CN117143942A - Synthesis method of 3 '-amino-2', 3 '-dideoxyguanosine 5' -triphosphate - Google Patents
Synthesis method of 3 '-amino-2', 3 '-dideoxyguanosine 5' -triphosphate Download PDFInfo
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- CN117143942A CN117143942A CN202311414822.XA CN202311414822A CN117143942A CN 117143942 A CN117143942 A CN 117143942A CN 202311414822 A CN202311414822 A CN 202311414822A CN 117143942 A CN117143942 A CN 117143942A
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- amino
- dideoxyguanosine
- kinase
- triphosphate
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- ILTYANYMUGEMNF-KVQBGUIXSA-N [[(2s,3s,5r)-3-amino-5-(2-amino-6-oxo-3h-purin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](N)C[C@@H]1N1C(N=C(N)NC2=O)=C2N=C1 ILTYANYMUGEMNF-KVQBGUIXSA-N 0.000 title claims abstract description 25
- 238000001308 synthesis method Methods 0.000 title abstract description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 24
- 108010092060 Acetate kinase Proteins 0.000 claims abstract description 21
- 108091000080 Phosphotransferase Proteins 0.000 claims abstract description 13
- 102000020233 phosphotransferase Human genes 0.000 claims abstract description 13
- HOQBPAWPBCAUGA-KVQBGUIXSA-N 2-amino-9-[(2r,4s,5s)-4-amino-5-(hydroxymethyl)oxolan-2-yl]-3h-purin-6-one Chemical compound O1[C@H](CO)[C@@H](N)C[C@@H]1N1C(NC(N)=NC2=O)=C2N=C1 HOQBPAWPBCAUGA-KVQBGUIXSA-N 0.000 claims abstract description 11
- 108010011356 Nucleoside phosphotransferase Proteins 0.000 claims abstract description 11
- LIPOUNRJVLNBCD-UHFFFAOYSA-N acetyl dihydrogen phosphate Chemical compound CC(=O)OP(O)(O)=O LIPOUNRJVLNBCD-UHFFFAOYSA-N 0.000 claims abstract description 10
- -1 3' -amino-dideoxyguanosine-5 ' -monophosphate Chemical compound 0.000 claims abstract description 9
- 239000002773 nucleotide Substances 0.000 claims abstract description 9
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 9
- 229910021645 metal ion Inorganic materials 0.000 claims abstract description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 6
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 6
- 230000008569 process Effects 0.000 claims abstract description 6
- 230000009471 action Effects 0.000 claims abstract description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 3
- 108020004202 Guanylate Kinase Proteins 0.000 claims description 22
- 102000006638 guanylate kinase Human genes 0.000 claims description 22
- 238000006243 chemical reaction Methods 0.000 claims description 18
- 108090000623 proteins and genes Proteins 0.000 claims description 17
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- 238000004108 freeze drying Methods 0.000 claims description 7
- 108010036383 guanosine kinase Proteins 0.000 claims description 7
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 6
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- 229910019142 PO4 Inorganic materials 0.000 claims description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 5
- 239000010452 phosphate Substances 0.000 claims description 5
- 230000003197 catalytic effect Effects 0.000 claims description 4
- YNIRKEZIDLCCMC-UHFFFAOYSA-K trisodium;phosphate;hydrate Chemical compound [OH-].[Na+].[Na+].[Na+].OP([O-])([O-])=O YNIRKEZIDLCCMC-UHFFFAOYSA-K 0.000 claims description 4
- 241000593583 Escherichia coli E1728 Species 0.000 claims description 3
- 230000035484 reaction time Effects 0.000 claims description 3
- 241000191070 Escherichia coli ATCC 8739 Species 0.000 claims description 2
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 claims description 2
- 102000001253 Protein Kinase Human genes 0.000 claims description 2
- 229910001425 magnesium ion Inorganic materials 0.000 claims description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical class OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 2
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 claims description 2
- WAEMQWOKJMHJLA-UHFFFAOYSA-N Manganese(2+) Chemical compound [Mn+2] WAEMQWOKJMHJLA-UHFFFAOYSA-N 0.000 claims 2
- 229910001437 manganese ion Inorganic materials 0.000 claims 2
- 241000204652 Thermotoga Species 0.000 claims 1
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 claims 1
- 229910001429 cobalt ion Inorganic materials 0.000 claims 1
- XLJKHNWPARRRJB-UHFFFAOYSA-N cobalt(2+) Chemical compound [Co+2] XLJKHNWPARRRJB-UHFFFAOYSA-N 0.000 claims 1
- 239000001177 diphosphate Substances 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 abstract description 17
- 108090000790 Enzymes Proteins 0.000 abstract description 16
- HDRRAMINWIWTNU-NTSWFWBYSA-N [[(2s,5r)-5-(2-amino-6-oxo-3h-purin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@H]1CC[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HDRRAMINWIWTNU-NTSWFWBYSA-N 0.000 abstract description 9
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
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- 102100040468 Guanylate kinase Human genes 0.000 description 7
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- 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 4
- 108020004414 DNA Proteins 0.000 description 4
- 239000006142 Luria-Bertani Agar Substances 0.000 description 4
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 4
- 239000001888 Peptone Substances 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- CVXBEEMKQHEXEN-UHFFFAOYSA-N carbaryl Chemical compound C1=CC=C2C(OC(=O)NC)=CC=CC2=C1 CVXBEEMKQHEXEN-UHFFFAOYSA-N 0.000 description 4
- 229960005286 carbaryl Drugs 0.000 description 4
- PHIQHXFUZVPYII-UHFFFAOYSA-N carnitine Chemical compound C[N+](C)(C)CC(O)CC([O-])=O PHIQHXFUZVPYII-UHFFFAOYSA-N 0.000 description 4
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- 239000011780 sodium chloride Substances 0.000 description 4
- BXZVVICBKDXVGW-NKWVEPMBSA-N Didanosine Chemical compound O1[C@H](CO)CC[C@@H]1N1C(NC=NC2=O)=C2N=C1 BXZVVICBKDXVGW-NKWVEPMBSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- WREGKURFCTUGRC-POYBYMJQSA-N Zalcitabine Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](CO)CC1 WREGKURFCTUGRC-POYBYMJQSA-N 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
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- 229960002363 thiamine pyrophosphate Drugs 0.000 description 3
- 235000008170 thiamine pyrophosphate Nutrition 0.000 description 3
- 239000011678 thiamine pyrophosphate Substances 0.000 description 3
- 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 description 3
- HBOMLICNUCNMMY-XLPZGREQSA-N zidovudine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](N=[N+]=[N-])C1 HBOMLICNUCNMMY-XLPZGREQSA-N 0.000 description 3
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- XNKLLVCARDGLGL-JGVFFNPUSA-N Stavudine Chemical compound O=C1NC(=O)C(C)=CN1[C@H]1C=C[C@@H](CO)O1 XNKLLVCARDGLGL-JGVFFNPUSA-N 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
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- 238000006209 dephosphorylation reaction Methods 0.000 description 2
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- 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 2
- 229910001629 magnesium chloride Inorganic materials 0.000 description 2
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- XKMLYUALXHKNFT-UHFFFAOYSA-N rGTP Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O XKMLYUALXHKNFT-UHFFFAOYSA-N 0.000 description 2
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- 239000001226 triphosphate Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 229960002555 zidovudine Drugs 0.000 description 2
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 2
- XKKCQTLDIPIRQD-JGVFFNPUSA-N 1-[(2r,5s)-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)CC1 XKKCQTLDIPIRQD-JGVFFNPUSA-N 0.000 description 1
- JTEGQNOMFQHVDC-RQJHMYQMSA-N 4-amino-1-[(2s,5r)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl]pyrimidin-2-one Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](CO)SC1 JTEGQNOMFQHVDC-RQJHMYQMSA-N 0.000 description 1
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 239000003298 DNA probe Substances 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 229940124639 Selective inhibitor Drugs 0.000 description 1
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- YYYHQQJYHWRQOG-NTSWFWBYSA-N [(2s,5r)-5-(2-amino-6-oxo-3h-purin-9-yl)oxolan-2-yl]methyl phosphono hydrogen phosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@H]1CC[C@@H](COP(O)(=O)OP(O)(O)=O)O1 YYYHQQJYHWRQOG-NTSWFWBYSA-N 0.000 description 1
- HDRRAMINWIWTNU-PRJDIBJQSA-N [[(5r)-5-(2-amino-6-oxo-3h-purin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@H]1CCC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HDRRAMINWIWTNU-PRJDIBJQSA-N 0.000 description 1
- 239000002259 anti human immunodeficiency virus agent Substances 0.000 description 1
- 239000000074 antisense oligonucleotide Substances 0.000 description 1
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- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 208000012839 conversion disease Diseases 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 229960002656 didanosine Drugs 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
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- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 229960001627 lamivudine Drugs 0.000 description 1
- JTEGQNOMFQHVDC-NKWVEPMBSA-N lamivudine Chemical compound O=C1N=C(N)C=CN1[C@H]1O[C@@H](CO)SC1 JTEGQNOMFQHVDC-NKWVEPMBSA-N 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
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- 239000011565 manganese chloride Substances 0.000 description 1
- 235000002867 manganese chloride Nutrition 0.000 description 1
- 229940099607 manganese chloride Drugs 0.000 description 1
- 229940099596 manganese sulfate Drugs 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- MIVBAHRSNUNMPP-UHFFFAOYSA-N manganese(2+);dinitrate Chemical compound [Mn+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O MIVBAHRSNUNMPP-UHFFFAOYSA-N 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
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- 238000010369 molecular cloning Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 150000008039 phosphoramides Chemical class 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 150000003230 pyrimidines Chemical class 0.000 description 1
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Classifications
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- 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
- C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
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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 relates to a synthesis method of 3 '-amino-2', 3 '-dideoxyguanosine 5' -triphosphate, which relates to the technical field of oligonucleotide medicines and comprises the following steps: nucleoside kinase catalyzes the production of 3' -amino-dideoxyguanosine-5 ' -monophosphate from 3' -amino-dideoxyguanosine; nucleotide kinase catalyzes the production of 3 '-amino-dideoxyguanosine-5' -biphosphoric acid from 3 '-amino-dideoxyguanosine-5' -monophosphate; 3 '-amino-dideoxyguanosine-5' -diphosphate is phosphorylated to 3 '-amino-2', 3 '-dideoxyguanosine 5' -triphosphate under the action of acetate kinase AcK, ATP and divalent metal ions; and the whole process uses ATP to dephosphorylate to generate ADP, and acetic acid kinase Ack catalyzes acetyl phosphate to dephosphorylate to generate acetic acid for phosphoric acid to generate ATP. The method provided by the invention has the advantages of simple operation, low production cost and the like, and fills in the synthesis of 3' -NH by using the biological enzyme method 2 Blank of ddGTP.
Description
Technical Field
The invention relates to the technical field of oligonucleotide medicines, in particular to a method for synthesizing 3 '-amino-2', 3 '-dideoxyguanosine 5' -triphosphate.
Background
Cancer is one of the leading causes of death worldwide. Traditional therapies, including surgery, radiation and chemotherapy, have increased the survival rate of many types of cancer over the past several decades. However, cancer recurrence and/or metastasis to distant organs remains a major challenge.
Oligonucleotide therapy, including antisense oligonucleotides, is expected to fill the blank therapeutic area because of its unique mechanism of regulating transcription and translation of disease genes at the RNA level. In addition, 6 oligonucleotide drugs are approved by the FDA in the last 3 years, and the pharmaceutical industry is attracted to the field of wide attention. Oligonucleotide analogue drugs are designed to increase their resistance to nucleases and binding and specificity to other substances. Wherein the N3 'and O5' modified phosphoramides are stable in both double and even triple strands and have a higher nuclease resistance than normal DNA or RNA. They can be used as DNA hybridization probes and primers for PCR amplification, and have high selectivity to low copy RNA sequences. However, 3' -aminonucleosides are currently very difficult to prepare and are also a major difficulty in the preparation of oligonucleotide-based drugs.
Currently, the most potent and selective inhibitors of anti-HIV drugs are 2',3' -dideoxycytidine, 2',3' -dideoxythymidine, 2',3' -unsaturated pyrimidines and 2',3' -dideoxynucleoside analogs. The most commonly used nucleoside derivatives include 3' -azido-3 ' -deoxythymidine (AZT, zidovudine), 2',3' -dideoxycytidine (ddC, zasitaglycone), 2',3' -dideoxyinosine (ddI, didanosine), 2',3' -dideoxy-2 ',3' -dideoxythymidine (d 4T, stavudine) and 2',3' -dideoxy-3 ' -thiacytidine (3 TC, lamivudine). Among the various inhibitors of Human Immunodeficiency Virus (HIV), dideoxynucleoside analogs, because 2',3' -dideoxynucleosides lack 3' -Ofi groups necessary for further growth of the DNA strand, insertion of 2',3' -dideoxynucleosides into the elongated DNA results in chain termination, preventing viral DNA synthesis during the reverse transcription phase, a step in the replication cycle of all retroviruses.
ddGTP (2 ',3' -dideoxyguanosine 5 '-triphosphate) is one of 2',3 '-dideoxyribonucleoside 5' -triphosphates (ddNTPs) that is used as a chain extension inhibitor for DNA polymerase for DNA sequencing. 3' -amino-2 ',3' -dideoxyguanosine 5' -triphosphate (3 ' -NH) 2 -ddGTP) is a derivative of ddGTP, and it has not been found that enzymatic synthesis of 3' -NH is possible 2 ddGTP, the study and process scale-up production of which is in a blank state.
The invention aims to provide 3 '-amino-2', 3 '-dideoxyguanosine triphosphate (3' -NH) 2 -ddGTP) 3 '-amino-5' -nucleoside triphosphate is a constituent unit of a nucleic acid (NP-DNA) other than an o-p internucleotide strand, wherein 3 '-OH is 3' -NH 2 Substitution, naturally has higher thermal stability than an isospecific duplex composed of DNA and RNA.
Disclosure of Invention
The invention aims to provide a synthesis method of 3' -amino-2 ',3' -dideoxyguanosine 5' -triphosphate, which adopts a one-step enzyme method, has the advantages of simple operation, low production cost and the like, and fills up the gap of synthesizing 3' -NH by a biological enzyme method 2 Blank of ddGTP.
In order to solve the technical problems, the invention adopts the following technical scheme:
with 3 '-amino-dideoxyguanosine (3' -NH) 2 -ddG) is used as a raw material, ATP is used as a phosphate donor, acetyl phosphate dephosphorization is used for providing Pi to ADP to form ATP, the ATP is formed to ADP, the cycle of adding Pi to the ADP to generate ATP is formed, thiamine pyrophosphate (TPP) is used as a coenzyme, and 3' -amino-2 ',3' -dideoxyguanosine 5' -triphosphate is synthesized by a one-pot enzyme method by using Nucleoside kinase (Nucleoside kinase), nucleotide kinase (nucleotide 5' -monophosphate kinase) and Acetate kinase (Acetate kinase)3’- NH 2 -ddGTP）。
Wherein, 3' -NH is synthesized by one-pot enzyme method 2 -ddGTP comprises the following steps:
(1) Nucleoside kinase catalyzes 3 '-amino-dideoxyguanosine (3' -NH) 2 -ddG) to 3' -amino-dideoxyguanosine-5 ' -monophosphate (3 ' -NH) 2 -ddGMP）；
(2) Nucleotide kinase catalysis of 3' -NH 2 -ddGMP formation of 3' -amino-dideoxyguanosine 5' -diphosphate (3 ' -NH) 2 -ddGDP）；
（3）3’-NH 2 The ddGDP is phosphorylated to form 3' -amino-2 ',3' -dideoxyguanosine 5' -triphosphate (3 ' -NH) under the action of acetate kinase AcK, ATP and divalent metal ions 2 -ddGTP）；
In the whole process, ATP is used for dephosphorization to generate ADP, and acetic acid kinase AcK catalyzes acetyl phosphate for dephosphorization to generate acetic acid for phosphoric acid to generate ATP;
wherein, nucleoside Kinase (Nucleoside Kinase) is derived from Guanylate Kinase (GK) of Escherichia coli E1728, GK is expressed in a recombinant vector through gene recombination, and is obtained through cell disruption, centrifugal filtration and freeze-drying, and the amino acid sequence of the Guanylate Kinase is shown as SEQ ID NO. 1;
wherein, the nucleotide Kinase NMPK is derived from Guanylate Kinase (GMK) of Escherichia coli ATCC 8739, the GMK is expressed in a recombinant vector through gene recombination, and is obtained through cell disruption, centrifugal filtration and freeze-drying, the Genebank ID is ACA75749.1, and the amino acid sequence is shown as SEQ ID NO. 2;
wherein, acetate kinase (AcK) is derived from Thermotoga maritima, genebank ID AKE29949.1, the amino acid sequence is shown in SEQ ID NO.3, and the Acetate kinase is obtained by gene recombination, expression in a recombinant vector, cell disruption, centrifugal filtration and freeze-drying. Acetate kinase (AcK) is a member of the phosphotransferase superfamily responsible for catalyzing the reversible reaction of acetyl phosphate and ADP to acetic acid and ATP.
The overall reaction scheme is shown in FIG. 1.
The pH value of the catalytic conversion in the step (1) to the step (3) is 6.0-9.0; the catalytic conversion temperature is 30-40 ℃; the reaction time is 2-28 h; the concentration of the ATP added during catalytic conversion is 1-20 mmol/L; the concentration of the added acetyl phosphate is 1-200 mmol/L; the concentration of divalent metal ions is 1 mmol/L-20 mmol/L; divalent metal ions include, but are not limited to, any one or more of manganese chloride, magnesium chloride, cobalt chloride, zinc chloride, manganese sulfate, manganese nitrate.
In the reaction, every 0.5-40 ml volume of the reaction system is used for preparing a substrate 3 '-amino-dideoxyguanosine (3' -NH) 2 -ddG) is controlled to be 0.2-20 mg, and guanosine kinase GK, guanylate kinase GMK and acetate kinase Ack are controlled to be 0.02-1 mg; the whole reaction system is an aqueous system, and buffer solution can be added into the aqueous system, and the buffer solution can be citric acid-sodium citrate buffer solution, disodium hydrogen phosphate-sodium hydroxide buffer solution or trimethylol methylamine salt buffer solution.
Among them, acetate kinase, guanosine kinase and guanylate kinase all belong to metal ion dependent enzymes, and the stability of the enzyme is higher when the divalent metal magnesium ion exists.
The acetyl phosphate plays a role in phosphate circulation, ATP is used for dephosphorylation to generate ADP in the whole process, and acetic acid kinase Ack catalyzes acetyl phosphate dephosphorylation to generate acetic acid for phosphate to generate ATP, so that the phosphate circulation is performed.
The synthesis method of the 3' -amino-2 ',3' -dideoxy guanosine 5' -triphosphate provided in the technical proposal has low material cost and easily obtained product in the whole technical route, and fills the current biological method for preparing the 3' -amino-2 ',3' -dideoxy guanosine 5' -triphosphate (3 ' -NH) 2 -ddGTP) provides a concept for preparing nucleosides and their phosphate derivatives by biological enzymes. And finally 3' -amino-2 ',3' -dideoxyguanosine 5' -triphosphate (3 ' -NH) 2 -ddGTP) conversion can reach about 30%.
Drawings
FIG. 1 is a synthetic scheme of 3 '-amino-2', 3 '-dideoxyguanosine 5' -triphosphate according to the invention;
FIG. 2 is a schematic illustration of the one pot enzymatic method of example 4 utilizing 3 '-amino-dideoxyguanosine (3'-NH 2 Preparation of 3' -amino-2 ',3' -dideoxyguanosine 5' -triphosphate (3 ' -NH) 2 -ddGTP), wherein the peak times of the corresponding substances are respectively: 3' -NH 2 -ddG (1.278min)，3’-NH 2 -ddGMP (1.800min)，3’-NH 2 -ddGDP (1.960min)，3’-NH 2 -ddGTP (2.625min)。
Detailed Description
The present invention will be specifically described with reference to examples below in order to make the objects and advantages of the present invention more apparent. It should be understood that the following text is intended to describe only one or more specific embodiments of the invention and does not limit the scope of the invention strictly as claimed.
Example 1: the embodiment is a recombinant strain construction and fermentation expression comprising Guanosine Kinase (GK) genes, and the specific steps are as follows:
1) Gene synthesis
The total synthesis of the Guanosine Kinase (GK) gene (SEQ ID NO. 1) from Escherichia coli E1728 was performed by Shanghai Jin Wei Intelligence bioengineering Co., ltd, and was ligated to pET28a vector to obtain recombinant vector pET28a-GK;
2) Construction of recombinant Strain E.coli BL21 (DE 3) pET-28 a-GK
Activating E.coli DH 5. Alpha./pET-28 a (+) strain (purchased from Novain Biotechnology Co., ltd.), adding 3-5. Mu.L of pET-28 a-GK (50 ng/. Mu.L) to 50-100. Mu.L of E.coli BL21 (DE 3) (which may be E.coli MG 1655) competent cells (purchased from Novain Biotechnology Co., ltd.), placing on ice for 20 min at 42℃for heat shock, continuing for 90 sec, rapidly returning to ice bath for 5 min, adding 800. Mu.L of non-resistant LB medium, culturing at 37℃at 200 rpm, culturing at 1 h, and then plating on a carba-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-GK.
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.
3) Construction of expression vector and inducible expression
The recombinant E.coli BL21 (DE 3) containing pET-28 a-GK obtained in 2) 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 GK recombinant vector were picked from the Canner plates and inoculated into 1 mL liquid LB medium containing 25. Mu.g/mL of Canner 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, performing induced expression at 25 ℃ for 20 h, centrifuging (4 ℃,4000 rpm,30 min), removing supernatant, washing with 30mL of 0.9% NaCl solution, and re-suspending to obtain GK target protein.
4) Preparation of crude enzyme powder
Collecting bacterial liquid containing GK target protein, washing twice with 50mmol/L Tris-HCl, suspending the bacterial liquid in 50mmol/L Tris-HCl buffer solution (pH 8.0), ultrasonically crushing cells in ice bath (amplitude transformer 6, power 500W, open 2s, closed 5s,30 min, centrifuging the ultrasonically crushed sample 12000 rpm at 4 ℃ for 30 min, taking supernatant, putting the supernatant into a freeze dryer at-80 ℃ for freeze drying for 24h, and grinding the obtained freeze-dried sample to obtain GK freeze-dried crude enzyme powder.
Example 2: the recombinant vector comprising guanylate kinase (GMK) gene, the recombinant strain comprising guanylate kinase (GMK) gene and the lyophilized crude enzyme powder comprising guanylate kinase (GMK) gene recombinant protein are prepared by the same method as that of Guanylate Kinase (GK).
Example 3: the embodiment is a recombinant strain construction and fermentation expression comprising acetate kinase AcK genes, and the specific steps are as follows:
1) Gene synthesis
According toThermotoga maritimaThe AcK gene of acetate kinase (genebank ID: AKE 29949.1) is of limited intellectual bioengineering by Shanghai Jin WeiThe company carries out total synthesis of genes and connects the genes to a pET28a vector to obtain a recombinant vector pET28a-AcK;
2) Construction of recombinant Strain E.coli BL21 (DE 3) pET-28a-AcK
Activating E.coli DH 5. Alpha./pET-28 a (+) strain (purchased from Novain Biotechnology Co., ltd.), adding 3-5. Mu.L of pET-28a-AcK (50 ng/. Mu.L) to 50-100. Mu.L of E.coli BL21 (DE 3) competent cells (which may also be E.coli MG 1655) (purchased from Novain Biotechnology Co., ltd.), placing on ice for 20 min at 42℃for heat shock, continuing for 90 sec, rapidly returning to ice bath for 5 min, adding 800. Mu.L of non-resistant LB medium, culturing at 37℃at 200 rpm, culturing at 1 h, and then plating on a card-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-AcK.
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.
3) Construction of expression vector and inducible expression
The recombinant E.coli BL21 (DE 3) containing pET-28a-Ack obtained in 2) 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 Ack recombinant vector were picked from the Canner plates and inoculated into 1 mL liquid LB medium containing 25. Mu.g/mL of Canner resistance and shake-cultured at 37℃for 12 h. Then, the culture was inoculated into fresh 1L liquid LB medium containing 25. Mu.g/mL of the carbaner resistance according to an inoculum size of 2% (v/v), when the culture was carried out at 37℃until the OD600 was about 0.6-0.8, IPTG (purchased from Shanghai Ala Biochemical technologies Co., ltd.) was added to a final concentration of 1.0 mmol/L,200 rpm, induced expression at 25℃of 20 h, and the supernatant was removed by centrifugation (4 ℃,4000 rpm,30 min), and the obtained bacterial sludge was resuspended by 30mL of a 0.9% NaCl solution for use.
4) Preparation of crude enzyme freeze-dried powder
Wherein, the preparation method of the acetate kinase Ack crude enzyme powder containing the Ack gene comprises the following steps:
after the bacterial solution was collected and washed twice with 50mmol/L Tris-HCl, the bacterial solution was resuspended in 50mmol/L Tris-HCl (pH 8.0) buffer and the cells were sonicated in an ice bath (horn 6, power 500W, 2s on, 5s off, 30 min). Centrifuging the ultrasonically crushed sample at 12000 rpm and 4 ℃ for 30 min; and (3) putting the supernatant into a freeze dryer at the temperature of minus 80 ℃ to freeze-dry for 24 hours, and grinding the obtained freeze-dried sample to prepare freeze-dried crude enzyme powder.
Example 4: this example uses 3 '-amino-dideoxyguanosine (3' -NH) with a one-pot enzymatic method 2 Preparation of 3 '-amino-dideoxyguanosine triphosphate (3' -NH) 2 -ddGTP), in particular:
2mL of Na was added to the reactor 2 HPO 4 Disodium hydrogen phosphate-sodium hydroxide buffer (50 mmol/L), 20mg of 3 '-amino-dideoxyguanosine (3' -NH) 2 -ddG), 2.2mg of ATP,2mg of magnesium chloride, 11.2mg of acetyl phosphate and 0.5mg of thiamine pyrophosphate, and controlling the pH of the reaction solution to 6.0-9.0 by using 5M NaOH; adding 0.05mg of acetate kinase Ack crude enzyme powder, 0.2mg of Guanosine Kinase (GK) crude enzyme powder and 0.2mg of guanylate kinase (GMK) crude enzyme powder, and starting the reaction;
controlling the temperature of the reaction liquid at 30 ℃ and the reaction rotating speed at 1000rpm; after a reaction time of 2 hours, 100. Mu.l of the reaction mixture was taken, inactivated by adding 50% methanol solution, and then centrifuged, and the resultant 3' -NH was measured by HPLC 2 Reaction conversion of ddGTP. Detection of 3' -NH by liquid phase 2 The ddGTP conversion was 31.3% and the HPLC profile is shown in FIG. 2.
Wherein 3' -NH is measured by liquid chromatography 2 ddGTP conversion is calculated as the ratio of the detected product peak Area:
3’-NH 2 conversion of ddGTP conv (3' -NH) 2 -ddGTP) %=Area[3’-NH 2 -ddGTP /(3’-NH 2 -ddGTP +3’-NH 2 -ddGDP +3’-NH 2 -ddGMP+3’-NH 2 -ddG] *100%
Wherein, HPLC detection conditions: the mobile phase was 10mM NH 4 OAc mobile phase a 5% acetonitrile, mobile phase B95% acetonitrile,the temperature is 40 ℃, the flow rate is 1.3mL/min, and the chromatographic column is Xbridge C18.
And (3) notes: the experimental procedure, without specific conditions noted in the examples, 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.
While the embodiments of the present invention have been described in detail with reference to the examples, the present invention is not limited to the above embodiments, and it will be apparent to those skilled in the art that various equivalent changes and substitutions can be made therein without departing from the principles of the present invention, and such equivalent changes and substitutions should also be considered to be within the scope of the present invention.
Claims (10)
1. A method for synthesizing 3 '-amino-2', 3 '-dideoxyguanosine 5' -triphosphate, which is characterized by comprising the following steps:
s1, catalyzing 3' -amino-dideoxyguanosine to generate 3' -amino-dideoxyguanosine-5 ' -monophosphate by nucleoside kinase;
s2, catalyzing 3 '-amino-dideoxyguanosine-5' -monophosphate by nucleotide kinase to generate 3 '-amino-dideoxyguanosine-5' -diphosphate;
s3.3 '-amino-dideoxyguanosine 5' -diphosphate is subjected to phosphate formation under the action of acetate kinase AcK, ATP and divalent metal ions to form 3 '-amino-2', 3 '-dideoxyguanosine 5' -triphosphate;
and the whole process uses ATP to dephosphorylate to generate ADP, and acetic acid kinase Ack catalyzes acetyl phosphate to dephosphorylate to generate acetic acid for phosphoric acid to generate ATP.
2. The method for synthesizing 3 '-amino-2', 3 '-dideoxyguanosine 5' -triphosphate according to claim 1, wherein: the nucleoside kinase in the step S1 is derived from guanosine kinase Escherichia coli E1728, and the guanosine kinase is expressed in a recombinant vector through gene recombination, is subjected to cell disruption, and is subjected to centrifugal filtration and freeze-drying to obtain the nucleoside kinase; the amino acid sequence of the nucleoside kinase is shown in SEQ ID NO. 1.
3. The method for synthesizing 3 '-amino-2', 3 '-dideoxyguanosine 5' -triphosphate according to claim 1, wherein: the nucleotide kinase in the step S2 is derived from Escherichia coli ATCC 8739 guanylate kinase, and the guanylate kinase is expressed in a recombinant vector through gene recombination, and is obtained through cell disruption, centrifugal filtration and freeze-drying; the amino acid sequence of the nucleotide kinase is shown in SEQ ID NO. 2.
4. The method for synthesizing 3 '-amino-2', 3 '-dideoxyguanosine 5' -triphosphate according to claim 1, wherein: the acetate kinase in step S3 is derived fromThermotoga maritima，Thermotoga maritimaThrough gene recombination, expression in recombinant vector, cell disruption, centrifugal filtration and freeze drying to obtain acetate kinase; the amino acid sequence of the acetate kinase is shown in SEQ ID NO. 3.
5. The method for synthesizing 3 '-amino-2', 3 '-dideoxyguanosine 5' -triphosphate according to claim 1, wherein: the pH value in the catalytic conversion process is 6.0-9.0, the temperature is 30-40 ℃, and the reaction time is 2-28 h.
6. The method for synthesizing 3 '-amino-2', 3 '-dideoxyguanosine 5' -triphosphate according to claim 1, wherein:
the ATP concentration is 1-20 mmol/L; the concentration of divalent metal ions is 1 mmol/L-20 mmol/L; the concentration of the acetyl phosphate is 1-200 mmol/L;
and the addition amount of 3' -amino-dideoxyguanosine is 0.2-20 mg and the addition amounts of nucleoside kinase, nucleotide kinase and acetate kinase are 0.02-1 mg in each 0.5-40 ml volume reaction system.
7. The method for synthesizing 3 '-amino-2', 3 '-dideoxyguanosine 5' -triphosphate according to claim 6, wherein: the divalent metal ion is one or more of manganese ion, magnesium ion, cobalt ion, zinc ion and manganese ion.
8. The method for synthesizing 3 '-amino-2', 3 '-dideoxyguanosine 5' -triphosphate according to claim 1, wherein: the whole reaction system is an aqueous system.
9. The method for synthesizing 3 '-amino-2', 3 '-dideoxyguanosine 5' -triphosphate according to claim 8, wherein: the aqueous system contains a buffer solution, wherein the buffer solution is a citric acid-sodium citrate buffer solution, a disodium hydrogen phosphate-sodium hydroxide buffer solution or a trimethylol methylamine salt buffer solution.
10. The method for synthesizing 3 '-amino-2', 3 '-dideoxyguanosine 5' -triphosphate according to claim 9, wherein: the buffer solution is disodium hydrogen phosphate-sodium hydroxide buffer solution, and the concentration is 10-200 mol/L.
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