Oligonucleotide production method

Abstract

The purpose of the present invention is to provide an oligonucleotide production method that makes it possible to reduce the amount of an organic solvent to be used and that is for obtaining an oligonucleotide with a desired yield. This problem is solved by, inter alia, the oligonucleotide production method characterized in that: a cleaning agent used in a synthesis step contains carbon dioxide and a base and / or contains carbon dioxide and an acid; and the carbon dioxide content contained in the cleaning agent is 50 v / v% or greater.

Description

Method for producing oligonucleotide

[0001] The present invention relates to a method for producing an oligonucleotide.

[0002] In the chemical synthesis of oligonucleotides, solid-phase synthesis using the phosphoramidite method is widely used. In the chemical synthesis of oligonucleotides, a large amount of organic solvents are used as cleaning agents in each step and after each step. Recently, from the perspective of environmental problems on Earth (e.g., global warming, etc.) and effects on the human body, reduction of the use of organic solvents has been demanded, and reduction is also sought for organic solvents used as cleaning agents in each step and after each step in the chemical synthesis of oligonucleotides. A method of performing a synthesis reaction of deoxyribonucleic acid, ribonucleic acid, and compounds containing them in a mixed fluid of chemical species such as supercritical fluids such as carbon dioxide, and obtaining ribonucleic acid, deoxyribonucleic acid, and compounds containing them has been studied (Patent Document 1). However, it has been found that it is difficult to obtain an oligonucleotide with a desired yield by simply replacing the cleaning agent used in each step of the chemical synthesis of oligonucleotides with a supercritical fluid such as carbon dioxide, and further improvement is required to further improve the yield of oligonucleotides.

[0003] Japanese Patent Application Laid-Open No. 11-206371

[0004] An object of the present invention is to provide a production method capable of reducing the organic solvent to be used and obtaining an oligonucleotide with a desired yield in a method for producing an oligonucleotide.

[0005] As a result of intensive studies to solve the above problems, the present inventors have found that in a method for producing an oligonucleotide, by using a cleaning agent containing carbon dioxide and a base and / or containing carbon dioxide and an acid for cleaning after each step, the organic solvent to be used can be reduced and an oligonucleotide with a desired yield can be obtained. Further studies were conducted and the present invention was completed.

[0006] In other words, the present invention relates to the following: (1) A method for producing an oligonucleotide comprising: (a) a step of removing a protecting group from a protected nucleoside that is directly or indirectly supported on a carrier and has a protecting group bonded to a hydroxyl group, thiol group, or amino group at the 3' or 5' position; (b) a step of bonding a nucleoside phosphoramidite to the hydroxyl group, thiol group, or amino group at the 3' or 5' position of the nucleoside from which the protecting group has been removed, which is directly or indirectly supported on a carrier; (c) a step of sulfidating or oxidizing the bond formed in step (b); and (d) a step of capping the unbonded hydroxyl group, thiol group, or amino group at the 3' or 5' position of the nucleoside that is directly or indirectly supported on a carrier, wherein the method comprises washing with a detergent containing carbon dioxide and a base after step (a), and / or washing with a detergent containing carbon dioxide and an acid after step (d), The method, characterized in that the carbon dioxide content in the detergent is 50 v / v% or more. (2) The method according to (1), wherein the base is at least one selected from the group consisting of pyridine, triethylamine, N,N-diisopropylamine, tetramethylethylenediamine, morpholine, N-methylmorpholine, imidazole, N-methylimidazole, diethylamine, diisopropylamine, dibutylamine, ethylamine, propylamine, isopropylamine, butylamine, and tert-butylamine. (3) The method according to (1) or (2), wherein the acid is at least one selected from the group consisting of acetic acid, nitric acid, sulfuric acid, lactic acid, phosphoric acid, and acrylic acid.

[0007] The method for producing oligonucleotides according to the present invention makes it possible to reduce the amount of organic solvent used and to obtain oligonucleotides in a desired yield.

[0008] Figure 1 shows the temperature-pressure diagram (phase diagram) of carbon dioxide.

[0009] The present invention will now be described in detail. Unless otherwise defined herein, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. All patents, applications, and other publications and information referenced herein are incorporated herein by reference in their entirety. In the event of any conflict between the publications referenced herein and the descriptions herein, the descriptions herein shall prevail.

[0010] In one aspect, the present invention relates to a method for producing oligonucleotides. In the present invention, the production of oligonucleotides is carried out using a so-called phosphoramidite method, in which nucleotides are added by a condensation reaction between a nucleoside phosphoramidite and a nucleoside, nucleotide, or oligonucleotide in the presence of a suitable activator.

[0011] In the present invention, a method for producing oligonucleotides includes, for example, the steps of: (a) removing a protecting group from a protected nucleoside that is directly or indirectly supported on a carrier and has a protecting group bonded to a hydroxyl group, thiol group, or amino group at the 3' or 5' position; (b) bonding a nucleoside phosphoramidite to the hydroxyl group, thiol group, or amino group at the 3' or 5' position of the nucleoside from which the protecting group has been removed, which is directly or indirectly supported on a carrier; (c) sulfiding or oxidizing the bond formed in step (b); and (d) capping the unbonded hydroxyl group, thiol group, or amino group at the 3' or 5' position of the nucleoside that is directly or indirectly supported on a carrier.

[0012] In the present invention, the method for producing oligonucleotides may include further steps in addition to (a) to (d) above. In the present invention, steps (a) to (d) do not have to be performed in the order of (a) → step (b) → step (c) → step (d), but may be performed in an order such as (a) → step (b) → step (d) → step (c).

[0013] In the present invention, a nucleoside refers to a compound in which a nucleoside base and a sugar are bonded, and may be a naturally occurring nucleoside such as adenosine, thymidine, guanosine, cytidine, and uridine, or a modified nucleoside. Modified nucleosides are not limited to these, but include, for example, those in which the hydroxyl group at the 3' or 5' position of the nucleoside is replaced with a thiol group or an amino group. The nucleoside base may be a naturally occurring base such as adenine, guanine, cytosine, thymine, and uracil, or a modified nucleoside base. The sugar portion of the nucleoside may be a naturally occurring deoxyribose or ribose, and may have a D configuration or an L configuration.

[0014] In the present invention, a nucleotide refers to a compound in which a nucleoside base, a sugar, and a phosphate group are bonded, and may be a naturally occurring nucleotide such as adenosine triphosphate, thymidine triphosphate, guanosine triphosphate, cytidine triphosphate, or uridine triphosphate, or a modified nucleotide. The nucleoside base portion of the nucleotide may be a naturally occurring base such as adenine, guanine, cytosine, thymine, and uracil, or a modified nucleoside base. The sugar portion of the nucleoside may be a naturally occurring deoxyribose or ribose, and may have a D or L configuration. The phosphate portion may be, for example, a phosphorothioate, phosphorodithioate, methylphosphonate, or methyl phosphate.

[0015] In the present invention, oligonucleotides refer to compounds having a structure in which a nucleoside base, a sugar, and a phosphate group are linked by a phosphodiester bond, and include naturally occurring oligonucleotides, such as 2'-deoxyribonucleic acid (hereinafter, "DNA") and ribonucleic acid (hereinafter, "RNA"), and nucleic acids containing a modified sugar moiety, a modified phosphate moiety, or a modified nucleobase. Modification of the sugar moiety includes replacing the ribose ring with a hexose, cyclopentyl, or cyclohexyl ring. Alternatively, the D-ribose ring of a naturally occurring nucleic acid may be replaced with an L-ribose ring, or the β-anomer of a naturally occurring nucleic acid may be replaced with an α-anomer. Oligonucleotides may also contain one or more non-basic moieties. Modified phosphate moieties include phosphorothioates, phosphorodithioates, methylphosphonates, and methyl phosphate. Such nucleic acid analogs are known to those skilled in the art. Oligonucleotides comprising the above two or more mixtures can be produced, for example, from oligonucleotides comprising a mixture of deoxyribo and ribonucleosides, particularly a mixture of deoxyribonucleosides and 2'-O-substituted ribonucleosides such as 2'-O-methyl or 2'-O-methoxyethylribonucleosides. Examples of oligonucleotides comprising a mixture of nucleosides include ribozymes.

[0016] In the present invention, nucleoside phosphoramidite refers to a nucleoside derivatized with an amidite. In the present invention, a nucleoside phosphoramidite is a nucleoside in which either the 3'-position hydroxyl group or the 5'-position hydroxyl group is phosphoramidized, and a protecting group is attached to the other. Examples of protecting groups include acetyl group, benzoyl group, isobutyl group, and dimethylformamidyl group. Methods for amiditization are generally known, but for example, it can be carried out by reacting a properly protected nucleoside with 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphodiamidite using 1H-tetrazole as an activator. The nucleoside phosphoramidite may be a monomer or an oligomer such as 2mer to 24mer.

[0017] In the present invention, a method for producing oligonucleotides may include a step of binding a nucleoside phosphoramidite to the hydroxyl group, thiol group, or amino group at the 3' or 5' position of a nucleoside directly or indirectly supported on a carrier, in the presence of an activator. In the present invention, the activator refers to an agent that activates the nucleoside phosphoramidite, and is used to react with a nucleoside, nucleotide, or oligonucleotide, and is also called an activator or coupling agent. In the present invention, activators commonly used in the phosphoramidite method can be used. The activators used in the present invention are not limited to these, but include, for example, 4,5-dicyanoimidazole, 5-(ethylthio)-1H-tetrazole, 5-(benzylthio)-1H-tetrazole, and saccharin 1-methylimidazole, and preferably 4,5-dicyanoimidazole.

[0018] In the present invention, "nucleoside directly supported on a carrier" refers to the nucleoside portion of a compound in which a nucleoside or nucleotide is bound to the reaction site of the carrier (the compound portion in which a nucleoside base and sugar are bound), and "nucleoside indirectly supported on a carrier" refers to the nucleoside portion in which a nucleotide is bound to the reaction site of the carrier via a compound such as a polynucleotide (the compound portion formed by the binding of a nucleoside base and sugar).

[0019] In one embodiment of the present invention, the present invention includes washing with a detergent containing carbon dioxide and a base after step (a). In one embodiment of the present invention, the present invention includes washing with a detergent containing carbon dioxide and an acid after step (d). In one embodiment of the present invention, the present invention includes washing with a detergent containing carbon dioxide and a base after step (a), and / or washing with a detergent containing carbon dioxide and an acid after step (d).

[0020] In the present invention, the carbon dioxide content in the detergent is 50 v / v% or more, more preferably 70 v / v% or more, even more preferably 90 v / v% or more, and particularly preferably 95 v / v% or more. The detergent may be prepared, for example, at -10°C and 6 MPa. As shown in the temperature-pressure diagram (phase diagram) of Figure 1, the state of carbon dioxide changes depending on the pressure and temperature conditions. In the present invention, carbon dioxide may be in the state of a supercritical fluid, gas, or liquid, but is preferably a supercritical fluid or gas.

[0021] In the present invention, the base is selected from the group consisting of pyridine, triethylamine, N,N-diisopropylamine, tetramethylethylenediamine, morpholine, N-methylmorpholine, imidazole, N-methylimidazole, diethylamine, diisopropylamine, dibutylamine, ethylamine, propylamine, isopropylamine, butylamine, and tert-butylamine, preferably pyridine, triethylamine, N-methylmorpholine, and N-methylimidazole, and more preferably pyridine. In the present invention, the base content in the detergent is preferably 0.1 v / v% to 50 v / v%, more preferably 0.1 v / v% to 30 v / v%, even more preferably 0.1 v / v% to 10 v / v%, and particularly preferably 0.1 v / v% to 5 v / v%.

[0022] In the present invention, the acid is selected from the group consisting of acetic acid, nitric acid, sulfuric acid, lactic acid, and acrylic acid, preferably acetic acid, sulfuric acid, and acrylic acid, and more preferably acetic acid. In the present invention, the acid content in the detergent is preferably 0.1 v / v% to 50 v / v%, more preferably 0.1 v / v% to 30 v / v%, even more preferably 0.1% to 10 v / v%, and particularly preferably 0.1 v / v% to 5 v / v%. In the present invention, the detergent may optionally contain components other than carbon dioxide, bases, and acids.

[0023] The present invention will be described in more detail with reference to the following examples, which are specific examples of the present invention and are not limited thereto. In Examples 1 to 4 and Comparative Examples 1 to 3, four sequences, 5'-TT-3', 5'-AT-3', 5'-CT-3', and 5'-GT-3', were synthesized on a succinate solid support containing a linker using the following reagents. In Examples 5 and 6, the 5'-TT-3' sequence was synthesized on a succinate solid support containing a linker using the following reagents. The succinate support was self-produced with a loading rate of 350 μmol / gram.

[0024] <Synthesis Reagents> (Deprotecting Agent) 3% Dichloroacetic Acid / Toluene (Nucleoside Phosphoramidites) N-Benzoyl-5'-O-(4,4'-Dimethoxytrityl)-2'-Deoxycytidine-3'-(2-Cyanoethyl-N,N-Diisopropyl)Phosphoramidite (dC) 5'-O-(4,4'-Dimethoxytrityl)-2'-Deoxythymidine-3'-(2-Cyanoethyl-N,N-Diisopropyl)Phosphoramidite (dT) N-Benzoyl-5'-O-(4,4'-Dimethoxytrityl)-2'-Deoxyadenosine-3'-(2-Cyanoethyl-N,N-Diisopropyl)Phosphoramidite (dA) N ２ -Isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine-3'-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (dG) (both manufactured by Hongen Biotech)

[0025] (Activating agent) 0.2 M 4,5-dimethylimidazole (manufactured by Tokyo Chemical Industry Co., Ltd.) / acetonitrile (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (Oxidizing agent) Iodine / water / pyridine (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (Capping agent) Acetic anhydride / pyridine / N-methylimidazole / acetonitrile (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)

[0026] <Test 1> 110 mg of the carrier was placed in the reaction column and set in a supercritical fluid apparatus. The synthesis reagents were then flowed through the reaction column according to the DNA synthesis program in the following order: (a) → (b) → (c) → (d). (Step (a)) Deprotection of the 5'-OH group of the nucleoside with a deprotecting agent (3% dichloroacetic acid / toluene) (Step (b)) Coupling reaction of the amidite with 0.2 M nucleoside phosphoramidite / acetonitrile and an activator (0.2 M 4,5-dimethylimidazole / acetonitrile) (Step (c)) Oxidation of the phosphite with an oxidizing agent (iodine / water / pyridine) (Step (d)) Capping of the unreacted 5'-OH group with a capping agent (acetic anhydride / pyridine / N-methylimidazole / acetonitrile)

[0027] Example 1 In Test 1, step (a) was performed using a washing agent prepared by mixing 99 v / v% liquefied carbon dioxide (-10°C, 6 MPa, purity: 99.9 v / v%) with 1 v / v% pyridine (manufactured by Fujifilm Wako Pure Chemical Industries), and washed for 60 minutes under supercritical fluid conditions of 40°C and 20 MPa. Steps (b), (c), and (d) were washed with acetonitrile (manufactured by Fujifilm Wako Pure Chemical Industries). After all solutions had been delivered, the carrier was removed from the container, filtered by suction, washed with 50 mL of acetonitrile, and dried.

[0028] Example 2 In Test 1, the washing in step (d) was performed using a washing agent prepared by mixing 99 v / v% liquefied carbon dioxide (-10°C, 6 MPa, purity: 99.9 v / v%) and 1 v / v% acetic acid (manufactured by Fujifilm Wako Pure Chemical Industries), and the washing was carried out for 60 minutes under supercritical fluid conditions of 40°C and 20 MPa. The washing in steps (a), (b), and (c) was performed with acetonitrile. After all solutions had been delivered, the carrier was removed from the container, filtered by suction, washed with 50 mL of acetonitrile, and dried.

[0029] Comparative Example 1 In Test 1, step (a) was performed using a cleaning agent consisting of liquefied carbon dioxide (-10°C, 6 MPa, purity: 99.9 v / v%), and the washing was carried out for 60 minutes under supercritical fluid conditions of 40°C and 20 MPa. Steps (b), (c), and (d) were performed using acetonitrile. After all solutions had been delivered, the carrier was removed from the container, filtered by suction, washed with 50 mL of acetonitrile, and dried.

[0030] Comparative Example 2 In Test 1, step (d) was performed using a cleaning agent consisting of liquefied carbon dioxide (-10°C, 6 MPa, purity: 99.9 v / v%), and the washing was carried out for 60 minutes under supercritical fluid conditions of 40°C and 20 MPa. Steps (a), (b), and (c) were washed with acetonitrile. After all solutions had been delivered, the carrier was removed from the container, filtered by suction, washed with 50 mL of acetonitrile, and dried.

[0031] <Test 2> 110 mg of the carrier was placed in the reaction column and set in the supercritical fluid apparatus. Synthesis reagents were flowed through the reaction column according to the DNA synthesis program in the order of steps (a) → (b) → (d) → (c).

[0032] Example 3 In Test 2, step (a) was performed using a cleaning agent consisting of liquefied carbon dioxide (-10°C, 6 MPa, purity: 99.9 v / v%) and pyridine (1 v / v%), and washed for 60 minutes under supercritical conditions of 40°C and 20 MPa. Step (d) was performed using a cleaning agent consisting of liquefied carbon dioxide (-10°C, 6 MPa, purity: 99.9 v / v%), and washed for 60 minutes under supercritical conditions of 40°C and 20 MPa. Steps (b) and (c) were washed with acetonitrile. After all solutions had been delivered, the carrier was removed from the container, filtered by suction, washed with 50 mL of acetonitrile, and dried.

[0033] Example 4 In Test 2, step (a) was performed using a cleaning agent consisting of 95 v / v% liquefied carbon dioxide (-10°C, 6 MPa, purity: 99.9 v / v%) and 5 v / v% pyridine, and was washed for 60 minutes at 20°C and atmospheric pressure. Step (d) was performed using a cleaning agent consisting of liquefied carbon dioxide (-10°C, 6 MPa, purity: 99.9 v / v%), and was washed for 60 minutes at 20°C and atmospheric pressure. Steps (b) and (c) were washed with acetonitrile. After all solutions had been delivered, the carrier was removed from the container, filtered by suction, washed with 50 mL of acetonitrile, and dried.

[0034] Comparative Example 3 In Test 2, step (a) was performed using a cleaning agent consisting of liquefied carbon dioxide (-10°C, 6 MPa, purity: 99.9 v / v%), and washing was carried out for 60 minutes under supercritical conditions of 40°C and 20 MPa. Step (d) was performed using a cleaning agent consisting of liquefied carbon dioxide (-10°C, 6 MPa, purity: 99.9 v / v%), and washing was carried out for 60 minutes under supercritical conditions of 40°C and 20 MPa. Steps (b) and (c) were washed with acetonitrile. After all solutions had been delivered, the carrier was removed from the container, filtered by suction, washed with 50 mL of acetonitrile, and dried.

[0035] Example 5 In Test 1, step (a) was performed using a washing agent prepared by mixing 60 v / v% liquefied carbon dioxide (-10°C, 6 MPa, purity: 99.9 v / v%) with 40 v / v% pyridine, and washing was carried out for 10 minutes under supercritical fluid conditions of 40°C and 20 MPa. Steps (b), (c), and (d) were washed with acetonitrile. After all solutions had been delivered, the carrier was removed from the container, filtered by suction, washed with 50 mL of acetonitrile, and dried.

[0036] Example 6 In Test 1, step (d) was performed using a cleaning agent prepared by mixing 60 v / v% liquefied carbon dioxide (-10°C, 6 MPa, purity: 99.9 v / v%) and 40 v / v% acetic acid, and washing was carried out for 10 minutes under supercritical fluid conditions of 40°C and 20 MPa. Steps (a), (b), and (c) were washed with acetonitrile. After all solutions had been delivered, the carrier was removed from the container, filtered by suction, washed with 50 mL of acetonitrile, and dried.

[0037] In Examples 7-10 and Comparative Examples 4-6, the four sequences 5'-UT-3', 5'-AT-3', 5'-CT-3', and 5'-GT-3' were synthesized on a succinate solid support containing a linker using the following reagents. The succinate support was in-house produced at a loading rate of 350 μmol / gram.

[0038] <Synthesis Reagents> (Deprotecting Agent) 3% Dichloroacetic Acid / Toluene (Nucleoside Phosphoramidites) N-Benzoyl-5'-O-[bis(4-methoxyphenyl)phenylmethyl]-2'-O-[(1,1-dimethylethyl)dimethylsilyl]-adenosine (rA) N-Acetyl-5'-O-[bis(4-methoxyphenyl)phenylmethyl]-2'-O-[(1,1-dimethylethyl)dimethylsilyl]-cytidine (rC) N-(2-methyl-1-oxopropyl)-5'-O-[bis(4-methoxyphenyl)phenylmethyl]-2'-O-[(1,1-dimethylethyl)dimethylsilyl]-guanosine (rG) 5'-O-[bis(4-methoxyphenyl)phenylmethyl]-2'-O-[(1,1-dimethylethyl)dimethylsilyl]-uridine (rU) All are (Hongen (Made by Biotech)

[0039] (Activator) 0.2 M 4,5-dimethylimidazole / acetonitrile (Oxidizing agent) Iodine / water / pyridine (Capping agent) Acetic anhydride / pyridine / N-methylimidazole / acetonitrile

[0040] <Test 3> 110 mg of the carrier was placed in the reaction column and set in a supercritical fluid apparatus. The synthesis reagents were then flowed through the reaction column according to the RNA synthesis program in the following order: (a) → (b) → (c) → (d). (a) Deprotection of the 5'-OH group of the nucleoside with a deprotecting agent (3% dichloroacetic acid / toluene) (b) Coupling reaction of the amidite with 0.2 M nucleoside phosphoramidite / acetonitrile and an activator (0.3 M 5-(ethylthio)-1H-tetrazole / acetonitrile) (c) Oxidation of the phosphite with an oxidizing agent (iodine / water / pyridine) (d) Capping of the unreacted 5'-OH group with a capping agent (acetic anhydride / pyridine / N-methylimidazole / acetonitrile)

[0041] Example 7 In Test 3, step (a) was performed using a washing agent prepared by mixing liquefied carbon dioxide (-10°C, 6 MPa, purity: 99.9 v / v%) and pyridine 1 v / v%, and washing was carried out for 60 minutes under supercritical fluid conditions of 40°C and 20 MPa. Steps (b), (c), and (d) were washed with acetonitrile. After all solutions had been delivered, the carrier was removed from the container, filtered by suction, washed with 50 mL of acetonitrile, and dried.

[0042] Comparative Example 4 In Test 3, step (a) was performed using a cleaning agent consisting of liquefied carbon dioxide (-10°C, 6 MPa, purity: 99.9 v / v%), and the washing was carried out for 60 minutes under supercritical fluid conditions of 40°C and 20 MPa. Steps (b), (c), and (d) were performed using acetonitrile. After all solutions had been delivered, the carrier was removed from the container, filtered by suction, washed with 50 mL of acetonitrile, and dried.

[0043] Example 8: In Test 3, for the cleaning in step (d), a cleaning agent prepared by mixing 99 v / v% of liquefied carbon dioxide gas (-10°C, 6 MPa, purity: 99.9 v / v%) and 1 v / v% of acetic acid was used, and it was used as a supercritical fluid under the conditions of 40°C and 20 MPa for 60 minutes of cleaning. For the cleaning in steps (a), (b) and (c), acetonitrile was used for cleaning. After the feeding of all solutions was completed, the carrier was taken out from the container, suction filtration was carried out, and it was washed with 50 mL of acetonitrile and then dried.

[0044] Comparative Example 5: In Test 3, for the cleaning in step (d), a cleaning agent consisting of liquefied carbon dioxide gas (-10°C, 6 MPa, purity: 99.9 v / v%) was used, and it was used as a supercritical fluid under the conditions of 40°C and 20 MPa for 60 minutes of cleaning. For the cleaning in steps (a), (b) and (c), acetonitrile was used for cleaning. After the feeding of all solutions was completed, the carrier was taken out from the container, suction filtration was carried out, and it was washed with 50 mL of acetonitrile and then dried.

[0045] <Test 4> 110 mg of the carrier was placed in the reaction column, set in the supercritical fluid device, and the synthetic reagents were flowed into the reaction column according to the RNA synthesis program in the order of step (a) → step (b) → step (d) → step (c).

[0046] Example 9: In Test 4, for the cleaning in step (a), a cleaning agent prepared by mixing 99 v / v% of liquefied carbon dioxide gas (-10°C, 6 MPa, purity: 99.9 v / v%) and 1 v / v% of pyridine was used, and it was used as a supercritical fluid under the conditions of 40°C and 20 MPa for 60 minutes of cleaning. For the cleaning in step (d), a cleaning agent consisting of liquefied carbon dioxide gas (-10°C, 6 MPa, purity: 99.9 v / v%) was used, and it was used as a supercritical fluid under the conditions of 40°C and 20 MPa for 60 minutes of cleaning. For the cleaning in steps (b) and (c), acetonitrile was used for cleaning. After the feeding of all solutions was completed, the carrier was taken out from the container, suction filtration was carried out, and it was washed with 50 mL of acetonitrile and then dried.

[0047] Example 10: In Test 4, for the cleaning in step (a), a cleaning agent obtained by mixing 95 v / v% of liquefied carbon dioxide gas (-10°C, 6 MPa, purity: 99.9 v / v%) and 5 v / v% of pyridine was used, and cleaning was performed for 60 minutes under the conditions of 20°C and normal pressure. For the cleaning in step (d), a cleaning agent composed of liquefied carbon dioxide gas (-10°C, 6 MPa, purity: 99.9 v / v%) was used, and cleaning was performed for 60 minutes under the conditions of 20°C and normal pressure. For the cleaning in steps (b) and (c), acetonitrile was used for cleaning. After the feeding of all solutions was completed, the carrier was taken out from the container, suction filtration was performed, and it was washed with 50 mL of acetonitrile and dried.

[0048] Comparative Example 6: In Test 4, for the cleaning in step (a), a cleaning agent composed of liquefied carbon dioxide gas (-10°C, 6 MPa, purity: 99.9 v / v%) was used, and cleaning was performed for 60 minutes as a supercritical fluid under the conditions of 40°C and 20 MPa. For the cleaning in step (d), a cleaning agent composed of liquefied carbon dioxide gas (-10°C, 6 MPa, purity: 99.9 v / v%) was used, and cleaning was performed for 60 minutes as a supercritical fluid under the conditions of 40°C and 20 MPa. For the cleaning in steps (b) and (c), acetonitrile was used for cleaning. After the feeding of all solutions was completed, the carrier was taken out from the container, suction filtration was performed, and it was washed with 50 mL of acetonitrile and dried.

[0049] <Evaluation method> (Measurement of absorbance of nucleic acid) For the carriers after nucleic acid synthesis obtained in Examples 1 to 10 and Comparative Examples 1 to 6, the following measurements were performed. Acetonitrile was transferred to a quartz cell without adding a measurement sample, and the absorbance at 412 nm was measured (blank measurement). Next, 5 mg of the measurement sample was dispersed in 50 mL of a 0.1 M acetonitrile solution of p-toluenesulfonic acid, and measurement was performed in the same manner as above. The actual DMT amount was determined by subtracting the blank measurement value from this value. The yield (%) of the measurement sample was calculated by the following formulas (1) and (2). Formula (1) DMT theoretical value (μmol / g) = amount of linker (μmol / g) / (1 + amount of linker (μmol / g) × molar mass of each base (g / mol)) Formula (2) actual DMT amount (μmol / g) / DMT theoretical value (μmol / g) × 100 = yield (%)

[0050] <Evaluation Results> (Yield) Table 1 summarizes the yields of the four sequences synthesized in Examples 1-4 and Comparative Examples 1-3, and the yield of the sequence (dT) synthesized in Examples 5 and 6. Example 1, in which a supercritical fluid made by mixing 99 v / v% liquefied carbon dioxide and 1 v / v% pyridine was used as the cleaning agent in step (a) of Test 1, showed a better yield than Comparative Example 1, in which a supercritical fluid made by mixing only liquefied carbon dioxide was used as the cleaning agent in step (a). Example 2, in which a supercritical fluid made by mixing 99 v / v% liquefied carbon dioxide and 1 v / v% acetic acid was used as the cleaning agent in step (d) of Test 1, showed a better yield than Comparative Example 2, in which a supercritical fluid made by mixing only liquefied carbon dioxide was used as the cleaning agent in step (d). In step (a) of Test 2, Example 3, which used a supercritical fluid made by mixing 99 v / v% liquefied carbon dioxide and 1 v / v% pyridine as the cleaning agent, and Example 4, which used a mixture of 95 v / v% liquefied carbon dioxide and 5 v / v% pyridine prepared at 20°C and atmospheric pressure as the cleaning agent, showed better yields than Comparative Example 3, which used a supercritical fluid made only from liquefied carbon dioxide in step (a) as the cleaning agent. In step (a) of Test 1, Example 5, which used a supercritical fluid made by mixing 60 v / v% liquefied carbon dioxide and 40 v / v% pyridine as the cleaning agent, showed better yields than Comparative Example 1, which used a supercritical fluid made only from liquefied carbon dioxide in step (a) as the cleaning agent. In Example 6, where a supercritical fluid made by mixing 60 v / v% liquefied carbon dioxide and 40 v / v% acetic acid was used as the cleaning agent in step (d) of Test 1, the yield was superior to that of Comparative Example 2, where only liquefied carbon dioxide was used as the supercritical fluid in step (d) as the cleaning agent.

[0051]

[0052] Table 2 shows the yields of the four sequences synthesized in Examples 7-10 and Comparative Examples 4-6. Example 7, in which a supercritical fluid made by mixing 99 v / v% liquefied carbon dioxide and 1 v / v% pyridine was used as the cleaning agent in step (a) of Test 3, showed a better yield than Comparative Example 4, in which a supercritical fluid made by mixing only liquefied carbon dioxide was used as the cleaning agent in step (a). Example 8, in which a supercritical fluid made by mixing 99 v / v% liquefied carbon dioxide and 1 v / v% acetic acid was used as the cleaning agent in step (d) of Test 3, showed a better yield than Comparative Example 5, in which a supercritical fluid made by mixing only liquefied carbon dioxide was used as the cleaning agent in step (d). In step (a) of Test 4, Example 9, which used a supercritical fluid prepared by mixing 99 v / v% liquefied carbon dioxide and 1 v / v% pyridine as the cleaning agent, and Example 10, which used a mixture of 95 v / v% liquefied carbon dioxide and 5 v / v% pyridine prepared at 20°C and atmospheric pressure as the cleaning agent, showed better yields than Comparative Example 6, which used a supercritical fluid prepared by mixing only liquefied carbon dioxide in step (a) as the cleaning agent.

[0053]

Claims

1. A method for producing an oligonucleotide comprising: (a) removing a protecting group from a protected nucleoside that is directly or indirectly supported on a carrier and has a protecting group bonded to a hydroxyl group, thiol group, or amino group at the 3' or 5' position; (b) bonding a nucleoside phosphoramidite to the hydroxyl group, thiol group, or amino group at the 3' or 5' position of the nucleoside from which the protecting group has been removed, which is directly or indirectly supported on a carrier; (c) sulfurizing or oxidizing the bond formed in step (b); and (d) capping the unbonded hydroxyl group, thiol group, or amino group at the 3' or 5' position of the nucleoside that is directly or indirectly supported on a carrier, wherein the method further comprises washing with a detergent containing carbon dioxide and a base after step (a), and / or washing with a detergent containing carbon dioxide and an acid after step (d), The method, characterized in that the carbon dioxide content in the cleaning agent is 50 v / v% or more.

2. The method according to claim 1, wherein the base is at least one selected from the group consisting of pyridine, triethylamine, N,N-diisopropylamine, tetramethylethylenediamine, morpholine, N-methylmorpholine, imidazole, N-methylimidazole, diethylamine, diisopropylamine, dibutylamine, ethylamine, propylamine, isopropylamine, butylamine, and tert-butylamine.

3. The method according to claim 1 or 2, wherein the acid is at least one selected from the group consisting of acetic acid, nitric acid, sulfuric acid, lactic acid, and acrylic acid.

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