WO2024098964A1 - 一种乙烯基膦酸修饰的mRNA帽类似物及其制备方法和应用 - Google Patents

一种乙烯基膦酸修饰的mRNA帽类似物及其制备方法和应用 Download PDF

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WO2024098964A1
WO2024098964A1 PCT/CN2023/119496 CN2023119496W WO2024098964A1 WO 2024098964 A1 WO2024098964 A1 WO 2024098964A1 CN 2023119496 W CN2023119496 W CN 2023119496W WO 2024098964 A1 WO2024098964 A1 WO 2024098964A1
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substituted
unsubstituted
alkyl
vinylphosphonic acid
mrna cap
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French (fr)
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黄磊
赵万年
沈奇
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江苏申基生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C07H19/207—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/02—Phosphorylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/02—Phosphorylation
    • C07H1/04—Introducing polyphosphoric acid radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention relates to the technical field of chemistry and bioengineering, and in particular to a vinylphosphonic acid-modified mRNA cap analogue, a preparation method and application thereof.
  • Eukaryotic mRNA carries a "cap” structure at its 5'-end, and it is well known that this "cap” structure plays an important role in translation.
  • the naturally occurring cap structure consists of 7-methylguanosine, which is linked to the 5'-end of the first transcribed nucleotide via a triphosphate bridge, resulting in 7G(5')ppp(5')N, where N is any nucleotide.
  • the mRNA cap plays an important role in gene expression.
  • the cap structure can protect mRNA from degradation by exonucleases, allow RNA to be transported from the nucleus to the cytoplasm, and participate in the assembly of the translation initiation complex.
  • m7G(5')ppp(5')G (mCAP) has been used as a primer for in vitro T7 or SP6 RNA polymerase transcription to obtain RNA with a cap structure at its 5' end.
  • RNA molecules that perform appropriately in various biological applications.
  • Such applications include research applications and commercial production of polypeptides, for example, polypeptides containing "non-natural" amino acids at specific sites are produced in cell-free translation systems, or polypeptides that require post-translational modification in terms of their activity or stability are produced in cultured cells. In the latter system, the synthesis is carried out significantly longer, and therefore more protein is produced.
  • the ability of the 5' end cap structure of mRNA to bind to the eukaryotic initiation factor (elF4E) determines the translation expression effect of the mRNA in the cell.
  • the existing cap analog structure is generally a natural cap structure, and its mRNA translation effect is unstable, with large differences in results for different sequences or different expression hosts.
  • the present application provides a vinylphosphonic acid-modified mRNA cap analog, a preparation method and an application thereof.
  • the mRNA cap analog uses vinylphosphonic acid in its structure to simulate the normal phosphonic acid function. Due to the improved structure, the mRNA cap analog modified with vinylphosphonic acid has a spatial conformational advantage and has better binding ability with the eukaryotic initiation factor (elF4E), which can ultimately improve the translation level of mRNA in cells.
  • elF4E eukaryotic initiation factor
  • a vinylphosphonic acid-modified mRNA cap analog comprising the following structure, or a pharmaceutically acceptable salt or stereoisomer thereof:
  • R 1 , R 2 , R 3 and R 4 are independently hydrogen, halogen, hydroxyl, substituted or unsubstituted alkyl, substituted or unsubstituted O-alkyl, substituted or unsubstituted S-alkyl, substituted or unsubstituted NH-alkyl, substituted or unsubstituted N-dihydrocarbyl, 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;
  • B 1 and B 2 are independently natural, modified, or non-natural nucleoside bases
  • Ra, Rb, and Rc are independently the following structures:
  • Y is O, OCH 2 , OCH, CH 2 , CH, C 2 H 3 or C 3 H 5 ;
  • Z is OH, SH, BH 3 , aryl, alkyl, O-alkyl or O-aryl;
  • X is O, CH 2 or NH;
  • W is H, OH, alkyl, O-alkyl, N-alkyl, S-alkyl, halogen; It indicates a double bond or a single bond, and is connected to the five-membered sugar ring; and in at least one of the structures of Ra, Rb, and Rc, Y is O, OCH, or CH.
  • R 1 , R 2 , R 3 and R 4 are independently hydrogen, halogen, hydroxy, substituted or unsubstituted alkyl, substituted or unsubstituted O-alkyl, substituted or unsubstituted S-alkyl, substituted or unsubstituted NH-alkyl.
  • the above-mentioned halogen is F, Cl or Br.
  • the number of carbon atoms in the above substituted or unsubstituted alkyl group is 0-5, preferably 1-5;
  • the number of carbon atoms in the substituted or unsubstituted alkyl group is 1, 2 or 3.
  • the substituted or unsubstituted alkyl group is a substituted or unsubstituted methyl group, a substituted or unsubstituted ethyl group, a substituted or unsubstituted n-propyl group, a substituted or unsubstituted isopropyl group, a substituted or unsubstituted n-butyl group, a substituted or unsubstituted isobutyl group, or a substituted or unsubstituted tert-butyl group.
  • the number of carbon atoms in the above substituted or unsubstituted O-alkyl group is 0-5, preferably 1-5;
  • the number of carbon atoms in the substituted or unsubstituted O-alkyl group is 1, 2 or 3.
  • the substituted or unsubstituted O-alkyl group is a substituted or unsubstituted methoxy group, a substituted or unsubstituted ethoxy group, or a substituted or unsubstituted propoxy group.
  • the number of carbon atoms in the substituted or unsubstituted S-alkyl group is 0-5, preferably 1-5;
  • the number of carbon atoms in the substituted or unsubstituted S-alkyl group is 1, 2 or 3.
  • the substituted or unsubstituted S-alkyl group is a substituted or unsubstituted methylthio group, a substituted or unsubstituted ethylthio group, or a substituted or unsubstituted propylthio group.
  • the number of carbon atoms in the above substituted or unsubstituted NH-alkyl is 0-5, preferably 1-5;
  • the number of carbon atoms in the substituted or unsubstituted NH-alkyl group is 1, 2, 3, 4 or 5.
  • the substituted or unsubstituted NH-alkyl group is a substituted or unsubstituted methylamino group, a substituted or unsubstituted ethylamino group, or a propylamino group.
  • the number of carbon atoms in the above substituted or unsubstituted N-dihydrocarbyl group is 0-5, preferably 1-5.
  • the substituent groups in the above alkyl, O-alkyl, S-alkyl, NH-alkyl, and N-dialkyl are independently selected from one or more of substituted or unsubstituted alkyl, substituted or unsubstituted O-alkyl, and substituted or unsubstituted amide.
  • the number of carbon atoms of the alkyl group is 1-5; the number of carbon atoms of the O-alkyl group is 1-5; the number of carbon atoms of the amide group is 1-5.
  • the alkyl group, O-alkyl group and amide group can be further substituted by a C1-C5 straight chain or branched chain alkyl group.
  • the hydrogen connected to the N atom in the amide group can be substituted by a C1-C5 straight chain or branched chain alkyl group.
  • the hydrogen connected to the N atom in the amide group can be substituted by one or more of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl and isopentyl.
  • the substituents in the above-mentioned alkyl, O-alkyl, S-alkyl, NH-alkyl, and N-dialkyl are independently selected from methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, formamide, acetamide, n-propionamide, isopropionamide, n-butyramide, isobutyramide, tert-butyramide, N-methylacetamido, N-ethylacetamido, N-n-propylacetamido, and N-isopropylacetamido.
  • R 1 , R 2 , R 3 and R 4 are independently hydrogen, F, hydroxyl, methoxy, ethoxy, propoxy, methoxymethyl, methoxyethoxy, acetamidomethyl, n-propionamidomethyl, isopropionamidomethyl, butyramidomethyl, N-methylacetamidomethyl, N-ethylacetamidomethyl, N-n-propylacetamidomethyl, N-isopropylacetamidomethyl.
  • B 1 and B 2 are independently adenine, guanine, cytosine, uracil or thymine.
  • Ra, Rb, and Rc are independently the following structures:
  • Y is O, CH, C 2 H 3 or C 3 H 5 ;
  • Z is OH or an alkyl group;
  • X is O or CH 2 ;
  • W is H, OH or an alkyl group;
  • the number of carbon atoms in the above alkyl group is 0-5.
  • the alkyl group has 1, 2 or 3 carbon atoms.
  • Y is CH.
  • R 1 , R 2 , and R 4 are all hydroxyl groups, R 3 is methoxy; B 1 is adenine, and B 2 is guanine;
  • Ra, Rb, and Rc have the following structure
  • the above-mentioned vinylphosphonic acid-modified mRNA cap analog has any of the following structures, or a pharmaceutically acceptable salt, stereoisomer, etc. thereof:
  • Ra, Rb, and Rc are of the following structure:
  • the vinylphosphonic acid-modified mRNA cap analog provided by the present invention may also be in the form of a salt, and the salt may be a sodium salt, a potassium salt, an ammonium salt, an organic amine salt, or the like, which are well known to those skilled in the art.
  • the cation of the ammonium salt is NH 4 + .
  • the organic amine salt includes but is not limited to triethylamine salt.
  • the mRNA cap analog modified with vinylphosphonic acid provided by the present invention can also be a stereoisomer of the above structure.
  • “Stereoisomer” refers to a compound having the same chemical structure but different spatial arrangements of atoms or groups.
  • the above stereoisomers include but are not limited to enantiomers, diastereomers, conformers (rotamers), geometric isomers (cis/trans), atropisomers, etc.
  • the carbon-carbon double bond of the above-mentioned vinylphosphonic acid-modified mRNA cap analog is at least one of E-type or Z-type.
  • a method for preparing a vinylphosphonic acid modified mRNA cap analog comprises the following steps: (1) synthesizing an imidazole salt of m7GDP or its modified analog; (2) preparing a dinucleotide linked by a phosphate bond; and (3) synthesizing a vinylphosphonic acid modified mRNA cap analog.
  • the method comprises the following steps: (1) synthesis of imidazolate salt of m7GDP or its modified analog: guanosine or its analog is subjected to diphosphorylation, N7 methylation, diphosphorylation and the like in sequence to synthesize imidazolate salt of m7GDP or its modified analog; (2) preparation of dinucleotide linked by phosphate ester bond: 2'OMe-A or its modified phosphoramidite monomer is coupled with protected guanosine or its modified analog under the action of tetrazole to form the first phosphate ester bond, the protecting group is removed by acid action, and then the second phosphate is introduced, and finally hydrolyzed to obtain dinucleotide linked by phosphate ester bond; (3) synthesis of mRNA cap analog modified by vinylphosphonic acid: imidazolate salt of m7GDP or its modified analog is reacted with dinucleotide linked by phosphate ester bond to prepare mRNA cap analog modified by vinylphosphonic
  • the specific steps include:
  • Petroleum ether/dichloromethane is slurried in a certain proportion, and the intermediate e is filtered to obtain the intermediate; e is dissolved in acetonitrile, 1.2 eq. of a phosphine reagent and 1.2 eq. of tetrazole are added and stirred to react.
  • 1.2 eq. of iodine pyridine solution is added to the reaction solution, which is then spin-dried after the monitoring reaction is completed.
  • 3 L of methanol and 3 L of concentrated ammonia water are added to the spin flask, and the reaction is carried out at room temperature for 4 hours. The reaction is monitored, and the reaction is spin-dried after the reaction is completed. 20 L of ultrapure water is added, and the solution enters a reverse ion osmosis device, and the solution is washed, concentrated, and freeze-dried to obtain a phosphate-linked dinucleotide.
  • the m7GDP or its modified analogue imidazole salt obtained in step (1) is dissolved in a DMF solution containing MnCl2 , and added to the DMF solution of the dinucleotide connected by the phosphate bond obtained in step (2), and the reaction is stirred at room temperature. After 24 hours, the reaction is terminated with a 0.25M EDTA solution; the mixture is loaded onto a DEAE Sephadex column. The product is linearly eluted with a 0-1.0M TEAB eluent.
  • the eluted product with an HPLC purity of >97% is collected, and the above separation liquid is concentrated, and then loaded onto a strong anion resin, and linearly eluted with a 0-1.0M sodium acetate eluent.
  • the eluted product with an HPLC purity of >98.5% is collected, and the high-purity eluents are combined, and the residual sodium acetate solution is removed by a nanofiltration device and concentrated to obtain the target product containing a vinylphosphonic acid-modified mRNA cap analog.
  • the invention discloses an application of a vinylphosphonic acid modified mRNA cap analog, which is used for mRNA capping under the T7 RNA polymerase system.
  • T7 RNA polymerase is a DNA-dependent RNA polymerase that has high specificity for the bacteriophage T7 promoter sequence. The enzyme synthesizes a large amount of RNA from the DNA inserted into the downstream of the transcription vector from the T7 promoter.
  • the IVT (in vitro transcription) reaction system catalyzed by T7 RNA polymerase is the most mature mRNA preparation system at present.
  • a 20ul IVT reaction system contains 50U of T7 RNA polymerase and 1ul of cap analog (100mM) to obtain the best transcription yield and capping efficiency.
  • the present invention provides a complex comprising the above-mentioned vinylphosphonic acid modified mRNA cap analog and a DNA template, wherein the DNA template includes a promoter region containing a transcription start site, and the transcription start site has a first nucleotide at nucleotide position +1 and a second nucleotide at nucleotide position +2; the B1 group in the structure of the vinylphosphonic acid modified mRNA cap analog is complementary to the first nucleotide base, and the B2 group in the structure of the vinylphosphonic acid modified mRNA cap analog is complementary to the second nucleotide base.
  • the present invention provides an RNA molecule, comprising the above-mentioned mRNA cap analog modified with vinylphosphonic acid.
  • the above-mentioned mRNA cap analog modified with vinylphosphonic acid is used as the cap structure or cap structure fragment of the above-mentioned RNA molecule.
  • the present invention provides a pharmaceutical composition comprising the above RNA molecule and a pharmaceutically acceptable carrier.
  • the present invention provides a vinylphosphonic acid modified mRNA cap analog, a preparation method and an application thereof.
  • the vinylphosphonic acid modified mRNA cap analog is suitable for mRNA produced by an in vitro co-transcription method using a DNA sequence as a template.
  • the DNA sequence can be derived from or modified from viruses, animals, plants and other species.
  • the mRNA produced by the mRNA cap analog has lower immunogenicity, higher protein translation efficiency and better stability.
  • the present invention has the following advantages:
  • the vinylphosphonic acid-modified mRNA cap analog of the present invention has a more stable molecular conformation due to the presence of a double bond structure, and is not easily recognized and hydrolyzed by nucleases.
  • this type of cap analog also shows better in vitro transcription yield and capping efficiency data; at the same time, the mRNA cap analog containing a double bond has a better binding ability with the eukaryotic initiation factor (elF4E), showing a better mRNA translation effect.
  • elF4E eukaryotic initiation factor
  • the vinylphosphonic acid modified cap structure designed by the present invention contains a vinyl group, which can effectively fix the spatial structure of the 5' end cap and improve the binding ability of the 5' end cap structure of mRNA with the eukaryotic initiation factor (elF4E), thereby improving the translation effect of mRNA.
  • elF4E eukaryotic initiation factor
  • Example 1 Synthesis of mRNA cap analogs modified with vinylphosphonic acid using intermediates A and B as raw materials
  • compound A is obtained by the following steps: 1) weigh 5g of guanosine and dissolve it in 70.0ml of trimethyl phosphate. The reaction solution is cooled to 0°C, phosphorus oxychloride (1.8eq.) is slowly added dropwise, and after stirring for 4 hours under an ice bath, water is added to quench the reaction, and the intermediate A1 is purified by reverse phase chromatography. 2) Intermediate A1, triphenylphosphine (1.0eq.), 2,2' disulfide dipyridine (2.0eq.), imidazole (8.0eq.) and triethylamine (1.0eq.) are dissolved in DMF and stirred for 10 hours.
  • intermediate A2 4M sodium perchlorate acetone solution is added to precipitate, and the filter cake is washed with acetone to obtain intermediate A2.
  • 3M sodium perchlorate acetone solution is added to precipitate, and the filter cake is washed with acetone to obtain intermediate A2.
  • 3) Take 10g of intermediate A2 and dissolve it in DMF, add tributylamine phosphate (3.0eq.) and zinc chloride (8.0eq.) and stir at room temperature for 5 hours. After the reaction, the intermediate A3 was purified by ion chromatography. 3) The intermediate A3 was dissolved in 20 times the volume of purified water, the reaction solution was cooled to 4°C, and dimethyl sulfate was slowly added dropwise. During the process, the pH was adjusted not to exceed 5 with 2M sodium hydroxide. The reaction was monitored by HPLC.
  • intermediate A4 was purified by ion chromatography. 5); Intermediate A4, triphenylphosphine (1.0 eq.), 2,2' disulfide dipyridine (2.0 eq.), imidazole (8.0 eq.) and triethylamine (1.0 eq.) were dissolved in DMF and stirred for 10 hours. After the reaction, 4M sodium perchlorate acetone solution was added to precipitate, and the filter cake was washed with acetone to obtain intermediate A.
  • Compound B is obtained by the following steps: 1) 10 g of guanosine is dissolved in 150 mL of pyridine, trimethylsilyl chloride (5.0 eq.) is added to the reaction solution, and then stirred at room temperature for 2 hours. The temperature is lowered to 0°C, isobutyryl chloride (1.5 eq.) is added dropwise to the reaction solution, and then reacted at room temperature for 3 hours. 20 mL of ammonia water and 500 mL of water are added dropwise to the reaction solution in an ice bath, and the aqueous phase is washed with dichloromethane and then recrystallized with hot water to obtain intermediate B1.
  • intermediate B3 10 g was dissolved in 100 mL of DMF solution, TBDMSCl (3.1 eq.) was added to the reaction solution and stirred at room temperature overnight. Saturated sodium carbonate aqueous solution was added and extracted with ethyl acetate to obtain intermediate B3 by crystallization.
  • intermediate B3 Take 8g of intermediate B3 and dissolve it in 80mL 80% acetic acid solution. After stirring at room temperature for 5 hours, add 400mL of ethyl acetate, and then adjust the pH to neutral with saturated sodium carbonate. Separate the organic phase, concentrate it, and then column chromatography to obtain intermediate B4.
  • intermediate B4 Take 5g of intermediate B4 and dissolve it in 50mL DMF. After adding DMSO (6.0eq.) and EDCI (3.0eq.) to the reaction solution, pyridine (1.0eq.) and trifluoroacetic acid (1.0eq.) are added dropwise to the reaction solution. After reacting at room temperature for 5 hours, the reaction solution is diluted with ethyl acetate, and the organic phase is washed with saturated sodium bicarbonate aqueous solution and water, respectively, and then concentrated. Column chromatography is performed to obtain intermediate B5.
  • Tetraethylmethylene diphosphate (1.5 eq.) was added dropwise to a suspension of NaH (60%, 1.0 eq.) in THF (80 mL) under ice bath, followed by stirring at 0°C for 0.5 hours.
  • a THF solution (40 mL) of intermediate B5 (3 g) was slowly added dropwise to the reaction solution. Stir at room temperature overnight.
  • Saturated aqueous ammonium chloride solution was added dropwise to the reaction solution under ice bath to quench the reaction, extracted with ethyl acetate and concentrated, and column chromatography was performed to obtain intermediate B6.
  • intermediate B6 2 g was dissolved in 40 mL of acetonitrile, triethylsilyl bromide (10.0 eq.) was added to the reaction solution, which was then heated to 75° C. overnight and concentrated. The crude product was prepared by reverse phase to obtain intermediate B7.
  • intermediate B9 0.6 g was dissolved in dichloromethane (12 mL), and tetrazole (1.1 eq.) and bis(2-cyanoethyl)-N,N-diisopropylphosphoramidite (1.1 eq.) were added to the reaction solution respectively. The mixture was reacted at room temperature for 5 hours. After the reaction was complete, the mixture was concentrated to obtain a crude product of intermediate B10.
  • Example 2 Synthesis of mRNA cap analogs modified with vinylphosphonic acid using intermediates C and D as raw materials
  • Compound C is obtained by the following steps: 1) 5 g of intermediate B7 is dissolved in H 2 O/TFA (1:1, 50 mL), stirred at room temperature for 1 hour, and the reaction is complete. Reverse phase chromatography is used for purification to obtain intermediate C1.
  • intermediate C3 2 g was dissolved in 40 mL of purified water, the reaction solution was cooled to 4° C. dimethyl sulfate was slowly added dropwise, and 2 M sodium hydroxide was used to adjust the pH to not more than 5 during the process. The reaction was monitored by HPLC. After the reaction was completed, intermediate C4 was purified by ion chromatography.
  • Compound D was obtained by the following steps: 5 kg of 2'OMe-rA phosphoramidite monomer was weighed into a single-mouth bottle, dissolved in 50 L of dichloromethane, and then 2.73 kg of 2',3'acetylguanosine was added, the temperature was lowered to 25 ⁇ 2°C, 880 g of tetrazole was added under nitrogen blowing, and the temperature was raised to 25 ⁇ 2°C for reaction. After the monitoring reaction is completed, 1.2 eq. of iodine pyridine solution is added to the reaction solution, and the reaction solution is dried after the monitoring reaction is completed. The concentrated ointment is dissolved in 4 L of dichloromethane, and 1.1 eq.
  • Example 3 Synthesis of mRNA cap analogs modified with vinylphosphonic acid using intermediates A and E as raw materials
  • compound E is obtained by the following steps: 1) 5 g of intermediate D1 is dissolved in 50 mL DMF, DMSO (6.0 eq.) and EDCI (3.0 eq.) are added to the reaction solution, and then pyridine (1.0 eq.) and trifluoroacetic acid (1.0 eq.) are added dropwise to the reaction solution. After reacting at room temperature for 5 hours, the reaction solution is diluted with ethyl acetate, the organic phase is washed with saturated sodium bicarbonate aqueous solution and water, respectively, and then concentrated, and column chromatography is performed to obtain intermediate E1.
  • Example 4 Synthesis of mRNA cap analogs modified with vinylphosphonic acid using intermediates B and C as raw materials
  • Example 5 Synthesis of mRNA cap analogs modified with vinylphosphonic acid using intermediates C and E as raw materials
  • Example 6 Synthesis of mRNA cap analogs modified with vinylphosphonic acid using intermediates A and F as raw materials
  • compound F is obtained by the following steps: 1) 5 g of intermediate B9 is dissolved in 50 mL DMF, DMSO (6.0 eq.) and EDCI (3.0 eq.) are added to the reaction solution, and then pyridine (1.0 eq.) and trifluoroacetic acid (1.0 eq.) are added dropwise to the reaction solution. After reacting at room temperature for 5 hours, the reaction solution is diluted with ethyl acetate, the organic phase is washed with saturated sodium bicarbonate aqueous solution and water, respectively, and then concentrated, and column chromatography is performed to obtain intermediate E1.
  • intermediate F2 1 g was dissolved in 20 mL of DMF, triethylsilyl bromide (10.0 eq.) was added to the reaction solution, which was then heated to 75° C. overnight and concentrated. The crude product was prepared by reverse phase to obtain intermediate F3.
  • Example 7 Synthesis of mRNA cap analogs modified with vinylphosphonic acid using intermediates C and F as raw materials
  • Example 8 Synthesis of mRNA cap analogs containing vinylphosphonic acid modification using intermediates D and G as raw materials (Compound 10)
  • compound G is obtained by the following steps:
  • Tetraethylmethylene diphosphate (80.0 mmol) was added dropwise to a suspension of NaH (60%, 53.0 mmol) in THF (50.0 mL) under ice bath, followed by stirring at 0°C for 0.5 hours.
  • a THF solution (70.0 mL) of intermediate H8 (53.0 mmol) was slowly added dropwise to the reaction solution. The mixture was stirred at room temperature overnight.
  • Saturated aqueous ammonium chloride solution was added dropwise to the reaction solution under ice bath to quench the reaction, extracted with ethyl acetate, concentrated, and subjected to column chromatography to obtain intermediate H9.
  • compound K is obtained by the following steps: Referring to the synthesis method of intermediates G4 to G, intermediates K2 to K are obtained in sequence. The reaction route flow is shown in equation (23):
  • intermediates O1, O2, O3, O4, O5 and O are obtained by referring to the synthesis methods of intermediates I9, I10, I11, I12, I13 and I respectively.
  • compound P is obtained by the following steps: Referring to the synthesis method of intermediates G4 to G, intermediates P2 to P are obtained in sequence. The reaction route flow is shown in equation (33):
  • Test Example 1 Determination of mRNA in vitro transcription yield and capping efficiency
  • LC-MS Liquid chromatography mass spectrometry
  • the magnetic beads are washed, and 100 ⁇ L of 75% methanol heated to 80°C is added to the washed magnetic beads.
  • the mixture is heated to 80°C on a heating plate for 3 minutes, and then placed on a magnetic stand to absorb the supernatant, and dried to 10 ⁇ l using an evaporating centrifuge at room temperature for 45 minutes.
  • the sample is then resuspended in 50 ⁇ l of 100 ⁇ M EDTA/1% MeOH and can be used for LC-MS analysis to determine the capping of RNA in the transcription reaction. Since the molecular weight of capped and non-capped bases is significantly different, the difference in molecular weight can be used to determine the capping rate of mRNA transcription initiated by different cap analogs. Specific results are shown in Table 4.
  • Examples 1-7 and compounds 10-13, 16, 17, 25, 31, and 34 can all transcribe the corresponding target mRNA through IVT.
  • the yields of Examples 1, 2, 3, compounds 10-13, 16, 17, 25, 31, and 34 are equivalent to those of Comparative Example 1
  • the capping efficiency of Examples 1, 2, 3, and compounds 10-13, 16, 17, 25, 31, and 34 is better than that of Comparative Example 1 or equivalent to that of Comparative Example 1.
  • the in vitro transcription yield and capping efficiency of Examples 4, 5, 6, and 7 are significantly lower than those of Comparative Example 1. This may be related to the structure of the cap analog.
  • the rigid structure of Examples 4, 5, 6, and 7 affects the binding of the cap analog to the template start transcription site, and ultimately affects the in vitro transcription yield and capping efficiency.
  • Test method First, dilute the sample with running buffer (50mM phosphate, 100mM sodium chloride, and 0.01% vol/vol Tween 20, pH 6.0 or 7.4).
  • the experimental samples include Examples 1-7, compounds 10-13, 16, 17, 25, 31, 34 and the cap analog sample of Comparative Example 1.
  • the injection time is 2min
  • the injection flow rate is 20ul/min
  • the dissociation time is 2.5min.
  • the residual sample is eluted with regeneration buffer (10mM HEPES, 150mM NaCl, and 0.01% vol/vol Tween 20, pH 7.4) for 30sec and the flow rate is set to 30ul/min.
  • the sensor graph is analyzed and the binding constant is calculated by Biacore T100 evaluation software (version2.0.2).
  • the cap analog structure when the cap analog structure contains a vinyl structure, it may have a certain improvement in improving the binding ability of the cap analog with eIF4E protein, but the rigid structure of Example 7 containing three vinyl structures affects the binding of the cap analog with eIF4E protein, and ultimately affects the in vitro transcription yield and capping efficiency.
  • Test method Using the eGFP coding sequence as a DNA template, in vitro transcription was initiated using the cap analogs of Examples 1-7, compounds 10-13, 16, 17, 25, 31, 34 and Comparative Example 1. The different mRNA products obtained were then used to transfect 293T cells. 293T cells were plated at (0.5-1) ⁇ 10 5 cells (24-well plate). During transfection, the cell density is generally 60-80% and 2 ⁇ g of mRNA is transfected per well. The transfection reagent is Lipofectamine MessengerMAX Transfection Reagent (Invitrogen) and is operated with reference to its instructions. The transfected cells were placed in a 37°C, CO 2 incubator and replaced with fresh complete culture medium 4-6 hours after transfection.
  • the cap analog structure when it contains a vinyl structure, it may have a promoting effect on improving the translation effect of the cap analog mRNA in cells, but the rigid structure containing three vinyl structures in Example 7 affects the binding of the cap analog to the elF4E protein, and ultimately affects the translation effect of the mRNA in cells.

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Abstract

本发明提供了一种乙烯基膦酸修饰的mRNA帽类似物及其制备方法和应用，本发明的乙烯基膦酸修饰的mRNA帽类似物，由于双键结构的存在使分子构象更加稳定，不容易被核酸酶识别并水解，并且该类帽类似物也表现出较好的体外转录产量以及加帽效率数据；同时，含有双键的mRNA帽类似物与真核起始因子(elF4E)有更好的结合能力，表现出更好的mRNA翻译效果。

Description

一种乙烯基膦酸修饰的mRNA帽类似物及其制备方法和应用
本申请要求于2022年11月08日提交中国专利局、申请号为202211395011.5、发明名称为“一种乙烯基膦酸修饰的mRNA帽类似物及其制备方法和应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及化学及生物工程技术领域，具体涉及一种乙烯基膦酸修饰的mRNA帽类似物及其制备方法和应用。
 背景技术
真核mRNA在其5'-末端带有“帽”结构，众所周知，此“帽”结构在翻译中起重要作用。天然存在的帽结构由7-甲基鸟苷组成，该鸟苷通过三磷酸桥连接到第一个转录核苷酸的5'-末端，从而导致7G(5')ppp(5')N，其中N是任何核苷酸。mRNA帽在基因表达中起重要作用。帽结构可以保护mRNA免受外切核酸酶的降解，使RNA可以从细胞核转运到细胞质，并参与翻译起始复合物的组装。m7G(5’)ppp(5’)G(mCAP)已用作体外T7或SP6RNA聚合酶转录的引物，以获得在其5'末端具有帽结构的RNA。
通过体外转录合成mRNA已经成为引入外源基因并进行表达蛋白的重要工具，并广泛应用于疾病的治疗和预防中，体外转录合成mRNA使得工作人员能够制备在各种生物学应用中表现适当的RNA分子。此类应用包括多肽的研究应用和商业生产，例如，在无细胞翻译体系中产生在特定位点包含“非天然”氨基酸的多肽，或在培养的细胞中产生就其活性或稳定性而言需要翻译后修饰的多肽。在后者体系中，合成进行显著更长的时间，并因此产生更多的蛋白质。
基于结构特点，现有技术中，mRNA的5’末端帽子结构与真核起始因子(elF4E)结合能力的高低决定了该mRNA在细胞内的翻译表达效果。现有的帽类似物结构一般是天然的帽子结构，其mRNA的翻译效果不稳定，对于不同的序列或者不同的表达宿主结果差异较大。
发明内容
本申请提供了一种乙烯基膦酸修饰的mRNA帽类似物及其制备方法和应用，该mRNA帽类似物结构中的使用乙烯基膦酸模拟正常的膦酸功能，由于结构的改进，乙烯基膦酸修饰的mRNA帽类似物具有空间构象的优势，和真核起始因子(elF4E)具有更好的结合能力，最终可以提高mRNA在细胞内的翻译水平。
一种乙烯基膦酸修饰的mRNA帽类似物，其包含以下结构，或其药学上可接受的盐、立体异构体：
其中，R1、R2、R3和R4独立的为氢、卤素、羟基、取代或未取代的烷基、取代或未取代的O-烷基、取代或未取代的S-烷基、取代或未取代的NH-烷基、取代或未取代的N-二烃基、取代或未取代的O-芳香基、取代或未取代的S-芳香基、取代或未取代的NH-芳香基、取代或未取代的O-芳烷基、取代或未取代的S-芳烷基、取代或未取代的NH-芳烷基；
B1、B2独立的为天然的、或修饰的、或非天然的核苷碱基；
Ra、Rb、Rc独立的为以下结构：
Y为O、OCH2、OCH、CH2、CH、C2H3或C3H5；Z为OH、SH、BH3、芳基、烷基、O-烷基或O-芳基；X为O、CH2或NH；W为H、OH、烷基、O-烷基、N-烷基、S-烷基、卤素；表示是双键或者单键，并与五元糖环连接；且Ra、Rb、Rc结构中至少有一个结构中Y为O、OCH或CH。
上述包含可替换为具有。
上述R1、R2、R3和R4独立的为氢、卤素、羟基、取代或未取代的烷基、取代或未取代的O-烷基、取代或未取代的S-烷基、取代或未取代的NH-烷基。
上述卤素为F、Cl或Br。
上述取代或未取代的烷基中碳原子个数为0-5；优选为1-5；
优选的，上述取代或未取代的烷基中碳原子个数为1、2或3。
优选的，上述取代或未取代的烷基为取代或未取代的甲基、取代或未取代的乙基、取代或未取代的正丙基、取代或未取代的异丙基、取代或未取代的正丁基、取代或未取代的异丁基、取代或未取代的叔丁基。
上述取代或未取代的O-烷基中碳原子个数为0-5；优选为1-5；
优选的，上述取代或未取代的O-烷基中碳原子个数为1、2或3。
优选的，上述取代或未取代的O-烷基为取代或未取代的甲氧基、取代或未取代的乙氧基、取代或未取代的丙氧基。
上述取代或未取代的S-烷基中碳原子个数为0-5；优选为1-5；
优选的，上述取代或未取代的S-烷基中碳原子个数为1、2或3。
优选的，上述取代或未取代的S-烷基为取代或未取代的甲硫基、取代或未取代的乙硫基、取代或未取代的丙硫基。
上述取代或未取代的NH-烷基中碳原子个数为0-5；优选为1-5；
优选的，上述取代或未取代的NH-烷基中碳原子个数为1、2、3、4或5。
优选的，上述取代或未取代的NH-烷基为取代或未取代的甲胺基、取代或未取代的乙胺基、丙胺基。
优选的，上述取代或未取代的N-二烃基中碳原子个数为0-5；优选为1-5。
上述烷基、O-烷基、S-烷基、NH-烷基、N-二烃基中的取代基团独立的选自取代或未取代的烷基、取代或未取代的O-烷基、取代或未取代的酰胺基中的一种或多种。
优选的，上述烷基的碳原子个数为1-5；上述O-烷基的碳原子个数为1-5；上述酰胺基的碳原子个数为1-5。优选的，上述烷基、O-烷基、酰胺基可进一步被C1-C5的直链或支链烷基取代。优选的，上述酰胺基团中的N原子连接的氢可被C1-C5的直链或支链烷基取代。优选的，上述酰胺基团中的N原子连接的氢可被甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正戊基、异戊基中的一个或多个取代。
优选的，上述烷基、O-烷基、S-烷基、NH-烷基、N-二烃基中的取代基团独立的选自甲基、乙基、正丙基、异丙基、甲氧基、乙氧基、正丙氧基、异丙氧基、甲酰胺基、乙酰胺基、正丙酰胺基、异丙酰胺基、正丁酰胺基、异丁酰胺基、叔丁酰胺基、N-甲基乙酰氨基、N-乙基乙酰氨基、N-正丙基乙酰氨基、N-异丙基乙酰氨基。
优选的，上述R1、R2、R3和R4独立的为氢、F、羟基、甲氧基、乙氧基、丙氧基、甲氧基甲基、甲氧基乙氧基、乙酰胺基甲基、正丙酰胺基甲基、异丙酰胺基甲基、丁酰胺基甲基、N-甲基乙酰氨基甲基、N-乙基乙酰氨基甲基、N-正丙基乙酰氨基甲基、N-异丙基乙酰氨基甲基。
上述B1、B2独立的为腺嘌呤、鸟嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶。
上述Ra、Rb、Rc独立的为以下结构：
其中Y为O、CH、C2H3或C3H5；Z为OH或烷基；X为O或CH2；W为H、OH或烷基；
上述烷基的碳原子个数为0-5。
上述烷基的碳原子个数为1、2或3。
Ra、Rb、Rc结构中至少有一个结构中Y为CH。
上述R1、R2、R4均为羟基，R3为甲氧基；B1为腺嘌呤、B2为鸟嘌呤；
Ra、Rb、Rc中有一个或两个为以下结构
[根据细则91更正 27.02.2024]
可选的，上述乙烯基膦酸修饰的mRNA帽类似物具有以下任一结构，或其药学上可接受的盐、立体异构体等：
优选的，上述Ra、Rb、Rc中只有一个为以下结构
本发明提供的一种乙烯基膦酸修饰的mRNA帽类似物还可以是盐的形式，所述盐可以是钠盐、钾盐、铵盐、有机胺盐等本领域技术人员熟知的盐。
所述铵盐的阳离子为NH4 +。
所述有机胺盐包括但不限于三乙胺盐。
本发明提供的一种乙烯基膦酸修饰的mRNA帽类似物还可以是上述结构的立体异构体。“立体异构体”是指具有相同化学构造，但原子或基团在空间上排列方式不同的化合物。上述立体异构体包括但不限于对映异构体、非对映异构体、构象异构体(旋转异构体)、几何异构体(顺/反)、阻转异构体等。
在可选的实施方案中，上述乙烯基膦酸修饰的mRNA帽类似物的碳碳双键为E型或Z型中的至少一种。
一种乙烯基膦酸修饰的mRNA帽类似物的制备方法，包括以下步骤：(1)m7GDP或其修饰类似物的咪唑盐的合成；(2)磷酸酯键连接的二核苷酸的制备；(3)乙烯基膦酸修饰的mRNA帽类似物合成。
具体的包括以下步骤：(1)m7GDP或其修饰类似物的咪唑盐的合成：对鸟苷或其类似物依次进行二磷酸化、N7的甲基化、二磷酸的咪唑化反应等合成m7GDP或其修饰类似物的咪唑盐；(2)磷酸酯键连接的二核苷酸的制备：通过2’OMe-A或其修饰亚磷酰胺单体与保护的鸟苷或其修饰类似物，在四氮唑的作用下偶联形成第一个磷酸酯键，通过酸作用，脱除保护基，然后引入第二个磷酸，最终水解得到磷酸酯键连接的二核苷酸；(3)乙烯基膦酸修饰的mRNA帽类似物合成：m7GDP或其修饰类似物的咪唑盐和磷酸酯键连接的二核苷酸反应制备乙烯基膦酸修饰的mRNA帽类似物。
具体步骤包括：
(1)m7GDP或其修饰类似物的咪唑盐的合成：
1)称取鸟苷或其类似物，溶解在磷酸三甲酯中，反应液冷却至0℃，缓慢滴加三氯氧磷(1.8eq.)，冰浴下搅拌4小时后，加水淬灭反应，反相色谱纯化得中间体a。2)将中间体a，三苯基膦(1.0eq.)，2,2’二硫二吡啶(2.0eq.)，咪唑(8.0eq.)和三乙胺(1.0eq.)溶解在DMF中充分搅拌10小时，反应结束后加入4M的高氯酸钠丙酮溶液中析出，滤饼用丙酮充分洗涤得中间体b。3)取中间体b溶解DMF中，加入磷酸三丁胺(3.0eq.)和氯化锌(8.0eq.)后室温搅拌5小时。反应结束后离子色谱纯化得到中间体c。3)将中间体c溶解在20倍体积的纯化水中，反应液冷却至4℃，缓慢滴加硫酸二甲酯，过程中用2M的氢氧化钠调节pH不超过5，HPLC监测反应，反应结束后离子色谱纯化得到中间体d。5)；将中间体d，三苯基膦(1.0eq.)，2,2’二硫二吡啶(2.0eq.)，咪唑(8.0eq.)和三乙胺(1.0eq.)溶解在DMF中充分搅拌10小时，反应结束后加入4M的高氯酸钠丙酮溶液中析出，滤饼用丙酮充分洗涤得到m7GDP或其修饰类似物的咪唑盐。
(2)磷酸酯键连接的二核苷酸的制备：
称取2’OMe-rA或其修饰亚磷酰胺单体于单口瓶中，用二氯甲烷溶解，再加入的2’,3’乙酰基鸟苷或其类似物，降温至25±2℃，氮气鼓吹下加入四氮唑，升温至25±2℃反应。监测反应结束后，将1.2eq.的碘吡啶溶液加入到反应液中，监测反应结束后旋干，浓缩后的油膏溶解在二氯甲烷中，加入1.1eq.的三氟乙酸，监测反应结束后，旋干，石油醚/二氯甲烷按一定比例打浆，过滤得中间体e；将e溶解在乙腈中，加入1.2eq.的膦试剂、1.2eq.的四氮唑充分搅拌反应，监测反应结束后，再加入1.2eq.的碘吡啶溶液加入到反应液中，监测反应结束后旋干，在旋瓶中加入3L甲醇和3L浓氨水，室温反应4小时，监测反应，反应结束后旋干，加入20L超纯水，进入反向离子渗透设备，洗涤浓缩，冻干得磷酸酯键连接的二核苷酸。
(3)乙烯基膦酸修饰的mRNA帽类似物合成：
将步骤(1)得到的m7GDP或其修饰类似物的咪唑盐溶解在含有MnCl2的DMF溶液中，并添加到步骤(2)得到的磷酸酯键连接的二核苷酸的DMF溶液中，在室温下搅拌反应，24小时后，用0.25M EDTA溶液终止反应；将混合物装载到DEAESephadex柱上。将产物使用0-1.0M的TEAB洗脱液线性梯度洗脱。收集HPLC纯度＞97％的洗脱产物，浓缩完以上分离液，再装载到强阴离子树脂，使用0-1.0M的醋酸钠洗脱液线性梯度洗脱，收集HPLC纯度＞98.5％的洗脱产物，合并高纯度洗脱液，通过纳滤设备去除残留的醋酸钠溶液并浓缩得目标产物含乙烯基膦酸修饰的mRNA帽类似物。
一种乙烯基膦酸修饰的mRNA帽类似物的应用，该乙烯基膦酸修饰的mRNA帽类似物用于T7RNA聚合酶体系下的mRNA加帽。T7RNA聚合酶是一种依赖DNA的RNA聚合酶，其对噬菌体T7启动子序列有高特异性。该酶从T7启动子插入到转录载体下游的DNA上合成大量RNA。T7RNA聚合酶催化的IVT(体外转录)反应体系是目前最成熟的mRNA制备体系。
通常20ul的IVT反应体系中含有50U的T7RNA聚合酶，同时搭配1ul的帽类似物(100mM)可以获得最佳的转录产量以及加帽效率。
本发明提供了一种复合体，其包含上述的一种乙烯基膦酸修饰的mRNA帽类似物和DNA模板，其中所述DNA模板包括含有转录起始位点的启动子区，所述转录起始位点具有在核苷酸位置+1处的第一核苷酸和在核苷酸位置+2处的第二核苷酸；所述乙烯基膦酸修饰的mRNA帽类似物结构中的B1基团与所述第一核苷酸碱基互补，所述乙烯基膦酸修饰的mRNA帽类似物结构中的B2基团与所述第二核苷酸碱基互补。
本发明提供了一种RNA分子，包含上述乙烯基膦酸修饰的mRNA帽类似物。上述乙烯基膦酸修饰的mRNA帽类似物作为上述RNA分子的帽结构或帽结构片段。
本发明提供了一种药物组合物，包含上述RNA分子，以及药学上可接受的载体。
本发明提供了一种乙烯基膦酸修饰的mRNA帽类似物及其制备方法和应用，该乙烯基膦酸修饰的mRNA帽类似物适用于以DNA序列为模板利用体外共转录方法生产的mRNA，该DNA序列可以来源或改造自病毒、动物、植物等物种，同时其生产的mRNA具有更低的免疫原性、更高的蛋白翻译效率、更好的稳定性。
本发明相比现有技术具有以下优点：
1、与现有帽结构类似物Cleancap相比，本发明的乙烯基膦酸修饰的mRNA帽类似物，由于双键结构的存在使分子构象更加稳定，不容易被核酸酶识别并水解，并且该类帽类似物也表现出较好的体外转录产量以及加帽效率数据；同时，含有双键的mRNA帽类似物与真核起始因子(elF4E)有更好的结合能力，表现出更好的mRNA翻译效果。
2、本发明设计的乙烯基膦酸修饰帽子结构，含有乙烯基基团，可以有效地固定5’末端帽子的空间结构，并且提高mRNA的5’末端帽子结构与真核起始因子(elF4E)结合能力，进而可以提高mRNA的翻译效果。
 具体实施方式
下面将结合本发明实施例，对本发明的技术方案进行清楚、完整的描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
术语解释：本发明中“eq.”为当量。
各实施例中所使用的原料名称及来源参见下表1：
表1
实施例1：以中间体A和B为原料的乙烯基膦酸修饰的mRNA帽类似物的合成方法
将中间体A(2mol)溶解在含有MnCl2(20mol)的DMF溶液中，随后将中间体B(1.8mol)加入到反应液中。在室温下搅拌反应。24小时后，用10L的0.25M EDTA溶液终止反应。将混合物装载到DEAE Sephadex柱(30×500cm)上。将产物使用0-1.0M的TEAB洗脱液线性梯度洗脱。浓缩得目标产物，反应路线流程，如方程式(1)所示：
其中，化合物A通过以下步骤得到：1)称取5g鸟苷，溶解在70.0ml的磷酸三甲酯中，反应液冷却至0℃，缓慢滴加三氯氧磷(1.8eq.)，冰浴下搅拌4小时后，加水淬灭反应，反相色谱纯化得中间体A1。2)将中间体A1，三苯基膦(1.0eq.)，2,2’二硫二吡啶(2.0eq.)，咪唑(8.0eq.)和三乙胺(1.0eq.)溶解在DMF中充分搅拌10小时，反应结束后加入4M的高氯酸钠丙酮溶液中析出，滤饼用丙酮充分洗涤得中间体A2。3)取10g中间体A2溶解DMF中，加入磷酸三丁胺(3.0eq.)和氯化锌(8.0eq.)后室温搅拌5小时。反应结束后离子色谱纯化得到中间体A3。3)将中间体A3溶解在20倍体积的纯化水中，反应液冷却至4℃，缓慢滴加硫酸二甲酯，过程中用2M的氢氧化钠调节pH不超过5，HPLC监测反应，反应结束后离子色谱纯化得到中间体A4。5)；将中间体A4，三苯基膦(1.0eq.)，2,2’二硫二吡啶(2.0eq.)，咪唑(8.0eq.)和三乙胺(1.0eq.)溶解在DMF中充分搅拌10小时，反应结束后加入4M的高氯酸钠丙酮溶液中析出，滤饼用丙酮充分洗涤得中间体A。
化合物A反应路线流程，如方程式(2)所示：
其中，化合物B通过以下步骤得到：1)取10g鸟苷溶解在150mL吡啶中，将三甲基氯硅烷(5.0eq.)加入到反应液中，随后室温搅拌2小时。降温至0℃，将异丁酰氯(1.5eq.)滴加到反应液中，随后室温反应3小时。冰浴下依次将20mL氨水，500mL水滴加到反应液中，二氯甲烷洗涤水相后水相用热水重结晶得到中间体B1。
2)取10g中间体B1溶解在150mL吡啶中，降温至0℃，将4,4’-二甲氧基三苯基氯甲烷(1.2eq.)的吡啶溶液(50mL)滴加到反应液中。室温搅拌4小时，TLC监测反应至完全。冰浴下用5％的NaHCO3水溶液淬灭反应。乙酸乙酯萃取后用正己烷结晶得到中间体B2。
3)取10g中间体B2溶解在100mL的DMF溶液中，TBDMSCl(3.1eq.)加入到反应液中后室温搅拌过夜。加入饱和碳酸钠水溶液后用乙酸乙酯萃取，结晶得到中间体B3。
4)取8g中间体B3溶解在80mL 80％的乙酸溶液中，室温搅拌5小时后加入400mL的乙酸乙酯，随后用饱和碳酸钠调节pH至中性。分离有机相浓缩后柱层析得到中间体B4。
5)取5g中间体B4溶解在50mL DMF中，DMSO(6.0eq.)和EDCI(3.0eq.)加入到反应液后再将吡啶(1.0eq.)和三氟乙酸(1.0eq.)滴加到反应液中。室温反应5小时后用乙酸乙酯稀释反应液，有机相分别用饱和碳酸氢钠水溶液和水洗涤后浓缩，柱层析得到中间体B5。
6)将四乙基亚甲基二磷酸脂(1.5eq.)冰浴下滴加到NaH(60％，1.0eq.)的THF(80mL)悬浊液中，随后0℃搅拌0.5小时。将中间体B5(3g)的THF溶液(40mL)缓慢滴加到反应液中。室温搅拌过夜。冰浴下将饱和氯化铵水溶液滴加到反应液中淬灭反应，乙酸乙酯萃取后浓缩，柱层析得到中间体B6。
7)取2g中间体B6溶解在40mL乙腈中，将三乙基溴硅烷(10.0eq.)加入到反应液中，随后加热到75℃过夜后浓缩，粗品用反相制备得到中间体B7。
8)取1g中间体B7和DMT-2'OMe-A(Bz)(1.2eq.)溶解在DMF中，将DCC(2.5eq.)加到反应液中室温过夜。反应液浓缩后柱层析得到中间体B8。
9)取1g中间体B8溶解在80％的醋酸水溶液中，室温搅拌过夜，反应完全，浓缩后纯化得到中间体B9。
10)取0.6g中间体B9溶解在二氯甲烷中(12mL)，分别将四氮唑(1.1eq.)和双(2-氰乙基)-N,N-二异丙基亚磷酰胺(1.1eq.)加入到反应液中，室温反应5小时，反应完全后浓缩得到中间体B10的粗品。
11)将中间体B10的粗品溶解在20mL的氨水中，加热至50℃过夜，反应结束后浓缩，反相色谱纯化得到中间体B。
化合物B反应路线流程，如方程式(3)所示：
实施例2：以中间体C和D为原料的乙烯基膦酸修饰的mRNA帽类似物的合成方法
以中间体C和D为原料，参考实施例1目标产物的合成方法得到实施例2的乙烯基膦酸修饰的mRNA帽类似物。反应路线流程，如方程式(4)所示：
其中，化合物C通过以下步骤得到：1)取5g中间体B7溶解在H2O/TFA(1：1，50mL)中，室温搅拌1小时，反应完全。反相色谱纯化得到中间体C1。
2)取3g中间体C1与三苯基膦(1.5eq.)，2,2’二硫二吡啶(1.5eq.)，三乙胺(2.5eq.)，咪唑(8.0eq.)溶解在20.0mL的DMF中，室温搅拌10小时，反应完全。反应液加入4M的高氯酸钠丙酮溶液中析出，滤饼用丙酮充分洗涤得到中间体C2。
3)称取2g中间体C2溶解DMF中，加入磷酸三乙胺(3.0eq.)和氯化锌(8.0eq.)后，室温搅拌8小时后，反应完全。反相色谱纯化得到中间体C3。
4)取2g中间体C3溶解在40mL纯化水中，反应液冷却至4℃，缓慢滴加硫酸二甲酯，过程中用2M的氢氧化钠调节pH不超过5，HPLC监测反应，反应结束后离子色谱纯化得到中间体C4。
5)参考中间体C2的合成方法得到中间体C。
化合物C反应路线流程，如方程式(5)所示：
其中，化合物D通过以下步骤得到：称取5kg的2’OMe-rA亚磷酰胺单体于单口瓶中，用50L的二氯甲烷溶解，再加入2.73kg的2’,3’乙酰基鸟苷，降温至25±2℃，氮气鼓吹下加入880g四氮唑，升温至25±2℃反应。监测反应结束后，将1.2eq.的碘吡啶溶液加入到反应液中，监测反应结束后旋干，浓缩后的油膏溶解在4L二氯甲烷中，加入1.1eq.的三氟乙酸，监测反应结束后，旋干，石油醚/二氯甲烷按一定比例打浆，过滤得中间体D1；将D1溶解在4L乙腈中，加入1.2eq.的膦试剂、1.2eq.的四氮唑充分搅拌反应，监测反应结束后，再加入1.2eq.的碘吡啶溶液加入到反应液中，监测反应结束后旋干，在旋瓶中加入3L甲醇和3L浓氨水，室温反应4小时，监测反应，反应结束后旋干，加入20L超纯水，进入反向离子渗透设备，洗涤浓缩，冻干得中间体D，反应路线流程，如方程式(6)所示：
实施例3：以中间体A和E为原料的乙烯基膦酸修饰的mRNA帽类似物的合成方法
以中间体A和E为原料，参考实施例1目标产物的合成方法得到实施例3的乙烯基膦酸修饰的mRNA帽类似物。反应路线流程，如方程式(7)所示：
其中，化合物E通过以下步骤得到：1)取5g中间体D1溶解在50mL DMF中，DMSO(6.0eq.)和EDCI(3.0eq.)加入到反应液后再将吡啶(1.0eq.)和三氟乙酸(1.0eq.)滴加到反应液中。室温反应5小时后用乙酸乙酯稀释反应液，有机相分别用饱和碳酸氢钠水溶液和水洗涤后浓缩，柱层析得到中间体E1。
2)将四乙基亚甲基二磷酸脂(1.5eq.)冰浴下滴加到NaH(60％，1.0eq.)的THF(80mL)悬浊液中，随后0℃搅拌0.5小时。将中间体E1(3g)的THF溶液(40mL)缓慢滴加到反应液中。室温搅拌过夜。冰浴下将饱和氯化铵水溶液滴加到反应液中淬灭反应，乙酸乙酯萃取后浓缩，柱层析得到中间体E2。
3)取1g中间体E2溶解在20mL乙腈中，将三乙基溴硅烷(10.0eq.)加入到反应液中，随后加热到75℃过夜后浓缩，粗品用反相制备得到中间体E3。
4)取0.3g中间体E3溶解在6mL氨水中，加热至50℃反应过夜。反应结束后浓缩，反相色谱纯化得到中间体E。反应路线流程，如方程式(8)所示：
实施例4：以中间体B和C为原料的乙烯基膦酸修饰的mRNA帽类似物的合成方法
以中间体B和C为原料，参考实施例1目标产物的合成方法得到实施例4的乙烯基膦酸修饰的mRNA帽类似物。反应路线流程，如方程式(9)所示：
实施例5：以中间体C和E为原料的乙烯基膦酸修饰的mRNA帽类似物的合成方法
以中间体C和E为原料，参考实施例1目标产物的合成方法得到实施例5的乙烯基膦酸修饰的mRNA帽类似物。反应路线流程，如方程式(10)所示：
实施例6：以中间体A和F为原料的乙烯基膦酸修饰的mRNA帽类似物的合成方法
以中间体A和F为原料，参考实施例1目标产物的合成方法得到实施例6的乙烯基膦酸修饰的mRNA帽类似物。反应路线流程，如方程式(11)所示：
其中，化合物F通过以下步骤得到：1)取5g中间体B9溶解在50mL DMF中，DMSO(6.0eq.)和EDCI(3.0eq.)加入到反应液后再将吡啶(1.0eq.)和三氟乙酸(1.0eq.)滴加到反应液中。室温反应5小时后用乙酸乙酯稀释反应液，有机相分别用饱和碳酸氢钠水溶液和水洗涤后浓缩，柱层析得到中间体E1。
2)将四乙基亚甲基二磷酸脂(1.5eq.)冰浴下滴加到NaH(60％，2.0eq.)的THF(80mL)悬浊液中，随后0℃搅拌0.5小时。将中间体E1(3g)的THF溶液(40mL)缓慢滴加到反应液中。室温搅拌过夜。冰浴下用冰水淬灭反应，室温搅拌2小时后浓缩。粗品用反相色谱纯化得到中间体F2。
3)取1g中间体F2溶解在20mLDMF中，将三乙基溴硅烷(10.0eq.)加入到反应液中，随后加热到75℃过夜后浓缩，粗品用反相制备得到中间体F3。
4)取0.3g中间体F3溶解在H2O/TFA(1：1，30mL)中，室温搅拌1小时，反应完全。反相色谱纯化得到中间体F。反应路线流程，如方程式(12)所示：
实施例7：以中间体C和F为原料的乙烯基膦酸修饰的mRNA帽类似物的合成方法
以中间体C和F为原料，参考实施例1目标产物的合成方法得到实施例7的乙烯基膦酸修饰的mRNA帽类似物。反应路线流程，如方程式(13)所示：
实施例8：以中间体D和G为原料的含乙烯基膦酸修饰的mRNA帽类似物的合成方法(化合物10)
以中间体D和G为原料，参考实施例1目标产物的合成方法得到实施例8的含乙烯基膦酸修饰的mRNA帽类似物。反应路线流程，如方程式(14)所示：
其中，化合物G通过以下步骤得到：
1)分别参考化合物B1、B2、B3、B4、B5、B6的合成方法得到中间体G2、G3、G4、G5、G6、G7。
2)取8g中间体G7溶解在80mL乙腈中，将三乙基溴硅烷(10.0eq.)加入到反应液中，随后室温搅拌过夜后浓缩得到中间体G8的粗品，无需纯化直接用于下一步反应。
3)将中间体G8粗品溶解在THF(10V)中，室温下加入TBAF(3.0eq.)。室温搅拌3小时。减压浓缩得到中间体G9的粗品，无需纯化直接用于下一步反应。
4)取中间体G8的粗品溶解在浓氨水中(10V)中，室温搅拌过夜。反应结束后减压浓缩，反相色谱纯化得到中间体G10。
5)分别参考化合物C2、C3、C4、C的合成方法得到中间体G11、G12、G13、G。反应路线流程，如方程式(15)所示：
实施例9：以中间体D和H为原料的含乙烯基膦酸修饰的mRNA帽类似物的合成方法(化合物11)
以中间体D和H为原料，参考实施例1目标产物的合成方法得到实施例9的含乙烯基膦酸修饰的mRNA帽类似物。反应路线流程，如方程式(16)所示：
其中，化合物H通过以下步骤得到：
(1)取化合物H1(0.18mol)和咪唑(0.36mol)溶解在DMF(3.5L)。冰浴下将叔丁基二苯基氯硅烷(0.2mol)加入到反应液中。室温搅拌5小时。将反应液倒入冰水中，乙酸乙酯萃取(500.0mL x 2)。有机相用饱和氯化钠洗涤后干燥，减压浓缩得到中间体H2，无需纯化直接用于下一步反应。
(2)冰盐浴下将醋酸酐(0.45mol)和吡啶(0.8mol)缓慢加到CrO3(0.45mol)的二氯甲烷(900.0mL)悬浮液中。室温搅拌15分钟后将中间体H2(0.15mol)加入到反应液中。室温搅拌1小时后将反应液倒入到冰的乙酸乙酯中，抽滤，滤液减压浓缩后经柱层析纯化得到中间体H3。
(3)将甲基三苯基溴化膦(0.15mol)冰浴下滴加到NaH(60％，0.12mol)的THF(500.0mL)悬浊液中，0℃搅拌0.5小时。将中间体H3(0.12mol)的THF溶液(300.0mL)缓慢滴加到反应液中。室温搅拌过夜。冰浴下将饱和氯化铵水溶液滴加到反应液中淬灭反应，乙酸乙酯萃取后浓缩，柱层析纯化得到中间体H4。
(4)取中间体H4(0.1mol)溶解在无水THF(420.0mL)中，控温-5～0℃，氮气氛围下滴加到硼烷二甲硫醚溶液(0.2mol，10M)中，滴加完毕后保温搅拌6小时。随后将氢氧化钠水溶液(0.7mol，350.0mL)和过氧化氢(80.0mL，30％水溶液)依次滴加到反应液中。恢复室温搅拌2小时。加入乙酸乙酯(200.0mL x 2)萃取。有机相依次用水、饱和食盐水洗涤，无水硫酸钠干燥，减压浓缩有机相，柱层析纯化得中间体H5。
(5)将中间体H5(85.0mmol)的THF(200.0mL)溶液冰浴下滴加到NaH(60％，95.0mmol)的THF(170.0mL)悬浊液中，0℃搅拌0.5小时。将碘甲烷(100.0mmol)滴加到反应液中。室温搅拌3小时。冰浴下将饱和氯化铵水溶液滴加到反应液中淬灭反应，乙酸乙酯萃取后浓缩，柱层析纯化得到中间体H6。
(6)取中间体H6(70.0mmol)溶解在无水THF(300.0mL)中，将TBAF的THF溶液(1M,0.3mol)加入后室温搅拌6小时。反应液用水洗涤，水相再用乙酸乙酯萃取一次。合并有机相，干燥后浓缩。柱层析纯化后得到中间体H7。
(7)取中间体H7(60.0mmol)溶解在DMF(130.0mL)中，DMSO(0.36mol)和EDCI(0.18mol)加入到反应液后再将吡啶(60.0mmol)和三氟乙酸(60.0mmol)滴加到反应液中。室温反应5小时后用乙酸乙酯稀释反应液，有机相分别用饱和碳酸氢钠水溶液和水洗涤后浓缩，柱层析得到中间体H8。
(8)将四乙基亚甲基二磷酸脂(80.0mmol)冰浴下滴加到NaH(60％，53.0mmol)的THF(50.0mL)悬浊液中，随后0℃搅拌0.5小时。将中间体H8(53.0mmol)的THF溶液(70.0mL)缓慢滴加到反应液中。室温搅拌过夜。冰浴下将饱和氯化铵水溶液滴加到反应液中淬灭反应，乙酸乙酯萃取后浓缩，柱层析得到中间体H9。
(9)取中间体H9(44.0mmol)溶解在醋酸(80.0mL)中，室温下加入醋酸酐(132.0mmol)后降温至10℃以下，将浓硫酸(6.6mmol)滴加到反应液中。室温搅拌过夜。反应液用DCM(150.0mL)稀释后，依次用水、饱和碳酸氢钠水溶液、饱和食盐水洗涤，无水硫酸钠干燥，减压浓缩有机相得中间体H10，无需纯化直接用于下一步反应。
(10)称取化合物H10(40.0mmol)和化合物H11(40.0mmol)悬浮在DCE(300.0mL)中，将BSA(0.1mol)加入到反应液中。反应升温至70℃搅拌1小时后，降温至0℃，将TMSOTf(0.12mol)滴加到反应液中。滴加完毕后升温至70℃搅拌3小时。降温至室温后，反应液用饱和碳酸氢钠水溶液洗涤，有机相减压浓缩后经柱层析纯化得到中间体H12。
(11)参考中间体G8的合成方法得到中间体H13。
(12)参考中间体G10的合成方法得到中间体H14。
(13)分别参考中间体G11、G12、G13和G的合成方法得到中间体H15、H16、H17和H。反应路线流程，如方程式(17)所示：
实施例10：以中间体D和I为原料的含乙烯基膦酸修饰的mRNA帽类似物的合成方法(化合物12)
以中间体D和I为原料，参考实施例1目标产物的合成方法得到实施例10的含乙烯基膦酸修饰的mRNA帽类似物。反应路线流程，如方程式(18)所示：
其中，化合物I通过以下步骤得到：
1)取中间体H5(0.33mol)，三苯基膦(0.66mol)和DIAD(0.66mol)溶解在THF(1.5L)中。将DPPA(0.66mol)滴加到反应液中。室温搅拌过夜。减压浓缩除去溶剂。经柱层析纯化得到中间体I1。
2)参考中间体H7的合成方法得到中间体I2。
3)参考中间体H8的合成方法得到中间体I3。
4)参考中间体H9的合成方法得到中间体I4。
5)参考中间体H10的合成方法得到中间体I5。
6)参考中间体H12的合成方法得到中间体I6。
7)参考中间体H14的合成方法得到中间体I7。
8)取中间体I7(55.0mmol)溶解在THF(500.0mL)中。将水(50.0mL)和三苯基膦(82.5mmol)加入后升温至50℃搅拌过夜。浓缩除去溶剂。粗品经柱层析纯化后得到中间体I8。
9)取中间体I8(50.0mmol)溶解在DCM(100.0mL)和吡啶中(100.0mL)。降温至-10℃，滴加乙酰氯(55.0mmol)后搅拌3小时。加入甲醇(3.0mL)淬灭反应。减压浓缩后经柱层析纯化得到中间体I9。
10)参考中间体H13的合成方法得到中间体I10。
11)参考中间体H15的合成方法得到中间体I11。
12)参考中间体H16的合成方法得到中间体I12。
13)参考中间体H17的合成方法得到中间体I13。
14)参考中间体H的合成方法得到中间体I。反应路线流程，如方程式(19)所示：
实施例11：以中间体D和J为原料的含乙烯基膦酸修饰的mRNA帽类似物的合成方法(化合物13)
以中间体D和J为原料，参考实施例1目标产物的合成方法得到实施例11的含乙烯基膦酸修饰的mRNA帽类似物。反应路线流程，如方程式(20)所示：
其中，化合物J通过以下步骤得到：
1)取中间体J1(80.0mmol)溶解在吡啶(520.0mL)中，室温下将硫代氯甲酸苯酯(0.24mol)加入到反应液后室温搅拌过夜。减压浓缩除去溶剂后经柱层析纯化得到中间体J2。
2)取中间体J2(75.0mmol)和AIBN(15.0mol)溶解在甲苯(590.0mL)中。反应液加热至回流后将三丁基氢化锡(0.23mol)加入。继续搅拌2小时。减压浓缩除去溶剂后，柱层析纯化得到中间体J3。
3)取中间体J3(42.0mmol)溶解在DCM(270.0mL)中。室温下将三氯乙酸(0.13mol)加入到反应液中，室温搅拌2小时。反应液分别用水，饱和碳酸氢钠洗涤。有机相减压浓缩后柱层析纯化得到中间体J4。
4)参考中间体G6的合成方法得到中间体J5。
5)参考中间体G7的合成方法得到中间体J6。
6)参考中间体G8的合成方法得到中间体J7。
7)参考中间体I7的合成方法得到中间体J8。
8)参考中间体I11的合成方法得到中间体J9。
9)参考中间体I12的合成方法得到中间体J10。
10)参考中间体I13的合成方法得到中间体J11。
11)参考中间体I的合成方法得到中间体J。反应路线流程，如方程式(21)所示：
实施例12：以中间体D和K为原料的含乙烯基膦酸修饰的mRNA帽类似物的合成方法(化合物15)
以中间体D和K为原料，参考实施例1目标产物的合成方法得到实施例12的含乙烯基膦酸修饰的mRNA帽类似物。反应路线流程，如方程式(22)所示：
其中，化合物K通过以下步骤得到：参考中间体G4～G的合成方法依次得到中间体K2～K。反应路线流程，如方程式(23)所示：
实施例13：以中间体I和L为原料的含乙烯基膦酸修饰的mRNA帽类似物的合成方法(化合物17)
以中间体I和L为原料，参考实施例1目标产物的合成方法得到实施例13的含乙烯基膦酸修饰的mRNA帽类似物。反应路线流程，如方程式(24)所示：
其中，化合物L通过以下步骤得到：参考实施例2中中间体D的合成方法得到中间体L。反应路线流程，如下方程式(25)：
实施例14：以中间体E和M为原料的含乙烯基膦酸修饰的mRNA帽类似物的合成方法(化合物25)
以中间体E和M为原料，参考实施例1目标产物的合成方法得到实施例14的含乙烯基膦酸修饰的mRNA帽类似物。反应路线流程，如方程式(26)所示：
其中，化合物M通过以下步骤得到：
1)参考中间体I5的合成方法得到中间体M1。
2)参考中间体I6的合成方法得到中间体M2。
3)参考中间体I7的合成方法得到中间体M3。
4)参考中间体I8的合成方法得到中间体M4。
5)参考中间体I9的合成方法得到中间体M5。
6)参考中间体H7的合成方法得到中间体M6。
7)参考中间体A1的合成方法得到中间体M7。
8)参考中间体K9的合成方法得到中间体M8。
9)参考中间体K10的合成方法得到中间体M9。
10)参考中间体K11的合成方法得到中间体M10。
11)参考中间体K的合成方法得到中间体M。反应路线流程，如方程式(27)所示：
实施例15：以中间体D和N为原料的含乙烯基膦酸修饰的mRNA帽类似物的合成方法(化合物31)
以中间体D和N为原料，参考实施例1目标产物的合成方法得到实施例15的含乙烯基膦酸修饰的mRNA帽类似物。反应路线流程，如方程式(28)所示：
其中，化合物N通过以下步骤得到：
1)参考中间体M4的合成方法得到中间体N1。
2)参考中间体M5的合成方法得到中间体N2。
3)参考中间体M3的合成方法得到中间体N3。
4)参考中间体M5的合成方法得到中间体N4。
5)参考中间体K6的合成方法得到中间体N5。
6)参考中间体M8的合成方法得到中间体N6。
7)参考中间体M9的合成方法得到中间体N7。
8)参考中间体M10的合成方法得到中间体N8。
9)参考中间体M的合成方法得到中间体N。
实施例16：以中间体D和O为原料的含乙烯基膦酸修饰的mRNA帽类似物的合成方法(化合物34)
以中间体D和O为原料，参考实施例1目标产物的合成方法得到实施例16的含乙烯基膦酸修饰的mRNA帽类似物。反应路线流程，如方程式(30)所示：
其中，化合物O通过以下步骤得到：分别参考中间体I9、I10、I11、I12、I13和I的合成方法得到中间体O1、O2、O3、O4、O5和O。
实施例17：以中间体D和P为原料的含乙烯基膦酸修饰的mRNA帽类似物的合成方法(化合物16)
以中间体D和P为原料，参考实施例1目标产物的合成方法得到实施例17的含乙烯基膦酸修饰的mRNA帽类似物。反应路线流程，如方程式(32)所示：
其中，化合物P通过以下步骤得到：参考中间体G4～G的合成方法依次得到中间体P2～P。反应路线流程，如方程式(33)所示：
对比例1：m7GpppA2’OMepG
m7GpppA2’OMepG的合成方法参考上述实施例的合成方法(所使用的原料参考各实施例中的制备方法)，反应路线流程，如方程式(34)所示：
各实施例所得到的帽类似物以及对比例所得到的帽类似物结构如下表2所示，
表2
测试例1：mRNA体外转录产量及加帽效率的测定
利用乙烯基膦酸修饰的mRNA帽类似物的体外合成：先用NotI线性化质粒，4℃酶切过夜；DNA模板抽提；体外转录合成mRNA，分别使用实施例1-7、化合物10-13、16、17、25、31、34的乙烯基膦酸修饰的mRNA帽类似物及对比例1的帽类似物作为帽结构。
反应体系如表3：
表3
备注：在实验过程中，首先计算好体系所需物料体积，然后进行加样。首先在体系中加入无菌无酶水，随后依次加入10X buffer、NTPs、帽类似物，混匀后轻轻离心，随后加入核酸酶抑制剂、无机焦磷酸酶、T7RNA聚合酶、线性化DNA模板，充分混匀后轻轻离心，于37℃下孵育。2小时后加入DNase I 1U，37℃继续孵育30分钟以去除DNA模板，然后进行RNA纯化，通常使用磁珠纯化方法。纯化的mRNA用无菌无酶水进行溶解，随后利用Nanodrop One进行定量检测。
液相色谱质谱法(LC-MS)被用来检测不同起始帽类似物的mRNA的IVT加帽率；首先需要设计一段与转录产物mRNA起始碱基匹配的具有标记的DNA探针，通常的标记为biotin标记，将链霉亲和素标记的磁珠清洗后与合成的DNA探针、mRNA及10×RNase H reaction buffer室温室温孵育30分钟，边孵育边缓慢混匀，随后加入20ul RNase H(5U/ul)孵育37度3h，每半个小时混匀一次。孵育结束后对磁珠进行清洗，清洗完成后的磁珠加入100μL加热到80℃的75％甲醇，混合物在加热板上加热到80℃，保持3分钟，然后放置磁力架上吸取上清，使用蒸发离心机在室温下干燥45分钟至10μl。然后将样品重新悬浮在50μl的100μM EDTA/1％MeOH中，即可用于LC-MS分析，确定转录反应中RNA的加帽情况。由于加帽与非加帽的碱基在分子量上有明显区别，利用分子质量差别即可判定不同帽类似物起始的mRNA转录的加帽率。具体结果见表4。
表4
由实验结果可知，实施例1-7以及化合物10-13、16、17、25、31、34均能够通过IVT转录出相应的目标mRNA。其中实施例1、2、3、化合物10-13、16、17、25、31、34与对比例1的产量相当，同时实施例1、2、3、化合物10-13、16、17、25、31、34的加帽效率优于对比例1或与对比例1的加帽效率相当。实施例4、5、6、7的体外转录产量以及加帽效率明显低于对比例1。这可能是和帽类似物结构相关，实施例4、5、6、7的刚性结构影响了帽类似物与模板起始转录位点的结合，最终也影响了体外转录产量以及加帽效率。
测试例2：真核起始因子(elF4E)结合能力测试
测试方法：首先用运行缓冲液(50mM磷酸盐,100mM氯化钠,and 0.01％vol/vol Tween 20,pH 6.0 or 7.4)稀释样本，此实验样本有实施例1-7、化合物10-13、16、17、25、31、34以及对比例1帽类似物样本。进样时间为2min，进样流速20ul/min，解离时间为2.5min。残留样本用再生缓冲液(10mM HEPES,150mM NaCl,and 0.01％vol/vol吐温20,pH 7.4))洗脱下来，时间为30sec，流速设为30ul/min。最后通过Biacore T100评价软件(version2.0.2)分析生成传感图并计算结合常数。
表5
由上表5的实验数据可知，实施例1、2、3、4、5、6以及化合物10-13、16、17、25、31、34与elF4E蛋白的结合常数优于对比例1，其中，实施例1、2、3、化合物10-13、16、17、25、31、34与elF4E蛋白的结合常数显著优于对比例1，说明实施例1、2、3、化合物10-13、16、17、25、31、34与elF4E蛋白的结合能力最强。同时我们也发现实施例7与elF4E蛋白的结合常数相比于对比例1最低，说明实施例7与elF4E蛋白的结合能力最弱。因此，当帽类似物结构中含有乙烯基结构，可能对提高帽类似物与elF4E蛋白的结合能力有一定的提高，但是实施例7的含有三个乙烯基结构的刚性结构影响了帽类似物与elF4E蛋白的结合，最终也影响了体外转录产量以及加帽效率。
测试例3：mRNA翻译效率
测试方法：采用eGFP编码序列为DNA模板，利用实施例1-7、化合物10-13、16、17、25、31、34及对比例1的帽类似物为起始进行体外转录。随后将获得的不同的mRNA产物进行293T细胞的转染。293T细胞以(0.5-1)×105个细胞进行铺板(24孔板)。转染时细胞密度一般60-80％为佳，每孔转染2μg mRNA，转染试剂选用Lipofectamine MessengerMAX Transfection Reagent(Invitrogen)并参考其使用方法进行操作。转染后的细胞放置在37℃，CO2孵育箱中，转染4-6小时后，更换为新鲜的完全培养基。在37℃的CO2培养箱箱中孵育24小时以后，荧光显微镜观察其中GFP的荧光强度，并且更具荧光强度计算实施例1-7、化合物10-13、16、17、25、31、34相对于对比例1的荧光强度比例。
表6
由上表6的实验数据可知，实施例1、2、3、4、5、6、化合物10-13、16、17、25、31、34的帽类似物转录的mRNA在细胞内翻译效果相比于对比例1好，其中，实施例1、2、3、化合物10-13、16、17、25、31、34的翻译效果显著优于对比例1，说明实施例1、2、3、化合物10-13、16、17、25、31、34的帽类似物转录的mRNA在细胞内翻译效果最强。同时我们也发现实施例7的翻译效果相比于对比例1最低。因此，当帽类似物结构中含有乙烯基结构，可能对提高帽类似物mRNA在细胞内翻译效果有促进作用，但是实施例7的含有三个乙烯基结构的刚性结构影响了帽类似物与elF4E蛋白的结合，最终也影响了mRNA在细胞内翻译效果。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。

Claims (14)

  1. 一种乙烯基膦酸修饰的mRNA帽类似物，其特征在于，其包含以下结构，或其药学上可接受的盐、立体异构体：
    其中，R1、R2、R3和R4独立的为氢、卤素、羟基、取代或未取代的烷基、取代或未取代的O-烷基、取代或未取代的S-烷基、取代或未取代的NH-烷基、取代或未取代的N-二烃基、取代或未取代的O-芳香基、取代或未取代的S-芳香基、取代或未取代的NH-芳香基、取代或未取代的O-芳烷基、取代或未取代的S-芳烷基、取代或未取代的NH-芳烷基；
    当所述R1为取代或未取代的O-烷基时，可通过所述O-烷基中的烷基碳原子与R1所在的核糖基团中的4’碳原子连接形成桥环；
    B1、B2独立的为天然的、或修饰的、或非天然的核苷碱基；
    Ra、Rb、Rc独立的为以下结构：
    Y为O、OCH2、OCH、CH2、CH、C2H3或C3H5；Z为OH、SH、BH3、芳基、烷基、O-烷基或O-芳基；X为O、CH2或NH；W为H、OH、烷基、O-烷基、N-烷基、S-烷基、卤素；表示是双键或者单键，并与五元糖环连接；
    且Ra、Rb、Rc结构中至少有一个结构中Y为O、OCH或CH。
  2. 根据权利要求1所述的一种乙烯基膦酸修饰的mRNA帽类似物，其特征在于：所述R1、R2、R3和R4独立的为氢、卤素、羟基、取代或未取代的烷基、取代或未取代的O-烷基、取代或未取代的S-烷基、取代或未取代的NH-烷基。
  3. 根据权利要求1-2任一项所述的一种乙烯基膦酸修饰的mRNA帽类似物，其特征在于：所述取代或未取代的O-烷基中碳原子个数为0-5。
  4. 根据权利要求1-2任一项所述的一种乙烯基膦酸修饰的mRNA帽类似物，其特征在于：所述取代或未取代的烷基中碳原子个数为0-5；
    所述取代或未取代的S-烷基中碳原子个数为0-5；
    所述取代或未取代的NH-烷基中碳原子个数为0-5；
    所述取代或未取代的N-二烃基中碳原子个数为0-5；
    所述取代或未取代的烷基、取代或未取代的O-烷基、取代或未取代的S-烷基、取代或未取代的NH-烷基、取代或未取代的N-二烃基中的取代基团独立的选自取代或未取代的烷基、取代或未取代的O-烷基、取代或未取代的酰胺基中的一种或多种。
  5. 根据权利要求1所述的一种乙烯基膦酸修饰的mRNA帽类似物，其特征在于：所述B1、B2独立的为腺嘌呤、鸟嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶。
  6. 根据权利要求1所述的一种乙烯基膦酸修饰的mRNA帽类似物，其特征在于：所述Ra、Rb、Rc独立的为以下结构：
    其中Y为O、CH、C2H3或C3H5；Z为OH或烷基；X为O或CH2；W为H、OH或烷基；
    且Ra、Rb、Rc结构中至少有一个结构中Y为CH。
  7. 根据权利要求1所述的一种乙烯基膦酸修饰的mRNA帽类似物，其特征在于：所述R1、R2、R4均为羟基，R3为甲氧基；B1为腺嘌呤、B2为鸟嘌呤；
    Ra、Rb、Rc中有一个或两个为以下结构
  8. [根据细则91更正 27.02.2024]
    根据权利要求1所述的一种乙烯基膦酸修饰的mRNA帽类似物，其特征在于，所述乙烯基膦酸修饰的mRNA帽类似物具有以下任一结构，或其药学上可接受的盐、立体异构体：
  9. 权利要求1-8任一项所述的一种乙烯基膦酸修饰的mRNA帽类似物的制备方法，其特征在于，包括以下步骤：(1)m7GDP或其修饰类似物的咪唑盐的合成；(2)磷酸酯键连接的二核苷酸的制备；(3)乙烯基膦酸修饰的mRNA帽类似物合成。
  10. 根据权利要求9所述的一种乙烯基膦酸修饰的mRNA帽类似物的制备方法，其特征在于，包括以下步骤：(1)m7GDP或其修饰类似物的咪唑盐的合成：对鸟苷或其类似物依次进行二磷酸化、N7的甲基化、二磷酸的咪唑化反应等合成m7GDP或其修饰类似物的咪唑盐；(2)磷酸酯键连接的二核苷酸的制备：通过2’OMe-A或其修饰亚磷酰胺单体与保护的鸟苷或其修饰类似物，在四氮唑的作用下偶联形成第一个磷酸酯键，通过酸作用，脱除保护基，然后引入第二个磷酸，最终水解得到磷酸酯键连接的二核苷酸；(3)乙烯基膦酸修饰的mRNA帽类似物合成：m7GDP或其修饰类似物的咪唑盐和磷酸酯键连接的二核苷酸反应制备乙烯基膦酸修饰的mRNA帽类似物。
  11. 权利要求1-8任一项所述的一种乙烯基膦酸修饰的mRNA帽类似物的应用，其特征在于：该乙烯基膦酸修饰的mRNA帽类似物用于T7 RNA聚合酶体系下的mRNA加帽。
  12. 一种复合体，其包含权利要求1-8任一项所述的一种乙烯基膦酸修饰的mRNA帽类似物和DNA模板，其中所述DNA模板包括含有转录起始位点的启动子区，所述转录起始位点具有在核苷酸位置+1处的第一核苷酸和在核苷酸位置+2处的第二核苷酸；所述乙烯基膦酸修饰的mRNA帽类似物结构中的B1基团与所述第一核苷酸碱基互补，所述乙烯基膦酸修饰的mRNA帽类似物结构中的B2基团与所述第二核苷酸碱基互补。
  13. 一种RNA分子，其特征在于，其包含权利要求1-8任一项所述的一种乙烯基膦酸修饰的mRNA帽类似物。
  14. 一种药物组合物，其特征在于，其包含权利要求13所述的一种RNA分子，以及药学上可接受的载体。
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