Cap analogs and uses thereof

Abstract

Cap analogs are provided.SOLUTION: A cap analog represented by formula (I): Wherein B1 and B2 are each a natural or modified base; E and F are each 0 or 1; R1 is H, OH, alkyl, O-alkyl, halogen, or is oxygen and the oxygen forms a bridged ring with C at the 2' and 4' positions; R2 is H, OH, an alkyl group, or an O-alkyl group; R3 is OCH2CH3, OCH2OCH3, or OCH2CH2CH3; R4 is hydrogen or a hydroxyl group; X1, X2 and X3, Y1, Y2 and Y3 are each O.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to the fields of chemical and biological engineering, and more specifically to a series of cap analogs useful for mRNA synthesis by in vitro transcription (IVT), and the use of cap analogs in mRNA synthesis. [Background technology]

[0002] In vitro transcription of mRNA has become an important tool for introducing exogenous genes to express proteins, and has been widely applied in the treatment and prevention of diseases. The immune response induced by exogenous mRNA affects the expression of target proteins through different pathways.

[0003] N1-2'O-methylation is crucial for the inhibition of RIG-I activation, and knockout of N1-2'O-methyltransferase in cells also leads to immune stimulation of endogenous RNA. 7 The synergistic effect of the G-cap reduces the binding affinity between RNA and RIG-I.

[0004] During mRNA transcription, IFIT1 and the translation factor EIF4E can competitively bind to mRNA templates. Endogenous and exogenous mRNAs bearing cap structures and 2'O-methylation are bound to EIF4E, initiating transcription. However, if the first silipopeptide residue of an exogenous mRNA lacks 2'O-methylation, it is recognized by IFIT1, preventing its binding to cellular factors and inhibiting transcriptional progression. However, N1 methylation alone is not sufficient to protect all endogenous mRNAs from IFIT1, and IFIT1 recognition of 2'O-methylation at the N1 and N2 silipopeptides indicates a competition between the methyl group and these protein residues.

[0005] Therefore, there is a strong demand in this field for the development of novel cap analogs that can effectively suppress the occurrence of immune responses caused by mRNA synthesized by in vitro transcription. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention aims to provide a novel cap analog having a structure represented by general formula (I) and uses thereof. [Means for solving the problem]

[0007] A first aspect of the present invention provides a cap analogue represented by formula (I): [ka] wherein B1 and B2 are each a natural or modified base; E and F are each 0 or 1; R1 is H, OH, an alkyl group, an O-alkyl group, a halogen atom, or an oxygen atom, and the oxygen atom forms a bridge with the C atoms at the 3' and 5' positions; R2 is H, OH, an alkyl group, an O-alkyl group, or a halogen; R3 is O-R5-R6; R4 is hydrogen, a hydroxy group, an O-methyl group, or O-R5-R6; R5 is a substituted or unsubstituted C 1-20 is an alkyl group; R6 is a substituted or unsubstituted O-alkyl group, a substituted or unsubstituted S-alkyl group, a substituted or unsubstituted NH-alkyl group, a substituted or unsubstituted N-dialkyl group, a substituted or unsubstituted O-aryl group, a substituted or unsubstituted S-aryl group, a substituted or unsubstituted NH-aryl group, a substituted or unsubstituted O-aralkyl group, a substituted or unsubstituted S-aralkyl group, a substituted or unsubstituted NH-aralkyl group, or hydrogen (when R5 is a substituted or unsubstituted C 2-20 alkyl group); X1, X2 and X3 are each O, CH2 or NH; Y1, Y2 and Y3 are O, S, Se or BH3, respectively.

[0008] In another embodiment, R3 is OCH2CH3, OCH2OCH3, or OCH2CH2OCH3.

[0009] In another embodiment, R4 is a hydroxy group, OCH2CH3, OCH2OCH3, or OCH2CH2OCH3.

[0010] In another embodiment, B1 and B2 are each adenine, N6-methyladenine, guanine, uracil, or thymine.

[0011] In another embodiment, the cap analog is m7 GpppA 2’O-ethyl p.g., m7 GpppA 2’O-ethyl pA, m7 GpppA 2’O-ethyl p.c., m7 GpppA 2’O-ethyl pU, m7 GpppC 2’O-ethyl pA, m7 GpppC 2’O-ethyl p.g., m7 GpppC 2’O-ethyl p.c., m7 GpppC 2’O-ethyl pU, m7 GpppG 2’O-ethyl pA, m7 GpppG 2’O-ethyl p.c., m7 GpppG 2’O-ethyl p.g., m7 GpppG 2’O-ethyl pU, m7 GpppU 2’O-ethyl pA, m7 GpppU 2’O-ethyl p.c., m7 GpppU 2’O-ethyl PC, or m7 GpppU 2’O-ethyl pU.

[0012] In another embodiment, the cap analog is m7 G 3’Ome pppA 2’O-ethyl p.g., m7 G3’Ome pppA 2’O-ethyl pA, m7 G 3’Ome pppA 2’O-ethyl p.c., m7 G 3’Ome pppA 2’O-ethyl pU, m7 G 3’Ome pppC 2’O-ethyl pA, m7 G 3’Ome pppC 2’O-ethyl p.g., m7 G 3’Ome pppC 2’O-ethyl p.c., m7 G 3’Ome pppC 2’O-ethyl pU, m7 G 3’Ome pppG 2’O-ethyl pA, m7 G 3’Ome pppG 2’O-ethyl p.c., m7 G 3’Ome pppG 2’O-ethyl p.g., m7 G 3’Ome pppG 2’O-ethyl pU, m7 G 3’Ome pppU 2’O-ethyl pA, m7 G 3’Ome pppU 2’O-ethyl p.c., m7 G 3’Ome pppU 2’O-ethyl PC, or m7 G 3’Ome pppU 2’O-ethyl pU.

[0013] In another embodiment, the cap analog is m7 G 2’Ome pppA 2’O-ethyl p.g., m7 G 2’Ome pppA 2’O-ethyl pA, m7 G 2’Ome pppA 2’O-ethyl p.c., m7 G 2’Ome pppA 2’O-ethyl pU, m7 G 2’Ome pppC 2’O-ethylpA, m7 G 2’Ome pppC 2’O-ethyl p.g., m7 G 2’Ome pppC 2’O-ethyl p.c., m7 G 2’Ome pppC 2’O-ethyl pU, m7 G 2’Ome pppG 2’O-ethyl pA, m7 G 2’Ome pppG 2’O-ethyl p.c., m7 G 2’Ome pppG 2’O-ethyl p.g., m7 G 2’Ome pppG 2’O-ethyl pU, m7 G 2’Ome pppU 2’O-ethyl pA, m7 G 2’Ome pppU 2’O-ethyl p.c., m7 G 2’Ome pppU 2’O-ethyl PC, or m7 G 2’Ome pppU 2’O-ethyl pU.

[0014] In another embodiment, the cap analog is m7 Gppp (N6-methyladenine) 2’O-ethyl p.g., m7 Gppp (N6-methyladenine) 2’O-ethyl pA, m7 Gppp (N6-methyladenine) 2’O-ethyl p.c., m7 Gppp (N6-methyladenine) 2’O-ethyl pU, m7 G 2’Ome ppp (N6-methyladenine) 2’O-ethyl pA, m7 G 2’Ome ppp (N6-methyladenine) 2’O-ethyl p.g., m7 G 2’Ome ppp (N6-methyladenine) 2’O-ethyl p.c., m7 G 2’Ome ppp (N6-methyladenine)2’O-ethyl pU, m7 G 3’Ome ppp (N6-methyladenine) 2’O-ethyl pA, m7 G 3’Ome ppp (N6-methyladenine) 2’O-ethyl p.c., m7 G 3’Ome ppp (N6-methyladenine) 2’O-ethyl PC, or m7 G 3’Ome ppp (N6-methyladenine) 2’O-ethyl pU.

[0015] In another embodiment, the cap analog is m7 GpppA 2’O-MOE p.g., m7 GpppA 2’O-MOE pA, m7 GpppA 2’O-MOE p.c., m7 GpppA 2’O-MOE pU, m7 GpppC 2’O-MOE pA, m7 GpppC 2’O-MOE p.g., m7 GpppC 2’O-MOE p.c., m7 GpppC 2’O-MOE pU, m7 GpppG 2’O-MOE pA, m7 GpppG 2’O-MOE p.c., m7 GpppG 2’O-MOE p.g., m7 GpppG 2’O-MOE pU, m7 GpppU 2’O-MOE pA, m7 GpppU 2’O-MOE p.c., m7 GpppU 2’O-MOE PC, or m7 GpppU 2’O-MOE pU.

[0016] In another embodiment, the cap analog is m7 G 3’Ome pppA 2’O-MOE p.g.,m7 G 3’Ome pppA 2’O-MOE pA, m7 G 3’Ome pppA 2’O-MOE p.c., m7 G 3’Ome pppA 2’O-MOE pU, m7 G 3’Ome pppC 2’O-MOE pA, m7 G 3’Ome pppC 2’O-MOE p.g., m7 G 3’Ome pppC 2’O-MOE p.c., m7 G 3’Ome pppC 2’O-MOE pU, m7 G 3’Ome pppG 2O-’MOE pA, m7 G 3’Ome pppG 2’O-MOE p.c., m7 G 3’Ome pppG 2’O-MOE p.g., m7 G 3’Ome pppG 2’O-MOE pU, m7 G 3’Ome pppU 2’O-MOE pA, m7 G 3’Ome pppU 2’O-MOE p.c., m7 G 3’Ome pppU 2’O-MOE PC, or m7 G 3’Ome pppU 2’O-MOE pU.

[0017] In another embodiment, the cap analog is m7 G 2’Ome pppA 2’O-MOE p.g., m7 G 2’Ome pppA 2’O-MOE pA, m7 G 2’Ome pppA 2’O-MOE p.c., m7 G 2’Ome pppA 2’O-MOE pU, m7 G2’Ome pppC 2’O-MOE pA, m7 G 2’Ome pppC 2’O-MOE p.g., m7 G 2’Ome pppC 2’O-MOE p.c., m7 G 2’Ome pppC 2’O-MOE pU, m7 G 2’Ome pppG 2’O-MOE pA, m7 G 2’Ome pppG 2’O-MOE p.c., m7 G 2’Ome pppG 2’O-MOE p.g., m7 G 2’Ome pppG 2’O-MOE pU, m7 G 2’Ome pppU 2’O-MOE pA, m7 G 2’Ome pppU 2’O-MOE p.c., m7 G 2’Ome pppU 2’O-MOE PC, or m7 G 2’Ome pppU 2’O-MOE pU.

[0018] In another embodiment, the cap analog is m7 Gppp (N6-methyladenine) 2’O-MOE p.g., m7 Gppp (N6-methyladenine) 2’O-MOE pA, m7 Gppp (N6-methyladenine) 2’O-MOE p.c., m7 Gppp (N6-methyladenine) 2’O-MOE pU, m7 G 2’Ome ppp (N6-methyladenine) 2’O-MOE pA, m7 G 2’Ome ppp (N6-methyladenine) 2’O-MOE p.g., m7 G 2’Ome ppp (N6-methyladenine) 2’O-MOE p.c., m7 G2’Ome ppp (N6-methyladenine) 2’O-MOE pU, m7 G 3’Ome ppp (N6-methyladenine) 2’O-MOE pA, m7 G 3’Ome ppp (N6-methyladenine) 2’O-MOE p.c., m7 G 3’Ome ppp (N6-methyladenine) 2’O-MOE PC, or m7 G 3’Ome ppp (N6-methyladenine) 2’O-MOE pU.

[0019] In another embodiment, the structure of the cap analog is represented by Formula (Ia) or Formula (Ib):

[0020] [ka]

[0021] [ka]

[0022] wherein B1 and B2 are each a natural or modified base; R3 is O-R5-R6.

[0023] In another embodiment, R3 is OCH2CH3, OCH2OCH3, or OCH2CH2OCH3.

[0024] In another embodiment, B1 and B2 are each adenine, N6-methyladenine, guanine, uracil, or thymine.

[0025] A second aspect of the present invention is a polynucleotide encoding a target polypeptide, (a) at least one ORF region; (b) at least one Kozak sequence in the 5′UTR; (c) 3'UTR; and (d) a cap analog of the invention, as described above, having at least one 5' initial cap. The present invention provides a polynucleotide comprising:

[0026] A third aspect of the present invention provides a pharmaceutical composition comprising the polynucleotide of the present invention as described above and a pharmaceutically acceptable carrier.

[0027] In another embodiment, the carrier is selected from the group consisting of lipid nanoparticles (LNP), liposomes, polymeric nanoparticles, solid lipid nanoparticles or emulsions.

[0028] A fourth aspect of the present invention provides a method for producing a polynucleotide according to the present invention as described above, comprising the steps of: (1) Prepare the DNA template: (2) The polynucleotide according to the present invention as described above is obtained by carrying out an in vitro transcription reaction in a reaction system containing RNA polymerase, nucleoside triphosphate, and the cap analog according to the present invention as described above.

[0029] In another embodiment, the nucleoside triphosphate may be a nucleoside triphosphate of a natural base, a modified nucleoside triphosphate, or an unnatural nucleoside triphosphate; preferably, ATP, CTP, GTP, UTP, 5me-CTP, 5me-UTP, PseudoUTP, or N1-me-PseudoUTP. In another embodiment, the RNA polymerase is a phage-derived RNA polymerase.

[0030] In another embodiment, the RNA polymerase is selected from the group consisting of T7, SP6, or T3.

[0031] In another embodiment, the RNA polymerase may be a mutant of a phage-derived RNA polymerase having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to the native sequence. [Effects of the Invention]

[0032] According to the present invention, a novel cap analogue can be provided that can effectively suppress the occurrence of immune responses caused by mRNA synthesized by in vitro transcription. [Brief explanation of the drawings]

[0033] [Figure 1] 1 shows a synthetic route for the cap analog according to Example 1. [Figure 2] 1 shows a synthetic route for the cap analog according to Example 1. [Figure 3] 1 shows a synthetic route for the cap analog according to Example 1. [Figure 4] 1 shows the protein expression status of different cap analog mRNAs obtained in the examples. [Figure 5] 1 shows the relative binding ability of different cap analog mRNAs obtained in the examples to RIG-I. [Figure 6] 1 shows the relative binding affinities of different cap analog mRNAs and IFIT1 obtained in the examples. [Figure 7] The results of measuring immunogenicity induction by different cap analog mRNAs obtained in the examples are shown below. [Figure 8] 1 shows the protein expression status of the cap analogues m7GBeta-D-LNApppA2′O-MOEpG and m7GAlpha-L-LNApppA2′O-ethylpG mRNA obtained in the examples. [Figure 9] 1 shows the relative binding ability of RIG-I to different cap analogues m7GBeta-D-LNApppA2′O-MOEpG and m7GAlpha-L-LNApppA2′O-ethylpG mRNA obtained in the examples. [Figure 10]1 shows the relative binding ability of IFIT1 to different cap analogs m7GBeta-D-LNApppA2′O-MOEpG and m7GAlpha-L-LNApppA2′O-ethylpG mRNA obtained in the examples. [Figure 11] 1 shows the results of measuring immunogenicity induction by different cap analogs m7GBeta-D-LNApppA2′O-MOEpG and m7GAlpha-L-LNApppA2′O-ethylpG mRNA obtained in the examples. [Figure 12] HPLC spectra of the capping efficiency of different cap analogs obtained in the examples, where A represents m7GpppApG, B represents m7GpppA2'OmepG, C represents m7G3'OmepppA2'O-ethylpG, D represents m7G2'OmepppA2'O-ethylpG, E represents m7G3'OmepppA2'O-MOEpG, F represents m7G2'OmepppA2'O-MOEpG, G represents m7Gppp(N6-methyladenine)2'O-MOEpG), H represents m7G2'OmepppC2'O-ethylpC, and I represents m7G3'OmepppA2'O-MOEpU. [Figure 13] HPLC spectra of the capping efficiency of different cap analogs obtained in the examples, where A represents m7GpppApG, B represents m7GpppA2'OmepG (control), C represents m7GBeta-D-LNApppA2'O-MOEpG, and D represents m7GAlpha-L-LNApppA2'O-ethylpG. DETAILED DESCRIPTION OF THE INVENTION

[0034] The present inventors conducted extensive and in-depth research, synthesized and screened a series of tri-oligonucleotide polymers, and found for the first time that mRNA obtained by IVT using the cap analog of formula (I) significantly inhibits the binding of mRNA to IFIT1 and also significantly inhibits the activation of RIG-I, thereby enabling more rigorous and effective suppression of immune responses. Based on this finding, the present invention was completed.

[0035] The present invention provides cap analogs having a structure represented by formula (I):

[0036] [ka]

[0037] wherein B1 and B2 are each a natural or modified base; E and F are each 0 or 1; R1 is H, OH, an alkyl group, an O-alkyl group (including but not limited to an O-methyl group), a halogen, or an oxygen, which forms a bridge with the Cs at the 3' and 5' positions; R2 is H, OH, an alkyl group, an O-alkyl group (including but not limited to an O-methyl group), or a halogen; R3 is O-R5-R6; R4 is hydrogen, a hydroxy group, an O-methyl group, or O-R5-R6; R5 is a substituted or unsubstituted C 1-20 is an alkyl group; R6 is a substituted or unsubstituted O-alkyl group, a substituted or unsubstituted S-alkyl group, a substituted or unsubstituted NH-alkyl group, a substituted or unsubstituted N-dialkyl group, a substituted or unsubstituted O-aryl group, a substituted or unsubstituted S-aryl group, a substituted or unsubstituted NH-aryl group, a substituted or unsubstituted O-aralkyl group, a substituted or unsubstituted S-aralkyl group, a substituted or unsubstituted NH-aralkyl group; R5 is substituted or unsubstituted C 2-20When an alkyl group, R6 may be hydrogen; X1, X2 and X3 are each O, CH2 or NH; Y1, Y2 and Y3 are O, S, Se or BH3, respectively.

[0038] In one embodiment of the present invention, R6 is hydrogen and R5 is substituted or unsubstituted C 2-5 R and R are alkyl groups, each of which is an O-ethyl group, an O-propyl group, an O-isopropyl group, an O-butyl group, an O-isobutyl group, an O-t-butyl group, an O-pentyl group, or the like, but is not limited thereto; or R is a hydroxy group, and R is an O-ethyl group, an O-propyl group, an O-isopropyl group, an O-butyl group, an O-isobutyl group, an O-t-butyl group, an O-pentyl group, or the like, but is not limited thereto.

[0039] In one embodiment of the present invention, R5 is a substituted or unsubstituted C 1-5 alkyl group and R6 is a substituted or unsubstituted C 1-5R3 and R4 are each an O-methylene-O-methyl group, an O-ethylene-O-methyl group, an O-propylene-O-methyl group, an O-butylene-O-methyl group, an O-pentylene-O-methyl group, an O-methylene-O-ethyl group, an O-methylene-O-propyl group, an O-methylene-O-isopropyl group, an O-methylene-O-butyl group, an O-methylene-O-isobutyl group, an O-methylene-O-t-butyl group, an O-methylene-O-pentyl group, an O-ethylene-O-ethyl group, an O-ethylene-O-propyl group, an O-ethylene-O-isopropyl group, an O-ethylene-O-butyl group, an O-ethylene-O-isobutyl group, an O-ethylene-O-t-butyl group, an O-ethylene-O-pentyl group, an O-propylene-O-ethyl group, an O-propylene ...butyl group, an O-ethylene-O-pentyl group, an O-propylene-O-ethyl group, an O-propylene-O-butyl group, an O-ethylene-O-butyl group, an O-ethylene-O- O-butylene-O-propyl, O-propylene-O-isopropyl, O-propylene-O-butyl, O-propylene-O-isobutyl, O-propylene-O-t-butyl, O-propylene-O-pentyl, O-butylene-O-ethyl, O-butylene-O-propyl, O-butylene-O-isopropyl, O-butylene-O-butyl, O-butylene-O-isobutyl, O-butylene-O-t-butyl, O-butylene-O-pentyl, O-pentylene-O-ethyl, O-pentylene-O-propyl, O-pentylene-O-isopropyl, O-pentylene-O-butyl, O-pentylene-O-isobutyl, O-pentylene-O-t-butyl, O-pentylene-O-pentyl, and the like;Alternatively, R4 is a hydroxy group, and R3 is an O-methylene-O-methyl group, an O-ethylene-O-methyl group, an O-propylene-O-methyl group, an O-butylene-O-methyl group, an O-pentylene-O-methyl group, an O-methylene-O-ethyl group, an O-methylene-O-propyl group, an O-methylene-O-isopropyl group, an O-methylene-O-butyl group, an O-methylene-O-isobutyl group, an O-methylene-O-t-butyl group, an O-methylene-O-pentyl group, an O-ethylene-O-ethyl group, an O-ethylene-O-propyl group, an O-ethylene-O-isopropyl group, an O-ethylene-O-butyl group, an O-ethylene-O-isobutyl group, an O-ethylene-O-t-butyl group, an O-ethylene-O-pentyl group, an O-propylene-O-ethyl group, an O-propylene -O-propyl group, O-propylene-O-isopropyl group, O-propylene-O-butyl group, O-propylene-O-isobutyl group, O-propylene-O-t-butyl group, O-propylene-O-pentyl group, O-butylene-O-ethyl group, O-butylene-O-propyl group, O-butylene-O-isopropyl group, O-butylene-O-butyl group, O-butylene-O-isobutyl group, O-butylene-O-t-butyl group, O-butylene-O-pentyl group, O-pentylene-O-ethyl group, O-pentylene-O-propyl group, O-pentylene-O-isopropyl group, O-pentylene-O-butyl group, O-pentylene-O-isobutyl group, O-pentylene-O-t-butyl group, O-pentylene-O-pentyl group, and the like, but are not limited to these;

[0040] Representative tri-oligonucleotide polymers among the cap analogs represented by formula (I) of the present invention are shown in the table below: [ka]

[0041] TIFF2026016395000007.tif165151 TIFF2026016395000008.tif198151 TIFF2026016395000009.tif165151 TIFF2026016395000010.tif206151

[0042] In one embodiment of the present invention, a cap analog is provided having a structure represented by formula (Ia) or (Ib) below, wherein formula (Ia) and formula (Ib) are in different conformations, with the proviso that formula (Ia) is in the β conformation and formula (Ib) is in the α conformation:

[0043] [ka]

[0044] [ka]

[0045] wherein B1 and B2 are each a natural or modified base; R3 is O-R5-R6. R5 is a substituted or unsubstituted C 1-20 is an alkyl group; R6 is a substituted or unsubstituted O-alkyl group, a substituted or unsubstituted S-alkyl group, a substituted or unsubstituted NH-alkyl group, a substituted or unsubstituted N-dialkyl group, a substituted or unsubstituted O-aryl group, a substituted or unsubstituted S-aryl group, a substituted or unsubstituted NH-aryl group, a substituted or unsubstituted O-aralkyl group, a substituted or unsubstituted S-aralkyl group, a substituted or unsubstituted NH-aralkyl group; R5 is substituted or unsubstituted C 2-20 When an alkyl group, R6 may also be hydrogen. In one embodiment of the present invention, in formula (Ia) or formula (Ib), R6 is hydrogen and R5 is substituted or unsubstituted C 2-5 The R3 is an alkyl group, and includes, but is not limited to, an O-ethyl group, an O-propyl group, an O-isopropyl group, an O-butyl group, an O-isobutyl group, an O-t-butyl group, an O-pentyl group, and the like.

[0046] In one embodiment of the present invention, in formula (Ia) or formula (Ib), R5 is a substituted or unsubstituted C 1-5 alkyl group and R6 is a substituted or unsubstituted C 1-5 With alkyl groups wherein R3 is an O-methylene-O-methyl group, an O-ethylene-O-methyl group, an O-propylene-O-methyl group, an O-butylene-O-methyl group, an O-pentylene-O-methyl group, an O-methylene-O-ethyl group, an O-methylene-O-propyl group, an O-methylene-O-isopropyl group, an O-methylene-O-butyl group, an O-methylene-O-isobutyl group, an O-methylene-O-t-butyl group, an O-methylene-O-pentyl group, an O-ethylene-O-ethyl group, an O-ethylene-O-propyl group, an O-ethylene-O-isopropyl group, an O-ethylene-O-butyl group, an O-ethylene-O-isobutyl group, an O-ethylene-O-t-butyl group, an O-ethylene-O-pentyl group, an O-propylene-O-ethyl group, an O-propylene ...butyl group, an O-ethylene-O-isobutyl group, an O-ethylene-O-t-butyl group, an O-ethylene-O-pentyl group, an O-propylene-O-ethyl group, an O-propylene-O-propyl group, an O-ethylene-O-propyl group, an O-ethylene-O-propyl group, an O-ethylene-O-butyl group, an O-ethylene-O-isobutyl group, an O O-butylene-O-isopropyl, O-propylene-O-butyl, O-propylene-O-isobutyl, O-propylene-O-t-butyl, O-propylene-O-pentyl, O-butylene-O-ethyl, O-butylene-O-propyl, O-butylene-O-isopropyl, O-butylene-O-butyl, O-butylene-O-isobutyl, O-butylene-O-t-butyl, O-butylene-O-pentyl, O-pentylene-O-ethyl, O-pentylene-O-propyl, O-pentylene-O-isopropyl, O-pentylene-O-butyl, O-pentylene-O-isobutyl, O-pentylene-O-t-butyl, O-pentylene-O-pentyl, and the like;Alternatively, R4 is a hydroxy group, and R3 is an O-methylene-O-methyl group, an O-ethylene-O-methyl group, an O-propylene-O-methyl group, an O-butylene-O-methyl group, an O-pentylene-O-methyl group, an O-methylene-O-ethyl group, an O-methylene-O-propyl group, an O-methylene-O-isopropyl group, an O-methylene-O-butyl group, an O-methylene-O-isobutyl group, an O-methylene-O-t-butyl group, an O-methylene-O-pentyl group, an O-ethylene-O-ethyl group, an O-ethylene-O-propyl group, an O-ethylene-O-isopropyl group, an O-ethylene-O-butyl group, an O-ethylene-O-isobutyl group, an O-ethylene-O-t-butyl group, an O-ethylene-O-pentyl group, an O-propylene-O-ethyl group, an O-propylene -O-propyl group, O-propylene-O-isopropyl group, O-propylene-O-butyl group, O-propylene-O-isobutyl group, O-propylene-O-t-butyl group, O-propylene-O-pentyl group, O-butylene-O-ethyl group, O-butylene-O-propyl group, O-butylene-O-isopropyl group, O-butylene-O-butyl group, O-butylene-O-isobutyl group, O-butylene-O-t-butyl group, O-butylene-O-pentyl group, O-pentylene-O-ethyl group, O-pentylene-O-propyl group, O-pentylene-O-isopropyl group, O-pentylene-O-butyl group, O-pentylene-O-isobutyl group, O-pentylene-O-t-butyl group, O-pentylene-O-pentyl group, and the like, but are not limited to these;

[0047] The present invention further provides a method for preparing a cap analog having a structure represented by Formula (I), Formula (Ia), or Formula (Ib), for example, but not limited to, by a solid-phase oligonucleotide synthesis process comprising the steps of:

[0048] (1) 5'-O-DMT-2'-O-TBDMS phosphoramidite (rA Ac , rC Ac , rG dmf , U), 2'-O-MOE-3'-O-phosphoramidite (A 2’O-MOE Ac , C 2’O-MOE Ac, G 2’O-MOE dmf , U 2’O-MOE ), and 2'-O-ethyl-3'-O-phosphoramidite (A 2’O-Ethyl Ac , C 2’O-Ethyl Ac , G 2’O-Ethyl dmf , U 2’O-Ethyl ), bis-cyanoethyl-N,N-diisopropyl CED phosphoramidite, or other raw materials are used to synthesize pNpN dinucleotide using a solid-phase synthesizer;

[0049] (2) pNpN dinucleotide and phosphate imidazolate, 7me-2'-O-4'-C-locked guanosine 5'-diphosphate ( m7 G Beta-D-LNA DPIm) or 2'-O,4'-C-methylene-alpha-L-ribofuranosylguanosine 5'-diphosphate ( m7 G Alpha-L-LNA DPIm) or N7-methylguanosine 5'-diphosphate Phosphoric acid ( m7 GDPIm) to produce a cap analog having a structure represented by formula (I), formula (Ia), or formula (Ib).

[0050] In one embodiment of the present invention, the method further comprises purifying the resulting cap analog by, for example, but not limited to, DEAE-650s (Toyopearl) and a semi-preparative RT-HPLC column.

[0051] The present invention further provides a method for synthesizing mRNA by in vitro transcription using a cap analog having a structure represented by Formula (I), Formula (Ia), or Formula (Ib), which comprises the steps of: In the first step, template DNA is prepared; In the second step, the enzyme mixture is prepared and the reaction buffer is prepared; In the third step, an in vitro transcription reaction is carried out; In the fourth step, dephosphorylation is performed; In the fifth step, the reaction mixture is purified.

[0052] In the first step, the template typically consists of a linearized plasmid with a target sequence inserted into the multicloning site, and the target sequence must be preceded by a promoter for the corresponding polymerase, such as a T7, T3, or SP6 promoter. The plasmid must be linearized before use, and the DNA can be linearized by, for example, digestion with an appropriate restriction endonuclease commonly known in the art, followed by purification by, but not limited to, treatment with phenol-chloroform followed by ethanol precipitation. Restriction endonucleases commonly used for plasmid linearization are those that produce blunt ends or 5' overhangs (linearizing templates with enzymes that produce 3' overhangs will result in aberrant transcripts).

[0053] In the second step, the mixed enzyme may be prepared from an RNA polymerase, a nuclease inhibitor, a pyrophosphatase, or the like. The reaction buffer prepared in the second step includes, but is not limited to, Tris-HCl (pH 7.9), MgCl 2 , spermidine, dithiothreitol (DTT), and Triton X-100.

[0054] In the third step, an in vitro transcription reaction can be carried out using the cap analogs provided by the present invention.

[0055] In the fourth step, reagents that can be used for dephosphorylation treatment include, but are not limited to, Antarctic TAB5 phosphatase, shrimp alkaline phosphatase, calf alkaline phosphatase, and the like.

[0056] In the fifth step, the product can be purified by a method commonly used in the art, such as high performance liquid chromatography, lithium chloride precipitation, ammonium acetate precipitation, etc., but is not limited to these.

[0057] The present invention further provides polynucleotides comprising the provided cap analogs, which encode target polypeptides, as well as pharmaceutical compositions comprising the polynucleotides encoding the target polypeptides.

[0058] Unless otherwise stated, the following terms used in the specification and claims have the following meanings.

[0059] As used herein, a "cap analog" refers to a structure at the 5' end of mature mRNA formed by post-transcriptional modification in eukaryotes, i.e., a methylguanosine cap, also known as a m 7 This refers to the GPPPN structure, which can prevent degradation of mRNA from the 5' end and help RNA transcripts pass through selective pores in the nuclear membrane to enter the cytoplasm, enhancing translation and helping to complete the full splicing process.

[0060] As used herein, the terms "base" and "natural base" can be used interchangeably, and both are also called nucleic acid bases, nitrogenous bases, and are nitrogen-containing compounds that form nucleosides, which are the building blocks of nucleotides; they may be adenine (A), guanine (G), cytosine (C), uracil (U), or thymine (T).

[0061] The term "modified base" refers to a substance obtained by replacing one or two hydrogen atoms in a natural base, and includes, but is not limited to, N6-methyladenine. "Alkyl group" refers to an aliphatic hydrocarbon group. The alkyl group portion may be a saturated alkyl group (e.g., one that does not contain any unsaturated units such as carbon-carbon double bonds or carbon-carbon triple bonds) or an unsaturated alkyl group (one that contains at least one unsaturated unit). The alkyl group portion may be saturated or unsaturated, branched, or straight-chain. There may be 1 to 8 carbon atoms (wherever it appears, numerical ranges such as "1 to 8" refer to each integer within the stated range; e.g., "1 to 8 carbon atoms" refers to alkyl groups containing 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to 8 carbon atoms, although the present definition includes the appearance of the term "alkyl group" even when there is no specified numerical range). The alkyl groups in the compounds described herein may be designated as "C1-C6 alkyl groups" or similar. By way of example, a "C1-C6 alkyl group" refers to alkyl groups having 1, 2, 3, 4, 5, or 6 carbon atoms in the alkyl chain. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, hexyl, and the like.

[0062] "Aralkyl" refers to the group -alkyl-aryl, where alkyl and aryl are as defined herein.

[0063] "Halogen" refers to fluorine, chlorine, bromine, and iodine.

[0064] "Alkoxy" refers to an "O-alkyl" group, where alkyl is as defined herein.

[0065] "Aromatic" means a planar ring having a delocalized π-electron system containing 4n+2 π-electrons, where n is an integer. An aromatic ring may consist of five, six, seven, eight, nine, ten, or more atoms. An aromatic ring is optionally substituted. The term "aromatic" includes carbocyclic aryl groups ("aryl groups" such as phenyl) and heterocyclic aryl groups (or "heteroaryl groups" or "heteroaromatic rings") (e.g., pyridine).

[0066] By "substituted," we mean that the referenced group may be substituted with one or more additional groups independently selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl, heteroaliphatic, hydroxyl, alkoxy, alkylthio, arylthio, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl, cyano, halo, carbonyl, thiocarbonyl, nitro, haloalkyl, fluoroalkyl, and amino, including mono- and di-substituted amine groups, and protected derivatives thereof. where the substituents are selected from the group consisting of halogen, CF3, OH, CN, NO2, SO3H, SO2NH2, SO2Me, NH2, COOH, CONH2, alkoxy groups, -N(CH3)2, and alkyl groups.

[0067] The term "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers or gel-like substances that must be sufficiently pure and sufficiently low in toxicity for human use. Here, "compatible" refers to the ability of each component in the composition to be mixed with the other components without significantly reducing the efficacy of the compound of the present invention. Some examples of pharmaceutically acceptable excipients or carriers include cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyhydric alcohols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., Tween®), wetting agents (e.g., sodium dodecyl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0068] The main advantage of the present invention is that the cap analogs provided in the present invention can effectively suppress the occurrence of immune responses.

[0069] It should be understood that, within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically disclosed below (e.g., in the Examples) may be combined with each other to form new or preferred technical solutions. For convenience, detailed descriptions will be omitted here. In the following description, the specific aspects, characteristics, and advantages of the above-mentioned compounds, methods, and pharmaceutical compositions will be described in detail to make the content of the present invention clearer. It should be understood that the following detailed description and examples are for reference only. After reading the content of the description of the present invention, those skilled in the art may make various changes and modifications to the present invention, and these changes and modifications are also within the scope of the claims of the present invention.

[0070] The present invention will be more specifically explained in the following examples. However, these examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention in any way. In the following examples, experimental methods for which specific conditions are not described were generally carried out under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, parts and percentages are by weight. All organic solvents used in the reactions in the examples were dried using methods known in the art.

[0071] The LC-MS analytical methods involved in measuring capping efficiency in the examples below were as follows: Instrument: Waters UPLC H-Class Column: ACQUITY UPLC Oligonucleotide BEH C18, 2.1*150mm Column temperature: 75℃ Mobile phase A: 200 mM hexafluoroisopropanol + 8.15 mM triethylamine pH 7.9 Mobile phase B: methanol Flow rate: 0.3mL / min gradient:

[0072] TIFF2026016395000013.tif50120

[0073] MS device: Waters SQ Detector 2 Ion source: ESI Ionization mode: ES - M / Z: 600~3000

[0074] Example 1 Brief description of the synthetic route to cap analogues Synthesis of cap analogs: Starting material 5'-O-DMT-2'-O-TBDMS phosphoramidite (rA Ac , rC Ac , rG dmf , U) and 2'-O-MOE-3'-O-phosphoramidite (A 2’O-MOE Ac , C 2’O-MOE Ac, G 2’O-MOE dmf , U 2’O-MOE ) and 2'-O-ethyl-3'-O-phosphoramidite (A 2’O-Ethyl Ac , C 2’O-Ethyl Ac , G 2’O-Ethyl dmf , U 2’O-Ethyl ) was obtained from Hongene, and bis-cyanoethyl-N,N-diisopropyl CED phosphoramidite was obtained from ChemGenes. pNpN dinucleotide was synthesized using a solid-phase synthesis system (12) and then fused to 7me-2'-O-4'-C-locked guanosine 5'-diphosphate ( m7 G Beta-D-LNA DPIm) or 2'-O,4'-C-methylene-alpha-L-ribofuranosylguanosine 5'-diphosphate ( m7 G Alpha-L-LNA DPIm) or N7-methylguanosine 5'-diphosphate ( m7 The starting capped oligonucleotide composition was reacted with GDPIm to produce the initial capped oligonucleotide composition, which was then purified using DEAE-650s (Toyopearl) and a semi-preparative RT-HPLC column to obtain the purified product. The synthesis process is shown in Figures 1-3.

[0075] Example 2 Synthesis of cap analog-containing mRNA (1) Preparation of template DNA The template typically consists of a linearized plasmid with the target sequence inserted into the multiple cloning site, and the target sequence must be preceded by a promoter for the corresponding polymerase, such as the T7, T3, or SP6 promoter. The plasmid must be linearized before use; the DNA is linearized by digestion with an appropriate restriction endonuclease and then purified by an appropriate method, such as phenol-chloroform treatment followed by ethanol precipitation. The restriction endonuclease used to linearize the plasmid should preferably be one that produces blunt ends or one that produces a 5' overhang (linearizing the template with an enzyme that produces a 3' overhang will result in aberrant transcripts).

[0076] (2) Preparation of mixed enzymes TIFF2026016395000014.tif36115

[0077] (3) Preparation of 10x reaction buffer TIFF2026016395000015.tif44137

[0078] (4) T7 in vitro transcription reaction TIFF2026016395000016.tif85122

[0079] The cap analogs are: m7 GpppApG, m7 GpppA 2’Ome p.g., m7 G 3’Ome pppA 2’O-ethyl p.g., m7 G 2’Ome pppA 2’O-ethyl p.g., m7 G 3’Ome pppA 2’O-MOE p.g., m7 G 2’Ome pppA 2’O-MOE p.g., m7 Gppp (N6-methyladenine) 2’O-MOE pG), m7 G 2’Ome pppC 2’O-ethyl p.c., m7 G 3’OmepppA 2’O-MOE pU. The reaction was carried out at 37°C for 2 hours. 1 U of DNase I was added, and the mixture was reacted at 37°C for 30 minutes.

[0080] (5) Treatment with Antarctic phosphatase: The reaction solution was purified using Thermo MEGAclear® transcript clean-up kit, and 2 μl of 5 U / μl Antarctic phosphatase was added to the purified mRNA, mixed uniformly, and then treated at 37°C for 1 hour. After the treatment, the amount of mRNA produced was analyzed using a spectrophotometer, and the results are shown in Table 1.

[0081] (6) Purification of reaction mixture The RNA was purified by HPLC using a gradient of buffer B (0.1 M triethylamine-acetic acid pH 7.0 and 25% acetonitrile) and buffer A (0.1 M triethylamine-acetic acid pH 7.0) from 30% to 60% buffer A at 1 ml / min for approximately 30 minutes. The RNA-containing fractions were combined, precipitated with isopropanol, and quantified spectrophotometrically.

[0082] (7) Measurement of capping efficiency The percentage of capped mRNA was analyzed by liquid chromatography-mass spectrometry (LC-MS) for each of the various initial capped oligonucleotides tested. In this experiment, a DNA probe complementary to the mRNA was used. When the RNA and DNA hybridized, RNase H specifically hydrolyzed the phosphodiester bonds of the RNA. The action of RNase H resulted in small RNA fragments with uniform 3' ends. The molecular weight and percentage of these RNA fragments were analyzed by LC-MS to determine the RNA capping status during the transcription reaction. Since the presence or absence of a 5'-end cap can be determined by molecular weight, the capping efficiency can be determined by analyzing the molecular weight and percentage of short RNAs containing 5' ends. Furthermore, the capped nucleotides added, as calculated from the molecular weight, are the capped nucleotides obtained by the synthetic route described in Example 1 above. The capping efficiency results are shown in Table 1 and Figure 12.

[0083] (8) Identification of protein expression In this example, luciferase mRNA (Gaussia luciferase) with different initiation cap structures (the structure of the initiation cap nucleotide included is: m7 GpppApG, m7 GpppA 2’Ome p.g., m7 G 3’Ome pppA 2’O-ethyl p.g., m7 G 2’Ome pppA 2’O-ethyl p.g., m7 G 3’Ome pppA 2’O-MOE p.g., m7 G 2’Ome pppA 2’O-MOE p.g., m7 Gppp (N6-methyladenine) 2’O-MOE p.g., m7 G 2’Ome pppC 2’O-ethyl p.c., m7 G 3’Ome pppA 2’O-MOE pU) in HeLa cells (ATCC HeLa cells were transfected with 4 × 10 5Cells were plated in a 6-well plate at a density of 1000 / well, and transfection was performed when the cell density reached approximately 80%. Each well was transfected with 2 μg of mRNA, using Lipofectamine MessengerMAX transfection reagent (Invitrogen) as the transfection reagent. The transfection process was performed according to the manufacturer's instructions. 24 hours after transfection, the luciferase signal intensity was detected using a microplate reader. Luciferase fluorescence measurement: First, the transfected cell culture medium was transferred to a 1.5 mL centrifuge tube and centrifuged. 10 μl of the cell supernatant was added to a 96-well plate, and 50 μl of 10 ng / mL substrate was added. The fluorescence intensity was measured using a Synergy HI (BioTek) device. Expression measurements were repeated six times for each mRNA, and the expression results were as follows: m7 The values ​​are relative to the results for GpppApG-cap. Data are expressed as mean ± standard deviation, and the results obtained are shown in Table 1. Protein expression determination of the unpurified mRNA was also completed at the same time, and the results are shown in Table 1 and Figure 4.

[0084] TIFF2026016395000017.tif115165

[0085] (9) Measurement of the binding ability of mRNA to RIG-I and IFIT1 In this example, luciferase mRNA (Gaussia luciferase) with different initiation cap structures (the structure of the initiation cap nucleotide included is: m7 GpppApG, m7 GpppA 2’Ome p.g., m7 G 3’Ome pppA 2’O-ethyl p.g., m7 G 2’Ome pppA 2’O-ethyl p.g., m7 G 3’Ome pppA 2’O-MOE p.g., m7 G 2’Ome pppA 2’O-MOE p.g., m7 Gppp (N6-methyladenine) 2’O-MOEp.g., m7 G 2’Ome pppC 2’O-ethyl p.c., m7 G 3’Ome pppA 2’O-MOE pU) in HeLa cells (ATCC CCL-2) were transfected, and 24 hours later, the cells were harvested. The intracellular proteins RIG-I and IFIT1 were co-immunoprecipitated with their associated RNAs by RNA co-immunoprecipitation. Finally, their mRNAs were reverse transcribed and subjected to real-time quantitative PCR. Their relative abundance was directly proportional to their binding ability to RIG-I and IFIT1.

[0086] 4 × 10 HeLa cells 5 Cells were plated in a 6-well plate at a density of 1 / well and transfected when the cell density reached approximately 80%. Each well was transfected with 2 μg of mRNA, using Lipofectamine MessengerMAX transfection reagent (Invitrogen). The transfection procedure was performed according to the manufacturer's instructions. After 24 hours, the cells were harvested, added with fixative, and incubated at room temperature for 10 minutes. An appropriate concentration of glycine was then added to terminate the reaction, and the cells were collected by centrifugation. An appropriate amount of lysis solution was added to the cells, incubated on ice for 30 minutes, and centrifuged to collect the supernatant. An appropriate amount (2-4 ng) of RIG-I and IFIT1 antibodies (Abcam) was added to the supernatant and incubated overnight on a shaker at 4°C. 20 μl of Protein A / G magnetic beads were then added and incubated for 2 hours on a shaker at 4°C. The beads were washed three times with a magnetic grid for 5 minutes each time. Then, 1 mL of TRIzol reagent was added to the beads to extract RNA and convert the RNA to cDNA. Finally, the expression of related genes was detected by real-time quantitative fluorescent PCR, with β-actin as the internal reference gene. The detection was repeated three times for each gene, and the expression results for each gene were as follows: m7 The values ​​are relative to the results for GpppApG cap. Data are expressed as mean ± standard deviation, and the results obtained are shown in Table 2, Figures 5 and 6.

[0087] TIFF2026016395000018.tif115162

[0088] (10) Immunogenicity measurement In this example, luciferase mRNA (Gaussia luciferase) with different initiation cap structures (the structure of the initiation cap nucleotide included is: m7 GpppApG, m7 GpppA 2’Ome p.g., m7 G 3’Ome pppA 2’O-ethyl p.g., m7 G 2’Ome pppA 2’O-ethyl p.g., m7 G 3’Ome pppA 2’O-MOE p.g., m7 G 2’Ome pppA 2’O-MOE p.g., m7 Gppp (N6-methyladenine) 2’O-MOE p.g., m7 G 2’Ome pppC 2’O-ethyl p.c., m7 G 3’Ome pppA 2’O-MOE pU) in HeLa cells (ATCC CCL-2) and 24 hours later, the cells were harvested and the expression of the inflammatory factors within the cells was detected, the relative abundance of which is directly proportional to the immunogenicity of the mRNA. 4 × 10 HeLa cells 5Cells were plated in a 6-well plate at a density of 1000 / well, and transfection was performed when the cell density reached approximately 80%. Each well was transfected with 2 μg of mRNA, using Lipofectamine MessengerMAX transfection reagent (Invitrogen) as the transfection reagent. The transfection process was performed according to the manufacturer's instructions. After 24 hours, the cells were harvested, RNA was extracted using TRIzol, and the RNA was reverse transcribed into cDNA. Finally, the expression of intracellular inflammatory factors was detected by real-time quantitative fluorescent PCR, with β-actin as the internal reference gene. Detection was repeated three times for each gene, and the expression results for each gene were as follows: m7 The values ​​are relative to the results for GpppApG cap. Data are expressed as mean ± standard deviation, and the results are shown in Table 3 and Figure 7.

[0089] TIFF2026016395000019.tif115165

[0090] Example 3 Cap Analog m7 G Beta-D-LNA pppA 2’O-MOE pG and m7 G Alpha-L-LNA pppA 2’O-ethyl Preparation and expression of pG mRNA (1) Preparation of template DNA The template typically consists of a linearized plasmid with the target sequence inserted into the multiple cloning site, and the target sequence must be preceded by a promoter for the corresponding polymerase, such as the T7, T3, or SP6 promoter. The plasmid must be linearized before use; the DNA is linearized by digestion with an appropriate restriction endonuclease and then purified by an appropriate method, such as phenol-chloroform treatment followed by ethanol precipitation. The restriction endonuclease used to linearize the plasmid should preferably be one that produces blunt ends or one that produces a 5' overhang (linearizing the template with an enzyme that produces a 3' overhang will result in aberrant transcripts).

[0091] (2) Preparation of mixed enzymes TIFF2026016395000020.tif36125

[0092] (3) Preparation of 10x reaction buffer TIFF2026016395000021.tif44137

[0093] (4) T7 in vitro transcription reaction TIFF2026016395000022.tif85122

[0094] The initiating cap oligonucleotides are: m7 GpppApG, m7 GpppA 2’Ome pG (control), m7 G Beta-D-LNA pppA 2’O-MOE pG and m7 G Alpha-L-LNA pppA 2’O-ethyl pG. The reaction was carried out at 50°C for 2 hours. 1 U of DNase I was added, and the mixture was reacted at 37°C for 30 minutes.

[0095] (5) Treatment with Antarctic phosphatase: The reaction solution was purified using Thermo MEGAclear® transcript clean-up kit, and 2 μl of 5 U / μl Antarctic phosphatase was added to the purified mRNA, mixed uniformly, and then treated at 37°C for 1 hour. After the treatment, the amount of mRNA produced was analyzed using a spectrophotometer, and the results are shown in Table 4.

[0096] (6) Purification of reaction mixture The RNA was purified by HPLC using a gradient of buffer B (0.1 M triethylamine-acetic acid pH 7.0 and 25% acetonitrile) and buffer A (0.1 M triethylamine-acetic acid pH 7.0) from 30% to 60% buffer A at 1 ml / min for approximately 30 minutes. The RNA-containing fractions were combined, precipitated with isopropanol, and quantified spectrophotometrically.

[0097] (7) Measurement of capping efficiency For each of the various initiation cap oligonucleotides tested, the percentage of capped mRNA was analyzed by liquid chromatography-mass spectrometry (LC-MS). In this experiment, a DNA probe complementary to the mRNA was used. When the RNA and DNA hybridized, RNase H specifically hydrolyzed the phosphodiester bonds of the RNA. The action of RNase H resulted in small RNA fragments with uniform 3' ends. The molecular weight and percentage of these RNA fragments were analyzed by LC-MS to determine the RNA capping status during the transcription reaction. Since the presence or absence of a 5'-end cap is indicated by its molecular weight, the capping efficiency can be determined by analyzing the molecular weight and percentage of short RNAs containing the 5' end. Furthermore, the cap added, as calculated from the molecular weight, is the cap nucleotide described above. The capping efficiency results are shown in Table 4 and Figure 13.

[0098] (8) Identification of protein expression In this example, luciferase mRNA (Gaussia luciferase) with different initiation cap structures (the structure of the initiation cap nucleotide included is: m7 GpppApG, m7 GpppA 2’Ome p.g., m7 G Beta-D-LNA pppA 2’O-MOE pG and m7 G Alpha-L-LNA pppA 2’O-ethyl Hela cells (ATCC CCL-2) were transfected with pG, and 24 hours later, the luciferase signal intensity was detected using a microplate reader, and the fluorescence signal intensity was directly proportional to the expression abundance of the protein.

[0099] 4 × 10 HeLa cells 5Cells were plated in a 6-well plate at a density of 1000 / well, and transfection was performed when the cell density reached approximately 80%. Each well was transfected with 2 μg of mRNA, using Lipofectamine MessengerMAX transfection reagent (Invitrogen) as the transfection reagent. The transfection process was performed according to the manufacturer's instructions. 24 hours after transfection, the luciferase signal intensity was detected using a microplate reader. Luciferase fluorescence measurement: First, the transfected cell culture medium was transferred to a 1.5 mL centrifuge tube and centrifuged. 10 μl of the cell supernatant was added to a 96-well plate, and 50 μl of 10 ng / mL substrate was added. The fluorescence intensity was measured using a Synergy HI (BioTek) device. Expression measurements were repeated six times for each mRNA, and the expression results were as follows: m7 The values ​​are relative to the results for GpppApG-cap. Data are expressed as mean ± standard deviation, and the results obtained are shown in Table 4. Protein expression determination of the unpurified mRNA was also completed at the same time, and the results are shown in Table 4 and Figure 8.

[0100] TIFF2026016395000023.tif98165

[0101] (9) Measurement of the binding ability of mRNA to RIG-I and IFIT1 In this example, luciferase mRNA (Gaussia luciferase) with different initiation cap structures (the structure of the initiation cap nucleotide included is: m7 GpppApG, m7 GpppA 2’Ome p.g., m7 G Beta-D-LNA pppA 2’O-MOE pG and m7 G Alpha-L-LNA pppA 2’O-ethylHela cells (ATCC CCL-2) were transfected with IFIT1 pG and harvested 24 hours later. The intracellular proteins RIG-I and IFIT1 were co-immunoprecipitated with their associated RNAs by RNA co-immunoprecipitation. Finally, their mRNAs were reverse transcribed and subjected to real-time quantitative PCR. Their relative abundance was directly proportional to their binding ability to RIG-I and IFIT1.

[0102] 4 × 10 HeLa cells 5 Cells were plated in a 6-well plate at a density of 1 / well and transfected when the cell density reached approximately 80%. Each well was transfected with 2 μg of mRNA, using Lipofectamine MessengerMAX transfection reagent (Invitrogen). The transfection procedure was performed according to the manufacturer's instructions. After 24 hours, the cells were harvested, added with fixative, and incubated at room temperature for 10 minutes. An appropriate concentration of glycine was then added to terminate the reaction, and the cells were collected by centrifugation. An appropriate amount of lysis solution was added to the cells, incubated on ice for 30 minutes, and centrifuged to collect the supernatant. An appropriate amount (2-4 ng) of RIG-I and IFIT1 antibodies (Abcam) was added to the supernatant and incubated overnight on a shaker at 4°C. 20 μl of Protein A / G magnetic beads were then added and incubated for 2 hours on a shaker at 4°C. The beads were washed three times with a magnetic grid for 5 minutes each time. Then, 1 mL of TRIzol reagent was added to the beads to extract RNA, and the RNA was reverse transcribed into cDNA. Finally, the expression of related genes was detected by real-time quantitative fluorescent PCR, with β-actin as the internal reference gene. The detection was repeated three times for each gene, and the expression results for each gene were as follows: m7 The values ​​are relative to the results for GpppApG cap. Data are expressed as mean ± standard deviation, and the results obtained are shown in Table 5, Figures 9 and 10.

[0103] TIFF2026016395000024.tif77165

[0104] (10) Immunogenicity measurement In this example, luciferase mRNA (Gaussia luciferase) with different initiation cap structures (the structure of the initiation cap nucleotide included is: m7 GpppApG, m7 GpppA 2’Ome p.g., m7 G Beta-D-LNA pppA 2’O-MOE pG and m7 G Alpha-L-LNA pppA 2’O-ethyl Hela cells (ATCC CCL-2) were transfected with IgG (pG), and 24 hours later, the cells were harvested and the expression of inflammatory factors within the cells was detected, the relative abundance of which is directly proportional to the immunogenicity of the mRNA. 4 × 10 Hela cells were transfected. 5 Cells were plated in a 6-well plate at a density of 1000 / well, and transfection was performed when the cell density reached approximately 80%. Each well was transfected with 2 μg of mRNA, using Lipofectamine MessengerMAX transfection reagent (Invitrogen) as the transfection reagent. The transfection process was performed according to the manufacturer's instructions. After 24 hours, the cells were harvested, RNA was extracted using TriZol, and the RNA was reverse transcribed into cDNA. Finally, the expression of intracellular inflammatory factors was detected by real-time quantitative fluorescent PCR, with β-actin as the internal reference gene. Detection was repeated three times for each gene, and the expression results for each gene were as follows: m7 The values ​​are relative to the results for GpppApG cap. Data are expressed as mean ± standard deviation, and the results are shown in Table 6 and Figure 11.

[0105] TIFF2026016395000025.tif93167

[0106] The above description is only a preferred embodiment of the present invention and does not limit the scope of the essential technical content of the present invention. The essential technical content of the present invention is broadly defined in the claims of the application, and any technical substance or method completed by another person that is exactly the same as that defined in the claims of the application or a modification with equivalent effects is also deemed to be within the scope of the claims.

Claims

1. A cap analog represented by formula (I): In the formula, B １ and B ２ are each a natural or modified base; E and F are each 0 or 1; R １ is H, OH, an alkyl group, an O-alkyl group, a halogen, or is oxygen, and said oxygen forms a bridge with the Cs at the 3' and 5' positions; R ２ is H, OH, an alkyl group, an O-alkyl group or a halogen; R ３ is O-R ５ -R ６ and R ４ is hydrogen, a hydroxy group, an O-methyl group, or O-R ５ -R ６ and R ５ is a substituted or unsubstituted C １－２０ is an alkyl group; R ６ is a substituted or unsubstituted O-alkyl group, a substituted or unsubstituted S-alkyl group, a substituted or unsubstituted NH-alkyl group, a substituted or unsubstituted N-dialkyl group, a substituted or unsubstituted O-aryl group, a substituted or unsubstituted S-aryl group, a substituted or unsubstituted NH-aryl group, a substituted or unsubstituted O-aralkyl group, a substituted or unsubstituted S-aralkyl group, a substituted or unsubstituted NH-aralkyl group, or hydrogen (R ５ is substituted or unsubstituted C ２－２０ alkyl group); X １ , X ２ and X ３ are O and CH ２ or NH; Y １ , Y ２ and Y ３ are O, S, Se or BH ３ is.

2. R ３ is OCH ２ CH ３ , OCH ２ OCH ３ or OCH ２ CH ２ OCH ３ The cap analog of claim 1 , wherein

3. R ４ is a hydroxy group, OCH ２ CH ３ , OCH ２ OCH ３ or OCH ２ CH ２ OCH ３ The cap analog of claim 1 , wherein

4. B １ and B ２ The cap analog of claim 1, wherein each of

5. The cap analog is ｍ７ GpppA ２’Ｏ－ｅｔｈｙｌ p.g., ｍ７ GpppA ２’Ｏ－ｅｔｈｙｌ pA, ｍ７ GpppA ２’Ｏ－ｅｔｈｙｌ pC, ｍ７ GpppA ２’Ｏ－ｅｔｈｙｌ pU, ｍ７ GpppC ２’Ｏ－ｅｔｈｙｌ pA, ｍ７ GpppC ２’Ｏ－ｅｔｈｙｌ p.g., ｍ７ GpppC ２’Ｏ－ｅｔｈｙｌ pC, ｍ７ GpppC ２’Ｏ－ｅｔｈｙｌ pU, ｍ７ GpppG ２’Ｏ－ｅｔｈｙｌ pA, ｍ７ GpppG ２’Ｏ－ｅｔｈｙｌ pC, ｍ７ GpppG ２’Ｏ－ｅｔｈｙｌ p.g., ｍ７ GpppG ２’Ｏ－ｅｔｈｙｌ pU, ｍ７ GpppU ２’Ｏ－ｅｔｈｙｌ pA, ｍ７ GpppU ２’Ｏ－ｅｔｈｙｌ pC, ｍ７ GpppU ２’Ｏ－ｅｔｈｙｌ PC, or ｍ７ GpppU ２’Ｏ－ｅｔｈｙｌ From pU The cap analog of claim 1 , selected from the group consisting of:

6. The cap analog is ｍ７ G ３’Ｏｍｅ pppA ２’Ｏ－ｅｔｈｙｌ p.g., ｍ７ G ３’Ｏｍｅ pppA ２’Ｏ－ｅｔｈｙｌ pA, ｍ７ G ３’Ｏｍｅ pppA ２’Ｏ－ｅｔｈｙｌ pC, ｍ７ G ３’Ｏｍｅ pppA ２’Ｏ－ｅｔｈｙｌ pU, ｍ７ G ３’Ｏｍｅ pppC ２’Ｏ－ｅｔｈｙｌ pA, ｍ７ G ３’Ｏｍｅ pppC ２’Ｏ－ｅｔｈｙｌ p.g., ｍ７ G ３’Ｏｍｅ pppC ２’Ｏ－ｅｔｈｙｌ pC, ｍ７ G ３’Ｏｍｅ pppC ２’Ｏ－ｅｔｈｙｌ pU, ｍ７ G ３’Ｏｍｅ pppG ２’Ｏ－ｅｔｈｙｌ pA, ｍ７ G ３’Ｏｍｅ pppG ２’Ｏ－ｅｔｈｙｌ pC, ｍ７ G ３’Ｏｍｅ pppG ２’Ｏ－ｅｔｈｙｌ p.g., ｍ７ G ３’Ｏｍｅ pppG ２’Ｏ－ｅｔｈｙｌ pU, ｍ７ G ３’Ｏｍｅ pppU ２’Ｏ－ｅｔｈｙｌ pA, ｍ７ G ３’Ｏｍｅ pppU ２’Ｏ－ｅｔｈｙｌ pC, ｍ７ G ３’Ｏｍｅ pppU ２’Ｏ－ｅｔｈｙｌ PC, or ｍ７ G ３’Ｏｍｅ pppU ２’Ｏ－ｅｔｈｙｌ The cap analog of claim 1, characterized in that it is selected from the group consisting of pU.

7. The cap analog is ｍ７ G ２’Ｏｍｅ pppA ２’Ｏ－ｅｔｈｙｌ p.g., ｍ７ G ２’Ｏｍｅ pppA ２’Ｏ－ｅｔｈｙｌ pA, ｍ７ G ２’Ｏｍｅ pppA ２’Ｏ－ｅｔｈｙｌ pC, ｍ７ G ２’Ｏｍｅ pppA ２’Ｏ－ｅｔｈｙｌ pU, ｍ７ G ２’Ｏｍｅ pppC ２’Ｏ－ｅｔｈｙｌ pA, ｍ７ G ２’Ｏｍｅ pppC ２’Ｏ－ｅｔｈｙｌ p.g., ｍ７ G ２’Ｏｍｅ pppC ２’Ｏ－ｅｔｈｙｌ pC, ｍ７ G ２’Ｏｍｅ pppC ２’Ｏ－ｅｔｈｙｌ pU, ｍ７ G ２’Ｏｍｅ pppG ２’Ｏ－ｅｔｈｙｌ pA, ｍ７ G ２’Ｏｍｅ pppG ２’Ｏ－ｅｔｈｙｌ pC, ｍ７ G ２’Ｏｍｅ pppG ２’Ｏ－ｅｔｈｙｌ p.g., ｍ７ G ２’Ｏｍｅ pppG ２’Ｏ－ｅｔｈｙｌ pU, ｍ７ G ２’Ｏｍｅ pppU ２’Ｏ－ｅｔｈｙｌ pA, ｍ７ G ２’Ｏｍｅ pppU ２’Ｏ－ｅｔｈｙｌ pC, ｍ７ G ２’Ｏｍｅ pppU ２’Ｏ－ｅｔｈｙｌ PC, or ｍ７ G ２’Ｏｍｅ pppU ２’Ｏ－ｅｔｈｙｌ The cap analog of claim 1, characterized in that it is selected from the group consisting of pU.

8. The cap analog is ｍ７ Gppp (N6-methyladenine) ２’Ｏ－ｅｔｈｙｌ p.g., ｍ７ Gppp (N6-methyladenine) ２’Ｏ－ｅｔｈｙｌ pA, ｍ７ Gppp (N6-methyladenine) ２’Ｏ－ｅｔｈｙｌ pC, ｍ７ Gppp (N6-methyladenine) ２’Ｏ－ｅｔｈｙｌ pU, ｍ７ G ２’Ｏｍｅ ppp (N6-methyladenine) ２’Ｏ－ｅｔｈｙｌ pA, ｍ７ G ２’Ｏｍｅ ppp (N6-methyladenine) ２’Ｏ－ｅｔｈｙｌ p.g., ｍ７ G ２’Ｏｍｅ ppp (N6-methyladenine) ２’Ｏ－ｅｔｈｙｌ pC, ｍ７ G ２’Ｏｍｅ ppp (N6-methyladenine) ２’Ｏ－ｅｔｈｙｌ pU, ｍ７ G ３’Ｏｍｅ ppp (N6-methyladenine) ２’Ｏ－ｅｔｈｙｌ pA, ｍ７ G ３’Ｏｍｅ ppp (N6-methyladenine) ２’Ｏ－ｅｔｈｙｌ pC, ｍ７ G ３’Ｏｍｅ ppp (N6-methyladenine) ２’Ｏ－ｅｔｈｙｌ PC, or ｍ７ G ３’Ｏｍｅ ppp (N6-methyladenine) ２’Ｏ－ｅｔｈｙｌ The cap analog of claim 1, characterized in that it is selected from the group consisting of pU.

9. The cap analog is ｍ７ GpppA ２’Ｏ－ＭＯＥ p.g., ｍ７ GpppA ２’Ｏ－ＭＯＥ pA, ｍ７ GpppA ２’Ｏ－ＭＯＥ pC, ｍ７ GpppA ２’Ｏ－ＭＯＥ pU, ｍ７ GpppC ２’Ｏ－ＭＯＥ pA, ｍ７ GpppC ２’Ｏ－ＭＯＥ p.g., ｍ７ GpppC ２’Ｏ－ＭＯＥ pC, ｍ７ GpppC ２’Ｏ－ＭＯＥ pU, ｍ７ GpppG ２’Ｏ－ＭＯＥ pA, ｍ７ GpppG ２’Ｏ－ＭＯＥ pC, ｍ７ GpppG ２’Ｏ－ＭＯＥ p.g., ｍ７ GpppG ２’Ｏ－ＭＯＥ pU, ｍ７ GpppU ２’Ｏ－ＭＯＥ pA, ｍ７ GpppU ２’Ｏ－ＭＯＥ pC, ｍ７ GpppU ２’Ｏ－ＭＯＥ PC, or ｍ７ GpppU ２’Ｏ－ＭＯＥ The cap analog of claim 1, characterized in that it is selected from the group consisting of pU.

10. The cap analog is ｍ７ G ３’Ｏｍｅ pppA ２’Ｏ－ＭＯＥ p.g., ｍ７ G ３’ Ｏｍｅ pppA ２’Ｏ－ＭＯＥ pA, ｍ７ G ３’Ｏｍｅ pppA ２’Ｏ－ＭＯＥ pC, ｍ７ G ３’Ｏｍｅ pppA ２’Ｏ－ＭＯＥ pU, ｍ７ G ３’Ｏｍｅ pppC ２’Ｏ－ＭＯＥ pA, ｍ７ G ３’Ｏｍｅ pppC ２’Ｏ－ＭＯＥ p.g., ｍ７ G ３’Ｏｍｅ pppC ２’Ｏ－ＭＯＥ pC, ｍ７ G ３’Ｏｍｅ pppC ２’Ｏ－ＭＯＥ pU, ｍ７ G ３’Ｏｍｅ pppG ２Ｏ－’ＭＯＥ pA, ｍ７ G ３’Ｏｍｅ pppG ２’Ｏ－ＭＯＥ pC, ｍ７ G ３’Ｏｍｅ pppG ２’Ｏ－ＭＯＥ p.g., ｍ７ G ３’Ｏｍｅ pppG ２’Ｏ－ＭＯＥ pU, ｍ７ G ３’Ｏｍｅ pppU ２’Ｏ－ＭＯＥ pA, ｍ７ G ３’Ｏｍｅ pppU ２’Ｏ－ＭＯＥ pC, ｍ７ G ３’Ｏｍｅ pppU ２’Ｏ－ＭＯＥ PC, or ｍ７ G ３’Ｏｍｅ pppU ２’Ｏ－ＭＯＥ The cap analog of claim 1, characterized in that it is selected from the group consisting of pU.

11. The cap analog is ｍ７ G ２’Ｏｍｅ pppA ２’Ｏ－ＭＯＥ p.g., ｍ７ G ２’Ｏｍｅ pppA ２’Ｏ－ＭＯＥ pA, ｍ７ G ２’Ｏｍｅ pppA ２’Ｏ－ＭＯＥ pC, ｍ７ G ２’Ｏｍｅ pppA ２’Ｏ－ＭＯＥ pU, ｍ７ G ２’Ｏｍｅ pppC ２’Ｏ－ＭＯＥ pA, ｍ７ G ２’Ｏｍｅ pppC ２’Ｏ－ＭＯＥ p.g., ｍ７ G ２’Ｏｍｅ pppC ２’Ｏ－ＭＯＥ pC, ｍ７ G ２’Ｏｍｅ pppC ２’Ｏ－ＭＯＥ pU, ｍ７ G ２’Ｏｍｅ pppG ２’Ｏ－ＭＯＥ pA, ｍ７ G ２’Ｏｍｅ pppG ２’Ｏ－ＭＯＥ pC, ｍ７ G ２’Ｏｍｅ pppG ２’Ｏ－ＭＯＥ p.g., ｍ７ G ２’Ｏｍｅ pppG ２’Ｏ－ＭＯＥ pU, ｍ７ G ２’Ｏｍｅ pppU ２’Ｏ－ＭＯＥ pA, ｍ７ G ２’Ｏｍｅ pppU ２’Ｏ－ＭＯＥ pC, ｍ７ G ２’Ｏｍｅ pppU ２’Ｏ－ＭＯＥ PC, or ｍ７ G ２’Ｏｍｅ pppU ２’Ｏ－ＭＯＥ The cap analog of claim 1, characterized in that it is selected from the group consisting of pU.

12. The cap analog is ｍ７ Gppp (N6-methyladenine) ２’Ｏ－ＭＯＥ p.g., ｍ７ Gppp (N6-methyladenine) ２’Ｏ－ＭＯＥ pA, ｍ７ Gppp (N6-methyladenine) ２’Ｏ－ＭＯＥ pC, ｍ７ Gppp (N6-methyladenine) ２’Ｏ－ＭＯＥ pU, ｍ７ G ２’Ｏｍｅ ppp (N6-methyladenine) ２’Ｏ－ＭＯＥ pA, ｍ７ G ２’Ｏｍｅ ppp (N6-methyladenine) ２’Ｏ－ＭＯＥ p.g., ｍ７ G ２’Ｏｍｅ ppp (N6-methyladenine) ２’Ｏ－ＭＯＥ pC, ｍ７ G ２’Ｏｍｅ ppp (N6-methyladenine) ２’Ｏ－ＭＯＥ pU, ｍ７ G ３’Ｏｍｅ ppp (N6-methyladenine) ２’Ｏ－ＭＯＥ pA, ｍ７ G ３’Ｏｍｅ ppp (N6-methyladenine) ２’Ｏ－ＭＯＥ pC, ｍ７ G ３’Ｏｍｅ ppp (N6-methyladenine) ２’Ｏ－ＭＯＥ PC, or ｍ７ G ３’Ｏｍｅ ppp (N6-methyladenine) ２’Ｏ－ＭＯＥ The cap analog of claim 1, characterized in that it is selected from the group consisting of pU.

13. The cap analog according to claim 1, characterized in that the structure of the cap analog is represented by formula (I-a) or formula (I-b): In the formula, B １ and B ２ are each a natural or modified base; R ３ is O-R ５ -R ６ is.

14. R ３ is OCH ２ CH ３ , OCH ２ OCH ３ or OCH ２ CH ２ OCH ３ The cap analog of claim 13, wherein:

15. B １ and B ２ and are adenine, N6-methyladenine, guanine, uracil, or thymine, respectively.

16. A polynucleotide encoding a target polypeptide, (a) at least one ORF region; (b) at least one 5′UTR of Kozak sequence; (c) a 3′UTR; and (d) a cap analog of any one of claims 1 to 15, having at least one 5' initial cap; A polynucleotide comprising:

17. A pharmaceutical composition comprising the polynucleotide of claim 16 and a pharmaceutically acceptable carrier.

18. 18. The pharmaceutical composition according to claim 17, wherein the carrier is selected from the group consisting of lipid nanoparticles (LNPs), liposomes, polymeric nanoparticles, solid lipid nanoparticles, or emulsions.

19. (1) Prepare the DNA template: (2) Obtaining the polynucleotide according to claim 16 by performing an in vitro transcription reaction in a reaction system containing an RNA polymerase, a nucleoside triphosphate, and the cap analog according to any one of claims 1 to 15. The method for producing a polynucleotide according to claim 16, comprising:

20. The method according to claim 19, wherein the RNA polymerase is a phage-derived RNA polymerase.

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