Oligonucleotides for RNA synthesis for 5'-capping

Abstract

This invention relates to a novel oligonucleotide primer for synthesizing 5'-capped RNA. The novel oligonucleotide primer for RNA capping provided in this invention, as Chemical Formula 1, can be effectively utilized in the fields of nucleic acid therapeutics or vaccines.

Description

Technical Field

[0001] This invention relates to a novel oligonucleotide for the synthesis of 5′-capped RNA. Background Technology

[0002] 5′ capping of RNA is the first step in the processing of pre-mRNA in eukaryotic cells, involving the formation of a cap at the 5′ end of the transcribed RNA. In eukaryotic mRNA molecules, a cap is formed at the 5′ end via a 5′ triphosphate bond to 7-methylguanosine (m... 7 The 5′ cap (cap-0) attached to the nucleus is mediated by methyltransferases in vivo. Eukaryotic mRNAs may have additional variations, including methylation of the 2′ hydroxyl group of the first ribose at the 5′ end (cap-1) and methylation of the 2′ hydroxyl group of the second ribose (cap-2). This 5′ capping process of mRNA provides resistance to degrading enzymes such as 5′ exonucleases as it leaves the nucleus and reaches the cytoplasm.

[0003] For medical or research purposes, genes can be artificially expressed by transfecting capped mRNA encoding a specific gene into eukaryotes or by microinjecting mRNA into cells or embryos. However, when uncapped RNA is used in this process, the RNA degrades rapidly and exhibits immunogenicity and significantly reduced protein translation efficiency. Therefore, it is crucial to correctly perform 5′ capping for in vitro mRNA synthesis.

[0004] For mRNA synthesis, the process of first transcribing RNA with a 5′-triphosphate group in vitro, followed by 5′ capping using a capping enzyme, has been found to be both expensive and inefficient. Therefore, a synthetic method has been developed that involves preparing an oligonucleotide with a 5′-capped structure and using the oligonucleotide as a primer to initiate in vitro transcription. For example, international publications WO2008 / 016473 and WO2013 / 059475 disclose dinucleotide mRNA cap analogs for the synthesis of 5′-capped RNA, and international publication WO2017 / 053297 discloses trinucleotide mRNA cap analogs for the synthesis of 5′-capped RNA.

[0005] Because capped RNA transcripts can be applied in therapeutic and / or preventative fields requiring protein synthesis, such as nucleic acid therapeutics and vaccines, it is necessary to develop oligonucleotide primers for 5′-capping to efficiently generate RNA transcripts. There is an urgent need to develop RNA cap analogs that, for example, can be manufactured more economically and efficiently through a simpler manufacturing process than conventional methods, efficiently perform the required transcription reaction, improve RNA production yield, reduce the generation of unwanted heterogeneous products, do not require additional enzymatic reactions, or enable large-scale RNA synthesis. Furthermore, if a cap structure is provided that can maintain mRNA stability in vivo, improve translation efficiency, or reduce side effects (such as immunogenicity), it could potentially lead to widespread application in the field of RNA therapeutics or vaccines when used as practical RNA therapeutics or vaccines. Summary of the Invention

[0006] Technical issues

[0007] One object of the present invention is to provide a novel oligonucleotide primer for RNA capping, its preparation method and its use.

[0008] Another object of the present invention is to provide an RNA molecule prepared using an oligonucleotide primer for RNA capping and its uses.

[0009] Technical solution

[0010] The inventors have provided a novel oligonucleotide primer for RNA capping, its preparation method, and its usage method. Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All patent and non-patent literature described herein in its entirety is incorporated herein by reference.

[0011] Novel oligonucleotide primers for RNA capping

[0012] This invention provides a compound represented by the following chemical formula 1, or a stereoisomer thereof, or a salt thereof:

[0013] [Chemical Formula 1]

[0014]

[0015] In the above chemical formula 1,

[0016] B1 and B2 are each independently natural, non-natural, or modified nucleoside bases;

[0017] X1 is -OH, -O(C) 1-4 Alkyl groups or halogens;

[0018] X2 is -H or connected to X1 to form an LNA ring {where at least one H of the LNA ring can be -(C 1-4 Alkyl), -OH or -O (C 1-4 Alkyl) substitution};

[0019] Y1 is -O(C 1-4 Alkyl), -O(C) 1-4 Alkyl)O(C 1-4 alkyl), -CH2O(C 1-4 Alkyl groups or halogens;

[0020] Y2 is -H or connected to Y1 to form an LNA ring {where at least one H of the LNA ring can be -(C 1-4 Alkyl), -OH or -O (C 1-4 Alkyl) substitution};

[0021] Z1 and Z2 are independently -OH and -O(C) 1-4 Alkyl), -O(C) 1-4 Alkyl)O(C 1-4 alkyl), -CH2O(C 1-4 Alkyl groups or halogens;

[0022] Z3 is -H or connected to Z1 to form an LNA ring {where at least one H of the LNA ring can be -(C 1-4 Alkyl), -OH or -O (C 1-4 Alkyl) substitution};

[0023] n is an integer from 0 to 3;

[0024] m is an integer from 1 to 10 {where when m is 2 or greater, each of B2, Z1 and Z2 can be different from each other};

[0025] R1 and R2 are each independently -H or -(C 1-4 Alkyl); and

[0026] R3 is -(C 1-4 alkyl).

[0027] In this invention, natural nucleoside bases and non-natural or modified nucleoside bases can be used as the aforementioned nucleoside bases.

[0028] In this invention, the base rings most commonly found in naturally occurring nucleosides are purine and pyrimidine rings. Naturally occurring purine rings include, for example, adenine. Guanine and N6-methyladenine Naturally occurring pyrimidine rings include, for example, cytosine. Thymine 5-Methylcytosine and uracil

[0029] According to one embodiment of the invention, the non-natural or modified nucleoside base may be an isomer of the natural nucleoside base, or may be substituted by at least one of the following: at least one -H of the aforementioned natural nucleoside base is replaced by -(C 1-4 Alkyl), -O(C) 1-4 Alkyl) or halogen substituted, at least one =CH- is substituted with =N-, and at least one =O is substituted with =S.

[0030] Furthermore, in this invention, the non-natural or modified nucleoside bases can be bases derived from nucleoside analogs having artificial bases that can be recognized by RNA polymerase, serving as substitutes for one of the natural NTPs (e.g., ATP, UTP, CTP, and GTP) or other specific NTPs (see 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.).

[0031] Furthermore, in this invention, the non-natural or modified nucleoside bases can be derived from halogen-substituted purines (e.g., 6-fluoropurine), halogen-substituted pyrimidines, N... 6 -Ethyl adenine, N 4 The bases of -(alkyl)-cytosine, 5-ethylcytosine, etc., but not limited to these.

[0032] Additionally, in this invention, another example of the aforementioned non-natural or modified nucleoside bases may include 8-azanine, pseudouridine (ψ), or 5-methylcytidine (m). 5 C), 1-methyl-pseuuridine (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 5C), 5-methoxy-uridine (mo) 5 U), 5-methoxyuridine (mo) 5 U) and 5-methyl-cytidine (m 5 C), 2′-O-methyluridine, 2′-O-methyluridine 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), but non-natural or modified nucleoside bases are not limited to this.

[0033] In addition, examples of natural, non-natural or modified nucleoside bases that can be used in this invention can be found in the references (see International Publication WO2018 / 144775, International Publication WO2018 / 144082, etc.).

[0034] According to one embodiment of the present invention, B1 and B2 can each be independently...

[0035]

[0036] Furthermore, according to one embodiment of the present invention,

[0037] X1 can be -OH; and

[0038] X2 can be -H.

[0039] Furthermore, according to one embodiment of the present invention,

[0040] Y1 can be -O(C) 1-4 alkyl) or -O(C 1-4 Alkyl)O(C 1-4 Alkyl) or -halogen; and

[0041] Y2 can be -H or connected to Y1 to form an LNA ring {where at least one H of the LNA ring can be -(C 1-4 Alkyl) substitution}.

[0042] Furthermore, according to one embodiment of the present invention,

[0043] Z1 can be -OH or -O(C) 1-4 alkyl);

[0044] Z2 can be -OH; and

[0045] Z3 can be -H.

[0046] Furthermore, according to one embodiment of the present invention, n can be 0 or 1.

[0047] Furthermore, according to one embodiment of the present invention, m can be 1.

[0048] Furthermore, according to one embodiment of the present invention,

[0049] R1 and R2 can each be -H independently; and

[0050] R3 can be -(C 1-4 alkyl).

[0051] Specifically, the compound represented by chemical formula 1 can be any one of the following groups of compounds:

[0052] (1)

[0053] (2)

[0054] (3)

[0055] (4)

[0056] (5)

[0057] (6)

[0058] (7)

[0059] (8)

[0060] (9)

[0061] (10)

[0062] (11) and

[0063] (12)

[0064] According to one embodiment of the present invention, the compound represented by chemical formula 1 may be at least one selected from the group consisting of:

[0065] 2-Amino-9-((2R,3R,4S,5R)-5-(((((((((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purine-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purine-7-onium (oligonucleotide 1);

[0066] 2-Amino-9-((2R,3R,4S,5R)-5-(((((((((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purine-9-yl)-4-(2-methoxyethoxy)tetrahydrofuran-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-purine-7-onium (oligonucleotide 2);

[0067] 2-Amino-9-((2R,3R,4S,5R)-5-(((((((((1S,3R,4R,6S,7S)-7-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-3- (6-amino-9H-purin-9-yl)-6-methyl-2,5-dioxabicyclo[2.2.1]hept-1-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-onium (oligonucleotide 3);

[0068] 2-Amino-9-((2R,3R,4S,5R)-5-(((((((((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(5-amino-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)( hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purine-9-yl)-4-methoxytetrahydrofuran-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-purine-7-onium (oligonucleotide 4);

[0069] 2-Amino-9-((2R,3R,4S,5R)-5-(((((((((1R,3R,4R,7S)-7-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-3-(6-amino-9H-purine-9-yl)-2,5-dioxabicyclo[2.2.1]hept-1-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-purine-7-onium (oligonucleotide 5);

[0070] 2-Amino-9-((2R,3R,4S,5R)-5-(((((((((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purine-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-purine-7-onium (oligonucleotide 6);

[0071] 2-Amino-9-((2R,3R,4S,5R)-5-(((((((((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(5-amino-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)( hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purine-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purine-7-onium (oligonucleotide 7);

[0072] 2-Amino-9-((2R,3R,4S,5R)-5-(((((((((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(5-amino-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-3,4-dihydroxytetrahydrofuran-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-onium (oligonucleotide 8);

[0073] 2-Amino-9-((2R,3R,4S,5R)-5-(((((((((2R,3R,4R,5R)-5-(6-amino-9H-purine-9-yl)-3-(((((2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purine-7-onium (oligonucleotide 9);

[0074] 2-Amino-9-((2R,3R,4S,5R)-5-(((((((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purine-9-yl)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purine-9-yl)-3,4-di Hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-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-purine-7-onium (oligonucleotide 10);

[0075] 2-Amino-9-((2R,3R,4S,5R)-5-(((((((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purine-9-yl)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purine-9-yl)-3,4- Dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purine-7-onium (oligonucleotide 11); and

[0076] 2-Amino-9-((2R,3R,4S,5R)-5-(((((((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purine-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purine-7-onium (oligonucleotide 12).

[0077] According to one embodiment of the present invention, the compound represented by chemical formula 1 may be an oligonucleotide primer for RNA capping.

[0078] In this invention, the term "stereoisomer" means a compound or salt thereof of the present invention having the same chemical formula or molecular formula but different spatial arrangements. Each of these stereoisomers and mixtures thereof are also included within the scope of the present invention. Additionally, unless otherwise stated, solid line bonds (-) connecting asymmetric carbon atoms may include wedge bonds representing the absolute arrangement of the stereocenter. or wedge-shaped dotted line key

[0079] In this invention, the term "salt" refers to salts commonly used in the pharmaceutical industry. Specifically, a salt can be an alkali addition salt. Examples of salts may include inorganic ionic salts prepared using, for example, calcium, potassium, sodium, or magnesium; amino acid salts prepared using arginine, lysine, histidine, etc.; and amine salts prepared using trimethylamine, triethylamine, ammonia, pyridine, methylpyridine, etc.; however, the types of salts referred to in this invention are not limited to these listed salts.

[0080] RNA capped using novel oligonucleotide primers

[0081] The oligonucleotide primers for RNA capping described above can be used to generate RNA molecules containing oligonucleotide primers. Therefore, the present invention provides an RNA molecule containing oligonucleotide primers for RNA capping represented by Chemical Formula 1.

[0082] The oligonucleotide primers for RNA capping according to the invention can be extended by RNA polymerase via incorporating NTPs into the 3′ end. In vitro transcription can be initiated in a transcription system under the control of a promoter containing essential components such as a DNA template (e.g., a DNA plasmid), RNA polymerase, 5′-nucleotide triphosphate, and a suitable buffer. Here, the oligonucleotide primers are complementary to the DNA template at the start site.

[0083] In this invention, the term promoter refers to a specific region on the DNA template that induces and controls the initiation of transcription of a particular DNA sequence (e.g., a gene). The promoter is located on the same strand of the DNA and upstream (towards the 5′ region of the sense strand). The promoter is typically adjacent to or partially overlaps with the DNA sequence to be transcribed. The positions of the nucleotides in the promoter are designed based on the transcription start site (position +1) where transcription of the DNA begins. The promoter oligonucleotide primer is complementary to the start site of the promoter sequence (wherein some embodiments it is at positions +1 and +2, and in the case of a promoter tetramer, at positions +1, +2, and +3).

[0084] In one embodiment, an oligonucleotide primer for RNA capping is attached to the 5′ upstream end of an RNA molecule to form a 5′-capped RNA. The resulting RNA molecule includes, but is not limited to, mRNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and small Cajalbody-specific RNA (scaRNA).

[0085] In one embodiment, the RNA molecule may be mRNA and may contain at least one coding sequence (CDS). Furthermore, the RNA molecule may contain a polyA sequence and / or a polyadenylation signal. The polyA sequence may be composed entirely or mostly of adenine nucleotides or their analogues or derivatives. The polyA sequence may be a tail adjacent to the 3′ untranslated region of the nucleic acid.

[0086] The present invention provides a method for synthesizing 5′-capped RNA molecules, comprising: (S-1) mixing a DNA template, an oligonucleotide primer for RNA capping represented by chemical formula 1 as described above, and an RNA polymerase; and (S-2) performing transcription of the polynucleotide template by incubating the mixture.

[0087] Meanwhile, methods for the promoter-controlled synthesis of RNA using oligonucleotide primers, RNA polymerase, and 5′-nucleoside triphosphate (NTP) DNA templates are known in the art to which this invention pertains. Therefore, RNA molecules containing the oligonucleotide primers represented by Chemical Formula 1 provided in this invention can be used for RNA capping, thereby enabling the synthesis of RNA molecules.

[0088] According to one embodiment of the present invention, the DNA template may be double-stranded linear DNA, partially double-stranded linear DNA, circular double-stranded DNA, DNA plasmid, PCR amplicon, or, in addition, a modified nucleic acid capable of reacting appropriately with RNA polymerase.

[0089] According to one embodiment of the invention, RNA molecule synthesis is performed in vitro. For large-scale transcription in vitro, single-subunit phage polymerases derived from T7, T3, SP6, K1-5, K1E, K1F, or K11 phages can be used. This family of polymerases does not require accessory proteins and has a simple, minimal promoter sequence of about 17 nucleotides, with minimal restrictions on the initiating nucleotide sequence. An example of an RNA polymerase suitable for use in the invention is T7 RNA polymerase (T7 RNAP), but those skilled in the art will understand that the invention can also be implemented using other RNA polymerases. Unlike DNA polymerases, T7 RNAP initiates RNA synthesis in the absence of a primer. The first step of initiation is called de novo RNA synthesis, in which the RNA polymerase recognizes a specific sequence on the DNA template, selects the first pair of triphosphate nucleotides complementary to the template residues at positions +1 and +2, and catalyzes the formation of a phosphodiester bond to form a dinucleotide.

[0090] Medical uses of capped RNA

[0091] The 5′-capped RNA molecule according to the present invention, as described above, can be effectively used for medical purposes.

[0092] According to one embodiment of the invention, the 5′-capped RNA molecule produced according to the invention can be used as a nucleic acid therapeutic agent or vaccine. For example, the nucleic acid therapeutic agent or vaccine can be used as an RNA vaccine (a vaccine for the prevention of cancer or infectious diseases). The above-mentioned nucleic acid therapeutic agent or vaccine can be administered to a subject and translated in vivo to produce the desired peptide.

[0093] According to one embodiment of the present invention, the above-mentioned 5′-capped RNA molecule can be introduced into cells to produce proteins that can treat medical conditions of cells or have therapeutic effects on cells.

[0094] According to one embodiment of the present invention, the above-mentioned nucleic acid therapeutic agent or vaccine may further include a vector capable of introducing RNA molecules together with RNA molecules into target cells.

[0095] According to one embodiment of the present invention, the present invention provides a peptide translated from a 5′-capped RNA molecule as described above.

[0096] According to one embodiment of the present invention, a cell in which a 5′-capped RNA molecule as described above is introduced is provided. The cell may be a somatic cell of the subject or a cell line capable of being cultured in vitro.

[0097] According to one embodiment of the present invention, the present invention provides a peptide produced by a cell in which a 5′-capped RNA molecule is introduced.

[0098] In this invention, pharmaceutical compositions for use as RNA therapeutics or vaccines can be formulated to treat specific conditions by injection or by other suitable routes known to those skilled in the art. Injectable compositions for parenteral administration typically contain an active compound in a suitable solution and / or pharmaceutical carrier such as sterile saline. The above compositions can also be formulated as suspensions in lipids or phospholipids, liposome suspensions, or aqueous emulsions.

[0099] Methods for preparing compositions and / or formulations for RNA therapeutics or vaccines are known to those skilled in the art [see Remington's Pharmaceutical Sciences (19th edition, Williams & Wilkins, 1995)]. The composition to be administered will contain a specific amount of the selected compound, in a pharmaceutically safe and effective quantity, for increasing the expression of the desired protein in target cells or tissues.

[0100] 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% of the compound; in some embodiments, the pharmaceutical composition contains up to about 10% of the compound; in some embodiments, the pharmaceutical composition contains up to about 5% of the compound; and in some embodiments, the pharmaceutical composition contains up to about 1% (w / v) of the compound. The selection of a suitable concentration depends on factors such as the required dose, frequency, and delivery method of the active agent.

[0101] For treatment of subjects such as mammals or humans, the dosage is determined based on factors such as the subject's weight and overall health, the condition being treated, and the severity of symptoms. The dosage and concentration are determined to produce the desired benefit while avoiding any undesirable side effects. For human patients, typical doses of the subject compound range from about 0.0005 to 500 mg / day, and in some embodiments from about 1 to 100 mg / day. For example, higher dosage regimens may include, for example, 50 to 100, 75 to 100, or 50 to 75 mg / day, while lower dosage regimens may include, for example, 1 to 50, 25 to 50, or 1 to 25 mg / day.

[0102] Beneficial effects

[0103] The novel oligonucleotides according to the present invention can be used to synthesize 5′-capped RNA, thereby improving not only RNA production processes (e.g., synthesis yield, synthesis scale, purity, etc.), but also the efficacy of nucleic acid therapeutics or vaccines utilizing oligonucleotides (e.g., RNA stability and / or protein expression efficiency) and reducing side effects (e.g., immunogenicity). Therefore, the present invention can be effectively used in the field of nucleic acid therapeutics or vaccines. Attached Figure Description

[0104] Figure 1 The electrophoresis results of the in vitro transcribed mRNA are shown.

[0105] Figure 2 The results of GFP mRNA expression were shown using a fluorescence microscope. Detailed Implementation

[0106] The structure and effects of the present invention will be described in more detail below through embodiments and experimental examples. These embodiments and experimental examples are for illustrative purposes only, but the scope of the present invention is not limited by these embodiments and experimental examples.

[0107] Preparation Example 1: Preparation of dihydrogen phosphate ((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxygen) (-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 ester, 2-triethylamine salt

[0108] Step 1. Preparation of phosphate (2R,3R,4R,5R)-5-(6-benzoylamino-9H-purine-9-yl)-4-fluoro-2-(hydroxy) (3aR,4R,6R,6aR)-6-(2-isobutyrylamino-6-oxo-1,6-)tetrahydrofuran-3-yl(2-cyanoethyl)(((3aR,4R,6R,6aR)-6-(2-isobutyrylamino-6-oxo-1,6-) Dihydro-9H-purin-9-yl)-2,2-dimethyltetrahydrofurano[3,4-d][1,3]m-dioxacyclopenten-4-yl)methyl) ester

[0109] N-(9-((3aR,4R,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofurano[3,4-d][1,3]m-dioxacyclopenten-4-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (4 g, 10.17 mmol) was dissolved in acetonitrile. After cooling to 0 to 5 °C, (2R,3R,4R,5R)-5-(6-benzoylamino-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphonamide (8.91 g, 10.17 mmol) and 1H-tetrazole (2.85 g, 40.7 mmol) were added sequentially. After 10 minutes, the resulting mixture was slowly warmed to room temperature and stirred for 1 hour. The reaction solution was cooled to 0-5°C, and then I₂ (0.1 M) (7.74 g, 30.5 mmol) was added to a THF / distilled water / pyridine (66:33:1) solution (101.6 mL). The reaction solution was slowly warmed to room temperature and stirred for 1 hour. After the reaction was complete, the reaction solution was diluted with DCM, 10% sodium thiosulfate pentahydrate solution was added, and the organic and aqueous layers were separated. The organic layer was dehydrated with Na₂SO₄ and concentrated under reduced pressure.

[0110] The concentrated residue was dissolved in DCM (200 mL). After cooling to 0–5 °C, TFA (10.17 mL) diluted in DCM (817 mL) was slowly added, and the mixture was stirred for 10 minutes. The temperature of the reaction solution was slowly increased to room temperature, and the mixture was stirred for 10 minutes. The reaction solution was cooled to 0–5 °C, and the pH was adjusted to 8–9 while a saturated NaHCO3(aq) solution was slowly added. The organic and aqueous layers were separated, and the organic layer was dried over Na2SO4 and filtered. After concentration under reduced pressure and purification by column chromatography, the desired product (5.62 g) was obtained as a yellow solid.

[0111] LC-MS(ESI, m / z) = 882.1(M+H) + ).

[0112] Step 2. Preparation of phosphate (2R,3R,4R,5R)-5-(6-benzoylamino-9H-purine-9-yl)-2-(((bis(2- (cyanoethoxy)phosphoryl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl(2-cyanoethyl)(((3aR,4R,6R,6aR)- 6-(2-Isobutyrylamino-6-oxo-1,6-dihydro-9H-purine-9-yl)-2,2-dimethyltetrahydrofurano[3,4-d][1, 3] m-dioxacyclopenten-4-yl)methyl) ester

[0113] The compound obtained in step 1 above, namely (2R,3R,4R,5R)-5-(6-benzoylamino-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl(2-cyanoethyl)(((3aR,4R,6R,6aR)-6-(2-isobutyrylamino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2,2-dimethyltetrahydrofurano[3,4-d][1,3]m-dioxacyclopenten-4-yl)methyl) ester (5.6 g, 6.35 mmol), was dissolved in DCM (21.2 mL). After cooling to 0 to 5 °C, bis(2-cyanoethyl)diisopropylphosphonamide (3.32 mL, 12.70 mmol) and 1H-tetrazole (0.890 g, 12.70 mmol) were added sequentially. After 10 minutes, the resulting mixture was slowly warmed to room temperature and stirred for 1 hour. The reaction solution was cooled to 0–5 °C, and then I₂ (0.1 M) (4.84 g, 19.05 mmol) was added in THF / distilled water / pyridine (66:33:1) (63.6 mL). The reaction solution was slowly warmed to room temperature and stirred for 1 hour. After the reaction was complete, the reaction solution was diluted with DCM, 10% sodium thiosulfate pentahydrate solution was added, and the organic and aqueous layers were separated. The organic layer was dehydrated with Na₂SO₄, concentrated under reduced pressure, and purified by column chromatography to give the desired product (5.78 g) as a yellow solid.

[0114] LC-MS(ESI, m / z) = 1086.1(M+H) + ).

[0115] Step 3. Preparation of dihydrogen phosphate ((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxygen) (-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 ester, 2-triethylamine salt

[0116] The compound obtained in step 2 above, namely (2R,3R,4R,5R)-5-(6-benzoylamino-9H-purin-9-yl)-2-(((bis(2-cyanoethoxy)phosphoryl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl(2-cyanoethyl)(((3aR,4R,6R,6aR)-6-(2-isobutyrylamino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2,2-dimethyltetrahydrofurano[3,4-d][1,3]m-dioxacyclopenten-4-yl)methyl) ester (2.89 g, 2.71 mmol), was dissolved in 1M HCl aqueous solution (162 mL, 162 mmol) and stirred at room temperature for 16 hours. After cooling the reaction solution to 0 to 5°C, ammonia water (42.2 mL, 1083 mmol) was slowly added. The reaction solution was slowly warmed to room temperature and then heated to 50 to 55°C for 23 hours. The reaction solution was washed twice with DCM (150 mL each time), and the aqueous layer was purified with DEAE Sephadex resin to give the desired product (1.98 g) as a white solid.

[0117] LC-MS(ESI, m / z) = 695.1(M+H) + ).

[0118] Preparation Example 2: Preparation of 2-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(((hydroxy((1H-imidazolium)) (azol-1-yl)phosphoryl)oxy)phosphoryl)oxy)methyl)tetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro- 1H-purine-7-onium sodium salt

[0119] Step 1. Preparation of 2-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(((hydroxy(phosphono)phosphine) Acyl)oxy)methyl)tetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purine-7-onium.triethylamine salt

[0120] 5 g (7.74 mmol) of trihydro((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl 2-triethylamine salt of diphosphate was dissolved in pure water (155 mL), and the pH of the solution was adjusted to 4.5 using acetic acid. After the slow addition of dimethyl sulfate (5.18 mL, 54.2 mmol) for 1 minute, the resulting solution was stirred at room temperature for 1 hour. Here, the pH of the solution was adjusted to 4 to 4.5 using 5N NaOH aqueous solution. After the reaction was complete, the mixture was washed with DCM (200 mL × 3 times), and the aqueous layer was adjusted to pH 5.5 to 6 using 1M TEAB. The reaction product was purified using DEAE Sephadex resin to give the desired product (3.03 g) as a white solid.

[0121] LC-MS (ESI, m / z) = 456.0 (MH) + ).

[0122] Step 2. Preparation of 2-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(((hydroxy((1H-imidazol- 1-yl)phosphoryl)oxy)phosphoryl)oxy)methyl)tetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purine Alon-7-onium sodium salt

[0123] The 2-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(((hydroxy(phosphono)phospho)oxy)methyl)tetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purine-7-onium triethylamine salt (3.03 g, 5.42 mmol) and imidazole (3.69 g, 54.2 mmol) obtained in step 1 above were dissolved in DMF (38.7 mL). After the addition of 2,2′-dithiodipyridine (5.97 g, 27.1 mmol), the mixture was stirred at room temperature for 10 hours. After the reaction was complete, the reaction solution was cooled to -10 to -20 °C, and 0.1 M sodium perchlorate was added dropwise to acetone (108 mL, 10.83 mmol). The obtained solid was filtered and dried to obtain the desired product (2.33 g), which was a white solid.

[0124] LC-MS (ESI, m / z) = 507.0 (MH) + ).

[0125] Preparation Example 3: Preparation of 2-amino-9-((2R,3R,4S,5R)-3-dihydroxy-5-(((hydroxy((1H-imidazol- 1-yl)phosphoryl)oxy)phosphoryl)oxy)methyl)-4-methoxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9- Sodium dihydro-1H-purine-7-onium

[0126] The desired product (159 mg) was obtained by reacting trihydro((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purine-9-yl)-4-hydroxy-3-methoxytetrahydrofuran-2-yl)methyl-2-triethylamine salt (190 mg, 0.331 mmol) in a manner similar to that used in Preparation Example 2, to a white solid (159 mg).

[0127] LC-MS(ESI, m / z) = 522.0(M+H) + ).

[0128] Example 1: Preparation of 2-amino-9-((2R,3R,4S,5R)-5-(((((((((2R,3R,4R,5R)-3- (((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran- 2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purine-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy (hydroxy)phosphoryl)oxy (hydroxy)phosphoryl)oxy (hydroxy)phosphoryl)oxy (hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydro Furan-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purine-7-onium,3-triethylamine salt (oligonucleotide 1)

[0129] The compounds obtained in Preparation Example 1 (20 mg, 0.029 mmol) and Preparation Example 2 (23.18 mg, 0.041 mmol) were dissolved in DMSO (0.29 mL), and MgCl2 (3.84 mg, 0.041 mmol) was added. The mixture was then stirred at room temperature for 7 hours. After the reaction was complete, the reaction mixture was diluted in 1.44 mL of 0.25 mM EDTA 2Na salt solution (aq). After confirming complete dissolution, the reaction mixture was diluted in 144 mL of pure water and purified using DEAE Sephadex resin to give the desired product (22.4 mg) as a white solid.

[0130] LC-MS (ESI, m / z) = 1132.6 (MH) + ).

[0131] Example 2: Preparation of 2-amino-9-((2R,3R,4S,5R)-5-(((((((((2R,3R,4R,5R)-3- (((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran- 2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purine-9-yl)-4-(2-methoxyethoxy)tetrahydro Furan-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)- 3,4-Dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purine-7-onium.3-triethylamine salt (oligonucleotide) Acid 2)

[0132] The dihydrogen phosphate ((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-(2-methoxyethoxy)tetrahydrofuran-2-yl)methyl ester, 2-triethylamine salt (92 mg, 0.123 mmol) obtained by synthesis in a manner similar to that of Preparation Example 2, and the compound obtained by Preparation Example 2 (91.3 mg, 0.172 mmol) were reacted in the same manner as in Example 1 to give the desired product (73 mg), which was a white solid.

[0133] LC-MS(ESI, m / z)=1187.7(M-3H + )

[0134] Example 3: Preparation of 2-amino-9-((2R,3R,4S,5R)-5-(((((((((1S,3R,4R,6S,7S)-7- (((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran- 2-yl)methoxy)(hydroxy)phosphoryl)oxy)-3-(6-amino-9H-purine-9-yl)-6-methyl-2,5-dioxabicyclo [2.2.1] Hept-1-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-purine-7-onium,3-triethylamine salt (Oligonucleotide 3)

[0135] The dihydrogen phosphate ((1S,3R,4R,6S,7S)-7-(((((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)-3-(6-amino-9H-purin-9-yl)-6-methyl-2,5-dioxabicyclo[2.2.1]hept-1-yl)methyl 2-triethylamine salt (30 mg, 0.034 mmol) obtained by synthesis in a manner similar to that of Preparation Example 2, and the compound obtained in Preparation Example 2 (35 mg, 0.069 mmol) were reacted in the same manner as in Example 1 to give the desired product (6 mg) as a white solid.

[0136] LC-MS (ESI, m / z) = 1156.1 (MH) + )

[0137] Example 4: Preparation of 2-amino-9-((2R,3R,4S,5R)-5-(((((((((2R,3R,4R,5R)-3- (((((2R,3S,4R,5R)-5-(5-amino-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidine-3- )-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino) - 9H-purine-9- 4-Methoxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy) Acyl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purine-7-onium .3 Triethylamine salt (oligonucleotide 4)

[0138] The dihydrogen phosphate ((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(5-amino-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purine-9-yl)-4-methoxytetrahydrofuran-2-yl)methyl ester.2-triethylamine salt (17 mg, 0.024 mmol) obtained by synthesis in a manner similar to that of Preparation Example 2, and the compound obtained by Preparation Example 2 (24.43 mg, 0.048 mmol) were reacted in the same manner as in Example 1 to give the desired product (3.8 mg) as a white solid.

[0139] LC-MS(ESI, m / z)=1145.8(M-2H + )

[0140] Example 5: Preparation of 2-amino-9-((2R,3R,4S,5R)-5-(((((((((1R,3R,4R,7S)-7- (((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran- 2-yl)methoxy)(hydroxy)phosphoryl)oxy)-3-(6-amino-9H-purine-9-yl)-2,5-dioxabicyclo[2.2.1] Heptyl-1-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3, 4-Dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purine-7-onium,3-triethylamine salt (oligonucleotide) 5)

[0141] The dihydrogen phosphate ((1R,3R,4R,7S)-7-(((((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)-3-(6-amino-9H-purin-9-yl)-2,5-dioxabicyclo[2.2.1]hept-1-yl)methyl ester.2-triethylamine salt (50 mg, 0.055 mmol) obtained by synthesis in a manner similar to that of Preparation Example 2, and the compound obtained by Preparation Example 2 (112.0 mg, 0.221 mmol) were reacted in the same manner as in Example 1 to give the desired product (8 mg) as a white solid.

[0142] LC-MS (ESI, m / z) = 1143.0 (MH) + )

[0143] Example 6: Preparation of 2-amino-9-((2R,3R,4S,5R)-5-(((((((((2R,3R,4R,5R)-3- (((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran- 2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purine-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy (hydroxy)phosphoryl)oxy (hydroxy)phosphoryl)oxy (hydroxy)phosphoryl)oxy (hydroxy)phosphoryl)oxy)methyl)-3-hydroxy-4-methoxy (2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purine-7-onium,3-triethylamine salt (oligonucleotide 6)

[0144] The compound obtained in Preparation Example 1 (30 mg, 0.03 mmol) and the compound obtained in Preparation Example 3 (32.7 mg, 0.06 mmol) were reacted in the same manner as in Example 1 to give the desired product (9 mg), which was a white solid.

[0145] LC-MS(ESI, m / z) = 1149.1(M+H) + )

[0146] Example 7: Preparation of 2-amino-9-((2R,3R,4S,5R)-5-(((((((((2R,3R,4R,5R)-3- (((((2R,3S,4R,5R)-5-(5-amino-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidine-3- )-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purine-9- 4-Fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl) 2-( ... Ononium-3-triethylamine salt (oligonucleotide 7)

[0147] The dihydrogen phosphate ((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(5-amino-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purine-9-yl)-4-fluorotetrahydrofuran-2-yl)methyl ester.2-triethylamine salt (20 mg, 0.029 mmol) obtained by synthesis in a manner similar to that of Preparation Example 3 (31.3 mg, 0.058 mmol) was reacted in the same manner as in Example 1 to give the desired product (16 mg) as a white solid.

[0148] LC-MS(ESI, m / z)=1147.1(M-2H + )

[0149] Example 8: Preparation of 2-amino-9-((2R,3R,4S,5R)-5-(((((((((2R,3R,4R,5R)-3- (((((2R,3S,4R,5R)-5-(5-amino-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidine-3- )-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purine-9- 4-Fluorotetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl) (3,4-Dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purine-7-onium. Triethylamine salt (oligonucleotide 8)

[0150] The dihydrogen phosphate ((2R,3R,4R,5R)-3-(((((2R,3S,4R,5R)-5-(5-amino-7-oxo-6,7-dihydro-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purine-9-yl)-4-fluorotetrahydrofuran-2-yl)methyl ester.2-triethylamine salt (20 mg, 0.029 mmol) obtained by synthesis in a manner similar to that of Preparation Example 2, and the compound obtained by Preparation Example 2 (30.5 mg, 0.058 mmol) were reacted in the same manner as in Example 1 to give the desired product (14.5 mg) as a white solid.

[0151] LC-MS(ESI, m / z)=1133.1(M-2H + )

[0152] Example 9: Preparation of 2-amino-9-((2R,3R,4S,5R)-5-(((((((((2R,3R,4R,5R)-5-(6-amino 2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-3, 4-Dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-fluorotetrahydrofuran-2-yl)methoxy)(hydroxy (3,4-dihydroxytetrahydrofuran) 2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purine-7-onium,3-triethylamine salt (oligonucleotide 9)

[0153] The dihydrogen phosphate ((2R,3R,4R,5R)-5-(6-amino-9H-purine-9-yl)-3-(((((2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-fluorotetrahydrofuran-2-yl)methyl ester.2-triethylamine salt (50 mg, 0.058 mmol) obtained by synthesis in a manner similar to that of Preparation Example 2, and the compound obtained in Preparation Example 2 (61.8 mg, 0.117 mmol) were reacted in the same manner as in Example 1 to give the desired product (34.6 mg) as a white solid.

[0154] LC-MS(ESI, m / z)=1092.8(M-2H + )

[0155] Example 10: Preparation of 2-amino-9-((2R,3R,4S,5R)-5-(((((((((2R,3R,4R,5R)-5-(2-amino 2-amino-6-oxo-1,6-dihydro-9H-purine-9-yl)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-di) (H-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-fluorotetrahydrofuran (2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3- Hydroxy-4-methoxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purine-7-onium,3-triethylamine salt (oligomeric) Nucleotide 10)

[0156] The dihydrogen phosphate ((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-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-fluorotetrahydrofuran-2-yl)methyl ester.2-triethylamine salt (20 mg, 0.02 mmol) obtained by synthesis in a manner similar to that of Preparation Example 3 (21.47 mg, 0.039 mmol) was reacted in the same manner as in Example 1 to give the desired product (15.2 mg) as a white solid.

[0157] LC-MS(ESI, m / z) = 1165.0(M+H) + )

[0158] Example 11: Preparation of 2-amino-9-((2R,3R,4S,5R)-5-(((((((((2R,3R,4R,5R)-5-(2-amino 2-amino-6-oxo-1,6-dihydro-9H-purine-9-yl)-3-(((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-di) (H-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-fluorotetrahydrofuran (2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3, 4-Dihydroxytetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purine-7-onium,3-triethylamine salt (oligonucleotide) 11)

[0159] The dihydrogen phosphate ((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-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-fluorotetrahydrofuran-2-yl)methyl ester.2-triethylamine salt (50 mg, 0.049 mmol) obtained by synthesis in a manner similar to that of Preparation Example 2, and the compound obtained by Preparation Example 2 (52.3 mg, 0.099 mmol) were reacted in the same manner as in Example 1 to give the desired product (26.3 mg) as a white solid.

[0160] LC-MS(ESI, m / z)=1147.9(M-2H + )

[0161] Example 12: Preparation of 2-amino-9-((2R,3R,4S,5R)-5-(((((((2R,3R,4R,5R)-3- (((((2R,3S,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purine-9-yl)-3,4-dihydroxytetrahydrofuran- 2-yl)methoxy)(hydroxy)phosphoryl)oxy)-5-(6-amino-9H-purine-9-yl)-4-fluorotetrahydrofuran-2-yl)methoxy (hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7-methyl- 6-O-6,9-dihydro-1H-purine-7-onium,3-triethylamine salt (oligonucleotide 12)

[0162] The compound obtained in Preparation Example 1 (40 mg, 0.047 mmol) and 2-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(((hydroxy(1H-imidazol-1-yl)phosphoryl)oxy)methyl)tetrahydrofuran-2-yl)-7-methyl-6-oxo-6,9-dihydro-1H-purine-7-onium sodium salt (42.6 mg, 0.095 mmol) obtained by a method similar to that in Preparation Example 2 were reacted in the same manner as in Example 1 to give the desired product (6.5 mg) as a white solid.

[0163] LC-MS(ESI, m / z)=1052.1(M-2H + )

[0164] Experimental Example 1: Preparation of GFP DNA template

[0165] To obtain a DNA template for mRNA synthesis, Bionics Co., Ltd. synthesized a plasmid gene with a T7 RNA promoter, 5′UTR, EGFP, 3′UTR, and poly A sequence. Then, to modify the promoter sequence according to the cap material, PCR (polymerase chain reaction) was performed as follows: the plasmid gene was mixed with primeSTAR HS premix (Takara Bio Inc., catalog #R040A), 2 μmol of forward primer (SEQ ID NO: 1), and 2 μmol of reverse primer (SEQ ID NO: 2), followed by cloning the existing plasmid gene using XbaI (Takara Bio Inc., catalog #1093A) and BamHI (Takara Bio Inc., catalog #1010A) restriction enzymes to construct a plasmid gene. The constructed plasmid gene was linearized using the SamI (Takara Bio Inc., catalog #1085A) restriction enzyme to obtain a GFP DNA transcription template (SEQ ID NO: 3) using the AccuPrep PCR purification kit (Bioneer Inc., catalog #K-3037).

[0166] Experimental Example 2: In vitro transcription of GFP mRNA

[0167] 2-1: Uncapped GFP In vitro transcription of mRNA

[0168] For the synthesis of mRNA without a cap structure at the 5′ end, a mixture of 40 mM Tris HCl (pH 7.9), 25 mM magnesium chloride, 2 mM spermidine, 2 mM ATP, 2 mM UTP, 2 mM CTP, 2 mM GTP, 5% DMSO (Sigma Aldrich catalog #472301-500 mL), 5 mg / mL GFP DNA transcription template, 800 U / mL recombinant RNase inhibitor protein (Takara BioInc., catalog #2316A), 2 U / mL yeast inorganic pyrophosphatase (Thermo Fisher Scientific Inc., catalog #EF0221), and 2500 U / mL T7 RNA polymerase (Dyne bio Inc., catalog #dy1670) was prepared.

[0169] The transcription reaction mixture was incubated at 37°C for 5 hours. 40 mM Tris·HCl (pH 7.5), 8 mM magnesium chloride, 5 mM DTT, and 5000 U / mL recombinant DNase I (Takara Bio Inc., catalog #2270A) were added to the reaction mixture, and the mixture was incubated at 37°C for 1 hour. The resulting mRNA was purified using the Zymo Research RNA clean & concentrator-25 kit (catalog #1017) according to the manufacturer's instructions.

[0170] mRNA synthesis is achieved by using Lonza GelStar. TM Electrophoretic confirmation of nucleic acid gel staining solution (catalog #5 0535) on 1% agarose gel ( Figure 1 ). Leverage Thermo Scientific TM Nanodrop TM One UV-Vis spectrometer was used to perform UV analysis of the synthesized mRNA. As a result, GFP mRNA containing approximately 1066 nt of poly A(120) was confirmed.

[0171] 2-2: In vitro transcription of GFP mRNA via co-transcriptional capping with ARCA cap analogues

[0172] For the synthesis of mRNA using an ARCA cap, a mixture of 40 mM Tris HCl (pH 7.9), 25 mM magnesium chloride, 2 mM spermidine, 2 mM ATP, 2 mM UTP, 2 mM CTP, 0.4 mM GTP, 1.6 mM ARCA (TriLink BioTechnologies, catalog #N-7003), 5% DMSO (Sigma Aldrich, catalog #472301-500 mL), 5 mg / mL GFP DNA transcription template, 800 U / mL recombinant RNase inhibitor protein (Takara Bio Inc., catalog #2316A), 2 U / mL yeast inorganic pyrophosphatase (Thermo Fisher Scientific Inc., catalog #EF0221), and 2500 U / mL T7 RNA polymerase (Dyne bioInc., catalog #dy1670) was prepared.

[0173] The transcription reaction mixture was incubated at 37°C for 5 hours. 40 mM Tris·HCl (pH 7.5), 8 mM magnesium chloride, 5 mM DTT, and 5000 U / mL recombinant DNase I (Takara Bio Inc., catalog #2270A) were added to the reaction mixture, and the mixture was incubated at 37°C for 1 hour. The resulting mRNA was purified using the Zymo Research RNA Cleaner & Concentrator-25 Kit (catalog #1017) according to the manufacturer's instructions.

[0174] mRNA synthesis is achieved by using Lonza GelStar. TM Electrophoretic confirmation of nucleic acid gel staining solution (catalog #5 0535) on 1% agarose gel ( Figure 1 ). Leverage Thermo Scientific TM Nanodrop TM One UV-Vis spectrometer was used to measure the UV analysis of the synthesized mRNA. As a result, it was confirmed that a GFP mRNA capped with a poly A(120) dimer containing approximately 1067 nt of ARCA was produced.

[0175] 2-3: In vitro transcription of GFP mRN via co-transcriptional capping as described in the examples

[0176] For the synthesis reaction of mRNA using the compounds of the various embodiments of the present invention, 40 mM Tris HCl (pH 7.9), 25 mM magnesium chloride, 2 mM spermidine, 2 mM ATP, 2 mM UTP, 2 mM CTP, 2 mM GTP, 3.2 mM of the compound of the embodiment, 5% DMSO (Sigma Aldrich catalog #472301-500 mL), 5 mg / mL GFP DNA transcription template, 800 U / mL recombinant RNase inhibitor protein (Takara Bio Inc., catalog #2316A), 2 U / mL yeast inorganic pyrophosphatase (Thermo Fisher Scientific Inc., catalog #EF0221) and 2500 U / mL T7 RNA polymerase (Dyne bio Inc., catalog #dy1670) were mixed.

[0177] The transcription reaction mixture was incubated at 37°C for 5 hours. 40 mM Tris·HCl (pH 7.5), 8 mM magnesium chloride, 5 mM DTT, and 5000 U / mL recombinant DNase I (Takara Bio Inc., catalog #2270A) were added to the reaction mixture, and the mixture was incubated at 37°C for 1 hour. The resulting mRNA was purified using the Zymo Research RNA Cleaner & Concentrator-25 Kit (catalog #1017) according to the manufacturer's instructions.

[0178] mRNA synthesis is achieved by using Lonza GelStar. TM Electrophoretic confirmation of nucleic acid gel staining solution (catalog #5 0535) on 1% agarose gel ( Figure 1 ). Leverage Thermo Scientific TM Nanodrop TM UV analysis of the synthesized mRNA was measured using a One UV-Vis spectrometer. The results confirmed the production of capped GFP mRNA containing approximately 1067 nt of polyA(120) according to the example.

[0179] Experimental Example 3: Translation of mRNA in HeLa cells

[0180] Translational activity of mRNAs produced via in vitro transcription was evaluated in the human cervical cancer cell line (HeLa). HeLa cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin at 37°C under a 5% CO2 atmosphere. 1 × 102 6HeLa cells were plated in 6-well plates at 100 cells / well. The next day, cells were transfected using transfection reagents (messengerMAX lipofectamine; Invitrogen, catalog #LMRNA003): Tube A was prepared by diluting 7.5 μL of the transfection reagent in 125 μL of Opti-MEM (Life Technologies) and incubating at room temperature for 10 minutes, following the manufacturer's instructions; tube B was prepared by diluting 5 μg of the prepared mRNA in 125 μL of Opti-MEM. The solutions in tubes A and B were mixed and incubated at room temperature for 5 minutes. During incubation, the cell culture medium was replaced with DMEM containing 10% FBS and no penicillin / streptomycin. The incubated mixture was then used to transfect the cells. After incubation at 37°C for 3 to 4 hours in a 5% CO2 atmosphere, the cell culture medium was replaced with DMEM supplemented with 10% FBS and 1% penicillin / streptomycin. The expression of the fluorescent protein was confirmed using a fluorescence microscope 96 hours after transfection.

[0181] As a result, the mRNA produced using the compounds of the examples exhibited excellent fluorescent activity. On the other hand, the capless mRNA, serving as the control group, showed no fluorescent activity whatsoever. Furthermore, all mRNAs produced using the compounds of the examples showed superior protein expression rates compared to mRNA synthesized using an ARCA cap. Figure 2 ).

[0182] Experiment Example 4: Western blot analysis

[0183] After transfection, the culture medium was removed and the cells were washed once with PBS. Cells were lysed at 4°C for 10 minutes after treatment with 200 μL of cell lysis buffer (RIPA + phosphatase inhibitor + PMSF). After recovering the cell lysis buffer, the cells were centrifuged at 12,000 rpm at 4°C for 10 minutes. The supernatant was then recovered, mixed with 5x SDS-sample loading buffer, and boiled at 100°C for 5–10 minutes. Electrophoresis was performed by running a 10% SDS-PAGE gel at 120V for 10 minutes and then at 170V for 1 hour. Samples were transferred using an Xcell 2 blot module (Invitrogen) at 30V for 1 hour. The resulting products were then blocked with 5% BSA-PBST at room temperature for 1 hour. The products were then reacted overnight at 4°C with rabbit anti-GFP antibody diluted 1:1000 in 5% BSA-PBST. The resulting products were then washed three times with PBST for 10 minutes each time. The HRP-conjugated rabbit secondary antibody was diluted 1:10000 in 2.5% BSA-PBST and treated at room temperature for 1 hour. The resulting product was then washed three times with PBST for 10 minutes each time. After ECL treatment, the product was confirmed by chemiluminescence, and its GFP expression level compared to β-actin was analyzed (Table 1).

[0184] [Table 1]

[0185] Comparison No hat ARCA Example 1 Example 6 Example 7 relative value 1.0 1.11 4.09 8.67 8.30 4.51

[0186] Experimental Example 5: Fluorescence-Activated Cell Sorting (FACS) Analysis

[0187] After transfection, the culture medium was removed, and any residual products were washed once with PBS. Following trypsin-EDTA treatment, the samples were incubated at 37°C for approximately 2–3 minutes until cell detachment began. The samples were then subjected to neutralization with culture medium (HG DMEM + 10% FBS + 1% penicillin / streptomycin) followed by centrifugation at 1200 rpm for 2 minutes at room temperature. The supernatant was removed, 1 mL of PBS was dispensed, and aggregated cells were released by pipetting. GFP signal (FITC) was measured using FACS (BDFACSDiva 8.0.3) [confirmed by forward scattering (FSC) and side scattering (SSC), shown in Table 2].

[0188] [Table 2]

[0189] Material % Parent - P3 relative value Negative 0.4 1.0 No hat 4.9 12.3 Example 1 35.6 89.0 Example 6 23.2 58 Example 7 24.3 60.8 Example 8 12.2 30.5 ARCA 9.4 23.5

[0190] As described above, specific aspects of the invention have been described in detail. It will be apparent to those skilled in the art that these specific descriptions are merely preferred exemplary embodiments and experimental examples, and that the scope of the invention is not limited thereto. Therefore, the essential scope of the invention will be defined by the appended claims and their equivalents.

Claims

1. A compound represented by the following chemical formula 1, its stereoisomer, or a salt thereof: [Chemical Formula 1] In the above chemical formula 1, B1 and B2 are independent of each other. , or ; X1 is -OH; X2 is -H; Y1 is -halogen and Y2 is -H; Z1 and Z2 are each independently -OH or -O(C) 1-4 alkyl); Z3 is -H; n is 1; m is 1; R1 and R2 are each independently -H or -(C 1-4 Alkyl); and R3 is -(C 1-4 alkyl).

2. The compound, its stereoisomer, or a salt thereof according to claim 1, wherein... Z1 is -OH or -O(C) 1-4 alkyl); Z2 is -OH; and Z3 is -H.

3. The compound according to claim 1, its stereoisomers, or salts thereof, wherein... R1 and R2 are each independently -H; and R3 is -(C 1-4 alkyl).

4. Choose compounds, their stereoisomers, or their salts from the group consisting of the following compounds: (1) ； (3) ； (5) ； (6) ； (7) ； (8) ； (10) ;and (11) 。 5. An RNA molecule comprising a compound, a stereoisomer thereof, or a salt thereof, as an oligonucleotide primer for capping RNA.

6. The RNA molecule according to claim 5, wherein... The oligonucleotide primers used for RNA capping are attached to the 5' upstream end of the RNA molecule.

7. The RNA molecule according to claim 5, wherein... The RNA molecule is an mRNA containing at least one coding sequence (CDS).

8. A method for synthesizing the RNA molecule according to claim 5, the method comprising: (S-1) A mixed DNA template, an oligonucleotide primer for RNA capping, a compound according to any one of claims 1 to 4, its stereoisomer or a salt thereof, and an RNA polymerase; and (S-2) Transcription of polynucleotide templates is performed by incubating the mixture.

9. The method for synthesizing RNA molecules according to claim 8, wherein... The method is performed in vitro.

10. A peptide for RNA molecule translation according to claim 5.

11. A cell in which the RNA molecule according to claim 5 is introduced.

12. A peptide produced by a cell according to claim 11.

13. A nucleic acid therapeutic agent comprising the RNA molecule according to claim 5.

14. The nucleic acid therapeutic agent according to claim 13, wherein The therapeutic agent contains a carrier capable of introducing the RNA molecule into target cells.

15. A vaccine comprising the RNA molecule according to claim 5.

16. The vaccine according to claim 15, wherein The vaccine contains a vector capable of introducing the RNA molecule into target cells.

17. The vaccine according to claim 15, wherein The vaccine is used to prevent cancer or infectious diseases.

Images