Novel modified trinucleotide capping derivative, and mRNA into which capping derivative is introduced

Abstract

The present invention relates to a novel modified trinucleotide capping derivative, and mRNA into which the capping derivative is introduced. The derivatives of the present invention in which a methylene bridge heterocyclic compound and arylmethyl are individually introduced into the N7-position of the 5'- terminal guanosine base have higher efficiency than capping materials used in existing mRNA vaccines, and thus can be efficiently used in the development of mRNA vaccines and the development of various mRNA-based therapeutic agents.

Description

Novel modified trinucleotide capping derivatives and mRNAs with capping derivatives

[0001] The present invention relates to a novel modified trinucleotide capping derivative and an mRNA into which a capping derivative has been introduced, and more particularly, to mRNA structures into which a trinucleotide-based capping material has been introduced, in which the N7 position of the 5'-terminal guanosine base has been modified with a methylene bridged heterocyclic compound and arylmethyl instead of the existing methyl.

[0002]

[0003] mRNA vaccines are playing a pivotal role in treating the COVID-19 pandemic, which is spreading globally. The capping material protects the mRNA vaccine from degradation. Specifically, to express a functionally important protein from DNA genetic information, transcription is required in the nucleus, where mRNA with a complementary sequence is synthesized based on a DNA template strand. This synthesized mRNA is then transported out of the nucleus and translated into the cytoplasm by ribosomes, where it synthesizes a specific protein.

[0004] In particular, N7 methylation in the CAP structure adds a methyl group to the seventh nitrogen atom of guanosine at the 5' end of mRNA. This methylation performs several important functions that significantly enhance the biological efficacy of mRNA. The N7 methylated 5' cap structure enhances the stability of the mRNA molecule, facilitates its binding to the ribosome, and helps initiate the translation process by promoting the binding of translation initiation factors eIF4E and eIF4G. Furthermore, the N7 methylated cap structure plays a crucial role in the efficient transport of mRNA from the nucleus to the cytoplasm and serves as a marker of cellular mRNA to avoid recognition and degradation by the innate immune system. Overall, the N7 methylated 5' cap is a critical feature for the stability and function of mRNA in eukaryotic cells, affecting the life cycle of RNA and its ability to efficiently produce proteins.

[0005] There are two capping methods used. The first is 5' capping, which involves post-transcriptional processing of the 5' end of the RNA product of DNA transcription. After transcription, the 5' end of the RNA transcript contains a free triphosphate group because it is the first nucleotide added to the chain. The capping process replaces the triphosphate group with another structure called a "cap." The cap is added by the enzyme guanyl transferase, which catalyzes the reaction between the 5' end of the RNA transcript and a guanine triphosphate (GTP) molecule. The second capping process used in mRNA vaccines is achieved through in vitro transcription (IVT), a technique that allows for simultaneous capping during mRNA transcription. The cap is added during mRNA synthesis, rather than as a separate step after transcription. This method simplifies the capping process, increasing capping efficiency and enabling simple processes and high-yield mRNA production. The 5' capping structure, a protein initiator, plays a crucial role in protein expression by forming a translation initiation complex with eukaryotic translation initiation factor 4E (eIF4E). It also serves as a marker of intracellular mRNA to avoid recognition and degradation by the innate immune system.

[0006] For about 20 years, dinucleotide-based ARCA cap compounds have been used as chemical capping materials through IVT, but after the actual COVID-19 outbreak, trinucleotide-based CleanCap, which has a much higher antibody expression efficiency, is being used as the main component of Pfizer's mRNA vaccine.

[0007] Developed by TriLink, CleanCap has a chemically modified trinucleotide structure and is reported to be the most effective capping material developed to date for expressing antigen proteins in mRNA vaccines. TriLink holds an exclusive patent for this capping material.

[0008] However, domestically, no technology has been developed to increase the antigen protein expression efficiency of mRNA vaccines, like CleanCap, preventing independent acquisition of mRNA vaccines. Therefore, the inventors synthesized mRNA using a capping technology that simultaneously transcribes and caps via IVT, due to its efficiency.

[0009]

[0010] Accordingly, the present inventors have developed a capping material substituted with a new N7-methylene bridged heterocyclic compound, which is different from the CleanCap modified nucleotide, and a capping material substituted with a new N7-arylmethyl (benzyl (BzCap1) and derivatives, which can increase the antigen protein expression efficiency of an mRNA vaccine like CleanCap, thereby completing the present invention.

[0011]

[0012]

[0013] In order to solve the above problem, a trinucleotide cap analogue comprising a compound of the following chemical formula 1 of the present invention:

[0014] [Chemical Formula 1]

[0015]

[0016] In the compound of the above chemical formula 1, R1 is a heterocyclic compound connected to the N7 position of the N7 guanosine base by a methylene bridge,

[0017] In the compound of the above chemical formula 1, X1 and X2 are H or CH3, and X3, X4 and X5 are each selected from the group consisting of O, S and Se,

[0018] R2 is a derivative containing Benzyl,

[0019] The above heterocyclic compound is a functional group selected from among saturated or partially saturated 5- to 10-membered heteroaromatic ring compounds containing 1 to 2 heteroatoms selected from O, N, and S,

[0020] The above heteroaromatic ring compound is any one functional group selected from the group consisting of furan, pyrrole, thiophen, imidazole, pyrazole, oxazol, isoxazole, thiazole, pyridine, pyrazine, pyrimidine, pyridazine, and triazine.

[0021] The above heterocyclic compound may contain 1 to 2 heteroatoms selected from O, N, and S, and may be any one functional group selected from a saturated or partially saturated 5- to 10-membered aromatic ring group. Specific examples of heterocyclic compounds may include thiophene, pyridyl, imidazole, tetrahydrofuranyl, 2,3-dihydrofuranyl, 2,5-dihydrofuranyl, pyrrolidinyl, 2,3-dihydropyrrolidinyl, 2,5-dihydropyrrolidinyl, tetrahydro-2H-pyranyl, 3,4-dihydro-2H-pyranyl, 4H-pyranyl, piperidinyl, 1,2,3,4-tetrahydropyridinyl, 1,4-dihydropyridinyl, piperazinyl, N-protected piperazinyl, morpholino, etc. The N-protecting group of piperazinyl may typically include an alkyl group, an alkylcarbonyl group, or an alkylsulfonyl group.

[0022] In the present invention, the trinucleotide cap analogue may be characterized by the following chemical formula 2 or chemical formula 3.

[0023] [Chemical Formula 2]

[0024]

[0025] [Chemical Formula 3]

[0026]

[0027] A trinucleotide cap analog comprising a compound of the following chemical formula [4-1], [4-2] or [4-3]:

[0028] [Chemical Formula 4-1]

[0029]

[0030] [Chemical Formula 4-2]

[0031]

[0032] [Chemical Formula 4-3]

[0033]

[0034] In the compound of the above chemical formula [4-1], [4-2] or [4-3],

[0035] The above R1 is a compound including a compound of chemical formula 5 linked to methyl (CH2) at the N7 position,

[0036] The above X1 and X2 are each H or CH3,

[0037] The above Y1 is 0 or 1,

[0038] The above Y2 is O or S,

[0039] The above Z is H or C1-C3 alkylbenzyl,

[0040] [Chemical Formula 5]

[0041]

[0042] In the above chemical formula 5, R2, R3, R4, R5 and R6 are each independently any one functional group selected from the group consisting of H, OH, Halo, methyl, alkyl, alkoxy, nitro, carboxylic, azide, amino and cyano.

[0043] Additionally, the present invention provides an mRNA capped at the 5' end with the trinucleotide cap analogue.

[0044] The present invention also provides a composition or kit for producing mRNA capped at the 5' end, comprising a trinucleotide cap analog.

[0045] The present invention also provides a pharmaceutical composition for expressing a desired peptide or protein, comprising an mRNA capped at the 5' end with a trinucleotide cap analog and a pharmaceutically acceptable carrier.

[0046]

[0047] The derivatives of the present invention, which introduce a methylene bridged heterocyclic compound and an arylmethyl group at the N7 position of the 5' terminal guanosine base, introduce a heterocyclic group and an arylmethyl (benzyl) group connected by a methylene bridge at the N7 position of the 5' terminal guanosine base, respectively, and are more efficient than the capping materials used in existing mRNA vaccines, and thus can be usefully used in the development of mRNA vaccines and various mRNA-based therapeutic agents.

[0048]

[0049] Figure 1 shows trinucleotide-based capping materials ThioCap1 and PyCap1, in which a methylene bridged thiophene group and a methylene bridged pyridine group are introduced at the 5'-terminal guanosine N7 position (R1), respectively.

[0050] Figure 2 shows the ThioCap1 material. 1 This is a graph showing the H NMR spectrum.

[0051] Figure 3 is a diagram of the ThioCap1 material. 31 This is a graph showing the P NMR spectrum.

[0052] Figure 4 is a diagram of the PyCap1 material. 1 This is a graph showing the H NMR spectrum.

[0053] Figure 5 is a diagram of the PyCap1 material. 31 This is a graph showing the P NMR spectrum.

[0054] Figure 6 confirms transcription of the capping material (ThioCap1) of the present invention using T7 RNA polymerase (Lane1: Luciferase Template + T7RNAPol + rNTPs + CleanCap (Commertial Cap1); Lane2: Luciferase Template + T7RNAPol + rATP + rGTP + rCTP + PseudoUTP + CleanCap (Commertial Cap1); Lane3: Luciferase Template + T7RNAPol + rNTPs + ThioCap1; Lane4: Luciferase Template + T7RNAPol + rATP + rGTP + rCTP + PseudoUTP + ThioCap1).

[0055] Figure 7 shows the results of confirming the capping efficiency of the capping material (ThioCap1) of the present invention by treating with RNase H using T7 RNA polymerase (Lane1: Luciferase Template + T7RNAPol + rNTPs -> gDNA + RNase H; Lane2: Luciferase Template + T7RNAPol + rNTPs + ARCA -> gDNA + RnaseH; Lane3: Luciferase Template + T7RNAPol + rNTPs + CleanCap -> gDNA + RNase H; Lane4: Luciferase Template + T7RNAPol + rNTPs + ThioCap1 -> gDNA + RNase H).

[0056] Figure 8 is a graph comparing the expression of the antigen protein (Luciferase) of Luciferase mRNA (Luc mRNA ThioCap1) to which the capping material of the present invention (ThioCap1) has been introduced and the existing CleanCap (Luc mRNA Cap1).

[0057] Figure 9 confirms transcription of the capping material (PyCap1) of the present invention using T7 RNA polymerase (Lane1: Luciferase Template + T7RNAPol + rNTPs + CleanCap (Commertial Cap1); Lane2: Luciferase Template + T7RNAPol + rNTPs + PyCap1).

[0058] Figure 10 shows the capping efficiency of the capping material (PyCap1) of the present invention using RNA polymerase, confirmed by treatment with RNase H.

[0059] Figure 11 is a graph comparing the expression of the antigen protein (Luciferase) of Luciferase mRNA (Luc mRNA PyCap1) to which the capping material of the present invention (PyCap1) has been introduced and the existing CleanCap (Luc mRNA Cap1).

[0060] Figure 12 is a chemical formula showing a trinucleotide-based capping material (BzCap1) in which an arylmethyl (benzyl) group is introduced at the 5'-terminal guanosine N7 position (R1).

[0061] Figure 13 is a schematic diagram showing the synthetic process of N7-(arylmethyl)guanosine 5'-diphosphoroimidazolide analogue.

[0062] Figure 14 is a schematic diagram showing the synthesis procedure of dinucleotide pAmpG.

[0063] Figure 15 is a schematic diagram showing the synthetic procedure of a guanosine N7-(aryl-2-ylmethyl) trinucleotide mRNA cap1 analog.

[0064] Figure 16 is a schematic diagram showing the synthetic procedure of N7-(aryl-2-ylmethyl) trinucleotide diphosphate cap1 analogues.

[0065] Figure 17 is a schematic diagram showing the synthetic procedure of N7-(aryl-2-ylmethyl) β-phosphorothioate trinucleotide Cap1 analogue.

[0066] Fig. 18 is 1 H NMR spectrum of Guanosine N7-benzyl trinucleotide cap analog.

[0067] Fig. 19 is 31 P NMR spectrum of Guanosine N7-benzyl trinucleotide cap analog.

[0068] Fig. 20 is 1 H NMR spectrum of Guanosine N7-benzyl trinucleotide diphosphate cap analog

[0069] Fig. 21 is 31 P NMR spectrum of Guanosine N7-benzyl trinucleotide diphosphate cap analog

[0070] 도 22는 ESI-MS of Guanosine N7-benzyl trinucleotide diphosphate cap analog.

[0071] 도 23은 1 H NMR spectrum of Guanosine N7-4-Cl-benzyl trinucleotide diphosphate cap analog.

[0072] 도 24는 31 P NMR spectrum of Guanosine N7-4-Cl-benzyl trinucleotide diphosphate cap analog.

[0073] 도 25는 ESI-MS of Guanosine N7-4-Cl-benzyl trinucleotide diphosphate cap analog.

[0074] 도 26은 1 H NMR spectrum of Guanosine N7, adenosine N6-dibenzyl trinucleotide cap analog.

[0075] 도 27은 31 P NMR spectrum of Guanosine N7, adenosine N6-dibenzyl trinucleotide cap analog.

[0076] 도 28은 ESI-MS of Guanosine N7, adenosine N6-dibenzyl trinucleotide cap analog.

[0077] 도 29는 1 H NMR spectrum of Guanosine N7-4-Cl-benzyl trinucleotide cap analog.

[0078] 도 30은 31 P NMR spectrum of Guanosine N7-4-Cl-benzyl trinucleotide cap analog.

[0079] 도 31은 1 H NMR spectrum of Guanosine N7-benzyl β-phosphorothioate trinucleotide cap analog(D1).

[0080] 도 32는 31 P NMR spectrum of Guanosine N7-benzyl β-phosphorothioate trinucleotide cap analog (D1).

[0081] 도 33은 1 H NMR spectrum of Guanosine N7-benzyl β-phosphorothioate trinucleotide cap analog (D2).

[0082] 도 34는 31 P NMR spectrum of Guanosine N7-benzyl β-phosphorothioate trinucleotide cap analog (D2).

[0083] 도 35는 1 H NMR spectrum of Guanosine N7-4-Cl-benzyl β-phosphorothioate trinucleotide cap analog (D1).

[0084] 도 36은 31 P NMR spectrum of Guanosine N7-4-Cl-benzyl β-phosphorothioate trinucleotide cap analog (D1).

[0085] 도 37은 1 H NMR spectrum of Guanosine N7-4-Cl-benzyl β-phosphorothioate trinucleotide cap analog (D2).

[0086] 도 38은 31P NMR spectrum of Guanosine N7-4-Cl-benzyl β-phosphorothioate trinucleotide cap analog (D2).

[0087] Fig. 39 is 1 H NMR spectrum of Inosine N7-benzyl trinucleotide cap analog.

[0088] Fig. 40 is 31 P NMR spectrum of Inosine N7-benzyl trinucleotide cap analog.

[0089] Fig. 41 is 1 H NMR spectrum of L-Guanosine N7-benzyl trinucleotide cap analog.

[0090] Fig. 42 is 31 P NMR spectrum of L-Guanosine N7-benzyl trinucleotide cap analog.

[0091] Figure 43 is a gel photograph showing the results of an experiment in which a capping material (BzCAP1) was transcribed using T7 RNA polymerase and the capping efficiency during in vitro transcription was confirmed by treatment with RNase H. The capping efficiency was compared with that of existing ARCA and CleanCap.

[0092] Figure 44 shows the transcription of various arylmethyl-modified trinucleotide-based capping materials of the present invention using T7 RNA polymerase.

[0093] Lane 1: Luciferase mRNA+ m7 GpppA m pG

[0094] Lane 2: Luciferase mRNA+ Bn7 GpppA m pG

[0095] Lane 3: Luciferase mRNA+ Bn7 GppA m pG

[0096] Lane 4: Luciferase mRNA+ 4-ClBn7 GppA m pG

[0097] Lane 5: Luciferase mRNA+ Bn7 Gppp Bn6 A m pG

[0098] Lane 6: Luciferase mRNA+ 4-ClBn7 GpppA m pG

[0099] Lane 7: Luciferase mRNA+ Bn7 GppspA m pG (D1)

[0100] Lane 8: Luciferase mRNA+ Bn7 GppspA m pG (D2)

[0101] Lane 9: Luciferase mRNA+ 4-ClBn7 GppspA m pG (D1)

[0102] Lane 10: Luciferase mRNA+ 4-ClBn7 GppspA m pG (D2)

[0103] Lane 11: Luciferase mRNA+ Bn7 IpppA m pG

[0104] Lane 12: Luciferase mRNA+ Bn7 LGpppA m pG

[0105] Figure 45 shows the capping efficiency of the various arylmethyl-modified trinucleotide-based capping materials of the present invention using RNA polymerase, as confirmed by RNase H treatment.

[0106] Lane 1: Luciferase mRNA+ m7 GpppA m pG

[0107] Lane 2: Luciferase mRNA+ Bn7 GpppA m pG

[0108] Lane 3: Luciferase mRNA+ Bn7 GppA m pG

[0109] Lane 4: Luciferase mRNA+ 4-ClBn7 GppA m pG

[0110] Lane 5: Luciferase mRNA+ Bn7 Gppp Bn6 A m pG

[0111] Lane 6: Luciferase mRNA+ 4-ClBn7 GpppA m pG

[0112] Lane 7: Luciferase mRNA+ Bn7 GppspA m pG (D1)

[0113] Lane 8: Luciferase mRNA+ Bn7 GppspA m pG (D2)

[0114] Lane 9: Luciferase mRNA+ 4-ClBn7 GppspA m pG (D1)

[0115] Lane 10: Luciferase mRNA+ 4-ClBn7 GppspA m pG (D2)

[0116] Lane 11: Luciferase mRNA+ Bn7 IpppA m pG

[0117] Lane 12: Luciferase mRNA+ Bn7 LGpppA m pG

[0118] Figure 46 is a graph confirming the expression of an antigen protein (Luciferase) using a trinucleotide-based capping material in which the N7 position of the 5' terminal guanosine is modified with various arylmethyls.

[0119] Lane 1: Luciferase mRNA+ m7 GpppA m pG

[0120] Lane 2: Luciferase mRNA+ Bn7 GpppA m pG

[0121] Lane 3: Luciferase mRNA+ Bn7 GppA m pG

[0122] Lane 4: Luciferase mRNA+ 4-ClBn7 GppA m pG

[0123] Lane 5: Luciferase mRNA+ Bn7 Gppp Bn6 A m pG

[0124] Lane 6: Luciferase mRNA+ 4-ClBn7 GpppA m pG

[0125] Lane 7: Luciferase mRNA+ Bn7 GppspA m pG (D1)

[0126] Lane 8: Luciferase mRNA+ Bn7 GppspA m pG (D2)

[0127] Lane 9: Luciferase mRNA+ 4-ClBn7 GppspA m pG (D1)

[0128] Lane 10: Luciferase mRNA+ 4-ClBn7 GppspA m pG (D2)

[0129] Lane 11: Luciferase mRNA+ Bn7 IpppA m pG

[0130] Lane 12: Luciferase mRNA+ Bn7 LGpppA m pG

[0131]

[0132] The present invention is described in detail below.

[0133] The present inventors have prepared a novel modified trinucleotide capping derivative and an mRNA into which a capping derivative has been introduced, and specifically, have developed various modified capping compounds in which a methylene bridged heterocyclic group and an arylmethyl (benzyl) group have been introduced at the N7 position of the 5'-terminal guanosine base, so as to increase the antigen protein expression efficiency of an mRNA vaccine like CleanCap and to increase the efficiency more than the CleanCap modified nucleotide, and have confirmed that the mRNA structures into which various methylene bridged heterocyclic groups and / or arylmethyl (benzyl) groups have been introduced have superior antigen protein expression efficiency than the capping materials used in existing mRNA vaccines, thereby completing the present invention.

[0134] The present invention is characterized by designing and synthesizing novel modified trinucleotide capping materials and verifying their potential for use as mRNA vaccines. Novel compounds were synthesized through organic chemical reactions, in which the N7 position of the 5'-terminal guanosine base in the CleanCap trinucleotide structure was modified with a methylene bridged thiophene compound, a methylene bridged pyridine compound, and an arylmethyl (benzyl) compound instead of the conventional methyl group. The synthetic process and results are presented.

[0135] It was confirmed that the capping material can be recognized by T7 RNA polymerase and introduced into RNA with an efficiency similar to or higher than ARCA or CleanCap during the transcription process. The existing ARCA showed a capping efficiency of about 50% and CleanCap showed a capping efficiency of about 86%, but ThioCap1 showed a capping efficiency of about 92% or more, BzCap1 and BzCap1 analogs showed a capping efficiency of about 90% or more, and PyCap1 capping material also showed a capping efficiency of about 88% or more.

[0136] In addition, it was confirmed that the Luciferase mRNA (Luc mRNA ThioCap 1) introduced with ThioCap1 developed in the present invention, the Luciferase mRNA (Luc mRNA PyCap 1) introduced with PyCap1, the Luciferase mRNA (Luc mRNA BzCap 1) introduced with BzCap1, and the Luciferase mRNA introduced with BzCap1 analogs showed higher antigen protein expression efficiency than the mRNA (Luc mRNA Cap 1) introduced with the existing CleanCap (see Figs. 8, 11, and 46).

[0137] It was confirmed that the Luciferase mRNA introduced with BzCap1 and BzCap1 analog developed in the present invention showed higher antigen protein expression efficiency than the mRNA introduced with the existing ARCA cap (cap 0) and CleanCap (Cap 1 commercial).

[0138] Therefore, through the above results, the trinucleotide cap analogs according to the present invention can be usefully utilized in the development of new mRNA vaccines and various mRNA-based therapeutic fields, as they are more efficient than the capping materials used in existing mRNA vaccines by introducing a methylene bridged heterocyclic compound and an arylmethyl group at the N7 position of the 5' terminal guanosine base, respectively.

[0139] A trinucleotide cap analog comprising a compound of the following chemical formula 1:

[0140] [Chemical Formula 1]

[0141]

[0142] In the compound of the above chemical formula 1, R1 is a heterocyclic compound connected to the N7 position of the N7 guanosine base by a methylene bridge,

[0143] In the compound of the above chemical formula 1, X1 and X2 are H or CH3, and X3, X4 and X5 are each selected from the group consisting of O, S and Se,

[0144] R2 is a derivative containing Benzyl,

[0145] The above heterocyclic compound is a functional group selected from among saturated or partially saturated 5- to 10-membered heteroaromatic ring compounds containing 1 to 2 heteroatoms selected from O, N, and S,

[0146] The above heteroaromatic ring compound may be any one functional group selected from the group consisting of furan, pyrrole, thiophen, imidazole, pyrazole, oxazol, isoxazole, thiazole, pyridine, pyrazine, pyrimidine, pyridazine, and triazine.

[0147] The 'heterocyclic compound' in the present invention is a heteroatom selected from O, N, and S.

[0148] It means a saturated or partially saturated 5- to 10-membered aromatic ring group containing 1 to 2 heteroatoms. Specific examples of such heterocyclic compounds may include thiophene, pyridyl, imidazole, tetrahydrofuranyl, 2,3-dihydrofuranyl, 2,5-dihydrofuranyl, pyrrolidinyl, 2,3-dihydropyrrolidinyl, 2,5-dihydro-pyrrolidinyl, tetrahydro-2H-pyranyl, 3,4-dihydro-2H-pyranyl, 4H-pyranyl, piperidinyl, 1,2,3,4-tetrahydropyridinyl, 1,4-dihydropyridinyl, piperazinyl, N-protected piperazinyl, morpholino, etc. The N-protecting group of piperazinyl may typically include an alkyl group, an alkylcarbonyl group, or an alkylsulfonyl group.

[0149] A trinucleotide cap analog comprising a compound of the following chemical formula [4-1], [4-2] or [4-3]:

[0150] [Chemical Formula 4-1]

[0151]

[0152] [Chemical Formula 4-2]

[0153]

[0154] [Chemical Formula 4-3]

[0155]

[0156] In the compound of the above chemical formula [4-1], [4-2] or [4-3],

[0157] The above R1 is a compound including a compound of chemical formula 5 linked to methyl (CH2) at the N7 position,

[0158] The above X1 and X2 are each H or CH3,

[0159] The above Y1 is 0 or 1,

[0160] The above Y2 is O or S,

[0161] The above Z is H or C1-C3 alkylbenzyl,

[0162] [Chemical Formula 5]

[0163]

[0164] In the above chemical formula 5, R2, R3, R4, R5 and R6 are each independently any one functional group selected from the group consisting of H, OH, Halo, methyl, alkyl, alkoxy, nitro, carboxylic, azide, amino and cyano.

[0165] The above R1 may be benzyl or chlorobenzyl, but is not limited thereto.

[0166] Additionally, the above Z may be methylbenzyl.

[0167] The substituents used to define the compounds according to the present invention are described in more detail as follows.

[0168] In the present invention, ‘halo’ or ‘halogen atom’ are terms that can be used interchangeably, and mean chloro, fluoro, bromo, and iodo.

[0169] In the present invention, 'alkyl' refers to a straight-chain, branched-chain, or cyclic aliphatic saturated hydrocarbon group having 1 to 5 carbon atoms. Specific examples of such alkyl groups include a methyl group, an ethyl group, a normal propyl group, an isopropyl group, a cyclopropyl group, and the like.

[0170] In the present invention, 'aryl' refers to a single-ring, aromatic hydrocarbon group having six carbon atoms. Specific examples of such aryl groups include a phenyl group, etc.

[0171] The present invention relates to a trinucleotide cap analogue comprising a compound of the following chemical formula 1:

[0172] [Chemical Formula 1]

[0173]

[0174] In the above chemical formula 1, R1 is a heterocyclic group connected to the N7 position by a CH2 (methylene bridge), and specifically, R1 provides a trinucleotide cap analogue (ThioCap1) that is a methylene bridged thiophene and a trinucleotide cap analogue (PyCap1) that is a methylene bridged pydidine.

[0175] The trinucleotide cap analogue according to the present invention may be characterized by the following chemical formula 2 or chemical formula 3.

[0176] [Chemical Formula 2]

[0177]

[0178] [Chemical Formula 3]

[0179]

[0180] The trinucleotide cap analogue according to the present invention may be Thio-Cap1 (2-mTHP7GpppAmpG) or Pyridyl-Cap1 (Py-Cap1, 2-mPy7GpppAmpG).

[0181] The present invention relates to a trinucleotide cap analogue comprising a compound of the following chemical formula [4-1], [4-2] or [4-3]:

[0182] [Chemical Formula 4-1]

[0183]

[0184] [Chemical Formula 4-2]

[0185]

[0186] [Chemical Formula 4-3]

[0187]

[0188] In the above chemical formula, R1 is chemical formula 6 connected to CH2 (methyl) at the N7 position,

[0189] [Chemical Formula 6]

[0190]

[0191] In the above formula,

[0192] R1 provides a trinucleotide cap analogue wherein R1 is arylmethyl (specifically benzyl).

[0193] Also, in the above chemical formula, R1 is chemical formula 7 connected to CH2 (methyl) at the N7 position,

[0194] [Chemical Formula 7]

[0195]

[0196] In the above formula,

[0197] R1 provides a trinucleotide cap analogue wherein R1 is arylmethyl (specifically, chlorobenzyl).

[0198] Trinucleotide cap analogues according to the present invention include benzyl-cap1 (Bz-Cap1, Bn7GpppAmpG), chlorobenzyl-cap1 (ClBz-Cap1, 4-ClBn7GpppAmpG), el-benzyl-cap1 (LBz-Cap1, Bn7LGpppAmpG), dibenzyl-cap1 (DiBz-Cap1, Bn7GpppBn6AmpG), inosine-cap1 (Ino-Cap1, Bn7GpppBn6AmpG), benzyldiphosphate-cap1 (Bzdipho-Cap1, Bn7GppAmpG), chlorobenzyldiphosphate-cap1 (ClBzdipho-Cap1, 4-ClBn7GppAmpG), benzylthiophosphate-cap1 (Bzthiopho1-Cap1, Bn7GppspAmpG(D1)), Benzylthiophosphate 2-Cap1 (Bzthiopho2-Cap1, Bn7GppspAmpG (D2)), chlorobenzylthiophosphate 1-Cap1 (ClBzthiopho1-Cap1, 4-ClBn7GppspAmpG)(D1)) and chlorobenzyldiphosphate 2-Cap1 (ClBzthiopho2-Cap1, 4-ClBn7GppspAmpG)(D2)).

[0199] Specifically, the cap analogue may be the compound of Formula 1 and Formulae [4-1] to [4-3] itself, or may further comprise, in addition to the compound of Formula 1 and Formulae [4-1] to [4-3], a hybridization sequence that may be complementary to a sequence on a DNA template at the initiation site. The length of the hybridization sequence of the cap analogue for use in the methods and compositions provided herein depends on several factors, including the identity of the template nucleotide sequence and the temperature at which such primer hybridizes with the DNA template or is used during in vitro transcription. The desired length of the hybridization nucleotide sequence of the cap analogue for use in transcription can be readily determined by one of ordinary skill in the art through routine experimentation. For example, the length of the hybridization nucleotide may be determined based on the desired hybridization specificity or selectivity.

[0200] Using the above cap analogs, 5' capped mRNA can be produced.

[0201] One aspect of the present invention provides a method for producing a 5' capped mRNA, comprising incorporating a cap analogue comprising a compound of the above chemical formula 1 and chemical formulae [4-1] to [4-3] during the synthesis of mRNA.

[0202] In addition, one aspect of the present invention provides a composition for producing 5'-capped mRNA, comprising a cap analogue comprising a compound of the above chemical formula 1 and chemical formulae [4-1] to [4-3].

[0203] In addition, one aspect of the present invention provides a use of a cap analogue comprising a compound of the above chemical formula 1 and chemical formulae [4-1] to [4-3] for the synthesis of mRNA.

[0204] In addition, one aspect of the present invention provides an mRNA 5'-capped with a cap analogue comprising a compound of the above chemical formula 1 and chemical formulae [4-1] to [4-3].

[0205] In addition, a method for increasing protein expression efficiency using the cap analogue and a method for increasing protein expression efficiency using a 5'-capped mRNA structure using the cap analogue are provided. In this case, the protein may be an antigen protein.

[0206] The method for producing a 5'-capped mRNA using a cap analogue comprising the compound of the above chemical formula 1 and chemical formulae [4-1] to [4-3] can be performed according to any method known in the art.

[0207] In addition, one aspect of the present invention is that 5'-capped mRNA can be produced by a co-transcription capping method, which is an mRNA synthesis method in which a chemically synthesized cap analog is simultaneously introduced during in vitro mRNA synthesis so that the cap analog constitutes the 5' end. Specifically, the method may include the steps of introducing a cap analog, which is a compound of Chemical Formula 1 and Chemical Formulae [4-1] to [4-3], into a mixture containing RNA polymerase under conditions in which transcription of a polynucleotide template is performed by the RNA polymerase, and incubating the mixture for a time sufficient to allow transcription of the template.

[0208] The present invention provides a pharmaceutical composition for expressing a desired peptide or protein, comprising an mRNA capped at the 5' end with a trinucleotide cap analog and a pharmaceutically acceptable carrier.

[0209] Depending on the type of the desired peptide or protein, the desired disease treatment or prevention effect can be achieved in vivo. Therefore, the peptide or protein can be used to treat or prevent any disease that can be treated or prevented by expression. Diseases that can be treated or prevented by expression of a specific peptide or protein are known, and the pharmaceutical composition can be used to induce expression of the peptide or protein and prevent or treat the desired disease.

[0210] In addition, one aspect of the present invention provides a pharmaceutical use of the 5' capped mRNA with the cap analogue for the prevention or treatment of any disease that is effective by in vivo expression of a peptide or protein.

[0211] The pharmaceutical composition and treatment or prevention method described above may be used for gene replacement therapy, genome editing, cancer immunotherapy, or treatment or prevention using a vaccine. In one specific example, the pharmaceutical composition is an mRNA vaccine.

[0212] The pharmaceutical composition may be formulated for administration by injection or by any other suitable route known to those skilled in the art for treating or preventing a specific condition. The injectable composition may include a pharmaceutically acceptable carrier, such as sterile saline. The injectable composition may also be formulated as a suspension in a lipid or phospholipid, a liposomal suspension, or an aqueous emulsion. Methods for formulating the pharmaceutical composition are well known to those skilled in the art.

[0213] In one specific example, the pharmaceutical composition may contain mRNA comprising the cap analogue as an active ingredient at a concentration of about 0.01% to 1%. The concentration may vary depending on the frequency of administration, dosage, administration method, etc.

[0214] In one specific example, the pharmaceutical composition can be administered to a mammal, specifically a human, and the dosage may vary depending on the subject's health condition, severity of the disease, body weight, age, race, etc., and an expert in the art can determine an appropriate dosage. In one specific example, the dosage for a human is in the range of 0.0001 to 100 mg / day, and more specifically, in the range of about 0.1 to 50 mg / day.

[0215] The 5'-capped mRNA with a cap analog according to the above aspect can be introduced into cells in vivo or in vitro to express proteins or peptides.

[0216] Methods for expressing protein peptides in cells in vivo or in vitro using the above mRNA are known in the art, and proteins or peptides can be appropriately expressed in cells according to such conventional methods.

[0217] Hereinafter, examples are presented to aid understanding of the present invention. The following examples are merely illustrative of the present invention, and it will be apparent to those skilled in the art that various changes and modifications are possible within the scope and technical spirit of the present invention. It is also natural that such changes and modifications fall within the scope of the appended claims.

[0218]

[0219]

[0220] <Example>

[0221] A. N7-methylene bridged heterocyclic compound substituted CleanCap analogues

[0222] <Example 1> Synthesis of trinucleotide capping material

[0223] Regarding the synthesis of a new modified trinucleotide capping material and its use as an mRNA vaccine, a new compound (ThioCap1) in which the N7 position of the 5'-terminal guanosine base of the trinucleotide structure of CleanCap is modified with a methylene bridged heterocyclic compound, more specifically, methylene bridged thiophene, instead of the existing methyl, and a new compound (PyCap1) in which the N7 position is modified with methylene bridged thiophene were synthesized through an organic chemical reaction (Fig. 1).

[0224]

[0225] <1-1> Reagent Information

[0226] Using a Bruker AV-400 spectrometer 1 H and 13 C, 31 P NMR spectra were recorded using D2O or DMSO-d6 as the solvent and tetramethylsilane as the internal standard. UV-Vis spectra were recorded at room temperature using a Cary series UV-Vis spectrophotometer (Agilent Technologies) and a 1-cm-long quartz cuvette, and the absorbance change was measured immediately after irradiating the sample solution in the cuvette with UV light. Fluorescence emission spectra were recorded at room temperature using a PF-65000 spectrofluorometer.

[0227]

[0228] <1-2> Synthesis method of N7-(hetroaryl-2-ylmethy) guanosine 5'-diphosphoroimidazolide analogues

[0229] ① Synthesis of triethylammonium salt of guanosine 5′-diphosphate

[0230]

[0231] To synthesize triethylammonium salt of guanosine 5′-diphosphate (GDP.TEA), triethylamine (1.7 mL, 12 mmol), imidazole (817 mg, 12 mmol), and 2,2′-dithiodipyridine (1.3 g, 6 mmol) were added to a stirred solution of triethylamine (930 mg, 2 mmol) of guanosine 5′-phosphate (GMP.TEA) in anhydrous dimethyl sulfoxide (4 mL), and the mixture was stirred for 5 minutes. After that, triphenylphosphine (1.56 g, 6 mmol) in anhydrous dimethyl sulfoxide (4 mL) was added, and stirring was continued at room temperature for 5 hours. After the reaction was completed, the mixture was slowly poured into a mixture of sodium perchlorate (0.5 g) in acetone (50 mL). After cooling at 4°C for 30 minutes, the mixture was centrifuged at 5,000 rpm for 8 minutes, and the supernatant was discarded. To remove traces of imidazole and triphenylphosphine, the solid was triturated with fresh acetone (20 mL), the mixture was cooled, and centrifuged again. This process was repeated once more, and the precipitate was dried in a vacuum oven at room temperature.

[0232] The obtained guanosine 5′-phosphoroimidazolide (NaGMP-IM) was dissolved in dimethylformamide (10 mL), and a 1 M solution of tributylammonium orthophosphate in dimethylformamide (5 mL) was added dropwise to the stirred mixture for 30 min. Finally, zinc chloride (200 mg, 1.5 mmol) was added, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was extracted with chloroform (3 × 20 mL) mixed with water (50 mL). The resulting solution was concentrated using a rotary evaporator with a bath temperature of 37°C. The resulting product was applied to an anion exchange resin for purification.

[0233] Chromatographic purification was performed using diethylaminoethyl weak anion exchange (DEAE) Sepharose fast-flow resin. The desired compound was eluted with CombiFlash EZ Prep using a 4-bed volume gradient of 0–30% with 1 M TEAB (triethylammonium bicarbonate) buffer, pH 7.5, at a flow rate of 3 mL / min. Fractions containing GDP.TEA were pooled, concentrated using a rotary evaporator (bath temperature 37°C), and then dried in a freeze dryer to obtain GDP.TEA as a fine white powder (951 mg, 64%). The NMR results of GDP.TEA are shown below.

[0234]

[0235] GDP.TEA. (951 mg, 64%)

[0236] 1 H NMR (400 MHz, DO) δ = 7.99 (s, 1H), 5.81 (d, J = 6.0 Hz, 1H), 4.63 (t, J = 5.2 Hz, 1H), 4.51 (t, J = 5.6 Hz, 1H), 4.22 (m, 1H), 4.14 (m, 1H), 4.05 (m, 1H); 31 P NMR (162 MHz, DO) δ = -6.47 (d, J = 23.5 Hz, 1P), -10.99 (d, J = 22.5 Hz, 1P).

[0237]

[0238] ② General synthetic process of N7-(hetroaryl-2-ylmethyl) guanosine 5′-diphosphate analogues

[0239]

[0240] The triethylamine salt of guanosine 5′-diphosphate (0.5 mmol, 1 equiv) was dispersed in DMSO (3 mL), and the appropriate 2-bromomethyl heteroaryl (2.5 mmol, 5 equiv) was added. The mixture was stirred at room temperature overnight. 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 a mixture of sodium perchlorate (0.5 g) in acetone (50 mL). After cooling at 4°C for 30 min, the mixture was centrifuged at 5,000 rpm for 8 min, and the supernatant was discarded. The solid was triturated with fresh acetone (20 mL), the mixture was cooled, and centrifuged again. This process was repeated once more, and the precipitate was dried under vacuum at room temperature. This process was repeated once more, and the precipitate was vacuum-dried at room temperature. The crude product solution, with a total volume of 15–20 mL, was separated using Sepharose high-flow resin as described above. The fractions containing the product were collected, concentrated using a rotary evaporator (water temperature 37°C), co-evaporated three times with ethanol, and dried in a freeze dryer.

[0241] According to the above method, N7-(thiophen-2-ylmethyl) guanosine 5′-diphosphate triethylammonium salt and N7-(pyridin-2-ylmethyl) guanosine 5′-diphosphate triethylammonium salt were synthesized using 2-bromomethyl thiophene and 2-bromomethyl pyridine, respectively, and the NMR results are shown below.

[0242]

[0243] N7-(thiophen-2-ylmethyl)guanosine 5′-diphosphate triethylammonium salt: N7-2-mTHP-GDP.TEA (155 mg, 42%).

[0244] 1H NMR (400 MHz, D2O) δ = 7.36 (d, J = 5.2 Hz, 1H), 7.27 (d, J = 3.6 Hz, 1H), 6.96 (t, J = 5.2 Hz,1H), 5.94 (d, J = 3.6 Hz, 1H), 5.80 (dd, J1 = 18.0 Hz, J2 = 15.2 Hz, 2H), 4.62 (t, J = 3.6 Hz,1H), 4.48 (t, J = 5.6 Hz, 1H), 4.32-4.27 (m, 1H), 4.26-4.14 (m, 2H); 31 P NMR (162 MHz, D2O) δ꺜 = -8.34 (d, J = 22.4 Hz, 1P), -11.10 (d, J = 22.5 Hz, 1P).

[0245]

[0246] N7-(pyridin-2-ylmethyl) guanosine 5′-diphosphate triethylammonium salt: N7-2mPy-GDP.TEA (213 mg, 58 %)

[0247] 1 H NMR (400 MHz, D2O) δ = 8.35 (d, J = 5.6 Hz, 1H), 7.70 (td, J1 = 8 Hz, J2 = 2 Hz, 1H), 7.35-7.29 (m, 2H), 6.04 (d, J = 3.2 Hz, 1H), 5.75 (d, J = 5.2 Hz, 2H), 4.64 (dd, J1 = 4.8 Hz, J2 = 3.2 Hz, 1H), 4.55 (dd, J1 = 6.0 Hz, J2 = 4.8 Hz, 1H), 4.36 (dd, J1 = 6.0 Hz, J2 = 2.4 Hz, 1H), 4.22 (dd, J1 = 5.2 Hz, J2 = 2.4 Hz, 1H); 31 P NMR (162 MHz, D2O) δ = -6.34 (d, J = 22.5 Hz, 1P), -10.95 (d, J = 23.5 Hz, 1P).

[0248]

[0249] ③ General synthetic process of N7-(hetroaryl-2-ylmethyl) guanosine 5′-diphosphoroimidazolide analogues

[0250]

[0251] To a stirred solution of triethylammonium salt of N7-(heteroaryl-2-ylmethyl) guanosine 5'-diphosphate (0.3 mmol) in anhydrous dimethyl sulfoxide (3 mL) were added triethylamine (251 μL, 1.8 mmol), imidazole (123 mg, 1.8 mmol), and 2,2'-dithiodipyridine (198 mg, 0.9 mmol), and the mixture was stirred for 5 minutes, followed by the addition of triphenylphosphine (236 mg, 0.9 mmol) in anhydrous dimethyl sulfoxide (2 mL). Stirring was continued at room temperature for 5 hours. After completion of the reaction, the mixture was slowly poured into a mixture of sodium perchlorate (250 mg) in acetone (25 mL). After cooling at 4°C for 30 minutes, the mixture was centrifuged, and the supernatant was discarded. To remove traces of imidazole and triphenylphosphine, the solid was triturated with fresh acetone (10 mL), the mixture was cooled, and centrifuged again. This process was repeated once more, and the precipitate was dried in a vacuum oven using P2O5 at room temperature.

[0252] According to the above method, N7-(thiophen-2-ylmethyl) guanosine 5'-diphosphate and N7-(pyridine-2-ylmethyl) guanosine 5'-diphosphate were used respectively 7 -(thiophen-2-ylmethyl)guanosine 5′-diphosphoroimidazolide and N 7 -(pyridin-2-ylmethyl) guanosine 5′-diphosphoroimidazolide was synthesized and its NMR results are shown below.

[0253]

[0254] <N7-(thiophen-2-ylmethyl) guanosine 5′-diphosphoroimidazolide: N7-2-mTHP-GDP-IM>

[0255] 1 H 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 P NMR (162 MHz, D2O) δ = -11.70 (d, J = 24.5 Hz, 1P), -19.90 (d, J = 21.5 Hz, 1P).

[0256] <N7-(pyridin-2-ylmethyl) guanosine 5′-diphosphoroimidazolide: N7-2mPy-GDP-IM>

[0257] 1 H 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 = 5.6 Hz, 1H), 4.32-4.27 (m, 1H), 4.17 (m, 1H), 4.04 (m, 1H); 31P NMR (162 MHz, DO) δ = -11.84 (d, J = 20.6 Hz, 1P), -19.92 (d, J = 21.5 Hz, 1P).

[0258]

[0259] <1-3> Synthesis method of dinucleotide, pAmpG

[0260]

[0261] ① Synthesis process of 5′-O-DMT-2′-O-methyl Adenosylyl (n-bz)-{3′-OP-[2-cyanoethyl] → 5′}-2′,3′-diacetyl Guanosine (n-ibu) 3

[0262] DMT-2'-O-methyl adenosine (n-bz) CED phosphoramidite 1 (1.29 g, 1.45 mmol) and 2',3'-dicacetyl guanosine (n-ibu) 2 (0.58 g, 1.32 mmol) were dried under high vacuum for 16 h. Anhydrous DMF (1.7 mL) and 0.45 M tetrazole were added to acetonitrile (7.3 mL, 3.30 mmol), and the mixture was stirred at room temperature for 3 h under an argon (nitrogen) atmosphere. After the consumption of compound 1 was confirmed by TLC, 70% t-butyl hydroperoxide (0.91 mL, 6.60 mmol) was added to water after cooling in an ice-salt bath for 30 min. The reaction mixture was stirred at room temperature for 1 h. After confirming the conversion of the intermediate 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).

[0263]

[0264] ② Synthesis process of 2′-O-Methyl Adenosylyl (n-bz)-{3′-OP-[2-cyanoethyl] → 5′}-2′,3′-diacetyl Guanosine (n-ibu) 4

[0265] 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 h. After confirming the conversion of compound 3 by TLC, 5% NaHCO3 solution (30 mL) was added and stirred for 10 min. The mixture was extracted with dichloromethane and evaporated. The resulting residue was purified by silica gel column chromatography to obtain product 4 (0.74 g, 0.79 mmol, 60%).

[0266]

[0267] 1 H NMR (DMSO, 400MHz): δ 12.12 (s, 1H), 11.59 (d, 1H), 11.26 (d, 1H), 7.93 (d, d, 2H), 8.28 (d, 1H), 8.06 (d, 2H), 7.66 (t, 1H), 7.57 (d, 2H), 6.19(d, 1H), 6.14 (dd, 1H), 5.84 (q, 1H), 5.54 (dd, 1H), 5. 39 (t, 1H), 5.20 (m, 1H), 4.83 (m, 1H), 4.48 (m, 3H), 4.30 (m, 3H), 3.64 (m, 2H), 3.37 (d, 3H), 2.97 (q, 2H), 2.78 (m, 1H), 2.15 (d, 3H), 2.03 (d, 3H), 1.13 (m, 6H); 31P NMR (DMSO, 162 MHz): δ -2.43 (d, 1P);

[0268]

[0269] ③ Synthesis process of 5′-O-Phosphoryl-2′-O-methyl Adenosylyl-{3′-OP→ 5′} Guanosine, (pAmpG) 5

[0270] Dinucleotide 4 (0.74 g, 0.7891 mmol) was dried under high vacuum for 16 h. 0.45 M tetrazole (3.51 mL, 1.58 mmol) and bis-(2-cyanoethyl)-N,N-diisopropyl-phosphoramidite (0.41 mL, 1.58 mmol) in acetonitrile were added, and the mixture was stirred at room temperature for 3 h under an argon (nitrogen) atmosphere. After confirming the consumption of compound 4, the reaction mixture was cooled in an ice-salt bath for 30 min, and 70% t-butyl hydroperoxide in water (17.2 mL, 3.16 mmol) was added. The mixture was stirred at room temperature for 1 h. After confirming the conversion of the intermediate by TLC, it was extracted with 2 x 200 mL of ethyl acetate and washed with 2 x 100 mL of 5% NaHCO3 solution. The extracted organic solutions were combined and evaporated. The resulting residue was dissolved in 25 mL of methanol and 25 mL of concentrated ammonia solution and stirred at 55°C for 7 h. After the deprotection was complete, the reaction mixture was evaporated together with methanol. The resulting residue was dissolved in water, adjusted to pH 5.5, and loaded onto a DEAE-Sepharose column. Solvent A: 1 M TEAB buffer (pH 7.5–8), solvent B: water. The fractions containing the product were collected, evaporated, and dried in a freeze dryer to obtain 5 (446 mg, 0.4908 mmol, 62%) as a fine white powder containing triethylammonium salt.

[0271]

[0272] Dinucleotide (pAmpG) TEA salt

[0273] 1H NMR (DO, 400 MHz): δ 8.53 (s, 1H), 8.10 (s, 1H), 7.89 (s, 1H), 6.06 (d, 1H), 5.77 (d, 1H), 4.88 (m, 1H), 4.50 (t, 1H), 4.42 (m, 2H), 4.27 (m, 1H), 4.12 (t, 2H), 3.92 (m, 2H), 3.37 (s, 3H); 31 P NMR (DO, 162 MHz): δ 3.69 (s, 1P), -0.84 (s, 1P)

[0274]

[0275] <1-4> General synthetic process of trinucleotide-based capping materials (ThioCap1 and PyCap1) with N7-methylene bridged heterocyclic group introduced

[0276]

[0277] To a solution of pAmpG triethylammonium salt (0.07 mmol, 63 mg) in DMSO (50 mM) were added N7-(heteroaryl-2-ylmethyl)guanosine 5'-diphosphoroimidazolide disodium salt (3.00 eq, 0.2 mmol) and zinc chloride (20.0 eq). After stirring at 37°C for 3 days, the reaction mixture was stopped by adding 500 mM EDTA aqueous solution (pH 8.0) (EDTA: molar equivalent of ZnCl2 1.30 eq). The mixture was diluted with water and purified by DEAE Sepharose fast-flow resin, and then further purified using reverse-phase HPLC (Shimadzu instrument; YMC-Actus Triart C8 column (separation, 250 × 20.0 mm ID); solvent A, 50 mM TEAA buffer (pH 6.0) containing 0.5% CH3CN; solvent B, CH3CN; linear gradient 5–80% B (25 min); flow rate, 10 mL / min; detection wavelength, 254 nm). The fractions containing the target compound were collected, concentrated, and lyophilized to obtain the target N7-(hetroaryl-2-ylmethyl) Cap1 analog as the triethylammonium salt. The product was redissolved in methanol (2.00 mL). 190 mM NaClO4 in acetone (24.0 mL) was added, and the resulting suspension was centrifuged (4,000 rpm, 20 min). The supernatant was discarded, and the precipitate was resuspended in acetone. The suspension-centrifugation process was repeated 3–4 more times. The precipitate was dried under reduced pressure to obtain the target N7-(heteroaryl-2-ylmethyl) Cap1 analogue as a sodium salt.

[0278] N according to the above method 7 -(thiophen-2-ylmethyl)guanosine 5′-diphosphoroimidazolide and N 7-(pyridin-2-ylmethyl) guanosine 5′-diphosphoroimidazolide, respectively, were used to introduce a trinucleotide-based capping material (ThioCap1( 2-mTHP7 GpppAmpG) and PyCap1( 2-mPy7 GpppAmpG)) was synthesized and its NMR analysis results are shown below.

[0279]

[0280] <Guanosine N7-(thiophen-2-ylmethyl) trinucleotide Cap1 analog>

[0281] < 2-mTHP7 GpppAmpG>

[0282]

[0283] 1 H NMR (400 MHz, DO) 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 = 5.2 Hz, 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); 31P 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 = 19.6 Hz, 1P).

[0284]

[0285] <Guanosine N7-(pyridin-2-ylmethyl) trinucleotide Cap1 analog>

[0286] < 2-mPy7 GpppAmpG>

[0287]

[0288] 1 H 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).

[0289]

[0290] <Example 2> Introduction of trinucleotide capping into mRNA and analysis of capping efficiency

[0291] It was analyzed that the trinucleotide capping material manufactured in Example 1 can be recognized by T7 RNA polymerase with high efficiency during the transcription process and introduced into mRNA.

[0292] As a result, the transcription efficiency of the capping material was similar for CleanCap (cap 1 commercial) and ThioCap1, and it was confirmed that the developed ThioCap1 can be recognized by T7 RNA polymerase and introduced into RNA with higher efficiency than ARCA or CleanCap (cap 1 commercial) during the transcription process. The existing ARCA showed a capping efficiency of about 50%, and CleanCap (cap 1 commercial) showed a capping efficiency of about 86%, but ThioCap1 showed a capping efficiency of about 92% or more, and PyCap1 showed a capping efficiency of about 88% or more (Fig. 7 and Fig. 10). In the case of ARCA, the expression efficiency of the antigen protein (Luciferase) was not comparatively measured due to the very low capping efficiency.

[0293]

[0294] <Example 3> Expression analysis of antigen protein using capping material substituted with a methylene bridge heterocyclic group

[0295] The mRNA translation process was confirmed by introducing a capping material manufactured by modifying the N7 position of the guanosine base at the 5' terminal with a methylene bridged thiophen compound instead of the existing methyl. Luciferase mRNA was used to confirm the expression level of the antigen protein, and the expression amount of the antigen protein was analyzed using HEK 293 cells. In the case of luciferase mRNA, two mRNAs were compared: mRNA using natural rNTP and mRNA introduced with pseudouridine triphosphate, which was used instead of rUTP to avoid hyperimmunity. In both cases, ThioCap1-introduced mRNA (Luc mRNA ThioCAP1) showed more efficient antigen protein expression than CleanCap-introduced mRNA (Luc mRNA CAP1), and PyCap1-introduced mRNA (Luc mRNA PyCAP1) also showed more efficient antigen protein expression than CleanCap-introduced mRNA (Luc mRNA CAP1) (Fig. 8 and Fig. 11).

[0296]

[0297] ① Preparation of luciferase DNA template

[0298] A linear dsDNA template containing the start codon from the luciferase plasmid to the poly-A tail was prepared by PCR. During successive PCRs, the T7 promoter and Kozak sequence were added to the luciferase dsDNA template, which was ultimately used for the transcription reaction. The primers used were: 1st forward primer: 5'- ATG GAA GAC GCC AAA AAC ATA AAG -3' (SEQ ID NO: 1); 2nd forward primer: TAA TAC GAC TCA CTA TAG GGC CAC CAT GGA AGA CGC CAA AAA CAT (SEQ ID NO: 2); and 3rd reverse primer: AAT CGC GCC TAG GCG CGC CCG TAC GGC TCT TC-3' (SEQ ID NO: 3).

[0299]

[0300] ② Preparation of luciferase mRNA

[0301] The concentration of luciferase template was measured using a nanodroplet spectrophotometer. For each transcription reaction, 100 ng of 1 μL template was placed in a microcentrifuge tube. Then, 1.5 μL of 10X transcription buffer (400 mM Tris-HCl, 60 mM MgCl2, 10 mM DTT, 20 mM spermidine) and 1.5 μL of 100 mM DTT were added to the tube. rATP, rCTP, and rUTP were used (1 μL, 10 mM), respectively. rGTP was used for cap-dependent transcription of luciferase (1 μL, 2.5 mM). The ratio of capping agent was maintained at 4-fold higher than rGTP to provide more than 80% capped mRNA. Therefore, 1 μL of capping agent (10 mM) was added. RNase inhibitor (0.5 μL, 40 U / μL) and T7 RNA polymerase (1 μL, 50 U / μL) were added. Nuclease-free water (4.5–5.5 μL) was added to make the final reaction volume 15 μL. For modified rNTPs, stocks were prepared at 10 mM concentrations and the corresponding derivatives were added instead of rCTP and rUTP. The reaction was incubated at 37°C for 1 h, after which 1.8 μL 10X DNase I buffer (10 mM Tris-HCl, 2.5 mM MgCl2, 0.5 mM CaCl2) and 1 μL DNase I (2 U / μL) were added to the reaction and incubated at 37°C for an additional 15 min to remove all DNA template.

[0302]

[0303] ③ Purification of transcribed mRNA

[0304] To remove any dsRNA and transcriptional byproducts that may have formed, dsRNA was separated by electrophoresis. Next, cellulose (10 μL, 0.2 g / mL) was added to the electrophoretically separated mRNA and gently shaken for 10 minutes. The mixture was then transferred to an RNA purification column along with the ethanol and binding buffer provided with the column, followed by a final centrifugation at 13,000 rpm for 1 minute. RNA preparation buffer and wash buffer were then successively added to the column and centrifuged. This step removed all proteins and unincorporated rTNP from the suspension. Finally, a new tube was attached, 10 μL of nuclease-free water was added to the column, and centrifugation was performed at 13,000 rpm for 2 minutes. Any dsDNA bound to the cellulose remained in the column, leaving only pure single-stranded luciferase mRNA to pass through. This sample was collected and stored at 70°C for further use.

[0305]

[0306] ④ Analysis of capping efficiency of transcribed mRNA

[0307] 2 μg of capped and transcribed mRNA was annealed by heating to 70°C with 3 μg of gDNA and RNase H buffer and then slowly cooling to 25°C. Next, thermally stable RNase H was used to cleave a short sequence containing the capping material from the long mRNA. o The reaction was carried out for 1 hour in C. The short sequences containing the cleaved capping material were analyzed by electrophoresis using a 16% denaturing PAGE gel. The short sequences cleaved from mRNA without the introduction of the capping material (uncapped mRNA) showed a faster gel migration, while the short sequences cleaved from mRNA containing the capping material (capped mRNA) showed a relatively slower gel migration.

[0308]

[0309] ⑤ Cell culture and preparation for transfection

[0310] HEK 293 cells were cultured in 60 mL culture plates containing DMEM containing 10% FBS and 1% pen / stripe. The day before transfection, cells were detached using trypsin and resuspended in fresh medium. Cells were seeded in 96-well plates. The following day, when cells reached 70% confluence, the transfection mixture was prepared and transfected.

[0311]

[0312] ⑥ Quantification and transformation of mRNA

[0313] Purified mRNA was quantified using a Nanodrop spectrophotometer, and the transcription yield for each rNTP combination was compared. For cell transfection, 500 ng of each mRNA was collected and mixed with 5 μL of Opti-MEM. 0.5 μL of lipofectamine was mixed with 5 μL of Opti-MEM in a separate tube and incubated at room temperature for 10 minutes. The mRNA and lipofectamine mixture was then transferred to the same tube and incubated for an additional 5 minutes at room temperature. Finally, the transfection mixture was added to the cells and incubated for 24 hours.

[0314]

[0315] ⑦ Luciferase expression analysis

[0316] After the incubation period, the medium was removed, and the cells were lysed using 50 μL of 1X lysis buffer. The cell lysate was transferred to a microcentrifuge tube. For the luciferase luminescence assay, 100 μL of luciferin substrate was collected for each sample in a 96-well white plate. Then, 20 μL of each cell lysate sample was added and mixed by pipetting. Finally, the luminescence of luciferase was recorded using a luminometer.

[0317] As a result, it was confirmed that the capping material according to the present invention can be recognized by T7 RNA polymerase and introduced into RNA with higher efficiency than ARCA or CleanCap during the transcription process, and while the existing ARCA showed a capping efficiency of about 50%, it was confirmed that ThioCap1 showed a capping efficiency of about 90% or more, and PyCap1 showed a capping efficiency of about 88% or more (Figs. 7 and 10).

[0318] Furthermore, the mRNA translation process using the capping material of the present invention was confirmed. Luciferase mRNA was used to determine the level of antigen protein expression, and the amount of antigen protein expression was confirmed using HEK 293 cells. For luciferase mRNA, two mRNAs were compared: one using natural rNTPs and the other using pseudouridine triphosphate instead of rUTP to avoid hyperimmunity.

[0319] As a result, mRNA with pseudouridine triphosphate introduced to avoid hyperimmunity showed higher expression efficiency of antigen protein than mRNA using rNTP.

[0320] In addition, it was confirmed that Luciferase mRNA (Luc mRNA ThioCap1) and PyCap1-introduced mRNA (Luc mRNA PyCAP1) developed in the present invention showed higher antigen protein expression efficiency than mRNA (Luc mRNA Cap1) introduced with existing CleanCap (cap 1 commercial) in both mRNA using rNTP and mRNA containing pseudouridine instead of rUTP (Figs. 8 and 11).

[0321]

[0322] B. N7-arylmethyl (benzyl(BzCap1))-substituted CleanCap analogues

[0323] <Example 1> Synthesis of trinucleotide capping material

[0324] Synthesis of a new modified trinucleotide capping material and its use as an mRNA vaccine, wherein the N7 position of the 5' terminal guanosine base in the trinucleotide structure of CleanCap is modified with arylmethyl, more specifically benzyl and 4-Clorobenzyl instead of the existing methyl. Bn7 GpppA m pG, Bn7 GppA m pG, 4-ClBn7 GppA m pG, Bn7 Gppp Bn6 A m pG, 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 pG) were synthesized through organic chemical reactions (Fig. 12).

[0325]

[0326] <1-1> Reagent Information

[0327] Using a Bruker AV-400 spectrometer 1 H and 13 C, 31P NMR spectra were recorded using D2O or DMSO-d6 as the solvent and tetramethylsilane as the internal standard. UV-Vis spectra were recorded at room temperature using a Cary series UV-Vis spectrophotometer (Agilent Technologies) and a 1-cm-long quartz cuvette, and the absorbance change was measured immediately after irradiating the sample solution in the cuvette with UV light. Fluorescence emission spectra were recorded at room temperature using a PF-65000 spectrofluorometer.

[0328]

[0329] <1-2> Synthesis method of N7-(arylmethyl) guanosine 5'-diphosphoroimidazolide analogues

[0330] ① Synthesis of triethylammonium salt of guanosine 5′-diphosphate

[0331]

[0332] To synthesize triethylammonium salt of guanosine 5′-diphosphate (GDP.TEA), triethylamine (1.7 mL, 12 mmol), imidazole (817 mg, 12 mmol), and 2,2′-dithiodipyridine (1.3 g, 6 mmol) were added to a stirred solution of triethylamine (930 mg, 2 mmol) of guanosine 5′-phosphate (GMP.TEA) in anhydrous dimethyl sulfoxide (4 mL), and the mixture was stirred for 5 minutes. After that, triphenylphosphine (1.56 g, 6 mmol) in anhydrous dimethyl sulfoxide (4 mL) was added, and stirring was continued at room temperature for 5 hours. After the reaction was completed, the mixture was slowly poured into a mixture of sodium perchlorate (0.5 g) in acetone (50 mL). After cooling at 4°C for 30 minutes, the mixture was centrifuged at 5,000 rpm for 8 minutes, and the supernatant was discarded. To remove traces of imidazole and triphenylphosphine, the solid was triturated with fresh acetone (20 mL), the mixture was cooled, and centrifuged again. This process was repeated once more, and the precipitate was dried in a vacuum oven at room temperature.

[0333] The obtained guanosine 5′-phosphoroimidazolide (NaGMP-IM) was dissolved in dimethylformamide (10 mL), and a 1 M solution of tributylammonium orthophosphate in dimethylformamide (5 mL) was added dropwise to the stirred mixture for 30 min. Finally, zinc chloride (200 mg, 1.5 mmol) was added, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was extracted with chloroform (3 × 20 mL) mixed with water (50 mL). The resulting solution was concentrated using a rotary evaporator with a bath temperature of 37°C. The resulting product was applied to an anion exchange resin for purification.

[0334] Chromatographic purification was performed using diethylaminoethyl weak anion exchange (DEAE) Sepharose fast-flow resin. The desired compound was eluted with CombiFlash EZ Prep using a 4-bed volume gradient of 0–30% with 1 M TEAB (triethylammonium bicarbonate) buffer, pH 7.5, at a flow rate of 3 mL / min. Fractions containing GDP.TEA were pooled, concentrated using a rotary evaporator (bath temperature 37°C), and then dried in a freeze dryer to obtain GDP.TEA as a fine white powder (951 mg, 64%). The NMR results of GDP.TEA are shown below.

[0335]

[0336] GDP.TEA. (951 mg, 64%)

[0337] 1 H NMR (400 MHz, DO) δ = 7.99 (s, 1H), 5.81 (d, J = 6.0 Hz, 1H), 4.63 (t, J = 5.2 Hz, 1H), 4.51 (t, J = 5.6 Hz, 1H), 4.22 (m, 1H), 4.14 (m, 1H), 4.05 (m, 1H); 31 P NMR (162 MHz, DO) δ = -6.47 (d, J = 23.5 Hz, 1P), -10.99 (d, J = 22.5 Hz, 1P).

[0338]

[0339] ② Synthesis of triethylammonium salt of guanosine 5′-diphosphate

[0340] According to the method of the above Example <1-2>, triethylammonium salt of inosine 5′-diphosphate (IDP.TEA, 830 mg, 57%) and triethylammonium salt of L-guanosine 5′-diphosphate (LGDP.TEA, 892 mg, 60%) were synthesized using inosine monophosphate (IMP) and L-guanosine monophosphate (LGMP), respectively, and the NMR results are shown below.

[0341] IDP.TEA. (830 mg, 57%).

[0342] 1 H NMR (400 MHz, DO) δ = 8.41 (s, 1H), 8.13 (s, 1H), 6.05 (d, J = 5.2 Hz, 1H), 4.68 (overlapped with HO, m, 1H), 4.54 (t, J = 4.0 Hz, 1H), 4.29 (bs, 1H), 4.21-4.07 (m, 2H); 31P NMR (162 MHz, DO) δ = -7.47 (d, J = 22.5 Hz, 1P), -11.07 (d, J = 22.3 Hz, 1P).

[0343] LGDP.TEA. (892 mg, 60%).

[0344] 1 H NMR (400 MHz, DO) 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, DO) δ = -6.35 (d, J = 22.5 Hz, 1P), -10.90 (d, J = 22.4 Hz, 1P).

[0345]

[0346] <1-3> Synthesis method of N7-(aryl-2-ylmethyl) guanosine 5'-mono or diphosphate analogues

[0347]

[0348] The triethylamine salt of guanosine 5'-diphosphate (0.5 mmol, 1 equiv) was dispersed in DMSO (3 mL), and the appropriate 2-bromomethyl aryl (2.5 mmol, 5 equiv) was added. The mixture was stirred at room temperature overnight. After the reaction was complete, the mixture was slowly poured into a mixture of acetone (50 mL) and sodium perchlorate (0.5 g). After cooling at 4°C for 30 min, the mixture was centrifuged at 5,000 rpm for 8 min, and the supernatant was discarded. The solid was triturated with fresh acetone (20 mL), the mixture was cooled, and centrifuged again. This process was repeated once more, 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 a DEAE Sepharose fast-flow resin as described previously. Fractions containing the product were collected, concentrated using a rotary evaporator (water temperature 37°C), co-evaporated three times with ethanol, and dried in a freeze dryer.

[0349] According to the above method, N7-benzyl guanosine 5′-monophosphate triethylammonium salt, N7-4-chlorobenzyl guanosine 5′-monophosphate triethylammonium salt, N7-benzyl guanosine 5′-diphosphate triethylammonium salt, N7-4-chlorobenzyl guanosine 5′-diphosphate triethylammonium salt, and N7-benzyl inosine 5′-diphosphate triethylammonium salt were synthesized using benzyl, 4-chlorobenzyl, and benzyl inosine, respectively, and the NMR results are shown below.

[0350]

[0351] N7-benzyl guanosine 5′-monophosphate triethylammonium salt: N7-Bn-GMP.TEA (270 mg, 82 %).

[0352] 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 = 2.3 Hz, 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.

[0353]

[0354] N7-4-cholobenzyl guanosine 5′-monoahosphate triethylammonium salt: N7-ClBn-GMP.TEA (260 mg, 75 %).

[0355] 1 H NMR (400 MHz, D2O) δ = 7.28 (q, J =8.1 Hz, 4H), 6.03 (d, J = 3.4 Hz, 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).

[0356]

[0357] N7-benzyl guanosine 5′-diphosphate triethylammonium salt: N7-Bn-GDP.TEA (247 mg, 67 %).

[0358] 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= 3.2 Hz, 1H), 4.53 (t, J = 5.2 Hz, 1H), 4.30 (m, 1H), 4.18-4.24 (m, 2H); 31 P NMR (162 MHz, D2O) δ = -6.48 (d, J = 23.5 Hz, 1P), -10.98 (d, J = 23.5 Hz, 1P).

[0359]

[0360] N7-4-chlorobenzyl guanosine 5′-diphosphate triethylammonium salt: N7-4-Cl-Bn-GDP.TEA (272 mg, 71 %).

[0361] 1 H NMR (400 MHz, D2O) δ꺜 = 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); 31 P NMR (162 MHz, D2O) δ꺜 = -8.88 (d, J = 21 Hz, 1P), -11.08 (d, J = 23 Hz, 1P).

[0362]

[0363] N7 -benzyl inosine 5′-diphosphate triethylammonium salt: N7-Bn-IDP.TEA

[0364] 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).

[0365]

[0366] <1-4> N7-(aryl-2-ylmethyl) guanosine 5'-mono or diphosphoro-imidazolide 유사체의 합성 방법

[0367]

[0368] To a stirred solution of triethylammonium salt of N7-(aryl-2-ylmethyl) guanosine 5'-diphosphate (0.3 mmol) in anhydrous dimethyl sulfoxide (3 mL) were added triethylamine (251 μL, 1.8 mmol), imidazole (123 mg, 1.8 mmol), and 2,2'-dithiodipyridine (198 mg, 0.9 mmol), and the mixture was stirred for 5 minutes, then triphenylphosphine (236 mg, 0.9 mmol) in anhydrous dimethyl sulfoxide (2 mL) was added. Stirring was continued at room temperature for 5 hours. After completion of the reaction, the mixture was slowly poured into a mixture of sodium perchlorate (250 mg) in acetone (25 mL). After cooling at 4°C for 30 minutes, the mixture was centrifuged, and the supernatant was discarded. To remove traces of imidazole and triphenylphosphine, the solid was triturated with fresh acetone (10 mL), the mixture was cooled, and centrifuged again. This process was repeated once more, and the precipitate was dried in a vacuum oven using P2O5 at room temperature.

[0369] According to the above method, N7-benzyl guanosine 5′-monophosphoroimidazolide, N7-4-Cl-benzyl guanosine 5′-monophosphoroimidazolide, N7-benzyl guanosine 5′-diphosphoroimidazolide, N7-4-Cl-benzyl guanosine 5′-diphosphoroimidazolide, and N7-benzyl inosine 5′-diphosphoroimidazolide were synthesized using N7-(benzyl-2-ylmethyl) guanosine 5′-diphosphate, N7-(4-chlorobenzyl-2-ylmethyl) guanosine 5′-diphosphate, and N7-benzyl inosine 5′-diphosphoroimidazolide, respectively, and the NMR results are shown below.

[0370]

[0371] N7-benzyl guanosine 5′-monophosphoroimidazolide: N7-Bn-GMP-IM (378mg, 93%)

[0372] 1 H NMR (400 MHz, D2O) = 7.74 (s, 1H), 7.39 (br s, 5H), 7.05 (d, J =1.2 Hz, 1H), 6.86 (s, 1H), 5.93 (d, J = 3.8 Hz, 1H), 5.63 (dd, J1 = 14.8 Hz, J2 = 14.8 Hz, 2H), 4.63 (t, J = 4.1 Hz, 1H), 4.32 (t, J = 5.0 Hz, 1H), 4.28-4.26 (m, 1H), 4.16-4.12 (m, 1H), 4.07-4.01 (m,1H); 31 P NMR (162 MHz, D2O) = -8.02 (s, 1P).

[0373] Electrospray ionization (ESI) mass spectrometry: Calculated m / z for C 20 H 22 N7O7P- ([M]): m / z 503.41; found([M-2]): 501.2.

[0374]

[0375] N7-4-Cl-benzyl guanosine 5′-monophosphoroimidazolide: N7-4-Cl-Bn-GMP-IM (392mg, 91%)

[0376] 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); 31P NMR (162 MHz, D2O): = -8.04 (s, 1P).

[0377] Electrospray ionization (ESI) mass spectrometry: Calculated m / z for C 20 H 21 ClN7O7P- ([M]): m / z 537.85; found([M-2]): 535.1.

[0378]

[0379] N7-benzyl guanosine 5′-diphosphoroimidazolide: N7-Bn-GDP-IM (153mg, 81%)

[0380] 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 = 21.4 Hz, 1P).

[0381]

[0382] N7-4-Cl-benzyl guanosine 5′-diphosphoroimidazolide: N7-4-Cl-Bn-GDP-IM

[0383] 1H 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 = 14.8 Hz, 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).

[0384]

[0385] <N7-benzyl inosine 5′-diphosphoroimidazolide: N7-Bn-IDP-IM>

[0386] 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 = 14.8 Hz, 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).

[0387]

[0388] <1-5> 디뉴클레오티드, pAmpG의 합성 방법

[0389]

[0390] ① Synthesis process of 5′-O-DMT-2′-O-methyl Adenosylyl (n-bz)-{3′-OP-[2-cyanoethyl] → 5′}-2′,3′-diacetyl Guanosine (n-ibu) 3

[0391] DMT-2'-O-methyl adenosine (n-bz) CED phosphoramidite 1 (1.29 g, 1.45 mmol) and 2',3'-dicacetyl guanosine (n-ibu) 2 (0.58 g, 1.32 mmol) were dried under high vacuum for 16 h. Anhydrous DMF (1.7 mL) and 0.45 M tetrazole were added to acetonitrile (7.3 mL, 3.30 mmol), and the mixture was stirred at room temperature for 3 h under an argon (nitrogen) atmosphere. After the consumption of compound 1 was confirmed by TLC, 70% t-butyl hydroperoxide (0.91 mL, 6.60 mmol) was added to water after cooling in an ice-salt bath for 30 min. The reaction mixture was stirred at room temperature for 1 h. After the conversion of the intermediate was confirmed 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).

[0392]

[0393] ② Synthesis process of 2′-O-Methyl Adenosylyl (n-bz)-{3′-OP-[2-cyanoethyl] → 5′}-2′,3′-diacetyl Guanosine (n-ibu) 4

[0394] 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 h. After confirming the conversion of compound 3 by TLC, 5% NaHCO3 solution (30 mL) was added and stirred for 10 min. The mixture was extracted with dichloromethane and evaporated. The resulting residue was purified by silica gel column chromatography to obtain product 4 (0.74 g, 0.79 mmol, 60%).

[0395] 1 H NMR (DMSO, 400MHz): δ 12.12 (s, 1H), 11.59 (d, 1H), 11.26 (d, 1H), 7.93 (d, d, 2H), 8.28 (d, 1H), 8.06 (d, 2H), 7.66 (t, 1H), 7.57 (d, 2H), 6.19(d, 1H), 6.14 (dd, 1H), 5.84 (q, 1H), 5.54 (dd, 1H), 5. 39 (t, 1H), 5.20 (m, 1H), 4.83 (m, 1H), 4.48 (m, 3H), 4.30 (m, 3H), 3.64 (m, 2H), 3.37 (d, 3H), 2.97 (q, 2H), 2.78 (m, 1H), 2.15 (d, 3H), 2.03 (d, 3H), 1.13 (m, 6H); 31 P NMR (DMSO, 162 MHz): δ -2.43 (d, 1P);

[0396]

[0397] ③ Synthesis process of 5′-O-Phosphoryl-2′-O-methyl Adenosylyl-{3′-OP→ 5′} Guanosine, (pAmpG) 5

[0398] Dinucleotide 4 (0.74 g, 0.7891 mmol) was dried under high vacuum for 16 h. 0.45 M tetrazole (3.51 mL, 1.58 mmol) and bis-(2-cyanoethyl)-N,N-diisopropyl-phosphoramidite (0.41 mL, 1.58 mmol) in acetonitrile were added, and the mixture was stirred at room temperature for 3 h under an argon (nitrogen) atmosphere. After confirming the consumption of compound 4, the reaction mixture was cooled in an ice-salt bath for 30 min, and 70% t-butyl hydroperoxide in water (17.2 mL, 3.16 mmol) was added. The mixture was stirred at room temperature for 1 h. After confirming the conversion of the intermediate by 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 h. After complete deprotection, the reaction mixture was evaporated with methanol. The resulting residue was dissolved in water, adjusted to pH 5.5, and loaded onto a DEAE-Sepharose column. Solvent A: 1 M TEAB buffer (pH 7.5–8), solvent B: water. The fractions containing the product were collected, evaporated, and dried in a freeze dryer to obtain 5 (446 mg, 0.4908 mmol, 62%) as a fine white powder containing triethylammonium salt. (Figure 14)

[0399]

[0400] Dinucleotide (pAmpG) TEA salt

[0401] 1H 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): δ 3.69 (s, 1P), -0.84 (s, 1P)

[0402]

[0403] Dinucleotide (pAmpG) imidazole sodium salt: pAmpG-IM.Na (378 mg, 93%)

[0404] 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 = 5.0 Hz, 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).

[0405]

[0406] <1-6> N7-(aryl)-2-ylmethyl) guanosine 5′-(2-thiodiphosphate)의 일반 합성 과정

[0407]

[0408] Completely dried imidazolized N7 modified guanosine monophosphate in DMSO (50 mM) ( Bn7 GMP-IM. Na) or 4-ClBn7 GMP-IM. Na (0.67 mmol) was added and dissolved. Then, dried thiophosphate TEA salt [(Et3NH), PSO3 3- ] (1 M in DMF) (4.5 equiv) was added and stirred at room temperature for 10 minutes, after which zinc chloride (8 equiv) and DMSO solution were added and stirred for 2 hours. The reaction mixture was then slowly poured into a cooled solution of sodium perchlorate (400 mg) and acetone (45 ml), centrifuged, the supernatant was discarded, and the precipitate was resuspended in acetone. The suspension-centrifugation process was repeated 3-4 times. The precipitate was dried over P2O5 in a vacuum desiccator, dissolved in water (15-20 ml), added EDTA Na4 (1.5 equiv) and extracted with chloroform (3X50 ml). The organic layer was then discarded, and the aqueous layer was purified with DEAE Sepharose fast flow resin as described above, and the fractions containing the product were collected, concentrated using a rotary evaporator, and dried using a freeze dryer to obtain a fine powder.

[0409]

[0410] < Bn7 GDPβS.TEA (320mg, 56%)>

[0411] 1 H NMR (400 MHz, DO) = 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, DO) = 32.86 (d, J = 32.1 Hz, 1P), -11.88 (d, J = 33.0 Hz, 1P).

[0412]

[0413] < 4-ClBn7 GDPβS. TEA (280 mg, 47%)>

[0414] 1 H NMR (400 MHz, DO) = 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 (162 MHz, DO) = 33.33 (d, J = 32.2 Hz, 1P), -11.81 (d, J = 31.9 Hz, 1P).

[0415]

[0416] <1-7> Method for the synthesis of N7-Arylmethyl substituted CleanCap analogues

[0417] To a solution of dinucleotide pAmpG triethylammonium salt (0.07 mmol, 63 mg) in DMSO (50 mM) were added N7-(arylmethyl)guanosine 5'-diphosphoroimidazolide disodium salt (16) (3.00 eq., 0.2 mmol) and zinc chloride (20.0 eq.). After stirring at 37°C for 3 days, 500 mM EDTA aq. (pH 8.0) (EDTA: 1.30 eq. per molar amount of ZnCl2) was added to the reaction mixture. The mixture was diluted with water and purified through DEAE Sepharose Fast Flow resin, followed by further purification using reverse-phase HPLC (instrument, Shimadzu; column, YMC-Actus Triart C8 (Preparative, 250x20.0 mm ID). The fractions containing the target compound were collected, concentrated, and lyophilized to provide the target N7-arylmethyl substituted CleanCap analog as a triethylammonium salt. The product was redissolved in methanol (2.00 mL). 190 mM NaClO4 in acetone (24.0 mL) was added to the mixture, and the resulting suspension was centrifuged (4,000 rpm, 20 min). The supernatant was discarded, and the precipitate was resuspended in acetone. The suspension-centrifugation process was repeated 3 to 4 more times. The precipitate was dried under reduced pressure to obtain the N7-arylmethyl substituted CleanCap analog.

[0418] N7-Bn-GpppA of N7-benzyl CleanCap analogues m The NMR analysis results of pG (26 mg, 29%) are shown below.

[0419] 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).

[0420]

[0421] <1-8> Guanosine N7-(aryl-2-ylmethyl) trinucleotide mRNA Cap1 유사체의 일반 합성 과정

[0422]

[0423] DMSO 2 ml에 pA mTo a solution of pG triethylammonium salt (0.07 mmol, 63 mg) was added N7-(aryl-2-ylmethyl)guanosine 5'-diphosphoroimidazolide disodium salt (3.00 eq, 0.2 mmol) and zinc chloride (20.0 eq). After stirring at 37°C for 3 days, the reaction mixture was stopped by adding 500 mM EDTA aqueous solution (pH 8.0) (EDTA: molar equivalent of ZnCl2 1.30 eq). The mixture was diluted with water and purified by DEAE Sepharose fast-flow resin, and then further purified using reverse-phase HPLC (Shimadzu instrument; YMC-Actus Triart C8 column (separation, 250 × 20.0 mm ID); solvent A, 50 mM TEAA buffer (pH 6.0) containing 0.5% CH3CN; solvent B, CH3CN; linear gradient 5–80% B (25 min); flow rate, 10 mL / min; detection wavelength, 254 nm). The fractions containing the target compound were collected, concentrated, and lyophilized to obtain the target N7-(aryl-2-ylmethyl) Cap1 analog as the triethylammonium salt. The product was redissolved in methanol (2.00 mL). 190 mM NaClO4 in acetone (24.0 mL) was added, and the resulting suspension was centrifuged (4,000 rpm, 20 min). The supernatant was discarded, and the precipitate was resuspended in acetone. The suspension-centrifugation process was repeated 3–4 more times. The precipitate was dried under reduced pressure to obtain the target N7-(aryl-2-ylmethyl) Cap1 analogue as a sodium salt (Figure 4).

[0424] Guanosine N7-benzyl trinucleotide Cap1( was prepared using N7-(benzyl-2-ylmethyl) guanosine 5′-diphosphoroimidazolide and N7-(4-Cl-benzyl-2-ylmethyl) guanosine 5′-diphosphoroimidazolid according to the above method. Bn7 GpppA mpG), Guanosine N7-4-Cl-benzyl trinucleotide Cap1 ( 4-ClBn7 GpppA m pG), L-Guanosine N7-benzyl trinucleotide Cap1( Bn7 LGpppA m pG), L-Guanosine N 7 , adenosine N 6 - dibenzyl trinucleotide Cap1 ( Bn7 Gppp Bn6 A m pG), Inosine N7-benzyl trinucleotide Cap1 ( Bn7 IpppA m pG) was synthesized and the NMR analysis results are shown below.

[0425]

[0426] <Guanosine N7-benzyl trinucleotide Cap1( Bn7 GpppA m pG)>

[0427]

[0428] 1 H NMR (400 MHz, DO) 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); 31P 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).

[0429]

[0430] <Guanosine N7-4-Cl-benzyl trinucleotide Cap1( 4-ClBn7 GpppA m pG)>

[0431]

[0432] 1 H 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).

[0433]

[0434] <L-Guanosine N7-benzyl trinucleotide Cap1( Bn7 LGpppA m pG)>

[0435]

[0436] 1 H 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 = 14.8 Hz, 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).

[0437]

[0438] <Guanosine N 7 , adenosine N 6 - dibenzyl trinucleotide Cap1( Bn7 Gppp Bn6 A m pG)>

[0439]

[0440] 1H 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); 31 P 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).

[0441]

[0442] <Inosine N7-benzyl trinucleotide Cap1( Bn7 IpppA m pG)>

[0443]

[0444] 1H NMR (400 MHz, DO) 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 HO, 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, DO) 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).

[0445]

[0446] <1-9> General synthetic process of N7-(aryl-2-ylmethyl) trinucleotide diphosphate Cap1 substance (BzCap1)

[0447]

[0448] Dinucleotide (pA) in a dry DMSO solution in an inert atmosphere m pG) In a solution containing triethylammonium salt (0.073 mmol, 65 mg) Bn7 GMP-IM (0.219 mmol, 142 mg, 3 equivalents) or 4-Cl-Bn7GMP-IM (0.219 mmol, 124 mg, 3 equiv) was added. Zinc chloride (20.0 equiv) dissolved in dry DMSO was added and stirred at 37°C for 3 days. The reaction mixture was then quenched by the addition of aqueous EDTA solution (pH 7.5) (EDTA: molar equivalent of ZnCl2 1.30 equiv). The mixture was diluted with water and purified using reverse-phase HPLC (prep pure 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). Fractions containing the target compound were collected and stored in a freeze dryer, and the product was identified through NMR analysis.

[0449]

[0450] <Guanosine N7-benzyl trinucleotide diphosphate Cap1( Bn7 GppA m pG)>

[0451]

[0452] 1 H NMR (400 MHz, DO) = 9.22 (s, 1H), 8.24 (s, 1H), 8.10 (d, J = 1.1 Hz, 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);31 P NMR (162 MHz, DO) = -0.79 (s, 1P), -11.09 (d, J = 21.2 Hz, 1P), -11.43 (d, J = 21.33 Hz, 1P).

[0453]

[0454] <Guanosine N7-4-Cl-benzyl trinucleotide diphosphate Cap1( 4-ClBn7 GppA m pG)>

[0455]

[0456] 1 H NMR (400 MHz, DO) = 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, DO) = -0.83 (s, 1P), -11.07 (d, J = 21.1 Hz, 1P), -11.4341 (d, J = 21.0 Hz, 1P).

[0457]

[0458] <1-10> Synthesis of N7-(aryl-2-ylmethyl)β-phosphorothioate trinucleotide Cap1 analogue

[0459]

[0460] Dinucleotide (pA) in a dry DMSO solution in an inert atmosphere mIn a solution containing sodium salt (0.075 mmol, 60 mg, 1 equivalent) of pG-IM Bn7 GDPβS.TEA (0.225 mmol, 192.14 mg, 3 equivalents) or 4-Cl-Bn7 GDPβS.TEA (0.225 mmol, 200 mg, 3 equiv) was added. Zinc chloride (20.0 equiv) dissolved in dry DMSO was added and stirred at 37°C for 3 days. The reaction mixture was then quenched by the addition of aqueous EDTA solution (pH 7.5) (EDTA: molar equivalent of ZnCl2 1.30 equiv). The mixture was diluted with water and purified using reverse-phase HPLC (prep pure 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 partial stereoisomers (D1 and D2) of the target compound were provided and separated based on retention time during HPLC purification. The product with the longest retention time was designated D2, and the product with the shortest retention time was designated D1. Fractions containing target compounds D1 and D2 were collected and stored in a freeze dryer, and the products were identified by NMR analysis.

[0461]

[0462] <Guanosine N7-benzyl β-phosphorothioate trinucleotide Cap1( Bn7 Gpp s pA m pG)(D1)>

[0463]

[0464] 1H 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).

[0465]

[0466] < Bn7 Gpp s pA m pG (D2)>

[0467]

[0468] 1H 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 = 4.0 Hz, 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).

[0469]

[0470] <Guanosine N7-4-Cl-benzyl β-phosphorothioate trinucleotide Cap1 ( 4-ClBn7 Gpp s pA m pG)(D1)>

[0471]

[0472] 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 = 3.9 Hz, 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).

[0473]

[0474] < 4-ClBn7 Gpp s pA m pG)(D2)>

[0475]

[0476] 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, DO) = 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).

[0477]

[0478] <Example 2> Introduction of trinucleotide caps into mRNA and analysis of capping efficiency

[0479] It was analyzed that the trinucleotide capping materials manufactured in Example 1 can be recognized by T7 RNA polymerase with high efficiency during the transcription process and introduced into mRNA.

[0480] As a result, it was confirmed that mRNA with a capping material that modified the guanosine N7 position at the 5' end to arylmethyl instead of the existing methyl showed an antigen protein expression level similar to or higher than that of the existing CleanCap (cap 1 commercial) and the previously reported ARCA cap during the translation process.

[0481] In addition, it was confirmed that the capping material can be recognized by T7 RNA polymerase and introduced into RNA with higher efficiency than ARCA during the transcription process. While the existing ARCA showed a capping efficiency of approximately 50% and CleanCap (cap 1 commercial) showed a capping efficiency of approximately 85%, it was confirmed that BzCap1 and BzCap analogs showed a capping efficiency of approximately 90% or more (see Figure 45).

[0482]

[0483] <Example 3> Expression analysis of antigen protein using capping material substituted with arylmethyl group

[0484] The mRNA translation process was confirmed by introducing a capping material manufactured by modifying the N7 position of the guanosine base at the 5' end with an arylmethyl compound instead of the existing methyl. Luciferase mRNA was used to confirm the expression level of the antigen protein, and the expression amount of the antigen protein was analyzed using HEK 293 cells. In the case of Luciferase mRNA, it was confirmed that the antigen protein expression efficiency of BzCap1 and BzCap analogs was expressed more efficiently than that of CleanCap in mRNA using natural rNTP (see Figure 46).

[0485]

[0486] ① Preparation of luciferase DNA template

[0487] A linear dsDNA template containing the start codon from the luciferase plasmid to the poly-A tail was prepared by PCR. During successive PCRs, the T7 promoter and Kozak sequence were added to the luciferase dsDNA template, which was ultimately used for the transcription reaction. The primers used were: 1st forward primer: 5'- ATG GAA GAC GCC AAA AAC ATA AAG -3' (SEQ ID NO: 1); 2nd forward primer: TAA TAC GAC TCA CTA TAG GGC CAC CAT GGA AGA CGC CAA AAA CAT (SEQ ID NO: 2); and 3rd reverse primer: AAT CGC GCC TAG GCG CGC CCG TAC GGC TCT TC-3' (SEQ ID NO: 3).

[0488]

[0489] ② Preparation of luciferase mRNA

[0490] The concentration of luciferase template was measured using a nanodroplet spectrophotometer. For each transcription reaction, 100 ng of 1 μL template was placed in a microcentrifuge tube. Then, 1.5 μL of 10X transcription buffer (400 mM Tris-HCl, 60 mM MgCl2, 10 mM DTT, 20 mM spermidine) and 1.5 μL of 100 mM DTT were added to the tube. rATP, rCTP, and rUTP were used (1 μL, 10 mM), respectively. rGTP was used for cap-dependent transcription of luciferase (1 μL, 2.5 mM). The ratio of capping agent was maintained at 4-fold higher than rGTP to provide more than 80% capped mRNA. Therefore, 1 μL of capping agent (10 mM) was added. RNase inhibitor (0.5 μL, 40 U / μL) and T7 RNA polymerase (1 μL, 50 U / μL) were added. Nuclease-free water (4.5–5.5 μL) was added to bring the final reaction volume to 15 μL. For modified rNTPs, stocks were prepared at 10 mM concentrations and the corresponding derivatives were added instead of rCTP and rUTP. The reaction was incubated at 37°C for 1 h, after which 1.8 μL 10X DNase I buffer (10 mM Tris-HCl, 2.5 mM MgCl2, 0.5 mM CaCl2) and 1 μL DNase I (2 U / μL) were added to the reaction and incubated at 37°C for an additional 15 min to remove all DNA template.

[0491]

[0492] ③ Purification of transcribed mRNA

[0493] To remove any dsRNA and transcription byproducts that may have formed, cellulose was added to the reaction (10 μL, 0.2 g / mL) and gently shaken for 10 minutes. Next, the mixture was transferred to an RNA purification column along with the ethanol and binding buffer provided with the column, and finally centrifuged at 13,000 rpm for 1 minute. RNA preparation buffer and wash buffer were then successively added to the column and centrifuged. At the end of this step, all proteins and unincorporated rTNP were removed as a suspension. Finally, a new tube was attached, 10 μL of nuclease-free water was added to the column, and centrifuged at 13,000 rpm for 2 minutes. Any dsDNA bound to the cellulose remained on the column. Only pure single-stranded GFP or luciferase mRNA passed through the column. This sample was collected and stored at 70°C for further use.

[0494]

[0495] ④ Analysis of capping efficiency of transcribed mRNA

[0496] 2 μg of capped and transcribed mRNA was annealed by heating to 70°C with 3 μg of gDNA and RNase H buffer and then slowly cooling to 25°C. Next, thermally stable RNase H was used to cleave a short sequence containing the capping material from the long mRNA. o The reaction was carried out for 1 hour in C. The short sequences containing the cleaved capping material were analyzed by electrophoresis using a 16% denaturing PAGE gel. The short sequences cleaved from mRNA without the introduction of the capping material (uncapped mRNA) showed a faster gel migration, while the short sequences cleaved from mRNA containing the capping material (capped mRNA) showed a relatively slower gel migration.

[0497]

[0498] ⑤ Cell culture and preparation for transfection

[0499] HEK 293 cells were cultured in 60 mL culture plates containing DMEM containing 10% FBS and 1% pen / stripe. The day before transfection, cells were detached using trypsin and resuspended in fresh medium. Cells were seeded in 96-well plates. The following day, when cells reached 70% confluence, the transfection mixture was prepared and transfected.

[0500]

[0501] ⑥ Quantification and transformation of mRNA

[0502] Purified mRNA was quantified using a Nanodrop spectrophotometer, and the transcription yield for each rNTP combination was compared. For cell transfection, 500 ng of each mRNA was collected and mixed with 5 μL of Opti-MEM. 0.5 μL of lipofectamine was mixed with 5 μL of Opti-MEM in a separate tube and incubated at room temperature for 10 minutes. The mRNA and lipofectamine mixture was then transferred to the same tube and incubated for an additional 5 minutes at room temperature. Finally, the transfection mixture was added to the cells and incubated for 24 hours.

[0503]

[0504] ⑦ Luciferase expression analysis

[0505] After the incubation period, the medium was removed, and the cells were lysed using 50 μL of 1X lysis buffer. The cell lysate was transferred to a microcentrifuge tube. For the luciferase luminescence assay, 100 μL of luciferin substrate was collected for each sample in a 96-well white plate. Then, 20 μL of each cell lysate sample was added and mixed by pipetting. Finally, the luminescence of luciferase was recorded using a luminometer.

[0506]

[0507] As a result, it was confirmed that the capping materials with various types of aryl methyl introduced according to the present invention can be recognized by T7 RNA polymerase and introduced into RNA with higher efficiency than ARCA or CleanCap during the transcription process, and the existing ARCA showed a capping efficiency of about 50% and CleaCap showed a capping efficiency of 89%, but the trinucleotide capping materials with various types of aryl methyl introduced ( Bn7 GpppA m pG, Bn7 GppA m pG, 4-ClBn7 GppA m pG, Bn7 Gppp Bn6 A m pG, 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 In the case of pG), it was confirmed that a capping efficiency of approximately 90% or more was shown (see Figure 45).

[0508] In addition, we confirmed the mRNA translation process in which a capping material was introduced, in which the N7 position of the guanosine base at the 5' end was modified with an arylmethyl compound instead of the existing methyl.

[0509] Luciferase mRNA was used to determine the level of antigen protein expression, and the expression level of the antigen protein was confirmed using HEK 293 cells. For luciferase mRNA, mRNA using natural rNTPs was used.

[0510]

[0511] In addition, the trinucleotide capping materials developed in the present invention are more effective than mRNAs introduced with existing ARCA cap (cap 0) and CleanCap (cap 1 commercial). Bn7 GpppA m pG, Bn7 GppA m pG, 4-ClBn7 GppA m pG, Bn7 Gppp Bn6 A m pG, 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 It was confirmed that Luciferase mRNA introduced with pG) showed higher antigen protein expression efficiency (see Figure 46).

[0512]

[0513] The present invention relates to a novel modified trinucleotide capping derivative and an mRNA to which a capping derivative has been introduced. The derivatives of the present invention, each introducing a methylene bridged heterocyclic compound and an arylmethyl group at the N7 position of the 5'-terminal guanosine base, provide derivatives that are more efficient than the capping materials used in existing mRNA vaccines. The novel modified trinucleotide capping derivative and the mRNA to which the capping derivative has been introduced can be usefully used in the development of new mRNA vaccines and various mRNA-based therapeutics, and thus can be utilized in various fields such as industrial processes.

[0514] Therefore, the modified trinucleotide capping derivative and the mRN into which the capping derivative is introduced according to one embodiment of the present invention can be considered to have industrial applicability.

Claims

1. A trinucleotide cap analogue comprising a compound of the following chemical formula 1: [Chemical Formula 1] In the compound of the above chemical formula 1, R1 is a heterocyclic compound connected to the N7 position of the N7 guanosine base by a methylene bridge, In the compound of the above chemical formula 1, X1 and X2 are H or CH3, and X3, X4 and X5 are each selected from the group consisting of O, S and Se, R2 is a derivative containing Benzyl, The above heterocyclic compound is a functional group selected from among saturated or partially saturated 5- to 10-membered heteroaromatic ring compounds containing 1 to 2 heteroatoms selected from O, N, and S, The above heteroaromatic ring compound is a functional group selected from the group consisting of furan, pyrrole, thiophen, imidazole, pyrazole, oxazol, isoxazole, thiazole, pyridine, pyrazine, pyrimidine, pyridazine, and triazine.

2. In paragraph 1, The trinucleotide cap analogue is a trinucleotide cap analogue having the following chemical formula 2 or chemical formula 3: [Chemical Formula 2] [Chemical Formula 3] 3. mRNA capped at the 5' end with a trinucleotide cap analogue of paragraph 1.

4. A composition for producing mRNA capped at the 5' end, comprising the trinucleotide cap analog of paragraph 1.

5. A pharmaceutical composition for expressing a desired peptide or protein, comprising an mRNA capped at the 5' end, including a trinucleotide cap analogue of paragraph 1, and a pharmaceutically acceptable carrier.

6. A trinucleotide cap analogue comprising a compound of the following chemical formula [4-1], [4-2], or [4-3]: [Chemical Formula 4-1] [Chemical Formula 4-2] [Chemical Formula 4-3] In the compound of the above chemical formula [4-1], [4-2] or [4-3], The above R1 is a compound including a compound of chemical formula 5 linked to methyl (CH2) at the N7 position, The above X1 and X2 are each H or CH3, The above Y1 is 0 or 1, The above Y2 is O or S, The above Z is H or C1-C3 alkylbenzyl, [Chemical Formula 5] In the above chemical formula 5, R2, R3, R4, R5 and R6 are each independently any one functional group selected from the group consisting of H, OH, Halo, methyl, alkyl, alkoxy, nitro, carboxylic, azide, amino and cyano.

7. In paragraph 6, A trinucleotide cap analogue, wherein the above R1 is benzyl or chlorobenzyl.

8. In paragraph 6, A trinucleotide cap analogue wherein the above Z is methylbenzyl.

9. An mRNA capped at the 5' end with a trinucleotide cap analogue of clause 6.

10. A composition for producing mRNA capped at the 5' end, comprising a trinucleotide cap analogue of clause 6.

11. A pharmaceutical composition for expressing a desired peptide or protein, comprising an mRNA capped at the 5' end, comprising a trinucleotide cap analogue of clause 6, and a pharmaceutically acceptable carrier.

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