Method for producing oligonucleotides and reagent kit for synthesizing oligonucleotides

Abstract

In the production of oligonucleotides using pseudo-solid-phase protecting groups, high-purity oligonucleotides can be obtained. [Solution] A nucleic acid compound having a pseudo-solid protecting group bonded to the 3' or 5' position of a nucleoside, nucleotide, or oligonucleotide is reacted with a solution containing an alkali metal hydroxide, and the pseudo-solid protecting group is removed from the nucleic acid compound by hydrolysis.

Description

[Technical Field]

[0001] This disclosure relates to a method for producing oligonucleotides and a reagent kit for the synthesis of oligonucleotides. [Background technology]

[0002] The artificial synthesis methods for nucleic acid molecules (deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)) used in nucleic acid drugs and other applications are broadly classified into so-called solid-phase synthesis and liquid-phase synthesis. In solid-phase synthesis, nucleic acid molecules with a predetermined sequence are synthesized by sequentially linking nucleotides on a solid support such as polymer beads. In liquid-phase synthesis, nucleic acid molecules are synthesized by sequentially linking nucleotides in solution. Some liquid-phase synthesis methods use highly lipid-soluble substances as solubilizing supports to synthesize nucleic acid molecules in solution.

[0003] An example of such a solubilizing support is the alkoxyphenyl derivative disclosed in Patent Document 1. The alkoxyphenyl derivative disclosed in Patent Document 1 functions as a pseudo-solid-phase protecting group when bound to a nucleoside or nucleotide, and is used in the production of oligonucleotides (oligonucleotide-protected products) in which nucleotides with protected nucleic acid bases and phosphate groups are sequentially linked. According to the alkoxyphenyl derivative disclosed in Patent Document 1, the simple and rapid synthesis of oligonucleotide-protected products is possible. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Patent No. 6770553 [Overview of the project] [Problems that the invention aims to solve]

[0005] However, when using alkoxyphenyl derivatives disclosed in Patent Document 1 or other conventionally known pseudo-solid-phase protecting groups, there was a problem in that it was difficult to obtain the target oligonucleotide with high purity. Therefore, the present disclosure aims to provide a method for producing oligonucleotides that can be obtained with high purity in the production of oligonucleotides using a pseudo-solid-phase protecting group, and a reagent kit for the synthesis of oligonucleotides. [Means for solving the problem]

[0006] As a result of diligent research to achieve the above objective, the present inventors have discovered that by using a solution containing alkali metal hydroxide, the pseudo-solid-phase protecting group can be removed from an oligonucleotide to which the pseudo-solid-phase protecting group is attached, thereby obtaining the target oligonucleotide in high purity, and have completed this disclosure.

[0007] In other words, one aspect of this disclosure is as follows: <1> A method for producing an oligonucleotide, comprising the step of reacting a nucleic acid compound having a pseudo-solid protecting group bonded to the 3' or 5' position of a nucleoside, nucleotide, or oligonucleotide with a solution containing an alkali metal hydroxide, thereby removing the pseudo-solid protecting group from the nucleic acid compound by a hydrolysis reaction. <2> The nucleic acid compound is represented by the following general formula (X): <1> A method for producing the alkyl group described above. [ka] [In general formula (X), Base represents a nucleic acid base that may be modified. If there are two or more Bases, the multiple Bases may be the same or different. In general formula (X), B represents a hydrogen atom, a halogen atom, or an optionally protected hydroxyl group. If there are two or more B's, the multiple B's may be the same or different. In general formula (X), W is either a hydrogen atom or a 5' protecting group. In general formula (X), R X R represents an alkyl group having 1 to 7 carbon atoms, which has an ethyl group, allyl group, aryl group, or electron-withdrawing group at the 2nd position. X If there are two or more of them, then there are multiple R X They may be the same or different. In general formula (X), Q represents an oxygen atom or a sulfur atom. If there are two or more Qs, the multiple Qs may be the same or different. In the general formula (X), y1 represents any integer greater than or equal to 0. In general formula (X), * represents the site to which the pseudo-solid-phase protecting group is bonded. <3> The aforementioned pseudo-solid protecting group is a group formed by the bonding of the carboxyl group in the following general formula (Y) to the 3' or 5' position of the nucleoside, nucleotide, or oligonucleotide. <1> or <2> A method for producing the alkyl group described above. [ka] [In general formula (Y), R Y Each of these independently represents a linear or branched alkyl group with 10 to 40 carbon atoms. In the general formula (Y), m represents an integer from 1 to 5, and if m is 2 or greater, there are multiple R Y O may be the same or different. In the general formula (Y), X is an oxygen atom, a sulfur atom, NH, or NR. N It represents. In the general formula (Y), n represents an integer between 1 and 4. In general formula (Y), R N [This represents a linear or branched alkyl group with 1 to 6 carbon atoms.] <4> The aforementioned pseudo-solid phase protecting group is -LY L A group represented by -Z or -Z, <1> or <2> A method for producing the alkyl group described above. [In the formula, L is given by formula (a1): [ka] (wherein ** represents the bonding position with the 3'- or 5'-position of the oligonucleotide; * is Y L represents the bonding position with; L1 represents an optionally substituted divalent hydrocarbon group having 1 to 22 carbon atoms, an oxygen atom, -NR- (R represents a hydrogen atom, an alkyl group or an aralkyl group); L2 represents a single bond or a formula: * C(R 3a )(R 3b )-O-R 1** , a formula: * C(=O)N(R 2 )-R 1 -N(R 3a ) ** or a formula: * C(=O)N(R 2 )-R 1 -C(R 3a )(R 3b ) ** (wherein * represents the bonding position with L1, ** is CR c R d represents the bonding position with; R 1 represents an optionally substituted alkylene group having 1 to 22 carbon atoms, R 2 and R 3a each independently represent a hydrogen atom or an optionally substituted alkyl group having 1 to 22 carbon atoms or R 2 and R 3a may together form an optionally substituted alkylene bond having 1 to 22 carbon atoms, R 3b represents a hydrogen atom or an optionally substituted alkyl group having 1 to 22 carbon atoms) represents a group; R c and R d each independently represent a hydrogen atom, an optionally substituted alkyl group having 1 to 22 carbon atoms or R c and R d may together form a single carbonyl group) represents a group (linker); In the formula, Y LThis represents a single bond, an oxygen atom, -NR- (where R represents a hydrogen atom, an alkyl group, or an aralkyl group), or a sulfur atom; In the equation, Z is given by equation (a2): [ka] (In the formula, * Y L This indicates the bonding position with; R 4 This indicates a hydrogen atom, or R b If the group is represented by the following formula (a3), then R 6 They may form a single bond or an -O- together with ring B, or they may form a fluorenyl group or a xanthenyl group together with ring B; k R 5 Each of the following independently represents an organic group having an aliphatic hydrocarbon group with 10 or more carbon atoms; k represents an integer from 1 to 4; and ring A represents k ORs. 5 In addition, the substituents may be selected from the group consisting of halogen atoms, C1-C6 alkyl groups which may be substituted with halogen atoms, and C1-C6 alkoxy groups which may be substituted with halogen atoms; R a R represents a phenyl group which may be substituted with a hydrogen atom or a halogen atom; b is a hydrogen atom, or formula (a3): [ka] (In the formula, * indicates the join position; j indicates an integer from 0 to 4; j R 7 These independently represent organic groups having an aliphatic hydrocarbon group with 10 or more carbon atoms; R 6 This indicates a hydrogen atom, or R 4 They may form a single bond or an -O- together with ring A, or form a fluorenyl group or a xanthenyl group together with ring A; and ring B may have j OR 7 In addition, the substituents may be selected from the group consisting of halogen atoms, C1-C6 alkyl groups which may be substituted with halogen atoms, and C1-C6 alkoxy groups which may be substituted with halogen atoms; Ra and R b These may combine to form a single carbonyl group. Formula (a2'): [ka] (In the formula, * Y L The bond position is indicated by; the definition of each other symbol is the same as that of formula (a2). ) or formula (a2''): [ka] (In the formula, * Y L The bond positions are shown; ring A' consists of k OR 5 In addition, the group may further have substituents selected from the group consisting of halogen atoms, C1-C6 alkyl groups which may be substituted with halogen atoms, and C1-C6 alkoxy groups which may be substituted with halogen atoms; the definition of each symbol is the same as that of formula (a2). <5> The pseudo-solid protecting group is a group formed by the bonding of -OM in the following general formula (1) to the 3' or 5' position of the nucleoside, nucleotide, or oligonucleotide. <1> or <2> A method for producing the alkyl group described above. General formula (1): MO-(O=)CC(=O)-W1-X1-Y1-Z1...(1) [In general formula (1), M represents a hydrogen atom or an alkali metal atom, W1 is an oxygen atom, a sulfur atom, or NR Z (N indicates a nitrogen atom, R Z (This represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.) X1 represents a single bond, an alkylene group having 1 to 12 carbon atoms (which may have an ether bond (-O-)), an arylene group (which may be substituted with an alkylene group having 1 to 3 carbon atoms), or a divalent heterocyclic group. Y1 is a single bond, ether bond, -OCO-, -NR ZCO-(R Z (The above is the same), indicates -CO- or -SCO-, Z1 represents an alkoxyphenyl group or alkoxyalkyl group having 10 to 30 carbon atoms. <6> The aforementioned pseudo-solid-phase protecting group is a group represented by general formula (II), <1> or <2> A method for producing the alkyl group described above. [ka] [In general formula (II), * indicates the binding position to the 3' or 5' position of the nucleoside, nucleotide, or oligonucleotide. R X1 Each of these is independently an alkyl group having 1 to 40 carbon atoms, an alkenyl group having 2 to 40 carbon atoms, an alkynyl group having 2 to 40 carbon atoms, or a group of the formula: -L 7 -OR X4 (In the formula, L 7 R is an alkylene group having 1 to 40 carbon atoms, an alkenylene group having 2 to 40 carbon atoms, or an alkynylene group having 2 to 40 carbon atoms. X4 (where is an alkyl group having 1 to 40 carbon atoms, an alkenyl group having 2 to 40 carbon atoms, or an alkynyl group having 2 to 40 carbon atoms), and at least one R X1 The number of carbon atoms is 10 or more. s is an integer from 1 to 5. L 11 This is an alkylene group having 1 to 6 carbon atoms, an alkenylene group having 2 to 6 carbon atoms, or an alkylene group having 2 to 6 carbon atoms. L 12 This is a single bond, -O- or -N(R X2 )-(wherein, R X2 (represented by a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, a haloalkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, or a haloalkenyl group having 2 to 6 carbon atoms) L 13 This is a single bond, -C(O)-, or an alkylene group having 1 to 6 carbon atoms. L 12 When it is a single bond, L 13 It is a single bond or -C(O)-. <7>The method for producing an oligonucleotide according to <1> or <2>, wherein the pseudo solid-phase protecting group is a group formed by bonding a carboxy group in a compound represented by the general formula (I') to the 3'-position or 5'-position of the nucleoside, the nucleotide, or the oligonucleotide.

Chemical formula

[0008] <13> A reagent kit for the synthesis of oligonucleotides, comprising: a compound that binds to the 3' or 5' position of a nucleoside, nucleotide, or oligonucleotide to form a pseudo-solid-phase protecting group; an alkali metal hydroxide; and at least one substrate selected from the group consisting of nucleosides, nucleotides, and oligonucleotides to which the compound binds. <14> The aforementioned pseudo-solid-phase protecting group forms a nucleic acid compound represented by the following general formula (X): <13> A reagent kit for synthesizing oligonucleotides as described above. [ka] [In general formula (X), Base represents a nucleic acid base that may be modified. If there are two or more Bases, the multiple Bases may be the same or different. In general formula (X), B represents a hydrogen atom, a halogen atom, or an optionally protected hydroxyl group. If there are two or more B's, the multiple B's may be the same or different. In general formula (X), W is either a hydrogen atom or a 5' protecting group. In general formula (X), R X R represents an alkyl group having 1 to 7 carbon atoms, which has an ethyl group, allyl group, aryl group, or electron-withdrawing group at the 2nd position. X If there are two or more of them, then there are multiple R X They may be the same or different. In general formula (X), Q represents an oxygen atom or a sulfur atom. If there are two or more Qs, the multiple Qs may be the same or different. In the general formula (X), y1 represents any integer greater than or equal to 0. In general formula (X), * represents the site to which the pseudo-solid-phase protecting group is bonded. <15> The aforementioned pseudo-solid protecting group is a group formed by the bonding of the carboxyl group in the following general formula (Y) to the 3' or 5' position of the nucleoside, nucleotide, or oligonucleotide. <13> or <14> A reagent kit for synthesizing oligonucleotides as described above. [ka] [In general formula (Y), R Y Each of these independently represents a linear or branched alkyl group with 10 to 40 carbon atoms. In the general formula (Y), m represents an integer from 1 to 5, and if m is 2 or greater, there are multiple R Y O may be the same or different. In the general formula (Y), X is an oxygen atom, a sulfur atom, NH, or NR. N It represents. In the general formula (Y), n represents an integer between 1 and 4. In general formula (Y), RN [This represents a linear or branched alkyl group with 1 to 6 carbon atoms.] <16> The aforementioned pseudo-solid phase protecting group is -LY L A group represented by -Z or -Z, <13> or <14> A reagent kit for synthesizing oligonucleotides as described above. [In the formula, L is given by formula (a1): [ka] (In the formula, ** This indicates the binding position with the 3' or 5' position of the oligonucleotide; * Y L The bond position is indicated; L1 indicates a substituted or otherwise divalent hydrocarbon group having 1 to 22 carbon atoms, an oxygen atom, or -NR- (where R indicates a hydrogen atom, an alkyl group, or an aralkyl group); L2 indicates a single bond or formula: * C(R 3a )(R 3b )-OR 1** ,formula: * C(=O)N(R 2 )-R 1 -N(R 3a ) ** Alternatively, formula: * C(=O)N(R 2 )-R 1 -C(R 3a )(R 3b ) ** (In the formula, * This indicates the binding site with L1. ** CR c R d The binding position with R is shown, 1 R represents an alkylene group having 1 to 22 carbon atoms, which may be substituted. 2 and R 3a Each independently represents a hydrogen atom or an optionally substituted C1-C22 alkyl group, or R 2 and R 3a Together, they may form an alkylene bond with 1 to 22 carbon atoms, which may be substituted. 3bR represents a group represented by a hydrogen atom or an alkyl group having 1 to 22 carbon atoms, which may be substituted; c and R d Each of these is independently a hydrogen atom, an optionally substituted alkyl group having 1 to 22 carbon atoms, or R c and R d It represents a group (linker) that may be formed by combining with a single carbonyl group; In the formula, Y L This represents a single bond, an oxygen atom, -NR- (where R represents a hydrogen atom, an alkyl group, or an aralkyl group), or a sulfur atom; In the equation, Z is given by equation (a2): [ka] (In the formula, * Y L This indicates the bonding position with; R 4 This indicates a hydrogen atom, or R b If the group is represented by the following formula (a3), then R 6 They may form a single bond or an -O- together with ring B, or they may form a fluorenyl group or a xanthenyl group together with ring B; k R 5 Each of the following independently represents an organic group having an aliphatic hydrocarbon group with 10 or more carbon atoms; k represents an integer from 1 to 4; and ring A represents k ORs. 5 In addition, the substituents may be selected from the group consisting of halogen atoms, C1-C6 alkyl groups which may be substituted with halogen atoms, and C1-C6 alkoxy groups which may be substituted with halogen atoms; R a R represents a phenyl group which may be substituted with a hydrogen atom or a halogen atom; b is a hydrogen atom, or formula (a3): [ka] (In the formula, * indicates the join position; j indicates an integer from 0 to 4; j R 7 These independently represent organic groups having an aliphatic hydrocarbon group with 10 or more carbon atoms; R6 This indicates a hydrogen atom, or R 4 They may form a single bond or an -O- together with ring A, or form a fluorenyl group or a xanthenyl group together with ring A; and ring B may have j OR 7 In addition, the substituents may be selected from the group consisting of halogen atoms, C1-C6 alkyl groups which may be substituted with halogen atoms, and C1-C6 alkoxy groups which may be substituted with halogen atoms; R a and R b These may combine to form a single carbonyl group. Formula (a2'): [ka] (In the formula, * Y L The bond position is indicated by; the definition of each other symbol is the same as that of formula (a2). ) or formula (a2''): [ka] (In the formula, * Y L The bond positions are shown; ring A' consists of k OR 5 In addition, the group may further have substituents selected from the group consisting of halogen atoms, C1-C6 alkyl groups which may be substituted with halogen atoms, and C1-C6 alkoxy groups which may be substituted with halogen atoms; the definition of each symbol is the same as that of formula (a2). <17> The pseudo-solid protecting group is a group formed by the bonding of -OM in the following general formula (1) to the 3' or 5' position of the nucleoside, nucleotide, or oligonucleotide. <13> or <14> A reagent kit for synthesizing oligonucleotides as described above. General formula (1): MO-(O=)CC(=O)-W1-X1-Y1-Z1...(1) [In general formula (1), M represents a hydrogen atom or an alkali metal atom, W1 is an oxygen atom, a sulfur atom, or NR Z (N indicates a nitrogen atom, R Z (This represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.) X1 represents a single bond, an alkylene group having 1 to 12 carbon atoms (which may have an ether bond (-O-)), an arylene group (which may be substituted with an alkylene group having 1 to 3 carbon atoms), or a divalent heterocyclic group. Y1 is a single bond, ether bond, -OCO-, -NR Z CO-(R Z (The above is the same), indicates -CO- or -SCO-, Z1 represents an alkoxyphenyl group or alkoxyalkyl group having 10 to 30 carbon atoms. <18> The aforementioned pseudo-solid-phase protecting group is a group represented by general formula (II), <13> or <14> A reagent kit for synthesizing oligonucleotides as described above. [ka] [In general formula (II), * indicates the binding position to the 3' or 5' position of the nucleoside, nucleotide, or oligonucleotide. R X1 Each of these is independently an alkyl group having 1 to 40 carbon atoms, an alkenyl group having 2 to 40 carbon atoms, an alkynyl group having 2 to 40 carbon atoms, or a group of the formula: -L 7 -OR X4 (In the formula, L 7 R is an alkylene group having 1 to 40 carbon atoms, an alkenylene group having 2 to 40 carbon atoms, or an alkynylene group having 2 to 40 carbon atoms. X4 (where is an alkyl group having 1 to 40 carbon atoms, an alkenyl group having 2 to 40 carbon atoms, or an alkynyl group having 2 to 40 carbon atoms), and at least one R X1 The number of carbon atoms is 10 or more. s is an integer from 1 to 5. L 11 This is an alkylene group having 1 to 6 carbon atoms, an alkenylene group having 2 to 6 carbon atoms, or an alkylene group having 2 to 6 carbon atoms. L 12This is a single bond, -O- or -N(R X2 )-(wherein, R X2 (represented by a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, a haloalkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, or a haloalkenyl group having 2 to 6 carbon atoms) L 13 This is a single bond, -C(O)-, or an alkylene group having 1 to 6 carbon atoms. L 12 When it is a single bond, L 13 It is a single bond or -C(O)-. <19> The pseudo-solid protecting group is a group formed by the bonding of a carboxyl group in a compound represented by general formula (I') to the 3' or 5' position of the nucleoside, nucleotide, or oligonucleotide. <13> or <14> A reagent kit for synthesizing oligonucleotides as described above. [ka] [In general formula (I'), ring D is a 5-6 member heteroaryl having 1-3 heteroatoms independently selected from phenyl, an 8-10 member bicyclic aryl, oxygen, nitrogen, and sulfur, or a 7-10 member bicyclic heteroaryl having 1-4 heteroatoms independently selected from oxygen, nitrogen, and sulfur. Y2 is H, halogen, OR 1A , NR 2A R 3A , SR 4A CR 5A R 6A R 7A , or a hydrophobic group comprising one or more aliphatic hydrocarbon groups having 10 or more carbon atoms, R 1A , R 2A , R 3A , R 4A , R 5A , R 6A , and R 7A These are, independently, alkyl groups with 1 to 6 carbon atoms, alkenyl groups with 1 to 6 carbon atoms, alkynyl groups with 1 to 6 carbon atoms, phenyl groups, OR groups. 8A ,-OC(O)R 8A , -C(O)OR 8A , NR8A R 9A , -NR 8A COR 9A ,-CONR 8A R 9A , a 3-7 member saturated or partially unsaturated monocyclic carbocyclyl, or a 3-7 member saturated or partially unsaturated monocyclic heterocyclyl having 1-2 heteroatoms independently selected from oxygen, nitrogen, and sulfur, R 8A and R 9A Each instance is independently either H or an alkyl group having 1 to 6 carbon atoms. P1 is NO2 or a silyl hydroxyl protecting group. R1 and R2 are independently H, a C1-C6 alkyl group, or a phenyl group, and the C1-C6 alkyl and phenyl groups can be optionally substituted with R3 (1-3), and R3 is a C1-C30 alkoxy group. e is an integer from 0 to 6. f is an integer between 0 and 6. <20> The synthesis reagent kit for oligonucleotides according to claim 13, wherein the pseudo-solid-phase protecting group is a group represented by the following general formula (1). [ka] [In general formula (1), ring E represents a condensed polycyclic aromatic hydrocarbon ring or an aromatic heterocycle, Y A *-OCR 2A -**, *-NRCR 2A -**, or *-SCR 2A -** represents R 2A Each of these independently represents a hydrogen atom, an alkyl group, an aromatic group, or an aromatic group-substituted alkyl group. k1 represents an integer from 1 to 5. The dashed lines and * indicate bonding locations with other structures, and ** indicates bonding locations with ring E. R A Each of these is independently an aliphatic hydrocarbon group or an organic group having an aliphatic hydrocarbon group, and k1 R A The number of carbon atoms in at least one aliphatic hydrocarbon group is 12 or more. Ring E is Y A and R A In addition, it may have further substituents. <21> The pseudo-solid protecting group is a group formed by the bonding of the carboxyl group of a compound selected from the group consisting of compounds represented by any of the following formulas (A) to (F) to the 3' or 5' position of the nucleoside, nucleotide, or oligonucleotide. <13> ~ <20> A reagent kit for synthesizing oligonucleotides as described in one of the following. [ka] [ka] [ka] [ka] [ka] [ka] <22> The nucleic acid compound represented by the above general formula (X) is R X The allyl group is <14> ~ <21> A reagent kit for synthesizing oligonucleotides as described in one of the following. <23> The alkali metal hydroxides mentioned above are lithium hydroxide or sodium hydroxide. <13> ~ <22> A reagent kit for synthesizing oligonucleotides as described in one of the following. [Effects of the Invention]

[0009] According to one aspect of this disclosure, the pseudo-solid-phase protecting group can be removed from an oligonucleotide to which the pseudo-solid-phase protecting group is attached, thereby obtaining a target oligonucleotide with high purity. [Brief explanation of the drawing]

[0010] [Figure 1] This is a characteristic diagram showing the HPLC measurement results before and after the reaction that produces compound 5 from compound 4. [Figure 2] This is a characteristic diagram showing the HPLC measurement results before and after the reaction that produces compound 7 from compound 6. [Figure 3] This is a characteristic diagram showing the HPLC measurement results before and after the reaction that produces compound 10 from compound 9. [Figure 4] This is a characteristic diagram showing the HPLC measurement results before and after the reaction that produces compound 12 from compound 11. [Figure 5] This is a characteristic diagram showing the HPLC measurement results before and after the reaction that produces compound 14 from compound 13 and compound 3. [Figure 6] This is a characteristic diagram showing the HPLC measurement results before and after the reaction in which compound 6 is converted to compound 7 using an aqueous sodium hydroxide solution. [Figure 7] This is a characteristic diagram showing the HPLC measurement results before and after the reaction in which compound 7 is produced from compound 6 using a lithium borohydride solution. [Figure 8] This is a characteristic diagram showing the 1H-NMR spectrum of compound 16 and the 1H-NMR spectrum of a commercially available Fujimat. [Figure 9] This is a characteristic diagram showing the mass spectrometry (FD-MS) spectrum of compound 16. [Figure 10] This is a characteristic diagram showing the mass spectrometry spectrum (FD-MS) of a commercially available Fujimat. [Modes for carrying out the invention]

[0011] In explaining this disclosure, specific examples will be given, but the content is not limited to the following, as long as it does not deviate from the intent of this disclosure, and may be modified as appropriate.

[0012] In this disclosure, a numerical range represented by "~" means a range that includes the numbers written before and after "~" as the lower and upper limits, respectively. In numerical ranges described in stages in this disclosure, the upper or lower limit of one numerical range may be replaced with the upper or lower limit of another numerical range described in stages. Furthermore, in numerical ranges described in this disclosure, the upper or lower limit of that range may be replaced with the values ​​shown in the examples. In this disclosure, the amount of each component in a composition means the total amount of any multiple substances present in the composition, unless otherwise specified, if there are multiple substances corresponding to each component in the composition. In this disclosure, a preferred combination of embodiments is a more preferred embodiment. In this disclosure, the term "process" includes not only independent processes but also processes that cannot be clearly distinguished from other processes, as long as their intended purpose is achieved.

[0013] <Method for manufacturing oligonucleotides> The present disclosure's method for producing oligonucleotides includes a step of reacting a nucleic acid compound having a pseudo-solid protecting group bonded to the 3' or 5' position of a nucleoside, nucleotide, or oligonucleotide with a solution containing an alkali metal hydroxide, thereby removing the pseudo-solid protecting group from the nucleic acid compound by a hydrolysis reaction. According to the present disclosure's method for producing oligonucleotides, the pseudo-solid protecting group can be removed from the nucleic acid compound, and the desired oligonucleotide can be obtained with high purity.

[0014] Here, the target oligonucleotide refers to an oligonucleotide having a pre-designed sequence. Therefore, obtaining the target oligonucleotide in high purity means obtaining an oligonucleotide with a pre-designed sequence in high purity. Obtaining the target oligonucleotide in high purity means, for example, that the solution after the reaction in which the pseudo-solid-phase protecting group is removed from the nucleic acid compound by hydrolysis contains 80% or more of the target oligonucleotide among all nucleic acid components, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 97% or more. The proportion of the target oligonucleotide among all nucleic acid components in the solution can be calculated using high-performance liquid chromatography (HPLC) with the solution, as described in the examples below. Furthermore, the method for producing oligonucleotides in this disclosure allows for the high yield of oligonucleotides obtained by removing the pseudo-solid-phase protecting group from the nucleic acid compound by hydrolysis. That is, according to the method for producing oligonucleotides in this disclosure, oligonucleotides obtained by removing the pseudo-solid-phase protecting group from nucleic acid molecules contained in a solution can be recovered in high yield.

[0015] The alkali metal hydroxide used in the method for producing oligonucleotides in this disclosure means at least one hydroxide selected from the group consisting of lithium, sodium, potassium, cesium, and francium hydroxides. In particular, in the method for producing oligonucleotides in this disclosure, it is preferable to use at least one hydroxide selected from the group consisting of lithium hydroxide (lithium hydroxide) and sodium hydroxide (sodium hydroxide), and it is more preferable to use lithium hydroxide (lithium hydroxide).

[0016] In the step of removing a pseudo-solid-phase protecting group from a nucleic acid compound, the nucleic acid compound is reacted with a solution containing an alkali metal hydroxide. The concentration of the alkali metal hydroxide is not particularly limited, but can be, for example, 0.1 M to 10 M, preferably 0.1 M to 5 M, more preferably 0.5 M to 5 M, even more preferably 0.5 M to 3 M, and still more preferably 0.8 M to 1.5 M. The reaction time is not particularly limited, but can be 10 minutes to 6 hours, preferably 15 minutes to 5 hours, more preferably 20 minutes to 4 hours, and even more preferably 30 minutes to 3 hours. The reaction of removing a pseudo-solid-phase protecting group from a nucleic acid compound can be stopped by adding a weak acid aqueous solution, such as saturated ammonium chloride aqueous solution, acetic acid aqueous solution, hydrogen fluoride aqueous solution, carbonic acid aqueous solution, hydrogen sulfide aqueous solution, oxalic acid aqueous solution, phosphoric acid aqueous solution, etc.

[0017] (Pseudo-solid protective group) The method for producing oligonucleotides according to this disclosure is not particularly limited, and conventionally known pseudo-solid protecting groups can be used. A pseudo-solid protecting group is a molecule that functions as a scaffold for oligonucleotide synthesis in a liquid-phase synthesis method, similar to a solid support in a solid-phase synthesis method of oligonucleotides, and is a molecule that has a hydrophobic group and is soluble in a non-polar solvent. In the method for producing oligonucleotides according to this disclosure, the pseudo-solid protecting group is usually attached to the hydroxyl group at the 3' position of the deoxyribose or ribose that constitutes the oligonucleotide, but the pseudo-solid protecting group may also be attached to the hydroxyl group at the 5' position of the deoxyribose or ribose.

[0018] [Pseudo-solid phase protecting group disclosed in Japanese Patent Publication No. 6770553] The pseudo-solid-phase protecting group disclosed in Japanese Patent Publication No. 6770553 can be a group formed by bonding the carboxyl group in the following general formula (Y) to the 3' or 5' position of the nucleoside, nucleotide, or oligonucleotide.

[0019] [ka]

[0020] In general formula (Y), R Y Each of these independently represents a linear or branched alkyl group with 10 to 40 carbon atoms. In the general formula (Y), m represents an integer from 1 to 5, and if m is 2 or greater, there are multiple R groups. Y O may be the same or different. In general formula (Y), X is an oxygen atom, a sulfur atom, NH, or NR. N This represents the general formula (Y), where n is an integer from 1 to 4. N represents a linear or branched alkyl group having 1 to 6 carbon atoms.

[0021] Compounds represented by general formula (Y) and pseudo-solid-phase protecting groups formed by said compounds are disclosed in Japanese Patent Publication No. 6770553. In the oligonucleotide production method of this disclosure, derivatives of the compound represented by general formula (Y) can also be used as compounds for forming pseudo-solid-phase protecting groups, as long as they do not impair the effects. Examples of derivatives include compounds in which the OH group portion of the carboxyl group (-COOH) of general formula (Y) is replaced by a substituent. Examples of substituents include substituents with high leaving ability that facilitate bonding with nucleosides or nucleotides, such as halogen atoms like F, Cl, Br, and I, or alkyl ester structures substituted with alkoxy groups. In addition, known leaving groups may be used as substituents as appropriate.

[0022] In the general formula (Y), R Y Each of these independently represents a linear or branched alkyl group having 10 to 40 carbon atoms, which may be substituted. Y The alkyl group may be a linear or branched alkyl group having 13 to 30 carbon atoms, or a linear or branched alkyl group having 15 to 20 carbon atoms, with a linear or branched alkyl group having 18 carbon atoms being particularly preferred. Y If multiple R are present, each may independently be a linear or branched alkyl group, but a linear alkyl group is preferred.Y Because the alkyl group has 10 to 40 carbon atoms, it readily binds to the hydroxyl group at the 3' position of deoxyribose or ribose, and also improves solubility and separation efficiency.

[0023] In the general formula (Y), m represents an integer from 1 to 5. If m is 2 or greater, there are multiple R Y O may be the same or different. In particular, m is preferably an integer between 2 and 4, and more preferably m is 3. Also, R Y The oxygen group is preferably attached to the 3rd, 4th, or 5th position when the carbonyl group (C=O group) directly attached to the phenyl group is considered to be at position 1. When m=3, it is preferably attached to positions 3, 4th, and 5. Furthermore, by setting the value of m within the above range, the solubility of the oligonucleotide with the pseudo-solid-phase protecting group attached in nonpolar solvents can be adjusted.

[0024] In general formula (Y), X is an oxygen atom or a sulfur atom, but is an NH group or an NR group. N However, this is also acceptable. In particular, it is preferable that X is an oxygen atom. N R represents a linear or branched, optionally substituted, alkyl group having 1 to 6 carbon atoms. N Examples of such groups include methyl group, ethyl group, 1-propyl group, 1-methyl-1-ethyl group, 1-butyl group, 1-methyl-1-propyl group, 1,1-dimethyl-1-ethyl group, 2-methyl-1-propyl group, 1-pentyl group, and 2-pentyl group.

[0025] In the general formula (Y), n represents an integer from 1 to 4. In particular, n is preferably 1 or 2.

[0026] In particular, as a pseudo-solid-phase protecting group, a group formed by the compound represented by formula (B) among the compounds represented by the general formula (Y) above can be used.

[0027] [ka]

[0028] Furthermore, in the method for producing oligonucleotides of this disclosure, the pseudo-solid protecting group is not limited to a group formed by a compound represented by general formula (Y), but may be a group formed by, for example, the following compounds. For example, a pseudo-solid protecting group comprising a group formed by a compound represented by formula (A) disclosed in Japanese Patent Publication No. 2022-68360 can be used. Furthermore, a pseudo-solid protecting group comprising a group formed by a compound represented by formula (C) disclosed in Japanese Patent Publication No. 7433684 can be used. Furthermore, a pseudo-solid protecting group comprising a group formed by a compound represented by formula (D) disclosed in International Publication No. 2018 / 203574 can be used. Furthermore, a pseudo-solid protecting group comprising a group formed by a compound represented by formula (E) disclosed in Japanese Patent Publication No. 2023-551647 can be used. Furthermore, a pseudo-solid protecting group comprising a group formed by a compound represented by formula (F) disclosed in International Publication No. 2021 / 039935 can be used.

[0029] [Pseudo-solid phase protective group disclosed in Japanese Patent Publication No. 2022-68360] Here, the pseudo-solid phase protecting group disclosed in Japanese Patent Publication No. 2022-68360 is -LY L It is represented by -Z or -Z. In the formula, L is given by formula (a1):

[0030] [ka]

[0031] During the ceremony, ** This indicates the binding position with the 3' or 5' position of the oligonucleotide; * Y L The bond position is indicated; L1 represents a substituted or substituted divalent hydrocarbon group having 1 to 22 carbon atoms, an oxygen atom, or -NR- (where R represents a hydrogen atom, an alkyl group, or an aralkyl group); and L2 represents a single bond or formula: * C(R 3a )(R3b )-OR 1** ,formula: * C(=O)N(R 2 )-R 1 -N(R 3a ) ** Alternatively, formula: * C(=O)N(R 2 )-R 1 -C(R 3a )(R 3b ) ** (In the formula, * This indicates the binding site with L1. ** CR c R d The binding position with R is shown, 1 R represents an alkylene group having 1 to 22 carbon atoms, which may be substituted. 2 and R 3a Each independently represents a hydrogen atom or an optionally substituted C1-C22 alkyl group, or R 2 and R 3a Together, they may form an alkylene bond with 1 to 22 carbon atoms, which may be substituted. 3b R represents a group represented by a hydrogen atom or an alkyl group having 1 to 22 carbon atoms, which may be substituted; c and R d Each of these is independently a hydrogen atom, an optionally substituted alkyl group having 1 to 22 carbon atoms, or R c and R d It represents a group (linker) that may be formed by combining with a single carbonyl group; Y L This represents a single bond, an oxygen atom, -NR- (where R represents a hydrogen atom, an alkyl group, or an aralkyl group), or a sulfur atom; Z is given by equation (a2):

[0032] [ka]

[0033] (In the formula, * Y L This indicates the bonding position with; R 4This indicates a hydrogen atom, or R b If the group is represented by the following formula (a3), then R 6 They may form a single bond or an -O- together with ring B, or they may form a fluorenyl group or a xanthenyl group together with ring B; k R 5 Each of the following independently represents an organic group having an aliphatic hydrocarbon group with 10 or more carbon atoms; k represents an integer from 1 to 4; and ring A represents k ORs. 5 In addition, the substituents may be selected from the group consisting of halogen atoms, C1-C6 alkyl groups which may be substituted with halogen atoms, and C1-C6 alkoxy groups which may be substituted with halogen atoms; R a R represents a phenyl group which may be substituted with a hydrogen atom or a halogen atom; b is a hydrogen atom, or formula (a3):

[0034] [ka]

[0035] (In the formula, * indicates the join position; j indicates an integer from 0 to 4; j R 7 These independently represent organic groups having an aliphatic hydrocarbon group with 10 or more carbon atoms; R 6 This indicates a hydrogen atom, or R 4 They may form a single bond or an -O- together with ring A, or form a fluorenyl group or a xanthenyl group together with ring A; and ring B may have j OR 7 In addition, the substituents may be selected from the group consisting of halogen atoms, C1-C6 alkyl groups which may be substituted with halogen atoms, and C1-C6 alkoxy groups which may be substituted with halogen atoms; R a and R b These may combine to form a single carbonyl group. (The group represented by formula (a2'):

[0036] [ka]

[0037] (In the formula, * Y L The bond position is indicated by; the definition of each other symbol is the same as that of formula (a2). ) or formula (a2''): [ka]

[0038] (In the formula, * Y L The bond positions are shown; ring A' consists of k OR 5 In addition, the group may further have substituents selected from the group consisting of halogen atoms, C1-C6 alkyl groups which may be substituted with halogen atoms, and C1-C6 alkoxy groups which may be substituted with halogen atoms; the definition of each symbol is the same as that of formula (a2).

[0039] In particular, as a pseudo-solid-phase protecting group disclosed in Japanese Patent Publication No. 2022-68360, we can mention a group formed by the bonding of the carboxyl group of a compound represented by the following formula (A) to the 3' or 5' position of the nucleoside, nucleotide, or oligonucleotide.

[0040] [ka]

[0041] [Pseudo-solid phase protecting group disclosed in Japanese Patent Publication No. 7433684] The pseudo-solid-phase protecting group disclosed in Japanese Patent Publication No. 7433684 is a group formed by a compound represented by the general formula (1): MO-(O=)CC(=O)-W1-X1-Y1-Z1···(1). The pseudo-solid-phase protecting group of general formula (1) has an oxalic acid structure "MO-(O=)CC(=O)-W1" at the molecular terminus attached to the 3'-hydroxyl group of a nucleoside or oligonucleotide.

[0042] In general formula (1), the "-OM" that constitutes one end of the oxalic acid structure represents a bonding group that attaches to the 3'-hydroxyl group of a nucleoside or oligonucleotide. "M" represents a hydrogen atom or an alkali metal atom. The type of alkali metal is not particularly limited and includes, for example, lithium, sodium, potassium, rubidium, cesium, francium, etc. Potassium is preferred.

[0043] In general formula (1), "W1" which constitutes the other end of the oxalic acid structure is an oxygen atom, a sulfur atom, or NR Z This indicates that "N" represents a nitrogen atom. Z " represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms (the same applies hereinafter). Among "W1", an oxygen atom is preferred.

[0044] Examples of C1-C6 alkyl groups include linear or branched alkyl groups such as methyl, ethyl, propyl, isopropyl, butyl, pentyl, and hexyl groups. Among C1-C6 alkyl groups, linear alkyl groups are preferred, and methyl groups are more preferred. Note that -W1-X1-(W1 is NR) Z (This indicates) a divalent heterocyclic group may be formed.

[0045] In general formula (1), "X1" (-X1-) represents a single bond; as a divalent group, it represents an alkylene group having 1 to 12 carbon atoms (which may have an ether bond (-O-)), an arylene group (which may be substituted with an alkylene group having 1 to 3 carbon atoms), or a divalent heterocyclic group.

[0046] Examples of alkylene groups having 1 to 12 carbon atoms include linear or branched alkylene groups such as methylene, ethylene, propylene, butylene, isobutylene, tert-butylene, sec-butylene, pentylene, isopentylene, neopentylene, hexylene, isohexylene, 3-methylpentylene, heptylene, octylene, nonylene, decylene, undecylene, and dodecylene.

[0047] Alkylene groups with 1 to 12 carbon atoms that have an ether bond are -(CH2) p -O-(CH2) p - Represented by a group (a dialkylene group having an ether bond). p is an integer from 1 to 6, preferably an integer from 1 to 3. Examples include the -CH2CH2-O-CH2CH2- group (a diethylene group having an ether bond).

[0048] An arylene group is a monocyclic or bicyclic to hexacyclic aromatic hydrocarbon (e.g., benzene, naphthalene, anthracene, phenanthrene, dihydrophenanthrene, or fluorene) from which two hydrogen atoms directly bonded to the carbon atoms constituting the ring have been removed. An example is the phenylene group (-C6H4-).

[0049] Arylene groups substituted with C1-C3 alkylene groups (or having C1-C3 alkylene groups) are groups in which two hydrogen atoms directly bonded to the carbon atoms constituting the above ring are each replaced by C1-C3 alkylene groups. For example, -(CH2) q -C6H4-(CH2) q - Groups (such as dimethylene-substituted phenylene groups and xylene groups in which the methyl group is replaced by a methylene group) are examples. q represents an integer from 1 to 3.

[0050] A divalent heterocyclic group is a group obtained by removing two hydrogen atoms directly bonded to the ring-constituting atoms (preferably carbon atoms and / or nitrogen atoms) from a monocyclic or bicyclic to hexacyclic heterocyclic compound (e.g., pyridine, pyrimidine, pyrazine, pyrrolidine, piperidine, piperazine, etc.). Examples include a divalent piperidine ring structure-containing group (hereinafter also referred to as "-(piperi)-") and a divalent piperazine ring structure-containing group (hereinafter also referred to as "-(pipera)-"). Note that a divalent piperazine ring structure-containing group is denoted as -W1-X1-(W1 is NR). Z It may consist of (which is also referred to as "-N(pipera)-" below).

[0051] In general formula (1), "Y1" (-Y1-) is a single bond; an ether bond (-O-), -OCO- (-OC(=O)-), -NR Z CO- (-(R Z )NC(=O)-), -CO- (-C(=O)-) or -SCO- (-SC(=O)-) as a divalent group.

[0052] In general formula (1), "Z1" (-Z1) represents an alkoxyphenyl group or an alkoxyalkyl group having an alkoxy group with 10 to 30 carbon atoms. The alkoxyphenyl group having an alkoxy group with 10 to 30 carbon atoms represents a phenyl group in which a hydrogen atom directly bonded to a carbon atom constituting the benzene ring is substituted with an alkoxy group with 10 to 30 carbon atoms. For example, a dialkoxyphenyl group to which two alkoxy groups are bonded, a trialkoxyphenyl group to which three alkoxy groups are bonded, etc. can be mentioned. Among these, a trialkoxyphenyl group is preferable. The alkoxyalkyl group having an alkoxy group with 10 to 30 carbon atoms represents an alkyl group in which a hydrogen atom directly bonded to a carbon atom constituting a linear or branched alkyl group with 1 to 6 carbon atoms (the same as above) is substituted with an alkoxy group with 10 to 30 carbon atoms. For example, an alkoxymethyl group, an alkoxyethyl group, an alkoxypropyl group, a dialkoxyisopropyl group, an alkoxybutyl group, an alkoxypentyl group, an alkoxyhexyl group can be mentioned. Among these, a dialkoxyisopropyl group to which two alkoxy groups are bonded is preferable.

[0053] In addition, as the pseudo-solid phase protecting group, a group formed by a compound represented by formula (C) among the compounds represented by the above general formula (1) disclosed in Japanese Patent No. 7433684 can be used.

[0054] [Chemical formula]

[0055] [Pseudo-solid phase protecting group disclosed in International Publication No. 2018 / 203574] The pseudo-solid-phase protecting group disclosed in International Publication No. 2018 / 203574 is a group represented by general formula (II).

[0056] [ka]

[0057] In the formula, * indicates the bond position with the hydroxyl group, and R X1 Each of these is independently an alkyl group having 1 to 40 carbon atoms, an alkenyl group having 2 to 40 carbon atoms, an alkynyl group having 2 to 40 carbon atoms, or a group of the formula: -L 7 -OR X4 (In the formula, L 7 R is an alkylene group having 1 to 40 carbon atoms, an alkenylene group having 2 to 40 carbon atoms, or an alkynylene group having 2 to 40 carbon atoms. X4 (where is an alkyl group having 1 to 40 carbon atoms, an alkenyl group having 2 to 40 carbon atoms, or an alkynyl group having 2 to 40 carbon atoms), and at least one R X1 The number of carbon atoms is 10 or more, s is an integer from 1 to 5, and L 11 L is an alkylene group having 1 to 6 carbon atoms, an alkenylene group having 2 to 6 carbon atoms, or an alkynylene group having 2 to 6 carbon atoms. 12 This is a single bond, -O- or -N(R X2 )-(wherein, R X2 (represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, a haloalkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, or a haloalkenyl group having 2 to 6 carbon atoms), L 13 L is a single bond, -C(O)-, or an alkylene group having 1 to 6 carbon atoms. 12 When it is a single bond, L 13 It is a single bond or -C(O)-.

[0058] Typical examples of pseudo-solid protecting groups represented by general formula (II) include, for example, the 3,4,5-tris(octadecyloxy)benzyloxysuccinyl group, (3-{3,4,5-tris(octadecyloxy)benzyloxycarbonyl}propanoyl group), 3,4,5-tris(octadecyloxy)benzoyl group, 4-oxo-4-(2,4,6-tris(octadecyloxy)phenyl)butyryl group, and 2-(3,4,5-tris(octadecyloxy)benzamide)ethoxysuccinyl group (3-[2-{3,4,5- Tris(octadecyloxy)benzamide}ethoxycarbonyl]propanoyl group), 2-(N-methyl-3,4,5-tris(octadecyloxy)benzamide)ethoxysuccinyl group (3-[2-{N-methyl-3,4,5-tris(octadecyloxy)benzamide}ethoxycarbonyl]propanoyl group), (N-methyl-3,4,5-tris(octadecyloxy)benzamide)acetyl group, ((3,4,5-tris(octadecyloxy)benzoyl)oxy)methyl group, 2-(N-methyl-3,4,5- Tris(octadecyloxy)benzamide)ethoxysuccinyloxymethyl group ({(3-[2-{N-methyl-3,4,5-tris(octadecyloxy)benzamide}ethoxycarbonyl]propanoyl)oxy}methyl group), 5-(3,4,5-tris(octadecyloxy)benzoyloxy)-4-oxo-pentanoyl group, 5-(2,4,6-tris(octadecyloxy)benzoyloxy)-4-oxo-pentanoyl group, 4-oxo-6-(3,4,5-tris(octadecyloxy)phenyl) Examples include the xanoyl group, (E)-4-oxo-6-(3,4,5-tris(octadecyloxy)phenyl)-5-hexenoyl group, diphenyl(3,4,5-tris(octadecyloxy)phenyl)methyl group, diphenyl(3,4-di(docosyloxy)phenyl)methyl group, diphenyl(3,5-di(docosyloxy)phenyl)methyl group, bis(4-(docosyloxy)phenyl)phenylmethyl group, and (4-((12-(octadecyloxy)dodecyl)oxy)phenyl)diphenylmethyl group.

[0059] Furthermore, as a pseudo-solid-phase protecting group, a group formed by the compound represented by formula (D) among the compounds represented by the general formula (II) disclosed in International Publication No. 2018 / 203574 can be used.

[0060] [ka]

[0061] [Pseudo-solid phase protective group disclosed in Japanese Patent Publication No. 2023-551647] The pseudo-solid-phase protecting group disclosed in Japanese Patent Publication No. 2023-551647 is a group formed by a compound of general formula (I').

[0062] [ka]

[0063] In the formula, ring D is a 5-6 member heteroaryl having 1-3 heteroatoms independently selected from phenyl, an 8-10 member bicyclic aryl, oxygen, nitrogen, and sulfur, or a 7-10 member bicyclic heteroaryl having 1-4 heteroatoms independently selected from oxygen, nitrogen, and sulfur, and Y2 is a hydrogen atom, halogen, OR 1A , NR 2A R 3A , SR 4A CR 5A R 6A R 7A , or a hydrophobic group containing one or more aliphatic hydrocarbon groups having 10 or more carbon atoms, R 1A , R 2A , R 3A , R 4A , R 5A , R 6A , and R 7A These are, independently, alkyl groups with 1 to 6 carbon atoms, alkenyl groups with 1 to 6 carbon atoms, alkynyl groups with 1 to 6 carbon atoms, phenyl groups, OR groups. 8A ,-OC(O)R 8A , -C(O)OR 8A , NR 8A R 9A , -NR 8ACOR 9A 、 -CONR 8A R 9A 、 a 3- to 7-membered saturated or partially unsaturated monocyclic carbocyclyl, or a 3- to 7-membered saturated or partially unsaturated monocyclic heterocyclyl having 1 to 2 heteroatoms independently selected from oxygen, nitrogen, and sulfur, where R 8A and R 9A are, each occurrence, independently a hydrogen atom or an alkyl having 1 to 6 carbon atoms, P1 is NO2 or a silyl hydroxyl protecting group, R1 and R2 are independently a hydrogen atom, an alkyl having 1 to 6 carbon atoms, or phenyl, the alkyl having from 1 to 6 carbon atoms and phenyl are optionally substituted by 1 to 3 R3s, R3 is an alkoxy having 1 to 30 carbon atoms, e is an integer from 0 to 6, and f is an integer from 0 to 6. [[ID=eleven]] [[ID=twelve]]

[0064] [[ID=thirteen]] [[ID=fourteen]]In the compound of general formula (I’), P1 can be selected from the group consisting of -O-TBDMS, -O-TIPS, -O-TBDPS, -O-TBoDPS, and -O-TBDAS. [[ID=fifteen]] [[ID=sixteen]]

[0065] [[ID=seventeen]] [[ID=eighteen]] [[ID=nineteen]]

Chemical formula

[0066] [[ID=twenty-five]] [[ID=twenty-six]]Also, as the pseudo-solid phase protecting group, a group formed from the compound represented by formula (E) among the compounds represented by the above general formula (I’) disclosed in Japanese Patent Application Laid-Open No. 2023-551647 can be used. [[ID=twenty-seven]] [[ID=twenty-eight]]

[0067] [[ID=twenty-nine]] [[ID=thirty]] [[ID=thirty-one]]

Chemical formula

[0068] [[ID=thirty-seven]] [[ID=thirty-eight]][Pseudo-solid phase protecting group disclosed in International Publication No. 2021 / 039935][[ID=thirty-nine]] [[ID=forty]]The pseudo-solid phase protecting group disclosed in International Publication No. 2021 / 039935 is a group represented by the following general formula (1). [[ID=forty-one]] [[ID=forty-two]]

[0069] [[ID=forty-three]] [[ID=forty-four]] [[ID=forty-five]]

Chemical formula

[0070] In general formula (1), ring E represents a fused polycyclic aromatic hydrocarbon ring or an aromatic heterocycle, and Y A *-OCR 2A -**, *-NRCR 2A -**, or *-SCR 2A -** represents R 2A Each of these independently represents a hydrogen atom, an alkyl group, an aromatic group, or an aromatic group-substituted alkyl group, k1 represents an integer from 1 to 5, the dashed portion and * represent the bond position with other structures, ** represents the bond position with ring E, and R A Each of these is independently an aliphatic hydrocarbon group or an organic group having an aliphatic hydrocarbon group, and k1 R A The number of carbon atoms in at least one aliphatic hydrocarbon group in is 12 or more, and ring E is Y A and R A In addition, it may have further substituents.

[0071] Furthermore, as a pseudo-solid-phase protecting group, a group formed by the compound represented by formula (F) among the compounds represented by the general formula (1) disclosed in International Publication No. 2021 / 039935 can be used.

[0072] [ka]

[0073] As explained above, examples of pseudo-solid-phase protecting groups include groups formed by the bonding of a carboxyl group of a compound selected from the group consisting of compounds represented by any of the above formulas (A) to (F) to the 3' or 5' position of the nucleoside, nucleotide, or oligonucleotide. These compounds also bond to the carboxyl group in the molecule to the 3' or 5' position of the nucleoside, nucleotide, or oligonucleotide.

[0074] (nucleic acid compound) In the method for producing oligonucleotides according to this disclosure, the nucleic acid compound is one in which the above-described pseudo-solid protecting group is linked to the 3' or 5' position of a nucleoside, nucleotide, or oligonucleotide. When the pseudo-solid protecting group is linked to the 3' position of a nucleoside, nucleotide, or oligonucleotide, the 3' position of the next nucleoside, nucleotide, or oligonucleotide is linked to the 5' position of the nucleoside, nucleotide, or oligonucleotide linked to the pseudo-solid protecting group, and thereafter, nucleosides, nucleotides, or oligonucleotides are sequentially linked from the 5' position toward the 3' position.

[0075] In other words, the nucleic acid compounds relating to this disclosure can be represented by the following general formula (X).

[0076] [ka]

[0077] In general formula (X), Base represents an optionally modified nucleic acid base. If there are two or more Bases, the multiple Bases may be the same or different. In general formula (X), B represents a hydrogen atom, a halogen atom, or an optionally protected hydroxyl group. If there are two or more Bs, the multiple Bs may be the same or different. In general formula (X), W is a hydrogen atom or a 5' protecting group. In general formula (X), R X R represents an alkyl group having 1 to 7 carbon atoms, which has an ethyl group, allyl group, aryl group, or electron-withdrawing group at the 2nd position. X If there are two or more of them, then there are multiple R X Q may be the same or different. In general formula (X), Q represents an oxygen atom or a sulfur atom. If there are two or more Qs, the multiple Qs may be the same or different. In general formula (X), y1 represents any integer greater than or equal to 0. In general formula (X), * represents the site to which the pseudo-solid protecting group described above is bonded.

[0078] The term "nucleic acid base" is not particularly limited as long as it is used in the synthesis of nucleic acids. Examples include pyrimidine bases such as cytosyl, uracil, and thyminyl groups, and purine bases such as adenyl and guanyl groups. Furthermore, "nucleic acid bases that may be modified" includes, in addition to the groups mentioned above, modified nucleic acid bases in which the nucleic acid base is substituted with 1 to 3 substituents at any position (e.g., halogen atoms, alkyl groups, aralkyl groups, alkoxy groups, acyl groups, alkoxyalkyl groups, hydroxyl groups, amino groups, monoalkylamino groups, dialkylamino groups, carboxyl groups, cyano groups, nitro groups, etc.) (e.g., 8-bromoadenyl group, 8-bromoguanyl group, 5-bromocytosyl group, 5-iodocytosyl group, 5-bromouracil group, 5-iodouracil group, 5-fluorouracil group, 5-methylcytosyl group, 8-oxoguanyl group, hypoxanthinyl group, etc.).

[0079] Furthermore, the term "nucleic acid bases that may be modified" also includes nucleic acid bases to which protecting groups are attached. For example, in nucleic acid bases having an amino group, such as an adenyl group, a guanyle group, or a cytosyl group, the amino group may be protected, or the amino group of the nucleic acid base may be protected by a protecting group that can withstand deprotection conditions at the 5' position. Specific examples of such "amino group protecting groups" include, for example, pivaloyl group, pivaloyloxymethyl group, trifluoroacetyl group, phenoxyacetyl group, 4-isopropylphenoxyacetyl group, 4-tert-butylphenoxyacetyl group, acetyl group, benzoyl group, isobutyryl group, dimethylformamidinyl group, and 9-fluorenylmethyloxycarbonyl group. Among these, phenoxyacetyl group, 4-isopropylphenoxyacetyl group, acetyl group, benzoyl group, isobutyryl group, and dimethylformamidinyl group are preferred. Furthermore, the carbonyl group of the nucleic acid base may be protected. For example, the carbonyl group can be protected by reacting it with phenol, 2,5-dichlorophenol, 3-chlorophenol, 3,5-dichlorophenol, 2-formylphenol, 2-naphthol, 4-methoxyphenol, 4-chlorophenol, 2-nitrophenol, 4-nitrophenol, 4-acetylaminophenol, pentafluorophenol, 4-pivaloyloxybenzyl alcohol, 4-nitrophenethyl alcohol, 2-(methylsulfonyl)ethanol, 2-(phenylsulfonyl)ethanol, 2-cyanoethanol, 2-(trimethylsilyl)ethanol, dimethylcarbamate chloride, diethylcarbamate chloride, ethylphenylcarbamate chloride, 1-pyrrolidinecarboxylic acid chloride, 4-morpholinecarboxylic acid chloride, diphenylcarbamate chloride, etc. However, the carbonyl group may not necessarily need to be introduced. As protecting groups for nucleic acid bases, the protecting groups described in WO2013 / 122236 (groups having linear or branched alkyl groups with 5 to 30 carbon atoms and / or linear or branched alkenyl groups with 5 to 30 carbon atoms) can also be used.

[0080] In general formula (X), with respect to B, the term "a hydroxyl group that may be protected" means that a protecting group is bonded to the hydroxyl group. The protecting group for the hydroxyl group shown as B is not particularly limited, and examples include methyl, benzyl, p-methoxybenzyl, tert-butyl, methoxymethyl, methoxyethyl, 2-tetrahydropyranyl, ethoxyethyl, cyanoethyl, cyanoethoxymethyl, phenylcarbamoyl, 1,1-dioxothiomorpholine-4-thiocarbamoyl, acetyl, pivaloyl, benzoyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, [(triisopropylsilyl)oxy]methyl (Tom), and 1-(4-chlorophenyl)-4-ethoxypiperidine-4-yl (Cpep) group. Among these, triethylsilyl group, triisopropylsilyl group, or tert-butyldimethylsilyl group are preferred, and tert-butyldimethylsilyl group is particularly preferred from the viewpoint of economy and ease of availability.

[0081] Furthermore, in general formula (X), B may be an organic group that bridges the carbon atom at position 4. Such organic groups are not particularly limited as long as they bridge the 2nd and 4th positions of the nucleoside, but examples include alkylene groups having 2 to 7 carbon atoms. These alkylene groups may be, for example, -O-, -NR B -(R here B (where represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), -S-, -CO-, -COO-, -OCONR B -(R here B (represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), -CONR B -(R here B The linker may be interrupted at one or more locations (preferably one or two locations) by a linker selected from a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.

[0082] In general formula (X), W is a hydrogen atom or a 5' protecting group. That is, when a pseudo-solid protecting group is linked to the 3' position in the synthesized oligonucleotide, the 5' position on the opposite side of the molecule may remain a hydroxyl group or a 5' protecting group may be linked thereto. The 5' protecting group is not particularly limited as long as it can be deprotected under acidic conditions and used as a protecting group for a hydroxyl group, but examples include trityl groups, 9-(9-phenyl)xanthenyl groups, 9-phenylthioxanthenyl groups, di(C1-C6 alkoxy)trityl groups such as 1,1-bis(4-methoxyphenyl)-1-phenylmethyl group (dimethoxytrityl group), and mono(C1-C18 alkoxy)trityl groups such as 1-(4-methoxyphenyl)-1,1-diphenylmethyl group (monomethoxytrityl group). Among these, monomethoxytrityl groups and dimethoxytrityl groups are preferred from the viewpoint of ease of deprotection and availability, and dimethoxytrityl groups are more preferred.

[0083] In general formula (X), R X R is a substance that acts as a protecting group for the phosphate group, and is not particularly limited as long as it can be deprotected under basic conditions, but it represents an ethyl group, allyl group, aryl group, or an alkyl group having 1 to 7 carbon atoms and having an electron-withdrawing group, with a cyano group at the 2 position. X If there are two or more of them, then there are multiple R X They may be the same or different. In particular, in general formula (X), R X It is preferable that it is an allyl group. X When is an allyl group, in the process for removing the pseudo-solid protecting group, R X Since it is not removed and has excellent reaction stability, and can be easily removed under specific reaction conditions such as palladium catalyst or sodium iodide, it is easy to synthesize high-purity oligonucleotides. X When the group is an allyl group, the phosphate esterification reaction can proceed more easily compared to a benzyl group, which can be removed under specific conditions, because it has less steric hindrance.

[0084] In the general formula (X), y1 represents any integer greater than or equal to 0, but preferably any integer greater than or equal to 1. In particular, y1 may be any integer between 1 and 30, any integer between 1 and 20, any integer between 1 and 10, any integer between 1 and 6, or any integer between 1 and 4.

[0085] (Process) The present disclosure's method for producing oligonucleotides involves first attaching a pseudo-solid protecting group to the 3' position of a nucleoside corresponding to the 3' end base of a pre-designed base sequence, or preparing a nucleoside with a pseudo-solid protecting group attached to its 3' position. In this case, oligonucleotides are synthesized sequentially from the 3' end to the 5' end of the pre-designed base sequence. Alternatively, a pseudo-solid protecting group may be attached to the 5' position of a nucleoside corresponding to the 5' end base of a pre-designed base sequence, and oligonucleotides are synthesized sequentially from the 5' end to the 3' end of the pre-designed base sequence. The following description will focus on a method in which a pseudo-solid protecting group is attached to the 3' position of a nucleoside corresponding to the 3' end base of a pre-designed base sequence, and oligonucleotides are synthesized sequentially from the 3' end to the 5' end of the designed base sequence.

[0086] Specifically, the hydroxyl group at the 3' position of a nucleoside, to which a 5' protecting group such as a 4,4-dimethoxytrityl group is bonded to the 5' position hydroxyl group, is bonded to a compound that acts as a pseudo-solid protecting group by a condensation reaction. The condensing agent is not particularly limited, but 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride or 1,1'-carbonyldiimidazole can be used. This allows a pseudo-solid protecting group to be linked to the 3' position of the nucleoside.

[0087] Next, the 5' protecting group can be removed by making a solution containing a nucleoside with a pseudo-solid protecting group linked to the 3' position acidic. Then, a coupling reaction is carried out using the phosphoramidite of the nucleoside corresponding to the next base by the so-called phosphoramidite method. After the coupling reaction, capping may be performed as needed. Furthermore, the phosphite ester bond generated in the coupling reaction is converted to a phosphate ester bond by an oxidation reaction. Then, by repeating the deprotection of the 5' position in the synthesized chain, the coupling reaction using phosphoramidite, the capping reaction, and the oxidation reaction, an oligonucleotide (nucleic acid compound) having the designed base sequence and with a pseudo-solid protecting group linked to the 3' position can be synthesized. It should be noted that the method for producing oligonucleotides in this disclosure is not limited to the method of coupling one base at a time as described above, but can also be used to couple oligonucleotides having a predetermined base sequence.

[0088] Next, a nucleic acid compound with a pseudo-solid protecting group linked to its 3' position is reacted with a solution containing an alkali metal hydroxide to remove only the pseudo-solid protecting group from the nucleic acid compound by hydrolysis. One embodiment of reacting a nucleic acid compound with a pseudo-solid protecting group linked to its 3' position with a solution containing an alkali metal hydroxide is to immerse the nucleic acid compound in the solution containing the alkali metal hydroxide. In the oligonucleotide production method of this disclosure, the use of an alkali metal hydroxide in this step can suppress the decomposition of the target oligonucleotide, and as a result, the synthesized oligonucleotide can be obtained with high purity. As described above, in the oligonucleotide production method of this disclosure, the pseudo-solid protecting group can be selectively removed from the nucleic acid compound by using a solution containing an alkali metal hydroxide.

[0089] Furthermore, the oligonucleotide manufacturing method of this disclosure allows for the efficient removal of pseudo-solid protecting groups from nucleic acid compounds, thereby improving the recovery rate of the target oligonucleotide. Moreover, since the pseudo-solid protecting groups can be removed from nucleic acid compounds by hydrolysis according to the oligonucleotide manufacturing method of this disclosure, the removed pseudo-solid protecting groups can be easily recovered and reused. That is, the oligonucleotide manufacturing method of this disclosure may include a step of recovering the removed pseudo-solid protecting groups after the step of removing the pseudo-solid protecting groups. In this case, since the pseudo-solid protecting groups are removed from the oligonucleotide by hydrolysis, the chemical structure of the pseudo-solid protecting groups can be maintained. Therefore, according to the oligonucleotide manufacturing method of this disclosure, the pseudo-solid protecting groups can be reused, the removed pseudo-solid protecting groups do not become waste reagents, and the costs associated with the pseudo-solid protecting groups can be kept low.

[0090] Furthermore, in the oligonucleotide production method of this disclosure, a protecting group attached to a nucleic acid base, a 5' protecting group attached to the 5' end of the oligonucleotide, etc., can be removed before or after the reaction to remove a pseudo-solid-phase protecting group from the nucleic acid compound.

[0091] <Synthesis reagent kit for oligonucleotides> On the other hand, the oligonucleotide synthesis reagent kit of the present disclosure comprises a compound that acts as a pseudo-solid protecting group by bonding to the 3' or 5' position of a nucleoside, nucleotide, or oligonucleotide; an alkali metal hydroxide; and at least one substrate selected from the group consisting of nucleosides, nucleotides, and oligonucleotides to which the compound integrates. In other words, the oligonucleotide synthesis reagent kit of the present disclosure is a kit containing reagents used in the above-described method for producing the oligonucleotide of the present disclosure.

[0092] The oligonucleotide synthesis reagent kit of this disclosure may include reagents containing a compound that acts as a pseudo-solid-phase protecting group, reagents containing an alkali metal hydroxide, and reagents containing at least one substrate selected from the group consisting of nucleosides, nucleotides, and oligonucleotides. Each reagent can be a solution obtained by dissolving the compound that acts as a pseudo-solid-phase protecting group, the alkali metal hydroxide, or the substrate in a suitable solvent. The compounds that act as a pseudo-solid-phase protecting group, the alkali metal hydroxide, and the substrate included in the oligonucleotide synthesis reagent kit of this disclosure may be those described above. [Examples]

[0093] The methods for producing oligonucleotides according to this disclosure will be described in more detail below with reference to examples, but the technical scope of this disclosure is not limited to the following examples.

[0094] (High-performance liquid chromatography measurement: HPLC) Column: Inertsil WP300 Diol (5μm, 4.6Φ × 150mm) Mobile phase: n-hexane, chloroform / methanol (1 / 1) Gradient: 0-11 min; 5 to 30% (chloroform / methanol) or 0-22 min; 5 to 40% (chloroform / methanol) Oven temperature: 30℃ Flow rate: 1.0mL / min Injection volume: 5μL Detection method: UV (λ=254nm)

[0095] (NMR measurement) A nuclear magnetic resonance spectrometer (ECZ400S, manufactured by JEOL Ltd.) was used.

[0096] (Mass spectrometry measurement) ESI-MS: Mass spectrometer (Waters Acquity UPLC H-class PLUS Bio System / Vion IMS Qtof) was used. FD-MS: A mass spectrometer (JMS-T200GC, manufactured by JEOL Ltd.) was used.

[0097] [Example 1] Synthesis of oligonucleotides (1) Synthesis of compound 1

[0098] [ka]

[0099] According to the method described in Japanese Patent Publication No. 7296636, 5'-O-(4,4'-dimethoxytrityl)thymidine (1.91 g, 3.51 mmol) and Fujimat (2.89 g, 2.93 mmol (registered trademark, manufactured by Fujimoto Chemical Products Co., Ltd.)) were placed in a round-bottom flask, tetrahydrofuran (THF) (29.3 mL) was added, and the mixture was stirred at room temperature. 1-methylimidazole (1.28 mL, 16.1 mmol) was added, and the mixture was stirred for a further 10 minutes. COMU (2.76 g, 6.44 mmol) was added, and the mixture was stirred for 3 hours. The reaction mixture was added dropwise to 120 mL of acetonitrile, and the precipitated solid was filtered, washed with acetonitrile, and then dried to obtain compound 1 (4.43 g, 100% yield).

[0100] (2) Synthesis of compound 2

[0101] [ka]

[0102] Compound 1 (4.43 g, 2.93 mmol) was placed in a round-bottom flask, and dichloromethane (117 mL) was added and the mixture was stirred at room temperature. Pyrrole (1.02 mL, 14.7 mmol) was added and the mixture was stirred for 10 minutes, then trifluoroacetic acid (0.29 mL, 3.81 mmol) was added and the mixture was stirred for 2 hours. The reaction mixture was added dropwise to 160 mL of an acetone-acetonitrile 1:3 (v / v) mixture, and the precipitated solid was filtered. The solid was further washed with acetonitrile and dried to obtain Compound 2 (3.59 g, 100% yield).

[0103] (3) Synthesis of compound 4

[0104] [ka]

[0105] Compound 2 (2.0 g, 1.65 mmol) and Compound 3 (2.42 g, 3.30 mmol) were placed in a round-bottom flask, anhydrous chloroform (27.6 mL) was added, and the mixture was stirred at room temperature. 5-benzylthiotetrazole (0.64 g, 3.31 mmol) was added, and the mixture was stirred for 1 hour. Then, pyridine (0.67 mL, 8.27 mmol) and [(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thione (1.02 g, 4.96 mmol) were added, and the mixture was stirred for 1 hour. 140 mL of acetonitrile was added dropwise to the reaction mixture, and the precipitated solid was filtered. The solid was washed with acetonitrile and then further washed with methanol. The solid was dried to obtain Compound 4 (2.93 g, 95% yield).

[0106] (4) Synthesis of compound 5

[0107] [ka]

[0108] Compound 4 (1.0 g, 0.53 mmol) was placed in a round-bottom flask and THF (21 mL) was added. The mixture was stirred at room temperature, and 1.61 mL of 1 M lithium hydroxide aqueous solution was added dropwise. After stirring for 1 hour, saturated ammonium chloride aqueous solution was added to stop the reaction. Then, distilled water and dichloromethane were added for extraction, and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure using an evaporator. The solid was crystallized by adding acetonitrile to the concentrate, and the resulting solid was removed by filtration. Compound 5 (0.46 g, yield 96%) was obtained by concentrating the acetonitrile. The HPLC measurement results before and after the reaction are shown in Figure 1, and the purity of compound 5 was 99 area%. In HPLC, peaks before a retention time of 2.5 minutes were excluded from the purity calculation as they were solvent-derived peaks.

[0109] 1 H-NMR (400MHz, CDCl3): δ7.57(d,1H),7.40-7.24(m,10H),6.85(d,4H),6.40-6.29(m,2H),5.90-5.79(m,1H),5.39-5.18(m,4H),4.61-4. 45(m,3H),4.31-4.07(m,4H),3.79(s,6H),3.45-3.24(m,2H),2.64(d t,1H),2.43-2.34(m,2H),2.20-2.14(m,1H),1,91(d,3H)1.48(d,3H)

[0110] [Example 2] Synthesis of oligonucleotides (1) Synthesis of compound 7

[0111] [ka]

[0112] According to the method of Example 1, N 4 Compound 7 was obtained using compound 3 from -benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine. The deprotection yield at the 3' position was 80%. The HPLC measurements before and after the reaction are shown in Figure 2, and the HPLC purity was 95 area%.

[0113] [Example 3] Synthesis of oligonucleotides Synthesis of compound 10

[0114] [ka]

[0115] According to the method of Example 1, N 4 Compound 10 was obtained using compound 9 from -benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine. The deprotection yield at the 3' position was 75%. The HPLC measurements before and after the reaction are shown in Figure 3, and the HPLC purity was 90 area%.

[0116] [Example 4] Synthesis of oligonucleotides Synthesis of compound 12

[0117] [ka]

[0118] Compound 12 was obtained from compound 9 using compound 11 according to Example 1(3,4). The deprotection yield at the 3' position was 65%. The HPLC measurement results before and after the reaction are shown in Figure 4, and the HPLC purity was 90 area%.

[0119] [Example 5] Synthesis of oligonucleotides Synthesis of compound 14

[0120] [ka]

[0121] From compound 6, a crude purified product of compound 14 was obtained using compound 13 and compound 3 according to Example 1 (3 and 4). Subsequently, purification was performed using a silica gel column (ethyl acetate / methanol = 100 / 0 to 95 / 5). The yield of the 3' deprotection reaction and silica gel column purification was 25%. HPLC measurements before and after the reaction are shown in Figure 5, and the HPLC purity was 99 area%. 1 H-NMR (400MHz, CDCl3): δ8.21-8.02(m,2H),7.93(t,6H),7.55-7.23(m,15H),6.84(d,6H),6.39-6.15(m,4H),5.96- 5.77(m,3H),5.43-5.20(m,9H),4.61-4.23(m,16H),3.78(s,6H),2.91-2.16(m,11H),1.90-1.86(m,3H),1.43(s,3H) ESI-MS: m / z 1801.4 [MH] -

[0122] [Example 6] Synthesis of oligonucleotides Compound 7 was synthesized under the same conditions as in Example 2, except that a 1M sodium hydroxide aqueous solution was used instead of a 1M lithium hydroxide aqueous solution in the 3' deprotection reaction. The yield of the 3' deprotection reaction was 98%. The HPLC measurement results before and after the reaction are shown in Figure 6, and the HPLC purity was 94 area%.

[0123] [Comparative Example 1] Synthesis of oligonucleotides The 3'-position deprotection reaction of Example 2 was carried out according to the conditions described in Japanese Patent No. 7296636. Specifically, a mixed solution of compound 6 with THF (9 mL)-2-propanol (1 mL) was cooled to 0°C. Under cooling, 4 mol / L lithium borohydride-THF solution (0.125 mL, 0.50 mmol) was added, and the mixture was stirred at the same temperature for 30 minutes. An aqueous ammonium chloride solution was added to the reaction mixture and extracted with dichloromethane. The organic layer was concentrated, and the residue was subjected to HPLC analysis, which showed an HPLC purity of 20%, as shown in Figure 7.

[0124] [Example 7] Recovery of pseudo-solid-phase protecting group (1) Deprotection reaction

[0125] [ka]

[0126] Compound 6 (0.8 g) was placed in a round-bottom flask and THF (16 mL) was added. The mixture was stirred at room temperature, and 1.21 mL of 1 M lithium hydroxide aqueous solution was added dropwise. After stirring, saturated ammonium chloride aqueous solution was added to stop the reaction. The mixture was then concentrated under reduced pressure using an evaporator. Extraction was performed by adding saturated ammonium chloride aqueous solution and chloroform, and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure using an evaporator. The solid was crystallized by adding acetonitrile to the concentrate, and the resulting solid was washed with acetonitrile and methanol and recovered by filtration to obtain compound 15 (0.32 g, yield 80%).

[0127] (2) Recovery of the carrier

[0128] [ka]

[0129] Next, compound 15 (0.1 g) was placed in a round-bottom flask and chloroform (9.0 mL) was added. The mixture was stirred at room temperature, and 0.1 M hydrochloric acid (5.0 mL) was added dropwise. After stirring vigorously for 1 minute, the aqueous layer was decanted. The above procedure was repeated once more, and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure using an evaporator. Methanol was added to the concentrate to crystallize the solid, and the resulting solid was washed with tanol and recovered by filtration to obtain compound 16 (0.088 g, yield 88%). As shown in Figure 8, compound 16 1 The H-NMR spectrum is from a commercially available Fujimat. 1 The 1H-NMR spectrum was consistent. In Figure 8, the upper panel shows the results of compound 16 recovered in this example. 1 The H-NMR spectrum (the peak around 3.5 ppm is the solvent peak) is shown in the lower panel, and the commercially available Fujimat spectrum is shown. 1 The 1H-NMR spectrum is shown. Furthermore, the mass spectrometry spectrum of compound 16 (Figure 9) was consistent with the mass spectrometry spectrum obtained using a commercially available Fujimat (Figure 10).

[0130] 1 H-NMR (400MHz, CDCl3): δ7.30(s,2H),4.86(s,2H),4.06-3.94(m,6H),1.87-1.66(m,6H),1.53-1.18(m,90H),0.88(t,3H) FD-MS:m / z 984.9 [M] +

Claims

1. A method for producing an oligonucleotide, comprising the step of reacting a nucleic acid compound having a pseudo-solid protecting group bonded to the 3' or 5' position of a nucleoside, nucleotide, or oligonucleotide with a solution containing an alkali metal hydroxide, thereby removing the pseudo-solid protecting group from the nucleic acid compound by a hydrolysis reaction.

2. The nucleic acid compound is represented by the following general formula (X), and the method for producing an oligonucleotide according to claim 1. 【Chemistry 1】 [In general formula (X), Base represents a nucleic acid base that may be modified. If there are two or more Bases, the multiple Bases may be the same or different. In general formula (X), B represents a hydrogen atom, a halogen atom, or an optionally protected hydroxyl group. If there are two or more B's, the multiple B's may be the same or different. In general formula (X), W is a hydrogen atom or a 5' protecting group. In general formula (X), R Ｘ R represents an alkyl group having 1 to 7 carbon atoms, having an ethyl group, allyl group, aryl group, or electron-withdrawing group with a cyano group at the 2nd position. Ｘ If there are two or more, then there are multiple R Ｘ They may be the same or different. In the general formula (X), Q represents an oxygen atom or a sulfur atom. If there are two or more Qs, the multiple Qs may be the same or different. In the general formula (X), y1 represents any integer greater than or equal to 0. In general formula (X), * represents the site to which the pseudo-solid-phase protecting group is bonded.

3. The method for producing an oligonucleotide according to claim 1, wherein the pseudo-solid protecting group is a group formed by bonding the carboxyl group in the following general formula (Y) to the 3' or 5' position of the nucleoside, the nucleotide, or the oligonucleotide. 【Chemistry 2】 [In general formula (Y), R Ｙ Each of these independently represents a linear or branched alkyl group having 10 to 40 carbon atoms. In the general formula (Y), m represents an integer from 1 to 5, and if m is 2 or greater, there are multiple R Ｙ O may be the same or different. In the general formula (Y), X is an oxygen atom, a sulfur atom, NH, or NR Ｎ It represents. In the general formula (Y), n represents an integer from 1 to 4. In general formula (Y), R Ｎ [This represents a linear or branched alkyl group having 1 to 6 carbon atoms.]

4. The aforementioned pseudo-solid phase protecting group is -L-Y Ｌ A method for producing an oligonucleotide according to claim 1, wherein the group is represented by -Z or -Z. [In the formula, L is given by formula (a1): 【Transformation 3】 (wherein, ＊＊ represents the bonding position with the 3'- or 5'-position of the oligonucleotide; ＊ is Y Ｌ represents the bonding position with; L １ represents an optionally substituted divalent hydrocarbon group having 1 to 22 carbon atoms, an oxygen atom, -NR- (R represents a hydrogen atom, an alkyl group or an aralkyl group); L ２ represents a single bond or a formula: ＊ C(R ３ａ )(R ３ｂ )-O-R １＊＊ , a formula: ＊ C(=O)N(R ２ )-R １ -N(R ３ａ ) ＊＊ or a formula: ＊ C(=O)N(R ２ )-R １ -C(R ３ａ )(R ３ｂ ) ＊＊ (wherein, ＊ represents the bonding position with L １ , ＊＊ represents the bonding position with CR ｃ R ｄ represents the bonding position with; R １ represents an optionally substituted alkylene group having 1 to 22 carbon atoms, R ２ and R ３ａ each independently represent a hydrogen atom or an optionally substituted alkyl group having 1 to 22 carbon atoms or R ２ and R ３ａ may together form an optionally substituted alkylene bond having 1 to 22 carbon atoms, R ３ｂ represents a hydrogen atom or an optionally substituted alkyl group having 1 to 22 carbon atoms) represents a group; R ｃ and R ｄ each independently represent a hydrogen atom, an optionally substituted alkyl group having 1 to 22 carbon atoms or R ｃ and R ｄ may together form a single carbonyl group) represents a group (linker); In the formula, Y Ｌ This represents a single bond, an oxygen atom, -NR- (where R represents a hydrogen atom, an alkyl group, or an aralkyl group), or a sulfur atom; In the equation, Z is given by equation (a2): 【Chemistry 4】 (In the formula, ＊ Y Ｌ This indicates the bonding position with; R ４ This indicates a hydrogen atom, or R ｂ If the group is represented by the following formula (a3), then R ６ They may form a single bond or an -O- together with ring B, or form a fluorenyl group or a xanthenyl group together with ring B; k R ５ Each independently represents an organic group having an aliphatic hydrocarbon group with 10 or more carbon atoms; k represents an integer from 1 to 4; and ring A represents k OR ５ In addition, it may further have substituents selected from the group consisting of halogen atoms, C1-C6 alkyl groups which may be substituted with halogen atoms, and C1-C6 alkoxy groups which may be substituted with halogen atoms; R ａ R represents a phenyl group which may be substituted with a hydrogen atom or a halogen atom; ｂ is a hydrogen atom, or formula (a3): 【Transformation 5】 (In the formula, ＊ indicates the joint position; j indicates an integer from 0 to 4; j R ７ Each independently represents an organic group having an aliphatic hydrocarbon group with 10 or more carbon atoms; R ６ This indicates a hydrogen atom, or R ４ They may form a single bond or an -O- together with ring A, or form a fluorenyl group or a xanthenyl group together with ring A; and ring B may have j OR ７ In addition, it may further have substituents selected from the group consisting of halogen atoms, C1-C6 alkyl groups which may be substituted with halogen atoms, and C1-C6 alkoxy groups which may be substituted with halogen atoms; R ａ and R ｂ These may come together to form a single carbonyl group. (The group represented by ) Formula (a2'): 【Transformation 6】 (In the formula, ＊ Y Ｌ The bond position is indicated by; the definition of each other symbol is the same as that of formula (a2). ) or formula (a2''): 【Transformation 7】 (In the formula, ＊ Y Ｌ The bond position is shown; ring A' has k OR ５ In addition, the group may further have substituents selected from the group consisting of halogen atoms, C1-C6 alkyl groups which may be substituted with halogen atoms, and C1-C6 alkoxy groups which may be substituted with halogen atoms; the definition of each symbol is the same as that of formula (a2).

5. The method for producing an oligonucleotide according to claim 1, wherein the pseudo-solid protecting group is a group formed by bonding -OM in the following general formula (1) to the 3' or 5' position of the nucleoside, nucleotide, or oligonucleotide. General formula (1): MO-(O=)CC(=O)-W １ -X １ -Y １ -Z １ ... (1) [In general formula (1), M represents a hydrogen atom or an alkali metal atom, W １ is an oxygen atom, a sulfur atom or NR Ｚ (N represents a nitrogen atom, R Ｚ (represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), X １ This represents a single bond, an alkylene group having 1 to 12 carbon atoms (which may have an ether bond (-O-)), an arylene group (which may be substituted with an alkylene group having 1 to 3 carbon atoms), or a divalent heterocyclic group. Y １ These are single bonds, ether bonds, -OCO-, and -NR Ｚ CO-(R Ｚ (The same as above), indicates -CO- or -SCO-, Z １ This represents an alkoxyphenyl group or alkoxyalkyl group having an alkoxy group with 10 to 30 carbon atoms.

6. The method for producing an oligonucleotide according to claim 1, wherein the pseudo-solid-phase protecting group is a group represented by general formula (II). 【Transformation 8】 [In general formula (II), * indicates the binding position to the 3' or 5' position of the nucleoside, nucleotide, or oligonucleotide.] R Ｘ１ Each of these is independently an alkyl group having 1 to 40 carbon atoms, an alkenyl group having 2 to 40 carbon atoms, an alkynyl group having 2 to 40 carbon atoms, or a group of the formula: -L ７ -O-R Ｘ４ (In the formula, L ７ R is an alkylene group having 1 to 40 carbon atoms, an alkenylene group having 2 to 40 carbon atoms, or an alkynylene group having 2 to 40 carbon atoms. Ｘ４ (where is an alkyl group having 1 to 40 carbon atoms, an alkenyl group having 2 to 40 carbon atoms, or an alkynyl group having 2 to 40 carbon atoms), and at least one R Ｘ１ The number of carbon atoms is 10 or more. s is an integer from 1 to 5. L １１ This is an alkylene group having 1 to 6 carbon atoms, an alkenylene group having 2 to 6 carbon atoms, or an alkylene group having 2 to 6 carbon atoms. L １２ is a single bond, -O- or -N(R) Ｘ２ )-(wherein, R Ｘ２ (represented by a hydrogen atom, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C2-C6 alkenyl group, or a C2-C6 haloalkenyl group), L １３ This is a single bond, -C(O)-, or an alkylene group having 1 to 6 carbon atoms. L １２ When L is a single bond, １３ L is a single bond or -C(O)-.

7. The method for producing an oligonucleotide according to claim 1, wherein the pseudo-solid protecting group is a group formed by bonding a carboxyl group in a compound represented by general formula (I') to the 3' or 5' position of the nucleoside, nucleotide, or oligonucleotide. 【Chemistry 9】 [In general formula (I'), ring D is a 5-6 member heteroaryl having 1-3 heteroatoms independently selected from phenyl, an 8-10 member bicyclic aryl, oxygen, nitrogen, and sulfur, or a 7-10 member bicyclic heteroaryl having 1-4 heteroatoms independently selected from oxygen, nitrogen, and sulfur. Y ２ is a hydrophobic group containing a hydrogen atom, a halogen, OR １Ａ , NR ２Ａ R ３Ａ , SR ４Ａ , CR ５Ａ R ６Ａ R ７Ａ , or an aliphatic hydrocarbon group having 10 or more carbon atoms, and R １Ａ , R ２Ａ , R ３Ａ , R ４Ａ , R ５Ａ , R ６Ａ , and R ７Ａ are each independently alkyl having 1 to 6 carbon atoms, alkenyl having 1 to 6 carbon atoms, alkynyl having 1 to 6 carbon atoms, phenyl, OR ８Ａ , -OC(O)R ８Ａ , -C(O)OR ８Ａ , NR ８Ａ R ９Ａ , -NR ８Ａ COR ９Ａ , -CONR ８Ａ R ９Ａ , a 3- to 7-membered saturated or partially unsaturated monocyclic carbocyclic ring, or a 3- to 7-membered saturated or partially unsaturated monocyclic heterocyclic ring having 1 to 2 heteroatoms independently selected from oxygen, nitrogen, and sulfur, and R ８Ａ and R ９Ａ are, each time they appear, independently a hydrogen atom or alkyl having 1 to 6 carbon atoms, P １ NO ２ or a silylhydroxyl protecting group, R １ and R ２ These are independently a hydrogen atom, a C1-C6 alkyl group, or a phenyl group, and the C1-C6 alkyl group and phenyl group have 1-3 R ３ It can be freely replaced by R ３ These are alkoxy compounds having 1 to 30 carbon atoms. e is an integer from 0 to 6. f is an integer between 0 and 6.

8. The method for producing an oligonucleotide according to claim 1, wherein the pseudo-solid-phase protecting group is a group represented by the following general formula (1). 【Chemistry 10】 [In general formula (1), ring E represents a fused polycyclic aromatic hydrocarbon ring or an aromatic heterocycle, Y Ａ *-OCR ２Ａ -**, *-NRCR ２Ａ -**, or *-SCR ２Ａ - Represents **, R ２Ａ Each of these independently represents a hydrogen atom, an alkyl group, an aromatic group, or an aromatic group-substituted alkyl group. k １ This represents an integer from 1 to 5, The dashed lines and asterisks (*) indicate bonding locations with other structures, while the double asterisks (**) indicate bonding locations with ring E. R Ａ Each of these is independently an aliphatic hydrocarbon group or an organic group having an aliphatic hydrocarbon group, k １ Individual R Ａ The number of carbon atoms in at least one aliphatic hydrocarbon group is 12 or more. Ring E is Y Ａ and R Ａ In addition, it may have further substituents.

9. The method for producing an oligonucleotide according to claim 1, wherein the pseudo-solid protecting group is a group formed by bonding the carboxyl group of a compound selected from the group consisting of compounds represented by any of the following formulas (A) to (F) to the 3' or 5' position of the nucleoside, nucleotide, or oligonucleotide. 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 【Chemistry 15】 【Chemistry 16】

10. The nucleic acid compound represented by the above general formula (X) is R Ｘ A method for producing an oligonucleotide according to claim 2, wherein the group is an allyl group.

11. The method for producing an oligonucleotide according to claim 1, wherein the alkali metal hydroxide is lithium hydroxide or sodium hydroxide.

12. A method for producing an oligonucleotide according to claim 1, further comprising the step of recovering the removed pseudo-solid protecting group after the step of removing the pseudo-solid protecting group.

13. A reagent kit for the synthesis of oligonucleotides, comprising: a compound that binds to the 3' or 5' position of a nucleoside, nucleotide, or oligonucleotide to form a pseudo-solid-phase protecting group; an alkali metal hydroxide; and at least one substrate selected from the group consisting of nucleosides, nucleotides, and oligonucleotides to which the compound binds.

14. The aforementioned pseudo-solid-phase protecting group forms a nucleic acid compound represented by the following general formula (X), as described in claim 13, as a reagent kit for the synthesis of oligonucleotides. 【Chemistry 17】 [In general formula (X), Base represents a nucleic acid base that may be modified. If there are two or more Bases, the multiple Bases may be the same or different. In general formula (X), B represents a hydrogen atom, a halogen atom, or an optionally protected hydroxyl group. If there are two or more B's, the multiple B's may be the same or different. In general formula (X), W is a hydrogen atom or a 5' protecting group. In general formula (X), R Ｘ R represents an alkyl group having 1 to 7 carbon atoms, having an ethyl group, allyl group, aryl group, or electron-withdrawing group with a cyano group at the 2nd position. Ｘ If there are two or more, then there are multiple R Ｘ They may be the same or different. In the general formula (X), Q represents an oxygen atom or a sulfur atom. If there are two or more Qs, the multiple Qs may be the same or different. In the general formula (X), y1 represents any integer greater than or equal to 0. In general formula (X), * represents the site to which the pseudo-solid-phase protecting group is bonded.

15. The reagent kit for the synthesis of oligonucleotides according to claim 13, wherein the pseudo-solid-phase protecting group is a group formed by bonding the carboxyl group in the following general formula (Y) to the 3' or 5' position of the nucleoside, nucleotide, or oligonucleotide. [Chemistry 18] [In general formula (Y), R Ｙ Each of these independently represents a linear or branched alkyl group having 10 to 40 carbon atoms. In the general formula (Y), m represents an integer from 1 to 5, and if m is 2 or greater, there are multiple R Ｙ O may be the same or different. In the general formula (Y), X is an oxygen atom, a sulfur atom, NH, or NR Ｎ It represents. In the general formula (Y), n represents an integer from 1 to 4. In general formula (Y), R Ｎ [This represents a linear or branched alkyl group having 1 to 6 carbon atoms.]

16. The aforementioned pseudo-solid phase protecting group is -L-Y Ｌ A reagent kit for the synthesis of oligonucleotides according to claim 13, wherein the group is represented by -Z or -Z. [In the formula, L is given by formula (a1): 【Chemistry 19】 (In the formula, ＊＊ This indicates the binding position with the 3' or 5' position of the oligonucleotide; ＊ Y Ｌ This indicates the bonding position with L １ L represents a divalent hydrocarbon group having 1 to 22 carbon atoms, which may be substituted, an oxygen atom, and -NR- (where R represents a hydrogen atom, an alkyl group, or an aralkyl group); L ２ This indicates a single bond or formula: ＊ C(R ３ａ ) (Caution ３ｂ )-O-R １＊＊ ,formula: ＊ C(=O)N(R) ２ )-R １ -N(R) ３ａ ) ＊＊ Alternatively, the formula: ＊ C(=O)N(R) ２ )-R １ -C(R ３ａ ) (Caution ３ｂ ) ＊＊ (In the formula, ＊ is, L １ It shows the bonding position with, ＊＊ CR ｃ R ｄ The bond position with R is shown, １ R represents an alkylene group having 1 to 22 carbon atoms, which may be substituted. ２ and R ３ａ Each independently represents a hydrogen atom or an optionally substituted C1-C22 alkyl group, or R ２ and R ３ａ Together, they may form an alkylene bond with 1 to 22 carbon atoms, which may be substituted. ３ｂ R represents a group represented by a hydrogen atom or an alkyl group having 1 to 22 carbon atoms, which may be substituted; ｃ and R ｄ Each is independently a hydrogen atom, an optionally substituted alkyl group having 1 to 22 carbon atoms, or R ｃ and R ｄ It represents a group (linker) that may be formed by combining with a single carbonyl group; In the formula, Y Ｌ This represents a single bond, an oxygen atom, -NR- (where R represents a hydrogen atom, an alkyl group, or an aralkyl group), or a sulfur atom; In the equation, Z is given by equation (a2): 【Chemistry 20】 (In the formula, ＊ Y Ｌ This indicates the bonding position with; R ４ This indicates a hydrogen atom, or R ｂ If the group is represented by the following formula (a3), then R ６ They may form a single bond or an -O- together with ring B, or form a fluorenyl group or a xanthenyl group together with ring B; k R ５ Each independently represents an organic group having an aliphatic hydrocarbon group with 10 or more carbon atoms; k represents an integer from 1 to 4; and ring A represents k OR ５ In addition, it may further have substituents selected from the group consisting of halogen atoms, C1-C6 alkyl groups which may be substituted with halogen atoms, and C1-C6 alkoxy groups which may be substituted with halogen atoms; R ａ R represents a phenyl group which may be substituted with a hydrogen atom or a halogen atom; ｂ is a hydrogen atom, or formula (a3): 【Chemistry 21】 (In the formula, ＊ indicates the joint position; j indicates an integer from 0 to 4; j R ７ Each independently represents an organic group having an aliphatic hydrocarbon group with 10 or more carbon atoms; R ６ This indicates a hydrogen atom, or R ４ They may form a single bond or an -O- together with ring A, or form a fluorenyl group or a xanthenyl group together with ring A; and ring B may have j OR ７ In addition, it may further have substituents selected from the group consisting of halogen atoms, C1-C6 alkyl groups which may be substituted with halogen atoms, and C1-C6 alkoxy groups which may be substituted with halogen atoms; R ａ and R ｂ These may come together to form a single carbonyl group. (The group represented by ) Formula (a2'): 【Chemistry 22】 (In the formula, ＊ Y Ｌ The bond position is indicated by; the definition of each other symbol is the same as that of formula (a2). ) or formula (a2''): 【Chemistry 23】 (In the formula, ＊ Y Ｌ The bond position is shown; ring A' has k OR ５ In addition, the group may further have substituents selected from the group consisting of halogen atoms, C1-C6 alkyl groups which may be substituted with halogen atoms, and C1-C6 alkoxy groups which may be substituted with halogen atoms; the definition of each symbol is the same as that of formula (a2).

17. The reagent kit for the synthesis of oligonucleotides according to claim 13, wherein the pseudo-solid-phase protecting group is a group formed by the bonding of -OM in the following general formula (1) to the 3' or 5' position of the nucleoside, nucleotide, or oligonucleotide. General formula (1): MO-(O=)CC(=O)-W １ -X １ -Y １ -Z １ ... (1) [In general formula (1), M represents a hydrogen atom or an alkali metal atom, W １ is an oxygen atom, a sulfur atom or NR Ｚ (N represents a nitrogen atom, R Ｚ (represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), X １ This represents a single bond, an alkylene group having 1 to 12 carbon atoms (which may have an ether bond (-O-)), an arylene group (which may be substituted with an alkylene group having 1 to 3 carbon atoms), or a divalent heterocyclic group. Y １ These are single bonds, ether bonds, -OCO-, and -NR Ｚ CO-(R Ｚ (The same as above), indicates -CO- or -SCO-, Z １ This represents an alkoxyphenyl group or alkoxyalkyl group having an alkoxy group with 10 to 30 carbon atoms.

18. The synthesis reagent kit for oligonucleotides according to claim 13, wherein the pseudo-solid-phase protecting group is a group represented by general formula (II). 【Chemistry 24】 [In general formula (II), * indicates the binding position to the 3' or 5' position of the nucleoside, nucleotide, or oligonucleotide.] R Ｘ１ Each of these is independently an alkyl group having 1 to 40 carbon atoms, an alkenyl group having 2 to 40 carbon atoms, an alkynyl group having 2 to 40 carbon atoms, or a group of the formula: -L ７ -O-R Ｘ４ (In the formula, L ７ R is an alkylene group having 1 to 40 carbon atoms, an alkenylene group having 2 to 40 carbon atoms, or an alkynylene group having 2 to 40 carbon atoms. Ｘ４ (where is an alkyl group having 1 to 40 carbon atoms, an alkenyl group having 2 to 40 carbon atoms, or an alkynyl group having 2 to 40 carbon atoms), and at least one R Ｘ１ The number of carbon atoms is 10 or more. s is an integer from 1 to 5. L １１ This is an alkylene group having 1 to 6 carbon atoms, an alkenylene group having 2 to 6 carbon atoms, or an alkylene group having 2 to 6 carbon atoms. L １２ is a single bond, -O- or -N(R) Ｘ２ )-(wherein, R Ｘ２ (represented by a hydrogen atom, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C2-C6 alkenyl group, or a C2-C6 haloalkenyl group), L １３ This is a single bond, -C(O)-, or an alkylene group having 1 to 6 carbon atoms. L １２ When it is a single bond, L １３ It is a single bond or a -C(O)- bond.

19. The reagent kit for the synthesis of oligonucleotides according to claim 13, wherein the pseudo-solid protecting group is a group formed by the bonding of a carboxyl group in a compound represented by general formula (I') to the 3' or 5' position of the nucleoside, nucleotide, or oligonucleotide. 【Chemistry 25】 [In general formula (I'), ring D is a 5-6 member heteroaryl having 1-3 heteroatoms independently selected from phenyl, an 8-10 member bicyclic aryl, oxygen, nitrogen, and sulfur, or a 7-10 member bicyclic heteroaryl having 1-4 heteroatoms independently selected from oxygen, nitrogen, and sulfur. Y ２ H, halogen, OR １Ａ , NR ２Ａ R ３Ａ , SR ４Ａ CR ５Ａ R ６Ａ R ７Ａ , or a hydrophobic group comprising one or more aliphatic hydrocarbon groups having 10 or more carbon atoms, R １Ａ , R ２Ａ , R ３Ａ , R ４Ａ , R ５Ａ , R ６Ａ , and R ７Ａ Each of these independently consists of an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 1 to 6 carbon atoms, an alkynyl group having 1 to 6 carbon atoms, a phenyl group, and an OR group. ８Ａ , -OC(O)R ８Ａ , -C(O)OR ８Ａ , NR ８Ａ R ９Ａ , -NR ８Ａ COR ９Ａ , -CONR ８Ａ R ９Ａ , a 3-7 member saturated or partially unsaturated monocyclic carbocyclyl, or a 3-7 member saturated or partially unsaturated monocyclic heterocyclyl having 1-2 heteroatoms independently selected from oxygen, nitrogen, and sulfur, R ８Ａ and R ９Ａ Each instance is independently either H or an alkyl group having 1 to 6 carbon atoms. P １ NO ２ or a silylhydroxyl protecting group, R １ and R ２ These are independently H, an alkyl group having 1 to 6 carbon atoms, or a phenyl group, and the alkyl group having 1 to 6 carbon atoms and the phenyl group have 1 to 3 R atoms. ３ It can be freely replaced by R ３ These are alkoxy compounds having 1 to 30 carbon atoms. e is an integer from 0 to 6. f is an integer between 0 and 6.

20. The synthesis reagent kit for oligonucleotides according to claim 13, wherein the pseudo-solid-phase protecting group is a group represented by the following general formula (1). 【Chemistry 26】 [In general formula (1), ring E represents a fused polycyclic aromatic hydrocarbon ring or an aromatic heterocycle, Y Ａ *-OCR ２Ａ -**, *-NRCR ２Ａ -**, or *-SCR ２Ａ - Represents **, R ２Ａ Each of these independently represents a hydrogen atom, an alkyl group, an aromatic group, or an aromatic group-substituted alkyl group. k １ This represents an integer from 1 to 5, The dashed lines and asterisks (*) indicate bonding locations with other structures, while the double asterisks (**) indicate bonding locations with ring E. R Ａ Each of these is independently an aliphatic hydrocarbon group or an organic group having an aliphatic hydrocarbon group, k １ Individual R Ａ The number of carbon atoms in at least one aliphatic hydrocarbon group is 12 or more. Ring E is Y Ａ and R Ａ In addition, it may have further substituents.

21. The oligonucleotide synthesis reagent kit according to claim 13, wherein the pseudo-solid protecting group is a group formed by bonding the carboxyl group of a compound selected from the group consisting of compounds represented by any of the following formulas (A) to (F) to the 3' or 5' position of the nucleoside, nucleotide, or oligonucleotide. 【Chemistry 27】 【Chemistry 28】 【Chemistry 29】 【Transformation 30】 【Chemistry 31】 【Chemistry 32】

22. The nucleic acid compound represented by the above general formula (X) is R Ｘ A synthesis reagent kit for oligonucleotides according to claim 14, wherein the group is an allyl group.

23. The alkyl group synthesis reagent kit according to claim 13, wherein the alkali metal hydroxide is lithium hydroxide or sodium hydroxide.

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