A capped analog, methods of making and using the same

Abstract

The application provides a capped analog, a preparation method and application thereof. Specifically, the application provides a capped analog shown in formula I. The capped analog of the application is first discovered. The capped analog of the application can significantly improve the yield of synthesized mRNA and the capping efficiency of mRNA.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a capped analogue, its preparation method, and its application. Background Technology

[0002] mRNA technology is widely used in the vaccine field and has potential applications in cancer treatment and the treatment of genetic diseases. Eukaryotic mRNA has a cap structure at its 5' end, which is crucial for enhancing biological activity and reducing immunogenicity. Currently, mRNA capping technology systems are relatively complex, suffering from problems such as poor recognition efficiency of capping enzymes leading to low capping efficiency, low mRNA synthesis yield, and high immunogenicity. Further development of novel capping analogs is needed.

[0003] Therefore, there is an urgent need in this field to develop new capping analogs to improve capping efficiency and mRNA yield. Summary of the Invention

[0004] The purpose of this invention is to provide novel capping analogs to improve capping efficiency and mRNA yield.

[0005] In a first aspect, the present invention provides a capped analogue of Formula I:

[0006]

[0007] Among them, B 1 and B 2 Each is an independent natural or modified base;

[0008] R 1 Selected from H, OH, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, O-C1-C6 alkyl, or halogen;

[0009] R 2 Selected from H, OH, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, O-C1-C6 alkyl, or halogen;

[0010] R 3 Selected from H, OH, substituted or unsubstituted O-C1-C6 alkyl, substituted or unsubstituted S-C1-C6 alkyl, substituted or unsubstituted -NH-C1-C6 alkyl, substituted or unsubstituted O-aryl, substituted or unsubstituted S-aryl, substituted or unsubstituted -NH-aryl, substituted or unsubstituted O-aralkyl, substituted or unsubstituted S-aralkyl, substituted or unsubstituted -NH-aralkyl, or halogen;

[0011] R 4 Selected from H, OH or halogens;

[0012] X1 X 2 and X 3 Each can be independently O, CH2, or NH;

[0013] Y 1 Y 2 and Y 3 Each can be independently represented as O, S, Se, or BH3.

[0014] In another preferred embodiment, B 1 and B 2 Each can be independently adenine, N6-methyladenine, guanine, uracil, or thymine.

[0015] In another preferred embodiment, R 1 It is selected from H, OH, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, O-C1-C4 alkyl, or halogen.

[0016] In another preferred embodiment, R 1 Selected from OH.

[0017] In another preferred embodiment, R 2 It is selected from H, OH, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, O-C1-C4 alkyl, or halogen.

[0018] In another preferred embodiment, R 2 Selected from OH or O-C1-C4 alkyl groups.

[0019] In another preferred embodiment, R 2 Selected from OH and O-CH3.

[0020] In another preferred embodiment, R 3 Selected from H, OH, substituted or unsubstituted O-C1-C4 alkyl, substituted or unsubstituted S-C1-C4 alkyl, substituted or unsubstituted -NH-C1-C4 alkyl, or halogen;

[0021] In another preferred embodiment, R 3 Selected from O-CH3.

[0022] In another preferred embodiment, R 4 Selected from OH.

[0023] In another preferred example, X 1 X 2 and X 3 Each is independently represented by O.

[0024] In another preferred embodiment, Y 1 Y 2 and Y 3 Each is independently represented by O.

[0025] In another preferred embodiment, the capped analogue is a specific compound prepared in the examples, with the preferred compounds selected from the group consisting of:

[0026]

[0027] A second aspect of the present invention provides a polynucleotide encoding a target polypeptide, the polynucleotide comprising: (a) at least one ORF region;

[0028] (b)5'UTR;

[0029] (c)3'UTR; and

[0030] (d) At least one 5' start capped capped analogue as described in the first aspect of the invention.

[0031] A third aspect of the present invention provides a composition comprising the polynucleotide as described in the second aspect of the present invention.

[0032] In another preferred embodiment, the composition includes at least one delivery medium.

[0033] In another preferred embodiment, the delivery medium includes nanoparticles, liposomes, exosomes, microvesicles, cell-penetrating peptides, electroporation devices, or gene guns.

[0034] In another preferred embodiment, the composition comprises a pharmaceutical composition.

[0035] In another preferred embodiment, the pharmaceutical composition comprises (a) a polynucleotide as described in the second aspect of the invention; and (b) a pharmaceutically acceptable carrier.

[0036] In another preferred embodiment, the carrier is selected from lipid nanoparticles (LNPs), liposomes, polymeric nanoparticles, solid lipid nanoparticles, or emulsions.

[0037] In another preferred embodiment, the component (a) accounts for 10-99.9 wt% of the total weight of the pharmaceutical composition, more preferably 20-99 wt%, and even more preferably 50-90 wt%.

[0038] In another preferred embodiment, the composition comprises a non-oral formulation.

[0039] In another preferred embodiment, the formulation comprises an injection.

[0040] In another preferred embodiment, the composition (such as a pharmaceutical composition) is administered to mammals via intravenous injection or local injection.

[0041] In another preferred embodiment, the mammal includes humans or non-human mammals.

[0042] In another preferred embodiment, the non-human mammal includes rodents such as mice, rats, or primates such as monkeys.

[0043] A fourth aspect of the present invention provides a method for preparing the polynucleotide described in the second aspect of the present invention, comprising the steps of:

[0044] (a) Provide a DNA template;

[0045] (b) The DNA template is subjected to an in vitro transcription reaction to obtain the polynucleotide described in the second aspect of the present invention, wherein the reaction system of the in vitro transcription reaction contains RNA polymerase, nucleoside triphosphate and the capping analogue described in the first aspect of the present invention.

[0046] In another preferred embodiment, the nucleoside triphosphate may be a naturally occurring nucleoside triphosphate, a modified nucleoside triphosphate, or a non-natural nucleoside triphosphate; preferably ATP, CTP, GTP, UTP, 5me-CTP, 5me-UTP, PseudoUTP, or N1-me-PseudoUTP.

[0047] In another preferred embodiment, the RNA polymerase is an RNA polymerase derived from a bacteriophage. Preferably, the RNA polymerase is a T7 bacteriophage, T3 bacteriophage, SP6 bacteriophage, or K11 bacteriophage RNA polymerase or a variant thereof.

[0048] In another preferred embodiment, the RNA polymerase is selected from T7, SP6, or T3.

[0049] In another preferred embodiment, the RNA polymerase may be a mutant of a bacteriophage-derived RNA polymerase having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% of its amino acid sequence identical to the native sequence.

[0050] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Detailed Implementation

[0051] Through extensive and in-depth research, the inventors unexpectedly discovered a novel capping analogue as shown in Formula I. This capping analogue significantly increases the yield of synthesized mRNA while also significantly improving the capping efficiency of mRNA. Based on this discovery, the present invention was completed.

[0052] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art.

[0053] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0054] In this specification, the terms "substantially" or "truly" are used to indicate that the standard deviation from the theoretical model or theoretical data is within 5%, preferably 3%, and more preferably 1%.

[0055] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0056] In this specification, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes both the scenario in which the event occurs and the scenario in which the event does not occur.

[0057] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0058] Before further describing the invention, it should be understood that the invention is not limited to the specific embodiments described herein; it should also be understood that the terminology used herein is for description only and not for limiting specific embodiments.

[0059] Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. For the purposes of this invention, the following terms are defined to be consistent with their common understanding in this art.

[0060] As used herein, the term “in vitro transcription” (IVT) refers to a cell-free reaction in which a double-stranded DNA (dsDNA) template is copied by a DNA-directed RNA polymerase to produce a product containing RNA molecules copied from the template.

[0061] As used herein, the term "DNA template" refers to the dsDNA molecule transcribed in an IVT reaction. The DNA template has a promoter (e.g., T7, T3, or SP6 promoter) recognized by RNA polymerase upstream of the region being transcribed.

[0062] As used herein, the term "RNA product" refers to the product of an IVT reaction. The RNA product of IVT contains a mixture of RNA molecules and, depending on how transcription is completed, may include double-stranded RNA (dsRNA) molecules. The molecular events that generate dsRNA molecules in an IVT reaction are unknown, but they can be detected using, for example, dsRNA-specific antibodies or liquid chromatography (e.g., HPLC).

[0063] The term "capped analogue" includes natural caps, such as 7mG, and any compound represented by the following formula I:

[0064]

[0065] Among them, B 1 and B 2 Each is an independent natural or modified base;

[0066] R 1 Selected from H, OH, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, O-C1-C6 alkyl, or halogen;

[0067] R 2 Selected from H, OH, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, O-C1-C6 alkyl, or halogen;

[0068] R 3 Selected from H, OH, substituted or unsubstituted O-C1-C6 alkyl, substituted or unsubstituted S-C1-C6 alkyl, substituted or unsubstituted -NH-C1-C6 alkyl, substituted or unsubstituted O-aryl, substituted or unsubstituted S-aryl, substituted or unsubstituted -NH-aryl, substituted or unsubstituted O-aralkyl, substituted or unsubstituted S-aralkyl, substituted or unsubstituted -NH-aralkyl, or halogen;

[0069] R 4 Selected from H, OH or halogens;

[0070] X 1 X 2 and X 3 Each can be independently O, CH2, or NH;

[0071] Y 1 Y 2 and Y3 Each can be independently represented as O, S, Se, or BH3.

[0072] In some implementations, the capping analogues are selected from the following table:

[0073]

[0074]

[0075] This article provides methods for capping RNA in an IVT reaction via co-transcription (i.e., using cap analogs) and achieving a capping efficiency of at least 95.0%.

[0076] As used in this article, "base" and "natural base" can be used interchangeably. Both can also be called nucleobases or nitrogenous bases. They are nitrogenous compounds that form nucleosides, which are components of nucleotides. They can be adenine (A), guanine (G), cytosine (C), uracil (U), or thymine (T).

[0077] As used in this article, "modified base" refers to a substance obtained by replacing one or more hydrogen atoms of a natural base, such as, but not limited to, N6-methyladenine.

[0078] The terms “polynucleotide,” “nucleotide sequence,” “nucleic acid sequence,” “nucleic acid molecule,” and “nucleic acid” are used interchangeably and include DNA, RNA, or their hybrids, which can be double-stranded or single-stranded.

[0079] In some implementations, the polynucleotide comprises a capped analogue.

[0080] This invention also provides a method for preparing the formulation.

[0081] In some embodiments, the method of preparing the formulation may include combining an RNA product obtained by transcription of template DNA as described above with a pharmaceutically acceptable excipient to produce a composition.

[0082] In some embodiments, the method for preparing the formulation includes:

[0083] (a) Using the above method, transcribe template DNA with RNA polymerase to produce a capped RNA product with or without modification; and (b) combine the RNA product with a pharmaceutically acceptable excipient; wherein the method is performed without a post-transcriptional capping step.

[0084] In some embodiments, the method may include administering the formulation to a mammalian subject at an effective therapeutic dose, wherein the subject may be a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Alternatively, the capped RNA may be administered to a non-mammal subject or to eukaryotic or prokaryotic cells within the body or administered in vivo.

[0085] In some embodiments, the capped RNA may be naked or formulated with suitable excipients for administration to a subject, such as a human. Formulations may include liquid formulations (solutions, suspensions, dispersions), topical formulations (gels, ointments, drops, creams), and liposome formulations (e.g., as per US 9,629,804 B2; US2012 / 0251618 A1; WO 2014 / 152211).

[0086] The formulation may include encapsulating RNA in viral particles. (Those described in US2016 / 0038432A1).

[0087] In some embodiments, the capped RNA product can be delivered to cells by packaging it into nanoparticles (e.g., cationic lipids and polymers) or non-viral vectors (e.g., protamine). Direct introduction of RNA into cells can also be achieved using transfection, microinjection, electroporation, or acoustic pore effects. RNA delivery (local or systemic) and packaging (with or without modification) can be performed at temperatures optimal for the delivery method or the formulation used (e.g., those described in US 9,629,804 B2; US2012 / 0251618 A1; WO 2014 / 152211; US2016 / 0038432 A1; US2016 / 0032316 A1; US ​​9,597,413 B2; US2012 / 0258176).

[0088] The methods and compositions provided herein can be used for the in vitro synthesis of capped RNA products encoding proteins (e.g., antigens for vaccines) for use in cancer immunotherapy (e.g., as per US 8,217,016 B2; US 2012 / 0009221A1; US ​​2013 / 0202645A1; US ​​9,587,003 B2; Sahin et al.).

[0089] (2014): those described in Nature Reviews DrugDiscovery 13, 759–80), or allergic tolerance (e.g. those described in Sahin (2014), see above), or recombinant or naturally occurring proteins used to produce protein replacement therapy (e.g., in US2016 / 0032316 A1; US ​​8,680,069);

[0090] PCT / US2013 / 031821; PCT / US2014 / 028330; US 9,181,321; US9,220,792B2; US 9,233,141B2; those described in Sahin (2014) (see above), complementary therapies (e.g., those described in Sahin (2014) (see above), cell reprogramming (e.g., those described in US2011 / 0143436A1; US ​​8,802,438; US 9,371,544; WO2009 / 077134 A2; those described in Sahin (2014) (see above), and genome editing / engineering (as described in Sahin (2014) (see above). Introducing capped RNA into target cells can alter cell phenotype by producing proteins or affecting the expression of targets in cells.

[0091] Group definition

[0092] As used herein, the term “substituted or unsubstituted” means that the group may be unsubstituted, or that the H in the group is substituted by one or more (e.g., 1-10, preferably 1-5, more preferably 1-3, most preferably 1-2) substituents.

[0093] The term "independently" means that at least two groups (or ring systems) in a structure with the same or similar value ranges can have the same or different meanings under specific circumstances. For example, if substituent X and substituent Y are independently hydrogen, halogen, hydroxyl, cyano, alkyl, or aryl, then when substituent X is hydrogen, substituent Y can be hydrogen, halogen, hydroxyl, cyano, alkyl, or aryl; similarly, when substituent Y is hydrogen, substituent X can be hydrogen, halogen, hydroxyl, cyano, alkyl, or aryl.

[0094] The terms “contain” and “include” are used in their open, non-restrictive sense.

[0095] The term "alkyl" refers to a monovalent, straight-chain or branched alkane group consisting only of carbon and hydrogen atoms, without unsaturation, and linked to other segments by a single bond, including (but not limited to) methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, and tert-butyl. For example, "C1-6 "Alkyl" refers to a saturated monovalent straight-chain or branched hydrocarbon group containing 1 to 6 carbon atoms, and so on.

[0096] The "alkoxy" group refers to the "O-alkyl" group, where alkyl is defined as above in this article.

[0097] The term "alkenyl" refers to a monovalent, straight-chain or branched alkane group consisting only of carbon and hydrogen atoms, containing at least one double bond, and connected to other segments via a single bond. This includes (but is not limited to) vinyl, propenyl, allyl, isopropenyl, butenyl, and isobutenyl groups. For example, "C..." 2-6 "Alkenyl" refers to a monovalent straight-chain or branched hydrocarbon group containing 2 to 6 carbon atoms and having at least one carbon-carbon double bond (>C=C<), and so on.

[0098] The term "alkynyl" refers to a monovalent, straight-chain or branched alkane group composed only of carbon and hydrogen atoms, containing at least one carbon-carbon triple bond, and connected to other segments by a single bond. This includes (but is not limited to) ethynyl, propynyl, butynyl, and pentyynyl groups. For example, "C..." 2-6 "Alkyne" refers to a monovalent straight-chain or branched hydrocarbon group containing 2 to 6 carbon atoms and having at least one carbon-carbon triple bond, and so on.

[0099] The term "heterocyclic group" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic, e.g., fused, bridged, or spirocyclic) non-aromatic group whose ring atoms consist of a carbon atom and at least one heteroatom selected from N, O, and S, wherein the S atom is optionally substituted to form S(=O), S(=O)2, or S(=O)(=NR). x ), R x Independently selected from H or C 1-4 Alkyl group. If the valence requirement is met, the heterocyclic group can be connected to the rest of the molecule via any one ring atom. For example, the term "3-8 membered heterocyclic group" as used in this invention refers to a heterocyclic group having 3 to 8 ring atoms. For example, the heterocyclic group can be ethylene oxide, aziridine propane, aziridine butane, oxadiazine, tetrahydrofuranyl, dioxadiazopentenyl, pyrrolyl, pyrrolidone, imidazoalkyl, pyrazolyl, tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, dithiaalkyl, or trithiaalkyl.

[0100] The term "aryl" refers to a monocyclic or fused polycyclic aromatic hydrocarbon group having a conjugated π-electron system. For example, aryl can be phenyl, naphthyl, anthracene, phenanthryl, acenaphthene, azulene, fluorenyl, indene, pyrene, etc.

[0101] "Aryl" refers to -alkyl-aryl, where alkyl and aryl are as defined above in this document.

[0102] The term "heteroaryl" refers to an aromatic group that is a monocyclic or fused polycyclic ring with a conjugated π-electron system, whose ring atom consists of a carbon atom and at least one heteroatom selected from N, O, and S. If the valence requirement is met, the heteroaryl group can be connected to the rest of the molecule through any one of its ring atoms. Examples of heteroaryl groups include thiophene, furanyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl and its benzo[a] derivatives, pyrrolopyridinyl, pyrrolopyrazinyl, pyrazolopyridinyl, imidazopyridinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, purine, etc.

[0103] The term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0104] The compounds of this invention may contain one or more asymmetric centers and thus appear as racemates, racemic mixtures, single enantiomers, diastereomers, and single diastereomers. The asymmetric centers that can exist depend on the nature of the various substituents on the molecule. Each such asymmetric center will independently produce two optical isomers, and all possible optical isomers, diastereomer mixtures, and pure or partially pure compounds are included within the scope of this invention. This invention encompasses all isomeric forms of the compounds.

[0105] The main advantages of this invention include:

[0106] (1) The present invention provides a novel capping analog as shown in Formula I. The capping analog of the present invention significantly improves the yield of synthesized mRNA and also significantly improves the capping efficiency of mRNA.

[0107] (2) The capped analogs of the present invention are simple and efficient to synthesize, have a high capping rate, and exhibit high mRNA expression efficiency and better protein expression with said capped analogs, and have good application prospects.

[0108] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0109] Unless otherwise specified, all reagents and materials used in the embodiments of this invention are commercially available products.

[0110] Example 1 Chemical Synthesis of Capped Analog I

[0111]

[0112] Step 1: Synthesis of compounds 1-3

[0113] In 200 mL of 1,2-dichloromethane, N-(6-oxoylide-1,7-dihydropurin-2-yl)acetamide (10.00 g, 51.77 mmol, 1.0 eq) and N,O-bis(trimethylsilyl)acetamide (21.06 g, 130.54 mmol, 2.0 eq) were added. The reaction solution was heated at 80 °C for 0.5 h until clear, then cooled to room temperature. Tin tetrachloride (28.32 g, 108.72 mmol, 2.1 eq) was added, and the mixture was stirred for 0.5 h. Add acetic acid-[(2S,3R,4R,5R)-2,3,4-triacetoxytetrahydrofuran-5-yl]methyl ester (18.13 g, 56.95 mmol, 1.1 eq), stir the reaction solution at room temperature for 24 hours, then quench the reaction with 20 mL of methanol. Dilute with saturated sodium bicarbonate aqueous solution, filter with diatomaceous earth, separate the organic phase, concentrate under reduced pressure, and perform column chromatography (dichloromethane / methanol = 40 / 1 to 20 / 1) to give compounds 1-3 (8.00 g, yield 34.2%). MS: m / z [M+H] + =452.1.

[0114] Step 2: Synthesis of compounds 1-4

[0115] Compounds 1-3 (6 g, 13.29 mmol, 1.0 eq) were added to 80 mL of ammonia-methanol (7 M), stirred at room temperature for 24 hours, and filtered to give a white solid. Recrystallization from water gave compound 2-amino-7-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-1,6-dihydropurine-6-one (1.40 g, yield 35.3%). MS: m / z [M+H] + =284.1.

[0116] Step 3: Synthesis of compounds 1-5

[0117] Compounds 1-4 (1.2 g, 4.24 mmol, 1.0 eq) were dissolved in triethyl phosphate (15 mL), and phosphorus oxychloride (1.95 g, 12.72 mmol, 1.0 eq) was slowly added dropwise under ice bath conditions. The reaction solution was stirred at 0°C for 3 hours, then quenched with triethylammonium bicarbonate (1 M), diluted with 300 mL of water, washed with dichloromethane, and passed through an ion-exchange column (DEAE Sephadex) to give compounds 1-5 (1.2 g, triethylamine salt, yield 50.1%). MS: m / z [M+H] + =364.0.

[0118] Step 4: Synthesis of compounds 1-6

[0119] Compounds 1-5 (1.51 g, 2.67 mmol, 1.0 eq), imidazole (1.82 g, 26.7 mmol, 10.0 eq), triphenylphosphine (3.50 g, 13.35 mmol, 5.0 eq), and 2,2'-dithiopyridine (2.94 g, 13.35 mmol, 5.0 eq) were added sequentially to 15 mL of DMF. Under nitrogen protection, the mixture was stirred at room temperature for 16 hours. The reaction solution was then slowly added dropwise to an acetone solution of sodium perchlorate (0.2 M), precipitating a white solid. The solid was ultrafiltered, the organic phase separated, and the solid was diluted with acetone. This process was repeated three times to obtain a white solid compound 1-6 (1.00 g, sodium salt, yield 85.9%). MS: m / z [M+H] + =414.1.

[0120] Step 5: Synthesis of compounds 1-7

[0121] Compounds 1-7 (400.0 mg, 0.92 mmol, 1.0 eq), anhydrous zinc chloride (0.25 g, 1.84 mmol, 2.0 eq), and triethylamine phosphate (1.10 g, 5.52 mmol, 5.0 eq) were added sequentially to 15 mL of DMF. Under nitrogen protection, the mixture was stirred at room temperature for 16 hours. After dilution with 20 mL of water, the solution was washed three times with dichloromethane. The aqueous phase was then passed through an ion-exchange column (DEAE Sephadex) to give compounds 1-7 (0.25 g, triethylamine salt, yield 36.4%). MS: m / z [M+H] + =444.0.

[0122] Step 6: Synthesis of compounds 1-8

[0123] Compound 1-7 (500.0 mg, 0.67 mmol, 1.0 eq) was added to 25 mL of water. The pH was adjusted to 4.0–4.5 with 0.2 M sodium hydroxide solution. Dimethyl sulfate (0.425 g, 3.35 mmol, 5 eq) was slowly added dropwise, maintaining the pH between 4.0 and 4.5 during the addition. After the addition was complete, the reaction was allowed to proceed at room temperature for 0.5 hours. The reaction solution was washed with dichloromethane, and the aqueous phase was passed through an ion exchange column (DEAE Sephadex) to give compound 1-8 (310.0 mg, triethylamine salt, yield 61.1%). MS: m / z [M+H] + =458.0.

[0124] Step 7: Synthesis of compounds 1-9

[0125] Compounds 1-9 were prepared according to the synthetic method of compounds 1-6 (0.30 g, sodium salt, yield 78.0%). MS: m / z [M+H] +=757.1.

[0126] Step 8: Synthesis of Compound 1

[0127] Add 1-8 (22.8 mg, 0.03 mmol, 1.0 eq), 1-9 (20.0 mg, 0.025 mmol, 1.0 eq), and anhydrous zinc chloride (34.0 mg, 0.25 mmol, 10 eq) to 3 mL of DMF. Under nitrogen protection, stir at room temperature for 24 hours. Dilute the reaction solution with 20 mL of water, wash with dichloromethane, pass the aqueous phase through an ion exchange column (DEAE Sephadex), and repeatedly lyophilize the purified product to give compound 1 (3.9 mg, triethylamine salt, yield 13.6%). MS: m / z [M+H] + =1146.1.

[0128] Example 3: External Assessment of Similar Objects with Caps

[0129] For ease of comparison, the capping method of CleanCapAG (Trilink, N-7113-1) is used as a comparison.

[0130] The DNA coding sequence of eGFP (SEQ ID NO.1) was used as the PCR template, and the specific sequence is as follows:

[0131]

[0132] Using an in vitro transcription kit (NEB, E2040S), CleanCap AG (Trilink, N-7113-1), and Pseudouridine (Trilink, N-1019-1), the required components for transcription were added according to the proportions recommended by the NEB kit. The mixture was then incubated at 37°C for 3 hours for transcription.

[0133] The specific sample addition ratios are as follows:

[0134]

[0135] After incubation, 2 μL of NEB Dnase I (NEB M0303L) was added to each transcription reaction mixture, and the mixture was incubated at 37°C for 0.5 h to digest the DNA template. Then, the final tailing reaction was performed using the polyA tailing kit (Life, AM1350). The reaction system was prepared according to the instructions, and the mixture was incubated at 37°C for 1 h to complete the tailing reaction.

[0136] The transcribed mRNA was extracted using an RNA recovery kit (Novizan, RC101-01), following the same procedure as described in the kit. After extraction, the mRNA was eluted in RNase-free water, diluted to approximately 250 ng / µl, aliquoted, and stored at -80°C. The yield of in vitro transcribed mRNA was quantified using Nanodrop, and the results are as follows:

[0137] sample Yield (μg) Example 1 140 Comparative Example 1 115.4

[0138] Analysis shows that when using the capped composition containing compound 1 from Example 1 for in vitro transcription to synthesize mRNA, the mRNA yield is significantly increased, reaching 140 μg, while the mRNA yield using the capped composition described in Comparative Example 1 is only 115.4 μg. The experimental results show that using the composition of this application for in vitro transcription increases the mRNA yield by more than 21%, indicating that the capped composition prepared in Example 1 has a better transcription effect.

[0139] (5) Comparison of capping rates

[0140] Liquid chromatography-mass spectrometry (LC-MS) was used to detect the IVT capping rate of mRNAs with different initiation cap analogs. First, a labeled DNA probe matching the initiation base of the transcript mRNA needs to be designed. The usual label is biotin. After washing the streptavidin-labeled magnetic beads, the probe was incubated with the synthesized DNA probe (mGmGmUmGmAmAmCmAmGmCmUmCmCdTdCdGdCmCmCmUmUmGmCmUmCmAmCmAmU, where m indicates that the nucleotide to the right of m has been 2'-O-methylated, dT refers to deoxyribothymine, dC refers to deoxyribocytosine, and dG refers to deoxyriboguanine), eGFP mRNA, and 10×RNase H reaction buffer at room temperature for 30 minutes, while slowly mixing. Then, 20 μL of RNase H (5 U / μL) was added and incubated at 37 degrees Celsius for 3 hours, mixing every half hour. After incubation, the magnetic beads were washed, and then 100 μL of 75% methanol heated to 80 °C was added. The mixture was heated to 80 °C on a hot plate for 3 minutes, and then the supernatant was aspirated and dried using an evaporative centrifuge at room temperature for 45 minutes to 10 μL. The sample was then resuspended in 50 μL of 100 μM EDTA / 1% MeOH for LC-MS analysis to determine the capping status of RNA during transcription. Since capped and uncapped bases have significant molecular weight differences, the capping rate of mRNA transcription initiated by different capping analogs can be determined using the molecular weight difference. Specific results are as follows:

[0141] sample Capping rate (%) Example 1 95.0 Comparative Example 1 92.7

[0142] Analysis shows that when using the capping composition of this application for in vitro transcription synthesis of mRNA, the capping efficiency of mRNA can reach 95.0%, while the capping efficiency of the composition of Comparative Example 1 is only 92.7%.

[0143] (5) Cellular protein expression

[0144] In this example, the eGFP coding sequence (SEQ ID NO.1) was used as a PCR template. In vitro transcription was performed using both Comparative Example 1 and the capped composition containing Compound 1 of Example 1 of this invention as the starting material. The resulting different mRNA products were then transfected into 293T cells. The transfection method is as follows:

[0145] 293T cells (purchased from ATCC) were seeded at 100,000 cells / well. Two 1.5ml EP tubes and two 15ml centrifuge tubes were prepared, labeled A and B respectively. Opti-MEM and mRNA were added to tube B, and Opti-MEM and Lipo3000 were added to tube A. For each transfection system, 300ng of mRNA was added to 15ul of Opti-MEM, and 1ul of Lipo3000 was added to 15ul of Opti-MEM. The mixture in tube B was vortexed for 2 seconds and then centrifuged briefly. 15μL of solution A was added to solution B, and the mixture was gently pipetted 5 times to mix. After standing at room temperature for 20 minutes, 30μL of the mixture was added dropwise to a 48-well plate. The culture dish was shaken left and right for 5 seconds each time, and then rotated 90° three times, shaking left and right for 5 seconds each time. After incubation for 24 hours, the fluorescence intensity was measured and calculated using a fluorescence microscope. The results are as follows:

[0146]

[0147] Analysis shows that the translation efficiency of mRNA protein obtained by in vitro transcription using the capping composition of this application is about 1.23 times that of Comparative Example 1, and the mRNA obtained by this capping method is more suitable for intracellular expression.

[0148] It should be understood that although this disclosure has been described in conjunction with a detailed description thereof, the foregoing description is intended to be illustrative and not to limit the scope of this disclosure as defined by the appended claims. Other aspects, advantages, and modifications are also within the scope of the following claims.

[0149] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A capped analogue as shown in Formula I: （I）； in, B 1 and B 2 Each is an independent, naturally occurring base; R 1 Selected from H, OH, or halogens; R 2 Selected from H, OH, or halogens; R 3 Selected from H, OH, or methoxy groups; R 4 Selected from H, OH or halogens; X 1 X 2 and X 3 Each is independently represented by O; Y 1 Y 2 and Y 3 Each is independently represented by O.

2. The capped analogue as described in claim 1, characterized in that, B 1 and B 2 Each can be independently adenine, N6-methyladenine, guanine, uracil, or thymine.

3. A polynucleotide encoding a target polypeptide, characterized in that, The polynucleotide comprises: (a) At least one ORF region; (b)5'UTR; (c)3'UTR; and (d) At least one 5' start capped analogue as described in claim 1.

4. A composition, characterized in that, It contains the polynucleotide as described in claim 3.

5. A method for preparing the polynucleotide according to claim 3, characterized in that, Including the following steps: (a) Provide a DNA template; (b) Performing an in vitro transcription reaction on the DNA template to obtain the polynucleotide of claim 3, wherein the reaction system of the in vitro transcription reaction contains RNA polymerase, nucleoside triphosphate and the capping analog of claim 1.

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