Nucleic acid synthesis using segmented amidites

Abstract

The purpose of the present invention is to provide a method for synthesizing an oligonucleotide by using a segment-type amidite. An oligonucleotide production method comprising at least one coupling step for coupling a nucleoside phosphoramidite with a thiol group or a hydroxyl group at the 3' or 5' of a nucleoside or a nucleotide in the presence of an activator, wherein, in the at least one coupling step, said nucleoside phosphoramidite has (a) two or more nucleoside portions or (b) at least one nucleoside portion and a linker portion, and said activator has a structure represented by formula (1) (in formula (1), X represents an organic base) or by formula (2) (in formula (2), R1 and R2 are each independently selected from the group consisting of H, straight chain or branched chain C1-7 alkyl groups and optionally substituted aromatic groups).

Description

[Technical field]

[0001] The present invention relates to a method for synthesizing oligonucleotides using segmented amidites. [Background technology]

[0002] The phosphoramidite method is widely used in the chemical synthesis of nucleic acids such as DNA oligonucleotides and RNA oligonucleotides. In this method, an oligonucleotide having a desired sequence is typically synthesized by sequentially adding appropriately protected nucleosides one by one. However, the reaction does not occur with a 100% probability, and the added nucleoside may be decomposed during the synthesis reaction, so that an oligonucleotide (Nn)mer having a length that is 1 or more (n) shorter than the desired length (N) may inevitably be generated. After synthesis, the oligonucleotide is purified by chromatography or the like. However, since the chromatographic mobility of the (N)mer, which is an oligonucleotide having the desired length, and the (N-1)mer, which is one nucleotide shorter than the desired length (N), are close, the presence of the (N-1)mer has a significant effect on the purification efficiency of the oligonucleotide and the purity of the purified product. Patent documents 1 and 2 and non-patent document 1 describe methods for suppressing the production of (N-1)mer oligonucleotides by utilizing nucleoside phosphoramidites (segmented amidites) having two or three nucleoside moieties in the synthesis, but these methods are not sufficiently efficient. In addition, Patent Document 3 describes a saccharin derivative as an activator that can be used more safely than 1H-tetrazole in the synthesis of oligonucleotides. However, Patent Document 3 does not fully examine the performance of the saccharin derivative as an activator. In addition, Patent Document 4 describes an activator containing at least one pyridinium salt and at least one substituted imidazole, an imidazolium salt, and a benzimidazolium salt as an activator that replaces 1H-tetrazole. However, Patent Document 4 does not describe the use of a salt with saccharin as an activator. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] WO 02 / 20543 [Patent Document 2] Special table number 2017-514479 [Patent Document 3] U.S. Patent No. 7,501,505 [Patent Document 4] U.S. Patent No. 6,642,373 [Non-patent literature]

[0004] [Non-Patent Document 1] RNA synthesis via dimer and trimer phosphoramidite block coupling [Tetrahedron Letters 52 (2011) 2575-2578] Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a method for synthesizing oligonucleotides using segmented amidites. [Means for solving the problem]

[0006] The present inventors have found an activator useful in a method for synthesizing oligonucleotides using segmented amidites during intensive research into the method. Based on this finding, further research has led to the completion of the present invention.

[0007] That is, the present invention relates to the following. [1] A method for producing an oligonucleotide, comprising the steps of: carrying out one or more coupling steps in the presence of an activator to attach a nucleoside phosphoramidite to a 3' or 5' hydroxyl or thiol group of a nucleotide or nucleoside; In at least one coupling step, the nucleoside phosphoramidite (a) a nucleoside phosphoramidite having two or more nucleoside moieties, or (b) a nucleoside phosphoramidite having one or more nucleoside moieties and a linker moiety; and The activator has the following formula: [ka] During the ceremony, X is an organic base. Or the following expression: [ka] During the ceremony, R 1 and R 2 are each independently H, straight or branched chain C 1~7 selected from the group consisting of alkyl groups, and optionally substituted aromatic groups; The method according to any one of claims 1 to 4, wherein

[0008] [2] X is N-methylimidazole, pyridine or 3-methylpyridine; R1 is H, C n H 2n+1 or a benzyl group, R2 is H, CH3, or C6H5; n is 1, 2 or 3; The method described in [1]. [3] The method according to [1] or [2], wherein the activator is 5-mercapto-1-methyltetrazole (1-Me-MCT), 5-mercapto-1-phenyltetrazole (1-Ph-MCT), saccharin 1-methylimidazole (SMI), or 5-ethylthio-1H-tetrazole (ETT). [4] In at least the last coupling step of two or more coupling steps, Nucleoside phosphoramidites (a) a nucleoside phosphoramidite having two or more nucleoside moieties, or (b) a nucleoside phosphoramidite having one or more nucleoside moieties and a linker moiety; The method according to any one of [1] to [3], [5] In at least the last coupling step of two or more coupling steps, The nucleoside phosphoramidite is a nucleoside phosphoramidite having three nucleoside moieties; The method according to any one of [1] to [4]. [6] In at least one of the two or more coupling steps, The nucleoside phosphoramidite is a nucleoside phosphoramidite having one nucleoside moiety; The method according to any one of [1] to [5]. [7] In only the last coupling step, which is performed two or more times, Nucleoside phosphoramidites (a) a nucleoside phosphoramidite having three nucleoside moieties, or (b) a nucleoside phosphoramidite having one or more nucleoside moieties and a linker moiety; and In the other coupling steps, The nucleoside phosphoramidite is a nucleoside phosphoramidite having one nucleoside moiety; The method according to any one of [1] to [6].

[0009] [8] A nucleoside phosphoramidite having two or more nucleoside moieties is represented by the following formula (I): [ka] During the ceremony, X 1 are each independently -O- or -S-; X 2 are each independently -O- or -S-; X 3 are each independently -O-, -S-, -CH2-, or -(CH2)2-; R 1 is a protecting group; R 2 are each independently -H, -NHR 6 , halogen, -CN, -CF3, or a hydroxyl group protected by an acyl-, ether- or silyl-protecting group; R 3 each independently represents -OCH2CH2CN, -SCH2CH2CN, a substituted or unsubstituted aliphatic group, -OR 7 or -SR 7 and; R 4 and R 5 are each independently a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl group; or R 4 and R 5 together with the nitrogen to which they are attached form a heterocycloalkyl group or a heteroaromatic group; R6 are each independently -H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl group, or a protecting group; R 7 are each independently a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, or a substituted or unsubstituted aralkyl group; B 1 , B 2 and B. 3 are each independently -H or a protected or unprotected base; and n is 0 or a positive integer; or a stereoisomer thereof.

[0010] [9] The method according to [8], wherein n is 0 or 1.

[10] R 2 The method according to [8] or [9], wherein:

[11] R 3 The method according to any one of [8] to

[10] , wherein

[0011]

[12] A nucleoside phosphoramidite having one or more nucleoside moieties and a linker moiety is represented by the following formula (II): [ka] During the ceremony, X 1 are each independently -O- or -S-; X 2 are each independently -O- or -S-; X 3 are each independently -O-, -S-, -CH2-, or -(CH2)2-; L is a linker; R 2 are each independently -H, -NHR 6, halogen, -CN, -CF3, or a hydroxyl group protected by an acyl-, ether- or silyl-protecting group; R 3 each independently represents -OCH2CH2CN, -SCH2CH2CN, a substituted or unsubstituted aliphatic group, -OR 7 or -SR 7 and; R 4 and R 5 are each independently a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl group; or R 4 and R 5 together with the nitrogen to which they are attached form a heterocycloalkyl or heteroaromatic group; R 6 are each independently -H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl group, or a protecting group; R 7 are each independently a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, or a substituted or unsubstituted aralkyl group; B 1 and B. 2 are each independently -H or a protected or unprotected base; and n is 0 or a positive integer; or a stereoisomer thereof.

[0012]

[13] A method for producing an oligonucleotide, comprising the steps of: carrying out one or more coupling steps in the presence of an activator to attach a nucleoside phosphoramidite to a 3' or 5' hydroxyl or thiol group of a nucleotide or nucleoside; In at least one coupling step, the nucleoside phosphoramidite (a) a nucleoside phosphoramidite having two or three nucleoside moieties, or (b) a nucleoside phosphoramidite having one or more nucleoside moieties and a linker moiety; and The activator has the following formula: [ka] During the ceremony, X is an organic base. Or the following expression: [ka] During the ceremony, R 1 and R 2 are each independently H, straight or branched chain C 1~7 The method according to any one of claims 1 to 4, wherein the alkyl group is selected from the group consisting of an alkyl group, and an optionally substituted aromatic group.

[0013]

[14] X is N-methylimidazole, pyridine or 3-methylpyridine; R1 is H, C n H 2n+1 or a benzyl group, R2 is H, CH3, or C6H5; n is 1, 2 or 3; The method described in

[13] .

[15] A method for producing an oligonucleotide, comprising the steps of: carrying out one or more coupling steps in the presence of an activator to attach a nucleoside phosphoramidite to a 3' or 5' hydroxyl or thiol group of a nucleotide or nucleoside; In at least one coupling step, the nucleoside phosphoramidite (a) a nucleoside phosphoramidite having two or more nucleoside moieties, or (b) a nucleoside phosphoramidite having one or more nucleoside moieties and a linker moiety; and The HOMO energy (au) of the activator is −0.21407 to −0.16858 in acetonitrile; and The orbital coefficient of the activator is 0.31531 to 0.59405 in acetonitrile. The method.

[16] The method according to

[15] , wherein the pKa of the activator is 3.65 to 7 in water.

[0014]

[17] A method for producing an oligonucleotide, comprising the steps of: carrying out one or more coupling steps in the presence of an activator to attach a nucleoside phosphoramidite to a 3' or 5' hydroxyl or thiol group of a nucleotide or nucleoside; In at least one coupling step, a nucleoside phosphoramidite is (a) a nucleoside phosphoramidite having two or three nucleoside moieties, or (b) a nucleoside phosphoramidite having one or more nucleoside moieties and a linker moiety; The HOMO energy (au) of the activator is −0.22024 to −0.16858 in acetonitrile; and The orbital coefficient of the activator is 0.31531 to 0.59405 in acetonitrile. The method.

[18] The method according to

[17] , wherein the pKa of the activator is 3.65 to 7 in water.

[19] A method for producing an oligonucleotide, comprising the steps of: carrying out one or more coupling steps in the presence of an activator to attach a nucleoside phosphoramidite to a 3' or 5' hydroxyl or thiol group of a nucleotide or nucleoside; In at least one coupling step, a nucleoside phosphoramidite is (a) a nucleoside phosphoramidite having two or three nucleoside moieties, or (b) a nucleoside phosphoramidite having one or more nucleoside moieties and a linker moiety; The pKa of the activator is 3.65 to 7 in water. The method.

[20] A method for producing an oligonucleotide, comprising the steps of: carrying out one or more coupling steps in the presence of an activator to attach a nucleoside phosphoramidite to a 3' or 5' hydroxyl or thiol group of a nucleotide or nucleoside; In at least one coupling step, a nucleoside phosphoramidite is (a) a nucleoside phosphoramidite having two or more nucleoside moieties, or (b) a nucleoside phosphoramidite having one or more nucleoside moieties and a linker moiety; The pKa of the activator is 4.3 to 7 in water. The method. Effect of the Invention

[0015] By using the activator of the present invention, oligonucleotides can be synthesized with high efficiency using nucleoside phosphoramidites having two or more nucleoside moieties, i.e., segmented amidites, or nucleoside phosphoramidites having one or more nucleoside moieties and a linker moiety. In addition, by using nucleoside phosphoramidites having two or more nucleoside moieties, i.e., segmented amidites, in the last coupling step, it is possible to suppress the production of (N-1)mers, which have a chromatographic mobility close to that of the desired target oligonucleotide and are difficult to remove by purification. In addition, using the activator of the present invention as an activator also contributes to suppressing the decomposition of segmented amidites during the synthesis process. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] 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, in the event of any discrepancy between the publications referenced herein and the description of this specification, the description of this specification shall prevail.

[0017] In one aspect, the present invention relates to a method for producing an oligonucleotide, the method comprising carrying out one or more coupling steps of binding a nucleoside phosphoramidite to a nucleotide or nucleoside having an unsubstituted hydroxyl group or an unsubstituted thiol group in the presence of an activator. In the present invention, oligonucleotides are prepared using the so-called phosphoramidite method, in which addition of nucleotides is carried out by condensation reaction of nucleoside phosphoramidite with nucleoside in the presence of a suitable activating agent in solution or on a solid support.

[0018] In the present invention, an oligonucleotide refers to a compound having a structure in which a base, a sugar, and a phosphate are linked by a phosphodiester bond, and includes naturally occurring oligonucleotides such as 2'-deoxyribonucleic acid (hereinafter "DNA") and ribonucleic acid (hereinafter "RNA"), and 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 a naturally occurring nucleic acid may be replaced with an L-ribose ring, or the β-anomer of a naturally occurring nucleic acid may be replaced with an α-anomer. The oligonucleotide may also contain one or more non-basic moieties. Modified phosphate moieties include phosphorothioates, phosphorodithioates, methylphosphonates, and methyl phosphates. 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 deoxyribo- 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.

[0019] In the present invention, the term "nucleoside phosphoramidite (segment-type amidite)" refers to a nucleoside derivatized with an amidite. The amiditization can be carried out, for example, by using 1H-tetrazole as an activator and reacting a suitably protected nucleoside with 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite.

[0020] In the present invention, a nucleoside refers to a compound in which a base and a sugar are bonded, and may be a naturally occurring nucleoside such as adenosine, thymidine, guanosine, cytidine, uridine, or a modified nucleoside. The base may be a naturally occurring base such as adenine, guanine, cytosine, thymine, and uracil, or a modified base. The sugar portion of the nucleoside may be a naturally occurring deoxyribose or ribose, and may be in the D-configuration or the L-configuration.

[0021] In the present invention, an activator is used to react a nucleoside phosphoramidite with a nucleotide or a nucleoside, and is also called an activating agent or a coupling agent. In one embodiment of the invention, the activator has the following formula: [ka] During the ceremony, X is an organic base which forms a salt complex with saccharin, preferably N-methylimidazole, pyridine or 3-methylpyridine; Or the following expression: [ka] During the ceremony, R 1 and R 2 are each independently H, straight or branched chain C 1~7 selected from the group consisting of alkyl groups, and optionally substituted aromatic groups; It has a structure represented by the following formula: Preferably, R 1 and R 2 are each independently H, a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a phenyl group, or a benzyl group, and more preferably, R1 is H, C n H 2n+1 or a benzyl group, R2 is H, CH3, or C6H5, and n is 1, 2, or 3.

[0022] In one embodiment of the invention, the activator is, for example, 5-mercapto-1-methyltetrazole (1-Me-MCT): [ka] 5-Mercapto-1-phenyltetrazole (1-Ph-MCT): [ka] 5-Benzylthiotetrazole (BTT): [ka] Saccharin 1-methylimidazole (SMI): [ka] 5-Ethylthio-1H-tetrazole (ETT) [ka] 4,5-Dicyanoimidazole (DCI): [ka] or benzimidazole trifluoromethanesulfonate (BIT): [ka] and preferably 1-Me-MCT, 1-Ph-MCT, SMI, ETT, or BTT, and more preferably 1-Me-MCT, 1-Ph-MCT, or SMI.

[0023] In one embodiment of the present invention, the HOMO energy (au) of the activator of the present invention in acetonitrile is -0.22024 to -0.16858. For example, the HOMO energy (au) of the activator of the present invention in acetonitrile is -0.21995 to -0.16858, -0.21407 to -0.16858, -0.19928 to -0.16858, -0.18904 to -0.16858, or -0.18741 to -0.16858, preferably -0.21407 to -0.16858 (au), and more preferably -0.18904 to -0.16858 (au). In one embodiment of the present invention, the orbital coefficient of the activator of the present invention is 0.31531 to 0.59405 in acetonitrile. For example, the orbital coefficient of the activator of the present invention is 0.31531 to 0.59405, 0.39931 to 0.59405, 0.51802 to 0.59405, or 0.53787 to 0.59405 in acetonitrile, and preferably 0.39931 to 0.59405.

[0024] In one embodiment of the present invention, the HOMO energy (au) of the activator is −0.22024 to −0.16858 in acetonitrile, and the orbital coefficient of the activator is 0.31531 to 0.59405 in acetonitrile. In one embodiment of the present invention, the HOMO energy (au) of the activator is −0.21407 to −0.16858 in acetonitrile, and the orbital coefficient of the activator is 0.31531 to 0.59405 in acetonitrile. The HOMO energy and orbital coefficients can be determined by a quantum chemistry calculation program, for example, by optimization calculation using the Becke-type 3-parameter density functional method (B3LYP) in the quantum chemistry calculation program Gaussian 16 manufactured by Gaussian.

[0025] In one embodiment of the present invention, the pKa of the activator of the present invention in water at 25° C. is 3.65 to 7.0, for example, 3.86 to 7.0, 4.1 to 7.0, 4.3 to 7.0, 4.5 to 7.0, 5.0 to 7.0, 5.5 to 7.0, 6.0 to 7.0, or 6.5 to 7.0.

[0026] In one embodiment of the present invention, in at least one coupling step in the method for producing the oligonucleotide, the nucleoside phosphoramidite is (a) a nucleoside phosphoramidite having two or more nucleoside moieties, or (b) a nucleoside phosphoramidite having one or more nucleoside moieties and a linker moiety.

[0027] In one embodiment of the present invention, in at least one coupling step in the method for producing the oligonucleotide, the nucleoside phosphoramidite is (a) a nucleoside phosphoramidite having two or three nucleoside moieties, or (b) a nucleoside phosphoramidite having one or more nucleoside moieties and a linker moiety. It is presumed that nucleoside phosphoramidites having two or three nucleoside moieties have higher reaction rates due to their smaller molecular size and therefore higher mobility and faster reaction rates than nucleoside phosphoramidites having four or more nucleoside moieties.

[0028] In one embodiment of the present invention, in at least the last of the two or more coupling steps in the method for producing the oligonucleotide, the nucleoside phosphoramidite is (a) a nucleoside phosphoramidite having two or more nucleoside moieties, or (b) a nucleoside phosphoramidite having one or more nucleoside moieties and a linker moiety.

[0029] In one embodiment of the present invention, in at least the last coupling step of the two or more coupling steps in the method for producing the oligonucleotide, the nucleoside phosphoramidite is a nucleoside phosphoramidite having three nucleoside moieties.

[0030] In one embodiment of the present invention, in at least one of the two or more coupling steps in the method for producing the oligonucleotide, the nucleoside phosphoramidite is a nucleoside phosphoramidite having one nucleoside moiety.

[0031] In one embodiment of the present invention, in only the last coupling step of two or more coupling steps in the method for producing the oligonucleotide, the nucleoside phosphoramidite is (a) a nucleoside phosphoramidite having three nucleoside moieties, or (b) a nucleoside phosphoramidite having one or more nucleoside moieties and a linker moiety, and in the other coupling steps, the nucleoside phosphoramidite is a nucleoside phosphoramidite having one nucleoside moiety.

[0032] In the present invention, nucleoside phosphoramidites having two or more nucleoside moieties can be prepared, for example, as described in WO 2019 / 212061. In one embodiment of the present invention, the nucleoside phosphoramidite having two or more nucleoside moieties has the following formula (I): [ka] or a stereoisomer thereof.

[0033] In formula (I), X 1 are each independently -O- or -S-. In formula (I), X 2 are each independently -O- or -S-. In formula (I), X3 are each independently -O-, -S-, -CH2- or -(CH2)2-.

[0034] In formula (I), R 1 is a protecting group, preferably an acid labile protecting group or a trialkylsilyl group such as t-butyldimethylsilyl or triisopropylsilyl. An acid labile protecting group is a protecting group that can be removed by contacting the group with a protic acid or a Lewis acid. Acid labile protecting groups are known to those skilled in the art. Examples of acid labile protecting groups include substituted or unsubstituted trityl groups, substituted or unsubstituted tetrahydropyranyl groups, substituted or unsubstituted tetrahydrofuranyl groups, or pixyl groups. The trityl group is usually substituted with an electron donating group such as an alkoxy group. In a more preferred embodiment, R 1 is substituted or unsubstituted trityl, 9-phenylxanthenyl (hereinafter "pixyl"), or tetrahydropyranyl (hereinafter "THP"). In an even more preferred embodiment, R 1 is unsubstituted trityl, monoalkoxytrityl, dialkoxytrityl, trialkoxytrityl, THP or pixyl. 1 is 4,4'-dimethoxytrityl. In formula (I), R 2 are each independently H, NHR 6 , halogen, CN, CF3, or a hydroxyl group protected by an acyl-based protecting group, an ether-based protecting group, or a silyl-based protecting group. Examples of halogen include F, Cl, Br, and I. Examples of acyl-based protecting groups include acetyl, benzoyl, and pivaloyl. Examples of ether-based protecting groups include benzyl, p-methoxybenzyl (PMB), and allyl. Examples of silyl-based protecting groups include t-butyldimethylsilyl (TBS), t-butyldiphenylsilyl (TBDPS), t-triisopropylsilyl (TIPS), triethylsilyl (TES), and trimethylsilyl (TMS). -H is preferred. In formula (I), R3 each independently represents -OCH2CH2CN, -SCH2CH2CN, a substituted or unsubstituted aliphatic group, -OR 7 or -SR 7 and preferably -OCH2CH2CN. Substituted or unsubstituted aliphatic groups include, but are not limited to, for example, 4-cyanobut-2-enylthio, 4-cyanobut-2-enyloxy, allylthio, allyloxy, crotylthio, or crotyloxy.

[0035] In formula (I), R 4 and R 5 are each independently a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl group; or R 4 and R 5 Together with the nitrogen to which they are attached, form a heterocycloalkyl group or a heteroaromatic group. Substituted or unsubstituted aliphatic groups include, but are not limited to, methyl, ethyl, isopropyl, etc., preferably isopropyl. Substituted or unsubstituted aromatic groups include, but are not limited to, phenyl, benzyl, naphthyl, 2-pyrenylmethyl, preferably phenyl, benzyl. Substituted or unsubstituted aralkyl groups include, but are not limited to, 2-fluorophenylmethoxypiperidin-4-yl, etc. Heterocycloalkyl groups include, but are not limited to, pyrrolidino, morpholino, etc., preferably morpholino.

[0036] In formula (I), R 6are each independently any one of -H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl group, or a protecting group including an acyl group. The substituted or unsubstituted aliphatic group includes, but is not limited to, for example, methyl, ethyl, allyl, 1-pentenyl, and 2-methoxyethyl, and is preferably methyl, allyl, and 2-methoxyethyl. The substituted or unsubstituted aromatic group includes, but is not limited to, for example, phenyl, benzyl, naphthyl, and 2-pyrenylmethyl, and is preferably phenyl and benzyl. The substituted or unsubstituted aralkyl group includes, but is not limited to, for example, 2-fluorophenylmethoxypiperidin-4-yl, and the like. The protecting group is, for example, t-butyldimethylsilyl, trifluoroacetyl, tert-butoxycarbonyl, benzyloxycarbonyl, phthaloyl, and p-toluenesulfonyl.

[0037] In formula (I), R 7 is a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, or a substituted or unsubstituted aralkyl group. Substituted or unsubstituted aliphatic groups include, but are not limited to, THP, 4-methoxytetrahydropyranyl, etc. Substituted or unsubstituted aromatic groups include, but are not limited to, o-chlorophenyl or p-chlorophenyl, etc. Substituted or unsubstituted aralkyl groups include, but are not limited to, 2-fluorophenylmethoxypiperidin-4-yl, etc.

[0038] In formula (I), B 1 , B 2 and B. 3are each independently H or a protected or unprotected base. Examples of protected or unprotected bases include, but are not limited to, naturally occurring bases such as adenine, guanine, cytosine, thymine, and uracil, as well as 7-deazaguanine, 7-deaza-8-azaguanine, 5-propynylcytosine, 5-propynyluracil, 7-deazaadenine, 7-deaza-8-azaadenine, 7-deaza-6-oxopurine, 6-oxopurine, 3-deazaadeno and modified bases such as sucrine, 2-oxo-5-methylpyrimidine, 2-oxo-4-methylthio-5-methylpyrimidine, 2-thiocarbonyl-4-oxo-5-methylpyrimidine, 4-oxo-5-methylpyrimidine, 2-aminopurine, 5-fluorouracil, 2,6-diaminopurine, 8-aminopurine, 4-triazolo-5-methylthymine, and 4-triazolo-5-methyluracil. In formula (I), n is 0 or a positive integer, preferably an integer of 0 or more and 4 or less, and more preferably 0 or 1.

[0039] In one embodiment of the invention, a nucleoside phosphoramidite having one or more nucleoside moieties and a linker moiety is a nucleoside phosphoramidite in which the linker is attached via a phosphorus atom at the 5' position of the nucleoside, e.g., via a phosphite, phosphate, thiophosphate, dithiophosphate.

[0040] In one embodiment of the invention, nucleoside phosphoramidites having one or more nucleoside moieties and a linker moiety may be prepared starting from phosphoramidites having a linker moiety such as, but not limited to, those available from Glen Research: PC Linker Phosphoramidite (3-(4,4'-dimethoxytrityl)-1-(2-nitrophenyl)-propan-1-yl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite) [ka]

[0041] 5'-Aminooxy-Modifier-11-CE Phosphoramidite (10-[N-Dimethoxytrityl-aminooxyethyl]-triethylene glycol-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite) [ka]

[0042] α-Tocopherol-TEG Phosphoramidite (1-Dimethoxytrityloxy-3-O-[(9-DL-α-tocopheryl)-triethylene glycol-1-yl]-glyceryl-2-O-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite) [ka]

[0043] 5'-DBCO-TEG Phosphoramidite (10-(6-oxo-6-(dibenzo[b,f]azacyclooct-4-yn-1-yl)-caproamide-N-ethyl)-O-triethylene glycol-1-[(2-cyanoethyl)-(N,N-diyl) isopropyl)]-phosphoramidite) [ka]

[0044] 5'-Cholesteryl-TEG Phosphoramidite (10-O-[1-Propyl-3-N-carbamoylcholesteryl]-triethylene glycol-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite) [ka]

[0045] DNP-TEG Phosphoramidite (1-Dimethoxytrityloxy-3-O-[N-(2,4-dinitrophenyl)-3-N-aminopropyl-(triethylene glycol)]-glyceryl-2-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite) [ka]

[0046] Cholesteryl-TEG Phosphoramidite (1-Dimethoxytrityloxy-3-O-(N-cholesteryl-3-aminopropyl)-triethylene glycol-glyceryl-2-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite) [ka]

[0047] 5'-Amino-Modifier TEG CE-Phosphoramidite (10-(O-trifluoroacetamido-N-ethyl)-triethylene glycol-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite) [ka]

[0048] Spacer Phosphoramidite 18 (18-O-Dimethoxytritylhexaethylene glycol, 1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite) [ka]

[0049] Spacer Phosphoramidite 9 (9-O-Dimethoxytrityl-triethylene glycol, 1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite) [ka]

[0050] PC Amino-Modifier Phosphoramidite ([(6-Trifluoroacetylamidocaproamidomethyl)-1-(2-nitrophenyl)-ethyl]-2-cyanoethyl-(N,N-diisopropyl)-phosphoramidite) [ka]

[0051] Thiol-Modifier C6 SS (1-O-Dimethoxytrityl-Hexyl-Disulfide, 1'-[(2-Cyanoethyl)-(N,N-Diisopropyl)]-Phosphoramidite) [ka]

[0052] 5'-Carboxy-Modifier C10 (10-Carboxy-decyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite, N-hydroxysuccinimide ester) [ka]

[0053] 5'-Thiol-Modifier C6 (S-Trityl-6-mercaptohexyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite) [ka]

[0054] Dithiol Serinol Phosphoramidite (3-Dimethoxytrityloxy-2-(3-((R)-α-Lipoamido)propanamido)propyl-1-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite) [ka]

[0055] 5'-Maleimide-Modifier Phosphoramidite (2-(1,7-dimethyl-3,5-dioxo-10-oxa-4-azatricyclo[5.2.1.02,6]dec-8-en-4-yl)-ethyl-1-O-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite) [ka]

[0056] PC Biotin Phosphoramidite (1-[2-nitro-5-(6-(N-(4,4'-dimethoxytrityl))-biotinamidocaproamidomethyl)phenyl]-ethyl-[2-cyanoethyl-(N,N-diisopropyl)]-phosphoramidite) [ka]

[0057] Protected BiotinLC Serinol Phosphoramidite (3-Dimethoxytrityloxy-2-(3-((4-t-butylbenzoyl)-biotinyl-3-aminopropyl)-diethyleneglycolyl-propylamido-glycanoylamido)propyl-1-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite) [ka]

[0058] 6-Fluorescein Serinol Phosphoramidite (3-Dimethoxytrityloxy-2-(3-(6-carboxy-(di-O-pivaloyl-fluorescein)propanamido)propyl)-1-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite) [ka]

[0059] Protected Biotin Serinol Phosphoramidite (3-Dimethoxytrityloxy-2-(3-((4-t-butylbenzoyl)-biotinyl)propanamido)propyl-1-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite) [ka]

[0060] 1-Ethynyl-dSpacer CE Phosphoramidite (5'-O-Dimethoxytrityl-1'-ethynyl-2'-deoxyribose-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite) [ka]

[0061] Azobenzene Phosphoramidite (3-O-(dimethoxytrityl)-2-N-(4-carboxyazobenzene)-D-threonine-1-yl-O-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite) [ka]

[0062] 5'-I-dT-CE Phosphoramidite (5'-iodo-2'-deoxythymidine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite) [ka]

[0063] Psoralen C6 Phosphoramidite (6-[4'-(hydroxymethyl)-4,5',8-trimethylsolerane]-hexyl-1-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite) [ka]

[0064] Psoralen C2 Phosphoramidite (2-[4'-(hydroxymethyl)-4,5',8-trimethylsolerane]-ethyl-1-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite) [ka]

[0065] 3-Cyanovinylcarbazole Phosphoramidite (CNVK) (5'-O-(4,4'-dimethoxytrityl)-1'-(3-cyanovinylcarbazol-9-yl)-2'-deoxy-β-D-ribofuranosyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite) [ka]

[0066] In one embodiment of the present invention, a nucleoside phosphoramidite having one or more nucleoside moieties and a linker moiety is represented by the following formula (II): [ka] or a stereoisomer thereof.

[0067] In formula (II), X 1 are each independently -O- or -S-. In formula (II), X 2 are each independently -O- or -S-. In formula (II), X 3 are each independently -O-, -S-, -CH2- or -(CH2)2-. In formula (II), L is a linker. Examples of the linker include, but are not limited to, [ka]

[0068] [ka]

[0069] [ka]

[0070] [ka]

[0071] [ka] etc.

[0072] In formula (II), R 2 are each independently -H, -NHR 6, halogen, -CN, -CF3, or a hydroxyl group protected by an acyl-based protecting group, an ether-based protecting group, or a silyl-based protecting group. Examples of halogen include F, Cl, Br, and I. Examples of acyl-based protecting groups include acetyl, benzoyl, and pivaloyl. Examples of ether-based protecting groups include benzyl, p-methoxybenzyl (PMB), and allyl. Examples of silyl-based protecting groups include t-butyldimethylsilyl (TBS), t-butyldiphenylsilyl (TBDPS), t-triisopropylsilyl (TIPS), triethylsilyl (TES), and trimethylsilyl (TMS). -H is preferred. In formula (II), R 3 each independently represents -OCH2CH2CN, -SCH2CH2CN, a substituted or unsubstituted aliphatic group, -OR 7 or -SR 7 and preferably -OCH2CH2CN. Substituted or unsubstituted aliphatic groups include, but are not limited to, for example, 4-cyanobut-2-enylthio, 4-cyanobut-2-enyloxy, allylthio, allyloxy, crotylthio, or crotyloxy.

[0073] In formula (II), R 4 and R 5 are each independently a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl group; or R 4 and R 5 together with the nitrogen to which they are attached form a heterocycloalkyl group or a heteroaromatic group. Substituted or unsubstituted aliphatic groups include, but are not limited to, methyl, ethyl, isopropyl, and the like, preferably isopropyl. Substituted or unsubstituted aromatic groups include, but are not limited to, phenyl, benzyl, toluyl, anyl, and the like, preferably phenyl, benzyl. Heterocycloalkyl groups include, but are not limited to, pyrrolidino, morpholino, and the like.

[0074] In formula (II), R 6 are each independently any one of -H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl group, or a protecting group including an acyl group. The substituted or unsubstituted aliphatic group includes, but is not limited to, for example, methyl, ethyl, allyl, 1-pentenyl, and 2-methoxyethyl, and is preferably methyl, allyl, and 2-methoxyethyl. The substituted or unsubstituted aromatic group includes, but is not limited to, for example, phenyl, benzyl, naphthyl, and 2-pyrenylmethyl, and is preferably phenyl and benzyl. The protecting group is, for example, t-butyldimethylsilyl.

[0075] In formula (II), R 7 is a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, or a substituted or unsubstituted aralkyl group. Substituted or unsubstituted aliphatic groups include, but are not limited to, THP, 4-methoxytetrahydropyranyl, etc. Substituted or unsubstituted aromatic groups include, but are not limited to, o-chlorophenyl or p-chlorophenyl, etc. Substituted or unsubstituted aralkyl groups include, but are not limited to, 2-fluorophenylmethoxypiperidin-4-yl, etc.

[0076] In formula (II), B 1 and B. 2are each independently H or a protected or unprotected base. Examples of protected or unprotected bases include, but are not limited to, naturally occurring bases such as adenine, guanine, cytosine, thymine, and uracil, as well as 7-deazaguanine, 7-deaza-8-azaguanine, 5-propynylcytosine, 5-propynyluracil, 7-deazaadenine, 7-deaza-8-azaadenine, 7-deaza-6-oxopurine, 6-oxopurine, 3-deazaadeno and modified bases such as thymine, 2-oxo-5-methylpyrimidine, 2-oxo-4-methylthio-5-methylpyrimidine, 2-thiocarbonyl-4-oxo-5-methylpyrimidine, 4-oxo-5-methylpyrimidine, 2-aminopurine, 5-fluorouracil, 2,6-diaminopurine, 8-aminopurine, 4-triazolo-5-methylthymine, and 4-triazolo-5-methyluracil. 1 and B. 2 At least one of the is adenine. In formula (II), n is 0 or a positive integer, preferably an integer of 0 or more and 4 or less, and more preferably 0 or 1. EXAMPLES

[0077] 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 be limiting.

[0078] Example 1 Comparison of sequential and segmental methods (1) Synthesis of oligonucleotides dT NittoPhase (registered trademark) HL dT300 (manufactured by Nitto Denko Corporation)) was filled in a reaction column in an amount equivalent to 1573 μmol, and a DNA oligonucleotide of 17mer (5' CCG ATT AAG CGA AGC TT 3') was synthesized using a nucleic acid synthesizer AKTA oligopilot plus100 (manufactured by Cytiva, formerly GE Healthcare Japan). Then, the reaction column was filled with the solid phase carrier with 17mer DNA in an amount equivalent to 205 μmol, and the nucleic acid was synthesized using the nucleic acid synthesizer AKTA oligopilot plus100 under three conditions: sequential method in which dC, dT, and dA are condensed in order using 5-ethylthio-1H-tetrazole (ETT) as an activator, segment method 1 in which dATC is condensed with 1.8 equivalents of amidite and a condensation time of 5 minutes, and segment method 2 in which dATC is condensed with 3.0 equivalents of amidite and a condensation time of 10 minutes. Other synthesis reagents used were the commonly used deprotection reagent, capping reagent and oxidation solution. dATC was prepared as described in WO 2019 / 212061. The solid support to which the DNA oligonucleotide was bound was immersed in 28% ammonia water to excise the DNA oligonucleotide from the solid support. A part of this liquid was diluted with water to prepare a DNA oligonucleotide sample solution. The remaining solution was used as a crude solution.

[0079] (2) Purification of oligonucleotides The obtained crude solution was purified using a purification device AKTA pure 25 (Cytiva, formerly GE Healthcare Japan) (purification conditions: column; 25 cm × 10 mm CV = 19.6 mL GE source 15Q resin, buffer; adjusted with NaOH, NaCl, and ultrapure water). (3) Analysis of DNA oligonucleotides The DNA oligonucleotide sample solutions before and after purification were measured by high performance liquid chromatography (HPLC) (measurement conditions: column; Waters XBridge OST C18 2.5 μm 50 × 4.6 mm, UV detection; 260 nm, Buffer A; HFIP / TEA in Water, Buffer B; methanol).

[0080] (4) Results The analytical results of the DNA oligonucleotide sample solution before purification are shown in Table 1. [Table 1] Here, impurity I refers to an impurity that appears near the N-1mer in HPLC measurement, impurity II refers to an impurity that appears near the N-2mer in HPLC measurement, and impurity III refers to an impurity that appears near the N-3mer in HPLC measurement, and so forth.

[0081] When synthesized by the sequential method, the contents of impurities I and II were 2.9% and 2.8%, respectively, whereas when synthesized by segment method 1, the contents of impurities I and II were 0.6% and 0.4%, respectively, and when synthesized by segment method 2, the contents were 0.7% and 1.8%, respectively. When synthesized by the segment method, the contents of impurities I and II could be significantly reduced compared to when synthesized by the sequential method.

[0082] The analytical results of the purified DNA oligonucleotide sample solution are shown in Table 2. [Table 2]

[0083] The purity of the target oligonucleotide was 90.46% in the stepwise method, whereas it was 92.34% in the segment method 1 and 92.53% in the segment method 2. The segment method gave a higher purity of the target oligonucleotide than the stepwise method. The FLP (full-length product, purity x total OD) was 9302 in the stepwise method, whereas it was 10702 in the segment method 1 and 10724 in the segment method 2. The segment method gave a higher yield of the target oligonucleotide than the stepwise method.

[0084] Example 2. Comparison of reaction rates (1) Synthesis of DNA oligonucleotides A reaction column was filled with NittoPhase (registered trademark) HL UnyLinker 350 (manufactured by Nitto Denko Corporation) in an amount equivalent to 2935 μmol, and a 17mer (5′ CCG ATT AAG CGA AGC TT 3′) DNA oligonucleotide was synthesized using a nucleic acid synthesizer, AKTA oligopilot plus100. Then, the 17mer DNA-attached solid support was filled in a reaction column in an amount equivalent to 90 μmol, and dGCC was condensed with 1.8 amidite equivalents and 5 minutes of condensation time using 5-mercapto-1-methyltetrazole (1-Me-MCT), 5-mercapto-1-phenyltetrazole (1-Ph-MCT), 5-benzylthiotetrazole (BTT), saccharin 1-methylimidazole (SMI), 5-ethylthio-1H-tetrazole (ETT), 4,5-dicyanoimidazole (DCI), 5-[3,5-bis(trifluoromethyl)phenyl]-1H-tetrazole (Activator42), or benzimidazole trifluoromethanesulfonate (BIT) as an activator using a nucleic acid synthesizer AKTA oligopilot plus10. All activators were dissolved in acetonitrile and adjusted to 0.25 M. Other synthesis reagents were 3% DCA in toluene as a deprotecting agent, pyridine in water, iodine as an oxidizing agent, pyridine in acetonitrile, N-methylimidazole, acetic anhydride or isobutyric anhydride as a capping agent, and TBA in acetonitrile as an amine wash reaction solution. dGCC was prepared as described in WO 2019 / 212061. The solid phase carrier to which the DNA oligonucleotide was bound was immersed in ammonia water to cleave the DNA oligonucleotide from the solid phase carrier. Activator42 has the following structure: [ka] has.

[0085] (2) Synthesis of RNA oligonucleotides NittoPhase (registered trademark) HL rU250 (Kinovate Life Science) was filled in a reaction column in an amount equivalent to 327 μmol, and a 17mer synthesis (5' CCG AUU AAG CGA AGC UU 3') RNA oligonucleotide was synthesized using a nucleic acid synthesizer AKTA oligopilot plus100. Then, a 17mer RNA-attached solid phase carrier was filled in a reaction column in an amount equivalent to 85 μmol, and rAUC was condensed with 2.0 amidite equivalents and 15 minutes of condensation time using 5-mercapto-1-methyltetrazole (1-Me-MCT), saccharin 1-methylimidazole (SMI), or 5-[3,5-bis(trifluoromethyl)phenyl]-1H-tetrazole (Activator42) as an activator using a nucleic acid synthesizer AKTA oligopilot plus10. All activators were dissolved in acetonitrile and adjusted to 0.25M. Other synthesis reagents were 3% DCA in toluene as a deprotecting agent, pyridine in water and iodine as oxidizing agents, pyridine in acetonitrile, N-methylimidazole, and acetic anhydride as capping agents, and TBA in acetonitrile as an amine wash reaction solution. rAUC was prepared as described in WO 2019 / 212061. The solid support bound with the RNA oligonucleotide was immersed in AMA reagent (28-30% ammonia water: methylamine aqueous solution = 1:1), filtered, and washed with DMSO. Then, TEA.3HF was slowly added dropwise in an ice bath and the solid support was shaken to excise the RNA oligonucleotide.

[0086] (3) Response rate analysis After cleavage, each oligonucleotide sample solution was measured by high performance liquid chromatography (HPLC) (measurement conditions: column; Waters XBridge OST C18 2.5 μm 50×4.6 mm, UV detection; 260 nm, Buffer A; HFIP / TEA in Water, Buffer B; methanol). In DNA synthesis, the reaction rate was calculated by taking the sum of the areas of the peaks detected up to 1.1 minutes after the peak of impurity III was observed in the HPLC measurement results as 100% and subtracting the peak area (%) of impurity III. In RNA synthesis, the reaction rate was calculated by taking the sum of the areas of the peaks detected up to 1.4 minutes after the peak of impurity III was observed in the HPLC measurement results as 100% and subtracting the peak area (%) of impurity III.

[0087] (4) Results The results for DNA synthesis are shown in Table 3, and the results for RNA synthesis are shown in Table 4. The HOMO energy and orbital coefficients of the activators used in this experiment are also shown in Tables 3 and 4. The HOMO energy and orbital coefficients were calculated by optimization calculations using the Becke-type 3-parameter density functional method (B3LYP) using the quantum chemical calculation program Gaussian 16 manufactured by Gaussian. Specifically, the structure of the active species (BIT is a neutral species, the others are anionic species) when the activator performs a nucleophilic attack on the amidite, i.e., the initial structure of the activator, was created using the Web-based calculation support program WebMO to create the initial structure of the molecule before calculation, and 6-31G(d) was entered as the basis function, -1 for anions and 0 for neutral species for charges, singlet for multiplicity, and acetonitrile for the solvent, and the HOMO energy and orbital coefficients were obtained by performing structure optimization and orbital calculation in that order. A simple structural correction of the initial structure to avoid collisions between atoms in the molecular model was appropriately performed by executing the Mechanics Optimize function of the Cleanup function in WebMO.

[0088] [Table 3] [Table 4]

[0089] From Tables 3 and 4, it can be seen that activators that showed higher reaction rates than Activator42 had higher HOMO energy levels and / or larger orbital coefficients. It is believed that the higher HOMO energy level of the activator reduces the energy difference between the HOMO energy level of the activator and the LUMO energy level of the segmented amidite, making the reaction easier to occur. In addition, the larger orbital coefficient of the activator increases the orbital overlap between the nitrogen atom of the activator and the phosphorus atom of the segmented amidite, making the reaction easier to occur.

[0090] Example 3 Comparison of the decomposition of segmented amidites (1) Synthesis of oligonucleotides using dGCC as a segmented amidite The 17mer DNA-attached solid phase carrier obtained in Example 2(1) was packed in a reaction column in an amount equivalent to 90 μmol, and dGCC was condensed with 1.8 amidite equivalents for 10 minutes using a nucleic acid synthesizer AKTA oligopilot plus10, with 0.6 M 5-mercapto-1-methyltetrazole (1-Me-MCT), 0.5 M 5-mercapto-1-phenyltetrazole (1-Ph-MCT), 0.25 M 5-benzylthiotetrazole (BTT), 0.25 M saccharin 1-methylimidazole (SMI), 0.6 M 5-ethylthio-1H-tetrazole (ETT), or 0.25 M 5-[3,5-bis(trifluoromethyl)phenyl]-1H-tetrazole (Activator42) as an activator. Other synthesis reagents were 3% DCA in toluene as a deprotecting agent, pyridine in water and iodine as oxidizing agents, pyridine in acetonitrile, N-methylimidazole, and acetic anhydride as capping agents, and TBA in acetonitrile as an amine wash reaction solution. dGCC was prepared as described in WO 2019 / 212061. The solid phase carrier to which the DNA oligonucleotide was bound was immersed in ammonia water at 55° C. for 12 to 16 hours to cleave the DNA oligonucleotide from the solid phase carrier.

[0091] (2) Analysis of DNA oligonucleotides The excised DNA oligonucleotide sample solution was measured by high performance liquid chromatography (HPLC) (measurement conditions: column; Waters XBridge OST C18 2.5 μm 50 × 4.6 mm, UV detection; 260 nm, Buffer A; 100 mM HFIP / 7 mM TEA in Water, pH 8.0, Buffer B; methanol, temperature; 60 ° C.). The peak area (%) of impurity II was calculated based on the sum of the areas of the peaks detected up to 1.1 minutes after the peak of impurity III was observed in the HPLC measurement results, taken as 100%.

[0092] (4) Results The results are shown in Table 5. Table 5 also shows the pKa values ​​in water of the activators used in this experiment. [Table 5] When synthesis was performed using Activator 42, the peak area of ​​impurity II was 9.089%, and significant decomposition of the segmented amidite was observed. On the other hand, when other activators were used, the peak area of ​​impurity II was about 1-2%, and almost no decomposition was observed. It is speculated that impurity II is likely generated by decomposition of the phosphate diester bond connecting the oligonucleotide and the segmented amidite by acid.

Claims

1. 1. A method for producing an oligonucleotide, comprising: carrying out one or more coupling steps in the presence of an activator to attach a nucleoside phosphoramidite to a 3' or 5' hydroxyl or thiol group of a nucleotide or nucleoside; In at least one coupling step, the nucleoside phosphoramidite (a) a nucleoside phosphoramidite having two or more nucleoside moieties, or (b) a nucleoside phosphoramidite having one or more nucleoside moieties and a linker moiety; and The activator has the following formula: 【Chemistry 1】 During the ceremony, X is an organic base; The method according to any one of claims 1 to 4, wherein

2. X is N-methylimidazole, pyridine or 3-methylpyridine; The method of claim 1.

3. The method of claim 1 or 2, wherein the activator is saccharin 1-methylimidazole (SMI).

4. In at least the last one of the two or more coupling steps, Nucleoside phosphoramidites (a) a nucleoside phosphoramidite having two or more nucleoside moieties, or (b) a nucleoside phosphoramidite having one or more nucleoside moieties and a linker moiety; The method according to any one of claims 1 to 3, wherein

5. In at least the last one of the two or more coupling steps, The nucleoside phosphoramidite is a nucleoside phosphoramidite having three nucleoside moieties; The method according to any one of claims 1 to 4.

6. In at least one of the two or more coupling steps, The nucleoside phosphoramidite is a nucleoside phosphoramidite having one nucleoside moiety; The method according to any one of claims 1 to 5.

7. In only the last coupling step, which is carried out two or more times, Nucleoside phosphoramidites (a) a nucleoside phosphoramidite having three nucleoside moieties, or (b) a nucleoside phosphoramidite having one or more nucleoside moieties and a linker moiety; and In the other coupling steps, The nucleoside phosphoramidite is a nucleoside phosphoramidite having one nucleoside moiety; The method according to any one of claims 1 to 6.

8. The nucleoside phosphoramidite having two or more nucleoside moieties is represented by the following formula (I): 【Chemistry 2】 (I) During the ceremony, X １ are each independently -O- or -S-; X ２ are each independently -O- or -S-; X ３ are each independently -O-, -S-, or -CH ２ - or - (CH ２ ) ２ - and R １ is a protecting group; R ２ are each independently -H, -NHR ６ , halogen, -CN, -CF ３ or a hydroxyl group protected by an acyl-, ether- or silyl-protecting group; R ３ Each independently represents -OCH ２ CH ２ CN, -SCH ２ CH ２ CN, a substituted or unsubstituted aliphatic group, -OR ７ or -SR ７ and R ４ and R ５ are each independently a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl group; or R ４ and R ５ together with the nitrogen to which they are attached form a heterocycloalkyl group or a heteroaromatic group; R ６ are each independently -H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl group, or a protecting group; R ７ are each independently a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, or a substituted or unsubstituted aralkyl group; B １ , B ２ and B ３ are each independently -H or a protected or unprotected base; and n is 0 or a positive integer; The method according to any one of claims 1 to 7, wherein the nucleoside phosphoramidite is represented by the following formula:

9. The method of claim 8 , wherein n is 0 or 1.

10. R ２ The method of claim 8 or 9, wherein is -H.

11. R ３ Ga-OCH ２ CH ２ The method according to any one of claims 8 to 10, wherein the compound is CN.

12. A nucleoside phosphoramidite having one or more nucleoside moieties and a linker moiety is represented by the following formula (II): 【Chemistry 3】 (II) During the ceremony, X １ are each independently -O- or -S-; X ２ are each independently -O- or -S-; X ３ are each independently -O-, -S-, or -CH ２ - or - (CH ２ ) ２ - and L is a linker; R ２ are each independently -H, -NHR ６ , halogen, -CN, -CF ３ or a hydroxyl group protected by an acyl-, ether- or silyl-protecting group; R ３ Each independently represents -OCH ２ CH ２ CN, -SCH ２ CH ２ CN, a substituted or unsubstituted aliphatic group, -OR ７ or -SR ７ and R ４ and R ５ are each independently a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl group; or R ４ and R ５ together with the nitrogen to which they are attached form a heterocycloalkyl or heteroaromatic group; R ６ are each independently -H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl group, or a protecting group; R ７ are each independently a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, or a substituted or unsubstituted aralkyl group; B １ and B ２ are each independently -H or a protected or unprotected base; and n is 0 or a positive integer; The method according to any one of claims 1 to 11, wherein the nucleoside phosphoramidite is represented by the following formula:

13. 1. A method for producing an oligonucleotide, comprising: carrying out one or more coupling steps in the presence of an activator to attach a nucleoside phosphoramidite to a 3' or 5' hydroxyl or thiol group of a nucleotide or nucleoside; In at least one coupling step, the nucleoside phosphoramidite (a) a nucleoside phosphoramidite having two or three nucleoside moieties, or (b) a nucleoside phosphoramidite having one or more nucleoside moieties and a linker moiety; and The activator has the following formula: 【Chemistry 4】 During the ceremony, X is an organic base; The method according to any one of claims 1 to 4, wherein

14. X is N-methylimidazole, pyridine or 3-methylpyridine; The method of claim 13.

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