Oligonucleotide for 5'-capped RNA synthesis

Abstract

The present invention relates to a novel oligonucleotide primer used for the synthesis of 5'-capped RNA. The novel oligonucleotide primer for RNA capping, represented by chemical formula 1 in the present invention, can be usefully utilized in the fields of nucleic acid therapeutic agents or vaccines.

Description

Oligonucleotides for 5'-capped RNA synthesis

[0001] The present invention relates to a novel oligonucleotide used in the synthesis of 5'-capped RNA.

[0002] RNA 5'-capping is the first step in pre-mRNA processing in eukaryotic cells, where the 5' end of the transcribed RNA is capped. In eukaryotes, the 5' end of the mRNA molecule contains 7-methylguanosine (m 7 G) A 5' cap (cap-0) is formed by a 5' triphosphate bond, which is mediated in vivo by methyltransferase enzymes. Eukaryotic mRNAs may have additional modifications, including methylation of the 2' hydroxyl group of the first ribose sugar at the 5' end (cap-1) and methylation of the 2' hydroxyl group of the second ribose sugar (cap-2). This 5' capping process of mRNA provides resistance to degrading enzymes such as 5' exonucleases when exporting mRNA from the nucleus to the cytoplasm.

[0003] For medical or research purposes, capped mRNA encoding a specific gene can be transfected into eukaryotic organisms or microinjected into cells or embryos to artificially express that gene. However, if uncapped RNA is used in this process, it is rapidly degraded, is immunogenic, and protein translation efficiency is drastically reduced. Therefore, proper 5' capping is crucial for in vitro mRNA synthesis.

[0004] For mRNA synthesis, the process of first transcribing RNA with a 5'-triphosphate group in vitro and then performing 5' capping using a capping enzyme has proven to be not only costly but also inefficient. Accordingly, synthetic methods have been developed that prepare oligonucleotides with a 5'-capped structure and use them as primers to initiate in vitro transcription. For example, International Publication Nos. WO2008 / 016473 and WO2013 / 059475 disclose dinucleotide mRNA cap analogs for synthesizing 5'-capped RNA, and International Publication No. WO2017 / 053297 discloses trinucleotide mRNA cap analogs for synthesizing 5'-capped RNA.

[0005] Capped RNA transcripts can be applied to therapeutic and / or preventive fields requiring protein synthesis, such as nucleic acid therapeutics and vaccines. Therefore, the development of 5'-capping oligonucleotide primers for their efficient production is essential. For example, there is an urgent need for RNA cap analogs that are simpler and more cost-effective than conventional methods in manufacturing, efficiently promote the desired transcription reaction, increase RNA production yield, reduce the production of undesirable heterologous products, eliminate the need for additional enzymatic reactions, or enable large-scale RNA synthesis. Furthermore, if a cap structure can be provided that can maintain mRNA stability in vivo, improve translation efficiency, or reduce side effects such as immunogenicity when applied as an actual RNA therapeutic or vaccine, universal utility in the RNA therapeutic or vaccine field can be achieved.

[0006] The purpose of the present invention is to provide a novel RNA capping oligonucleotide primer, a method for producing the same, and a use thereof.

[0007] Another object of the present invention is to provide an RNA molecule prepared using the above-described oligonucleotide primer for RNA capping and its use.

[0008] To achieve the above objectives, the present inventors provide novel RNA capping oligonucleotide primers, methods for their preparation, and methods for their use. Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. All patent and non-patent literature cited throughout the disclosure herein is incorporated by reference in its entirety.

[0009] Novel RNA capping oligonucleotide primers

[0010] The present invention provides an oligonucleotide primer compound for RNA capping represented by the following chemical formula 1, a stereoisomer thereof, or a salt thereof:

[0011] [Chemical Formula 1]

[0012]

[0013] In the above chemical formula,

[0014] B1 and B2 are each independently a natural, unnatural, or modified nucleoside base;

[0015] X is -OH, -O(C 1-4 alkyl), or -halo;

[0016] Y is -halo;

[0017] Z1 and Z2 are each independently -OH, -O(C 1-4 alkyl), or -halo;

[0018] R1 and R2 are each independently -H or -(C 1-4 alkyl);

[0019] R3 is -(C 1-4 alkyl).

[0020] According to a specific example of the present invention, the oligonucleotide primer compound for RNA capping, a stereoisomer thereof, or a salt thereof may be in the following range:

[0021] B1 and B2 are each independently a natural, unnatural, or modified nucleoside base;

[0022] X is -OH, -O(C 1-4 alkyl), or -halo;

[0023] Y is -halo;

[0024] Z1 is -OH, -O(C 1-4 alkyl), or -halo;

[0025] Z2 is -OH or -halo;

[0026] R1 and R2 are each independently -H or -(C 1-4 alkyl);

[0027] R3 is -(C 1-4 alkyl).

[0028] According to a specific example of the present invention, the oligonucleotide primer compound for RNA capping, a stereoisomer thereof, or a salt thereof may be in the following range:

[0029] B1 and B2 are each independently a natural, unnatural, or modified nucleoside base;

[0030] X is -OH, -O(C 1-4 alkyl), or -halo;

[0031] Y is -halo;

[0032] Z1 is -OH or -O(C 1-4 alkyl);

[0033] Z2 is -OH or -halo;

[0034] R1 and R2 are each independently -H or -(C 1-4 alkyl);

[0035] R3 is -(C 1-4 alkyl).

[0036] According to a specific example of the present invention, the oligonucleotide primer compound for RNA capping, a stereoisomer thereof, or a salt thereof may be in the following range:

[0037] B1 and B2 are each independently a natural, unnatural, or modified nucleoside base;

[0038] X is -OH or -halo;

[0039] Y is -halo;

[0040] Z1 is -OH, -O(C 1-4 alkyl), or -halo;

[0041] Z2 is -OH or -halo;

[0042] R1 and R2 are each independently -H or -(C 1-4 alkyl);

[0043] R3 is -(C 1-4 alkyl).

[0044] According to a specific example of the present invention, the oligonucleotide primer compound for RNA capping, a stereoisomer thereof, or a salt thereof may be in the following range:

[0045] B1 and B2 are each independently a natural, unnatural, or modified nucleoside base;

[0046] X is -OH or -halo;

[0047] Y is -halo;

[0048] Z1 is -OH or -O(C 1-4 alkyl);

[0049] Z2 is -OH or -halo;

[0050] R1 and R2 are each independently -H or -(C 1-4 alkyl);

[0051] R3 is -(C 1-4 alkyl).

[0052] In the present invention, the nucleoside base may be a natural nucleoside base as well as a non-natural or modified nucleoside base.

[0053] In the present invention, the most commonly found base rings in naturally occurring nucleosides are purine and pyrimidine rings. Naturally occurring purine rings include, for example, adenine ( ), guanine( ), and N 6 -methyladenine( ) include. Naturally occurring pyrimidine rings include, for example, cytosine ( ), thymine( ), 5-methylcytosine( ), uracil ( ) is included.

[0054] According to a specific example of the present invention, the unnatural or modified nucleoside base is an isomer of the natural nucleoside base or at least one -H of the natural nucleoside base is -(C 1-4 alkyl), -O(C 1-4 It may be substituted with one or more selected from the group consisting of substitution with alkyl) or -halo, substitution with one or more =CH-, substitution with =N-, and substitution with one or more =O.

[0055] Also in the present invention, the unnatural or modified nucleoside base may be a base derived from a nucleoside analogue comprising an artificial base recognizable by RNA polymerase as a substitute for one of the natural NTPs (e.g., ATP, UTP, CTP and GTP) or another specific NTP (see, e.g., Loakes, D., Nucleic Acids Res., 29:2437-2447 (2001); Crey-Desbiolles, C., et al., Nucleic Acids Res., 33:1532-1543 (2005); Kincaid, K., et al., Nucleic Acids Res., 33:2620-2628 (2005); Preparata, FP, Oliver, JS, J. Comput. Biol. 753-765 (2004); and Hill, F., et al., Proc Natl Acad. Sci. USA, 95:4258-4263 (1998)], etc.).

[0056] In addition, in the present invention, the unnatural or modified nucleoside base is a halogen-substituted purine (e.g., 6-fluoropurine), a halogen-substituted pyrimidine, N 6 -Ethyl adenine, N 4 - It may be a base derived from (alkyl)-cytosine, 5-ethylcytosine, etc., but is not limited thereto.

[0057] In addition, in the present invention, another example of the unnatural or modified nucleoside base is 8-azaguanosine, pseudouridine (ψ), 5-methyl-cytidine (m 5 C), 1-methyl-pseudouridine (m 1 ψ), 1-methyl-pseudouridine (m 1 ψ) and 5-methyl-cytidine (m 5 C), 2-thiouridine (s 2 U), 2-thiouridine (s 2 U) and 5-methyl-cytidine (m 5 C), 5-methoxy-uridine (mo 5 U), 5-methoxy-uridine (mo5 U) and 5-methyl-cytidine (m 5 C), 2'-O-methyl uridine, 2'-O-methyl uridine and 5-methyl-cytidine (m 5 C), N 6 -methyl-adenosine (m 6 A) or N 6 -methyl-adenosine (m 6 A) and 5-methyl-cytidine (m 5 C) including, but not limited to.

[0058] In addition, examples of natural, unnatural or modified nucleoside bases that can be used in the present invention can be found in International Publication No. WO2018 / 144775 or International Publication No. WO2018 / 144082.

[0059] According to a specific example of the present invention, B1 and B2 are each independently , , , , , or It can be. In one aspect, the above B1 is and B2 is , or It may include, but is not limited to.

[0060] Additionally, according to a specific example of the present invention, Y may be -F.

[0061] Additionally, according to a specific example of the present invention, both R1 and R2 can be -H.

[0062] Additionally, according to a specific example of the present invention, R3 may be -methyl.

[0063] Specifically, the compound represented by the above chemical formula 1 may be any one selected from the group consisting of the following compounds:

[0064] (1) ;

[0065] (2) ;

[0066] (3) ;

[0067] (4) ;

[0068] (5) ;

[0069] (6) ;

[0070] (7) ;

[0071] (8) ;

[0072] (9) ; and

[0073] (10) .

[0074] According to a specific example of the present invention, the compound represented by the chemical formula 1 may be at least one selected from the group consisting of the following compounds:

[0075] 2-Amino-9-((2R,3R,4S,5R)-5-((((((((2R,3R,4R,5R)-3-((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-( 6-amino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium (oligonucleotide 1);

[0076] 2-Amino-9-((2R,3R,4S,5R)-5-((((((((2R,3R,4R,5R)-3-((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6 -amino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium (oligonucleotide 2);

[0077] 2-Amino-9-((2R,3R,4S,5R)-5-((((((((2R,3R,4R,5R)-3-((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5- (6-amino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium (oligonucleotide 3);

[0078] 2-Amino-9-((2R,3R,4S,5R)-5-((((((((2R,3R,4R,5R)-3-((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5- (6-amino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium (oligonucleotide 4);

[0079] 2-Amino-9-((2R,3R,4R,5R)-5-((((((((2R,3R,4R,5R)-3-((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6 -amino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium (oligonucleotide 5);

[0080] 2-Amino-9-((2R,3R,4R,5R)-5-((((((((2R,3R,4R,5R)-3-((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6 -amino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium (oligonucleotide 6);

[0081] 2-Amino-9-((2R,3R,4R,5R)-5-((((((((2R,3R,4R,5R)-3-((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-a Mino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium (oligonucleotide 7);

[0082] 2-Amino-9-((2R,3S,4S,5R)-5-((((((((2R,3R,4R,5R)-3-((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-fluoro-5 -(6-(methylamino)-9H-purin-9-yl)tetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium (oligonucleotide 8);

[0083] 2-Amino-9-((2R,3S,4S,5R)-5-((((((((2R,3R,4R,5R)-3-((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-a (Mino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium (oligonucleotide 9); and

[0084] 2-Amino-9-((2R,3R,4R,5R)-5-((((((((2R,3R,4R,5R)-3-((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-fluoro-5 -(6-(methylamino)-9H-purin-9-yl)tetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium (oligonucleotide 10).

[0085] According to a specific example of the present invention, the compound represented by the above chemical formula 1 may be an oligonucleotide primer for RNA capping.

[0086] In the present invention, the term "stereoisomer" means a compound of the present invention or a salt thereof having the same chemical formula or molecular formula but having a steric difference. Each of these stereoisomers and mixtures thereof are also included in the scope of the present invention. In addition, unless otherwise stated, a solid bond connecting an asymmetric carbon atom ( ) is a wedge-shaped ( ) or wedge-shaped dotted line joint ( ) may be included.

[0087] In the present invention, the term "salt" refers to a salt commonly used in the pharmaceutical industry. Specifically, it may be a base addition salt. For example, there are inorganic ion salts made of calcium, potassium, sodium, or magnesium; amino acid salts made of arginine, lysine, histidine, and the like; and amine salts made of trimethylamine, triethylamine, ammonia, pyridine, picoline, and the like; but the types of salts meant in the present invention are not limited to these listed salts.

[0088] RNA capped with novel oligonucleotide primers

[0089] The above-described RNA capping oligonucleotide primer can be used to produce an RNA molecule comprising the same. Accordingly, the present invention provides an RNA molecule comprising the RNA capping oligonucleotide primer represented by Chemical Formula 1.

[0090] The oligonucleotide primer for RNA capping according to the present invention can be extended by RNA polymerase through the incorporation of NTPs at the 3'-end. In vitro transcription can be initiated under the control of a promoter in a transcription system containing essential components such as a DNA template (e.g., a DNA plasmid), RNA polymerase, a nucleoside 5'-triphosphate, and a suitable buffer. Here, the oligonucleotide primer is complementary to the DNA template at the initiation site.

[0091] In the present invention, a promoter refers to a specific region of a dsDNA template that induces 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 (toward the 5' region of the sense strand) of the DNA. The promoter is typically immediately adjacent to or partially overlapping with the DNA sequence to be transcribed. The nucleotide positions within the promoter are designed relative to the transcription start site where DNA transcription begins (position +1). The initiator oligonucleotide primer is complementary to the initiation site of the promoter sequence (which in certain embodiments is at positions +1 and +2, and in the case of an initiation tetramer, at positions +1, +2, and +3).

[0092] In one embodiment, the oligonucleotide primer for RNA capping is attached to the 5' end of an RNA molecule to form a 5'-capped RNA. The RNA molecule thus formed includes, but is not limited to, mRNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and small Cajal body-specific RNA (scaRNA).

[0093] In one embodiment, the RNA molecule is mRNA and may comprise one or more coding sequences (CDS). Additionally, the RNA molecule may comprise a polyA sequence and / or a polyadenylation signal. The polyA sequence may be composed entirely or predominantly of adenine nucleotides or analogs or derivatives thereof. The polyA sequence may be a tail positioned adjacent to the 3' untranslated region of the nucleic acid.

[0094] The present invention also provides a method for synthesizing a 5'-capped RNA molecule, comprising the steps of (S-1) mixing a DNA template, an oligonucleotide primer for RNA capping represented by the above-described chemical formula 1, and an RNA polymerase; and (S-2) incubating the mixture to perform transcription of the polynucleotide template.

[0095] Meanwhile, methods for DNA-templated and promoter-controlled synthesis of RNA using oligonucleotide primers, RNA polymerase, and nucleoside 5'-triphosphates (NTPs) are well known in the art. Therefore, an RNA molecule comprising the RNA capping oligonucleotide primer represented by Chemical Formula 1 provided in the present invention can be synthesized.

[0096] According to a specific example of the present invention, the DNA template may be a double-stranded linear DNA, a partially double-stranded linear DNA, a circular double-stranded DNA, a DNA plasmid, a PCR amplicon, or, in addition, a modified nucleic acid capable of appropriately reacting with RNA polymerase.

[0097] According to a specific embodiment of the present invention, the synthesis of the RNA molecule is performed in vitro. To perform large-scale transcription in vitro, single-subunit phage polymerases derived from T7, T3, SP6, K1-5, K1E, K1F, or K11 bacteriophages can be used. Polymerases of this family have simple minimal promoter sequences of ~17 nucleotides that do not require auxiliary proteins and have minimal constraints on the initiation nucleotide sequence. An example of an RNA polymerase that can be used in the present invention is T7 RNA polymerase (T7 RNAP), but those skilled in the art will appreciate that the present invention can also be practiced using other RNA polymerases. Unlike DNA polymerases, T7 RNAP initiates RNA synthesis in the absence of a primer. The first step in initiation is called de novo RNA synthesis, where RNA polymerase recognizes a specific sequence on the DNA template, selects the first pair of nucleotide triphosphates complementary to the template residues at positions +1 and +2, and catalyzes the formation of a phosphodiester bond to form a dinucleotide.

[0098] Medical uses of capped RNA

[0099] The 5'-capped RNA molecule according to the present invention as described above can be usefully utilized for pharmaceutical purposes.

[0100] According to a specific embodiment of the present invention, the 5'-capped RNA molecule produced according to the present invention can be utilized as a nucleic acid therapeutic agent or vaccine. For example, the nucleic acid therapeutic agent or vaccine can be used as an RNA vaccine (for preventing cancer or infectious diseases). The nucleic acid therapeutic agent or vaccine can be administered to a subject and translated in vivo to produce a desired peptide.

[0101] According to a specific embodiment of the present invention, the 5'-capped RNA molecule can be introduced into a cell to produce a protein that can treat a medical condition of the cell or have a therapeutic effect on the cell.

[0102] According to a specific embodiment of the present invention, the nucleic acid therapeutic agent or vaccine may further comprise, together with the RNA molecule, a carrier capable of introducing the RNA molecule into a target cell.

[0103] According to a specific embodiment of the present invention, the present invention provides a peptide translated from the above-described 5'-capped RNA molecule.

[0104] According to a specific embodiment of the present invention, the present invention provides a cell into which the above-described 5'-capped RNA molecule has been introduced. The cell may be a somatic cell of a subject, or may be a cell line capable of being cultured in vitro.

[0105] In addition, according to a specific embodiment of the present invention, the present invention provides a peptide produced in a cell into which a 5'-capped RNA molecule has been introduced.

[0106] In the present invention, pharmaceutical compositions for RNA therapeutics or vaccines may be formulated for administration by injection or other suitable routes known to those skilled in the art for treating specific conditions. Injectable compositions for parenteral administration typically contain the active compound in a suitable solution and / or pharmaceutical carrier, such as sterile physiological saline. The compositions may also be formulated as suspensions in lipids or phospholipids, liposomal suspensions, or aqueous emulsions.

[0107] Methods for preparing compositions and / or formulations for RNA therapeutics or vaccines are known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences (19 thEd., Williams & Wilkins, 1995). The composition to be administered will contain a specific amount of a compound selected in a pharmaceutically safe and effective amount to increase the expression of the target protein in the target cell or tissue.

[0108] In some embodiments, the pharmaceutical composition contains at least 0.1% (w / v) of the compound as described above, in some embodiments, the pharmaceutical composition contains more than 0.1%, in some embodiments, the pharmaceutical composition contains no more than about 10%, in some embodiments, the pharmaceutical composition contains no more than about 5%, and in some embodiments, the pharmaceutical composition contains no more than about 1% (w / v). The selection of a suitable concentration will depend on factors such as the desired dose, frequency, and method of delivery of the active agent.

[0109] When treating a subject, such as a mammal or a human, the dosage is determined based on factors such as the subject's body weight and overall health, the condition being treated, and the severity of symptoms. The dosage and concentration that provides the desired benefit while avoiding any undesirable side effects are determined. A typical dosage of the subject compound is in the range of about 0.0005 to 500 mg / day for a human patient, and in some embodiments, in the range of about 1 to 100 mg / day. For example, higher dosage regimens can include, for example, 50 to 100, 75 to 100, or 50 to 75 mg / day, and lower dosage regimens can include, for example, 1 to 50, 25 to 50, or 1 to 25 mg / day.

[0110] The novel oligonucleotides of the present invention can be utilized in the synthesis of 5'-capped RNA to not only improve RNA production processes (e.g., synthesis yield, synthesis scale, purity, etc.), but also enhance the efficacy of nucleic acid therapeutics or vaccines utilizing them (e.g., RNA stability and / or protein expression efficiency) and reduce side effects (e.g., reduced immunogenicity). Therefore, the present invention can be usefully utilized in the field of nucleic acid therapeutics or vaccines.

[0111] Hereinafter, the composition and effects of the present invention will be described in more detail through examples and experimental examples. These examples and experimental examples are intended solely to illustrate the present invention, and the scope of the present invention is not limited by these examples and experimental examples.

[0112] Manufacturing Example 1: Preparation of ((2R,3R,4R,5R)-3-((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methyl dihydrogen phosphate.2-triethylamine salt

[0113] Step 1. Preparation of (2R,3R,4R,5R)-4-fluoro-2-(hydroxymethyl)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl benzoate

[0114] N-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (1.5 g, 2.281 mmol) was dissolved in pyridine (11.4 mL), and the mixture was cooled to 0 to 5 °C. Benzoyl chloride (0.582 mL, 5.02 mmol) was added, and after 15 minutes, the temperature was raised to 40 °C. The reaction mixture was stirred for 2 hours, and then ethyl acetate (100 mL) was added at room temperature. The organic layer was washed with an aqueous solution of copper sulfate and an aqueous solution of saturated sodium bicarbonate, dehydrated over sodium sulfate, and concentrated under reduced pressure.

[0115] After cooling the concentrated residue to 0 to 5 °C, a 1% trifluoroacetic acid / dichloromethane (228 mL) solution was slowly added. After 15 minutes, the reaction solution was slowly warmed to room temperature and stirred for 15 minutes. After cooling the reaction solution to 0 to 5 °C, a saturated sodium bicarbonate solution was slowly added to adjust the pH of the aqueous layer to 8 to 9. The organic layer and the aqueous layer were separated, and the organic layer was dried with sodium sulfate and filtered. After concentration under reduced pressure, the residue was separated by column chromatography to obtain the title compound (0.825 g) as a white solid.

[0116] LC-MS (ESI, m / z) = 460.1 (M+H + ).

[0117] Step 2. Preparation of (2R,3R,4R,5R)-2-(((((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-fluoro-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl benzoate

[0118] (2R,3R,4R,5R)-4-fluoro-2-(hydroxymethyl)-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl benzoate (0.41 g, 0.892 mmol) obtained in step 1 above was dissolved in acetonitrile (3 mL). After cooling to 0 to 5 ℃, 4Å molecular sieve (82 mg), (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)menoxy)methyl)-4-fluorotetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (0.86 g, 0.982 mmol) and 1H-tetrazole (0.25 g, 3.57 mmol) were added in sequence. After 10 minutes, the temperature was slowly increased to room temperature and stirred for 1 hour.

[0119] After cooling the reaction mixture to 0 to 5 °C, a solution of iodine (0.679 g, 2.68 mmol) dissolved in tetrahydrofuran / distilled water / pyridine (66:33:1) (8.92 mL) was added. The mixture was slowly warmed to room temperature and stirred for 30 minutes. After completion of the reaction, dichloromethane and a 10% sodium thiosulfate pentahydrate solution were added, and the organic and aqueous layers were separated. The organic layer was dehydrated with sodium sulfate and concentrated under reduced pressure.

[0120] After cooling the concentrated residue to 0 to 5 °C, a 2% trifluoroacetic acid / dichloromethane (44.6 mL) solution was slowly added and stirred for 30 minutes. After the reaction was completed, a saturated sodium bicarbonate solution was slowly added to adjust the pH to 8 to 9. The organic and aqueous layers were separated, and the organic layer was dried with sodium sulfate and filtered. After concentration under reduced pressure, the residue was separated by column chromatography to obtain the title compound (0.714 g) as a white solid.

[0121] LC-MS (ESI, m / z) = 948.1 (M+H + ).

[0122] Step 3. Preparation of (2R,3R,4R,5R)-2-(((((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-(((bis(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-fluoro-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl benzoate

[0123] (2R,3R,4R,5R)-2-(((((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-fluoro-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl benzoate (0.71 g, 0.749 mmol) obtained in step 2 above was dissolved in dichloromethane (2.5 mL). After cooling to 0 to 5 ℃, 4Å molecular sieve (142 mg), bis(2-cyanoethyl) diisopropylphosphoramidite (0.587 mL, 2.247 mmol), and 1H-tetrazole (0.105 g, 12.70 mmol) were added sequentially. After 10 minutes, the temperature was slowly increased to room temperature and stirred for 2 hours.

[0124] After cooling the reaction mixture to 0 to 5 °C, a solution of iodine (0.57 g, 2.247 mmol) in tetrahydrofuran / distilled water / pyridine (66:33:1) (7.5 mL) was added. The mixture was slowly warmed to room temperature and stirred for 30 minutes. After completion of the reaction, dichloromethane and a 10% sodium thiosulfate pentahydrate solution were added, and the organic layer and the aqueous layer were separated. The organic layer was dehydrated with sodium sulfate, concentrated under reduced pressure, and separated by column chromatography to obtain the title compound (0.684 g) as a yellow solid.

[0125] LC-MS (ESI, m / z) = 1134.0 (M+H + ).

[0126] Step 4. Preparation of ((2R,3R,4R,5R)-3-((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methyl dihydrogen phosphate.2-triethylamine salt

[0127] To (2R,3R,4R,5R)-2-(((((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-(((bis(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-fluoro-5-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl benzoate (0.684 g, 0.603 mmol) obtained in step 3 above, aqueous ammonia (9.4 mL, 241 mmol) was added at room temperature. After heating to 50 to 55 °C, the mixture was stirred for 40 hours. After washing the reaction solution with dichloromethane, the aqueous layer was purified with DEAE Sephadex resin to obtain the title compound (0.38 g) as a white solid.

[0128] LC-MS (ESI, m / z) = 697.0 (M+H + ).

[0129] Manufacturing Example 2. Preparation of ((2R,3R,4R,5R)-3-((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methyl dihydrogen phosphate.2-triethylamine salt

[0130] A reaction was performed in a similar manner to Preparation Example 1 using N-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3-methoxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (0.5 g, 0.747 mmol) to obtain the title compound (0.244 g) as a white solid.

[0131] LC-MS (ESI, m / z) = 709.0 (M+H + ).

[0132] Manufacturing Example 3. Preparation of 2-amino-9-((2R,3R,4S,5R)-3-hydroxy-5-(((hydroxy((hydroxy(1H-imidazol-1-yl)phosphoryl)oxy)phosphoryl)oxy)methyl)-4-methoxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.sodium salt

[0133] Step 1. Preparation of ((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-hydroxy-3-methoxytetrahydrofuran-2-yl)methyl dihydrogen phosphate.triethylamine salt

[0134] 2-Amino-9-((2R,3R,4S,5R)-3-hydroxy-5-(hydroxymethyl)-4-methoxytetrahydrofuran-2-yl)-1,9-dihydro-6H-purin-6-one (2 g, 6.73 mmol) was added trimethyl phosphate (33.5 mL, 289 mmol) and stirred at room temperature for 20 minutes. After the reaction solution was cooled to 0 to 5 °C, chlorophosphoric acid (0.815 mL, 8.75 mmol) was slowly added dropwise and stirred at 2 to 5 °C for 6 hours. The pH of the reaction solution was adjusted to 4 to 5 using a 1 M aqueous triethylamine bicarbonate solution, and the aqueous layer was washed with dichloromethane. The aqueous layer was purified with DEAE Sephadex resin to obtain the title compound (2.9 g) as a white solid.

[0135] LC-MS (ESI, m / z) = 378.0 (M+H + ).

[0136] Step 2. Preparation of ((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-hydroxy-3-methoxytetrahydrofuran-2-yl)methyl hydrogen (1H-imidazol-1-yl)phosphonate, sodium salt

[0137] To ((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-hydroxy-3-methoxytetrahydrofuran-2-yl)methyl dihydrogen phosphate.triethylamine salt (2.9 g, 6.06 mmol) obtained in the above step 1 was added dimethylformamide (43.3 mL), and 1H-imidazole (4.13 g, 60.6 mmol), triphenylphosphine (7.95 g, 30.3 mmol), and 2,2'-dithiodipyridine (6.68 g, 30.3 mmol) were added. The reaction solution was stirred at room temperature for 17 hours and then cooled to -40 to -78 °C. A solution of sodium perchlorate (1.484 g) dissolved in acetone (121 mL) was slowly added, and the resulting solid was filtered and dried to obtain the title compound (2.4 g) as a white solid.

[0138] LC-MS (ESI, m / z) = 428.1 (M+H + ).

[0139] Step 3. Preparation of ((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-hydroxy-3-methoxytetrahydrofuran-2-yl)methyl trihydrogen diphosphate.2-triethylamine salt

[0140] ((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-hydroxy-3-methoxytetrahydrofuran-2-yl)methyl hydrogen (1H-imidazol-1-yl)phosphonate. Sodium salt (2.36 g, 5.25 mmol) obtained in step 2 above was dissolved in dimethylformamide (30 mL), and zinc chloride (1.432 g, 10.51 mmol) was slowly added. After stirring for 30 minutes under nitrogen gas, 1 M tributylammonium phosphate / dimethylformamide (29.9 mL) was added. The reaction solution was stirred at room temperature for 6 hours, and then diluted with 25 mM EDTA disodium salt aqueous solution and distilled water. The aqueous layer was washed with dichloromethane and purified with DEAE Sephadex resin to obtain the title compound (2.145 g) as a white solid.

[0141] LC-MS (ESI, m / z) = 458.0 (M+H + ).

[0142] Step 4. Preparation of 2-amino-9-((2R,3R,4S,5R)-3-hydroxy-5-(((hydroxy(phosphonooxy)phosphoryl)oxy)methyl)-4-methoxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.triethylamine salt

[0143] ((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-hydroxy-3-methoxytetrahydrofuran-2-yl)methyl trihydrogen diphosphate.2-triethylamine salt (2.145 g, 3.25 mmol) obtained in the above step 3 was dissolved in purified water (65 mL) and the pH of the solution was adjusted to 4 to 5 with acetic acid. Dimethyl sulfate (2.175 mL, 22.76 mmol) was slowly added over 1 minute and stirred at room temperature for 7 hours. At this time, the pH of the solution was adjusted to 4 to 4.5 using 5N sodium hydroxide aqueous solution. The reaction solution was washed with dichloromethane, and the aqueous layer was purified with DEAE Sephadex resin to obtain the title compound (1 g) as a white solid.

[0144] LC-MS (ESI, m / z) = 470.0 (M-2H + ).

[0145] Step 5. Preparation of 2-amino-9-((2R,3R,4S,5R)-3-hydroxy-5-(((hydroxy((hydroxy(1H-imidazol-1-yl)phosphoryl)oxy)phosphoryl)oxy)methyl)-4-methoxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.sodium salt

[0146] To 2-amino-9-((2R,3R,4S,5R)-3-hydroxy-5-(((hydroxy(phosphonooxy)phosphoryl)oxy)methyl)-4-methoxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.triethylamine salt (1 g, 1.744 mmol) obtained in the above step 4 was added dimethylformamide (12.46 mL), and 1H-imidazole (1.187 g, 17.44 mmol), triphenylphosphine (2.287 g, 8.72 mmol), and 2,2'-dithiodipyridine (1.921 g, 8.72 mmol) were added. The reaction solution was stirred at room temperature for 18 hours and then cooled to -40 to -78 °C. A solution of sodium perchlorate (0.427 g) dissolved in acetone (35 mL) was slowly added, and the resulting solid was filtered and dried to obtain the title compound (0.91 g) as a white solid.

[0147] LC-MS (ESI, m / z) = 520.0 (M-2H + ).

[0148] Manufacturing Example 4. Preparation of 2-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(((hydroxy((hydroxy(1H-imidazol-1-yl)phosphoryl)oxy)phosphoryl)oxy)methyl)tetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.sodium salt

[0149] ((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl trihydrogen diphosphate.2-triethylamine salt (5 g, 7.74 mmol) was used to obtain the title compound (2.33 g) as a white solid through a similar method as in steps 4 and 5 of Preparation Example 3.

[0150] LC-MS (ESI, m / z) = 505.8 (M-2H + ).

[0151] Manufacturing Example 5. Preparation of 2-amino-9-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(((hydroxy((hydroxy(1H-imidazol-1-yl)phosphoryl)oxy)phosphoryl)oxy)methyl)tetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.sodium salt

[0152] A reaction was conducted in a similar manner to Preparation Example 3 using 2-amino-9-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,9-dihydro-6H-purin-6-one (1 g, 3.51 mmol) to obtain the title compound (354 mg) as a white solid.

[0153] LC-MS (ESI, m / z) = 508.0 (M-2H + ).

[0154] Manufacturing Example 6. Preparation of 2-amino-9-((2R,3S,4S,5R)-4-fluoro-3-hydroxy-5-(((hydroxy((hydroxy(1H-imidazol-1-yl)phosphoryl)oxy)phosphoryl)oxy)methyl)tetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.sodium salt

[0155] A reaction was conducted in a similar manner to Preparation Example 3 using 2-amino-9-((2R,3S,4S,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,9-dihydro-6H-purin-6-one (3.0 g, 10.52 mmol) to obtain the title compound (552.1 mg) as a white solid.

[0156] LC-MS (ESI, m / z) = 508.0 (M-2H + ).

[0157] Example 1. 2-Amino-9-((2R,3R,4S,5R)-5-((((((((2R,3R,4R,5R)-3-(((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino Preparation of -9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.3-triethylamine salt (oligonucleotide 1)

[0158] The compound obtained in Preparation Example 1 (110 mg, 0.122 mmol) and the compound obtained in Preparation Example 3 (107 mg, 0.196 mmol) were dissolved in DMSO (1.224 mL). Magnesium chloride (15.15 mg, 0.159 mmol) was added and stirred at room temperature for 7 hours. After the reaction was completed, the mixture was diluted in 6 mL of a 25 mM EDTA disodium salt solution. After confirming complete dissolution, the mixture was diluted in 6 mL of purified water. The title compound (74.3 mg) was obtained as a white solid by purification with DEAE Sephadex resin.

[0159] LC-MS (ESI, m / z) = 1148.1 (M-2H + ).

[0160] Example 2. 2-Amino-9-((2R,3R,4S,5R)-5-((((((((2R,3R,4R,5R)-3-(((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino Preparation of -9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.3-triethylamine salt (oligonucleotide 2)

[0161] The compound (80 mg, 0.088 mmol) obtained in the above Preparation Example 2 and the compound (76 mg, 0.114 mmol) obtained in the above Preparation Example 3 were reacted in the same manner as in Example 1 to obtain the title compound (52.5 mg) as a white solid.

[0162] LC-MS (ESI, m / z) = 1160.0 (M-2H + ).

[0163] Example 3. 2-Amino-9-((2R,3R,4S,5R)-5-((((((((2R,3R,4R,5R)-3-(((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-a Preparation of (mino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.3-triethylamine salt (oligonucleotide 3)

[0164] The compound (110 mg, 0.122 mmol) obtained in the above Preparation Example 1 and the compound (136 mg, 0.159 mmol) obtained in the above Preparation Example 4 were reacted in the same manner as in Example 1 to obtain the title compound (64 mg) as a white solid.

[0165] LC-MS (ESI, m / z) = 1134.0 (M-2H + ).

[0166] Example 4. 2-Amino-9-((2R,3R,4S,5R)-5-((((((((2R,3R,4R,5R)-3-((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amido Preparation of (9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.3-triethylamine salt (oligonucleotide 4)

[0167] The compound (91 mg, 0.1 mmol) obtained in the above Preparation Example 2 and the compound (111 mg, 0.13 mmol) obtained in the above Preparation Example 4 were reacted in the same manner as in Example 1 to obtain the title compound (44.4 mg) as a white solid.

[0168] LC-MS (ESI, m / z) = 1146.0 (M-2H + ).

[0169] Example 5. 2-Amino-9-((2R,3R,4R,5R)-5-((((((((2R,3R,4R,5R)-3-(((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino Preparation of (9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.3-triethylamine salt (oligonucleotide 5)

[0170] The compound (70 mg, 0.078 mmol) obtained in the above Preparation Example 1 and the compound (76 mg, 0.101 mmol) obtained in the above Preparation Example 5 were reacted in the same manner as in Example 1 to obtain the title compound (17.5 mg) as a white solid.

[0171] LC-MS (ESI, m / z) = 1136.0 (M-2H + ).

[0172] Example 6. 2-Amino-9-((2R,3R,4R,5R)-5-((((((((2R,3R,4R,5R)-3-((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino- Preparation of (9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.3-triethylamine salt (oligonucleotide 6)

[0173] The compound (73 mg, 0.08 mmol) obtained in the above Preparation Example 2 and the compound (78 mg, 0.104 mmol) obtained in the above Preparation Example 5 were reacted in the same manner as in Example 1 to obtain the title compound (21.4 mg) as a white solid.

[0174] LC-MS (ESI, m / z) = 1148.0 (M-2H + ).

[0175] Example 7. 2-Amino-9-((2R,3R,4R,5R)-5-((((((((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9 Preparation of (H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.3-triethylamine salt (oligonucleotide 7)

[0176] ((2R,3R,4R,5R)-3-((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methyl dihydrogen phosphate.2-triethylamine salt (100 mg, 0.112 mmol) and the compound obtained in Preparation Example 5 (94 mg, 0.145 mmol) were reacted in the same manner as in Example 1 to obtain the title compound (50.5 mg) as a white solid.

[0177] LC-MS (ESI, m / z) = 1133.8 (M-2H + ).

[0178] Example 8. 2-Amino-9-((2R,3S,4S,5R)-5-((((((((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-fluoro-5-(6- Preparation of (methylamino)-9H-purin-9-yl)tetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.3-triethylamine salt (oligonucleotide 8)

[0179] ((2R,3R,4R,5R)-3-((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-fluoro-5-(6-(methylamino)-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl dihydrogen phosphate.2-triethylamine salt (100 mg, 0.109 mmol) and the compound obtained in Preparation Example 6 (108.5 mg, 0.142 mmol) were reacted in the same manner as in Example 1 to obtain the title compound (32.1 mg) as a white solid.

[0180] LC-MS (ESI, m / z) = 1147.9 (M-2H + ).

[0181] Example 9. 2-Amino-9-((2R,3S,4S,5R)-5-((((((((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9 Preparation of (H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.3-triethylamine salt (oligonucleotide 9)

[0182] ((2R,3R,4R,5R)-3-((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purin-9-yl)-4-fluorotetrahydrofuran-2-yl)methyl dihydrogen phosphate.2-triethylamine salt (100 mg, 0.112 mmol) and the compound obtained in Preparation Example 6 (110 mg, 0.145 mmol) were reacted in the same manner as in Example 1 to obtain the title compound (57.1 mg) as a white solid.

[0183] LC-MS (ESI, m / z) = 1134.1 (M-2H + ).

[0184] Example 10. 2-Amino-9-((2R,3R,4R,5R)-5-((((((((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-fluoro-5-(6 Preparation of -(methylamino)-9H-purin-9-yl)tetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purin-7-ium.3 sodium salt (oligonucleotide 10)

[0185] ((2R,3R,4R,5R)-3-((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-fluoro-5-(6-(methylamino)-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl dihydrogen phosphate.2-triethylamine salt (1.157 g, 1.27 mmol) and the compound obtained in Preparation Example 5 (0.947 g, 1.778 mmol) were reacted in the same manner as in Example 1, and purified with AKTA AVANT to obtain the title compound (300 mg) as a white solid.

[0186] LC-MS (ESI, m / z) = 1149.2 (M-1H + ).

[0187] Experimental Example 1: In vitro transcription of eGFP mRNA

[0188] 1-1: In vitro transcription of non-capped eGFP mRNA

[0189] For mRNA synthesis reaction without a cap structure at the 5' end, 40 mM Tris-HCl (pH 7.9), 16.5 mM magnesium acetate, 2 mM spermidine, 5 mM ATP, 5 mM 1mP UTP, 5 mM CTP, 5 mM GTP, 5% DMSO (Sigma-Aldrich catalog # 472301-500ML), 5 mg / mL eGFP DNA transcription template, 800 U / mL recombinant RNase inhibitor protein (Takara catalog # 2316A), 2 U / mL yeast inorganic pyrophosphatase (Thermo Scientific catalog # EF0221), and 40 KU / mL T7 RNA polymerase (DyneBio catalog # dy1670) were mixed.

[0190] The transcription reaction mixture was incubated at 38°C for 4 hours. 400 U / mL recombinant DNase I (Takara catalog # 2270A) was added to the reaction mixture and incubated at 37°C for 1 hour. The synthesized RNA was purified using RNA Clean & Concentrator TM -25 (Zymo Research, catalog #R1017) was used for purification and concentration was measured using a Thermo Scientific Nanodrop one instrument.

[0191] 1-2: In vitro transcription of eGFP mRNA through co-transcriptional capping of the example

[0192] For mRNA synthesis reaction using the example compound of the present invention, 40 mM Tris-HCl (pH 7.9), 16.5 mM magnesium acetate, 2 mM spermidine, 5 mM ATP, 5 mM 1mP UTP, 5 mM CTP, 5 mM GTP, 4 mM example compound, 5% DMSO (Sigma Aldrich catalog # 472301-500ML), 5 mg / mL eGFP DNA transcription template, 800 U / mL recombinant RNase inhibitor protein (Takara catalog # 2316A), 2 U / mL yeast inorganic pyrophosphatase (Thermo Scientific catalog # EF0221), 40 KU / mL T7 RNA polymerase (DyneBio catalog # dy1670) were mixed.

[0193] The transcription reaction mixture was incubated at 37°C for 5 hours. 400 U / mL recombinant DNase I (Takara catalog # 2270A) was added to the reaction mixture and incubated at 37°C for 1 hour. The synthesized RNA was purified using RNA Clean & Concentrator TM -25 (Zymo Research, catalog #R1017) was used for purification and concentrations were measured using a Thermo Scientific Nanodrop one instrument (Table 1).

[0194]

[0195] Experimental Example 2: Western Blot Analysis

[0196] The translational activity of each eGFP protein mRNA produced by in vitro transcription was evaluated in human hepatoma cells (Huh-7; Human Hepatocellular Carcinoma Cells, Korea Cell Line Bank, catalog #60104). Huh-7 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin at 37°C in an atmosphere of 5% CO2. 1x10 6Huh-7 cells were plated in 6-well plates at 10 μL / well. The following day, cells were transfected using a transfection reagent (lipofectamine 3000; Invitrogen, catalog #L3000015). According to the manufacturer's instructions, 6 μL of the transfection reagent was diluted in 125 μL of complex medium (Opti-MEM; Life technologies) in tube A and incubated for 10 minutes at room temperature. In tube B, 3 μg of the prepared mRNA was diluted in 125 μL of Opti-MEM. The solutions in tubes A and B were mixed and incubated for 5 minutes at room temperature. During the incubation, the cell culture medium was replaced with Opti-MEM supplemented with 10% FBS and without penicillin / streptomycin. The incubated mixture was then used for transfection. Expression of eGFP protein was confirmed by Western blot 24 hours after transfection.

[0197] After the transfection experiment, the medium was removed, and the cells were lysed by treating with 200 μL of cell lysis buffer (50 mM Tris-HCl, pH 8.0, 2 mM EDTA, 1% NP-40, 1x complete protease inhibitor cocktail solution). The cell lysate was collected and centrifuged at 12,000 rpm, 4°C, for 10 minutes. The supernatant was collected, mixed with 5x SDS-sample loading buffer, and boiled at 100°C for 5–10 minutes. The samples were run on a 10% SDS PAGE-gel at 120 V for 10 minutes and 170 V for 1 hour. The gel was transferred to a PVDF (polyvinylidene difluoride) membrane and blocked with blocking buffer (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 0.1% Tween-20, 5% w / v non-fat dried milk) for 1 h at room temperature. The membrane was then incubated with primary antibody overnight at 4°C. Subsequently, the membrane was washed three times for 10 min with TBST (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 0.1% Tween-20). HRP-conjugated secondary antibody (goat anti-Mouse IgG; Invitrogen, catalog #G-21040) was diluted in blocking buffer and treated with the membrane for 1 h at room temperature. The membrane was then washed three times for 10 min with TBST. The results were confirmed using Western Lightning Plus (PerkinElmer Life Science) (Table 2).

[0198]

[0199] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and experimental examples and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An oligonucleotide primer compound for RNA capping represented by the following chemical formula 1, a stereoisomer thereof, or a salt thereof: [Chemical Formula 1] In the above chemical formula, B1 and B2 are each independently a natural, unnatural, or modified nucleoside base; X is -OH, -O(C 1-4 alkyl), or -halo; Y is -halo; Z1 and Z2 are each independently -OH, -O(C 1-4 alkyl), or -halo; R1 and R2 are each independently -H or -(C 1-4 alkyl); R3 is -(C 1-4 alkyl).

2. In paragraph 1, B1 and B2 are each independently a natural, unnatural, or modified nucleoside base; X is -OH, -O(C 1-4 alkyl), or -halo; Y is -halo; Z1 is -OH, -O(C 1-4 alkyl), or -halo; Z2 is -OH or -halo; R1 and R2 are each independently -H or -(C 1-4 alkyl); R3 is -(C 1-4 alkyl); An oligonucleotide primer compound for RNA capping, a stereoisomer thereof, or a salt thereof.

3. In paragraph 1, B1 and B2 are each independently a natural, unnatural, or modified nucleoside base; X is -OH, -O(C 1-4 alkyl), or -halo; Y is -halo; Z1 is -OH or -O(C 1-4 alkyl); Z2 is -OH or -halo; R1 and R2 are each independently -H or -(C 1-4 alkyl); R3 is -(C 1-4 alkyl); An oligonucleotide primer compound for RNA capping, a stereoisomer thereof, or a salt thereof.

4. In paragraph 1, B1 and B2 are each independently a natural, unnatural, or modified nucleoside base; X is -OH or -halo; Y is -halo; Z1 is -OH, -O(C 1-4 alkyl), or -halo; Z2 is -OH or -halo; R1 and R2 are each independently -H or -(C 1-4 alkyl); R3 is -(C 1-4 alkyl); An oligonucleotide primer compound for RNA capping, a stereoisomer thereof, or a salt thereof.

5. In paragraph 1, B1 and B2 are each independently a natural, unnatural, or modified nucleoside base; X is -OH or -halo; Y is -halo; Z1 is -OH or -O(C 1-4 alkyl); Z2 is -OH or -halo; R1 and R2 are each independently -H or -(C 1-4 alkyl); R3 is -(C 1-4 alkyl); An oligonucleotide primer compound for RNA capping, a stereoisomer thereof, or a salt thereof.

6. In any one of paragraphs 1 to 5, The above unnatural or modified nucleoside base is an isomer of the above natural nucleoside base or at least one -H of the above natural nucleoside base is -(C 1-4 alkyl), -O(C 1-4 alkyl) or -halo, at least one =CH- is substituted with =N-, and at least one =O is substituted with =S; An oligonucleotide primer compound for RNA capping, a stereoisomer thereof, or a salt thereof.

7. In any one of paragraphs 1 to 5, B1 and B2 are each independently , , , , , or person; An oligonucleotide primer compound for RNA capping, a stereoisomer thereof, or a salt thereof.

8. In paragraph 7, B1 is and; B2 is , or person; An oligonucleotide primer compound for RNA capping, a stereoisomer thereof, or a salt thereof.

9. In paragraph 1, A compound selected from the group consisting of the following compounds, a stereoisomer thereof, or a salt thereof: (1) ; (2) ; (3) ; (4) ; (5) ; (6) ; (7) ; (8) ; (9) ; and (10) .

10. An RNA molecule comprising an oligonucleotide primer for RNA capping according to any one of claims 1 to 9.

11. In paragraph 10, An oligonucleotide primer for capping RNA is an RNA molecule attached to the 5' top of the RNA molecule.

12. In paragraph 10, An RNA molecule is an mRNA molecule that contains one or more coding sequences (CDS). 13.(S-1) A step of mixing a DNA template, an oligonucleotide primer for RNA capping according to any one of claims 1 to 9, and an RNA polymerase; and (S-2) A step of incubating the mixture to perform transcription of the polynucleotide template; A method for synthesizing an RNA molecule according to claim 10, comprising:

14. In paragraph 13, A synthetic method in which the synthesis of RNA molecules is performed in vitro.

15. An in vitro cell into which an RNA molecule according to Article 10 has been introduced.

16. A nucleic acid therapeutic agent comprising an RNA molecule according to Article 10.

17. In paragraph 16, A nucleic acid therapeutic agent comprising a carrier capable of introducing RNA molecules into target cells.

18. A vaccine comprising an RNA molecule according to Article 10.

19. In paragraph 18, A vaccine comprising a vector capable of introducing RNA molecules into target cells.

20. In paragraph 18, Vaccines to prevent cancer or infectious diseases.

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