Method for producing oligonucleotides

Abstract

The purpose of the present invention is to provide a method that is for producing oligonucleotides, that curbs the amount of nucleoside phosphoramidites remaining in a reaction container in a synthesis step, and that reduces the usage amount of nucleoside phosphoramidites. This method for producing oligonucleotides comprises: (a) a step for detaching a protecting group from a protected nucleoside that is directly or indirectly supported on a carrier and that has the protecting group bonded to a hydroxyl group, a thiol group, or an amino group located at the 3'-position or the 5'-position; (b) a step for, in the presence of an activating reagent, forming a bond between a nucleoside phosphoramidite and the hydroxyl group, the thiol group, or the amino group located at the 3'-position or the 5'-position of the nucleoside from which the protecting group had been detached and which is directly or indirectly supported on the carrier; (c) a step for sulfurizing or oxidizing the bond formed in the step (b); and (d) a step for capping an unbound hydroxyl group, thiol group, or amino group located at the 3'-position or the 5'-position in the nucleoside directly or indirectly supported on the carrier. The solution temperature in any one of steps (a)-(d) is 0-20°C.

Description

Methods for producing oligonucleotides

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

[0002] The phosphoramidite method is widely used in the chemical synthesis of nucleic acids such as DNA oligonucleotides and RNA oligonucleotides. In the phosphoramidite method, oligonucleotides are typically synthesized by sequentially adding nucleoside phosphoramidites to nucleosides, nucleotides, or oligonucleotides in the presence of a suitable activator. In the phosphoramidite method, nucleoside phosphoramidites are generally used in an amount of 1.5 to 10.0 times the theoretical amount. Since nucleoside phosphoramidites are expensive among synthetic raw materials, reducing the amount of nucleoside phosphoramidite used is expected to significantly reduce production costs.

[0003] However, a method for obtaining oligonucleotides with good purity while reducing the amount of nucleoside phosphoramidite used has not been known until now. Regarding oligonucleotide synthesis reactions, Patent Document 1 describes that each reaction temperature is about 0°C to about 27°C, and Patent Document 2 describes that the reaction temperature is preferably 0°C to 100°C, more preferably 20°C to 50°C, and particularly preferably 20°C to 30°C. However, there is no description of a method for obtaining oligonucleotides with good purity while reducing the amount of nucleoside phosphoramidite used by suppressing the amount of nucleoside phosphoramidite remaining in the reaction vessel during the synthesis step.

[0004] JP 60-67494 A and WO 2017 / 111137 A

[0005] An object of the present invention is to provide a method for producing an oligonucleotide, which can reduce the amount of nucleoside phosphoramidite used by suppressing the amount of nucleoside phosphoramidite remaining in a reaction vessel.

[0006] The present inventors, while conducting extensive research into methods for producing oligonucleotides, discovered that by lowering the temperature of the solution in the oligonucleotide production process, the amount of nucleoside phosphoramidite remaining in the reaction vessel during the synthesis process can be suppressed, thereby reducing the amount of nucleoside phosphoramidite used. Based on this finding, they continued their research and completed the present invention.

[0007] That is, 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 support and has a protecting group bound to a hydroxyl group, a thiol group, or an amino group at the 3'- or 5'-position, (b) a step of binding a nucleoside phosphoramidite to the hydroxyl group, the thiol group, or the amino group at the 3'- or 5'-position of the nucleoside that is directly or indirectly supported on a support and from which the protecting group has been removed, in the presence of an activating agent, (c) a step of sulfurizing or oxidizing the bond formed by step (b), and (d) a step of capping any unbound hydroxyl group, the thiol group, or the amino group at the 3'- or 5'-position of the nucleoside that is directly or indirectly supported on a support, wherein the temperature of the solution in any of steps (a) to (d) is 0 to 20°C.

[0008] [2] The method according to [1], wherein the amount of the nucleoside phosphoramidite used in the step (b) is 1.0 to 2.0 times the equivalent of the supported nucleoside. [3] The method according to [1] or [2], wherein the temperature of the solution in any of the steps (a) to (d) is 5 to 15°C.

[0009] [4] The method according to any one of [1] to [3], wherein the activator is selected from the group consisting of 4,5-dicyanoimidazole, 5-(ethylthio)-1H-tetrazole, 5-(benzylthio)-1H-tetrazole, and saccharin 1-methylimidazole.

[0010] By keeping the solution temperature low in each step of the oligonucleotide production, it is possible to suppress the amount of nucleoside phosphoramidite remaining in the reaction vessel during the synthesis step, and oligonucleotides can be obtained with good purity while reducing the amount of nucleoside phosphoramidite used, which is an expensive material.

[0011] Although not wishing to be bound by a particular theory, it is believed that the nucleoside phosphoramidite remaining in the reaction vessel occurs when the nucleoside phosphoramidite binds to a hydroxyl group formed by the unintentional elimination of the 2-cyanoethyl (CNET) protecting group at the phosphate site of the oligonucleotide during the coupling step, and the nucleoside phosphoramidite is unable to participate in the chain elongation reaction (reaction with the 5'- or 3'-terminal hydroxyl group of the nucleoside directly or indirectly supported on the support). By lowering the temperature of the solution, the elimination of the CNET protecting group from the nucleotide during the process is suppressed, and the amount of nucleoside phosphoramidite consumed by binding to the hydroxyl group of the oligonucleotide formed by the elimination of the CNET protecting group is reduced, thereby realizing a reduction in the amount of nucleoside phosphoramidite used.

[0012] Fig. 1 shows the in-column residual rate and purity in Comparative Example 1, Example 1, Example 2, Example 3, and Example 4. Fig. 2 shows the in-column residual rate and purity in Comparative Example 2, Example 5, and Example 6. Fig. 3 shows the in-column residual rate and purity in Comparative Example 3 and Example 7. Fig. 4 shows the in-column residual rate and purity in Comparative Example 4, Example 8, and Example 9.

[0013] The present invention will be described in detail below. Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning 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 addition, if there is any discrepancy between the publications referenced herein and the description in this specification, the description in this specification shall prevail.

[0014] In one aspect, the present invention relates to a method for producing an oligonucleotide. In the present invention, the oligonucleotide is produced using the so-called phosphoramidite method, in which nucleotides are added by a condensation reaction between a nucleoside phosphoramidite and a nucleoside, a nucleoside, or an oligonucleotide in the presence of a suitable activator. In the present invention, the method for producing an oligonucleotide may include, for example, (a) a step of removing (deprotecting) a protecting group from a protected nucleoside that is directly or indirectly supported on a carrier and has a protecting group bound to a hydroxyl group, thiol group, or amino group at the 3' or 5' position, (b) a step of coupling a nucleoside phosphoramidite to the 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 and from which the protecting group has been removed, (c) a step of sulfurizing or oxidizing the bond formed by step (b), and (d) a step of capping the unbound 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. In the present invention, the method for producing an oligonucleotide may include further steps in addition to the above steps (a) to (d). In one embodiment of the present invention, a method for producing an oligonucleotide comprises: (a) a step of removing (deprotecting) a protecting group from a protected nucleoside, which is directly or indirectly supported on a carrier and has a protecting group bound to a hydroxyl group, a thiol group, or an amino group at the 3'- or 5'-position; (b) a step of coupling a nucleoside phosphoramidite to the hydroxyl group, the thiol group, or the amino group at the 3'- or 5'-position of the nucleoside, which is directly or indirectly supported on a carrier and from which the protecting group has been removed, in the presence of an activating agent; (c) a step of sulfurizing or oxidizing the bond formed by step (b); and (d) a step of capping any unbound hydroxyl group, the thiol group, or the amino group at the 3'- or 5'-position of the nucleoside, which is directly or indirectly supported on a carrier.

[0015] 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, or uridine, or a modified nucleoside. Modified nucleosides include, but are not limited to, nucleosides in which the hydroxyl group at the 3' or 5' position of the nucleoside is substituted with a thiol group or an amino group. The nucleoside base may be a naturally occurring base such as adenine, guanine, cytosine, thymine, or uracil, or a modified nucleoside base. The sugar portion of the nucleoside may be naturally occurring deoxyribose or ribose, and may be in the D-configuration or the L-configuration.

[0016] In the present invention, a nucleotide refers to a compound comprising a nucleoside base, a sugar, and a phosphate bond, 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, or uracil, or a modified nucleoside base. The sugar portion of the nucleoside may be naturally occurring deoxyribose or ribose, and may be in the D- or L-configuration. The phosphate portion may be, for example, phosphorothioate, phosphorodithioate, methylphosphonate, or methylphosphate.

[0017] As used herein, the term "oligonucleotide" refers to a compound having a structure in which nucleoside bases, sugars, and phosphates are linked by phosphodiester bonds. This term includes naturally occurring oligonucleotides, such as 2'-deoxyribonucleic acid (hereinafter "DNA") and ribonucleic acid (hereinafter "RNA"), as well as nucleic acids containing modified sugar moieties, modified phosphate moieties, or modified nucleobases. Modifications to the sugar moiety include replacing the ribose ring with a hexose, cyclopentyl, or cyclohexyl ring. Alternatively, the D-ribose ring of naturally occurring nucleic acids may be replaced with an L-ribose ring, or the β-anomer of naturally occurring nucleic acids may be replaced with an α-anomer. Oligonucleotides may also contain one or more abasic moieties. Modified phosphate moieties include phosphorothioates, phosphorodithioates, methylphosphonates, and methylphosphates. Such nucleic acid analogs are known to those skilled in the art. Oligonucleotides comprising mixtures of two or more of the above can be prepared, for example, from oligonucleotides comprising mixtures of deoxyribonucleosides and ribonucleosides, particularly mixtures of deoxyribonucleosides and 2'-O-substituted ribonucleosides such as 2'-O-methyl or 2'-O-methoxyethyl ribonucleosides. Examples of oligonucleotides comprising mixtures of nucleosides include ribozymes.

[0018] In the present invention, a nucleoside phosphoramidite refers to a nucleoside derivatized with an amidite. In the present invention, a nucleoside phosphoramidite has either the 3'- or 5'-hydroxyl group of the nucleoside phosphoramidite converted to a phosphoramidite, and the other hydroxyl group is bound to a protecting group. The amiditization can be carried out, for example, by using 1H-tetrazole as an activating agent and reacting a suitably protected nucleoside with 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite. The nucleoside phosphoramidite may be a monomer or an oligomer, such as a 2-mer to 24-mer.

[0019] In the present invention, an activating agent refers to an agent that activates a nucleoside phosphoramidite, and is used to react with a nucleoside, nucleotide, or oligonucleotide. It is also referred to as an activator or coupling agent. In the present invention, activating agents commonly used in the phosphoramidite method can be used. Examples of activating agents that can be used in the present invention include, but are not limited to, 4,5-dicyanoimidazole, 5-(ethylthio)-1H-tetrazole, 5-(benzylthio)-1H-tetrazole, and saccharin 1-methylimidazole, with 4,5-dicyanoimidazole being preferred.

[0020] In the present invention, "a nucleoside directly supported on a carrier" refers to the nucleoside portion (a compound portion in which a nucleoside base and a sugar are bonded) of a compound in which a nucleoside or nucleotide is bonded to a reaction site on a carrier, and "a nucleoside indirectly supported on a carrier" refers to the nucleoside portion (a compound portion in which a nucleoside base and a sugar are bonded) of a compound in which a nucleotide is bonded to a reaction site on a carrier via a compound such as a polynucleotide.

[0021] In one embodiment of the present invention, there is provided a method for producing an oligonucleotide, the method comprising: (a) a step of removing a protecting group from a protected nucleoside, which is supported directly or indirectly on a support and has a protecting group bound to a hydroxyl group, a thiol group, or an amino group at the 3'- or 5'-position; (b) a step of coupling a nucleoside phosphoramidite to the hydroxyl group, the thiol group, or the amino group at the 3'- or 5'-position of the nucleoside, which is supported directly or indirectly on a support and from which the protecting group has been removed, in the presence of an activating agent; (c) a step of sulfurizing or oxidizing the bond formed by step (b); and (d) a step of capping any unbound hydroxyl group, the thiol group, or the amino group at the 3'- or 5'-position of the nucleoside, which is supported directly or indirectly on a support. In the method, the temperature of the solution in any of steps (a) to (d) is 0 to 20°C, and preferably 5 to 20°C. Specifically, the temperature is 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, or 20°C, or may be within a range between any two of these values. Examples of methods for controlling the temperature of the solution include a method in which a solution previously adjusted to a desired temperature is supplied to each step, a method in which the solution is supplied to each step and then controlled to the desired temperature by cooling the reaction vessel, or the like, and a method in which a pipe is provided in the reaction vessel and a refrigerant or the like is passed through the pipe to control the temperature to the desired level. In the present invention, the temperature at which a solution previously adjusted to a desired temperature is supplied to each step is referred to as the supply liquid temperature.

[0022] In one embodiment of the present invention, the amount of the activating agent used in step (b) is not particularly limited, but is preferably 2.0 to 15.0 times the equivalent amount of the nucleoside phosphoramidite used in the step, for example. Specifically, the equivalents are 2.0 times, 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 5.5 times, 6.0 times, 6.5 times, 7.0 times, 7.5 times, 8.0 times, 8.5 times, 9.0 times, 9.5 times, 10.0 times, 10.5 times, 11.0 times, 11.5 times, 12.0 times, 12.5 times, 13.0 times, 13.5 times, 14.0 times, 14.5 times, and 15.0 times, and may be within a range between any two of these values.

[0023] In one embodiment of the present invention, the amount of the activator used in step (b) is not particularly limited, but is preferably 10.0 to 25.0 times the equivalent of the supported nucleoside, specifically 10.0 times equivalent, 10.5 times equivalent, 11.0 times equivalent, 11.5 times equivalent, 12.0 times equivalent, 12.5 times equivalent, 13.0 times equivalent, 13.5 times equivalent, 14.0 times equivalent, 14.5 times equivalent, 15.0 times equivalent, 15.5 times equivalent, 16.0 times equivalent, 16.5 times equivalent, 17.0 times equivalent, 17.5 times equivalent, 18.0 times equivalent, 18.5 times equivalents, 19.0 times equivalents, 19.5 times equivalents, 20.0 times equivalents, 20.5 times equivalents, 21.0 times equivalents, 21.5 times equivalents, 22.0 times equivalents, 22.5 times equivalents, 23.0 times equivalents, 23.5 times equivalents, 24.0 times equivalents, 24.5 times equivalents, 25.0 times equivalents, and may be within a range between any two of these values.

[0024] In one embodiment of the present invention, the amount of the nucleoside phosphoramidite used in step (b) is not particularly limited, but is preferably 1.0 to 2.0 times the equivalent of the supported nucleoside, specifically 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 times the equivalent, or may be within a range between any two of these values.

[0025] As used herein, the term "amount of nucleoside phosphoramidite remaining in a reaction vessel" refers to the amount of nucleoside phosphoramidite remaining in a reaction vessel during the synthesis of an oligonucleotide, for example, according to the theory described above, etc. The amount of phosphoramidite remaining in a reaction vessel can be measured, for example, by collecting the waste liquid from the oxidation step or sulfurization step in each synthesis cycle, and optionally the washing liquid after the coupling reaction during the synthesis cycle, and subjecting the amount of DMTr (4,4'-dimethoxytriphenylmethyl group) protecting group of the nucleoside phosphoramidite to HPLC analysis.

[0026] The present invention will be described in more detail with reference to the following examples, which illustrate specific embodiments of the present invention and are not intended to limit the scope of the present invention. (1) Synthesis of DNA Oligonucleotides Porous resin beads (NittoPhase® HL UnyLinker 350) were placed in a synthesis column (volume 12.6 ml) so that the synthesis scale (total reaction sites on the beads) was 480 μmol, and the column was set in an AKTA Oligopilotplus 100 synthesizer (manufactured by Cytiva). Nucleoside phosphoramidite and 4,5-dicyanoimidazole (DCI) as an activator were added in the amounts shown in Table 1, and a coupling reaction (condensation time: 5 minutes) was carried out under the conditions shown in Table 1. All activators were dissolved in acetonitrile to a concentration of 0.7 M. Other synthesis reagents used included 3% DCA in toluene as a deprotecting agent, 0.2 M xanthan hydride in pyridine as a sulfurizing agent, a mixed solution of lutidine, N-methylimidazole, and acetic anhydride in acetonitrile as a capping agent, and TBA in acetonitrile (8:2 ratio) as an amine wash reaction solution. A 24-mer DNA oligonucleotide (5'-TCGACGTATTGACGTATTGACGTA-3', all phosphite esters sulfurized: SEQ ID NO: 1) was synthesized, and the terminal DMTr protecting group was removed. The porous resin beads to which the DNA oligonucleotide was bound were dried. The porous resin beads were then immersed in ammonia water to cleave the DNA oligonucleotide from the beads, and the base amino groups were deprotected, resulting in a filtrate in which the DNA oligonucleotide was dissolved.

[0027] (2) Measurement of Purity of Synthesized DNA Oligonucleotides The oligonucleotide sample filtrate was adjusted to 5 OD and measured by high performance liquid chromatography (HPLC) under the following conditions. The sum of the peak areas from the detection of the main component up to about 10 minutes was taken as 100%, and the peak area (%) of the main component was taken as the synthetic purity (full-length: area%). Column: Waters, ACQUITY UPLC Oligonucleotide BEH C18 Column, 130 Å, 1.7 μm, 2.1 mm × 100 mm UV detection: 260 nm Mobile phase A: 400 mM HFIP / 15 mM TEA aqueous solution Mobile phase B: Methanol Column temperature: 60°C

[0028] (3) Measurement of the amount of nucleoside phosphoramidite remaining in the column. Nucleoside phosphoramidites bound to the phosphate moieties of the oligonucleotides were released by the sulfurizing agent during the sulfurization step and discharged together with the waste liquid. Therefore, all waste liquid from the sulfurization step in each of the 24 synthesis cycles of 24mer DNA, i.e., a total of 24 cycles, was collected. After the coupling reaction in the 24th synthesis cycle, the column was thoroughly washed with 5 column volumes (63 ml) of acetonitrile, which was also collected as waste liquid. The amount of nucleoside phosphoramidite was estimated from the absolute amount of the DMTr protecting group on the nucleoside phosphoramidite. The nucleoside phosphoramidite was hydrolyzed to obtain the DMTr protecting group. After diluting appropriately with a 0.1 M solution of paratoluenesulfonic acid monohydrate (pTSA) in acetonitrile, the absolute amount of the DMTr protecting group was measured by HPLC under the following conditions. Column: Waters Atlantis T3, 130 Å, 3.0 μm, 2.1 mm × 150 mm MS detection: ESI-Posi. m / z 303 Mobile phase A: 0.1% formic acid aqueous solution Mobile phase B: acetonitrile Column temperature: 40°C

[0029] The residual rate of nucleoside phosphoramidite in the column was calculated by dividing the absolute amount of DMTr in the waste liquid (corresponding to the amount of bound nucleoside phosphoramidite) by the amount of nucleoside phosphoramidite introduced into the reaction (synthesis scale × nucleoside phosphoramidite equivalent).

[0030] (4) Results The results are shown in Table 1. With respect to the % relative to the comparative example, the values ​​of Examples 1 to 4 indicate the residual rates of Examples 1 to 4 when the residual rate in the column of Comparative Example 1 is set to 1, and the values ​​of Examples 5 and 6 indicate the residual rates of Examples 5 and 6 when the residual rate in the column of Comparative Example 2 is set to 1. If the % relative to the comparative example is less than 1, it indicates that the amount remaining in the column is suppressed compared to the comparative example.

[0031] Figure 1 shows the in-column residual rate and purity for Examples 1, 2, 3, and Comparative Example 1, in which 1.7 equivalents of nucleoside phosphoramidite and 3.5 times the amount of activator relative to the amount of nucleoside phosphoramidite were used, and the feed solution temperatures were 4°C, 8°C, 15°C, and 22.5°C, respectively, and for Example 4, in which 1.7 equivalents of nucleoside phosphoramidite and 14 times the amount of activator relative to the amount of nucleoside phosphoramidite were used, and the feed solution temperature was 15°C. The in-column residual rate decreased when the feed solution temperature was 20°C or lower. The purity of the synthesized DNA oligonucleotide was highest when the feed solution temperature was 15°C, and the purity of the synthesized DNA oligonucleotide in Example 4 was even higher.

[0032] Figure 2 shows the in-column retention rate and purity for Example 5 and Comparative Example 2, in which 1.2 equivalents of nucleoside phosphoramidite and 3.5 times the amount of activator relative to the amount of nucleoside phosphoramidite were used, and the feed solution temperatures were 15°C and 22.5°C, respectively, and for Example 6, in which 1.2 equivalents of nucleoside phosphoramidite and 14 times the amount of activator relative to the amount of nucleoside phosphoramidite were used, and the feed solution temperature was 15°C. As shown in Figure 2, when the feed solution temperature was 20°C or below, the in-column retention rate decreased, and the purity of the synthesized DNA oligonucleotides was high. In Example 6, the purity of the synthesized DNA oligonucleotides was even higher.

[0033] (5) Synthesis of RNA Oligonucleotide Porous resin beads (NittoPhase® HL 250-2'OMeA) were placed in a synthesis column (volume 12.6 ml) so that the synthesis scale (total reaction sites on the beads) was 352 μmol, and the column was set in an AKTA oligopilotplus100 synthesizer (manufactured by Cytiva). Nucleoside phosphoramidite and 5-(ethylthio)-1H-tetrazole (ETT) as an activator were added in the amounts shown in Table 2, and a coupling reaction (condensation time: 10 minutes) was carried out under the conditions shown in Table 2. All activators were dissolved in acetonitrile to a concentration of 0.6 M. Other synthesis reagents included 3% DCA in toluene as a deprotecting agent, iodine solution (0.05 mol / L; solvent: pyridine:water (9:1) ratio) as an oxidizing agent, a mixed solution of lutidine, N-methylimidazole, and acetic anhydride in acetonitrile as a capping agent, and TBA in acetonitrile (8:2 ratio) as an amine wash reaction solution. A 24-mer 2'OMe RNA oligonucleotide (5'-UCGACGUAUUGACGUAUUGACGUA-3', all phosphite esters oxidized; SEQ ID NO: 2) was synthesized, and the terminal DMTr protecting group was removed. The porous resin beads to which the RNA oligonucleotide was bound were dried. The porous resin beads were then immersed in ammonia water to cleave the RNA oligonucleotide from the beads, followed by deprotection of the base amino groups, yielding a filtrate in which the 2'OMe RNA oligonucleotide was dissolved.

[0034] (6) Measurement of Purity of Synthesized RNA Oligonucleotides The oligonucleotide sample filtrate adjusted to 5 OD was measured by high performance liquid chromatography (HPLC) under the following conditions. The sum of the peak areas from the detection of the main component up to about 10 minutes was taken as 100%, and the peak area (%) of the main component was taken as the synthetic purity (full-length: area%). Column: Waters, ACQUITY UPLC Oligonucleotide BEH C18 Column, 130 Å, 1.7 μm, 2.1 mm × 100 mm UV detection: 260 nm Mobile phase A: 400 mM HFIP / 15 mM TEA aqueous solution Mobile phase B: Methanol Column temperature: 60 °C

[0035] (7) Measurement of the amount of nucleoside phosphoramidite remaining in the column. Nucleoside phosphoramidites attached to the phosphate moieties of the oligonucleotides were cleaved by the oxidizing agent during the oxidation step and discharged together with the waste liquid. Therefore, all waste liquid from the oxidation step in each of the 24 synthesis cycles of 24mer 2'OMeRNA, i.e., a total of 24 cycles, was collected. After the coupling reaction in the 24th synthesis cycle, the column was thoroughly washed with 5 column volumes (63 ml) of acetonitrile, which was also collected as waste liquid. The amount of nucleoside phosphoramidite was estimated from the absolute amount of the DMTr protecting group on the nucleoside phosphoramidite. The nucleoside phosphoramidite was hydrolyzed to obtain the DMTr protecting group. After appropriate dilution with a 0.1 M solution of paratoluenesulfonic acid monohydrate (pTSA) in acetonitrile, the absolute amount of the DMTr protecting group was measured by HPLC under the following conditions. Column: Waters Atlantis T3, 130 Å, 3.0 μm, 2.1 mm × 150 mm MS detection: ESI-Posi. m / z 303 Mobile phase A: 0.1% formic acid aqueous solution Mobile phase B: acetonitrile Column temperature: 40°C

[0036] The residual rate of nucleoside phosphoramidite in the column was calculated by dividing the absolute amount of DMTr in the waste liquid (corresponding to the amount of bound nucleoside phosphoramidite) by the amount of nucleoside phosphoramidite introduced into the reaction (synthesis scale × nucleoside phosphoramidite equivalent).

[0037] (8) Results The results are shown in Table 2. With respect to the percentage relative to the comparative example, the value of Example 7 indicates the residual rate of Example 7 when the residual rate in the column of Comparative Example 3 is set to 1, and the values ​​of Examples 8 and 9 indicate the residual rates of Examples 8 and 9 when the residual rate in the column of Comparative Example 4 is set to 1. If the percentage relative to the comparative example is less than 1, it indicates that the amount remaining in the column is suppressed compared to the comparative example.

[0038] The column retention rate and purity for Example 7 and Comparative Example 3, in which 2.0 equivalents of nucleoside phosphoramidite and 3.3 times the amount of activator relative to the amount of nucleoside phosphoramidite were used, and the feed temperature was 15°C and 22.5°C, respectively, are shown in Figure 3. The column retention rate of the synthesized RNA oligonucleotide decreased to 16.7% when the feed temperature was 15°C, improving the purity of the RNA oligonucleotide. The column retention rate and purity for Example 8 and Comparative Example 4, in which 1.4 equivalents of nucleoside phosphoramidite and 3.3 times the amount of activator relative to the amount of nucleoside phosphoramidite were used, and the feed temperature was 15°C and 22.5°C, respectively, and for Example 9, in which 1.4 equivalents of nucleoside phosphoramidite and 12 times the amount of activator relative to the amount of nucleoside phosphoramidite were used, and the feed temperature was 15°C, are shown in Figure 4. 4, when the temperature of the feed solution was lowered to 20°C or below, the residual fraction in the column decreased, and the purity of the synthesized RNA oligonucleotides showed high values. In Example 9, the purity of the synthesized RNA oligonucleotides showed even higher values. Therefore, compared to Comparative Example 3, it was found that in Example 9, even for modified RNA nucleoside phosphoramidites, the purity could be improved by reducing the residual fraction in the column, even when the amount of RNA nucleoside phosphoramidite input was significantly reduced.

Claims

1. 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 support and has a protecting group bound to a hydroxyl group, a thiol group, or an amino group at the 3'-position or the 5'-position; (b) binding a nucleoside phosphoramidite to the hydroxyl group, thiol group, or amino group at the 3'- or 5'-position of the nucleoside, which has been deprotected and is directly or indirectly supported on a support, in the presence of an activating agent; (c) sulfurizing or oxidizing the bond formed by step (b); and (d) capping the free hydroxyl group, thiol group, or amino group at the 3'-position or 5'-position of the nucleoside directly or indirectly supported on the carrier; Including, The method as described above, wherein the temperature of the solution in any one of steps (a) to (d) is 5 to 19°C.

2. 2. The method of claim 1, wherein the amount of the nucleoside phosphoramidite used in step (b) is 1.0 to 2.0 times the equivalent of the supported nucleoside.

3. 3. The method according to claim 1, wherein the temperature of the solution in any one of steps (a) to (d) is 5 to 15°C.

4. 3. The method of claim 1, wherein the activating agent is selected from the group consisting of 4,5-dicyanoimidazole, 5-(ethylthio)-1H-tetrazole, 5-(benzylthio)-1H-tetrazole, and saccharin 1-methylimidazole.