Oligonucleotide cap analog and composition comprising same

Abstract

The present invention relates to an oligonucleotide cap analog and a composition comprising same, and provides a trinucleotide or tetranucleotide-based mRNA Cap1 analog having a structure in which an imidazolylmethyl group is introduced at the N2 position of 5'-terminal guanosine and an alkyl, arylmethyl, or heteroarylmethyl group is substituted at the N7 position. The modified cap analog can be introduced co-transcriptionally during an in vitro transcription process, and exhibits improved capping efficiency and translation efficiency compared to conventional structures.

Description

Oligonucleotide cap analogue and composition containing the same

[0001] The present invention relates to oligonucleotide cap analogs and compositions containing the same, and more specifically, to oligonucleotide-based mRNA Cap1 analogs comprising selective structural modification of a guanosine base and / or adjacent nucleotides and compositions containing the same. The present invention particularly relates to modified capping materials usable in the process of in vitro transcription, and to oligonucleotide cap analogs and compositions containing the same for producing mRNA with improved protein expression efficiency.

[0002]

[0003] With the recent rapid advancement of mRNA-based vaccine and therapeutic technologies, various technologies are being developed to improve the structural stability and protein expression efficiency of synthetic mRNA produced through in vitro transcription. In particular, the 5′ cap structure located at the 5′ end of mRNA is known to play an important role in ribosome recognition, translation initiation efficiency, mRNA stability, and the regulation of innate immune responses.

[0004] Natural eukaryotic mRNA has a structure in which 7-methylguanosine (m7G) is linked via 5′-5′ triphosphate bonds and exists in the form of Cap0 (m7GpppN) or Cap1 (m7GpppNm). Among these, the Cap1 structure is a form with added 2′-O-methylation, which is reported to reduce recognition by innate immune receptors and improve translation efficiency.

[0005] In this regard, various dinucleotide or trinucleotide-based capping materials usable in in vitro transcription processes have been developed, and commercial capping reagents such as Anti-Reverse Cap Analog (ARCA) and CleanCap™ (m7GpppAmpG) are representative examples. Trinucleotide-based capping structures are known to provide higher capping and translation efficiency by directly mimicking the Cap1 structure.

[0006] However, most conventional capping structures rely on simple methylation (m7G) at the N7 position, and research on structural modifications at other positions of guanosine bases, such as the N2 position, has been relatively limited. Furthermore, existing capping structures have room for further improvement in terms of translation efficiency and immunosuppressive effects, and certain applications require enhanced protein expression characteristics or regulated immunosuppressive characteristics.

[0007] In particular, as the commercialization of mRNA-based vaccines and therapeutics expands, functional improvement through the precise chemical design of cap structures is recognized as an important technical challenge, as even minute differences in translation efficiency can directly affect immunogenicity, effective dose, productivity, and safety.

[0008] Therefore, there is a need to develop a novel cap structure that can improve translation efficiency, stability, or immunomodulatory properties by selectively modifying specific locations of guanosine bases while maintaining the existing Cap1 structure.

[0009] [Project ID] 1711160184

[0010] [Assignment No.] 2022M3E5F1016552

[0011] [Ministry Name] Ministry of Science and ICT

[0012] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0013] [Research Project Name] Development of Basic and Core Technologies for Next-Generation Infectious Disease Vaccines

[0014] [Research Project Title] Site-specific introduction into mRNA vaccine constructs to form an immune response

[0015] Development of new modified nucleotides capable of cleavage

[0016] [Name of Project Performing Organization] Jeonbuk National University

[0017] [Research Period] 2021.01.01 ~ 2022.12.31

[0018]

[0019] The technical problem that the present invention aims to solve is to provide an oligonucleotide cap analog that is structurally distinct from the existing Cap1 structure and applicable to in vitro transcription, as an mRNA cap analog comprising selective structural modification of a guanosine base and / or adjacent nucleotides.

[0020] Furthermore, the technical objective of the present invention is to provide mRNA with improved protein expression efficiency, capping efficiency, immunomodulation, expression persistence, purification process, and organ-specific drug delivery by providing a composition comprising the above-mentioned cap analog.

[0021]

[0022] The technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned technical problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below.

[0023]

[0024] To achieve the above technical problem, one embodiment of the present invention provides an oligonucleotide cap analog represented by the following chemical formula 1.

[0025] [Chemical Formula 1] G*-p n1 -N1-(pN) n2

[0026] (In the above Chemical Formula 1, G* is a modified guanosine in which a substituted or unsubstituted C1-C10 alkyl, or a substituted or unsubstituted C2-C10 alkenyl, C2-C10 alkynyl, C6-C20 aryl, or C3-C20 heteroaryl group connected by a methylene bridge (-CH2-) is bonded at the N7 position of guanosine (G), and a hydrogen, a substituted or unsubstituted C1-C10 alkyl, or a substituted or unsubstituted C2-C10 alkenyl, C2-C10 alkynyl, C6-C20 aryl, or C3-C20 heteroaryl group connected by a methylene bridge (-CH2-) is bonded at the N2 position;

[0027] p n1 The phosphate group is a phosphate group, wherein the bridging atom between phosphorus atoms within the phosphate group or the non-bridging atom bonded to the phosphorus atom is each independently selected from oxygen (O), sulfur (S), or NH, and n1 is 3 or 4;

[0028] N1 is a first nucleotide (A (adenosine), C (cytidine), G (guanosine), U (uracil)) to which an amino group or a nitrogen atom within the ring is unsubstituted, or a C1-C10 alkyl group substituted or unsubstituted at the said amino group or nitrogen atom, or a substituted or unsubstituted C2-C10 alkenyl, C2-C10 alkynyl, C6-C20 aryl, or C3-C20 heteroaryl group connected by a methylene bridge (-CH2-);

[0029] pN is a nucleotide linked by a phosphate group, and n2 is 1 or 2)

[0030] In an embodiment of the present invention, the substituent bonded to the N7 position of the G* may be benzyl, 4-fluorobenzyl, 4-bromobenzyl, 4-iodobenzyl, 3-methoxybenzyl, furan-2-ylmethyl, thiophene-2-ylmethyl, pyridine-2-ylmethyl, ethyl, allyl, propargyl, or a deuterated methyl group.

[0031] In an embodiment of the present invention, the substituent bonded to the N2 position of the G* may be imidazolylmethyl.

[0032] In an embodiment of the present invention, when N1 is adenosine (A), cytidine (C), or guanosine (G), the substituent bonded to the amino group may be methyl, deuteride methyl, benzyl, pyridylmethyl, fluorobenzyl, or trifluoromethylphenyl.

[0033] In an embodiment of the present invention, the 2' or 3' position of each nucleotide ribose included in G*, N1 and pN may each be independently selected from a hydroxyl group (-OH), a halogen, a substituted or unsubstituted C1-C10 alkoxy group, a haloalkyl group, an amino group, an acetamidoethyl group, a methoxyethyl group, a substituted or unsubstituted C1-C10 acyloxy group, and a substituted or unsubstituted C1-C10 sulfonyloxy group.

[0034] To achieve the above technical problem, another embodiment of the present invention provides a capped mRNA in which an oligonucleotide cap analog is introduced at the 5' end.

[0035] To achieve the above technical problem, another embodiment of the present invention provides a composition for expressing a target protein comprising capped mRNA.

[0036] To achieve the above technical objectives, another embodiment of the present invention provides a pharmaceutical composition for the prevention or treatment of a disease comprising capped mRNA and a pharmaceutically acceptable carrier.

[0037] In an embodiment of the present invention, the pharmaceutical composition may be for use in a vaccine, an anticancer agent, an immunotherapy agent, an antibody therapy agent, a cell therapy agent, or a gene therapy agent. To achieve the above technical objective, another embodiment of the present invention provides a composition for producing 5'-terminally capped mRNA comprising an oligonucleotide cap analog.

[0038]

[0039] The present invention relates to a modified oligonucleotide-based mRNA Cap1 analog and a composition containing the same. The oligonucleotide-based Cap1 analog according to the present invention can provide high capping efficiency in an in vitro transcription reaction, is easy to purify, exhibits low dsRNA production, and the generated mRNA exhibits high expression efficiency and persistence in cells and mice, is immunomodulatory, and may be capable of organ-specific expression.

[0040] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the composition of the invention described in the description or claims of the present invention.

[0041]

[0042] Figure 1 shows a trinucleotide cap analog with modified N2 and N7 positions.

[0043] Figure 2 shows a trinucleotide-based capping material IM2,m7GpppAmpG in which an imidazole compound linked by methylene is introduced at the 5′ terminal guanosine N2 position (R3) and a methyl group is introduced at the N7 position (R1).

[0044] Figure 3 shows a trinucleotide-based capping material IM2,Bn7GpppAmpG in which an imidazole compound linked by methylene is introduced at the 5′ terminal guanosine N2 position (R3) and a benzyl linked by a methylene bridge is introduced at the N7 position (R1).

[0045] Figure 4 shows a trinucleotide-based capping material IM2,Thio7GpppAmpG in which an imidazole compound linked by methylene is introduced at the 5′ terminal guanosine N2 position (R3) and a methylene-bridged thiophene is introduced at the N7 position (R1).

[0046] Figure 5 is a schematic diagram showing the synthesis process of an N7-(methyl)guanosine 5′-diphosphoromidazolid analog.

[0047] Figure 6 is a schematic diagram showing the synthesis procedure of trinucleotide cap analogs modified at the N2 and N7 positions.

[0048] Figure 7 is a graph confirming the expression of antigen proteins (Luciferase) with ARCA (Dinucleotide Cap) and CleanCap (m7GpppAmpG), with modified mRNA introduced with a trinucleotide-based capping material (IM2,Bn7GpppAmpG) in which a benzyl group connected by a methylene bridge at the 5′ terminal guanosine N7 position and an imidazole group connected by a methylene bridge at the N2 position are introduced.

[0049] Figure 8 is an experimental graph comparing the Luciferase mRNA transcription efficiency of trinucleotide cap analogs modified at the N2 and N7 positions.

[0050] Figure 9 is an experimental graph comparing the Luciferase mRNA capping efficiency of trinucleotide cap analogs modified at the N2 and N7 positions.

[0051] Figure 10 is an experimental graph comparing the translation efficiency of Luciferase mRNA of trinucleotide cap analogs modified at the N2 and N7 positions.

[0052] Figures 11 to 30 are schematic diagrams of compound synthesis.

[0053] Figures 31 to 46 are structural diagrams of the synthesized compounds.

[0054] Figure 47 is an agarose gel electrophoresis of IVT mRNA containing various trinucleotide caps and tetranucleotide cap analogs at the 5′ end.

[0055] Figure 48 shows the translation characteristics of gel-cut purified IVT mRNA containing various trinucleotide caps and tetranucleotide cap analogs at the 5′ end.

[0056] Figure 49 is a 20% urea modified gel analysis of the capping efficiency of various cap analogs.

[0057] Figure 50 shows the separation data of mRNA with introduced capping material by RP-HPLC.

[0058] Figure 51 is an experiment confirming innate immunity of mRNA with a modified cap introduced.

[0059] Figure 52 is an mRNA immunoassay with a modified cap introduced.

[0060] Figure 53 shows the evaluation of antigen protein expression and persistence of mRNA with a modified cap introduced.

[0061] FIGS. 54 to 56 are drawings showing the results of analyzing organ distribution based on luciferase expression in each organ after administering a cap analog according to an embodiment of the present invention containing benzyl-capped mRNA to CD-1 mice via intraperitoneal (IP) or intravenous injection (IV).

[0062] Figure 57 is a diagram showing the RP-HPLC purification results of benzyl-capped mRNA and a chromatogram showing the capped mRNA product peak.

[0063] Figure 58 is a diagram showing the results of analyzing the presence of dsRNA using a dot-blot with a J2 antibody using RP-HPLC fractionation and a cellulose-based separation method.

[0064] Figure 59 is a diagram showing the efficiency of decapped mRNA.

[0065] Fig. 60 is a structural diagram for CD3-7Gpppm6AmpG.

[0066]

[0067] The present invention will be described in detail below.

[0068]

[0069] The present invention relates to an oligonucleotide cap analogue that is introduced to the 5' end of mRNA to improve the protein expression efficiency, capping efficiency, immunomodulation, expression persistence, purification process, and organ-specific drug delivery efficiency of the mRNA. More specifically, the present invention provides an oligonucleotide cap analogue represented by the following chemical formula 1.

[0070] [Chemical Formula 1]

[0071] G*-p n1 -N1-(pN) n2

[0072] (in the above Chemical Formula 1, G* is a modified guanosine in which a substituted or unsubstituted C1-C10 alkyl, or a substituted or unsubstituted C2-C10 alkenyl, C2-C10 alkynyl, C6-C20 aryl, or C3-C20 heteroaryl group connected by a methylene bridge (-CH2-) is bonded at the N7 position of guanosine (G), and a hydrogen, a substituted or unsubstituted C1-C10 alkyl, or a substituted or unsubstituted C2-C10 alkenyl, C2-C10 alkynyl, C6-C20 aryl, or C3-C20 heteroaryl group connected by a methylene bridge (-CH2-) is bonded at the N2 position; p n1is a phosphate group, wherein the bridging atom between the phosphorus atoms within the phosphate group or the non-bridging atom bonded to the phosphorus atom is each independently selected from oxygen (O), sulfur (S), or NH, and n1 is 3 or 4; N1 is a first nucleotide (A (adenosine), C (cytidine), G (guanosine), U (uracil)) to which an amino group or a ring nitrogen atom is unsubstituted, or a C1-C10 alkyl group substituted or unsubstituted to the amino group or nitrogen atom, or a substituted or unsubstituted C2-C10 alkenyl, C2-C10 alkynyl, C6-C20 aryl, or C3-C20 heteroaryl group connected by a methylene bridge (-CH2-); pN is a nucleotide connected by a phosphate group, and n2 is 1 or 2)

[0073] In the above chemical formula 1, G* represents modified guanosine and may have a structure in which a substituent is introduced at a specific position of the guanosine. The G* is a portion corresponding to guanosine in the 5' terminal cap structure of mRNA and may regulate interactions with cap-binding proteins or affect the protein expression efficiency, capping efficiency, immunomodulation, expression persistence, purification process, and organ-specific drug delivery efficiency of mRNA.

[0074] Specifically, the substituent bonded to the N7 position of the above G* may be a benzyl, 4-fluorobenzyl, 4-bromobenzyl, 4-iodobenzyl, 3-methoxybenzyl, furan-2-ylmethyl, thiophene-2-ylmethyl, pyridine-2-ylmethyl, ethyl, allyl, propargyl, or deuterated methyl group.

[0075] The above substituents are introduced at the N7 position of guanosine to regulate the chemical properties of the cap structure and may affect interactions with cap-binding proteins or the stability and translation efficiency of mRNA. For example, substituents containing benzyl or substituted benzyl groups can increase hydrophobic interactions through the aromatic ring structure, thereby regulating binding characteristics with cap-binding proteins and providing resistance to decapsases, which can improve protein expression efficiency.

[0076] In addition, substituents containing heteroaryl groups, such as furan-2-ylmethyl, thiophene-2-ylmethyl, or pyridine-2-ylmethyl, can improve translation efficiency by controlling the physicochemical properties of cap analogs through changes in electronic properties or polarity. Meanwhile, alkyl or unsaturated hydrocarbon substituents, such as ethyl, allyl, or propargyl, can control the stability or biological activity of cap analogs by providing structural diversity.

[0077] In addition, substituents containing deuterium methyl groups can improve stability against chemical or enzymatic degradation due to isotope effects, and accordingly, can improve the stability of mRNA into which cap analogs have been introduced or the protein expression efficiency.

[0078] The substituent bonded to the N2 position of the above G* may be imidazolylmethyl. The imidazolylmethyl group is a substituent containing an imidazole ring and can exhibit various electronic and chemical properties through a heterocyclic structure containing a nitrogen atom.

[0079] By introducing the aforementioned imidazolylmethyl group at the N2 position of guanosine, the stereochemical or electronic properties of the cap structure of the oligonucleotide cap analog of the present invention can be controlled, and the interaction characteristics with the cap-binding protein can be influenced. Additionally, the imidazole ring may possess hydrogen bond-forming ability and electron-donating properties, thereby altering intermolecular interactions around the cap structure.

[0080] The above p n1 represents a phosphate linkage and may include a polyphosphate structure in which multiple phosphate groups are connected to each other. The phosphate linkage serves to connect the modified guanosine (G*) and the first nucleotide (N1), and the chemical stability or enzymatic recognition characteristics of the cap analog may be controlled depending on the length or structure of the phosphate linkage.

[0081] The above N1 refers to a first nucleotide, and the first nucleotide may be adenosine (A), cytidine (C), guanosine (G), or uracil (U). The above N1 may be a modified nucleotide in which a substituent is introduced to the base or ribose portion as needed. For example, if the above first nucleotide is adenosine (A), the amino group at the N6 position of adenosine may be substituted, and if it is cytidine (C), the amino group at the N4 position of cytidine may be substituted. Also, if it is guanosine (G), the amino group at the N2 position of guanosine may be substituted, and if it is uracil (U), the nitrogen atom at the N3 position of uracil may be substituted.

[0082] More specifically, when N1 is adenosine (A), cytidine (C), or guanosine (G), the substituent bonded to the amino group may be methyl, deuteride methyl, benzyl, pyridylmethyl, fluorobenzyl, or trifluoromethylphenyl.

[0083] Substituents such as those mentioned above can control the electronic properties or stereochemical structure of a base by being introduced into the amino group of the nucleotide base. For example, small alkyl substituents, such as methyl groups, can increase the hydrophobicity of the base and induce relatively small stereochemical changes in the molecular structure. In addition, aromatic substituents, such as benzyl, fluorobenzyl, or trifluoromethylphenyl, can alter intermolecular interactions through the aromatic ring structure and control the stereochemical environment around the cap structure.

[0084] In addition, substituents containing heteroaryl groups, such as pyridylmethyl, can alter electronic properties or polarity through a ring structure containing nitrogen atoms, thereby controlling the physicochemical properties of oligonucleotide cap analogs.

[0085] The above (pN) n2 represents one or more nucleotide residues connected by phosphate bonds. The nucleotide residues may include ribonucleotides or modified nucleotides, and depending on the value of n2, one or two additional nucleotides may be included.

[0086] Therefore, the oligonucleotide cap analog of the present invention comprises modified guanosine (G*), a phosphate group (p n1 ), first nucleotide (N1) and additional nucleotide residue ((pN) n2 It has an oligonucleotide structure including ), and said structure can mimic or replace the 5' end cap structure of mRNA.

[0087] The 2' or 3' position of each nucleotide ribose included in G*, N1 and pN may be substituted with various functional groups. Each of the substituents may be independently selected from a hydroxyl group (-OH), a halogen, a substituted or unsubstituted C1-C10 alkoxy group, a haloalkyl group, an amino group, an acetamidoethyl group, a methoxyethyl group, a substituted or unsubstituted C1-C10 acyloxy group, and a substituted or unsubstituted C1-C10 sulfonyloxy group.

[0088] Substituents such as those mentioned above can be introduced into the 2' or 3' positions of the ribose backbone to regulate the chemical stability or enzymatic recognition properties of nucleotides. For example, substituents such as alkoxy groups, acyloxy groups, or sulfonyloxy groups can improve the degradation stability of nucleotides by altering the physicochemical properties of the ribose structure. Additionally, substituents such as halogens or haloalkyl groups can affect the chemical stability or intermolecular interactions of nucleotides by altering the electronic properties of the molecule.

[0089] In addition, substituents such as amino groups can regulate intermolecular interactions through their ability to form hydrogen bonds, thereby regulating the structural characteristics or biological activity of oligonucleotide cap analogs.

[0090] In relation to other embodiments of the present invention described below, detailed descriptions of contents that overlap with those previously mentioned have been omitted.

[0091] Another embodiment of the present invention provides a capped mRNA in which the oligonucleotide cap analog is introduced at the 5' end.

[0092] The capped mRNA may have a structure in which an oligonucleotide cap analog according to the present invention is connected to the 5' end of the mRNA, and the cap analog may mimic or replace the 5' cap structure of natural mRNA. Generally, mRNA in eukaryotic cells has a cap structure at the 5' end, and the cap structure can play an important role in maintaining the stability of the mRNA, promoting the initiation of translation through ribosome binding, and inhibiting degradation by exonucleases.

[0093] The capped mRNA to which the oligonucleotide cap analog according to the present invention has been introduced can maintain or enhance the function of the cap structure, thereby improving the intracellular protein expression efficiency, capping efficiency, immunomodulation, expression persistence, purification process, and organ-specific drug delivery of the mRNA. In addition, the cap analog can regulate the interaction characteristics with a cap-binding protein, thereby increasing the expression level of the target protein.

[0094] Another embodiment of the present invention provides a composition for expressing a target protein comprising the capped mRNA.

[0095] The above composition for expressing the target protein comprises capped mRNA in which an oligonucleotide cap analog according to the present invention is introduced at the 5' end, thereby inducing the expression of the corresponding protein when the capped mRNA is delivered into a cell.

[0096] The above-mentioned target protein may be a protein having a specific function or physiological activity, for example, a therapeutic protein, an antigen protein, an enzyme protein, a regulatory protein, or other proteins having biological activity. The above-mentioned capped mRNA can be translated within a cell to express the above-mentioned target protein, and accordingly, can be utilized for various biological or medical purposes.

[0097] Another embodiment of the present invention provides a pharmaceutical composition for the prevention or treatment of a disease comprising capped mRNA and a pharmaceutically acceptable carrier.

[0098] The above-mentioned pharmaceutically acceptable carrier is a material used for the formulation and delivery of a pharmaceutical composition, and may be a material that is non-toxic in vivo and pharmaceutically acceptable. For example, the carrier may include a buffer solution, physiological saline, an aqueous solvent, a lipid, a lipid nanoparticle, a polymer carrier, or other delivery systems for nucleic acid delivery.

[0099] The above pharmaceutical composition may include all therapeutic agents related to the synthesis or replacement of proteins and may not be limited to a specific therapeutic agent. Furthermore, the above pharmaceutical composition may be, more specifically, for use as a vaccine, an anticancer agent, an immunotherapy agent, or a gene therapy agent. For example, if the above pharmaceutical composition is for use as a vaccine, the capped mRNA may include a nucleotide sequence encoding an antigen protein of a specific pathogen, and the mRNA may be translated in cells within the body to induce an immune response by expressing the antigen protein. Accordingly, the above pharmaceutical composition may be utilized as a vaccine for the prevention or treatment of infectious diseases.

[0100] In addition, if the above pharmaceutical composition is for use as a cancer treatment, the capped mRNA may include a nucleotide sequence encoding a tumor-associated antigen, a tumor-specific antigen, or a protein having anticancer activity. Inhibition of tumor growth or elimination may be induced through an immune response induced by the expression of the mRNA or through the expression of an anticancer protein.

[0101] Meanwhile, if the above pharmaceutical composition is for use as an immunotherapeutic agent, the capped mRNA may include a nucleotide sequence encoding a protein that regulates an immune response, such as a cytokine, chemokine, or immune regulatory protein, and accordingly may regulate or enhance the immune response of the subject.

[0102] In addition, if the above pharmaceutical composition is for gene therapy, the capped mRNA may include a nucleotide sequence encoding a protein capable of supplementing or replacing the function of a deficient or mutated gene associated with a specific disease. Accordingly, the above pharmaceutical composition can be usefully applied to the treatment of various genetic diseases or other diseases.

[0103] Additionally, the pharmaceutical composition may further include stabilizers, preservatives, buffers, adjuvants, or other pharmaceutically acceptable additives as needed.

[0104] Another embodiment of the present invention provides a composition for producing 5'-terminally capped mRNA comprising an oligonucleotide cap analog.

[0105] For example, the mRNA manufacturing composition may additionally include RNA polymerase, nucleoside triphosphates (NTPs), reaction buffer, or other components necessary for the transcription reaction in addition to the oligonucleotide cap analog. Additionally, the composition may further include auxiliary components to improve capping efficiency or transcription efficiency.

[0106] Capped mRNA prepared using the composition described above can exhibit enhanced stability and translation efficiency and can effectively induce the expression of a target protein. Therefore, the mRNA preparation composition of the present invention can be usefully utilized in the manufacture of protein expression systems, mRNA vaccines, or other mRNA-based therapeutic agents.

[0107]

[0108] Hereinafter, in order to specifically explain the present invention, it will be described in detail with reference to examples.

[0109]

[0110] Experimental method

[0111] 1. Synthesis of trinucleotide capping materials

[0112] This invention relates to the synthesis of a novel modified trinucleotide capping material and its use as an mRNA vaccine. Specifically, it involves introducing a heterocyclic compound in which the N2 position of the guanosine base at the 5′ end of the CleanCap trinucleotide structure is connected by a methylene bridge (e.g., imidazole), and modifying the N7 position by replacing the existing methyl or methyl bases with aryl (benzyl) or heterocyclic (methylthiophene) compounds connected by a methylene bridge to produce a novel capping material (IM2 m7 GpppA m pG, IM2 Bn7 GpppA m pG, IM2 thio7 GpppA m pGs were synthesized through organic chemical reactions (Figs. 1 to 6).

[0113]

[0114] 1-1. Reagent Information

[0115] Using a Bruker AV-400 spectrometer 1 H and 13 C, 31P NMR spectra were recorded using D2O or DMSO-d6 as the solvent and tetramethylsilane as the internal standard. UV-Vis spectra were recorded at room temperature using a Cary series UV-Vis spectrophotometer (Agilent Technologies) and a 1 cm quartz cuvette, and changes in absorbance were measured immediately after irradiating the sample solution with UV light in the cuvette. Fluorescence emission spectra were recorded at room temperature using a PF-65000 spectrofluorometer.

[0116]

[0117] 1-2. Method for Synthesizing N7-(arylmethyl)guanosine 5′-diphosphoroimidazolide Analogues

[0118] ① Synthesis of the triethylammonium salt of guanosine 5′-diphosphate

[0119] To synthesize the triethylammonium salt of guanosine 5′-diphosphate (GDP.TEA), triethylamine (1.7 mL, 12 mmol), imidazole (817 mg, 12 mmol), and 2,2′-dithiodipyridine (1.3 g, 6 mmol) were added to a stirred solution of guanosine 5′-phosphate (GMP.TEA) in anhydrous dimethyl sulfoxide (4 mL), and the mixture was stirred for 5 minutes. Then, triphenylphosphine (1.56 g, 6 mmol) was added to anhydrous dimethyl sulfoxide (4 mL), and stirring was continued at room temperature for 5 hours. After the reaction was complete, the mixture was slowly poured into a mixture of sodium perchlorate (0.5 g) in acetone (50 mL). After cooling at 4°C for 30 minutes, the mixture was centrifuged at 5,000 rpm for 8 minutes, and the supernatant was discarded. To remove trace amounts of imidazole and triphenylphosphine, the solid was ground with fresh acetone (20 mL), the mixture was cooled, and then centrifuged again. This process was repeated one more time, and the precipitate was dried in a vacuum oven at room temperature (Fig. 5).

[0120] The guanosine 5′-phosphoromidazolade (NaGMP-IM) obtained in this way was dissolved in dimethylformamide (10 mL) and added dropwise to a mixture of 1 M tributylammonium orthophosphate solution in dimethylformamide (5 mL) stirred for 30 minutes. Finally, zinc chloride (200 mg, 1.5 mmol) was added, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was extracted with chloroform (3 × 20 mL) soaked in water (50 mL). The resulting solution was concentrated using a rotary evaporator with a water bath temperature of 37°C. The resulting product was applied to an anion exchange resin for purification.

[0121] Chromatographic purification was performed using DEAE (diethylaminoethyl weak anion exchange) Sepharose high-speed flow resin. The desired compound was eluted using CombiFlash EZ Prep with 4 bed volumes having a 0–30% gradient of pH 7.5 and 1 M TEAB (triethylammonium bicarbonate) buffer at a flow rate of 3 mL / min. The GDP.TEA-containing fractions were collected, concentrated using a rotary evaporator (water bath temperature 37°C), and then dried in a freeze-dryer to obtain fine white powder GDP.TEA (951 mg, 64%). The NMR results of GDP.TEA are shown below.

[0122] GDP.TEA. (951 mg, 64 %)

[0123] 1 H NMR (400 MHz, DO) δ = 7.99 (s, 1H), 5.81 (d, J = 6.0 Hz, 1H), 4.63 (t, J = 5.2 Hz, 1H), 4.51 (t, J = 5.6 Hz, 1H), 4.22 (m, 1H), 4.14 (m, 1H), 4.05 (m, 1H); 31P NMR (162 MHz, DO) δ = -6.47 (d, J = 23.5 Hz, 1P), -10.99 (d, J = 22.5 Hz, 1P).

[0124]

[0125] 1-3. Method for Synthesizing N7-(aryl-2-ylmethyl)guanosine 5′-mono or diphosphate Analogs

[0126] Triethylamine salt of guanosine 5′-diphosphate (0.5 mmol, 1 equivalent) was dispersed in DMSO (3 mL), and an appropriate amount of 2-bromomethylaryl / heteroaryl or methyl iodide (2.5 mmol, 5 equivalents) was added. The mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was slowly poured into a mixture of sodium perchlorate (0.5 g) mixed in acetone (50 mL). After cooling at 4°C for 30 minutes, the mixture was centrifuged at 5,000 rpm for 8 minutes, and the supernatant was discarded. The solid was ground with fresh acetone (20 mL), the mixture was cooled, and centrifuged again. This process was repeated once more, and the precipitate was vacuum dried at room temperature. A crude product solution with a total volume of 15–20 mL was separated using a Sepharose (DEAE Sepharose) high-flow resin as previously described. The fraction containing the product was collected and concentrated using a rotary evaporator (water temperature 37°C), then co-evaporated with ethanol three times and dried in a freeze dryer (Fig. 11).

[0127] N7-methyl guanosine 5′-diphosphate triethylammonium salt, N7-benzyl guanosine 5′-diphosphate triethylammonium salt, and N7-(thiophen-2-ylmethyl) guanosine 5′-diphosphate triethylammonium salt were synthesized using methyl, benzyl, and thiophen-2-ylmethyl, respectively, according to the above method, and the NMR results are shown below.

[0128]

[0129] *

[0130] *N7-methyl guanosine 5′-diphosphate triethylammonium salt(N7-m-GDP.TEA (182 mg, 48 %))

[0131] 1 H NMR (400 MHz, DO) δ = 5.97 (d, J = 2.4 Hz, 1H), 4.57 (dd, J1= 9.2 Hz, J2= 7.6 Hz, 1H), 4.48 (t, J = 4.8 Hz, 1H), 4.31-4.28 (m, 1H), 4.27-4.22 (m, 1H), 4.20-4.13 (m, 1H), 4.03 (s, 1H); 31 P NMR (162 MHz, DO) δ = -7.68 (d, J = 22.7 Hz, 1P), -11.07 (d, J = 24.3 Hz, 1P).

[0132] N7-benzyl guanosine 5′-diphosphate triethylammonium salt(N7-Bn-GDP.TEA (247 mg, 67%))

[0133] 1H NMR (400 MHz, D2O) δ = 7.23-7.38 (m, 5H), 5.99 (d, J = 3.6 Hz, 1H), 5.64 (d.d, J1= 21.6 Hz, J2= 15.2 Hz, 2H), 4.61 (dd, J1= 5.2 Hz, J2= 3.2 Hz, 1H), 4.53 (t, J = 5.2 Hz, 1H), 4.30 (m, 1H), 4.18-4.24 (m, 2H); 31 P NMR (162 MHz, D2O) δ = -6.48 (d, J = 23.5 Hz, 1P), -10.98 (d, J = 23.5 Hz, 1P).

[0134] N7-(thiophen-2-ylmethyl) guanosine 5′-diphosphate triethylammonium salt(N7-2-mTP-GDP.TEA (155 mg, 42 %))

[0135] 1 H NMR (400 MHz, D2O) δ = 7.36 (d, J = 5.2 Hz, 1H), 7.27 (d, J = 3.6 Hz, 1H), 6.96 (t, J = 5.2 Hz,1H), 5.94 (d, J = 3.6 Hz, 1H), 5.80 (dd, J1= 18.0 Hz, J2= 15.2 Hz, 2H), 4.62 (t, J = 3.6 Hz,1H), 4.48 (t, J = 5.6 Hz, 1H), 4.32-4.27 (m, 1H), 4.26-4.14 (m, 2H); 31 P NMR (162 MHz, D2O) δ = -8.34 (d, J = 22.4 Hz, 1P), -11.10 (d, J = 22.5 Hz, 1P).

[0136]

[0137] 1-4. N2-1H-imidazol-1-yl methyl, N7-substituted guanosine 5′ diphosphate 유사체의 합성 방법

[0138] The triethylammonium salt of N7-arylmethyl / heteroarylmethylguanosine 5′-diphosphate (0.5 mmol) and imidazole (2.5 mmol) was added to a stirred solution of water (3 mL), formaldehyde (2.5 mmol) was added, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was loaded directly onto a DEAE Sepharose column (solvent A: water, solvent B: 1 M TEAB buffer (pH 7.5-8)). The fraction containing the product was pooled, evaporated, and dried in a freeze dryer to obtain a fine white powder containing the triethylammonium salt (Fig. 12).

[0139] N2-1H-imidazol-1-yl methyl, N7-methyl guanosine 5′-diphosphate(1-mIM2, ​​m7GDP)

[0140] 1 H NMR (400 MHz, DO) δ = 7.86 (s, 1H), 7.26 (s, 1H), 6.90 (s, 1H), 6.07 (d, J = 3.2 Hz, 1H), 5.50 (q, J = 14.0 Hz, 2H), 4.55 (t, J = 3.2) Hz, 1H), 4.48 (t, J = 4.8 Hz, 1H), 4.31-4.26 (m, 1H), 4.23-4.17 (m, 2H), 4.01 (s, 3H); 31 P NMR (162 MHz, DO) δ = -6.42 (d, J = 26.4 Hz, 1P), -11.06 (d, J = 23.5 Hz, 1P).

[0141] N2-1H-imidazol-1-yl methyl, N7-benzyl guanosine 5′-diphosphate(1-mIM2, ​​Bn7GDP)

[0142] 1H NMR (400 MHz, D2O) δ = 8.00 (s, 1H), δ = 7.38 (s, 1H), 7.37-7.27 (m, 5H), δ = 6.98 (s, 1H), 6.12 (d, 1H), 5.66 (d.d, 2H), 5.55 (dd, 2H), 4.66 (m, 1H), 4.53 (t, 1H), 4.34 (m, 1H), δ = 4.24 (m, 1H); 31 P NMR (162 MHz, D2O) δ = -6.44 (d, J = 21.5 Hz, 1P), -10.92 (d, J = 21.3 Hz, 1P).

[0143] N2-1H-imidazol-1-yl methyl, N7-(thiophen-2-ylmethyl) guanosine 5′-diphosphate(1-mIM2, 2-mTP7GDP)

[0144] 1 H NMR (400 MHz, D2O) δ = 8.27 (s, 1H), 7.37 (s, 1H), 7.33 (d, J = 6.4 Hz, 1H), 7.22 (d, J = 3,6 Hz, 1H), 7.07 (s, 1H), 6.93 (T, J = 5.2 Hz, 1H), 6.01 (d, J = 3.6 Hz, 1H), 5.80 (q, J = 24.8 Hz, 2H), 5.58 (q, J = 16.8 Hz, 2H), 4.61 (t, J = 4.8 Hz, 1H), 4.46 (t, J = 10 Hz, 1H), 4.29 (m, 1H), 4.22-4.13 (m, 2H); 31 P NMR (162 MHz, D2O) δ = -7.07 (d, J = 22.4 Hz, 1P), -10.97 (d, J = 22.7 Hz, 1P).

[0145]

[0146] 1-5. Method for Synthesizing N2-1H-imidazol-1-yl methyl, N7-substituted guanosine 5′ diphosphoro-imidazolide Analogue

[0147] Triethylamine (251 μL, 1.8 mmol), imidazole (123 mg, 1.8 mmol), and 2,2'-dithiodipyridine (198 mg, 0.9 mmol) were added to a stirred solution of the triethylammonium salt of N7-(aryl-2-ylmethyl) guanosine 5′-diphosphate (0.3 mmol) in dimethyl anhydride (3 mL), and the mixture was stirred for 5 minutes. Then, triphenylphosphine (236 mg, 0.9 mmol) was added to dimethyl anhydride (2 mL). Stirring was continued for 5 hours at room temperature. After the reaction was complete, the mixture was slowly poured into a mixture of sodium perchlorate (250 mg) in acetone (25 mL). After cooling at 4°C for 30 minutes, the mixture was centrifuged and the supernatant was discarded. To remove trace amounts of imidazole and triphenylphosphine, the solid was ground with fresh acetone (10 mL), the mixture was cooled, and then centrifuged again. This process was repeated one more time, and the precipitate was dried in a vacuum oven using P2O5 at room temperature (Fig. 13).

[0148] N2-1H-imidazole-1-ylmethyl, N7-methyl guanosine 5′-diphosphate, N2-1H-imidazole-1-ylmethyl, N7-benzyl guanosine 5′-diphosphate, and N2-1H-imidazole-1-ylmethyl, N7-(thiophene-2-ylmethyl)guanosine 5′-diphosphate were synthesized according to the above method, and the NMR results are shown below.

[0149] N2-1H-imidazol-1-yl methyl, N7-methyl guanosine 5′-diphosphoroimidazolide (1-mIM2, ​​m7-GDP-IM)

[0150] 1H NMR (400 MHz, D2O) δ = 7.91 (s, 1H), 7.84 (s, 1H), 7.27 (s, 1H), 7.20 (s, 1H), 6.93 (s, 1H), 6.84 (s, 1H), 6.03 (d, J = 3.6 Hz, 1H), 5.51 (q, J = 14.0 Hz, 2H), 4.49 (t, J = 4.4 Hz, 1H), 4.28-4.22 (m, 2H), 4.19-4.14 (m, 1H), 4.05 (m, 1H), 3.96 (s, 3H); 31 P NMR (162 MHz, D2O) δ = -11.92 (d, J = 21.5 Hz, 1P), -19.95 (d, J = 17.6 Hz, 1P).

[0151] N2-1H-imidazol-1-yl methyl, N7-benzyl guanosine 5′-diphosphoroimidazolide(1-mIM2, Bn7-GDP-IM)

[0152] 1 H NMR (400 MHz, D2O) δ = 7.98 (s, 1H), 7.85 (s, 1H),7.39-7.29 (m, 5H), 7.21 (s, 1H), 6.97 (s, 1H), 6.85 (s, 1H), 6.06 (d, 1H), 5.59 (dd, 2H), 5.53 (dd, 2H), 4.58 (t, 1H), 4.32 (m, 2H), 4.20 (m, 1H), 4.06 (m, 1H); 31 P NMR (162 MHz, D2O) δ = -11.68 (d, J = 21.7 Hz, 1P), -19.87 (d, J = 21.5 Hz, 1P).

[0153] N2-1H-imidazol-1-yl methyl, N7-(thiophen-2-ylmethyl) guanosine 5′-diphosphoroimidazolide(1-mIM2, 2-mTP7GDP-IM)

[0154] 1H NMR (400 MHz, DO) δ = 7.88 (s, 1H), 7.79 (s, 1H), 7.34 (d, J = 6.4 Hz, 1H), 7.25 (s, 1H), 7.22 (d, J = 4.0 Hz, 1H), 7.16 (s, 1H), 6.94 (t, J = 5.2 Hz, 1H), 6.90 (s, 1H), 6.80 (s, 1H), 5.99 (d, J = 3.6 Hz, 1H), 5.75 (q, J = 24.8 Hz, 2H), 5.48 (q, J = 23.2 Hz, 2H), 4.50 (t, J = 4.0 Hz, 1H), 4.27-4.22 (m, 2H), 4.18-4.12 (m, 1H), 4.04-3.97 (m, 1H); 31 P NMR (162 MHz, DO) δ = -11.74 (d, J = 20.4 Hz, 1P), -19.91 (d, J = 21.5 Hz, 1P).

[0155]

[0156] 1-6. Synthesis Method of Dinucleotide, pAmpG

[0157] ① Synthesis process of 5′-O-DMT-2′-O-methyl Adenosylyl (n-bz)-{3′-OP-[2-cyanoethyl] → 5′}-2′,3′-diacetyl Guanosine (n-ibu) 3

[0158] DMT-2'-O-methyl adenosine (n-bz) CED phosphoramidite 1 (1.29 g, 1.45 mmol) and 2',3'-dicacetyl guanosine (n-ibu) 2 (0.58 g, 1.32 mmol) were dried under high vacuum for 16 hours. Anhydrous DMF (1.7 mL) and 0.45 M tetrazole were added to acetonitrile (7.3 mL, 3.30 mmol) and stirred at room temperature for 3 hours under an argon (nitrogen) atmosphere. After confirming the consumption of Compound 1 by TLC, the mixture was cooled in an ice-salt bath for 30 minutes, after which 70% t-butyl hydroperoxide (0.91 mL, 6.60 mmol) was added to water. The reaction mixture was stirred at room temperature for 1 hour. After confirming the conversion of the intermediate by TLC. It was extracted with 2×200 mL of ethyl acetate and washed with 2×100 mL of 5% NaHCO3 solution. The extracted organic solution was combined with sodium sulfate, dried, and evaporated to obtain crude solid 3 (1.77 g) (Fig. 14).

[0159]

[0160] ② Synthesis process of 2′-O-Methyl Adenosylyl (n-bz)-{3′-OP-[2-cyanoethyl] → 5′}-2′,3′-diacetyl Guanosine (n-ibu) 4

[0161] Crude Dinucleotide 3 solid (1.77 g) was dissolved in 3% trichloroacetic acid in dichloromethane (31.2 mL, 5.72 mmol). The mixture was stirred at room temperature for 1 hour. After confirming the conversion of Compound 3 by TLC, 5% NaHCO3 solution (30 mL) was added and stirred for 10 minutes. The mixture was extracted with dichloromethane and evaporated. Subsequently, the resulting residue was purified by silica gel column chromatography to obtain Product 4 (0.74 g, 0.79 mmol, 60%).

[0162] 1 H NMR (DMSO, 400MHz): δ 12.12 (s, 1H), 11.59 (d, 1H), 11.26 (d, 1H), 7.93 (d, d, 2H), 8.28 (d, 1H), 8.06 (d, 2H), 7.66 (t, 1H), 7.57 (d, 2H), 6.19(d, 1H), 6.14 (dd, 1H), 5.84 (q, 1H), 5.54 (dd, 1H), 5. 39 (t, 1H), 5.20 (m, 1H), 4.83 (m, 1H), 4.48 (m, 3H), 4.30 (m, 3H), 3.64 (m, 2H), 3.37 (d, 3H), 2.97 (q, 2H), 2.78 (m, 1H), 2.15 (d, 3H), 2.03 (d, 3H), 1.13 (m, 6H); 31 P NMR (DMSO, 162 MHz): δ -2.43 (d, 1P);

[0163]

[0164] ③ 5′-O-Phosphoryl-2′-O-methyl Adenosylyl-{3′-OP→ 5′} Guanosine, (pA m pG) 5의 합성과정

[0165] Dinucleotide 4 (0.74 g, 0.7891 mmol) was dried under high vacuum for 16 hours. 0.45 M tetrazole (3.51 mL, 1.58 mmol) and bis-(2-cyanoethyl)-N,N-diisopropyl-phosphoramidite (0.41 mL, 1.58 mmol) in acetonitrile were added, and the mixture was stirred at room temperature for 3 hours under an argon (nitrogen) atmosphere. After confirming the consumption of Compound 4, the reaction mixture was cooled in an ice-salt bath for 30 minutes, and 70% t-butyl hydroperoxide (17.2 mL, 3.16 mmol) in water was added. The mixture was stirred at room temperature for 1 hour. After confirming the conversion of the intermediate by TLC, it was extracted with 2 x 200 mL of ethyl acetate and washed with 2 x 100 mL of 5% NaHCO3 solution. The extracted organic solutions were combined and evaporated. The resulting residue was dissolved in 25 mL of methanol and 25 mL of concentrated ammonia solution and stirred at 55°C for 7 hours. After deprotection was complete, the reaction mixture was evaporated with methanol. The resulting residue was dissolved in water, adjusted to pH 5.5, and loaded onto a DEAE-Sepharose column (Solvent A: 1 M TEAB buffer (pH 7.5–8), Solvent B: Water). The fraction containing the product was collected, evaporated, and dried in a freeze-dryer to obtain a fine white powder 5 (446 mg, 0.4908 mmol, 62%) containing triethylammonium salt.

[0166] Dinucleotide (pAmpG) TEA salt

[0167] 1H NMR (DO, 400 MHz): δ 8.53 (s, 1H), 8.10 (s, 1H), 7.89 (s, 1H), 6.06 (d, 1H), 5.77 (d, 1H), 4.88 (m, 1H), 4.50 (t, 1H), 4.42 (m, 2H), 4.27 (m, 1H), 4.12 (t, 2H), 3.92 (m, 2H), 3.37 (s, 3H); 31 P NMR (DO, 162 MHz): δ 3.69 (s, 1P), -0.84 (s, 1P)

[0168]

[0169] 1-7. General Synthesis Process of Guanosine N2-1H-imidazol-1-yl methyl, N7-substituted trinucleotide mRNA Cap1 Analog

[0170] pA in 4ml of DMSO mN2-1H-imidazole-1-ylmethyl, N7-substituted guanosine 5′-diphosphoro-imidazolaide derivative (3.00 equivalents, 0.2 mmol) and zinc chloride (20.0 equivalents) were added to a solution containing pG triethylammonium salt (0.07 mmol, 63 mg). After stirring at 37°C for 3 days, a 500 mM aqueous EDTA solution (pH 8.0) (EDTA: 1.30 equivalents per molar amount of ZnCl2) was added to stop the reaction mixture. The mixture was diluted with water and purified using DEAE Sepharose high-flow resin, followed by further purification using reverse-phase HPLC (Shimadzu instrument; YMC-Actus Triart C8 column (for separation, 250 × 20.0 mm ID); solvent A, 50 mM TEAA buffer containing 0.5% CH3CN (pH 6.0); solvent B, CH3CN; linear gradient 5-80% B (25 min); flow rate, 10 mL / min; detection wavelength, 254 nm). The fraction containing the target compound was collected, concentrated, and freeze-dried to obtain the target N2-1H-imidazole-1-ylmethyl, N7-substituted trinucleotide mRNA Cap1 analog as a triethylammonium salt (Fig. 15).

[0171] According to the above method, a trinucletide-based capping material having a benzyl group connected by a methyl and methylene bridge at the 5′-terminal guanosine N7 position and a thiophene group connected by a methylene bridge, and an imidazole group connected by a methylene bridge at the N2 position introduced ( IM2,m7 GpppAmpG, IM2,Bn7 GpppAmpG, IM2,thio7 GpppAmpG) was synthesized, and the results of its NMR analysis are shown below (Figs. 16 and 17).

[0172] Guanosine N2-1H-imidazol-1-yl methyl, N7-methyl trinucleotide Cap1 analog(1-mIM2, ​​m7GpppAmpG)

[0173] 1H NMR (400 MHz, D2O) δ = 9.06 (s, 1H), 8.85 (s, 1H), 8.25 (s, 1H), 7.95 (s, 1H), 7.84 (s, 1H), 7.63 (s, 1H), 7.32 (s, 1H), 5.93-5.84 (m, 2H), 5.75-5.56 (m, 3H), 4.81 (bs, 1H), 4.63 (t, J = 5.2 Hz, 1H), 4.45-4.05 (m, 13H), 3.90 (s, 3H), 3.33 (s, 3H); 31 P NMR (162 MHz, D2O) δ = -0.92 (s, 1P), -11.46 (t,J= 14.5 Hz, 2P), -22.81 (t,J= 18.6 Hz, 1P).

[0174] Guanosine N2-1H-imidazol-1-yl methyl, N7-benzyl trinucleotide Cap1 유사체 (1-mIM2, Bn7GpppAmpG)

[0175] 1 H NMR (400 MHz, D2O) δ = 8.86 (s, 1H), 8.25 (s, 1H), 8.00 (s, 1H), 7.84 (s, 1H), 7.53 (s, 1H), 7.31 (s, 1H), 7.09 (bs, 2H), 7.00 (bs, 3H), 5.94 (d, 1H), 5.83 (d, 1H), 5.70 (d, 1H), 5.66 (dd, 2H), 5.37 (bs, 2H), 4.82 (m, 1H), 4.67 (t, 2H), 4.53 (t, 1H), 4.40 (m, 2H), 4.37-4.09 (m, 9H), 3.29 (s, 3H); 31 P NMR (162 MHz, D2O) δ = -0.81 (s, 1P), -11.25 (d,J= 19.6 Hz, 1P), -11.52 (d,J= 17.6 Hz, 1P), -22.80 (t,J= 17.8 Hz, 1P).

[0176] Guanosine N2-1H-imidazol-1-yl methyl, N7-(thiophen-2-ylmethyl) trinucleotide Cap1 analog (1-mIM2, ​​2-mTP7GpppAmpG)

[0177] 1 H NMR (400 MHz, DO) δ = 9.38 (s, 1H), 8.85 (s, 1H), 8.26 (s, 1H), 8.01 (s, 1H), 7.84 (s, 1H), 7.52 (s, 1H), 7.29 (s, 1H), 7.06 (m, 1H), 6.66 (bs, 1H), 5.91 (d,J= 4.0 Hz, 1H), 5.83 (d,J= 6.0 Hz, 1H), 5.72-5.62 (m, 3H), 5.57 (bs, 2H), 4.66 (bs, 1H), 4.48 (t,J= 4.4 Hz, 1H), 4.41-4.35 (m, 3H), 4.34-4.28 (m, 3H), 4.24-4.21 (m, 1H), 4.18-4.06 (m, 6H), 3.29 (s, 3H); 31 P NMR (162 MHz, D2O) δ = -0.91 (s, 1P), -11.51 (q,J= 24.1 Hz, 2P), -22.93 (t,J= 19.9 Hz, 1P).

[0178]

[0179] 2. Analysis of antigen protein expression using a capping substance in which N7-benzyl was introduced to N2-Imidazol

[0180] 2-1. Preparation of Luciferase DNA Template

[0181] A linear dsDNA template containing start codons up to the poly-A tail in the luciferase plasmid was prepared by PCR. During serial PCR, the T7 promoter and kozak sequences were added to the luciferase dsDNA template, which was ultimately used for the transcription reaction. The primers used were: primary forward primer: 5′- ATG GAA GAC GCC AAA AAC ATA AAG -3' (Sequence No. 1), secondary forward primer: TAA TAC GAC TCA CTA TAG GGC CAC CAT GGA AGA CGC CAA AAA CAT (Sequence No. 2); reverse primer: AAT CGC GCC TAG GCG CGC CCG TAC GGC TCT TC-3' (Sequence No. 3).

[0182]

[0183] 2-2. Preparation of Luciferase mRNA

[0184] The concentration of the luciferase template was measured using a nanodroplet spectrophotometer. For each transcription reaction, 100 ng of 1 μL of template was placed in a microcentrifuge tube. Then, 1.5 μL of 10X transcription buffer (400 mM Tris-HCl, 60 mM MgCl2, 10 mM DTT, 20 mM spermidine) and 1.5 μL of 100 mM DTT were added to the tube. rATP, rCTP, and rUTP were used, respectively (1 μL, 10 mM). rGTP was used for the cap-dependent transcription of luciferase (1 μL, 2.5 mM). The ratio of the capping agent was maintained four times higher than that of rGTP to provide high capping efficiency. Therefore, 1 μL of the capping agent (10 mM) was added. RNase inhibitor (0.5 μL, 40 U / μL) and T7 RNA polymerase (1 μL, 50 U / μL) were added. Nuclease-free water (4.5–5.5 μL) was added to make the final volume for the reaction 15 μL. The reaction was carried out at 37°C for 1 hour, and after 1 hour, 1.8 μL of 10X DNase I buffer (10 mM Tris-HCl, 2.5 mM MgCl2, 0.5 mM CaCl2) and 1 μL of DNase I (2 U / μL) were added to the reaction, and the mixture was incubated at 37°C for another 15 minutes to remove all DNA templates.

[0185]

[0186] 2-3. Purification of transcribed mRNA

[0187] To remove any dsRNA and transcription byproducts that may have formed, cellulose (10 μL, 0.2 g / mL) was added to the reaction and gently shaken for 10 minutes. Next, the mixture was transferred to an RNA purification column along with the ethanol and binding buffer provided with the column, and finally centrifuged at 13,000 rpm for 1 minute. Then, RNA preparation buffer and wash buffer were added to the column in succession, and centrifuged. At the end of this step, any rTNPs that were not integrated with proteins were removed as a suspension. Finally, a new tube was attached, 10 μL of nuclease-free water was added to the column, and centrifuged at 13,000 rpm for 2 minutes. All dsDNA bound to the cellulose remains in the column. Only pure single-stranded GFP or luciferase mRNA passes through the column. This sample was collected and stored at 70°C for further application.

[0188]

[0189] 2-4. Analysis of Capping Efficiency of Transcribed mRNA

[0190] 2 µg of capped and transcribed mRNA was annealed with 3 µg of gDNA and RNase H buffer by raising to 70°C and then slowly cooling to 25°C. Next, thermally stable RNase H was used to cleave the short sequence containing the capping material from the long mRNA 37 o The reaction was carried out at C for 1 hour. The results of the short sequences containing the cleaved capping material were analyzed by electrophoresis using a 16% denaturing PAGE gel. Short sequences cleaved from uncapped mRNA showed faster gel migration, while short sequences cleaved from capped mRNA showed relatively slower gel migration.

[0191]

[0192] 2-5. Cell Culture and Preparation for Transfection

[0193] HEK 293 cells were cultured in 60 mL culture plates of DMEM containing 10% FBS and 1% Pen / Strip. The day before transfection, cells were detached using trypsin and resuspended in fresh medium. Cells were seeded into 96-well plates. The next day, when the cells were 70% confluent, the transfection mixture was prepared and transfection was performed.

[0194]

[0195] 2-6. Quantification of mRNA and Transformation

[0196] The purified mRNA was quantified using a nanodrop spectrophotometer, and the transcription yields for each rNTP combination were compared. For cell infection, 500 ng of each mRNA was taken and mixed with 5 μL of Opti-MEM. 0.5 μL of Lipofectamine was mixed with 5 μL of Opti-MEM in another tube and incubated at room temperature for 10 minutes. Then, the mRNA and Lipofectamine mixture was transferred to the same tube and incubated at room temperature for another 5 minutes. Finally, the transfection mixture was added to the cells and incubated for 24 hours.

[0197]

[0198] 3. Chemical Synthesis

[0199] 3-1. General Information and Devices Used

[0200] All reagents used in this invention were commercially available and used as is without further purification. ¹H NMR, ¹³C NMR, and ³¹P NMR spectra were measured using a Bruker AV-400 spectrometer; D₂O, CDCl₃-d6, or DMSO-d6 were used as solvents, and tetramethylsilane was used as an internal standard. UV-Vis spectra were measured at room temperature using a Cary Series UV-Vis spectrophotometer (Agilent Technologies) in a quartz cuvette with an optical path length of 1 cm. Changes in absorbance were measured immediately after UV irradiation of the sample solution within the cuvette. Fluorescence emission spectra were measured at room temperature using a PF-65000 fluorescence spectrophotometer.

[0201]

[0202] 3-2. Synthesis of 3′-OMe-Guanosine-5′-monophosphate triethylammonium salt

[0203] 3′-MeO-Guanosine (1 g, 3.3 mmol, 1 equivalent) dried the previous night was dissolved in 0.3 M trimethyl phosphate (6 mL) and stirred at 40°C for 30 minutes. After 30 minutes, the reaction temperature was lowered to -10°C, and POCl₃ (1.2 equivalents) was slowly added dropwise. A color change in the reaction slurry was observed during the dropwise addition, and the reaction was continued for 4 hours under the same temperature conditions. Subsequently, a 0.5 M triethylammonium bicarbonate (Et₃NH₂CO₃, TEAB) solution (20 volume equivalents) was added to quench the reaction, and the mixture was stirred for 30 minutes from 0°C to room temperature. The resulting solution was stored for lyophilization. The freeze-dried compound was dissolved in water (total volume 15-20 mL) and the pH was adjusted to 7.0 to 7.5. It was then purified using DEAE Sepharose fast flow resin according to the method described below. Chromatographic purification was performed using DEAE (diethylaminoethyl, weakly basic anion exchange) Sepharose fast flow resin. The target compound was eluted using a CombiFlash EZ Prep device at a flow rate of 3 mL / min under elution conditions corresponding to four times the resin bed volume, using a gradient of 0% to 20% of 1 M TEAB (triethylammonium bicarbonate) buffer solution (pH 7.5). After combining the fractions containing the product, the mixture was concentrated using a rotary evaporator (water bath temperature 37°C), co-evaporated three times with ethanol, and then dried using a freeze-dryer to obtain the final product (Fig. 18).

[0204] ¹H NMR (400 MHz, D₂O): δ 8.13 (s, 1H), 5.83 (d, J = 16.0 Hz, 1H), 4.84 (t, J = 8.0 Hz, 1H), 4.35 (s, 1H), 4.11-4.09 (m, 1H), 3.90 (s, 2H), 3.47 (s, 3H)

[0205] ³¹P NMR (162 MHz, D₂O): δ 23.82 (s, 1P)

[0206]

[0207] 3-3. Synthesis of 3′-OMe-Guanosine-5′-diphosphate triethylammonium salt

[0208] While stirring the triethylammonium salt of 3′-OMe-guanosine 5′-monophosphate (GMP·TEA) (1200 mg, 2 mmol) dissolved in dimethyl sulfoxide (4 mL), triethylamine (1.7 mL, 12 mmol), imidazole (817 mg, 12 mmol), and 2,2′-dithiodipyridine (1.3 g, 6 mmol) were added. After stirring the mixture for 5 minutes, triphenylphosphine (1.56 g, 6 mmol) dissolved in dimethyl sulfoxide (4 mL) was added. The mixture was then stirred at room temperature for 5 hours (Fig. 19).

[0209] After the reaction was completed, the reaction mixture was slowly poured into a mixture of sodium perchlorate (0.5 g) dissolved in acetone (50 mL). After cooling at 4°C for 30 minutes, the mixture was centrifuged at 5,000 rpm for 8 minutes and the supernatant was removed.

[0210] To remove the residues of imidazole and triphenylphosphine, the precipitate was ground with fresh acetone (20 mL), followed by cooling and centrifugation. This process was repeated one more time, and the final precipitate was vacuum dried at room temperature.

[0211] The 3′-OMe-guanosine 5′-phosphoroimidazolide (Na·3′-OMe-GMP-IM) obtained in this way was dissolved in dimethyl sulfoxide (10 mL), and a 1 M tributylammonium orthophosphate solution (5 mL) in dimethylformamide was added dropwise over 30 minutes while vigorously stirring. Then, anhydrous zinc chloride (200 mg, 1.5 mmol) was added, and the reaction mixture was stirred overnight at room temperature.

[0212] Water (50 mL) was added to the reaction mixture to terminate the reaction, and the mixture was extracted with chloroform (3 × 20 mL). The resulting solution was concentrated using a rotary evaporator (water bath temperature 37°C). The concentrated substance was purified using an anion exchange resin.

[0213] Chromatographic purification was performed using DEAE (diethylaminoethyl, weakly basic anion exchange) Sepharose fast flow resin. The target compound was eluted using a CombiFlash EZ Prep device at a flow rate of 3 mL / min, with an elution condition corresponding to four times the resin bed volume using a gradient of 0% to 30% of 1 M TEAB (triethylammonium bicarbonate) buffer solution (pH 7.5). After combining the fractions containing 3′-OMe-GDP·TEA, the product was concentrated using a rotary evaporator (water bath temperature 37°C), co-evaporated with ethanol three times, and finally dried in a freeze-dryer to obtain a fine white powder product.

[0214] ¹H NMR (400 MHz, D₂O): δ 8.08(s, 1H), 5.83(d, J = 8.0 Hz, 1H), 4.86(t, J = 4.0 Hz, 1H), 4.38(s, 1H), 4.17-4.16(m, 1H), 4.12(t, J = 4.0 Hz, 2H), 3.48(s, 3H)

[0215] ³¹P NMR (162 MHz, D₂O): δ -6.34 (d, J = 22.68 Hz, 1P), -10.94 (d, J = 22.6 Hz, 1P)

[0216]

[0217] 3-4. General method for the synthesis of N7-alkyl, alkenyl, alkynyl, (aryl-2-ylmethyl) and (heteroaryl-2-ylmethyl) guanosine 5′-monophosphate, guanosine 5′-diphosphate, or 3′-OMe-guanosine 5′-diphosphate derivatives

[0218] To a solution in which a triethylammonium salt (0.4 mmol, 1 equivalent) of guanosine 5′-monophosphate, guanosine 5′-diphosphate, or 3′-OMe-guanosine 5′-diphosphate was suspended in dimethyl sulfoxide (DMSO, 2 mL), a suitable CD₃I, alkyl bromide, alkenyl bromide, alkenyl bromide, and 2-bromomethylaryl or 2-bromomethyl heteroaryl compound (2 mmol, 5 equivalents) was added. The mixture was stirred overnight at 37°C to 40°C (Fig. 20).

[0219] After the reaction was completed, the reaction mixture was slowly dropwise added using a 1 mL micropipette to a pre-cooled sodium perchlorate (0.4 g) and acetone (50 mL) solution. Then, a 0.5 M triethylammonium bicarbonate (Et₃NH₂CO₃, TEAB) solution (20 vol-eqv) was added to terminate the reaction, and the mixture was stirred for 30 minutes at a temperature from 0°C to room temperature. Afterward, the obtained solution was stored for freeze-drying.

[0220] The freeze-dried compound was dissolved in water (total volume 15-20 mL), the pH was adjusted to 7 to 7.5, and then purified using DEAE Sepharose Fast Flow resin. Chromatographic purification was performed using DEAE (diethylaminoethyl, weakly basic anion exchange) Sepharose fast flow resin. The target compound was eluted using a CombiFlash EZ Prep device at a flow rate of 3 mL / min under elution conditions corresponding to four times the resin volume (bed volume) using a gradient condition of 0% to 20% of 1 M TEAB (triethylammonium bicarbonate) buffer solution (pH 7.5). After combining the fractions containing the product, the solution was concentrated using a rotary evaporator (water bath temperature 37°C), co-evaporated three times with ethanol, and then dried in a freeze-dryer.

[0221]

[0222] 3-4-1. N7-Benzyl Guanosine 5′-Monophosphate Triethylammonium Salt

[0223] N7-Bn-GMP·TEA(270 mg, 82%)

[0224] ¹H NMR (400 MHz, D₂O): δ 7.39 (m, 5H), 6.05 (d, J = 4 Hz, 1H), 5.64 (s, 2H), 4.68 (d, J = 4.7 Hz, 1H), 4.46 (t, J = 4.9 Hz, 1H), 4.40 (q, J = 2.3 Hz, 1H), 4.18-4.13 (m, 1H), 4.06-4.01 (m, 1H)

[0225] ³¹P NMR (162 MHz, D₂O):δ 2.30(s, 1P)

[0226] ESI MS: Calculated value m / z for C₁7H₁9N5O8P 452.34 ([M]), measured value 451.2 ([M-1])

[0227] (The above spectrum data is described in the applicant's previous patent literature)

[0228]

[0229] 3-4-2. N7-Deuteride Methyl (CD₃) Guanosine 5′-Diphosphate Triethylammonium Salt

[0230] N7-CD₃-GDP·TEA (174 mg, 81%)

[0231] ¹H NMR (400 MHz, D₂O): δ 5.96 (d, J = 4.0 Hz, 1H), 4.58-4.56 (m, 1H), 4.47 (t, J = 4.0 Hz, 1H), 4.30-4.23 (m, 2H), 4.18-4.13 (m, 1H)

[0232] ³¹P NMR (162 MHz, D₂O): δ -8.03 (d, J = 21.0 Hz, 1P), -11.11 (d, J = 22.6 Hz, 1P)

[0233]

[0234] 3-4-3. N7-ethylguanosine 5′-diphosphate triethylammonium salt

[0235] N7-Ethyl-GDP·TEA (187 mg, 86%)

[0236] ¹H NMR (400 MHz, D₂O): δ 5.97 (d, J = 4.0 Hz, 1H), 4.59 (t, J = 4.0 Hz, 1H), 4.50 (t, J = 8.0 Hz, 1H), 4.42 (dd, J₁ = 4.0 Hz, J₂ = 8.0 Hz, 2H), 4.28(s, 1H), 4.20(s, 2H), 1.45(t, J = 8.0 Hz, 3H)

[0237] ³¹P NMR (162 MHz, D₂O): δ -6.81 (d, J = 22.6 Hz, 1P), -11.05 (d, J = 24.3 Hz, 1P)

[0238]

[0239] 3-4-4. N7-allyl guanosine 5′-diphosphate triethylammonium salt

[0240] N7-Allyl-GDP·TEA (168 mg, 77%)

[0241] ¹H NMR (400 MHz, D₂O): δ 6.09-6.00 (m, 1H), 5.98 (d, J = 4.0 Hz, 1H), 5.28-5.17 (m, 2H), 5.02-5.01 (m, 2H), 4.58 (t, J = 4.0 Hz, 1H), 4.50(t, J = 4.0 Hz, 1H), 4.42-4.27(m, 2H), 4.19(s, 1H)

[0242] ³¹P NMR (162 MHz, D₂O):δ -7.23(d, J = 22.6 Hz, 1P), -10.97(d, J = 22.6 Hz, 1P

[0243]

[0244] 3-4-5. N7-Propargyl Guanosine 5′-Diphosphate Triethylammonium Salt

[0245] N7-Propargyl-GDP·TEA (147 mg, 46%)

[0246] ¹H NMR (400 MHz, D₂O): δ 5.99 (d, J = 4.30 Hz, 1H), 5.26 (d, J = 4.0 Hz, 2H), 4.62 (d, J = 4.0 Hz, 1H), 4.50 (t, J = 8.0 Hz, 1H), 4.30(t, J₁ = 3.40 Hz, J₂ = 4.0 Hz, 1H), 4.23-4.17(m, 2H), 2.93(s, 1H)

[0247] ³¹P NMR (162 MHz, D₂O): δ -7.56 (d, J = 22.68 Hz, 1P), -11.08 (d, J = 22.68 Hz, 1P)

[0248]

[0249] 3-4-6. N7-Benzyl Guanosine 5′-Diphosphate Triethylammonium Salt

[0250] N7-Bn-GDP·TEA(198 mg, 86%)

[0251] ¹H NMR (400 MHz, D₂O): δ 7.33 (s, 5H), 5.97 (d, J = 3.6 Hz, 1H), 5.60 (s, 2H), 4.63 (t, J = 4.0 Hz, 1H), 4.52 (t, J = 4.0 Hz, 1H), 4.30(s, 1H), 4.22(s, 2H)

[0252] ³¹P NMR (162 MHz, D₂O): δ -7.24 (d, J = 17.8 Hz, 1P), -10.98 (d, J = 21.0 Hz, 1P)

[0253]

[0254] 3-4-7. N7-4-fluorobenzyl guanosine 5′-diphosphate triethylammonium salt

[0255] N7-4-F-Bn-GDP·TEA (155 mg, 66%)

[0256] ¹H NMR (400 MHz, D₂O): δ 7.41-7.37 (m, 2H), 7.04 (t, J = 8.0 Hz, 2H), 5.96 (d, J = 4.0 Hz, 1H), 5.58 (d, J = 8.0 Hz, 2H), 4.60 (t, J = 4.0 Hz, 1H), 4.52(t, J = 4.0 Hz, 1H), 4.29(s, 1H), 4.22(m, 2H)

[0257] ³¹P NMR (162 MHz, D₂O): δ -6.64 (d, J = 22.6 Hz, 1P), -10.97 (d, J = 24.3 Hz, 1P)

[0258]

[0259] 3-4-8. N7-4-Bromobenzyl Guanosine 5′-Diphosphate Triethylammonium Salt

[0260] N7-4-Br-Bn-GDP·TEA (120 mg, 49%)

[0261] ¹H NMR (400 MHz, D₂O): δ 7.71 (d, J = 8.0 Hz, 1H), 7.15 (d, J = 8.0 Hz, 1H), 6.01 (d, J = 4.0 Hz, 1H), 5.59 (s, 2H), 4.54 (t, J = 4.0 Hz, 1H), 4.32(m, 1H), 4.28(s, 1H), 4.24(s, 1H)

[0262] ³¹P NMR (162 MHz, D₂O): δ -7.18 (d, J = 24.3 Hz, 1P), -10.95 (d, J = 24.3 Hz, 1P)

[0263]

[0264] 3-4-9. N7-4-iodobenzyl guanosine 5′-diphosphate triethylammonium salt

[0265] N7-4-I-Bn-GDP·TEA (132 mg, 52%)

[0266] ¹H NMR (400 MHz, D₂O): δ 7.69 (d, J = 8.0 Hz, 1H), 7.11 (d, J = 8.0 Hz, 1H), 5.98 (s, 1H), 5.57 (d, J = 8.0 Hz, 2H), 4.57 (s, 1H), 4.51(t, J = 4.0 Hz, 1H), 4.28(s, 1H), 4.20(s, 1H)

[0267] ³¹P NMR (162 MHz, D₂O): δ -6.36 (d, J = 22.6 Hz, 1P), -10.97 (d, J = 24.3 Hz, 1P)

[0268]

[0269] 3-4-10. N7-3-Methoxybenzyl Guanosine 5′-Diphosphate Triethylammonium Salt

[0270] N7-3-MeO-Bn-GDP·TEA (126 mg, 53%)

[0271] ¹H NMR (400 MHz, D₂O): δ 7.27 (t, J = 8.0 Hz, 1H), 6.96-6.87 (m, 3H), 5.97 (d, J = 4.0 Hz, 1H), 5.60 (d, J = 8.0 Hz, 2H), 4.60 (t, J = 8.0 Hz, 1H), 4.51(t, J = 4 Hz, 1H), 4.21-4.19(m, 2H), 3.73(s, 3H)

[0272] ³¹P NMR (162 MHz, D₂O): δ -6.47 (d, J = 24.3 Hz, 1P), -11.01 (d, J = 22.6 Hz, 1P)

[0273]

[0274] 3-4-11. N7-(thiophene-2-ylmethyl)guanosine 5′-diphosphate triethylammonium salt

[0275] N7-2mTHP-GDP·TEA (155 mg, 42%)

[0276] ¹H NMR (400 MHz, D₂O): δ 7.36 (d, J = 5.2 Hz, 1H), 7.27 (d, J = 3.6 Hz, 1H), 6.96 (t, J = 5.2 Hz, 1H), 5.94 (d, J = 3.6 Hz, 1H), 5.80 (dd, J₁ = 18.0 Hz, J₂ = 15.2 Hz, 2H), 4.62(t, J = 3.6 Hz, 1H), 4.48(t, J = 5.6 Hz, 1H), 4.32-4.27(m, 1H), 4.26-4.14(m, 2H)

[0277] ³¹P NMR (162 MHz, D₂O): δ -8.34 (d, J = 22.4 Hz, 1P), -11.10 (d, J = 22.5 Hz, 1P)

[0278]

[0279] 3-4-12. N7-(furan-2-ylmethyl)guanosine 5′-diphosphate triethylammonium salt

[0280] N7-(furan-2-ylmethyl)-GDP·TEA (104 mg, 48%)

[0281] ¹H NMR (400 MHz, D₂O): δ 7.41 (m, 1H), 6.58 (d, J = 4.0 Hz, 1H), 6.36-6.35 (m, 1H), 5.93 (d, J = 4.0 Hz, 2H), 5.61 (d, J = 4.0 Hz, 2H), 4.62(t, J = 4.0 Hz, 1H), 4.42(t, J = 4.0 Hz, 1H), 4.31-4.29(m, 1H), 4.25-4.20(m, 1H)

[0282] ³¹P NMR (162 MHz, D₂O): δ -10.42 (d, J = 21.0 Hz, 1P), -11.27 (d, J = 19.4 Hz, 1P)

[0283]

[0284] 3-4-13. N7-(pyridine-2-ylmethyl)guanosine 5′-diphosphate triethylammonium salt

[0285] N7-2mPy-GDP·TEA(213 mg, 58%)

[0286] ¹H NMR (400 MHz, D₂O): δ 8.35 (d, J = 5.6 Hz, 1H), 7.70 (td, J₁ = 8 Hz, J₂ = 2 Hz, 1H), 7.35-7.29 (m, 2H), 6.04 (d, J = 3.2 Hz, 1H), 5.75(d, J = 5.2 Hz, 2H), 4.64(dd, J₁ = 4.8 Hz, J₂ = 3.2 Hz, 1H), 4.55(dd, J₁ = 6.0 Hz, J₂ = 4.8 Hz, 1H), 4.36(dd, J₁ = 6.0 Hz, J₂ = 2.4 Hz, 1H), 4.22(dd, J₁ = 5.2 Hz, J₂ = 2.4 Hz, 1H)

[0287] ³¹P NMR (162 MHz, D₂O): δ -6.34 (d, J = 22.5 Hz, 1P), -10.95 (d, J = 23.5 Hz, 1P)

[0288]

[0289] 3-4-14. 3′-OMe-guanosine N7-benzyl 5′-diphosphate triethylammonium salt

[0290] N7-Bn-3′-OMe-GDP·TEA (198 mg, 86%)

[0291] ¹H NMR (400 MHz, D₂O): δ 9.35(s, 1H), 7.38(s, 5H), 6.00(s, 1H), 5.61(s, 2H), 4.83(s, 1H), 4.45(s, 1H), 4.27(s, 1H), 4.15(s, 2H), 3.44(s, 3H)

[0292] ³¹P NMR (162 MHz, D₂O): δ -10.81 (d, J = 19.44 Hz, 2P), -11.37 (d, J = 21.06 Hz, 1P)

[0293]

[0294] 3-5. Chemical Synthesis of Dinucleotides pAmpG, pm6AmpG, and pAmoepG

[0295] 3-5-1. Synthesis of the dinucleotide pAmpG

[0296] The synthesis method is shown in Fig. 21.

[0297]

[0298] 3-5-2. Synthesis of 5′-O-DMT-2′-O-methyl adenosyl(n-bz)-{3′-OP-[2-cyanoethyl]→5′}-2′,3′-diacetyl guanosine(n-ibu): 3

[0299] DMT-2′-O-methyl adenosine (n-bz) CED phosphoramidite 1 (1.29 g, 1.45 mmol) and 2′,3′-diacetyl guanosine (n-ibu) 2 (0.58 g, 1.32 mmol) were dried under high vacuum conditions for 16 hours. Then, anhydrous DMF (1.7 mL) and a 0.45 M tetrazole solution in acetonitrile (7.3 mL, 3.30 mmol) were added, and the mixture was stirred at room temperature for 3 hours under an argon (or nitrogen) atmosphere. After confirming the consumption of Compound 1 using TLC, the reaction mixture was cooled in an ice-salt bath for 30 minutes. Next, 70% t-butyl hydroperoxide in water (0.91 mL, 6.60 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. After confirming the conversion of the intermediate using TLC, it was extracted with ethyl acetate (2 × 200 mL) and washed with a 5% NaHCO₃ aqueous solution (2 × 100 mL). The organic layers were combined, dried with sodium sulfate, and the solvent was evaporated to obtain crude solid 3 (1.77 g).

[0300]

[0301] 3-5-3. Synthesis of 2′-O-methyl adenosyl(n-bz)-{3′-OP-[2-cyanoethyl]→5′}-2′,3′-diacetyl guanosine(n-ibu): 4

[0302] A crude dinucleotide 3 solid (1.77 g) was dissolved in a 3% trichloroacetic acid solution (31.2 mL, 5.72 mmol) in dichloromethane. The mixture was stirred at room temperature for 1 hour. After confirming the conversion of compound 3 using TLC, a 5% NaHCO₃ aqueous solution (30 mL) was added and stirred for 10 minutes. Subsequently, the mixture was extracted with dichloromethane, and the solvent was evaporated.

[0303] Next, the resulting residue was purified by silica gel column chromatography to obtain product 4 (0.74 g, 0.79 mmol, 60%).

[0304] ¹H NMR (DMSO, 400 MHz): δ 12.12 (s, 1H), 11.59 (d, J = 4.0 Hz, 1H), 11.26 (d, J = 4.0 Hz, 1H), 7.93 (dd, J₁ = 4.0 Hz, J₂ = 4.0 Hz, 2H), 8.28 (d, J = 4.0 Hz, 1H), 8.06 (d, J = 4.0 Hz, 2H), 7.66 (t, J = 4.0 Hz, 1H), 7.57 (d, J = 4.0 Hz, 2H), 6.19 (d, J = 4.0 Hz, 1H), 6.14 (dd, J₁ = 4.0 Hz, J₂ = 4.0 Hz, 1H), 5.84(q, J₁ = 4.3 Hz, J₂ = 4.3 Hz, 1H), 5.54(dd, J₁ = 8.0 Hz, J₂ = 8.0 Hz, 1H), 5.39(t, J = 4.2 Hz, 1H), 5.20(m, 1H), 4.83 (m, 1H), 4.48 (m, 3H), 4.30 (m, 3H), 3.64 (m, 2H), 3.37 (d, J = 4.0 Hz, 3H), 2.97 (q, J₁ = 4.4 Hz, J₂ = 4.4 Hz, 2H), 2.78(m, 1H), 2.15(d, J = 4.0 Hz, 3H), 2.03(d, J = 4.6 Hz, 3H), 1.13(m, 6H)

[0305] ³¹P NMR (DMSO, 162 MHz):δ -2.43 (d, J = 9.7 Hz, 1P)

[0306]

[0307] 3-5-4. Synthesis of 5′-O-phosphoryl-2′-O-methyladenosilyl-{3′-OP→5′} guanosine (pAmpG): 5

[0308] Dinucleotide 4 (0.74 g, 0.7891 mmol) was dried under high vacuum conditions for 16 hours. Then, 0.45 M tetrazole solution in acetonitrile (3.51 mL, 1.58 mmol) and bis-(2-cyanoethyl)-N,N-diisopropyl-phosphoramidite (0.41 mL, 1.58 mmol) were added, and the mixture was stirred at room temperature for 3 hours under an argon (or nitrogen) atmosphere.

[0309] After confirming whether compound 4 was consumed, the reaction mixture was cooled in an ice-salt bath for 30 minutes, and 70% t-butyl hydroperoxide (17.2 mL, 3.16 mmol) in water was added. This was stirred at room temperature for 1 hour.

[0310] After confirming the conversion of the intermediate using TLC, the mixture was extracted with ethyl acetate (2 × 200 mL) and washed with a 5% NaHCO₃ aqueous solution (2 × 100 mL). The extracted organic layers were combined, and the solvent was evaporated.

[0311] Next, the resulting residue was dissolved in methanol (25 mL) and an aqueous solution of concentrated ammonia (25 mL) and stirred at 55°C for 7 hours. After the deprotection reaction was completed, the reaction mixture was co-evaporated with methanol.

[0312] The generated residue was dissolved in water, the pH was adjusted to 5.5, and then loaded onto a DEAE-Sepharose column. Solvent A is 1 M TEAB buffer solution (pH 7.5-8), and solvent B is water.

[0313] The fractions containing the product were combined, concentrated, and freeze-dried to obtain 5 (446 mg, 0.4908 mmol, 62%) of fine white powder in the form of triethylammonium salt.

[0314]

[0315] 3-5-5. Triethylammonium (TEA) salt of dinucleotide (pAmpG)

[0316] ¹H NMR (D₂O, 400 MHz): δ 8.48 (s, 1H), 8.08 (s, 1H), 7.86 (s, 1H), 6.02 (d, J = 4.0 Hz, 1H), 5.75 (d, J = 8.0 Hz, 1H), 4.88-4.82(m, 1H), 4.46-4.41(m, 2H), 4.38(s, 1H), 4.26(t, J = 4.0 Hz, 1H), 4.12(s, 2H), 3.92(d, J = 4.0 Hz, 2H), 3.37(s, 3H)

[0317] ³¹P NMR (D₂O, 162 MHz):δ 2.88(s, 1P), -0.86(s, 1P)

[0318]

[0319] 3-5-6. Method for Synthesizing the Dinucleotide pm6AmpG

[0320] The synthesis method is shown in Fig. 22.

[0321]

[0322] 3-5-7. Synthesis of 5′-O-DMT-2′-O-methyl-N6-methyladenosilyl(n-bz)phosphoramidite (5′-O-DMT-m6Am PAC): 1

[0323] This compound was synthesized by modifying the method reported by Sikorski et al. (2020).

[0324] A suspension of DMT-2′-O-methyl adenosine (n-bz) CED phosphoramidite (1.50 g) was prepared in dichloromethane (DCM), stirred at room temperature for 10 minutes, and then methyl iodide was slowly added. Next, an aqueous solution of tetrabutylammonium bromide (0.1 M, 8 mL) was added, followed by the addition of an aqueous solution of 1 M NaOH (8 mL).

[0325] The above mixture was vigorously stirred at room temperature for 1 hour. After confirming the consumption of the starting material via TLC, the reaction mixture was diluted with water (100 mL) and diethyl ether (100 mL).

[0326] The organic layer was separated, and the aqueous layer was extracted twice with diethyl ether (50 mL). The organic layers were combined, dried with anhydrous Na₂SO₄, filtered, and the solvent evaporated. The resulting residue was purified by silica gel column chromatography to obtain the product. The compound (0.80 g) contained small amounts of unidentified impurities exhibiting chemical shifts of 12.7 ppm and 12.6 ppm on ³¹P NMR, and very small amounts of unidentified signals were observed at 8.4 ppm, 0.9 ppm, and -0.8 ppm. The process proceeded to the next step without further purification, and subsequent steps were performed according to a method substantially identical to the general synthesis procedure for dinucleotide pAmpG.

[0327]

[0328] 3-5-8. Synthesis of 5′-O-DMT-2′-O-methyl-N6-methyladenosilyl(n-bz)-{3′-OP-[2-cyanoethyl]→5′}-2′,3′-diacetylguanosine(n-ibu): 4

[0329] DMT-2′-O-methyl-N6-methyl adenosine (n-bz) CED phosphoramidite 2 (0.80 g, 0.88 mmol) and 2′,3′-diacetyl guanosine (n-ibu) 3 (0.46 g, 1.05 mmol) were dried under high vacuum conditions for 16 hours.

[0330] Next, 0.45 M tetrazole solution (4.5 mL, 2.02 mmol) in anhydrous DMF (1.7 mL) and acetonitrile was added, and the mixture was stirred at room temperature for 3 hours under an argon (or nitrogen) atmosphere. After confirming the consumption of compound 1 using TLC, the reaction mixture was cooled in an ice-salt bath for 30 minutes, and 70% t-butyl hydroperoxide (0.56 mL, 4.04 mmol) in water was added. The reaction mixture was stirred at room temperature for 1 hour. After confirming the conversion of the intermediate using TLC, the mixture was extracted with ethyl acetate (2 × 200 mL) and washed with a 5% NaHCO₃ aqueous solution (2 × 100 mL). The extracted organic layers were combined, dried with sodium sulfate, and the solvent was evaporated to obtain crude solid 4 (1.20 g).

[0331]

[0332] 3-5-9. Synthesis of 2′-O-methyl-N6-methyladenosilyl(n-bz)-{3′-OP-[2-cyanoethyl]→5′}-2′,3′-diacetylguanosine(n-ibu): 5

[0333] A crude dinucleotide 4 solid (1.20 g) was dissolved in a 3% trichloroacetic acid solution (31.2 mL, 3.52 mmol) in dichloromethane. This was stirred at room temperature for 1 hour.

[0334] After confirming whether compound 4 was converted using TLC, a 5% NaHCO₃ aqueous solution (30 mL) was added and stirred for 10 minutes.

[0335] Subsequently, the mixture was extracted with dichloromethane and the solvent was evaporated. Then, the resulting residue was purified by silica gel column chromatography to obtain product 5 (0.48 g).

[0336] ¹H NMR (DMSO, 400 MHz): δ 12.11 (s, 1H), 11.57 (s, 1H), 8.71 (d, J = 4.0 Hz, 1H), 8.59 (d, J = 8.0 Hz, 1H), 8.25 (d, J = 4.0 Hz, 1H), 7.33 (d, J = 4.0 Hz, 3H), 7.24 (d, J = 8.0 Hz, 2H), 6.13-6.08 (m, 2H), 5.82 (t, J = 4.0 Hz, 1H), 5.52 (t, J = 4.0 Hz, 1H), 5.32 (s, 1H), 5.14(s, 1H), 4.70(q, J₁ = 4.0 Hz, J₂ = 4.0 Hz, 1H), 4.46-4.41(m, 3H), 4.25(t, J = 8.0 Hz, 3H), 3.36(s, 3H), 3.58(d, J = 4.0 Hz, 2H), 3.31(s, 1H), 3.23(s, 4H), 2.94(q, J₁ = 4.0 Hz, J₂ = 4.0 Hz, 2H), 2.76(q, J₁ = 8.0 Hz, J₂ = 8.0 Hz, 1H), 2.12(d, J = 4.0 Hz, 3H), 2.01(d, J = 4.0 Hz, 3H), 1.12(s, 3H)

[0337] ³¹P NMR (DMSO, 162 MHz):δ -2.46 (d, J = 11.34 Hz, 1P)

[0338]

[0339] 3-5-10. Synthesis of 5′-O-phosphoryl-2′-O-methyl-N6-methyladenosilyl-{3′-OP→5′} guanosine (pm6AmpG): 6

[0340] Dinucleotide 5 (0.48 g, 0.50 mmol) was dried under high vacuum conditions for 16 hours. Then, 0.45 M tetrazole solution in acetonitrile (2.23 mL, 1.05 mmol) and bis-(2-cyanoethyl)-N,N-diisopropyl-phosphoramidite (0.25 mL, 1.00 mmol) were added, and the mixture was stirred at room temperature for 3 hours under an argon (or nitrogen) atmosphere. After checking whether compound 5 was consumed, the reaction mixture was cooled in an ice-salt bath for 30 minutes, and 70% t-butyl hydroperoxide in water (10.88 mL, 2.00 mmol) was added.

[0341] This was stirred at room temperature for 1 hour. After confirming the conversion of the intermediate using TLC, it was extracted with ethyl acetate (2 × 200 mL) and washed with a 5% NaHCO₃ aqueous solution (2 × 100 mL). The extracted organic layers were combined and the solvent was evaporated. Next, the resulting residue was dissolved in methanol (25 mL) and a concentrated ammonia aqueous solution (25 mL) and stirred at 55°C for 7 hours. After the deprotection reaction was completed, the reaction mixture was co-evaporated with methanol. The resulting residue was dissolved in water, the pH was adjusted to 5.5, and then loaded onto a DEAE-Sepharose column. Solvent A was 1 M TEAB buffer solution (pH 7.5-8), and solvent B was water. The fractions containing the product were combined, concentrated, and freeze-dried to obtain a fine white powder 6 (0.27 g, 62%) in the form of a triethylammonium salt.

[0342]

[0343] 3-5-11. Triethylammonium (TEA) salt of dinucleotide (pm6AmpG)

[0344] ¹H NMR (400 MHz, D₂O): δ 8.46 (s, 1H), 8.09 (s, 1H), 7.84 (s, 1H), 6.04 (d, J = 4.0 Hz, 1H), 5.73 (d, J = 8.0 Hz, 1H), 4.86-4.83 (m, 1H), 4.46(t, J = 4.0 Hz, 1H), 4.38(s, 1H), 4.26(s, 1H), 4.12(s, 2H), 3.98-3.89(m, 2H), 3.41(s, 3H), 3.01(s, 3H)

[0345] ³¹P NMR (162 MHz, D₂O):δ 3.50(s, 1P), -0.87(s, 1P)

[0346]

[0347] 3-5-12. Method for synthesizing dinucleotide pAmoepG (Fig. 23)

[0348]

[0349] 3-5-13. Synthesis of 5′-O-DMT-2′-methoxyethyl adenosyl(n-bz)-{3′-OP-[2-cyanoethyl]→5′}-2′,3′-diacetyl guanosine(n-ibu): 3

[0350] DMT-2′-methoxyethyl adenosine (n-bz) CED phosphoramidite 1 (1.55 g, 1.45 mmol) and 2′,3′-diacetyl guanosine (n-ibu) 2 (0.58 g, 1.32 mmol) were dried under high vacuum conditions for 16 hours. Then, 0.45 M tetrazole solution in anhydrous DMF (1.7 mL) and acetonitrile (7.3 mL, 3.30 mmol) were added, and the mixture was stirred at room temperature for 3 hours under an argon (or nitrogen) atmosphere. After confirming the consumption of Compound 1 using TLC, the reaction mixture was cooled in an ice-salt bath for 30 minutes, and 70% t-butyl hydroperoxide in water (0.91 mL, 6.60 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. After confirming whether the intermediate was converted using TLC, it was extracted with ethyl acetate (2 × 200 mL) and washed with a 5% NaHCO₃ aqueous solution (2 × 100 mL). The extracted organic layers were combined, dried with sodium sulfate, and then the solvent was evaporated to obtain crude solid 3 (1.80 g).

[0351]

[0352] 3-5-14. Synthesis of 2′-Methoxyethyl Adenosyl(n-bz)-{3′-OP-[2-Cyanoethyl]→5′}-2′,3′-Diacetyl Guanosine(n-ibu): 4

[0353] A crude dinucleotide 3 solid (1.80 g) was dissolved in a 3% trichloroacetic acid solution (31.2 mL, 5.72 mmol) in dichloromethane. The mixture was stirred at room temperature for 1 hour. After confirming the conversion of compound 3 using TLC, a 5% NaHCO₃ aqueous solution (30 mL) was added and stirred for 10 minutes. Subsequently, the mixture was extracted with dichloromethane and the solvent was evaporated. Then, the resulting residue was purified by silica gel column chromatography to obtain product 4 (0.75 g).

[0354]

[0355] 3-5-15. Synthesis of 5′-O-phosphoryl-2′-methoxyethyl adenosylyl-{3′-OP→5′} guanosine (pAmoepG): 5

[0356] Dinucleotide 4 (0.75 g, 0.7891 mmol) was dried under high vacuum conditions for 16 hours. Then, 0.45 M tetrazole solution in acetonitrile (3.51 mL, 1.58 mmol) and bis-(2-cyanoethyl)-N,N-diisopropyl-phosphoramidite (0.41 mL, 1.58 mmol) were added, and the mixture was stirred at room temperature for 3 hours under an argon (or nitrogen) atmosphere.

[0357] After checking whether compound 4 was consumed, the reaction mixture was cooled in an ice-salt bath for 30 minutes, and 70% t-butyl hydroperoxide (17.2 mL, 3.16 mmol) in water was added.

[0358] This was stirred at room temperature for 1 hour. After confirming the conversion of the intermediate using TLC, it was extracted with ethyl acetate (2 × 200 mL) and washed with a 5% NaHCO₃ aqueous solution (2 × 100 mL). The extracted organic layers were combined and the solvent was evaporated. Next, the resulting residue was dissolved in methanol (25 mL) and a concentrated ammonia aqueous solution (25 mL) and stirred at 55°C for 7 hours. After the deprotection reaction was completed, the reaction mixture was co-evaporated with methanol. The resulting residue was dissolved in water, the pH was adjusted to 5.5, and then loaded onto a DEAE-Sepharose column. Solvent A was 1 M TEAB buffer solution (pH 7.5-8), and solvent B was water. The fractions containing the product were combined, concentrated, and freeze-dried to obtain a fine white powder 5 (500 mg, 60%) in the form of a triethylammonium salt.

[0359]

[0360] 3-5-16. Triethylammonium (TEA) salt of dinucleotide (pAmoepG)

[0361] ¹H NMR (400 MHz, D₂O): δ 8.43 (s, 1H), 8.11 (s, 1H), 7.89 (s, 1H), 6.02 (d, J = 8.0 Hz, 1H), 5.75 (d, J = 4.0 Hz, 1H), 4.81 (s, 1H), 4.52(s, 1H), 4.42(s, 2H), 4.25(s, 1H), 4.12(s, 2H), 3.94(s, 2H), 3.75-3.71(m, 1H), 3.58-3.53(m, 1H), 3.37-3.28(m, 2H), 3.02(s, 3H)

[0362] ³¹P NMR (162 MHz, D₂O):δ 1.91(s, 1P), -0.77(s, 1P)

[0363]

[0364] 3-6. Chemical Synthesis of Trinucleotide pAmpAmpG

[0365] 3-6-1. Method for Synthesizing Trinucleotide pAmpAmpG

[0366] The synthesis method is shown in Fig. 24.

[0367]

[0368] 3-6-2. Synthesis of 5′-O-DMT-2′-O-methyl adenosyl(n-bz)-{3′-OP-[2-cyanoethyl]→5′}-2′,3′-diacetyl guanosine(n-ibu): 3

[0369] DMT-2′-O-methyl adenosine (n-bz) CED phosphoramidite 1 (1.29 g, 1.45 mmol) and 2′,3′-diacetyl guanosine (n-ibu) 2 (0.58 g, 1.32 mmol) were dried under high vacuum conditions for 16 hours. Then, 0.45 M tetrazole solution in anhydrous DMF (1.7 mL) and acetonitrile (7.3 mL, 3.30 mmol) was added, and the mixture was stirred at room temperature for 3 hours under an argon (or nitrogen) atmosphere. After confirming the consumption of compound 1 using TLC, the reaction mixture was cooled in an ice-salt bath for 30 minutes, and 70% t-butyl hydroperoxide in water (0.91 mL, 6.60 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. After confirming the conversion of the intermediate using TLC, it was extracted with ethyl acetate (2 × 200 mL) and washed with a 5% NaHCO₃ aqueous solution (2 × 100 mL). The extracted organic layers were combined, dried with sodium sulfate, and the solvent was evaporated to obtain crude solid 3 (1.77 g).

[0370]

[0371] 3-6-3. Synthesis of 2′-O-methyl adenosyl(n-bz)-{3′-OP-[2-cyanoethyl]→5′}-2′,3′-diacetyl guanosine(n-ibu): 4

[0372] A crude dinucleotide 3 solid (1.77 g) was dissolved in a 3% trichloroacetic acid solution (31.2 mL, 5.72 mmol) in dichloromethane. The mixture was stirred at room temperature for 1 hour. After confirming the conversion of compound 3 using TLC, a 5% NaHCO₃ aqueous solution (30 mL) was added and stirred for 10 minutes. Subsequently, the mixture was extracted with dichloromethane and the solvent was evaporated. Then, the resulting residue was purified by silica gel column chromatography to obtain product 4 (0.74 g, 0.79 mmol, 60%).

[0373] ¹H NMR (DMSO, 400 MHz): δ 12.12 (s, 1H), 11.59 (d, J = 4.0 Hz, 1H), 11.26 (d, J = 4.0 Hz, 1H), 7.93 (dd, J₁ = 4.0 Hz, J₂ = 4.0 Hz, 2H), 8.28 (d, J = 4.0 Hz, 1H), 8.06 (d, J = 4.0 Hz, 2H), 7.66 (t, J = 4.0 Hz, 1H), 7.57 (d, J = 4.0 Hz, 2H), 6.19 (d, J = 4.0 Hz, 1H), 6.14 (dd, J₁ = 4.0 Hz, J₂ = 4.0 Hz, 1H), 5.84(q, J₁ = 4.3 Hz, J₂ = 4.3 Hz, 1H), 5.54(dd, J₁ = 8.0 Hz, J₂ = 8.0 Hz, 1H), 5.39(t, J = 4.2 Hz, 1H), 5.20(m, 1H), 4.83 (m, 1H), 4.48 (m, 3H), 4.30 (m, 3H), 3.64 (m, 2H), 3.37 (d, J = 4.0 Hz, 3H), 2.97 (q, J₁ = 4.4 Hz, J₂ = 4.4 Hz, 2H), 2.78(m, 1H), 2.15(d, J = 4.0 Hz, 3H), 2.03(d, J = 4.6 Hz, 3H), 1.13(m, 6H)

[0374] ³¹P NMR (DMSO, 162 MHz):δ -2.43 (d, J = 9.7 Hz, 1P)

[0375]

[0376] 3-6-4. Synthesis of 5′-O-DMT-2′-O-methyl adenosyl(n-bz)-{3′-OP-[2-cyanoethyl]→2′-O-methyl adenosyl(n-bz)-{3′-OP-[2-cyanoethyl]→5′}-2′,3′-diacetyl guanosine(n-ibu): 5

[0377] DMT-2′-O-methyl adenosine(n-bz) CED phosphoramidite 1 (1.29 g, 1.45 mmol) and compound 4 (0.74 g, 0.79 mmol) were dried under high vacuum conditions for 16 hours.

[0378] Next, 0.45 M tetrazole solution (7.3 mL, 3.30 mmol) in anhydrous DMF (1.7 mL) and acetonitrile was added, and the mixture was stirred at room temperature for 3 hours under an argon (or nitrogen) atmosphere. After confirming the consumption of compound 1 using TLC, the reaction mixture was cooled in an ice-salt bath for 30 minutes, and 70% t-butyl hydroperoxide (0.91 mL, 6.60 mmol) in water was added. The reaction mixture was stirred at room temperature for 1 hour. After confirming the conversion of the intermediate using TLC, the mixture was extracted with ethyl acetate (2 × 200 mL) and washed with a 5% NaHCO₃ aqueous solution (2 × 100 mL). The extracted organic layers were combined, dried with sodium sulfate, and the solvent was evaporated to obtain crude solid 5 (1.6 g).

[0379]

[0380] 3-6-5. Synthesis of 2′-O-methyl adenosyl(n-bz)-{3′-OP-[2-cyanoethyl]→2′-O-methyl adenosyl(n-bz)-{3′-OP-[2-cyanoethyl]→5′}-2′,3′-diacetyl guanosine(n-ibu): 6

[0381] A crude trinucleotide 5 solid (1.6 g) was dissolved in a 3% trichloroacetic acid solution (31.2 mL, 5.72 mmol) in dichloromethane. The mixture was stirred at room temperature for 1 hour. After confirming the progress of the reaction using TLC, a 5% NaHCO₃ aqueous solution (30 mL) was added and stirred for 10 minutes. Subsequently, the mixture was extracted with dichloromethane and the solvent was evaporated. Then, the resulting residue was partially purified by silica gel column chromatography to obtain crude product 6 (0.90 g).

[0382]

[0383] 3-6-6. Synthesis of 5′-O-phosphoryl-2′-O-methyladenosilyl-{3′-OP→5′}-2′-O-methyladenosine-{3′-OP→5′}guanosine (pAmpAmpG): 7

[0384] Trinucleotide 6 (0.90 g) was dried under high vacuum conditions for 16 hours.

[0385] Next, 0.45 M tetrazole solution in acetonitrile (4 mL, 1.80 mmol) and bis-(2-cyanoethyl)-N,N-diisopropyl-phosphoramidite (0.76 mL, 1.80 mmol) were added, and the mixture was stirred at room temperature for 3 hours under an argon (or nitrogen) atmosphere. After confirming that Compound 4 had been consumed, the reaction mixture was cooled in an ice-salt bath for 30 minutes, and 70% t-butyl hydroperoxide in water (18.5 mL, 3.47 mmol) was added. This mixture was stirred at room temperature for 1 hour. After confirming the conversion of the intermediate using TLC, the mixture was extracted with ethyl acetate (2 × 200 mL) and washed with a 5% NaHCO₃ aqueous solution (2 × 100 mL). The extracted organic layers were combined, and the solvent was evaporated. Next, the resulting residue was dissolved in methanol (25 mL) and an aqueous solution of concentrated ammonia (25 mL) and stirred at 55°C for 7 hours. After the deprotection reaction was completed, the reaction mixture was co-evaporated with methanol.

[0386] The generated residue was dissolved in water, the pH was adjusted to 5.5, and then loaded onto a DEAE-Sepharose column. Solvent A is 1 M TEAB buffer solution (pH 7.5-8), and solvent B is water.

[0387] The fractions containing the product were combined, concentrated, and freeze-dried to obtain a fine white powder 7 (580 mg, 60%) in the form of a triethylammonium salt.

[0388]

[0389] 3-6-7. Trinucleotide pAmpAmpG.TEA

[0390] ¹H NMR (400 MHz, D₂O):δ 8.48(s, 1H), 8.27(s, 1H), 8.07(s, 1H), 7.90(s, 1H), 7.83(s, 1H), 6.05(d, J = 4.0 Hz, 1H), 5.93(d, J = 8.0 Hz, 1H), 5.77(d, J = 8.0 Hz, 1H), 4.90-4.84(m, 2H), 4.54(t, J = 8.0 Hz, 1H), 4.99-4.95(m, 2H), 4.40(s, 2H), 4.36(t, J = 4.0 Hz, 1H), 4.25(s, 1H), 4.18(s, 2H), 4.12-4.08(m, 2H), 3.90(s, 2H), 3.40(d, J = 4.0 Hz, 6H)

[0391] ³¹P NMR (162 MHz, D₂O): δ 3.73(s, 1P), -0.93(s, 1P), -1.09(s, 1P)

[0392]

[0393] 3-7. General method for the synthesis of 3′-OMe-guanosine 5′-monophosphoromidazolid, N7-benzyl guanosine 5′-monophosphoromidazolid, N7-modified guanosine 5′-diphosphoromidazolid, and dinucleotide 5′-phosphoromidazolid sodium salt

[0394] Triethylamine (3 equivalents), imidazole (10 equivalents), and 2,2′-dithiodipyridine (4 equivalents) were added to a stirred solution of triethylammonium salt of 3′-OMe-guanosine 5′-monophosphate or N7-modified guanosine 5′-monophosphate / diphosphate or triethylammonium salt of dinucleotide pAmpG (1 equivalent) in anhydrous dimethyl sulfoxide (2 mL), and the mixture was stirred for 5 minutes (Fig. 25).

[0395] Next, triphenylphosphine (4 equivalents) dissolved in anhydrous dimethyl sulfoxide (2 mL) was added. The mixture was stirred continuously overnight at room temperature. After the reaction was complete, the reaction mixture was slowly poured into a pre-cooled mixture containing sodium perchlorate (300 mg) in acetone (25 mL). After cooling at -20°C for 30 minutes, the mixture was centrifuged and the supernatant was removed. To remove the residues of imidazole and triphenylphosphine, the solid was ground again with cold acetone (10 mL) and centrifuged. The above process was repeated twice.

[0396] Next, the generated precipitate was dried in a vacuum oven in the presence of P₂O₅ at room temperature. The reaction was confirmed by NMR to show 100% conversion, and the product was obtained with a yield of over 90%.

[0397]

[0398] 3-7-1. 3′-OMe-guanosine 5′-monophosphoroimidazolid sodium salt

[0399] The synthesis method is shown in Fig. 26.

[0400]

[0401] 3′-OMeGMPIM.Na(355 mg, 91%)

[0402] ¹H NMR (400 MHz, D₂O): δ 7.87(s, 1H), 7.76(s, 1H), 7.09(s, 1H), 6.89(s, 1H), 5.78(s, 1H), 4.85(s, 1H), 4.26(s, 1H), 4.00(s, 3H), 3.41(s, 3H)

[0403] ³¹P NMR (162 MHz, D₂O):δ 7.79(s, 1P)

[0404]

[0405] 3-7-2. N7-Benzyl Guanosine 5′-Monophosphoromidazolid Sodium Salt

[0406] The synthesis method is shown in Fig. 27.

[0407]

[0408] N7-Bn-GMP-IM(378 mg, 93%)

[0409] ¹H NMR (400 MHz, D₂O): δ 7.74(s, 1H), 7.39(br s, 5H), 7.05(d, J = 1.2 Hz, 1H), 6.86(s, 1H), 5.93(d, J = 3.8 Hz, 1H), 5.63(dd, J₁ = 14.8 Hz, J₂ = 14.8 Hz, 2H), 4.63(t, J = 4.1 Hz, 1H), 4.32(t, J = 5.0 Hz, 1H), 4.28-4.26(m, 1H), 4.16-4.12(m, 1H), 4.07-4.01(m, 1H)

[0410] ³¹P NMR (162 MHz, D₂O):δ -8.02(s, 1P)

[0411]

[0412] 3-7-3. N7-Benzyl Guanosine 5′-Diphosphoromidazolid Sodium Salt

[0413] The synthesis method is shown in Fig. 28.

[0414]

[0415] N7-BnGDPIM.Na(285 mg, 91%)

[0416] ¹H NMR (400 MHz, D₂O): δ 7.79 (s, 1H), 7.33 (s, 5H), 7.18 (s, 1H), 6.87 (s, 1H), 5.94 (d, J = 4.0 Hz, 1H), 5.55 (d, J = 4.0 Hz, 2H), 4.54 (t, J = 4.2 Hz, 1H), 4.30-4.25 (m, 2H), 4.18-4.13 (m, 1H), 4.05-4.00 (m, 1H)

[0417] ³¹P NMR (162 MHz, D₂O): δ -11.76 (d, J = 21.06 Hz, 1P), -19.88 (d, J = 21.06 Hz, 1P)

[0418]

[0419] 3-7-4. Dinucleotide (pAmpG) 5′-Monophosphoromidazolid Sodium Salt

[0420] The synthesis method is shown in Fig. 29.

[0421]

[0422] pAmpG-IM.Na(378 mg, 93%)

[0423] ¹H NMR (400 MHz, D₂O): δ 8.10 (d, J = 4.0 Hz, 2H), 7.89 (s, 1H), 7.75 (s, 1H), 7.07 (s, 1H), 6.86 (s, 1H), 5.95 (d, J = 4.0 Hz, 1H), 5.80 (d, J = 4.0 Hz, 1H), 4.85-4.81 (m, 1H), 4.45 (t, J = 4.0 Hz, 1H), 4.40 (t, J = 4.0 Hz, 1H), 4.30 (d, J = 8.0 Hz, 2H), 4.14 (s, 2H), 3.97(s, 1H), 3.91-3.87(m, 1H), 3.41(s, 3H)

[0424] ³¹P NMR (162 MHz, D₂O):δ -0.81(s, 1P), -8.15(s, 1P)

[0425]

[0426] 3-8. General method for the synthesis of guanosine N7-modified or guanosine N7 and adenosine N6 double-modified or 3′-OMe guanosine N7-modified or N7-guanosine and adenosine 2′-methoxyethyl modified trinucleotide mRNA cap analogs and N7-modified tetranucleotide mRNA cap analogs (Fig. 31)

[0427] The starting material (2.50 equivalents of the imidazole portion and 1.00 equivalents of the other portion) was dried overnight using a freeze dryer and replaced with nitrogen gas. Anhydrous zinc chloride (20.0 equivalents) and anhydrous DMSO solution were added to the solid starting material under an inert atmosphere.

[0428] After stirring at 37°C for 3 days, the reaction mixture was poured into a pre-cooled solution containing anhydrous NaClO₄ and acetone. The solution was left at -20°C for 30 minutes, then centrifuged at 8,000 rpm for 5 minutes, and the supernatant was removed. The solid was ground again with cold acetone (15 mL) and centrifuged. This process was repeated three more times, and the resulting precipitate was dried under high vacuum at room temperature. Subsequently, the reaction was terminated by adding a 500 mM aqueous EDTA solution (pH 8.0) (1.30 equivalents of EDTA per 1 mole of ZnCl₂). The mixture was diluted with water and purified using DEAE Sepharose fast flow resin with 1 M TEAB buffer, followed by further purification using reverse-phase HPLC.

[0429] The reversed-phase HPLC conditions are as follows: Instrument, Shimadzu; Column, YMC-Actus Triart C8 (Preparative, 250 × 20.0 mm ID); Solvent A, 50 mM TEAA buffer (pH 6.0, containing 0.5% CH₃CN); Solvent B, CH₃CN; Linear gradient 5-80% B (25 min); Flow rate 10 mL / min; Detection wavelength 254 nm.

[0430]

[0431] 3-8-1. Guanosine N7-CD₃ trinucleotide cap analog

[0432] The ¹H NMR and ³¹P NMR data of CD₃7GpppAmpG show a single methyl signal corresponding to CD₃ substitution at the N7 position and a typical triphosphate pattern (approx. 0, -11, -22 ppm regions), which is consistent with the structurally N7-alkylated guanosine-adenosine-guanosine trimer cap structure (Fig. 31).

[0433] 1H NMR (400 MHz, DO) δ = 8.27 (s, 1H), 7.85 (s, 1H), (s, 1H), 5.89 (d, J = 4.0 Hz, 1H), 5.78 (d, J = 4.6 Hz, 1H), 5.73 (d, J = 4.0 Hz, 1H), 4.47 (t, J = 4.2 Hz, 1H), 4.41 (t, J = 4.0 Hz, 2H), 4.33 (t, J = 4.2 Hz, 1H), 4.30 (s, 2H), 4.26 (s, 3H), 4.19 (s, 3H), 4.15-4.11(m,3H), 3.32(s,3H); 31P NMR (162 MHz, DO) δ = -0.87 (s, 1P), -11.25 (d, J = 19.4 Hz, 1P), -11.53 (d, J = 17.8 Hz, 1P), -22.80 (d, J = 17.8 Hz, 1P).

[0434]

[0435] 3-8-2. Guanosine N7-Ethyl trinucleotide cap analog

[0436] Ethyl7GpppAmpG shows terminal methyl (δ= 1.3 ppm) and methylene signals of the ethyl group, and three phosphate signals characteristic of triphosphate are observed in ³¹P NMR, which is interpreted as an N7-ethyl-substituted trinucleotide cap structure (Fig. 32).

[0437] 1H NMR (400 MHz, DO) δ = 8.25 (s, 1H), 8.02 (s, 1H), 7.84 (s, 1H), 5.88 (d, J = 8.0 Hz, 1H), 5.77 (d, J = 4.0 Hz, 1H), 5.72 (d, J = 4.0) Hz, 1H), 4.48 (t, J = 8.0 Hz, 1H), 4.39 (d, J = 4.4 Hz, 2H), 4.36 (t, J = 4.0 Hz, 1H), 4.35 (t, J = 4.2 Hz, 3H), 4.29 (s, 3H), 4.25-4.10 (m, 6H), 3.34 (s, 3H), 1.38 (t, J = 8.0 Hz, 3H); 31P NMR (162 MHz, DO) δ = -0.95 (s, 1P), -11.42 (t, J = 16.2 Hz, 1P), -22.83 (t, J = 17.8 Hz, 1P).

[0438]

[0439] 3-8-3. Guanosine N7-Allyl trinucleotide cap analog

[0440] Allyl7GpppAmpG has a vinyl proton signal (δ= 6.0-5.3 ppm) of the allyl group and a phosphate chain signal was confirmed in ³¹P NMR, confirming that it is consistent with an N7-allyl substitution cap structure (Fig. 33).

[0441]

[0442] *1H NMR (400 MHz, DO) δ = 8.30 (s, 1H), 8.10 (s, 1H), 7.92 (s, 1H), 6.06-5.99 (m, 1H), 5.93 (d, J = 4.0 Hz, 1H), 5.84-5.81 (m, 2H), 5.35 (q, J1= 4.0 Hz, J2= 4.0 Hz 2H), 4.97 (s, 1H), 4.86-4.83 (m, 1H), 4.57 (t, J = 4.0 Hz, 2H), 4.47 (t, J = 4.4 Hz, 2H), 4.41 (t, J = 4.0 Hz, 1H), 4.34 (s, 4H), 4.24-4.18 (m, 4H), 3.45 (s, 3H); 31P NMR (162 MHz, DO) δ = -0.87 (s, 1P), -10.94 (t, J = 9.7 Hz, 2P), -22.03 (t, J = 17.8 Hz, 1P).

[0443]

[0444] 3-8-4. Guanosine N7-Propargyl trinucleotide cap analog

[0445] In Propargyl7GpppAmpG, the alkyne proton signal of the propargyl group is confirmed, and the phosphate peak pattern also matches the triphosphate cap structure (Fig. 34).

[0446] 1H NMR (400 MHz, DO) δ 8.37 (s, 1H), 8.13 (s, 1 2H), 7.87 (s, 1H), 6.03 (d, J = 4.0 Hz, 1H), 5.76 (d, J = 4.0 Hz, 1H), 4.86 (t, J = 4.0) Hz, 2H), 4.41 (d, J = 16.0 Hz, 6H), 4.28 (s, 3H), 4.13 (s, 4H), 4.01 (s, 2H), 3.42 (s, 3H), 3.33 (s, 1H),;31P NMR (162 MHz, DO) δ -0.97 (s, 1P), -11.19 (d, J = 19.44 Hz, 1P), -11.45 (d, J = 17.82 Hz, 1P), -22.59 (t, J = 17.82 Hz, 1P).

[0447]

[0448] 3-8-5. 3'-OMe-Guanosine N7-Benzyl trinucleotide cap

[0449] In Bn7,3'-O-mGpppAmpG, the aromatic proton (δ= 7.2 ppm) of the benzyl group and the 3'-OMe signal were simultaneously detected, and the ³¹P NMR results showed three phosphate signals, confirming that it is a triphosphate structure (Fig. 35).

[0450] 1H NMR (400 MHz, DO) δ 9.31 (s, 1H), 8.33 (s, 1H), 8.06 (s, 1H), 7.85 (s, 1H), 7.24 (d, J = 8.0 Hz, 2H), 7.17 (d, J = 8.0 Hz, 3H), 5.85 (d, J = 8.0 Hz, 1H), 5.76 (d, J = 4.0 Hz, 1H), 5.72 (d, J = 8.0 Hz, 1H), 5.47 (s, 2H), 4.82 (s, 1H), 4.37 (d, J = 12.0 Hz, 5H), 4.25 (s, 2H), 4.18 (s, 1H), 4.10 (t, J = 16.0 Hz, 6H), 3.40 (s, 3H), 3.30 (s, 3H); 31P NMR (162 MHz, DO) δ -0.88 (s, 1P), -11.47 (d, J1 = 19.44 Hz, J2 = 17.82 Hz, 2P), -23.03 (t, J = 17.82 Hz, 1P).

[0451]

[0452] 3-8-6. Guanosine N7-Benzyl, adenosine 2'-Methoxyethyl diphosphate trinucleotide cap analog

[0453] Bn7GppAmoepG exhibits a signal pattern with two ³¹P NMRs corresponding to diphosphate, and a methoxyethyl signal was confirmed due to adenosine 2'-MOE substitution (Fig. 36).

[0454] 1H NMR (400 MHz, DO) δ 9.16 (s, 1H), 8.18 (s, 1H), 8.05 (s, 1H), 7.89 (s, 1H), 7.21 (s, 5H), 5.85 (d, J = 4.0 Hz, 1H), 5.77-5.73 (m, 2H), 5.41 (t, J1 = 12.0 Hz, J2 = 16.0 Hz, 2H), 4.50 (s, 1H), 4.37 (d, J = 16.0 Hz, 5H), 4.26 (d, J = 12.0 Hz, 1H), 4.14 (d, J = 28.0) Hz, 5H), 3.66 (s, 1H), 3.47 (s, 1H), 3.34-3.23 (m, 2H), 2.98 (s, 3H);31P NMR (162 MHz, DO) δ -0.75 (s, 1P), -11.12 (d, J = 21.06 Hz, 1P), -11.12 (d, J = 22.68 Hz, 1P).

[0455]

[0456] 3-8-7. Guanosine N7-Benzyl, adenosine 2'-Methoxyethyl triphosphate trinucleotide cap analog

[0457] It was confirmed that Bn7GpppAmoepG simultaneously exhibits a signal with three triphosphate groups and an MOE substitution signal, and that a benzyl aromatic signal is clearly observed (Fig. 37).

[0458] 1H NMR (400 MHz, DO) δ 9.30 (s, 1H), 8.31 (s, 1H), 7.20 (d, J = 2.80 Hz, 5H), 5.89 (s, 1H), 5.82 (s, 1H), 5.73 (s, 1H), 5.47 (s, 2H), 4.80 (s, 2H), 4.57 (s, 1H), 4.42 (s, 4H), 4.26 (d, J = 2.0 Hz, 4H), 4.17 (s, 1H), 4.11 (s, 3H), 3.65 (s, 1H), 3.44 (s, 1H), 3.29 (s, 1H), 3.23 (s, 1H), 2.98 (s, 3H); 31P NMR (162 MHz, DO) δ -0.75 (s, 1P), -11.24 (d, J = 19.44 Hz, 1P), -11.60 (d, J = 16.20 Hz, 1P), -22.93 (t, J = 17.82 Hz, 1P).

[0459]

[0460] 3-8-8. Guanosine N7-4-F-benzyl trinucleotide cap analog 4-FBn7 GpppAmpG

[0461] Each compound exhibits a specific signal of the corresponding substituent (including ¹F NMR) and, in common, maintains a triphosphate pattern in ³¹P NMR, confirming that it is an N7-substituted guanosine-based trinucleotide cap structure (Fig. 38).

[0462] 1H NMR (400 MHz, D2O) δ = 8.26 (s, 1H), 8.02 (s, 1H), 7.84 (s, 1H), 7.28 (m, 2H), 6.89 (t, J = 8.0 Hz, 2H), 5.83 (d, J = 4.0 Hz, 1H), 5.80 (d, J = 4.0 Hz, 1H), 5.73 (d, J = 8.0 Hz, 1H), 5.42 (d, J = 4.0 Hz, 2H), 4.40 (q, J1 = 4.0 Hz, J2 = 4.0 Hz, 3H), 4.32-4.27 (m, 4H), 4.21-4.08 (m, 6H), 3.32 (s, 3H); 31P NMR (162 MHz, D2O) δ = -0.87 (s, 1P), -11.25 (d, J = 19.4 Hz, 1P), -11.53 (d, J = 17.8 Hz, 1P), -22.80 (s, 1P); 19F NMR (376 MHz, D2O) δ = -113.2 (s, 1F).

[0463]

[0464] 3-8-9. Guanosine N7-4-Br-benzyl trinucleotide cap analog

[0465] 4-BrBn7GpppAmpG (도 39)

[0466] 1H NMR (400 MHz, D2O) δ = 8.25 (s, 1H), 8.01 (s, 1H), 7.23 (s, 1H), 7.22 (d, J = 8.0 Hz, 2H), 7.10 (d, J = 8.0 Hz, 2H), 5.82 (d, J = 4.0 Hz, 1H), 5.71 (d, J = 4.0 Hz, 1H), 5.42 (q, J1 = 16.0 Hz, J2 = 16.0 Hz, 2H), 4.57 (s,1H), 4.54 (t, J1 = 4.0 Hz, 3H), 4.39 ((d, J1 = 4.0 Hz, 2H), 4.28 ((d, J1 = 4.0 Hz, 2H), 44.27-4.08(m,7H), 3.32 (s, 3H); 31P NMR (162 MHz, D2O) δ = -0.87 (s, 1P), -11.25 (d, J = 19.4 Hz, 1P), -11.53 (d, J = 17.8 Hz, 1P), -22.86 (s, 1P).

[0467]

[0468] 3-8-10. Guanosine N7-4-I-benzyl trinucleotide cap analog

[0469] 4-IBn7GpppAmpG (도 40)

[0470] 1H NMR (400 MHz, D2O) δ = 8.27 (s, 1H), 8.04 (s, 1H), 7.84 (s, 1H), 7.41 (d, J = 8.0 Hz, 2H), 6.96 (d, J = 8.0 Hz, 2H), 5.83 (dd, J1 = 4.0 Hz, J2 = 4.0 Hz, 2H), 5.72 (d, J = 8.0 Hz, 1H), 5.41 (d, J = 12.0 Hz, 2H), 4.40 (s,3H), 4.30(s, 2H), 4.26 (s, 2H), 4.13 (s, 2H), 4.12 (s, 4H), 3.29 (s, 3H); 31P NMR (162 MHz, D2O) δ = -0.84 (s, 1P), -11.24 (d, J = 19.4 Hz, 1P), -11.55 (d, J = 17.8 Hz, 1P), -22.88 (d, J = 17.8 Hz, 1P).

[0471]

[0472] 3-8-11. Guanosine N7-3-OMe-benzyl trinucleotide cap analog

[0473] 3-OMe-Bn7GpppAmpG (도 41)

[0474] 1H NMR (400 MHz, D2O) δ = 8.27 (s, 1H), 8.01 (s, 1H), 7.83 (s, 1H), 7.09 (t, J = 8.0 Hz, 1H), 6.80 (s, 1H), 6.67 (d, J = 8.0 Hz, 1H), 5.82 (t, J = 4.0 Hz, 2H), 5.71 (d, J = 4.0 Hz, 1H), 4.43 (d, J = 8.0 Hz, 2H), 4.41 (t, J = 8.0 Hz, 4H), 4.29 (s,5H), 4.19-4.06 (m,6H), 3.63 (s, 3H), 3.29 (s, 3H); 31P NMR (162 MHz, D2O) δ = -0.82 (s, 1P), -11.24 (d, J = 19.4 Hz, 1P), -11.55 (d, J = 17.8 Hz, 1P), -22.89 (d, J = 19.4 Hz, 1P).

[0475]

[0476] 3-8-12. Guanosine N7- (Furan-2-ylmethyl) trinucleotide cap analog

[0477] 2-mFurn7GpppAmpG (도 42)

[0478] 1H NMR (400 MHz, D2O) δ = 8.33 (s, 1H), 8.09 (s, 1H), 7.86 (s, 1H), 7.37 (s, 1H), 6.52 (d, J = 4.0 Hz, 1H), 6.29 (s, 1H), 5.88 (d, J = 4.0 Hz, 1H), 5.77 (d, J = 4.0 Hz, 1H), 5.73 (d, J = 4.0 Hz, 1H), 5.54 (s, 2H), 4.40 (s, 3H), 4.32 (s, 2H), 4.26 (s, 4H), 4.15 (d, J = 8.0 Hz, 2H), 4.11 (s,4H), 3.31 (s, 3H); 31P NMR (162 MHz, D2O) δ = -0.90 (s, 1P), -11.38 (d, J = 19.4 Hz, 1P), -11.58 (d, J = 17.8 Hz, 1P), -22.99 (s, 1P).

[0479]

[0480] 3-8-13. Guanosine N7-Benzyl, adenosine N6-Methyl trinucleotide cap analog

[0481] Bn7Gpppm6AmpG (도 43)

[0482] 1H NMR (400 MHz, D2O) δ = 8.21 (s, 1H), 8.01 (s,1H), 7.82 (s,1H), 7.28-7.15 (m, 5H), 5.84 (d, J = 4.8 Hz, 1H), 5.78 (d, J = 4.0 Hz, 1H), 5.71 (d, J = 8.0 Hz, 1H), 5.41 (d, J = 4.0 Hz, 2H), 4.83-4.81 (m, 2H), 4.53 (t, J = 4.0 Hz, 1H), 4.38 (s, 3H), 4.31-4.26 (m, 4H), 4.22-4.11 (m, 5H), 3.33 (s, 3H), 3.00 (s, 3H); 31P NMR (162 MHz, D2O) δ = -0.88 (s, 1P), -11.28 (d, J = 17.8 Hz, 1P), -11.55 (d, J = 17.8 Hz, 1P), -22.88 (d, J = 17.8 Hz, 1P).

[0483]

[0484] 3-8-14. Guanosine N7-Pyridine, adenosine N6-Benzyl trinucleotide cap analog

[0485] 2-m.Py7GpppBn6AmpG (도 44)

[0486] 1H NMR (400 MHz, D2O) δ 8.33 (s, 1H), 8.22 (d, J = 4 Hz, 1H), 8.04 (s, 1H), 7.85 (s, 1H), 7.69 (t, J = 8.0 Hz, 1H), 7.33 (t, J = 12.0 Hz, 6H), 7.28-7.18 (m 2H), 5.89 (d, J = 4.0 Hz, 1H), 5.84 (d, J = 4.0 Hz, 1H), 5.73 (d, J = 4.0 Hz, 1H), 5.6 (s, 2H), 4.61 (s, 1H), 4.39 (d, J = 16.0 Hz, 4H), 4.26 (d, J = 12.0 Hz, 3H), 4.15 (t, J = 8.0 Hz, 5H), 3.30 (s, 3H);31P NMR (162 MHz, D2O) δ -0.83 (s, 1P), -11.45 (t, J = 25.92 Hz, 2P), -22.93 (d, J = 16.2 Hz, 1P).

[0487]

[0488] 3-8-15. Guanosine N7-Thiophene, adenosine N6-Benzyl trinucleotide cap analog

[0489] 2-m.Thio7GpppBn6AmpG (도 45)

[0490] 1H NMR (400 MHz, D2O) δ 9.25 (s, 1H), 8.34 (s, 1H), 8.03(s, 1H), 7.87 (s, 1H), 7.26 (s, 1H), 7.16 (s, 1H), 7.08 (s, 1H), 6.75 (s, 1H), 5.89 (s, 1H), 5.75 (s, 1H), 5.63 (s, 1H), 4.85 (s, 2H), 4.58 (d, J = 4.0 Hz, 1H), 4.40 (s, 4H), 4.26 (s, 1H), 4.12 (s, 6H), 3.32 (s, 3H). 31P NMR (162 MHz, D2O) δ -0.83 (s, 1P), -11.38 (s, 2P), -22.81 (s, 1P).

[0491]

[0492] 3-8-16. Guanosine N7- Benzyl tetranucleotide cap

[0493] Bn7GpppAmpAmpG (도 46)

[0494] 1H NMR (400 MHz, D2O) δ 8.32 (s, 1H), 8.25 (s, 1H), 8.09 (s, 1H), 7.89 (s, 1H), 7.84 (s, 1H), 7.24 (d, J = 4.0 Hz, 2H), 7.18 (d, J = 8.0 Hz, 3H), 5.94 (d, J = 4.0 Hz, 1H), 5.79 (d, J = 4.0 Hz, 2H), 5.73 (d, J = 8.0 Hz, 1H), 5.46 (s, 1H), 4.43-4.37 (m, 4H), 4.34 (t, J = 8.0 Hz, 1H), 4.26 (s, 3H), 4.12 (s, 8H), 3.44 (s, 3H), 3.36 (s, 3H);31P NMR (162 MHz, D2O) δ -0.92 (s, 1P), -1.14 (s, 1P), -11.28 (d, J = 17.82 Hz, 1P), -11.53 (d, J = 17.82 Hz, 1P), -22.82 (d, J = 17.82 Hz, 1P).

[0495]

[0496] 3-8-17. Guanosine N7-CD3, adenosine N6-Methyl trinucleotide cap

[0497] CD3-7 Gppp m6 A m pG(도 60)

[0498] 1H NMR (400 MHz, D2O) δ = 8.93 (s, 1H), 8.26 (s,1H), 8.07 (s,1H), 7.87 (s,1H), 5.93 (d, J = 8.0 Hz, 1H), 5.80 (d, J = 4.0 Hz, 1H), 5.75 (d, J = 4.0 Hz, 1H), 4.86-4.82 (m, 1H), 4.49 (t, J = 4.0 Hz, 1H), 4.42 (t, J = 4.0 Hz, 3H), 4.38 (t, J = 8.0 Hz, 1H), 4.33 (d, J = 4.0 Hz, 1H), 4.29 (s, 3H), 4.18 (s, 1H), 4.13 (s, 3H), 3.37 (s, 3H), 3.05 (s, 3H); 31P NMR (162 MHz, D2O) δ = -0.88 (s, 1P), -11.54 (t, J = 19.4 Hz, 2P), -23.06 (d, J = 17.8 Hz, 1P).

[0499]

[0500] 2. mRNA 합성 및 정제

[0501] For each in vitro transcription reaction, 100–200 ng of DNA template (1 μL) was added to a microcentrifuge tube. Subsequently, 1.5 μL of 10× transcription buffer (400 mM Tris-HCl, 60 mM MgCl₂, 10 mM DTT, and 20 mM spermidine) and 1.5 μL of 10 mM DTT were added. 1 μL each of 10 mM solutions of rATP, rCTP, and rUTP were added. To improve mRNA capping efficiency, the modified cap analog was used in a 4-fold molar excess relative to rGTP, and accordingly, 1 μL of 10 mM cap analog was included in the reaction. Afterward, RNase inhibitor (0.5 μL, 40 U / μL) and T7 RNA polymerase (1 μL, 50 U / μL) were added, and the final reaction volume was adjusted to 15 μL using nuclease-free water.

[0502] A positive control reaction was performed in parallel using the existing m7GpppAmpG under the same concentration and molar ratio conditions. The transcription reaction was carried out at 37°C for 2 hours, after which DNase I (1 μL, 2 U / μL) was added and the reaction was further carried out at 37°C for 15 minutes to remove residual DNA template.

[0503] The integrity of the in vitro transcribed RNA was confirmed by agarose gel electrophoresis. The mRNA product was separated on a 0.8% agarose gel, exfoliated using a sterile blade, and purified using the AccuPrep® PCR / Gel Purification Kit according to the manufacturer's instructions. The concentration and purity of the purified mRNA were measured using a Calibri+ LB 916 microvolume spectrophotometer (Ver. 25B05) (Fig. 47).

[0504]

[0505] 3. Measurement of translational activity using cultured mammalian cells

[0506] HEK293, RAW264.7, B16F10, and CT26 cells were maintained under standard cell culture conditions in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. One day prior to transduction, cells were isolated using 0.25% trypsin-EDTA (containing phenol red, Thermo Fisher Scientific), resuspended in fresh medium, and seeded into 96-well plates. Transduction was performed when the cell density reached approximately 80% confluence.

[0507] For each transduction reaction, 500 ng of mRNA was diluted in 5 μL of Opti-MEM (Thermo Fisher Scientific). Simultaneously, 0.5 μL of Lipofectamine MessengerMAX (Thermo Fisher Scientific) was diluted in 5 μL of Opti-MEM and reacted at room temperature for 5 minutes. The diluted mRNA solution and the Lipofectamine solution were mixed and gently stirred, then centrifuged briefly and reacted at room temperature for 10 minutes to form a complex. The formed transduction mixture was directly added to the cells and cultured for 24 hours.

[0508] After culture, the medium was removed, and 40 μL of 1× lysis buffer prepared from the Cell Culture Lysis 5× Reagent of the Luciferase Assay System (Promega) was added to lyse the cells. The cell lysate was recovered in a microcentrifuge tube. To measure luciferase activity, 100 μL of luciferin substrate was dispensed into a white 96-well plate, followed by the addition of 20 μL of each cell lysate. Subsequently, luminescence values ​​were measured using a luminometer (Fig. 48).

[0509]

[0510] 4. Evaluation of whole-length mRNA capping efficiency

[0511] The capping efficiency of each cap analog introduced during the in vitro transcription process was evaluated individually for each transcript. To analyze the capping efficiency of the modified mRNA, an RNase H-mediated cleavage assay targeting the 5′ end of the transcript was performed. For this purpose, a short DNA oligonucleotide (5′-GTC TTC CAT GGT GGC CCC-3′) complementary to the 5′ coding region (UTR) was designed, and the oligonucleotide was configured to induce site-specific cleavage of the mRNA at the 5′ cap adjacent region.

[0512] For the hybridization reaction, 10 μL (approx. 10 μg) of purified mRNA was mixed with 1.5 μL of hybridization buffer (500 mM Tris-HCl, 750 mM KCl, 30 mM MgCl₂, and 10 mM DTT) and 2 μL of DNA oligonucleotide (10 μM). The mixture was heated at 95°C for 5 minutes, then slowly cooled to room temperature over 30 minutes, and further reacted at room temperature for 10-15 minutes to induce double-strand formation. Subsequently, 2 μL of thermostable RNase H was added and the cleavage reaction was performed by reacting at 50°C for 1 hour, followed by heating at 65°C for 20 minutes to inactivate the enzyme.

[0513] To analyze the generated RNA fragments, 15 μL of the digestion solution was mixed with 3 μL of 6× loading dye (8 M urea, 50% formamide, 20 mM EDTA, 0.03% bromophenol blue, and 0.03% xylene cyanol) and heat-denatured at 95°C for 3 minutes. Subsequently, electrophoresis was performed on a 15% denatured polyacrylamide gel containing 1× TBE buffer and 7 M urea. After electrophoresis, the gel was stained with ethidium bromide at room temperature for 5 minutes, and images were acquired using a trans-illuminator.

[0514] The intensity of the bands corresponding to the cleavage RNA fragments generated from different capping analogs was quantified by densitometric analysis using GelAnalyzer software (ver. 23.1.1). Capping efficiency was calculated as a relative value based on mRNA capped with commercially available conventional m7GpppAmpG (Fig. 49).

[0515]

[0516] 5. Characterization of MSCap: HPLC Purification Using Hydrophobic Interactions

[0517] The cap incorporation efficiency of trinucleotide cap analogs was evaluated in an in vitro transcription (IVT) system using a T7 promoter-based DNA template encoding GFP and Firefly luciferase. The commercial clean cap trinucleotide analog m7GpppAmpG was used as a control. To increase the cap incorporation efficiency, the rGTP concentration was lowered to 2.5 mM, while the remaining rNTPs and cap analogs were maintained at 10 mM, and the reaction was performed at a cap-to-rGTP ratio (cap:rGTP = 4:1).

[0518] RNA integrity analysis using HPLC revealed that mRNA capped with MSCap (referring to all capping analogs corresponding to the embodiments of the present invention) exhibited a significantly longer retention time than mRNA capped with uncapped RNA (5′-triphosphate form) or a commercially available capping analog (m7GpppAmpG). This “hydrophobic effect” is attributed to aryl or heteroaryl substituents at the guanosine N7 position and was consistently observed in GFP mRNA of approximately 800 nt in length. Consequently, capped RNA and uncapped RNA could be clearly separated via RP-HPLC, and the capping efficiency could be directly calculated for each sample. Importantly, this indicates that the RP-HPLC separation of capped GFP mRNA can be efficiently performed in the process thanks to these characteristics of MSCap.

[0519]

[0520] 6. Immunogenicity

[0521] After verifying translation efficiency using luciferase and a GFP reporter, promising cap structures that enhance mRNA translation characteristics were evaluated using HEK293 cells, and the effects of 5′ cap structures on the regulation of immunogenicity as well as protein synthesis were investigated. The 5′ cap structure of mRNA plays a crucial dual role in both translation and innate immune recognition. The conventional m7GpppAmpG cap supports ribosome binding but is easily recognized by immune sensors, inducing the expression of IFN-α and IFN-β, which consequently inhibits protein synthesis. To test this hypothesis, luciferase mRNAs containing various modified cap structures were synthesized.

[0522] · Bn7GpppAmpG,

[0523] · 4-ClBn7GpppAmpG,

[0524] · 2-mTP7GpppAmpG,

[0525] · Bn7GpppBn6AmpG,

[0526] · Bn7GppspAmpG (D2),

[0527] · 4-ClBn7GppspAmpG (D1)

[0528] mRNA containing the above cap structure was produced and evaluated in a co-culture system with HEK293 cells, RAW 264.7 macrophages, and T cells.

[0529]

[0530] 7. Purification of Benzyl-capped mRNA and Analysis of dsRNA Production Amount

[0531] RP-HPLC purification and dot-blot analysis were performed to purify benzyl-capped mRNA incorporating the cap analog of the present invention and to confirm the generation of dsRNA. mRNA synthesized via in vitro transcription (IVT) was purified using reverse phase-high performance liquid chromatography (RP-HPLC). RP-HPLC analysis was performed under the following conditions. The mobile phase used was 0.1 M triethylammonium acetate (TEAA) at pH 7.0, and acetonitrile was used as the solvent. The proportion of solvent B was gradually increased from 0% to 30% over 30 minutes. The analysis temperature was maintained at 55°C, and the flow rate was set to 1 mL / min. 1 μg of each GFP mRNA sample was injected, and RNA elution was monitored using a UV detector at 260 nm.

[0532] Each HPLC fraction was separated and collected, then lyophilized for about 6 to 7 hours to ensure complete drying. Subsequently, each fraction was resuspended in 20 μL of solution, and 10 μL of this was used for dot-blot analysis.

[0533] dsRNA detection was performed using dot-blot analysis with the dsRNA-specific antibody J2 antibody. In addition, the presence of dsRNA in each HPLC fraction was analyzed to confirm the fractional distribution of dsRNA.

[0534]

[0535] result

[0536] 1. Introduction of trinucleotide cappings into mRNA and analysis of capping efficiency

[0537] It was analyzed that the prepared trinucleotide capping materials could be recognized by T7 RNA polymerase with high efficiency during the transcription process and introduced into mRNA (Fig. 8). As a result, it was confirmed that mRNA introduced with the capping material, in which N7-benzyl was introduced to N2-Imidazol, exhibited antigen protein expression levels similar to or higher than those of the existing m7GpppAmpG during translation (Figs. 7 and 10). Furthermore, it was confirmed that the capping material could be recognized by T7 RNA polymerase with efficiency similar to that of m7GpppAmpG during the transcription process and introduced into RNA. Additionally, while m7GpppAmpG showed a capping efficiency of approximately 89%, the developed capping materials showed a capping efficiency of over 90% (Fig. 9).

[0538]

[0539] 2. Analysis of antigen protein expression using a capping substance in which N7-benzyl was introduced to N2-Imidazol

[0540] The mRNA translation process was verified by introducing a heterocyclic compound formed by connecting the N2 position of the 5′-terminal guanosine base with a methylene bridge, and introducing a capping material prepared by modifying the N7 position with benzyl and methylthiophene. Luciferase mRNA was used to confirm the expression level of the antigen protein, and the expression amount of the antigen protein was analyzed using HEK 293 cells. In the case of Luciferase mRNA, in mRNA using natural rNTPs IM2,m7 GpppA m pG , IM2,Bn7 GpppA m pG, IM2,thio7 GpppA m It was confirmed that the antigen protein expression efficiency of pG was more efficient than that of m7GpppAmpG (Figs. 7 and 10).

[0541]

[0542] 3. Luciferase Expression Analysis

[0543] After the culture period, the medium was removed, and the cells were lysed using 50 μL of 1X lysis buffer. The cell lysates were transferred to microcentrifuge tubes. For luciferase luminescence analysis, 100 μL of luciferin substrate was taken for each sample in a 96-well white plate. Then, 20 μL of each cell lysate sample was added and mixed by pipetting. Finally, the luminescence of luciferase was recorded using a luminometer.

[0544] As a result, it was confirmed that the capping material according to the present invention can be recognized by T7 RNA polymerase with high efficiency similar to m7GpppAmpG during the transcription process and introduced into RNA, and it was confirmed that the existing m7GpppAmpG showed a capping efficiency of 89%, and various trinucleotide capping materials (IM2m7GpppAmpG, IM2Bn7GpppAmpG, IM2thio7GpppAmpG) also showed a high capping efficiency of about 90% or more (Figs. 8 and 9).

[0545] In addition, the mRNA translation process was verified by introducing an imidazole group connected by a methylene bridge to the N2 position of the 5′-terminal guanosine base, and an aryl or heterocyclic compound connected by a methyl or methylene bridge to the N7 position of the 5′-terminal guanosine base. Luciferase mRNA was used to determine the expression level of the antigen protein, and the expression amount of the antigen protein was confirmed using HEK 293 cells. In the case of Luciferase mRNA, mRNA containing natural rNTPs was used.

[0546] Regarding the expression levels of Luciferase protein, it was confirmed that in the case of mRNA with natural rNTPs introduced, the expression level increased by approximately three times in the case of the capping material with N7-benzyl introduced to N2-Imidazol compared to the case of mRNA with m7GpppAmpG introduced. In the case of mRNA with modified pseudo-uridine introduced instead of uridine, it was also confirmed that the expression level of Luciferase protein increased by approximately two times in the case of the capping material with N7-benzyl introduced to N2-Imidazol compared to the mRNA with m7GpppAmpG introduced (Figs. 7 and 10).

[0547]

[0548] 4. Results of agarose gel electrophoresis of IVT mRNA containing various trinucleotide / tetranucleotide cap analogs

[0549] Figure 47 shows the results of agarose gel electrophoresis performed on IVT mRNA with various trinucleotide cap analogs and tetranucleotide cap analogs introduced at the 5′ end. As shown in Figure 47, mRNA transcribed using each cap analog exhibited a stable band pattern on the gel, confirming that the transcription reaction proceeded normally. In addition, no significant degradation products or abnormal migration patterns were observed, confirming that the cap analogs of the present invention do not impair the quality and structural stability of mRNA under in vitro transcription conditions.

[0550]

[0551] 5. Cell line-specific translation characteristics of IVT mRNA containing various cap analogs

[0552] Figure 48 shows the results of measuring protein expression levels after gel-cut purification of IVT mRNA prepared to include various trinucleotide cap analogs and tetranucleotide cap analogs at the 5′ end, followed by introduction into different cell lines. Specifically, Figure 48 A) shows translation characteristics in HEK293 cells, B) in B16F10 cells, C) in RAW264.7 cells, and D) in CT26 cells. As shown in Figure 48, protein expression of mRNA containing the modified cap analogs of the present invention was confirmed in each of the cell lines, suggesting that the cap analogs of the present invention can effectively function in translation in various cellular environments. Furthermore, various MSCaps with different cap structures showed significantly higher expression efficiency than the existing commercial m7GpppAmpG, indicating that the MSCap cap analogs of the present invention can be utilized as design elements to regulate protein expression characteristics according to cell type or purpose.

[0553]

[0554] 6. Results of Capping Efficiency Analysis of Various Cap Analogs Using 20% ​​Urea Modified Gel

[0555] Figure 49 shows the results of analyzing the 5′ end capping efficiency of mRNA prepared using various capping analogs through 20% urea-denatured gel electrophoresis. As shown in Figure 49, mRNA with the introduced capping analog exhibited a migration pattern distinct from uncapped mRNA, which allows for the confirmation of the introduction of the capping and the relative capping efficiency (Table 1). In particular, even when using the modified trinucleotide capping analog of the present invention, efficiency similar to or higher than that of the existing m7GpppAmpG capping was observed, indicating that the capping analog of the present invention can be efficiently introduced to the 5′ end under in vitro transcription conditions. Therefore, it is confirmed that the capping analog of the present invention is a useful material applicable to the mRNA manufacturing process in terms of capping efficiency.

[0556] Cap AnaloguesCapping EfficiencyUncapped mRNA0% m7 GpppAmpG92.00% CD3-7 GpppAmpG95.50% Ethyl-7 GpppA m pG92% Allyl-7 GpppA m pG96%Proprgyl-7GpppA m pG90.86% Bn7,m3' GpppA m pG95.60% Bn7 GppA moe pG86.01% Bn7 GpppA moe pG88.14% 4-FBn7 GpppA m pG92% 4-BrBn7 GpppA m pG93.19 4-IBn7 GpppA m pG95.26% 3-MeOBn7 GpppA m pG92.92% 2-mFuran7 GpppA mpG94.38% Bn7 Gppp m6 A m pG90.94 2-mThio 7Gppp Bn6 A m pG88.00% 2-mPy7 Gppp Bn6 A m pG94.50% Bn7 GpppA m pA m pG96.09%

[0557] 7. Characterization Results of MSCap The analysis results showed that m7GpppAmpG exhibited a capping efficiency of approximately 92%, while MSCap-series trinucleotide analogs achieved high capping efficiencies of over 90% or equivalent. This demonstrated that the newly synthesized modified Cap 1 analogs exhibited transcription performance equivalent to or superior to that of m7GpppAmpG.

[0558] These results highlight the improved capping efficiency of the synthesized modified cap, and some analogs demonstrated performance superior to m7GpppAmpG, proving their potential as next-generation tools for mRNA synthesis.

[0559] In addition, it was observed that mRNA capped with MSCap had a reduced content of dsRNA impurities compared to transcripts capped with m7GpppAmpG.

[0560] This effect was consistently observed regardless of the purification method used (e.g., oligo(dT)₂ affinity chromatography or cellulose-based purification), and the same trend was observed in GFP mRNA of approximately 800 nt in length. These results demonstrate that MSCap is a powerful technological tool capable of producing high-quality mRNA with both excellent resolution and low dsRNA content.

[0561]

[0562] 8. Securing High-Purity mRNA by RP HPLC Separation

[0563] mRNA synthesized using MSCap according to the present invention exhibits significant differences in physicochemical properties compared to uncapped mRNA present in the reaction mixture. In particular, MSCap has increased hydrophobicity due to an aryl or heteroaryl substituent introduced at the N7 position of guanosine, and accordingly, the separation characteristics between capped mRNA and uncapped mRNA are significantly improved (Fig. 50).

[0564] As a result, the difference in retention time between capped mRNA and uncapped mRNA in reverse-phase high-performance liquid chromatography (reverse-phase HPLC) is greatly increased, making it possible to achieve much clearer peak separation compared to using conventional cap structures (Cap0, Cap1, or CleanCap, etc.).

[0565] These characteristics allow for the effective removal of uncapped mRNA when MSCap is applied, enabling the securing of high-purity capped mRNA with minimized incomplete transcripts that are highly likely to trigger immune responses. Furthermore, as the separation between capped and uncapped mRNA increases, previously required multi-step purification processes can be simplified, allowing high-purity mRNA to be obtained through single or minimal-step HPLC processes. Additionally, because MSCap-based mRNA exhibits clear separation characteristics, consistent product quality can be reliably secured even in large-scale production processes, while improving accuracy and facilitating quality control during HPLC-based quantitative analysis.

[0566] In addition, the MSCap according to the present invention can be utilized as a key purification technology in the manufacturing process of mRNA vaccines and therapeutic agents. In particular, since uncapped mRNA can induce unnecessary immune responses by activating innate immune receptors such as TLR3 and TLR7 / 8, it is very important to remove it.

[0567] Using MSCap allows for the simultaneous optimization of the isolation and purification of capped mRNA through simple structural improvements, enabling the production of high-quality mRNA without the need for additional processes.

[0568] In particular, MSCap can serve as a platform technology that goes beyond simply improving capping efficiency to 혁신적으로 improve the purification process itself, providing a significant competitive advantage in terms of reducing production costs and enhancing quality compared to existing technologies.

[0569]

[0570] 8. Immunogenicity

[0571] The existing commercial Cap1, m7GpppAmpG, induced strong IFN-α and IFN-β reactions, whereas the aryl and heteroaryl substituent caps maintained high luciferase activity while showing relatively weak interferon expression (Fig. 51).

[0572] In particular, benzyl-substituted analogs exhibited characteristics of low immunogenicity and high expression efficiency, demonstrating the potential to maintain translation efficiency while reducing innate immune activation.

[0573] Synthesizing these results, it was proven that the 5′ cap structure of mRNA is a key determinant of translation efficiency and immunogenicity. It was also confirmed that the cap structure plays a role in increasing protein synthesis efficiency while simultaneously reducing the innate immune response, and that strong interactions with initiation factors such as eIF4E are important for improving the overall performance of mRNA therapeutics. Furthermore, the expression of IFN-γ, a marker of the adaptive immune system, was slightly elevated, confirming that the immune system was more stable overall compared to the existing commercial Cap1 (Fig. 52).

[0574] Subsequently, after verifying the luciferase protein expression and immune response profile in vitro (at the cell level), luciferase expression was reconfirmed by cross-validating the results in vivo (CD-1 mouse model).

[0575]

[0576] 9. Evaluation of antigen protein expression and persistence (In vivo)

[0577] To evaluate the expression characteristics of mRNA into which the capping derivative of the present invention was introduced in vivo, luciferase reporter mRNA was administered via the intraperitoneal (IP) route, and luminescence intensity was measured using whole-body bioluminescence (IVIS, in vivo imaging system) at 6 hours, 7 days, 14 days, and thereafter (Fig. 53). When compared under the same LNP composition, the MSCap-introduced mRNA administered via the IP route generally showed higher expression signals compared to the control group capped with commercial Cap1 at all measurement times. Even at the initial time (6 hours), it showed strong expression of about 1.8-2.1 times; however, as time progressed, while the luminescence signal of the commercial Cap1 group decreased rapidly, the MSCap group decreased gradually, maintaining an average high expression of more than 3 times even after 14 days of administration. These results show that the capping derivative of the present invention improves resistance to mRNA decapping and intracellular stability (Fig. 59), significantly improving not only translation efficiency but also long-term expression persistence. In addition, no weight loss or toxic reactions were observed in any of the experimental groups during the experimental period, confirming that the capping derivative of the present invention possesses both biocompatibility and safety.

[0578]

[0579] 10. Analysis of in vivo expression and tissue distribution of MSCap mRNA

[0580] To evaluate the in vivo expression efficiency and tissue distribution characteristics of MSCap according to the present invention, mRNA encoding luciferase was prepared with different cap structures and administered to CD1 mice via intravenous (IV) or intraperitoneal (IP) injection. Six hours after administration, the liver, spleen, heart, and kidney were isolated, and the expression levels were evaluated through RT-PCR and bioluminescence analysis.

[0581] As a result, the highest expression was observed in the liver across all experimental groups; however, mRNA coated with MSCap exhibited generally higher expression compared to the existing commercial cap1 structure, showing the greatest increase specifically in the liver. Additionally, a tendency for increased expression in the kidneys and spleen was confirmed depending on the type of MSCap.

[0582] While the existing cap structure exhibited a liver-centered expression pattern, MSCap demonstrated an effect of expanding tissue distribution characteristics by increasing expression in various tissues such as the kidney and spleen. Even with IP administration, a significant distribution of Benzyl-capped mRNA was confirmed in the kidney, confirming that MSCap-based mRNA is translated differently in terms of tissue distribution within the body.

[0583] From these results, it was confirmed that MSCap not only improves protein expression efficiency in an in vivo environment but also has the effect of improving tissue distribution characteristics, and is a useful technical means for developing mRNA therapeutics applicable to various diseases (Figs. 54 to 56).

[0584]

[0585] 11. Purification of Benzyl-capped mRNA and Analysis of dsRNA Production Amount

[0586] As a result of RP-HPLC analysis, a major product peak corresponding to capped mRNA was identified in both mRNA containing the m7GpppAmpG cap and benzyl-capped mRNA, which allowed for the effective purification of the mRNA. Upon collecting and analyzing each fraction, a major product fraction containing capped mRNA was identified, and this fraction was used for subsequent dsRNA analysis.

[0587] As a result of performing dot-blot analysis using the J2 antibody, dsRNA signals were observed in the HPLC fraction between approximately 23 and 26 minutes in mRNA using the existing commercial cap1 (m7GpppAmpG), and relatively strong dsRNA bands were detected in some fractions. On the other hand, in the case of benzyl-capped mRNA, dsRNA signals appeared very weak under the same analysis conditions, and dsRNA signals were hardly detected, especially in the major product fraction.

[0588] In addition, a similar trend was observed in the results of further confirming the presence of dsRNA using a cellulose-based separation method. While a strong signal was detected by a dsRNA-specific antibody (J2) in crude mRNA, the dsRNA signal was significantly lower in benzyl-capped mRNA, showing a tendency for reduced dsRNA production compared to the case using commercial cap1 (m7GpppAmpG). These results indicate that the benzyl-capped mRNA of the present invention can reduce the formation of dsRNA byproducts compared to the conventional cap structure and thus can lower innate immunity (Figs. 57 and 58).

Claims

1. Oligonucleotide cap analog represented by the following chemical formula 1: [Chemical Formula 1] G*-p n1 -N1-(pN) n2 (In the above Chemical Formula 1, G* is a modified guanosine in which a substituted or unsubstituted C1-C10 alkyl, or a substituted or unsubstituted C2-C10 alkenyl, C2-C10 alkynyl, C6-C20 aryl, or C3-C20 heteroaryl group connected by a methylene bridge (-CH2-) is bonded at the N7 position of guanosine (G), and a hydrogen, a substituted or unsubstituted C1-C10 alkyl, or a substituted or unsubstituted C2-C10 alkenyl, C2-C10 alkynyl, C6-C20 aryl, or C3-C20 heteroaryl group connected by a methylene bridge (-CH2-) is bonded at the N2 position; p n1 The phosphate group is a phosphate group, wherein the bridging atom between phosphorus atoms within the phosphate group or the non-bridging atom bonded to the phosphorus atom is each independently selected from oxygen (O), sulfur (S), or NH, and n1 is 3 or 4; N1 is a first nucleotide (A (adenosine), C (cytidine), G (guanosine), U (uracil)) to which an amino group or a nitrogen atom within the ring is unsubstituted, or a C1-C10 alkyl group substituted or unsubstituted at the said amino group or nitrogen atom, or a substituted or unsubstituted C2-C10 alkenyl, C2-C10 alkynyl, C6-C20 aryl, or C3-C20 heteroaryl group connected by a methylene bridge (-CH2-); pN is a nucleotide linked by a phosphate group, and n2 is 1 or 2) 2. A cap analog of claim 1, wherein the substituent bonded to the N7 position of the G* is benzyl, 4-fluorobenzyl, 4-bromobenzyl, 4-iodobenzyl, 3-methoxybenzyl, furan-2-ylmethyl, thiophene-2-ylmethyl, pyridine-2-ylmethyl, ethyl, allyl, propargyl, or a deuterated methyl group.

3. A cap analogue according to claim 1, wherein the substituent bonded to the N2 position of the G* is imidazolylmethyl.

4. A cap analogue according to claim 1, wherein N1 is adenosine (A), cytidine (C), or guanosine (G), and the substituent bonded to the amino group is methyl, dimethylated benzyl, pyridylmethyl, fluorobenzyl, or trifluoromethylphenyl.

5. A cap analogue of claim 1, wherein the 2' or 3' position of each nucleotide ribose included in G*, N1 and pN is independently selected from any one of a hydroxyl group (-OH), a halogen, a substituted or unsubstituted C1-C10 alkoxy group, a haloalkyl group, an amino group, an acetamidoethyl group, a methoxyethyl group, a substituted or unsubstituted C1-C10 acyloxy group, and a substituted or unsubstituted C1-C10 sulfonyloxy group.

6. Capped mRNA having an oligonucleotide cap analog according to claim 1 introduced at the 5' end.

7. A composition for expressing a target protein comprising the capped mRNA of claim 6.

8. A pharmaceutical composition for the prevention or treatment of a disease, comprising the capped mRNA of claim 6 and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition is for use as a vaccine, an anticancer agent, an immunotherapy agent, or a gene therapy agent.

10. A composition for producing 5'-terminally capped mRNA comprising an oligonucleotide cap analog of Claim 1.

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