Novel modified trinucleotide capped derivatives and mRNA incorporating capped derivatives

Abstract

The invention relates to a novel modified trinucleotide capped derivative and mRNA introduced with the capped derivative, a methylene bridged heterocyclic compound and a derivative of aryl methyl are respectively introduced at the N7 position of 5 '-terminal guanosine base, and the efficiency is superior to that of a capped substance used in an existing mRNA vaccine; therefore, the present invention can be effectively used in the fields of development of mRNA vaccines and development of various mRNA-based therapeutic agents.

Description

Technical Field

[0001] This invention relates to novel modified trinucleotide capping derivatives and mRNAs incorporating capping derivatives. Specifically, it relates to mRNA structures incorporating a capping substance of aryl methyl groups by modifying the N7 position of the 5'-terminal guanosine base from an existing methyl group to a methylene-bridged heterocyclic compound or an aryl methyl trinucleotide. Background Technology

[0002] mRNA vaccines have played a crucial role in treating the global COVID-19 pandemic. Capping substances protect mRNA vaccines from degradation. Specifically, in order to express functionally important specific proteins from DNA genetic information, transcription is required—the synthesis of mRNA with complementary sequences based on a DNA template strand in the cell nucleus. This synthesized mRNA is then transported out of the nucleus and, in the cytoplasm, undergoes translation via ribosomes to synthesize specific proteins.

[0003] In particular, within the CAP structure, N7 methylation refers to the addition of a methyl group to the seventh nitrogen atom of the guanosine monophosphate at the 5' end of mRNA. This methylation plays several important roles in significantly enhancing the biological effects of mRNA. The 5' capping structure of N7 methylation improves the stability of mRNA molecules, promotes their binding to ribosomes, and facilitates the binding of translation initiation factors eIF4E and eIF4G, thereby helping to initiate the translation process. Furthermore, the N7 methylated capping structure plays a crucial role in the efficient transport of mRNA from the nucleus to the cytoplasm and serves as a marker for intracellular mRNA, preventing its recognition and degradation by the innate immune system. In summary, the 5' capping of N7 methylation is an important feature of mRNA stability and function in eukaryotic cells, influencing the RNA lifecycle and the ability to efficiently produce proteins.

[0004] There are two methods for capping. The first is 5' capping of the 5' end of the RNA product transcribed from DNA after post-transcriptional processing. After transcription, the 5' end of the RNA transcript contains a free triphosphate group because it is the first nucleotide included in the strand. The capping process replaces the triphosphate group with a different structure called a "cap." This cap is added by guanylyl transferase. This enzyme catalyzes the reaction between the 5' end of the RNA transcript and the guanosine triphosphate (GTP) molecule. The second capping process used in mRNA vaccines is performed through in vitro transcription (IVT), a technique that allows mRNA transcription and capping to occur simultaneously. The cap is added during mRNA synthesis, rather than as an additional step after transcription. This method simplifies the capping process, improves capping efficiency, and enables simple processes and high-yield mRNA production. The 5' capped structure, as a protein expression initiation factor, plays an important role in protein expression by forming a translation initiation complex with eukaryotic translation initiation factor 4E (eIF4E). In addition, it also serves as a marker for intracellular mRNA to prevent it from being recognized and degraded by the innate immune system.

[0005] For about 20 years, dinucleotide-based ARCA cap compounds have been used as chemical capping substances via IVT, but in fact, after the COVID-19 pandemic, trinucleotide-based CleanCap, which has higher antibody expression efficiency, has been used as the main component of Pfizer's mRNA vaccine.

[0006] TriLink's CleanCap, a chemically modified trinucleotide structure, is the most efficient capping compound developed to date for expressing mRNA vaccine antigen proteins. TriLink holds a separate and exclusive patent on this capping compound.

[0007] However, technologies like CleanCap, which can improve the expression efficiency of mRNA vaccine antigen proteins, have not yet been developed in China, making it impossible to independently obtain mRNA vaccines. Therefore, the inventors synthesized mRNA using a capping technique that simultaneously performs transcription and capping via IVT, due to its high efficiency. Summary of the Invention

[0008] Technical issues

[0009] Therefore, the inventors have developed a novel capping substance substituted with a novel N7-methylene-bridged heterocyclic compound that can improve the expression efficiency of mRNA vaccine antigen proteins like CleanCap, and which is different from the nucleotide modified by CleanCap, as well as a novel capping substance substituted with N7-arylmethyl (benzyl (BzCap1)) and its derivatives, thereby completing the present invention.

[0010] Technical solution

[0011] To address the aforementioned problems, the present invention provides a trinucleotide cap analogue comprising the following compound represented by Chemical Formula 1:

[0012] [Chemical Formula 1]

[0013] ,

[0014] In the compound of Formula 1, R1 is a methylene-bridged heterocyclic compound in which the N7 position of the N7 guanosine base is linked by a methylene bridge.

[0015] In the compounds of chemical formula 1, X1 and X2 are H or CH3, and X3, X4 and X5 are selected from the group consisting of O, S and Se, respectively.

[0016] R2 is a derivative that includes benzyl.

[0017] The heterocyclic compound comprises one or two heteroatoms selected from O, N, and S, and is any functional group selected from saturated or partially saturated 5- to 10-membered heteroaromatic compounds.

[0018] The heterocyclic aromatic compound is selected from any functional group of the group consisting of furan, pyrrole, thiophene, imidazole, pyrazole, oxazole, isoxazole, thiazole, pyridine, pyrazine, pyrimidine, pyridazine, and triazine.

[0019] The heterocyclic compound may comprise one or two heteroatoms selected from O, N, and S, and may be any functional group selected from saturated or partially saturated 5- to 10-membered aromatic cyclic groups. Specific examples of heterocyclic compounds may include thiophene, pyridyl, imidazole, tetrahydrofuranyl, 2,3-dihydrofuranyl, 2,5-dihydrofuranyl, pyrrolidinyl, 2,3-dihydropyrrolidinyl, and 2,5-dihydropyrrolidyl. The compounds include pyranyl, tetrahydro-2H-pyranyl, 3,4-dihydro-2H-pyranyl, 4H-pyranyl, piperidinyl, 1,2,3,4-tetrahydropyridinyl, 1,4-dihydropyridinyl, piperazinyl, N-protected piperazinyl, and morpholino. The N-protecting group of the piperazinyl group can typically include alkyl, alkyl carbonyl, and alkyl sulfonyl groups.

[0020] In this invention, the trinucleotide cap analogue may be represented by the following chemical formula 2 or chemical formula 3:

[0021] [Chemical Formula 2]

[0022] ,

[0023] [Chemical Formula 3]

[0024] .

[0025] Trinucleotide cap analogs, including compounds represented by the following chemical formulas [4-1], [4-2], or [4-3]:

[0026] [Chemical Formula 4-1]

[0027] ,

[0028] [Chemical Formula 4-2]

[0029] ,

[0030] [Chemical Formula 4-3]

[0031] .

[0032] In the compounds represented by chemical formula [4-1], chemical formula [4-2], or chemical formula [4-3], R1 is a compound comprising the compound represented by chemical formula 5, which is attached to the N7 position via a methylene group (CH2).

[0033] X1 and X2 are H or CH3, respectively.

[0034] Y1 is either 0 or 1.

[0035] Y2 is O or S.

[0036] Z is H or C1-C3 alkylbenzyl.

[0037] [Chemical Formula 5]

[0038] ,

[0039] In the chemical formula 5, R2, R3, R4, R5 and R6 are each independently selected from any functional group in the group consisting of H, hydroxyl (OH), halogen, methyl, alkyl, alkoxy, nitro, carboxylic, azide, amino and cyano.

[0040] Furthermore, the present invention provides mRNA with a 5' cap using the aforementioned trinucleotide cap analog.

[0041] Furthermore, the present invention provides compositions or kits for preparing mRNA with a 5' end capped by a trinucleotide cap analog.

[0042] Furthermore, the present invention provides pharmaceutical compositions for the expression of target peptides or proteins, comprising 5'-capped mRNA and a pharmaceutically acceptable vector, said mRNA comprising a trinucleotide-capped analog.

[0043] The effects of the invention

[0044] The present invention introduces a methylene-bridged heterocyclic compound and an aryl methyl derivative at the N7 position of the 5'-terminal guanosine base. Since a heterocyclic group and an aryl methyl (benzyl) group connected by a methylene bridge are introduced at the N7 position of the 5'-terminal guanosine base, the efficiency is superior to that of the capping substances used in existing mRNA vaccines. Therefore, it can be beneficially applied to the development of mRNA vaccines and various mRNA-based therapeutic agents. Attached Figure Description

[0045] Figure 1 The trinucleotide capped compounds ThioCap1 and PyCap1 are shown with methylene-bridged thiophene and methylene-bridged pyridine groups, respectively, introduced at the 5'-terminal guanosine N7 position (R1).

[0046] Figure 2 It displays the ThioCap1 substance. 1 A chart of H NMR spectra.

[0047] Figure 3 It displays the ThioCap1 substance. 31 A chart of P NMR spectra.

[0048] Figure 4 It displays the PyCap1 substance. 1 A chart of H NMR spectra.

[0049] Figure 5 It displays the PyCap1 substance. 31 A chart of P NMR spectra.

[0050] Figure 6 The electrophoretic diagrams showing the transcription of the capping substance (ThioCap1) of the present invention confirmed by T7 RNA polymerase are shown (lane 1: luciferase template + T7 RNA polymerase + rNTPs + CleanCap (commercially available Cap1); lane 2: luciferase template + T7 RNA polymerase + rATP + rGTP + rCTP + pseudouridine triphosphate + CleanCap (commercially available Cap1); lane 3: luciferase template + T7 RNA polymerase + rNTPs + ThioCap1; lane 4: luciferase template + T7 RNA polymerase + rATP + rGTP + rCTP + pseudouridine triphosphate + ThioCap1).

[0051] Figure 7The electrophoresis diagram shows the efficiency of the capping material (ThioCap1) of the present invention, confirmed by T7 RNA polymerase after treatment with RNase H (lane 1: luciferase template + T7 RNA polymerase + rNTPs -> gDNA + RNase H; lane 2: luciferase template + T7 RNA polymerase + rNTPs + ARCA -> gDNA + RNase H; lane 3: luciferase template + T7 RNA polymerase + rNTPs + CleanCap -> gDNA + RNase H; lane 4: luciferase template + T7 RNA polymerase + rNTPs + ThioCap1 -> gDNA + RNase H).

[0052] Figure 8 This is a graph comparing the expression of the antigen protein (luciferase) of the luciferase mRNA (Luc mRNAThioCap1) incorporating the capping substance (ThioCap1) of this invention with that of the existing CleanCap (Luc mRNA Cap1).

[0053] Figure 9 The electrophoresis diagrams showing the transcription of the capped material (PyCap1) of the present invention confirmed by T7 RNA polymerase are shown (lane 1: luciferase template + T7 RNA polymerase + rNTPs + CleanCap (commercially available Cap1); lane 2: luciferase template + T7 RNA polymerase + rNTPs + PyCap1).

[0054] Figure 10 The image shows an electrophoresis diagram of the capping efficiency of the capping material (PyCap1) of the present invention, confirmed by RNA polymerase after treatment with RNase H.

[0055] Figure 11 This is a graph comparing the expression of the antigen protein (luciferase) of the luciferase mRNA (Luc mRNAPyCap1) with the capping substance (PyCap1) of this invention and the existing CleanCap (Luc mRNA Cap1).

[0056] Figure 12 It shows the chemical formula of a trinucleotide capped substance (BzCap1) with an aryl methyl (benzyl) functional group introduced at the 5'-terminal guanosine N7 position (R1).

[0057] Figure 13 This is a schematic diagram showing the synthesis process of N7-(arylmethyl)guanosine 5'-diphosphorylimidazole analog.

[0058] Figure 14 This is a schematic diagram showing the synthesis procedure of the dinucleotide pAmpG.

[0059] Figure 15 This is a schematic diagram showing the synthesis procedure of guanosine N7-(aryl-2-ylmethyl)trinucleotide mRNA cap1 analog.

[0060] Figure 16 This is a schematic diagram showing the synthetic procedure for N7-(aryl-2-ylmethyl)trinucleotide diphosphate cap1 analogs.

[0061] Figure 17 This is a schematic diagram showing the synthetic procedure for N7-(aryl-2-ylmethyl)β-thiophosphate trinucleotide Cap1 analog.

[0062] Figure 18 It is a cap analog of guanosine N7-benzyl trinucleotide. 1 H NMR spectrum.

[0063] Figure 19 It is a cap analog of guanosine N7-benzyl trinucleotide. 31 P NMR spectrum.

[0064] Figure 20 It is a cap analog of guanosine N7-benzyltrinucleotide diphosphate. 1 H NMR spectrum.

[0065] Figure 21 It is a cap analog of guanosine N7-benzyltrinucleotide diphosphate. 31 P NMR spectrum.

[0066] Figure 22 It is an ESI-MS of a guanosine N7-benzyltrinucleotide diphosphate cap analog.

[0067] Figure 23 It is a cap analog of guanosine N7-4-Cl-benzyl trinucleotide diphosphate. 1 H NMR spectrum.

[0068] Figure 24 It is a cap analog of guanosine N7-4-Cl-benzyl trinucleotide diphosphate. 31 P NMR spectrum.

[0069] Figure 25 It is an ESI-MS of a guanosine N7-4-Cl-benzyltrinucleotide diphosphate cap analog.

[0070] Figure 26 It is a cap analog of guanosine N7 and adenosine N6-dibenzyl trinucleotide. 1 H NMR spectrum.

[0071] Figure 27 It is a cap analog of guanosine N7 and adenosine N6-dibenzyl trinucleotide. 31 P NMR spectrum.

[0072] Figure 28 It is an ESI-MS of guanosine N7 and adenosine N6-dibenzyl trinucleotide cap analogs.

[0073] Figure 29 It is a cap analog of guanosine N7-4-Cl-benzyl trinucleotide. 1 H NMR spectrum.

[0074] Figure 30 It is a cap analog of guanosine N7-4-Cl-benzyl trinucleotide. 31 P NMR spectrum.

[0075] Figure 31 It is a guanosine N7-benzyl β-thiophosphate trinucleotide cap analog (D1) 1 H NMR spectrum.

[0076] Figure 32 It is a guanosine N7-benzyl β-thiophosphate trinucleotide cap analog (D1) 31 P NMR spectrum.

[0077] Figure 33 It is a guanosine N7-benzyl β-thiophosphate trinucleotide cap analog (D2) 1 H NMR spectrum.

[0078] Figure 34 It is a guanosine N7-benzyl β-thiophosphate trinucleotide cap analog (D2) 31 P NMR spectrum.

[0079] Figure 35 It is a guanosine N7-4-Cl-benzyl β-thiophosphate trinucleotide cap analog (D1) 1 H NMR spectrum.

[0080] Figure 36 It is a guanosine N7-4-Cl-benzyl β-thiophosphate trinucleotide cap analog (D1) 31 P NMR spectrum.

[0081] Figure 37 It is a guanosine N7-4-Cl-benzyl β-thiophosphate trinucleotide cap analog (D2) 1 H NMR spectrum.

[0082] Figure 38It is a guanosine N7-4-Cl-benzyl β-thiophosphate trinucleotide cap analog (D2) 31 P NMR spectrum.

[0083] Figure 39 It is an analogue of inosine N7-benzyl trinucleotide. 1 H NMR spectrum.

[0084] Figure 40 It is an analogue of inosine N7-benzyl trinucleotide. 31 P NMR spectrum.

[0085] Figure 41 It is an L-guanosine N7-benzyl trinucleotide cap analog. 1 H NMR spectrum.

[0086] Figure 42 It is an L-guanosine N7-benzyl trinucleotide cap analog. 31 P NMR spectrum.

[0087] Figure 43 This is a gel image showing the results of RNase H treatment on the transcription of the capping substance (BzCAP1) using T7 RNA polymerase and the capping efficiency during in vitro transcription. The capping efficiency was compared with existing methods such as ARCA and CleanCap.

[0088] Figure 44 The image shows an electrophoretic diagram of the transcription of various arylmethyl-modified trinucleotide capping substances of the present invention, confirmed by T7 RNA polymerase.

[0089] Lane 1: Luciferase mRNA+ m7 GpppAmpG

[0090] Lane 2: Luciferase mRNA+ Bn7 GpppAmpG

[0091] Lane 3: Luciferase mRNA+ Bn7 GppAmpG

[0092] Lane 4: Luciferase mRNA+ 4-ClBn7 GppAmpG

[0093] Lane 5: Luciferase mRNA+ Bn7 Gppp Bn6 AmpG

[0094] Lane 6: Luciferase mRNA+ 4-ClBn7 GpppAmpG

[0095] Lane 7: Luciferase mRNA+ Bn7 GppspAmpG (D1)

[0096] Lane 8: Luciferase mRNA+ Bn7 GppspAmpG (D2)

[0097] Lane 9: Luciferase mRNA+ 4-ClBn7 GppspAmpG (D1)

[0098] Lane 10: Luciferase mRNA+ 4-ClBn7 GppspAmpG (D2)

[0099] Lane 11: Luciferase mRNA+ Bn7 IpppAmpG

[0100] Lane 12: Luciferase mRNA+ Bn7 LGpppAmpG

[0101] Figure 45 The capping efficiency of various arylmethyl-modified trinucleotide capping substances of the present invention was confirmed by RNA polymerase after treatment with RNase H.

[0102] Lane 1: Luciferase mRNA+ m7 GpppAmpG

[0103] Lane 2: Luciferase mRNA+ Bn7 GpppAmpG

[0104] Lane 3: Luciferase mRNA+ Bn7 GppAmpG

[0105] Lane 4: Luciferase mRNA+ 4-ClBn7 GppAmpG

[0106] Lane 5: Luciferase mRNA+ Bn7 Gppp Bn6 AmpG

[0107] Lane 6: Luciferase mRNA+ 4-ClBn7 GpppAmpG

[0108] Lane 7: Luciferase mRNA+ Bn7 GppspAmpG (D1)

[0109] Lane 8: Luciferase mRNA+ Bn7 GppspAmpG (D2)

[0110] Lane 9: Luciferase mRNA+ 4-ClBn7 GppspAmpG (D1)

[0111] Lane 10: Luciferase mRNA+ 4-ClBn7 GppspAmpG (D2)

[0112] Lane 11: Luciferase mRNA+ Bn7 IpppAmpG

[0113] Lane 12: Luciferase mRNA+ Bn7 LGpppAmpG

[0114] Figure 46 This is a chart that confirms the expression of antigen proteins (luciferase) after using capping substances of trinucleotides modified with various arylmethyl groups at the N7 position of the 5'-terminal guanosine.

[0115] Lane 1: Luciferase mRNA+ m7 GpppAmpG

[0116] Lane 2: Luciferase mRNA+ Bn7 GpppAmpG

[0117] Lane 3: Luciferase mRNA+ Bn7 GppAmpG

[0118] Lane 4: Luciferase mRNA+ 4-ClBn7 GppAmpG

[0119] Lane 5: Luciferase mRNA+ Bn7 Gppp Bn6 AmpG

[0120] Lane 6: Luciferase mRNA+ 4-ClBn7 GpppAmpG

[0121] Lane 7: Luciferase mRNA+ Bn7 GppspAmpG (D1)

[0122] Lane 8: Luciferase mRNA+ Bn7 GppspAmpG (D2)

[0123] Lane 9: Luciferase mRNA+ 4-ClBn7 GppspAmpG (D1)

[0124] Lane 10: Luciferase mRNA+ 4-ClBn7 GppspAmpG (D2)

[0125] Lane 11: Luciferase mRNA+ Bn7 IpppAmpG

[0126] Lane 12: Luciferase mRNA+ Bn7 LGpppAmpG Detailed Implementation

[0127] The present invention will now be described in detail.

[0128] The inventors prepared novel modified trinucleotide capping derivatives and mRNAs incorporating these capping derivatives. Specifically, they developed various modified capping compounds that, by introducing a methylene-bridged heterocyclic group and an aryl methyl group at the N7 position of the 5' guanosine base, respectively, can improve the antigen protein expression efficiency of mRNA vaccines, similar to CleanCap, and even higher than CleanCap-modified nucleotides. This invention was ultimately completed by confirming that mRNA structures incorporating various methylene-bridged heterocyclic groups and / or aryl methyl groups exhibit superior antigen protein expression efficiency compared to capping substances used in existing mRNA vaccines.

[0129] The present invention is characterized by the design and synthesis of novel modified trinucleotide capping substances and the confirmation of their potential use as mRNA vaccines. The present invention synthesizes new compounds through organic chemical reactions that modify the N7 position of the 5' guanosine base in the CleanCap trinucleotide structure from the existing methyl group to methylene-bridged thiophene, methylene-bridged pyridin, and arylmethyl compounds, and the synthesis process and results are shown.

[0130] It has been confirmed that during transcription, capping substances can be recognized and introduced into RNA by T7 RNA polymerase with similar or higher efficiency to ARCA or CleanCap. The capping efficiency of existing ARCA is about 50%, CleanCap is about 86%, while ThioCap1 has a capping efficiency of more than 92%, BzCap1 and BzCap1 analogs have a capping efficiency of more than 90%, and PyCap1 capping substances also show a capping efficiency of more than 88%.

[0131] Furthermore, it has been confirmed that compared with existing CleanCap mRNA (Luc mRNA Cap 1), luciferase mRNA incorporating ThioCap1 developed in this invention (Luc mRNA ThioCap 1), luciferase mRNA incorporating PyCap1 (Luc mRNA PyCap 1), luciferase mRNA incorporating BzCap1 (Luc mRNA BzCap 1), and luciferase mRNA incorporating BzCap1 analogs all exhibit higher antigen protein expression efficiency (see [link to original document]). Figure 8 , Figure 11 and Figure 46 ).

[0132] It has been confirmed that luciferase mRNA incorporating BzCap1 and its analogues developed in this invention exhibits higher antigen protein expression efficiency compared to mRNAs incorporating existing ARCA cap (cap 0) and CleanCap (cap 1 commercial).

[0133] Therefore, based on the aforementioned results, the trinucleotide cap analog of the present invention, by introducing a methylene-bridged heterocyclic compound and an aryl methyl functional group at the N7 position of the 5'-terminal guanosine base, respectively, is more efficient than the capping substances used in existing mRNA vaccines, and thus can be effectively applied to the development of novel mRNA vaccines and various mRNA-based therapeutic fields.

[0134] Trinucleotide cap analogs, including the compounds shown in Formula 1 below:

[0135] [Chemical Formula 1]

[0136] ,

[0137] In the compound of Formula 1, R1 is a methylene-bridged heterocyclic compound in which the N7 position of the N7 guanosine base is linked by a methylene bridge.

[0138] In the compounds of chemical formula 1, X1 and X2 are H or CH3, and X3, X4 and X5 are selected from the group consisting of O, S and Se, respectively.

[0139] R2 is a derivative that includes a benzyl group.

[0140] The heterocyclic compound comprises one or two heteroatoms selected from O, N, and S, and is any functional group selected from saturated or partially saturated 5- to 10-membered heteroaromatic ring compounds.

[0141] The heterocyclic aromatic compound may be any functional group selected from the group consisting of furan, pyrole, thiophen, imidazole, pyrazole, oxazol, isoxazole, thiazole, pyridine, pyrazine, primidine, pyridazine, and triazine.

[0142] In this invention, "heterocyclic compound" refers to a 5- to 10-membered aromatic cyclic group comprising one or two heteroatoms selected from O, N, and S, and which is saturated or partially saturated. Specifically, such heterocyclic compounds may include: thiophene, pyridyl, imidazole, tetrahydrofuranyl, 2,3-dihydrofuranyl, 2,5-dihydrofuranyl, pyrrolidinyl, 2,3-dihydropyrrolidinyl, and 2,5-dihydropyrrolidinyl. The group of piperazinyl groups includes dinyl, tetrahydro-2H-pyranyl, 3,4-dihydro-2H-pyranyl, 4H-pyranyl, piperidinyl, 1,2,3,4-tetrahydropyridinyl, 1,4-dihydropyridinyl, piperazinyl, N-protected piperazinyl, and morpholino. The N-protecting group of the piperazinyl group can typically include alkyl, alkyl carbonyl, and alkyl sulfonyl groups.

[0143] Trinucleotide cap analogs, including compounds represented by the following chemical formulas [4-1], [4-2], or [4-3]:

[0144] [Chemical Formula 4-1]

[0145] ,

[0146] [Chemical Formula 4-2]

[0147] ,

[0148] [Chemical Formula 4-3]

[0149] .

[0150] In the compounds represented by chemical formula [4-1], chemical formula [4-2], or chemical formula [4-3],

[0151] R1 is a compound of formula 5 that includes a compound linked at the N7 position via a methyl group (CH2).

[0152] X1 and X2 are H or CH3, respectively.

[0153] Y1 is either 0 or 1.

[0154] Y2 is O or S.

[0155] Z is H or C1-C3 alkylbenzyl.

[0156] [Chemical Formula 5]

[0157] ,

[0158] In the chemical formula 5, R2, R3, R4, R5 and R6 are each independently selected from any functional group in the group consisting of H, hydroxyl (OH), halogen, methyl, alkyl, alkoxy, nitro, carboxylic, azide, amino and cyano.

[0159] R1 can be benzyl or chlorobenzyl, but is not limited to these.

[0160] Furthermore, Z can be methyl benzyl.

[0161] A more detailed description of the substituents used to define the compounds of this invention is provided below.

[0162] In this invention, "halogen" or "halogen atom" are interchangeable terms, referring to chlorine, fluorine, bromine, and iodine.

[0163] In this invention, "alkyl" refers to a straight-chain, branched, or cyclic aliphatic saturated hydrocarbon group having 1 to 5 carbon atoms. Specific examples of such alkyl groups may include methyl, ethyl, n-propyl, isopropyl, cyclopropyl, etc.

[0164] In this invention, "aryl" refers to a monocyclic aromatic hydrocarbon group having 6 carbon atoms. Specific examples of these aryl groups may include phenyl, etc.

[0165] This invention provides a trinucleotide cap analogue comprising the following compound represented by Chemical Formula 1:

[0166] [Chemical Formula 1]

[0167] ,

[0168] In the chemical formula 1, R1 is a heterocyclic group connected at the N7 position via CH2 (methylene bridge). Specifically, R1 is provided as a methylene-bridged thiophene trinucleotide cap analog (ThioCap1) and R1 is a methylene-bridged pyridine trinucleotide cap analog (PyCap1).

[0169] The present invention is characterized in that the trinucleotide cap analogue of the present invention can be represented by the following chemical formula 2 or chemical formula 3.

[0170] [Chemical Formula 2]

[0171] ,

[0172] [Chemical Formula 3]

[0173] .

[0174] The trinucleotide cap analogue of the present invention can be thio-Cap1 (Thio-Cap1, 2-mTHP7 GpppA m pG) or pyridyl-Cap1 (Py-Cap1, pG) or pyridyl-Cap1 (Py-Cap1, 2-mPy7 GpppA m pG).

[0175] This invention provides a trinucleotide cap analogue, comprising compounds represented by the following chemical formulas [4-1], [4-2], or [4-3]:

[0176] [Chemical Formula 4-1]

[0177] ,

[0178] [Chemical Formula 4-2]

[0179] ,

[0180] [Chemical Formula 4-3]

[0181] ,

[0182] In the chemical formula, R1 is chemical formula 6, which is linked at the N7 position via CH2 (methyl).

[0183] [Chemical Formula 6]

[0184] ,

[0185] In the above formula,

[0186] R1 is an arylmethyl group (specifically benzyl).

[0187] Furthermore, the present invention provides a trinucleotide cap analogue in which R1 is a chemical formula of formula 7 linked at the N7 position by CH2 (methyl).

[0188] [Chemical Formula 7]

[0189] ,

[0190] In the above formula,

[0191] R1 is arylmethyl (specifically chlorobenzyl).

[0192] The trinucleotide cap analogue of the present invention can be benzyl-Cap1 (Bz-Cap1, Bn7 GpppA m pG), chlorobenzyl-Cap1 (ClBz-Cap1, 4-ClBn7 GpppA m pG), L-benzyl-Cap1 (LBz-Cap1, Bn7 LGpppA m pG), dibenzyl-Cap1 (DiBz-Cap1, Bn7 Gppp Bn6 A m pG), inosine-Cap1 (Ino-Cap1, Bn7 Ippp Bn6 A m pG), benzyl diphosphate-Cap1 (Bzdipho-Cap1, Bn7 GppA m pG), chlorobenzyl diphosphate-Cap1 (ClBzdipho-Cap1, 4-ClBn7 GppA m pG), benzyl thiophosphate 1-Cap1 (Bzthiopho1-Cap1, Bn7 Gpp s pA m pG(D1)), benzyl thiophosphate 2-Cap1 (Bzthiopho2-Cap1, Bn7 Gpp s pA m pG(D2)), chlorobenzyl thiophosphate 1-Cap1 (ClBzthiopho1-Cap1, 4-ClBn7 Gpp s pA m pG)(D1) and chlorobenzyl diphosphate 2-Cap1 (ClBzthiopho2-Cap1, ... 4-ClBn7 Gpp s pA m pG)(D2)).

[0193] Specifically, the cap analogue can be the compound itself represented by Chemical Formula 1 and Chemical Formulas [4-1] to [4-3], and may also include a hybridization sequence at the starting site that is complementary to a sequence on the DNA template, in addition to the compounds represented by Chemical Formula 1 and Chemical Formulas [4-1] to [4-3]. The length of the hybridization sequence of the cap analogue used in the methods and compositions provided by this invention depends on several factors, such as the physical form of the template nucleotide sequence and the temperature used during hybridization on the DNA template or in in vitro transcription. The desired length of the cap analogue hybridization nucleotide sequence used for transcription can be readily determined by those skilled in the art through routine experiments. For example, the length of the hybridization nucleotide can be determined based on the desired hybridization specificity or selectivity.

[0194] Using the cap analogue, 5' capped mRNA can be prepared.

[0195] One aspect of the present invention provides a method for preparing 5' capped mRNA, comprising adding a cap analog comprising the compounds of chemical formula 1 and the compounds shown in chemical formulas [4-1] to [4-3] during mRNA synthesis.

[0196] Furthermore, one aspect of the present invention provides a 5' capped mRNA preparation composition comprising a capped analogue containing the compounds of the stated chemical formula 1 and those represented by chemical formulas [4-1] to [4-3].

[0197] Furthermore, one aspect of the present invention provides the use of cap analogs comprising the compounds of chemical formula 1 and those shown in chemical formulas [4-1] to [4-3] for mRNA synthesis.

[0198] Furthermore, one aspect of the present invention provides 5' capped mRNA using cap analogs comprising the compounds shown in Chemical Formula 1 and Chemical Formulas [4-1] to [4-3].

[0199] Furthermore, the present invention provides a method for improving protein expression efficiency using the cap analogue, and a method for improving protein expression efficiency using a 5' capped mRNA structure derived from the cap analogue. In this case, the protein may be an antigen protein.

[0200] The method for preparing 5' capped mRNA using capped analogs comprising the compounds shown in Chemical Formula 1 and Chemical Formulas [4-1] to [4-3] can be carried out according to any method known in the art.

[0201] Furthermore, one aspect of the present invention is that 5'-capped mRNA can be prepared by a co-transcriptional capping method, which involves simultaneously introducing a chemically synthesized cap analogue during in vitro mRNA synthesis, such that the cap analogue forms the 5' end. Specifically, the method may include: introducing a cap analogue of the compound shown in Formula 1 and Formulas [4-1] to [4-3] into a mixture comprising RNA polymerase under conditions where RNA polymerase is transcribing on a polynucleotide template; and culturing the mixture for a sufficient time to allow template transcription.

[0202] The present invention provides a pharmaceutical composition for the expression of a target peptide or protein, comprising a 5'-capped mRNA and a pharmaceutically acceptable vector.

[0203] Depending on the type of target peptide or protein, therapeutic or preventative effects against a target disease can be achieved in vivo. Therefore, it can be used to treat or prevent any disease caused by peptide or protein expression. For diseases known to be treatable or preventable by expressing specific types of peptides or proteins, the pharmaceutical composition can be used to induce the expression of the peptide or protein, thereby preventing or treating the target disease.

[0204] Furthermore, one aspect of the invention provides a medical use of the 5'-capped mRNA of the capped analogue to prevent or treat any disease that can produce therapeutic effects through the expression of peptides or proteins in vivo.

[0205] The pharmaceutical composition and treatment or prevention method described herein can be used for gene replacement therapy, genome editing, cancer immunotherapy, or treatment or prevention using vaccines. In one specific embodiment, the pharmaceutical composition is an mRNA vaccine.

[0206] The pharmaceutical composition can be formulated for injection or administration via other suitable routes known to those skilled in the art to treat or prevent a specific disease. Injectable compositions include pharmaceutically acceptable carriers, such as sterile saline. Injectable compositions can also be formulated as suspensions in lipids or phospholipids, liposome suspensions, or aqueous emulsions. Methods for formulating the pharmaceutical composition are well known to those skilled in the art.

[0207] In one specific example, the pharmaceutical composition may include an active ingredient, namely mRNA comprising the cap analogue, at a concentration of approximately 0.01% to 1%. The concentration may vary depending on the frequency of administration, dosage, route of administration, etc.

[0208] In one specific example, the pharmaceutical composition can be administered to mammals, specifically humans, at a dosage that may vary depending on the individual's health condition, disease severity, weight, age, ethnicity, etc., and an appropriate dosage can be determined by those skilled in the art. In one specific example, the human dosage ranges from 0.0001 to 100 mg / day, more specifically from about 0.1 to 50 mg / day.

[0209] Capped mRNAs of the cap analogue 5' according to one aspect can be introduced into cells in vivo or in vitro to express proteins or peptides.

[0210] Methods for expressing proteins or peptides in vivo or in vitro cells using the mRNA are known in the art, and proteins or peptides can be appropriately expressed in cells according to these conventional methods.

[0211] The following embodiments are provided to aid in understanding the present invention. These embodiments are merely illustrative of the invention, and it is obvious to those skilled in the art that various changes and modifications can be made within the scope and technical concept of the present invention, and such changes and modifications naturally fall within the scope of the appended claims.

[0212] <Example>

[0213] A. CleanCap analogues substituted with N7-methylene-bridged heterocyclic compounds

[0214] <Example 1> Synthesis of Trinucleotide Capping Substances

[0215] This embodiment aims to synthesize novel modified trinucleotide capping substances for application in mRNA vaccines. Specifically, novel compounds (ThioCap1) and (PyCap1) were synthesized through organic chemical reactions. In the CleanCap trinucleotide structure, the N7 position of the 5' terminal guanosine base was replaced by a methylene-bridged heterocyclic compound. More specifically, a methylene-bridged thiophene replaced the original methyl group, which was then replaced by a methylene-bridged pyridine. Figure 1 ).

[0216] <1-1> Reagent Information

[0217] Recorded using a Bruker AV-400 spectrometer 1 H, 13 C and 31P NMR spectra were obtained using D₂O or DMSO-d₆ as solvents and tetramethylsilane as an internal standard. UV-Vis spectra were recorded at room temperature using a Cary series UV-Vis spectrophotometer (Agilent Technologies) and 1 cm quartz cuvettes, with absorbance changes measured immediately after UV irradiation of the sample solution in the cuvettes. Fluorescence emission spectra were recorded at room temperature using a PF-65000 spectrofluorometer.

[0218] <1-2> Synthetic methods for N7-(heteroaryl-2-ylmethyl)guanosine 5'-diphosphatidylimidazole analogs

[0219] ① Synthesis of guanosine 5'-diphosphate triethylammonium salt

[0220]

[0221] To synthesize triethylamine guanosine 5'-bisphosphate (GDP.TEA), triethylamine (930 mg, 2 mmol) of guanosine 5'-phosphate (GMP.TEA) was dissolved in anhydrous dimethyl sulfoxide (4 mL). Triethylamine (1.7 mL, 12 mmol), imidazole (817 mg, 12 mmol), and 2,2′-dithiopyridine (1.3 g, 6 mmol) were added to this stirred solution. After stirring the mixture for 5 minutes, triphenylphosphine (1.56 g, 6 mmol) was added to the 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 0.5 g sodium perchlorate and acetone (50 mL). After cooling at 4 °C for 30 minutes, the mixture was centrifuged at 5000 rpm for 8 minutes, and the supernatant was discarded. To remove trace amounts of imidazole and triphenylphosphine, the solid was pulverized with fresh acetone (20 mL), the mixture was cooled, and then centrifuged again. This process was repeated once, and the precipitate was dried at room temperature in a vacuum oven.

[0222] The obtained guanosine 5'-phosphorylimidazolium sodium (NaGMP-IM) was dissolved in dimethylformamide (10 mL) and added dropwise to a 1 M tributylamine 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 at a water bath temperature of 37 °C. The final product was purified using an anion exchange resin.

[0223] Chromatographic purification was performed using a diethylaminoethyl weak anion exchange (DEAE) Sepharose high-flow-rate resin. The desired compound was eluted using CombiFlash EZ Prep at a flow rate of 3 mL / min with a 0-30% gradient (four bed volumes) of 1 M triethylammonium bicarbonate (TEAB) buffer (pH 7.5). The fraction containing GDP.TEA was collected, concentrated using a rotary evaporator (water bath temperature 37°C), and then lyophilized to obtain a fine white powder of GDP.TEA (951 mg, 64%). The NMR results for GDP.TEA are shown below.

[0224] GDP.TEA. (951 mg, 64%)

[0225] 1 H NMR (400 MHz, D2O) 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); 31 P NMR (162 MHz, D2O) δ = -6.47 (d, J = 23.5 Hz, 1P), -10.99 (d, J = 22.5 Hz, 1P).

[0226] ② General synthetic process of N7-(heteroaryl-2-ylmethyl)guanosine 5′-bisphosphate analogs

[0227]

[0228] Guanosine 5′-diphosphate triethylamine salt (0.5 mmol, 1 equivalent) was dispersed in DMSO (3 mL), and an appropriate amount of 2-bromomethylheteroaryl (2.5 mmol, 5 equivalent) was added. The mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was diluted with water (20 mL) and extracted with diethyl ether (3 × 10 mL). After the reaction was complete, the mixture was slowly poured into 50 mL of acetone mixed with 0.5 g of sodium perchlorate. After cooling at 4 °C for 30 min, the mixture was centrifuged at 5000 rpm for 8 min, and the supernatant was discarded. The solid was crushed with fresh acetone (20 mL), the mixture was cooled, and centrifuged again. This process was repeated once, and the precipitate was dried under vacuum at room temperature. The crude product solution with a total volume of 15–20 mL was separated using the aforementioned Sepharose fast-flow resin. The fraction containing the product was collected, concentrated using a rotary evaporator (water temperature 37°C), then co-evaporated three times with ethanol, and dried in a freeze dryer.

[0229] According to the method described above, N7-(thiophen-2-ylmethyl)guanosine 5′-diphosphate triethylammonium salt and N7-(pyridin-2-ylmethyl)guanosine 5′-diphosphate triethylammonium salt were synthesized using 2-bromomethylthiophene and 2-bromomethylpyridine, respectively, and their NMR results are shown below.

[0230] N7-(thiophen-2-ylmethyl) guanosine 5′-diphosphate triethylammoniumsalt: N7-2-mTHP-GDP.TEA (155 mg, 42 %).

[0231] 1H NMR (400 MHz, D2O) δ = 7.36 (d, J = 5.2 Hz, 1H), 7.27 (d, J = 3.6Hz, 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).

[0232] N7-(pyridin-2-ylmethyl) guanosine 5′-diphosphate triethylammoniumsalt: N7-2mPy-GDP.TEA (213 mg, 58 %)

[0233] 1 H NMR (400 MHz, D2O) δ = 8.35 (d, J = 5.6 Hz, 1H), 7.70 (td, J1 = 8Hz, 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 = 5.2Hz, J2 = 2.4 Hz, 1H); 31 P NMR (162 MHz, D2O) δ = -6.34 (d, J = 22.5 Hz, 1P), -10.95 (d, J = 23.5 Hz, 1P).

[0234] ③ General synthetic process of N7-(heteroaryl-2-ylmethyl)guanosine 5'-diphosphorylimidazole analogs

[0235]

[0236] To a solution of N7-(heteroaryl-2-ylmethyl)guanosine 5'-bisphosphate (0.3 mmol) triethylammonium salt, stirred in anhydrous dimethyl sulfoxide (3 mL), triethylamine (251 μL, 1.8 mmol), imidazole (123 mg, 1.8 mmol), and 2,2'-dithiodipyridine (198 mg, 0.9 mmol) were added. After stirring the mixture for 5 minutes, triphenylphosphine (236 mg, 0.9 mmol) dissolved in anhydrous dimethyl sulfoxide (2 mL) was added. Stirring was continued for 5 hours at room temperature. After the reaction was complete, the mixture was slowly poured into 25 mL of acetone mixed with a solution of 250 mg of sodium perchlorate. 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 crushed with fresh acetone (10 mL), the mixture was cooled, and then centrifuged again. Repeat this process once, drying the precipitate at room temperature in a vacuum oven using P2O5.

[0237] According to the method described above, N7-(thiophene-2-ylmethyl)guanosine 5'-diphosphate and N7-(pyridin-2-ylmethyl)guanosine 5'-diphosphate were respectively used to synthesize N 7 -(thiophene-2-ylmethyl)guanosine 5'-diphosphatidylimidazole and N 7 -(pyridin-2-ylmethyl)guanosine 5'-diphosphonylimidazole, its NMR results are shown below.

[0238] <N7-(thiophen-2-ylmethyl) guanosine 5′-diphosphoroimidazolide: N7-2-mTHP-GDP-IM>

[0239] 11H NMR (400 MHz, D2O) δ = 7.99 (s, 1H), 7.53 (dd, J1 = 5.8 Hz, J2 = 1.6 Hz, 1H), 7.40 (dd, J1 = 3.2 Hz, J2 = 1.2 Hz, 1H), 7.36 (dd, J = 1.6 Hz, 1H), 7.12 (dd, J1 = 5.2 Hz, J2 = 3.6 Hz, 1H), 7.06 (bs, 1H), 6.03 (d, J = 4.4 Hz, 1H), 5.91 (d, J = 4.4 Hz, 2H), 4.69 (t, J = 4.0 Hz, 1H), 4.45 - 4.38 (m, 2H), 4.30 (m, 1H), 4.17 (m, 1H); 31 31P NMR (162 MHz, D2O) δ = -11.70 (d, J = 24.5 Hz, 1P), -19.90 (d, J = 21.5 Hz, 1P).

[0240] <N7-(pyridin-2-ylmethyl) guanosine 5′-diphosphoroimidazolide: N7-2mPy-GDP-IM>

[0241] 1 1H NMR (400 MHz, D2O) δ = 8.35 (d, J = 7.6 Hz, 1H), 7.83 - 7.77 (m, 2H), 7.33 (d, J = 8.0 Hz, 2H), 7.18 (bs, 1H), 6.88 (bs, 1H), 6.02 (d, J = 4.0 Hz, 1H), 5.69 (dd, J1 = 22.0 Hz, J2 = 15.6 Hz, 2H), 4.60 (t, J = 4.8 Hz, 1H), 4.35 (t, J = �.6 Hz, 1H), 4.32 - 4.27 (m, 1H), 4.17 (m, 1H), 4.04 (m, 1H);[[ID=]) 31 31P NMR (162 MHz, D2O) δ = -11.84 (d, J = 20.6 Hz, 1P), -19.92 (d, J = 21.5 Hz, 1P).

[0242] <1 - 3> Synthesis method of dinucleotide and pAmpG

[0243]

[0244] ① Synthesis of 5′-O-DMT-2′-O-methyladenosyl(n-bz)-{3′-OP-[2-cyanoethyl] → 5′}-2′,3′-diacetylguanosine(n-ibu)3

[0245] DMT-2'-O-methyladenosine (n-bz), CED phosphorousamide 1 (1.29 g, 1.45 mmol), and 2',3'-diacetylguanosine (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 tetrazolium were added to acetonitrile (7.3 mL, 3.30 mmol), and the mixture was stirred at room temperature for 3 hours under an argon (nitrogen) atmosphere. After confirming the consumption of compound 1 by thin-layer chromatography (TLC), the mixture was cooled in an ice-salt bath for 30 minutes, and then 70% tert-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 intermediate conversion by TLC, the mixture 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).

[0246] ② Synthesis of 2′-O-methyladenosyl(n-bz)-{3′-OP-[2-cyanoethyl] → 5′}-2′,3′-diacetylguanosine(n-ibu)4

[0247] 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 TLC confirmation of the conversion of compound 3, 30 mL of 5% NaHCO3 solution was added and the mixture was stirred for 10 minutes. The mixture was extracted with dichloromethane and evaporated. The resulting residue was then purified by silica gel column chromatography to give product 4 (0.74 g, 0.79 mmol, 60%).

[0248] 1H 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); 31P NMR (DMSO, 162 MHz): δ -2.43 (d, 1P);

[0249] ③ Synthesis of 5′-O-phosphoryl-2′-O-methyladenosyl-{3′-OP→5′}guanosine, (pAmpG)5

[0250] Dinucleotide 4 (0.74 g, 0.7891 mmol) was dried under high vacuum for 16 hours. 0.45 M tetrazolium (3.51 mL, 1.58 mmol) and bis-(2-cyanoethyl)-N,N-diisopropylphosphonamide (0.41 mL, 1.58 mmol) were added to acetonitrile, 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% tert-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 intermediate conversion by thin-layer chromatography (TLC), the mixture 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, the reaction mixture was evaporated together with methanol. The resulting residue was dissolved in water, the pH was adjusted to 5.5, and the solution was loaded onto a DEAE-Sepharose column. Solvent A: 1M TEAB buffer (pH 7.5–8), and solvent B: water were used. The fraction containing the product was collected, evaporated, and dried in a freeze dryer to obtain a fine white powder containing triethylammonium salt, 5 (446 mg, 0.4908 mmol, 62%).

[0251] Dinucleotide (pAmpG) TEA salt

[0252] 1 H NMR (D2O, 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 (D2O, 162 MHz): δ3.69 (s, 1P), -0.84 (s, 1P)

[0253] <1-4> General Synthetic Procedures for Trinucleotide Capped Substances (ThioCap1 and PyCap1) Introducing N7-Methylene Bridged Heterocyclic Groups

[0254]

[0255] pAmpG triethylammonium salt (0.07 mmol, 63 mg) was dissolved in DMSO (50 mM). N7-(heteroaryl-2-ylmethyl)guanosine 5'-diphosphonylimidazolium disodium salt (3.00 equivalents, 0.2 mmol) and zinc chloride (20.0 equivalents) were added to this solution. After stirring at 37°C for 3 days, the reaction was terminated by adding 500 mM EDTA aqueous solution (pH 8.0) (EDTA:ZnCl2 molar equivalents were 1.30 equivalents). The mixture was diluted with water and purified using DEAE Sepharose high-flow-rate resin, followed by further purification by reversed-phase HPLC (Shimadzu instrument; YMC-Actus Triart C8 column (for separation, 250 × 20.0 mm I.D.); 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 lyophilized to obtain the target N7-(heteroaryl-2-ylmethyl)Cap1 analog as a triethylammonium salt. The product was redissolved in methanol (2.00 mL). Acetone (24.0 mL) containing 190 mM NaClO4 was added, and the resulting suspension was centrifuged (4000 rpm, 20 min). The supernatant was discarded, and the precipitate was resuspended in acetone. Repeat the suspension-centrifugation process 3-4 times. Dry the precipitate under reduced pressure to obtain the target N7-(heteroaryl-2-ylmethyl)Cap1 analog in the form of sodium salt.

[0256] According to the method described above, N is used respectively. 7 -(thiophene-2-ylmethyl)guanosine 5'-diphosphatidylimidazole and N 7 ThioCap1 (a trinucleotide capping compound with an N7-methylene-bridged heterocyclic group was synthesized using 5'-diphosphorylimidazole (-(pyridin-2-ylmethyl)guanosine 5'-diphosphorylimidazole). 2-mTHP7 GpppAmpG) and PyCap1 2-mPy7 The NMR analysis results of GpppAmpG are shown below.

[0257] <Guanosine N7-(thiophen-2-ylmethyl) trinucleotide Cap1 analog>

[0258] < 2-mTHP7 GpppAmpG>

[0259]

[0260] 1 H NMR (400 MHz, D2O) δ = 9.29 (s, 1H), 8.34 (s, 1H), 8.11 (s, 1H),7.91 (s, 1H), 7.29 (d, J = 5.2 Hz, 1H), 7.21 (d, J = 3.6 Hz, 1H), 6.88 (dd,J1 = 5.2 Hz, J2 = 3.6 Hz, 1H), 5.90 (d, J = 6.0 Hz, 1H), 5.81 (d, J = 4.4 Hz,1H), 5.78 (d, J = 6.0 Hz, 1H), 5.70 (s, 2H), 4.88 (m, 1H), 4.76 (t, J = 5.2Hz, 1H), 4.58 (t, J = 4.4 Hz, 1H), 4.48-4.40 (m, 3H), 4.39-4.28 (m, 4H),4.25-4.12 (m, 5H), 3.36 (s, 3H); 31 P NMR (162 MHz, D2O) δ = -0.84 (s, 1P), -11.30 (d, J = 19.6 Hz, 1P), -11.51 (d, J = 18.6 Hz, 1P), -22.93 (t, J = 19.6Hz, 1P).

[0261] <Guanosine N7-(pyridin-2-ylmethyl) trinucleotide Cap1 analog>

[0262] < 2-mPy7 GpppAmpG>

[0263]

[0264] 1H NMR (400 MHz, D2O) δ = 8.38 (s, 1H), 8.31 (d, J = 4.8 Hz, 1H), 8.13(s, 1H), 7.90 (s, 1H), 7.77 (t, J = 7.6 Hz, 1H), 7.40 (d, J = 8.0 Hz, 1H),7.28 (t, J = 5.6 Hz, 1H), 5.95 (d, J = 6.0 Hz, 1H), 5.93 (d, J = 4.0 Hz, 1H),5.78 (d, J = 6.0 Hz, 1H), 5.72 (s, 2H), 4.88 (m, 1H), 4.80 (t, J = 5.6 Hz,1H), 4.67 (t, J = 3.6 Hz,1H), 4.50 (t, J = 4.8 Hz,1H), 4.47-4.42 (m, 2H), 4.41-4.28 (m, 4H), 4.27-4.13 (m, 5H), 3.36 (s, 3H); 31 P NMR (162 MHz, D2O) δ =-0.82 (s, 1P), -11.29 (d, J = 19.6 Hz, 1P), -11.50 (d, J = 18.6 Hz, 1P), -22.81 (t, J = 18.6 Hz, 1P).

[0265] <Example 2> Introduction of Trinucleotide Capping Substances into mRNA and Analysis of Capping Efficiency

[0266] The trinucleotide capping material prepared in Example 1 was analyzed to determine whether it could be efficiently recognized by T7 RNA polymerase and introduced into mRNA during transcription.

[0267] The results showed that, in terms of transcription efficiency, CleanCap (cap 1 commercial) and ThioCap1 were similar. During transcription, the developed ThioCap1 demonstrated higher capping efficiency in terms of recognition by T7 RNA polymerase and introduction of RNA than ARCA or CleanCap (cap 1 commercial). The existing ARCA has a capping efficiency of approximately 50%, CleanCap (cap 1 commercial) approximately 86%, while ThioCap1 achieved a capping efficiency of over 92%, and PyCap1 achieved a capping efficiency of over 88%. Figure 7 and Figure 10Due to the extremely low capping efficiency of ARCA, the expression efficiency of its antigen protein (luciferase) was not compared and determined.

[0268] <Example 3> Antigen protein expression analysis using capping substances substituted with methylene-bridged heterocyclic groups.

[0269] The translation process following the introduction of a capping substance into mRNA by modifying the N7 position of the 5'-terminal guanosine base from the original methyl group to a methylene-bridged thiophene compound was confirmed. To confirm the expression level of the antigen protein, luciferase mRNA was used, and the expression level of the antigen protein was analyzed using HEK 293 cells. For luciferase mRNA, two mRNAs were compared: one using native rNTPs and the other using pseudouridine triphosphate to replace the rUTP to avoid over-immunization. In both cases, the mRNA with ThioCap1 (Luc mRNA ThioCAP1) showed more effective antigen protein expression than the mRNA with CleanCap (Luc mRNACAP1), and the mRNA with PyCap1 (Luc mRNA PyCAP1) also showed more effective antigen protein expression than the mRNA with CleanCap (Luc mRNACAP1). Figure 8 and Figure 11 ).

[0270] ① Preparation of luciferase DNA template

[0271] Linear dsDNA templates, including the start codon from the luciferase plasmid to the poly-A tail, were prepared by PCR. During serial PCR, the T7 promoter and Kozak sequence were added to the luciferase dsDNA template, which was then used for transcription. The primers used included: 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); and reverse primer: AAT CGC GCC TAG GCG CGC CCG TAC GGC TCT TC-3' (Sequence No. 3).

[0272] ② Preparation of luciferase mRNA

[0273] The concentration of luciferase template was measured using a nanodrop spectrophotometer. For each transcription reaction, 1 μL of 100 ng template was added to 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. (1 μL, 10 mM) of rATP, rCTP, and rUTP were used, respectively. For cap-dependent transcription by luciferase, rGTP (1 μL, 2.5 mM) was used. The proportion of capping material was maintained 4 times higher than rGTP to provide more than 80% capped mRNA. Therefore, 1 μL of capping material (10 mM) was added. An RNase inhibitor (0.5 μL, 40 U / μL) and T7 RNA polymerase (1 μL, 50 U / μL) were also added. Nuclease-free water (4.5–5.5 μL) was added to bring the final reaction volume to 15 μL. For the modified rNTPs, a 10 mM stock solution was prepared, and rCTP and rUTP were replaced with their respective derivatives. The reaction was incubated at 37 °C for 1 hour. 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 mixture, and the mixture was further incubated at 37 °C for 15 minutes to remove all DNA template.

[0274] ③ Purification of transcribed mRNA

[0275] To remove any dsRNA and transcriptional byproducts that might form, dsRNA was separated by electrophoresis. In the next step, cellulose (10 μL, 0.2 g / mL) was added to the electrophoretically separated mRNA, and the mixture was gently agitated for 10 minutes. The mixture was then transferred to an RNA purification column containing the ethanol and binding buffer provided with the column, and centrifuged at 13000 rpm for 1 minute. Next, RNA preparation buffer and wash buffer were continuously added to the column, followed by centrifugation. After this step, all proteins and unintegrated rTNPs were removed by suspension. Finally, a new centrifuge tube was attached, 10 μL of nuclease-free water was added to the column, and the column was centrifuged at 13000 rpm for 2 minutes. All cellulose-bound dsDNA remained in the column; only pure single-stranded luciferase mRNA passed through. The sample was collected and stored at 70°C for subsequent use.

[0276] ④ Analysis of mRNA capping efficiency

[0277] 2 μg of capped transcribed mRNA was heated to 70 °C with 3 μg of gDNA and RNase H buffer, and then slowly cooled to 25 °C for annealing. Next, the mixture was reacted at 37 °C for 1 hour using heat-stable RNase H to cleave the short sequence containing the capping material from the long mRNA. The cleaved short sequences containing the capping material were analyzed by electrophoresis on a 16% denaturing PAGE gel. Short sequences cleaved from uncapped mRNA showed faster gel migration, while those cleaved from capped mRNA showed relatively slower gel migration.

[0278] ⑤ Cell culture and preparation for transfection

[0279] HEK 293 cells were cultured in 60 mL DMEM dishes containing a mixture of 10% FBS and 1% penicillin / strep. The day before transfection, cells were isolated using trypsin and resuspended in fresh medium. Cells were seeded in 96-well plates. The next day, when cells reached 70% confluence, the transfection mixture was prepared and transfection was performed.

[0280] ⑥ Quantification and transfection of mRNA

[0281] Purified mRNA was quantified using a nanodrop spectrophotometer, and the transcription yields of various rNTP combinations were compared. For cell infection, 500 ng of each mRNA was 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 min. The mRNA and Lipofectamine mixture was then transferred to the same tube and incubated for another 5 min at room temperature. Finally, the transfection mixture was added to the cells and incubated for 24 h.

[0282] ⑦ Luciferase expression analysis

[0283] After the culture period, the culture 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 added to each well of a white 96-well plate. Then, 20 μL of each cell lysate sample was added and mixed with a pipette. Finally, the luciferase luminescence was recorded using a luminometer.

[0284] The results showed that the capping substance in this invention can be recognized and introduced into RNA by T7 RNA polymerase during transcription with a higher efficiency than ARCA or CleanCap. The capping efficiency of existing ARCA is about 50%, while the capping efficiency of ThioCap1 reaches more than 90%, and the capping efficiency of PyCap1 also reaches more than 88%. Figure 7 and Figure 10 ).

[0285] Furthermore, the translation process of the mRNA containing the capping substance of this invention was confirmed. To confirm the expression level of the antigen protein, luciferase mRNA was used, and the expression level of the antigen protein was confirmed using HEK 293 cells. For luciferase mRNA, two mRNAs were compared: one using native rNTPs and the other using pseudouridine triphosphate to replace rUTPs to avoid over-immunization.

[0286] The results showed that, compared with mRNA using rNTPs, mRNA incorporating pseudouridine triphosphates, used to avoid over-immunization, exhibited higher antigen protein expression efficiency.

[0287] Furthermore, it was confirmed that compared to the existing CleanCap (cap 1 commercial) mRNA (Luc mRNA Cap1), the luciferase mRNA (Luc mRNA ThioCap1) incorporating ThioCap1 developed in this invention and the mRNA (Luc mRNA PyCAP1) incorporating PyCap1 both exhibited higher antigen protein expression efficiency in mRNAs using rNTPs and mRNAs containing pseudouridine replacing rUTPs. Figure 8 and Figure 11 ).

[0288] B. CleanCap analogues substituted with N7-arylmethyl (benzyl (BzCap1))

[0289] <Example 1> Synthesis of Trinucleotide Capping Substances

[0290] This embodiment aims to synthesize novel modified trinucleotide capping substances for application in mRNA vaccines. This study synthesized several novel compounds through organic chemical reactions. Bn7 GpppA m pG, Bn7 GppA m pG, 4-ClBn7 GppA m pG, Bn7 Gppp Bn6 A mpG, 4-ClBn7 GpppA m pG, Bn7 Gpp s pA m pG(D1) Bn7 Gpp s pA m pG(D2) 4-ClBn7 Gpp s pA m pG(D1) 4-ClBn7 Gpp s pA m pG, Bn7 IpppA m pG and Bn7 LGpppA m These compounds modify the N7 position of the 5'-terminal guanosine base in the CleanCap trinucleotide structure from the original methyl group to an arylmethyl group, and more specifically, to a benzyl group and a 4-chlorobenzyl group. Figure 12 ).

[0291] <1-1> Reagent Information

[0292] Recorded using a Bruker AV-400 spectrometer 1 H, 13 C and 31 P NMR spectra were obtained using D₂O or DMSO-d₆ as solvents and tetramethylsilane as an internal standard. UV-Vis spectra were recorded at room temperature using a Cary series UV-Vis spectrophotometer (Agilent Technologies) and 1 cm quartz cuvettes, with absorbance changes measured immediately after UV irradiation of the sample solution in the cuvettes. Fluorescence emission spectra were recorded at room temperature using a PF-65000 spectrofluorometer.

[0293] <1-2> Synthetic methods for N7-(arylmethyl)guanosine 5'-diphosphatidylimidazole analogs

[0294] ① Synthesis of guanosine 5'-diphosphate triethylammonium salt

[0295]

[0296] To synthesize triethylammonium guanosine 5'-bisphosphate (GDP.TEA), a solution of triethylamine (930 mg, 2 mmol) of guanosine 5'-phosphate (GMP.TEA) was stirred in anhydrous 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 anhydrous dimethyl sulfoxide (4 mL) was added, and stirring continued at room temperature for 5 hours. After the reaction was complete, the mixture was slowly poured into acetone (50 mL) mixed with 0.5 g of sodium perchlorate. After cooling at 4 °C for 30 minutes, the mixture was centrifuged at 5000 rpm for 8 minutes, and the supernatant was discarded. To remove trace amounts of imidazole and triphenylphosphine, the solid was pulverized with fresh acetone (20 mL), and the mixture was cooled and centrifuged again. Repeat this process once, drying the precipitate at room temperature in a vacuum oven.

[0297] The obtained guanosine 5'-phosphorylimidazolium sodium (NaGMP-IM) was dissolved in dimethylformamide (10 mL) and added dropwise to a 1 M tributylamine 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 at a water bath temperature of 37 °C. The final product was purified using an anion exchange resin.

[0298] Chromatographic purification was performed using a diethylaminoethyl weak anion exchange (DEAE) Sepharose high-flow-rate resin. The desired compound was eluted using CombiFlash EZ Prep at a flow rate of 3 mL / min with a 0-30% gradient (four bed volumes) of 1 M triethylammonium bicarbonate (TEAB) buffer (pH 7.5). The fraction containing GDP.TEA was collected, concentrated using a rotary evaporator (water bath temperature 37°C), and then lyophilized to obtain a fine white powder of GDP.TEA (951 mg, 64%). The NMR results for GDP.TEA are shown below.

[0299] GDP.TEA. (951 mg, 64%)

[0300] 1H NMR (400 MHz, D2O) 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); 31 P NMR (162 MHz, D2O) δ = -6.47 (d, J = 23.5 Hz, 1P), -10.99 (d, J = 22.5 Hz, 1P).

[0301] ② Synthesis of triethylammonium salts of 5'-inosine diphosphate and L-5'-guanosine diphosphate

[0302] According to the method in Examples <1-2>, 5'-bisphosphate inosine triethylammonium salt (IDP.TEA, 830 mg, 57%) and L-5'-bisphosphate guanosine triethylammonium salt (LGDP.TEA, 892 mg, 60%) were synthesized using inosine monophosphate (IMP) and L-guanosine monophosphate (LGMP), respectively, and their NMR results are shown below.

[0303] IDP.TEA. (830 mg, 57%).

[0304] 1 H NMR (400 MHz, D2O) δ = 8.41 (s, 1H), 8.13 (s, 1H), 6.05 (d, J = 5.2Hz, 1H), 4.68 (overlapped with H2O, m, 1H), 4.54 (t, J = 4.0 Hz, 1H), 4.29(bs, 1H), 4.21-4.07 (m, 2H); 31P NMR (162 MHz, D2O) δ = -7.47 (d, J = 22.5Hz, 1P), -11.07 (d, J = 22.3 Hz, 1P).

[0305] LGDP.TEA. (892 mg, 60%).

[0306] 1H NMR (400 MHz, D2O) δ = 8.08 (s, 1H), 5.89 (d, J = 5.6 Hz, 1H), 4.74(t, J = 5.2 Hz, 1H), 4.59 (t, J = 4.0 Hz, 1H), 4.29 (m, 1H), 4.20 (m, 1H),4.13 (m, 1H); 31P NMR (162 MHz, D2O) δ = -6.35 (d, J = 22.5 Hz, 1P), -10.90(d, J = 22.4 Hz, 1P).

[0307] <1-3> Synthetic methods for N7-(aryl-2-ylmethyl)guanosine 5'-monophosphate or diphosphate analogs

[0308]

[0309] Guanosine 5'-diphosphate triethylamine salt (0.5 mmol, 1 equivalent) was dispersed in DMSO (3 mL), and an appropriate amount of 2-bromomethylaryl (2.5 mmol, 5 equivalent) was added. The mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was slowly poured into 50 mL of acetone and mixed with 0.5 g of sodium perchlorate. After cooling at 4 °C for 30 min, the mixture was centrifuged at 5000 rpm for 8 min, and the supernatant was discarded. The solid was crushed with fresh acetone (20 mL), the mixture was cooled, and centrifuged again. This process was repeated once, and the precipitate was dried under vacuum at room temperature. The crude product solution with a total volume of 15–20 mL was separated using the aforementioned Sepharose (DEAESepharose) fast-flow resin. The fraction containing the product was collected, concentrated using a rotary evaporator (water temperature 37 °C), then co-evaporated three times with ethanol, and dried in a freeze dryer.

[0310] According to the method described above, N7-benzylguanosine 5′-monophosphate triethylammonium salt, N7-4-chlorobenzylguanosine 5′-monophosphate triethylammonium salt, N7-benzylguanosine 5′-diphosphate triethylammonium salt, N7-4-chlorobenzylguanosine 5′-diphosphate triethylammonium salt, and N7-benzyl inosine 5′-diphosphate triethylammonium salt were synthesized using benzyl, 4-chlorobenzyl, and benzyl inosine, respectively. The NMR results are shown below.

[0311] N7-benzyl guanosine 5′-monophosphate triethylammonium salt: N7-Bn- GMP.TEA (270 mg, 82%).

[0312] 1 H 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 = 2.3Hz, 1H), 4.18-4.13 (m, 1H), 4.06-4.01 (m, 1H); 31 P NMR (162 MHz, D2O): δ =2.30 (s, 1P). ESI MS: Calculated m / z for C 17 H 19 N5O8P ([M]): m / z 452.34; found([M-1]): 451.2.

[0313] N7-4-cholobenzyl guanosine 5′-monoahosphate triethylammonium salt: N7-ClBn-GMP.TEA (260 mg, 75 %).

[0314] 1H NMR (400 MHz, D2O) δ = 7.28 (q, J =8.1 Hz, 4H), 6.03 (d, J = 3.4Hz, 1H), 5.58 (s, 1H), 4.63 (t, J = 4.1 Hz, 1H), 4.47 (t, J = 5.0 Hz, 1H),4.35 (s, 1H), 4.12 (d, J = 12.2 Hz, 1H), 4.01-3.97 (m, 1H); 31 P NMR (162 MHz,D2O): δ = 3.70 (s, 1P).

[0315] N7-benzyl guanosine 5′-diphosphate triethylammonium salt: N7-Bn- GDP.TEA (247 mg, 67%).

[0316] 1 H 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 = 3.2Hz, 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).

[0317] N7-4-chlorobenzyl guanosine 5′-diphosphate triethylammonium salt: N7- 4-Cl-Bn-GDP.TEA (272 mg, 71 %).

[0318] 1 H NMR (400 MHz, D2O) δ = 7.38 (s, 4H), 6.02 (d, J = 4 Hz, 1H), 5.62(s, 2H), 4.68 (t, J = 8 Hz, 1H), 4.52 (t, J = 8 Hz, 1H), 4.38-4.34 (m, 1H),4.31-4.25 (m, 1H), 4.24-4.18 (m, 1H); 31P NMR (162 MHz, D2O) δ = -8.88 (d, J =21 Hz, 1P), -11.08 (d, J = 23 Hz, 1P).

[0319] N7 -benzyl inosine 5′-diphosphate triethylammonium salt: N7-Bn-IDP.TEA

[0320] 1 H NMR (400 MHz, D2O) δ = 8.18 (s, 1H), 7.40-7.27 (m, 5H), 6.14 (d, J= 3.2 Hz, 1H), 5.75 (q, J = 15.2 Hz, 2H), 4.61 (t, J = 4.8 Hz, 1H), 4.52 (t,J = 6.0 Hz, 1H), 4.32 (m, 1H), 4.24 (m, 2H); 31 P NMR (162 MHz, D2O) δ = -6.35(d, J = 23.5 Hz, 1P), -11.00 (d, J = 23.5 Hz, 1P).

[0321] <1-4> Synthetic methods for N7-(aryl-2-ylmethyl)guanosine 5'-monophosphate or diphosphatidylimidazole analogs

[0322]

[0323] To a solution of N7-(aryl-2-ylmethyl)guanosine 5'-bisphosphate (0.3 mmol) triethylammonium salt, stir in anhydrous dimethyl sulfoxide (3 mL), add triethylamine (251 μL, 1.8 mmol), imidazole (123 mg, 1.8 mmol), and 2,2'-dithiodipyridine (198 mg, 0.9 mmol). After stirring the mixture for 5 minutes, add triphenylphosphine (236 mg, 0.9 mmol) dissolved in anhydrous dimethyl sulfoxide (2 mL). Continue stirring at room temperature for 5 hours. After the reaction is complete, slowly pour the mixture into 25 mL of acetone and mix with 250 mg of sodium perchlorate. After cooling at 4 °C for 30 minutes, centrifuge the mixture and discard the supernatant. To remove trace amounts of imidazole and triphenylphosphine, pulverize the solid with fresh acetone (10 mL), cool the mixture, and centrifuge again. Repeat this process once, drying the precipitate at room temperature in a vacuum oven using P2O5.

[0324] According to the method described above, N7-benzylguanosine 5′-monophosphoroimidazolide, N7-4-chlorobenzylguanosine 5′-monophosphoroimidazolide, N7-benzylguanosine 5′-diphosphoroimidazolide, and N7-4-chlorobenzylguanosine 5′-diphosphoroimidazolide were synthesized using N7-(benzyl-2-ylmethyl)guanosine 5′-bisphosphate, N7-(4-chlorobenzyl-2-ylmethyl)guanosine 5′-bisphosphate, and N7-(benzylinosine-2-ylmethyl)guanosine 5′-bisphosphate, respectively. The NMR results for N7-benzyl inosine 5′-diphosphoroimidazolide and N7-benzyl inosine 5′-diphosphoroimidazolide are shown below.

[0325] N7-benzyl guanosine 5′-monophosphoroimidazolide: N7-Bn-GMP-IM (378mg, 93%)

[0326] 1 H NMR (400 MHz, D2O) = 7.74 (s, 1H), 7.39 (br s, 5H), 7.05 (d, J =1.2Hz, 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); 31 P NMR (162 MHz, D2O) = -8.02(s, 1P).

[0327] Electrospray ionization (ESI) mass spectrometry: Calculated m / z forC 20 H 22N7O7P- ([M]): m / z 503.41; found([M-2]): 501.2.

[0328] N7-4-Cl-benzyl guanosine 5′-monophosphoroimidazolide: N7-4-Cl-Bn-GMP- IM (392mg, 91%)

[0329] 1 H NMR (400 MHz, D2O) = 7.76 (s, 1H), 7.34 (s, 4H), 7.05 (s, 1H), 6.88(s, 1H), 5.92 (d, J = 3.4 Hz, 1H), 5.58 (dd, J1 = 14.8 Hz, J2 = 15 Hz, 2H),4.61 (t, J = 4.5Hz, 1H), 4.31-4.28 (m, 2H), 4.13-4.04 (m, 2H); 31 P NMR (162MHz, D2O): = -8.04 (s, 1P).

[0330] Electrospray ionization (ESI) mass spectrometry: Calculated m / z forC 20 H 21 ClN7O7P- ([M]): m / z 537.85; found([M-2]): 535.1.

[0331] N7-benzyl guanosine 5′-diphosphoroimidazolide: N7-Bn-GDP-IM (153mg, 81%)

[0332] 1H NMR (400 MHz, D2O) δ = 7.80 (s, 1H), 7.24-7.41 (bs, 5H), 7.19 (s,1H), 6.88 (s, 1H), 5.97 (d, J = 4.0 Hz, 1H), 5.59 (dd, J1 = 21.2 Hz, J2 =14.4 Hz, 2H), 4.54 (t, J = 4.0 Hz, 1H), 4.25-4.33 (m, 2H), 3.99-4.20 (m, 2H);31P NMR (162 MHz, D2O) δ = -11.76 (d, J = 21.5 Hz, 1P), -19.88 (d, J = 21.4Hz, 1P).

[0333] N7-4-Cl-benzyl guanosine 5′-diphosphoroimidazolide: N7-4-Cl-Bn-GDP-IM

[0334] 1 H NMR (400 MHz, D2O) δ = 7.82 (s, 1H), 7.32 (bs, 4H), 7.20 (bs, 1H), 6.89 (bs, 1H), 5.97 (d, J = 4.0 Hz, 1H), 5.54 (dd, J1 = 23.6 Hz, J2 = 14.8Hz, 2H), 4.55 (t, J = 4.0 Hz, 1H), 4.31 (d, J = 4.8 Hz, 2H), 4.20 (m, 1H), 4.06 (m, 1H); 31 P NMR (162 MHz, D2O) δ = -11.72 (d, J = 25.6 Hz, 1P), -19.80 (d, J = 20.6 Hz, 1P).

[0335] <N7-benzyl inosine 5′-diphosphoroimidazolide: N7-Bn-IDP-IM>

[0336] 1 H NMR (400 MHz, D2O) δ = 8.17 (s, 1H), 7.77 (s, 1H), 7.37-7.26 (m,5H), 7.17 (s, 1H), 6.86 (s, 1H), 6.10 (d, J = 3.2 Hz, 1H), 5.66 (q, J = 14.8Hz, 2H), 4.35 (t, J = 4.0 Hz, 1H), 4.31-4.25 (m, 2H), 4.17 (m, 1H), 4.03 (m,1H); 31 P NMR (162 MHz, D2O) δ = -11.76 (d, J = 29.3 Hz, 1P), -19.88 (d, J =21.4 Hz, 1P).

[0337] <1-5> Synthesis of pAmpG dinucleotide

[0338] ① Synthesis of 5′-O-DMT-2′-O-methyladenosyl(n-bz)-{3′-OP-[2-cyanoethyl] → 5′}-2′,3′-diacetylguanosine(n-ibu)3

[0339] DMT-2'-O-methyladenosine (n-bz), CED phosphoramidide 1 (1.29 g, 1.45 mmol), and 2',3'-diacetylguanosine (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 tetrazolium were added to acetonitrile (7.3 mL, 3.30 mmol), and the mixture was stirred at room temperature for 3 hours under an argon (nitrogen) atmosphere. After confirming the consumption of compound 1 by thin-layer chromatography (TLC), the mixture was cooled in an ice-salt bath for 30 minutes, and then an aqueous solution of 70% tert-butyl hydroperoxide (0.91 mL, 6.60 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. After confirming the intermediate conversion by TLC, the mixture 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).

[0340] ② Synthesis of 2′-O-methyladenosyl(n-bz)-{3′-OP-[2-cyanoethyl] → 5′}-2′,3′-diacetylguanosine(n-ibu)4

[0341] 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 TLC confirmation of the conversion of compound 3, 30 mL of 5% NaHCO3 solution was added and the mixture was stirred for 10 minutes. The mixture was extracted with dichloromethane and evaporated. The resulting residue was then purified by silica gel column chromatography to give product 4 (0.74 g, 0.79 mmol, 60%).

[0342] 1H 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);

[0343] ③ Synthesis of 5′-O-phosphoryl-2′-O-methyladenosyl-{3′-OP→5′}guanosine, (pAmpG)5

[0344] Dinucleotide 4 (0.74 g, 0.7891 mmol) was dried under high vacuum for 16 hours. 0.45 M tetrazolium (3.51 mL, 1.58 mmol) and bis-(2-cyanoethyl)-N,N-diisopropylphosphonamide (0.41 mL, 1.58 mmol) were added to acetonitrile, 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% tert-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 intermediate conversion by thin-layer chromatography (TLC), the mixture 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, the reaction mixture was evaporated together with methanol. The resulting residue was dissolved in water, the pH was adjusted to 5.5, and the solution was loaded onto a DEAE-Sepharose column. Solvent A: 1M 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 containing triethylammonium salt (5 oz., 446 mg, 0.4908 mmol, 62%). Figure 14 ).

[0345] Dinucleotide (pAmpG) TEA salt

[0346] 1 H NMR (D2O, 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 (D2O, 162 MHz): δ3.69 (s, 1P), -0.84 (s, 1P)

[0347] Dinucleotide (pAmpG) imidazole sodium salt pAmpG-IM.Na (378 mg, 93%)

[0348] 1 H NMR (400 MHz, D2O) = 8.10 (d, J = 6.84 Hz, 2H), 7.89 (s, 1H), 7.75(s, 1H), 7.07 (s, 1H), 6.86 (s, 1H), 5.96 (d, J = 5.4 Hz, 1H), 5.80 (d, J =5.24 Hz, 1H), 4.85 - 4.81 (m, 1H), 4.45 (t, J = 4.68 Hz, 1H), 4.40 (t, J = 5.0Hz, 1H), 4.31 - 4.29 (m, 2H), 4.14 (s, 2H) 3.98-3.96 (m, 1H), 3.91-3.87 (m, 1H) 3.41 (s, 3H); 31 P NMR (162 MHz, D2O) = -0.81 (s, 1P), -8.15 (s, 1P).

[0349] <1-6> General Synthesis of N7-(aryl-2-ylmethyl)guanosine 5′-(2-thiodiphosphate)

[0350]

[0351] In DMSO (50 mM), completely dried imidazole-modified N7-modified guanosine monophosphate was added.Bn7 GMP-IM. Na) or 4-ClBn7 GMP-IM. Na (0.67 mmol) was dissolved. Then, dried thiophosphate TEA salt [(Et3NH), PSO3] was added. 3- Add 1M in DMF (4.5 equivalents) and stir at room temperature for 10 minutes. Then add zinc chloride (8 equivalents) and DMSO solution, and continue stirring for 2 hours. Afterward, slowly pour the reaction mixture into a cold solution containing sodium perchlorate (400 mg) and acetone (45 ml), centrifuge, decant the supernatant, and resuspend the precipitate in acetone. Repeat the suspension-centrifugation process 3-4 times. Dry the precipitate in a vacuum desiccator with P2O5, dissolve it in water (15-20 ml), add EDTA Na4 (1.5 equivalents), and extract with chloroform (3 x 50 ml). Then discard the organic layer, purify the aqueous layer using DEAE Sepharose fast-flow resin as described above, collect the fraction containing the product, concentrate it using a rotary evaporator, and then freeze-dry it to obtain a fine powder.

[0352] < Bn7 GDPβS.TEA (320mg, 56%)>

[0353] 1 H NMR (400 MHz, D2O) = 7.40 - 7.36 (m, 5H), 6.04 (d, J = 3.7 Hz, 1H), 5.71 (s, 2H), 4.66 (dd, J1 = 2.72 Hz, J2 = 4.96 Hz, 2H), 4.36-4.34 (m, 1H),4.28-4.27 (m, 2H); 31 P NMR (162 MHz, D2O) = 32.86 (d, J = 32.1 Hz, 1P), -11.88 (d, J = 33.0 Hz, 1P).

[0354] < 4-ClBn7 GDPβS. TEA (280mg, 47%)>

[0355] 1H NMR (400 MHz, D2O) = 7.32 (q, J = 8.5 Hz, 4H), 6.05 (d, J = 3.4 Hz,1H), 5.65 (dd, J1 = 14.9 Hz, J2 = 14.9 Hz, 2H), 4.74 (t, J = 4.4 Hz, 1H), 4.66 (d, J = 5.1 Hz, 1H), 4.38-4.33 (m, 2H), 4.29-4.24 (m, 1H); 31 P NMR (162MHz, D2O) = 33.33 (d, J = 32.2 Hz, 1P), -11.81 (d, J = 31.9 Hz, 1P).

[0356] <1-7> Synthetic methods for N7-arylmethyl-substituted CleanCap analogs

[0357] The dinucleotide pAmpG triethylammonium salt (0.07 mmol, 63 mg) was dissolved in DMSO (50 mM), and N7-(arylmethyl)guanosine 5'-diphosphonylimidazolium disodium salt (16) (3.00 equivalents, 0.2 mmol) and zinc chloride (20.0 equivalents) were added to the solution. After stirring at 37 °C for 3 days, 500 mM EDTA aqueous solution (pH 8.0) (EDTA:ZnCl2 molar equivalent was 1.30 equivalents) was added to the reaction mixture. The mixture was diluted with water and purified by DEAE Sepharose fast flow resin, followed by further purification by reversed-phase HPLC (instrument: Shimadzu; column: YMC-Actus Triart C8 (preparative type, 250 x 20.0 mm ID)). The fraction containing the target compound was collected, concentrated, and lyophilized to obtain the target N7-arylmethyl-substituted CleanCap analog in the form of triethylammonium salt. The product was redissolved in methanol (2.00 mL). A solution of 190 mM NaClO4 in acetone (24.0 mL) was added to the mixture, and the mixture was centrifuged (4000 rpm, 20 min) to obtain the resulting suspension. The supernatant was discarded, and the precipitate was resuspended in acetone. This suspension-centrifugation process was repeated 3–4 times. The precipitate was dried under reduced pressure to obtain the N7-arylmethyl-substituted CleanCap analog.

[0358] The NMR analysis results of the N7-benzylCleanCap analog N7-Bn-GpppAmpG (26 mg, 29%) are shown below.

[0359] 1H NMR (400 MHz, D2O) δ = 8.19 (s, 1H), 7.93 (s, 1H), 7.77 (s, 1H),7.01-7.23 (m, 5H), 5.75 (d, J = 5.6 Hz, 1H), 5.69 (d, J = 4.4 Hz, 1H), 5.66(d, J = 6.4 Hz, 1H), 5.44 (dd, J1 = 26.0 Hz, J2 = 14.4 Hz, 2H), 4.71-4.80 (m,2H), 4.38 (t, J = 4.4 Hz, 1H), 4.34 (t, J = 6.0 Hz, 1H), 4.29 (t, J = 5.2 Hz,2H), 4.24 (t, J = 4.4 Hz, 1H), 4.14-4.21 (m, 3H), 3.96-4.14 (m, 5H), 3.25 (s,3H); 31 P NMR (162 MHz, D2O) δ = -0.86 (s, 1P), -11.23 (d, J = 19.4 Hz, 1P), -11.43 (d, J = 17.7 Hz, 1P), -22.70 (t, J = 17.7 Hz, 1P).

[0360] <1-8> General Synthesis Process of Guanosine N7-(aryl-2-ylmethyl)trinucleotide mRNA Cap1 Analog

[0361]

[0362] pAmpG triethylammonium salt (0.07 mmol, 63 mg) was dissolved in 2 ml DMSO. N7-(aryl-2-ylmethyl)guanosine 5'-diphosphonylimidazolium disodium salt (3.00 equivalents, 0.2 mmol) and zinc chloride (20.0 equivalents) were added to this solution. After stirring at 37°C for 3 days, the reaction was terminated by adding 500 mM EDTA aqueous solution (pH 8.0) (EDTA:ZnCl2 molar equivalents were 1.30 equivalents). The mixture was diluted with water and purified using DEAE Sepharose high-flow-rate resin, followed by further purification by reversed-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 lyophilized to obtain the target N7-(aryl-2-ylmethyl)Cap1 analog as a triethylammonium salt. The product was redissolved in methanol (2.00 mL). 190 mM NaClO4 was added to acetone (24.0 mL), and the resulting suspension was centrifuged (4000 rpm, 20 min). The supernatant was discarded, and the precipitate was resuspended in acetone. Repeat the suspension-centrifugation process 3-4 times. Dry the precipitate under reduced pressure to obtain the target N7-(aryl-2-ylmethyl)Cap1 analogue in sodium salt form. Figure 4 ).

[0363] According to the method described above, guanosine N7-benzyl trinucleotide Cap1 was synthesized using N7-(benzyl-2-ylmethyl)guanosine 5′-diphosphorylimidazole and N7-(4-chlorobenzyl-2-ylmethyl)guanosine 5′-diphosphorylimidazole. Bn7 GpppAmpG), guanosine N7-4-chlorobenzyl trinucleotide Cap1 ( 4-ClBn7 GpppAmpG), L-guanosine N7-benzyltrinucleotide Cap1 ( Bn7 LGpppAmpG), L-guanosine N 7 adenosine N 6 -Dibenzyltrinucleotide Cap1 ( Bn7 Gppp Bn6 AmpG), inosine N7-benzyltrinucleotide Cap1 ( Bn7 The NMR analysis results of IpppAmpG are shown below.

[0364] <Guanosine N7-benzyl trinucleotide Cap1(Bn7 GpppA m pG)>

[0365]

[0366] 1 H NMR (400 MHz, D2O) δ = 8.19 (s, 1H), 7.93 (s, 1H), 7.77 (s, 1H),7.01-7.23 (m, 5H), 5.75 (d, J = 5.6 Hz, 1H), 5.69 (d, J = 4.4 Hz, 1H), 5.66(d, J = 6.4 Hz, 1H), 5.44 (dd, J1 = 26.0 Hz, J2 = 14.4 Hz, 2H), 4.71-4.80 (m,2H), 4.38 (t, J = 4.4 Hz, 1H), 4.34 (t, J = 6.0 Hz, 1H), 4.29 (t, J = 5.2 Hz,2H), 4.24 (t, J = 4.4 Hz, 1H), 4.14-4.21 (m, 3H), 3.96-4.14 (m, 5H), 3.25 (s,3H); 31 P NMR (162 MHz, D2O) δ = -0.86 (s, 1P), -11.23 (d, J = 19.4 Hz, 1P), -11.43 (d, J = 17.7 Hz, 1P), -22.70 (t, J = 17.7 Hz, 1P).

[0367] <Guanosine N7-4-Cl-benzyl trinucleotide Cap1( 4-ClBn7 GpppA m pG)>

[0368]

[0369] 1H NMR (400 MHz, D2O) δ = 8.34 (s, 1H), 8.09 (s, 1H), 7.90 (s, 1H),7.17 (d, J = 8.8 Hz, 2H), 7.05 (d, J = 8.4 Hz, 2H), 5.89 (d, J = 5.2 Hz, 2H),5.76 (d, J = 5.6 Hz, 1H), 5.45 (dd, J1 = 20.4 Hz, J2 = 14.8 Hz, 2H), 4.89 (m,1H), 4.75 (t, J = 5.6 Hz, 1H), 4.62 (t, J = 4.4 Hz, 1H), 4.47-4.43 (m, 3H),4.41-4.32 (m, 3H), 4.32-4.22 (m, 3H), 4.22-4.13 (m, 3H), 3.36 (s, 3H); 31 P NMR(162 MHz, D2O) δ = -0.82 (s, 1P), -11.19 (d, J = 19.6 Hz, 1P), -11.47 (d, J =18.6 Hz, 1P), -22.83 (t, J = 19.4 Hz, 1P).

[0370] <L-Guanosine N7-benzyl trinucleotide Cap1( Bn7 LGpppA m pG)>

[0371]

[0372] 1H NMR (400 MHz, D2O) δ = 9.29 (s, 1H), 8.17 (s, 1H), 8.02 (s, 1H),7.82 (s, 1H), 7.28 (m, 2H), 7.20 (m, 3H), 5.88 (d, J = 5.2 Hz, 1H), 5.82 (d,J = 3.6 Hz, 1H), 5.71 (d, J = 6.0 Hz, 1H), 5.46 (dd, J1 = 22.4 Hz, J2 = 14.8Hz, 2H), 4.79 (m, 1H), 4.52 (t, J = 4.4 Hz, 1H), 4.41-4.34 (m, 3H), 4.31-4.19(m, 5H), 4.18-4.03 (m, 5H), 3.24 (s, 3H); 31 P NMR (162 MHz, D2O) δ = -0.93 (s,1P), -11.33 (d, J = 18.6 Hz, 1P), -11.54 (d, J = 17.5 Hz, 1P), -22.80 (t, J =18.6 Hz, 1P).

[0373] <Guanosine N 7 , adenosine N 6 - dibenzyl trinucleotide Cap1( Bn7 Gppp Bn6 A m pG)>

[0374]

[0375] 1 H NMR (400 MHz, D2O) δ = 8.32 (s, 1H), 8.00 (s, 1H), 7.85 (s, 1H),7.26-7.05 (m, 10H), 5.88 (d, J = 6.0 Hz, 1H), 5.77 (d, J = 4.8 Hz, 1H), 5.73(d, J = 8.0 Hz, 1H), 5.42 (bs, 2H), 4.85 (m, 2H), 4.59 (m, 2H), 4.44-4.36 (m,4H), 4.32-4.22 (m, 4H), 4.20-4.08 (m, 5H),3.30 (s, 3H); 31P NMR (162 MHz, D2O)δ = -0.83 (s, 1P), -11.31 (d, J = 18.6 Hz, 1P), -11.51 (d, J = 17.6 Hz, 1P), -23.00 (t, J = 17.5 Hz, 1P).

[0376] <Inosine N7-benzyl trinucleotide Cap1( Bn7 IpppA m pG)>

[0377]

[0378] 1 H NMR (400 MHz, D2O) δ = 8.31 (s, 1H), 8.21 (s, 1H), 8.03 (s, 1H), 7.84 (s, 1H), 7.29-7.22 (m, 2H), 7.17-7.08 (m, 3H), 6.07 (d, J = 4.0 Hz, 1H), 5.85 (d, J = 6.0 Hz, 1H), 5.72 (d, J = 6.0 Hz, 1H), 5.62 (bs, 2H), 4.75 (overlapped with H2O, m, 1H), 4.63 (t, J = 4.4 Hz, 1H), 4.44 (t, J = 5.9 Hz,1H), 4.41 (t, J = 5.0 Hz, 1H), 4.38-4.31 (m, 3H), 4.31-4.22 (m, 2H), 4.22-4.06 (m, 6H), 3.30 (s, 3H); 31 P NMR (162 MHz, D2O) δ = -0.87 (s, 1P), -11.27 (d, J = 19.2 Hz, 1P), -11.54 (d, J = 18.7 Hz, 1P), -22.94 (t, J = 19.8 Hz, 1P).

[0379] <1-9> General Synthesis Process of N7-(aryl-2-ylmethyl)trinucleotide diphosphate Cap1 (BzCap1)

[0380]

[0381] In an anhydrous DMSO solution under an inert atmosphere, triethylammonium dinucleotide (pAmpG) salt (0.073 mmol, 65 mg) was added, followed by... Bn7 GMP-IM (0.219 mmol, 142 mg, 3 equivalents) or 4-Cl-Bn7 GMP-IM (0.219 mmol, 124 mg, 3 equivalents). Zinc chloride (20.0 equivalents) dissolved in anhydrous DMSO solution was added, and the mixture was stirred at 37°C for 3 days. The reaction was then terminated by adding an aqueous EDTA solution (pH 7.5) (EDTA: ZnCl2 molar equivalent was 1.30 equivalents). The mixture was diluted with water and purified by reversed-phase HPLC (preparative purification C18 AQ 100 Ao, 250 × 20.0 mm ID; solvent A: 0.05 M ammonium acetate buffer (pH 5.5); solvent B: 33% CH3CN in 0.05 M ammonium acetate buffer; linear gradient 0–80% B (70 min); flow rate: 10 mL / min; detection wavelength: 254 nm). The fraction containing the target compound was collected, stored in a freeze dryer, and the product was confirmed by NMR analysis.

[0382] <Guanosine N7-benzyl trinucleotide diphosphate Cap1( Bn7 GppA m pG)>

[0383]

[0384] 1 H NMR (400 MHz, D2O) = 9.22 (s, 1H), 8.24 (s, 1H), 8.10 (d, J = 1.1Hz, 1H), 7.92 (s, 1H), 7.26 (s, 5H), 5.88 (d, J = 5.7 Hz, 1H), 5.82 (d, J =4.4 Hz, 1H), 5.78 (d, J = 14.8 Hz, 1H), 5.46 (dd, J1 = 12.0 Hz, J2 = 12.0 Hz, 2H), 4.87-4.83 (m, 1H), 4.56 (t, J = 4.8 Hz, 1H), 4.45-4.39 (m, 4H), 4.33-4.30 (m, 5H), 4.16 (br s, 4H), 3.36 (s, 3H); 31P NMR (162 MHz, D2O) = -0.79(s, 1P), -11.09 (d, J = 21.2 Hz, 1P), -11.43 (d, J = 21.33 Hz, 1P).

[0385] <Guanosine N7-4-Cl-benzyl trinucleotide diphosphate Cap1( 4-ClBn7 GppA m pG)>

[0386]

[0387] 1 H NMR (400 MHz, D2O) = 9.29 (s, 1H), 8.22 (s, 1H), 8.06 (s, 1H), 7.86(s, 1H), 7.14 (d, J = 8.3 Hz, 2H), 7.08 (d, J = 8.2 Hz, 2H), 5.83-5.82 (m,2H), 5.72 (d, J = 5.6 Hz, 1H), 5.37 (dd, J1 = 12.0 Hz, J2 = 16.0 Hz, 2H), 4.53-4.52 (m, 1H), 4.38 (s, 4H), 4.30-4.26 (m, 5H), 4.12 (br s, 5H), 3.32 (s,3H); 31 P NMR (162 MHz, D2O) = -0.83 (s, 1P), -11.07 (d, J = 21.1 Hz, 1P), -11.4341 (d, J = 21.0 Hz, 1P).

[0388] <1-10> Synthesis of Cap1 analogue of N7-(aryl-2-ylmethyl)β-thiophosphate trinucleotide

[0389]

[0390] In an inert atmosphere of anhydrous DMSO solution, sodium dinucleotide (pAmpG-IM) salt (0.075 mmol, 60 mg, 1 equivalent) was added, followed by the addition of... Bn7 GDPβS.TEA (0.225 mmol, 192.14 mg, 3 equivalents) or 4-Cl-Bn7GDPβS.TEA (0.225 mmol, 200 mg, 3 equivalents). Zinc chloride (20.0 equivalents) dissolved in anhydrous DMSO solution was added, and the mixture was stirred at 37°C for 3 days. The reaction was then terminated by adding an aqueous EDTA solution (pH 7.5) (EDTA: ZnCl2 molar equivalent was 1.30 equivalents). The mixture was diluted with water and purified using reversed-phase HPLC (preparative purification C18 AQ 100 Ao, 250 × 20.0 mm ID; solvent A: 0.05 M ammonium acetate buffer (pH 5.5); buffer B: 33% CH3CN in 0.05 M ammonium acetate buffer; linear gradient 0–80% B (110 min); flow rate: 3 mL / min; detection wavelength: 254 nm). Two diastereomers of the target compound (D1 and D2) were provided and separated by retention time during HPLC purification. The product with the longer retention time was labeled D2, and the product with the shorter retention time was labeled D1. Fractions containing target compounds D1 and D2 were collected and stored in a freeze dryer. The products were confirmed by NMR analysis.

[0391] <Guanosine N7-benzyl β-phosphorothioate trinucleotide Cap1( Bn7 Gpp s pA m pG)(D1)>

[0392]

[0393] 1 H NMR (400 MHz, D2O) = 9.35 (s, 1H), 8.40 (s, 1H), 8.04 (s, 1H), 7.84(s, 1H), 7.20-7.11 (m, 5H), 5,86 (t, J = 4.0 Hz, 2H), 5.73 (d, J = 8.0 Hz,1H), 5.47 (dd, J1 = 16.0 Hz, J2 = 12.0 Hz, 2H), 4.88 (br s, 1H), 4.76 (t, J =8.0 Hz, 1H), 4.62 (t, J = 4.0 Hz, 5H), 4.46 (t, J = 4.0 Hz, 2H), 4.42-4.41(m, 3H), 4.31 (br s, 2H), 4.25 (br s, 1H), 4.19 (br s, 3H), 4.11 (br s, 2H), 3.28 (s, 3H);31 P NMR (162 MHz, D2O) = 29.68 (d, J = 25.9 Hz, 1P), -0.81 (s,1P), -12.30 (t, J = 25.9 Hz, 2P).

[0394] < Bn7 Gpp s pA m pG (D2)>

[0395]

[0396] 1 H NMR (400 MHz, D2O) = 8.16 (s, 1H), 8.00 (s, 1H), 7.85 (s, 1H), 7.17(br s, 5H), 5,81 (d, J = 8.0 Hz, 1H), 5.75 (d, J = 4Hz, 1H), 5.72 (d, J = 4.0Hz, 1H), 5.37 (dd, J1 = 12.0 Hz, J2 = 16.0 Hz, 2H), 4.82-4.78 (m, 1H), 4.65(t, J = 8.0 Hz, 1H), 4.49 (t, J = 4.0 Hz, 1H), 4.38 (t, J = 4.0 Hz, 1H),4.35-4.33 (m, 2H), 4.27-4.26 (m, 4H), 4.11 (br s, 5H), 3.31 (s, 3H); 31 P NMR(162 MHz, D2O) = 29.60 (t, J = 24.3 Hz, 1P), -0.93 (s, 1P), -12.11 (d, J =24.3 Hz, 1P), -12.46 (d, J = 25.9 Hz, 1P).

[0397] <Guanosine N7-4-Cl-benzyl β-phosphorothioate trinucleotide Cap1 ( 4 -ClBn7 Gpp s pA m pG)(D1)>

[0398]

[0399] 1H NMR (400 MHz, D2O) = 9.49 (s, 1H), 8.49 (s, 1H), 8.14 (s, 1H), 7.91(s, 1H) 7.17 (d, J = 8.4 Hz, 1H), 7.05 (d, J = 8.5 Hz, 1H), 5.95 (d, J = 3.9Hz, 1H), 5.91 (d, J = 6.0 Hz, 1H), 5.79 (d, J = 5.8 Hz, 1H), 5.51 (dd, J1 =14.8 Hz, J2 = 14.7 Hz, 2H), 4.96-4.92 (m, 1H), 4.80 (t, J = 5.5 Hz, 1H),4.53-4.45 (m, 4H), 4.39-4.38 (m, 2H), 4.31-4.18 (m, 7H), 3.34 (s, 3H); 31 P NMR(162 MHz, D2O) = 29.54 (t, J = 62.0 Hz, 1P), -0.75 (s, 1P), -12.22 (t, 2P, J= 22.9 Hz, 2P).

[0400] < 4-ClBn7 Gpp s pA m pG)(D2)>

[0401]

[0402] 1 H NMR (400 MHz, D2O) = 9.42 (s, 1H), 8.37 (s, 1H), 8.08 (s, 1H), 7.90(s, 1H) 7.19 (d, J = 7.6 Hz, 2H), 7.08 (d, J = 7.6 Hz, 2H), 5.90-5.87 (m,2H), 5.77 (br s, 1H), 5.59 (dd, J1 = 13.4 Hz, J2 = 15.0 Hz, 2H), 4.96 (br s,1H), 4.48-4.41 (m, 5H), 4.36 (br s, 2H), 4.30 (br s, 1H), 4.24-4.18 (m, 6H),3.36 (s, 3H); 31P NMR (162 MHz, D2O) = 29.75 (t, J = 25.3 Hz, 1P), -0.86 (s,1P), -11.92 (d, J = 25.3 Hz, 1P) and -12.29 (d, J = 24.6 Hz, 1P).

[0403] <Example 2> Introduction of Trinucleotide Capping Substances into mRNA and Analysis of Capping Efficiency

[0404] The trinucleotide capping material prepared in Example 1 was analyzed to determine whether it could be efficiently recognized by T7 RNA polymerase and introduced into mRNA during transcription.

[0405] The results showed that by introducing a capping substance into the mRNA by modifying the original methyl group at the 5' end of the guanosine N7 position to an aryl methyl group, the antigen protein expression level during translation was similar to or higher than that of the existing CleanCap (cap 1 commercial) and the previously reported ARCAcap.

[0406] Furthermore, it was confirmed that the capping substance can be recognized and introduced into RNA by T7 RNA polymerase during transcription with a higher efficiency than ARCA. The capping efficiency of existing ARCA is approximately 50%, while CleanCap (cap 1 commercial) is approximately 85%. However, BzCap1 and its analogues achieve a capping efficiency of over 90% (see [link to study]). Figure 45 ).

[0407] <Example 3> Analysis of antigen protein expression using arylmethyl-modified capping substances

[0408] Translation was confirmed by introducing a capping substance into mRNA, prepared by modifying the N7 position of the 5'-terminal guanosine base from the original methyl group to an aryl methyl compound. To confirm the expression level of the antigen protein, luciferase mRNA was used, and the expression level of the antigen protein was analyzed using HEK 293 cells. For luciferase mRNA, it was confirmed that BzCap1 and its analogues were more efficient at expressing antigen proteins than CleanCap in mRNA using native rNTPs (see [link to documentation]). Figure 46 ).

[0409] ① Preparation of luciferase DNA template

[0410] Linear dsDNA templates, including the start codon from the luciferase plasmid to the poly-A tail, were prepared by PCR. During serial PCR, the T7 promoter and Kozak sequence were added to the luciferase dsDNA template, which was then used for transcription. The primers used included: 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); and reverse primer: AAT CGC GCC TAG GCG CGC CCG TAC GGC TCT TC-3' (Sequence No. 3).

[0411] ② Preparation of luciferase mRNA

[0412] The concentration of luciferase template was measured using a nanodrop spectrophotometer. For each transcription reaction, 1 μL of 100 ng template was added to 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. (1 μL, 10 mM) of rATP, rCTP, and rUTP were used, respectively. For cap-dependent transcription by luciferase, rGTP (1 μL, 2.5 mM) was used. The proportion of capping material was maintained 4 times higher than rGTP to provide more than 80% capped mRNA. Therefore, 1 μL of capping material (10 mM) was added. An RNase inhibitor (0.5 μL, 40 U / μL) and T7 RNA polymerase (1 μL, 50 U / μL) were also added. Nuclease-free water (4.5–5.5 μL) was added to bring the final reaction volume to 15 μL. For the modified rNTPs, a 10 mM stock solution was prepared, and rCTP and rUTP were replaced with their respective derivatives. The reaction was incubated at 37 °C for 1 hour. 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 mixture, and the mixture was further incubated at 37 °C for 15 minutes to remove all DNA template.

[0413] ③ Purification of transcribed mRNA

[0414] To remove any dsRNA and transcriptional byproducts that may form, cellulose (10 μL, 0.2 g / mL) was added to the reaction mixture and gently shaken for 10 minutes. Next, the mixture was transferred to an RNA purification column containing the ethanol and binding buffer provided with the column, and centrifuged at 13000 rpm for 1 minute. Then, RNA preparation buffer and wash buffer were continuously added to the column and centrifuged. After this step, all proteins and unintegrated rTNPs were removed by suspension. Finally, a new centrifuge tube was attached, 10 μL of nuclease-free water was added to the column, and centrifuged at 13000 rpm for 2 minutes. All cellulose-bound dsDNA remained in the column. Only pure single-stranded GFP or luciferase mRNA passed through the column. The sample was collected and stored at 70°C for subsequent use.

[0415] ④ Analysis of mRNA capping efficiency

[0416] 2 μg of capped transcribed mRNA was heated to 70 °C with 3 μg of gDNA and RNase H buffer, and then slowly cooled to 25 °C for annealing. Next, the mixture was reacted at 37 °C for 1 hour using heat-stable RNase H to cleave the short sequence containing the capping material from the long mRNA. The cleaved short sequences containing the capping material were analyzed by electrophoresis on a 16% denaturing PAGE gel. Short sequences cleaved from uncapped mRNA showed faster gel migration, while those cleaved from capped mRNA showed relatively slower gel migration.

[0417] ⑤ Cell culture and preparation for transfection

[0418] HEK 293 cells were cultured in 60 mL DMEM dishes containing a mixture of 10% FBS and 1% penicillin / strep. The day before transfection, cells were isolated using trypsin and resuspended in fresh medium. Cells were seeded in 96-well plates. The next day, when cells reached 70% confluence, the transfection mixture was prepared and transfection was performed.

[0419] ⑥ Quantification and transfection of mRNA

[0420] Purified mRNA was quantified using a nanodrop spectrophotometer, and the transcription yields of various rNTP combinations were compared. For cell infection, 500 ng of each mRNA was 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 min. The mRNA and Lipofectamine mixture was then transferred to the same tube and incubated for another 5 min at room temperature. Finally, the transfection mixture was added to the cells and incubated for 24 h.

[0421] ⑦ Luciferase expression analysis

[0422] After the culture period, the culture 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 added to each well of a white 96-well plate. Then, 20 μL of each cell lysate sample was added and mixed with a pipette. Finally, the luciferase luminescence was recorded using a luminometer.

[0423] The results showed that the aryl methyl capping substances introduced in this invention can be recognized and introduced into RNA by T7 RNA polymerase with a higher efficiency than ARCA or CleanCap during transcription. The existing ARCA capping efficiency is about 50%, while CleanCap is about 89%, but the introduction of various aryl methyl trinucleotide capping substances ( Bn7 GpppAmpG, Bn7 GppAmpG, 4-ClBn7 GppAmpG, Bn7 Gppp Bn6 AmpG, 4-ClBn7 GpppAmpG, Bn7 GppspAmpG(D1). Bn7 GppspAmpG(D2). 4- ClBn7 GppspAmpG(D1). 4-ClBn7 GppspAmpG, Bn7 IpppAmpG and Bn7 The capping efficiency of LGpppAmpG reached over 90% (see [link]). Figure 45 ).

[0424] Furthermore, the translation process following the introduction of a capped substance into mRNA by modifying the N7 position of the 5'-terminal guanosine base from the original methyl group to an arylmethyl compound was confirmed.

[0425] To confirm the expression level of the antigen protein, luciferase mRNA was used, and the expression level of the antigen protein was analyzed using HEK 293 cells. For luciferase mRNA, mRNA containing native rNTPs was used.

[0426] Furthermore, it has been confirmed that, compared to mRNAs with existing ARCA cap (cap 0) and CleanCap (cap 1 commercial), the introduction of various trinucleotide capping substances developed in this invention (…) Bn7 GpppAmpG, Bn7 GppAmpG, 4- ClBn7 GppAmpG, Bn7 Gppp Bn6 AmpG, 4-ClBn7 GpppAmpG, Bn7 GppspAmpG(D1). Bn7 GppspAmpG(D2). 4- ClBn7 GppspAmpG(D1). 4-ClBn7 GppspAmpG, Bn7 IpppAmpG and Bn7 LGpppAmpG's luciferase mRNA showed higher antigen protein expression efficiency (see [link]). Figure 46 ).

[0427] Industrial applicability

[0428] This invention relates to novel modified trinucleotide capping derivatives and mRNAs incorporating these capping derivatives. Specifically, it provides a method for introducing a methylene-bridged heterocyclic compound and an aryl methyl derivative at the N7 position of the 5' guanosine base, achieving higher efficiency than capping substances used in existing mRNA vaccines. The novel modified trinucleotide capping derivatives and mRNAs incorporating these capping derivatives can be effectively applied to the development of novel mRNA vaccines and various mRNA-based therapeutics, thus possessing broad application potential in industrial processes and other fields.

[0429] Therefore, the modified trinucleotide capping derivative and the mRNA incorporating the capping derivative according to an embodiment of the present invention have industrial applicability.

Claims

1. A trinucleotide cap analogue, characterized in that, Including the following compounds represented by chemical formula 1: Chemical Formula 1: ， In the compound of chemical formula 1, R1 is a heterocyclic compound in which the N7 position of the N7 guanosine base is bridged by a methylene group. In the compounds of chemical formula 1, X1 and X2 are H or CH3, and X3, X4 and X5 are selected from the group consisting of O, S and Se, respectively. R2 is a derivative that includes a benzyl group. The heterocyclic compound comprises one or two heteroatoms selected from O, N, and S, and is any functional group selected from saturated or partially saturated 5- to 10-membered heteroaromatic ring compounds. The heteroaromatic ring compound is selected from any functional group in the group consisting of furan, pyrrole, thiophene, imidazole, pyrazole, oxazole, isoxazole, thiazole, pyridine, pyrazine, pyrimidine, pyridazine, and triazine.

2. The trinucleotide cap analogue according to claim 1, characterized in that, The trinucleotide cap analogue is represented by the following chemical formula 2 or chemical formula 3: Chemical formula 2: ， Chemical formula 3: 。 3. An mRNA capped at the 5' end using the trinucleotide cap analogue of claim 1.

4. A composition for preparing mRNA comprising a 5' end capped with a trinucleotide cap analog as described in claim 1.

5. A pharmaceutical composition for the expression of a target peptide or protein, characterized in that, The mRNA includes a 5' capped mRNA and a pharmaceutically acceptable vector, wherein the mRNA includes the trinucleotide capped analog of claim 1.

6. A trinucleotide cap analogue, characterized in that, This includes compounds represented by the following chemical formulas 4-1, 4-2, or 4-3: Chemical formula 4-1: ， Chemical formula 4-2: ， Chemical formula 4-3: ， In the compounds represented by chemical formula 4-1, chemical formula 4-2 or chemical formula 4-3, R1 is a compound comprising a compound of formula 5 linked by a methyl group at the N7 position. X1 and X2 are H or CH3, respectively. Y1 is either 0 or 1. Y2 is O or S. Z is H or C1-C3 alkylbenzyl. Chemical formula 5: ， In the chemical formula 5, R2, R3, R4, R5 and R6 are each independently selected from any functional group in the group consisting of H, hydroxyl, halogen, methyl, alkyl, alkoxy, nitro, carboxyl, azide, amino and cyano.

7. The trinucleotide cap analogue according to claim 6, characterized in that, R1 is benzyl or chlorobenzyl.

8. The trinucleotide cap analogue according to claim 6, characterized in that, Z is methyl benzyl.

9. An mRNA capped at the 5' end using the trinucleotide cap analog of claim 6.

10. A composition for preparing mRNA comprising a 5' end capped with a trinucleotide cap analog of claim 6.

11. A pharmaceutical composition for the expression of a target peptide or protein, characterized in that, The mRNA includes a 5' capped mRNA and a pharmaceutically acceptable vector, wherein the mRNA includes the trinucleotide capped analog of claim 6.

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