CN117587086A - Method for preparing N1-methyl pseudouridine triphosphate - Google Patents
Method for preparing N1-methyl pseudouridine triphosphate Download PDFInfo
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- CN117587086A CN117587086A CN202410064420.XA CN202410064420A CN117587086A CN 117587086 A CN117587086 A CN 117587086A CN 202410064420 A CN202410064420 A CN 202410064420A CN 117587086 A CN117587086 A CN 117587086A
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- methyl pseudouridine
- kinase
- triphosphate
- enzyme
- methyl
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- UVBYMVOUBXYSFV-XUTVFYLZSA-N 1-methylpseudouridine Chemical compound O=C1NC(=O)N(C)C=C1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 UVBYMVOUBXYSFV-XUTVFYLZSA-N 0.000 title claims abstract description 73
- 239000001226 triphosphate Substances 0.000 title claims abstract description 47
- 235000011178 triphosphate Nutrition 0.000 title claims abstract description 47
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 26
- 238000006243 chemical reaction Methods 0.000 claims abstract description 65
- 102000004190 Enzymes Human genes 0.000 claims abstract description 38
- 108090000790 Enzymes Proteins 0.000 claims abstract description 38
- 238000006555 catalytic reaction Methods 0.000 claims abstract description 33
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 claims abstract description 27
- LIPOUNRJVLNBCD-UHFFFAOYSA-N acetyl dihydrogen phosphate Chemical compound CC(=O)OP(O)(O)=O LIPOUNRJVLNBCD-UHFFFAOYSA-N 0.000 claims abstract description 19
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 claims abstract description 18
- 229910019142 PO4 Inorganic materials 0.000 claims abstract description 18
- 239000010452 phosphate Substances 0.000 claims abstract description 18
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims abstract description 16
- 239000000758 substrate Substances 0.000 claims abstract description 15
- 238000004519 manufacturing process Methods 0.000 claims abstract description 13
- 238000005580 one pot reaction Methods 0.000 claims abstract description 8
- 239000003054 catalyst Substances 0.000 claims abstract description 6
- 239000012467 final product Substances 0.000 claims abstract description 3
- 101710148009 Putative uracil phosphoribosyltransferase Proteins 0.000 claims description 28
- 102000007410 Uridine kinase Human genes 0.000 claims description 28
- 108010092060 Acetate kinase Proteins 0.000 claims description 26
- 108091000080 Phosphotransferase Proteins 0.000 claims description 21
- 102000020233 phosphotransferase Human genes 0.000 claims description 20
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 16
- DJJCXFVJDGTHFX-UHFFFAOYSA-N Uridinemonophosphate Natural products OC1C(O)C(COP(O)(O)=O)OC1N1C(=O)NC(=O)C=C1 DJJCXFVJDGTHFX-UHFFFAOYSA-N 0.000 claims description 15
- FOGRQMPFHUHIGU-UHFFFAOYSA-N Uridylic acid Natural products OC1C(OP(O)(O)=O)C(CO)OC1N1C(=O)NC(=O)C=C1 FOGRQMPFHUHIGU-UHFFFAOYSA-N 0.000 claims description 15
- 229960002363 thiamine pyrophosphate Drugs 0.000 claims description 15
- 235000008170 thiamine pyrophosphate Nutrition 0.000 claims description 15
- 239000011678 thiamine pyrophosphate Substances 0.000 claims description 15
- YXVCLPJQTZXJLH-UHFFFAOYSA-N thiamine(1+) diphosphate chloride Chemical compound [Cl-].CC1=C(CCOP(O)(=O)OP(O)(O)=O)SC=[N+]1CC1=CN=C(C)N=C1N YXVCLPJQTZXJLH-UHFFFAOYSA-N 0.000 claims description 15
- DJJCXFVJDGTHFX-XVFCMESISA-N uridine 5'-monophosphate Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1 DJJCXFVJDGTHFX-XVFCMESISA-N 0.000 claims description 15
- 159000000003 magnesium salts Chemical class 0.000 claims description 13
- 239000005515 coenzyme Substances 0.000 claims description 8
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 8
- 239000003381 stabilizer Substances 0.000 claims description 8
- 230000035484 reaction time Effects 0.000 claims description 7
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- 239000001177 diphosphate Substances 0.000 claims description 6
- YIXJRHPUWRPCBB-UHFFFAOYSA-N magnesium nitrate Chemical compound [Mg+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O YIXJRHPUWRPCBB-UHFFFAOYSA-N 0.000 claims description 6
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 claims description 4
- 235000011180 diphosphates Nutrition 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 3
- 239000008363 phosphate buffer Substances 0.000 claims description 2
- MOBMOJGXNHLLIR-GBNDHIKLSA-N pseudouridine 5'-phosphate Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(O)=O)O[C@H]1C1=CNC(=O)NC1=O MOBMOJGXNHLLIR-GBNDHIKLSA-N 0.000 claims description 2
- 238000010276 construction Methods 0.000 abstract description 6
- 230000009286 beneficial effect Effects 0.000 abstract description 4
- 239000002777 nucleoside Substances 0.000 abstract description 4
- 230000007613 environmental effect Effects 0.000 abstract description 3
- 150000003833 nucleoside derivatives Chemical class 0.000 abstract description 3
- 238000010170 biological method Methods 0.000 abstract description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 abstract 2
- AFINAILKDBCXMX-PBHICJAKSA-N (2s,3r)-2-amino-3-hydroxy-n-(4-octylphenyl)butanamide Chemical compound CCCCCCCCC1=CC=C(NC(=O)[C@@H](N)[C@@H](C)O)C=C1 AFINAILKDBCXMX-PBHICJAKSA-N 0.000 abstract 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 abstract 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 abstract 1
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 24
- 239000000243 solution Substances 0.000 description 19
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- 108020000553 UMP kinase Proteins 0.000 description 13
- 239000000843 powder Substances 0.000 description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 239000012071 phase Substances 0.000 description 12
- 102100029150 Uridine-cytidine kinase 2 Human genes 0.000 description 11
- 108020004999 messenger RNA Proteins 0.000 description 11
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- 102100036409 Activated CDC42 kinase 1 Human genes 0.000 description 10
- 101000928956 Homo sapiens Activated CDC42 kinase 1 Proteins 0.000 description 10
- 230000001580 bacterial effect Effects 0.000 description 10
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- 108700021021 mRNA Vaccine Proteins 0.000 description 9
- 229940126582 mRNA vaccine Drugs 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 229930185560 Pseudouridine Natural products 0.000 description 8
- PTJWIQPHWPFNBW-UHFFFAOYSA-N Pseudouridine C Natural products OC1C(O)C(CO)OC1C1=CNC(=O)NC1=O PTJWIQPHWPFNBW-UHFFFAOYSA-N 0.000 description 8
- 101710166980 Uridylate kinase Proteins 0.000 description 8
- WGDUUQDYDIIBKT-UHFFFAOYSA-N beta-Pseudouridine Natural products OC1OC(CN2C=CC(=O)NC2=O)C(O)C1O WGDUUQDYDIIBKT-UHFFFAOYSA-N 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- PTJWIQPHWPFNBW-GBNDHIKLSA-N pseudouridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1C1=CNC(=O)NC1=O PTJWIQPHWPFNBW-GBNDHIKLSA-N 0.000 description 8
- 239000003643 water by type Substances 0.000 description 8
- 230000003197 catalytic effect Effects 0.000 description 7
- 230000005847 immunogenicity Effects 0.000 description 7
- 238000010587 phase diagram Methods 0.000 description 7
- 241000588724 Escherichia coli Species 0.000 description 6
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 6
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 238000004108 freeze drying Methods 0.000 description 5
- 239000008055 phosphate buffer solution Substances 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- XTWYTFMLZFPYCI-KQYNXXCUSA-N 5'-adenylphosphoric acid Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XTWYTFMLZFPYCI-KQYNXXCUSA-N 0.000 description 4
- XTWYTFMLZFPYCI-UHFFFAOYSA-N Adenosine diphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(O)=O)C(O)C1O XTWYTFMLZFPYCI-UHFFFAOYSA-N 0.000 description 4
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- 208000012839 conversion disease Diseases 0.000 description 4
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- 239000002609 medium Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 3
- 241000204652 Thermotoga Species 0.000 description 3
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 3
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- 238000011161 development Methods 0.000 description 3
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- 230000002779 inactivation Effects 0.000 description 3
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 3
- 108060006633 protein kinase Proteins 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 229940035893 uracil Drugs 0.000 description 3
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 3
- 229940045145 uridine Drugs 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 229940125606 CureVac COVID-19 vaccine Drugs 0.000 description 2
- 239000006142 Luria-Bertani Agar Substances 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 2
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000003125 aqueous solvent Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
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- CVXBEEMKQHEXEN-UHFFFAOYSA-N carbaryl Chemical compound C1=CC=C2C(OC(=O)NC)=CC=CC2=C1 CVXBEEMKQHEXEN-UHFFFAOYSA-N 0.000 description 2
- 229960005286 carbaryl Drugs 0.000 description 2
- PHIQHXFUZVPYII-UHFFFAOYSA-N carnitine Chemical compound C[N+](C)(C)CC(O)CC([O-])=O PHIQHXFUZVPYII-UHFFFAOYSA-N 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
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- 125000003835 nucleoside group Chemical group 0.000 description 2
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- 108700022172 2019-nCoV Vaccine mRNA-1273 Proteins 0.000 description 1
- ACNXXUCYLYKAPB-UHFFFAOYSA-N 5-bromo-6-pyrrolidin-1-yl-1h-pyrimidine-2,4-dione Chemical compound OC1=NC(O)=C(Br)C(N2CCCC2)=N1 ACNXXUCYLYKAPB-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
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- 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 description 1
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- 206010061218 Inflammation Diseases 0.000 description 1
- 229940026207 Moderna COVID-19 vaccine Drugs 0.000 description 1
- 229940026233 Pfizer-BioNTech COVID-19 vaccine Drugs 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- OLRONOIBERDKRE-XUTVFYLZSA-N [[(2r,3s,4r,5s)-3,4-dihydroxy-5-(1-methyl-2,4-dioxopyrimidin-5-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound O=C1NC(=O)N(C)C=C1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 OLRONOIBERDKRE-XUTVFYLZSA-N 0.000 description 1
- PGAVKCOVUIYSFO-UHFFFAOYSA-N [[5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound OC1C(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)OC1N1C(=O)NC(=O)C=C1 PGAVKCOVUIYSFO-UHFFFAOYSA-N 0.000 description 1
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- 239000000411 inducer Substances 0.000 description 1
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- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000037360 nucleotide metabolism Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- HWGNBUXHKFFFIH-UHFFFAOYSA-I pentasodium;[oxido(phosphonatooxy)phosphoryl] phosphate Chemical compound [Na+].[Na+].[Na+].[Na+].[Na+].[O-]P([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O HWGNBUXHKFFFIH-UHFFFAOYSA-I 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
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Classifications
-
- 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
- C12P19/305—Pyrimidine nucleotides
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for preparing N1-methyl pseudouridine triphosphate, which belongs to the technical field of biochemistry, and the method provided by the invention is used for obtaining an enzyme catalyst through construction of recombinant strains, taking N1-methyl pseudouridine as a substrate, taking adenosine triphosphate as a phosphate donor, adding acetyl phosphate to provide a phosphate group, constructing phosphoric acid circulation, and carrying out enzyme catalytic reaction by adopting a one-pot method to obtain a final product N1-methyl pseudouridine triphosphate. The method provided by the invention has the advantages of high conversion rate of the substrate N1-methyl pseudouridine, low cost and easiness in obtaining, fills the blank of preparing the N1-methyl pseudouridine triphosphate by the biological method at present, provides a thought for preparing the nucleoside and the phosphoric acid derivative thereof by biological enzyme, adopts a one-pot enzyme catalysis method, has the advantages of simple operation, mild reaction condition, low cost, high yield, environmental friendliness and no pollution, and is beneficial to industrialized amplified production.
Description
Technical Field
The invention belongs to the technical field of biochemistry, and particularly relates to a method for preparing N1-methyl pseudouridine triphosphate.
Background
In the current field of vaccine development and production, mRNA vaccines are rapidly evolving. Compared with the traditional inactivated vaccine and attenuated vaccine, the mRNA vaccine has the advantages of short production period, higher safety and the like. More importantly, mRNA vaccines can be rapidly updated for iteration in development, and thus are superior in coping with ever-emerging viruses such as new coronavirus mutants.
However, the development of mRNA vaccines also faces some difficulties, one of the biggest challenges being the immunogenicity of mRNA. mRNA as a foreign substance, when entering the human body, activates the immune system of the human body, and thus causes an inflammatory reaction caused by the mRNA itself, and may cause the mRNA to be cleared by the immune system without functioning. The introduction of naturally modified nucleotides is one of the effective means to reduce the immunogenicity of mRNA vaccines.
Pseudouridine (Pseudouridine) is the most abundant modified nucleoside on RNA, also known as the "fifth nucleoside" of RNA. In 2005, katalin Karik et al found that the introduction of pseudouridine into RNA reduced its immunogenicity, and that the immunogenicity of RNA decreased with increasing proportion of pseudouridine introduced. In 2008, katalin Karik et al also found that complete replacement of uridine mRNA with pseudouridine not only greatly reduced mRNA immunogenicity, but also improved mRNA stability and enhanced translation ability. In 2015, oliwia Andries et al found that complete replacement of uridine with N1-methyl pseudouridine reduced mRNA immunogenicity and enhanced mRNA protein expression capacity more than complete replacement of uridine with pseudouridine.
These studies suggest that the introduction of pseudouridine or N1-methyl pseudouridine into mRNA may be effective in reducing the immunogenicity of mRNA vaccines, enhancing the stability of mRNA, and enhancing its protein expression ability.
Two new types of coronal mRNA vaccines have been marketed: mRNA-1273 (Moderna) and BNT162b2 (pyro-Biontech) each replaced Uridine Triphosphate (UTP) with N1-methyl pseudouridine triphosphate (N1-Met- φTP). While the new crown mRNA vaccine CVnCoV of the german vaccine CureVac was very low in potency, only 48%, this result has led to a broad proposition, presumably due to the lack of modified nucleosides for CVnCoV. It follows that the use of pseudouridine or N1-methyl pseudouridine substitution would be a trend towards mRNA vaccine production.
N1-methyl pseudouridine triphosphate (N1-Met- φTP) is difficult to obtain and expensive, such as N1-methyl pseudouridine triphosphate sodium salt of 100 mM, sold at a price of about 20 μL/935 yuan. Currently, N1-methyl pseudouridine triphosphate is generally synthesized chemically. The chemical synthesis method is a multi-step process, and in order to realize regioselectivity and stereoselectivity, the process of protecting and deprotecting the group is needed, the operation steps are complex, the cost is high, toxic and harmful substances are easy to generate, and the environment is polluted. The enzymatic synthesis is more and more paid attention to due to the factors of high regioselectivity, stereoselectivity, mild reaction conditions, green environmental protection, no pollution and the like, but the enzymatic synthesis method of the N1-methyl pseudouridine triphosphate is not yet seen at present, and meanwhile, the enzymatic synthesis method also has high substrate conversion rate so as to develop the application of the N1-methyl pseudouridine triphosphate in preparation and production.
Therefore, the field needs to develop a method for preparing N1-methyl pseudouridine triphosphate, which is simple and easy to operate, accords with the production principle of green chemistry, can ensure better substrate conversion rate while protecting ecological environment, and is beneficial to the large-scale production and application of N1-methyl pseudouridine triphosphate.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for preparing N1-methyl pseudouridine triphosphate, which takes N1-methyl pseudouridine as a substrate, adds an enzyme catalyst and a phosphate donor, constructs a phosphate cycle, adopts a one-pot method to carry out an enzyme catalytic reaction to obtain a final product N1-methyl pseudouridine triphosphate, and the enzyme catalytic reaction comprises the following steps:
the first stage: in a liquid reaction system, N1-methyl pseudouridine is used as a substrate, adenosine triphosphate and acetyl phosphate are used as phosphate donors, thiamine pyrophosphate is used as coenzyme, magnesium salt is used as a kinase stabilizer, and enzyme catalytic reaction is carried out under the catalysis of uridine kinase to generate N1-methyl pseudouridine;
and a second stage: n1-methyl pseudouridylic acid generated in the first stage is used as a substrate, adenosine triphosphate and acetyl phosphate are used as phosphate donors, thiamine pyrophosphate is used as coenzyme, magnesium salt is used as a kinase stabilizer, and enzyme catalysis reaction is carried out under the catalysis of uridylic acid kinase to generate N1-methyl pseudouridylic acid diphosphate;
and a third stage: taking N1-methyl pseudouridine diphosphate generated in the second stage as a substrate, taking adenosine triphosphate and acetyl phosphate as phosphate donors, taking thiamine pyrophosphate as coenzyme, taking magnesium salt as a kinase stabilizer, and performing enzyme catalytic reaction under the catalysis of acetate kinase to generate N1-methyl pseudouridine triphosphate;
wherein, the Genbank serial number of the uridine kinase is EGM3941553.1, the Genbank serial number of the uridine kinase is WP_000224577.1, and the Genbank serial number of the acetate kinase is AKE29949.1.
In a specific embodiment, the concentration of N1-methyl pseudouridine is 10-50 mmol/L.
Preferably, the concentration of N1-methyl pseudouridine is 38.7 mmol/L.
In a specific embodiment, the concentration of the adenosine triphosphate is 1-10 mmol/L.
Preferably, the concentration of the adenosine triphosphate is 2-7 mmol/L; more preferably, the concentration of adenosine triphosphate is 4 mmol/L.
In a specific embodiment, the concentration of the acetyl phosphate is 1-200 mmol/L.
Preferably, the concentration of the acetylphosphoric acid is 10-190 mmol/L; more preferably, the concentration of the acetylphosphoric acid is 20-180 mmol/L; most preferably, the concentration of the acetyl phosphate is 160 mmol/L.
In a specific embodiment, the thiamine pyrophosphate concentration is 0.1% -2% of the N1-methyl pseudouridine concentration.
Preferably, the thiamine pyrophosphate concentration is 1% of the N1-methylpseuduride concentration.
In a specific embodiment, the concentration of the magnesium salt is 1 mmol/L to 20 mmol/L, and the magnesium salt is any one or a combination of more of magnesium chloride, magnesium sulfate and magnesium nitrate.
Preferably, the concentration of the magnesium salt is 10mmol/L, and the magnesium salt is magnesium chloride.
In a specific embodiment, the buffer solution of the liquid reaction system is phosphate buffer solution with the concentration of 50 mmol/L.
In a specific embodiment, the temperature of the enzyme-catalyzed reaction is 30-40 ℃, the reaction time of the enzyme-catalyzed reaction is 2-28 h, and the pH of the enzyme-catalyzed reaction is 6.0-9.0.
Preferably, the temperature of the enzyme catalytic reaction is 30 ℃, the reaction time of the enzyme catalytic reaction is 4 hours, and the pH of the enzyme catalytic reaction is 7.0-8.0; more preferably, the pH of the enzyme-catalyzed reaction is 7.0.
In a specific embodiment, in the liquid reaction system, the uridine kinase, the uridylic acid kinase and the acetate kinase exist in the following forms: an enzyme in free form, an immobilized enzyme or an enzyme in bacterial form.
In another aspect of the present invention, there is provided a reaction system comprising an aqueous solvent, a substrate N1-methyl pseudouridine, an enzyme catalyst, adenosine triphosphate, acetyl phosphate, a coenzyme, and a kinase stabilizer;
the enzyme catalyst comprises uridine kinase, uridylate kinase and acetate kinase, wherein the Genbank serial number of the uridine kinase is EGM3941553.1, the Genbank serial number of the uridylate kinase is WP-000224577.1, and the Genbank serial number of the acetate kinase is AKE29949.1.
In a specific embodiment, the coenzyme is thiamine pyrophosphate and the kinase stabilizer is any one or a combination of more of magnesium chloride, magnesium sulfate and magnesium nitrate.
In one embodiment, the aqueous solvent is a buffer.
In a specific embodiment, the buffer is phosphate buffer at a concentration of 50 mmol/L.
The reaction system provided by the invention can carry out enzymatic reaction to prepare the N1-methyl pseudouridine triphosphate.
In the reaction system, the concentration of N1-methyl pseudouridine, the concentration of adenosine triphosphate, the concentration of acetylphosphoric acid, the concentration of thiamine pyrophosphate and the concentration of magnesium salt refer to the initial parameters and/or the feeding parameters of the reaction system.
Technical terminology
Uridine kinase UK is a pyrimidine ribonucleoside kinase, widely existing in microorganisms, animals and humans, and is an important catalyst in nucleotide metabolism compensation pathways. Uridine kinase UK can catalyze the phosphorylation of N1-methyl pseudouridine (N1-Met-phi) to N1-methyl pseudouridine (N1-Met-phi MP), but adenosine triphosphate ATP is required as a phosphate donor.
In the present application, the uridine kinase UK used is derived from E.coliEscherichia coliThrough gene recombination, expression in recombinant vector, cell disruption and purification, and freeze drying. Hereinafter referred to as ec uk, genbank accession number EGM3941553.1.
Uridylate kinase UMPK is a monomeric enzyme that is ubiquitous in organisms, and is widely found in microorganisms, plants and animals, and catalyzes the transfer of the gamma-phosphate of adenosine triphosphate ATP to N1-methyl pseudouridylate (N1-Met-phi MP) to form N1-methyl pseudouridylate-5' -diphosphate (N1-Met-phi DP).
In the present application, the uridylic acid kinase used is derived fromEscherichia albertiiThrough gene recombination, expression in recombinant vector, cell disruption and purification, and freeze drying. Hereinafter Eaumpk, genbank accession number WP_000224577.1.
Acetate kinase ACK is a member of the phosphotransferase superfamily responsible for catalyzing the reversible reaction of acetyl phosphate and adenosine diphosphate ADP to acetate and adenosine triphosphate ATP. In the present invention, gamma-phosphate transfer of adenosine triphosphate ATP to N1-methyl pseudouridine 5 '-diphosphate (N1-Met-phi DP) may also be catalyzed to form N1-methyl pseudouridine 5' -triphosphate (N1-Met-phi TP).
In the present application, usedIs derived from Thermotoga maritimaThermotoga maritimaThrough gene recombination, expression in recombinant vector, cell disruption and purification, and freeze drying. Hereinafter referred to as TmACK, which has Genbank accession number AKE29949.1.
In the reaction system of the present application, uric acid kinase, uridylic acid kinase and acetate kinase may be produced by constructing recombinant plasmids and recombinant engineering bacteria using the above-mentioned wet bacterial cells, crude enzyme solution, crude enzyme powder or pure enzyme, etc., and may be commercially available products, or by referring to the "molecular cloning laboratory Manual".
Through extensive and intensive studies, the inventors of the present application have unexpectedly developed an enzyme-catalyzed preparation method of N1-methyl pseudouridine triphosphate through a large number of screening experiments, in which uric acid kinase, uridylic acid kinase and acetate kinase exhibit high conversion rates to substrates of the reaction.
The beneficial effects of this application include:
1. the N1-methyl pseudouridine triphosphate is prepared by using the screened uridine kinase EcUK, uridylate kinase EaUMPK and acetate kinase TmACK and the preparation method, the substrate N1-methyl pseudouridine has high conversion rate, low cost and easy obtainment, fills the blank of preparing the N1-methyl pseudouridine triphosphate by the current biological method, and provides ideas for preparing nucleosides and phosphate derivatives thereof by biological enzymes.
2. The application adopts the one-pot enzyme catalysis method, has the advantages of simple operation, mild reaction condition, low cost, high yield, environmental protection and no pollution, and is beneficial to industrialized amplified production.
Drawings
FIG. 1 is a liquid phase diagram of a method for producing N1-methyl pseudouridylic acid (N1-Met- φMP) by screening uridine kinase from different sources in example 2, wherein the abscissa indicates time (min) and the ordinate indicates Absorbance (AU);
FIG. 2 is a liquid phase diagram of the preparation of N1-methyl pseudouridine triphosphate (N1-Met- φTP) using uridine kinase EcUK, uridylic acid kinase EauPK and acetate kinase TmACK in examples 2-5, wherein the abscissa indicates time (minutes) and the ordinate indicates Absorbance (AU);
FIG. 3 is a synthetic scheme for the preparation of N1-methyl pseudouridine triphosphate (N1-Met- φTP) using a one-pot enzymatic method in example 5.
Detailed Description
The following description of the present invention will be made clearly and fully, and it is apparent that the embodiments described are some, but not all, of the embodiments of the present invention. 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 experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor LaboratoryPress, 1989) or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. The experimental materials involved in the invention are all commercial reagents unless specified, and are available from commercial sources. In the examples below, IPTG was isopropyl- β -D-thiogalactoside as inducer. ATP is adenosine triphosphate. ADP is adenosine diphosphate.
Example 1: acquisition of enzyme powders of uridine kinase UK, uridylate kinase UMPK and acetate kinase Ack
1) Construction and transformation of recombinant vectors
Taking the construction of recombinant strain E.coli BL21 (DE 3) pET-28a-UK as an example:
the gene synthesis of uridine kinase UK genes from different sources is carried out by Shanghai Jin Wei intelligent bioengineering Co., ltd, and the UK genes are connected to a pET28a vector to obtain a recombinant vector pET28a-UK;
the uridine kinase UK genes of different origins are shown in table 1 below:
TABLE 1
Activating E.coli DH 5. Alpha./pET-28 a (+) strain (purchased from Novain Biotechnology Co., ltd.), adding 3-5. Mu.L of pET-28a-UK (50 ng/. Mu.L) to 50-100. Mu.L of E.coli BL21 (DE 3) competent cells (purchased from Novain Biotechnology Co., ltd.), placing on ice for 20 min, heat-shocking at 42℃for 90 sec, rapidly returning to ice bath for 5 min, adding 800. Mu.L of non-resistant LB culture solution, culturing at 37℃at 200 rpm, plating on a Carna-resistant LB agar plate medium, and culturing overnight at 37℃for 12 h to obtain recombinant E.coli BL21 (DE 3) containing pET-28 a-UK.
The formula of the non-resistance LB culture solution is as follows: yeast powder 5 g/L, sodium chloride 10 g/L, peptone 10 g/L;
the formula of the LB agar plate medium containing the carbaryl resistance is as follows: yeast powder 5 g/L, sodium chloride 10 g/L, peptone 10 g/L, agar powder 20 g/L, and cascara 25. Mu.g/mL.
The construction and transformation of the recombinant vector of uridylic acid kinase UMPK and acetate kinase ACK are the same as those of the recombinant vector of uridylic acid kinase UK.
Among them, the uridylic acid kinase UMPK genes of different sources are shown in Table 2:
TABLE 2
The acetate kinase ACK genes from different sources are shown in table 3:
TABLE 3 Table 3
2) Induction expression of expression vectors
The recombinant E.coli BL21 (DE 3) containing pET-28a-UK obtained in 1) was directly plated on a solid LB plate containing 25. Mu.g/mL of the carbaryl resistance, and cultured at 37℃for 12-14 h to obtain a monoclonal colony. Single colonies of E.coli BL21 (DE 3) containing the UK recombinant vector were picked from the Carna plates and inoculated into 1 mL liquid LB medium containing 25. Mu.g/mL Carna resistance and shake cultured at 37℃for 12 h. Then inoculating into fresh 1L liquid LB medium containing 25 mug/mL of Canada resistance according to 2% (v/v) inoculum size, culturing at 37 ℃ until OD600 is about 0.6-0.8, adding IPTG (purchased from Shanghai Ala Biochemical technology Co., ltd.) to a final concentration of 1.0 mmol/L,200 rpm, carrying out induced expression at 25 ℃ for 20 h, centrifuging (4 ℃,4000 rpm,30 min), removing supernatant, washing with 30mL of 0.9% NaCl solution, and resuspending to obtain bacterial liquid containing UK target protein.
In the present invention, the recombinant vectors of uridylic acid kinase UMPK and acetate kinase ACK are expressed in the same manner as the expression vector of uridylic acid kinase UK. By adopting the same method, bacterial solutions containing UK target proteins, bacterial solutions containing UMPK target proteins and bacterial solutions containing ACK target proteins can be obtained.
3) Preparation of lyophilized enzyme powder
In the invention, the bacterial liquid containing the UK target protein, the bacterial liquid containing the UMPK target protein and the bacterial liquid containing the ACK target protein are prepared by freeze-drying enzyme powder in the following modes.
Firstly, bacterial liquid is collected, after washing twice with 50mmol/L Tris-HCl, bacterial body is suspended in 50mmol/L Tris-HCl (pH8.0) buffer solution, cells are broken by ultrasound in ice bath (amplitude transformer 6, power 500W, open for 2s, close for 5s,30 minutes, a sample after ultrasonic breaking is centrifuged for 30 minutes at 12000 rpm and 4 ℃, after that, supernatant is taken and put into a freeze dryer at-80 ℃ for freeze-drying for 24 hours, and the obtained freeze-dried sample is ground to obtain freeze-dried crude enzyme powder.
Example 2: screening of uridine kinase UK of different origins
The freeze-dried enzyme powders of uridine kinase UK from different sources obtained in example 1 were subjected to catalytic reaction, 2 mL phosphate buffer solution (phosphate concentration: 50mmol/L,20 mg N1-methyl pseudouridine (N1-Met-phi)), 4.2 mg ATP,14 mg uracil, 2mg magnesium chloride, 0.2 mg thiamine pyrophosphate and 22 mg acetyl phosphate were added to a reaction flask (8 mL), and the pH of the reaction solution was controlled to 7.0 with 5M NaOH; 2mg of uridine kinase from different sources is added to start the reaction; controlling the temperature of the reaction liquid at 30 ℃ and the reaction rotating speed at 1000rpm; after the reaction time of 4 hours, 100. Mu.l of the reaction mixture was taken, inactivated by adding 50% methanol solution, and then centrifugally filtered, and the reaction conversion of the product N1-methyl pseudouridylic acid (N1-Met-. Phi.MP) was measured by HPLC.
Wherein, the conversion rate of N1-methyl pseudouridylic acid (N1-Met-phi MP) measured by liquid chromatography is calculated by the ratio of the detected product peak Area:
conversion of N1-methyl pseudouridylic acid (N1-Met- φMP) conv N1-Met- φMP% = Area [ N1-Met- φMP/(N1-Met- φMP+N 1-Met- φMP) ]. 100%.
Wherein, high Performance Liquid Chromatograph (HPLC) was purchased from agilent technologies, inc, HPLC detection conditions: the mobile phase was 10mM NH 4 OAc mobile phase a:5% acetonitrile, mobile phase B:95% acetonitrile, temperature 40 ℃, flow rate 1.3 mL/min, column Xbridge C18 (available from Waters/Waters, U.S.A.).
The conversion of uridine kinase UK from each source was as follows in table 4:
TABLE 4 Table 4
The results show that the method has the advantages of,Escherichia colithe catalytic conversion rate of the source uridine kinase UK is higher, the conversion rate is 89.5%, and the catalytic production of N1-methyl pseudouridylic acid (N1-Met-phi MP) is shown in the figure 1. The highest conversion rate of N1-methyl pseudouridylic acid (N1-Met-phi MP) is 89.5 percent through liquid phase detection.
Example 3: screening for uridylic acid kinase UMPK of different sources
The preparation of uridylate kinase UMPK vector construction expression and freeze-dried crude enzyme powder was carried out in the same manner as in example 1, the uridylate kinase UMPK enzyme powder of different sources obtained in example 1 was subjected to catalytic reaction, 2 mL phosphate buffer solution (phosphate concentration: 50mmol/L,20 mg N1-methyl pseudouridylate, 4.2 mg ATP,14 mg uracil, 2mg magnesium chloride, 0.2 mg thiamine pyrophosphate and 22 mg acetyl phosphate were added to a reaction flask (8 mL), and the pH of the reaction solution was controlled to 7.0 with 5M NaOH; 2mg of uridylic acid kinase of different origin was added,Thermotoga maritima1mg of acetate kinase of origin,Escherichia coli2mg of uridine kinase from source, starting the reaction; the temperature of the reaction solution is controlled at 30 ℃,the reaction rotating speed is controlled at 1000rpm; after the reaction time of 4 hours, 100. Mu.L of the reaction solution was taken, and after the inactivation by adding 50% methanol solution, the reaction was centrifugally filtered, and the reaction conversion of the product N1-methyl pseudouridine triphosphate (N1-Met-phi. TP) was measured by high performance liquid chromatography HPLC.
Wherein, the conversion rate of N1-methyl pseudouridine triphosphate (N1-Met-phi TP) measured by liquid chromatography is calculated by the ratio of the detected product peak Area:
conversion of N1-methyl pseudouridine triphosphate (N1-Met-phitp) conv N1-Met-phitp% = Area [ N1-Met-phitp/(N1-Met-phimp+n 1-Met-phidp+n 1-Met-phitp) ].
Wherein, high Performance Liquid Chromatograph (HPLC) was purchased from agilent technologies, inc, HPLC detection conditions: the mobile phase was 10mM NH4OAc mobile phase A:5% acetonitrile, mobile phase B:95% acetonitrile, temperature 40 ℃, flow rate 1.3 mL/min, column Xbridge C18 (available from Waters/Waters, U.S.A.).
The conversion results for uridylate kinase UMPK from each source are shown in Table 5 below:
TABLE 5
As shown in FIG. 2, isEscherichia albertiiThe liquid phase diagram of the catalytic production of N1-methyl pseudouridine triphosphate (N1-Met-phi TP) by using the source uridylate kinase UMPK can be seen by combining the liquid phase diagram with the above table 5,Escherichia albertiithe catalytic conversion rate of the source uridylate kinase UMPK is higher, and the conversion rate is 13.4%.
Example 4: screening of acetate kinase ACKs of different origins
The carrier construction expression of acetate kinase ACK and the preparation of freeze-dried crude enzyme powder are the same as in example 1, acetate kinase ACK enzyme powder from different sources obtained in example 1 is subjected to catalytic reaction, 2 mL phosphate buffer solution with the phosphate concentration of 50mmol/L,20 mg of N1-methyl pseudouridylic acid, 4.2 mg of ATP,14 mg of uracil, 2mg of magnesium chloride, 0.2 mg of thiamine pyrophosphate and 22 mg of acetyl phosphate are added into a reaction bottle (8 mL), and the pH of the reaction solution is controlled at 7.0 by 5M NaOH; adding inEscherichia albertii2mg of uridylic acid kinase from source, 1mg of acetate kinase from different sources,Escherichia coli2mg of uridine kinase from source, starting the reaction; controlling the temperature of the reaction liquid at 30 ℃ and the reaction rotating speed at 1000rpm; after the reaction time of 4 hours, 100. Mu.L of the reaction solution was taken, and after the inactivation by adding 50% methanol solution, the reaction was centrifugally filtered, and the reaction conversion of the product N1-methyl pseudouridine triphosphate (N1-Met-phi. TP) was measured by high performance liquid chromatography HPLC.
Wherein, the conversion rate of N1-methyl pseudouridine triphosphate (N1-Met-phi TP) measured by liquid chromatography is calculated by the ratio of the detected product peak Area:
conversion of N1-methyl pseudouridine triphosphate (N1-Met- φTP) conv N1-Met- φTP% = Area [ N1-Met- φTP/(N1-Met- φ+N 1-Met- φMP+N 1-Met- φDP+N 1-Met- φTP) ]
Wherein, high Performance Liquid Chromatograph (HPLC) was purchased from agilent technologies, inc, HPLC detection conditions: the mobile phase was 10mM NH4OAc mobile phase A:5% acetonitrile, mobile phase B:95% acetonitrile, temperature 40 ℃, flow rate 1.3 mL/min, column Xbridge C18 (from Waters/Waters, U.S.A.)
The conversion results of acetate kinase ACK from each source are shown in table 6 below:
TABLE 6
As shown in FIG. 2, isThermotoga maritimaThe liquid phase diagram of the catalytic production of N1-methyl pseudouridine triphosphate (N1-Met-phi TP) by the source acetate kinase ACK, combined with the liquid phase diagram and the results shown in the table 6 above, shows that,Thermotoga maritimathe catalytic conversion rate of the acetic acid kinase ACK is higher, and the conversion rate is 13.4%.
Example 5: one-pot enzyme method for preparing N1-methyl pseudouridine triphosphate
According to the screening results of examples 2-4, uridine kinase EcUK, uridylate kinase EauPK and acetate kinase TmACK with optimal conversion rate are obtained, and one-pot enzyme catalysis is carried out to prepare N1-methyl pseudouridine triphosphate, and the synthetic route is shown in figure 3.
2. 2 mL phosphate buffer solution, 20 mg of N1-methyl pseudouridylic acid, 4.2 mg of ATP,14 mg of uracil, 2. 2mg of magnesium chloride, 0.2. 0.2 mg of thiamine pyrophosphate and 22. 22 mg of acetyl phosphate are added into a reaction kettle, the phosphate concentration is controlled to be 50mmol/L, and the pH value of the reaction solution is controlled to be 7.0 by 5M NaOH; adding uridine kinase UK 2mg, acetate kinase TmACK 1.2 mg and uridylate kinase EaUMPK 4 mg to start reaction; controlling the temperature of the reaction liquid at 30-40 ℃ and the reaction rotating speed at 600-1000 rpm; taking 100 mu l of reaction solution after the reaction time is 2-28 h, adding 50% methanol solution for inactivation, centrifuging and filtering, and measuring the reaction conversion rate of the product N1-methyl pseudouridine triphosphate (N1-Met-phi TP) by HPLC.
Wherein, the conversion rate of N1-methyl pseudouridine triphosphate (N1-Met-phi TP) measured by liquid chromatography is calculated by the ratio of the detected product peak Area:
conversion of N1-methyl pseudouridine triphosphate (N1-Met- φTP) conv N1-Met- φTP% = Area [ N1-Met- φTP/(N1-Met- φ+N 1-Met- φMP+N 1-Met- φDP+N 1-Met- φTP) ]
Wherein, high Performance Liquid Chromatograph (HPLC) was purchased from agilent technologies, inc, HPLC detection conditions: the mobile phase was 10mM NH4OAc mobile phase A:5% acetonitrile, mobile phase B:95% acetonitrile, temperature 40 ℃, flow rate 1.3 mL/min, column Xbridge C18 (from Waters/Waters, U.S.A.)
As shown in FIG. 2, a liquid phase diagram of the catalytic production of N1-methyl pseudouridine triphosphate (N1-Met- φTP) using uridine kinase EcUK, uridylate kinase EauPK and acetate kinase TmACK with optimal conversion is shown. The conversion of N1-methyl pseudouridine triphosphate (N1-Met-phi TP) was 13.4% by liquid phase detection.
In summary, the above embodiments are only preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, but any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (8)
1. The method for preparing the N1-methyl pseudouridine triphosphate is characterized by taking N1-methyl pseudouridine as a substrate, adding an enzyme catalyst and a phosphate donor, constructing a phosphate cycle, and carrying out an enzyme catalytic reaction by adopting a one-pot method to obtain a final product N1-methyl pseudouridine triphosphate, wherein the enzyme catalytic reaction comprises the following steps:
the first stage: in a liquid reaction system, N1-methyl pseudouridine is used as a substrate, adenosine triphosphate and acetyl phosphate are used as phosphate donors, thiamine pyrophosphate is used as coenzyme, magnesium salt is used as a kinase stabilizer, and enzyme catalytic reaction is carried out under the catalysis of uridine kinase to generate N1-methyl pseudouridine;
and a second stage: n1-methyl pseudouridylic acid generated in the first stage is used as a substrate, adenosine triphosphate and acetyl phosphate are used as phosphate donors, thiamine pyrophosphate is used as coenzyme, magnesium salt is used as a kinase stabilizer, and enzyme catalysis reaction is carried out under the catalysis of uridylic acid kinase to generate N1-methyl pseudouridylic acid diphosphate;
and a third stage: taking N1-methyl pseudouridine diphosphate generated in the second stage as a substrate, taking adenosine triphosphate and acetyl phosphate as phosphate donors, taking thiamine pyrophosphate as coenzyme, taking magnesium salt as a kinase stabilizer, and performing enzyme catalytic reaction under the catalysis of acetate kinase to generate N1-methyl pseudouridine triphosphate;
wherein, the Genbank serial number of the uridine kinase is EGM3941553.1, the Genbank serial number of the uridine kinase is WP_000224577.1, and the Genbank serial number of the acetate kinase is AKE29949.1.
2. The method for producing N1-methyl pseudouridine triphosphate according to claim 1, wherein the concentration of N1-methyl pseudouridine is 10-50 mmol/L.
3. The method for preparing N1-methyl pseudouridine triphosphate according to claim 1, wherein the concentration of adenosine triphosphate is 1-10 mmol/L.
4. The method for preparing N1-methyl pseudouridine triphosphate according to claim 1, wherein the concentration of the acetyl phosphate is 1-200 mmol/L.
5. The method for preparing N1-methyl pseudouridine triphosphate according to claim 1, wherein the thiamine pyrophosphate concentration is 0.1% -2% of the N1-methyl pseudouridine concentration.
6. The method for preparing N1-methyl pseudouridine triphosphate according to claim 1, wherein the concentration of the magnesium salt is 1 mmol/L to 20 mmol/L, and the magnesium salt is any one or a combination of more of magnesium chloride, magnesium sulfate and magnesium nitrate.
7. The method for preparing N1-methyl pseudouridine triphosphate according to claim 1, wherein the buffer of the liquid reaction system is phosphate buffer with a concentration of 50 mmol/L.
8. The method for preparing N1-methyl pseudouridine triphosphate according to claim 1, wherein the temperature of the enzyme-catalyzed reaction is 30-40 ℃, the reaction time of the enzyme-catalyzed reaction is 2-28 h, and the pH of the enzyme-catalyzed reaction is 6.0-9.0.
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