. The precipitated insoluble material was filtered off, and the solvent of the filtrate was distilled off. Benzene was added to the residue, the insoluble material deposited again was filtered off, and the solvent in the filtrate was distilled off.

0.16 g (0.2 mmo of this residue
l) was dissolved in DMF (4 ml) and triethylamine 1 was added.
5 μl (0.11 mmol) and aminopropyl-CPG
(1.0 g, 85.7 μmol / g amount of amino group was introduced) was added, and the mixture was allowed to stand at room temperature for 24 hours. CPG
The carrier was collected by filtration, washed with methylene chloride, and dried under reduced pressure.
This CPG carrier has a solution of caps A and B (Japan Millipore
Limited) was added in an amount of 5 ml each and left for 5 minutes. It was washed with pyridine and methylene chloride in this order and dried under reduced pressure to obtain the target compound.

The amount of the compound of Example 1 introduced into the target compound was quantified by the following method. That is, the target compound 11.
A deblocking solution (3% dichloroacetic acid / methylene chloride solution, Nippon Millipore Limited) was added to 4 mg, and the mixture was shaken for 3 minutes, and the total amount was adjusted to 20 ml with methylene chloride. After distilling out 0.4 ml of the solvent, perchloric acid was added to the residue.
Add an ethanol solution (3: 2 v / v, 3 ml), and add 50
The absorbance of the dimethoxytrityl cation at 0 nm was measured (ε = 71700).

Introduction amount of dimethoxytrityl group = 53.1
μmol / g

[0103]

Example 3 2- [2-O- (4,4′-dimethoxytrityloxy)
Si) ethylsulfonyl] ethyl 2,2,2-trichloro
Rhoethoxy carbonate 2- [2-O- (4,4'-dimethoxytrityloxy) ethylsulfonyl] ethanol (Tetrahedron Lett.
Pyridine was added to 1.1 g (2.4 mmol) of 27, 4705 (1986)), and the mixture was evaporated under reduced pressure and dried. This was dissolved in methylene chloride (12 ml), and 2,2,2-trichloroethoxycarbonyl chloride 0.35 ml (2.6 mmo
1) was added and stirred for 2 hours. After confirming the completion of the reaction by TLC, the reaction solution was diluted with methylene chloride-5% aqueous sodium hydrogen carbonate (100 m).
(l: 100 ml), and the mixture was separated. The organic layer was washed with 5% aqueous sodium hydrogen carbonate, the organic layers were collected and dried over sodium sulfate (anhydrous). The solvent was distilled off, and the residue was subjected to reverse phase column chromatography (Preparative C18, Waters, φ2.
2 x 7.0 cm, solvent: 60% acetonitrile-water), and 1.35 g (89%) of the target compound in an amorphous form.
%)Obtained.

1H-NMR (270 MHz, CDCl ₃ , TMS) δppm: 7.41-
6.83 (13H, m, Ph), 4.75 (2H, s, -CH ₂ CCl ₃ ), 4.70-4.66 (2H,
t, CH ₂ C H ₂ OC0, J = 5.94Hz), 3.80 (6H, s, CH _3- ), 3.68-3.
64 (2H, t, DMT-O-CH ₂ C H _2- , J = 5.28Hz), 3.61-3.56 (2H, t, C H
₂ CH ₂ OCO-, J = 6.27Hz), 3.23-3.19 (2H, t, DMT-OC H ₂ CH _2- ,
(J = 5.28Hz)

[0105]

Example 4

[0106]

[Chemical 14]

0.126 g of the compound of Example 3 (0.2
Aminopropyl-CPG dissolved in DMF (5 ml) and washed with 15 μl (0.11 mmol) of triethylamine and 1% triethylamine / DMF.
(1.0 g, 85.7 μmol / g amount of amino group was introduced) was added, and the mixture was allowed to stand at room temperature for 4 days. The CPG carrier was collected by filtration, washed with methylene chloride and dried under reduced pressure. 5 ml each of Cap A and B solutions (Japan Millipore Limited) were added to this CPG carrier and left for 5 minutes. Pyridine,
It was washed with methylene chloride in this order and dried under reduced pressure to obtain the target compound.

The amount of the compound of Example 3 introduced into the target compound was quantified by the following method. 11.4 mg of the target compound,
A deblocking solution (3% dichloroacetic acid / methylene chloride solution, Nippon Millipore Limited) was added, and the mixture was shaken for 3 minutes, and the total amount was adjusted to 20 ml with methylene chloride. After distilling off 0.4 ml of the solvent, a perchloric acid-ethanol (3: 2 = v / v) solution (3 ml) was added to the residue to give 500 n.
The absorbance of the dimethoxytrityl cation at m was measured (ε = 71700).

Introduction amount of dimethoxytrityl group = 24.0
μmol / g

[0110]

Example 5 Triethylammonium 5′-O-dimethoxytriti
Le-2-N-isobutyryl 2'-deoxyguanosine
-3' -O-phenyl phosphate 1,2,4-triazole 0.8 g (11.55 mmo
Pyridine was added to l), and the mixture was evaporated under reduced pressure and dried. This was suspended in 10 ml of dioxane, and 0.52 ml (3.5 mmol) of phenyl phosphorodichloride was added. This was stirred under ice-cooling and triethylamine 1.0 ml (7.3
(5 mmol) was added, the mixture was returned to room temperature and stirred for 2 hours. The generated hydrochloride was filtered off, and a phenylphosphorobistriazolidate / dioxane solution (0.35 mmol /
ml).

5'-O-dimethoxytrityl-2-N-
Isobutyryl 2'-deoxyguanosine (Methods Enz
ymol.65,610 (1980)) 0.15 g (0.23 mmol)
1 ml (0.35 mmol) of a phenyl phosphorobistriazolidate / dioxane solution was added to and the mixture was stirred at room temperature for 3 hours. About 10 ml of 30% pyridine water was added to the reaction solution, and the mixture was extracted with methylene chloride (30 ml), and then the organic layer was washed with 0.1 M triethylamine hydrogen carbonate aqueous solution (TEA
It was washed with B) and the solvent was distilled off. The residue was dissolved in methylene chloride (1 ml) and n-containing 1% triethylamine was added.
Trituration with hexane: ether (1: 1 v / v, 30 ml) gave 81 mg (40%) of the desired compound.

1H-NMR (270 MHz, CDCl ₃ , TMS) δppm: 12.07 (1
H, brs, NH), 9.67 (1H, brs, NH), 7.80-7.64 (1H, m, H8), 7.47-
6.91 (18H, m, Ph), 6.12-6.10 (1H, m, H1 '), 5.48-5.45 (1H, m,
H N + Et ₃ ) 4.64-4.58 (1H, m, H3 '), 4.28 (1H, m, H4'), 3.74 (6H,
s, OCH ₃ ), 3.38-3.18 (2H, m, H5 '), 2.98-2.89 (6H, m, NCH ₂ ),
2.73-2.51 (2H, m, H2 ' ), 2.34-2.29 (1H, m, C H (CH 3) 2), 1.25-
1.19 (9H, m, CH ₂ C H ₃ ), 1.12-0.99 (6H, m, CH (C H ₃ ) ₂ )

[0113]

Example 6

[0114]

[Chemical 15]

95 mg (5 μmol) of the compound of Example 2
Was placed in a column with a filter, 5 ml of deblocking solution (Nippon Millipore Limited) was added, and the mixture was treated for 1 minute. It was washed with 5 ml of methylene chloride and treated again with the deblocking solution for 1 minute. The mixture was washed with 5 ml of methylene chloride and pyridine in that order, pyridine was added, the mixture was evaporated, and CPG was dried. To this, a solution prepared by dissolving 20 mg of the compound of Example 5 in 0.5 ml of pyridine was added, the solvent was distilled off, and the residue was dried. To this, 2,4,6-trimethylbenzenesulfonyl-3-nitrotriazole 40 mg / pyridine 0.5
The ml solution was added and heated at 40 ° C. for 30 minutes. After washing 3 times with 5 ml of pyridine, 2.5 ml of Cap A solution (Japan Millipore Limited) and 2.5 ml of Cap B solution (Japan Millipore Limited) were added and treated for 3 minutes. The target compound was obtained by washing 3 times with 5 ml of methylene chloride.

[0116]

Example 7 5'-0-[(3,4-dibenzyloxy) benzyl]
Thymidine 3'-0-[(1,1-dimethylethyl) dimethylsilyl]
Thymidine (Can.J.Chem., 56,2768 (1978)) 713mg (2mm
ol) was dissolved in 4 mL of tetrahydrofuran, 175 mg (4 mmol) of 55% sodium hydride was added under an argon atmosphere, and the mixture was stirred at 60 ° C. for 2 hours. After returning to room temperature, a solution prepared by dissolving 678 mg (2 mmol) of (3,4-dibenzyloxy) benzyl chloride in 1 mL of tetrahydrofuran was added dropwise, 149.9 mg (1 mmol) of sodium iodide was added, and the mixture was stirred at room temperature. After 16 hours, the solvent was distilled off under reduced pressure, the residue was dissolved in 50 mL of ethyl acetate, and 50 mL of 0.01
It was washed twice with an N-ammonium chloride aqueous solution and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure, and the residue was washed with 1
3'-0-[(1,1-dimethylethyl) dimethylsilyl] -5'-0 by applying to a silica gel column of 00 g (230-400 mesh) and eluting with 0.5-3% methanol-methylene chloride. A mixture containing-[(3,4, -dibenzyloxy) benzyl] thymidine was obtained.
This was dissolved in 1.93 mL of tetrahydrofuran, 1.93 mL of a tetrahydrofuran solution (1M) of tetrabutylammonium fluoride was added, and the mixture was stirred at room temperature. After 30 minutes, the solvent was distilled off under reduced pressure, the residue was dissolved in 50 mL of ethyl acetate, washed twice with 50 mL of saturated saline, and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure, and the residue was washed with 3
It was applied to a silica gel column of 0 g (230-400 mesh) and eluted with 1 to 3% methanol-methylene chloride to obtain 345.4 mg (23.7%) of the target compound.

1H-NMR (270 MHz, CDCl ₃ , TMS) δppm: 8.26 (1H,
s, NH), 7.50 (1H, s, H6), 7.47-7.28,6.91-6.79 (13H, m, Ph),
6.36 (1H, t, H1 ', J = 6.75Hz), 5.17 (4H, s, PhCH ₂ ), 4.47,4.45
(2H, 2s, PhCH ₂ OC H ₂ ), 4.41-4.36 (1H, m, H3 '), 4.04-4.01 (1
H, m, H4 '), 3.72-3.56 (2H, m, H5'), 2.31-2.05 (2H, m, H2 '),
1.62 (3H, s, CH ₃ ).

[0118]

Example 8 5'-0-[(3,4-dibenzyloxy) benzyl] thymi
Gin-3'-0- (2-cyanoethyl N, N-diiso
Propyl) phosphoramidite 271.7 mg (0.499 mmol) of the compound of Example 7 was treated with pyridine to give 3
After being azeotropically dried and dissolved in 10 mL of tetrahydrofuran, 1.39 mL (8 mmol) of diisopropylethylamine and 0.822 mL (4 mmol) of 2-cyanoethyl N, N-diisopropylchlorophosphoramidite were added, and under an argon atmosphere, Stir at room temperature. After 30 minutes, the precipitate was filtered off and the solvent was evaporated under reduced pressure. The residue was dissolved in 100 mL of ethyl acetate and treated with 50 mL of ice-cold 10% Na2CO3 water.
It was washed twice and dried over anhydrous sodium sulfate. After evaporating the solvent under reduced pressure, apply the residue to a column containing 40 g (70-230 mesh) of silica gel and elute with methylene chloride: ethyl acetate: triethylamine (45:45:10, v / v / v). This gave 286.9 mg (77%) of the desired compound.

1H-NMR (270 MHz, CDCl3, TMS) δ ppm: 8.
07 (1H, bs, NH), 7.53, 7.51 (1
H, 2s, H6), 7.45-7.25, 6.92-
6.81 (13H, m, Ph), 6.37 (1H, t,
H1 ′, J = 6.60 Hz), 5.15 (4H, s, P
_{hCH 2), 4.62-4.53 (1H,} m, H
3 '), 4.47 (2H, s, PhCH 2 OC H 2),
4.22, 4.14 (1H, 2bs, H4 '), 3.9
0-3.53 (6H, m, H5 ' , POCH 2, PNC
H), 2.68-2.53 (2H, m , NCCH 2),
2.47-1.05 (2H, m, H2 '), 1.58
(3H, s, CH ₃ ), 1.17 (12H, d, (C H
₃ ) ₂ CH, J = 5.94 Hz).

[0120]

[Example 9]

[0121]

[Chemical 16]

CycloneTM Plus DNA / RNA Synthesizer from Milligen / Biosearch (Japan Millipore Limited), with the reagents for synthesis and 1 program
A 5.0 μmol amidite cartridge was connected. However, at this time, instead of the β-cyanoethylamidite solution corresponding to thymidine, Example 8 prepared at about 35 mM was used.
An acetonitrile solution of the compound was used, and 95 mg (5 μmol) of the compound of Example 6 was used as a solid-phase carrier and 15.0 μmol
Used as an empty column for use. TGGGAG
The following nucleotide sequence was input, and after the last T was linked, a program was set up in which no acid treatment was performed to obtain a derivative in which the oligonucleotide was constructed on controlled pore glass (CPG).

This was dried under reduced pressure, then taken out from the column, immersed in about 10 ml of 29% ammonia water and sealed, and heated at 60 ° C. for 5 hours. CPG was removed by filtration and washed twice with 10 ml of water. The filtrate and the washing solution were combined, the solvent was concentrated to about half, washed with 30 mL of ethyl acetate three times, and the aqueous solution was concentrated under reduced pressure to about 3 ml. Reverse phase column chromatography (Preparativ
e C18, Waters 1.5 x 15 cm; 50 mM triethylamine hydrogencarbonate aqueous solution (TEAB), pH 7.5; 15-50% acetonitrile; linear gradient; 254 nm) to obtain 67 OD (260 nm) of an amorphous target compound. This compound was analyzed by reverse phase HPLC (Inertsil ODS, 6x150 mm;
1M triethylamine acetate aqueous solution (TEAA), pH 7.5; 1
0-60% acetonitrile / 20min; linear gradi
ent; 1 ml / min; 260 nm), and was eluted at 19.51 min.

UVmax: 255 nm

[0125]

Example 10

[0126]

[Chemical 17]

CycloneTM Plus DNA / RNA Synthesizer from Milligen / Biosearch (Japan Millipore Limited) is supplied with the synthetic reagents and 1
A 5.0 μmole midite cartridge was connected. However, at this time, instead of the β-cyanoethylamidite solution corresponding to guanosine, 5′-O prepared to about 35 mM was prepared.
-Dimethoxytrityl-2-N-isobutyryl-2'-
A deoxyguanosine-3'-O- (methyl N, N-diisopropyl) phosphoramidite (Sigma) solution in acetonitrile was used. As a solid phase carrier, 95 mg (5 μmol) of the compound of Example 6 was used as an empty column for 15.0 μmol. I used it after all. Enter the base sequence GX
After binding, the target compound was obtained by activating a program set so that no acid treatment was performed.

[0128]

[Embodiment 11]

[0129]

[Chemical 18]

95 mg (5 μmol) of the compound of Example 10
Synthesis was performed on the packed column in the same manner as in Example 9. Reversed phase column chromatography (Preparative C1
8, Waters 1.5x 15 cm; 50 mM TEAB, pH 7.5; 20-
50% acetonitrile; linear gradient; 254 nm) to obtain an amorphous target compound at 50 OD (260 nm). This compound was applied on reverse phase HPLC (Inertsil ODS, 6 x 150m
m; 0.1 M TEAA, pH 7.5; 10-60% acetonitrile / 20mi
n; linear gradient; 1 ml / min; 260 nm)
It was eluted at 9.15 min. UVmax: 254 nm In the same manner as described above, the compounds shown in Tables 1 and 2 below were obtained.

[0131]

[Table 1]

[0132]

[Table 2]

[0133]

Example 23

[0134]

[Chemical 19]

1.60 g (2.87 m) of the compound of Example 1
mol) was dissolved in DMF (18 ml), and pentachlorophenol 0.96 g (3.6 mmol), 1,3-
Dicyclohexylcarbodiimide 0.96 g (4.
5 mmol) was added and the mixture was stirred for 18 hours. The precipitated insoluble material was filtered off, and the solvent of the filtrate was distilled off. Benzene was added to the residue, the insoluble material deposited again was filtered off, and the solvent in the filtrate was distilled off.

Of this residue, 1.0 g (0.92 m
(mol) was dissolved in DMF (20 ml) and triethylamine (75 μl, 0.55 mmol) was added to Cosmo Seal 1
50NH2-300 (3.0 g, amino group 450 μm
ol / g amount introduced, Nakarai Tesque Co., Ltd.
Was added and the mixture was allowed to stand at room temperature for about 20 hours. The carrier was collected by filtration, washed with methylene chloride and dried under reduced pressure. 10 ml each of Cap A and B solutions (Nippon Millipore Limited) are added to this carrier.
The mixture was left for 10 minutes. It was washed with pyridine and methylene chloride in this order and dried under reduced pressure to obtain the target compound.

The amount of the compound of Example 1 introduced into the target compound was quantified by the following method. To 5.4 mg of the target compound, a deblocking solution (3% dichloroacetic acid / methylene chloride solution,
(Japan Millipore Limited) was added and shaken for 3 minutes, and the total amount was adjusted to 20 ml with methylene chloride. 0.4 of them
After distilling off the solvent of ml, a perchloric acid-ethanol solution (3: 2 v / v, ml) was added to the residue, and the absorbance of the dimethoxytrityl cation at 500 nm was measured (ε = 71).
700).

Introduction amount of dimethoxytrityl group = 51.8
μmol / g

[0139]

Example 24

[0140]

[Chemical 20]

1.60 g (2.87 m) of the compound of Example 1
mol) was dissolved in DMF (18 ml), and pentachlorophenol 0.96 g (3.6 mmol), 1,3-
Dicyclohexylcarbodiimide 0.96 g (4.
5 mmol) was added and the mixture was stirred for 18 hours. The precipitated insoluble material was filtered off, and the solvent of the filtrate was distilled off. Benzene was added to the residue, the insoluble material deposited again was filtered off, and the solvent in the filtrate was distilled off.

Of this residue 0.48 g (0.6 m
mol) was dissolved in DMF (15 ml) and dimethylaminopyridine 110 mg (0.9 mmol) and p-alkoxybenzyl alcohol resin (1.0 g, amino group 0.9 mmol / g amount introduced, domestic chemical ( Strain)) was added and the mixture was allowed to stand at room temperature for 25 hours. The carrier was collected by filtration, washed with methylene chloride, methanol and ether in this order and then dried under reduced pressure. 10 ml of each of Cap A and B solutions (Nippon Millipore Limited) were added to this carrier and left for 15 minutes. It was washed with methylene chloride, methanol and ether in this order and dried under reduced pressure to obtain the target compound.

The amount of the compound of Example 1 introduced into the target compound was quantified by the following method. 10.6 mg of the target compound,
A deblocking solution (3% dichloroacetic acid / methylene chloride solution, Nippon Millipore Limited) was added and shaken for 3 minutes, and the total amount was adjusted to 20 ml with methylene chloride. After distilling off 0.4 ml of the solvent, a perchloric acid-ethanol solution (3: 2 v / v, 6 ml) was added to the residue, and the absorbance of the dimethoxytrityl cation at 500 nm was measured (ε = 71700).

Introduction amount of dimethoxytrityl group = 302 μ
mol / g

[0145]

Example 25

[0146]

[Chemical 21]

Poly (oxyethylene) diglycolic acid
9.9 g (3.3 mmol) was dissolved in pyridine and evaporated under reduced pressure, the residue was dissolved in 10 ml of dichloromethane, and 1,3-dicyclohexylcarbodiimide was stirred under ice cooling.
226 mg (1.1 mmol) was added. After 15 minutes, 2- {2-O- dissolved in 2 ml of dichloromethane.
(4,4′-dimethoxytrityloxy) ethylsulfonyl} ethanol (Tetrahedron Lett. 27, 4705 (1
986)) 0.5 g (1.1 mmol) and 0.13 g (1.1 mmol) of dimethylaminopyridine were added and the mixture was allowed to stand at room temperature
Stir for time. After confirming the disappearance of the raw materials by TLC, the precipitate was filtered off and concentrated to about 50 ml. This solution was dropped into 500 ml of ice-cooled ether to obtain a powder. This was dissolved in ethylene chloride and purified with a 200 g silica gel column (solvent, 20% methanol-methylene chloride),
2.77 g of the desired compound was obtained.

1 H-NMR (270 MHz, CDCl ₃ , TMS) δ ppm:
7.43-7.09, 6.88-6.81 (m, P
_{h), 4.64-4.58 (m, -SO} 2 CH 2 C H
_{2 O-CO-), 3.65 (} s, -OCH 2 CH 2 O-), 3.53-
_{3.50 (m, DMT-OCH 2} C H 2 SO 2 -), 3.49-3.46 (m, -SO 2 C H 2 CH 2 O-C
O-), 3.34-3.31 (m, DMT -O-C H
₂ CH ₂ SO ₂ -)

[0149]

Example 26

[0150]

[Chemical formula 22]

0.7 g of the compound of Example 25 (about 0.2 m
(mol) in 6 ml of methylene chloride and cooled with ice
-Dicyclohexylcarbodiimide 64 mg (0.
3 mmol) and pentachlorophenol (63 mg,
After adding a solution of 0.24 mmol) dissolved in 2 ml of DMF, the mixture was returned to room temperature and stirred for 7 days. After the precipitate was filtered off, the solvent of the filtrate was distilled off and the residue was dissolved in 10 ml of methylene chloride and dropped into 100 ml of ether under ice cooling to obtain a powder.
This was dissolved in 6 ml of DMF, and 37 mg (0.3 mmol) of dimethylaminopyridine and p-alkoxybenzyl alcohol resin (0.25 g, the amino group was 0.9 m).
The amount of mol / g introduced, Kokusan Kagaku Co., Ltd., was added, and the mixture was left at room temperature for 24 hours. The carrier was recovered by filtration, washed with pyridine, methylene chloride and ether, and dried under reduced pressure. 10 ml each of Cap A and B solutions (Nippon Millipore Limited) were added to this carrier and left for 30 minutes. It was washed with methylene chloride, methanol and ether in this order and dried under reduced pressure to obtain the target compound.

The amount of the compound of Example 1 introduced into the target compound was quantified by the following method. To 6.7 mg of the target compound, a deblocking solution (3% dichloroacetic acid / methylene chloride solution,
(Japan Millipore Limited) was added and shaken for 3 minutes, and the total amount was adjusted to 20 ml with methylene chloride. 0.4 of them
After distilling off the solvent of ml, a perchloric acid-ethanol solution (3: 2 v / v, 3 ml) was added to the residue, and the absorbance of the dimethoxytrityl cation at 500 nm was measured (ε = 7).
1700).

Introduction amount of dimethoxytrityl group = 16 μm
ol / g

Claims (1)

[Claims] 1. A general formula: [In the formula, R ¹ , R ² and R ³ each independently represent the same or different and represent a hydrogen atom or an alkoxy group having 1 to 4 carbon atoms, and X ¹ represents an —S— group, an —SO— group or SO ₂ - represents a group, a is - (CH ₂₎ h - group (h is an integer of 1 to _{16), - (CH 2)} h O- group (h is an integer of 1 to 16), _{- (CH 2) j -O-} CO- (CH 2)
k - group (j is a positive integer, k is 0 or a positive integer, and j + k is 2 to _{16), - (CH 2)} j
_{-NH-CO- (CH 2) k} - group (j is a positive integer, k is 0 or a positive integer, and j + k is 2 to 1
6 _{is), - (CH 2) j} -S-CO- (CH 2) k
- group (j is a positive integer, k is 0 or a positive integer, and j + k is 2 to _{16), - (CH 2)} n
_{-O-CO-CH 2 (OCH} 2 CH 2) p-OCH 2 -
A group (n is a positive integer, p is 0 or a positive integer, and n + p is 2 to 100), Y ¹
Represents a hydroxyl group, a phenyloxy group which may have a substituent, or an ethyloxy group which may be substituted with a halogen. ] To the compound represented by the general formula: [In the formula, X ² represents an oxygen atom, a sulfur atom or an NH group, and HX ² -P (circled) represents a polymer substance. ] It is obtained by reacting a polymer substance represented by the formula (However, when Y ¹ in the formula (2) is a hydroxyl group, it is reacted with a carboxylic acid activator and then with a polymer substance (3)). The general formula: [Wherein R ¹ , R ² , R ³ , X ¹ , A, X ² and HX ²
-P (circled) has the same meaning as described above. ] The polymer substance (4) represented by
A general formula characterized by extending the A chain [In the formula, R ⁴ , R ⁵ , R ⁶ and Z group (in the formula, R ⁴ , R ⁵
And R ⁶ are independently the same or different and each is a hydrogen atom,
Represents an alkyl group having 1 to 4 carbon atoms, an aryl group which may have a substituent or an anthraquinonyl group which may have a substituent, and Z represents a carbon atom or a silicon atom,
R ⁵ , R ⁶ and Z together represent a fluorenyl group or a xanthenyl group, or R ⁴ , R ⁵ , R ⁶ and Z together represent a hydrogen atom) and R ⁷ represents Hydrogen atom, an aralkyl group having 1 to 4 carbon atoms which may have a substituent, an aryl group which may have a substituent or an aralkyl group which may have a substituent, Y ²
Are the same or different and represent an oxygen atom, a sulfur atom or an NH group, Y ³ is an oxygen atom, a sulfur atom, an NH group, an alkylene group having 1 to 4 carbon atoms or a phenylene group, and X ³ is an oxygen or a sulfur atom. And D represents an oligonucleotide having a chain length of 2 to 30. However, D does not include hydroxyl groups at the 5'end and 3'end of each oligonucleotide. ]
The manufacturing method of the compound represented by.