Capping agent for nucleic acid synthesis, capping solution for nucleic acid synthesis, and method for producing nucleic acid

Abstract

The present invention addresses the problem of providing: a phosphoramidite-type capping agent for nucleic acid synthesis, the capping agent having excellent solubility, stability, and capping efficiency; a solution of the capping agent; and a method for producing a nucleic acid using the capping agent. A capping agent for nucleic acid synthesis according to the present invention comprises a compound represented by general formula (1). In general formula (1), Cy forms, together with an adjacent -O-P-O- group, an aliphatic heterocyclic group including a 5- or 6-membered ring and optionally having a substituent, and R1 and R2 each independently represent an aliphatic ring group or an aromatic ring group including a 5- or 6-membered ring and optionally having a substituent.

Description

Capping agent for nucleic acid synthesis, capping solution for nucleic acid synthesis, and method for producing nucleic acids

[0001] The present invention relates to a capping agent for nucleic acid synthesis, a capping solution for nucleic acid synthesis, and a method for producing nucleic acids.

[0002] Nucleic acids are typically synthesized by repeating a series of reaction cycles: (1) deprotection (detritylation) of a nucleoside or oligonucleotide, (2) coupling reaction, (3) capping reaction of unreacted products, and (4) oxidation or sulfurization reaction of the coupling product. In recent years, nucleic acid synthesis using the "phosphoamidite method," which uses phosphoramidites in the coupling reaction (2), has become mainstream. A specific example of the reaction cycle in the phosphoramidite method is shown below.

[0003]

[0004] (3) In capping reactions, the most common method is to cap the 5'-hydroxyl group of the unreacted product by acetylation using acetic anhydride. However, in capping by acetylation, an excess amount of acetic anhydride is used relative to the nucleic acid, and it is known that the following side reactions occur, leading to problems such as a decrease in purity and reaction yield due to an increase in impurities.

[0005]

[0006] To address the aforementioned problems, various phosphoramidite-type capping agents have been developed since around 1994 as alternatives to acetic anhydride. Specific examples of phosphoramidite-type capping agents are shown below.

[0007]

[0008] These phosphoramidite-type capping agents prevent the reduction in purity and reaction yield caused by side reactions that were problematic with acetylation-based capping.

[0009] D. Yu et al. , Tetrahedron Lett. , 1994, 35, 8565

[0010] In this context, when the present inventors investigated the capping agent described in Non-Patent Document 1, it became clear that uncapped products (uncapped products) may be observed after capping. In other words, it became clear that the capping efficiency may be insufficient. Furthermore, since capping agents are usually used by dissolving them in organic solvents (such as acetonitrile), excellent solubility in organic solvents (hereinafter also simply referred to as "solubility") is required. In addition, excellent stability during storage (hereinafter also simply referred to as "stability") is required for capping agents.

[0011] Therefore, in view of the above circumstances, the present invention aims to provide a phosphoramidite-type capping agent for nucleic acid synthesis and a solution thereof that are excellent in solubility, stability and capping efficiency, as well as a method for producing nucleic acids using the same.

[0012] As a result of diligent research into the above-mentioned problems, the inventors discovered that these problems can be solved by using a phosphoramidite with a specific structure, leading to the present invention. Specifically, the inventors found that the above-mentioned problems can be solved by the following configuration.

[0013] (1) A capping agent for nucleic acid synthesis comprising a compound represented by the general formula (1) described later. (2) The capping agent for nucleic acid synthesis according to (1) above, wherein the substituents that Cy can take are alkyl groups or phenyl groups. (3) R １ and R ２ A nucleic acid synthesis capping agent according to (1) or (2) above, wherein the substituent that can be taken is an alkyl group. (4) A nucleic acid synthesis capping agent according to any one of (1) to (3) above, wherein Cy, together with the adjacent -O-P-O- group, forms an unsubstituted 1,3,2-dioxaphosphorane ring or a 1,3,2-dioxaphospholinane ring. (5) R １ and R ２(1) to (4) above, wherein the capping agent for nucleic acid synthesis is an unsubstituted cyclopentane ring group, a cyclohexane ring group, a cyclopentadiene ring group, or a phenyl group. (6) A capping solution for nucleic acid synthesis comprising the capping agent for nucleic acid synthesis and an organic solvent, as described in any of (1) to (5) above. (7) A method for producing nucleic acids, comprising repeatedly performing a reaction cycle comprising: a deprotection step of deprotecting a hydroxyl-protected nucleoside or oligonucleotide; a coupling reaction step of obtaining a coupling product by coupling the nucleoside or oligonucleotide after the deprotection step with a phosphoramidite; a capping reaction step of capping the unreacted product from the coupling reaction step using the capping solution described in (6) above; and an oxidation reaction or sulfurization reaction step of oxidizing or sulfurizing the coupling product.

[0014] As shown below, the present invention provides a phosphoramidite-type capping agent for nucleic acid synthesis and a solution thereof, which are excellent in solubility, stability, and capping efficiency, as well as a method for producing nucleic acids using the same.

[0015] Before and after storage of Cap1 and DDP ３１ P NMR spectrum HPLC chart for comparing capping efficiency for primary hydroxyl groups HPLC chart for comparing capping efficiency for secondary hydroxyl groups HPLC chart for comparing base treatment HPLC chart of 12-mer oligonucleotide containing DNA bases (A, G, C, T) HPLC chart of 12-mer oligonucleotide (reverse) containing DNA bases (A, G, C, T)

[0016] The following describes the present invention's capping agent for nucleic acid synthesis, capping solution for nucleic acid synthesis, and method for producing nucleic acids. In this specification, numerical ranges expressed using "~" mean a range that includes the values ​​written before and after "~" as the lower and upper limits. In this specification, adenine in DNA (deoxyribonucleic acid) bases may be represented as "A", guanine as "G", cytosine as "C", and thymine as "T". In this specification, "nucleic acid" means a chain-like compound (oligonucleotide) in which nucleotides are linked by phosphodiester bonds, and includes DNA (deoxyribonucleic acid), RNA (ribonucleic acid), etc. In this 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.

[0017] [1] Capping agent for nucleic acid synthesis The capping agent for nucleic acid synthesis of the present invention (hereinafter also referred to as "the capping agent of the present invention") is a capping agent for nucleic acid synthesis comprising a compound represented by the general formula (1) described later.

[0018] The capping agent of the present invention has the above-mentioned structure and is therefore thought to be able to solve the problems described above. The reason for this is not clear, but it is presumed to be as follows. As shown in general formula (1) described later, the ester moiety of the phosphoramidite in the capping agent of the present invention has a cyclic structure. Therefore, it is thought to have low steric hindrance and high reactivity to the hydroxyl group of the unreacted product in the coupling reaction (excellent capping efficiency). Furthermore, as shown in general formula (1) described later, the substituent of the amine moiety in the capping agent of the present invention is cyclic, so it is thought to have excellent solubility in organic solvents and stability during storage.

[0019] The general formula (1) will be explained below.

[0020]

[0021] In the general formula (1), Cy, together with the adjacent -O-P-O- groups, forms an optionally substituted 5- or 6-membered aliphatic heterocyclic group, and R １ and R ２ each independently represents an optionally substituted 5- or 6-membered aromatic or aliphatic ring group.

[0022] [Cy] As described above, in the general formula (1), Cy, together with the adjacent -O-P-O- groups, forms an optionally substituted 5- or 6-membered aliphatic heterocyclic group. Among them, for the reason that the effects of the present invention are more excellent, it is preferable to form an optionally substituted 5-membered aliphatic heterocyclic group together with the adjacent -O-P-O- groups. The above Cy preferably forms a 1,3,2-dioxaphospholane ring or a 1,3,2-dioxaphosphorinane ring (especially a 1,3,2-dioxaphospholane ring) which may have a substituent together with the adjacent -O-P-O- groups, and more preferably forms an unsubstituted 1,3,2-dioxaphospholane ring or a 1,3,2-dioxaphosphorinane ring (especially a 1,3,2-dioxaphospholane ring). The following structural formulas represent a 1,3,2-dioxaphospholane ring group (the left structural formula) and a 1,3,2-dioxaphosphorinane ring group (the right structural formula).

[0023]

[0024] Examples of the above substituent include the substituent W described later. Among them, for the reason that the effects of the present invention are more excellent, it is preferably an alkyl group (especially having 1 to 4 carbon atoms) or a phenyl group.

[0025] [R １ and R ２ As described above, in the general formula (1), R １ and R ２ each independently represents an optionally substituted 5- or 6-membered aromatic or aliphatic ring group. Among them, for the reason that the effects of the present invention are more excellent, it is preferably an unsubstituted 5- or 6-membered aromatic or aliphatic ring group, and more preferably an unsubstituted 5- or 6-membered aliphatic ring group. The above R１ and R ２ For reasons that the effects of the present invention are superior, each of these groups is preferably a cyclopentane ring group, a cyclohexane ring group, a cyclopentadiene ring group, or a phenyl group, which may each have a substituent; more preferably an unsubstituted cyclopentane ring group, a cyclohexane ring group, a cyclopentadiene ring group, or a phenyl group; even more preferably an unsubstituted cyclopentane ring group or a cyclohexane ring group; and particularly preferably an unsubstituted cyclohexane ring group. Examples of the substituents include substituent W, which will be described later. Among these, alkyl groups (particularly having 1 to 4 carbon atoms) are preferred for reasons that the effects of the present invention are superior.

[0026] [Preferred Embodiments] The capping agent of the present invention is preferably a compound represented by the following general formula (1A) for reasons that the effects of the present invention are superior.

[0027]

[0028] In general formula (1A), R １ and R ２ Each independently represents a 5- or 6-membered aromatic ring group or aliphatic ring group which may have substituents, and R ３ and R ４ Each of these independently represents a hydrogen atom or a substituent, and n represents 2 or 3.

[0029] R in general formula (1A) １ and R ２ The definition, specific examples, and preferred embodiments of R in the general formula (1) described above are given by R １ and R ２ It is the same as this.

[0030] As described above, in general formula (1A), R ３ and R ４ Each of these independently represents a hydrogen atom or a substituent. Among these, a hydrogen atom is preferred because it provides superior effects for the present invention. Note that there are multiple R ３ R can be the same or different, and there may be multiple Rs. ４These may be the same or different. Examples of the substituents include substituent W, which will be described later. Among these, alkyl groups (particularly having 1 to 4 carbon atoms) or phenyl groups are preferred for the reasons that the effects of the present invention are superior.

[0031] As described above, in general formula (1A), n represents 2 or 3. Among these, 2 is preferred because it provides superior effects for the present invention.

[0032] [Specific Examples] Specific examples of the capping agent of the present invention are listed below. However, the invention is not limited to these examples.

[0033]

[0034] [Substituent W] The substituent W used herein is described below. Substituents W include, for example, halogen groups (halogen atoms), alkyl groups (e.g., methyl groups; including cycloalkyl groups, bicycloalkyl groups, and tricycloalkyl groups), alkenyl groups (including cycloalkenyl groups and bicycloalkenyl groups), alkynyl groups, aryl groups, heterocyclic groups (may also be called heterocyclic groups), cyano groups, hydroxyl groups (may also be called hydroxyl groups), nitro groups, carboxyl groups, alkoxy groups, aryloxy groups, silyloxy groups, heterocyclic oxy groups, acyloxy groups, carbamoyl groups, carbamoyloxy groups, alkoxycarbonyloxy groups, aryloxycarbonyloxy groups, amino groups (including anilino groups), ammonia groups, and dialkyl groups. Mino group, acylamino group, aminocarbonylamino group, alkoxycarbonylamino group, aryloxycarbonylamino group, sulfamoylamino group, alkyl or arylsulfonylamino group, mercapto group, alkylthio group, arylthio group, heterocyclic thio group, sulfamoyl group, sulfo group, alkyl or arylsulfinyl group, alkyl or arylsulfonyl group, acyl group, aryloxycarbonyl group, alkoxycarbonyl group, aryl or heterocyclic azo group, imide group, phosphino group, phosphinyl group, phosphinyloxy group, phosphinylamino group, phosphono group, silyl group, hydrazino group, ureido group, boronic acid group (-B(OH)) ２ ), phosphat group (-OPO(OH)２ ), sulfate group (-OSO ３ Examples include H), other known substituents, etc.

[0035] [Manufacturing Method] The method for manufacturing the capping agent of the present invention is not particularly limited and can be manufactured by combining known methods. For example, it can be manufactured by referring to the synthesis route of Cap1 described later.

[0036] [2] Capping solution for nucleic acid synthesis The capping solution for nucleic acid synthesis of the present invention (hereinafter also referred to as "the capping solution of the present invention") is a capping solution for nucleic acid synthesis that contains the capping agent and organic solvent of the present invention described above. The capping solution of the present invention may contain components other than the capping agent and organic solvent of the present invention.

[0037] [Capping agent] The capping agent of the present invention is as described above.

[0038] [Content] In the capping solution of the present invention, the content of the capping agent of the present invention can be, for example, 0.01 to 5 M (mol / L), and is preferably 0.05 to 3 M, and more preferably 0.1 to 2 M, for the reason that the effects of the present invention are superior.

[0039] [Organic solvent] The organic solvent is not particularly limited and examples include acetonitrile, tetrahydrofuran (THF), toluene, dichloromethane, etc. However, for the reasons that the effects of the present invention are superior, it is preferably an amphiphilic organic solvent, more preferably acetonitrile, more preferably THF, and even more preferably acetonitrile.

[0040] [Moisture Content] The capping solution of the present invention is preferably substantially water-free (anhydrous). Here, "substantially water-free (anhydrous)" means that the moisture content of the capping solution of the present invention is 100 ppm by mass or less relative to the total mass of the capping solution of the present invention. The moisture content of the capping solution of the present invention is more preferably 50 ppm by mass or less relative to the total mass of the capping solution of the present invention for the reason that the effects of the present invention (especially stability) are better. [3] Method for Producing Nucleic Acids The method for producing nucleic acids of the present invention (hereinafter also referred to as "the method for producing nucleic acids of the present invention") is a method for producing nucleic acids that includes a coupling reaction step of obtaining a coupling product by a coupling reaction and a capping reaction step of capping the unreacted product of the coupling reaction step using the capping solution of the present invention described above. The method for producing nucleic acids of the present invention may include other steps.

[0041] [Coupling Reaction Step] The coupling reaction step is a step in which a coupling product is obtained by a coupling reaction. Specific examples of the above coupling reactions include the H-phosphonate method, the phosphotryester method (phosphate triester method), the phosphodiester method (phosphate diester method), and the phosphoramidite method. Among these, the phosphoramidite method is preferred because it has a high nucleic acid synthesis capacity and yields high-purity nucleic acids. An example of a coupling reaction step using the phosphoramidite method is the coupling reaction step in a preferred embodiment described later.

[0042] [Capping Reaction Step] The capping reaction step is a step in which the unreacted product from the coupling reaction step described above is capped using the capping solution of the present invention described above. Since the unreacted product that did not couple in the coupling reaction step described above has a hydroxyl group at its 5'-terminus (position 5'), this hydroxyl group of the unreacted product is reacted with the capping agent of the present invention described above to form a capped product. Since the capped product does not undergo a subsequent coupling reaction, the generation of nucleic acids lacking specific bases can be prevented, and separation and purification in the final stage can be facilitated. An example of a capping reaction step in the case of a coupling reaction step using the phosphoramidite method is the capping reaction step in a preferred embodiment described later.

[0043] [Preferred Embodiments] A preferred embodiment of the manufacturing method of the present invention is, for example, a method for producing nucleic acids that involves repeatedly performing a reaction cycle including the following steps (1) to (4). This method is the same as a conventionally known nucleic acid synthesis method using the phosphoramidite method, except that the capping solution of the present invention is used in the capping reaction step. (1) A deprotection step in which a hydroxyl group protected nucleoside or oligonucleotide is deprotected. (2) A coupling reaction step in which the nucleoside or oligonucleotide after the deprotection step is coupled with a phosphoramidite to obtain a coupling product. (3) A capping reaction step in which the unreacted product from the coupling reaction step is capped using the capping solution of the present invention described above. (4) An oxidation reaction or sulfurization reaction step in which the coupling product is oxidized or sulfurized.

[0044] [Deprotection Step] The deprotection step is a step of deprotecting a nucleoside or oligonucleotide whose hydroxyl group at the 5'-terminus (5' position) is protected. This enables the following coupling reaction. Any conventionally known nucleoside can be used. The oligonucleotide is usually an oligonucleotide obtained by repeatedly performing the above reaction cycle. The protecting group that protects the hydroxyl group is preferably a protecting group that can be removed by an acid, and for reasons that the effects of the present invention are superior, it is preferably a protecting group that can be removed by a Brønsted acid such as trichloroacetic acid or dichloroacetic acid, a trityl protecting group (e.g., 4,4'-dimethoxytrityl group (DMTr group)) or a silyl protecting group (e.g., trimethylsilyl group) is more preferred, and a trityl protecting group is even more preferred. The hydroxyl-protected nucleoside or oligonucleotide used in the deprotection step is usually bound to a carrier. The support is preferably a solid-phase synthesis support that allows for easy removal of excess reagents used during nucleic acid synthesis by washing. Specific examples include glass-based porous support; porous polymer support such as polystyrene-based porous support or acrylamide-based porous support. A glass-based porous support refers to a porous support containing glass as a component, such as particulate porous glass particles (CPG: Controlled Pore Glass). Methods for deprotecting hydroxyl-protected nucleosides or oligonucleotides include using dichloroacetic acid-toluene solution, trichloroacetic acid-dichloromethane solution, etc.

[0045] [Coupling Reaction Step] The coupling reaction step is a step in which a coupling reaction is carried out between the nucleoside or oligonucleotide after the deprotection step described above and a phosphoramidite to obtain a coupling product. Conventionally known phosphoramidites can be used. It is preferable to use an activator in the coupling reaction. Examples of activators include 1H-tetrazole, 5-methylthio-1H-tetrazole (MTT), 5-ethylthio-1H-tetrazole (ETT), 5-nitrophenyl-1H-tetrazole (NPT), 5-benzylthio-1H-tetrazole (BTT), 5-[3,5-bis(trifluoromethyl)phenyl]-1H-tetrazole, 4,5-dicyanoimidazole (DCI), N-methylbenzimidazolium=trifluoromethanesulfonic acid, N-(phenyl)imidazolium=trifluoromethanesulfonic acid, etc. Among these, ETT, BTT, and DCI are preferred because they provide superior effects for the present invention. Furthermore, the activator described above may be used in solution form dissolved in a solvent, such as acetonitrile.

[0046] [Capping Reaction Step] This step involves capping the unreacted product from the coupling reaction step described above using the capping solution of the present invention described above. In the coupling reaction step, it is preferable to use an activator together with the capping solution of the present invention for better performance. Specific examples and preferred embodiments of the activator are the same as those described in the coupling reaction step described above.

[0047] [Oxidation or Sulfurization Reaction Step] The oxidation or sulfurization reaction step is a step in which the phosphite ester bond in the coupling product described above is oxidized or sulfurized. As a result, the coupling product becomes a phosphate ester or a thiophosphate ester. Examples of oxidizing agents include iodine-pyridine aqueous solution and iodine-pyridine-tetrahydrofuran aqueous solution. Furthermore, the oxidizing agent described above may also contain iodides such as hydrogen iodide and alkali iodides (e.g., potassium iodide). Examples of sulfiding agents include phenylacetyl disulfide (PADS), 3H-1,2-benzodithiol-3-one-1,1-dioxide (Beaucage reagent), 5-phenyl-3H-1,2,4-dithiazoline-3-one (POS), 5-amino-3H-1,2,4-dithiazoline-3-thion (ADTT), and [(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thion (DDTT). The above-mentioned sulfiding agents may also be used in solution form dissolved in a solvent, such as acetonitrile and pyridine.

[0048] [Other steps] The preferred embodiment of the manufacturing method of the present invention described above may include steps other than those described above, such as a cutting step and a purification step.

[0049] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0050] [Synthesis of Capping Agents] Each capping agent was synthesized as follows. Cap1 is the compound represented by the general formula (1) above (specific compound) (where Cy, together with the adjacent -O-P-O- group, forms an unsubstituted 1,3,2-dioxaphosphorane ring, R １ and R ２ (both represent an unsubstituted cyclohexane ring group). On the other hand, Cap2 corresponds to R in general formula (1). １ and R ２Since both are compounds with chain-like (branched) aliphatic groups, they do not fall under the category of specified compounds. Furthermore, Cap3 is a compound in which the Cy in general formula (1) does not form a ring, so it does not fall under the category of specified compounds.

[0051] [Cap1] Under an argon stream, a solution of dicyclohexylamine (3.2 mL, 15.8 mmol) in anhydrous diethyl ether (10 mL) was added to a solution of 2-chloro-1,3,2-dioxaphosphorane (0.7 mL, 7.91 mmol) in anhydrous diethyl ether (10 mL) under ice cooling, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction suspension was filtered through Celite and the solvent was removed by vacuum distillation. A hexane solution of the crude product was added to silica gel (DIOL MB100-40 / 75, Fuji Silysia Chemical) and the solvent was removed by vacuum distillation. The product was then purified by silica gel column chromatography (hexane, DIOL MB100-40 / 75, Fuji Silysia Chemical) to obtain Cap1 (where Et represents the ethyl group in the reaction equation) (1.90 g, 89%) as a white solid (Mp. (melting point): 54-56°C).

[0052]

[0053] NMR (nuclear magnetic resonance) measurements, IR (infrared absorption spectroscopy) measurements, and HRMS (high-resolution mass spectrometry) measurements were performed on Cap1. The results are shown below.

[0054] １ H NMR (500MHz, CDCl ３ ): δ 4.17-4.09 (m, 2H), 3.91-3.83 (m, 2H), 2.94-2.86 (m, 2H), 1.78-1.72 (m, 8H), 1.60-1.50 (m, 6H), 1.26-1.18 (m, 4H), 1.12-1.03 (m, 2H). １３ C NMR (126MHz, CDCl ３ ): δ 63.9, 63.8, 53.84, 53.77, 35.63, 35.57, 26.9, 25.6. ３１ P NMR (202 MHz, CDCl ３ ): δ 144.3. IR(ATR)cm －１ :2925,2850. HRMS(EI): calcd for C １４ ​H ２６ NO ２ P[M] 271.1701, found 271.1701.

[0055] [Cap2] Under an argon stream, a solution of diisopropylamine (4.5 mL, 31.6 mmol) in anhydrous diethyl ether (50 mL) was added to a solution of 2-chloro-1,3,2-dioxaphosphorane (1.4 mL, 15.8 mmol) in anhydrous diethyl ether (10 mL) under ice cooling, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction suspension was filtered through Celite and the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel chromatography (hexane, DIOL MB100-40 / 75, Fuji Silysia Chemical) to obtain Cap2 (in the reaction formula, Et represents the ethyl group and iPr represents the isopropyl group) (1.1 g, 36%) as a colorless transparent liquid. １ H NMR, １３ 1C NMR and ３１ The P NMR spectrum was consistent with the literature value (Chem. Eur. J. 2001, 7, 1455-1467).

[0056]

[0057] [Cap3] Under an argon stream, diethyl chlorophosphite (0.92 mL, 6.39 mmol) and dicyclohexylamine (1.3 mL, 6.39 mmol) were added to a tetrahydrofuran (60 mL) solution of N,N-diisopropylethylamine (2.2 mL, 12.8 mmol) under ice cooling, and the mixture was stirred at room temperature for 3 hours. After the reaction was complete, the reaction suspension was filtered through Celite and the solvent was removed by vacuum distillation. Silica gel (DIOL MB100-40 / 75, Fuji Silysia Chemical) was added to a hexane solution of the crude product and the solvent was removed by vacuum distillation. The product was then purified by silica gel column chromatography (hexane, DIOL MB100-40 / 75, Fuji Silysia Chemical) to obtain Cap3 (in the reaction formula, Et represents the ethyl group and iPr represents the isopropyl group) (684 mg, 36%) as a colorless transparent liquid.

[0058]

[0059] NMR, IR, and HRMS measurements were performed on Cap3. The results are shown below.

[0060] １ H NMR (500MHz, CDCl ３ ): δ 3.75-3.58 (m, 4H), 3.11-3.03 (m, 2H), 1.75-1.45 (m, 14H), 1.33-1.23 (m, 10H), 1.11-1.02 (m, 2H). １３ C NMR (126MHz, CDCl ３ ): δ 58.9, 58.8, 51.8, 51.7, 35.62, 35.56, 26.7, 25.7, 17.03, 16.96. ３１ P NMR (202 MHz, CDCl ３ ): δ 144.0. IR(ATR)cm －１ :2926,2852. HRMS(EI): calcd for C １６ H ３２ NO ２ P [M] 301.2171, found 301.2172.

[0061] [Evaluation] The following evaluations were conducted on various capping agents.

[0062] [Solubility] Cap1 to Cap3 and commercially available diethyl N,N-diisopropyl phosphoramidite (DDP; the Cy in general formula (1) does not form a ring, and R １ and R ２ The solubility of Cap2 and Cap3 (which do not fall under the category of specific compounds because both are compounds with chain-like (branched) aliphatic groups) in an organic solvent (acetonitrile) was investigated. As a result, Cap2 and Cap3 showed low solubility in acetonitrile, making it difficult to prepare a 0.1 M acetonitrile solution, which is commonly used in solid-phase synthesis methods (phosphoamidite method) using automated DNA synthesizers. On the other hand, Cap1 and DDP were able to be prepared in acetonitrile solutions of over 2.0 M, indicating excellent solubility.

[0063] [Stability] The stability of Cap1 and DDP was evaluated. Specifically, Cap1 and DDP were stored at room temperature for 3 months in containers filled with argon, and then these...​３１ The P NMR spectrum was measured. ３１ Figure 1 shows the P NMR spectral data. In DDP, the 144.2 ppm phosphorus peak disappeared after 3 months. On the other hand, in Cap1, the 144.4 ppm phosphorus peak remained almost unchanged even after 3 months, indicating superior stability.

[0064] [Capping efficiency]

[0065] <Capping efficiency for primary hydroxyl groups> Using dT-CPG (thymidine in which the hydroxyl group at the 5' position is protected by a dimethoxytrityl group (DMTr group), bound to porous glass particles (CPG carrier) via a spacer), T-9mer oligonucleotides (T) are synthesized by an automated DNA synthesis apparatus. ９ The compound was synthesized. The synthesis scale was 0.2 μmol, and the procedure was carried out under trityl-OFF conditions (the DMTr group was deprotected at the end with a 3 w / v% trichloroacetic acid-dichloromethane solution). A commercially available T phosphoramidite (with the hydroxyl group at the 5' position protected by a DMTr group) was used as a 0.1 M anhydrous acetonitrile solution. 5-ethylthio-1H-tetrazole (ETT, 0.25 M anhydrous acetonitrile solution) was used as the activator, and the coupling time was 25 seconds.

[0066] Next, using (1) to (3) below, the primary hydroxyl group at the 5'-terminus of T-9mer was capped, followed by coupling of T with phosphoramidite, oxidation of the phosphite ester, and deprotection of the DMTr group. The water content of (1) to (3) was 100 ppm by mass or less relative to the total mass of each solution.

[0067] (1) CapA (a solution of acetic anhydride (capping agent) / tetrahydrofuran = 9.1 / 90.9 (volume ratio)) and CapB (a solution of tetrahydrofuran / 1-methylimidazole (activator) / pyridine = 8:1:1 (volume ratio)) (2) A 0.1 M anhydrous acetonitrile solution of DDP (capping agent) and a 0.25 M anhydrous acetonitrile solution of ETT (activator) (3) A 0.1 M anhydrous acetonitrile solution of Cap1 (capping agent) and a 0.25 M anhydrous acetonitrile solution of ETT (activator)

[0068] The capping times were as follows: (1) CapA and CapB for 45 seconds (using normal capping conditions), (2) DDP and ETT, and (3) Cap1 and ETT for 25 seconds (using coupling conditions in the normal phosphoramidite method, as they are phosphoramidite-type capping agents).

[0069] Subsequently, as a base treatment, the oligonucleotides were cleaved from the CPG support by treatment with a 28% aqueous ammonia solution at room temperature for 2 hours. After removing the ammonia from the cleaved solution by vacuum concentration, the solution was analyzed by reverse-phase HPLC (high-performance liquid chromatography). The HPLC analysis conditions are shown below ((a) to (f)).

[0070] (a) Eluent Solution A: 0.1 M triethylammonium acetate buffer (pH 7.0) Solution B: Acetonitrile (b) Gradient Solution B: 5-15% (30 min) (c) Column Waters Xbridge ＴＭ Shield RP18 2.5 μm (4.6 × 50 mm) (d) Flow rate 1.0 mL / min (e) Column temperature 40°C (f) Detection UV (ultraviolet) detector (254 nm)

[0071] The obtained HPLC chart is shown in Figure 2. The yield of oligonucleotides was calculated from the peak area ratio of the HPLC.

[0072] In the above synthesis, as capping progresses, T ９ Alternatively, capped-T ９ Or both (both are capped products) are produced, and if capping does not proceed, T １０ (Uncapped product) is generated.

[0073] (1) When capping is performed using (capping agent: acetic anhydride), T ９ It was produced with a yield of 96%, T １０ 2% was generated. When capping was performed using (2) (capping agent: DDP), capped-T ９ It was produced with a yield of 92%, T １０It was produced with a yield of 7%. When capping was performed using (3) (capping agent: Cap1), capped-T ９ and T ９ These were obtained with yields of 88% and 10%, respectively. １０ It was not observed.

[0074] As described above, Cap1 showed superior capping efficiency compared to DDP. It also showed superior capping efficiency compared to acetic anhydride.

[0075]

[0076] <Capping efficiency for secondary hydroxyl groups> Using dT-CPG, an oligonucleotide (T) is obtained by condensing a reverse synthesis phosphoramidite (reverse T) with T only at the 5'-terminus using an automated DNA synthesizer. Ｒ T ８ , T Ｒ = Reverse T, in the reaction equation below, T Ｒ (Reverse T, represented by italicized T) was synthesized. The synthesis scale was 0.2 μmol, and the procedure was carried out under trityl OFF conditions. Commercially available T phosphoramidite was used as a 0.1 M anhydrous acetonitrile solution. ETT (0.25 M anhydrous acetonitrile solution) was used as the activator, and the coupling time was 25 seconds.

[0077] Next, using each of the above (1) to (3), T Ｒ T ８ After capping the secondary hydroxyl group of the reversed T, coupling with phosphoramidite, oxidation of the phosphite ester, and deprotection of the DMTr group were performed. The capping time was the same as the <capping efficiency for primary hydroxyl groups> described above.

[0078] Subsequently, the sample was excised and HPLC analysis was performed, similar to the method described above for <capping efficiency for primary hydroxyl groups>.

[0079] The obtained HPLC chart is shown in Figure 3. The yield of oligonucleotides was calculated from the peak area ratio of the HPLC.

[0080] In the above synthesis, as capping progresses, T Ｒ T ８or capped-T Ｒ T ８ Or both of them (both are capped forms) are generated, and TT is generated if capping does not proceed Ｒ T ８ (uncapped form) is generated.

[0081] When capping is carried out using (1) (capping agent: acetic anhydride), T Ｒ T ８ is generated with a yield of 83%, and TT Ｒ T ８ is generated with a yield of 16%. When capping is carried out using (2) (capping agent: DDP), capped-T Ｒ T ８ is generated with a yield of 91%, and TT Ｒ T ８ is generated with a yield of 6%. When capping is carried out using (3) (capping agent: Cap1), capped-T Ｒ T ８ and T Ｒ T ８ are obtained with yields of 78% and 19% respectively, and TT Ｒ T ８ was not observed. <00,00277>

[0082] As described above, Cap1 showed excellent capping efficiency compared to DDP. Also, it showed excellent capping efficiency compared to acetic anhydride. <0000,280>

[0084] [Comparison of Base Treatment] Except for changing the base treatment to conditions A and B below, the procedure from the synthesis of oligonucleotides to HPLC analysis was carried out in the same manner as when capping was performed using (3) of <Capping Efficiency for Primary Hydroxyl Groups> described above. In condition A, the oligonucleotides were treated with a 50 mM potassium carbonate methanol solution at room temperature for 4 hours to cleave from the CPG support, and then 0.1 M triethylammonium acetate buffer (pH 7.0) was added to the cleaved solution, methanol was removed by vacuum concentration, and the solution was analyzed by reverse-phase HPLC. In condition B, the oligonucleotides were treated with a 50 mM potassium carbonate ethylene glycol solution at room temperature for 4 hours to cleave from the CPG support, and then NAP ＴＭ After removing the 50 mM potassium carbonate ethylene glycol solution using a -5 column, the solution was analyzed by reverse-phase HPLC.

[0085] The obtained HPLC chart is shown in Figure 4. The yield of oligonucleotides was calculated from the peak area ratio of the HPLC.

[0086] Under condition A (treatment with a methanol solution of 50 mM potassium carbonate), T is compared to treatment with a 28% aqueous ammonia solution. ９ Increased capped-T ９ A decrease was observed. Under condition B (treatment with 50 mM potassium carbonate ethylene glycol solution), T ９ Only generates capped-T ９ It was not observed.

[0087] [Synthesis of nucleic acids]

[0088] [Synthesis and Purification of 12-mer Oligonucleotides Containing DNA Bases (A, G, C, T)] Using dT-CPG, 12-mer oligonucleotides containing DNA bases (A, G, C, T) were synthesized using an automated DNA synthesizer. The synthesis scale was 0.2 μmol, and the process was carried out under trityl OFF conditions (deprotecting the DMTr group with a 3 w / v% trichloroacetic acid-dichloromethane solution at the end) and trityl ON conditions (not deprotecting the DMTr group at the end). Commercially available phosphoramidites Bz-dA (phosphoramidite of 2'-deoxyadenosine in which the amino group is protected with a benzoyl group and the hydroxyl group at the 5' position is protected with a DMTr group), iBu-dG (phosphoramidite of 2'-deoxyguanosine in which the amino group is protected with an isobutyryl group and the hydroxyl group at the 5' position is protected with a DMTr group), Ac-dC (phosphoramidite of 2'-deoxycytidine in which the amino group is protected with an acetyl group and the hydroxyl group at the 5' position is protected with a DMTr group), and T were used as 0.1 M anhydrous acetonitrile solutions. 5-ethylthio-1H-tetrazole (ETT, 0.25 M anhydrous acetonitrile solution) was used as the activator, and the coupling time was 25 seconds. Capping was performed using either (1) or (3) below. The capping time was 45 seconds for (1) and 25 seconds for (3). The water content of (1) and (3) is 100 ppm by mass or less relative to the total mass of each solution.

[0089] (1) CapA (a solution of acetic anhydride (capping agent) / tetrahydrofuran = 9.1 / 90.9 (volume ratio)) and CapB (a solution of tetrahydrofuran / 1-methylimidazole (activator) / pyridine = 8:1:1 (volume ratio)) (3) A 0.1 M anhydrous acetonitrile solution of Cap1 (capping agent) and a 0.25 M anhydrous acetonitrile solution of ETT (activator)

[0090] Subsequently, the oligonucleotides obtained under trityl-OFF conditions were cleaved from the CPG support by treatment with 28% ammonia aqueous solution at 55°C for 16 hours, and then ammonia was removed by vacuum concentration. The solution was then analyzed by reverse-phase HPLC. The oligonucleotides obtained under trityl-ON conditions were cleaved from the CPG support by treatment with 28% ammonia aqueous solution at 55°C for 16 hours, and then ammonia was removed by vacuum concentration. A simple purification was performed using Sep-Pak®, and the solution was then analyzed by reverse-phase HPLC. The HPLC analysis conditions were the same as those described above for <capping efficiency for primary hydroxyl groups>.

[0091] The obtained HPLC chart is shown in Figure 5.

[0092] When capping was performed using (1) (capping agent: acetic anhydride) and when capping was performed using (3) (capping agent: Cap1), no by-products originating from capping were observed in either case, demonstrating that the target 12-mer oligonucleotide could be efficiently synthesized. Furthermore, simple purification using Sep-Pak removed peaks other than the target oligonucleotide, yielding high-purity target 12-mer oligonucleotide.

[0093]

[0094] [Synthesis and Purification of 12-mer Oligonucleotides (Reverse) Containing DNA Bases (A, G, C, T)] Except for using phosphoramidite for reversing, the synthesis and purification of 12-mer oligonucleotides (reverse) containing DNA bases (A, G, C, T) was performed according to the same procedure as described above in [Synthesis and Purification of 12-mer Oligonucleotides (A, G, C, T)], and HPLC analysis was performed.

[0095] The obtained HPLC chart is shown in Figure 6.

[0096] When capping was performed using (1) (capping agent: acetic anhydride) and when capping was performed using (3) (capping agent: Cap1), no by-products originating from capping were observed in either case, demonstrating that the target 12-mer oligonucleotide could be efficiently synthesized. Furthermore, simple purification using Sep-Pak removed peaks other than the target oligonucleotide, yielding high-purity target 12-mer oligonucleotide.

[0097]

Claims

1. A capping agent for nucleic acid synthesis comprising a compound represented by the following general formula (1). In general formula (1), Cy, together with the adjacent -O-P-O- group, forms a 5- or 6-membered aliphatic heterocyclic group which may have substituents, and R １ and R ２ Each of these independently represents a 5- or 6-membered aromatic ring group or aliphatic ring group which may have substituents.

2. The nucleic acid synthesis capping agent according to claim 1, wherein the substituents that Cy may take are alkyl groups or phenyl groups.

3. The aforementioned R １ and R ２ The capping agent for nucleic acid synthesis according to claim 1, wherein the substituent that can be taken is an alkyl group.

4. The capping agent for nucleic acid synthesis according to claim 1, wherein the Cy, together with the adjacent -O-P-O- group, forms an unsubstituted 1,3,2-dioxaphosphorane ring or a 1,3,2-dioxaphospholinane ring.

5. The aforementioned R １ and R ２ The capping agent for nucleic acid synthesis according to claim 1, wherein the group is an unsubstituted cyclopentane ring group, a cyclohexane ring group, a cyclopentadiene ring group, or a phenyl group.

6. A capping solution for nucleic acid synthesis comprising the nucleic acid synthesis capping agent and organic solvent described in claim 1.

7. A method for producing nucleic acids, comprising repeatedly performing a reaction cycle comprising: a deprotection step of deprotecting a hydroxyl-protected nucleoside or oligonucleotide; a coupling reaction step of obtaining a coupling product by coupling the nucleoside or oligonucleotide after the deprotection step with a phosphoramidite; a capping reaction step of capping the unreacted product from the coupling reaction step using the capping solution described in claim 6; and an oxidation reaction or sulfurization reaction step of oxidizing or sulfurizing the coupling product.

Images