Method for producing oligonucleotide

Abstract

The purpose of the present invention is to provide a method for producing an oligonucleotide, wherein the amount of nucleoside phosphoramidite remaining in a reaction vessel is suppressed and the amount of used nucleoside phosphoramidite is reduced. The abovementioned problem is solved by, inter alia, a method for producing an oligonucleotide, the method comprising: (a) a step for detaching a protective group from a protected nucleoside where the protective group is bonded to a hydroxyl group, a thiol group, or an amino group at the 3' position or the 5' position; (b) a step for, in the presence of an activator, bonding a nucleoside phosphoramidite to the hydroxyl group, thiol group, or amino group at the 3' position or the 5' position of the nucleoside from which the protective group has been detached; (c) a step for sulfurizing or oxidizing the bond formed by step (b); and (d) a step for capping the hydroxyl group, thiol group, or amino group at the unbonded 3' position or 5' position of the nucleoside, wherein the capping of step (d) includes the operation of causing the nucleoside and a capping solution to come into contact for only a prescribed period of time [T] (minutes), the capping solution includes N-methyl imidazole at a blending rate of [N] (%) (the room temperature volume percentage (%) of unblended N-methyl imidazole with respect to the unblended room temperature total volume (100%) of reagents blended as capping solution components), and the product ([N] ×[T]) of [N] and [T] is 0.5 to 3.0.

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 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 high purity while reducing the amount of nucleoside phosphoramidite used has not been known. Patent Document 1 describes a method for synthesizing oligonucleotides via a specific intermediate on a cleavable support having an azidomethyl moiety, in response to the difficulty in scaling up oligonucleotide synthesis, which often requires excessive amounts of expensive reagents. Patent Document 2 describes a synthesis method using multi-base nucleotides, in response to the difficulty in scaling up nucleic acid synthesis at low cost, due to the low material utilization rate of commercially available column synthesizers. Patent Document 3 describes a method for synthesizing oligonucleotides using a highly lipophilic phosphoramidite, a type of alkoxyphosphoramidite compound that can be used as a capping reagent, in response to the limitation in scale-up and the need for excessive amounts of reaction reagents and raw materials in solid-phase synthesis. However, none of Patent Documents 1 to 3 focuses on the conditions of the capping step, let alone the relationship between the capping step and the amount of nucleoside phosphoramidite remaining in the reaction vessel. Patent Documents 4 to 7 and Non-Patent Documents 1 and 2 describe various capping steps using a capping solution containing 1-methylimidazole, but these documents also do not consider the relationship between the capping step and the amount of nucleoside phosphoramidite remaining in the reaction vessel. Patent Document 8 describes that in the synthesis of thiolated oligonucleotides, a process that excluded the capping step was superior to a comparative example in which the capping step was performed, but does not describe the relationship between the capping step and the amount of nucleoside phosphoramidite remaining in the reaction vessel.

[0004] International Publication No. 2021 / 198883 International Publication No. 2019 / 158007 Patent Registration No. 7229539 International Publication No. 2022 / 132681 Chinese Patent Publication No. 115010769 International Publication No. 2002 / 014340 International Publication No. 2014 / 028739 Special Publication No. 2019-518778

[0005] BioTechniques 22(4): 752-756 (April 1997) Molecules 2023, 28(7), 3265

[0006] 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.

[0007] The present inventors have conducted extensive research into methods for producing oligonucleotides and have found that by relaxing the conditions for the capping step, the amount of nucleoside phosphoramidite remaining in the reaction vessel during the synthesis step can be reduced, thereby enabling a reduction in the amount of nucleoside phosphoramidite used. Based on this finding, further research has led to the completion of the present invention.

[0008] 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, the protecting group being 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, a thiol group, or an amino group at the 3'- or 5'-position of the nucleoside from which the protecting group has been removed, in the presence of an activator; (c) a step of sulfurizing or oxidizing the bond formed by step (b); and (d) a step of capping an unbound hydroxyl group, a thiol group, or an amino group at the 3'- or 5'-position of the nucleoside, wherein the capping in step (d) comprises an operation of contacting the nucleoside with a capping solution for a predetermined time [T] (minutes), The method as described above, wherein the capping solution contains N-methylimidazole at a blending ratio [N] (%) (wherein the ratio (%) of the volume of N-methylimidazole at room temperature before blending is relative to the total volume (100%) of each reagent blended as a component of the capping solution at room temperature before blending), and the product of [N] and [T] ([N] x [T]) is 0.5 to 3.0.

[0009] [2] The method according to [1], wherein the oligonucleotide has a sulfurized internucleoside bond. [3] The method according to [1] or [2], wherein [N] is 1.1 to 8.0 (%). [4] The method according to any one of [1] to [3], wherein [T] is 0.1 to 0.5 (minutes). [5] The method according to any one of [1] to [4], wherein the temperature of the solution in any one of steps (a) to (d) is 0 to 20°C.

[0010] [6] The method according to any one of [1] to [5], wherein the amount of the activating agent used in step (b) is 10.0 to 15.0 equivalents of the amount of the nucleoside phosphoramidite used in step (b). [7] The method according to any one of [1] to [6], wherein the amount of the activating agent used in step (b) is 10.0 to 25.0 equivalents of the nucleoside supported in step (b). [8] The method according to any one of [1] to [7], wherein the activating agent in step (b) is selected from the group consisting of 4,5-dicyanoimidazole and 5-(ethylthio)-1H-tetrazole.

[0011] By relaxing the conditions for the capping step in a method for producing an oligonucleotide, 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.

[0012] While not wishing to be bound by any 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 a 2-cyanoethyl (CNET) protecting group from the phosphate moiety of the oligonucleotide during the coupling step, making it unable to participate in the chain elongation reaction (reaction with the 5'- or 3'-terminal hydroxyl group of the nucleoside). By relaxing the conditions of the capping step, the binding of the nucleoside phosphoramidite to the hydroxyl group of the oligonucleotide formed by the elimination of the CNET protecting group is suppressed, reducing the amount of nucleoside phosphoramidite consumed in the reaction vessel and achieving a reduction in the amount of nucleoside phosphoramidite used. Note that the phenomenon of nucleoside phosphoramidite remaining in the reaction vessel is not limited to any particular carrier or reaction vessel.

[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 a nucleotide is added by a condensation reaction between a nucleoside phosphoramidite and a nucleoside, a nucleotide, or an oligonucleotide in the presence of a suitable activator.

[0015] 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, which 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 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.

[0016] In the present invention, the method for producing an oligonucleotide may include further steps in addition to the steps (a) to (d) above. In one embodiment of the present invention, the method for producing an oligonucleotide includes: (a) a step of removing (deprotecting) a protecting group from a protected nucleoside, the protecting group being 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 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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. Preferred are 4,5-dicyanoimidazole and 5-(ethylthio)-1H-tetrazole.

[0022] The remaining nucleoside phosphoramidite in the reaction vessel is not a phenomenon limited by whether the protected nucleoside or nucleosides described in steps (a), (b), and (d) are supported on a support or not. In the present invention, the protected nucleoside or nucleosides described in steps (a), (b), and (d) may or may not be supported on a support, but it is preferable that they are directly or indirectly supported on a support. In the present invention, "nucleoside directly supported on a support" refers to the nucleoside portion (the compound portion in which a nucleoside base and a sugar are bonded) of a compound in which a nucleoside or nucleotide is bonded to the reaction site of a support, and "nucleoside indirectly supported on a support" refers to the nucleoside portion (the compound portion in which a nucleoside base and a sugar are bonded) of a compound in which a nucleotide is bonded to the reaction site of a support via a compound such as a polynucleotide. The phenomenon of nucleoside phosphoramidite remaining in a reaction vessel is not limited to a specific carrier or a specific reaction vessel, and the type and shape of the carrier and reaction vessel used in the present invention are not particularly limited. The phenomenon of nucleoside phosphoramidite remaining in a reaction vessel is not limited to either solid-phase synthesis or liquid-phase synthesis, and the present invention can be applied to either solid-phase synthesis or liquid-phase synthesis, but application to solid-phase synthesis is preferred. In the present invention, the "carrier" is not particularly limited in type or shape as long as it can directly or indirectly support nucleosides, but for example, a carrier for solid-phase synthesis or a carrier for liquid-phase synthesis is preferred. In the present invention, examples of carriers for solid-phase synthesis include polymer-based carriers, glass-based carriers, and silicon-based carriers, with polymer-based carriers and glass-based carriers being more preferred. In the case of polymer-based carriers, the type is not limited, but polystyrene-based carriers are more preferred. The shape of the carrier for solid-phase synthesis can be selected from any shape, such as particles, rods, or plates, and they may have a porous structure, with porous particles being preferred. The support for solid-phase synthesis is preferably a polymer-based or glass-based porous particle, and in the case of a polymer-based support, the type is not limited, but polystyrene-based porous particles are more preferred. In the present invention, the support for liquid-phase synthesis is preferably a soluble support, a pseudo-solid-phase support, or a pseudo-solid-phase protecting group.In the present invention, there is no particular limitation on which site of a nucleoside or nucleotide binds to the reactive site on the support. The binding site can be selected appropriately depending on the type of support for solid-phase synthesis or liquid-phase synthesis. In the present invention, the type and shape of the "reaction vessel" are not particularly limited as long as they can hold the support and reaction components for a certain period of time. A vessel (e.g., a column) made of a material selected from the group consisting of glass, stainless steel, polyether ether ketone, etc., and combinations thereof can be suitably used.

[0023] In the method for producing an oligonucleotide according to the present invention, the conditions for the capping step are relaxed. The relaxed conditions for the capping step can be expressed as an index by the product ([N] x [T]) of the predetermined contact time [T] (minutes) in the operation of contacting a nucleoside with a capping solution and the compounding ratio [N] (%) of N-methylimidazole contained in the capping solution (where [N] x [T] is the ratio (%) of the room temperature volume of N-methylimidazole before compounding to the total room temperature volume (100%) of each reagent compounded as a component of the capping solution before compounding). The relaxed conditions for the capping step mean, for example, that the product ([N] x [T]) of [N] and [T] is approximately 0.5 to 3.0.

[0024] In one embodiment of the present invention, a method for producing an oligonucleotide comprises: (a) a step of removing a protecting group from a protected nucleoside, the protecting group being 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, a thiol group, or an amino group at the 3'- or 5'-position of the nucleoside from which the protecting group has been removed, in the presence of an activator; (c) a step of sulfurizing or oxidizing the bond formed by step (b); and (d) a step of capping an unbound hydroxyl group, a thiol group, or an amino group at the 3'- or 5'-position of the nucleoside, wherein the capping in step (d) comprises an operation of contacting the nucleoside with a capping solution for a predetermined time [T] (minutes), The capping solution contains N-methylimidazole at a blending ratio [N] (%) (wherein the ratio (%) of the volume of N-methylimidazole at room temperature before blending to the total volume (100%) of each reagent blended as a capping solution component at room temperature before blending), and the product of [N] and [T] ([N] x [T]) is 0.5 to 3.0.

[0025] The contact of the nucleoside with the capping solution can be carried out according to a conventional method. In a typical contact method, multiple types of solutions, such as capping solution A and capping solution B, are mixed immediately before flowing into a reaction vessel (e.g., a column) in a flow channel, but these solutions may also be mixed in the reaction vessel. In the present invention, the contact time refers to the time required for the mixed capping solution to pass through the reaction vessel.

[0026] In one embodiment of the present invention, the product of [N] and [T] ([N] × [T]) is not particularly limited as long as the conditions for the capping step are relaxed, but is preferably, for example, 0.5 to 3.0. Specifically, the product is 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0, or may be within a range between any two of these values.

[0027] In one embodiment of the present invention, the blending ratio [N] (%) of N-methylimidazole contained in the capping solution is not particularly limited, but may be, for example, 1.0 to 30% and preferably 1.1 to 8.0%. Specifically, the blending ratio may be 1.1%, 1.4%, 1.5%, 2.0%, 2.5%, 3.0%, 3.3%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, or 8.0%, or may be within a range between any two of these values.

[0028] In one embodiment of the present invention, [T] is not particularly limited, but may be, for example, 0.05 to 3 (minutes), preferably 0.1 to 0.5 (minutes). Specifically, [T] is 0.1 (minutes), 0.15 (minutes), 0.2 (minutes), 0.25 (minutes), 0.3 (minutes), 0.35 (minutes), 0.4 (minutes), 0.45 (minutes), or 0.5 (minutes), and may be within a range between any two of these values.

[0029] In one aspect of the present invention, the type of internucleoside bond that the oligonucleotide produced has is not particularly limited, but for example, the oligonucleotide produced preferably has sulfurized internucleoside bond.For example, it is preferred that at least one of the bonds formed by step (b) is sulfurized, specifically, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, or 30 or more may be sulfurized.More preferably, 6 or more of the bonds formed by step (b) may be sulfurized. More preferably, 12 or more of the bonds formed in step (b) may be sulfurized. Even more preferably, 17 or more of the bonds formed in step (b) may be sulfurized. Even more preferably, 18 or more of the bonds formed in step (b) may be sulfurized. In one embodiment of the present invention, the type of internucleoside bond contained in the produced oligonucleotide is not particularly limited, and for example, the produced oligonucleotide may have an oxidized internucleoside bond, but preferably has a sulfurized internucleoside bond. For example, it is preferable that at least one site of the bond formed by step (b) is sulfurized. Specifically, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 74% or more, 75% or more, 78% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% of the sites may be sulfurized. More preferably, 25% or more of the sites of the bonds formed by step (b) may be sulfurized. More preferably, 50% or more of the sites of the bonds formed by step (b) may be sulfurized. Even more preferably, 74% or more of the sites of the bonds formed by step (b) may be sulfurized. Even more preferably, 78% or more of the sites of the bonds formed by step (b) may be sulfurized.More preferably, 100% of the bonds formed in step (b) may be sulfurized. The structure of the 3'-terminal of the oligonucleotide is not limited independently of the type of internucleoside bond contained in the oligonucleotide. However, when the 3'-terminal has a phosphate structure, the phosphate structure may or may not be sulfurized, but is preferably sulfurized.

[0030] 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 equivalents of the supported nucleoside, more preferably about 1.1 to about 1.7 equivalents, and even more preferably about 1.2 equivalents. Specifically, the amount may be 1.0 equivalent, 1.1 equivalent, 1.11 equivalent, 1.12 equivalent, 1.13 equivalent, 1.14 equivalent, 1.15 equivalent, 1.16 equivalent, 1.17 equivalent, 1.18 equivalent, 1.19 equivalent, 1.2 equivalent, 1.3 equivalent, 1.4 equivalent, 1.5 equivalent, 1.6 equivalent, 1.7 equivalent, 1.8 equivalent, 1.9 equivalent, or 2.0 equivalents, or may be within a range between any two of these values.

[0031] 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 equivalents, more preferably 10.0 to 15.0 equivalents, of the amount of nucleoside phosphoramidite used in the step. Specifically, the amount is 2.0 equivalents, 2.5 equivalents, 3.0 equivalents, 3.5 equivalents, 4.0 equivalents, 4.5 equivalents, 5.0 equivalents, 5.5 equivalents, 6.0 equivalents, 6.5 equivalents, 7.0 equivalents, 7.5 equivalents, 8.0 equivalents, 8.5 equivalents, 9.0 equivalents, 9.5 equivalents, 10.0 equivalents, 10.5 equivalents, 11.0 equivalents, 11.5 equivalents, 12.0 equivalents, 12.5 equivalents, 13.0 equivalents, 13.5 equivalents, 14.0 equivalents, 14.5 equivalents, 15.0 equivalents, or within a range between any two of these values.

[0032] In one embodiment of the present invention, the amount of the activating agent used in step (b) is not particularly limited, but is preferably, for example, 10.0 to 25.0 equivalents of the supported nucleoside. Specifically, the amount is 10.0 equivalents, 10.5 equivalents, 11.0 equivalents, 11.5 equivalents, 12.0 equivalents, 12.5 equivalents, 13.0 equivalents, 13.5 equivalents, 14.0 equivalents, 14.5 equivalents, 15.0 equivalents, 15.5 equivalents, 16.0 equivalents, 16.5 equivalents, 17.0 equivalents, 17.5 equivalents, 18.0 equivalents, 18.5 equivalents, 19.0 equivalents, 19.5 equivalents, 20.0 equivalents, 20.5 equivalents, 21.0 equivalents, 21.5 equivalents, 22.0 equivalents, 22.5 equivalents, 23.0 equivalents, 23.5 equivalents, 24.0 equivalents, 24.5 equivalents, 25.0 equivalents, and may be within a range between any two of these values.

[0033] In one embodiment of the present invention, the temperature of the solution in any of steps (a) to (d) is not particularly limited, but is preferably 0 to 30° C., more preferably 0 to 20° C., and even more preferably 5 to 20° C. Specifically, the temperature may be 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., 20° C., 21° C., 22° C., 22.5° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., or 30° 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 controlled 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, a method in which a pipe is provided inside the reaction vessel and a refrigerant or the like is passed through the pipe to control the temperature to the desired level, etc. In the present invention, the temperature at which the solution previously controlled to a desired temperature is supplied to each step is referred to as the supply liquid temperature.

[0034] In one embodiment of the present invention, the components further contained in the capping solution are not particularly limited, but are preferably, for example, a base, an acetylating agent, or a solvent.

[0035] In one embodiment of the present invention, the type of base that can be contained in the capping solution component is not particularly limited, but is preferably, for example, 2,6-lutidine or pyridine. The base content (%) (wherein the ratio (%) of the room-temperature volume of the base before addition to the total room-temperature volume of each reagent added as a capping solution component (100%)) is not particularly limited, but is preferably, for example, 3 to 30%, and more preferably 5 to 25%. Specifically, the base content may be 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%, or may be within a range between any two of these values.

[0036] In one embodiment of the present invention, the type of acetylating agent that can be included in the capping solution components is not particularly limited, but is preferably, for example, acetic anhydride. The blending ratio (%) of the acetylating agent (where the ratio (%) of the room temperature volume of the acetylating agent before blending to the total room temperature volume (100%) of each reagent blended as the capping solution components) is not particularly limited, but is preferably, for example, 2 to 20%, more preferably 2.5 to 15%, and even more preferably 5 to 15%. Specifically, the blending ratio may be 2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or may be within a range between any two of these values.

[0037] In one embodiment of the present invention, the type of solvent that can be contained in the capping solution components is not particularly limited, but for example, organic solvents are preferred, and examples of organic solvents include acetonitrile and tetrahydrofuran.

[0038] 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 nucleoside 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.

[0039] The present invention will now be described in more detail with reference to the following examples, which illustrate particular embodiments of the present invention and are not intended to limit the invention thereto.

[0040] <Materials and Methods> The oligonucleotides used in the Examples and Comparative Examples are as follows.

[0041] 1. SEQ ID NO: 1 (1) Synthesis of DNA oligonucleotide (SEQ ID NO: 1) Porous resin beads (NittoPhase® HL UnyLinker350) 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 oligopilotplus100 synthesizer (manufactured by Cytiva). Nucleoside phosphoramidite and 4,5-dicyanoimidazole (DCI) as an activating agent were added in the amounts shown in Tables 2 to 7, and a coupling reaction (condensation time: 5 minutes) was carried out under the conditions shown in Tables 2 to 7. All activating agents 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 or pyridine, 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', the phosphite ester (internucleoside linkage) and the 3'-terminal phosphate are all 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.

[0042] (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

[0043] (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 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

[0044] The residual rate of nucleoside phosphoramidite in the column was calculated by dividing the absolute amount of DMTr in the waste solution (corresponding to the amount of bound nucleoside phosphoramidite) by the amount of nucleoside phosphoramidite introduced into the reaction (synthesis scale × nucleoside phosphoramidite equivalent). This waste solution refers to the waste solution discharged in the sulfurization step or oxidation step.

[0045] 2. SEQ ID NO: 2 (1) Synthesis of DNA oligonucleotide (SEQ ID NO: 2) Porous resin beads (NittoPhase® HL UnyLinker350) 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 oligopilotplus100 synthesizer (manufactured by Cytiva). Nucleoside phosphoramidite and 4,5-dicyanoimidazole (DCI) as an activating agent were added in the amounts shown in Table 6, and a coupling reaction (condensation time: 10 minutes) was carried out under the conditions shown in Table 6. All activating agents were dissolved in acetonitrile to a concentration of 0.7 M. Other synthesis reagents used 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 DNA oligonucleotide (5'-TCGACGTATTGACGTATTGACGTA-3', the phosphite ester (internucleoside linkage) and the 3'-terminal phosphate were all oxidized; SEQ ID NO: 2) 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. A filtrate containing the DNA oligonucleotide was obtained.

[0046] (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

[0047] (3) 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 of each of the 24 synthesis cycles of 24mer DNA, i.e., a total of 24 cycles, was collected. After the coupling reaction of 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

[0048] The residual rate of nucleoside phosphoramidite in the column was calculated by dividing the absolute amount of DMTr in the waste solution (corresponding to the amount of bound nucleoside phosphoramidite) by the amount of nucleoside phosphoramidite introduced into the reaction (synthesis scale × nucleoside phosphoramidite equivalent). This waste solution refers to the waste solution discharged in the sulfurization step or oxidation step.

[0049] 3. SEQ ID NO: 3 (1) Synthesis of DNA oligonucleotide (SEQ ID NO: 3) Porous resin beads (NittoPhase® HL UnyLinker350) 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 oligopilotplus100 synthesizer (manufactured by Cytiva). Nucleoside phosphoramidite and 4,5-dicyanoimidazole (DCI) as an activating agent were added in the amounts shown in Table 7, and a coupling reaction (condensation time: 5 minutes) was carried out under the conditions shown in Table 7. All activating agents were dissolved in acetonitrile to a concentration of 0.7 M. Other synthesis reagents used were 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'-CGTACGTACGTACGTACGTACGTA-3', the phosphite ester (internucleoside linkage) and the 3'-terminal phosphate are all sulfurized: SEQ ID NO: 3) 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 porous resin beads, and the base amino groups were deprotected, resulting in a filtrate in which the DNA oligonucleotide was dissolved.

[0050] (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

[0051] (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 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

[0052] The residual rate of nucleoside phosphoramidite in the column was calculated by dividing the absolute amount of DMTr in the waste solution (corresponding to the amount of bound nucleoside phosphoramidite) by the amount of nucleoside phosphoramidite introduced into the reaction (synthesis scale × nucleoside phosphoramidite equivalent). This waste solution refers to the waste solution discharged in the sulfurization step or oxidation step.

[0053] 4. SEQ ID NO: 4 (1) Synthesis of DNA oligonucleotide (SEQ ID NO: 4) Porous resin beads (NittoPhase® HL UnyLinker350) 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 oligopilotplus100 synthesizer (manufactured by Cytiva). Nucleoside phosphoramidite and 4,5-dicyanoimidazole (DCI) as an activating agent were added in the amounts shown in Table 7, and a coupling reaction (condensation time: 5 minutes) was carried out under the conditions shown in Table 7. All activating agents were dissolved in acetonitrile to a concentration of 0.7 M. Other synthesis reagents used were 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 21-mer DNA oligonucleotide (5'-TATACCGATTAAGCGAAGTTT-3', the phosphite ester (internucleoside linkage) and the 3'-terminal phosphate were all sulfurized; SEQ ID NO: 4) 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 porous resin beads, and the base amino groups were deprotected. A filtrate containing the DNA oligonucleotide was obtained.

[0054] (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

[0055] (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 21 synthesis cycles of 21mer DNA was collected. After the coupling reaction in the 21st 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

[0056] The residual rate of nucleoside phosphoramidite in the column was calculated by dividing the absolute amount of DMTr in the waste solution (corresponding to the amount of bound nucleoside phosphoramidite) by the amount of nucleoside phosphoramidite introduced into the reaction (synthesis scale × nucleoside phosphoramidite equivalent). This waste solution refers to the waste solution discharged in the sulfurization step or oxidation step.

[0057] 5. SEQ ID NO: 5 (1) Synthesis of DNA oligonucleotide (SEQ ID NO: 5) Porous resin beads (NittoPhase® HL UnyLinker350) 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 oligopilotplus100 synthesizer (manufactured by Cytiva). Nucleoside phosphoramidite and 4,5-dicyanoimidazole (DCI) as an activating agent were added in the amounts shown in Table 7, and a coupling reaction (condensation time: 5 minutes) was carried out under the conditions shown in Table 7. All activating agents were dissolved in acetonitrile to a concentration of 0.7 M. Other synthesis reagents used were 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 25-mer DNA oligonucleotide (5'-TTTGAAGCGAATTAGCCATATCGTA-3', the phosphite ester (internucleoside linkage) and the 3'-terminal phosphate are all sulfurized; SEQ ID NO: 5) 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 porous resin beads, and the base amino groups were deprotected. A filtrate containing the DNA oligonucleotide was obtained.

[0058] (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

[0059] (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 25 synthesis cycles of 25mer DNA, i.e., a total of 25 synthesis cycles, was collected. After the coupling reaction in the 25th 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

[0060] The residual rate of nucleoside phosphoramidite in the column was calculated by dividing the absolute amount of DMTr in the waste solution (corresponding to the amount of bound nucleoside phosphoramidite) by the amount of nucleoside phosphoramidite introduced into the reaction (synthesis scale × nucleoside phosphoramidite equivalent). This waste solution refers to the waste solution discharged in the sulfurization step or oxidation step.

[0061] 6. SEQ ID NO: 6 (1) Synthesis of RNA oligonucleotide (SEQ ID NO: 6) Porous resin beads (NittoPhase® HL UnyLinker350) 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 oligopilotplus100 synthesizer (manufactured by Cytiva). Nucleoside phosphoramidite and 5-(ethylthio)-1H-tetrazole (ETT) as an activator were added in the amounts shown in Table 7, and a coupling reaction (condensation time: 10 minutes) was carried out under the conditions shown in Table 7. All activators were dissolved in acetonitrile to a concentration of 0.6 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 23-mer 2'OMe RNA oligonucleotide (5'-GGUUUAACGUACGUACGUACGUA-3', the phosphite ester (internucleoside linkage) and the 3'-terminal phosphate are all sulfurized; SEQ ID NO: 6) 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 porous resin beads, and the base amino groups were deprotected, resulting in a filtrate in which the 2'OMe RNA oligonucleotide was dissolved.

[0062] (2) Measurement of Purity of Synthesized RNA 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 approximately 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

[0063] (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 23 synthesis cycles of 23mer 2'OMeRNA was collected. After the coupling reaction in the 23rd 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

[0064] The residual rate of nucleoside phosphoramidite in the column was calculated by dividing the absolute amount of DMTr in the waste solution (corresponding to the amount of bound nucleoside phosphoramidite) by the amount of nucleoside phosphoramidite introduced into the reaction (synthesis scale × nucleoside phosphoramidite equivalent). This waste solution refers to the waste solution discharged in the sulfurization step or oxidation step.

[0065] 7. SEQ ID NO: 7 (1) Synthesis of DNA oligonucleotide (SEQ ID NO: 7) Porous resin beads (NittoPhase® HL UnyLinker350) 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 oligopilotplus100 synthesizer (manufactured by Cytiva). Nucleoside phosphoramidite and 4,5-dicyanoimidazole (DCI) as an activator were added in the amounts shown in Table 6, and a coupling reaction (condensation time: 5 minutes for cycles including a sulfurization step, 10 minutes for cycles including an oxidation step) was carried out under the conditions shown in Table 6. All activators were dissolved in acetonitrile to a concentration of 0.7 M. Other synthetic reagents used included 3% DCA in toluene as a deprotecting agent, 0.2 M xanthan hydride in pyridine as a sulfurizing agent, iodine solution (0.05 mol / L; the solvent was pyridine:water (9:1)) 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) as an amine wash reaction solution. A 24-mer DNA oligonucleotide (5'-TCGACGTATTGACGTATTGACGTA-3', in which the phosphites (internucleoside linkages) at positions 1 to 17 from the 3' end and the 3'-terminal phosphate were sulfurized, and the phosphites at positions 18 to 23 from the 3' end were oxidized; SEQ ID NO: 7) was synthesized, and the terminal DMTr protecting group was removed. The porous resin beads to which the DNA oligonucleotides were bound were dried. Thereafter, the porous resin beads were immersed in aqueous ammonia, the DNA oligonucleotide was cleaved from the porous resin beads, and the basic amino groups were deprotected to obtain a filtrate in which the DNA oligonucleotide was dissolved.

[0066] (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

[0067] (3) Measurement of the amount of nucleoside phosphoramidite remaining in the column. Nucleoside phosphoramidites bound to the phosphate moieties of oligonucleotides are released by the sulfurizing or oxidizing agent during the sulfurizing or oxidation step and discharged together with the waste liquid. Therefore, all waste liquid from the sulfurizing or oxidation step in each of the 24 synthesis cycles of 24mer DNA, i.e., a total of 24 synthesis cycles, was collected. After the coupling reaction of 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

[0068] The residual rate of nucleoside phosphoramidite in the column was calculated by dividing the absolute amount of DMTr in the waste solution (corresponding to the amount of bound nucleoside phosphoramidite) by the amount of nucleoside phosphoramidite introduced into the reaction (synthesis scale × nucleoside phosphoramidite equivalent). This waste solution refers to the waste solution discharged in the sulfurization step or oxidation step.

[0069] 8. SEQ ID NO: 8 (1) Synthesis of DNA oligonucleotide (SEQ ID NO: 8) Porous resin beads (NittoPhase® HL UnyLinker350) 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 oligopilotplus100 synthesizer (manufactured by Cytiva). Nucleoside phosphoramidite and 4,5-dicyanoimidazole (DCI) as an activator were added in the amounts shown in Table 6, and a coupling reaction (condensation time: 5 minutes for cycles including a sulfurization step, 10 minutes for cycles including an oxidation step) was carried out under the conditions shown in Table 6. 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, iodine solution (0.05 mol / L; the solvent was pyridine:water (9:1)) 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) as an amine wash reaction solution. A 24-mer DNA oligonucleotide (5'-TCGACGTATTGACGTATTGACGTA-3', in which the phosphites (internucleoside bonds) at positions 3 to 20 from the 3' end were sulfurized, and the phosphites (internucleoside bonds) at positions 1, 2, 21 to 23 from the 3' end and the 3'-terminal phosphate were oxidized; SEQ ID NO: 8) was synthesized, and the terminal DMTr protecting groups were removed. The porous resin beads to which the DNA oligonucleotides were bound were dried. Thereafter, the porous resin beads were immersed in aqueous ammonia, the DNA oligonucleotide was cleaved from the porous resin beads, and the basic amino groups were deprotected to obtain a filtrate in which the DNA oligonucleotide was dissolved.

[0070] (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

[0071] (3) Measurement of the amount of nucleoside phosphoramidite remaining in the column. Nucleoside phosphoramidites bound to the phosphate moieties of oligonucleotides are released by the sulfurizing or oxidizing agent during the sulfurizing or oxidation step and discharged together with the waste liquid. Therefore, all waste liquid from the sulfurizing or oxidation step in each of the 24 synthesis cycles of 24mer DNA, i.e., a total of 24 synthesis cycles, was collected. After the coupling reaction of 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

[0072] The residual rate of nucleoside phosphoramidite in the column was calculated by dividing the absolute amount of DMTr in the waste solution (corresponding to the amount of bound nucleoside phosphoramidite) by the amount of nucleoside phosphoramidite introduced into the reaction (synthesis scale × nucleoside phosphoramidite equivalent). This waste solution refers to the waste solution discharged in the sulfurization step or oxidation step.

[0073] <Results and Discussion> 1. Evaluation of the optimum range of [N] x [T]

[0074] The evaluation results of Examples 1 to 7 and Comparative Examples 1 to 3, including the in-column residual rate (%) and final product purity (%), are shown in Table 2. The in-column residual rates of Examples 1 to 7 and Comparative Examples 2 and 3 were also calculated and shown as a percentage (%), relative to the in-column residual rate of Comparative Example 1, which was the comparative capping condition ([N] × [T] = 5.0), taken as 100%. Values ​​less than 100% indicate a suppression of the in-column residual rate compared to Comparative Example 1. The difference (points) between the purity of the final product of Examples 1 to 7 and Comparative Examples 2 and 3 and the purity of the final product of Comparative Example 1 was also calculated and shown. Positive values ​​indicate an improvement in the purity of the final product compared to Comparative Example 1.

[0075] In Examples 1 to 7, where the value of [N] × [T] was in the range of 0.5 to 3.0, the ratio of the in-column residual rate to that of Comparative Example 1 was less than 100%, which indicated that the in-column residual rate was suppressed. In addition, the increase in the purity of the final product compared to Comparative Example 1 was a positive value, which indicated that the purity of the final product was improved.

[0076] On the other hand, in both Comparative Example 2, in which the value of [N] × [T] was 0.4, and Comparative Example 3, in which capping was not performed, the ratio of the in-column residual rate to Comparative Example 1 was 100% or more, indicating that the in-column residual rate was not suppressed. Furthermore, the increase in the final product purity compared to Comparative Example 1 was a negative value, indicating that the purity of the final product was not improved.

[0077] From the above, it was found that in the production of oligonucleotides, rather than not performing capping, performing capping under mild capping conditions, such as an [N] × [T] value of 0.5 to 3.0, can suppress the rate of nucleoside phosphoramidite remaining in the column and further improve the purity of the final synthetic oligonucleotide product.

[0078] 2. Evaluation of the amount of nucleoside phosphoramidite input

[0079] The evaluation results for Examples 5, 20, and 8 and Comparative Examples 1, 13, and 4, including the in-column residual rate (%) and final product purity (%), are shown in Table 3. The in-column residual rates for Examples 5, 20, and 8 were also calculated and shown as a percentage (%) relative to the in-column residual rate for Comparative Examples 1, 13, and 4, which correspond to the comparative capping conditions ([N] x [T] = 5.0), taken as 100%. Values ​​less than 100% indicate a suppressed in-column residual rate compared to the corresponding comparative example. The difference (points) between the final product purity for Examples 5, 20, and 8 and the final product purity for Comparative Examples 1, 13, and 4 was also calculated and shown. Positive values ​​indicate an improved final product purity compared to the corresponding comparative example.

[0080] In Example 5 (nucleoside phosphoramidite input amount 1.2 equivalents), Example 20 (nucleoside phosphoramidite input amount 1.6 equivalents), and Example 8 (nucleoside phosphoramidite input amount 1.7 equivalents), where the [N] x [T] value was in the range of 0.5 to 3.0, the ratio of the residual rate in the column to the comparative example was less than 100%, indicating that the residual rate in the column was suppressed.Furthermore, the increase in the final product purity compared to the comparative example was a positive value, indicating that the final product purity was improved.However, these effects were more pronounced when the nucleoside phosphoramidite input amount was 1.2 equivalents than when it was 1.7 equivalents. In particular, the effect of improving the purity of the final product compared to the comparative examples was clearly related to the amount of nucleoside phosphoramidite input. The effect was greater when the amount of nucleoside phosphoramidite input was 1.6 equivalents (+0.7 points) than when it was 1.7 equivalents (+0.1 points), and the effect was even greater when it was 1.2 equivalents (+4.4 points). This phenomenon will be discussed below. First, comparing Comparative Examples 1, 13, and 4, Comparative Example 4, which uses a higher amount of nucleoside phosphoramidite input, has a high final product purity of 85.8%, but also a high in-column residual rate of 33.3%. This indicates that a high purity final product can be obtained by inputting an excess amount of nucleoside phosphoramidite, but also indicates that a large amount of nucleoside phosphoramidite is not used in the synthesis of the final product and remains in the column. On the other hand, in Comparative Example 13 and Comparative Example 1, the amount of nucleoside phosphoramidite input was reduced compared to Comparative Example 4, but this method, which focuses only on economical aspects, is undesirable because it reduces the purity of the final product to 84.8% or 80.2%. From the above, it can be seen that in order to obtain a final product with high purity economically, it is necessary to effectively utilize the nucleoside phosphoramidite input, that is, to suppress the residual rate in the column.Therefore, the present invention was applied in each example, and the effect of the invention was to suppress the residual fraction in the column in Examples 5, 20, and 8, but the contribution to the increased purity of the final product was remarkable when the amount of nucleoside phosphoramidite input was small (increased purity was observed at 1.2 to 1.7 equivalents, but the effect of increasing purity was particularly great at 1.2 to 1.6 equivalents). In Example 20 (input amount of nucleoside phosphoramidite: 1.6 equivalents) or Example 5 (input amount of nucleoside phosphoramidite: 1.2 equivalents), even though the input amount of nucleoside phosphoramidite was reduced to less than 1.7 equivalents, the effect of the present invention increased the purity of the final product to 85.5% (Example 20) or 84.6% (Example 5), respectively. According to the present invention, the residual rate in the column is suppressed and the input nucleoside phosphoramidite is effectively utilized in the synthesis of the final product, which means that both quality and economy can be achieved.

[0081] 3. Evaluation of Feed Solution Temperature and (Activator / Nucleoside Phosphoramidite) Input Ratio

[0082] The evaluation results for Examples 5, 8, 9, 10, 21, and 22 and Comparative Examples 1, 4, 5, and 6, including the in-column residual rate (%) and final product purity (%), are shown in Table 4. The in-column residual rates for Examples 5, 8, 9, and 10 were also calculated and shown as a percentage (%), relative to the in-column residual rate for Comparative Examples 1, 4, 5, and 6, which correspond to the comparative capping conditions ([N] x [T] = 5.0), taken as 100%. Values ​​less than 100% indicate a suppressed in-column residual rate compared to the corresponding comparative example. The difference (points) between the final product purity for Examples 5, 8, 9, and 10 and the final product purity for Comparative Examples 1, 4, 5, and 6 was also calculated and shown. Positive values ​​indicate an improved final product purity compared to the corresponding comparative example.

[0083] The values ​​of [N] x [T] were in the range of 0.5 to 3.0 in Example 5 (nucleoside phosphoramidite input amount 1.2 equivalents, total process feed solution temperature 22.5°C, (activator / nucleoside phosphoramidite) input amount ratio 3.5 times), Example 8 (nucleoside phosphoramidite input amount 1.7 equivalents, total process feed solution temperature 22.5°C, (activator / nucleoside phosphoramidite) input amount ratio 3.5 times), and Example 9 (nucleoside phosphoramidite input amount 1.2 equivalents, total process feed solution temperature 15°C, In Example 10 (nucleoside phosphoramidite input amount 1.7 equivalents, supply liquid temperature 15°C throughout the entire process, (activator / nucleoside phosphoramidite) input amount ratio 14), the in-column residual rate relative to the comparative example was less than 100%, indicating that the in-column residual rate had been suppressed, and further, the increase in the final product purity relative to the comparative example was a positive value, indicating that the final product purity had improved. In addition, in Example 5 (nucleoside phosphoramidite input amount 1.2 equivalents, feed solution temperature throughout the entire process 22.5°C, (activator / nucleoside phosphoramidite) input amount ratio 3.5 times), the residual ratio in the column was 14.3%, and the final product purity was 84.6%, while in Example 21 (nucleoside phosphoramidite input amount 1.2 equivalents, feed solution temperature throughout the entire process 22.5°C, (activator / nucleoside phosphoramidite) input amount ratio 14 times) and Example 9 (nucleoside phosphoramidite input amount 1.2 equivalents, feed solution temperature throughout the entire process 15°C, The residual rate in the column (activator / nucleoside phosphoramidite input ratio 14 times) was further suppressed to 12.8% (Example 21) and 13.8% (Example 9), and the purity of the final product improved to 85.7% (Example 21) and 90.4% (Example 9). This indicates that by lowering the temperature of the feed solution and / or increasing the input ratio of activator / nucleoside phosphoramidite, the residual rate in the column can be further suppressed and the purity of the final product can be further improved compared to when capping conditions are relaxed alone.In Example 8 (nucleoside phosphoramidite input amount 1.7 equivalents, total process feed solution temperature 22.5°C, (activator / nucleoside phosphoramidite) input amount ratio 3.5 times), the column residual rate was 25.6% and the final product purity was 85.9%, while in Example 22 (nucleoside phosphoramidite input amount 1.7 equivalents, total process feed solution temperature 22.5°C, (activator / nucleoside phosphoramidite) input amount ratio 14 times) and Example 10 (nucleoside phosphoramidite input amount 1.7 equivalents, total process feed solution temperature 15°C, ( Similarly, the residual rate in the column of (activator / nucleoside phosphoramidite) input ratio (14 times) was suppressed to 17.9% (Example 22) and 21.1% (Example 10), and the purity of the final product increased to 87.1% (Example 22) and 92.5% (Example 10). This indicates that by lowering the temperature of the feed solution and / or increasing the (activator / nucleoside phosphoramidite) input ratio, the residual rate in the column can be further suppressed and the purity of the final product can be improved compared to when capping conditions are relaxed alone.

[0084] From the above, it was found that in the production of oligonucleotides, the effect of suppressing the residual rate of nucleoside phosphoramidite in the column by performing capping under mild capping conditions, such as an [N] × [T] value of 0.5 to 3.0, and further the effect of improving the purity of the final synthetic oligonucleotide product, can be further enhanced by lowering the temperature of the feed solution and / or increasing the (activator / nucleoside phosphoramidite) input ratio.

[0085] 4. Evaluation of the effects of capping solution components other than NMI

[0086] Table 5 shows the column residual rates (%) and final product purity (%) evaluation results for Examples 5, 11 to 13, 23, and 24, and Comparative Examples 1, 14, and 15. The column residual rates for Examples 5 and 11 to 13 were calculated and reported as a percentage (%) relative to the column residual rate for Comparative Example 1, which had an acetic anhydride blending rate of 10% and comparative capping conditions ([N] × [T] = 5.0). The column residual rates for Examples 23 and 24 were calculated and reported as a percentage (%) relative to the column residual rate for Comparative Examples 14 and 15, which had acetic anhydride blending rates of 5% and 2.5%, respectively, and comparative capping conditions ([N] × [T] = 5.0). Values ​​less than 100% indicate a reduced column residual rate compared to the comparative examples. The difference (points) between the column residual rate for Examples 5 and 11 to 13 and the column residual rate for Comparative Example 1 was also calculated and reported. The difference (points) between the purity of the final product in Examples 23 and 24 and that of Comparative Example 14 or 15 was also calculated and shown. Positive values ​​indicate an improvement in the purity of the final product compared to the corresponding Comparative Example.

[0087] In Example 5 (lutidine blended at 15%), Example 11 (lutidine blended at 5%), Example 12 (lutidine blended at 22.5%), and Example 13 (pyridine blended at 15%), which had an acetic anhydride blending ratio of 10% and a value of [N] × [T] in the range of 0.5 to 3.0, the ratio of the residual rate in the column to the comparative example (Comparative Example 1 with an acetic anhydride blending ratio of 10%) was less than 100%, which showed that the residual rate in the column was suppressed as an effect of the invention. Furthermore, the increase in the purity of the final product as compared to the comparative example (Comparative Example 1) was a positive value, which showed that the purity of the final product was improved as an effect of the invention. In Example 23 (5% acetic anhydride blend, 15% lutidine blend) and Example 24 (2.5% acetic anhydride blend, 15% lutidine blend), where the acetic anhydride blending ratio was reduced to 5% or 2.5%, and the [N] x [T] value was in the range of 0.5 to 3.0, the ratio of the in-column residual rate to the comparative example (Comparative Example 14, where the acetic anhydride blending ratio was 5%, or Comparative Example 15, where the acetic anhydride blending ratio was 2.5%) was less than 100%, demonstrating the suppression of in-column residual rate as an effect of the present invention. Furthermore, the increase in the final product purity as compared to the comparative example (Comparative Example 14 or 15) was a positive value, demonstrating the improvement of the final product purity as an effect of the present invention. These effects were not significantly different among Examples 5, 11-13, 23, and 24, in which the blending ratio of capping solution components other than N-methylimidazole was varied. Furthermore, in Comparative Examples 14 (acetic anhydride blending rate of 5%) and 15 (acetic anhydride blending rate of 2.5%), the blending rate of acetic anhydride was reduced from that of Comparative Example 1 (acetic anhydride blending rate of 10%). However, while the purity of the final product in Comparative Example 1 was 80.2%, the purities of the final products in Comparative Examples 14 and 15 were 79.6% and 80.4%, respectively, and the reduction in the blending rate of acetic anhydride did not improve the purity of the final product. In other words, it was confirmed that the present invention, which controls the value of [N] × [T] within the range of 0.5 to 3.0 rather than reducing the blending rate of acetic anhydride, is effective in improving the purity of the final product.

[0088] From the above, it was found that in the production of oligonucleotides, the effect of suppressing the residual rate of nucleoside phosphoramidite in the column by performing capping under mild capping conditions, such as an [N] × [T] value of 0.5 to 3.0, and further the effect of improving the purity of the final synthetic oligonucleotide product, are hardly affected by capping solution components other than N-methylimidazole.

[0089] 5. Evaluation of the effect of sulfuration on oligonucleotides

[0090] The evaluation results for Examples 5, 8, 14, 15, 25, and 26 and Comparative Examples 1, 4, 7, 8, 16, and 17, including the in-column residual rate (%) and final product purity (%), are shown in Table 6. The in-column residual rates for Examples 5, 8, 14, 15, 25, and 26 were also calculated and reported as a percentage of the in-column residual rate for Comparative Examples 1, 4, 7, 8, 16, and 17, which correspond to the comparative capping conditions ([N] x [T] = 5.0), taken as 100%. Values ​​less than 100% indicate a suppressed in-column residual rate compared to the corresponding comparative example. The difference (points) between the final product purity for Examples 5, 8, 14, 15, 25, and 26 and the final product purity for Comparative Examples 1, 4, 7, 8, 16, and 17 was also calculated and reported. Positive values ​​indicate an improved final product purity compared to the corresponding comparative example.

[0091] Example 5 (1.2 equivalents of nucleoside phosphoramidite input, SEQ ID NO: 1 (sulfuration at all linking positions P)), Example 8 (1.7 equivalents of nucleoside phosphoramidite input, SEQ ID NO: 1 (sulfuration at all linking positions P)), Example 14 (1.2 equivalents of nucleoside phosphoramidite input, SEQ ID NO: 2 (no sulfuration at all linking positions P)), Example 15 (1.7 equivalents of nucleoside phosphoramidite input, SEQ ID NO: 2 (no sulfuration at all linking positions P)), Example 2 (1.7 equivalents of nucleoside phosphoramidite input, SEQ ID NO: 2 (no sulfuration at all linking positions P)), where the value of [N] × [T] is in the range of 0.5 to 3.0. In Example 5 (nucleoside phosphoramidite input amount 1.2 equivalents, SEQ ID NO: 7 (of a total of 23 linkage Ps, 17 linkage Ps at the ends ((17 / 23) x 100 = 74%) were sulfurized)), and Example 26 (nucleoside phosphoramidite input amount 1.2 equivalents, SEQ ID NO: 8 (of a total of 23 linkage Ps, 18 linkage Ps at the center ((18 / 23) x 100 = 78%) were sulfurized)), the ratio of in-column residual rate to the comparative example was less than 100%, indicating that the in-column residual rate was suppressed. However, in Examples 5, 8, 25, and 26, which were sulfurized conditions, the increase in the final product purity compared to the comparative example was positive, and the final product purity was improved, but in Examples 14 and 15, which were sulfurized conditions, the increase was not positive, and the final product purity was not improved.

[0092] From the above, it was found that in the production of oligonucleotides, capping under mild capping conditions, such as an [N] × [T] value of 0.5 to 3.0, can suppress the rate of nucleoside phosphoramidite remaining in the column. It was also found that sulfurizing the oligonucleotide can improve the purity of the final synthetic oligonucleotide product. In particular, it was found that the purity of the final synthetic oligonucleotide product can be improved by sulfurizing 74% or more of the linking moieties P in the oligonucleotide, sulfurizing 78% or more of the linking moieties P, sulfurizing 17 or more of the linking moieties P, or sulfurizing 18 or more of the linking moieties P.

[0093] 6. Evaluation of oligonucleotide sequences

[0094] The evaluation results for Examples 5, 16 to 19 and Comparative Examples 1, 9 to 12, including the in-column residual rate (%) and final product purity (%), are shown in Table 7. The in-column residual rates for Examples 5, 16 to 19 were also calculated and shown as a percentage (%), relative to the in-column residual rate for Comparative Examples 1, 9 to 12, which correspond to the comparative capping conditions ([N] × [T] = 5.0), taken as 100%. Values ​​less than 100% indicate a suppressed in-column residual rate compared to the corresponding Comparative Example. The difference (points) between the final product purity for Examples 5, 16 to 19 and the final product purity for Comparative Examples 1, 9 to 12 was also calculated and shown. Positive values ​​indicate an improved final product purity compared to the corresponding Comparative Example.

[0095] In Example 5 (SEQ ID NO: 1, activator DCI), Example 16 (SEQ ID NO: 3, activator DCI), Example 17 (SEQ ID NO: 4, activator DCI), Example 18 (SEQ ID NO: 5, activator DCI), and Example 19 (SEQ ID NO: 6, activator ETT), where the [N] x [T] value was in the range of 0.5 to 3.0, the ratio of in-column retention rate to the comparative example was less than 100%, indicating that the in-column retention rate was suppressed.Furthermore, the increase in the final product purity compared to the comparative example was a positive value, indicating that the purity of the final product was improved.These effects did not differ significantly among Examples 5, 16 to 19, which varied the nucleic acid species, sequence, chain length, presence or absence of modification, and activator species of the oligonucleotide.

[0096] From the above, it was found that in the production of oligonucleotides, the effect of suppressing the residual rate of nucleoside phosphoramidites in the column and further improving the purity of the final synthetic oligonucleotide product by carrying out capping under mild capping conditions, such as an [N] × [T] value of 0.5 to 3.0, is hardly affected by the nucleic acid species, sequence, chain length, presence or absence of modification, or type of activator of the oligonucleotide.

Claims

1. A method for producing an oligonucleotide, comprising: (a) removing a protecting group from a protected nucleoside, the protecting group being bound to a hydroxyl group, a thiol group or an amino group at the 3'- or 5'-position; (b) binding a nucleoside phosphoramidite to the hydroxyl group, a thiol group or an amino group at the 3'- or 5'-position of the nucleoside from which the protecting group has been removed, in the presence of an activator; (c) sulfurizing or oxidizing the bond formed by step (b); and (d) capping the unbound hydroxyl group, thiol group or amino group at the 3'- or 5'-position of the nucleoside, wherein the capping in step (d) comprises contacting the nucleoside with a capping solution for a predetermined time [T] (minutes), The above-mentioned method, wherein the capping solution contains N-methylimidazole at a blending ratio [N] (%) (wherein the ratio (%) of the room temperature volume of N-methylimidazole before mixing to the total room temperature volume (100%) of each reagent mixed as a component of the capping solution before mixing), and the product of [N] and [T] ([N] x [T]) is 0.5 to 3.

0.

2. The method of claim 1, wherein said oligonucleotide has sulfurized internucleoside linkages.

3. The method according to claim 1 or 2, wherein [N] is 1.1 to 8.0(%).

4. The method according to any one of claims 1 to 3, wherein [T] is 0.1 to 0.5 (min).

5. The method according to any one of claims 1 to 4, wherein the temperature of the solution in any one of steps (a) to (d) is 0 to 20°C.

6. The method of any one of claims 1 to 5, wherein the amount of the activating agent used in step (b) is 10.0 to 15.0 equivalents of the amount of the nucleoside phosphoramidite used in step (b).

7. The method of any one of claims 1 to 6, wherein the amount of activating agent used in step (b) is 10.0 to 25.0 equivalents of the supported nucleoside in step (b).

8. The method of any one of claims 1 to 7, wherein the activating agent in step (b) is selected from the group consisting of 4,5-dicyanoimidazole and 5-(ethylthio)-1H-tetrazole.

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