CN117567528B - Cap analogue, synthesis method thereof and mRNA - Google Patents
Cap analogue, synthesis method thereof and mRNA Download PDFInfo
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- CN117567528B CN117567528B CN202410052590.6A CN202410052590A CN117567528B CN 117567528 B CN117567528 B CN 117567528B CN 202410052590 A CN202410052590 A CN 202410052590A CN 117567528 B CN117567528 B CN 117567528B
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- 238000001308 synthesis method Methods 0.000 title abstract description 15
- 108020004999 messenger RNA Proteins 0.000 title abstract description 14
- 238000006243 chemical reaction Methods 0.000 claims abstract description 87
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims abstract description 66
- 239000000047 product Substances 0.000 claims abstract description 31
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims abstract description 23
- 238000000034 method Methods 0.000 claims abstract description 18
- 238000006366 phosphorylation reaction Methods 0.000 claims abstract description 16
- 239000002994 raw material Substances 0.000 claims abstract description 13
- 239000013067 intermediate product Substances 0.000 claims abstract description 9
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 67
- 238000003756 stirring Methods 0.000 claims description 32
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 27
- RIOQSEWOXXDEQQ-UHFFFAOYSA-N triphenylphosphine Chemical compound C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-N 0.000 claims description 18
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 claims description 18
- INQOMBQAUSQDDS-UHFFFAOYSA-N iodomethane Chemical compound IC INQOMBQAUSQDDS-UHFFFAOYSA-N 0.000 claims description 16
- CYTQBVOFDCPGCX-UHFFFAOYSA-N trimethyl phosphite Chemical compound COP(OC)OC CYTQBVOFDCPGCX-UHFFFAOYSA-N 0.000 claims description 13
- 230000002194 synthesizing effect Effects 0.000 claims description 10
- -1 methylene phosphorus dichloride Chemical compound 0.000 claims description 9
- 235000005074 zinc chloride Nutrition 0.000 claims description 9
- 239000011592 zinc chloride Substances 0.000 claims description 9
- 238000010791 quenching Methods 0.000 claims description 7
- 230000000171 quenching effect Effects 0.000 claims description 7
- HAXFWIACAGNFHA-UHFFFAOYSA-N aldrithiol Chemical compound C=1C=CC=NC=1SSC1=CC=CC=N1 HAXFWIACAGNFHA-UHFFFAOYSA-N 0.000 claims description 6
- 239000011261 inert gas Substances 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical class [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 claims description 3
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 3
- IOPIMTCNBFTMDS-UHFFFAOYSA-N C=[P] Chemical compound C=[P] IOPIMTCNBFTMDS-UHFFFAOYSA-N 0.000 claims 1
- 238000003786 synthesis reaction Methods 0.000 abstract description 8
- 230000015572 biosynthetic process Effects 0.000 abstract description 6
- 238000013518 transcription Methods 0.000 abstract description 3
- 230000035897 transcription Effects 0.000 abstract description 3
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 abstract 2
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 abstract 1
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 abstract 1
- 229940029575 guanosine Drugs 0.000 abstract 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 39
- 238000000746 purification Methods 0.000 description 16
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 15
- 238000001514 detection method Methods 0.000 description 11
- 239000002777 nucleoside Substances 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 9
- 239000007791 liquid phase Substances 0.000 description 9
- 150000003833 nucleoside derivatives Chemical class 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 8
- 229910052757 nitrogen Inorganic materials 0.000 description 8
- 239000007795 chemical reaction product Substances 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 238000005070 sampling Methods 0.000 description 7
- 230000000694 effects Effects 0.000 description 6
- XHXFXVLFKHQFAL-UHFFFAOYSA-N phosphoryl trichloride Chemical compound ClP(Cl)(Cl)=O XHXFXVLFKHQFAL-UHFFFAOYSA-N 0.000 description 6
- 239000002253 acid Substances 0.000 description 4
- KKTCWAXMXADOBB-UHFFFAOYSA-N azanium;hydrogen carbonate;hydrate Chemical class [NH4+].O.OC([O-])=O KKTCWAXMXADOBB-UHFFFAOYSA-N 0.000 description 4
- 125000004432 carbon atom Chemical group C* 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 239000001177 diphosphate Substances 0.000 description 4
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 4
- 235000011180 diphosphates Nutrition 0.000 description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 3
- 239000003054 catalyst Substances 0.000 description 3
- 238000009749 continuous casting Methods 0.000 description 3
- VAYGXNSJCAHWJZ-UHFFFAOYSA-N dimethyl sulfate Chemical compound COS(=O)(=O)OC VAYGXNSJCAHWJZ-UHFFFAOYSA-N 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 239000001226 triphosphate Substances 0.000 description 3
- 235000011178 triphosphate Nutrition 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical class OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 2
- 108010010677 Phosphodiesterase I Proteins 0.000 description 2
- 238000007069 methylation reaction Methods 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- OGHAROSJZRTIOK-KQYNXXCUSA-O 7-methylguanosine Chemical group C1=2N=C(N)NC(=O)C=2[N+](C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OGHAROSJZRTIOK-KQYNXXCUSA-O 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 208000012839 conversion disease Diseases 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- SMZORVKKJCIPCG-UHFFFAOYSA-N iodo(trimethyl)-$l^{4}-sulfane Chemical compound CS(C)(C)I SMZORVKKJCIPCG-UHFFFAOYSA-N 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- OJVWCPKLFCTIPB-UHFFFAOYSA-N n,n-dibutylbutan-1-amine;phosphoric acid Chemical compound OP(O)([O-])=O.CCCC[NH+](CCCC)CCCC OJVWCPKLFCTIPB-UHFFFAOYSA-N 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 125000004430 oxygen atom Chemical group O* 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- DQWPFSLDHJDLRL-UHFFFAOYSA-N triethyl phosphate Chemical compound CCOP(=O)(OCC)OCC DQWPFSLDHJDLRL-UHFFFAOYSA-N 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- 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
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Saccharide Compounds (AREA)
Abstract
The invention relates to the technical field of chemical synthesis, in particular to a cap analogue, a synthesis method thereof and mRNA. The synthesis method of the cap analogue comprises the following steps: s1, taking guanosine as a raw material, reacting the obtained product with imidazole to obtain an intermediate product after a methyl reaction and a phosphorylation reaction, and preparing the cap analogue by using the intermediate product. The stability of each intermediate is high in the synthesis process, the yield of the obtained cap analogue product is high, and the cap analogue is applied to mRNA, so that the stability of the mRNA is improved, and the transcription efficiency and capping rate are further improved.
Description
Technical Field
The invention relates to the technical field of chemical synthesis, in particular to a cap analogue, a synthesis method thereof and mRNA.
Background
"cap" structure (m 7G 5'] ppp 5' ] N, where N is any nucleotide) it is typically composed of a 7-methylguanosine moiety, linked to the nucleoside at position 5 of the RNA strand via a 5-5 triphosphate bridge. The unique cap structure plays a critical role in providing resistance to 5 '-exonuclease, which helps to protect mRNA from rapid degradation by 5' -exonuclease activity. Many studies are currently made on cap analogues, but intermediate products are not stable during the synthesis of cap analogues at present, so that the cap analogue yields obtained are low.
Disclosure of Invention
The present invention is directed to solving at least one of the technical problems existing in the related art. Therefore, the invention provides a synthesis method of cap analogues, which has strong stability of each intermediate in the synthesis process, and the obtained cap analogues have high yield.
In one aspect of the present invention, there is provided a method of synthesizing a cap analogue comprising the steps of:
s1, toIs used as raw material and synthesized by a methyl reaction>；
S2, byIs synthesized by phosphorylation reaction as raw material>；
S3, makingReacting with imidazole to obtain an intermediate product, reacting said intermediate product with +.>Reacting to obtain the cap analogue.
Further, step S1 includes:
diluting methyl iodide with N, N-dimethylformamide three times at 0-10deg.C, adding into the mixture for 0.5-1 hrAnd then carrying out a methyl reaction for 16-24h at 28-32 ℃ in a mixed system of N, N-dimethylformamide.
Further, the method comprises the steps of,the molar ratio of methyl iodide to methyl iodide is 1: (1.4 to 1.6).
Further, step S2 includes:
s21, adding methylene phosphorus dichloride to the mixture under the protection of inert gas at the temperature of-5 to 5 DEG CIn a mixed system of trimethyl phosphite and trimethyl phosphite, carrying out phosphorylation reaction for 1-3 h at room temperature;
s22, quenching the product obtained in the step S21 by using a saturated ammonium bicarbonate aqueous solution at the temperature of 0-10 ℃ until the PH of the system is 7, and keeping the temperature and continuously stirring for 0.5h.
Further, in step S21,the molar ratio of the phosphorus oxychloride to the methylene phosphorus dichloride is (1-3): (2-4);
the volume ratio of the molar quantity to trimethyl phosphite is 1mol: (1.5 to 2.5) L.
Further, step S3 includes:
s31, makeReacting with imidazole, the steps are as follows:
adding triethylamine, imidazole, triphenylphosphine and dipyridine disulfide under the protection of inert gasAnd stirring and reacting in a mixed system of N, N-dimethylformamide for 1-3 h.
Further, the method comprises the steps of,the molar ratio of imidazole to imidazole is 1: (2-4);
the mol ratio of triethylamine, imidazole, triphenylphosphine and dithiodipyridine is (2-4): (2-4): (1-3): (1-3).
Further, step S3 further includes:
s32: at the temperature of 8-12 ℃, adding into the product obtained in the step S31And zinc chloride, and then stirring at room temperature for reaction for 16-24 hours to obtain the cap analogue;
and zinc chloride in a molar ratio of 1: (4-6).
In another aspect of the invention, the invention provides a cap analogue synthesized using the synthesis method described previously.
In another aspect of the invention, the invention provides an mRNA comprising a cap analogue as described previously.
The above technical solutions in the embodiments of the present invention have at least one of the following technical effects:
in the synthesis method of the cap analogue, the initial raw material is nucleoside, the nucleoside is subjected to a methyl reaction, and then the product after the methyl reaction is subjected to a phosphorylation reaction, so that the methyl is carried out before the nucleoside is connected with phosphoric acid, the damage of materials caused by unstable structure of the diphosphate is avoided, the methyl can be more stable in the product, and the methyl cannot fall off during the phosphorylation reaction.
The invention is thatIn the product, the two phosphoric acid groups are connected by the C atom in the biphosphoric acid structure, so that the stability of the biphosphoric acid structure is improved, and the biphosphoric acid structure is more stable and not easy to damage in the subsequent synthesis and purification process. Further, in the final cap analogue product, the structure of the triphosphate bridge uses C atoms for connecting phosphate groups, and the cap analogue is applied to mRNA, so that the stability of the mRNA is improved, and the transcription efficiency and capping rate are further improved.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the invention, and other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the results of HPLC purification in step S1 of example 1 of the present invention.
FIG. 2 is a graph showing the results of HPLC purification at step S2 in example 1 of the present invention.
FIG. 3 is a graph showing the results of HPLC detection of the system after completion of the reaction in step S3 in example 1 of the present invention.
FIG. 4 is a graph showing the results of HPLC purification at step S4 in example 1 of the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions in the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The following examples are illustrative of the invention but are not intended to limit the scope of the invention.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the embodiments of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
In one aspect of the present invention, there is provided a method of synthesizing a cap analogue, the method comprising the steps of: s1, toIs used as raw material and synthesized by a methyl reaction>The method comprises the steps of carrying out a first treatment on the surface of the S2, in->Is synthesized by phosphorylation reaction as raw material>The method comprises the steps of carrying out a first treatment on the surface of the S3, make->Reacting with imidazole to obtain an intermediate product, reacting said intermediate product withReacting to obtain the cap analogue.
It will be appreciated that the structural formula of the cap analogue of the present invention comprises。
In the synthesis method of the cap analogue, the initial raw material is nucleoside, the nucleoside is subjected to a methyl reaction, and then the product after the methyl reaction is subjected to a phosphorylation reaction, methyl is carried out before the nucleoside is connected with phosphoric acid, the nucleoside is purified, the damage of materials caused by unstable structure of the diphosphate is avoided, the methyl can be more stable in the product, and the methyl cannot fall off during the phosphorylation reaction.
Compared with the prior art that nucleoside is subjected to phosphorylation reaction and then the product of the phosphorylation reaction is subjected to methylation reaction, the synthesis process has more methylation reaction sites, and the reaction system has more impurities compared with the phosphorylation reaction, so that higher time and material waste are caused. The inventor of the invention surprisingly found that by adjusting the sequence of the methyl reaction and the phosphorylation reaction, the methyl reaction is carried out on the nucleoside first, and then the phosphorylation reaction is carried out on the product after the methyl reaction, the stability of the product can be effectively improved, and the waste of time and materials can be reduced.
The invention is thatIn the product, the structure of the biphosphoric acid uses C atoms to connect two phosphoric acid groups, compared with the traditional structure of connecting two phosphoric acid groups by O atoms, the inventionThe product increases the stability of the structure of the diphosphate, so that the diphosphate is more stable and not easy to damage in the subsequent synthesis and purification process. Further, in the final cap analogue product, the structure of the triphosphate bridge uses C atoms for connecting phosphate groups, and the cap analogue is applied to mRNA, so that the stability of the mRNA is improved, and the transcription efficiency and capping rate are further improved.
In some embodiments of the invention, step S1 comprises: diluting methyl iodide with N, N-dimethylformamide three times at 0-10deg.C, and adding into the mixture for 0.5-1 hrAnd then carrying out a methyl reaction for 16-24h at 15-30 ℃ in a mixed system of N, N-dimethylformamide.
The inventors have unexpectedly found that the use of methyl iodide for the methylol reaction is better than dimethyl sulfate and trimethyl sulphur iodide.
In some embodiments of the present invention,the molar ratio of methyl iodide to methyl iodide is 1: (1.4 to 1.6), for example, 1:1.4, 1:1.5, or 1:1.6. Relative to the above molar ratio range, whenWhen the molar ratio of the methyl iodide to the reaction substrate is less than 1:1.6, the methyl iodide is excessive to cause the rapid increase of the two methyl byproducts, so that the purity of the reaction system is reduced; when->When the molar ratio of the catalyst to methyl iodide is greater than 1:1.4, the reaction is carried out at the bottomExcess material remains, resulting in a decrease in purity of the reaction system.
In some embodiments of the invention, step S2 comprises: s21, adding methylene phosphorus dichloride to the mixture under the protection of inert gas at the temperature of-5 to 5 DEG CIn a mixed system of trimethyl phosphite and trimethyl phosphite, carrying out phosphorylation reaction for 1-3 h (for example, 1h, 2h or 3h and the like) at room temperature; s22, quenching the product obtained in the step S21 by using a saturated ammonium bicarbonate aqueous solution at the temperature of 0-10 ℃ until the pH value of the system is 7, and continuously stirring for 0.5h.
In some embodiments of the present invention, in step S21,the molar ratio of the phosphorus oxychloride to the methylene phosphorus dichloride is (1-3): (2-4), for example, may be 1:2, 1:3, 1:4, 2:2, 2:3, 2:4, 3:2, 3:3, or 3:4, etc.;the volume ratio of the molar quantity to trimethyl phosphite is 1mol: (1.5 to 2.5) L, for example, may be 1mol:1.5L, 1mol:2L or 1mol:2.5L, etc.
In some embodiments of the present invention, step S3 includes: s31, makeReacting with imidazole, the steps are as follows: adding triethylamine, imidazole, triphenylphosphine and dipyridine disulfide under the protection of inert gasIn the mixed system of N, N-dimethylformamide, stirring reaction is carried out for 1-3 h (for example, 1h, 2h or 3h, etc.).
In some embodiments of the present invention,the molar ratio of imidazole to imidazole is 1: (2-4), for example, may be 1:2, 1:3 or 1:4, etc.; the mol ratio of triethylamine, imidazole, triphenylphosphine and dithiodipyridine is (2-4): (2-4): (1-3): (1-3), for example, may be 3:3:2:2, 2:2:1:1, or 4:4:3:3, etc.
In some embodiments of the present invention, step S3 further comprises: s32: adding the catalyst to the product obtained in the step S31 under the condition of 8-12 ℃ (for example, the catalyst can be at 8 ℃, 10 ℃ or 12 ℃), and the likeAnd zinc chloride, and then stirring at room temperature for reaction for 16-24h (for example, 16h, 18h, 20h, 22h or 24h, etc.), thereby obtaining the cap analogue;and zinc chloride in a molar ratio of 1: (4-6), for example, may be 1:4, 1:5, or 1:6.
In some embodiments of the invention, the synthetic route and specific synthetic steps for the cap analogs are as follows:
s1, adding 100mL of DMF (N, N-dimethylformamide) into a 500mL reaction bottle, adding 15 mmole of M1 under stirring, cooling the system to 6 ℃, slowly adding 22.5 mmole of methyl iodide, heating the system obviously, reacting the system at 30 ℃ for 20 hours after the addition, displaying yellow clear state of the system, monitoring the sample feeding mass spectrum of the system, and carrying out HPLC purification after the reaction is finished.
S2, 100mL of trimethyl phosphite is added into a 500mL reaction bottle, 50mmol of M2 is added under stirring, nitrogen is exchanged for the system, 75mmol of methylene phosphorus dichloride is added at 0 ℃, the temperature of the system is obviously raised, and the system is stirred for 2 hours at room temperature after the addition is finished. Sampling and sending to liquid phase detection reaction to show that the reaction is completed. Post-treating the reaction product: quenching reaction is carried out on the reaction product by using saturated ammonium bicarbonate water solution at the temperature of 0 ℃ until the PH of the system is 7, stirring is carried out for 0.5h after the addition, and HPLC purification is carried out on the reaction system.
S3, adding 20mL of DMF (dimethyl formamide) into a 100mL reaction bottle, adding 10 mmole of M3 under stirring, changing nitrogen into a system, adding 30 mmole of triethylamine, 30 mmole of imidazole, 20 mmole of triphenylphosphine and 20 mmole of dithiodipyridine, reacting for 2h, sampling the system after the reaction is finished, carrying out liquid phase detection reaction, and directly carrying out the next reaction continuous casting on the system until the reaction is finished.
S4, cooling the system in the reaction bottle in the step S3 to 10 ℃, adding 20 mmoles of M5 and 100 mmoles of zinc chloride into the system, stirring at room temperature for reaction for 16-24 hours, sampling, carrying out liquid phase detection reaction, and purifying the system by HPLC after the reaction is finished, thus obtaining the cap analogue product.
In the description herein, room temperature refers to a temperature of 15 to 30 ℃.
In another aspect of the invention, the invention provides a cap analogue synthesized using the synthesis method described previously.
It should be noted that the cap analogue is identical to the previous description and will not be described in detail here.
In another aspect of the invention, the invention provides an mRNA comprising a cap analogue as described above.
It will be appreciated that in the mRNA of the present invention, the cap analogue of the present invention is attached at the 5' end.
The invention is further illustrated below with reference to specific examples, which are given solely for the purpose of illustration and are not to be construed as limiting the invention.
Examples
Example 1
The synthesis method of the cap analogue comprises the following steps:
s1, adding 100mL of DMF (N, N-dimethylformamide) into a 500mL reaction flask, and adding 15mmol under stirringThe system is white and slightly turbid, the system is cooled to 0-10 ℃, 22.5mmol of methyl iodide is slowly added, the temperature of the system is obviously raised, the system reacts for 20 hours at 30 ℃ after the addition, the system is yellow and clear, the system sends a sample to detect HPLC (high performance liquid chromatography) 66.53%, and H is carried out after the reaction is finishedThe purification by PLC (high performance liquid chromatography) is shown in FIG. 1, wherein the purification result is 95.44% by HPLC and 58.97% by Yield by YIeld.
S2, adding 100mL of trimethyl phosphite into a 500mL reaction bottle, adding 50mmol of the product obtained in the step S1 under stirring, replacing nitrogen with the system, adding 75mmol of methylene phosphorus dichloride at 0 ℃, heating the system obviously, and stirring for 2 hours at room temperature after the addition is finished. The sample was taken for liquid phase detection reaction and HPLC 93.76% indicated the reaction was complete. Post-treating the reaction product: the reaction product is quenched by saturated ammonium bicarbonate water solution at 0 ℃ until the pH value of the system is 7, stirring is carried out for 0.5h after the addition, and the reaction system is subjected to HPLC purification, wherein the purification result is shown in figure 2, HPLC is 96.32%, and YIeld is 76.48%.
S3, adding 20mL of DMF into a 100mL reaction bottle, adding 10mmol of the product obtained in the step S2 under stirring, changing nitrogen into the system, adding 30mmol of triethylamine, 30mmol of imidazole, 20mmol of triphenylphosphine and 20mmol of dithiodipyridine, reacting for 2h, sampling the system after the reaction is finished, carrying out liquid phase detection reaction, wherein the detection result is shown in figure 3, HPLC is 92.26%, showing that the reaction is finished, and directly carrying out the next reaction continuous casting.
S4, cooling the system in the reaction bottle in the step S3 to 10 ℃, and adding 20mmol into the systemAnd 100mmol zinc chloride, stirring at room temperature for reaction for 20h, sampling and sending to liquid phase detection reaction, wherein HPLC is 59.80% after reaction, and purifying by HPLC to obtain cap analogue product, the result is shown in FIG. 4, HPLC is 98.56%, and YIeld is 40.22%.
Comparative example 1
The synthesis method of the cap analogue is basically the same as example 1, except for the following steps:
s1, 500mL reaction flask was charged with 100mL trimethyl phosphite and 50mmol was added under stirringAdding 75mmol of methylene phosphorus dichloride at 0 ℃ after nitrogen exchange, stirring for 2 hours at room temperature, and reacting with HPLC 90.32%And (3) carrying out post-treatment on the product: quenching reaction is carried out on the reaction product by using saturated ammonium bicarbonate water solution at the temperature of 0 ℃ until the PH of the system is 7, stirring is carried out for 0.5h after the addition, and HPLC purification is carried out on the reaction system. 97.73 percent of HPLC and 81.32 percent of YIeld.
S2, adding 100mL of DMF (N, N-dimethylformamide) into a 500mL reaction bottle, adding 15mmol of the product obtained in the step S1 under stirring, cooling the system to 6 ℃, slowly adding 22.5mmol of methyl iodide, reacting the system at 30 ℃ for 20h after the addition, monitoring the sample feeding of the system, and performing HPLC purification after the reaction is completed, wherein the HPLC is 32.13%. 94.11% of HPLC and 20.03% of YIeld.
Step S3 and step S4 are the same as in example 1.
The effect of the comparative example is that the impurity of the reaction system is increased by firstly adding phosphoric acid and then adding methyl, the product is damaged, and the total yield of the two steps is reduced from 45.10% to 16.29%.
Comparative example 2
The synthesis method of the cap analogue is basically the same as that of example 1, except that step S2 is modified as follows:
100mL of triethyl phosphate is added into a 500mL reaction bottle, 15mmol of the product obtained in the step S1 is added under stirring, the system is clarified, the temperature is reduced to 0-5 ℃, 30mmol of phosphorus oxychloride is added into the system, after stirring for 4 hours, the reaction is detected to be finished, 89.43 percent of HPLC is carried out, 50mL of water is added for quenching, and HPLC purification is carried out, wherein 96.19 percent of HPLC and 80.11 percent of YIeld are carried out
100mL of DMF (N, N-dimethylformamide) is added into a 500mL reaction bottle, 10mmol of the product obtained in the step S21 is added under stirring, the nitrogen is replaced by the system, 30mmol of triethylamine, 30mmol of imidazole, 20mmol of triphenylphosphine and 20mmol of dithiodipyridine are added for reaction for 2h, and the system after the reaction is finished is sampled and sent to a liquid phase detection reaction, which shows that the reaction is completed with HPLC 80.40%, and the system is directly subjected to the next reaction continuous casting.
100mL of DMF (N, N-dimethylformamide) is added into a 500mL reaction bottle, 10mmol of the product obtained in the step S22 is added under stirring, nitrogen is replaced by a system, 30mmol of tributylamine phosphate is added, the catalytic amount of zinc chloride is added, the reaction is stirred for 16-24h, the reaction HPLC is detected to be 68.32%, 50mL of water is added after the reaction is finished, and HPLC purification is performed to obtain 94.11% and 43.52% of YIeld.
Step S3 and step S4 are the same as in example 1.
The cap analogues obtained in this comparative example have the structural formula:
comparative example 3
The synthesis method of the cap analogue is basically the same as that of example 1, except for step S1
S1, adding 100mL of DMF (N, N-dimethylformamide) into a 500mL reaction flask, and adding 50mmol under stirringThe system is white and slightly turbid, the system is cooled to 0-10 ℃, 100mmol of dimethyl sulfate is slowly added, the system is heated, the system reacts for 20 hours at 30 ℃ after the addition, the system is yellow and clear, the system is sent to monitor HPLC (high performance liquid chromatography) and the reaction is completed.
The effect of this comparative example is: methyl raw material is remained on dimethyl sulfate and system impurities are more.
Comparative example 4
The synthesis method of the cap analogue is basically the same as that of example 1, except for step S2:
s2, adding 100mL of trimethyl phosphite into a 500mL reaction bottle, adding 50mmol of the product obtained in the step S1 under stirring, replacing nitrogen with the system, adding 75mmol of methylene phosphorus dichloride at 0 ℃, obviously heating the system, and stirring for 2 hours at the temperature of 0-5 ℃ after the addition is finished. Sampling and sending to liquid phase detection reaction, wherein HPLC (high performance liquid chromatography) 52.61% shows that the residual reaction raw materials are more than or equal to 40%, and the reaction time is prolonged for 2 hours; sampling again and sending to liquid phase detection reaction, HPLC (high performance liquid chromatography) 71.39% shows that the residual reaction raw materials are more than or equal to 15%, and carrying out post-treatment on reaction products: quenching reaction is carried out on the reaction product by using saturated ammonium bicarbonate water solution at the temperature of 0 ℃ until the PH of the system is 7, stirring is carried out for 0.5h after the addition, and HPLC purification is carried out on the reaction system. HPLC 95.10% and YIeld 60.38%.
The effect of this comparative example is: when the temperature control reaction is carried out, the low temperature can lead to slow reaction, and excessive raw materials remain, thereby influencing the reaction conversion rate and further reducing the reaction yield; thus, the reaction effect at room temperature (15-30 ℃ C.) is better.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (8)
1. A method of synthesizing a cap analogue, comprising the steps of:
s1, toIs used as raw material and synthesized by a methyl reaction>；
S2, byIs synthesized by phosphorylation reaction as raw material；
S3, makingWith imidazole to obtain intermediate productThe intermediate product and +.>Reacting to obtain the cap analogue +.>。
2. The method of synthesizing a cap analogue according to claim 1, wherein step S1 comprises:
diluting methyl iodide with N, N-dimethylformamide three times at 0-10deg.C, and adding into the mixture for 0.5-1 hrAnd then carrying out a methyl reaction for 16-24h at 15-30 ℃ in a mixed system of N, N-dimethylformamide.
3. The method for synthesizing a cap analogue according to claim 2, wherein,the molar ratio of methyl iodide to methyl iodide is 1:1.4 to 1.6.
4. The method of synthesizing a cap analogue according to claim 1, wherein step S2 comprises:
s21, adding methylene phosphorus dichloride to the mixture under the protection of inert gas at the temperature of-5 to 5 DEG CIn a mixed system of trimethyl phosphite and trimethyl phosphite, carrying out phosphorylation reaction for 1-3 h at room temperature;
s22, quenching the product obtained in the step S21 by using a saturated ammonium bicarbonate aqueous solution at the temperature of 0-10 ℃ until the PH of the system is 7, and keeping the temperature and continuously stirring for 0.5h.
5. The method for synthesizing a cap analogue according to claim 4, wherein in step S21,molar ratio to methylene phosphorus dichloride1-3: 2-4;
the volume ratio of the molar quantity to trimethyl phosphite is 1mol: 1.5-2.5L.
6. The method of synthesizing a cap analogue according to claim 1, wherein step S3 comprises:
s31, makeReacting with imidazole, the steps are as follows:
adding triethylamine, imidazole, triphenylphosphine and dipyridine disulfide under the protection of inert gasAnd stirring and reacting in a mixed system of N, N-dimethylformamide for 1-3 h.
7. The method for synthesizing a cap analogue according to claim 6, wherein,the molar ratio of imidazole to imidazole is 1: 2-4;
the molar ratio of triethylamine, imidazole, triphenylphosphine and dithiodipyridine is 2-4: 2-4: 1-3: 1-3.
8. The method of synthesizing a cap analogue according to claim 6 or 7, wherein step S3 further comprises:
s32: at the temperature of 8-12 ℃, adding into the product obtained in the step S31And zinc chloride, and then stirring at room temperature for reaction for 16-24 hours to obtain the cap analogue;
and zinc chloride in a molar ratio of 1: 4-6.
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