Segment for oligonucleotide synthesis, method for producing same, and method for synthesizing oligonucleotide using same

Abstract

The present invention provides a segment for oligonucleotide synthesis that can be more easily purified and supplied in large quantities, a method for producing the same, and a method for synthesizing an oligonucleotide. A segment for oligonucleotide synthesis is synthesized via a phosphoramidite (1) using an amiditizing reagent. TIFF2022065127000032.tif69133

Description

[Technical Field]

[0001] The present invention relates to a segment for oligonucleotide synthesis, a method for producing the same, and a method for synthesizing an oligonucleotide using the same. [Background technology]

[0002] In recent years, attention has been focused on nucleic acid drugs based on natural or unnatural oligonucleotides. To obtain nucleic acid drugs designed to achieve the desired effect, chemical synthesis methods are used.

[0003] Conventional oligonucleotide synthesis primarily involves using monomer amidites as starting materials and carrying out stepwise coupling reactions one base at a time to extend the length of the nucleotide (see Non-Patent Document 1). In oligonucleotide synthesis, extending one base at a time requires not only a coupling reaction in which the monomer amidite reacts with the 5'-hydroxyl group of the nucleoside, but also a step of oxidizing or sulfurizing the trivalent phosphorus and a step of deprotecting the protecting group of the 5'-hydroxyl group of the nucleoside in preparation for the next coupling reaction. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Caruthers et al., Bioactive Molecules, 3, pp. 3-21 (1987) Summary of the Invention [Problem to be solved by the invention]

[0005] However, when using raw materials used in conventional methods, the yield of each coupling reaction in the above oligonucleotide synthesis does not necessarily reach 100%, making it difficult to synthesize oligonucleotides of a certain length with high efficiency.

[0006] The oxidation / sulfurization and deprotection steps that follow the coupling reaction also do not always proceed 100%, so the yield of oligonucleotides of the desired length decreases as the length of the desired oligonucleotide increases.

[0007] Furthermore, in the final stage of obtaining an oligonucleotide having the desired length, a purification step is required to remove by-products and reagent residues generated in each of the above steps. Typical by-products generated in the synthesis of oligonucleotide N-mers in a synthesis method in which extension is performed base by base include (N-1)-mers, which are one base shorter, and (N-2)-mers, which are two bases shorter, generated in the coupling step. These (N-1)-mers and (N-2)-mers have very similar structures and physical properties to the target N-mer. Therefore, in the stage of purifying the N-mer using chromatography or the like, the difference in mobility between the target N-mer and the by-products, such as (N-1)-mers and (N-2)-mers, is small. This poses a problem: the purification burden required to accurately separate the N-mer from other N-mers is heavy.

[0008] In addition, the commonly used method of oligonucleotide synthesis is solid-phase synthesis, in which nucleosides with their 3'-terminus anchored to a solid support are subjected to chain elongation in the 5'-direction. This method requires the experimenter to either purchase commercially available monomer amidites for solid-phase synthesis or prepare them themselves. Commercially available monomer amidites are generally expensive, posing a barrier to large-scale synthesis of the desired oligonucleotides. Furthermore, if the experimenter prepares the monomer amidites themselves, they must be prepared with high purity and high yield. This is because if the monomer nucleotides are not highly pure, they are likely to affect the coupling reaction during oligonucleotide synthesis.

[0009] The present invention has been made in view of the above circumstances, and aims to provide a segment for oligonucleotide synthesis that can be more easily purified and supplied in large quantities, a method for producing the same, and a method for synthesizing an oligonucleotide using the same. [Means for solving the problem]

[0010] In order to solve the above problems, the segment for oligonucleotide synthesis and the method for producing the same of the present invention, as well as the method for synthesizing an oligonucleotide using the same, employ the following means. A first aspect of the present invention is a compound represented by the following formula (I): [ka] It is a segment for oligonucleotide synthesis represented by the formula:

[0011] In the formula (I), B is independently a protected or unprotected nucleoside base; R 1 is a protecting group; R 2 , R 3 , R 4 is OCH2CH=CH2; R 5 is a substituted or unsubstituted aliphatic group or a substituted or unsubstituted aromatic group; X is independently =O or =S; Y is independently H, NHR 6 , halogen, CN, CF3, or a hydroxyl group protected by an acyl-based protecting group, an ether-based protecting group, or a silyl-based protecting group; R 6 is H, an aliphatic group, or an aromatic group; Z is independently H, an alkyl, an O-alkyl, an N-alkyl, or a halogen; can be The value of (m+n) is an integer between 3 and 23 inclusive.

[0012] In the first embodiment, when B in the formula (I) is a nucleoside base protected by a protecting group, the protecting group may be an acyl-based protecting group.

[0013] In the first aspect, in the formula (I), the R 1is a protecting group removable under acidic conditions or a trialkylsilyl group, the Y is preferably H or a hydroxyl group protected with a t-butyldimethylsilyl group, the Z is preferably H, and the R 5 may be an isopropyl group.

[0014] The present invention reference The embodiment is represented by the following formula (I): [ka] The present invention relates to a method for producing a segment for oligonucleotide synthesis represented by the formula:

[0015] In the formula (I), B is independently a protected or unprotected nucleoside base; R 1 is a protecting group; R 2 , R 3 , R 4 is OCH2CH=CH2; R 5 is a substituted or unsubstituted aliphatic group or a substituted or unsubstituted aromatic group; X is independently =O or =S; Y is independently H, NHR 6 , halogen, CN, CF3, or a hydroxyl group protected by an acyl-based protecting group, an ether-based protecting group, or a silyl-based protecting group; R 6 is H, an aliphatic group, or an aromatic group; Z is independently H, an alkyl, an O-alkyl, an N-alkyl, or a halogen. the law of nature The value of (m+n) is an integer between 3 and 23 inclusive.

[0016] The manufacturing method includes: (a) Formula (II): [ka] A nucleoside represented by R 2 P{N(R 5 )2}2 structure, and a trivalent phosphorus compound having the following formula (III): [ka] to react with a nucleoside having the structure of the following formula (IV): [ka] preparing a compound having the structure (b) reacting a compound having the structure of formula (IV) with R 3 P{N(R 5 a trivalent phosphorus compound having the structure of formula (III)2, and a nucleoside having the structure of formula (III) above, or a nucleoside having the structure of formula (V): [ka] or a compound having the following formula (VI): [ka] and reacting the compound with a compound having the structure of the following formula (VII): [ka] preparing a compound having the structure (c) optionally repeating step (b) at least once; (d) The obtained intermediate is reacted with R 4 P{N(R 5 )2}2 with a trivalent phosphorus compound having the structure of formula (I), to prepare a segment having the structure of formula (I), (e) If necessary, following the step (a), a step of reacting the compound represented by formula (IV) obtained in the step (a), or following the step (b), a step of reacting the compound represented by formula (VII) obtained in the step (b), with an oxidizing agent or a sulfurizing agent.

[0017] In the second embodiment, when B in the formula (I) is a nucleoside protected by a protecting group, the protecting group may be an acyl-based protecting group.

[0018] In the second embodiment, in the formula (I), the R 1is a protecting group removable under acidic conditions or a trialkylsilyl group, the Y is preferably H or a hydroxyl group protected with a t-butyldimethylsilyl group, the Z is preferably H, and the R 5 may be an isopropyl group.

[0019] The second embodiment may, if necessary, include a step of reacting the compound represented by formula (IV) obtained in step (a) or the compound represented by formula (VII) obtained in step (b) with an oxidizing agent or a sulfurizing agent.

[0020] A third aspect of the present invention is a method for synthesizing an oligonucleotide using the segment for oligonucleotide synthesis represented by the above formula (I).

[0021] The synthesis method includes: (a) a condensation step of the amidite portion of the oligonucleotide synthesis segment represented by formula (I) with a hydroxyl group of a nucleoside or nucleotide; (b) an oxidation step of the phosphorous bond portion obtained in the condensation step; and (c) a deprotection step of the terminal protecting group of the oligonucleotide synthesis segment condensed with the nucleoside or nucleotide in the condensation step.

[0022] In the third embodiment, each of the steps may be carried out in a solution.

[0023] In the third embodiment, each of the steps may be carried out on a solid phase carrier. [Effects of the Invention]

[0024] The oligonucleotide synthesis segment of the present invention can reduce the number of steps compared to the stepwise synthesis of oligonucleotides using commonly used monomer amidites, thereby improving the yield of oligonucleotides of the desired length compared to the yield obtained by conventional methods.

[0025] Furthermore, when an N-mer oligonucleotide is synthesized using the oligonucleotide synthesis segment of the present invention, by-products having lengths of N-1 to N-3 are not generated, which reduces the burden of purifying the target N-mer oligonucleotide, resulting in easier purification and the ability to supply the target product in larger quantities. [Brief explanation of the drawings]

[0026] [Figure 1] FIG. 1 shows an ESI-MS spectrum of the dT trimer amidite, which is a segment for oligonucleotide synthesis obtained in Example 1. [Figure 2] FIG. 1 shows the 31P NMR spectrum of the dT trimer amidite, which is a segment for oligonucleotide synthesis obtained in Example 1. [Figure 3] FIG. 1 shows the LC spectrum of an oligothymidine 18-mer synthesized using the oligonucleotide synthesis segment obtained in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0027] An embodiment for obtaining a segment for oligonucleotide synthesis according to the present invention will be described below. The oligonucleotide synthesis segment in this embodiment is represented by the following formula (I): [ka] It has a structure represented by the following formula: In the formula (I), B is independently a protected or unprotected nucleoside base; R 1 is a protecting group; R 2 , R 3 , R 4 are independently OCH2CH2CN, SCH2CH2CN, OCH2CH=CH2, OCH3; R 5is a substituted or unsubstituted aliphatic group or a substituted or unsubstituted aromatic group; X is independently an unshared electron pair, ═O, or ═S; Y is independently H, NHR 6 , halogen, CN, CF3, or a hydroxyl group protected by an acyl-based protecting group, an ether-based protecting group, or a silyl-based protecting group; R 6 is H, an aliphatic group, or an aromatic group; Z is independently H, an alkyl, an O-alkyl, an N-alkyl, or a halogen; can be The value of (m+n) is an integer between 3 and 23 inclusive.

[0028] In this embodiment, the oligonucleotide synthesis segment is obtained by (1) starting with a nucleoside in which the 5'-hydroxyl group and, if necessary, the nucleoside base moiety are protected with protecting groups, but the 3'-hydroxyl group is unprotected (hereinafter referred to as a "5'-protected-3'-unprotected nucleoside"), phosphorylating the 3'-hydroxyl group to obtain a 3'-phosphoramidite as an intermediate in the reaction system, (2) reacting the resulting intermediate with the 5'-hydroxyl group of a nucleoside in which the nucleoside base moiety is protected with protecting groups, if necessary, but both the 3'-hydroxyl and 5'-hydroxyl groups are unprotected (hereinafter referred to as a "3',5'-unprotected nucleoside"), and reacting the remaining unprotected 3'-hydroxyl group at the 3'-terminus with a trivalent phosphorylating agent to form a phosphoramidite. Repeating (2) the required number of times (n times) synthesizes an (n+1)-mer nucleotide phosphoramidite.

[0029] In contrast, when synthesizing nucleotide dimers or trimers using conventional methods, it was necessary to prepare a nucleoside in advance by protecting the 3'-hydroxyl group of a 5'-protected, 3'-unprotected nucleoside, followed by a reaction to remove only the 5'-hydroxyl protecting group, thereby protecting the 3'-hydroxyl group and, if necessary, the nucleoside base moiety with a protecting group, resulting in an unprotected 5'-hydroxyl group (hereinafter referred to as a "5'-unprotected, 3'-protected nucleoside"). In contrast, in the present embodiment, a nucleoside in which both the 5'-hydroxyl and 3'-hydroxyl groups are unprotected, which requires one fewer protecting group than conventional methods, can be used for segment synthesis. Therefore, 3',5'-unprotected nucleosides, which are one of the main raw materials for segment synthesis, can be prepared in fewer steps than conventional methods, allowing for larger-scale synthesis at lower cost and in a shorter time.

[0030] Nucleoside bases in this embodiment include natural bases such as adenyl, guanyl, cytosinyl, thyminyl, and uracil, as well as modified bases such as 5-methylcytosinyl, 5-fluorouracil, 7-methylguanyl, and 7-deazaadenyl. The amino groups in these nucleoside bases include benzyl-, allyl-, carbamate-, and acyl-protecting groups. Preferably, acyl-protecting groups such as acetyl, benzoyl, phenoxyacetyl, and isopropylcarbonyl are used.

[0031] The aliphatic group in this embodiment is a saturated or unsaturated, linear or branched C-C 18 Hydrocarbons, saturated or unsaturated, cyclic C3-C 18 The aliphatic and aromatic groups include hydrocarbons. Preferably, they are saturated or unsaturated C1-C8 hydrocarbons or C3-C8 cyclic hydrocarbons. In this embodiment, the aromatic group includes a carbocyclic aromatic ring such as a phenyl group, a carbocyclic aromatic ring such as a naphthyl group, or a carbocyclic aromatic ring fused to a non-carbon aromatic ring. In this embodiment, the aliphatic and aromatic groups may be substituted with saturated or unsaturated C1-C8 hydrocarbons or C3-C8 cyclic hydrocarbons, halogen, cyano, nitro, aromatic rings, or other substituents.

[0032] In this embodiment, the protecting group for the 5'-, 3'-, or 2'-hydroxyl group includes a protecting group removable under acidic conditions, an acyl protecting group, and a silyl protecting group. Protecting groups removable under acidic conditions include ether protecting groups including a substituted or unsubstituted trityl group, a pixyl group, and a substituted or unsubstituted tetrahydropyranyl (THP) group, with a representative protecting group being the 4,4'-dimethoxytrityl group. Silyl protecting groups include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, and triphenylsilyl. Acyl protecting groups include an acetyl group and a benzoyl group. Alternatively, a nucleoside bonded so as to bridge the 4'- and 2'-positions can also be used as a starting material. In this case, the 4'-position and the 2'-position can form a bond (4'-position)-LO-(2'-position), and an example of L is a C1-C6 alkylene group (wherein a carbon atom in the middle may be substituted with an oxygen atom or a nitrogen atom bonded to an alkyl group).

[0033] In step (1) above, the 3'-hydroxyl group of a 5'-protected or 3'-unprotected nucleoside is reacted with a trivalent phosphorylating agent to generate a 3'-phosphoramidite in situ. The resulting 3'-phosphoramidite is then added, without isolation or purification, to an activating agent to activate the amidite moiety, followed by reaction with a 3',5'-unprotected nucleoside to extend the nucleotide sequence by one base. This is then further reacted with a trivalent phosphorylating agent to generate a 3'-phosphoramidite. Representative trivalent phosphorylating agents include, but are not limited to, NCCH2CH2OP[N(i-C3H7)2]2 and CH2=CHCH2OP[N(i-C3H7)2]2. Representative activating agents include, but are not limited to, 1H-tetrazole, S-ethylthio-1H-tetrazole, dicyanoimidazole, and salts of sulfonic acids with azoles or tertiary amines. The reaction is carried out in a dry solvent such as dichloromethane, acetonitrile, tetrahydrofuran, DMF, or toluene.

[0034] In step (1), a trivalent phosphorylating agent (1.05-1.2 equivalents of 5'-protected and 3'-unprotected nucleoside) and an activating agent (0.4-0.7 equivalents of 5'-protected and 3'-unprotected nucleoside) are added to a solution (0.2-0.4 M) of 5'-protected and 3'-unprotected nucleoside, and the mixture is stirred at room temperature for 2-5 hours. The resulting 3'-phosphoramidite is purified on silica gel and then subjected to the subsequent step (2).

[0035] In step (2), the 3'-phosphoramidite obtained in step (1) is added to a 3',5'-unprotected nucleoside (1.3-2.0 equivalents of 5'-protected and 3'-unprotected nucleoside) and an activating agent (2-3 equivalents of 5'-protected and 3'-unprotected nucleoside). The reaction is allowed to proceed at room temperature for 0.5-1.5 hours. After silica gel filtration and concentration of the filtrate, a nucleotide intermediate with an unprotected hydroxyl group at the 3'-terminus is obtained. The yield is approximately 60-95%. A solution of this nucleotide intermediate (0.05-0.4 M) is then reacted with a trivalent phosphorylating agent (1.2-2.0 equivalents of 5'-protected and 3'-unprotected nucleoside) and an activating agent (0.5-1.0 equivalent of 5'-protected and 3'-unprotected nucleotide) to obtain the trimer nucleotide 3'-phosphoramidite. If necessary, step (2) can be further carried out to obtain a tetramer or larger of nucleotides. When synthesizing a tetramer or larger of a segment, as an alternative to the method of extending the segment one base at a time, segments that are already dimers or larger can be condensed together to extend the segment by two or more bases at a time.

[0036] Synthesis of oligonucleotides using the oligonucleotide synthesis segment represented by formula (I) can be performed in solution (hereinafter referred to as "liquid phase synthesis") or on a solid support (hereinafter referred to as "solid phase synthesis"). When synthesis is performed by liquid phase synthesis, a silyl-based protecting group or an aliphatic-containing protecting group is introduced into the 3'-hydroxyl group of the 3'-terminal nucleoside to increase solubility in the reaction solvent, and the resulting nucleoside is subjected to the following repeated steps: (a) condensation with the oligonucleotide synthesis segment represented by formula (I), (b) oxidation or sulfurization of the phosphorous bond obtained in the condensation step, and (c) deprotection of the terminal protecting group of the oligonucleotide synthesis segment condensed with the nucleoside or nucleotide in the condensation step. When synthesis is performed by solid-phase synthesis, the following steps are repeated: (a) condensation with the oligonucleotide synthesis segment represented by formula (I), (b) capping of the unreacted 5'-hydroxyl group of the nucleoside or nucleotide on the solid support, (c) oxidation of the phosphite bond obtained in the condensation step, and (d) deprotection of the terminal protecting group of the oligonucleotide synthesis segment condensed with the nucleoside or nucleotide in the condensation step. In both methods, the desired oligonucleotide can be obtained by subsequent treatment under basic conditions.

[0037] Specifically, whether using liquid-phase or solid-phase synthesis, the first step of oligonucleotide synthesis involves activating the 3'-terminal amidite of the oligonucleotide synthesis segment represented by formula (I) with an activator and condensing it with a nucleoside or nucleotide with an unprotected 5'-hydroxyl group. Examples of activators that can be used include commonly used activators for activating trivalent phosphorus, such as 1H-tetrazole, S-ethylthio-1H-tetrazole, dicyanoimidazole, and salts of sulfonic acid with azoles or tertiary amines, but are not limited to these. The time required for this condensation reaction is generally about 1 to 30 minutes, depending on the scale of the reaction.

[0038] Next, in the second step of oligonucleotide synthesis, the intermediate obtained in the above condensation reaction is reacted with an oxidizing or sulfurizing agent to give a pentavalent phosphate or thiophosphate nucleotide.

[0039] Next, in the third step of oligonucleotide synthesis, the intermediate obtained in the oxidation or sulfurization reaction is reacted with an anhydrous acidic solution to obtain a nucleotide with an unprotected 5'-hydroxyl group.

[0040] In the oligonucleotide synthesized using the segment for oligonucleotide synthesis of this embodiment, the protecting groups of the nucleoside bases, the protecting groups of the 5'-, 3'-, or 2'-hydroxyl groups, and the protecting groups of the phosphate groups in the phosphate bonds are deprotected under deprotection conditions corresponding to the protecting groups used.

[0041] The following examples describe and illustrate one embodiment of the present invention. According to the procedures shown in Examples 1 and 2, a tetramer phosphoramidite, which is one of the segments for oligonucleotide synthesis represented by formula (I), was synthesized. According to the procedures shown in Example 3, a pentamer phosphoramidite, which is one of the segments for oligonucleotide synthesis represented by formula (I), was synthesized. Furthermore, according to the procedures shown in Example 4, an 18-mer oligonucleotide was synthesized using a trimer phosphoramidite, which is one example of a compound represented by formula (I).

[0042] [Example 1] Synthesis of tetrameric phosphoramidite (3) (Step 1: DMTr-T p(OCH2CH2CN) T (OCH2CH2CN)(N(i-C3H7)2) Synthesis of (1) [ka]

[0043] To a solution of 5'-O-DMTrthymidine (16.3 g, 30.0 mmol) in dichloromethane (90 mL), the amiditizing reagent NCCH2CH2OP[N(i-C3H7)2]2 (10.5 mL, 33.0 mmol), diisopropylamine (2.12 mL, 15.0 mmol), and 1H-tetrazole (1.05 g, 15.0 mmol) were added, in that order, at room temperature. After stirring at room temperature for 3 hours, the reaction mixture was added to a solution of thymidine (10.9 g, 45.0 mmol) and 1H-tetrazole (5.25 g, 75.0 mmol) in dimethylformamide (DMF) (90 mL) and stirred at room temperature for 1 hour. The reaction mixture was then added to 180 mL of dichloromethane, loaded onto 340 g of silica gel, and washed with dichloromethane. The resulting filtrate was concentrated under reduced pressure to obtain the intermediate. The intermediate was dissolved in dichloromethane (180 mL), and the amiditizing reagents NCCH2CH2OP[N(i-C3H7)2]2 (12.4 mL, 39.0 mmol) and 1H-tetrazole (1.05 g, 15.0 mmol) were added in this order at room temperature. After stirring for 3 hours at room temperature, the product was purified by silica gel column chromatography using dichloromethane-IPA as the eluent to obtain the desired phosphoramidite 1 (24.2 g, 74.4% yield). MS: 1108.4 (MNa + )

[0044] (Step 2: DMTr-T p(OCH2CH2CN) T p(OCH2CH2CN) T (OCH2CH2CN)(N(i-C3H7)2) Synthesis of (2) [ka]

[0045] A dichloromethane solution (23 mL) of the above phosphoramidite 1 (10.0 g, 9.21 mmol) was added dropwise to a DMF solution (30 mL) of thymidine (2.90 g, 12.0 mmol) and 1H-tetrazole (1.94 g, 27.6 mmol) and stirred at room temperature for 1 hour. Dichloromethane (46 mL) was added to the reaction solution, which was then loaded onto 340 g of silica gel and washed with dichloromethane. The filtrate was concentrated and dissolved in dichloromethane (81 mL), and the amiditizing reagent NCCH2CH2OP[N(i-C3H7)2]2 (3.19 mL, 10.6 mmol) and 1H-tetrazole (0.4 g, 5.70 mmol) were added in that order at room temperature. After stirring at room temperature for 3 hours, the product was dissolved in dichloromethane (163 mL) and purified by silica gel column chromatography using dichloromethane-IPA as the eluent to obtain the desired phosphoramidite 2 (9.65 g, yield 73.4%). The ESI-MS spectrum of the obtained phosphoramidite 2 is shown in Figure 1. 31 The P NMR spectra are shown in Figure 2. MS: 1448.5 (MNa + )

[0046] (Step 3: DMTr-T p(OCH2CH2CN) T p(OCH2CH2CN) T p(OCH2CH2CN) T p(OCH2CH2CN)(N(i-C3H7)2) Synthesis of (3) [ka]

[0047] A dichloromethane solution (70 mL) of the above phosphoramidite 2 (10.0 g, 7.01 mmol) was added to a DMF solution (90 mL) of thymidine (2.21 g, 9.11 mmol) and 1H-tetrazole (1.47 g, 21.2 mmol) and stirred at room temperature for 1 hour. Dichloromethane (140 mL) was added to the reaction solution, which was then loaded onto 340 g of silica gel and washed with dichloromethane. The filtrate was concentrated and dissolved in dichloromethane (70 mL), and the amiditizing reagent NCCH2CH2OP[N(i-C3H7)2]2 (2.89 mL, 9.11 mmol) and 1H-tetrazole (0.340 g, 4.90 mmol) were added in that order at room temperature. After stirring at room temperature for 3 hours, the product was dissolved in dichloromethane (140 mL) and purified by silica gel column chromatography using dichloromethane-IPA as the eluent to obtain the desired tetrameric phosphoramidite 3 (9.40 g, 75.9% yield). MS: 1791.6(MNa + )

[0048] [Example 2] Synthesis of tetrameric phosphoramidite (6) (Step 4: DMTr-T p(OCH2CH2CN) C (OCH2CH2CN)(N(i-C3H7)2) Synthesis of (4) [ka]

[0049] To a solution of 5'-O-DMTrthymidine (16.3 g, 30.0 mmol) in dichloromethane (90 mL), the amiditizing reagent NCCH2CH2OP[N(i-C3H7)2]2 (10.5 mL, 33.0 mmol), diisopropylamine (2.12 mL, 15.0 mmol), and 1H-tetrazole (1.05 g, 15.0 mmol) were added in this order at room temperature. After stirring at room temperature for 3 hours, the reaction mixture was added to a solution of N4-benzoylcytidine (12.9 g, 39.0 mmol) and 1H-tetrazole (6.31 g, 90.0 mmol) in DMF (90 mL) and stirred at room temperature for 1 hour. After adding 180 mL of dichloromethane to the reaction solution, the amiditizing reagent NCCH2CH2OP[N(i-C3H7)2]2 (12.4 mL, 39.0 mmol) and 1H-tetrazole (1.05 g, 15.0 mmol) were added in this order at room temperature. After stirring at room temperature for 3 hours, the mixture was loaded onto 340 g of silica gel and washed with dichloromethane. The filtrate was concentrated under reduced pressure to give the intermediate (yield 70-90%). The intermediate was dissolved in dichloromethane (180 mL) and purified by silica gel column chromatography using dichloromethane-IPA as the eluent to give the desired phosphoramidite 4 (27.1 g, yield 77.0%). MS: 1197.4(MNa + )

[0050] (Step 5: dG p(OCH2CH2CN) T(5) [ka]

[0051] To a solution of 2-cyanoethyl 5'-O-DMTr-N2-isobutyryl-guanosine 3'-(N,N'-diisopropyl)phosphoramidite (30.0 g, 35.7 mmol) and thymidine (11.2 g, 46.4 mmol) in dichloromethane (180 mL), 1H-tetrazole (7.50 g, 107 mmol) was added and stirred at room temperature for 1 hour. Phenylacetyl disulfide (32.4 g, 107 mmol) was added to the reaction solution and stirred for an additional hour. After adding 180 mL of dichloromethane, the mixture was loaded onto 340 g of silica gel and washed with dichloromethane. The filtrate was concentrated under reduced pressure to give the product (yields: 70-90%). The product was dissolved in dichloromethane (180 mL) and purified by silica gel column chromatography using dichloromethane-IPA as the eluent to give the intermediate (yields: 70-90%). To the intermediate (15 g, 15.3 mmol), 1000 mL of a 3% dichloroacetic acid solution in dichloromethane was added at 0°C and stirred at room temperature for 30 minutes. The reaction mixture was loaded onto 1500 g of silica gel and washed with ethyl acetate. The mixture was further purified by silica gel column chromatography using dichloromethane-IPA as an eluent to obtain the desired dimer 5 (22.1 g, 87.3% yield). MS: 723.2 (MNa + )

[0052] (Step 6: DMTr-T p(OCH2CH2CN) C (OCH2CH2CN) G p(OCH2CH2CN) T (OCH2CH2CN)(N(i-C3H7)2) Synthesis of (6) [ka]

[0053] A dichloromethane solution (50 mL) of compound 4 (10.0 g, 8.51 mmol) was added dropwise to a DMF solution (45 mL) of compound 5 (7.26 g, 10.2 mmol) and 1H-tetrazole (1.79 g, 25.5 mmol) and stirred at room temperature for 1 hour. Dichloromethane (90 mL) was added to the reaction solution, which was then loaded onto 340 g of silica gel and washed with dichloromethane. The filtrate was concentrated and dissolved in dichloromethane (85 mL), and the amiditizing reagent NCCHCHOP[N(i-C3H7)2]2 (3.19 mL, 10.6 mmol) and 1H-tetrazole (0.420 g, 5.96 mmol) were added in this order at room temperature. After stirring at room temperature for 3 hours, the product was dissolved in dichloromethane (170 mL) and purified by silica gel column chromatography using dichloromethane-IPA as an eluent to obtain the desired phosphoramidite 6 (9.42 g, yield 63.1%). MS: 1974.6 (MNa + )

[0054] Example 3: Synthesis of pentameric amidite (15) (Step 7: DMTr-A Bz p(OCH2CH=CH2) Synthesis of (8) [ka]

[0055] To a solution (20 mL) of 5'-O-DMTr-N-5-benzoyl 2'-deoxyadenosine (7) (3.3 g, 5.0 mmol) in dichloromethane-acetonitrile (1:1), the amiditizing reagent CH2=CHCH2OP[N(i-C3H7)2]2 (2.3 mL, 7.5 mmol) and 1H-tetrazole (0.25 g, 3.5 mmol) were added in two portions at 0 °C. After stirring at room temperature for 12 h, dichloromethane (60 mL) was added and the mixture was purified by silica gel column chromatography using hexane-ethyl acetate as the eluent to give the desired phosphoramidite 8 (4.0 g, 94% yield).

[0056] Step 8: Synthesis of 3´-TBS-T(9) [ka]

[0057] Imidazole (33, 500 mmol) and tert-butyldimethylsilyl chloride (25 g, 170 mmol) were added in two portions to a DMF solution (200 mL) of thymidine (10 g, 41 mmol). After stirring at room temperature for 3 hours, 5 mL of methanol was added to the reaction mixture, which was then added dropwise to distilled water (1000 mL). The residue was filtered and then dissolved in THF. Trifluoroacetic acid (40 mL) and distilled water (40 mL) were added at 0°C, and the mixture was stirred for 1 hour. The reaction mixture was added dropwise to distilled water (500 mL) and extracted with dichloromethane (500 mL). The resulting crude product was purified by silica gel column chromatography using hexane-ethyl acetate as the eluent to obtain the desired 3'-protected thymidine 9 (13 g, 88% yield).

[0058] (Step 9:T p(OCH2CH=CH2) T OTBS Synthesis of (10) [ka]

[0059] To a solution of phosphoramidite monomer 8 (1.3 g, 1.0 mmol) and 3'-protected thymidine 9 (0.36 g, 1.0 mmol) in dichloromethane-acetonitrile (1:1, 10 mL) was added 5-ethylthio-1H-tetrazole (0.62 g, 4.0 mmol). After stirring at room temperature for 30 min, a solution of tert-butyl hydroperoxide (TBHP) in decane (0.60 mL, 3.0 mmol) was added and stirred for an additional 30 min. The reaction mixture was concentrated, and the resulting crude product was treated with a 3% dichloroacetic acid solution in dichloromethane (20 mL) at room temperature for 15 min. The reaction mixture was then purified by silica gel column chromatography using dichloromethane-methanol as the eluent to give the desired 5'-unprotected dAT dimer 10 (0.65 g, 58% yield).

[0060] (Step 10: DMTr-A Bzp(OCH2CH=CH2) A Bz p(OCH2CH=CH2) T OTBS Synthesis of (11) [ka]

[0061] To a solution of phosphoramidite monomer 8 (0.83 g, 0.99 mmol) and 5´-unprotected dAT dimer 10 (0.62 g, 0.76 mmol) in dichloromethane-acetonitrile (10 mL) was added 5-ethylthio-1H-tetrazole (0.47 g, 3.0 mmol). After stirring at room temperature for 30 min, a decane solution of TBHP (0.46 mL, 2.3 mmol) was added and stirred at room temperature for an additional 30 min. The reaction mixture was concentrated, and the resulting crude product was purified by silica gel column chromatography using dichloromethane-methanol as the eluent to give the desired 5´-DMTr-dAAT trimer 11 (0.65 g, 58% yield).

[0062] (Step 11: DMTr-A Bz p(OCH2CH=CH2) A Bz p(OCH2CH=CH2) T (OCH2CH=CH2)(N(i-C3H7)2) Synthesis of (12) [ka]

[0063] A solution of tetrabutylammonium fluoride in THF (1.2 mL, 1.2 mmol) was added to 5'-DMTr-dAAT trimer 11 (0.94 g, 0.60 mmol) at 0 °C. After stirring for 4 h, the reaction mixture was concentrated, and the resulting crude product was purified by silica gel column chromatography using dichloromethane-methanol as the eluent and dried overnight under vacuum. A portion of this intermediate (0.71 g, 0.49 mmol) was used to prepare a dichloromethane-acetonitrile solution (5.0 mL), and then the amiditizing reagent CH2=CHCH2OP[N(i-C3H7)2]2 (0.21 mL, 0.74 mmol) and 1H-tetrazole (0.024 g, 0.34 mmol) were added in two portions at 0 °C. After stirring at room temperature for 12 hours, dichloromethane (5.0 mL) was added and the mixture was purified by silica gel column chromatography using hexane-ethyl acetate as the eluent to obtain the desired 5'-DMTr protected trimer phosphoramidite 12 (0.39 g, overall yield 66%).

[0064] (Step 12: DMTr-A Bz p(OCH2CH=CH2) A Bz p(OCH2CH=CH2) A Bz p(OCH2CH=CH2) T OTBS Synthesis of (13) [ka]

[0065] To a solution of 5'-DMTr-protected phosphoramidite trimer 12 (0.54 g, 0.33 mmol) and 5'-unprotected dimer 10 (0.24 g, 0.30 mmol) in dichloromethane-acetonitrile (6 mL) was added 5-ethylthio-1H-tetrazole (0.19 g, 1.2 mmol). After stirring at room temperature for 90 min, a decane solution of TBHP (0.20 mL, 0.9 mmol) was added and the mixture was stirred at room temperature for an additional 30 min. The reaction mixture was concentrated, and the resulting crude product was purified by silica gel column chromatography using dichloromethane-methanol as the eluent to give the desired 5',3'-protected dAATAT pentamer 13 (0.67 g, 94% yield).

[0066] (Step 13: DMTr-A Bz p(OCH2CH=CH2) A Bz p(OCH2CH=CH2) A Bz p(OCH2CH=CH2) T OH Synthesis of (14) [ka]

[0067] To the 5',3'-protected dAATAT pentamer 13 (0.67 g, 0.28 mmol), a THF solution of tetrabutylammonium fluoride (0.56 mL, 0.56 mmol) was added at 0 °C. After stirring for 4 h, the reaction mixture was concentrated, and the resulting crude product was purified by silica gel column chromatography using dichloromethane-methanol as the eluent to give the desired 5'-protected, 3'-unprotected pentamer 14 (0.51 g, 80% overall yield).

[0068] (Step 14: DMTr-A Bz p(OCH2CH=CH2) A Bz p(OCH2CH=CH2) A Bz p(OCH2CH=CH2) T (OCH2CH=CH2)(N(i-C3H7)2) Synthesis of (15) [ka]

[0069] To a solution of 5'-protected, 3'-unprotected dAATAT pentamer 14 (0.46 g, 0.21 mmol) in dichloromethane-acetonitrile (2.0 mL), the amiditizing reagent CH2=CHCH2OP[N(i-C3H7)2]2 (0.89 μL, 0.31 mmol) and 1H-tetrazole (0.010 g, 0.14 mmol) were added in two portions at 0 °C. After stirring at room temperature for 12 h, dichloromethane (5.0 mL) was added and the mixture was purified by silica gel column chromatography using hexane-ethyl acetate as the eluent to give the desired pentameric phosphoramidite 15 (0.41 g, 81% overall yield).

[0070] Example 4 (dT 18 Solid-phase synthesis of An oligonucleotide (dT18-mer) was synthesized using the synthetic segment (tetramer phosphoramidite) 3 obtained in Example 1 and ChemGene's universal linker CPG1000 Å (1.0 μmol) as a starting material in an NTS M-2-MX oligonucleotide solid-phase synthesizer according to the following protocol. Synthesis Cycle: (Condensation) 0.1M tetramer phosphoramidite 3 / acetonitrile solution (14.4 μL) 0.25M 5-benzylthiotetrazole / acetonitrile solution (24.8μL) Reaction time: 2 minutes (Capping) 10% acetic anhydride / THF solution (320 μL) 0.2M N-methylimidazole / acetonitrile solution Reaction time: 1 minute (Oxidation) 0.05M iodine / water / pyridine solution (320 μL) (Detritylated) 10% dichloroacetic acid / toluene solution (765 μL) Reaction time: 2 minutes (Cleavage / Deprotection) Concentrated Ammonia Water Reaction time: 60 minutes, room temperature

[0071] The LC spectrum of the resulting dT18 mer is shown in FIG.

[0072] As described above, the oligonucleotide synthesis segment of this embodiment allows the use of amidites of a length equal to or greater than the length of a nucleoside trimer in oligonucleotide synthesis. Therefore, when synthesizing an oligonucleotide of a desired length of N-mer, it is not possible to produce (N-1)mers or (N-2)mers, which are one base shorter or two bases shorter than N-mers, as are produced in the coupling step of monomer amidites in commonly used oligonucleotide synthesis. Therefore, when purifying the N-mer using chromatography or the like in the final stage of oligonucleotide synthesis, the difference in mobility between the desired N-mer and by-products of lengths equal to or less than the (N-3)mer is large, and therefore the purification load required to separate the N-mer from other N-mers can be reduced.

[0073] Furthermore, the oligonucleotide synthesis segment of this embodiment can reduce the number of steps required to synthesize the same N-mer oligonucleotide compared to the conventional method of extending the oligonucleotide one base at a time, thereby improving the yield of oligonucleotides of the desired length.

[0074] Furthermore, in producing the oligonucleotide synthesis segment of this embodiment, a partially oxidized / sulfurized pentavalent phosphate bond can be formed at the segment stage. Therefore, even when only some phosphate bonds in an oligonucleotide are oxidized / sulfurized differently from the phosphate bonds in other portions, an oligonucleotide containing the desired modified phosphate bond can be synthesized more simply without changing the oligonucleotide synthesis procedure.

[0075] Furthermore, in the method for producing an oligonucleotide synthesis segment according to this embodiment, a nucleoside in which only the 5'-hydroxyl group is protected, or a nucleoside in which both the 5'-hydroxyl group and the 3'-hydroxyl group are unprotected, is directly reacted with a phosphorylating agent for preparing an amidite. This eliminates the need to purchase a commercially available 3'-amidite monomer or to prepare it in advance. This allows for the mass production of desired oligonucleotide synthesis segments compared to conventional methods.

[0076] Furthermore, the oligonucleotide synthesis segment of this embodiment can be used not only for the large-scale synthesis of relatively short oligonucleotides by liquid-phase synthesis, but also for the synthesis of long oligonucleotides by solid-phase synthesis. Therefore, since by-products having lengths of N-1 or N-2 are not produced, the purification burden after synthesis of long N-mer oligonucleotides in particular can be reduced, and the desired N-mer oligonucleotide can be obtained with simpler purification.

Claims

1. The following formula (I): 【Chemistry 1】 (In the formula, B is independently a nucleoside base, protected by a protecting group or unprotected; When B is a nucleoside base protected by a protecting group, the protecting group is an acyl protecting group; R １ is a protecting group; R ２ , R ３ , R ４ is OCH ２ CH=CH ２ and R ５ is a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group; X is independently =0, or =S; Y is independently H, NHR ６ , halogens, CN, CF ３、 or a hydroxyl group protected by an acyl-based protecting group, an ether-based protecting group, or a silyl-based protecting group; R ６ is H, an aliphatic group, or an aromatic group; Z is independently H, alkyl, O-alkyl, N-alkyl, halogen; The value of (m+n) is an integer between 3 and 23. A segment for oligonucleotide synthesis represented by the formula:

2. In the formula (I), The R １ is a protecting group or a trialkylsilyl group that can be removed under acidic conditions, Y is H or a hydroxyl group protected by a t-butyldimethylsilyl group, Z is H, The R ５ The segment for oligonucleotide synthesis according to claim 1, wherein is an isopropyl group.

3. A method for synthesizing an oligonucleotide using the oligonucleotide synthesis segment represented by formula (I) according to claim 1, comprising: (a) condensing the amidite moiety of the oligonucleotide synthesis segment represented by formula (I) with a hydroxyl group of a nucleoside or nucleotide; (b) an oxidation or sulfurization step of the phosphorous bond obtained in the condensation step; (c) a step of deprotecting the terminal protecting group of the oligonucleotide synthesis segment condensed with the nucleoside or nucleotide in the condensation step; A synthesis method comprising:

4. The method of claim 3 , wherein each of the steps is carried out in solution.

5. The synthesis method according to claim 3, wherein each of the steps is carried out on a solid support.

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