Novel modified trinucleotide capping derivatives and mRNA into which the capping derivatives have been introduced

Abstract

This invention relates to novel modified trinucleotide capping derivatives and mRNA into which the capping derivatives have been introduced. The derivatives according to this invention, in which a methylene-bridged heterocyclic compound and an arylmethyl group are introduced at the N7 position of the 5' terminal guanosine base, respectively, exhibit higher efficiency than capping materials used in conventional mRNA vaccines, and can therefore be usefully used in the development of mRNA vaccines and various mRNA-based therapeutic agents.

Description

[Technical Field]

[0001] The present invention relates to novel modified trinucleotide capping derivatives and mRNA into which capping derivatives (capped derivatives) have been introduced. More specifically, it relates to mRNA structures into which a trinucleotide-based capping substance has been introduced, in which the N7 position of the 5' terminal guanosine base is modified with a methylene-bridged heterocyclic compound and an arylmethyl instead of the conventional methyl group. [Background technology]

[0002] mRNA vaccines play a crucial role in treating the coronavirus (COVID-19) pandemic, which is spreading worldwide. Capping substances protect mRNA vaccines from degradation. Specifically, in order to express functionally important proteins from DNA genetic information, a transcription process is necessary in the nucleus, in which mRNA with a complementary sequence based on the DNA template strand is synthesized. The synthesized mRNA is then transported out of the nucleus and undergoes a translation process in the cytoplasm via ribosomes to synthesize specific proteins.

[0003] In particular, N7 methylation in the CAP structure involves adding a methyl group to the seventh nitrogen atom of guanosine located at the 5′ end of mRNA. This methylation plays several important roles in significantly improving the biological effects of mRNA. The N7-methylated 5′ cap structure enhances the stability of the mRNA molecule, promotes binding to ribosomes, and facilitates the initiation of translation by promoting binding to the translation initiation factors eIF4E and eIF4G. Furthermore, the N7-methylated cap structure plays a crucial role in the efficient translocation of mRNA from the nucleus to the cytoplasm and functions as an intracellular marker for mRNA to avoid recognition and degradation by the innate immune system. Overall, the N7-methylated 5′ cap is an important feature for mRNA stability and function in eukaryotic cells, influencing the RNA lifecycle and the efficient production capacity of proteins.

[0004] Two methods are used for the mRNA capping process. The first method is 5′ capping by post-transcription treatment applied to the 5′ end of the RNA product of DNA transcription. After transcription is complete, the 5′ end of the RNA transcript contains a free triphosphate group because it is the first nucleotide incorporated into the chain. In the capping process, this triphosphate base is replaced with another structure called a "cap." The cap is added by the enzyme guanylate transferase. This enzyme catalyzes the reaction between the 5′ end of the mRNA transcript and a guanine triphosphate (GTP) molecule. The second method is in vitro transcription (IVT) capping, which is used in mRNA vaccines. In this technique, capping is possible simultaneously with mRNA transcription. That is, the cap is added during mRNA synthesis, rather than at a separate post-transcription stage. This method simplifies the capping process, increases capping efficiency, and enables high-yield mRNA production in a simple process. Furthermore, the 5′ cap structure, a protein expression initiation factor, plays a crucial role in protein expression by forming a translation initiation complex with eukaryotic translation initiation factor 4E (eIF4E). It also functions as an intracellular marker for mRNA, preventing it from being recognized and degraded by the innate immune system.

[0005] For about 20 years, dinucleotide-based ARCA cap compounds have been used as chemical capping substances for IVT (in vitro thrombosis). However, since the COVID-19 pandemic, trinucleotide-based CleanCap, which has significantly higher antibody expression efficiency, has been used as the main component of Pfizer's mRNA vaccine.

[0006] CleanCap, developed by TriLink, has a chemically modified trinucleotide structure and has been reported to be the most efficient capping material developed to date for the expression of antigen proteins in mRNA vaccines. TriLink holds the unique and exclusive patent for this capping material.

[0007] However, in South Korea, technologies capable of increasing the antigen protein expression efficiency of mRNA vaccines, such as CleanCap, have not been developed, and therefore, they have not been able to secure mRNA vaccines independently. Therefore, the inventors focused on this efficiency and synthesized mRNA using a capping technology in which transcription and capping occur simultaneously via IVT. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] The inventors have developed a cap material substituted with a novel N7-methylene-bridged heterocyclic compound different from CleanCap-modified nucleotides, which can enhance the antigen protein expression efficiency of mRNA vaccines, similar to CleanCap, and a cap material substituted with a novel N7-arylmethyl (benzyl (BzCap1)) and its derivatives, thereby completing this invention. [Means for solving the problem]

[0009] To solve the above problems, one aspect of the present invention provides a trinucleotide cap analog comprising the compound of the following chemical formula 1: [Chemical formula 1] [ka] In the compound of chemical formula 1, R1 is a methylene-bridged heterocyclic compound linked to the N7 position of the N7 guanosine base by a methylene bridge. In the compound of chemical formula 1, X1 and X2 are H or CH3, and X3, X4, and X5 are selected from the group consisting of O, S, and Se, respectively. R2 is a benzyl-containing derivative, The heterocyclic compound contains one to two heteroatoms selected from O, N, and S, and is a functional group selected from saturated or partially saturated 5-membered to 10-membered heteroaromatic ring compounds. The aforementioned heteroaromatic ring compound is one functional group selected from the group consisting of furan, pyrrole, thiophene, imidazole, pyrazole, oxazole, isoxazole, thiazole, pyridine, pyrazine, pyrimidine, pyridazine, and triazine.

[0010] The heterocyclic compound may contain one to two heteroatoms selected from O, N, and S, and may be any one functional group selected from saturated or partially saturated 5- to 10-membered aromatic ring groups. Specific examples of heterocyclic compounds include thiophene, pyridyl, imidazole, tetrahydrofuranyl group, 2,3-dihydrofuranyl group, 2,5-dihydrofuranyl group, pyrrolidinyl group, 2,3-dihydropyrrolidinyl group, 2,5-dihydropyrrolidinyl group, tetrahydro-2H-pyranyl group, 3,4-dihydro-2H-pyranyl group, 4H-pyranyl group, piperidinyl group, 1,2,3,4-tetrahydropyridinyl group, 1,4-dihydropyridinyl group, piperazinyl group, N-protected piperazinyl, and morpholino group. The N-protecting group of piperazinyl may typically include an alkyl group, an alkylcarbonyl group, or an alkylsulfonyl group.

[0011] In the present invention, the trinucleotide cap analog may be characterized by having the following chemical formula 2 or chemical formula 3. [Chemical formula 2] [ka] [Chemical formula 3] [ka]

[0012] Trinucleotide cap analogs comprising compounds of the following chemical formulas [4-1], [4-2], or [4-3]: [Chemical formula 4-1] [ka] [Chemical formula 4-2] [ka] [Chemical formula 4-3] [ka]

[0013] In the compounds of the aforementioned chemical formulas [4-1], [4-2], or [4-3], The aforementioned R1 is a compound containing a compound of chemical formula 5 linked at the N7 position by a methylene crosslink (-CH2-), The aforementioned X1 and X2 are H or CH3, respectively. The aforementioned Y1 is 0 or 1, The aforementioned Y2 is either O or S, The aforementioned Z is H or C1-C3 alkylbenzyl, [Chemical formula 5] [ka] In the above chemical formula 5, R2, R3, R4, R5, and R6 are each independently selected from the group consisting of H, OH, halo, methyl, alkyl, alkoxy, nitro, carboxyl, azide, amino, and cyano.

[0014] Furthermore, the present invention can produce mRNA capped at the 5' end with the aforementioned trinucleotide cap analog.

[0015] Furthermore, the present invention provides a composition or kit for the production of 5'-end capped mRNA, comprising a trinucleotide cap analog.

[0016] Furthermore, the present invention is The present invention provides a pharmaceutical composition for the expression of a target peptide or protein, comprising a 5'-terminated capped mRNA containing a trinucleotide cap analog and a pharmaceutically acceptable carrier. [Effects of the Invention]

[0017] The derivative according to the present invention, in which a methylene-bridged heterocyclic compound and an arylmethyl group are introduced at the N7 position of the 5'-terminal guanosine base, exhibits higher efficiency than conventional capping materials used in mRNA vaccines by introducing a methylene-bridged heterocyclic group and an arylmethyl group, respectively, at the N7 position of the 5'-terminal guanosine base. This makes it useful for the development of mRNA vaccines and various mRNA-based therapeutic agents. [Brief explanation of the drawing]

[0018] [Figure 1] This figure shows ThioCap1 and PyCap1, trinucleotide-based capping materials in which a methylene-bridged thiophene group and a methylene-bridged pyridine group are introduced at the N7 position (R1) of the 5' terminal guanosine, respectively. [Figure 2] This graph shows the 1H NMR spectrum of the ThioCap1 substance. [Figure 3] This graph shows the 31P NMR spectrum of the ThioCap1 substance. [Figure 4] This graph shows the 1H NMR spectrum of the PyCap1 substance. [Figure 5] This graph shows the 31P NMR spectrum of the PyCap1 substance. [Figure 6] This figure shows the results of confirming the transcription of the capping material (ThioCap1) of the present invention using T7 RNA polymerase (Lane 1: Luciferase template + T7RNAPol + rNTPs + CleanCap (commercially available Cap1); Lane 2: Luciferase template + T7RNAPol + rATP + rGTP + rCTP + pseudoUTP + CleanCap (commercially available Cap1); Lane 3: Luciferase template + T7RNAPol + rNTPs + ThioCap1; Lane 4: Luciferase template + T7RNAPol + rATP + rGTP + rCTP + pseudoUTP + ThioCap1). [Figure 7] This figure shows the results of confirming the capping efficiency of the capping material (ThioCap1) of the present invention using T7 RNA polymerase by RNase H treatment (Lane 1: Luciferase template + T7RNAPol + rNTPs → gDNA + RNase H; Lane 2: Luciferase template + T7RNAPol + rNTPs + ARCA → gDNA + RNase H; Lane 3: Luciferase template + T7RNAPol + rNTPs + CleanCap → gDNA + RNase H; Lane 4: Luciferase template + T7RNAPol + rNTPs + ThioCap1 → gDNA + RNase H). [Figure 8] This graph shows a comparison of the expression levels of luciferase antigen protein between luciferase mRNA (Luc mRNA ThioCap1) into which the capping material of the present invention (ThioCap1) has been introduced, and conventional CleanCap (Luc mRNA Cap1). [Figure 9] This figure shows the results of confirming the transcription of the cap material (PyCap1) of the present invention using T7 RNA polymerase (Lane 1: Luciferase template + T7RNAPol + rNTPs + CleanCap (commercially available Cap1); Lane 2: Luciferase template + T7RNAPol + rNTPs + PyCap1). [Figure 10] This figure shows the results of confirming the capping efficiency of the present invention's capping material (PyCap1) using RNA polymerase after RNase H treatment. [Figure 11] This graph compares the expression levels of the antigen protein (luciferase) between luciferase mRNA (Luc mRNA PyCap1) into which the capping material of the present invention (PyCap1) has been introduced, and conventional CleanCap (Luc mRNA Cap1). [Figure 12] This is the chemical formula for a trinucleotide-based capping substance (BzCap1) in which an arylmethyl group is introduced at the N7 position (R1) of the 5' terminal guanosine. [Figure 13] This is a schematic diagram showing the synthesis process of the N7-(arylmethyl)guanosine 5'-diphosphoruidazolide analog. [Figure 14] This is a schematic diagram showing the synthesis procedure for the dinucleotide pAmpG. [Figure 15] This is a schematic diagram showing the synthesis procedure for guanosine N7-(aryl-2-ylmethyl)trinucleotide mRNA Cap1 analog. [Figure 16] This is a schematic diagram showing the synthesis procedure for the N7-(aryl-2-ylmethyl)trinucleotide diphosphate Cap1 analog. [Figure 17] This is a schematic diagram showing the synthesis procedure for the N7-(aryl-2-ylmethyl)β-phosphorothioate trinucleotide Cap1 analog. [Figure 18] This figure shows the 1H NMR spectrum of a guanosine N7-benzyl trinucleotide cap analog. [Figure 19] This figure shows the 31P NMR spectrum of a guanosine N7-benzyltrinucleotide Cap analog. [Figure 20] This figure shows the 1H NMR spectrum of a guanosine N7-benzyl trinucleotide diphosphate Cap analog. [Figure 21]This figure shows the 31P NMR spectrum of a guanosine N7-benzyl trinucleotide diphosphate Cap analog. [Figure 22] This is ESI-MS of a guanosine N7-benzyl trinucleotide diphosphate Cap analog. [Figure 23] This figure shows the 1H NMR spectrum of guanosine N7-4-Cl-benzyl trinucleotide diphosphate cap analog. [Figure 24] This figure shows the 31P NMR spectrum of a guanosine N7-4-chlorobenzyl trinucleotide diphosphate Cap analog. [Figure 25] This is an ESI-MS of a guanosine N7-4-chlorobenzyl trinucleotide diphosphate Cap analog. [Figure 26] This figure shows the 1H NMR spectrum of a guanosine N7, adenosine N6-dibenzyl trinucleotide cap analog. [Figure 27] This figure shows the 31P NMR spectra of guanosine N7 and adenosine N6-dibenzyl trinucleotide Cap analogs. [Figure 28] This is an ESI-MS of guanosine N7 and adenosine N6-dibenzyl trinucleotide Cap analogs. [Figure 29] This figure shows the 1H NMR spectrum of guanosine N7-4-Cl-benzyl trinucleotide cap analog. [Figure 30] This figure shows the 31P NMR spectrum of a guanosine N7-4-chlorobenzyltrinucleotide Cap analog. [Figure 31]This figure shows the 1H NMR spectrum of guanosine N7-benzyl β-phosphorothioate trinucleotide cap analog (D1). [Figure 32] This figure shows the 31P NMR spectrum of guanosine N7-benzyl β-phosphorothioate trinucleotide Cap analog (D1). [Figure 33] This figure shows the 1H NMR spectrum of guanosine N7-benzyl β-phosphorothioate trinucleotide Cap analog (D2). [Figure 34] This figure shows the 31P NMR spectrum of guanosine N7-benzyl β-phosphorothioate trinucleotide Cap analog (D2). [Figure 35] This figure shows the 1H NMR spectrum of guanosine N7-4-Cl-benzyl β-phosphorothioate trinucleotide cap analog (D1). [Figure 36] This figure shows the 31P NMR spectrum of guanosine N7-4-chlorobenzyl β-phosphorothioate trinucleotide Cap analog (D1). [Figure 37] This figure shows the 1H NMR spectrum of guanosine N7-4-chlorobenzyl β-phosphorothioate trinucleotide Cap analog (D2). [Figure 38] This figure shows the 31P NMR spectrum of guanosine N7-4-chlorobenzyl β-phosphorothioate trinucleotide Cap analog (D2). [Figure 39] This figure shows the 1H NMR spectrum of an inosine N7-benzyl trinucleotide cap analog. [Figure 40]This figure shows the 31P NMR spectrum of an inosine N7-benzyltrinucleotide Cap analog. [Figure 41] This figure shows the 1H NMR spectrum of L-guanosine N7-benzyl trinucleotide cap analog. [Figure 42] This figure shows the 31P NMR spectrum of an L-guanosine N7-benzyltrinucleotide Cap analog. [Figure 43] This gel image shows experimental results confirming the transcription of the capping material (BzCap1) using T7 RNA polymerase and the capping efficiency during in vitro transcription by RNase H treatment, comparing the capping efficiency with that of conventional ARCA and CleanCap. [Figure 44] This figure shows the results of confirming the transcription of various arylmethyl-modified trinucleotide-based capping materials of the present invention using T7 RNA polymerase. Lane 1: Luciferase mRNA + m7GpppAmpG Lane 2: Luciferase mRNA + Bn7GpppAmpG Lane 3: Luciferase mRNA + Bn7GppAmpG Lane 4: Luciferase mRNA + 4-ClBn7GppAmpG Lane 5: Luciferase mRNA + Bn7GpppBn6AmpG Lane 6: Luciferase mRNA + 4-ClBn7GpppAmpG Lane 7: Lu Luciferase mRNA + Bn7GppspAmpG (D1) lane 8: Luciferase mRNA + Bn7GppspAmpG (D2) lane 9: Luciferase mRNA + 4-ClBn7GppspAmpG (D1) lane 10: Luciferase mRNA + 4-ClBn7GppspAmpG (D2) lane 11: Luciferase mRNA + Bn7IpppAmpG lane 12: Luciferase mRNA + Bn7LGpppAmpG [Figure 45]This figure shows the results of confirming the capping efficiency of various arylmethyl-modified trinucleotide-based capping materials of the present invention by RNase H treatment using RNA polymerase. Lane 1: Luciferase mRNA + m7GpppAmpG Lane 2: Luciferase mRNA + Bn7GpppAmpG Lane 3: Luciferase mRNA + Bn7GppAmpG Lane 4: Luciferase mRNA + 4-ClBn7GppAmpG Lane 5: Luciferase mRNA + Bn7GpppBn6AmpG Lane 6: Luciferase mRNA + 4-ClBn7GpppAmpG Lane 7: Luciferase mRNA+Bn7GppspAmpG(D1) lane 8: Luciferase mRNA+Bn7GppspAmpG(D2) lane 9: Luciferase mRNA+4-ClBn7GppspAmpG(D1) lane 10: Luciferase mRNA+4-ClBn7GppspAmpG(D2) lane 11: Luciferase mRNA+Bn7IpppAmpG lane 12: Luciferase mRNA+Bn7LGpppAmpG [Figure 46]This graph shows the results of confirming the expression of the antigen protein (luciferase) using trinucleotide-based capping materials modified with various arylmethyl groups at the N7 position of the 5' terminal guanosine. Lane 1: Luciferase mRNA + m7GpppAmpG Lane 2: Luciferase mRNA + Bn7GpppAmpG Lane 3: Luciferase mRNA + Bn7GppAmpG Lane 4: Luciferase mRNA + 4-ClBn7GppAmpG Lane 5: Luciferase mRNA + Bn7GpppBn6AmpG Lane 6: Luciferase mRNA + 4-ClBn7GpppAmpG Lane 7: Luciferase mRNA+Bn7GppspAmpG(D1) lane 8: Luciferase mRNA+Bn7GppspAmpG(D2) lane 9: Luciferase mRNA+4-ClBn7GppspAmpG(D1) lane 10: Luciferase mRNA+4-ClBn7GppspAmpG(D2) lane 11: Luciferase mRNA+Bn7IpppAmpG lane 12: Luciferase mRNA+Bn7LGpppAmpG [Modes for carrying out the invention]

[0019] The present invention will be described in detail below.

[0020] The inventors have produced novel modified trinucleotide capping derivatives and mRNA into which these capping derivatives have been introduced. Specifically, they have developed various modified capping compounds in which a methylene-bridged heterocyclic group and an arylmethyl group have been introduced at the N7 position of the 5' guanosine base, respectively, so as to be able to increase the expression efficiency of antigen proteins in mRNA vaccines, similar to CleanCap, but with higher efficiency than CleanCap-modified nucleotides. Furthermore, they have confirmed that mRNA structures into which various methylene-bridged heterocyclic groups and / or arylmethyl groups have been introduced exhibit higher antigen protein expression efficiency than capping materials used in conventional mRNA vaccines, thus completing the present invention.

[0021] The distinguishing feature of this invention lies in the design and synthesis of a novel modified trinucleotide cap material and the confirmation of its potential use as an mRNA vaccine. Specifically, novel compounds were synthesized by organic chemical reactions in which the N7 position of the 5' terminal guanosine base in the trinucleotide structure of CleanCap was modified with methylene-bridged thiophene, methylene-bridged pyridine, and arylmethyl compounds instead of the conventional methyl group. The synthesis process and results are shown.

[0022] The capping material was confirmed to be recognized by T7 RNA polymerase during the transcription process with an efficiency nearly equivalent to or greater than that of ARCA or CleanCap, and to be introduced into RNA. Furthermore, while conventional ARCA showed a capping efficiency of approximately 50% and CleanCap approximately 86%, ThioCap1 showed an efficiency of over 92%, BzCap1 and BzCap1 analogs showed an efficiency of over 90%, and PyCap1 also showed an efficiency of over 88%.

[0023] Furthermore, compared to conventional mRNA with CleanCap introduced (Luc mRNA Cap1), the luciferase mRNA with ThioCap1 introduced (Luc mRNA ThioCap1), the luciferase mRNA with PyCap1 introduced (Luc mRNA PyCap1), the luciferase mRNA with BzCap1 introduced (Luc mRNA BzCap1), and the luciferase mRNA with a BzCap1 analog introduced, all developed in this invention, were confirmed to exhibit higher antigen protein expression efficiency (see Figures 8, 11, and 46).

[0024] Compared to mRNA with conventional ARCA caps (Cap0) and CleanCap (Cap1 commercial), luciferase mRNA with BzCap1 and BzCap1 analogs developed in this invention was confirmed to exhibit higher antigen protein expression efficiency.

[0025] Therefore, based on the above results, the trinucleotide cap analog of the present invention, by introducing a methylene-bridged heterocyclic compound and an arylmethyl group, respectively, at the N7 position of the 5' terminal guanosine base, exhibits higher efficiency than cap materials used in conventional mRNA vaccines and can be usefully utilized in the development of novel mRNA vaccines and various mRNA-based therapeutic agents.

[0026] Trinucleotide cap analogs containing the compound of the following chemical formula 1: [Chemical formula 1] [ka] In the compound of chemical formula 1, R1 is a methylene-bridged heterocyclic compound linked to the N7 position of the N7 guanosine base by a methylene bridge. In the compound of chemical formula 1, X1 and X2 are H or CH3, and X3, X4, and X5 are selected from the group consisting of O, S, and Se, respectively. R2 is a derivative containing benzyl, The heterocyclic compound contains one to two heteroatoms selected from O, N, and S, and is a functional group selected from saturated or partially saturated 5-membered to 10-membered heteroaromatic ring compounds. The aforementioned heteroaromatic ring compound is one functional group selected from the group consisting of furan, pyrrole, thiophene, imidazole, pyrazole, oxazole, isoxazole, thiazole, pyridine, pyrazine, pyrimidine, pyridazine, and triazine.

[0027] In the present invention, "heterocyclic compound" means a saturated or partially saturated 5- to 10-membered aromatic ring group containing at least one or two heteroatoms selected from O, N, and S. Specific examples of such heterocyclic compounds include thiophene, pyridyl, imidazole, tetrahydrofuranyl group, 2,3-dihydrofuranyl group, 2,5-dihydrofuranyl group, pyrrolidinyl group, 2,3-dihydropyrrolidinyl group, 2,5-dihydropyrrolidinyl group, tetrahydro-2H-pyranyl group, 3,4-dihydro-2H-pyranyl group, 4H-pyranyl group, piperidinyl group, 1,2,3,4-tetrahydropyridinyl group, 1,4-dihydropyridinyl group, piperazinyl, N-protected piperazinyl, and morpholino group. Common N-protecting groups for piperazinyl include alkyl groups, alkylcarbonyl groups, and alkylsulfonyl groups.

[0028] Trinucleotide cap analogs comprising compounds of the following chemical formulas [4-1], [4-2], or [4-3]: [Chemical formula 4-1] [ka] [Chemical formula 4-2] [ka] [Chemical formula 4-3] [ka] In the compounds of the aforementioned chemical formulas [4-1], [4-2], or [4-3], The aforementioned R1 is a compound containing a compound of chemical formula 5 linked at the N7 position by a methylene crosslink (-CH2-), The aforementioned X1 and X2 are H or CH3, respectively. The aforementioned Y1 is 0 or 1, The aforementioned Y2 is either O or S, The aforementioned Z is H or C1-C3 alkylbenzyl, [Chemical formula 5] [ka] In the above chemical formula 5, R2, R3, R4, R5, and R6 are each independently selected from the group consisting of H, OH, halo, methyl, alkyl, alkoxy, nitro, carboxyl, azide, amino, and cyano.

[0029] The aforementioned R1 may be benzyl or chlorobenzyl, but is not limited thereto.

[0030] Furthermore, Z may be methylbenzyl.

[0031] The substituents used to define the compounds according to the present invention will be described in more detail as follows:

[0032] In this invention, "halo" or "halogen atom" are interchangeable terms and refer to chloro, fluoro, bromo, and iodine.

[0033] In this invention, "alkyl" refers to a linear, branched, or cyclic aliphatic saturated hydrocarbon group having 1 to 5 carbon atoms. Specific examples of such alkyl groups include methyl, ethyl, n-propyl, isopropyl, and cyclopropyl groups.

[0034] In this invention, "aryl" refers to a monocyclic aromatic hydrocarbon group having six carbon atoms. Specific examples of such aryl groups include the phenyl group.

[0035] The present invention relates to a trinucleotide cap analog comprising the compound of the following chemical formula 1: [Chemical formula 1] [ka] In the above chemical formula 1, R1 is a heterocyclic group linked to the N7 position by a methylene bridge (-CH2-). Specifically, the present invention provides a trinucleotide cap analog (ThioCap1) in which R1 is methylene bridged thiophene, and a trinucleotide cap analog (PyCap1) in which R1 is methylene bridged pyridine.

[0036] The trinucleotide cap analog according to the present invention may be characterized by being represented by the following chemical formula 2 or chemical formula 3. [Chemical formula 2] [ka] [Chemical formula 3] [ka]

[0037] The trinucleotide cap analog according to the present invention is Thio-Cap1, 2-mTHP7 GpppA m pG) or Pyridyl Cap 1 (Py-Cap1, 2-mPy7 GpppA m (pG) It may be possible.

[0038] The present invention relates to a trinucleotide cap analog comprising a compound of the following chemical formulas [4-1], [4-2], or [4-3]: [Chemical formula 4-1] [ka] [Chemical formula 4-2] [ka] [Chemical formula 4-3] [ka] In the above chemical formula, R1 is a chemical formula 6 linked by a methylene bridge (-CH2-) at the N7 position. [Chemical formula 6]

Chemical

[0039] Also, in the above chemical formula, R1 is a chemical formula 7 linked by a methylene bridge (-CH2-) at the N7 position. [Chemical formula 7]

Chemical

[0040] The trinucleotide cap analog according to the present invention is benzyl-cap 1 (Bz-Cap1, Bn7 GpppA m pG), chlorobenzyl-cap 1 (ClBz-Cap1, 4-ClBn7 GpppA m pG), L-benzyl-cap 1 (LBz-Cap1, Bn7 LGpppA m pG), dibenzyl-cap 1 (DiBz-Cap1, Bn7 Gppp Bn6 A m pG), inosine-cap 1 (Ino-Cap1, Bn7 IpppA m pG), benzyl diphosphate-cap 1 (Bzdipho-Cap1, Bn7 GppA m pG), chlorobenzyl diphosphate-cap 1 (ClBzdipho-Cap1, 4-ClBn7 GppA m pG), benzyl thiophosphate 1-cap 1 (Bzthiopho1-Cap1, Bn7 Gpp spA m pG(D1), benzylthiophosphate 2-Cap1, Bn7 Gpp s pA m pG(D2)), chlorobenzylthiophosphate 1-Cap1 (ClBzthiopho1-Cap1, 4-ClBn7 Gpp s pA m pG(D1)) and chlorobenzyl diphosphate 2-cap1 (ClBzthiopho2-Cap1, 4-ClBn7 Gpp s pA m pG(D2)) is also acceptable.

[0041] Specifically, the cap analogs may be the compounds of chemical formulas 1 and [4-1] to [4-3] themselves, or they may further include hybridize sequences that are complementary to the sequence on the DNA template at the start site, in addition to the compounds of chemical formulas 1 and [4-1] to [4-3]. The length of the hybridize sequence of the cap analog used in the methods and compositions disclosed herein depends on several factors, including the properties (entities) of the template nucleotide sequence, the temperature at which the primer hybridizes with the DNA template, or the temperature used during in vitro transcription. The desired length of the hybridize nucleotide sequence of the cap analog for use in transcription can be readily determined by ordinary experiments by those skilled in the art. For example, the length of the hybridize nucleotide can be determined based on the desired hybridize specificity or selectivity.

[0042] 5'-end capped mRNA can be produced using the aforementioned cap analogue.

[0043] One aspect of the present invention provides a method for producing 5'-end capped mRNA, which includes introducing cap analogs containing the compounds of chemical formula 1 and chemical formulas [4-1] to [4-3] during the mRNA synthesis process.

[0044] Furthermore, one aspect of the present invention provides a composition for producing 5'-terminated capped mRNA having a cap analog containing the compounds of chemical formula 1 and chemical formulas [4-1] to [4-3].

[0045] Furthermore, one aspect of the present invention provides an application for using cap analogs containing the compounds of chemical formula 1 and chemical formulas [4-1] to [4-3] in mRNA synthesis.

[0046] Furthermore, one aspect of the present invention provides 5'-ended capped mRNA using cap analogs containing compounds of chemical formula 1 and chemical formulas [4-1] to [4-3].

[0047] Furthermore, the present invention provides a method for improving protein expression efficiency using the cap analogue, and a method for improving protein expression efficiency using a 5'-end capped mRNA structure with the cap analogue. In this case, the protein may be an antigen protein.

[0048] A method for producing 5'-end capped mRNA using cap analogs containing the compounds of chemical formula 1 and chemical formulas [4-1] to [4-3] can be carried out according to any method known in the art.

[0049] Furthermore, 5'-terminated capped mRNA according to one aspect of the present invention can be produced by a method of mRNA synthesis in which a chemically synthesized cap analog is simultaneously introduced during in vitro mRNA synthesis, and the cap analog constitutes the 5' terminus, i.e., a co-transcription capping method. Specifically, this method may include the steps of introducing a cap analog, which is a compound of chemical formula 1 and chemical formulas [4-1] to [4-3], into a mixture containing RNA polymerase under conditions in which transcription of a polynucleotide template by RNA polymerase is performed, and incubating the mixture for a time sufficient to allow transcription of the template.

[0050] The present invention provides a pharmaceutical composition for the expression of a target peptide or protein, comprising a 5'-terminated capped mRNA containing a trinucleotide cap analog and a pharmaceutically acceptable carrier.

[0051] Depending on the type of peptide or protein used, therapeutic or preventive effects for the target disease can be obtained in vivo. Therefore, it can be used to treat or prevent any disease that can be treated or prevented by the expression of a peptide or protein. Diseases that can be treated or prevented by the expression of specific peptides or proteins are well known, and the pharmaceutical composition can be used to induce the expression of such peptides or proteins, thereby enabling the prevention or treatment of the target disease.

[0052] Furthermore, one aspect of the present invention provides a pharmaceutical application in which mRNA capped at the 5' end with the cap analog is used to prevent or treat any disease in which the effect is obtained by the in vivo expression of a peptide or protein.

[0053] The aforementioned pharmaceutical composition and treatment or prevention method can be used in gene replacement therapy, genome editing, cancer immunotherapy, or treatment or prevention using vaccines. As a specific example, the pharmaceutical composition is an mRNA vaccine.

[0054] The pharmaceutical composition may be formulated for administration by injection or by other suitable routes known to those skilled in the art for the treatment or prevention of specific medical conditions. The injectable composition may include, for example, sterile saline as a pharmaceutically acceptable carrier. The injectable composition may also be formulated as a suspension in lipids or phospholipids, a liposomal suspension, or an aqueous emulsion. Methods for formulating the pharmaceutical composition are widely known to those skilled in the art.

[0055] As a specific example, the pharmaceutical composition may contain mRNA containing the cap analog, which is the active ingredient, at a concentration of approximately 0.01% to 1%. The concentration may vary depending on the frequency of administration, the amount administered, and the method of administration.

[0056] As a specific example, the pharmaceutical composition can be administered to mammals, particularly humans, and the dosage will vary depending on the individual's health condition, the severity of the disease, weight, age, race, etc., and an expert in the relevant technical field can determine the appropriate dosage. As a specific example, the dosage for humans is in the range of 0.0001 to 100 mg / day, and more specifically, in the range of approximately 0.1 to 50 mg / day.

[0057] mRNA capped at the 5' end with the cap analogue described above can be introduced into cells in vivo or in vitro to express proteins or peptides.

[0058] Methods for expressing proteins or peptides in cells in vivo or in vitro using the aforementioned mRNA are known in the art, and proteins or peptides can be expressed in cells as appropriate according to these conventional general methods.

[0059] The following examples illustrate the present invention. These examples are merely illustrative, and it will be obvious to those skilled in the art that various changes or modifications are possible within the scope of the invention and the technical idea, and these changes or modifications also fall within the scope of the appended claims. [Examples]

[0060] <Examples> CleanCap analogs substituted with A.N7-methylene-bridged heterocyclic compounds <Example 1> Synthesis of Trinucleotide Capped Material This invention relates to the synthesis of novel modified trinucleotide capping materials and their use as mRNA vaccines. In the trinucleotide structure of CleanCap, the N7 position of the 5' terminal guanosine base was modified with a methylene-bridged heterocyclic compound, more specifically with methylene-bridged thiophene, instead of the conventional methyl group. Novel compounds (ThioCap1) and methylene-bridged pyridine were then synthesized by organic chemical reactions (Figure 1).

[0061] <1-1> Reagent Information Using the Bruker AV-400 spectrometer, 1 H and 13 C, 31 3P NMR spectra were recorded using D2O or DMSO-d6 as the solvent, with 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, measuring the absorbance change immediately after irradiation of the sample solution in the cuvette with ultraviolet light. Fluorescence emission spectra were recorded at room temperature using a PF-65000 spectrofluorometer.

[0062] <1-2> Synthesis method of N7-(hetroaryl-2-ylmethyl)guanosine 5'-diphosphoroimidazolide analog 1) Synthesis of triethylammonium salt of guanosine 5'-diphosphate [ka] To synthesize the triethylammonium salt of guanosine 5'-diphosphate (GDP.TEA), a solution was obtained by stirring triethylamine (930 mg, 2 mmol) of guanosine 5'-phosphate (GMP.TEA) in anhydrous dimethyl sulfoxide (4 mL). Triethylamine (1.7 mL, 12 mmol), imidazole (817 mg, 12 mmol), and 2,2'-dithiodipyridine (1.3 g, 6 mmol) were added to this solution, and the mixture was stirred for 5 minutes. Then, triphenylphosphine (1.56 g, 6 mmol) was added to anhydrous dimethyl sulfoxide (4 mL), and stirring was continued at room temperature for 5 hours. After the reaction was complete, the mixture was slowly poured into a mixture of acetone (50 mL) and sodium perchlorate (0.5 g). After cooling at 4°C for 30 minutes, the mixture was centrifuged at 5,000 rpm for 8 minutes, and the supernatant was removed. To remove trace amounts of imidazole and triphenylphosphine, the solid was pulverized with fresh acetone (20 mL), the mixture was cooled, and then centrifuged again. This process was repeated one more time, and the precipitate was dried at room temperature in a vacuum oven.

[0063] The guanosine 5'-phosphoroidazolide (NaGMP-IM) obtained in this way was dissolved in dimethylformamide (10 mL), and this solution was added dropwise to a mixture obtained by stirring a 1 M solution of tributylammonium orthophosphate in dimethylformamide (5 mL) for 30 minutes. Finally, zinc chloride (200 mg, 1.5 mmol) was added, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was poured into water (50 mL) and extracted with chloroform (20 mL x 3). The resulting solution was concentrated using a rotary evaporator at a water bath temperature of 37 °C. The result was applied to an anion exchange resin for purification.

[0064] Chromatographic purification was performed using a weak anion exchange type Sepharose fast-flow resin containing a DEAE (DiethylAminoEthyl) group. The target compound was eluted using a CombiFlash EZ Prep at a flow rate of 3 mL / min in a 4-bed volume under a 0-30% concentration gradient using 1 M TEAB (triethylammonium bicarbonate) buffer (pH 7.5). The GDP.TEA-containing fraction was collected, concentrated in a rotary evaporator (tank temperature 37°C), and then dried in a freeze-dryer to obtain fine white powder GDP.TEA (951 mg, 64%). The NMR results for GDP.TEA are shown below.

[0065] GDP.TEA (951 mg, yield 64%) 1 H NMR(400MHz,D2O)δ=7.99(s,1H),5.81(d,J=6.0Hz,1H),4.63(t,J=5.2Hz,1H),4.51(t,J=5.6Hz,1H),4.22(m,1H),4.14(m,1H),4.05(m,1H); 31 PNMR(162MHz,D2O)δ=-6.47(d,J=23.5Hz,1P),-10.99(d,J=22.5Hz,1P).

[0066] 2) General synthesis process of N7-(hetroaryl-2-ylmethyl)guanosine 5'-diphosphate analog [ka] 0.5 mmol, 1 equivalent of the triethylamine salt of guanosine 5'-bisphosphate was dispersed in 3 mL of dimethyl sulfoxide (DMSO), and a suitable 2-bromomethyl heteroaromatic compound (2.5 mmol, 5 equivalents) was added. The mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was diluted with water (20 mL) and extracted with diethyl ether (3 × 10 mL). After the reaction was complete, the mixture was slowly poured into a solution of acetone (50 mL) mixed with sodium perchlorate (0.5 g). After cooling at 4°C for 30 minutes, the mixture was centrifuged at 5,000 rpm for 8 minutes, and the supernatant was removed. The solid was pulverized with fresh acetone (20 mL), the mixture was cooled, and then centrifuged again. This process was repeated one more time, and the resulting precipitate was vacuum-dried at room temperature. A total volume of 15-20 mL of the crude product solution was separated using Sepharose Fast Flow resin as described above. The fraction containing the product was collected, concentrated using a rotary evaporator at a water temperature of 37°C, then evaporated three times with ethanol, and dried in a freeze-dryer.

[0067] Following the method described above, N7-(thiophen-2-ylmethyl)guanosine 5'-diphosphate triethylammonium salt and N7-(pyridin-2-ylmethyl)guanosine 5'-diphosphate triethylammonium salt were synthesized using 2-bromomethylthiophene and 2-bromomethylpyridine, respectively, and the NMR results are shown below.

[0068] N7-(thiophen-2-ylmethyl)guanosine 5'-diphosphate triethylammonium salt (N7-2-mTHP-GDP.TEA): 155 mg, 42%. 1H NMR(400MHz,D2O)δ=7.36(d,J=5.2Hz,1H),7.27(d,J=3.6Hz,1H),6.96(t,J=5.2Hz,1H),5.94(d,J=3.6Hz,1H),5.80 (dd,J1=18.0Hz,J2=15.2Hz,2H),4.62(t,J=3.6Hz,1H),4.48(t,J=5.6Hz,1H),4.32-4.27(m,1H),4.26-4.14(m,2H); 31 P NMR(162MHz,D2O)δ=-8.34(d,J=22.4Hz,1P),-11.10(d,J=22.5Hz,1P).

[0069] N7-(pyridine-2-ylmethyl)guanosine 5'-diphosphate triethylammonium salt (N7-2mPy-GDP.TEA): 213 mg, 58%. 1 H NMR(400MHz,D2O)δ=8.35(d,J=5.6Hz,1H),7.70(td,J1=8Hz,J2=2Hz,1H),7.35-7.29(m,2H),6.04(d,J=3.2Hz,1H),5.75(d,J=5.2Hz,2H),4. 64(dd,J1=4.8Hz,J2=3.2Hz,1H),4.55(dd,J1=6.0Hz,J2=4.8Hz,1H),4.36(dd,J1=6.0Hz,J2=2.4Hz,1H),4.22(dd,J1=5.2Hz,J2=2.4Hz,1H); 31 P NMR(162MHz,D2O)δ=-6.34(d,J=22.5Hz,1P),-10.95(d,J=23.5Hz,1P).

[0070] 3) General synthesis process of N7-(hetroaryl-2-ylmethyl)guanosine 5'-diphosphoroimidazolide analog [ka] To a solution prepared by stirring triethylammonium salt of N7-(heteroaryl-2-ylmethyl)guanosine 5'-diphosphate (0.3 mmol) in anhydrous dimethyl sulfoxide (3 mL), triethylamine (251 μL, 1.8 mmol), imidazole (123 mg, 1.8 mmol), and 2,2'-dithiodipyridine (198 mg, 0.9 mmol) were added, and the mixture was stirred for 5 minutes. Then, triphenylphosphine (236 mg, 0.9 mmol) was added to anhydrous dimethyl sulfoxide (2 mL). The mixture was stirred at room temperature for 5 hours. After the reaction was complete, the mixture was slowly poured into a mixture of acetone (25 mL) and sodium perchlorate (250 mg). After cooling at 4 °C for 30 minutes, the mixture was centrifuged and the supernatant was removed. To remove trace amounts of imidazole and triphenylphosphine, the solid was pulverized with fresh acetone (10 mL), and the mixture was cooled and centrifuged again. This process was repeated one more time, and the precipitate was dried in a vacuum oven at room temperature using phosphorus pentoxide (P2O5).

[0071] According to the above method, N7-(thiophen-2-ylmethyl)guanosine 5'-diphosphate and N7-(pyridine-2-ylmethyl)guanosine 5'-diphosphate were used to prepare N7-(thiophen-2-ylmethyl)guanosine 5'-diphosphoruidazolide (N 7 -(thiophen-2-ylmethyl)guanosine 5'-diphosphoroimidazolide) and N7-(pyridine-2-ylmethyl)guanosine 5'-diphosphoroimidazolide (N 7 We synthesized (pyridin-2-ylmethyl)guanosine 5′-diphosphoroimidazolide, and its NMR spectrum data is shown below.

[0072] <N7-(チオフェン-2-イルメチル)グアノシン5’-ジホスホロイミダゾリド(N7-(thiophen-2-ylmethyl)guanosine 5′-diphosphoroimidazolide):N7-2-mTHP-GDP-IM> 1H NMR(400MHz,D2O)δ=7.99(s,1H),7.53(dd,J1=5.8Hz,J2=1.6Hz,1H),7.40(dd,J1=3.2Hz,J2=1.2Hz,1H),7.36(dd,J=1.6Hz,1H),7.12(dd,J1=5.2 Hz,J2=3.6Hz,1H),7.06(bs,1H),6.03(d,J=4.4Hz,1H),5.91(d,J=4.4Hz ,2H),4.69(t,J=4.0Hz,1H),4.45-4.38(m,2H),4.30(m,1H),4.17(m,1H); 31 P NMR(162MHz,D2O)δ=-11.70(d,J=24.5Hz,1P),-19.90(d,J=21.5Hz,1P).

[0073] <N7-(ピリジン-2-イルメチル)グアノシン5’-ジホスホロイミダゾリド(N7-(pyridin-2-ylmethyl)guanosine 5′-diphosphoroimidazolide):N7-2mPy-GDP-IM> 1 H NMR(400MHz,D2O)δ=8.35(d,J=7.6Hz,1H),7.83-7.77(m,2H),7.33(d,J=8.0Hz,2H),7.18(bs,1H),6.88(bs,1H),6.02(d,J=4.0Hz,1 H),5.69(dd,J1=22.0Hz,J2=15.6Hz,2H),4.60(t,J=4.8Hz,1H),4.35(t,J=5.6Hz,1H),4.32-4.27(m,1H),4.17(m,1H),4.04(m,1H); 31 P NMR(162MHz,D2O)δ=-11.84(d,J=20.6Hz,1P),-19.92(d,J=21.5Hz,1P).

[0074] <1-3> Synthesis method of ジヌクレオチドpAmpG

change

[0075] 1) 5'-O-DMT-2'-O-methyladenosyl(n-bz)-{3'-OP-[2-cyanoethyl]→5'}-2',3'-diacetylguanosine(n-ibu)3 (5'-O-DMT-2'-O-methyl Adenosylyl (n-bz)-{3'-OP-[2-cyanoethyl]→5'}-2',3'-diacetyl Guanosine(n-ibu)3) synthesis process DMT-2'-O-methyl adenosine (n-bz)CED phosphoramidite 1 (1.29 g, 1.45 mmol) and 2',3'-dicacetyl guanosine (n-ibu) 2 (0.58 g, 1.32 mmol) were dried under high vacuum for 16 hours. Acetonitrile (7.3 mL, 3.30 mmol) was mixed with anhydrous DMF (1.7 mL) and 0.45 M tetrazole, and stirred at room temperature under an argon (or nitrogen) atmosphere for 3 hours. After confirming the consumption of compound 1 by TLC (Thin Layer Chromatography), the mixture was cooled in an ice-salt bath for 30 minutes, and 70% tert-butyl hydroperoxide (0.91 mL, 6.60 mmol) was added to water. The reaction mixture was stirred at room temperature for 1 hour. The conversion of the intermediate was confirmed by TLC, and the mixture was extracted with ethyl acetate (200 mL x 2) and washed with 5% NaHCO3 solution (100 mL x 2). The extracted organic solution was dried in contact with sodium sulfate and evaporated to obtain crude solid 3 (1.77 g).

[0076] 2) 2'-O-methyladenosilyl(n-bz)-{3'-OP-[2-cyanoethyl]→5'}-2',3'-diacetylguanosine(n-ibu)4 (2′-O-Methyl Adenosylyl(n-bz)-{3′-OP-[2-cyanoethyl]→5′}-2′,3′-diacetyl Guanosine(n-ibu)4) synthesis process Crude dinucleotide 3 solid (1.77 g) was dissolved in 3% trichloroacetic acid in dichloromethane (31.2 mL, 5.72 mmol). The mixture was stirred at room temperature for 1 hour. After confirming the conversion of compound 3 by TLC, 5% NaHCO3 solution (30 mL) was added and the mixture was stirred for 10 minutes. After extraction with dichloromethane, the mixture was evaporated. The resulting residue was then purified by silica gel column chromatography to obtain product 4 (0.74 g, 0.79 mmol, 60%).

[0077] 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,2) H),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,162MHz):δ -2.43(d,1P).

[0078] 3) 5'-O-phosphoryl-2'-O-methyladenosilyl-{3'-OP→5'}-guanosine (pAmpG)5 (5′-O-Phosphoryl-2′-O-methyl Adenosylyl-{3′-OP→5′}Guanosine,(pAmpG)5) synthesis process Dinucleotide 4 (0.74 g, 0.7891 mmol) was dried under high vacuum for 16 hours. 0.45 M tetrazole (3.51 mL, 1.58 mmol) and bis-(2-cyanoethyl)-N,N-diisopropyl-phosphoramidite (0.41 mL, 1.58 mmol) were added to acetonitrile, and the mixture was stirred at room temperature under an argon (nitrogen) atmosphere for 3 hours. After confirming the consumption of compound 4, the reaction mixture was cooled in an ice-salt bath for 30 minutes, and 70% tert-butyl hydroperoxide in water (17.2 mL, 3.16 mmol) was added. The mixture was stirred at room temperature for 1 hour. After confirming the transformation of the intermediate by TLC, the mixture was extracted with ethyl acetate (200 mL x 2) and washed with 5% NaHCO3 solution (100 mL x 2). The extracted organic solutions were combined and evaporated. The resulting residue was dissolved in 25 mL of methanol and 25 mL of concentrated ammonia water and stirred at 55°C for 7 hours. After the deprotection reaction was complete, the reaction mixture was evaporated together with methanol. The resulting residue was dissolved in water, adjusted to pH 5.5, and then loaded onto a DEAE-Sepharose column. Solvent A: 1 M TEAB buffer (pH 7.5-8), solvent B: water, the fraction containing the product was collected and evaporated, then dried in a freeze-dryer to obtain a fine white powder 5 (446 mg, 0.4908 mmol, 62%) containing triethylammonium salt.

[0079] Dinucleotide (pAmpG)TEA salt 1 H NMR(D2O,400MHz):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,162MHz):δ 3.69(s,1P),-0.84(s,1P)

[0080] <1-4> General synthesis process of trinucleotide-based capping materials (ThioCap1 and PyCap1) into which an N7-methylene bridged heterocyclic group has been introduced. [ka] To a solution of DMSO (50 mM) containing pAmpG triethylammonium salt (0.07 mmol, 63 mg), N7-(heteroaryl-2-ylmethyl)guanosine 5'-diphosphoruidazolide disodium salt (3.00 equivalents, 0.2 mmol) and zinc chloride (20.0 equivalents) were added. After stirring at 37°C for 3 days, the reaction was stopped by adding 500 mM EDTA aqueous solution (pH 8.0) (EDTA:molar equivalent of ZnCl2 1.30 equivalents). The mixture was diluted with water and purified using DEAE Sepharose Fast Flow resin, followed by further purification using reverse-phase HPLC (Shimadzu Corporation; YMC-Actus Triart C8 column (for separation, 250 × 20.0 mm I.D.); 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 fraction containing the target compound was collected, concentrated, and lyophilized to obtain the target N7-(heteroaryl-2-ylmethyl)Cap1 analog as triethylammonium salt. The product was redissolved in methanol (2.00 mL). 190 mM NaClO4 was added to acetone (24.0 mL), and the resulting suspension was centrifuged (4,000 rpm, 20 min). The supernatant was removed, and the precipitate was resuspended in acetone. The suspension and centrifugation process was repeated 3-4 more times. The precipitate was dried under reduced pressure to obtain the target N7-(heteroaryl-2-ylmethyl)Cap1 analog as a sodium salt.

[0081] By the above method, N7-(thiophen-2-ylmethyl)guanosine 5'-diphosphorusmidazolide (N 7-(thiophen-2-ylmethyl)guanosine 5'-diphosphoroimidazolide) and N7-(pyridine-2-ylmethyl)guanosine 5'-diphosphoroimidazolide (N 7 Using (pyridin-2-ylmethyl)guanosine 5'-diphosphoroimidazolide) respectively, a trinucleotide-based capping material (ThioCap1( 2-mTHP7 GpppAmpG) and PyCap1( 2-mPy7 GpppAmpG)) was synthesized, and the NMR analysis results are shown below.

[0082] <Guanosine N7-(thiophen-2-ylmethyl)trinucleotide Cap1 analog> < 2-mTHP7 GpppAmpG> [ka]

[0083] 1 H NMR(400MHz,D2O)δ=9.29(s,1H),8.34(s,1H),8.11(s,1H),7.91(s,1H),7.29(d,J=5.2Hz, 1H),7.21(d,J=3.6Hz,1H),6.88(dd,J1=5.2Hz,J2=3.6Hz,1H),5.90(d,J=6.0Hz,1H),5.81 (d,J=4.4Hz,1H),5.78(d,J=6.0Hz,1H),5.70(s,2H),4.88(m,1H),4.76(t,J=5.2Hz,1H),4 .58(t,J=4.4Hz,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(162MHz,D2O)δ=-0.84(s,1P),-11.30(d,J=19.6Hz,1P),-11.51(d,J=18.6Hz,1P),-22.93(t,J=19.6Hz,1P).

[0084] <Guanosine N7-(pyridin-2-ylmethyl)trinucleotide Cap1 analog> < 2-mPy7 GpppAmpG> [ka]

[0085] 1 H NMR(400MHz,D2O)δ=8.38(s,1H),8.31(d,J=4.8Hz,1H),8.13(s,1H),7.90(s,1H),7.77(t,J=7. 6Hz,1H),7.40(d,J=8.0Hz,1H),7.28(t,J=5.6Hz,1H),5.95(d,J=6.0Hz,1H),5.93(d,J=4.0Hz, 1H),5.78(d,J=6.0Hz,1H),5.72(s,2H),4.88(m,1H),4.80(t,J=5.6Hz,1H),4.67(t,J=3.6Hz,1 H),4.50(t,J=4.8Hz,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(162MHz,D2O)δ=-0.82(s,1P),-11.29(d,J=19.6Hz,1P),-11.50(d,J=18.6Hz,1P),-22.81(t,J=18.6Hz,1P).

[0086] <Example 2> Introduction of trinucleotide capping material into mRNA and analysis of capping efficiency We analyzed whether the trinucleotide capping material produced in Example 1 could be recognized by T7 RNA polymerase with high efficiency during the transcription process and introduced into mRNA.

[0087] As a result, the transcription efficiency of the capping material was almost the same for CleanCap (Cap1 commercial) and ThioCap1. In terms of capping efficiency during the transcription process, it was confirmed that the developed ThioCap1 was recognized by T7 RNA polymerase and introduced into RNA with higher efficiency than ARCA or CleanCap (Cap1 commercial). While conventional ARCA showed a capping efficiency of approximately 50% and CleanCap (Cap1 commercial) showed approximately 86%, ThioCap1 showed a capping efficiency of over 92%, and PyCap1 also showed a capping efficiency of over 88% (see Figures 7 and 10). Since ARCA had an extremely low capping efficiency, comparative measurements of the expression efficiency of the antigen protein (luciferase) were not performed.

[0088] <Example 3> Expression analysis of antigen protein using a cap substance substituted with a methylene crosslinked heterocyclic compound We investigated the translation process of mRNA into which a capping substance was introduced, obtained by modifying the N7 position of the 5' terminal guanosine base with a methylene-bridged thiophene compound instead of the conventional methyl group. To confirm the expression level of the antigen protein, we used luciferase mRNA and analyzed the expression level of the antigen protein using HEK293 cells. Two types of luciferase mRNA were compared: mRNA using natural rNTPs and mRNA into which pseudouridine triphosphate was introduced instead of rUTP to avoid excessive immune responses. In both cases, mRNA with ThioCap1 introduced (Luc mRNA ThioCAP1) showed higher antigen protein expression than mRNA with CleanCap introduced (Luc mRNA CAP1), and similarly, mRNA with PyCap1 introduced (Luc mRNA PyCAP1) also showed higher expression efficiency than mRNA with CleanCap introduced (Luc mRNA CAP1) (see Figures 8 and 11).

[0089] 1) Preparation of the luciferase DNA template A linear double-stranded DNA (dsDNA) template containing the start codon and poly-A tail was prepared from a luciferase plasmid by PCR. The T7 promoter and Kozak sequence were added to the luciferase dsDNA template by sequential PCR and ultimately used for transcription. The primers used were: primary forward primer: 5'-ATG GAA GAC GCC AAA AAC ATA AAG-3' (SEQ ID NO: 1), secondary forward primer: TAA TAC GAC TCA CTA TAG GGC CAC CAT GGA AGA CGC CAA AAA CAT (SEQ ID NO: 2), and reverse primer: AAT CGC GCC TAG GCG CGC CCG TAC GGC TCT TC-3' (SEQ ID NO: 3).

[0090] 2) Preparation of luciferase mRNA The concentration of the luciferase template was measured using a nanodrop spectrophotometer. In each transcription reaction, 1 μL of template (100 ng) was placed in a microcentrifuge tube. Then, 1.5 μL of 10× 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 luciferase cap-dependent transcription (1 μL, 2.5 mM). The ratio of capping material was maintained four times higher than that of rGTP to obtain mRNA that was capped more than 80%. Therefore, 1 μL of capping material (10 mM) was added. RNase inhibitors (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, a stock was prepared at a concentration of 10 mM, and the derivative was added in place of rCTP and rUTP. After culturing the reaction mixture at 37°C for 1 hour, 1.8 μL of 10× DNase I buffer (10 mM Tris-HCl, 2.5 mM MgCl2, 0.5 mM CaCl2) and 1 μL of DNase I (2 U / μL) were added to the reaction, and the mixture was cultured at 37°C for a further 15 minutes to remove all DNA templates.

[0091] 3) Purification of transcribed mRNA To remove any potentially formed dsRNA and transcription byproducts, dsRNA was separated by electrophoresis. Cellulose (10 μL, 0.2 g / mL) was then added to the mRNA separated by electrophoresis, and the mixture was gently shaken for 10 minutes. Next, the mixture was transferred to an RNA purification column with ethanol and binding buffer supplied with the column, and finally centrifuged at 13,000 rpm for 1 minute. RNA preparation buffer and wash buffer were then continuously added to the column, and centrifuged again. Upon completion of this step, all proteins and unbound rNTPs had migrated into the suspension and were removed as a suspension. Finally, a new tube was attached, 10 μL of nuclease-free water was added to the column, and centrifuged at 13,000 rpm for 2 minutes. All cellulose-bound double-stranded DNA (dsDNA) remained in the column, and only pure single-stranded luciferase mRNA passed through the column. This sample was collected and stored at 70°C for further application.

[0092] 4) Analysis of the capping efficiency of transcribed mRNA Two μg of capped transcription mRNA was heated to 70°C with three μg of gDNA and RNase H buffer, then slowly cooled to 25°C for annealing. Next, the mixture was reacted at 37°C for one hour using thermally stable RNase H to cleave the long mRNA into a short sequence containing the capping material. The cleaved short sequence containing the capping material was subjected to electrophoresis using a 16% denatured PAGE gel, and the results were analyzed. Uncapped mRNA showed faster gel migration of the cleaved short sequence, while capped mRNA showed relatively slower gel migration of the cleaved short sequence.

[0093] 5) Cell culture and preparation for phenotypic infection HEK293 cells were cultured in 60 mL culture plates in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% FBS and 1% Pen / Streptol. The day before phenotypic infection, cells were isolated using trypsin and resuspended in fresh medium. Cells were seeded into 96-well plates. The following day, when the cells reached 70% confluence, the phenotypic infection mixture was prepared and phenotypic infection was performed.

[0094] 6) Quantification and transformation of mRNA The purified mRNA was quantified using a nanodrop spectrophotometer, and the transcription yield for each rNTP combination was compared. For cell infection, 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 at room temperature for a further 5 minutes. Finally, the transformation mixture was added to the cells and incubated for 24 hours.

[0095] 7) Analysis of luciferase expression After the culture period, the culture medium was removed and the cells were lysed with 50 μL of 1× lysis buffer. The cell lysates were transferred to a microcentrifuge tube. For luciferase luminescence analysis, 100 μL of luciferin substrate was taken from each sample in a 96-well white plate. For luciferase luminescence analysis, 100 μL of luciferin substrate was dispensed into 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.

[0096] As a result, we 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. While conventional ARCA shows a capping efficiency of approximately 50%, ThioCap1 shows a capping efficiency of approximately 90% or more, and PyCap1 shows a capping efficiency of approximately 88% or more (see Figures 7 and 10).

[0097] Furthermore, the translation process of mRNA into which the capping substance according to the present invention was introduced was confirmed. To confirm the expression level of the antigen protein, luciferase mRNA was used, and the expression level of the antigen protein was confirmed using HEK293 cells. For luciferase mRNA, two types were compared: mRNA prepared using natural rNTPs and mRNA prepared by introducing pseudouridine triphot instead of rUTP to avoid excessive immune responses.

[0098] As a result, it was confirmed that mRNA into which pseudouridine triphosphate was introduced to avoid excessive immunity showed higher antigen protein expression efficiency than mRNA using natural rNTPs.

[0099] Furthermore, compared to conventional mRNA with CleanCap (Cap1 commercial) introduced (Luc mRNA Cap1), the luciferase mRNA with ThioCap1 introduced in this invention (Luc mRNA ThioCap1) and the mRNA with PyCap1 introduced (Luc mRNA PyCap1) were confirmed to show higher antigen protein expression efficiency in both mRNA using rNTPs and mRNA containing pseudouridine instead of rUTP (see Figures 8 and 11).

[0100] CleanCap analogs substituted with B.N7-arylmethyl (benzyl (BzCap1)) <Example 1> Synthesis of Trinucleotide Capped Material The present invention This relates to the synthesis of novel modified trinucleotide capping materials and their use as mRNA vaccines. In the trinucleotide structure of CleanCap, the N7 position of the 5' terminal guanosine base is modified with arylmethyl, more specifically benzyl and 4-chlorobenzyl, instead of the conventional methyl group. 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) was synthesized by an organic chemical reaction (see Figure 12).

[0101] <1-1> Reagent Information Using the Bruker AV-400 spectrometer, 1 H and 13 C, 31 3P NMR spectra were recorded using D2O or DMSO-d6 as the solvent, with 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, measuring the absorbance change immediately after irradiation of the sample solution in the cuvette with ultraviolet light. Fluorescence emission spectra were recorded at room temperature using a PF-65000 spectrofluorometer.

[0102] <1-2> Synthesis method of N7-(arylmethyl)guanosine 5'-diphosphoroimidazolide analog 1) Synthesis of triethylammonium salt of guanosine 5'-diphosphate [ka]

[0103] To synthesize the triethylammonium salt of guanosine 5'-diphosphate (GDP.TEA), a solution was obtained by stirring triethylamine (930 mg, 2 mmol) of guanosine 5'-phosphate (GMP.TEA) in anhydrous dimethyl sulfoxide (4 mL). Triethylamine (1.7 mL, 12 mmol), imidazole (817 mg, 12 mmol), and 2,2'-dithiodipyridine (1.3 g, 6 mmol) were added to the solution, and the mixture was stirred for 5 minutes. Then, triphenylphosphine (1.56 g, 6 mmol) was added to anhydrous dimethyl sulfoxide (4 mL), and the mixture was stirred at room temperature for 5 hours. After the reaction was complete, the mixture was gradually poured into a mixture of acetone (50 mL) and sodium perchlorate (0.5 g). After cooling at 4°C for 30 minutes, the mixture was centrifuged at 5,000 rpm for 8 minutes, and the supernatant was removed. To remove trace amounts of imidazole and triphenylphosphine, the solid was pulverized with fresh acetone (20 mL), the mixture was cooled, and then centrifuged again. This process was repeated one more time, and the precipitate was dried at room temperature in a vacuum oven.

[0104] The thus obtained guanosine 5'-phosphorimidazolide (NaGMP-IM) was dissolved in dimethylformamide (10 mL). Separately, the solution was added dropwise to a mixture obtained by stirring a 1 M solution of tributylammonium orthophosphate in dimethylformamide (5 mL) for 30 minutes. Finally, zinc chloride (200 mg, 1.5 mmol) was added, and the reaction mixture was stirred at room temperature overnight. After pouring the reaction mixture into water (50 mL), it was extracted with chloroform (3 × 20 mL). The resulting solution was concentrated using a rotary evaporator (rotary evaporator) at a water bath temperature of 37°C. The resultant was applied to an anion exchange resin for purification.

[0105] Purification by chromatography was carried out using a weak anion exchange type Sepharose Fast Flow resin having a DEAE (DiethylAminoEthyl) group. The target compound was eluted with CombiFlash EZ Prep using a 4 bed volume at a flow rate of 3 mL / min under the condition of a concentration gradient of 0 to 30% with 1 M TEAB (triethylammonium bicarbonate) buffer (pH 7.5). The GDP.TEA-containing fractions were collected, concentrated with a rotary evaporator (rotary evaporator) (bath temperature 37°C), and then dried with a freeze dryer to obtain GDP.TEA as a fine white powder (951 mg, 64%). The NMR results of GDP.TEA are shown below.

[0106] GDP.TEA. (951 mg, 64%) 1 H NMR (400 MHz, D2O) δ = 7.99 (s, 1H), 5.81 (d, J = 6.0 Hz, 1H), 4.63 (t, J = 5.2 Hz, 1H), 4.51 (t, J = 5.6 Hz, 1H), 4.22 (m, 1H), 4.14 (m, 1H), 4.05 (m, 1H); 31 P NMR (162 MHz, D2O) δ = -6.47 (d, J = 23.5 Hz, 1P), -10.99 (d, J = 22.5 Hz, 1P).

[0107] 2) Synthesis of triethylammonium salt of guanosine 5'-diphosphate According to the method of Example <1-2>, using inosine monophosphate (IMP) and L-guanosine monophosphate (LGMP) respectively, the triethylammonium salt of inosine 5'-diphosphate (IDP.TEA, 830 mg, 57%) and the triethylammonium salt of L-guanosine 5'-diphosphate (LGDP.TEA, 892 mg, 60%) were synthesized, and their NMR results are shown below.

[0108] IDP.TEA. (830mg, 57%) 1 H NMR (400 MHz, D2O) δ = 8.41 (s, 1H), 8.13 (s, 1H), 6.05 (d, J = 5.2 Hz, 1H), 4.68 (overlapped with H2O, m, 1H), 4.54 (t, J = 4.0 Hz, 1H), 4.29 (bs, 1H), 4.21 - 4.07 (m, 2H); 31P NMR (162 MHz, D2O) δ = -7.47 (d, J = 22.5 Hz, 1P), -11.07 (d, J = 22.3 Hz, 1P).

[0109] LGDP.TEA. (892mg, 60%) 1 H NMR (400 MHz, D2O) δ = 8.08 (s, 1H), 5.89 (d, J = 5.6 Hz, 1H), 4.74 (t, J = 5.2 Hz, 1H), 4.59 (t, J = 4.0 Hz, 1H), 4.29 (m, 1H), 4.20 (m, 1H), 4.13 (m, 1H); 31P NMR (162 MHz, D2O) δ = -6.35 (d, J = 22.5 Hz, 1P), -10.90 (d, J = 22.4 Hz, 1P).

[0110] <1-3>Method for synthesizing N7-(aryl-2-ylmethyl)guanosine 5'-mono or diphosphate analog (N7-(aryl-2-ylmethyl)guanosine 5'-mono or diphosphate analog)

Chemical formula

[0111] According to the above method, using benzyl, 4-chlorobenzyl, and benzylinosine, respectively, N7-benzyl guanosine 5'-monophosphate triethylammonium salt, N7-4-chlorobenzyl guanosine 5'-monophosphate triethylammonium salt, N7-benzyl guanosine 5'-diphosphate triethylammonium salt, N7-benzyl guanosine 5'-diphosphate triethylammonium salt, and N7-benzyl inosine 5'-diphosphate triethylammonium salt. We synthesized triethylammonium salt and the NMR results are shown below.

[0112] N7-benzyl guanosine 5'-monophosphate triethylammonium salt: N7-Bn-GMP.TEA (270 mg, 82%). 1 H NMR(400MHz,D2O)δ=7.39(m,5H),6.05(d,J=4Hz,1H),5.64(s,2H),4.68(d,J=4.7Hz,1H),4.46(t,J=4.9Hz,1H),4.40(q,J=2.3Hz,1H),4.18-4.13(m,1H),4.06-4.01(m,1H); 31 P NMR(162MHz,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.

[0113] N7-4-chlorobenzyl guanosine 5'-monoahosphate triethylammonium salt: N7-ClBn-GMP.TEA (260 mg, 75%). 1 H NMR(400MHz,D2O)δ=7.28(q,J =8.1Hz,4H),6.03(d,J=3.4Hz,1H),5.58(s,1H),4.63(t,J=4.1Hz,1H),4.47(t,J=5.0Hz,1H),4.35(s,1H),4.12(d,J=12.2Hz,1H),4.01-3.97(m,1H); 31 P NMR(162MHz,D2O):δ=3.70(s,1P).

[0114] N7-benzyl guanosine 5'-diphosphate triethylammonium salt: N7-Bn-GDP.TEA (247mg, 67%). 1 H NMR(400MHz,D2O)δ=7.23-7.38(m,5H),5.99(d,J=3.6Hz,1H),5.64(d.d,J1=21.6Hz,J2=15.2Hz,2H),4.61(dd,J1=5.2Hz,J2=3.2Hz,1H),4.53(t,J=5.2Hz,1H),4.30(m,1H),4.18-4.24(m,2H); 31 P NMR(162MHz,D2O)δ=-6.48(d,J=23.5Hz,1P),-10.98(d,J=23.5Hz,1P).

[0115] N7-4-chlorobenzyl guanosine 5'-diphosphate triethylammonium salt: N7-4-Cl-Bn-GDP.TEA (272 mg, 71%). 1 H NMR(400MHz,D2O)δ=7.38(s,4H),6.02(d,J=4Hz,1H),5.62(s,2H),4.68(t,J=8Hz, 1H),4.52(t,J=8Hz,1H),4.38-4.34(m,1H),4.31-4.25(m,1H),4.24-4.18(m,1H); 31 P NMR(162MHz,D2O)δ=-8.88(d,J=21Hz,1P),-11.08(d,J=23Hz,1P).

[0116] N7-benzylinosine 5'-diphosphate triethylammonium salt: N7-Bn-IDP.TEA 1 H NMR(400MHz,D2O)δ=8.18(s,1H),7.40-7.27(m,5H),6.14(d,J=3.2Hz,1H),5.75(q,J =15.2Hz,2H),4.61(t,J=4.8Hz,1H),4.52(t,J=6.0Hz,1H),4.32(m,1H),4.24(m,2H); 31 P NMR(162MHz,D2O)δ=-6.35(d,J=23.5Hz,1P),-11.00(d,J=23.5Hz,1P).

[0117] <1-4>N7-(アリール-2-イルメチル)グアノシン5'-モノはジホスホロ-イミダゾリド analog (N7-(aryl-2-ylmethyl)guanosine Synthesis method of 5'-mono or diphosphoro-imidazolide analog)

change

[0118] According to the above method, N7-(benzyl-2-ylmethyl)guanosine 5'-diphosphate, N7-(4-chlorobenzyl-2-ylmethyl)guanosine 5'-diphosphate, and N7-(benzylinosine-2-ylmethyl)guanosine 5'-diphosphate were used to produce N7-benzylguanosine 5'-monophosphoroimidazolide, N7-4-chlorobenzylguanosine 5'-monophosphoroimidazolide, and N7-benzylguanosine 5'-diphosphoroimidazolide. We synthesized 5'-diphosphoroimidazolide, N7-4-Cl-benzyl guanosine 5'-diphosphoroimidazolide, and N7-benzyl inosine 5'-diphosphoroimidazolide, and their NMR results are shown below.

[0119] N7-benzyl guanosine 5'-monophosphoroimidazolide: N7-Bn-GMP-IM (378 mg, 93%) 1 H NMR(400MHz,D2O)=7.74(s,1H),7.39(br s,5H),7.05(d,J =1.2Hz,1H),6.86(s,1H),5.93(d,J=3.8Hz,1H),5.63(dd,J1=14.8Hz,J2=14.8Hz,2H),4.63(t ,J=4.1Hz,1H),4.32(t,J=5.0Hz,1H),4.28-4.26(m,1H),4.16-4.12(m,1H),4.07-4.01(m,1H); 31 P NMR(162MHz,D2O)=-8.02(s,1P).

[0120] Electrospray ionization mass spectrometry (ESI-MS): C 20 H22 N7O7P - Calculated value ([M]): m / z 503.41; Found value ([M-2]): 501.2.

[0121] N7-4-Chlorobenzylguanosine 5'-Monophosphoroimidazolide: N7-4-Cl-Bn-GMP-IM (392 mg, 91%) . 1 H NMR(400MHz,D2O)=7.76(s,1H),7.34(s,4H),7.05(s,1H),6.88(s,1H),5.92(d,J=3.4Hz,1H),5 .58(dd,J1=14.8Hz,J2=15Hz,2H),4.61(t,J=4.5Hz,1H),4.31-4.28(m,2H),4.13-4.04(m,2H); 31 P NMR(162MHz,D2O):=-8.04(s,1P).

[0122] Electrospray ionization mass spectrometry (ESI-MS: Electrospray ionization mass spectrometry): C 20 H 21 ClN7O7P-([M]) Calculated value (Calculated): m / z 537.85; Found value ([M-2]): 535.1.

[0123] N7-benzyl guanosine 5'-diphosphoroimidazolide: N7-Bn-GDP-IM (153 mg, 81%) 1H NMR(400MHz,D2O)δ=7.80(s,1H),7.24-7.41(bs,5H),7.19(s,1H),6.88(s,1H),5.97(d,J=4.0Hz,1H), 5.59(dd,J1=21.2Hz,J2=14.4Hz,2H),4.54(t,J=4.0Hz,1H),4.25-4.33(m,2H),3.99-4.20(m,2H);31P NMR(162MHz,D2O)δ=-11.76(d,J=21.5Hz,1P),-19.88(d,J=21.4Hz,1P).

[0124] N7-4-chlorobenzyl guanosine 5'-diphosphoroimidazolide: N7-4-Cl-Bn-GDP-IM 1H NMR(400MHz,D2O)δ=7.82(s,1H),7.32(bs,4H),7.20(bs,1H),6.89(bs,1H),5.97(d,J=4.0Hz,1H),5.54 (dd,J1=23.6Hz,J2=14.8Hz,2H),4.55(t,J=4.0Hz,1H),4.31(d,J=4.8Hz,2H),4.20(m,1H),4.06(m,1H); 31 P NMR(162MHz,D2O)δ=-11.72(d,J=25.6Hz,1P),-19.80(d,J=20.6Hz,1P).

[0125] <N7-ベンジルイノシン5’-ジホスホロイミダゾリド(N7-benzyl inosine 5’-diphosphoroimidazolide):N7-Bn-IDP-IM> 1 H NMR(400MHz,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= 3.2Hz,1H),5.66(q,J=14.8Hz,2H),4.35(t,J=4.0Hz,1H),4.31-4.25(m,2H),4.17(m,1H),4.03(m,1H); 31 P NMR(162MHz,D2O)δ=-11.76(d,J=29.3Hz,1P),-19.88(d,J=21.4Hz,1P).

[0126] <1-5> Methods for synthesizing dinucleotides and pAmpG 1) 5'-O-DMT-2'-O-methyladenosilyl(n-bz)-{3'-OP-[2-cyanoethyl]→5'}-2',3'-diacetylguanosine(n-ibu)3 (5′-O-DMT-2′-O-methyl Adenosylyl(n-bz)-{3′-OP-[2-cyanoethyl]→5′}-2′,3′-diacetyl Guanosine(n-ibu)3) synthesis process DMT-2'-O-methyladenosine (n-bz)CED phosphoramidite 1 (1.29 g, 1.45 mmol) and 2',3'-diacetylguanosine (n-ibu) 2 (0.58 g, 1.32 mmol) were dried under high vacuum for 16 hours. Anhydrous DMF (1.7 mL) and 0.45 M tetrazole were added to acetonitrile (7.3 mL, 3.30 mmol) and stirred at room temperature under an argon (or nitrogen) atmosphere for 3 hours. After confirming the consumption of compound 1 by TLC, the mixture was cooled in an ice-salt bath for 30 minutes, and 70% tert-butyl hydroperoxide (0.91 mL, 6.60 mmol) was added to water. The reaction mixture was stirred at room temperature for 1 hour. After confirming the transformation 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 contacted with sodium sulfate, dried, and evaporated to obtain crude solid 3 (1.77 g).

[0127] 2) 2'-O-methyladenosilyl(n-bz)-{3'-OP-[2-cyanoethyl]→5'}-2',3'-diacetylguanosine(n-ibu)4 (2′-O-Methyl Adenosylyl(n-bz)-{3′-OP-[2-cyanoethyl]→5′}-2′,3′-diacetyl Guanosine(n-ibu)4) synthesis process Crude dinucleotide 3 solid (1.77 g) was dissolved in 3% trichloroacetic acid in dichloromethane (31.2 mL, 5.72 mmol). The mixture was stirred at room temperature for 1 hour. After confirming the conversion of compound 3 by TLC, 5% NaHCO3 solution (30 mL) was added and the mixture was stirred for 10 minutes. After extraction with dichloromethane, the mixture was evaporated. The resulting residue was then purified by silica gel column chromatography to obtain product 4 (0.74 g, 0.79 mmol, 60%).

[0128] 11H NMR (DMSO, 400 MHz): 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 31P NMR (DMSO, 162 MHz): δ -2.43 (d, 1P).

[0129] 3) Synthesis process of 5'-O-phosphoryl-2'-O-methyladenosyl-{3'-OP→5'}-guanosine (pAmpG)5 (5'-O-Phosphoryl-2′-O-methyl Adenosylyl-{3′-OP→5′}Guanosine, (pAmpG)5) (5′-O-Phosphoryl-2′-O-methyl Adenosylyl-{3′-OP→5′} Guanosine, (pAmpG)5) Synthesis process Dinucleotide 4 (0.74 g, 0.7891 mmol) was dried under high vacuum for 16 hours. 0.45 M tetrazole (3.51 mL, 1.58 mmol) and bis-(2-cyanoethyl)-N,N-diisopropyl-phosphoramidite (0.41 mL, 1.58 mmol) were added to acetonitrile, and the mixture was stirred at room temperature under an argon (nitrogen) atmosphere for 3 hours. After confirming the consumption of compound 4, the reaction mixture was cooled in an ice-salt bath for 30 minutes, and 70% tert-butyl hydroperoxide in water (17.2 mL, 3.16 mmol) was added. The mixture was stirred at room temperature for 1 hour. After confirming the conversion of the intermediate by TLC, the mixture was extracted with ethyl acetate (200 mL x 2) and washed with 5% NaHCO3 solution (100 mL x 2). The extracted organic solutions were combined and evaporated. The resulting residue was dissolved in methanol (25 mL) and concentrated ammonia water (25 mL) and stirred at 55°C for 7 hours. After deprotection was complete, the reaction mixture was evaporated with methanol. The resulting residue was dissolved in water, adjusted to pH 5.5, and loaded onto a DEAE-Sepharose column. Solvent A: 1 M TEAB buffer (pH 7.5-8), solvent B: water, the fraction containing the product was collected and evaporated, then dried in a freeze-dryer to obtain a fine white powder 5 (446 mg, 0.4908 mmol, 62%) containing triethylammonium salt (Figure 14).

[0130] Dinucleotide (pAmpG)TEA salt 1 H NMR (D2O, 400MHz): 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,162MHz):δ3.69(s,1P),-0.84(s,1P)

[0131] Dinucleotide (pAmpG)imidazole sodium salt :pAmpG-IM.Na (378mg, 93%) 1 H NMR(400MHz,D2O)=8.10(d,J=6.84Hz,2H),7.89(s,1H),7.75(s,1H),7.07(s,1H),6.86(s,1H),5.96(d,J=5.4Hz,1H),5.80(d,J=5.24Hz,1H),4.85 - 4.81(m,1H),4.45(t,J=4.68Hz,1H),4.40(t,J=5.0Hz,1H),4.31 - 4.29(m,2H),4.14(s,2H)3.98-3.96(m,1H),3.91-3.87(m,1H)3.41(s,3H); 31 P NMR(162MHz,D2O)=-0.81(s,1P),-8.15(s,1P).

[0132] <1-6> General synthesis process of N7-(aryl-2-ylmethyl)guanosine 5′-(2-thiodiphosphate) [ka] DMSO (50 mM) with completely dried imidazole-modified N7 guanosine monophosphate ( Bn7 GMP-IM.Na) or 4-ClBn7 GMP-IM.Na (0.67 mmol) was added to dissolve it. Then, the dried thiophosphate TEA salt [(Et3NH)3PSO3 3-4.5 equivalents of 1 M DMSO were added and stirred at room temperature for 10 minutes. Then, zinc chloride (8 equivalents) and DMSO solution were added and stirring continued for 2 hours. The reaction mixture was then slowly poured into a cooled solution of sodium perchlorate (400 mg) and acetone (45 mL). After centrifugation, the supernatant was removed and the precipitate was resuspended in acetone. The suspension-centrifugation process was repeated 3-4 more times. The precipitate was dried with P2O5 in a vacuum dryer, then dissolved in water (15-20 mL), EDTA Na4 (1.5 equivalents) was added, and it was extracted with chloroform (3 × 50 mL). The organic layer was then removed, and the aqueous layer was purified using DEAE Sepharose Fast Flow resin as described above. The fraction containing the product was collected, concentrated in a rotary evaporator, and then dried in a freeze-dryer to obtain a fine powder.

[0133] < Bn7 GDPβS.TEA (320mg, 56%) 1 H NMR(400MHz,D2O)=7.40-7.36(m,5H),6.04(d,J=3.7Hz,1H),5.71(s,2H),4 .66(dd,J1=2.72Hz,J2=4.96Hz,2H),4.36-4.34(m,1H),4.28-4.27(m,2H); 31 P NMR(162MHz,D2O)=32.86(d,J=32.1Hz,1P),-11.88(d,J=33.0Hz,1P).

[0134] < 4-ClBn7 GDPβS.TEA (280mg, 47%) 1 H NMR(400MHz,D2O)=7.32(q,J=8.5Hz,4H),6.05(d,J=3.4Hz,1H),5.65(dd,J1=14.9Hz,J2=14. 9Hz,2H),4.74(t,J=4.4Hz,1H),4.66(d,J=5.1Hz,1H),4.38-4.33(m,2H),4.29-4.24(m,1H); 31 P NMR(162MHz,D2O)=33.33(d,J=32.2Hz,1P),-11.81(d,J=31.9Hz,1P).

[0135] <1-7> Synthesis method of N7-Arylmethyl Substituted CleanCap analog To a solution of dinucleotide pAmpG triethylammonium salt (0.07 mmol, 63 mg) dissolved in DMSO (50 mM), N7-(arylmethyl)guanosine 5′-diphosphoruidazolide disodium salt (16) (3.00 equivalents, 0.2 mmol) and zinc chloride (20.0 equivalents) were added. After stirring at 37°C for 3 days, a 500 mM aqueous EDTA solution (pH 8.0) (EDTA: 1.30 equivalents per molar amount of ZnCl2) was added to the reaction mixture. The mixture was diluted with water and purified using DEAE Sepharose fast-flow resin, followed by further purification using reverse-phase HPLC (apparatus: Shimadzu Corporation, column: YMC-Actus Triart C8 (Preparative, 250 × 20.0 mm ID)). The fraction containing the target compound was collected, concentrated, and lyophilized to obtain the target N7-arylmethyl-substituted CleanCap analog as the 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 removed, 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 an N7-arylmethyl-substituted CleanCap analog.

[0136] The NMR analysis results for N7-Bn-GpppAmpG (26 mg, 29%), an analog of N7-benzylCleanCap, are shown below. 1H NMR(400MHz,D2O)δ=8.19(s,1H),7.93(s,1H),7.77(s,1H),7.01-7.23(m,5H),5.75(d ,J=5.6Hz,1H),5.69(d,J=4.4Hz,1H),5.66(d,J=6.4Hz,1H),5.44(dd,J1=26.0Hz,J2=1 4.4Hz,2H),4.71-4.80(m,2H),4.38(t,J=4.4Hz,1H),4.34(t,J=6.0Hz,1H),4.29(t,J =5.2Hz,2H),4.24(t,J=4.4Hz,1H),4.14-4.21(m,3H),3.96-4.14(m,5H),3.25(s,3H); 31 P NMR(162MHz,D2O)δ=-0.86(s,1P),-11.23(d,J=19.4Hz,1P),-11.43(d,J=17.7Hz,1P),-22.70(t,J=17.7Hz,1P).

[0137] <1-8> General synthesis process of Guanosine N7-(aryl-2-ylmethyl)trinucleotide mRNA Cap1 analog

change

[0138] According to the above method, using N7-(benzyl-2-ylmethyl)guanosine 5′-diphosphoroimidazolide and N7-(4-chlorobenzyl-2-ylmethyl)guanosine 5′-diphosphoroimidazolide, guanosine N7-benzyl trinucleotide Cap1 ( Bn7 GpppAm pG), 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 (L-Guanosine N7), Adenosine N 6 - Dibenzyl trinucleotide Cap1 (adenosine N 6 -dibenzyl trinucleotide Cap1)( Bn7 Gppp Bn6 A m pG), Inosine N7-benzyl trinucleotide Cap1 Bn7 IpppA m We synthesized pG and the NMR analysis results are shown below.

[0139] <Guanosine N7-benzyl trinucleotide Cap1> Bn7 GpppA m pG)> [ka]

[0140] 11H 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 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).

[0141] <Guanosine N7-4-Chloro-benzyl trinucleotide Cap1 ( 4-ClBn7 GpppA m pG)>

Chemical formula

[0142] [[ID=第十九]] 1 1H NMR (400 MHz, D2O) δ = 8.34 (s, 1H), 8.09 (s, 1H), 7.90 (s, 1H), 7.17 (d, J = 8.8 Hz, 2H), 7.05 (d, J = 8.4 Hz, 2H), 5.89 (d, J = 5.2 Hz, 2H), 5.76 (d, J = 5.6 Hz, 1H), 5.45 (dd, J1 = 20.4 Hz, J2 = 14.8 Hz, 2H), 4.89 (m, 1H), 4.75 (t, J = 5.6 Hz, 1H), 4.62 (t, J = 4.4 Hz, 1H), 4.47 - 4.43 (m, 3H), 4.41 - 4.32 (m, 3H), 4.32 - 4.22 (m, 3H), 4.22 - 4.13 (m, 3H), 3.36 (s, 3H); 31P NMR(162MHz,D2O)δ=-0.82(s,1P),-11.19(d,J=19.6Hz,1P),-11.47(d,J=18.6Hz,1P),-22.83(t,J=19.4Hz,1P).

[0143] <L-グアノシンN7-ベンジルトリヌクレオチドCap1(L-Guanosine N7-benzyl trinucleotide Cap1)( Bn7 LGpppA m pG)>

change

[0144] 1 H NMR(400MHz,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=5.2Hz,1H),5.82(d,J=3.6Hz,1H),5.71(d,J= 6.0Hz,1H),5.46(dd,J1=22.4Hz,J2=14.8Hz,2H),4.79(m,1H),4.52(t,J=4.4 Hz,1H),4.41-4.34(m,3H),4.31-4.19(m,5H),4.18-4.03(m,5H),3.24(s,3H); 31 P NMR(162MHz,D2O)δ=-0.93(s,1P),-11.33(d,J=18.6Hz,1P),-11.54(d,J=17.5Hz,1P),-22.80(t,J=18.6Hz,1P).

[0145] <グアノシンN7,アデノシンN 6 -ジベンジルトリヌクレオチドCap1(Guanosine N 7 adenosine N 6 -dibenzyl trinucleotide Cap1)( Bn7 GpppBn6A m pG)>

change

[0146] 1 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 31P NMR (162 MHz, D2O) δ = -0.83 (s, 1P), -11.31 (d, J = 18.6 Hz, 1P), -11.51 (d, J = 17.6 Hz, 1P), -23.00 (t, J = 17.5 Hz, 1P).

[0147] <Inosine N7-benzyl trinucleotide Cap1( Bn7 IpppA m pG)>

Chemical formula

[0148] 1 1H NMR (400 MHz, D2O) δ = 8.31 (s, 1H), 8.21 (s, 1H), 8.03 (s, 1H), 7.84 (s, 1H), 7.29 - 7.22 (m, 2H), 7.17 - 7.08 (m, 3H), 6.07 (d, J = 4.0 Hz, 1H), 5.85 (d, J = 6.0 Hz, 1H), 5.72 (d, J = 6.0 Hz, 1H), 5.62 (bs, 2H), 4.75 (overlapped with H2O, m, 1H), 4.63 (t, J = 4.4 Hz, 1H), 4.44 (t, J = 5.9 Hz, 1H), 4.41 (t, J = 5.0 Hz, 1H), 4.38 - 4.31 (m, 3H), 4.31 - 4.22 (m, 2H), 4.22 - 4.06 (m, 6H), 3.30 (s, 3H); 31P NMR(162MHz,D2O)δ=-0.87(s,1P),-11.27(d,J=19.2Hz,1P),-11.54(d,J=18.7Hz,1P),-22.94(t,J=19.8Hz,1P).

[0149] <1-9> General synthesis process of N7-(aryl-2-ylmethyl)trinucleotide diphosphate Cap1 material (BzCap1) [ka] Dinucleotide (pA) in a dry DMSO solution under an inert atmosphere m To a solution containing pG) triethylammonium salt (0.073 mmol, 65 mg) Bn7 GMP-IM (0.219 mmol, 142 mg, 3 equivalents) or 4-Cl-Bn7 GMP-IM (0.219 mmol, 124 mg, 3 equivalents) was added. Zinc chloride (20.0 equivalents) dissolved in dried DMSO solution was added, and the mixture was stirred at 37°C for 3 days. Subsequently, aqueous EDTA solution (pH 7.5) (EDTA:molar equivalent of ZnCl2 1.30 equivalents) was added to the reaction mixture to stop the reaction. 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: 0.05 M ammonium acetate buffer with 33% CH3CN (linear gradient 0-80% B (70 min); flow rate, 10 mL / min; detection wavelength, 254 nm)). The fraction containing the target compound was collected, stored in a freeze-dryer, and confirmed by NMR analysis of the product.

[0150] <Guanosine N7-benzyl trinucleotide diphosphate Cap1> Bn7 GppA m pG)> [ka]

[0151] 1 1H NMR (400 MHz, D2O) = 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 31P NMR (162 MHz, D2O) = -0.79 (s, 1P), -11.09 (d, J = 21.2 Hz, 1P), -11.43 (d, J = 21.33 Hz, 1P).

[0152] <Guanosine N7-4-chlorobenzyl trinucleotide diphosphate Cap1 ( 4-ClBn7 GppA m pG)>

Chemistry

[0153] 1 1H NMR (400 MHz, D2O) = 9.29 (s, 1H), 8.22 (s, 1H), 8.06 (s, 1H), 7.86 (s, 1H), 7.14 (d, J = 8.3 Hz, 2H), 7.08 (d, J = 8.2 Hz, 2H), 5.83 - 5.82 (m, 2H), 5.72 (d, J = 5.6 Hz, 1H), 5.37 (dd, J1 = 12.0 Hz, J2 = 16.0 Hz, 2H), 4.53 - 4.52 (m, 1H), 4.38 (s, 4H), 4.30 - 4.26 (m, 5H), 4.12 (br s, 5H), 3.32 (s, 3H); 31P NMR(162MHz,D2O)=-0.83(s,1P),-11.07(d,J=21.1Hz,1P),-11.4341(d,J=21.0Hz,1P).

[0154] <1-10> Synthesis of N7-(aryl-2-ylmethyl)β-phosphorothioate trinucleotide Cap1 analog [ka] Dinucleotide (pA) in a dry DMSO solution under an inert atmosphere m To a solution containing pG-IM) sodium salt (0.075 mmol, 60 mg, 1 equivalent), Bn7 GDPβS.TEA (0.225 mmol, 192.14 mg, 3 equivalents) or 4-Cl-Bn7 GDPβS.TEA (0.225 mmol, 200 mg, 3 equivalents) was added. Zinc chloride (20.0 equivalents) dissolved in dry DMSO solution was added, and the mixture was stirred at 37°C for 3 days. Subsequently, an aqueous EDTA solution (pH 7.5) (EDTA:molar equivalent of ZnCl2 1.30 equivalents) was added to the reaction mixture to stop the reaction. 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, a solution containing 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 stereoisomers of the target compound (D1 and D2) were obtained and separated based on residence time during HPLC purification. The product with the longer residence time was designated as D2, and the product with the shorter residence time was designated as D1. The fractions containing the target compounds D1 and D2 were collected and stored in a freeze-dryer, and the product was confirmed by NMR analysis.

[0155] <Guanosine N7-benzyl β-phosphorothioate trinucleotide Cap1(Guanosine N7-benzyl β-phosphorothioate trinucleotide Cap1)( Bn7 GppspA m pG)(D1)>

Chem.

[0156] 1 1H NMR(400MHz,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 = 4.0Hz,2H),5.73(d,J = 8.0Hz,1H),5.47(dd,J1 = 16.0Hz,J2 = 12.0Hz,2H),4.88(br s,1H),4.76(t,J = 8.0Hz,1H),4.62(t,J = 4.0Hz,5H),4.46(t,J = 4.0Hz,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 31P NMR(162MHz,D2O)=29.68(d,J = 25.9Hz,1P), - 0.81(s,1P), - 12.30(t,J = 25.9Hz,2P).

[0157] < Bn7 GppspA m pG(D2)>

Chem.

[0158] 11H 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 = 4 Hz, 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 31P 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).

[0159] <Guanosine N7-4-Chloro-benzyl β-phosphorothioate trinucleotide Cap1 ( 4-ClBn7 GppspA m pG)(D1)>

Chemical formula

[0160] 1 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); 31P NMR(162MHz,D2O)=29.54(t,J=62.0Hz,1P),-0.75(s,1P),-12.22(t,2P,J=22.9Hz,2P).

[0161] < 4-ClBn7 GppspA m pG(D2)> [ka]

[0162] 1 H NMR(400MHz,D2O)=9.42(s,1H),8.37(s,1H),8.08(s,1H),7.90(s,1H)7.19(d,J=7.6Hz,2H),7.08(d,J=7.6Hz,2H),5.90-5.87(m,2H),5.77(br s,1H),5.59(dd,J1=13.4Hz,J2=15.0Hz,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); 31 P NMR(162MHz,D2O)=29.75(t,J=25.3Hz,1P),-0.86(s,1P),-11.92(d,J=25.3Hz,1P)and-12.29(d,J=24.6Hz,1P).

[0163] <Example 2> Introduction of trinucleotide capping material into mRNA and analysis of capping efficiency We analyzed whether the trinucleotide capping material produced in Example 1 could be recognized by T7 RNA polymerase with high efficiency during the transcription process and introduced into mRNA.

[0164] As a result, it was confirmed that mRNA into which a cap substance was introduced, in which the guanosine N7 position at the 5′ end was modified with an arylmethyl compound instead of the conventional methyl compound, exhibited antigen protein expression levels during translation that were nearly equivalent to or higher than those of the conventional CleanCap (Cap1 commercial) and previously reported ARCA caps.

[0165] Furthermore, it was confirmed that the capping material was recognized by T7 RNA polymerase with higher efficiency than ARCA during the transcription process and could be introduced into RNA. While conventional ARCA showed a capping efficiency of approximately 50% and CleanCap (Cap1 commercial) showed approximately 85%, BzCap1 and BzCap analogs were found to show a relatively higher capping efficiency of over 90% (see Figure 45).

[0166] <Example 3> Antigen protein expression analysis using a cap substance substituted with an arylmethyl group We investigated the translation process of mRNA into which a cap substance was introduced, prepared by modifying the N7 position of the 5' end guanosine base with an arylmethyl compound instead of the conventional methyl group. To confirm the degree of antigen protein expression, we used luciferase mRNA and analyzed the expression level of the antigen protein using HEK293 cells. In the case of luciferase mRNA, it was confirmed that the antigen protein expression efficiency of BzCap1 and BzCap analogs was more efficient than that of CleanCap in mRNA using natural rNTPs (see Figure 46).

[0167] 1) Preparation of the luciferase DNA template A linear double-stranded DNA (dsDNA) template containing the start codon and poly-A tail was prepared from a luciferase plasmid by PCR. The T7 promoter and Kozak sequence were added to the luciferase dsDNA template by sequential PCR and ultimately used for transcription. The primers used were: primary forward primer: 5'-ATG GAA GAC GCC AAA AAC ATA AAG-3' (SEQ ID NO: 1), secondary forward primer: TAA TAC GAC TCA CTA TAG GGC CAC CAT GGA AGA CGC CAA AAA CAT (SEQ ID NO: 2); reverse primer: AAT CGC GCC TAG GCG CGC CCG TAC GGC TCT TC-3' (SEQ ID NO: 3).

[0168] 2) Preparation of luciferase mRNA The concentration of the luciferase template was measured using a nanodrop spectrophotometer. In each transcription reaction, 1 μL of template (100 ng) 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 luciferase cap-dependent transcription (1 μL, 2.5 mM). The ratio of capping material was maintained four times higher than that of rGTP to obtain mRNA that was capped more than 80%. Therefore, 1 μL of capping material (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, a stock was prepared at a concentration of 10 mM, and the derivative was added in place of rCTP and rUTP. After culturing the reaction mixture at 37°C for 1 hour, 1.8 μL of 10× DNase I buffer (10 mM Tris-HCl, 2.5 mM MgCl2, 0.5 mM CaCl2) and 1 μL of DNase I (2 U / μL) were added to the reaction, and the mixture was cultured at 37°C for a further 15 minutes to remove all DNA templates.

[0169] 3) Purification of transcribed mRNA To remove any potentially formed dsRNA and transcription byproducts, cellulose (10 μL, 0.2 g / mL) was added to the reaction and gently shaken for 10 minutes. The mixture was then transferred to an RNA purification column with ethanol and binding buffer supplied with the column, and finally centrifuged at 13,000 rpm for 1 minute. RNA preparation buffer and wash buffer were then continuously added to the column and centrifuged again. Upon completion of this step, all proteins and unbound rNTPs had migrated into the suspension and were removed as a suspension. Finally, a new tube was fitted, 10 μL of nuclease-free water was added to the column, and centrifuged at 13,000 rpm for 2 minutes. All double-stranded DNA (dsDNA) bound to the cellulose remained in 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 application.

[0170] 4) Capping efficiency analysis of transcribed mRNA Two μg of capped transcription mRNA was heated to 70°C with three μg of gDNA and RNase H buffer, then cooled to 25°C for annealing. Next, the mixture was reacted at 37°C for one hour using thermally stable RNase H to cleave the long mRNA into a short sequence containing the capping material. The cleaved short sequences containing the capping material were subjected to electrophoresis using a 16% denatured PAGE gel, and the results were analyzed. Uncapped mRNA showed faster gel migration of the cleaved short sequences, while capped mRNA showed relatively slower gel migration of the cleaved short sequences.

[0171] 5) Cell culture and preparation for phenotypic infection HEK293 cells were cultured in 60 mL culture plates in DMEM containing 10% FBS and 1% pen / strip. The day before phenotypic infection, cells were isolated using trypsin and resuspended in fresh medium. Cells were seeded into 96-well plates. The following day, when the cells reached 70% confluence, the phenotypic infection mixture was prepared and phenotypic infection was performed.

[0172] 6) Quantification and transformation of mRNA The purified mRNA was quantified using a nanodrop spectrophotometer, and the transcription yield for each rNTP combination was compared. For cell infection, 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 cultured at room temperature for 10 minutes. The mRNA and lipofectamine mixture was then transferred to the same tube and cultured for a further 5 minutes at room temperature. Finally, the transformation mixture was added to the cells and cultured for 24 hours.

[0173] 7) Analysis of luciferase expression After the culture period, the culture medium was removed and the cells were lysed with 50 μL of 1× lysis buffer. The cell lysates were transferred to a microcentrifuge tube. For luciferase luminescence analysis, 100 μL of luciferin substrate was taken from each sample in a 96-well white plate. Then, 20 μL of each cell lysate sample was added and mixed by pipetting. Finally, the luciferase luminescence was recorded using a luminometer.

[0174] As a result, it was confirmed that the capping materials into which various types of arylmethyl according to the present invention were introduced were recognized by T7 RNA polymerase with higher efficiency than ARCA or CleanCap during the transcription process and could be introduced into RNA. Here, while conventional ARCA showed a capping efficiency of approximately 50% and CleanCap showed approximately 89%, the trinucleotide capping materials into which various arylmethyl were introduced showed higher efficiency. Bn7 GpppA m pG, Bn7 GppA m pG, 4-ClBn7GppA 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 At pG, it was confirmed that the capping efficiency was over 90% (see Figure 45).

[0175] Furthermore, we confirmed the translation process of mRNA into which a capping substance was introduced, in which the N7 position of the guanosine base at the 5′ end was modified with an arylmethyl compound instead of the conventional methyl group.

[0176] To confirm the level of antigen protein expression, luciferase mRNA was used, and the expression level of the antigen protein was also confirmed using HEK293 cells. In the case of luciferase mRNA, mRNA derived from natural rNTPs was used.

[0177] Furthermore, the trinucleotide capping material developed in this invention is superior to mRNA with conventional ARCA caps (cap0) and CleanCaps (cap1 commercial) introduced. Bn7 GpppA m pG, Bn7 GppAmpG, 4-ClBn7 GppA m pG, Bn7 GpppBn6A m pG, 4-ClBn7 GpppA m pG, Bn7 GppspA m pG(D1), Bn7 GppspAm pG(D2), 4-ClBn7 GppspAmpG(D1), 4-ClBn7 GppspA m pG, Bn7 IpppA m pG and Bn7 LGpppA m It was confirmed that luciferase mRNA with pG introduced showed higher antigen protein expression efficiency (see Figure 46). [Industrial applicability]

[0178] This invention relates to novel modified trinucleotide capping derivatives and mRNA into which the capping derivatives have been introduced. The derivatives according to this invention, in which a methylene-bridged heterocyclic compound and an arylmethyl group are introduced at the N7 position of the 5′ terminal guanosine base, respectively, provide derivatives that exhibit higher efficiency than capping materials used in conventional mRNA vaccines. The novel modified trinucleotide capping derivatives and mRNA into which the capping derivatives have been introduced according to this invention can be usefully used in the development of new mRNA vaccines and various mRNA-based therapeutic agents, and have diverse applications in industrial processes and other fields.

[0179] Therefore, the modified trinucleotide capping derivative and the mRNA into which the capping derivative has been introduced according to one embodiment of the present invention can be said to have industrial applicability.

Claims

1. Trinucleotide cap analogs containing the compound of chemical formula 1 below: [Chemical formula 1] 【Chemistry 1】 In the compound of chemical formula 1, R １ It is a heterocyclic compound in which the N7 position of an N7 guanosine base is linked by a methylene bridge. In the compound of chemical formula 1, X １ and X ２ is H or CH ３ X ３ , X ４ and X ５ These are selected from the group consisting of O, S, and Se, respectively. R ２ It is a derivative containing benzyl, The heterocyclic compound contains one to two heteroatoms selected from O, N, and S, and is a functional group selected from saturated or partially saturated 5-membered to 10-membered heteroaromatic ring compounds. The aforementioned heteroaromatic ring compound is one functional group selected from the group consisting of furan, pyrrole, thiophene, imidazole, pyrazole, oxazole, isoxazole, thiazole, pyridine, pyrazine, pyrimidine, pyridazine, and triazine.

2. The trinucleotide cap analog described above is the trinucleotide cap analog according to claim 1, represented by the following chemical formula 2 or chemical formula 3. [Chemical formula 2] 【Chemistry 2】 [Chemical formula 3] 【Transformation 3】

3. mRNA capped at the 5' end with the trinucleotide cap analog described in claim 1.

4. A composition for producing 5'-end capped mRNA, comprising the trinucleotide cap analog described in claim 1.

5. A pharmaceutical composition for the expression of a target peptide or protein, comprising a 5'-terminated capped mRNA containing the trinucleotide cap analog described in claim 1 and a pharmaceutically acceptable carrier.

6. Trinucleotide cap analogs comprising compounds of the following chemical formulas [4-1], [4-2], or [4-3]: [Chemical formula 4-1] 【Chemistry 4】 [Chemical formula 4-2] 【Transformation 5】 [Chemical formula 4-3] 【Transformation 6】 In the compounds of the aforementioned chemical formulas [4-1], [4-2], or [4-3], Said R １ is a compound containing a compound of Chemical Formula 5 linked by a methylene bridge (—CH ２ —) at the N7 position, The aforementioned X １ and X ２ These are H or CH, respectively. ３ And, The aforementioned Y １ is 0 or 1, The aforementioned Y ２ is either O or S, The aforementioned Z is H or C １ -C ３ It is alkylbenzyl, [Chemical formula 5] 【Transformation 7】 In the aforementioned chemical formula 5, R ２ , R ３ , R ４ , R ５ and R ６ Each of these is independently one functional group selected from the group consisting of H, OH, halo, methyl, alkyl, alkoxy, nitro, carboxyl, azide, amino, and cyano.

7. The aforementioned R １ The trinucleotide cap analog according to claim 6, wherein is benzyl or chlorobenzyl.

8. The trinucleotide cap analog according to claim 6, wherein Z is methylbenzyl.

9. A trinucleotide cap analog of the mRNA with a 5' end capped according to claim 6.

10. A composition for producing 5'-terminated mRNA, comprising the trinucleotide cap analog described in claim 6.

11. A pharmaceutical composition for the expression of a target peptide or protein, comprising a 5'-terminated capped mRNA containing the trinucleotide cap analog described in claim 6 and a pharmaceutically acceptable carrier.

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