Novel mrna5' - terminal cap analogs, RNA molecules incorporating same, uses thereof, and methods of synthesizing RNA molecules or peptides

Abstract

The problem to be solved by the present invention is to provide new mRNA5' terminal cap analogs which will allow more efficient transcription of mRNAs capped therewith and higher expression levels of proteins encoded by such mRNAs than those obtained with mRNA5' terminal cap analogs of the prior art.SOLUTION: The present invention relates to new mRNA5' terminal cap analogs, RNA molecules containing them, their use, and methods for their in vitro synthesis, as well as methods for the synthesis of proteins or peptides in vitro or in cell cultures, including the translation of RNA molecules.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to novel mRNA 5'-end cap analogs, RNA molecules incorporating same, their uses, and methods for synthesizing RNA molecules in vitro, as well as methods for synthesizing proteins or peptides in vitro or in cells, which methods include translating the RNA molecules. [Background technology]

[0002] The 7-methylguanosine cap (m) present at the 5' end of eukaryotic mRNA 7 The 5' cap (G) plays a crucial role in many fundamental cellular processes, primarily by protecting mRNA from premature degradation and by serving as a molecular marker for proteins involved in mRNA transport and translation. [1] Therefore, chemical 5' cap modification paves the way for the design of molecular tools to selectively modulate cap-dependent processes and, consequently, mRNA metabolism. [2] The presence of a 5' cap is necessary under normal conditions to regulate mRNA and its efficient translation. Chemically synthesized m 7 The GpppG capped mRNA analog is used as a reagent for in vitro transcription of capped mRNA.[3]

[0003] In vitro transcribed (IVT) 5'-capped mRNA is a useful tool for studying mRNA translation, transport, and processing and represents an emerging class of promising therapeutic molecules. IVT mRNA finds application in the expression of proteins in eukaryotic cells, extracts, cell cultures, and even whole organisms. Finally, IVT RNA has attracted much attention in recent years as a tool for the safe delivery of exogenous proteins for anticancer vaccination, antiviral vaccination, and gene replacement therapy [4].

[0004] Synthesis of 5'-capped mRNA using cap analogs can be achieved by in vitro transcription. [3] This method, called co-transcriptional capping, involves the use of all four ribonucleoside 5'-triphosphates (NTPs: ATP, GTP, CTP, UTP) and m 7 RNA synthesis is carried out by RNA polymerase on a DNA matrix in the presence of a dinucleotide cap such as GpppG. The DNA template is designed so that the first transcribed nucleotide is G. The polymerase reacts with GTP or m 7 GpppG initiates transcription, thereby incorporating a single nucleotide into the 5' end of the resulting RNA. To improve the rate of incorporation of the cap analog (capping efficiency), the GTP concentration is lowered relative to other NTPs and the dinucleotide cap concentration is increased (4-10 times higher than GTP). Unfortunately, even with a high excess of dinucleotide cap over GTP, capping efficiency is less than 100%, rarely exceeding 90%. Uncapped mRNAs are significantly less stable and translationally active than capped mRNAs. Furthermore, they may induce unwanted immune responses in cells and reduce translation efficiency even for capped mRNAs

[11] . One method for removing uncapped mRNAs is to treat the post-transcription mixture with appropriate enzymes (e.g., a mixture of 5'-polyphosphatase and 5'-exonuclease) that degrade uncapped RNA and leave capped mRNA intact. Another limitation of the dinucleotide-based mRNA capping method is the incorporation of the dinucleotide cap back into the translationally inactive Gpppm mRNA. 7 The problem was solved by the discovery of "anti-reverse cap analogs" (ARCAs), which modify the 2' or 3' position of 7-methylguanosine (usually by replacing one of the OH groups with an OCH3 group) to prevent reverse incorporation. [5, 6]

[0005] Co-transcriptional capping has been shown to enable the incorporation of various modified cap structures at the 5' end of mRNA. These modified cap structures can serve as molecular beacons or confer new properties to mRNA molecules, such as improved translation efficiency and stability. Among other things, preferred cap analogs are those modified at the triphosphate bridge. [7] Substitution of even a single atom in the 5',5'-triphosphate bridge has been shown to significantly affect mRNA properties. For example, substitution of a single atom at the β position of the cap oligophosphate bridge, designated β-S-ARCA, significantly improved mRNA translation efficiency in vitro and in vivo. [8, 9] Meanwhile, substitution of a single O atom with a CH2 group at the α-β position reduced translation efficiency.

[10] These dramatic differences in the biological effects of different single-atom substitutions within the cap indicate the high sensitivity of the translation machinery to modifications of the oligophosphate chain, suggesting this is an important area for further exploration. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Moore M, "From birth to death: The complex lives of eukaryotic mRNAs", Science, 2005, 309(5740), pp. 1514-1518. [Non-patent document 2] Ziemniak M, Strenkowska M, Kowalska J, Jemielity J, "Potential therapeutic applications of RNA cap analogs", Future Medicinal Chemistry, 2013, 5 (10), pp. 1141-1172 [Non-patent document 3] Grudzien-Nogalska E, Stepinski J, Jemielity J, Zuberek J, Stolarski R, Rhoads RE, Darzynkiewicz E, Synthesis of anti-reverse cap analogs (ARCAs) and their applications in mRNA translation and stability. High-Throughput Methods, and Chemical-Based Approaches

Outdoor Tools 4

Direct Environment 5

Outdoor Configuration6

Outdoor Tools 10

Outdoor Content11

Outdoor Tools 12

[0007] The object of the present invention is to provide new mRNA 5'-end cap analogs that will enable the transcription efficiency of mRNAs capped with them to be increased and the expression level of proteins encoded by such mRNAs to be increased compared to mRNAs obtained using prior art mRNA 5'-end cap analogs. A particular object of the present invention is to provide new mRNA 5'-end cap analogs that will enable the easy separation of capped and uncapped mRNAs present in an in vitro reaction mixture without the need for enzymatic treatment. Unexpectedly, this invention has solved the above-defined problems. [Means for solving the problem]

[0008] The subject of the present invention is a compound of the formula:

[0009] [ka]

[0010] (In the formula, R 1 , R 2 , R 3 , R 4 is selected from the group consisting of H, CH3, and alkyl, and the substituents R with different numbers may be the same or different; n is 0 or 1, R 5 is benzyl, substituted benzyl, in particular mono- or di-substituted, preferably with a substituent selected from the group consisting of chlorine, fluorine, bromine, iodine, methyl, alkyl, nitro, carboxyl, azido, amino, hydroxyl, or any combination thereof, wherein the benzyl is selected from the group consisting of substituted benzyl, (1-naphthylmethyl), (2-naphthylmethyl), substituted naphthylmethyl, alkylaryl, aryl, which may be ortho-, meta-, or para-substituted; Base 1 and base 2 are

[0011] [ka]

[0012] are independently selected from the group comprising: X 1 , X 3 is selected from the group consisting of O, S, Se, and the substituents X with different numbers may be the same or different; X 2 , X 4 , X 5 is selected from the group consisting of O, S, Se, BH3, and X substituents with different numbers may be the same or different; X 6 is selected from the group comprising O, CH2, CF2, CCl2, Preferably, R 5 is benzyl, monosubstituted benzyl, disubstituted benzyl, 1-methylnaphthyl, or 2-methylnaphthyl; X1 , X 4 , X 5 , X 6 is O and X 2 and X 3 is O or S, and R 3 , R 4 is H) is a compound of

[0013] In a preferred embodiment, R 5 is selected from the group consisting of benzyl, chlorobenzyl, fluorobenzyl, bromobenzyl, iodobenzyl, methylbenzyl, alkylbenzyl, nitrobenzyl, carboxybenzyl, azidobenzyl, aminobenzyl, hydroxybenzyl, and difluorobenzyl.

[0014] Preferably, the compound is formula:

[0015] [ka]

[0016] Compound m 7 Gppp Bn6 A m p.g., formula:

[0017] [ka]

[0018] Compound m 7 Gppp 2MeBn6 A m p.g., formula:

[0019] [ka]

[0020] Compound m 7 Gppp 3Me Bn 6 Am p.g., formula:

[0021] [ka]

[0022] Compound m 7 Gppp 4MeBn6 A m p.g., formula:

[0023] [ka]

[0024] Compound m 7 Gppp 4FBn6 A m p.g., formula:

[0025] [ka]

[0026] Compound m 7 Gppp 3,4diFBn6 A m p.g., formula:

[0027] [ka]

[0028] Compound m 7 Gppp 1Naphm6 A m p.g., formula:

[0029] [ka]

[0030] Compound m 7 Gppp 2Naphm6 Am p.g., formula:

[0031] [ka]

[0032] Compound m 7 Gpp S p Bn6 A m p.g., formula:

[0033] [ka]

[0034] Compound m 7 Gppp 5'S,Bn6 A m p.g., formula:

[0035] [ka]

[0036] Compound m 7 Gppp Bn6 A m pGpG was selected from the group including

[0037] Preferably, the compounds according to the invention consist essentially of a single stereoisomer or comprise a mixture of at least two stereoisomers, a first diastereoisomer and a second diastereoisomer, which diastereoisomers are identical except that they have different stereochemical configurations around a stereogenic phosphorus atom, which is bonded to a sulfur atom, a selenium atom, or a borane group.

[0038] Another embodiment of the present invention is an RNA molecule containing at its 5' end a compound according to the invention as defined above.

[0039] A further embodiment of the present invention is a method for the in vitro synthesis of an RNA molecule according to the invention as defined above, comprising the step of reacting ATP, CTP, UTP and GTP, a compound according to the invention as defined above, and a polynucleotide template in the presence of an RNA polymerase under conditions that allow the RNA polymerase to synthesize RNA copies on the polynucleotide template, some of the RNA copies containing the compound according to the invention, such that an RNA molecule according to the invention is produced.

[0040] Another embodiment of the invention is a method for synthesizing a protein or peptide in vitro, comprising translating an RNA molecule according to the invention in a cell-free protein synthesis system, wherein the RNA molecule comprises an open reading frame under conditions that allow translation from the open reading frame of the RNA molecule to form the protein or peptide encoded by the open reading frame.

[0041] Another embodiment of the present invention is a method for synthesizing a protein or peptide in a living cell, comprising the step of incorporating into the cell an RNA molecule according to the present invention, said RNA molecule comprising an open reading frame under conditions that allow translation of the RNA molecule from the open reading frame with formation of the protein or peptide encoded by said open reading frame, wherein said cell is not contained within a patient's body.

[0042] Another embodiment of the present invention is a method for purifying an RNA molecule according to the present invention, comprising the steps of using a chromatographic method, preferably a reversed-phase HPLC method, wherein the column is equilibrated, a sample containing an RNA molecule according to the present invention is introduced into the chromatographic column, separation of the components of the sample occurs in a buffered aqueous / organic solvent system, and fractions containing the RNA molecule according to the present invention are collected, and the RNA molecule according to the present invention is separated from RNA molecules that do not contain a compound having a structure according to the present invention as defined above at their 5' ends.

[0043] Another embodiment of the present invention is the use of the compounds according to the invention in the in vitro synthesis of RNA molecules.

[0044] Another embodiment of the present invention is the use of an RNA molecule according to the invention in the in vitro synthesis of proteins or peptides.

[0045] Another embodiment of the present invention is a compound according to the present invention or an RNA molecule according to the present invention for use in medicine, pharmacology or diagnostics.

[0046] Surprisingly, it has been found that the trinucleotide or tetranucleotide analogues of the mRNA 5' end cap according to the present invention allow for the easy purification of in vitro transcribed mRNA by facilitating the separation of capped mRNA from uncapped mRNA by available chromatographic methods, in particular reversed-phase HPLC.

[0047] Equally surprising, the present invention enables the production of mRNAs with significantly higher protein expression efficiencies than mRNAs obtained using known mRNA 5'-end cap analogs.

[0048] Unexpectedly, these properties were obtained by introducing a hydrophobic substituent at the N6-adenosine position of trinucleotide or tetranucleotide cap analogs. This modification significantly alters the migration of the cap analog and capped mRNA on a reversed-phase packed chromatography column, allowing for the separation of capped and uncapped mRNA. Independently, this modification results in improved efficiency of mRNA expression in eukaryotic cells. Furthermore, this modification does not preclude, and therefore can be used in conjunction with, other previously identified cap modifications that improve mRNA properties, such as triphosphate bridge modifications or natural epigenetic modifications in the form of methylation at the 2'-O position of the first or second transcribed nucleotide.

[0049] All publications cited herein and references indicated herein are hereby incorporated by reference.

[0050] For a better understanding of the invention, a brief description of the drawings is provided in the examples and accompanying drawings. [Brief explanation of the drawings]

[0051] [Figure 1] FIG. 1 shows reverse-phase HPLC purification of mRNA obtained using various cap analogs. [Figure 2] FIG. 1 shows protein expression in 3T3-L1 cells from HPLC-purified mRNA. [Figure 3] Figure 1 shows protein expression in JAWSII cells from mRNA capped with selected analogs (m7GpppApG, m2 7,2'-OGpppSG, m7Gpppm6AmpG, m7GpppG, m2 7,2'-OGpppG, m2 7,2'-OGppSpG D1, m7GpppBn6AmpG) purified by HPLC. [Figure 4] FIG. 1 shows protein expression in JAWSII cells from mRNA capped with selected analogs (m7GpppAmpG, m7GpppBn6AmpG, m7Gppp3MeBn6AmpG, m7Gppp2MeBn6AmpG, m7Gppp4MeBn6AmpG, m7Gppp4FBn6AmpG, m7Gppp3,4diFBn6AmpG, m7Gppp1Naphm6AmpG, m7Gppp2Naphm6AmpG, m2 7,2'-OGpppG) purified by HPLC. DETAILED DESCRIPTION OF THE INVENTION

[0052] The term "alkyl" refers to a saturated, straight-chain or branched hydrocarbon substituent having the indicated number of carbon atoms, preferably 1 to 10. Examples of alkyl substituents are -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, -n-octyl, -n-nonyl, and -n-decyl. Representative branched (C1-C10) alkyls include -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, -neopentyl, -1-methylbutyl, -2-methylbutyl, -3-methylbutyl, -1,1-dimethylpropyl, -1,2-dimethylpropyl, -1-methylpentyl, -2-methylpentyl, -3-methylpentyl, -4-methylpentyl, -1-ethylbutyl, -2-ethylbutyl, -3-ethylbutyl, -1,1-dimethylbutyl, -1,2-dimethylbutyl, 1,3-dimethylbutyl, -2,2-dimethylbutyl, -2,3-dimethylbutyl, -3,3-dimethylbutyl, -1-methylhexyl, 2-methylhexyl, - Examples include 3-methylhexyl, -4-methylhexyl, -5-methylhexyl, -1,2-dimethylpentyl, -1,3-dimethylpentyl, -1,2-dimethylhexyl, -1,3-dimethylhexyl, -3,3-dimethylhexyl, 1,2-dimethylheptyl, -1,3-dimethylheptyl, and -3,3-dimethylheptyl.

[0053] The term "aryl" refers to an unsaturated, cyclic, aromatic, or heteroaromatic (i.e., containing heteroatoms in place of carbon) hydrocarbon substituent having the indicated number of carbon atoms, preferably 6 to 10. Examples of aryl are phenyl, naphthyl, anthracyl, phenanthryl, and pyridyl.

[0054] The term "alkylaryl" refers to an unsaturated hydrocarbon substituent composed of an alkyl portion and an aryl portion (as defined above) bonded together. Examples of alkylaryl are benzyl, phenylethyl, phenylpropyl, naphthylmethyl, naphthylethyl, and the like.

[0055] The term "heteroatom" means an atom selected from the group of oxygen, sulfur, nitrogen, phosphorus, and the like.

[0056] The term "HPLC" refers to high performance liquid chromatography, and solvents designated as "HPLC" solvents refer to solvents of sufficient purity for HPLC (high performance liquid chromatography) analysis.

[0057] The term "NMR" means nuclear magnetic resonance.

[0058] The following examples are provided solely to illustrate the present invention and to clarify certain aspects thereof, and are not intended to limit the invention or to equate its full scope as defined by the appended claims. In the following examples, standard materials and methods used in the art were used, or manufacturer's recommendations for particular materials and methods were followed, unless otherwise indicated. [Example]

[0059] Trinucleotide and tetranucleotide cap analogs were synthesized using a combination of solid-phase and solution-phase synthesis methods, as described in Examples 1-6, and then isolated using a two-step purification procedure. The starting point was an appropriately modified oligonucleotide: a dinucleotide (pA) on a high-load support using the phosphoramidite method, as described in Example 2. * pG, A * represents an adenosine substituted at the N6 position) or a trinucleotide (pA * The synthesis of the oligonucleotide (pGpG) was carried out in a similar manner.

[11] The corresponding phosphoramidite modified at the N6-position of adenosine was obtained by N-alkylation of commercially available adenosine phosphoramidite under phase-transfer catalysis conditions, as described in Example 1. The oligonucleotide was cleaved from the support, deprotected, and isolated by ion-exchange chromatography as a triethylammonium salt suitable for activation to P-imidazolide and subsequent coupling reaction in the presence of ZnCl2. The activated oligonucleotide was then converted to m 7This was subjected to a coupling reaction with GDP

[12] , and m7GpppA * (pG) k Analogs (wherein A * represents an N6-modified adenosine, where k is equal to 1 or 2) was obtained as described in Examples 3 and 6.

[0060] Example 3 illustrates the preparation of trinucleotide cap analogs modified at the N6 position of adenosine with groups such as benzyl, substituted benzyl, 1-naphthylmethyl, and 2-naphthylmethyl. Other trinucleotides not modified within the triphosphate bridge as described in claim 1 can be prepared using procedures similar to those described in Example 3, with the pA obtained as described in Example 2. * The synthesis can be performed using pG dinucleotides (appropriately modified at the N6 position of adenosine). Examples 4 and 5 demonstrate the preparation of trinucleotide cap analogs modified with a benzyl moiety at the N6 position of adenosine and containing further modifications within the 5',5'-triphosphate bridge. The synthesis of β-phosphorothioate analogs (Example 4) involves the use of p Bn6 A m pG must be converted to the corresponding P-imidazolide, and then m 7 The compounds were coupled with GDP-β-S

[12] . The compounds were isolated by ion exchange chromatography and further purified by RP HPLC to yield ammonium salts suitable for biological studies. In the case of the β-phosphorothioates, the products were isolated as a mixture of two stereoisomers, which could not be separated during the RP HPLC purification step. The synthesis of 5'-phosphorothioate cap analogs (Example 5) required two-step modification of 5'-deprotected oligonucleotides on solid support to incorporate a sulfur atom at the 5' position of adenosine. Other trinucleotides or tetranucleotides modified within the triphosphate bridge were synthesized using the strategy described in Examples 1-6, using the appropriate N6-modified adenosine phosphoramidite and the appropriate N6-modified oligonucleotide (pA) in combination with methods for triphosphate bridge modification described in the literature for dinucleotide cap analogs. * pG or pA *pGpG) can be used to synthesize it. 17、18、19、20、21

[0061] Example 6 illustrates the preparation of tetranucleotide cap analogs modified with a benzyl group at the N6 position of adenosine. Other tetranucleotides not modified within the triphosphate bridge as described in claim 1 can be prepared by synthesis of pA tetranucleotides appropriately modified at the N6 position of adenosine using procedures similar to those described in Example 6. * It can be synthesized using pGpG trinucleotides.

[0062] Transcripts incorporating the compound according to the invention or a benchmark (reference) compound at the 5' end were obtained by in vitro transcription in the presence of T7 RNA polymerase and a DNA template containing the Φ6.5 promoter sequence of this polymerase. To analyze the protein expression efficiency in mammalian cells, mRNA transcripts containing the compound according to the invention or a reference compound and encoding Gaussia luciferase as a reporter gene were obtained. The in vitro transcription reaction was carried out under the conditions described in Example 7. The obtained mRNA was pre-purified and analyzed by reverse-phase HPLC under the conditions described in Example 8. The results of this analysis were compared with the most structurally similar unmodified trinucleotide cap analog

[11] (m7Gppp m6 A m Similar analysis results for mRNA obtained using the trinucleotide cap analogs of the present invention are shown in Figure 1 alongside those for mRNA obtained using the trinucleotide cap analogs of the present invention (pG). Prior to testing for protein expression efficiency, mRNA capped with the trinucleotide cap analogs of the present invention was subjected to RP HPLC purification to remove double-stranded RNA impurities, as described in Example 8. In each case, mRNA was purified using either uncapped mRNA or a reference mRNA (mRNA). 7 GpppApG, m 7 GpppA m pG or m 7 Gppp m6 A mIncreased retention times of mRNAs were observed compared to those capped with 5'-pG (capped with 5'-pG), and the retention time of a given mRNA increased with increasing hydrophobicity of the substituent at the N6-adenosine position (Table 1). Reference mRNAs were also subjected to HPLC purification as described in Example 8, but they were previously subjected to a procedure for enzymatic removal of uncapped (5'-triphosphate) mRNA as described in Example 7. The resulting mRNAs capped with the compounds of the present invention or the reference compounds were introduced into mammalian cell lines (fibroblasts—3T3-L1 and dendritic cells—JAWS II) by transfection using lipofectamine, and Gaussia luciferase expression levels in the extracellular medium were then measured at appropriate intervals as described in Example 9. The results of these experiments are shown in Figures 2, 3, and 4, which show the overall (total) Gaussia luciferase expression levels obtained throughout the entire experiment (88 hours), which is the sum of the Gaussia luciferase expression levels obtained at individual time points.

[0063] Example 1 N 6 -modified 2'-O-methyladenosine 3'-O-phosphoramidite (A * Synthesis of -CEP 5'-O-Dimethoxytrityl-N6-phenoxyacetyl-2'-O-methyladenosine (5'-O-DMT-2'-O-Me-rA Pac The 3'-O-phosphoramidite (1 eq.) of ) and benzyl bromide (4 eq.) were dissolved in CHCl (to obtain a 0.1 M solution of the phosphoramidite) and mixed with a solution of tetrabutylammonium bromide (1 eq.) in 1 M NaOH (1 volume of the reaction mixture). The mixture was vigorously stirred for 30 min and then diluted with 50 mL of water and 50 mL of diethyl ether. The layers were separated, and the aqueous phase was extracted twice with diethyl ether (50 mL). The organic layers were combined, dried over anhydrous NaSO, and evaporated. The residue was dissolved in CHCl with triethylamine (0.5% v / v) and evaporated onto silica gel. The product was isolated by flash chromatography on a 20 g silica gel column using gradient elution (0 → 100% ethyl acetate in n-hexane) and, after evaporation, A* A mixture of diastereomers of -CEP phosphoramidite was obtained as a white foam.

[0064] [Table 1A]

[0065] [Table 1B]

[0066] [Table 1C]

[0067] Bn6 A m -CEP: 1 H NMR (500 MHz, CDCl3, 25℃): δ = 8.60 (s, 1H, H8), 8.58 (s, 1H, H8), 8.26 (s, 1H, H2), 8.19 (s, 1H, H2), 7.46-6.60 (m, 46H, ArH), 6.15 (d, 3 J H,H = 5.4 Hz, 1H, H1'), 6.13 (d, 3 J H,H = 5.0 Hz, 1H, H1'), 5.65 (s, 4H, CH 2 (bn6) ) 5.13 (s, 4H, CH 2 (Pac) ), 4.67 (m, 2H, H3'), 4.54 (m, 2H, H2'), 4.41 (m, 1H, H4'), 4.35 (m, 1H, H4'), 3.90 (m, 2H, OCH2CH2CN), 3.77 (s, 12H, OCH 3 DMT ), 3.72-3.52 (m, 8H, OCH2CH2CN, H5', CH iPr ), 3.48 (s, 6H, CH 3 2'-O ), 3.38 (dd, 2 J H,H = 10.6 Hz, 3 JH,H = 3.8 Hz, 2H, H5''), 2.63 (t, 3 J H,H = 6.3 Hz, 2H, OCH2CH2CN), 2.37 (t, 3 J H,H = 6.3 Hz, 2H, OCH2CH2CN), 1.19 (m, 24 H, CH 3 iPr ) ppm; 31 P NMR (202.5 MHz, CDCl3, H3PO4, 25℃): δ = 151.0 (s, 1P, P), 150.3 (s, 1P, P) ppm; 2MeBn6 A m -CEP: 1 H NMR (500 MHz, CDCl3, TMS, 25℃): δ = 8.62 (s, 1H, H8), 8.60 (s, 1H, H8), 8.24 (s, 1H, H2), 8.17 (s, 1H, H2), 7.45-6.62 (m, 44H, ArH), 6.13 (d, 3 J H,H = 4.8 Hz, 1H, H1'), 6.11 (d, 3 J H,H = 5.0 Hz, 1H, H1'), 5.63 (s, 4H, CH 2 (2MeBn) ) 5.15 (s, 4H, CH 2 (Pac) ), 4.65 (m, 1H, H3'), 4.57 (m, 2H, H3', H2'), 4.50 (m, 1H, H2'), 4.40 (m, 1H, H4'), 4.34 (m, 1H, H4'), 3.88 (m, 2H, OCH2CH2CN), 3.77 (4x s, 12H, OCH 3 (DMTr) ), 3.71-3.50 (m, 8H, OCH2CH2CN, H5', CH (iPr) ), 3.46 (6H, CH 3 (2'-O) ), 3.36 (m, 2H, H5''), 2.62 (t, 3 J H,H = 6.3 Hz, 2H, OCH2CH2CN), 2.37 (t, 3 JH,H = 6.4 Hz, 2H, OCH2CH2CN), 2.33 (s, 6H, CH 3 (2MeBn) ) 1.19 (m, 18 H, CH 3 (iPr) ), 1.07 (d, 3 J H,H = 6.8 Hz, 6H, CH 3 (iPr) ) ppm; 31 P NMR (202.5 MHz, CDCl3, H3PO4, 25℃): δ = 151.0 (s, 1P, P), 150.4 (s, 1P, P) ppm; 3MeBn6 A m -CEP: 1 H NMR (500 MHz, CDCl3, TMS, 25℃): δ = 8.60 (s, 1H, H8), 8.59 (s, 1H, H8), 8.26 (s, 1H, H2), 8.20 (s, 1H, H2), 7.46-6.61 (m, 44H, ArH), 6.15 (d, 3 J H,H = 5.2 Hz, 1H, H1'), 6.14 (d, 3 J H,H = 5.1 Hz, 1H, H1'), 5.62 (s, 4H, CH 2 (3MeBn) ) 5.14 (s, 4H, CH 2 (Pac) ), 4.66 (m, 1H, H3'), 4.60 (m, 1H, H3'), 4.58 (m, 1H, H2'), 4.54 (m, 1H, H2'), 4.41 (m, 1H, H4'), 4.36 (m, 1H, H4'), 3.89 (m, 2H, OCH2CH2CN), 3.77 (4x s, 12H, OCH 3 (DMTr) ), 3.73-3.50 (m, 8H, OCH2CH2CN, H5', CH (iPr) ), 3.48 (2x s, 6H, CH 3 (2'-O) ), 3.37 (m, 2H, H5''), 2.63 (t, 3 J H,H = 6.3 Hz, 2H, OCH2CH2CN), 2.37 (t, 3 JH,H = 6.3 Hz, 2H, OCH2CH2CN), 2.23 (s, 6H, CH 3 (3MeBn) ) 1.20 (m, 18 H, CH 3 (iPr) ), 1.08 (d, 3 J H,H = 6.8 Hz, 6H, CH 3 (iPr) ) ppm; 31 P NMR (202.5 MHz, CDCl3, H3PO4, 25℃): δ = 151.1 (s, 1P, P), 150.4 (s, 1P, P) ppm; 4MeBn6 A m -CEP: 1 H NMR (500 MHz, CDCl3, TMS, 25℃): δ = 8.59 (s, 1H, H8), 8.57 (s, 1H, H8), 8.26 (s, 1H, H2), 8.19 (s, 1H, H2), 7.45-6.61 (m, 44H, ArH), 6.15 (d, 3 J H,H = 5.1 Hz, 1H, H1'), 6.13 (d, 3 J H,H = 5.0 Hz, 1H, H1'), 5.61 (s, 4H, CH 2 (4MeBn) ) 5.12 (s, 4H, CH 2 (Pac) ), 4.67 (m, 1H, H3'), 4.60 (m, 1H, H3'), 4.58 (m, 1H, H2'), 4.54 (m, 1H, H2'), 4.41 (m, 1H, H4'), 4.36 (m, 1H, H4'), 3.89 (m, 2H, OCH2CH2CN), 3.77 (4x s, 12H, OCH 3 (DMTr) ), 3.73-3.50 (m, 8H, OCH2CH2CN, H5', CH (iPr) ), 3.48 (6H, CH 3 (2'-O) ), 3.36 (m, 2H, H5''), 2.63 (t, 3 J H,H = 6.3 Hz, 2H, OCH2CH2CN), 2.37 (t, 3 J H,H= 6.3 Hz, 2H, OCH2CH2CN), 2.22 (s, 6H, CH 3 (4MeBn) ) 1.20 (m, 18 H, CH 3 (iPr) ), 1.08 (d, 3 J H,H = 6.8 Hz, 6H, CH 3 (iPr) ) ppm; 31 P NMR (202.5 MHz, CDCl3, H3PO4, 25℃): δ = 151.0 (s, 1P, P), 150.4 (s, 1P, P) ppm; 1Naph6 A m -CEP: 1 H NMR (500 MHz, CDCl3, TMS, 25℃): δ = 8.59 (s, 1H, H8), 8.58 (s, 1H, H8), 8.24 (s, 1H, H2), 8.16 (s, 1H, H2), 8.14 (d, 3 J H,H = 8.4 Hz, 2H, ArH Naph ), 7.78 (d, 3 J H,H = 8.1 Hz, 2H, ArH Naph ), 7.63 (d, 3 J H,H = 8.2 Hz, 2H, ArH Naph ), 7.51 (m, 2H, ArH Naph ), 7.45 (m, 2H, ArH Naph ), 7.43-6.63 (m, 40H, ArH), 6.13 (s, 4H, CH 2 (1NaphCH2) ), 6.11 (d, 3 J H,H = 4.9 Hz, 1H, H1'), 6.10 (d, 3 J H,H = 5.0 Hz, 1H, H1'), 5.17 (s, 4H, CH 2 (Pac)), 4.63 (m, 1H, H3'), 4.56 (m, 2H, H3', H2'), 4.48 (m, 1H, H2'), 4.38 (m, 1H, H4'), 4.33 (m, 1H, H4'), 3.87 (m, 2H, OCH2CH2CN), 3.75 (4x s, 12H, OCH 3 (DMTr) ), 3.70-3.53 (m, 8H, OCH2CH2CN, H5', CH (iPr) ), 3.44 (6H, CH 3 (2'-O) ), 3.34 (m, 2H, H5''), 2.60 (t, 3 J H,H = 6.3 Hz, 2H, OCH2CH2CN), 2.34 (t, 3 J H,H = 6.3 Hz, 2H, OCH2CH2CN), 1.17 (d, 3 J H,H = 6.7 Hz, 18 H, CH 3 (iPr) ), 1.06 (d, 3 J H,H = 6.7 Hz, 6H, CH 3 (iPr) ) ppm; 31 P NMR (202.5 MHz, CDCl3, H3PO4, 25℃): δ = 151.0 (s, 1P, P), 150.4 (s, 1P, P) ppm; 2Naph6 A m -CEP: 1 H NMR (500 MHz, CDCl3, TMS, 25℃): δ = 8.52 (s, 1H, H8), 8.51 (s, 1H, H8), 8.21 (s, 1H, H2), 8.14 (s, 1H, H2), 7.66 (m, 4H, ArH Naph ), 7.60 (m, 4H, ArH Naph ), 7.39-6.55 (m, 42H, ArH), 6.07 (d, 3 J H,H = 4.8 Hz, 1H, H1'), 6.05 (d, 3 J H,H = 4.8 Hz, 1H, H1'), 5.74 (s, 4H, CH2 (1NaphCH2) ), 5.09 (s, 4H, CH 2 (Pac) ), 4.58 (m, 1H, H3'), 4.50 (m, 2H, H3', H2'), 4.44 (m, 1H, H2'), 4.32 (m, 1H, H4'), 4.27 (m, 1H, H4'), 3.79 (m, 2H, OCH2CH2CN), 3.69 (4x s, 12H, OCH 3 (DMTr) ), 3.64-3.44 (m, 8H, OCH2CH2CN, H5', CH (iPr) ), 3.38 (6H, CH 3 (2'-O) ), 3.28 (m, 2H, H5''), 2.53 (t, 3 J H,H = 6.3 Hz, 2H, OCH2CH2CN), 2.28 (t, 3 J H,H = 6.3 Hz, 2H, OCH2CH2CN), 1.10 (m, 18 H, CH 3 (iPr) ), 0.99 (d, 3 J H,H = 6.8 Hz, 6H, CH 3 (iPr) ) ppm; 31 P NMR (202.5 MHz, D2O, H3PO4, 25℃): δ = 151.0 (s, 1P, P), 150.4 (s, 1P, P) ppm; 4FBn6 A m -CEP: 1 H NMR (500 MHz, CDCl3, TMS, 25℃): δ = 8.61 (s, 1H, H8), 8.59 (s, 1H, H8), 8.28 (s, 1H, H2), 8.21 (s, 1H, H2), 7.45-6.59 (m, 43H, ArH), 6.16 (d, 3 J H,H = 4.7 Hz, 1H, H1'), 6.15 (d, 3 J H,H = 4.9 Hz, 1H, H1'), 5.57 (s, 4H, CH 2 (4FBn) ) 5.12 (s, 4H, CH 2 (Pac)), 4.69 (m, 1H, H3'), 4.64-4.60 (m, 2H, H3', H2'), 4.55 (m, 1H, H2'), 4.41 (m, 1H, H4'), 4.36 (m, 1H, H4'), 3.89 (m, 2H, OCH2CH2CN), 3.77 (4x s, 12H, OCH 3 (DMTr) ), 3.73-3.51 (m, 8H, OCH2CH2CN, H5', CH (iPr) ), 3.48 (6H, CH 3 (2'-O) ), 3.36 (m, 2H, H5''), 2.63 (t, 3 J H,H = 6.3 Hz, 2H, OCH2CH2CN), 2.38 (t, 3 J H,H = 6.3 Hz, 2H, OCH2CH2CN), 1.20 (m, 18 H, CH 3 (iPr) ), 1.08 (d, 3 J H,H = 6.8 Hz, 6H, CH 3 (iPr) ) ppm; 19 F NMR (470.6 MHz, D2O, NaF, 25℃): δ = -115.4 (m, 1F, F) ppm; 31 P NMR (202.5 MHz, CDCl3, H3PO4, 25℃): δ = 151.0 (s, 1P, P), 150.4 (s, 1P, P) ppm; 3,4diFBn6 A m -CEP: 1 H NMR (500 MHz, CDCl3, TMS, 25℃): δ = 8.62 (s, 1H, H8), 8.61 (s, 1H, H8), 8.30 (s, 1H, H2), 8.23 (s, 1H, H2), 7.46-6.58 (m, 42H, ArH), 6.17 (d, 3 J H,H = 4.9 Hz, 1H, H1'), 6.16 (d, 3 J H,H = 4.7 Hz, 1H, H1'), 5.53 (s, 4H, CH 2 (3,4diFBn)) 5.13 (s, 4H, CH 2 (Pac) ), 4.70 (m, 1H, H3'), 4.65-4.58 (m, 2H, H3', H2'), 4.54 (m, 1H, H2'), 4.41 (m, 1H, H4'), 4.36 (m, 1H, H4'), 3.88 (m, 2H, OCH2CH2CN), 3.77 (4xs, 12H, OCH 3 (DMTr) ), 3.73-3.51 (m, 8H, OCH2CH2CN, H5', CH (iPr) ), 3.48 (6H, CH 3 (2'-O) ), 3.37 (m, 2H, H5''), 2.63 (t, 3 J H,H = 6.3 Hz, 2H, OCH2CH2CN), 2.38 (t, 3 J H,H = 6.3 Hz, 2H, OCH2CH2CN), 1.20 (m, 18 H, CH 3 (iPr) ), 1.08 (d, 3 J H,H = 6.8 Hz, 6H, CH 3 (iPr) ) ppm; 19 F NMR (470.6 MHz, D2O, NaF, 25℃): δ = -137.8 (m, 1F, F), -140.0 (m, 1F, F) ppm; 31 P NMR (202.5 MHz, CDCl3, H3PO4, 25℃): δ = 150.9 (s, 1P, P), 150.4 (s, 1P, P) ppm;

[0068] Example 2 5'-phosphorylated dinucleotides and trinucleotides (pA * pG or pA * Synthesis of pGpG The synthesis of the dinucleotide was carried out manually using a 10 mL syringe fitted with a fritted filter. iBu3'-lcaa PrimerSupport 5G (GE Healthcare, 308 μmol / g) was placed in a syringe and washed with dry acetonitrile. In the coupling step, 1.2 to 1.5 equivalents of phosphoramidite (A) dissolved in 1 mL of anhydrous acetonitrile was added. *- CEP, G-CEP, or bis-(2-cyanoethyl)-N,N-diisopropylphosphoramidite) and 1.5 mL of 0.30 M 5-(benzylthio)-1-H-tetrazole in acetonitrile were shaken for 15 min in a syringe capped with a plunger. A solution of 3% (v / v) trichloroacetic acid in dichloromethane was used as the detritylation reagent, and pyridine / water (9:1) was used for oxidation. v / v 0.05 M iodine in acetonitrile was used. After the final cycle of synthesis, the RNA still on the solid support was treated with 20% (v / v) diethylamine in acetonitrile to remove the 2-cyanoethyl protecting group. Finally, the solid support was washed with acetonitrile and dried with argon. The product was cleaved from the solid support and purified with AMA (40% methylamine / 33% ammonium hydroxide, 1:1 v / v The product was deprotected at 55°C for 1 h, evaporated to dryness, and redissolved in DMSO (200 μL). The TBDMS group was removed using triethylammonium trihydrofluoride (TEA·3HF; 250 μL, 65°C for 3 h), after which the mixture was cooled and diluted with 0.25 M NaHCO3 in water (20 mL). The product was isolated by ion-exchange chromatography on DEAE Sephadex (gradient elution, 0–0.9 M TEAB) and, after evaporation, reached a pH of 1. * The triethylammonium salt of pG dinucleotide was obtained. Yields were estimated by UV absorbance at 260 nm, assuming an extinction coefficient ε = 27.1 L / mmol / cm for dinucleotides or 39.0 L / mmol / cm for trinucleotides.

[0069] [Table 2A]

[0070] [Table 2B]

[0071]

Table 2C

[0072] p 2MeBn6 A m pG (TEAH + ): 1 1H NMR (500 MHz, D2O, TMS, 25 °C): δ = 8.41 (s, 1H, H8 A ), 8.14 (s, 1H, H2 A ), 7.93 (s, 1H, H8 G ), 7.30 - 7.15 (m, 4H, ArH 2MeBn ), 6.11 (d, 3 J H,H = 5.1 Hz, 1H, H1' A ), 5.83 (d, 3 J H,H = 5.2 Hz, 1H, H1' G ), 4.92 (m, 1H, H3' A ), 4.76 (m, 2H, CH 2, Bn, overlapping with HDO), 4.72 (m, 1H, H2' G ), 4.50 - 4.47 (m, 2H, H2' A , H3' G ), 4.46 (s, 1H, H4' A ), 4.34 (s, 1H, H4' G ), 4.25 - 4.17 (m, 2H, H5' G , H5'' G ), 4.12 - 4.04 (m, 2H, H5' A , H5'' A ), 3.50 (s, 3H, CH 3, 2’-O[A] ), 3.36 - 3.04 (m, CH 2, TEAH+ ), 2.36 (s, 3H, CH 3, 2MeBn ), 1.27 (m, CH 3, TEAH+ ) ppm; 31P NMR (202.5 MHz, D2O, H3PO4, 25°C): δ = 1.38 (s, 1P, P). 5’A ), 0.04 ( s , 1P , P A-G ) ppm; p 4MeBn6 A m pG (TEAH + ): 1 H NMR (500 MHz, D2O, TMS, 25°C): δ = 8.39 (s, 1H, H8). A ), 8.14 ( s , 1H , H2 A ), 7.93 (s, 1H, H8 G ), 7.27 (d, 3 J H,H = 8.0 Hz, 2H, ArH 4MeBn(3&5) ), 7.18 (d, 3 J H,H = 7.9 Hz, 2H, ArH 4MeBn(2&6) ), 6.10 (d, 3 J H,H = 5.0 Hz, 1H, H1' A ), 5.83 (d, 3 J H,H = 5.2 Hz, 1H, H1' G ), 4.92 (m, 1H, H3' A ), 4.79 (m, 2H, CH 2, Bn, HDO and solvents), 4.72 (m, 1H, H2' G ), 4.50-4.45 (m, 3H, H2' A , H3 G , H4' A ), 4.34 (s, 1H, H4' G ), 4.25-4.17 (m, 2H, H5' G , H5 G ), 4.12-4.05 (m, 2H, H5' A , H5 A ), 3.50 (s, 3H, CH 3, 2’-O[A] ), 3.20 (q, 3 J H,H = 7.3 Hz, CH 2, TEAH+), 2.29 (s, 3H, CH 3, 2MeBn ), 1.28 (t, 3 J H,H = 7.3 Hz, CH 3, TEAH+ ) ppm; 31 P NMR (202.5 MHz, D2O, H3PO4, 25℃): δ = 1.06 (s, 1P, P 5’A ), 0.05 (s, 1P, P A-G ) ppm; p 1Naphm6 A m pG (TEAH + ): 1 H NMR (500 MHz, D2O, TMS, 25℃): δ = 8.30 (s, 1H, H8 A ), 8.07 (s, 1H, H2 A ), 8.04 (m, 1H, ArH Naph ), 7.91 (s, 1H, H8 G ), 7.88 (m, 1H, ArH Naph ), 7.80 (m, 1H, ArH Naph ), 7.57-7.40 (m, 4H, ArH Naph ), 6.05 (d, 3 J H,H = 5.0 Hz, 1H, H1' A ), 5.80 (d, 3 J H,H = 5.2 Hz, 1H, H1' G ), 5.17 (d, 2 J H,H = 14.9 Hz, 1H, CH 2, Naphm ), 5.05 (d, 2 J H,H = 14.9 Hz, 1H, CH 2, Naphm ), 4.92 (m, 1H, H3' A ), 4.71 (m, 1H, H2' G ), 4.50-4.45 (m, 3H, H2' A , H3' G , H4' A ), 4.34 (s, 1H, H4' G), 4.26-4.17 (m, 2H, H5' G , H5'' G ), 4.09 (m, 2H, H5' A , H5'' A ), 3.51 (s, 3H, CH 3, 2’-O[A] ), 3.33-3.03 (m, CH 2, TEAH+ ), 1.29-1.24 (m, CH 3, TEAH+ ) ppm; 31 P NMR (202.5 MHz, D2O, H3PO4, 25℃): δ = 1.39 (s, 1P, P 5’A ), 0.06 (s, 1P, P A-G ) ppm; p 2Naphm6 A m pG (TEAH + ): 1 H NMR (500 MHz, D2O, TMS, 25℃): δ = 8.37 (s, 1H, H8 A ), 8.02 (s, 1H, H2 A ), 7.88 (s, 1H, H8 G ), 7.78 (m, 2H, ArH Naph ), 7.71 (m, 2H, ArH Naph ), 7.43 (m, 3H, ArH Naph ), 5.97 (d, 3 J H,H = 4.7 Hz, 1H, H1' A ), 5.78 (d, 3 J H,H = 5.1 Hz, 1H, H1' G ), 4.94-4.86 (m, 3H, H3' A , CH 2, Naphm ), 4.67 (m, 1H, H2' G ), 4.49-4.43 (m, 3H, H2' A , H3' G , H4' A ), 4.33 (s, 1H, H4' G ), 4.25-4.16 (m, 2H, H5' G , H5''G ), 4.11 (m, 2H, H5' A , H5'' A ), 3.49 (s, 3H, CH 3, 2’-O[A] ), 3.35 - 3.03 (m, CH 2, TEAH+ ), 1.29 - 1.24 (m, CH 3, TEAH+ ) ppm; 31 P NMR (202.5 MHz, D2O, H3PO4, 25℃): δ = 1.31 (s, 1P, P 5’A ), 0.04 (s, 1P, P A-G ) ppm; p 4FBn6 A m pG (TEAH + ): 1 H NMR (500 MHz, D2O, TMS, 25℃): δ = 8.40 (s, 1H, H8 A ), 8.15 (s, 1H, H2 A ), 7.93 (s, 1H, H8 G ), 7.39 (m, 2H, ArH 4FBn ), 7.09 (m, 2H, ArH 4FBn ), 6.11 (d, 3 J H,H = 5.1 Hz, 1H, H1' A ), 5.83 (d, 3 J H,H = 5.2 Hz, 1H, H1' G ), 4.93 (m, 1H, H3' A ), 4.83 (m, 2H, CH 2, Bn, HDO and overlapping), 4.73 (m, 1H, H2' G ), 4.50 - 4.44 (m, 3H, H2' A , H3' G , H4' A ), 4.35 (s, 1H, H4' G ), 4.25 - 4.17 (m, 2H, H5' G , H5'' G ), 4.12 - 4.04 (m, 2H, H5' A , H5''A ), 3.50 (s, 3H, CH 3, 2’-O[A] ), 3.20 (q, 3 J H,H = 7.3 Hz, CH 2, TEAH+ ), 1.28 ( t, 3 J H,H = 7.3 Hz, CH 3, TEAH+ ) ppm; 31 P NMR (202.5 MHz, D2O, H3PO4, 25°C): δ = 1.08 (s, 1P, P). 5’A ), 0.06 ( s , 1P , P A-G ) ppm; p 3,4diFBn6 A m pG (TEAH + ): 1 H NMR (500 MHz, D2O, TMS, 25°C): δ = 8.42 (s, 1H, H8). A ), 8.14 ( s , 1H , H2 A ), 7.92 (s, 1H, H8 G ), 7.30–7.14 (m, 3H, ArH 3,4diFBn ), 6.12 (d, 3 J H,H = 5.1 Hz, 1H, H1' A ), 5.83 (d, 3 J H,H = 5.3 Hz, 1H, H1' G ), 4.93 (m, 1H, H3' A ), 4.83 (m, 2H, CH 2, Bn, HDO and solvents), 4.74 (m, 1H, H2' G ), 4.51-4.45 (m, 3H, H2' A , H3 G , H4' A ), 4.35 (s, 1H, H4' G ), 4.26-4.17 (m, 2H, H5' G , H5 G ), 4.12-4.05 (m, 2H, H5' A , H5 A), 3.50 (s, 3H, CH 3, 2’-O[A] ), 3.20 (q, 3 J H,H = 7.3 Hz, CH 2, TEAH+ ), 1.28 (t, 3 J H,H = 7.3 Hz, CH 3, TEAH+ ) ppm; 31 P NMR (202.5 MHz, D2O, H3PO4, 25℃): δ = 1.20 (s, 1P, P 5’A ), 0.06 (s, 1P, P A-G ) ppm; p Bn6 A m pGpG (TEAH + ): 1 H NMR (500 MHz, DO, TMS, 25 °C) δ = 8.39 (s, 1H), 8.09 (s, 1H), 7.95 (s, 1H), 7.87 (s, 1H), 7.47 - 7.29 (m, 5H), 6.07 (d, J = 5.3 Hz, 1H), 5.82 (d, J = 5.5 Hz, 1H), 5.76 (d, J = 5.0 Hz, 1H), 4.95 - 4.89 (m, 1H), 4.79 (1H, overlapped with HDO), 4.79 (1H, overlapped with HDO), 4.71 (t, J = 5.5 Hz, 1H), 4.48 (t, J = 5.3 Hz, 1H). 4.47 - 4.43 (m, 4H), 4.34 - 4.30 (m, 1H), 4.29 - 4.14 (m, 4H), 4.08 - 3.98 (m, 2H), 3.46 (s, 3H), 3.20 (q, J = 7.3 Hz), 1.28 (t, J = 7.3 Hz) ppm; 31 P NMR (202 MHz, D2O, H3PO4, 25℃) δ = 1.73 - 1.43 (m, 1P), 0.41 - 0.27 (m, 1P), -0.04 - -0.13 (m, 1P) ppm.

[0073] Example 3 Trinucleotide cap analogue m 7 GpppA * Synthesis of pG Step 1. Activation of pNpG: The dinucleotide 5'-phosphate was dissolved in DMF (to obtain a 0.05 M solution), followed by the addition of imidazole (16 equivalents), 2,2'-dithiodipyridine (6 equivalents), triethylamine (6 equivalents), and triphenylphosphine (6 equivalents). The mixture was stirred at room temperature for 2 hours. The product was precipitated by adding sodium perchlorate (10 equivalents) in acetonitrile (10 times the volume of DMF). The precipitate was centrifuged at 4°C, washed three times with cold acetonitrile, and dried under reduced pressure to obtain the sodium salt of the dinucleotide P-imidazolide (Im-pNpG).

[0074] Step 2. Triphosphate bridge formation: 7-methylguanosine 5'-diphosphate (m 7 GDP (1.5 equiv.) and Im-pNpG (1 equiv.) were suspended in DMF (to obtain a 0.05 M solution of p-imidazolide). ZnCl (8 equiv.) was then added, and the mixture was stirred at room temperature for 2 h. The reaction was quenched by adding a solution of NaEDTA (20 mg / mL, 8 equiv.) and NaHCO (10 mg / mL) in water, and the product was isolated by ion-exchange chromatography on DEAE Sephadex (gradient elution, 0–1.2 M TEAB) and, after evaporation, m 7 Gppp bn6 A m The triethylammonium salt of pG was obtained. Further purification by RP HPLC (C18) using a linear gradient of acetonitrile in aqueous buffer of CH3COONH4, pH 5.9, gave (after repeated lyophilization from water) m 7 GpppA * The ammonium salt of pG was obtained. The yield was estimated by UV absorbance at 260 nm, assuming an extinction coefficient ε = 35.0 L / mmol / cm.

[0075] [Table 3A]

[0076] [Table 3B]

[0077] m 7 Gppp Bn6 A m pG: 1 H NMR (500 MHz, D2O, TMS, 25℃): δ = 9.06 (s, 1H, H8 m7G ), 8.46 (s, 1H, H8 A ), 8.19 (s, 1H, H2 A ), 8.03 (s, 1H, H8 G ), 7.44-7.31 (m, 5H, ArH Bn ), 6.03 (d, 3 J H,H = 5.6 Hz, 1H, H1' A ), 5.88 (d, 3 J H,H = 3.7 Hz, 1H, H1' m7G ), 5.84 (d, 3 J H,H = 5.8 Hz, 1H, H1' G ), 4.93 (m, 1H, H3' A ), 4.81 (m, 1H, H2' G , overlapping with HDO), 4.85-4.74 (m, 2H, CH 2, Bn , overlapping with HDO), 4.59 (m, 1H, H2' m7G ), 4.53-4.49 (m, 2H, H4' A , H3' G ), 4.48-4.44 (m, 2H, H2' A , H3' m7G ), 4.38-4.16 (m, 8H, H4' m7G , H4' G , H5' m7G , H5'' m7G , H5' A , H5'' A , H5' G , H5'' G), 4.02 (s, 3H, CH 3, m7G ), 3.43 (s, 3H, CH 3, 2'-O ) ppm; 31 P NMR (202.5 MHz, D2O, H3PO4, 25℃): δ = 0.03 (s, 1P, P A-G ), -10.58 (m, 2P, P α , P γ ), -21.98 (t, 2 J P,P = 18.4 Hz, 1P, P β ) ppm; m 7 Gppp 2MeBn6 A m pG: 1 H NMR (500 MHz, D2O, TMS, 25℃): δ = 9.08 (s, 1H, H8 m7G ), 8.50 (s, 1H, H8 A ), 8.21 (s, 1H, H2 A ), 8.06 (s, 1H, H8 G ), 7.29-7.12 (m, 4H, ArH 2MeBn ), 6.04 (d, 3 J H,H = 5.5 Hz, 1H, H1' A ), 5.89 (d, 3 J H,H = 3.4 Hz, 1H, H1' m7G ), 5.85 (d, 3 J H,H = 5.6 Hz, 1H, H1' G ), 4.95 (m, 1H, H3' A ), 4.81 (m, 1H, H2' G , overlapping with HDO), 4.75 (m, 2H, CH 2, Bn , overlapping with HDO), 4.59 (m, 1H, H2' m7G ), 4.54-4.44 (m, 4H, H2' A , H3' m7G , H3' G , H4' A), 4.38-4.15 (m, 8H, H4' m7G , H4' G , H5' m7G , H5 m7G , H5' A , H5 A , H5' G , H5 G ), 4.02 (s, 3H, CH 3, m7G ), 3.43 (s, 3H, CH 3, 2'-O ), 2.34 (s, 3H, CH 3, 2MeBn ) ppm; 31 P NMR (202.5 MHz, D2O, H3PO4, 25°C): δ = 0.04 (s, 1P, P). A-G ), -10.56 (m, 2P, P). α , P γ ), -21.98 (m, 1P, P). β ) ppm; m 7 Gppp 3MeBn6 A m pG: 1 H NMR (500 MHz, D2O, TMS, 25°C): δ = 9.07 (s, 1H, H8). m7G ), 8.48 ( s , 1H , H8 A ), 8.20 (s, 1H, H2 A ), 8.04 ( s , 1H , H8 G ), 7.29–7.12 (m, 4H, ArH 3MeBn ), 6.03 (d, 3 J H,H = 5.5 Hz, 1H, H1' A ), 5.88 (d, 3 J H,H = 3.5 Hz, 1H, H1' m7G ), 5.84 (d, 3 J H,H = 5.7 Hz, 1H, H1' G ), 4.93 (m, 1H, H3' A ), 4.83 (m, 1H, H2' G , HDO and dioxide), 4.76 (m, 2H, CH2, Bn , overlapping with HDO), 4.58 (m, 1H, H2' m7G ), 4.52 - 4.44 (m, 4H, H2' A , H3' m7G , H3' G , H4' A ), 4.38 - 4.15 (m, 8H, H4' m7G , H4' G , H5' m7G , H5'' m7G , H5' A , H5'' A , H5' G , H5'' G ), 4.01 (s, 3H, CH 3, m7G ), 3.42 (s, 3H, CH 3, 2'-O ), 2.27 (s, 3H, CH 3, 2MeBn ) ppm; 31 P NMR (202.5 MHz, D2O, H3PO4, 25℃): δ = 0.02 (s, 1P, P A-G ), -10.60 (m, 2P, P α , P γ ), -22.00 (t, 2 J P,P = 18.4 Hz, 1P, P β ) ppm; m[[ID=5{0]] 7 Gppp 4MeBn6 A m pG: 1 H NMR (500 MHz, D2O, TMS, 25℃): δ = 9.07 (s, 1H, H8 m7G ), 8.49 (s, 1H, H8 A ), 8.19 (s, 1H, H2 A ), 8.06 (s, 1H, H8 i G ), 7.22 (m, 2H, ArH 4MeBn ), 7.11 (m, 2H, ArH 4MeBn ), 6.02 (d, 3 J H,H = 5.4 Hz, 1H, H1' A ), 5.88 (d, 3J H,H = 3.6 Hz, 1H, H1' m7G ), 5.84 (d, 3 J H,H = 5.6 Hz, 1H, H1' G ), 4.94 (m, 1H, H3' A ), 4.79 (m, 1H, H2' G , overlapping with HDO), 4.71 (m, 2H, CH 2, Bn ), 4.58 (m, 1H, H2' m7G ), 4.54 - 4.48 (m, 2H, H3' G , H4' A ), 4.45 (m, 2H, H2' A , H3' m7G ), 4.39 - 4.16 (m, 8H, H4'<° m7G , H4' G , H5' m7G , H5'' m7G , H5' A , H5'' A , H5' G , H5'' G ), 4.01 (s, 3H, CH 3, m7G ), 3.44 (s, 3H, CH 3, 2'-O ), 2.24 (s, 3H, CH 3, 2MeBn ) ppm; 31 P NMR (202.5 MHz, D2O, H3PO4, 25°C): δ = 0.02 (s, 1P, P A-G ), -10.56 (m, 2P, P α , P γ ), -21.92 (m, 1P, P β ) ppm; m 7 Gppp 1Naphm6 A m pG: 1 H NMR (500 MHz, D2O, TMS, 25°C): δ = 8.97 (s, 1H, H8 m7G ), 8.33 (s, 1H, H8 A ), 8.03 (s, 2H, H2 A , H8 It should be noted that there might be some inaccuracies in the original text such as the inconsistent format of some tags (e.g., <° m7G which might be a misprint). The translation is done based on the best understanding of the provided text.G ), 7.80 - 7.25 (m, 7H, ArH Naph ), 5.94 (m, 1H, H1')[[]] A ), 5.79 (d,[[]] 3 J[[]] H,H = 5.3 Hz, 1H, H1')[[]] G ), 5.73 (m, 1H, H1')[[]] m7G ), 4.97 (m, 1H, H3')[[]] A ), 4.83 (m, 2H, CH[[]] 2, Bn , overlapping with HDO), 4.73 (m, 1H, H2')[[]] G ), 4.54 (m, 1H, H4')[[]] A ), 4.51 - 4.17 (m, 12H, H2')[[]] m7G , H2')[[]] A , H3')[[]] m7G , H3')[[]] G , H4')[[]] m7G , H4')[[]] G , H5')[[]] m7G , H5'')[[]] m7G , H5')[[]] A , H5'')[[]] A , H5')[[]] G , H5'')[[]] G ), 3.84 (s, 3H, CH[[]] 3, m7G ), 3.50 (s, 3H, CH[[]] 3, 2'-O ) ppm;[[]] 31 P NMR (202.5 MHz, D2O, H3PO4, 25°C): δ = 0.02 (s, 1P, P[[]] A-G ), -10.56 (m, 2P, P[[]] α , P[[]] γ ), -21.85 (m, 1P, P[[]] β ) ppm;[[]] m[[]] 7 Gppp[[]] 2Naphm6 A[[]] m pG:[[]] 1 H NMR (500 MHz, D2O, TMS, 25°C): δ = 8.93 (s, 1H, H8[[]] m7G ), 8.46 (s, 1H, H8[[]] A ), 8.04 (s, 2H, H2[[]] A , H8[[]]G ), 7.72 - 7.19 (m, 7H, ArH Naph ), 5.89 (m, 1H, H1') A ), 5.79 (d,[[]] 3 J H,H = 5.3 Hz, 1H, H1')[[]] G ), 5.71 (m, 1H, H1')[[]] m7G ), 4.97 (m, 1H, H3')[[]] A ), 4.83 (m, 2H, CH 2, Bn , overlapping with HDO), 4.71 (m, 1H, H2')[[]] G ), 4.54 (m, 1H, H4')[[]] A ), 4.51 - 4.16 (m, 12H, H2' m7G , H2' A , H3' m7G , H3' G , H4' m7G , H4' G , H5' m7G , H5'' m7G , H5' A , H5'' A , H5' G , H5'' G ), 3.77 (s, 3H, CH 3, m7G ), 3.49 (s, 3H, CH 3, 2'-O ) ppm; 31 P NMR (202.5 MHz, D2O, H3PO, subscript 4, 25°C): δ = 0.03 (s, 1P, P A-G ), -10.56 (m, 2P, P α , P γ ), -21.75 (m, 1P, P β ) ppm; m 7 Gppp 4FBn6 A m pG: 1 H NMR (500 MHz, D2O, TMS, 25°C): δ = 9.08 (s, 1H, H8 m7G ), 8.47 (s, 1H, H8 A ), 8.19 (s, 1H, H2 A), 8.05 (s, 1H, H8 G ), 7.38 (m, 2H, ArH 4FBn ), 7.06 (m, 2H, ArH 4FBn ), 6.03 (d, 3 J H,H = 5.5 Hz, 1H, H1' A ), 5.88 (d, 3 J H,H = 3.3 Hz, 1H, H1' m7G ), 5.84 (d, 3 J H,H = 5.7 Hz, 1H, H1' G ), 4.94 (m, 1H, H3' A ), 4.81 (m, 1H, H2' G , overlapping with HDO), 4.84-4.74 (m, 2H, CH 2, Bn , overlapping with HDO), 4.58 (m, 1H, H2' m7G ), 4.54-4.48 (m, 2H, H3' G , H4' A ), 4.46 (m, 2H, H2' A , H3' m7G ), 4.39-4.15 (m, 8H, H4' m7G , H4' G , H5' m7G , H5'' m7G , H5' A , H5'' A , H5' G , H5'' G ), 4.02 (s, 3H, CH 3, m7G ), 3.43 (s, 3H, CH 3, 2'-O ) ppm; 19 F NMR (470.6 MHz, D2O, NaF, 25℃): δ = -115.35 (s, 1F, F) ppm; 31 P NMR (202.5 MHz, D2O, H3PO4, 25℃): δ = 0.04 (s, 1P, P A-G ), -10.58 (m, 2P, P α , P γ), -21.99 (m, 1P, P β ) ppm; m 7 Gppp 3,4diFBn6 A m pG: 1 H NMR (500 MHz, D2O, TMS, 25℃): δ = 9.09 (s, 1H, H8 m7G ), 8.48 (s, 1H, H8 A ), 8.19 (s, 1H, H2 A ), 8.08 (s, 1H, H8 G ), 7:30-7:14 (m, 3H, ArH 3,4diFBn ), 6.03 (d, 3 J H,H = 5.6 Hz, 1H, H1' A ), 5.88 (d, 3 J H,H = 3.6 Hz, 1H, H1' m7G ), 5.85 (d, 3 J H,H = 5.7 Hz, 1H, H1' G ), 4.94 (m, 1H, H3' A ), 4.81 (m, 1H, H2' G , overlapping with HDO), 4.80 (m, 2H, CH 2, Bn , overlapping with HDO), 4.59 (m, 1H, H2' m7G ), 4.52 (m, 1H, H4' A ), 4.50 (m, 1H, H3' G ), 4.48-4.44 (m, 2H, H2' A , H3' m7G ), 4.38-4.16 (m, 8H, H4' m7G , H4' G , H5' m7G , H5'' m7G , H5' A , H5'' A , H5' G , H5'' G ), 4.02 (s, 3H, CH 3, m7G ), 3.43 (s, 3H, CH3, 2'-O ) ppm; 19 F NMR (470.6 MHz, D2O, NaF, 25℃): δ = -138.22 (s, 1F, F), -140.39 (s, 1F, F) ppm; 31 P NMR (202.5 MHz, D2O, H3PO4, 25℃): δ = 0.03 (s, 1P, P A-G ), -10.58 (m, 2P, P α , P γ ), -21.97 (m, 1P, P β ) ppm;

[0078] Example 4 β-phosphorothioate trinucleotide cap analogue m 7 Gpp S p Bn6 A m Synthesis of pG Process 1.p Bn6 A m Activation of pG: Dinucleotide 5'-phosphate p Bn6 A m pG(615mOD 260nm , 22.7 μmol) was dissolved in DMF (400 μL), followed by the addition of imidazole (24.7 mg, 363 μmol), 2,2'-dithiodipyridine (30 mg, 136 μmol), triethylamine (9.5 μL, 68 μmol), and triphenylphosphine (35.7 mg, 136 μmol). The mixture was stirred at room temperature for 48 hours. The product was precipitated by the addition of a solution of lithium perchlorate (24.1 mg, 227 μmol) in acetonitrile (4.0 mL). The precipitate was centrifuged at 4°C, washed three times with cold acetonitrile, and dried under reduced pressure to give the dinucleotide P-imidazolide Im-p Bn6 A m The lithium salt of pG was obtained (19 mg).

[0079] Step 2. Triphosphate bridge formation: 7-methylguanosine β-thiodiphosphate m 7 GDP-β-S (378 mOD 260nm, 33.2 μmol; obtained as previously described and stored in TEAB at -20 °C)

[12] The residue was evaporated to dryness and suspended in DMF (890 μL). Then, ZnCl (24.1 mg, 177 μmol) and Im-p Bn6 A m PG (19 mg) was added and the mixture was stirred at room temperature for 2 h. The reaction was quenched by adding a solution of Na2EDTA (72.5 mg) and NaHCO3 (36 mg) in water (3.6 mL), and the product was isolated by ion exchange chromatography on DEAE Sephadex (gradient elution, 0-1.2 M TEAB) and evaporated to m 7 Gpp S p Bn6 A m The triethylammonium salt of pG was obtained. Further purification by RP HPLC (C18) using a linear gradient of acetonitrile in aqueous buffer of CH3COONH4, pH 5.9, gave (after repeated lyophilization from water) m 7 GppSp Bn6 A m P-diastereomeric mixture of pG (217 mOD 260nm , 6.8 μmol, 31%) was obtained as the ammonium salt.

[0080] m 7 Gpp S p Bn6 A m pG:RP HPLC (gradient elution, 0–50% MeOH in CH3COONH4, pH 5.9, 7.5 min, then isocratic):R t =8.311 min;HRMS ESI(-): m / z 1250.17041 (C 39 H 48 N 15 O 23 P4S- [MH]- calculated value 1250.17241); 1 H NMR (500 MHz, D2O, TMS, 25℃): δ = 9.13 (s, 1H, H8 m7G ), 8.55 (s, 1H, H8 A ), 8.22 (s, 1H, H2 A ), 8.07 (s, 1H, H8G ), 7.41-7.25 (m, 5H, ArH Bn ), 6.05 (2x d, 3 J H,H = 5.9 Hz, 1H, H1' A ), 5.91 (2x d, 3 J H,H = 3.7 Hz, 1H, H1' m7G ), 5.85 (d, 3 J H,H = 5.6 Hz, 1H, H1' G ), 5.00 (m, 1H, H3' A ), 4.81 (m, 1H, H2' G Overlapping with HDO), 4.85-4.73 (m, 2H, CH 2, Bn , overlapping with HDO), 4.63 (m, 1H, H2' m7G ), 4.58-4.49 (m, 4H, H2' A , H3' m7G , H3' G , H4' A ), 4.42-4.16 (m, 8H, H4' m7G , H4' G , H5' m7G , H5'' m7G , H5' A , H5'' A , H5' G , H5'' G ), 4.04-4.01 (2x s, 3H, CH 3, m7G ), 3.45-3.43 (2x s, 3H, CH 3, 2'-O ) ppm; 31 P NMR (202.5 MHz, D2O, H3PO4, 25℃): δ = 30.92 (m, 1P, P β ), 0.06-0.01 (2x s, 1P, P A-G ), -11.45 (m, 2P, P α , P γ ) ppm;

[0081] Example 5 5'-phosphorothiolate trinucleotide cap analog m 7 Gppp 5'S,Bn6 A m Synthesis of pG Process 1.p Bn6,5'S A m Synthesis of pG: The synthesis of the dinucleotide was carried out manually using a 10 mL syringe equipped with a fritted filter. iBu 3'-lcaa PrimerSupport 5G (GE Healthcare, 308 μmol / g, 650 mg) was placed in a syringe and washed with dry acetonitrile. For the coupling step, 1.2 to 1.5 equivalents of 3'-lcaa dissolved in 1 mL of anhydrous acetonitrile were added. Bn6 A m The phosphoramidite and 1.5 mL of 0.30 M 5-(benzylthio)-1-H-tetrazole in acetonitrile were shaken for 30 min in a plunger-capped syringe. A solution of 3% (v / v) trichloroacetic acid in dichloromethane was used as the detritylation reagent, and pyridine / water (9:1) was used for oxidation. v / v 0.05 M iodine in DMF was used. After the final cycle of synthesis, the support was treated with 20% (v / v) diethylamine in acetonitrile to remove the 2-cyanoethyl protecting group, washed with acetonitrile, and dried with argon. The dinucleotide, still on the solid support, was then converted to its 5'-iodine derivative by shaking the support with a solution of triphenoxymethylphosphonium iodide (1.08 g) in DMF (5 mL) for 15 min. The resin was then washed with DMF (15 mL) and acetonitrile (50 mL), dried, and transferred to a flask containing a cold solution of triethylammonium thiophosphate (approximately 0.15 M) and triethylamine (0.30 M) in DMF (1 mL). The slurry was shaken overnight at 2-4 °C, filtered, and washed with DMF (15 mL) and then acetonitrile (50 mL). The product was cleaved and deprotected with AMA (methylamine / ammonium hydroxide, 1:1). v / vThe product was deprotected using a 500-molecular-weight fraction (55°C, 1 hour) and isolated by ion exchange chromatography on DEAE Sephadex (gradient elution, 0-0.9 M TEAB). After evaporation, the product was purified by precipitation. 5'S,Bn6 A m The triethylammonium salt of pG was obtained (2400 mOD, 88.5 μmol).

[0082] Process 2.p Bn6,5'S A m Activation of pG: Dinucleotide 5'-phosphorothiolate p Bn6,5'S A m pG(2400mOD 260nm , 88.5 μmol) was dissolved in DMF (1.80 mL), followed by the addition of imidazole (96.3 mg, 1.42 mmol), 2,2'-dithiodipyridine (117 mg, 531 μmol), triethylamine (74.2 μL, 531 μmol), and triphenylphosphine (139 mg, 531 μmol). The mixture was stirred at room temperature for 2 hours. The product was precipitated by the addition of sodium perchlorate (108 mg, 885 μmol) in a cold acetonitrile solution (20 mL). The precipitate was centrifuged at 4 °C, washed three times with cold acetonitrile, and dried under reduced pressure to give the dinucleotide P-imidazolide Im-p bn6 A m The sodium salt of pG was obtained (92.2 mg).

[0083] Step 3. Triphosphate bridge formation: 7-methylguanosine-5'-diphosphate (m 7 GDP;1210mOD260 nm , 106 μmol) and Im-p 5'S,Bn6 A m pG (92.2 mg) was suspended in DMF (1.77 mL). ZnCl (144 mg, 1.06 mmol) was then added, and the mixture was stirred at room temperature for 2 h. The reaction was quenched by adding a solution (3 mL) of NaEDTA (20 mg / mL) and NaHCO (10 mg / mL) in water, and the product was isolated by ion-exchange chromatography on DEAE Sephadex (gradient elution, 0–1.2 M TEAB). After evaporation, the 2′-O-TBDMS protective cap was removed. 7 Gppp5'S,Bn6 A m p.g. TBDMS m mixed in 7 Gppp 5'S,Bn6 A m The triethylammonium salt of pG was obtained. To remove the TBDMS group, the solid was dissolved in DMSO (150 μL) and TEA (133 μL), followed by the addition of TEA·3HF (78 μL). The mixture was shaken at 60 °C for 1 h, and the reaction was quenched by the addition of water (15 mL). The product was isolated by ion-exchange chromatography on DEAE Sephadex (gradient elution, 0–1.2 M TEAB) and further purified by RP HPLC (C18) using a linear gradient of acetonitrile in aqueous buffer of CH3COONH4, pH 5.9, to give m (after repeated lyophilization from water). 7 Gppp 5'S,Bn6 A m The ammonium salt of pG was obtained (1530 mOD, 47.7 μmol).

[0084] m 7 Gppp 5'S,Bn6 A m pG:RP HPLC (gradient elution, 0–50% MeOH in CH3COONH4, pH 5.9, 7.5 min, then isocratic):R t =8.496 min;HRMS ESI(-): m / z 1250.17363 (C 39 H 48 N 15 O 23 P4S- [MH]- calculated value 1250.17241); 1 H NMR (500 MHz, D2O, TMS, 25℃): δ = 9.08 (s, 1H, H8 m7G ), 8.37 (s, 1H, H8 A ), 8.22 (s, 1H, H2 A ), 8.11 (s, 1H, H8 G ), 7.43-7.25 (m, 5H, ArH Bn ), 6.00 (d, 3 J H,H = 5.8 Hz, 1H, H1' A ), 5.89 (d,3 J H,H = 2.9 Hz, 1H, H1' m7G ), 5.86 (d, 3 J H,H = 5.5 Hz, 1H, H1' G ), 4.86 (m, 1H, H3' A ), 4.80 (m, 1H, H2' G , overlapping with HDO), 4.78 (m, 2H, CH 2, Bn , overlapping with HDO), 4.58 (m, 1H, H2' m7G ), 4.56-4.49 (m, 3H, H2' A , H3' G , H4' A ), 4.47 (m, 1H, H3' m7G ), 4.42-4.18 (m, 6H, H4' m7G , H4' G , H5' m7G , H5'' m7G , H5' G , H5'' G ), 4.02 (s, 3H, CH 3, m7G ), 3.40 (s, 3H, CH 3, 2'-O ), 3.33 (m, 2H, H5' A , H5'' A ) ppm; 31 P NMR (202.5 MHz, D2O, H3PO4, 25℃): δ = 8.19 (m, 1P, P α ), -0.09 (s, 1P, P A-G ), -10.59 (m, 1P, P γ ), -22.67 (m, 1P, P β ) ppm;

[0085] Example 6 Tetranucleotide cap analogs (m 7 GpppBn 6 A m Synthesis of pGpG Step 1. Synthesis of Im-pBn6AmpGpG: Bn6 A mpGpG triethylammonium salt (5366 mOD 260nm (138 μmol) was dissolved in DMF (2800 μL), followed by the addition of imidazole (150 mg, 2201 μmol), 2,2'-dithiodipyridine (182 mg, 826 μmol), triethylamine (84 μL, 826 μmol), and triphenylphosphine (216 mg, 826 μmol). The mixture was stirred at room temperature for 2 hours, and then the product was precipitated by the addition of a cold solution of sodium perchlorate (169 mg, 1376 μmol) in acetonitrile. The precipitate was centrifuged at 4°C, washed three times with cold acetonitrile, dried over PO under reduced pressure, and purified by Im-p Bn6 A m The sodium salt of pGpG was obtained (183 mg).

[0086] Im-p Bn6 A m pGpG: RP-HPLC (gradient elution, 0–50% MeOH in CH3COONH4, pH 5.9, 7.5 min, then isocratic): R t =8.865 minutes;

[0087] Process 2.m 7 Gppp Bn6 A m Synthesis of pGpG: Im-p bn6 A m pGpG sodium salt (5366 mOD 260nm , 138 μmol) was dissolved in DMF (2750 μL), and then m7GDP (1882 mOD 260nm , 99 mg, 165 μmol) and anhydrous ZnCl2 (225 mg, 1651 μmol) were added. The mixture was stirred at room temperature for 1 h, and then the reaction was quenched by adding Na2EDTA (676 mg) and NaHCO3 (338 mg) in water (27.5 mL). The product was isolated by ion-exchange chromatography on DEAE Sephadex (gradient elution, 0-1.2 M TEAB) and purified by RP HPLC (C18) using a linear gradient of acetonitrile in aqueous buffer of CH3COONH4, pH 5.9 (after repeated evaporation and lyophilization from water). 7 Gppp Bn6 Am The ammonium salt of pGpG was obtained (2151 mOD 260nm , 53.2 μmol, 27%). Yields were estimated by UV absorbance at 260 nm assuming an extinction coefficient ε = 40.5 L / mmol / cm).

[0088] m 7 Gppp Bn6 A m pGpG: RP-HPLC (gradient elution, 0–50% MeOH in CH3COONH4, pH 5.9, 7.5 min, then isocratic): R t =7.840 min;HRMS ESI(-): m / z 1579.24313 (C 49 H 60 N 20 O 31 P5- [MH]- calculated value 1579.24269); 1 H NMR (500 MHz, D2O, TMS, 70℃) δ = 9.55 (s, 1H), 8.96 (s, 1H), 8.72 (s, 1H), 8.55 (s, 1H), 8.53 (s, 1H), 7.92 - 7.76 (m, 6H), 6.50 (d, J = 6.2 Hz, 1H), 6.39 (d, J = 4.1 Hz, 1H), 6.33 (d, J = 5.4 Hz, 1H), 6.28 (d, J = 5.9 Hz, 1H), 5.42 - 5.31 (m, 5H), 5.30 - 5.25 (m, 1H), 5.18 (t, J = 5.3 Hz, 1H), 5.10 (t, J = 4.6 Hz, 1H), 4.98 - 4.88 (m, 6H), 4.73 - 4.61 (m, 7H), 4.51 (s, 3H), 3.82 (s, 3H) ppm; 31 P NMR { 1 H} (203 MHz, D2O, H3PO4, 70℃) δ = 0.82 (s, 1P), 0.40 (s, 1P), -10.06 (d, J = 19.0, 1P), -10.18 (d, J = 18.4 Hz, 1P), -21.52 (dd, J = 19.0, 18.4 Hz) ppm;

[0089] Example 7 Preparation of capped mRNA by in vitro transcription mRNA encoding Gaussia luciferase was generated using the pJET_T7_Gluc_128A plasmid digested with the restriction enzyme AarI (ThermoFisher Scientific) as a template. The plasmid was obtained by cloning the T7 promoter sequence and Gaussia luciferase coding sequence into pJET_luc_128A.

[13] A typical in vitro transcription reaction (20 μl) was incubated at 37°C for 2 hours and contained: RNA Pol buffer (40 mM Tris-HCl, pH 7.9, 10 mM MgCl2, 1 mM DTT, 2 mM spermidine), 10 U / μl T7 RNA polymerase, 1 U / μl RiboLock RNase inhibitor, 2 mM ATP / CTP / UTP, 0.5 mM GTP, 3 mM cap analog of interest, and 50 ng / μl digested plasmid as template. After 2 hours of incubation, 1 U / μl of DNase I was added, and incubation continued at 37°C for 30 minutes. Crude mRNA was purified using NucleoSpin Clean-up XS (Macherey-Nagel). The quality of the transcripts was checked on a native 1.2% 1x TBE agarose gel, and the concentration was determined spectrophotometrically. To remove uncapped RNA, the transcripts were treated with 5'-polyphosphatase (Epicentre) and Xrn1 (New England Biolabs). Briefly, mRNA was incubated with 5'-polyphosphatase (20 U / 5 μg of mRNA) in a proprietary buffer at 37°C for 30 minutes, after which the mRNA was purified using NucleoSpin RNA Clean-up XS. The purified mRNA was incubated with Xrn-1 (1 U / 1 μg of mRNA) in a dedicated buffer at 37°C for 60 minutes, and then the mRNA was purified using NucleoSpin RNA Clean-up XS.

[0090] Example 8 Purification of capped mRNA using HPLC Transcripts were purified using an Agilent Technologies Series 1200 HPLC with an RNASep™ Prep - RNA Purification Column (ADS Biotec). Separations were performed at 55°C as described

[14] . A linear gradient (Gradient A) of 35% to 55% Buffer B (0.1 M triethylammonium acetate, pH 7.0, and 25% acetonitrile) in Buffer A (0.1 M triethylammonium acetate, pH 7.0) over 22.1 min at a flow rate of 0.9 ml / min was used. Alternatively, a linear gradient (Gradient B) of 17.5% to 25.8% Buffer C (0.1 M triethylammonium acetate, pH 7.0, and 50% acetonitrile) in Buffer A (0.1 M triethylammonium acetate, pH 7.0) over 20 min at a flow rate of 0.9 ml / min was used. The retention times of the mRNAs according to the present invention, compared to uncapped mRNAs of the same sequence or mRNAs capped with reference analogs, are summarized in Table 1. After purification, mRNA molecules were recovered from the collected fractions by precipitation with isopropanol. The quality of the transcripts was verified on a native 1.2% 1x TBE agarose gel, and their concentrations were determined spectrophotometrically.

[0091] Importantly, use of the purification procedure described above resulted in the production of novel cap analogs (m 7 GpppA * The gradient A allowed the separation of Gaussia luciferase-encoding mRNA with capped RNA (pG-RNA) from uncapped RNA (ppp-RNA) of the same sequence. The retention time at which mRNA elution occurred depended on the type of substituent at the N6-adenosine position (Table 1). For example, the gradient A allowed the separation of the mRNA of the analyzed m 7 Gppp Bn6 A m The retention times of the pG-RNAGluc and ppp-RNAGluc transcripts were 19.4 and 18.2 min, respectively.

[0092] [Table 4]

[0093] Example 9 Protein expression analysis 3T3-L1 (mouse embryonic fibroblast-like cells, ATCC CL-173) were grown in DMEM (Gibco) supplemented with 10% FBS (Sigma), GlutaMAX (Gibco), and 1% penicillin / streptomycin (Gibco) at 5% CO2 and 37°C. The mouse immature dendritic cell line JAWS II (ATCC CRL-11904) was grown in RPMI 1640 (Gibco) supplemented with 10% FBS, sodium pyruvate (Gibco), 1% penicillin / streptomycin, and 5 ng / ml GM-CSF (PeproTech) at 5% CO2 and 37°C. In a typical experiment, 10 4 JAWS II cells and 4 x 10 3 3T3-L1 cells were seeded in 100 μl of antibiotic-free medium per well of a 96-well plate. Cells in each well were transfected for 16 hours using 10 μl of Opti-MEM (Gibco) mixed with 0.3 μl of Lipofectamine MessengerMAX Transfection Reagent (Invitrogen) and 25 ng of mRNA. To assess Gaussia luciferase expression at multiple time points, the medium was completely removed and replaced with fresh medium at each time point. To detect luminescence from Gaussia luciferase, 50 μl of 10 ng / ml h-coelenterazine (NanoLight) in PBS was added to 10 μl of cell culture medium, and luminescence was measured using a Synergy H1 (BioTek) microplate reader.

[0094] conclusion Examples 1-6 describe methods for preparing trinucleotide- and tetranucleotide-capped analogs according to the invention. Realizations of the invention whose synthesis is not described in the examples can be carried out by methods identical or very similar to those exemplified.

[0095] Examples 7 and 8 describe methods for the preparation and purification of mRNA obtained with the use of compounds according to the invention under conditions that allow the separation of capped from uncapped mRNA.

[0096] Figure 1 shows a representative chromatogram demonstrating such separation, compared with the corresponding results obtained for mRNA obtained using an unmodified reference trinucleotide. As demonstrated, it is not possible to separate capped and uncapped mRNA under identical conditions. Table 1 compares the chromatographic properties of mRNA modified with various cap analogs. mRNA modified with the cap analogs of the present invention eluted from the HPLC column with a longer retention time than mRNA that was uncapped or terminated with a reference cap analog known from the prior art. This suggests that the introduction of an appropriately hydrophobic substituent at the N6-adenosine position facilitates the purification of mRNA from uncapped contaminants. Furthermore, analysis of the data shown in Figure 1 (integration of the corresponding signals) allowed the calculation of the capping efficiency of mRNA obtained with the compounds of the present invention, which was 91.5%. This suggests that when using the capped trinucleotide analogs of the present invention, it is possible to obtain capping efficiencies comparable to those obtained with unmodified nucleotides of the present invention (capping efficiencies of approximately 90% obtained in the literature under similar conditions

[12] ).

[0097] Example 9 describes a method for analyzing protein expression in mammalian cells from mRNA according to the invention obtained using the compounds according to the invention. The analysis was carried out in two cell lines (fibroblasts - 3T3-L and dendritic cells - JAWS II) representing cells of different origins. In the case of mRNA obtained using the compounds according to the invention, enzymatic treatment was not carried out because it would be redundant.

[0098] The mRNA obtained with the use of the compounds according to the invention showed increased protein expression levels compared to the mRNA obtained with cap analogs representative of the prior art in at least one of the experimental variants tested.

[0099] Achieving increased protein expression has many applications in biotechnology and biopharmaceutical manufacturing (manufacturing human recombinant proteins) as well as mRNA-based gene therapy. Increasing protein expression in dendritic cells is particularly beneficial for therapeutic anti-cancer vaccine applications.

[15]

[0100] Increasing protein expression in cells from other tissues (eg, lung, liver, and other organs) is particularly beneficial for gene replacement therapy applications.

[16]

[0101] The therapeutic effect of mRNA according to the present invention can be expected to be achieved at a lower dose compared to mRNA obtained using prior art methods. Lowering the mRNA dosage reduces the risk of side effects associated with the toxicity of therapeutic mRNA and improves the probability of successful treatment. Furthermore, preparing mRNA using compounds according to the present invention is expected to enable the production of mRNA free of unwanted mRNA triphosphate impurities. (References) 1. Moore, M., From birth to death: The complex lives of eukaryotic mRNAs. Science 2005, 309 (5740), 1514-1518. 2. Ziemniak, M.; Strenkowska, M.; Kowalska, J.; Jemielity, J., Potential therapeutic applications of RNA cap analogs. Future Medicinal Chemistry 2013, 5 (10), 1141-1172. 3. Grudzien-Nogalska, E.; Stepinski, J.; Jemielity, J.; Zuberek, J.; Stolarski, R.; Rhoads, R. E.; Darzynkiewicz, E., Synthesis of anti-reverse cap analogs (ARCAs) and their applications in mRNA translation and stability. Translation Initiation: Cell Biology, High-Throughput Methods, and Chemical-Based Approaches 2007, 431, 203-227. 4. Sahin, U.; Kariko, K.; Tureci, O., mRNA-based therapeutics - developing a new class of drugs. Nature Reviews Drug Discovery 2014, 13 (10), 759-780. 5. Stepinski, J.; Waddell, C.; Stolarski, R.; Darzynkiewicz, E.; Rhoads, R. E., Synthesis and properties of mRNAs containing the novel "anti-reverse" cap analogs 7-methyl(3'-O-methyl)GpppG and 7-methyl(3'-deoxy)GpppG. Rna-a Publication of the Rna Society 2001, 7 (10), 1486-1495. 6. Jemielity, J.; Fowler, T.; Zuberek, J.; Stepinski, J.; Lewdorowicz, M.; Niedzwiecka, A.; Stolarski, R.; Darzynkiewicz, E.; Rhoads, R. E., Novel "anti-reverse" cap analogs with superior translational properties. Rna-a Publication of the Rna Society 2003, 9 (9), 1108-1122. 7. Jemielity, J.; Kowalska, J.; Rydzik, A. M.; Darzynkiewicz, E., Synthetic mRNA cap analogs with a modified triphosphate bridge - synthesis, applications and prospects. New Journal of Chemistry 2010, 34 (5), 829-844. 8. Grudzien-Nogalska, E.; Jemielity, J.; Kowalska, J.; Darzynkiewicz, E.; Rhoads, R. E., Phosphorothioate cap analogs stabilize mRNA and increase translational efficiency in mammalian cells. Rna-a Publication of the Rna Society 2007, 13 (10), 1745-1755. 9. Kuhn, A. N.; Diken, M.; Kreiter, S.; Selmi, A.; Kowalska, J.; Jemielity, J.; Darzynkiewicz, E.; Huber, C.; Tureci, O.; Sahin, U., Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo. Gene Therapy 2010, 17 (8), 961-971. 10. Grudzien, E.; Kalek, M.; Jemielity, J.; Darzynkiewicz, E.; Rhoads, R. E., Differential inhibition of mRNA degradation pathways by novel cap analogs. Journal of Biological Chemistry 2006, 281 (4), 1857-1867. 11. Sikorski, Pawel J; Warminski, Marcin; Kubacka, Dorota; Ratajczak, Tomasz; Nowis, Dominika; Kowalska, Joanna; Jemielity, Jacek; The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5' cap modulates protein expression in living cells. Nucleic Acids Research 2020, 305-1048. 12. J. Kowalska, M. Lewdorowicz, J. Zuberek, E. Grudzien-Nogalska, E. Bojarska, J. Stepinski, RE Rhoads, E. Darzynkiewicz, RE Davis, J. Jemielity, RNA 2008, 14, 1119-1131. 13. M. Warminski, PJ Sikorski, Z. Warminska, M. Lukaszewicz, A. Kropiwnicka, J. Zuberek, E. Darzynkiewicz, J. Kowalska, J. Jemielity, Bioconjugate Chemistry 2017, 28, 1978-1992. 14. D. Weissman, N. Pardi, H. Muramatsu, and K. Kariko, Methods Mol Biol 2013, 969, 43-54. 15. Norbert Pardi, Michael J. Hogan, Frederick W. Porter, Drew Weissman, N. Nature Reviews Drug Discovery vol. 17, 261-279 (2018). 16. Berraondo P, Martini PGV, Avila MA, et al Messenger RNA therapy for rare genetic metabolic diseases Gut 2019;68:1323-1330. 17. Anna Maria Rydzik et. al., Organic & Biomolecular Chemistry, Issue 22, 2009. 18. M. Kalek et al., Bioorganic & Medicinal Chemistry, Vol. 14, Issue 9, 1 May 2006, Pages 3223-3230 19. J. Kowalska et.al. (2009), Phosphoroselenoate Dinucleotides for Modification of mRNA 5' End. ChemBioChem, 10: 2469-2473. 20. J. Kowalska, et. al., Nucleic Acids Research, Volume 42, Issue 16, 15 September 2014, Pages 10245-10264, 21. A. Rydzik et.al., Nucleic Acids Research, Volume 45, Issue 15, 6 September 2017, Pages 8661-8675,

Claims

1. formula: 【Chemistry 1】 (In the formula, R 1 , R 2 , R 3 and R 4 is H, CH 3 and alkyl, wherein the R substituents with different numbers may be the same or different; n is 0 or 1, R 5 is benzyl, substituted benzyl, particularly mono- or di-substituted benzyl, preferably benzyl selected from the group consisting of substituted benzyl substituted with one of the following substituents: chlorine, fluorine, bromine, iodine, methyl, alkyl, nitro, carboxyl, azido, amine, hydroxyl, or a combination thereof, 1-naphthylmethyl, 2-naphthylmethyl, substituted naphthylmethyl, alkylaryl, aryl; base 1 and bases 2 teeth 【Chemistry 2】 are independently selected from the group consisting of X 1 , X 3 is selected from the group consisting of O, S, Se, and the substituents X with different numbers may be the same or different; X 2 , X 4 , and X 5 O, S, Se, BH 3 wherein the X substituents with different numbers may be the same or different; X 6 is O, CH 2 , CF 2 , CCl 2 is selected from the group consisting of In the formula, preferably R 5 is benzyl, monosubstituted benzyl, disubstituted benzyl, 1-naphthylmethyl, or 2-naphthylmethyl; and X 1 , X 4 , X 5 , X 6 is O and X 2 , X 3 is O or S, and R 3 , R 4 is H) Compound.

2. formula: 【Transformation 3】 Compound m 7 Gppp Bn6 A m p.g., formula: 【Chemistry 4】 Compound m 7 Gppp 2MeBn6 A m p.g., formula: 【Transformation 5】 Compound m 7 Gppp 3MeBn6 A m p.g., formula: 【Transformation 6】 Compound m 7 Gppp 4MeBn6 A m p.g., formula: 【Transformation 7】 Compound m 7 Gppp 4FBn6 A m p.g., formula: 【Transformation 8】 Compound m 7 Gppp 3,4diFBn6 A m p.g., formula: 【Chemistry 9】 Compound m 7 Gppp 1Naphm6 A m p.g., formula: 【Chemistry 10】 Compound m 7 Gppp 2Naphm6 A m p.g., formula: 【Chemistry 11】 Compound m 7 Gpp S p Bn6 A m p.g., formula: 【Chemistry 12】 Compound m 7 Gppp 5'S,Bn6 A m p.g., formula: 【Chemistry 13】 Compound m 7 Gppp Bn6 A m pGpG 2. The compound of claim 1, selected from the group consisting of:

3. 10. The compound of claim 1, consisting essentially of a single stereoisomer or comprising a mixture of at least two stereoisomers, a first diastereomer and a second diastereomer, which diastereomers are otherwise identical except for having different stereochemical configurations about a stereogenic phosphorus atom, which is bonded to a sulfur atom, a selenium atom, or a borane group.

4. 4. An RNA molecule incorporating a compound according to any one of claims 1 to 3 at its 5' end.

5. 10. A method for synthesizing in vitro an RNA molecule according to claim 4, comprising reacting ATP, CTP, UTP, GTP, a compound according to claims 1-3, and a polynucleotide template in the presence of an RNA polymerase under conditions conducive to transcription of the polynucleotide template by the RNA polymerase into RNA copies, some of the RNA copies incorporating a compound according to any one of claims 1-3, thereby producing the RNA molecule according to claim 4.

6. 10. A method for synthesizing a protein or peptide in vitro, comprising translating an RNA molecule of claim 4 in a cell-free protein synthesis system, wherein the RNA molecule comprises an open reading frame, under conditions that promote translation of the open reading frame of the RNA molecule into a protein or peptide encoded by the open reading frame.

7. 10. A method for synthesizing a protein or peptide in a living cell, comprising the step of introducing into the cell the RNA molecule of claim 4, wherein the RNA molecule comprises an open reading frame, under conditions that promote translation of the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame, wherein the cell is not contained within a patient's body.

8. A method for purifying the molecule of claim 4, comprising the steps of using a chromatographic method, preferably a reverse-phase HPLC method, wherein the column is equilibrated, a sample containing the molecule of claim 4 is introduced into the chromatographic column, the components of the sample are separated in a buffered aqueous / organic solvent system, fractions containing the molecule of claim 4 are collected, and the mRNA molecule of claim 4 is separated from other RNA molecules that do not have the structure defined in any one of claims 1 to 3 at their 5' ends.

9. 10. Use of a compound according to any one of claims 1 to 3 in the in vitro synthesis of RNA molecules.

10. 10. Use of an RNA molecule according to claim 4 in the in vitro synthesis of a protein or peptide.

11. A compound according to any one of claims 1 to 3 or an RNA molecule according to claim 4 for use in medicine, pharmacology or diagnostics.

Images