Nucleic acid synthesis method using segment-type amidite

Abstract

To provide a method for synthesizing an oligonucleotide by using a segment-type amidite.SOLUTION: A method for producing an oligonucleotide comprises performing at least one coupling step for coupling a nucleoside phosphoramidite to a hydroxyl group or a thiol group at the 3' or 5' of a nucleotide or a nucleoside in the presence of an activator, wherein, in the at least one coupling step, the nucleoside phosphoramidite is (a) a nucleoside phosphoramidite having two or more nucleoside portions or (b) a nucleoside phosphoramidite having at least one nucleoside portion and a linker portion, and the activator has a structure represented by formula (1), wherein in formula (1), X represents an organic base, or by formula (2), wherein 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.SELECTED DRAWING: None

Description

Technical Field

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

Background Art

[0002] In the chemical synthesis of nucleic acids such as DNA oligonucleotides and RNA oligonucleotides, the phosphoramidite method is widely used. 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. Thus, an oligonucleotide (N-n)mer having a length that is 1 or more (n) shorter than the desired length (N) can inevitably occur. After synthesis, the oligonucleotide is purified by chromatography or the like. However, since the (N)mer, which is an oligonucleotide having the desired length, and the (N-1)mer, which is 1 nucleotide shorter than the desired length (N), have similar chromatographic mobilities, the presence of the (N-1)mer greatly affects 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 generation of (N-1)mer oligonucleotides, which utilize nucleoside phosphoramidites (segmental amidites) having two or three nucleoside moieties in the synthesis. However, those methods were 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, in Patent Document 3, the performance of the saccharin derivative as an activator has not been sufficiently examined. Further, 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 to replace 1H-tetrazole. However, in Patent Document 4, the use of a salt with saccharin as an activator is not described.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] An object of the present invention is to provide a method for synthesizing an oligonucleotide using a segment-type amidite.

Means for Solving the Problems

[0006] While the inventors of the present invention were earnestly researching a method for synthesizing oligonucleotides using segmental amidites, they found an activator useful in the method. As a result of further research based on such findings, the present invention was completed.

[0007] That is, the present invention relates to the following. [1] A method for producing an oligonucleotide, comprising: performing one or more coupling steps of bonding a nucleoside phosphoramidite to a 3'- or 5'-hydroxyl group or thiol group of a nucleotide or nucleoside in the presence of an activator; in at least one coupling step, 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 and the activator is represented by the following formula: [Chemical formula] wherein X is an organic base, or is represented by the following formula: [Chemical formula] wherein R 1 and R 2 are each independently selected from the group consisting of H, a linear or branched C 1~7 alkyl group, and an optionally substituted aromatic group, and having the structure represented by the above, the method.

[0008] [2] X is N-methylimidazole, pyridine or 3-methylpyridine, R1 is H, C n H 2n+1 or a benzyl group, and R 2 is H, CH 3 , or C 6 H 5 and 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 one of the coupling steps performed two or more times, 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 and is the method according to any one of [1] to [3]. [5] In at least the last one of the coupling steps performed two or more times, the nucleoside phosphoramidite is a nucleoside phosphoramidite having three nucleoside moieties, and is the method according to any one of [1] to [4]. [6] In at least one of the coupling steps performed two or more times, the nucleoside phosphoramidite is a nucleoside phosphoramidite having one nucleoside moiety, and is the method according to any one of [1] to [5]. [7] Only in the last one of the coupling steps performed two or more times, the nucleoside phosphoramidite is (a) a nucleoside phosphoramidite having three nucleoside moieties, or (b) A nucleoside phosphoramidite having one or more nucleoside moieties and linker moieties and in other cycles of the coupling step, 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)

Chemical formula

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

[10] The method according to [8] or [9], wherein R 2 is -H.

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

[10] , wherein R 3 is -OCH 2 CH 2 CN.

[0011]

[12] A nucleoside phosphoramidite having one or more nucleoside moieties and a linker moiety is represented by the following formula (II)

Chemical formula

[11] , which is a nucleoside phosphoramidite represented by the formula or a stereoisomer thereof.

[0012]

[13] A method for producing an oligonucleotide, In the presence of an activator, performing one or more coupling steps of bonding a nucleoside phosphoramidite to a 3' or 5' hydroxyl group or a thiol group of a nucleotide or a nucleoside, in at least one coupling step, the nucleoside phosphoramidite being (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 being of the following formula:

Chemical formula

Chemical formula

[0013]

[14] X is N-methylimidazole, pyridine or 3-methylpyridine, R 1 is H, C n H 2n+1 or a benzyl group, R 2 is H, CH 3 or C 6 H 5 and n is 1, 2 or 3, The method according to

[13] .

[15] A method for producing an oligonucleotide, including performing one or more coupling steps of bonding a nucleoside phosphoramidite to a 3'- or 5'-hydroxyl group or thiol group of a nucleotide or nucleoside in the presence of an activator, in at least one coupling step, the nucleoside phosphoramidite being (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 (a.u.) of the activator being from -0.21407 to -0.16858 in acetonitrile, and the orbital coefficient of the activator being from 0.31531 to 0.59405 in acetonitrile, the method.

[16] The method according to

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

[0014]

[17] A method for producing an oligonucleotide, comprising performing one or more coupling steps of bonding a nucleoside phosphoramidite to a 3'- or 5'-hydroxyl group or thiol group of a nucleotide or nucleoside in the presence of an activator, in at least one coupling step, the nucleoside phosphoramidite being (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 (a.u.) of the activator being from -0.22024 to -0.16858 in acetonitrile, and the orbital coefficient of the activator being from 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 performing one or more coupling steps of binding a nucleoside phosphoramidite to a 3' or 5' hydroxyl group or thiol group of a nucleotide or nucleoside in the presence of an activator, wherein in at least one coupling step, 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, and the pKa of the activator is 3.65 to 7 in water. The above method.

[20] A method for producing an oligonucleotide, comprising performing one or more coupling steps of binding a nucleoside phosphoramidite to a 3' or 5' hydroxyl group or thiol group of a nucleotide or nucleoside in the presence of an activator, wherein in at least one coupling step, 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, and the pKa of the activator is 4.3 to 7 in water. The above method.

Advantages 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., segmental amidites, or nucleoside phosphoramidites having one or more nucleoside moieties and linker moieties. Further, in the last coupling step, by using a nucleoside phosphoramidite having two or more nucleoside moieties, i.e., a segmental amidite, the generation of (N-1)mer, which has a chromatographic mobility close to that of the desired oligonucleotide and is difficult to remove by purification, can be suppressed. Further, using the activator of the present invention as the activator also contributes to suppressing the decomposition of the segmental amidite in the synthesis process.

BEST MODE FOR CARRYING OUT THE INVENTION

[0016] Hereinafter, the present invention will be described in detail. Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, and other publications and information referred to in this specification are hereby incorporated by reference in their entirety. Also, in the case of any conflict between the publications referred to in this specification 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, which includes performing one or more coupling steps of bonding 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, the production of oligonucleotides is carried out by using the so-called phosphoramidite method in which the addition of nucleotides is carried out by a condensation reaction between a nucleoside phosphoramidite and a nucleoside in the presence of a suitable activator in solution or on a solid support.

[0018] In the present invention, an oligonucleotide refers to a compound having a structure in which bases, sugars, and phosphates are linked by phosphodiester bonds, 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. Modification of the sugar moiety includes replacing the ribose ring with a hexose, cyclopentyl, or cyclohexyl ring. Alternatively, the D-ribose ring of a naturally occurring nucleic acid may be replaced with an L-ribose ring, or the β-anomer of a naturally occurring nucleic acid may be replaced with an α-anomer. The oligonucleotide may also contain one or more non-basic moieties. Modified phosphate moieties include phosphorothioate, phosphorodithioate, methylphosphonate, and methyl phosphate. Such nucleic acid analogs are known to those skilled in the art. Oligonucleotides comprising a mixture of two or more of the above can be produced, for example, from oligonucleotides comprising a mixture of deoxyribo and ribonucleosides, particularly a mixture of deoxyribonucleosides and 2'-O-substituted ribonucleosides such as 2'-O-methyl or 2'-O-methoxyethyl ribonucleosides. Examples of oligonucleotides comprising a mixture of nucleosides include ribozymes.

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

[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, or 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 moiety of the nucleoside may be a naturally occurring deoxyribose or ribose, and may have a D configuration or an 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 referred to as an activating agent or a coupling agent. In one embodiment of the present invention, the activator has the following formula:

Chemical formula

Chemical formula

[0022] In one aspect of the present invention, the activator is, for example, 5-mercapto-1-methyltetrazole (1-Me-MCT): [Chemical formula] 5-mercapto-1-phenyltetrazole (1-Ph-MCT): [Chemical formula] 5-benzylthiotetrazole (BTT): [Chemical formula] saccharin 1-methylimidazole (SMI): [Chemical formula] 5-ethylthio-1H-tetrazole (ETT) [Chemical formula] 4,5-dicyanoimidazole (DCI): [Chemical formula] or benzimidazole trifluoromethanesulfonate (BIT): [Chemical formula] and preferably is 1-Me-MCT, 1-Ph-MCT, SMI, ETT, or BIT, more preferably 1-Me-MCT, 1-Ph-MCT, or SMI.

[0023] In one aspect of the present invention, the HOMO energy (a.u.) of the activator of the present invention is -0.22024 to -0.16858 in acetonitrile. For example, the HOMO energy (a.u.) of the activator of the present invention 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 in acetonitrile, preferably -0.21407 to -0.16858 (a.u.), and more preferably -0.18904 to -0.16858 (a.u.). In one aspect 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, preferably 0.39931 to 0.59405.

[0024] In one aspect of the present invention, the HOMO energy (a.u.) 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 aspect of the present invention, the HOMO energy (a.u.) 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 the orbital coefficient can be determined by a quantum chemical calculation program. For example, in the quantum chemical calculation program Gaussian16 manufactured by Gaussian, they can be determined by an optimization calculation using the Becke-type 3-parameter density functional method (B3LYP).

[0025] In one aspect 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 aspect 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 aspect 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. A nucleoside phosphoramidite having two or three nucleoside moieties is presumed to have a higher reaction rate because, compared with a nucleoside phosphoramidite having four or more nucleoside moieties, it has higher motility and a faster reaction rate due to its smaller molecular size.

[0028] In one aspect of the present invention, in at least the last one of the coupling steps performed two or more times 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 aspect of the present invention, in at least the last one of the coupling steps performed two or more times in the method for producing the oligonucleotide, the nucleoside phosphoramidite is a nucleoside phosphoramidite having three nucleoside moieties.

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

[0031] In one aspect of the present invention, in only the last one of the coupling steps performed two or more times 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, a nucleoside phosphoramidite having two or more nucleoside moieties can be prepared, for example, as described in International Publication No. 2019 / 212061. In one aspect of the present invention, a nucleoside phosphoramidite having two or more nucleoside moieties has the following formula (I)

Chemical formula

[0033] In formula (I), X 1 is each independently -O- or -S-. In formula (I), X 2 is each independently -O- or -S-. In formula (I), X3 is, independently of each other, -O-, -S-, -CH 2 - or -(CH 2 ) 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 by 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. Most preferably, R 1 is 4,4'-dimethoxytrityl. In formula (I), R 2 is, independently of each other, H, NHR 6 , halogen, CN, CF 3、or any one of the hydroxyl groups protected by an acyl protecting group, an ether protecting group or a silyl protecting group. The halogen is, for example, F, Cl, Br, and I. Examples of the acyl protecting group include acetyl, benzoyl, pivaloyl, etc. Examples of the ether protecting group include benzyl, p-methoxybenzyl (PMB), allyl, etc. Examples of the silyl protecting group include t-butyldimethylsilyl (TBS), t-butyldiphenylsilyl (TBDPS), t-triisopropylsilyl (TIPS), triethylsilyl (TES), trimethylsilyl (TMS), etc. Preferably, it is -H. In formula (I), R 3 are each independently -OCH 2 CH 2 CN, -SCH 2 CH 2 CN, a substituted or unsubstituted aliphatic group, -OR 7 or -SR 7 and preferably -OCH 2 CH 2 CN. Examples of the substituted or unsubstituted aliphatic group include, but are not limited to, 4-cyanobut-2-enylthio, 4-cyanobut-2-enyloxy, allylthio, allyloxy, crotylthio or crotyloxy, etc.

[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 5Together with the nitrogen to which they are attached, they form a heterocycloalkyl group or a heteroaromatic group. Examples of the substituted or unsubstituted aliphatic group include, but are not limited to, methyl, ethyl, isopropyl, etc., and isopropyl is preferred. Examples of the substituted or unsubstituted aromatic group include, but are not limited to, phenyl, benzyl, naphthyl, 2-pyrenylmethyl, and phenyl and benzyl are preferred. Examples of the substituted or unsubstituted aralkyl group include, but are not limited to, 2-fluorophenylmethoxypiperidin-4-yl, etc. Examples of the heterocycloalkyl group include, but are not limited to, pyrrolidino, morpholino, etc., and morpholino is preferred.

[0036] In formula (I), R 6 is 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 such as an acyl group. Examples of the substituted or unsubstituted aliphatic group include, but are not limited to, methyl, ethyl, allyl, 1-pentenyl, 2-methoxyethyl, and methyl, allyl, 2-methoxyethyl are preferred. Examples of the substituted or unsubstituted aromatic group include, but are not limited to, phenyl, benzyl, naphthyl, 2-pyrenylmethyl, and phenyl and benzyl are preferred. Examples of the substituted or unsubstituted aralkyl group include, but are not limited to, 2-fluorophenylmethoxypiperidin-4-yl, etc. The protecting group is, for example, t-butyldimethylsilyl, trifluoroacetyl, tert-butoxycarbonyl, benzyloxycarbonyl, phthaloyl, p-toluenesulfonyl.

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

[0038] In formula (I), B 1 、B 2 and B 3 are each independently H, or a protected or unprotected base. Examples of the protected or unprotected base include, but are not limited to, naturally occurring bases such as adenine, guanine, cytosine, thymine, and uracil, and modified bases such as 7-deazaguanine, 7-deaza-8-azaguanine, 5-propynylcytosine, 5-propynyluracil, 7-deazaadenine, 7-deaza-8-azaadenine, 7-deaza-6-oxopurine, 6-oxopurine, 3-deazaadenosine, 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, etc. In formula (I), n is 0 or a positive integer, preferably an integer of 0 or more and 4 or less, more preferably 0 or 1.

[0039] In one aspect of the present invention, a nucleoside phosphoramidite having one or more nucleoside moieties and a linker moiety is a nucleoside phosphoramidite in which the linker is bonded at the 5'-position of the nucleoside via a phosphorus atom, for example, via a phosphite ester, a phosphate ester, a thiophosphate ester, or a dithiophosphate ester.

[0040] In one aspect of the present invention, a nucleoside phosphoramidite having one or more nucleoside moieties and a linker moiety may be prepared using, as a raw material, a phosphoramidite having a linker moiety as shown below, which can be obtained, for example, from Glen Research: PC Linker Phosphoramidite (3-(4,4'-dimethoxytrityl)-1-(2-nitrophenyl)-propan-1-yl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite)

Chemical formula

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

Chemical formula

[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)

Chemical formula

[0043] 5'-DBCO-TEG Phosphoramidite (10-(6-oxo-6-(dibenzo[b,f]azacyclooct-4-yl)-caproamido-N-ethyl)-O-triethyleneglycol-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite) propyl)]-phosphoramidite)

Chem.

[0044] 5'-Cholesteryl-TEG Phosphoramidite (10-O-[1-propyl-3-N-carbamoylcholesteryl]-triethyleneglycol-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite)

Chem.

[0045] DNP-TEG Phosphoramidite (1-dimethoxytrityloxy-3-O-[N-(2,4-dinitrophenyl)-3-N-aminopropyl-(triethyleneglycol)]-glyceryl-2-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite)

Chem.

[0046] Cholesteryl-TEG Phosphoramidite (1-dimethoxytrityloxy-3-O-(N-cholesteryl-3-aminopropyl)-triethyleneglycol-glyceryl-2-O-(2-cyanoethyl)-(N,N,-diisopropyl)-phosphoramidite)

Chem.

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

Chem.

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

Chem.

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

Chem.

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

Chem.

[0051] Thiol-Modifier C6 S-S (1-O-dimethoxytrityl-hexyl-disulfide, 1’-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite)

Chem.

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

Chem.

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

Chem.

[0054] Dithiol Serinol Phosphoramidite (3-dimethoxytrityloxy-2-(3-((R)-α-lipoamide)propanamide)propyl-1-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite)

Chem.

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

Chem.

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

Chem.

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

Chem.

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

Chem.

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

Chem.

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

Chem.

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

Chem.

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

Chem.

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

Chem.

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

Chem.

[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)

Chem.

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

Chem.

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

Chem.

[0068]

Chem.

[0069] [Chemistry]

[0070] [Chemistry]

[0071] [Chemistry] etc. can be mentioned.

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

[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. Examples of the substituted or unsubstituted aliphatic group include, but are not limited to, methyl, ethyl, isopropyl, etc., and isopropyl is preferred. Examples of the substituted or unsubstituted aromatic group include, but are not limited to, phenyl, benzyl, toluyl, anil, etc., and phenyl and benzyl are preferred. Examples of the heterocycloalkyl group include, but are not limited to, pyrrolidino, morpholino, etc.

[0074] In formula (II), R 6 is 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 such as an acyl group. Examples of the substituted or unsubstituted aliphatic group include, but are not limited to, methyl, ethyl, allyl, 1-pentenyl, 2-methoxyethyl, and methyl, allyl, 2-methoxyethyl are preferred. Examples of the substituted or unsubstituted aromatic group include, but are not limited to, phenyl, benzyl, naphthyl, 2-pyrenylmethyl, and phenyl and benzyl are preferred. The protecting group is, for example, t-butyldimethylsilyl.

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

[0076] In formula (II), B 1 and B 2 are each independently H, or a protected or unprotected base. Examples of the protected or unprotected base include, but are not limited to, naturally occurring bases such as adenine, guanine, cytosine, thymine, and uracil, and modified bases such as 7-deazaguanine, 7-deaza-8-azaguanine, 5-propynylcytosine, 5-propynyluracil, 7-deazaadenine, 7-deaza-8-azaadenine, 7-deaza-6-oxopurine, 6-oxopurine, 3-deazaadenosine, 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 a preferred embodiment, at least one of B 1 and B 2 is adenine. In formula (II), n is 0 or a positive integer, preferably an integer of 0 or more and 4 or less, more preferably 0 or 1.

Examples

[0077] The present invention will be described in more detail with reference to the following examples, which show specific examples of the present invention, but the present invention is not limited thereto.

[0078] Example 1 Comparison between the sequential method and the segment method (1) Synthesis of oligonucleotides dT NittoPhase (registered trademark) HL dT300 (manufactured by Nitto Denko Corporation) was filled into a reaction column in an amount corresponding to 1573 μmol, and a 17-mer (5’ CCG ATT AAG CGA AGC TT 3’) DNA oligonucleotide was synthesized using a nucleic acid synthesizer AKTA oligopilot plus100 (manufactured by Cytiva, former GE Healthcare Japan). Then, a solid-phase carrier with 17-mer DNA attached was filled into the reaction column in an amount corresponding to 205 μmol, and using the nucleic acid synthesizer AKTA oligopilot plus100, with 5-ethylthio-1H-tetrazole (ETT) as an activator, it was synthesized under three conditions: the sequential method of condensing dC, dT, and dA in sequence, segment method 1 in which dATC was condensed at an amidite equivalent of 1.8 equivalents and a condensation time of 5 minutes, and segment method 2 in which dATC was condensed at an amidite equivalent of 3.0 equivalents and a condensation time of 10 minutes. Other synthetic reagents used were commonly used deprotection reagents, capping reagents, and oxidation solutions. dATC was prepared as described in International Publication No. 2019 / 212061. The solid-phase carrier to which the DNA oligonucleotide was bound was immersed in 28% aqueous ammonia, and the DNA oligonucleotide was cut out from the solid-phase carrier. A part of this solution 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 apparatus AKTA pure 25 (manufactured by Cytiva, former 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 Measurements by high performance liquid chromatography (HPLC) were performed on the DNA oligonucleotide sample solutions before and after purification (measurement conditions: column; Waters XBridge OST C18 2.5μm 50×4.6mm, UV detection; 260nm, BufferA; HFIP / TEA in Water, BufferB; methanol).

[0080] (4) Results The analysis results of the DNA oligonucleotide sample solution before purification are shown in Table 1.

Table 1

[0081] When synthesized by the sequential method, the contents of impurity I and impurity II were 2.9% and 2.8% respectively, while when synthesized by segment method 1, the contents of impurity I and impurity II were 0.6% and 0.4% respectively, and when synthesized by segment method 2, they were 0.7% and 1.8% respectively. When synthesized by the segment method, the contents of impurity I and impurity II could be significantly reduced compared with the case of synthesis by the sequential method.

[0082] The analysis results of the DNA oligonucleotide sample solution after purification are shown in Table 2.

Table 2

[0083] The purity of the target oligonucleotide is 90.46% in the sequential method, while it is 92.34% in segment method 1 and 92.53% in segment method 2. When synthesized by the segment method, the target oligonucleotide could be obtained with a higher purity compared to the case of synthesis by the sequential method. Also, the FLP (full-length product, purity × total OD) is 9302 in the sequential method, while it is 10702 in segment method 1 and 10724 in segment method 2. When synthesized by the segment method, the target oligonucleotide could be obtained with a higher yield compared to the case of synthesis by the sequential method.

[0084] Example 2 Comparison of reaction rates (1) Synthesis of DNA oligonucleotides The reaction column was packed with NittoPhase (trademark registered) HL UnyLinker 350 (manufactured by Nitto Denko Corporation) in an amount corresponding to 2935 μmol, and a 17-mer (5’ CCG ATT AAG CGA AGC TT 3’) DNA oligonucleotide was synthesized using a nucleic acid synthesizer AKTA oligopilot plus 100. Subsequently, the solid-phase carrier with 17-mer DNA was packed into the reaction column in an amount corresponding to 90 μmol, and with a nucleic acid synthesizer AKTA oligopilot plus 10, using 5-mercapto-1-methyltetrazole (1-Me-MCT), 5-mercapto-1-phenyltetrazole (1-Ph-MCT), 5-benzylthioltetrazole (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 activators, dGCC was condensed at an amidite equivalent number of 1.8 equivalents and a condensation time of 5 minutes. All the activators were dissolved in acetonitrile and adjusted to 0.25 M. Other synthetic reagents used were 3% DCA in toluene as a deprotecting agent, pyridine in water as an oxidizing agent, pyridine, N-methylimidazole, acetic anhydride or isobutyric anhydride in acetonitrile as a capping agent, and TBA in acetonitrile as an amine wash reaction solution. dGCC was prepared as described in International Publication No. 2019 / 212061. The solid-phase carrier to which the DNA oligonucleotide was bound was immersed in aqueous ammonia to cleave the DNA oligonucleotide from the solid-phase carrier. Note that Activator42 has the following structure: [Chemical formula] It has.

[0085] (2) Synthesis of RNA oligonucleotide The reaction column was packed with NittoPhase (trademark registered) HL rU250 (manufactured by Kinovate Life Science) in an amount corresponding to 327 μmol, and using a nucleic acid synthesizer AKTA oligopilot plus100, an RNA oligonucleotide of 17mer synthesis (5’ CCG AUU AAG CGA AGC UU 3’) was synthesized. Then, the solid-phase carrier with 17mer RNA was packed into the reaction column in an amount corresponding to 85 μmol, and using a nucleic acid synthesizer AKTA oligopilot plus10, as activators, 5-mercapto-1-methyltetrazole (1-Me-MCT), saccharin 1-methylimidazole (SMI), or 5-[3,5-bis(trifluoromethyl)phenyl]-1H-tetrazole (Activator42) was used to condense rAUC with 2.0 equivalents of amidite equivalent number and a condensation time of 15 minutes. All the activators were dissolved in acetonitrile and adjusted to 0.25 M. Other synthetic reagents used were 3% DCA in toluene as a deprotecting agent, pyridine in water as an oxidizing agent, pyridine, N-methylimidazole, acetic anhydride in acetonitrile as a capping agent, and TBA in acetonitrile as an amine wash reaction solution. rAUC was prepared as described in International Publication No. 2019 / 212061. The solid-phase carrier to which the RNA oligonucleotide was bound was immersed in an AMA reagent (28~30% aqueous ammonia: aqueous methylamine solution = 1:1), filtered through a filter, and washed with DMSO. Then, TEA.3HF was slowly added dropwise under an ice bath, and the RNA oligonucleotide was excised from the solid-phase carrier by shaking.

[0086] (3) Analysis of reaction rate For each oligonucleotide sample solution after excision, measurement was performed by high-performance liquid chromatography (HPLC) (measurement conditions: column; Waters XBridge OST C18 2.5 μm 50×4.6 mm, UV detection; 260 nm, BufferA; HFIP / TEA in Water, BufferB; methanol). In DNA synthesis, after the peak of impurity III was observed in the HPLC measurement results, the sum of the areas of the peaks detected by 1.1 minutes later was set as 100%, and the reaction rate was calculated by subtracting the peak area (%) of impurity III. In RNA synthesis, after the peak of impurity III was observed in the HPLC measurement results, the sum of the areas of the peaks detected by 1.4 minutes later was set as 100%, and the reaction rate was calculated by subtracting the peak area (%) of impurity III.

[0087] (4) Results The results of DNA synthesis are shown in Table 3, and the results of RNA synthesis are shown in Table 4. In addition, the HOMO energy and orbital coefficients of the activator used in this experiment are also shown in Table 3 and Table 4. The HOMO energy and orbital coefficients were obtained by optimization calculations using the quantum chemical calculation program Gaussian16 manufactured by Gaussian and adopting the Becke-type 3-parameter density functional method (B3LYP). Specifically, the structure of the active species (BIT is a neutral species, and the others are anion species) when the activator makes a nucleophilic attack on the amidite, that is, the initial structure of the activator, was created as the initial structure of the molecule before calculation using the web-based calculation support program WebMO. The basis function was set to 6-31G(d), the charge was -1 for anions, 0 for neutral species, the multiplicity was singlet, and acetonitrile was input as the solvent, and the structure optimization and orbital calculation were performed in this order to obtain the HOMO energy and orbital coefficients. To avoid atomic collisions in the molecular model, a simple structural correction of the initial structure was appropriately performed by executing the Mechanics Optimize of the Cleanup function in WebMO.

[0088] [Table 3] [Table 4]

[0089] From Tables 3 and 4, it can be seen that the activators showing a higher reaction rate than Activator42 had a high HOMO energy level and / or a large orbital coefficient. It is considered that due to the high HOMO energy level of the activator, the energy difference between the HOMO energy level of the activator and the LUMO energy level of the segmental amidite became smaller, making the reaction more likely to occur. Also, due to the large orbital coefficient of the activator, the orbital overlap between the nitrogen atom of the activator and the phosphorus atom of the segmental amidite became larger, making the reaction more likely to occur.

[0090] Example 3 Comparison of the decomposition of segmental amidites (1) Synthesis of oligonucleotides using dGCC as the segmental amidite The solid-phase support with 17mer DNA obtained in Example 2(1) was packed into the reaction column in an amount corresponding to 90 μmol, and 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-benzylthioltetrazole (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 the activator, dGCC was condensed at 1.8 equivalents of amidite equivalent and a condensation time of 10 minutes. Other synthetic reagents used were 3% DCA in toluene as the deprotecting agent, pyridine in water as the oxidizing agent, pyridine, N-methylimidazole, acetic anhydride in acetonitrile as the capping agent, and TBA in acetonitrile as the amine wash reaction solution. dGCC was prepared as described in International Publication No. 2019 / 212061. The solid-phase support to which the DNA oligonucleotide was bound was immersed in aqueous ammonia at 55 °C for 12 to 16 hours to cleave the DNA oligonucleotide from the solid-phase support.

[0091] (2) Analysis of DNA oligonucleotides For the DNA oligonucleotide sample solution after excision, measurement was performed by high performance liquid chromatography (HPLC) (measurement conditions: column; Waters XBridge OST C18 2.5μm 50×4.6mm, UV detection; 260nm, Buffer A; 100mM HFIP / 7mM TEA in Water, pH8.0, Buffer B; methanol, temperature; 60°C). After the peak of impurity III was observed in the HPLC measurement results, the peak area (%) of impurity II was calculated when the total area of the peaks detected by 1.1 minutes later was taken as 100%.

[0092] (4) Results The results are shown in Table 5. Also, the pKa value in water of the activator used in this experiment is shown in Table 5 together.

Table 5

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; Or the following formula: 【Chemistry 2】 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

2. X is N-methylimidazole, pyridine or 3-methylpyridine; R １ is H, C ｎ H ２ｎ＋１ or a benzyl group, R ２ is H, CH ３ , or C ６ H ５ and n is 1, 2 or 3; The method of claim 1.

3. 3. The method of claim 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 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 3】 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 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 , 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 4】 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 5】 During the ceremony, X is an organic base; Or the following formula: 【Chemistry 6】 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.

14. X is N-methylimidazole, pyridine or 3-methylpyridine; R １ is H, C ｎ H ２ｎ＋１ or a benzyl group, R ２ is H, CH ３ , or C ６ H ５ and n is 1, 2 or 3; The method of claim 13.

15. 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 HOMO energy (a.u.) of the activator is between −0.21407 and −0.16858 in acetonitrile; and The orbital coefficient of the activator is 0.31531 to 0.59405 in acetonitrile; The method.

16. 16. The method of claim 15, wherein the activator has a pKa of 3.65 to 7 in water.

17. 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, 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 (a.u.) of the activator is between −0.22024 and −0.16858 in acetonitrile; and The orbital coefficient of the activator is 0.31531 to 0.59405 in acetonitrile; The method.

18. The method of claim 17, wherein the pKa of the activator is from 3.65 to 7 in water.

19. 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, 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. 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, 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.

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