Linkers and solid supports for nucleic acid synthesis

Abstract

The present invention addresses the problem of providing a solid phase carrier for nucleic acid synthesis which enables, when a nucleic acid is synthesized using the same and cut out, easy separation of the nucleic acid from an adduct by HPLC, a compound which serves as a linker of the solid phase carrier, a compound which is a precursor of the linker, and a nucleic acid production method using the solid phase carrier. The compound of the present invention is represented by general formula (1). In general formula (1): Ar represents an optionally substituted tricyclic or tetracyclic aromatic ring; Z represents a hydrogen atom or a protecting group removable by an acid; and R1 to R4 independently represent a hydrogen atom, an alkyl group or an alkoxy group.

Description

[Technical field]

[0001] The present invention relates to a compound, a linker, a solid phase support, and a method for producing a nucleic acid. [Background technology]

[0002] Conventionally, a solid support having a 1,2-diol unit called a universal linker (hereinafter also referred to as a "universal support") has been used in the synthesis of nucleic acids (for example, Patent Document 1, Non-Patent Documents 1-2). The use of a universal support has the advantage that it is not necessary to prepare a solid support corresponding to the 3'-end of the nucleic acid. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] US Patent Application Publication No. 2004 / 0152905 [Non-patent literature]

[0004] [Non-Patent Document 1] Synthesis,2021,53,4440-4448 [Non-Patent Document 2] Tetrahedron,2021,92,132261 Summary of the Invention [Problem to be solved by the invention]

[0005] When nucleic acids are synthesized using a universal support, they must be excised from the universal support by treatment with an aqueous ammonia solution or the like. For example, the following scheme (where CPG: Controlled Pore Glass, Nu - As shown in the formula (III): nucleophile, R: hydrogen atom or substituent, the nucleic acid is excised.

[0006] [ka]

[0007] At this time, an adduct in which a 1,2-diol unit is added to the nucleic acid may remain, and in such a case, it is necessary to purify it by HPLC (high performance liquid chromatography) or the like.

[0008] Under these circumstances, the present inventors synthesized nucleic acids using the universal support described in Patent Document 1 and the like, and excised the nucleic acids, but it became clear that in some cases the nucleic acid peak and the adduct peak could not be sufficiently separated by HPLC.

[0009] In view of the above-mentioned circumstances, the present invention aims to provide a solid phase support for nucleic acid synthesis, which is a solid phase support that allows nucleic acid to be synthesized using the solid phase support and, when the nucleic acid is excised, to easily separate the nucleic acid from an adduct by HPLC; a compound that serves as a linker for the solid phase support; a compound that serves as a precursor of the linker; and a method for producing nucleic acid using the solid phase support. [Means for solving the problem]

[0010] As a result of intensive investigations into the above-mentioned problems, the present inventors have found that the above-mentioned problems can be solved by using a specific compound having a highly lipophilic tricyclic or tetracyclic aromatic ring, and have arrived at the present invention. That is, the present inventors have found that the above problems can be solved by the following configuration.

[0011] (1) A compound represented by the general formula (1) described below. (2) A compound represented by general formula (2) described below. (3) The compound according to the above (1) or (2), wherein in the general formula (1) or (2) described below, Ar is an anthracene ring, a phenanthrene ring, a tetracene ring, or a pyrene ring, each of which may have a substituent. (4) The compound according to any one of the above (1) to (3), wherein in the general formula (1) or (2) described below, Ar is a phenanthrene ring which may have a substituent. (5) The compound according to any one of the above (1) to (4), wherein in general formula (1) or (2) described below, Z is a protecting group that can be removed by an acid. (6) The compound according to (5) above, wherein the protecting group that can be removed by an acid is a trityl-based protecting group or a silyl-based protecting group. (7) In the general formula (1) or (2) described below, R 1 ~R 4 is a hydrogen atom. (8) The compound according to any one of the above (2) and (3) to (7) which cite at least the above (2), wherein in general formula (2) described later, L is an alkylene group which may have an oxygen atom, an arylene group which may have an oxygen atom, or a combination thereof. (9) A linker for a solid phase support for nucleic acid synthesis, which uses the compound according to any one of (1) to (8) above. (10) A solid support comprising a compound represented by general formula (3) described below. (11) The solid phase support according to (10) above, which is used for nucleic acid synthesis. (12) The solid phase support according to (10) or (11) above, wherein in general formula (3) described later, Ar is an anthracene ring, a phenanthrene ring, a tetracene ring, or a pyrene ring, each of which may have a substituent. (13) The solid phase support according to any one of (10) to (12) above, wherein in the general formula (3) described below, Ar is a phenanthrene ring which may have a substituent. (14) The solid phase support according to any one of (10) to (13) above, wherein in general formula (3) described below, Z is a protecting group that can be removed by an acid. (15) The solid phase support according to (14) above, wherein the protecting group that can be removed by an acid is a trityl-based protecting group or a silyl-based protecting group. (16) In the general formula (3) described below, R 1 ~R 4The solid phase support according to any one of (10) to (15) above, wherein is a hydrogen atom. (17) The solid phase support according to any one of (10) to (16) above, wherein in general formula (3) described later, L is an alkylene group optionally having an oxygen atom, an arylene group optionally having an oxygen atom, or a combination thereof. (18) The solid phase carrier according to any one of (10) to (17) above, wherein in the general formula (3) described below, Sp is a porous polymer carrier or a glass-based porous carrier. (19) A method for producing a nucleic acid, comprising a step of carrying out a nucleic acid synthesis reaction on the solid phase support according to any one of (10) to (18) above. (20) The method for producing a nucleic acid according to (19) above, wherein the nucleic acid synthesis reaction is carried out by a phosphoramidite method. Effect of the Invention

[0012] As shown below, according to the present invention, a solid phase carrier for nucleic acid synthesis, which is capable of easily separating the nucleic acid and the adduct by HPLC when the nucleic acid is synthesized and excised, a compound serving as a linker for the solid phase carrier, a compound serving as a precursor of the linker, and a method for producing nucleic acid using the solid phase carrier can be provided. The solid phase carrier of the present invention also has the effect that when the nucleic acid is synthesized and excised using the solid phase carrier, a peak of a cyclic phosphate derived from the linker is observed by HPLC. The solid phase carrier of the present invention also has the effect that it is difficult to decompose because of its stable structure. [Brief description of the drawings]

[0013] [Figure 1] 1 shows HPLC charts of Example 1 and Comparative Example 1. [Diagram 2] 1 shows HPLC charts of Example 1 and Comparative Example 1. [Diagram 3] 1 is an HPLC chart of Example 2. [Figure 4] 1 is an HPLC chart of Example 3. [Diagram 5] 1 is an HPLC chart of Example 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The compound, linker, solid phase carrier, and method for producing nucleic acid of the present invention will be described below. In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits. In addition, in this specification, adenine may be represented as "A", guanine as "G", cytosine as "C", and thymine as "T". In addition, when the nucleic acid is excised, the nucleic acid and the adduct can be easily separated by HPLC, a peak of the cyclic phosphate derived from the linker can be easily observed by HPLC, and decomposition is difficult. These facts may be collectively referred to as "the effects of the present invention are excellent."

[0015] [1] Compound (1) The compound (1) of the present invention is a compound represented by the following general formula (1). Compound (1) of the present invention is a suitable compound for a precursor of a linker of the solid phase support of the present invention described below. Hereinafter, compound (1) of the present invention is also referred to as a "specific linker precursor."

[0016] [ka]

[0017] In the general formula (1), Ar represents an optionally substituted tricyclic or tetracyclic aromatic ring, Z represents a hydrogen atom or a protecting group that can be removed by an acid, R 1 ~R 4 each independently represents a hydrogen atom, an alkyl group, or an alkoxy group.

[0018] [Ar] As described above, in the general formula (1), Ar represents an aromatic ring having three or four rings which may have a substituent. Here, the term "tricyclic or tetracyclic aromatic ring" refers to a ring in which three or four monocyclic aromatic rings are condensed. The aromatic ring is a ring in which the number of electrons contained in the π electron system is 4n+2 (n is an integer of 0 or more). The monocyclic aromatic ring is preferably a benzene ring, since the effects of the present invention are more excellent. Hereinafter, the "tricyclic or tetracyclic aromatic ring" is also referred to as the "specific aromatic ring".

[0019] Specific examples of the specific aromatic ring include an anthracene ring, a phenanthrene ring, a chrysene ring, a pyrene ring, a triphenylene ring, and a tetracene ring. Among these, an anthracene ring, a phenanthrene ring, a tetracene ring, or a pyrene ring is preferred, and a phenanthrene ring is more preferred, because the effects of the present invention are more excellent.

[0020] The substituent that the specific aromatic ring may have is not particularly limited, and specific examples thereof include the substituent W described below. The above-mentioned substituent is preferably an alkyl group, an alkoxy group, a dialkylamino group, or a halogeno group (halogen atom) because the effects of the present invention are more excellent.

[0021] [Z] As described above, Z represents a hydrogen atom or a protecting group that can be removed by an acid. Among them, a protecting group that can be removed by an acid is preferred because the effects of the present invention are more excellent.

[0022] The protecting group that can be removed by the acid is not particularly limited, but in order to obtain better effects of the present invention, a protecting group that can be removed by a Brønsted acid such as trichloroacetic acid or dichloroacetic acid is preferable, a trityl-based protecting group or a silyl-based protecting group is more preferable, and a trityl-based protecting group is even more preferable.

[0023] The trityl-based protecting group may be, for example, any substituent (e.g., C 1-6 Alkoxy group, C 1-6Examples of the trityl protecting group include a trityl group which may be substituted with a substituent selected from an alkyl group, a halogen atom, etc. (two or more substituents may be taken together to form a ring). Specific examples of the trityl protecting group include a trityl group (triphenylmethyl group (Tr)), a monomethoxytrityl group (e.g., a 4-methoxyphenyldiphenylmethyl group (MMTr)), a dimethoxytrityl group (e.g., a 4,4'-dimethoxyphenylphenylmethyl group (DMTr)), a 9-phenylxanthen-9-yl group (a pixyl group), etc. The trityl protecting group is preferably a 4,4'-dimethoxyphenylphenylmethyl group (DMTr) because the effect of the present invention is more excellent.

[0024] The silyl-based protecting group may be, for example, any substituent (e.g., C 1-6 Alkoxy group, C 1-6 Examples of the silyl protecting group include a silyl group tri-substituted with a substituent selected from an alkyl group, a phenyl group, etc., and specific examples thereof include a trimethylsilyl group, a triethylsilyl group, an isopropyldimethylsilyl group, a tert-butyldimethylsilyl group, a dimethylmethoxysilyl group, a methyldimethoxysilyl group, a tert-butyldiphenylsilyl group, etc. The silyl protecting group is preferably a trimethylsilyl group because the effect of the present invention is more excellent.

[0025] In this specification, "C 1-6 Examples of the "alkyl group" include linear, branched, or cyclic alkyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, tert-pentyl, cyclopentyl, hexyl, and cyclohexyl. Of these, methyl and ethyl groups are preferred because they provide better effects for the present invention.

[0026] In addition, in this specification, "C 1-6Examples of the "alkoxy group" include linear, branched, or cyclic alkoxy groups such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentyloxy, isopentyloxy, sec-pentyloxy, tert-pentyloxy, cyclopentyloxy, hexyloxy, and cyclohexyloxy. Among these, methoxy and tert-butoxy are preferred because they provide better effects in the present invention.

[0027] In the present specification, examples of the "halogen atom" include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. Among these, a fluorine atom, a chlorine atom, and a bromine atom are preferred because they provide better effects for the present invention.

[0028] [R 1 ~R 4 ] As described above, in the general formula (1), R 1 ~R 4 each independently represents a hydrogen atom, an alkyl group (e.g., having 1 to 10 carbon atoms, preferably having 1 to 6 carbon atoms, and more preferably having 1 to 4 carbon atoms), or an alkoxy group (e.g., having 1 to 10 carbon atoms, preferably having 1 to 6 carbon atoms, and more preferably having 1 to 4 carbon atoms). R 1 ~R 4 is preferably a hydrogen atom because the effects of the present invention are more excellent.

[0029] [Substituent W] The substituent W in this specification will be described. Examples of the substituent W include a halogeno group (halogen atom), an alkyl group (e.g., a tert-butyl group) (including a cycloalkyl group, a bicycloalkyl group, and a tricycloalkyl group), an alkenyl group (including a cycloalkenyl group and a bicycloalkenyl group), an alkynyl group, an aryl group, a heterocyclic group (which may also be called a heterocyclic group), a cyano group, a hydroxy group, a nitro group, a carboxy group, an alkoxy group, an aryloxy group, a silyloxy group, a heterocyclic oxy group, an acyloxy group, a carbamoyl group, a carbamoyloxy group, an alkoxycarbonyloxy group, an aryloxycarbonyloxy group, an amino group (including an anilino group), an ammonio group, a dialkylamino group, an acylamino group, an aminocarbonylamino group, an alkoxy Examples of the substituent include a carbonylamino group, an aryloxycarbonylamino group, a sulfamoylamino group, an alkyl or arylsulfonylamino group, a mercapto group, an alkylthio group, an arylthio group, a heterocyclic thio group, a sulfamoyl group, a sulfo group, an alkyl or arylsulfinyl group, an alkyl or arylsulfonyl group, an acyl group, an aryloxycarbonyl group, an alkoxycarbonyl group, an aryl or heterocyclic azo group, an imido group, a phosphino group, a phosphinyl group, a phosphinyloxy group, a phosphinylamino group, a phosphono group, a silyl group, a hydrazino group, a ureido group, a boronic acid group (-B(OH)2), a phosphato group (-OPO(OH)2), a sulfato group (-OSOH), and other known substituents.

[0030] [Manufacturing method] The method for producing the specific linker precursor is not particularly limited, and the specific linker precursor can be produced by combining known methods. For example, the specific linker precursor can be produced by referring to the synthetic route of compound 1 or compound 2 in Example 1 described later. Specifically, for example, a precursor compound of compound 1 is obtained by reacting a 3-4 ring aromatic ring compound having a substituent W such as bromophenanthrene and a halogeno group with furan which may have a substituent in the presence of a strong base such as sodium amide. Then, compound 1 can be produced by converting an olefin into a diol using an oxidizing agent such as osmium tetroxide. In addition, a specific linker precursor (a compound represented by general formula (1) in which Z is a protecting group which can be removed by an acid) can be produced by protecting one of the hydroxy groups of compound 1 with a protecting group which can be removed by an acid.

[0031] [2] Compound (2) The compound (2) of the present invention is a compound represented by the following general formula (2). Compound (2) of the present invention is a suitable compound for a linker of the solid phase support of the present invention described below. Hereinafter, compound (2) of the present invention is also referred to as a "specific linker."

[0032] [ka]

[0033] In the general formula (2), Ar represents an optionally substituted tricyclic or tetracyclic aromatic ring, Z represents a hydrogen atom or a protecting group that can be removed by an acid, and R 1 ~R 4 each independently represents a hydrogen atom, an alkyl group, or an alkoxy group, and L represents a divalent hydrocarbon group which may have an oxygen atom.

[0034] Ar, Z, and R 1 ~R 4 Specific examples and preferred embodiments are the same as those of the above-mentioned general formula (1).

[0035] [L] As described above, in general formula (2), L represents a divalent hydrocarbon group which may contain an oxygen atom. The divalent hydrocarbon group is not particularly limited, and specific examples thereof include a divalent alkylene group (eg, having 1 to 10 carbon atoms) and a divalent aromatic hydrocarbon group. For reasons of achieving better effects of the present invention, L is preferably an alkylene group which may have an oxygen atom, an arylene group which may have an oxygen atom, or a combination thereof, is preferably a divalent alkylene group, and is more preferably an ethylene group.

[0036] [Manufacturing method] The method for producing the specific linker is not particularly limited, and the specific linker can be produced by combining known methods. For example, the specific linker can be produced by referring to the synthetic route of compound 3 in Example 1 described later. Specifically, for example, the specific linker can be produced by reacting compound 2 with a carboxylic acid anhydride such as succinic anhydride in the presence of a base.

[0037] [3] Solid phase support The solid phase support of the present invention is a solid phase support (hereinafter also referred to as a "specific support") consisting of a compound represented by the following general formula (3). The specific support is preferably used as a solid phase support (universal support) for nucleic acid synthesis. The compound (specific carrier) represented by general formula (3) may be a compound in which one group (residue) obtained by removing Sp from general formula (3) is bonded to Sp, or a compound in which multiple of the above-mentioned residues are bonded to Sp.

[0038] [ka]

[0039] In the general formula (3), Ar represents an optionally substituted tricyclic or tetracyclic aromatic ring, Z represents a hydrogen atom or a protecting group that can be removed by an acid, and R 1 ~R 4 each independently represents a hydrogen atom, an alkyl group, or an alkoxy group, L represents a divalent hydrocarbon group, and Sp represents a solid phase carrier.

[0040] Ar, Z, R 1 ~R 4 Specific examples and preferred embodiments of L are the same as those in the above general formulae (1) and (2).

[0041] [Sp] As described above, in the general formula (3), Sp represents a solid phase carrier. Hereinafter, the solid phase carrier represented by Sp is also referred to as "Sp carrier". The Sp carrier may have a spacer (e.g., an alkylene group) for binding to the carboxy group of the specific linker described above.

[0042] The Sp support is preferably a support for solid-phase synthesis from which excess reagents used during nucleic acid synthesis can be easily removed by washing. Specific examples include glass-based porous supports; porous polymer supports such as polystyrene-based porous supports and acrylamide-based porous supports. The Sp carrier is preferably a glass-based porous carrier or a porous polymer carrier (particularly a polystyrene-based porous carrier) because the effects of the present invention are more excellent.

[0043] The glass-based porous carrier refers to a porous carrier containing glass as a constituent, and examples thereof include, but are not limited to, particulate porous glass particles (CPG), etc. In the case of synthesizing a long-chain nucleotide, a CPG having a pore size of 20 to 400 nm, more preferably 50 to 200 nm, and even more preferably 100 nm is most preferably used.

[0044] A polystyrene-based porous carrier is a porous carrier made of a resin mainly composed of structural units of styrene or its derivatives. Examples of polystyrene-based porous carriers include porous carriers made of styrene-hydroxystyrene-divinylbenzene-based copolymer particles (see JP 2005-097545 A, JP 2005-325272 A, and JP 2006-342245 A), and porous carriers made of styrene-(meth)acrylonitrile-hydroxystyrene-divinylbenzene-based copolymers (see JP 2008-074979 A).

[0045] The acrylamide-based porous carrier is a porous carrier made of a resin mainly composed of structural units of acrylamide or its derivatives. Examples of the acrylamide-based porous carrier include a porous carrier made of an aromatic monovinyl compound-divinyl compound-(meth)acrylamide derivative copolymer. When the Sp carrier is an acrylamide-based solid phase carrier, if the content of the structural unit derived from the (meth)acrylamide derivative monomer is too small, the effect of preventing the decrease in the amount of synthesis of nucleic acid and the decrease in synthesis purity cannot be obtained, and if it is too large, it is difficult to form a porous resin. Therefore, it is preferably 0.3 to 4 mmol / g, more preferably 0.4 to 3.5 mmol / g, and even more preferably 0.6 to 3 mmol / g.

[0046] The shape of the Sp carrier is not particularly limited and may be any of a plate-like, particulate, fibrous, etc., but a particulate carrier is preferred because it can increase the efficiency of filling the synthesis reaction vessel and is less likely to break. In this specification, the term "particle" does not mean a strictly spherical shape, but rather means that the carrier may have a certain shape (for example, an approximately spherical shape such as an oval sphere, a polyhedral shape, a cylindrical shape, an irregular shape such as a confetti shape, etc.).

[0047] The size (volume) of the Sp carrier is not particularly limited, but if the average particle size of the porous particles measured by laser diffraction (scattering method) is smaller than 1 μm, the back pressure becomes too high or the liquid delivery speed becomes slow when packed in a column, while if the average particle size is larger than 1000 μm, the gaps between the carrier particles become large when packed in a column, making it difficult to efficiently pack the carrier particles into a column of a certain volume. Therefore, the size is preferably 1 to 1000 μm, more preferably 5 to 500 μm, and even more preferably 10 to 200 μm.

[0048] The specific surface area of ​​the Sp support measured by the multipoint BET method is not particularly limited, but is preferably 0.1 m 2 If the molecular weight is less than 500 m / g, the degree of swelling in an organic solvent is low, and the synthesis reaction tends to be difficult to occur. 2 If the pore size is larger than 0.1 to 500 m / g, the pore size becomes small, and the synthesis reaction tends to be difficult to occur. 2 / g, more preferably 10 to 300m 2 / g, more preferably 50 to 200m 2 / g.

[0049] The average pore size of the Sp carrier measured by mercury intrusion porosimetry is not particularly limited, but if the pore size is too small, the synthetic reaction site becomes small, making it difficult for the desired reaction to occur, or the nucleotide length tends to be less than the desired number, while if the pore size is too large, the hydroxyl group on the surface of the porous particle, which is the reaction site, and the substance involved in the reaction have less chance to come into contact with each other, so the yield tends to decrease. Therefore, the pore size is preferably 1 to 200 nm, more preferably 5 to 100 nm, and even more preferably 20 to 70 nm.

[0050] [Loading amount] As described above, the compound (specific carrier) represented by general formula (3) may be a compound in which one group (residue) obtained by removing Sp from general formula (3) is bound to Sp, or a compound in which multiple of the above residues are bound to Sp. The amount of the above residue bound to Sp (loading amount) is not particularly limited, but if it is too small, the yield of nucleic acid decreases, while if it is too large, the purity of nucleic acid decreases. Therefore, the molar amount of the above residue relative to 1 g of Sp is preferably 1 to 2000 μmol / g, more preferably 10 to 1000 μmol / g, even more preferably 20 to 100 μmol / g, and particularly preferably 30 to 50 μmol / g.

[0051] [Manufacturing method] The method for producing the solid phase carrier of the present invention is not particularly limited, but an example thereof includes a method of reacting the specific linker described above with a solid phase carrier (hereinafter also referred to as a "reactive solid phase carrier") that has a functional group (e.g., an amino group) that can react with the carboxy group of the linker described above and becomes an Sp carrier after the reaction.

[0052] Examples of reactive solid phase supports include CPG solid phase supports having a long-chain aminoalkyl spacer (lcaa-CPG solid phase supports), polystyrene-based porous supports having amino groups and / or hydroxy groups, and acrylamide-based porous supports having amino groups and / or hydroxy groups (especially hydroxy groups).

[0053] [4] Methods for producing nucleic acids The method for producing nucleic acid of the present invention (hereinafter also simply referred to as "the method for producing nucleic acid of the present invention") is a method for producing nucleic acid comprising a step of carrying out a nucleic acid synthesis reaction on the above-mentioned solid phase support (specific support) of the present invention. That is, the method for producing nucleic acid of the present invention is a method for producing nucleic acid using the specific support as a universal support.

[0054] [Nucleic acid synthesis reaction] As the nucleic acid synthesis reaction, for example, various known synthesis methods using an automatic nucleic acid synthesizer can be used. In this specification, the term "nucleic acid synthesis reaction" refers in particular to an elongation reaction of nucleotides constituting a nucleic acid. That is, an elongated oligonucleotide is obtained by sequentially binding nucleotides to a nucleoside, nucleotide, or oligonucleotide bound to a solid support. In addition, in the present specification, "nucleic acid" refers to a chain-like compound (oligonucleotide) in which nucleotides are linked by phosphodiester bonds, and includes DNA, RNA, etc. Nucleic acid may be either single-stranded or double-stranded, but is preferably single-stranded because it can be efficiently synthesized by a nucleic acid synthesizer. In the present specification, "nucleic acid" includes not only oligonucleotides containing purine bases such as adenine (A) and guanine (G) and pyrimidine bases such as thymine (T), cytosine (C), and uracil (U), but also modified oligonucleotides containing these modified nucleic acid bases.

[0055] Examples of nucleic acid synthesis reactions include the H-phosphonate method, the phosphoester method, and the phosphoramidite method. Among these, the phosphoramidite method is preferred because it has a high nucleic acid synthesis ability and can produce highly pure nucleic acid.

[0056] [Cut-out process] The production method of the present invention usually includes a step of excising the synthesized nucleic acid from a specific carrier, which is a universal support (excision treatment). The excision treatment is not particularly limited, but may be, for example, a method of treating with ammonia and / or amines. Examples of amines include methylamine, ethylamine, isopropylamine, ethylenediamine, diethylamine, triethylamine, etc. It is desirable to use ammonia and / or amines in a mixture with a solvent. Examples of the solvent include water, alcohols (e.g., methanol, ethanol, etc.). Two or more of these solvents may be mixed in an appropriate ratio.

[0057] Nucleic acid is obtained by the excision treatment. A specific example of the scheme for excising nucleic acid from a specific carrier is as described above. At this time, as described above, an adduct in which a 1,2-diol unit is added to a nucleic acid may remain. However, when the solid phase support of the present invention is used, the nucleic acid and the adduct can be easily separated by HPLC. EXAMPLES

[0058] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0059] [Example 1]

[0060] [Synthesis of specific carriers] PT-CPG (specific carrier) was synthesized as follows.

[0061] [ka]

[0062] <Synthesis of Compound 1>

[0063] (1) Under an argon atmosphere, furan (5.63 mL, 77.8 mmol) was added to a solution of 9-bromophenanthrene (2.00 g, 7.78 mmol) and sodium amide (910 mg, 23.3 mmol) in anhydrous tetrahydrofuran (15 mL), and the mixture was stirred at room temperature for 19 hours. After the reaction was completed, the reaction solution was cooled to 0°C and water was added. The mixture was then diluted with ethyl acetate, and the organic layer was washed twice with water and once with saturated saline, then dried over sodium sulfate, and the solvent was distilled under reduced pressure. The crude product was purified by silica gel column chromatography (hexane / ethyl acetate = 100:1 to 50:1) to obtain the precursor compound of compound 1 (1.10 g, as a mixture) as a light brown solid (Chem.Commun.2014,50,6869-6871.).

[0064] (2) Next, 50% N-methylmorpholine oxide aqueous solution (2.28 g, 4.56 mL, 19.5 mmol) and 0.1 M osmium tetroxide tert-butanol solution (77.8 μL, 0.00778 mmol) were added to a solution of the precursor compound (1.10 g, mixture) in acetone (15 mL), and the mixture was heated to reflux for 3 hours. After the reaction was completed, a saturated aqueous solution of sodium thiosulfate was added to the reaction solution, and extraction was performed five times with a mixed solution of chloroform and methanol (chloroform / methanol = 10:1). The collected organic layer was washed once with water and once with saturated saline, then dried over sodium sulfate, and the solvent was distilled under reduced pressure. The crude product was purified by silica gel chromatography (from chloroform only to chloroform / methanol = 100:1) to obtain compound 1 (a compound represented by the general formula (1). Here, Ar represents a phenanthrene ring, Z represents a hydrogen atom, and R 1 ~R 4 represents a hydrogen atom.) (specific linker precursor) (910 mg, two-step yield 42%) was obtained as a light brown solid.

[0065] 1 H-NMR(500MHz,DMSO-d6) δ 8.90-8.88(m,2H),8.10-8.08(m,2H),7.74-7.70(m,4H),5.75(s,2H),5.17-5.15(m,2H),3.70-3.67(m,2H). 13 C-NMR(125MHz,DMSO-d6) δ 138.6,129.7,127.3,126.7,125.6,124.5,123.8.IR(ATR)cm -1 :3355,3240.HRMS(FAB):calcd for C 18 H 14 NaO3[M+Na] + 301.0841, found 301.0828.

[0066] <Synthesis of compound 2> Under an argon atmosphere, 4,4'-dimethoxytrityl chloride (731 mg, 2.16 mmol) was added to a solution of compound 1 (500 mg, 1.80 mmol) in anhydrous pyridine (10 mL), and the mixture was stirred at room temperature for 20 hours. After completion of the reaction, the reaction solution was diluted with ethyl acetate, and the organic layer was washed twice with water and once with saturated saline, then dried over sodium sulfate, and the solvent was distilled under reduced pressure. The crude product was purified by silica gel column chromatography (hexane / ethyl acetate = 3:1) to obtain compound 2 (a compound represented by general formula (1), where Ar represents a phenanthrene ring, Z represents a DMTr group, and R represents a 1-methyl-2-phenylpropanediol group). 1 ~R 4 represents a hydrogen atom.) (specific linker precursor) (1.01 g, yield 97%) was obtained as a pale yellow solid.

[0067] 1 H-NMR(500MHz,CDCl3) δ 8.66-8.63(m,2H),7.91-7.89(m,1H),7.65-7.48(m,6H),7.39-7.26(m,5H),7.20(d,1H,J=7.0Hz),6.90-6 .86(m,4H),5.73(s,1H),5.12(s,1H),3.98(d,1H,J=5.0Hz),3.91-3.87(m,2H),3.83(s,3H),3.81(s,3H). 13 C-NMR(125MHz,CDCl3) δ 158.9,158.8,144.7,138.4,138.0,136.1,135.8,130.3,130.2,128.3,128.2,127.2,126.7,126.5,1 26.4,126.0,125.5,125.0,124.4,123.5,123.3,113.5,88.9,84.7,83.0,73.0,70.6,55.3.IR(ATR)cm -1 :3499,3004,2953,1607,1507.HRMS(ESI-TOF):calcd for C 39 H 32 NaO5[M+Na] + 603.2147,found 603.2149.

[0068] <Synthesis of compound 3> Under an argon atmosphere, triethylamine (1.19 mL, 8.61 mmol) was added to a solution of compound 2 (500 mg, 0.861 mmol) in anhydrous dichloromethane (10 mL). Succinic anhydride (345 mg, 3.44 mmol) was added and stirred at room temperature for 20 hours. After the reaction was completed, the reaction solution was extracted five times with a mixed solution of chloroform and methanol (chloroform / methanol = 10:1), and the collected organic layer was washed once with water and once with saturated saline, dried over sodium sulfate, and the solvent was distilled under reduced pressure. The crude product was purified by silica gel chromatography (from chloroform only to chloroform / methanol = 100:1) to obtain compound 3 (a compound represented by general formula (2). Here, Ar represents a phenanthrene ring, Z represents a DMTr group, and R represents a methyl group). 1 ~R 4 represents a hydrogen atom, and L represents an ethylene group.) (specific linker) (368 mg, yield 63%) was obtained as a light brown solid.

[0069] 1 H-NMR(500MHz,CDCl3) δ 8.63(d,2H,J=8.5Hz),7.95-7.93(m,1H),7.64-7.60(m,3H),7.55-7.49(m,3H),7.42-7.36(m,4H),7.31-7.21(m,4H),6.87-6. 82(m,4H),5.83(s,1H),5.26(s,1H),4.76(d,1H,J=6.0Hz),4.09(d,1H,J=6.0Hz),3.83(s,3H),3.81(s,3H),2.95-2.77(m,4H). 13 C-NMR(125MHz,CDCl3) δ 172.7,158.7,158.6,145.4,138.7,137.8,136.7,136.4,130.3,130.2,128.3,128.0,127.4,126.9,126.8,126 .7,126.4,125.8,125.4,125.0,123.4,123.3,113.4,88.1,84.7,83.6,82.6,73.9,73.1,55.3,29.3.IR(ATR)cm -1 :3649,3007,2929,1733,1607,1507.HRMS(ESI-TOF):calcd for C 43 H36 NaO8[M+Na] + 703.2308, found 703.2303.

[0070] <Synthesis of PT-CPG> Under an argon stream, diisopropylethylamine (6.80 μL, 40.0 μmol) was added to a suspension of lcaa (long-chain aminoalkyl spacer)-CPG (500 mg, amino group content: 40.0 μmol / g) in anhydrous acetonitrile (4 mL), and further compound 3 (13.6 mg, 20.0 μmol) and HBTU (Hexafluorophosphate Benzotriazole Tetramethyl Uronium) (7.58 mg, 20.0 μmol) were added. After stirring at room temperature for 1 hour, the reaction solution was filtered, the solid support (filtrate) was washed with acetonitrile, and further dried under vacuum overnight. The obtained solid was suspended in Cap A solution (tetrahydrofuran solution of acetic anhydride) and Cap B solution (tetrahydrofuran solution of methylimidazole) and stirred for 30 minutes. The reaction solution was filtered again, the solid support (filtrate) was washed with acetonitrile, and dried under vacuum overnight to obtain PT-CPG (compound represented by general formula (3). Here, Ar represents a phenanthrene ring, Z represents a DMTr group, R 1 ~R 4 represents a hydrogen atom, L represents an ethylene group, and Sp represents the one obtained by removing the amino group from the long-chain aminoalkyl spacer of lcaa-CPG.) (specific support). The loading amount of compound 3 in PT-CPG was quantified by the DMTr assay method and was 37 ± 1.3 μmol / g. The DMTr assay method is a method for indirectly quantifying the loading amount by treating the solid support with a deblocking solution (dichloromethane solution of 3 w / v% trichloroacetic acid) and measuring the absorbance (504 nm) of the amount of the deprotected DMTr (dimethoxytrityl) group.

[0071] 〔Synthesis of Oligonucleic Acid〕 Using PT-CPG as a universal support, a T-10mer oligonucleic acid (T 10) was synthesized (wherein T represents a thymine residue and CE represents a cyanoethyl group). The synthesis scale was 0.2 μmol, and the synthesis was carried out under trityl-OFF conditions (finally, the DMTr group was deprotected using a dichloromethane solution of 3 w / v% trichloroacetic acid). Commercially available phosphoramidite of T was prepared as a 0.1 M anhydrous acetonitrile solution and used. 5-ethylthio-1H-tetrazole (0.25 M anhydrous acetonitrile solution) was used as the activator, and the condensation time was 10 minutes only for the 3'-terminus and 25 seconds for the others.

[0072] [ka]

[0073] [Cleavage of oligonucleotide from universal support and removal of linker] The oligonucleotide was cleaved from the universal support and the associated linker was removed by treatment with 28% aqueous ammonia at room temperature for 2 hours (condition A).

[0074] [ka]

[0075] [Comparative Example 1] An oligonucleotide was synthesized and excised in the same manner as in Example 1, except that CUTAG-CPG (Sigma-Aldrich) (structure below, R: methyl group) (hereinafter also referred to simply as "CUTAG") was used instead of PT-CPG.

[0076] [ka]

[0077] [evaluation] For Example 1 and Comparative Example 1, the solutions after the cleavage treatment were analyzed by reverse phase HPLC.

[0078] [Condition 1] First, HPLC was performed under the conditions shown in Table 1 below.

[0079] [Table 1]

[0080] FIG. 1 shows the HPLC chart. As shown in FIG. 1, in Comparative Example 1, the oligonucleic acid (T 10 ), and adducts (T 10 On the other hand, in Example 1, only the peak of the oligonucleic acid was observed.

[0081] [Condition 2] The reason why the adduct peak was not observed in Example 1 was expected to be because the retention time of the adduct peak was long. Therefore, HPLC was measured for Example 1 under conditions that make separation more difficult. Specifically, the gradient was changed from "Solution B: 5-15%" to "Solution B: 8-18%". The HPLC conditions are shown in Table 2 below.

[0082] [Table 2]

[0083] The HPLC chart is shown in Figure 2. The chart including the washing period (after 30 minutes) (including wash) is also shown. For comparison, the chart of Comparative Example 1 (Solution B: 5-15%) is also shown.

[0084] 〔result〕 As shown in FIG. 1, in Comparative Example 1, the oligonucleic acid (T 10 ) peak and adduct (T 10-adduct) was observed at a close position. In contrast, as shown in FIG. 2, in the case of Example 1, even under conditions that make separation more difficult (liquid B: 8-18%), the oligonucleic acid peak and the adduct peak were sufficiently separated. In other words, it was shown that when nucleic acid is synthesized using the specific carrier of Example 1, when the nucleic acid is excised, the nucleic acid and the adduct can be easily separated by HPLC. Furthermore, in the case of Example 1, a peak of the cyclic phosphate (cp) derived from the linker was also observed in the HPLC chart (including wash).

[0085] [Comparison with Non-Patent Document 2] In the CPG3 shown in FIG. 3 of Non-Patent Document 2, an oligonucleotide (T 10 The HPLC chart of the solution after the cleavage treatment when the oligonucleotide (T 10 ) peak at about 10 minutes retention time, and adduct (T 10 -3) peak was observed at a retention time of approximately 24 minutes.

[0086] [ka]

[0087] In contrast, in the case of the above-mentioned Example 1, as shown in FIG. 10 ) peak at about 10 minutes retention time, and adduct (T 10 The peak of the β-adduct was observed at a retention time of about 27 minutes. In other words, the specific support of Example 1 described above has better peak separation than the universal support of Non-Patent Document 2.

[0088] [Example 2]

[0089] [Synthesis of Oligonucleotides] Using PT-CPG as a universal support, oligonucleotides (T9X) were synthesized with a nucleoside other than T only at the 3′ end (X) using an automatic DNA synthesizer. MeO U), LNA-T ( LNA T) was used. The synthesis scale was 0.2 μmol, and the synthesis was performed under trityl OFF conditions. Commercially available phosphoramidite of T was prepared as a 0.1 M anhydrous acetonitrile solution and used. 5-ethylthio-1H-tetrazole (0.25 M anhydrous acetonitrile solution) was used as the activator, and the condensation time was 10 minutes only for the 3' end and 25 seconds for the others.

[0090] [ka]

[0091] [Cleavage of oligonucleotide from universal support and removal of linker] The oligonucleotide was excised from the universal support and the associated linker was removed by treatment with a 28% aqueous ammonia solution at 55° C. for 8 hours (condition B).

[0092] [ka]

[0093] 〔evaluation〕 The solution after the cleavage treatment was analyzed by reverse phase HPLC under the same HPLC conditions as those in Condition 2 described above.

[0094] FIG. 3 shows the HPLC chart. As can be seen from Figure 3, a peak for the oligonucleic acid (T9X) and a peak for the cyclic phosphate (cp) derived from the linker were observed. However, no peak for the adduct (T9X-adduct) was observed, even though the analysis was performed under the same conditions (conditions 2) as in Figure 2. From this, it is considered that the oligonucleic acid was completely excised and that no adduct was present in the solution after the excision treatment.

[0095] [Example 3]

[0096] [Synthesis of Oligonucleotides] Phosphorothioated oligonucleotides (sONs) were synthesized using an automatic DNA synthesizer with PT-CPG as a universal support. The synthesis scale was 0.2 μmol, and the synthesis was performed under trityl OFF conditions. Ac C. Pac A. iPrPac The phosphoramidite of G was prepared as a 0.1M anhydrous acetonitrile solution and used. 5-Ethylthio-1H-tetrazole (0.25M anhydrous acetonitrile solution) was used as the activating agent, and the condensation time was 10 minutes for the 3' end only and 25 seconds for the rest. DDTT (dimethylamino-methylidene) amino)-3H-1,2,4-dithiazaoline-3-thione) (0.05M anhydrous pyridine / anhydrous acetonitrile solution) was used as the sulfurizing agent.

[0097] [ka]

[0098] [Cleavage of oligonucleotide from universal support and removal of linker] The oligonucleotides were excised from the universal support and the associated linker was removed by treatment with 28% aqueous ammonia at room temperature for 2 hours (condition A) or with 28% aqueous ammonia at 55°C for 3 hours (condition C).

[0099] [ka]

[0100] 〔evaluation〕 The solution after the cleavage treatment was analyzed by reverse phase HPLC under the HPLC conditions shown in Table 3 below (Condition 3).

[0101] [Table 3]

[0102] FIG. 4 shows the HPLC chart. As can be seen from Figure 4, the peak of phosphorothioated oligonucleotide (sON) and the peak of the adduct (sON-adduct) were separated. In addition, a peak of cyclic phosphate (cp) derived from the linker was also observed in the HPLC chart under condition C.

[0103] [Example 4]

[0104] [Synthesis of specific carriers] PT-PS(a) (specific carrier) and PT-PS(c) (specific carrier) were synthesized as follows.

[0105] [ka]

[0106] <PT-PS(a)> Under argon flow, Primer support TMDiisopropylethylamine (30.8 μL, 180 μmol) was added to a suspension of 3a (200 mg, amino group content: 90.0 μmol) in anhydrous acetonitrile (4 mL), and compound 3a (61.3 mg, 90.0 μmol) and HBTU (34.1 mg, 90.0 μmol) synthesized by the same procedure as compound 3 in Example 1 were added. After stirring at room temperature for 1 hour, the reaction solution was filtered, the solid phase carrier (filtered material) was washed with acetonitrile, and further dried in vacuum overnight. The obtained solid was suspended in Cap A solution (acetonitrile solution of acetic anhydride) and Cap B solution (acetonitrile solution of methylimidazole) and stirred for 30 minutes. The reaction solution was filtered again, and the solid phase carrier (filtered material) was washed with acetonitrile and dried in vacuum overnight to obtain PT-PS (a). The amount of compound 3a supported on PT-PS (a) was quantified by the DMTr assay method and was 349 ± 10 μmol / g.

[0107] <PT-PS(c)>

[0108] (Synthesis of compound 3c) Under an argon atmosphere, 2,2'-((oxybis(ethane-2,1-diyl))bis(oxy))diacetic acid (91.8 mg, 0.413 mmol) was added to anhydrous pyridine (3 mL) solution of compound 2 (200 mg, 0.344 mmol) synthesized by the same procedure as in Example 1. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (65.9 mg, 0.344 mmol) and 4-dimethylaminopyridine (4.20 mg, 0.00344 mmol) were added and stirred at room temperature for 20 hours. After the reaction was completed, extraction was performed five times with a mixed solution of chloroform and methanol (chloroform / methanol = 10:1). The collected organic layer was washed once with water and once with saturated saline, then dried over sodium sulfate, and the solvent was distilled under reduced pressure. The crude product was purified by silica gel chromatography (chloroform only to chloroform / methanol=20:1) to obtain compound 3c (105 mg, yield 39%) as a white solid.

[0109] 11H-NMR (500 MHz, CDCl3) δ 8.66 - 8.64 (m, 2H), 7.97 - 7.95 (m, 1H), 7.66 - 7.61 (m, 3H), 7.57 - 7.54 (m, 1H), 7.48 - 7.47 (m, 2H), 7.47 - 7.22 (m, 8H), 6.87 - 6.83 (m, 4H), 5.87 (s, 1H), 5.33 (s, 1H), 4.79 (d, 1H, J = 6.0 Hz), 4.48 and 4.24 (ABq, 1H, 1H, J = 16.5 Hz), 4.16 (s, 2H), 4.12 (d, 1H, J = 6.0 Hz), 3.86 - 3.72 (m, 14H). 13 13C-NMR (125 MHz, CDCl3) δ 171.4, 171.0, 158.7, 145.3, 138.7, 137.5, 136.6, 136.2, 130.4, 130.3, 128.3, 128.0, 127.0, 126.8, 126.5, 125.8, 125.3, 125.0, 124.5, 123.4, 113.4, 88.1, 83.6, 82.6, 77.3, 77.2, 77.0, 76.8, 73.9, 73.1, 71.5, 70.8, 70.7, 70.3, 69.0, 68.6, 55.3, 45.1, 9.2. IR (ATR) cm -1 : 2929, 1746, 1607, 1507. HRMS (ESI-TOF): calcd for C 47 H 43 O 11 [M - H] ? 783.2805, found 783.2809.

[0110] (Synthesis of PT-PS(c)) Under an argon stream, Primer support TMTo a suspension of 3c (100 mg, amino group content: 45.0 μmol) in anhydrous acetonitrile (2 mL), diisopropylethylamine (15.4 μL, 90.0 μmol) was added, and further compound 3c (35.3 mg, 45.0 μmol) and HBTU (17.1 mg, 45.0 μmol) were added. After stirring at room temperature for 1 hour, the reaction solution was filtered, the solid phase support (filtered material) was washed with acetonitrile, and further dried in vacuum overnight. The obtained solid was suspended in Cap A solution (acetonitrile solution of acetic anhydride) and Cap B solution (acetonitrile solution of methylimidazole) and stirred for 30 minutes. The reaction solution was filtered again, the solid phase support (filtered material) was washed with acetonitrile, and dried in vacuum overnight to obtain PT-PS (c). The amount of compound 3c supported on PT-PS (c) was quantified by the DMTr assay method* and was 296 ± 21 μmol / g.

[0111] [Synthesis of Oligonucleotides] Using PT-PS(a) and PT-PS(c) as universal supports, T-10mer oligonucleotides (T 10 ) was synthesized. The synthesis scale was 1.0 μmol, and the synthesis was performed under trityl OFF conditions. Commercially available phosphoramidite of T was prepared as a 0.1 M anhydrous acetonitrile solution and used. 5-ethylthio-1H-tetrazole (0.25 M anhydrous acetonitrile solution) was used as the activator, and the condensation time was 10 minutes for the 3' end only and 25 seconds for the others.

[0112] [ka]

[0113] [Cleavage of oligonucleotide from universal support and removal of linker] The oligonucleotide was cleaved from the universal support and the associated linker was removed by treatment with a 28% aqueous ammonia solution at 55° C. for 48 hours.

[0114] [ka]

[0115] 〔evaluation〕 The solution after the cleavage treatment was analyzed by reverse phase HPLC under the same HPLC conditions as those in Condition 2 described above.

[0116] FIG. 5 shows the HPLC chart. As can be seen from FIG. 10 ) peak was observed.

Claims

1. A compound represented by the following general formula (1): 【Chemistry 1】 In the general formula (1), Ar represents a tricyclic or tetracyclic aromatic ring which may have a substituent, Z represents a hydrogen atom or a protecting group which can be removed by an acid, R １ ~R ４ each independently represents a hydrogen atom, an alkyl group, or an alkoxy group.

2. A compound represented by the following general formula (2): 【Chemistry 2】 In the general formula (2), Ar represents a tricyclic or tetracyclic aromatic ring which may have a substituent, Z represents a hydrogen atom or a protecting group which can be removed by an acid, R １ ~R ４ each independently represents a hydrogen atom, an alkyl group, or an alkoxy group, and L represents a divalent hydrocarbon group which may have an oxygen atom.

3. The compound according to claim 1 or 2, wherein Ar is an anthracene ring, a phenanthrene ring, a tetracene ring, or a pyrene ring, each of which may have a substituent.

4. The compound according to claim 1 or 2, wherein Ar is a phenanthrene ring which may have a substituent.

5. The compound according to claim 1 or 2, wherein Z is a protecting group that can be removed by an acid.

6. The compound according to claim 5 , wherein the protecting group removable with an acid is a trityl-based protecting group or a silyl-based protecting group.

7. The R １ ~R ４ The compound according to claim 1 or 2, wherein is a hydrogen atom.

8. The compound according to claim 2 , wherein L is an alkylene group optionally having an oxygen atom, an arylene group optionally having an oxygen atom, or a combination thereof.

9. A linker for a solid phase support for nucleic acid synthesis, which comprises the compound according to claim 1 or 2.

10. A solid support comprising a compound represented by the following general formula (3): 【Chemistry 3】 In the general formula (3), Ar represents a tricyclic or tetracyclic aromatic ring which may have a substituent, Z represents a hydrogen atom or a protecting group which can be removed by an acid, R １ ~R ４ each independently represents a hydrogen atom, an alkyl group, or an alkoxy group; L represents a divalent hydrocarbon group which may have an oxygen atom; and Sp represents a solid phase carrier.

11. The solid phase support according to claim 10, which is used for nucleic acid synthesis.

12. The solid phase support according to claim 10 or 11, wherein Ar is an anthracene ring, a phenanthrene ring, a tetracene ring, or a pyrene ring, each of which may have a substituent.

13. The solid phase support according to claim 10 or 11, wherein Ar is a phenanthrene ring which may have a substituent.

14. The solid phase support according to claim 10 or 11, wherein Z is a protecting group that can be removed by an acid.

15. The solid phase support according to claim 14, wherein the protecting group that can be removed by an acid is a trityl-based protecting group or a silyl-based protecting group.

16. The R １ ~R ４ The solid phase support according to claim 10 or 11, wherein is a hydrogen atom.

17. The solid phase support according to claim 10 or 11, wherein L is an alkylene group optionally having an oxygen atom, an arylene group optionally having an oxygen atom, or a combination thereof.

18. The solid phase carrier according to claim 10 or 11, wherein the Sp is a porous polymer carrier or a glass-based porous carrier.

19. A method for producing a nucleic acid, comprising a step of carrying out a nucleic acid synthesis reaction on the solid phase carrier according to claim 10 or 11.

20. The method for producing a nucleic acid according to claim 19, wherein the nucleic acid synthesis reaction is carried out by a phosphoramidite method.

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