Compound, RNA synthesis initiator, and use thereof

Abstract

The present invention addresses the problem of providing a compound that can be used as an RNA synthesis initiator having a desired transcriptional activity, an RNA synthesis initiator containing the compound, a kit, RNA, and a method for producing RNA. The present invention provides a compound represented by formula (1). In the formula, each symbol other than X1, X2, and X has a definition stated in the description, X1, X2, and X each independently represent -O- or -S-, and each X, if existing in a number more than one, may be the same or different from each other. At least one of X1, X2, and X represents -S-.

Description

Compounds, RNA synthesis initiators, and their uses

[0001] The present invention relates to a compound that can be used as an initiator of RNA synthesis. The present invention further relates to an initiator of RNA synthesis, a kit, RNA, and a method for producing RNA.

[0002] Generally, a "cap structure" is added to the 5' end of mRNA, which has functions such as stabilization in vivo, improvement of translation activity (protein synthesis), and suppression of immunogenicity. Patent Document 1 describes a nucleotide compound having a Cap structure at the 5' end. Patent Document 1 also describes that capped mRNA can be synthesized with high efficiency by using this nucleotide compound in in vitro transcription synthesis. Patent Document 2 describes that translation activity is improved by introducing an S atom into the sugar / linker portion of the Cap structure.

[0003] Special table 2018-527015 publication Special table 2023-540562 publication

[0004] An object of the present invention is to provide a compound that can be used as an RNA synthesis initiator having desired transcription activity.A further object of the present invention is to provide an RNA synthesis initiator, a kit, RNA, and a method for producing RNA, each containing the compound.

[0005] As a result of extensive research to solve the above problems, the present inventors synthesized compounds in which the sugar moiety of the 5'-terminal Cap structure was changed to a thiosugar, and confirmed their transcriptional activity, finding that they exhibited the desired transcriptional activity. Furthermore, when capped mRNA was used to evaluate cellular and in vivo translation activity, they found that it exhibited high translation activity. The present invention was completed based on the above findings. Specifically, the present invention provides the following:

[0006] <1> A compound represented by the following formula (1): In the formula, B １ teeth, where * indicates the bond position, B ２ is a natural, modified, or non-natural nucleobase; B ３R may be the same or different when present in plural and is a natural, modified or non-natural nucleobase; １ and R ２ are each independently hydrogen, fluorine, —OH, or —O-alkyl; R ３ and R ４ are each independently hydrogen, fluorine, —OH, or —O-alkyl; R ４ When there are a plurality of X's, they may be the same or different; １ , X ２ and X are each independently -O- or -S-, and when a plurality of X's are present, they may be the same or different, provided that X １ , X ２ and at least one of X is —S—, and n is an integer of 0 to 7. <2> The compound according to <1>, which is a compound represented by the following formula (2): In the formula, n represents an integer of 0 to 2, and each R is independently —H, —CH ３ wherein each symbol other than n and R is as defined in <1>. <3> An RNA synthesis initiator comprising the compound according to <1> or <2>. <4> An RNA synthesis kit comprising the RNA synthesis initiator according to <3> and an RNA polymerase. <5> RNA comprising the RNA synthesis initiator according to <3>. <6> A method for producing RNA, comprising transcribing a template nucleic acid in the presence of the RNA synthesis initiator according to <3>.

[0007] According to the present invention, an RNA synthesis initiator capable of exerting a desired transcription activity can be provided.

[0008] Figure 1 shows the results of measuring the translation activity of capped mRNA in cells. Figure 2 shows the results of measuring the translation activity of capped mRNA in cells. Figure 3 shows the results of measuring the translation activity of capped mRNA in cells. Figure 4 shows the results of measuring the translation properties of capped mRNA in vivo in mice.

[0009] Hereinafter, embodiments of the present invention will be described in detail. In this specification, a numerical range indicated using "to" means a range that includes the numerical values ​​before and after "to" as the minimum and maximum values, respectively. In this specification, Me represents a methyl group, and Bn represents a benzyl group.

[0010] <Terminology> Nucleic acid refers to an oligonucleotide, a polynucleotide, or any fragment thereof, any ribo- or deoxyribo-derivative, and a naturally occurring or synthetic molecule containing natural and / or modified nucleotide residues and internucleotide linkages. Nucleic acid may be single-stranded, double-stranded, triple-stranded, or quadruple-stranded, and may be a sense or antisense strand. It refers to naturally occurring (e.g., genomic) or synthetic DNA or RNA, or any DNA- or RNA-like molecule. Alternative nucleic acid backbones include, but are not limited to, phosphorothioate, phosphoroselenoate, alkyl phosphotriester, aryl phosphotriester, alkyl phosphonate, aryl phosphonate, phosphoboronate, morpholino nucleic acid (MNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), etc.

[0011] An RNA synthesis initiator refers to a ribo-oligonucleotide, deoxyribo-oligonucleotide, or chimeric ribo- / deoxyribo-oligonucleotide, where the single strand may be naturally occurring or synthetic and typically contains a sequence of about 2 to about 10 nucleotides, about 3 to about 8 nucleotides, or about 3 to about 5 nucleotides. The RNA synthesis initiator may contain one or more modified groups. The RNA synthesis initiator may include RNA, DNA, and / or other modified nucleosides.

[0012] The RNA synthesis initiator can be used as an initiating capped oligonucleotide primer. The RNA synthesis initiator has an unmodified or open 3'-OH group and can be extended by RNA polymerase by incorporating an NTP at the 3' end of the primer. In vitro transcription can be initiated under the control of a promoter in a transcription system containing a DNA template (e.g., a DNA plasmid), RNA polymerase, nucleoside 5'-triphosphates, and an appropriate buffer.

[0013] Modification groups refer to any chemical group attached to the initiating primer at a position including the sugar, the nucleoside base, the triphosphate bridge and / or the internucleotide phosphate.

[0014] A label or detectable label refers to a compound or combination of compounds that can be bound to or associated with a molecule such that the molecule can be detected directly or indirectly by detecting the label. Detectable labels include radioisotopes (e.g., carbon, phosphorus, iodine, indium, sulfur, tritium, etc.), mass isotopes (e.g., H ２ , C １３ or N １５ ), a dye or fluorophore (eg, cyanine, fluorescein, or coumarin), a hapten (eg, biotin), or any compound that can be detected directly or indirectly.

[0015] In reference to the initiating capped oligonucleotide primer and the DNA template, complementary or complementarity refers to standard Watson / Crick base pairing rules. For example, 5'-A-G-T-C-3' is complementary to 3'-T-C-A-G-5'. Non-natural or synthetic nucleotides may be included in the nucleic acid. Non-natural or synthetic nucleotides include, but are not limited to, base- and sugar-modified nucleosides, nucleotides, and nucleic acids, such as inosine, 7-deazaguanosine, 2'-O-methylguanosine, 2'-fluoro-2'-deoxycytidine, pseudouridine, locked nucleic acids (LNA), and peptide nucleic acids (PNA). Complementarity need not be perfect; the duplex may contain mismatched base pairs, degenerates, or non-matching nucleotides.

[0016] Substantially complementary refers to two sequences that hybridize under stringent hybridization conditions. One skilled in the art will understand that substantially complementary sequences need not hybridize along their entire length.

[0017] Nucleosides include all naturally occurring nucleosides, including all forms of naturally occurring nucleoside bases and furanosides. The most common base rings found in naturally occurring nucleosides are purine and pyrimidine rings. Naturally occurring purine rings include, for example, adenine, guanine, and N-methyl-. ６ 2'-O-methyladenine. Naturally occurring pyrimidine rings include, for example, cytosine, thymine, 5-methylcytosine, pseudouracil. Naturally occurring nucleosides include, for example, but are not limited to, ribo, 2'-O-methyl, or 2'-deoxyribo derivatives of adenosine, guanosine, cytidine, thymidine, uridine, inosine, 7-methylguanosine, or pseudouridine.

[0018] Nucleoside analogs, modified nucleosides, or nucleoside derivatives include synthetic nucleosides. Nucleoside derivatives also include nucleosides with modified bases or / and modified sugar moieties, with or without protecting groups. Examples include 2'-deoxy-2'-fluorouridine, 5-fluorouridine, and the like. Other nucleoside derivatives include, for example, LNA nucleosides, halogen-substituted purines (e.g., 6-fluoropurine), halogen-substituted pyrimidines, N-substituted nucleosides, and the like. ６ -ethyladenine, N ４ -(alkyl)-cytosine, 5-ethylcytosine, and the like.

[0019] Modified NTPs refer to nucleoside 5'-triphosphates that have chemical groups attached at any position, including the sugar, base, or triphosphate chain.

[0020] A promoter refers to a region of a dsDNA template that directs and controls the initiation of transcription of a specific DNA sequence (e.g., a gene). The promoter is located on the same strand and upstream of the DNA (toward the 5' region of the sense strand). The promoter is usually immediately adjacent to (or partially overlaps with) the DNA sequence to be transcribed. The nucleotide position in the promoter is specified relative to the transcription start site (position +1), where transcription of the DNA begins. The initiation oligonucleotide primer is complementary to the start site of the promoter sequence (e.g., at positions +1 and +2, or in the case of an initiation tetramer, at positions +1, +2, and +3).

[0021] Transcription or transcription reaction refers to the method of enzymatically producing RNA that is complementary to DNA template, thereby producing several RNA copies of DNA sequence.The RNA molecule synthesized in transcription reaction is also called RNA transcript.Transcription reaction can be carried out using the compound of the present invention.The transcription of DNA template can be exponential, nonlinear or linear.DNA template can include double-stranded linear DNA, partially double-stranded linear DNA, circular double-stranded DNA, DNA plasmid, PCR amplicon, or modified nucleic acid template that is compatible with RNA polymerase.

[0022] Alkyl refers to a single bond of a hydrocarbon having 1 to 10 carbon atoms, and includes not only alkyl groups that do not have a substituent (unsubstituted alkyl groups) but also alkyl groups that have a substituent (substituted alkyl groups). The alkyl group may be linear or branched, and may be substituted with a halogen atom or the like. The number of carbon atoms in the alkyl group is preferably 1 to 6, more preferably 1 to 4, and even more preferably 1 or 2. Specific examples include methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, benzyl, etc.

[0023] <Compound> The compound of the present invention is a compound represented by the following formula (1). In the formula, B １ teeth, where * indicates the bond position, B ２ is a natural, modified, or non-natural nucleobase; B ３R may be the same or different when present in plural and is a natural, modified or non-natural nucleobase; １ and R ２ are each independently hydrogen, fluorine, —OH, or —O-alkyl; R ３ and R ４ are each independently hydrogen, fluorine, —OH, or —O-alkyl; R ４ When there are a plurality of X's, they may be the same or different; １ , X ２ and X are each independently -O- or -S-, and when a plurality of X's are present, they may be the same or different, provided that X １ , X ２ and at least one of X is —S—, and n is an integer from 0 to 7.

[0024] R １ and R ２ are each independently preferably —OH, —OCH ３ More preferably, R １ is —OH. ３ and R ４ are each independently preferably —OH or —OCH ３ More preferably, R ３ Ha-OCH ３ and R ４ is —OH. n is preferably an integer of 0 to 5, more preferably an integer of 0 to 3, and even more preferably an integer of 0 to 2.

[0025] In a preferred embodiment, the compound of the present invention is a compound represented by the following formula (2): In the formula, n represents an integer of 0 to 2, each R is independently -H or -alkyl, and each symbol other than n and R has the same meaning as in formula (1).

[0026] Examples of the compound of the present invention are shown below, but the compound of the present invention is not limited to the following.

[0027]

[0028]

[0029]

[0030]

[0031]

[0032] The compounds of the present invention can be synthesized by the methods described in the Examples below or by methods analogous thereto.

[0033] <RNA Synthesis Initiator> The present invention provides an RNA synthesis initiator comprising the compound of the present invention described above. The RNA synthesis initiator of the present invention can be used as an initiation capped oligonucleotide primer. The RNA synthesis initiator of the present invention may have a hybridization sequence that can be complementary to a sequence on a DNA template at the initiation site. The length of the hybridization sequence varies depending on several factors, including the identity of the template nucleotide sequence, the temperature at which this primer hybridizes with the DNA template during in vitro transcription, and the temperature. The desired length of the specific nucleotide sequence of an initiation capped oligonucleotide primer for use in transcription can be determined appropriately by one skilled in the art.

[0034] The nucleotide length of the RNA synthesis initiator is 2 to 9, optionally 2 to 7, or optionally 3 to 5, and is 3, for example.

[0035] By using an RNA synthesis initiator, the efficiency of transcription initiation can be enhanced compared to the efficiency of initiation with the usual GTP, ATP, CTP, or UTP. Transcription initiation is thought to be enhanced when RNA synthesis starts from an initiating capped oligonucleotide primer rather than from any NTP in the transcription mixture. Enhanced transcription initiation efficiency results in a higher yield of RNA transcripts.

[0036] In one example, RNA can be synthesized by using the RNA synthesis initiator of the present invention as an initiating capped oligonucleotide primer having a substitution or modification. The substitution and modification of the initiating capped oligonucleotide primer are preferably those that do not substantially impair RNA synthesis. Whether the initiating capped oligonucleotide primer having a substitution or modification can be used to obtain desired synthesis results can be determined by conventional test synthesis. The substitution or modification of the initiating capped oligonucleotide primer may include, for example, one or more modified nucleoside bases, one or more modified sugars, one or more modified internucleotide linkages, and / or one or more modified triphosphate bridges.

[0037] The modified initial capped oligonucleotide primer can be extended by RNA polymerase on a DNA template by incorporation of an NTP into the open 3'-OH group. The initial capped oligonucleotide primer may contain natural RNA and DNA nucleosides, modified nucleosides or nucleoside analogs.

[0038] In one example, the modification group may be a thermally labile group that dissociates from the modified initial capped oligonucleotide primer at an increasing rate as the temperature of the enzymatic reaction medium is increased. Examples of thermally labile groups on oligonucleotides and NTPs are described in Nucleic Acids Res., 36:e131 (2008), Collect. Symp. Ser., 10:259-263 (2008), and Analytical Chemistry, 81:4955-4962 (2009).

[0039] In one example, at least one NTP, which may have a modification, may be added to a transcription reaction to synthesize RNA. Preferably, the NTP modification does not substantially impair RNA polymerase-mediated RNA synthesis. The NTP modification may include, for example, one or more modified nucleoside bases, one or more modified sugars, or one or more modified 5'-triphosphates. The modified NTP may be incorporated at the 3' end of an initial capped oligonucleotide primer and may support further extension of the primer without blocking transcription.

[0040] In one example, the modification group of the initiating capped oligonucleotide primer can be a detectable label or an affinity label. When a detectable label or an affinity label is used, the RNA containing the detectable label or affinity label can be identified by size, mass, color, and / or affinity after transcription. In one example, the detectable label is a fluorescent dye and the affinity label is biotin. In one example, one or more components of the transcription reaction (the initiating capped oligonucleotide primer and / or NTP) can be labeled with a detectable label or an affinity label. In this case, the RNA can be identified by, for example, size, mass, affinity, or color after transcription. In one example, the detectable label is a fluorescent dye and the affinity label is biotin.

[0041] <Kit> The present invention provides an RNA synthesis kit comprising the RNA synthesis initiator of the present invention and an RNA polymerase. For example, the RNA synthesis kit may contain transcription reagents for synthesizing general RNA (e.g., FLuc mRNA). Specifically, the RNA synthesis kit comprises the RNA synthesis initiator of the present invention and an RNA polymerase, and may further optionally comprise one or more components selected from the group consisting of one or more unmodified NTPs, one or more modified NTPs, enzymes other than RNA polymerase, a reaction buffer, magnesium, a DNA template, a container for performing a transcription reaction, and instructions for performing RNA synthesis.

[0042] <Method for Producing RNA> According to the present invention, there is provided a method for producing RNA, which comprises transcribing a template nucleic acid in the presence of the RNA synthesis initiator of the present invention.

[0043] In eukaryotes, transcription of messenger RNA (mRNA) is carried out by RNA polymerase II. RNA polymerase II is a multisubunit enzyme. To perform large-scale in vitro transcription, single-subunit phage polymerases derived from T7, T3, SP6, K1-5, K1E, K1F, or K11 bacteriophages can generally be used. Polymerases of this family do not require accessory proteins and have simple minimal promoter sequences of approximately 17 nucleotides with minimal constraints on the initiation nucleotide sequence. Among the above, T7 RNA polymerase is preferably used.

[0044] T7 RNA polymerase can initiate RNA synthesis in the absence of a primer. The first step in initiation is called de novo RNA synthesis, in which the RNA polymerase recognizes a specific sequence on the DNA template, selects a first pair of nucleotide triphosphates that are complementary to the template residues at positions +1 and +2, and catalyzes the formation of a phosphodiester bond to form a dinucleotide. The initiating nucleotide has a lower affinity for the polymerase than those used in elongation.

[0045] T7 RNA polymerase is also known to be able to initiate transcription in the presence of short oligonucleotide primers.

[0046] <RNA> According to the present invention, there is provided an RNA comprising the RNA synthesis initiator of the present invention.

[0047] The RNA is preferably mRNA (messenger RNA). The RNA of the present invention can be produced by transcribing a template nucleic acid in the presence of the RNA synthesis initiator of the present invention.

[0048] <Uses> The RNA containing the RNA synthesis initiator of the present invention may be used for the treatment (e.g., therapy or prevention) of a disease. By administering the RNA containing the RNA synthesis initiator of the present invention to a subject having a disease, the disease can be treated (treated or prevented).

[0049] The RNA containing the RNA synthesis initiator of the present invention can be formulated into a pharmaceutical composition together with a pharmaceutically acceptable carrier and / or additive. The formulation can be appropriately carried out depending on the administration route.

[0050] Examples of pharmaceutically acceptable carriers and / or additives include, but are not limited to, carriers, excipients, binders, fillers, suspending agents, flavoring agents, sweeteners, disintegrating agents, dispersing agents, surfactants, lubricants, coloring agents, diluents, solubilizers, wetting agents, plasticizers, stabilizers, penetration enhancers, wetting agents, antifoaming agents, antioxidants, and preservatives.

[0051] The pharmaceutical composition may be administered orally or parenterally. Parenteral administration may include, but is not limited to, intravenous, subcutaneous, intradermal, intramuscular, intracolonic, intrarectal, or intraperitoneal administration. Examples of administration include, but are not limited to, intramuscular injection, subcutaneous injection, or intravenous injection.

[0052] For example, the pharmaceutical composition can be formulated for injection, which typically contains the RNA comprising the RNA synthesis initiator of the present invention in a suitable solution such as sterile saline and / or pharmaceutical carrier.

[0053] The pharmaceutical composition may be formulated as a suspension in lipids, phospholipids, liposome suspensions, or aqueous emulsions. In one example, the RNA containing the RNA synthesis initiator of the present invention may be encapsulated in lipid nanoparticles. That is, a pharmaceutical composition containing lipid nanoparticles encapsulating the RNA containing the RNA synthesis initiator of the present invention may be prepared and used.

[0054] The pharmaceutical composition preferably contains at least 0.1% (w / v) of RNA containing the RNA synthesis initiator of the present invention, more preferably 0.1 to 10% (w / v), even more preferably 0.1 to 5% (w / v), and particularly preferably 0.1 to 1% (w / v).

[0055] The dosage of RNA can be determined based on the subject's weight, the type of disease, the severity of symptoms, and the like. The dosage of RNA containing the RNA synthesis initiator of the present invention is about 0.0005 to 500 mg / day, preferably about 1 to 100 mg / day, in human patients. The dosage may be 50 to 100 mg / day, 75 to 100 mg / day, or 50 to 75 mg / day, or may be 1 to 50 mg / day, 25 to 50 mg / day, or 1 to 25 mg / day in human patients.

[0056] The following examples will further illustrate the embodiments of the present invention. The materials, amounts used, ratios, processing details, processing procedures, etc. shown in the following examples can be changed as appropriate without departing from the spirit of the embodiments of the present invention. Therefore, the scope of the embodiments of the present invention is not limited to the specific examples shown below. Unless otherwise specified, "parts" and "%" are based on mass.

[0057] Unless otherwise specified, purification by column chromatography was performed using an automatic purification system ISOLERA (Biotage) or a medium-pressure liquid chromatograph YFLC-Wprep2XY.N (Yamazen Corporation). Unless otherwise specified, the carrier used in silica gel column chromatography was SNAPKP-Sil Cartridge (Biotage) or Hi-Flash Column W001, W002, W003, W004, or W005 (Yamazen Corporation). The mixture ratio of the eluent used in column chromatography is a volume ratio. For example, "gradient elution of hexane:ethyl acetate = 50:50 to 0:100" means that the eluent of 50% hexane / 50% ethyl acetate was finally changed to an eluent of 0% hexane / 100% ethyl acetate. Furthermore, for example, "gradient elution of hexane:ethyl acetate = 50:50 to 0:100, gradient elution of methanol:ethyl acetate = 0:100 to 20:80" means that the eluent was changed from 50% hexane / 50% ethyl acetate to 0% hexane / 100% ethyl acetate, then to 0% methanol / 100% ethyl acetate, and finally to 20% methanol / 80% ethyl acetate.

[0058] The MS spectrum was measured using an ACQUITY SQD LC / MS System (manufactured by Waters Corporation, ionization method: ESI (ElectroSpray Ionization) method).

[0059] NMR spectra were measured using tetramethylsilane as an internal standard with a Bruker AV300 (manufactured by Bruker, 300 MHz) or a Bruker AV400 (manufactured by Bruker, 400 MHz), and all δ values ​​were expressed in ppm.

[0060] <Synthesis of 7-methylthioguanosine 5'-diphosphate imidazolide (diphosphate intermediate 1)>

[0061] Intermediate 1-1 was synthesized according to the method described in Japanese Patent No. 5970550 (FUJIFILM Corporation). Under a nitrogen atmosphere, N,O-bis(trimethylsilyl)acetamide (65.3 mL, 266 mmol) was added to a solution of 2-amino-6-chloropurine (15.0 g, 88.8 mmol) in toluene (1.2 L), and the mixture was stirred at 100°C for 1 hour. The reaction solution was returned to room temperature, and Intermediate 1-1 (42.0 g, 80.7 mmol) and trimethylsilyl triflate (29.2 mL, 21.7 mmol) were added, followed by stirring at 110°C for 2 hours. After confirming completion of the reaction, ethyl acetate (600 mL) and saturated aqueous sodium bicarbonate (600 mL) were added, and the organic layer was separated by extraction and washed with saturated brine (600 mL). The resulting organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: hexane / ethyl acetate) to obtain a white solid (24.0 g). Under a nitrogen atmosphere, a 7 mol / L ammonia methanol solution (54 mL) was added to the obtained white solid at room temperature, and the mixture was allowed to stand for two days. After confirming the completion of the reaction, the mixture was concentrated under reduced pressure. Ethyl acetate (72 mL) was added to the obtained residue, and the mixture was stirred at 90°C for 30 minutes. The white solid was then filtered and dried to obtain Intermediate 1-2 (10.3 g). ESI-MS (+) = 318.0

[0062] To Intermediate 1-2 (10.0 g) was added 50% aqueous formic acid (240 mL), and the mixture was stirred at 60°C for 6 hours. After confirming the completion of the reaction, the mixture was concentrated under reduced pressure. To the residue was added 7 mol / L ammonia methanol solution (250 mL), and the mixture was allowed to stand for 12 hours, and then concentrated under reduced pressure. Methanol (125 mL) was added to the resulting residue at room temperature and stirred, and then ethyl acetate (250 mL) was added and stirred for 30 minutes. The solid was filtered and dried to obtain Intermediate 1-3 (9.42 g). ESI-MS (+) = 300.4

[0063] Toluenesulfonic acid monohydrate (17.3 g, 91.2 mmol) was added to a mixture of intermediate 1-3 (9.10 g, 30.4 mmol), 2,2-dimethoxypropane (18.6 mL, 152 mmol), and N,N-dimethylformamide (180 mL), and the mixture was stirred at 50°C for 2 hours. Triethylamine (46 mL) was added to the reaction mixture under ice cooling, and the mixture was stirred and then concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: ethyl acetate / methanol) to give intermediate 1-4 (15.0 g). ESI-MS (+) = 340.4

[0064] Under a nitrogen atmosphere, N,N-dimethylformamide dimethyl acetal (6.1 mL, 45.6 mmol) was added to a mixture of Intermediate 1-4 (15.0 g) and N,N-dimethylformamide (100 mL) at room temperature, and the mixture was stirred for 7 hours. Ethyl acetate (600 mL) was added to the reaction mixture, and the precipitated solid was filtered and dried to obtain Intermediate 1-5 (5.0 g). ESI-MS (+) = 395.5

[0065] Under a nitrogen atmosphere, 1H-tetrazole (0.534 g, 7.62 mmol) and di-tert-butyl N,N-diisopropylphosphoramidite (1.41 g, 5.08 mmol) were added to a mixture of intermediate 1-5 (1.00 g, 2.54 mmol) and N,N-dimethylformamide (10 mL), and the mixture was stirred at room temperature for 1 hour. After confirming the completion of the reaction, 70% tert-butyl hydroperoxide (3.81 mmol) was added and the mixture was stirred for 1 hour. A saturated aqueous solution of sodium thiosulfate (4 mL) was added and the mixture was stirred for 30 minutes. Dichloromethane and water were added, and the organic layer was separated by extraction. The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: hexane / ethyl acetate → ethyl acetate / methanol) to obtain intermediate 1-6 (1.0 g) as a white solid. ESI-MS (+) = 587.5

[0066] To a mixture of intermediate 1-6 (1.00 g, 1.70 mmol), dichloromethane (2 mL), and water (3 mL), trifluoroacetic acid (3 mL) was added at room temperature, and the mixture was stirred for 9 hours. The reaction mixture was concentrated under reduced pressure and azeotroped with toluene. The residue was diluted with water and purified with a DEAE Sephadex column (eluent: water / 1 M triethylammonium bicarbonate) to obtain intermediate 1-7 (1.00 g) as a white solid. ESI-MS (+) = 380.3

[0067] Under a nitrogen atmosphere, triethylamine (0.348 mL, 2.50 mmol) and triphenylphosphine (0.656 g, 2.50 mmol) were added to a mixture of intermediate 1-7 (0.600 g, 1.25 mmol), imidazole (0.426 mg, 6.25 mmol), 2,2'-dipyridyl disulfide (0.551 g, 2.50 mmol), and N,N-dimethylformamide (6 mL) at room temperature, and the mixture was stirred for 3 hours. Under ice cooling, the reaction mixture was added dropwise to a solution of sodium perchlorate (1.2 g, 10.0 mmol) in acetone (50 mL). After the addition was complete, the mixture was stirred for 30 minutes and then centrifuged to remove the supernatant. Acetone (20 mL) was added to the residue, which was then washed with stirring, and the supernatant was then removed again by centrifugation. This washing procedure was repeated three times, and the mixture was then concentrated under reduced pressure to obtain intermediate 1-8 (0.353 g) as a pale yellow solid. ESI-MS(+)=430.2

[0068] Under a nitrogen atmosphere, zinc chloride (0.634 g) and a 1 mol / L N,N-dimethylformamide solution (4.7 mL) of tributylammonium phosphate were added to a mixture of intermediate 1-8 (0.319 g) and N,N-dimethylformamide (4.7 mL), and the mixture was stirred overnight. Dichloromethane (20 mL) and water (20 mL) were added to the reaction mixture, and the aqueous layer was separated by a separation operation. Ethylenediamine-N,N,N',N'-tetraacetic acid disodium salt dihydrate (1.73 g) was added to the aqueous layer and stirred, followed by purification on a DEAE Sephadex column (eluent: water / 1 M triethylammonium bicarbonate) to obtain intermediate 1-9 (0.230 g) as a white solid. Under a nitrogen atmosphere, iodomethane (0.12 mL) was added to a mixture of intermediate 1-9 (0.230 g) and dimethyl sulfoxide (2.0 mL), and the mixture was stirred overnight. Dichloromethane (20 mL) and water (20 mL) were added to the reaction mixture, and the aqueous layer was separated by a separation operation. The aqueous layer was loaded onto a DEAE Sephadex column and purified using water / 1 M triethylammonium bicarbonate as the eluent to obtain intermediate 1-10 (0.210 g) as a white solid.

[0069] Under a nitrogen atmosphere, triethylamine (0.174 mL) and triphenylphosphine (0.328 g) were added to a mixture of intermediate 1-10 (0.210 g), imidazole (0.213 g), 2,2'-dipyridyl disulfide (0.275), and N,N-dimethylformamide (6 mL) at room temperature, and the mixture was stirred for 5 hours. Under ice-cooling, the reaction mixture was added dropwise to a solution of sodium perchlorate (0.765 g) in acetone (50 mL). After the addition was complete, the mixture was stirred for 30 minutes and then centrifuged to remove the supernatant. Acetone (20 mL) was added to the residue, which was then washed with stirring, and the supernatant was removed again by centrifugation. This washing procedure was repeated three times, followed by concentration under reduced pressure to obtain diphosphate intermediate 1 (0.105 g) as a pale yellow solid. ESI-MS (+) = 524.2

[0070] <Synthesis of diphosphate intermediate 2 and diphosphate intermediate 3>

[0071] Diphosphate intermediate 2 and phosphate intermediate 3 were synthesized according to the method described in Japanese Patent No. 6,814,997.

[0072] <Synthesis of Dinucleotide 5' Phosphate Intermediate 3>

[0073] Dinucleotide 5' phosphate intermediate 3 was synthesized according to the method described in Org. Process Res. Dev. 2022.26.2771-2778.

[0074] <Synthesis of Dinucleotide 5' Phosphate Intermediate 4>

[0075] Under a nitrogen atmosphere, intermediate 1-1 (30.0 g, 57.6 mmol) and trimethylsilyl triflate (41.7 mL, 230 mmol) were added to a mixture of 6-chloropurine (9.80 g, 63.4 mmol), diazabicycloundecene (17.2 mL, 115 mmol), and acetonitrile (300 mL), and the mixture was stirred at 70°C for 2 hours. After confirming the completion of the reaction, ethyl acetate (600 mL) and saturated aqueous sodium bicarbonate (600 mL) were added at room temperature, and the organic layer was separated by extraction and washed with saturated brine (600 mL). The resulting organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: hexane / ethyl acetate) to obtain a white solid (13.0 g). ESI-MS (+) = 461.4

[0076] To a mixture of the obtained white solid (13.0 g) and methanol (33 mL), 7 mol / L ammonia methanol solution (30 mL) was added, and the mixture was left to stand overnight, followed by concentration under reduced pressure. Ethyl acetate (30 mL) was added to the obtained residue, and the mixture was stirred for 30 minutes. The white solid was filtered and dried to obtain Intermediate 4-1 (4.72 g). ESI-MS (+) = 303.1

[0077] Under a nitrogen atmosphere, 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (5.92 mL, 18.6 mmol) was added to a mixture of intermediate 4-1 (4.70 g, 15.5 mmol) and pyridine (47 mL), and the mixture was stirred overnight. After confirming the completion of the reaction, ethyl acetate (47 mL) and saturated aqueous sodium bicarbonate (47 mL) were added at room temperature, and the organic layer was separated by extraction and washed with saturated brine (600 mL). The resulting organic layer was dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: hexane / ethyl acetate) to obtain intermediate 4-2 (2.37 g) as a pale red solid. ESI-MS (+) = 545.5

[0078] Under a nitrogen atmosphere, silver(I) oxide (1.17 g, 5.04 mmol) was added to a mixture of intermediate 4-2 (0.53 g, 1.01 mmol), iodomethane (0.63 mL, 10.1 mmol), and dichloromethane (2.7 mL), and the mixture was stirred for 18 hours. After confirming the completion of the reaction, the solid matter was removed by filtration through Celite, and the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: hexane / ethyl acetate) to obtain intermediate 4-3 (0.45 g, 0.805 mmol) as a white solid. ESI-MS (+) = 559.4

[0079] A mixture of intermediate 4-3 (0.45 g, 0.805 mmol) and 7 mol / L ammonia methanol solution (4.5 mL) was stirred at 110°C for 1 hour using a microwave reactor. After returning to room temperature, the mixture was concentrated under reduced pressure to obtain intermediate 4-4. ESI-MS (+) = 540.4. Under a nitrogen atmosphere, benzoyl chloride (0.187 mL, 1.61 mmol) was added to a mixture of the obtained intermediate 4-4 and pyridine (4 mL), and the mixture was stirred overnight. After concentrating the reaction mixture under reduced pressure, ethyl acetate (10 mL) and saturated aqueous sodium bicarbonate (10 mL) were added to the residue, and the organic layer was separated by extraction and washed with saturated brine. The resulting organic layer was dried over sodium sulfate and then concentrated under reduced pressure. To a solution of the residue in tetrahydrofuran (8 mL), TBAF (tetrahydrofuran solution, 1 mol / L) (2.41 mL) was added and the mixture was stirred for 1 hour. After confirming the completion of the reaction, ethyl acetate and water were added, and the organic layer was separated by extraction and concentrated under reduced pressure. Potassium carbonate was added to a methanol solution of the residue and stirred, after which ethyl acetate and water were added, and the organic layer was separated by extraction and washed with saturated brine. The organic layer was dried over sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: hexane / ethyl acetate → ethyl acetate / methanol) to obtain intermediate 4-5 as a white solid. ESI-MS (+) = 402.3

[0080] Under a nitrogen atmosphere, dimethoxytrityl chloride (0.818 g, 2.42 mmol) was added to a mixture of the obtained intermediate 4-5 and pyridine (4 mL), and the mixture was stirred overnight. Methylene chloride (10 mL) and saturated aqueous sodium bicarbonate (10 mL) were added to the reaction mixture, and the organic layer was separated by extraction and washed with saturated brine. The obtained organic layer was dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: hexane / ethyl acetate) to obtain intermediate 4-6 (0.320 g) as a white solid. ESI-MS (+) = 704.5

[0081] Under a nitrogen atmosphere, 1H-tetrazole (0.0159 g, 0.227 mmol) was added to a mixture of intermediate 4-6 (0.160 g, 0.227 mmol), 2-cyanoethyl-N,N,N'-N'-tetraisopropylphosphoramidite (0.194 g, 0.682 mmol), and acetonitrile (2.3 mL) under ice cooling, and the mixture was stirred at room temperature for 1 hour. A 0.4 mol / L iodine solution (THF / pyridine / water = 78 / 20 / 2) was added to the reaction solution until the reaction solution turned red. After confirming the completion of the reaction, ethyl acetate (10 mL), water (10 mL), and sodium sulfite were added to the reaction solution. After stirring for a while, the organic layer was separated by extraction and washed with saturated brine. The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was then purified by silica gel column chromatography (eluent: hexane / ethyl acetate) to obtain Intermediate 4-7 (0.196 g, 0.217 mmol) as a white solid. ESI-MS (+) = 821.5 (hydrolyzate).

[0082] Under a nitrogen atmosphere, 1H-tetrazole (0.0456 g, 0.652 mmol) was added to a mixture of intermediate 4-7 (0.196 g, 0.217 mmol), N-isobutyryl-2-2',3'-acetylguanosine (CAS. 163586-86-5, 0.0950 g, 0.217 mmol), and acetonitrile (2.2 mL) under ice cooling, and the mixture was stirred at room temperature for 1 hour. A 0.4 mol / L iodine solution (THF / pyridine / water = 78 / 20 / 2) was added to the reaction solution until the reaction solution turned red. After confirming the completion of the reaction, ethyl acetate (10 mL), water (10 mL), and sodium sulfite were added to the reaction solution. After stirring for a while, the organic layer was separated by extraction and washed with saturated brine. The organic layer was dried over sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: hexane / ethyl acetate) to obtain Intermediate 4-8 as a white solid. ESI-MS (+) = 1256.8

[0083] Trifluoroacetic acid (0.125 mL) was added to a mixture of intermediate 4-8, dichloromethane (2.7 mL), and methanol (0.27 mL), and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate / methanol) to obtain intermediate 4-9 (0.128 g, 0.134 mmol) as a white solid. ESI-MS (+) = 954.5

[0084] Under a nitrogen atmosphere, 1H-tetrazole (0.0282 g, 0.403 mmol) was added to a mixture of intermediate 4-9 (0.128 g, 0.134 mmol), bis(2-cyanoethyl)-N,N-diisopropylphosphoramidite (0.105 mL, 0.403 mmol), and acetonitrile (1.3 mL) under ice cooling, and the mixture was stirred at room temperature for 1 hour. A 0.4 mol / L iodine solution (THF / pyridine / water = 78 / 20 / 2) was added to the reaction solution until the reaction solution turned red. After confirming the completion of the reaction, ethyl acetate (5 mL), water (5 mL), and sodium sulfite were added to the reaction solution. After stirring for a while, the organic layer was separated by extraction and washed with saturated brine. The organic layer was dried over sodium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: hexane / ethyl acetate) to obtain Intermediate 4-10 (0.180 g, 0.158 mmol) as a pale yellow solid. ESI-MS (+) = 1140.6

[0085] Under a nitrogen atmosphere, a mixture of intermediate 4-10 (0.180 g, 0.158 mmol), 7 mol / L ammonia methanol solution (2.5 mL), 1 mol / L methylamine methanol solution (2.5 mL), and 30% aqueous ammonia (5.0 mL) was stirred overnight. The reaction mixture was concentrated under reduced pressure, and then acetonitrile (5 mL) was added to the residue and stirred for 30 minutes. The solid was filtered and dried under reduced pressure to obtain dinucleotide 5' phosphate intermediate 4 (0.070 g) as a white solid. ESI-MS (+) = 723.3

[0086] <Synthesis of Dinucleotide 5' Phosphate Intermediate 5>

[0087] Intermediate 5-1 was synthesized in the same manner as Intermediate 4-8, using Intermediate 1-5 instead of N-isobutyryl-2-2',3'-acetylguanosine and 5'-dimethoxytrityl-N-benzoyl-adenosine-2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoromidite instead of Intermediate 4-7. ESI-MS (+) = 1197.8. Intermediate 5-2 was synthesized in the same manner as Intermediate 4-9, using Intermediate 5-1 instead of Intermediate 4-9. ESI-MS (+) = 895.5. Intermediate 5-3 was synthesized in the same manner as Intermediate 4-10, using Intermediate 5-2 instead of Intermediate 4-9. ESI-MS (+) = 1081.7.

[0088] Under a nitrogen atmosphere, a mixture of intermediate 5-3 (0.138 g, 0.128 mmol), 7 mol / L ammonia methanol solution (2.5 mL), 1 mol / L methylamine methanol solution (2.5 mL), and 30% aqueous ammonia (5.0 mL) was stirred overnight. The reaction mixture was concentrated under reduced pressure, and then 80% aqueous trifluoroacetic acid solution (2.6 mL) was added to the residue, followed by stirring at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and then acetonitrile (5 mL) was added to the residue, followed by stirring for 30 minutes. The solid was filtered and dried under reduced pressure to obtain dinucleotide 5' phosphate intermediate 5 (0.078 g) as a white solid. ESI-MS (+) = 723.4

[0089] <Synthesis of Dinucleotide 5' Phosphate Intermediate 6>

[0090] Under a nitrogen atmosphere, triethylamine (0.299 mL, 2.15 mmol) and benzylamine (0.234 mL, 2.15 mmol) were added to a mixture of intermediate 4-3 (0.800 g, 1.43 mmol), methanol (4 mL), and methylene chloride (4 mL), and the mixture was stirred overnight. Ethyl acetate and water were added to the reaction mixture, and the organic layer was separated by extraction and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: hexane / ethyl acetate → ethyl acetate / methanol), and intermediate 6-1 (1.0 g) was obtained as a colorless oil. ESI-MS (+) = 630.3

[0091] Benzoyl chloride (0.498 mL, 4.29 mmol) was added to a mixture of the obtained intermediate 6-1 and pyridine (5 mL) under a nitrogen atmosphere, and the mixture was stirred overnight. The reaction mixture was concentrated under reduced pressure, and then ethyl acetate and saturated aqueous sodium bicarbonate were added to the residue. The organic layer was separated by extraction and washed with saturated brine. The obtained organic layer was dried over sodium sulfate and then concentrated under reduced pressure. TBAF (tetrahydrofuran solution, 1 mol / L) (2.05 mL) was added to a solution of the residue (0.677 g) in tetrahydrofuran (4.7 mL), and the mixture was stirred for 1 hour. After confirming the completion of the reaction, ethyl acetate and water were added, and the organic layer was separated by extraction and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: hexane / ethyl acetate → ethyl acetate / methanol), and intermediate 6-2 was obtained as a yellow oil. ESI-MS (+) = 416.2

[0092] Under a nitrogen atmosphere, dimethoxytrityl chloride (0.378 g, 1.12 mmol) was added to a mixture of intermediate 6-2 (0.309 g, 0.744 mmol) and pyridine (3.7 mL), and the mixture was stirred overnight. Methylene chloride (10 mL) and saturated aqueous sodium bicarbonate (10 mL) were added to the reaction mixture, and the organic layer was separated by extraction and washed with saturated brine. The resulting organic layer was dried over sodium sulfate and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: hexane / ethyl acetate) to obtain intermediate 6-3 (0.531 g) as a white solid. ESI-MS (+) = 794.2

[0093] Dinucleotide 5' phosphate intermediate 6 was synthesized in five steps from intermediate 6-3 according to the synthesis method of dinucleotide 5' phosphate intermediate 4. ESI-MS (+) = 813.1

[0094] <Synthesis of Dinucleotide 5' Phosphate Intermediate 7>

[0095] Dinucleotide 5' phosphate intermediate 7 was synthesized in the same manner as dinucleotide 5' phosphate intermediate 6, except that a tetrahydrofuran solution of methylamine (1 mol / L) was used instead of benzylamine in the synthesis of intermediate 6-1. ESI-MS (+) = 737.2

[0096] <Synthesis of Dinucleotide 5' Phosphate Intermediate 8>

[0097] Under a nitrogen atmosphere, a 1 mol / L sodium hydroxide solution (5.5 mL) containing tetrabutylammonium bromide (0.173 g, 0.536 mmol) was added to a mixture of 5'-dimethoxytrityl-N-benzoyl-adenosine-2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoromidite (0.500 g, 0.536 mmol), benzyl bromide (0.254 mL, 2.14 mmol), and methylene chloride (5.4 mL) at room temperature, and the mixture was stirred for 30 minutes. After confirming the completion of the reaction, the organic layer was separated by a separation operation and washed with saturated brine. The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: hexane / ethyl acetate) to obtain intermediate 8-1 (0.699 g) as a yellow oil. ESI-MS (+) = 895.7 (detected as a hydrolyzed product).

[0098] Intermediate 8-2 was synthesized in the same manner as Intermediate 4-8, using Intermediate 8-1 instead of Intermediate 4-7. ESI-MS (+) = 1330.8 Intermediate 8-3 was synthesized in the same manner as Intermediate 4-9, using Intermediate 8-2 instead of Intermediate 4-8. ESI-MS (+) = 1028.7 Intermediate 8-4 was synthesized in the same manner as Intermediate 4-10, using Intermediate 8-3 instead of Intermediate 4-9. ESI-MS (+) = 1214.8 Dinucleotide 5' phosphate intermediate 8 was synthesized in the same manner as Dinucleotide 5' phosphate intermediate 4, using Intermediate 8-4 instead of Intermediate 4-10. ESI-MS (+) = 797.5

[0099] <Synthesis of Dinucleotide 5' Phosphate Intermediate 9>

[0100] The dinucleotide 5' phosphate intermediate was synthesized in the same manner as the dinucleotide 5' phosphate intermediate 8, except that methyl iodide was used instead of benzyl bromide in the synthesis of intermediate 6-1. ESI-MS (+) = 721.4

[0101] <Synthesis of Trinucleotide 5' Phosphate Intermediate 10>

[0102] Trinucleotide 5' phosphate intermediate 10 was synthesized in the same manner as dinucleotide 5' phosphate intermediate 8 according to the above synthesis scheme. SI-MS(+)=1066.2

[0103] <Synthesis of Compound 1-1>

[0104] To a mixture of diphosphate intermediate 1 (0.030 g), dinucleotide 5' phosphate intermediate 3 (0.043 g), 1-methylimidazole (0.030 mL), and dimethyl sulfide (1.0 mL) was added zinc chloride (15.4 mg) under a nitrogen atmosphere and stirred at 55°C for 2 hours. After standing overnight at room temperature, water (10 mL) and ethylenediamine-N,N,N',N'-tetraacetic acid disodium salt dihydrate were added and stirred, followed by crude purification on a DEAE Sephadex column (eluent: water / 1 M triethylammonium bicarbonate). After purification using a UPLC separation and purification system (column: Waters, CSH C18, eluent: 50 mM TEAA (pH 7.0) / 50 mM TEAA (pH 7.0):MeCN = 50:50), a mixture of the obtained white solid and water (0.2 mL) was added dropwise to an acetone solution (2 mL) of sodium perchlorate (20 mg) under ice-cooling. After the addition was completed, the mixture was stirred for 30 minutes and then centrifuged to remove the supernatant. Acetone (2 mL) was added to the residue, which was washed with stirring, and then centrifuged again to remove the supernatant. This washing procedure was repeated three times, followed by concentration under reduced pressure to obtain compound 1-1 (0.010 g) as a white solid. ESI-MS (+) = 1161.87

[0105] <Synthesis of Compounds 1-2 and 1-3>

[0106] Compound 1-2 was synthesized in the same manner as compound 1-1 using diphosphate intermediate 2 and dinucleotide 5' phosphate intermediate 4. ESI-MS (+) = 1162.3

[0107] Compound 1-3 was synthesized in the same manner as compound 1-1 using diphosphate intermediate 2 and dinucleotide 5' phosphate intermediate 5. ESI-MS (+) = 1162.0

[0108] <Synthesis of Compounds 7, 8, and 9> Compound 7 was synthesized in the same manner as for Compound 1-1, using diphosphate intermediate 3 and dinucleotide 5' phosphate intermediate 4. ESI-MS (+) = 1176.1

[0109] Compound 8 was synthesized in the same manner as compound 1-1 using diphosphate intermediate 3 and dinucleotide 5' phosphate intermediate 7. ESI-MS (+) = 1190.2

[0110] Compound 9 was synthesized in the same manner as compound 1-1 using diphosphate intermediate 2 and dinucleotide 5' phosphate intermediate 6. ESI-MS (+) = 1252.3

[0111] <Synthesis of Compound 15, Compound 16, and Compound 17>

[0112] Compound 15 was synthesized in the same manner as compound 1-1 using diphosphate intermediate 1 and dinucleotide 5' phosphate intermediate 8. ESI-MS (+) = 1252.3

[0113] Compound 16 was synthesized in the same manner as compound 1-1 using diphosphate intermediate 1 and dinucleotide 5' phosphate intermediate 9. ESI-MS (+) = 1176.7

[0114] Compound 17 was synthesized in the same manner as compound 1-1 using diphosphate intermediate 1 and trinucleotide 5' phosphate intermediate 10. ESI-MS (+) = 1521.9

[0115] <Comparative Compound 1>

[0116] Comparative compound 1 was obtained as a reagent from Fujifilm Wako Pure Chemical Industries, Ltd.

[0117] <In vitro transcription (IVT) synthesis of capped mRNA> Capped mRNA encoding the full-length spike protein analogue of SARS-CoV-2 (Lys986Pro, Val1987Pro) was prepared by polymerase chain reaction using Compound 1-1, Compound 1-2, and Compound 1-3.

[0118] The IVT reaction to prepare capped mRNA used a linearized DNA template containing the T7 promoter sequence and associated regulatory sequences, mRNA coding sequence (CDS), 3' and 5' untranslated regions (UTRs), and poly(A) tail. Water for injection, magnesium acetate, HEPES buffer (containing spermidine and DTT), T7 RNA polymerase, DNA template, RNase inhibitor, capping compounds (Compound 1-1 / Compound 1-2 / Compound 1-3), pyrophosphatase, and a ribonucleotide mixture (GTP, ATP, CTP, N1-methylpseudouridine triphosphate) were mixed and the IVT reaction was carried out at 38°C for 4 hours. To remove the DNA template, DNase was added to the reaction mixture, and the mixture was incubated at 38°C for 15 minutes. Lithium chloride was then added to the reaction mixture, and the precipitated mRNA was isolated by centrifugation. The yield and full-length purity of the capped mRNA were measured by UV spectroscopy and capillary gel electrophoresis. The yield of mRNA containing Compound 1-1 was 5.4 mg per mL of IVT reaction, with a full-length purity of 86%. The yields of mRNA containing Compound 1-2 and Compound 1-3 were 5.2 mg / mL and 4.9 mg / mL, respectively, with full-length purities of 85% and 87%. When IVT synthesis was performed using Comparative Compound 1 as the capping compound, the yield of capped mRNA was 5.1 mg / mL, with a full-length purity of 88%.

[0119] Capped mRNAs encoding human erythropoietin (hEPO) and firefly luciferase (Fluc) were prepared by polymerase chain reaction using comparative compounds 1, 1-1, 1-2, 1-3, 7, 8, and 9 in the same manner as in the "In vitro transcription (IVT) synthesis of capped mRNA" section above, and the full-length purity and dsRNA content were measured. The mRNAs prepared using compounds 1-1, 1-2, 1-3, and 7-9 had high full-length purity, low contents of dsRNA that induces natural immunity, and equivalent in vitro transcription activity to that of comparative compound 1 (Table 2). Although not particularly limited, compounds 16, 17, and 18 also have similar structural features and are therefore expected to provide high-quality mRNA.

[0120]

[0121] Translation of capped mRNA in cells: The translation activity of the prepared luciferase (FLuc) mRNA and human erythropoietin (hEPO) mRNA was evaluated in cultured cells (A549, HEK293, HeLa). Following the standard protocol for the reagent, Lipofectamine® MessengerMAX Reagent and the mRNA sample were mixed and added to the cells at 0.15 μL / well of Lipofectamine® MessengerMAX Reagent and 24 or 100 ng / well of the mRNA sample. Eight or 24 hours after addition, the protein expression level was measured using one of the following methods, depending on the protein species being expressed.

[0122] [Firefly luciferase (FLuc)] FLuc protein expression was assessed by measuring luminescence intensity after reaction with the substrate. Approximately 30 minutes before luminescence detection, 20 L of CellTiter-Fluor Cell Viability Ce solution was added per well, stirred, and the plate was placed in an incubator for 30 minutes. After heating, fluorescence measurement (excitation wavelength: 330 nm, emission wavelength: 505 nm) was performed using a microplate reader to measure intracellular protease activity. After measurement, 20 μL of supernatant was discarded from the 96-well plate, and 100 μL of Steady-Glo solution (prepared according to the reagent protocol) was added per well and stirred for 10 minutes in the dark. After stirring, a light-shielding sticker was affixed to the bottom of the plate, and luminescence measurement was performed using a microplate reader.

[0123] [Erythropoietin (hEPO)] EPO protein expression was assessed by measuring the EPO concentration in the culture supernatant using ELISA. After adding the mRNA sample, the culture supernatant was collected into a new 96-well plate 8 and 24 hours later and stored at -80°C until the day of ELISA measurement. After supernatant collection, culture medium was added to the 96-well plate to adjust the medium volume to 100 L per well, and then 20 μL of CellTiter-Fluor Cell Viability solution was added per well. After stirring, the plate was placed in an incubator and heated for 30 minutes. After heating, fluorescence measurement (excitation wavelength: 330 nm, emission wavelength: 505 nm) was performed using a microplate reader to measure intracellular protease activity. EPO-ELISA measurements were performed according to the Human EPO ELISA Kit protocol. The culture supernatant was appropriately diluted with water or PBS(-) and used as a measurement sample for ELISA, and the EPO concentration in the culture supernatant was calculated from the obtained calibration curve.

[0124] The mRNAs prepared using Compounds 1-1, 1-2, 1-3, and 7 were translated with the same efficiency as that of the comparative compound 1 (Figures 1 and 2). Furthermore, the mRNAs prepared using Compounds 8 and 9 had higher translation activity than the comparative compounds (Figure 3).

[0125] <In vivo evaluation in mice> Ionized lipid (Lipid-5, CAS: 2089251-33-0), neutral lipid, cholesterol (product name: Cholesterol HP; Nippon Fine Chemical Co., Ltd.), 1,2-dimyristoyl-rac-glycero-3-(methylpolyoxyethylene 2000) (hereinafter referred to as DMGPEG2000) (product name: SUNBRIGHT® GM-020; NOF Corporation) were dissolved in ethanol at a molar ratio of ionized lipid:neutral lipid:cholesterol:DMG-PEG2000 = 50:10:38.5:1.5 mol% to a total lipid concentration of 12.5 mmol / L to obtain an oil phase. mRNA (hEPO) capped with Comparative Compound 1, Compound 1-1, or Compound 8 was diluted with 50 mmol / L citrate buffer at pH 4.0 so that the weight ratio of total lipid concentration to mRNA concentration was approximately 20:1 to obtain an aqueous phase. The aqueous and oil phases were then mixed using a NanoAssembler (Precision Nanosystems) so that the volume ratio of aqueous phase to oil phase was 3:1. After neutralizing the mixture, the mixture was dialyzed using a dialysis cassette to obtain mRNA (hEPO)-encapsulated lipid nanoparticles.

[0126] LNPs encapsulating mRNA (hEPO) capped with comparative compound 1, compound 1-1, or compound 8 were prepared and administered intravenously once (0.1 mg / kg) to mice (ICR, 5 weeks old, female, 3 animals). Plasma hEPO concentrations were measured 6 and 24 hours later by ELISA (Human Erythropoietin ELISA Lit (Abcam)) according to the protocol. At both 6 and 24 hours after administration, the hEPO expression levels of compounds 1-1 and 8 were approximately 2-4 times higher than that of comparative compound 1, demonstrating high translation properties in vivo ( FIG. 4 ). This is presumably because the compounds have a non-natural thioribose structure, making them less recognizable by cap-degrading enzymes in vivo and highly resistant to degradation. Therefore, compounds 1-2, 1-3, the compounds listed in Table 1, 16, 17, and 18 are also expected to exhibit high translation properties due to similar effects.

Claims

1. A compound represented by the following formula (1): In the formula, B １ teeth, where * indicates the bond position, B ２ is a natural, modified, or non-natural nucleobase; B ３ R may be the same or different when present in plural and is a natural, modified or non-natural nucleobase; １ and R ２ are each independently hydrogen, fluorine, —OH, or —O-alkyl; R ３ and R ４ are each independently hydrogen, fluorine, —OH, or —O-alkyl; R ４ When there are a plurality of X's, they may be the same or different; １ , X ２ and X are each independently -O- or -S-, and when a plurality of X's are present, they may be the same or different, provided that X １ , X ２ and at least one of X is —S—, and n is an integer from 0 to 7.

2. The compound according to claim 1, which is a compound represented by the following formula (2): In the formula, n represents an integer of 0 to 2, and each R is independently —H, —CH ３ and each symbol other than n and R has the same meaning as in claim 1.

3. An RNA synthesis initiator comprising the compound according to claim 1 or 2.

4. An RNA synthesis kit comprising the RNA synthesis initiator according to claim 3 and an RNA polymerase.

5. RNA comprising the RNA synthesis initiator according to claim 3.

6. A method for producing RNA, comprising transcribing a template nucleic acid in the presence of the RNA synthesis initiator according to claim 3.

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