mRNA cap analogs and their uses

Abstract

One aspect relates to a compound of formula 1, a cap analog containing the compound, an mRNA 5'-capped with the cap analog, a method for producing an mRNA using the cap analog, a use of the cap analog in producing an mRNA, and a pharmaceutical composition for expressing a desired peptide or protein, the pharmaceutical composition containing an mRNA 5'-capped with the cap analog.

Description

[Technical field]

[0001] The present invention relates to a cap analog and a method for expressing a protein using the same, and more specifically, to an mRNA cap analog, a 5'-capped mRNA containing the same and a method for producing the same, a pharmaceutical composition for expressing a desired peptide or protein containing the 5'-capped mRNA, and a method for producing a desired peptide or protein using the 5'-capped mRNA. [Background technology]

[0002] RNA therapeutics have recently been presented as a new paradigm for the treatment of infectious diseases and cancer. In particular, the development of mRNA vaccines using in vitro transcription has been highlighted as the most promising approach to RNA virus infections that can overcome the limitations of conventional vaccines such as attenuated vaccines, inactivated vaccines, and synthetic antigen vaccines. RNA virus infections have relatively strong transmission and rapid mutation due to unstable structures, so rapid and safe vaccine development is required. mRNA vaccines can respond to such infectious diseases and shorten the vaccine development speed in that it takes about one month to derive candidate substances for mRNA vaccines. The mRNA introduced into the body is degraded within 24 hours, and unlike DNA vaccines, it does not enter the nucleus of the cell but is translated into proteins by ribosomes in the cytoplasm, so there is no risk of gene mutation and it has a great advantage of being safe.

[0003] The following components are required to synthesize mRNA molecules in vitro: a linearized DNA template, an RNA polymerase (mainly T7 RNA polymerase), four ribonucleoside triphosphates (NTPs) A, G, C, and U (or m1Ψ, 1-methylpseudouridine), and a cap analog. In some cases, poly(A) polymerase can be used to polyadenylate 200-250 adenosines at the 3' end, and a capping enzyme can be used instead of a cap analog to synthesize a 5' capping structure at the 5' end. Compared to other components that are relatively optimized, the 5' capping structure has a complex structure and plays an important role in the protein translation of mRNA molecules, and is the most expensive component in in vitro synthesis, so research into its efficient synthesis is actively underway.

[0004] In order for the mRNA to be efficiently translated into a coding protein, it must undergo a post-transcriptional maturation process. During this process, the 5' end of the mRNA molecule is supplemented with the 7-methylguanosine ( 7m The process in which the 5'-5'-triphosphate chain is linked to the 5'-cap structure (G) is called the 5' capping process, and the linked structure is called the 5' capping structure (G). 7m The 5' capping structure can protect the 5' end of mRNA from biodegradation by 5' exonuclease, and influences the movement of mRNA from the nucleus to the cytoplasm. In particular, the 5' capping structure is recognized by eukaryotic translation initiation factor 4E (eIF4E) and plays an important role in expressing proteins by forming a translation initiation complex.

[0005] In order to apply such capped mRNA to clinical applications, it must have a structure (Cap1 mRNA) in which the 2' hydroxyl group of the first nucleotide linked to 7-methylguanosine via a 5'-triphosphate chain is methylated. When the unmethylated Cap0 structure is applied, MDA5 (an intracellular sensor protein) that recognizes the Cap0 structure recognizes the 2' hydroxyl group when exogenous mRNA with the Cap0 structure is introduced into the body, and induces an immune inflammatory response, thereby impairing the binding of mRNA to eIF4E and inhibiting protein expression. On the other hand, exogenous Cap1 mRNA with 2' OH methylation does not induce an immune response even when introduced into the body because it is not recognized by MDA5, and therefore can induce relatively high protein expression, making it suitable for clinical use.

[0006] Commercialized mRNA vaccines based on the Cap1 mRNA structure mentioned above include Moderna's mRNA-1273 and Pfizer / BioNTech's BNT162b2. Both mRNA vaccines were developed to respond to the infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus Type 2 (SARS-CoV-2), an RNA virus that has caused a global pandemic. In the case of the BNT162b2 mRNA vaccine, in order to reduce production costs, a co-transcriptional capping method was adopted, which is an mRNA synthesis method in which a chemically synthesized cap analog is simultaneously introduced during mRNA synthesis in a test tube and the cap analog forms the 5' end, rather than the traditional capping method that uses a capping enzyme.

[0007] The cap analog used in BNT162b2 is Cleancap® AG (3'OMe), a third generation cap analog developed by TriLink, Inc. (U.S. Patent 10,913,768). 7m G (3’OMe) pppA (2’OMe)It is a trimer cap analog of pG structure, which is introduced during mRNA synthesis and can synthesize Cap1 mRNA structure without using enzymes. The trimer cap analog has one or more more bases than GTP, and therefore has stronger hydrogen bond strength during base pairing with DNA template strand for transcription initiation than GTP. Therefore, unlike the previous second generation cap analog (U.S. Patent 7,074,596) which is a dimer and competitively base pairs with guanosine triphosphate (GTP) with DNA template, the trimer cap analog can preferentially base pair with GTP, and thus the amount of cap analog input during mRNA synthesis in vitro can be reduced, and as a result, mRNA impurities with pppG 5' ends can also be reduced.

[0008] BNT162b2, which introduces a trimer cap analog, was temporarily faced with a shortage of raw materials due to the overwhelming demand for responding to the global Covid-19 pandemic. Furthermore, the cap analog remains the highest cost in mass-producing mRNA vaccines. Rapid and economical production and supply of cap analogs is necessary to produce mRNA vaccines to adequately respond to highly contagious RNA viral infections.

[0009] The currently commercially available manufacturing process for trimer cap analogs can be broadly divided into six steps as follows (U.S. Patent 10,913,768).

[0010] Reaction Scheme 1 [ka]

[0011] In the reaction formula 1, G represents guanosine, 7mG means 7-methylguanosine, N means any one of adenosine, cytidine, guanosine, and uridine, p means -P(=O)O2-, Me means methyl, and Im means imidazolide. In more detail, pN (2’OMe) If the step of synthesizing pN is included, a long process of at least 10 steps must be involved.

[0012] In particular, when Trilink's cap analogs are mass-produced, the main factor that increases the production costs and synthesis time in the entire manufacturing process is the purification step using ion exchange chromatography (mainly DEAE resin) that must be performed between the steps of the manufacturing process. After purifying the intermediates at each manufacturing step, a large amount of buffer solution (mainly triethylammonium bicarbonate (TEAB) buffer) used in column purification must be distilled in order to proceed to the next step in organic solvent. In the entire manufacturing process, pG (3’OMe) , p.p.G. (3’OMe) ,pp 7m G (3’Ome) , pN (2’OMe) pN, 7m G (3’OMe) pppN (2’OMe) Since purification must be performed at each stage of pN production, a total of five purification steps using ion exchange columns are required. Such lengthy steps, column purification, and distillation of the buffer solution increase the time and cost required for the production of the cap analog, making it less economical. Summary of the Invention [Problem to be solved by the invention]

[0013] One aspect is to provide a cap analog that can increase the efficiency of in vitro synthesis of 5'-capped mRNA molecules, not only increasing the efficiency of protein expression from capped mRNA, but also enabling economical production through reducing the number of synthesis and purification steps of the cap analog itself.

[0014] Another embodiment is to provide a 5' capped mRNA as the cap analog.

[0015] Yet another aspect is to provide a method for producing mRNA using the cap analogs.

[0016] Yet another aspect is to provide a composition or kit for producing 5'-capped mRNA, comprising the cap analog.

[0017] Yet another aspect is to provide a pharmaceutical composition for expression of a desired peptide or protein, comprising said cap analog.

[0018] Yet another embodiment is to provide a cell comprising a 5'-capped mRNA as the cap analog.

[0019] Yet another embodiment is to provide a cell comprising as said cap analog a protein or peptide translated from a 5' capped mRNA. [Means for solving the problem]

[0020] One embodiment provides a compound of formula 1, or a pharma- ceutically acceptable salt thereof: [ka]

[0021] where n=0, 1 or 2; Y1, Y2 and Y3 are each independently O or S; R1 is C 1~6 alkyl or CH2Ph, R2 and R3 are each independently H or a sulfonyl-containing group, with the proviso that at least one of R2 and R3 is a sulfonyl-containing group, said sulfonyl-containing group being independently selected from the group consisting of mesyl, esyl, triflyl, tresyl, tosyl, brosyl, nosyl, and dansyl; R4 and R5 are each independently OH or methoxy; R6 is OH or a mononucleotide or oligonucleotide having 1 to 7 bases; Z and Z' are each independently a natural, modified or non-natural nucleoside base.

[0022] Another embodiment provides a cap analog, which is a compound of Formula 1 above.

[0023] Yet another embodiment provides a 5'-capped mRNA as the cap analog.

[0024] Yet another aspect provides a method for producing a 5' capped mRNA comprising including said cap analog during mRNA synthesis.

[0025] Yet another embodiment provides a composition or kit for producing a 5'-capped mRNA comprising the cap analog.

[0026] Yet another aspect provides a pharmaceutical composition for expressing a desired peptide or protein, comprising a 5'-capped mRNA as the cap analog, and a pharma- ceutically acceptable carrier.

[0027] Yet another embodiment provides a cell comprising a 5'-capped mRNA as the cap analog.

[0028] Yet another embodiment provides a cell comprising as said cap analog a protein or peptide translated from a 5' capped mRNA. Effect of the Invention

[0029] In one embodiment, the cap analog, like TriLink's trimer cap analog, is used during mRNA synthesis to increase the efficiency of in vitro synthesis of 5'-capped mRNA molecules and increase the peptide or protein expression efficiency of the capped mRNA. In particular, it may be particularly useful for expression of genes where initial protein expression is important (e.g., expression of the nsp1 portion of self-amplifying RNA) since it may further increase early protein expression after transfection in a cap-dependent protein expression manner. Moreover, the cap analog can be synthesized economically in terms of time and cost through a reduction in the number of synthesis steps and purification steps, and may contribute to the synthesis of 5'-capped mRNA that is generally more economical, along with the increase in peptide or protein expression efficiency by the use of the capped mRNA.

[0030] Therefore, similar to currently commercialized trimer cap analogs, the cap analog can increase the efficiency of in vitro synthesis of 5'-capped mRNA molecules, significantly increase the peptide or protein expression efficiency of capped mRNA compared to conventional trimer cap analogs, and can be produced economically.

[0031] As such, the cap analogs have excellent advantages in terms of efficacy and production costs, and mRNAs containing the cap analogs disclosed herein can be very useful in treating or preventing diseases in mammals, including humans. [Brief description of the drawings]

[0032] [Figure 1]1 is a graph showing the relative in vitro transcription (IVT) yield (%) of compound (8) of Example 8 in accordance with one embodiment of the present invention, compared to 7mG(3'OMe)pppA(2'OMe)pG, after measuring the in vitro transcription (IVT) yield of capped luciferase mRNA using compound (8) of Example 8 or 7mG(3'OMe)pppA(2'OMe)pG as a cap analog. [Diagram 2] This is a graph showing the results of transfecting a HEK293T cell line with luciferase mRNA produced by in vitro transcription (IVT) reaction using compound (8) of Example 8 or 7mG(3'OMe)pppA(2'OMe)pG as a cap analogue, measuring luciferase activity over time, and quantifying the expression level of translated luciferase protein (mean ± standard deviation (n=3), ***: P<0.05, NS: not significant). [Diagram 3] FIG. 1 is a graph showing the results of calculating the total luciferase protein expression level from 1 hour to 48 hours after transfection with compound (8) of Example 8 or luciferase mRNA capped with 7mG(3'OMe)pppA(2'OMe)pG according to one embodiment of the present invention, as the AUC (area under the curve) value. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] The present invention will now be described in further detail.

[0034] All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the art in the field of the present invention unless otherwise defined. In addition, although preferred methods and samples are described in this specification, similar or equivalent methods and samples are also included in the scope of the present invention. In addition, numerical values ​​described in this specification are considered to include the meaning of "about" even if not expressly stated. The contents of all publications described as references in this specification are incorporated herein by reference in their entirety.

[0035] Definition of Terms As used herein, the following terms have the meanings defined below, unless otherwise specified.

[0036] The term "complementary" or "complementarity" as used herein in the context of a complex between a cap analog and a DNA template refers to standard Watson / Crick base pairing rules. For example, the sequence "5'-AGTC-3'" is complementary to the sequence "3'-TCAG-5'". The complementarity need not be perfect, and the duplicate may contain mismatched base pairs, degenerate or unmatched nucleotides. One of ordinary skill in the art may experimentally determine duplex stability by considering numerous variables, including, for example, the length of the oligonucleotide, the base composition of the oligonucleotide and its sequence, the occurrence of mismatched base pairs, ionic strength, hybridization buffer components, and reaction conditions.

[0037] Complementarity can be "complete" or "total" when all nucleotide bases of the two nucleic acid strands are matched by recognized base-pairing rules, "partial" when only a portion of the nucleotide bases of the cap analog and the DNA target are matched by recognized base-pairing rules, or "absent" when none of the nucleotide bases of the two nucleic acid strands are matched by recognized base-pairing rules.

[0038] The term "nucleoside" as used herein includes all naturally occurring nucleosides. The base rings most frequently found in naturally occurring nucleosides are the purine ring and the pyrimidine ring. Naturally occurring purine rings include, for example, adenine, guanine, and N6-methyladenine. Naturally occurring pyrimidine rings include, for example, cytosine, thymine, 5-methylcytosine, and uracil. Naturally occurring nucleosides include, but are not limited to, ribo, 2'-O-methyl, or 2'-deoxyribo derivatives of, for example, adenosine, guanosine, cytidine, thymidine, uridine, inosine, 7-methylguanosine, or uridine.

[0039] The terms "nucleoside analogs," "modified nucleosides," or "nucleoside derivatives" include synthetic nucleosides as described herein. The nucleoside derivatives also include nucleosides having modified base and / or sugar moieties, with or without protecting groups, such as 2'-deoxy-2'-fluorouridine, 5-fluorouridine, and the like. The compounds and methods provided herein include the above base rings and their synthetic analogs, as well as non-natural heterocyclic substituted base sugars and acyclic substituted base sugars. Other nucleoside derivatives that may be utilized in the present invention include, for example, LNA nucleosides, halogen substituted purines (e.g., 6-fluoropurine), halogen substituted pyrimidines, N6-ethyladenine, N4-(alkyl)-cytosine, 5-ethylcytosine, and the like (U.S. Patent No. 6,762,298).

[0040] The term "modified nucleoside base" refers to a base contained in the "modified nucleoside", such as a halogen-substituted purine (e.g., 6-fluoropurine), a halogen-substituted pyrimidine, N6-ethyladenine, N4-(alkyl)-cytosine, 5-ethylcytosine, and the like.

[0041] The term “C 1―n"Alkyl" means a linear or branched saturated hydrocarbon radical chain of 1 to n carbons. Specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, isopentyl, and n-hexyl.

[0042] In the process of synthesizing a substance, the term "in situ method" refers to carrying out the next step of reaction of the obtained product in the original vessel without additional purification process.

[0043] One embodiment relates to a compound of formula 1, or a pharma- ceutically acceptable salt thereof: chemical formula 1 [ka]

[0044] where n=0, 1 or 2; Y1, Y2 and Y3 are each independently O or S; R1 is C 1~6 alkyl or CH2Ph, R2 and R3 are each independently H or a sulfonyl-containing group, with the proviso that at least one of R2 and R3 is a sulfonyl-containing group, said sulfonyl-containing group being independently selected from the group consisting of mesyl, esyl, triflyl, tresyl, tosyl, brosyl, nosyl, and dansyl; R4 and R5 are each independently OH or methoxy; R6 is OH or a mononucleotide or oligonucleotide having 1 to 7 bases; Z and Z' are each independently a natural, modified or non-natural nucleoside base.

[0045] The pharmaceutically acceptable salt means the salt generally used in the pharmaceutical field, and may be specifically a base addition salt.The salt may be, for example, a monovalent metal salt, a divalent metal salt, an amine salt, or an amino acid salt.The monovalent metal salt may be, for example, a Na salt, a Li salt, or a K salt, the divalent metal salt may be, for example, a Ca salt, a Zn salt, or a Mg salt, the amine salt may be, for example, a trimethylamine salt, a triethylamine salt, an ammonia salt, a pyridine salt, or a picoline salt, and the amino acid salt may be, but is not limited to, an arginine salt, a lysine salt, or a histidine salt.

[0046] The pharma- ceutically acceptable salt may be in the form of a salt that allows the compound of Chemical Formula 1 to be in a stable, electrically neutral form in an aqueous liquid.

[0047] The compound of formula 1, or a pharma- ceutically acceptable salt thereof, may be, for example, a compound of formula 1a: chemical formula 1a [ka]

[0048] Where: n=0, 1 or 2; X1, X2, X3, and X4 are each independently absent, a monovalent metal ion, a divalent metal ion, or a combination thereof, wherein X1, X2, X3, and X4 are selected such that the compound of Formula 1 is electroneutral; Y1, Y2 and Y3 are each independently O or S; R1 is C 1~6 alkyl or CH2Ph, R2 and R3 are each independently H or a sulfonyl-containing group, with the proviso that at least one of R2 and R3 is a sulfonyl-containing group, said sulfonyl-containing group being independently selected from the group consisting of mesyl, esyl, triflyl, tresyl, tosyl, brosyl, nosyl, and dansyl; R4 and R5 are each independently OH or methoxy; R6 is OH or a mononucleotide or oligonucleotide having 1 to 7 bases; Z and Z' are each independently a natural, modified or non-natural nucleoside base.

[0049] In one embodiment, the monovalent metal ion is Na + , Li + and K + and the divalent metal ion is selected from the group consisting of Mg 2+ , Zn 2+ and Ca 2+ The compound is selected from the group consisting of:

[0050] In one embodiment, the sulfonyl-containing group is a mesyl, esyl, or tosyl group.

[0051] In one embodiment, the C 1―6 Alkyl is methyl, ethyl, propyl, isopropyl or t-butyl.

[0052] In one embodiment, Z and Z' in Formula 1 are each independently a natural purine or pyrimidine base moiety, or modified forms thereof. More specifically, Z and Z' are each independently selected from the group consisting of guanine, adenine, cytosine, thymine, uracil, and modified forms thereof, and in one embodiment, Z and Z' are adenine and guanine, respectively.

[0053] In one embodiment, in Formula 1, R6 is OH, a mononucleotide, or an oligonucleotide having 2 to 5 bases.

[0054] In one embodiment, the compound having the structure of formula 1a is In formula 1a, n=0, 1 or 2; X1, X2, X3 and X4 are each independently absent or Na; Y1, Y2 and Y3 are each independently O or S; R1 is C 1~6 is alkyl, R2 and R3 are each independently H or a sulfonyl-containing group, with the proviso that at least one of R2 and R3 is a sulfonyl-containing group, said sulfonyl-containing groups being each independently selected from the group consisting of mesyl, esyl, and tosyl; R4 and R5 are each independently OH or methoxy; R6 is OH or a mononucleotide or oligonucleotide having 1 to 3 bases; Z is a compound which is a natural, modified or unnatural nucleoside base.

[0055] In one embodiment, R4 is methoxy and R5 is OH.

[0056] In one embodiment, the compound of Formula 1 is a compound selected from the group consisting of the compounds listed in Table 1 below: [Table 1]

[0057] In the above abbreviated compound names, the G means guanosine, 7m G means 7-methylguanosine, A means adenosine, p means -P(=O)(OH)O-, Es means esyl, Ms means mesyl, Ts means tosyl and Me means methyl.

[0058] The compound of formula 1 can be prepared, for example, by the method illustrated in the Examples. A person skilled in the art can prepare the compound of formula 1 by appropriately modifying the method illustrated in the Examples below, and modifying the reaction conditions, reaction sequence, and reaction compounds.

[0059] A specific example of the compound of Formula 1 can be prepared by the method shown in Reaction Scheme 2a below. Reaction Scheme 2a [ka]

[0060] Illustrative specific preparation methods of the above reaction schemes are described in detail in the following Examples.

[0061] The above reaction scheme 2a may be briefly represented by the following abbreviations: [ka]

[0062] wherein G means guanosine, 7m G means 7-methylguanosine, A means adenosine, p means -P(=O)(OH)O-, TBS means tertiary butyldimethylsilyl, Me means methyl, Im means imidazolide and Ms means mesyl.

[0063] The above preparation method can prepare the compound of Formula 1 through four steps. Therefore, the compound of Formula 1 can be prepared more economically with fewer steps than the conventional known methods for preparing cap analogues.

[0064] The compound of Formula 1 according to one embodiment of the present invention can be used as a cap analogue. Thus, the compound of Formula 1, that is, the cap analogue, can be used to prepare 5'-capped mRNA.

[0065] Accordingly, one particular embodiment provides a cap analog that is a compound of Formula 1 above.

[0066] In addition, one particular embodiment provides a method for producing a 5'-capped mRNA, comprising including a cap analog, which is a compound of Formula 1, during mRNA synthesis.

[0067] In addition, a specific embodiment provides a composition or kit for producing 5'-capped mRNA, comprising a cap analog that is a compound of Chemical Formula 1.

[0068] In addition, a particular embodiment provides a use of the cap analog, which is the compound of Chemical Formula 1, in synthesizing mRNA.

[0069] In addition, a particular embodiment provides a 5'-capped mRNA as a cap analog, which is the compound of Chemical Formula 1.

[0070] The method for preparing 5'-capped mRNA using the cap analog of the compound of Formula 1 may be carried out by any method known in the art. In one embodiment, 5'-capped mRNA may be prepared by a co-transcriptional capping method, which is an mRNA synthesis method in which a chemically synthesized cap analog is simultaneously introduced during in vitro mRNA synthesis, and the cap analog constitutes the 5'-end. Specifically, the method may include introducing the cap analog of the compound of Formula 1 into a mixture containing RNA polymerase under conditions in which a polynucleotide template is transcribed by the RNA polymerase, and incubating the mixture for a time sufficient to allow transcription of the template.

[0071] The cap analog of Chemical Formula 1 according to one embodiment can increase the transcription efficiency of mRNA in vitro compared to the efficiency of initiation using standard GTP, standard ATP, standard CTP or standard UTP, and can subsequently increase the protein expression efficiency of the capped mRNA transcribed during the translation process. By increasing the transcription efficiency, the synthesis of mRNA can be increased by, for example, about 10%, about 20%, about 40%, about 60%, about 80%, about 90%, about 100%, about 150%, about 200% or about 500% compared to synthesizing mRNA by a conventional method.

[0072] The cap analogue, which is the compound of formula 1 according to the above embodiment, is a conventional trimeric cap analogue of Trilink Co., Ltd. ( 7m G (3’OMe) pppA (2’OMe) pG) can significantly increase the protein expression level of the 5'-capped mRNA molecule. More specifically, in the compound of Formula 1, the 3' OH residue is monosulfonylated and the 5'-capped mRNA molecule is 5'-capped with a cap analogue, whereas the conventional Trilink trinucleotide 3'-methoxy cap analogue ( 7m G (3’OMe) pppA (2’OMe) After transfection, early protein expression can be significantly increased by mRNA molecules 5'-capped with pG, thereby significantly increasing the overall protein expression level (see Experimental Examples).

[0073] The cap analog, which is the compound of Chemical Formula 1 according to one embodiment, has the advantage that it can be synthesized economically in terms of time and cost by reducing the number of synthesis steps and column purification steps compared to the conventional TriLink trimer cap analog.

[0074] Specifically, when preparing the compound of formula 1, an optimized starting material (e.g., G (2’TBS) By selecting the starting material (G), the conventional two-step process of converting guanosine to guanosine monophosphate and then to diphosphate can be shortened to a one-step process of immediately converting guanosine to diphosphate, and the next step is 7-methylation of guanosine in situ, thereby shortening the crystallization / purification process. (2’TBS) ) was mesylated at the 3'OH and 5'OH, and then 7-methylguanosine diphosphate (pp 7m G), 7-methylguanosine diphosphate (pp 7m The synthesis steps up to G) can be shortened by two steps compared to the conventional Trilink trimer cap analogue production method (method shown in reaction scheme 1 below). 7mG (3’OMe) pppN (2’OMe) The method for producing pN requires a total of five ion exchange column purifications in the following reaction scheme 1. (3’OMe) , p.p.G. (3’OMe) ,pp 7m G (3’Ome) , pN (2’OMe) pN, 7m G (3’OMe) pppN (2’OMe) pN) (see US Pat. No. 10,913,768), the cap analog containing the compound of formula 1 can be prepared by the following reaction scheme 2 (purification target: pp 7m G (3’OR) , pN (2’OMe) pN, 7m G (3’OR) pppN (2’OMe) pN), through a total of three ion exchange column purifications. Thus, the cap analog of the compound of Formula 1 according to one embodiment can be prepared using a significantly reduced number of purification steps and a shorter synthesis process than conventional cap analogs.

[0075] Reaction Scheme 1 [ka]

[0076] Reaction Scheme 2 [ka]

[0077] In the reaction schemes 1 and 2, G represents guanosine; 7m G represents 7-methylguanosine, N represents any one of adenosine, cytidine, guanosine, and uridine, p represents -P(=O)O2-, TBS represents tertiary butyldimethylsilyl, Me represents methyl, Im represents imidazolide, and R represents a substituent selected from the group consisting of tosyl, brosyl, nosyl, esyl, mesyl, triflyl, tresyl, and dansyl.

[0078] Therefore, the capped analog of the compound of Formula 1 according to the above embodiment has an advantage that the production time and production cost of the capped analog itself can be further reduced.

[0079] In one embodiment, the method for producing 5'-capped mRNA may further include adding at least one modified NTP to the transcription reaction. The modification of the at least one modified NTP does not substantially impair RNA polymerase-mediated synthesis of mRNA. The modified NTP may, for example, include one or more modified nucleoside bases, one or more modified sugars, one or more modified 5'-triphosphates. Such modified NTPs are incorporated onto the 3' end of the cap analog, which does not block transcription and supports additional extension of the primer. The modified group of the modified NTP may be a detectable label or a detectable marker. Thus, after transcription, the produced mRNA containing the detectable label or marker may be identified by size, mass, color, and / or affinity capture. In one embodiment, the detectable label or marker is a fluorescent dye and the affinity capture label is biotin.

[0080] In one embodiment, one or more components of the transcription reaction (cap analog and / or NTP) can be labeled with a detectable label or marker. Thus, after transcription, the mRNA molecule can be identified, for example, by size, mass, affinity capture, or color. For example, the detectable label is a fluorescent dye and the affinity capture label is biotin.

[0081] The 5'-capped mRNA production kit or composition thereof may include all transcription reagents for normal mRNA synthesis (e.g., FLuc mRNA). More specifically, the kit may include one or more reagents selected from the group consisting of a cap analog, a container labeled for transcription, a manual for performing mRNA synthesis, one or more unmodified NTPs, one or more modified NTPs (e.g., methylpseudouridine-5'-triphosphate), RNA polymerase, other enzymes, a reaction buffer, magnesium, and a DNA template.

[0082] The 5'-capped mRNA produced using a cap analog according to one embodiment contains the cap analog within its structure, and the 5'-capped mRNA can be administered to an organism and used to express a protein in the organism.

[0083] Thus, yet another aspect provides a method for expressing a desired peptide or protein in vivo, comprising administering to the body an mRNA containing the cap analog.

[0084] In another aspect, there is provided a pharmaceutical composition for expressing a desired peptide or protein, comprising an mRNA containing the cap analog and a pharma- ceutically acceptable carrier.

[0085] In the present application, the term "target peptide or protein" refers to any protein that requires expression in a living body, and in the present application, the mRNA capable of expressing the target peptide or protein is a cap analog according to one embodiment, which is 5'-capped and capable of expressing the target protein in a living body.

[0086] Depending on the type of the target peptide or protein, the pharmaceutical composition can have a therapeutic or preventive effect against the target disease in vivo. Therefore, the pharmaceutical composition can be used to treat or prevent any disease that can be treated or prevented by expression of a peptide or protein. Diseases that can be treated or prevented by expression of a specific type of peptide or protein are known, and the pharmaceutical composition can be used to induce expression of the peptide or protein and to prevent or treat the target disease.

[0087] Another aspect provides a pharmaceutical use of mRNA containing said cap analogue for the prevention or treatment of any disease which can be ameliorated by in vivo expression of a peptide or protein.

[0088] The pharmaceutical composition and the treatment or prevention method may be used for gene replacement therapy, genome editing, cancer immunotherapy, or treatment or prevention using a vaccine, etc. In one embodiment, the pharmaceutical composition is an mRNA vaccine.

[0089] The pharmaceutical compositions may be formulated for administration by injection or other suitable route known to those of ordinary skill in the art to treat or prevent a particular condition. Injectable compositions include a pharma- ceutically acceptable carrier, such as sterile saline. Injectable compositions may also be formulated as suspensions in lipids or phospholipids, liposomal suspensions, or aqueous emulsions. Methods for formulating the pharmaceutical compositions are well known to those of ordinary skill in the art.

[0090] In one embodiment, the pharmaceutical composition may contain the cap analog-containing mRNA as an active ingredient at a concentration of about 0.01% to 1%, which may vary depending on the frequency of administration, dosage, and administration method.

[0091] In one embodiment, the pharmaceutical composition can be administered to a mammal, specifically a human, and the dosage varies depending on the individual's health condition, severity of the disease, body weight, age, race, etc., and an expert in the art can determine the appropriate dosage. In one embodiment, the dosage for a human is in the range of 0.0001 to 100 mg / day, more specifically, in the range of about 0.1 to 50 mg / day.

[0092] An mRNA containing a cap analog according to one embodiment of the invention can be introduced into a cell in vivo or in vitro to express a protein or peptide.

[0093] Thus, another aspect provides a cell comprising an mRNA comprising a cap analog according to the previous aspect.

[0094] Yet another embodiment provides a cell comprising a protein or peptide translated from an mRNA comprising a cap analog according to the previous embodiment.

[0095] Methods for expressing proteins or peptides in cells in vivo or in vitro using the above-mentioned mRNA are known in the art, and proteins or peptides can be appropriately expressed in cells by such conventional general methods. EXAMPLES

[0096] The following examples are presented to aid in understanding the present invention. The following examples are merely illustrative of the present invention, and it is obvious to those skilled in the art that various changes and modifications are possible within the scope and technical spirit of the present invention, and it goes without saying that such changes and modifications are included in the scope of the appended claims.

[0097] Explanation of Abbreviations The abbreviations used below are as follows: TBDMS: tertiary butyldimethylsilyl MsCl: methanesulfonyl chloride TEA.3HF: Triethylamine trihydrofluoride THF: tetrahydrofuran ACN: Acetonitrile DMS: Dimethyl sulfate PW: Pure water DPS: 2,2'-dipyridyl disulfide TEA: Triethylamine PPh3: Triphenylphosphine DMF: Dimethylformamide DMTr: 4,4'-dimethoxytrityl DTT: Dithiothreitol

[0098] IVT: in vitro transcription Examples 1 to 3: 7-Methyl-3'-mesyloxy-guanosine 5'-diphosphate imidazolide (Im-pp 7m G (3’OMesyloxy) )(3) Manufacturing [ka]

[0099] Example 1: Preparation of 2'-O-tert-butyldimethylsilyl-3'mesyloxy-5'mesyloxyguanosine (1) In the case of 2'-O-tertiary butyldimethylsilylguanosine, it was obtained commercially and used as the starting material without further treatment. 3.97 g (9.99 mmol) of 2'-O-tertiary butyldimethylsilylguanosine was added to 91 ml of anhydrous pyridine, and then stirred for 10 minutes at 0°C while gradually adding 2.32 ml (29.97 mmol) of methanesulfonyl chloride. The reaction solution was then stirred at 10-20°C for 3 hours. 270 ml of distilled water was gradually added at 0°C, and then stirred at 5-10°C for 1 hour. The crystals formed at this time were filtered, washed with distilled water, and dried in vacuum to obtain 4.36 g (yield: 80%) of the target compound (1).

[0100] 1H NMR (400 MHz, DMSO-d6, 25℃): δ= 10.72 (s, 0.84H), 7.96 (s, 1H), 6.56 (1.65H), 5.80 (d, 1H), 5.15 (d, 1H), 4.96 (m, 1H), 4.56 ~ 4.62 (m, 3H, overlapped), 3.32 (m, 3H), 3.25 (m, 3H), 0.699 (m, 9H), -0.014 (m, 3H), -0.231 (m, 3H)

[0101] Example 2: Preparation of 7-methyl-3'-mesyloxyguanosine 5'-diphosphate triethylammonium salt (2) 4.2 g (7.59 mmol) of 2'-O-tertiary butyldimethylsilyl-3'mesyloxy-5'mesyloxyguanosine (1) was dissolved in 33.6 mL of anhydrous acetonitrile, 17.81 g (19.73 mmol) of tris(tetrabutylammonium) hydrogen pyrophosphate was added, and the mixture was stirred at room temperature for 48 hours. Then, 3.71 mL (22.77 mmol) of triethylamine trihydrofluoride was gradually added at room temperature while stirring for 1 hour. 42 mL of dichloromethane and 42 mL of purified water were added to the reaction solution for washing, and the aqueous layer was separated. 42 mL of dichloromethane was added to the separated aqueous layer for washing, and the aqueous layer was further separated. The pH of the separated aqueous layer was adjusted to 4.0 using glacial acetic acid. 28.79 mL (303.6 mmol) of dimethyl sulfate was gradually added to the reaction solution over 30 minutes, and the mixture was stirred at room temperature for 3 hours. At this time, the pH of the reaction solution was maintained at 4.0±0.5 using 1M sodium hydroxide. The reaction solution was extracted three times with 126 mL of dichloromethane to remove unreacted dimethyl sulfate, and then the aqueous layer was separated. The separated aqueous layer was titrated to pH 5.5 using 1M sodium hydroxide, and the target compound was separated using a DEAE Sepharose column (140x210 mm) and freeze-dried to obtain 1.68 g (yield 30.0%) of the triethylammonium salt (2) of the target compound.

[0102] 1H NMR (400 MHz, D2O, 25℃): δ= 6.11 (d, 1H), 5.44 (dd, 1H), 5.02 (m, 1H), 4.74 (m, D2O overlapped, 1H), 4.23 (m, 2H), 4.1 (s, 3H), 3.32 (s, 3H), 3.19 ~ 3.13 (m, 12H), 1.28 ~ 1.18 (m, 18H) 31 P NMR (162 MHz, D2O, 25℃): δ= -7.13 (d, 1P), -10.77 (d, 1P)

[0103] Example 3: Preparation of 7-methyl-3'-mesyloxyguanosine 5'-diphosphate imidazolide (3) 1.0 g (1.36 mmol) of 7-methyl-3'-mesyloxyguanosine 5'-diphosphate triethylammonium salt (2) was dissolved in 40 ml of dimethylformamide, and 0.93 g (13.6 mmol) of imidazole and 1.50 g (6.8 mmol) of 2,2'-dipyridyl disulfide were added. 0.189 mL (1.36 mmol) of triethylamine and 1.78 g (6.8 mmol) of triphenylphosphine were added to the reaction solution, and the mixture was stirred at room temperature for 2 hours. The reaction solution was added to a solution in which 0.333 g (2.72 mmol) of sodium perchlorate was dissolved in 320 mL of acetone, and the mixture was cooled to 4°C. The crystals formed were filtered, washed with cold acetone, and then dried in a vacuum to obtain 0.83 g (yield 100.0%) of the sodium salt of the target compound (3).

[0104] 1 H NMR (400 MHz, D2O, 25℃): δ= 7.999 (s, 1H), 7.32 (s, 1H), 7.07 (s, 1H), 6.06 (d, 1H), 5.29 (m, 1H), 4.90 (m, 1H), 4.67 (m, 1H), 4.21 ~ 4.08 (m, 2H), 4.05 (s, 3H), 3.29 (s, 3H) 31 P NMR (162 MHz, D2O, 25℃): δ= -11.41 (d, 1P), -19.61 (d, 1P)

[0105] Examples 4 to 7: Preparation of pA(2'OMe)pG(7) [ka]

[0106] Example 4: Preparation of N2-isobutyryl-2',3'-diacetoxy-guanosine (4) 2.4g (3.66mmol) of N2-isobutyryl-5'-O-DMT guanosine was dissolved in 12ml of dichloromethane, and then 1.47ml (18.3mmol) of pyridine and 1.78ml (18.3mmol) of acetic anhydride were added and stirred at room temperature for 5 hours. 24ml of ethyl acetate was added to the reaction solution for extraction, and then washed with 14.4ml of saturated aqueous sodium bicarbonate solution, 14.4ml of 20% aqueous citric acid solution, and 14.4ml of distilled water. The organic layer was dried with sodium sulfate, and the residue obtained by vacuum distillation was dried with nitrogen for one day. 36ml of 3% aqueous trichloroacetic acid solution was added to the dried product, and the mixture was reacted at room temperature for 3 hours, after which 24ml of methanol was added and the mixture was stirred for another 2 hours and 30 minutes. The reaction solution was distilled under reduced pressure, and 36ml of dichloromethane and 18ml of distilled water were added, followed by neutralization by adding saturated aqueous sodium bicarbonate solution. The organic layer was separated, dried with sodium sulfate, and then distilled under reduced pressure. The residue after vacuum distillation was dissolved in 12 ml of ethyl acetate and then slowly added to 108 ml of hexane at room temperature to crystallize. The crystals were filtered, washed three times with 12 ml of hexane, and then dried in vacuum to obtain 1.43 g of the target compound (4) (yield 89.6%).

[0107] LC-MS (ESI, m / z) = 438.16 [M + H + ]

[0108] Example 5: Preparation of (N2-isobutyryl-2',3'-diacetoxy-guanosinyl)-N6-benzoyl-2'-methoxy-adenosinylcyanoethyl phosphate (5) In the case of 5'-O-DMT-N6-benzoyl-2'-methoxy-adenosine amidite, it was obtained commercially and used as the starting material without further treatment. 1.43 g (3.28 mmol) of N2-isobutyryl-2',3'-diacetoxy-guanosine (4) and 3.78 g (4.26 mmol) of 5'-O-DMT-N6-benzoyl-2'-methoxy-adenosine amidite were dissolved in 28.4 mL of 1H-tetrazole (0.45 M in acetonitrile (12.79 mmol)) and stirred at room temperature for 1 h. 544 mg (2.15 mmol) of iodine was dissolved in 56.7 mL of tetrahydrofuran:distilled water:pyridine mixture (v:v:v, 7:2:1), and the solution was added to the reaction solution and stirred for 45 min. To the reaction solution, 7.2 ml of 10% sodium thiosulfate aqueous solution was added, followed by the addition of 43 ml of dichloromethane and 14.3 ml of distilled water, followed by separation of the organic layer. The separated organic layer was dried over sodium sulfate, distilled under reduced pressure, and then dried under nitrogen for one day. 21.5 ml of 3% aqueous trichloroacetic acid solution was added to the dried product, reacted at room temperature for 3 hours, then 14.3 ml of methanol was added, and the mixture was stirred for another 2 hours and 30 minutes. The reaction solution was distilled under reduced pressure, and 21.5 ml of dichloromethane and 10.7 ml of distilled water were added, followed by neutralization by adding a saturated aqueous solution of sodium bicarbonate. The separated organic layer was washed with distilled water, dried over sodium sulfate, and distilled under reduced pressure. The residue distilled under reduced pressure was dissolved in 28.6 ml of dichloromethane, and then slowly added to 86 ml of methyl tertiary butyl ether at room temperature to crystallize. The crystals formed were filtered and dried in vacuum for one day, yielding 2.7 g of the target compound (5) (yield 87.8%).

[0109] LC-MS (ESI, m / z) = 938.28 [M + H + ]

[0110] Example 6: p(OCE)2A bz (2'OMe)p(OCE)G ib Preparation of (2',3'OAc) (6) 2g (2.13mmol) of (N2-isobutyryl-2',3'-diacetoxy-guanosinyl)-N6-benzoyl-2'-methoxy-adenosinylcyanoethyl phosphate ester (5) and 1.11ml (4.26mmol) of bis(2-cyanoethyl)-N,N-diisopropylphosphoramidite were dissolved in 9.47mL of 1H-tetrazole (0.45M acetonitrile solution (4.26mmol)) and stirred at room temperature for 30 minutes. 811mg (3.2mmol) of iodine was dissolved in 30ml of a mixture of tetrahydrofuran (THH):distilled water:pyridine (v:v:v, 7:2:1), and the solution was added to the reaction solution and stirred for 30 minutes. 20ml of 10% sodium thiosulfate solution was added to the reaction solution, followed by addition of 100ml of dichloromethane and 40ml of distilled water, and the organic layer was separated. The separated organic layer was dried over sodium sulfate and distilled under reduced pressure. 27 ml of dichloromethane was added to the concentrated solution distilled under reduced pressure to dissolve it, and then it was slowly added to 133 ml of methyl tertiary butyl ether at room temperature to crystallize it. The crystals formed were filtered and dried in vacuum for one day to obtain 2.2 g of the target compound (6) (yield 92.2%).

[0111] LC-MS (ESI, m / z) = 1124.30 [M + H + ]

[0112] Example 7: Preparation of pA(2'OMe)pG(7) p(OCE)2A bz (2'OMe)p(OCE)G ib 2 g (1.78 mmol) of (2',3'OAc) (6) was added to a mixture of 44 mL of methanol and 44 mL of concentrated ammonia, and the mixture was stirred at 50-55°C for 24 hours. When the reaction was completed, the solvent was distilled under reduced pressure, and 20 mL of methanol was added to the concentrate, which was then distilled three times. The concentrate was dissolved in distilled water again, and purified using a DEAE Sepharose column and reverse phase chromatography to obtain 1.13 g (70% yield) of the triethylammonium salt of the target compound (7).

[0113] 1H NMR (400 MHz, D2O, 25℃): δ= 8.44 (s, 1H), 8.12 (s, 1H), 7.90 (s, 1H), 6.07 (m, 1H), 5.80 (m, 1H), 4.46 ~ 4.01 (m, 8H), 3.45 (m, 3H) LC-MS (ESI, m / z) = 707.13 [M + H+]

[0114] Example 8: 7m G (3’OMs) pppA (2’OMe) Preparation of pG(8) [ka]

[0115] 0.55 g of magnesium chloride was added and dissolved in 27.5 mL of dimethylformamide. 7m 0.6 g (0.99 mmol) of G(3'OMs)(3) and 0.5 g (0.55 mmol) of pA(2'OMe)pG(7) were added and stirred at room temperature for 24 hours. After the reaction was completed, 275 mL of 25 mM ethylenediaminetetraacetic acid solution was added dropwise to terminate the reaction, cooled to room temperature, and neutralized with 1 M sodium bicarbonate solution. The reaction solution was purified using a DEAE Sepharose column (140 x 210 mm), distilled, and dried in vacuum. The dried solid was dissolved in 2.5 mL of distilled water, and then added to a solution of 299 mg (2.44 mmol) of sodium perchlorate dissolved in 15 mL of acetone, cooled to 4°C, and the resulting crystals were filtered, washed with cold acetone, and dried in vacuum to obtain 0.43 g (yield 60.0%) of the sodium salt of the target compound (8).

[0116] 1H NMR (400 MHz, D2O, 25℃): δ= 8.28 (s, 1H), 8.05 (s, 1H), 7.89 (s, 1H), 5.92 (d, 1H), 5.82 (d, 1H), 5.77 (d, 1H), 5.33 (m, 1H), 4.59 ~ 4.12 (m, overlapped, 13H), 4.00 (s, 3H), 3.39 (s, 3H), 3.29 (s, 3H) 31 P NMR (162 MHz, D2O, 25℃): δ= -0.27 (s, 1P), -10.88 (dd, 2P), -22.29 (t, 1P). LC-MS (ESI, m / z) = 1224.13 [M + H + ]

[0117] Experimental example 1: In vitro transcription (IVT) of capped luciferase mRNA via co-transcriptional capping For the mRNA synthesis reaction using the compound of Example 8 as a cap analog, 100 ng / ml firefly luciferase, DNA transcription template (containing poly A 101), 5 mM ATP, CTP, GTP, me 1 A transcription reaction mixture was prepared containing φTP (1-N-Methyl-Pseudouridine 5'-Triphosphate), 4 mM of the compound of Example 8, 10 U / ml T7 RNA polymerase (New England Biolab # dy1670), 1 U / ml RNase inhibitor protein, 0.01 U / ml inorganic pyrophosphatase, 40 mM Tris HCl (pH 8.0), 20 mM magnesium acetate, 1 mM spermidine, and 10 mM DTT. As a comparative example, the trimer cap analog m7G from Trilink was used instead of the compound of Example 8. (3’OMe) pppA (2’OMe)pG was added under the same conditions to prepare transcription reaction mixtures. Each prepared transcription reaction mixture was reacted at 37°C for 1 to 4 hours. Then, to terminate the reaction, 10 mM Tris·HCl (pH 7.5), 2.5 mM magnesium chloride, 0.1 mM calcium chloride, and 100 U / mL DNaseI (Thermo Fisher catalog #2270A) were added to the reaction mixture and reacted at 25°C for 1 hour. The mRNA after the reaction was terminated was purified using Monarch® RNA Cleanup Kit from New England Biolabs or reverse-phase high performance liquid chromatography according to the manufacturer's instructions. The concentration of the purified mRNA was measured using Thermo Scientific Nanodrop, and the total amount of mRNA was calculated by multiplying the concentration by the reaction volume. Then, to derive the mRNA in vitro transcription (IVT) yield, the total amount of mRNA synthesized using each different cap analog was divided by the amount of L-DNA input to quantify the in vitro transcription (IVT) yield value by the cap analog. Specifically, an in vitro transcription (IVT) yield value of 100 means that 100 μg mRNA was synthesized from 1 μg L-DNA. It can be calculated by the following formula 1.

[0118] Then, Trilink's trimer cap analogue 7m G (3’OMe) pppA (2’OMe) The in vitro transcription (IVT) yield value (Formula 1) of pG was designated as 100%, and the in vitro transcription (IVT) yield value (Formula 1) of each example compound was determined as follows: 7m G (3’OMe) pppA (2’OMe) Divide by the pG yield value (Formula 1) and then multiply by 100. 7m G (3’OMe) pppA (2’OMe) The in vitro transcription (IVT) yield of Example 8 relative to pG was expressed as a percentage (%), which can be calculated by the following Equation 2.

[0119]

number

[0120]

number

[0121] Example 8 or 7m G (3’OMe) pppA (2’OMe) The in vitro transcription (IVT) yield values ​​for pG were run in triplicate and the significance was verified via unpaired t-tests, and the results are shown in Figure 1.

[0122] According to the results of FIG. 1, the in vitro transcription (IVT) yield of the compound of Example 8 is higher than that of the conventional third generation cap analog. 7m G (3’OMe) pppA (2’OMe) It was confirmed that the in vitro transcription (IVT) yield of pG was almost the same as that of pG. Thus, the compound according to one embodiment is a conventional third generation cap analog. 7m G (3’OMe) pppA (2’OMe) It was confirmed that it can be used to synthesize 5'-capped mRNA with a yield equivalent to that of pG.

[0123] Experimental example 2: Translation of capped luciferase mRNA in HEK293T cells In Experimental Example 1, the compound of Example 8 or 7m G (3’OMe) pppA (2’OMe) The translation activity of capped luciferase mRNA produced by in vitro transcription (IVT) of each pG was evaluated in human embryonic kidney 293T cell line (HEK293T). HEK293T cells were cultured in DMEM supplemented with 10% FBS, 1% penicillin / streptomycin at 37°C under 5% CO2 atmosphere. 5HEK293T cells were plated at 10 cells / well in a 6-well plate. The next day, cells were transfected with 100 ng of mRNA per well using transfection reagent (messengerMAX lipofectamine (Invitrogen catalog #LMRNA003)) (as recommended by the transfection reagent supplier, in tube A, 0.3 μL of transfection reagent was diluted in 5 μL of complex medium (Opti-MEM (Life technologies)) and incubated at room temperature for 10 minutes, and in tube B, 10 μL of Opti-MEM was diluted with the capped luciferase mRNA prepared above. 200ng was prepared by diluting. The solutions in tube A and tube B were mixed and incubated at room temperature for 5 minutes. The vehicle control group was incubated in the same manner, except for the capped luciferase mRNA. The incubated mixed solution was then used to transfect cells. After transfection, cells were harvested at 1, 4, 6, 12, 24, and 48 hours, and luciferase activity was measured using the Dual-Glo® Luciferase Assay kit (Promega catalog #E2910) according to the manufacturer's recommendations (75μl phosphate buffer solution (PBS) was added after removing the medium from the transfected cells). Then, 75μl of Dual-Glo® reagent was added and mixed, and incubated for 10 minutes, and firefly luciferase (firefly luciferase) was measured using a luminometer (Perkin Elmer). The luminescence of firefly luciferase was measured. Then, 75 μl of Dual-Glo® Stop&Glo® Reagent was added and incubated for 10 minutes, after which the luminescence of Renilla luciferase was measured again. The ratio of the luminescence of firefly luciferase to the luminescence of Renilla luciferase, which was the control group, was calculated, and the luciferase activity (relative light unit (RLU)) was normalized for each time period and shown in FIG. 2.All luciferase activity measurements were performed independently and repeated three times, and the significance was verified by one-way ANOVA and Tukey's post hoc tests. In addition, to estimate the total luciferase protein expression level from 1 hour to 48 hours after transfection, the AUC (area under the curve) was calculated using GraphPad Prism. To obtain the total peak area value, the luciferase value at each time was substituted on the Y axis and the corresponding time value was substituted on the X axis, and a curve as shown in Figure 2 was generated. Then, using the trapezoid rule of Prism, the mRNA capped with Example 8 compound and 7m G (3’OMe) pppA (2’OMe) The total luciferase area value with pG-capped mRNA was calculated and is shown in Figure 3. The formula for calculating the area value in Prism is as follows:

[0124]

number

[0125] The mRNA capped with the compound according to Example 8 was synthesized by conventional 7m G (3’OMe) pppA (2’OMe) The mRNA capped with pG showed significantly higher luciferase activity from early time points 6 to 12 hours after transfection (see FIG. 2). In addition, due to the increased protein expression at the early transfection time point, the total protein expression level was also significantly higher in the mRNA capped with Example 8 than in the mRNA capped with pG. 7m G (3’OMe) pppA (2’OMe) It was confirmed that the mRNA was higher than that capped with pG (see FIG. 3).

[0126] According to the results shown in Figures 2 and 3, it was found that a capping analog according to one embodiment in which a sulfonyl-containing group is introduced instead of a methyl group to the 3' hydroxyl group of methylguanosine has an improved protein expression enhancing effect compared to conventional commercialized cap analogs.

Claims

1. A compound of formula 1, or a pharma- ceutically acceptable salt thereof: 【Chemistry 1】 where n=0, 1 or 2; Y １ , Y ２ and Y ３ are each independently O or S; R １ is C １～６ Alkyl or CH ２ Ph, R ２ and R ３ are each independently H or a sulfonyl-containing group, with the proviso that R ２ and R ３ at least one of the sulfonyl-containing groups is independently selected from the group consisting of mesyl, esyl, triflyl, tresyl, tosyl, brosyl, nosyl, and dansyl; R ４ and R ５ are each independently OH or methoxy; R ６ is OH or a mononucleotide or oligonucleotide having 1 to 7 bases, Z and Z' are each independently a natural, modified or unnatural nucleoside base.

2. 2. The compound of claim 1 having the structure of formula 1a: Formula 1a 【Chemistry 2】 where n=0, 1 or 2; X １ , X ２ , X ３ and X ４ are each independently absent, a monovalent metal ion, a divalent metal ion, or a combination thereof; １ , X ２ , X ３ and X ４ is selected so that the compound of formula 1 is electroneutral; Y １ , Y ２ and Y ３ are each independently O or S; R １ is C １～６ Alkyl or CH ２ Ph, R ２ and R ３ are each independently H or a sulfonyl-containing group, with the proviso that R ２ and R ３ at least one of the sulfonyl-containing groups is independently selected from the group consisting of mesyl, esyl, triflyl, tresyl, tosyl, brosyl, nosyl, and dansyl; R ４ and R ５ are each independently OH or methoxy; R ６ is OH or a mononucleotide or oligonucleotide having 1 to 7 bases, Z and Z' are each independently a natural, modified or unnatural nucleoside base.

3. The monovalent metal ion is Na ＋ , Li ＋ and K. ＋ and the divalent metal ion is selected from the group consisting of Mg ２＋ , Zn ２＋ and Ca ２＋ 3. The compound of claim 2, selected from the group consisting of:

4. 3. The compound of claim 2, wherein Z and Z' are each independently selected from the group consisting of guanine, adenine, cytosine, thymine, uracil, and modified forms thereof.

5. n=0, 1 or 2; X １ , X ２ , X ３ and X ４ are each independently absent or Na; Y １ , Y ２ and Y ３ are each independently O or S; R １ is C １～６ is alkyl, R ２ and R ３ are each independently H or a sulfonyl-containing group, with the proviso that R ２ and R ３ at least one of the sulfonyl-containing groups is a sulfonyl-containing group, each sulfonyl-containing group being independently selected from the group consisting of mesyl, esyl, and tosyl; R ４ and R ５ are each independently OH or methoxy; R ６ is OH or a mononucleotide or oligonucleotide having 1 to 3 bases, 3. The compound of claim 2, wherein Z and Z' are each independently a natural nucleoside base.

6. 2. The compound of claim 1, selected from the group consisting of compounds of the formula: ７ｍＧ (3'OEsyl) pppA (2'OMe) pG 【Chemistry 3】 、 ７ｍＧ (3'OMesyl) pppA (2'OMe) pG 【Chemistry 4】 and ７ｍＧ (3'OTosyl) pppA (2'OMe) pG 【Chemistry 5】 。

7. An mRNA 5'-capped with a cap analog, the compound being a compound according to any one of claims 1 to 6.

8. 10. A method for producing a 5'-capped mRNA comprising including a cap analog, the compound of any one of claims 1 to 6, during mRNA synthesis.

9. A composition or kit for producing 5'-capped mRNA, comprising a cap analogue which is a compound according to any one of claims 1 to 6.

10. A pharmaceutical composition for expressing a desired peptide or protein, comprising an mRNA 5'-capped with a cap analogue, which is a compound described in any one of claims 1 to 6, and a pharma- ceutically acceptable carrier.

11. A cell comprising the 5'-capped mRNA of claim 7.

12. A cell comprising a protein or peptide translated from the 5'-capped mRNA of claim 7.

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