Nucleic acid synthesis method using segmented amidites

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, so that an oligonucleotide (N−n)mer having a length shorter by 1 or more (n) than the desired length (N) may 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, in which nucleoside phosphoramidites (segmental amidites) having two or three nucleoside moieties are used in the synthesis, but those methods are not sufficiently efficient methods. Furthermore, Patent Document 3 describes saccharin derivatives as activators that can be used more safely than 1H-tetrazole in the synthesis of oligonucleotides. However, Patent Document 3 does not adequately examine the performance of saccharin derivatives as activators. Furthermore, Patent Document 4 describes activators containing at least one pyridinium salt and at least one substituted imidazole, as well as imidazolium salts and benzimidazolium salts, as alternatives to 1H-tetrazole. However, Patent Document 4 does not describe the use of salts with saccharin as activators. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] International Publication No. 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] [Overview of the project] [Problems that the invention aims to solve]

[0005] The present invention aims to provide a method for synthesizing oligonucleotides using segmented amidites. [Means for solving the problem]

[0006] The inventors of this invention, while diligently researching methods for synthesizing oligonucleotides using segmented amidites, discovered a useful activator in this method. Based on this finding, they continued their research and completed the present invention.

[0007] In other words, the present invention relates to the following: [1] A method for producing oligonucleotides, The process includes one or more coupling steps in which a nucleoside phosphoramidite is attached to the 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 (a) Nucleoside phosphoramidites having two or more nucleoside moieties, (b) Nucleoside phosphoramidites having one or more nucleoside moieties and linker moieties And, The activator is given by the following formula: [ka] During the ceremony, X is an organic base. Alternatively, the following formula: [ka] During the ceremony, R 1 and R 2 These are H, and linear or branched C, respectively, independently. 1~7 Selected from the group consisting of alkyl groups and optionally substituted aromatic groups, The method having a structure represented by

[0008] [2]X is N-methylimidazole, pyridine, or 3-methylpyridine, R1 is H, Cn H 2n+1 or 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 of two or more coupling processes, Nucleoside phosphoramidites (a) Nucleoside phosphoramidites having two or more nucleoside moieties, (b) Nucleoside phosphoramidites having one or more nucleoside moieties and linker moieties The method described in any one of the following [1] to [3]. [5] In at least the last of two or more coupling steps, A nucleoside phosphoramidite is a nucleoside phosphoramidite having three nucleoside moieties. The method described in any one of the following [1] to [4]. [6] In at least one of the two or more coupling steps, A nucleoside phosphoramidite is a nucleoside phosphoramidite having one nucleoside moiety. The method described in any one of the following [1] to [5]. [7] In the last of the two or more coupling processes, Nucleoside phosphoramidites (a) Nucleoside phosphoramidite having three nucleoside moieties, or (b) Nucleoside phosphoramidites having one or more nucleoside moieties and linker moieties And, In 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)

Chemical formula

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

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

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

[10] , wherein is -OCH2CH2CN.

[0011]

[12] A nucleoside phosphoramidite having one or more nucleoside moieties and linker moieties is given by the following formula (II) [ka] During the ceremony, X 1 Each is independently -O- or -S-; X 2 Each is independently -O- or -S-; X 3 These are, independently, -O-, -S-, -CH2-, or -(CH2)2-; L is the linker; R 2 These are -H and -NHR, respectively, independently. 6, halogen, -CN, -CF3, or hydroxyl groups protected with acyl protecting groups, ether protecting groups, or silyl protecting groups; R 3 These are, independently, -OCH2CH2CN, -SCH2CH2CN, a substituted or unsubstituted aliphatic group, and -OR 7 or -SR 7 and; R 4 and R 5 Each of these is independently a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, or a substituted or unsubstituted alkalkyl group; or R 4 and R 5 They, together with the nitrogen to which they are bound, form a heterocycloalkyl group or heteroaromatic group; R 6 Each of these is independently -H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted alkalkyl group, or a protecting group; R 7 Each of these is independently a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, or a substituted or unsubstituted alkalkyl group; B 1 and B 2 Each of these is independently -H, or a protected or unprotected base; and n is 0 or a positive integer; The method according to any one of [1] to

[11] , wherein the nucleoside phosphoramidite shown is or a stereoisomer thereof.

[0012]

[13] A method for producing oligonucleotides, The process includes one or more coupling steps in which a nucleoside phosphoramidite is attached to the 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 (a) Nucleoside phosphoramidites having two or three nucleoside moieties, or (b) Nucleoside phosphoramidites having one or more nucleoside moieties and linker moieties And, The activator is given by the following formula: [ka] During the ceremony, X is an organic base. Alternatively, the following formula: [ka] During the ceremony, R 1 and R 2 These are H, and linear or branched C, respectively, independently. 1~7 The method having a structure represented by a group selected from the group consisting of alkyl groups and optionally substituted aromatic groups.

[0013]

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

[13] .

[15] A method for producing oligonucleotides, The process includes one or more coupling steps in which a nucleoside phosphoramidite is attached to the 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 (a) Nucleoside phosphoramidites having two or more nucleoside moieties, (b) Nucleoside phosphoramidites having one or more nucleoside moieties and linker moieties 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 aforementioned 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 oligonucleotides, The process includes one or more coupling steps in which a nucleoside phosphoramidite is attached to the 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 (a) Nucleoside phosphoramidites having two or three nucleoside moieties, or (b) A nucleoside phosphoramidite having one or more nucleoside moieties and linker moieties, 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 aforementioned 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 oligonucleotides, The process includes one or more coupling steps in which a nucleoside phosphoramidite is attached to the 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 (a) Nucleoside phosphoramidites having two or three nucleoside moieties, or (b) A nucleoside phosphoramidite having one or more nucleoside moieties and linker moieties, The pKa of the activator is 3.65 to 7 in water. The aforementioned method.

[20] A method for producing oligonucleotides, The process includes one or more coupling steps in which a nucleoside phosphoramidite is attached to the 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 (a) Nucleoside phosphoramidites having two or more nucleoside moieties, (b) A nucleoside phosphoramidite having one or more nucleoside moieties and linker moieties, The pKa of the activator is 4.3 to 7 in water. The aforementioned method. [Effects 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. Furthermore, by using nucleoside phosphoramidites having two or more nucleoside moieties, i.e., segmented amidites, in the final step of the coupling process, the formation of (N-1)mers, which have similar chromatographic mobility to the desired oligonucleotide and are difficult to remove by purification, can be suppressed. In addition, using the activator of the present invention as the activator also contributes to suppressing the degradation of segmented amidites during the synthesis process. [Modes for carrying out the invention]

[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 the event of any conflict between a publication referenced herein and the description herein, the description herein shall prevail.

[0017] In one aspect, the present invention relates to a method for producing oligonucleotides, comprising one or more coupling steps in which a nucleoside phosphoramidite is attached 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 produced using a so-called phosphoramidite method, in which nucleotides are added by a condensation reaction between a nucleoside phosphoramidite and a nucleoside in solution or on a solid support in the presence of a suitable activator.

[0018] In the present invention, oligonucleotides refer to compounds having a structure in which a base, sugar, and phosphate are linked by a phosphodiester bond, and include naturally occurring oligonucleotides, such as 2'-deoxyribonucleic acid (hereinafter, "DNA") and ribonucleic acid (hereinafter, "RNA"), and nucleic acids containing a modified sugar moiety, a modified phosphate moiety, or a modified nucleobase. Modification of the sugar moiety includes replacing the ribose ring with a hexose, cyclopentyl, or cyclohexyl ring. Alternatively, the D-ribose ring of a naturally occurring nucleic acid may be replaced with an L-ribose ring, or the β-anomer of a naturally occurring nucleic acid may be replaced with an α-anomer. Oligonucleotides may also contain one or more non-basic moieties. Modified phosphate moieties include phosphorothioates, phosphorodithioates, methylphosphonates, and methyl phosphate. Such nucleic acid analogs are known to those skilled in the art. Oligonucleotides comprising two or more of the above mixtures can be produced, for example, from oligonucleotides comprising a mixture of deoxyribo and ribonucleosides, particularly a mixture of deoxyribonucleosides and 2'-O-substituted ribonucleosides such as 2'-O-methyl or 2'-O-methoxyethyl ribonucleosides. Examples of oligonucleotides comprising a mixture of nucleosides include ribozymes.

[0019] In the present invention, nucleoside phosphoramidite (segmented amidite) refers to a nucleoside derivatized with amidite. Amiditation can be carried out, for example, by reacting a properly protected nucleoside with 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphodiamidite 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, or uracil, or a modified base. The sugar portion of the nucleoside may be a naturally occurring deoxyribose or ribose, and may have a D configuration or an L configuration.

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

[0022] In one embodiment of the present invention, the activator is, for example, 5-mercapto-1-methyltetrazole (1-Me-MCT): [ka] 5-Mercapto-1-phenyltetrazole (1-Ph-MCT): [ka] 5-Benzylthiotetrazol (BTT): [ka] Saccharin 1-methylimidazole (SMI): [ka] 5-ethylthio-1H-tetrazole (ETT) [ka] 4,5-dicyanoimidazole (DCI): [ka] Or benzimidazole trifluoromethanesulfonate (BIT): [ka] The material is 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 is -0.22024 to -0.16858 in acetonitrile. For example, the HOMO energy (au) 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 (au), and more preferably -0.18904 to -0.16858 (au). In one embodiment of the present invention, the orbital coefficients of the activator of the present invention are 0.31531 to 0.59405 in acetonitrile. For example, the orbital coefficients of the activator of the present invention are 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 coefficients of the activator are 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 coefficients of the activator are 0.31531 to 0.59405 in acetonitrile. The HOMO energy and orbital coefficients can be determined using quantum chemistry calculation programs. For example, they can be obtained using an optimization calculation employing the Becke-type three-parameter density functional theory (B3LYP) in Gaussian16, a quantum chemistry calculation program from Gaussian Inc.

[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 embodiment of the present invention, in a method for producing the oligonucleotide, 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.

[0027] In one embodiment of the present invention, in a method for producing the oligonucleotide, 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. Nucleoside phosphoramidites having two or three nucleoside moieties are presumed to have a higher reaction rate compared to nucleoside phosphoramidites having four or more nucleoside moieties, due to their smaller molecular size, resulting in higher mobility and faster reaction rates.

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

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

[0031] In one embodiment of the present invention, in a method for producing the oligonucleotide, in the last of two or more coupling steps, 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 International Publication No. 2019 / 212061. In one embodiment of the present invention, a nucleoside phosphoramidite having two or more nucleoside moieties is given by the following formula (I) [ka] It is a nucleoside phosphoramidite or its stereoisomer represented by [the specified formula].

[0033] In equation (I), X 1 Each of these is independently either -O- or -S-. In equation (I), X 2 Each of these is independently either -O- or -S-. In equation (I), X3 These are, independently, -O-, -S-, -CH2-, or -(CH2)2-.

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

[0035] In equation (I), R 4 and R 5 Each of these is independently a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, or a substituted or unsubstituted alkalkyl group; or R 4 and R 5 These groups, together with the nitrogen to which they are bonded, form heterocycloalkyl groups or heteroaromatic groups. Examples of substituted or unsubstituted aliphatic groups include, but are not limited to, methyl, ethyl, and isopropyl, with isopropyl being preferred. Examples of substituted or unsubstituted aromatic groups include, but are not limited to, phenyl, benzyl, naphthyl, and 2-pyrenylmethyl, with phenyl and benzyl being preferred. Examples of substituted or unsubstituted alphaalkyl groups include, but are not limited to, 2-fluorophenylmethoxypiperidine-4-yl. Examples of heterocycloalkyl groups include, but are not limited to, pyrrolidino and morpholino, with morpholino being preferred.

[0036] In equation (I), R 6Each of these is independently one of the following: -H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted alkalkyl group, or a protecting group including an acyl group. Examples of substituted or unsubstituted aliphatic groups, but not limited to these, include methyl, ethyl, allyl, 1-pentenyl, and 2-methoxyethyl, with methyl, allyl, and 2-methoxyethyl being preferred. Examples of substituted or unsubstituted aromatic groups, but not limited to these, include phenyl, benzyl, naphthyl, and 2-pyrenylmethyl, with phenyl and benzyl being preferred. Examples of substituted or unsubstituted alkalkyl groups, but not limited to these, include 2-fluorophenylmethoxypiperidine-4-yl. Examples of protecting groups include t-butyldimethylsilyl, trifluoroacetyl, tert-butoxycarbonyl, benzyloxycarbonyl, phthaloyl, and p-toluenesulfonyl.

[0037] In equation (I), R 7 This is a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, or a substituted or unsubstituted alphaalkyl group. Examples of substituted or unsubstituted aliphatic groups include, but are not limited to, THP and 4-methoxytetrahydropyranyl. Examples of substituted or unsubstituted aromatic groups include, but are not limited to, o-chlorophenyl or p-chlorophenyl. Examples of substituted or unsubstituted alphaalkyl groups include, but are not limited to, 2-fluorophenylmethoxypiperidine-4-yl.

[0038] In equation (I), B 1 B 2 and B 3Each of these is independently H, or a protected or unprotected base. Protected and 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-oxoprine, 6-oxoprine, and 3-deazaadeno Examples of modified bases include syn, 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 equation (I), n is 0 or a positive integer, preferably an integer between 0 and 4, and more preferably 0 or 1.

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

[0040] In one embodiment of the present invention, nucleoside phosphoramidites having one or more nucleoside moieties and linker moieties may be prepared from, but are not limited to, phosphoramidites having linker moieties such as those shown below, which are 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]azacycloocta-4-in-1-yl)-caproamide-N-ethyl)-O-triethylene glycol-1-[(2-cyanoethyl)-(N,N-di- (Sopropyl)-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-trifluoroacetylamidecaproamidemethyl)-1-(2-nitrophenyl)-ethyl]-2-cyanoethyl-(N,N-diisopropyl)-phosphoramidite) [ka]

[0051] Thiol-Modifier C6 SS (1-O-dimethoxytritylhexyl disulfide, 1'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite) [ka]

[0052] 5'-Carboxy-Modifier C10(10-Carboxydecyl-(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-Lipoamide)propanamide)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]deca-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))-biotinamidecaproamidemethyl)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-propylamide-glycanoylamide)propyl-1-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite) [ka]

[0058] 6-Fluorescein Serinol Phosphoramidite(3-Dimethoxytrityloxy-2-(3-(6-Carboxy-(di-O-pivaloylfluorescein)propanamide)propyl)-1-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite) [ka]

[0059] Protected Biotin Serinol Phosphoramidite (3-Dimethoxytrityloxy-2-(3-((4-t-butylbenzoyl)-biotinyl)propanamide)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-cyanovinylcarbazole-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 linker moieties is given by the following formula (II) [ka] It is a nucleoside phosphoramidite or its stereoisomer represented by [the specified formula].

[0067] In equation (II), X 1 Each of these is independently either -O- or -S-. In equation (II), X 2 Each of these is independently either -O- or -S-. In equation (II), X 3 These are, independently, -O-, -S-, -CH2-, or -(CH2)2-. In equation (II), L is a linker. While not limited to these, a linker can be any other linker, for example, [ka]

[0068] [ka]

[0069] [ka]

[0070] [ka]

[0071] [ka] These are some examples.

[0072] In equation (II), R 2 These are -H and -NHR, respectively, independently. 6The protecting group is one of the following: a halogen, -CN, -CF3, or a hydroxyl group protected by an acyl protecting group, an ether protecting group, or a silyl protecting group. Examples of halogens include F, Cl, Br, and I. Examples of acyl protecting groups include acetyl, benzoyl, and pivaloyl. Examples of ether protecting groups include benzyl, p-methoxybenzyl (PMB), and allyl. Examples of silyl protecting groups include t-butyldimethylsilyl (TBS), t-butyldiphenylsilyl (TBDPS), t-triisopropylsilyl (TIPS), triethylsilyl (TES), and trimethylsilyl (TMS). Preferably, it is -H. In equation (II), R 3 These are, independently, -OCH2CH2CN, -SCH2CH2CN, a substituted or unsubstituted aliphatic group, and -OR 7 or -SR 7 The aliphatic group is preferably -OCH2CH2CN. Examples of substituted or unsubstituted aliphatic groups include, but are not limited to, 4-cyanobuto-2-enylthio, 4-cyanobuto-2-enyloxy, allylthio, allyloxy, clotylthio, or clotyloxy.

[0073] In equation (II), R 4 and R 5 Each of these is independently a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, or a substituted or unsubstituted alkalkyl group; or R 4 and R 5 These groups, together with the nitrogen to which they are bonded, form heterocycloalkyl groups or heteroaromatic groups. Examples of substituted or unsubstituted aliphatic groups include, but are not limited to, methyl, ethyl, and isopropyl, with isopropyl being preferred. Examples of substituted or unsubstituted aromatic groups include, but are not limited to, phenyl, benzyl, toluyl, and anilyl, with phenyl and benzyl being preferred. Examples of heterocycloalkyl groups include, but are not limited to, pyrrolidino and morpholino.

[0074] In formula (II), R 6 is, independently of each other, 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 preferably methyl, allyl, 2-methoxyethyl. Examples of the substituted or unsubstituted aromatic group include, but are not limited to, phenyl, benzyl, naphthyl, 2-pyrenylmethyl, and preferably phenyl, 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. 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 2Each of these is independently H, or a protected or unprotected base. Protected and 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-oxoprine, 6-oxoprine, and 3-deazaadeno Examples of modified bases include syn, 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, B 1 and B 2 At least one of them is adenine. In equation (II), n is 0 or a positive integer, preferably an integer between 0 and 4, and more preferably 0 or 1. [Examples]

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

[0078] Example 1: Comparison of Sequential Method and Segmentation Method (1) Synthesis of oligonucleotides dT NittoPhase®HL dT300 (manufactured by Nitto Denko Corporation) was packed into a reaction column in an amount equivalent to 1573 μmol, and 17mer (5' CCG ATT AAG CGA AGC TT 3') DNA oligonucleotides were synthesized using a nucleic acid synthesizer AKTA oligopilot plus100 (manufactured by Cytiva, formerly GE Healthcare Japan). Subsequently, 17mer DNA-attached solid support was packed into the reaction column in an amount equivalent to 205 μmol, and synthesis was performed using the nucleic acid synthesizer AKTA oligopilot plus100 under three conditions: a sequential method in which dC, dT, and dA were condensed in order, a segmentation method 1 in which dATC was condensed with an amidite equivalent of 1.8 equivalents and a condensation time of 5 minutes, and a segmentation method 2 in which dATC was condensed with an amidite equivalent of 3.0 equivalents and a condensation time of 10 minutes. Other synthetic reagents used were commonly employed deprotection reagents, capping reagents, and oxidation solutions. dATC was prepared as described in International Publication No. 2019 / 212061. A solid support to which DNA oligonucleotides were bound was immersed in 28% aqueous ammonia, and the DNA oligonucleotides were excised from the solid support. A portion 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 an AKTA pure 25 purification system (Cytiva, formerly GE Healthcare Japan). (Purification conditions: Column; 25cm × 10mm CV = 19.6mL GE source 15Q resin, Buffer; prepared with NaOH, NaCl, and ultrapure water). (3) Analysis of DNA oligonucleotides 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 Table 1 shows the results of the analysis of the DNA oligonucleotide sample solution before purification. [Table 1] Here, impurity I refers to an impurity that appears near the N-1 mer in HPLC measurement, impurity II refers to an impurity that appears near the N-2 mer in HPLC measurement, and impurity III refers to an impurity that appears near the N-3 mer in HPLC measurement; the same applies hereafter.

[0081] When synthesized using the sequential method, the content of impurity I and impurity II was 2.9% and 2.8%, respectively. In contrast, when synthesized using segmentation method 1, the content of impurity I and impurity II was 0.6% and 0.4%, respectively, and when synthesized using segmentation method 2, the content was 0.7% and 1.8%, respectively. The content of impurity I and impurity II could be significantly reduced when synthesized using the segmentation method compared to when synthesized using the sequential method.

[0082] Table 2 shows the analysis results of the purified DNA oligonucleotide sample solution. [Table 2]

[0083] The purity of the target oligonucleotide was 90.46% using the sequential method, compared to 92.34% using segmentation method 1 and 92.53% using segmentation method 2. This indicates that the segmentation method yielded higher purity of the target oligonucleotide compared to the sequential method. Furthermore, the FLP (full-length product, purity × total OD) was 9302 using the sequential method, compared to 10702 using segmentation method 1 and 10724 using segmentation method 2. This suggests that the segmentation method yielded higher yields of the target oligonucleotide compared to the sequential method.

[0084] Example 2: Comparison of response rates (1) Synthesis of DNA oligonucleotides NittoPhase (trademark registered) HL UnyLinker 350 (manufactured by Nitto Denko Corporation) was packed into a reaction column in an amount equivalent to 2935 μmol, and 17mer (5' CCG ATT AAG CGA AGC TT 3') DNA oligonucleotides were synthesized using the AKTA oligopilot plus100 nucleic acid synthesizer. Subsequently, a solid support with 17mer DNA was packed into the reaction column in an amount equivalent to 90 μmol, and dGCC was condensed using an AKTA oligopilot plus10 nucleic acid synthesizer with 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 (Activator 42), or benzimidazole trifluoromethanesulfonate (BIT) as the activator, at a volume of 1.8 amidite equivalents and a condensation time of 5 minutes. All activators were dissolved in acetonitrile and prepared to a 0.25 M solution. Other synthetic reagents used included 3% DCA in toluene as a deprotecting agent, pyridine in water and iodine as oxidizing agents, pyridine in acetonitrile, N-methylimidazole, acetic anhydride, or isobutyric anhydride as capping agents, and TBA in acetonitrile as the amine wash reaction solution. dGCC was prepared as described in International Publication No. 2019 / 212061. A solid support to which DNA oligonucleotides were bound was immersed in ammonia water, and the DNA oligonucleotides were excised from the solid support. The Activator42 has the following structure: [ka] It has.

[0085] (2) Synthesis of RNA oligonucleotides A reaction column was packed with 327 μmol of NittoPhase (trademark) HL rU250 (manufactured by Kinovate Life Science), and 17mer RNA oligonucleotides (5' CCG AUU AAG CGA AGC UU 3') were synthesized using an AKTA oligopilot plus100 nucleic acid synthesizer. Subsequently, 85 μmol of 17mer RNA-attached solid support was packed into the reaction column, and rAUC was condensed using an AKTA oligopilot plus10 nucleic acid synthesizer with 5-mercapto-1-methyltetrazole (1-Me-MCT), saccharin 1-methylimidazole (SMI), or 5-[3,5-bis(trifluoromethyl)phenyl]-1H-tetrazole (Activator 42) as the activator, at a concentration of 2.0 amidite equivalents and a condensation time of 15 minutes. All activators were dissolved in acetonitrile and prepared to 0.25 M. Other synthetic reagents used included 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 the amine wash reaction solution. The rAUC was prepared as described in International Publication No. 2019 / 212061. A solid support to which RNA oligonucleotides were bound was immersed in AMA reagent (28-30% aqueous ammonia:methylamine aqueous solution = 1:1), filtered, and washed with DMSO. Then, TEA.3HF was slowly added dropwise under ice bath and shaken to excavate the RNA oligonucleotides from the solid support.

[0086] (3) Analysis of response rates Each oligonucleotide sample solution after cleavage 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 peak areas detected up to 1.1 minutes after the observation of the peak of impurity III 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 peak areas detected up to 1.4 minutes after the observation of the peak of impurity III 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. Tables 3 and 4 also show the HOMO energy and orbital coefficients of the activator used in this experiment. The HOMO energy and orbital coefficients were determined by optimization calculations using the Becke-type 3-parameter density functional theory (B3LYP) with Gaussian16, a quantum chemistry calculation program from Gaussian. Specifically, the structure of the active species (BIT is a neutral species, 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 computational support program WebMO. The initial structure of the molecule before calculation was created, and the basis function was 6-31G(d), the charge was -1 for anions and 0 for neutral species, the multiplicity was singlet, and the solvent was acetonitrile. The HOMO energy and orbital coefficients were determined by performing structural optimization followed by orbital calculations. Simple structural modifications of the initial structure to avoid interatomic collisions in the molecular model were performed as appropriate by executing the Mechanics Optimize function of the Cleanup function in WebMO.

[0088] [Table 3] [Table 4]

[0089] Tables 3 and 4 show that activators that showed a higher reaction rate than Activator 42 had a higher HOMO energy level and / or larger orbital coefficients. It is thought that the higher HOMO energy level of the activator reduced the energy difference between the HOMO energy level of the activator and the LUMO energy level of the segmented amidite, making the reaction more likely to occur. In addition, it is thought that the larger orbital coefficients of the activator increased the orbital overlap between the nitrogen atom of the activator and the phosphorus atom of the segmented amidite, making the reaction more likely to occur.

[0090] Example 3: Comparison of decomposition of segmented amidite (1) Synthesis of oligonucleotides using dGCC as segmented amidite The 17mer DNA-attached solid support obtained in Example 2(1) was packed into a reaction column in an amount equivalent to 90 μmol, and dGCC was condensed using an AKTA oligopilot plus10 nucleic acid synthesizer with 0.6M 5-mercapto-1-methyltetrazole (1-Me-MCT), 0.5M 5-mercapto-1-phenyltetrazole (1-Ph-MCT), 0.25M 5-benzylthiotetrazole (BTT), 0.25M saccharin 1-methylimidazole (SMI), 0.6M 5-ethylthio-1H-tetrazole (ETT), or 0.25M 5-[3,5-bis(trifluoromethyl)phenyl]-1H-tetrazole (Activator 42) as activators, at a volume of 1.8 amidite equivalents and a condensation time of 10 minutes. Other synthetic reagents used included 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 the amine wash reaction solution. dGCC was prepared as described in International Publication No. 2019 / 212061. A solid support to which DNA oligonucleotides were bound was immersed in ammonia water at 55°C for 12 to 16 hours, and the DNA oligonucleotides were excised from the solid support.

[0091] (2) Analysis of DNA oligonucleotides The DNA oligonucleotide sample solution after excision 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). After the peak of impurity III was observed in the HPLC measurement results, the peak area (%) of impurity II was calculated, with the sum of the peak areas detected up to 1.1 minutes later being taken as 100%.

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

Claims

1. A method for producing oligonucleotides, The process includes one or more coupling steps in which a nucleoside phosphoramidite is attached to the 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 (a) Nucleoside phosphoramidites having two or more nucleoside moieties, (b) Nucleoside phosphoramidites having one or more nucleoside moieties and linker moieties And in at least the last of the two or more coupling steps, the nucleoside phosphoramidite is a nucleoside phosphoramidite having three nucleoside moieties, The activator is given by the following formula: 【Chemistry 1】 During the ceremony, R1 and R2 are, independently, H, linear or branched C. １～７ Selected from the group consisting of alkyl groups and optionally substituted aromatic groups, The method having a structure represented by

2. R １ H, C ｎ H ２ｎ＋１ or benzyl group, R ２ H, CH ３ , or C ６ H ５ And, n is 1, 2, or 3. The method according to claim 1.

3. The method according to claim 1 or 2, wherein the activator is 5-mercapto-1-methyltetrazole (1-Me-MCT), 5-mercapto-1-phenyltetrazole (1-Ph-MCT), or 5-ethylthio-1H-tetrazole (ETT).

4. In at least one of the coupling processes that are performed two or more times, A nucleoside phosphoramidite is a nucleoside phosphoramidite having one nucleoside moiety. The method according to any one of claims 1 to 3.

5. In the last of the two or more coupling processes, A nucleoside phosphoramidite is a nucleoside phosphoramidite having three nucleoside moieties. In other coupling processes, A nucleoside phosphoramidite is a nucleoside phosphoramidite having one nucleoside moiety. The method according to any one of claims 1 to 4.

6. A nucleoside phosphoramidite having two or more nucleoside moieties is given by the following formula (I) 【Chemistry 2】 During the ceremony, X １ is, independently of each other, -O- or -S-; X ２ Each is independently -O- or -S-; X ３ These are, independently, -O-, -S-, and -CH. ２ - or - (CH ２ ) ２ - and; R １ is a protecting group; R ２ These are, independently, -H and -NHR ６ , halogen, -CN, -CF ３ , or a hydroxyl group protected with an acyl protecting group, an ether protecting group, or a silyl protecting group; R ３ Each of them is independent of -OCH ２ CH ２ CN, -SCH ２ CH ２ CN, substituted or unsubstituted aliphatic group, -OR ７ or -SR ７ And; R ４ and R ５ Each of these is independently a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, or a substituted or unsubstituted alkalkyl group; or R ４ and R ５ These, together with the nitrogen to which they are bound, form a heterocycloalkyl group or a heteroaromatic group; R ６ Each of these is independently -H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted alkalkyl group, or a protecting group; R ７ Each of these is independently a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, or a substituted or unsubstituted alkalkyl group; B １ , B ２ and B ３ Each of these is 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 5, wherein the nucleoside phosphoramidite represented by or a stereoisomer thereof.

7. The method according to claim 6, wherein n is 0 or 1.

8. R ２ The method according to claim 6 or 7, wherein is -H.

9. R ３ ga-OCH ２ CH ２ The method according to any one of claims 6 to 8, wherein the material is CN.

10. A nucleoside phosphoramidite having one or more nucleoside moieties and linker moieties is given by the following formula (II) 【Transformation 3】 During the ceremony, X １ Each is independently -O- or -S-; X ２ Each is independently -O- or -S-; X ３ These are, independently, -O-, -S-, and -CH. ２ - or - (CH ２ ) ２ - and; L is the linker; R ２ These are, independently, -H and -NHR ６ , halogen, -CN, -CF ３ , or a hydroxyl group protected with an acyl protecting group, an ether protecting group, or a silyl protecting group; R ３ Each of them is independent of -OCH ２ CH ２ CN, -SCH ２ CH ２ CN, substituted or unsubstituted aliphatic group, -OR ７ or -SR ７ And; R ４ and R ５ Each of these is independently a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, or a substituted or unsubstituted alkalkyl group; or R ４ and R ５ They, together with the nitrogen to which they are bound, form a heterocycloalkyl group or a heteroaromatic group; R ６ Each of these is independently -H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted alkalkyl group, or a protecting group; R ７ Each of these is independently a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, or a substituted or unsubstituted alkalkyl group; B １ and B ２ Each of these is 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 9, wherein the nucleoside phosphoramidite is represented by or a stereoisomer thereof.

11. A method for producing oligonucleotides, The process includes one or more coupling steps in which a nucleoside phosphoramidite is attached to the 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 (a) Nucleoside phosphoramidites having two or three nucleoside moieties, (b) Nucleoside phosphoramidites having one or more nucleoside moieties and linker moieties The coupling process is performed two or more times, and in at least the last of the two or more times the nucleoside phosphoramidite is a nucleoside phosphoramidite having three nucleoside moieties, and the activator is of the following formula: 【Chemistry 4】 During the ceremony, R1 and R2 are, independently, H, linear or branched C. １～7 The method having a structure represented by a group selected from the group consisting of alkyl groups and optionally substituted aromatic groups.

12. R １ H, C ｎ H ２ｎ＋１ or benzyl group, R ２ H, CH ３ , or C ６ H ５ And, n is 1, 2, or 3. The method according to claim 11.

Images