CN115362161A - 用于制备3’-o-氨基-2’-脱氧核糖核苷-5’-三磷酸的方法 - Google Patents
用于制备3’-o-氨基-2’-脱氧核糖核苷-5’-三磷酸的方法 Download PDFInfo
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- CN115362161A CN115362161A CN202180026121.1A CN202180026121A CN115362161A CN 115362161 A CN115362161 A CN 115362161A CN 202180026121 A CN202180026121 A CN 202180026121A CN 115362161 A CN115362161 A CN 115362161A
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- hydroxylamine
- triphosphate
- aryl
- deoxyribonucleoside
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- 238000004519 manufacturing process Methods 0.000 title description 3
- 235000011178 triphosphate Nutrition 0.000 claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 17
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 claims abstract description 11
- XYEOALKITRFCJJ-UHFFFAOYSA-N o-benzylhydroxylamine Chemical class NOCC1=CC=CC=C1 XYEOALKITRFCJJ-UHFFFAOYSA-N 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 10
- 125000002264 triphosphate group Chemical class [H]OP(=O)(O[H])OP(=O)(O[H])OP(=O)(O[H])O* 0.000 claims description 8
- 229910052736 halogen Inorganic materials 0.000 claims description 7
- 150000002367 halogens Chemical class 0.000 claims description 7
- UOKZUTXLHRTLFH-UHFFFAOYSA-N o-phenylhydroxylamine Chemical class NOC1=CC=CC=C1 UOKZUTXLHRTLFH-UHFFFAOYSA-N 0.000 claims description 7
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 claims description 6
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 claims description 6
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- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 claims description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 5
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- OJYSJFUYARGLPG-UHFFFAOYSA-N o-[(4-nitrophenyl)methyl]hydroxylamine Chemical compound NOCC1=CC=C([N+]([O-])=O)C=C1 OJYSJFUYARGLPG-UHFFFAOYSA-N 0.000 claims description 5
- CFMZSMGAMPBRBE-UHFFFAOYSA-N 2-hydroxyisoindole-1,3-dione Chemical compound C1=CC=C2C(=O)N(O)C(=O)C2=C1 CFMZSMGAMPBRBE-UHFFFAOYSA-N 0.000 claims description 4
- LRFVTYWOQMYALW-UHFFFAOYSA-N 9H-xanthine Chemical compound O=C1NC(=O)NC2=C1NC=N2 LRFVTYWOQMYALW-UHFFFAOYSA-N 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 239000005549 deoxyribonucleoside Substances 0.000 claims description 4
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- 125000005647 linker group Chemical group 0.000 claims description 4
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 4
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims description 4
- HWKGEDJHAMSGKI-UHFFFAOYSA-N o-(2-phenoxyethyl)hydroxylamine Chemical class NOCCOC1=CC=CC=C1 HWKGEDJHAMSGKI-UHFFFAOYSA-N 0.000 claims description 4
- QCGLSKQHZHTJMW-UHFFFAOYSA-N o-(2-phenylmethoxyethyl)hydroxylamine Chemical class NOCCOCC1=CC=CC=C1 QCGLSKQHZHTJMW-UHFFFAOYSA-N 0.000 claims description 4
- 229930024421 Adenine Natural products 0.000 claims description 3
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- 150000001875 compounds Chemical class 0.000 claims description 3
- 229940104302 cytosine Drugs 0.000 claims description 3
- UOISMTPJFYEVBW-UHFFFAOYSA-N o-[(2,3,4,5,6-pentafluorophenyl)methyl]hydroxylamine Chemical compound NOCC1=C(F)C(F)=C(F)C(F)=C1F UOISMTPJFYEVBW-UHFFFAOYSA-N 0.000 claims description 3
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- 229930010555 Inosine Natural products 0.000 claims description 2
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 claims description 2
- 125000004103 aminoalkyl group Chemical group 0.000 claims description 2
- 125000005335 azido alkyl group Chemical group 0.000 claims description 2
- YFIBSNDOVCWPBL-UHFFFAOYSA-N hexa-1,5-diyne Chemical group C#CCCC#C YFIBSNDOVCWPBL-UHFFFAOYSA-N 0.000 claims description 2
- 229960003786 inosine Drugs 0.000 claims description 2
- BZCDKOOXCPRISF-UHFFFAOYSA-N o-(2-phenylethyl)hydroxylamine Chemical class NOCCC1=CC=CC=C1 BZCDKOOXCPRISF-UHFFFAOYSA-N 0.000 claims description 2
- HJMRDUVCXLKHRV-UHFFFAOYSA-N o-(4-bromophenyl)hydroxylamine Chemical compound NOC1=CC=C(Br)C=C1 HJMRDUVCXLKHRV-UHFFFAOYSA-N 0.000 claims description 2
- FYFFQSVGQFTCMP-UHFFFAOYSA-N o-[(2,5-dichlorophenyl)methyl]hydroxylamine Chemical compound NOCC1=CC(Cl)=CC=C1Cl FYFFQSVGQFTCMP-UHFFFAOYSA-N 0.000 claims description 2
- WJWMBNQCKBRAHA-UHFFFAOYSA-N o-[(2-chlorophenyl)methyl]hydroxylamine Chemical compound NOCC1=CC=CC=C1Cl WJWMBNQCKBRAHA-UHFFFAOYSA-N 0.000 claims description 2
- OOJGDPODQAPHLV-UHFFFAOYSA-N o-[(2-methoxyphenyl)methyl]hydroxylamine Chemical compound COC1=CC=CC=C1CON OOJGDPODQAPHLV-UHFFFAOYSA-N 0.000 claims description 2
- MCNWHNCNFXEFLN-UHFFFAOYSA-N o-[(2-methylphenyl)methyl]hydroxylamine Chemical compound CC1=CC=CC=C1CON MCNWHNCNFXEFLN-UHFFFAOYSA-N 0.000 claims description 2
- OVMBDDNTFJQHST-UHFFFAOYSA-N o-[(3,4-dichlorophenyl)methyl]hydroxylamine Chemical compound NOCC1=CC=C(Cl)C(Cl)=C1 OVMBDDNTFJQHST-UHFFFAOYSA-N 0.000 claims description 2
- JDDDGXOQVRPABR-UHFFFAOYSA-N o-[(3,5-dichlorophenyl)methyl]hydroxylamine Chemical compound NOCC1=CC(Cl)=CC(Cl)=C1 JDDDGXOQVRPABR-UHFFFAOYSA-N 0.000 claims description 2
- NMYFFUUIFUDPRN-UHFFFAOYSA-N o-[(3-chlorophenyl)methyl]hydroxylamine Chemical compound NOCC1=CC=CC(Cl)=C1 NMYFFUUIFUDPRN-UHFFFAOYSA-N 0.000 claims description 2
- MVSMBIBGGPSEHQ-UHFFFAOYSA-N o-[(4-methoxyphenyl)methyl]hydroxylamine Chemical compound COC1=CC=C(CON)C=C1 MVSMBIBGGPSEHQ-UHFFFAOYSA-N 0.000 claims description 2
- TVLXOGBRADDHAM-UHFFFAOYSA-N o-[2-(trifluoromethyl)phenyl]hydroxylamine Chemical compound NOC1=CC=CC=C1C(F)(F)F TVLXOGBRADDHAM-UHFFFAOYSA-N 0.000 claims description 2
- OSGYPUBYTWDYMZ-UHFFFAOYSA-N o-[[4-(trifluoromethyl)phenyl]methyl]hydroxylamine Chemical compound NOCC1=CC=C(C(F)(F)F)C=C1 OSGYPUBYTWDYMZ-UHFFFAOYSA-N 0.000 claims description 2
- 229940075420 xanthine Drugs 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 abstract description 8
- 238000011109 contamination Methods 0.000 abstract description 2
- 238000002360 preparation method Methods 0.000 abstract description 2
- 239000000047 product Substances 0.000 description 15
- 108091033319 polynucleotide Proteins 0.000 description 8
- 102000040430 polynucleotide Human genes 0.000 description 8
- 239000002157 polynucleotide Substances 0.000 description 8
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 239000008346 aqueous phase Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 3
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- JDFUCZPRZYSQGR-KVQBGUIXSA-N [[(2r,3s,5r)-5-(2-amino-6-oxo-3h-purin-9-yl)-3-aminooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](ON)C[C@@H]1N1C(N=C(N)NC2=O)=C2N=C1 JDFUCZPRZYSQGR-KVQBGUIXSA-N 0.000 description 3
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 3
- 229910052794 bromium Inorganic materials 0.000 description 3
- 229910052801 chlorine Inorganic materials 0.000 description 3
- 239000000460 chlorine Substances 0.000 description 3
- 238000010511 deprotection reaction Methods 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 229910052731 fluorine Inorganic materials 0.000 description 3
- 239000011737 fluorine Substances 0.000 description 3
- 239000002777 nucleoside Substances 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 125000001424 substituent group Chemical group 0.000 description 3
- OLXZPDWKRNYJJZ-UHFFFAOYSA-N 5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-ol Chemical compound C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(CO)O1 OLXZPDWKRNYJJZ-UHFFFAOYSA-N 0.000 description 2
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 2
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 2
- CKRZKMFTZCFYGB-UHFFFAOYSA-N N-phenylhydroxylamine Chemical class ONC1=CC=CC=C1 CKRZKMFTZCFYGB-UHFFFAOYSA-N 0.000 description 2
- OFBQJSOFQDEBGM-UHFFFAOYSA-N Pentane Chemical compound CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 description 2
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- POCUPXSSKQAQRY-UHFFFAOYSA-N hydroxylamine;hydrate Chemical compound O.ON POCUPXSSKQAQRY-UHFFFAOYSA-N 0.000 description 2
- 238000000622 liquid--liquid extraction Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- -1 nucleoside triphosphates Chemical class 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 238000000638 solvent extraction Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- MXHRCPNRJAMMIM-ULQXZJNLSA-N 1-[(2r,4s,5r)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-tritiopyrimidine-2,4-dione Chemical compound O=C1NC(=O)C([3H])=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 MXHRCPNRJAMMIM-ULQXZJNLSA-N 0.000 description 1
- YKBGVTZYEHREMT-KVQBGUIXSA-N 2'-deoxyguanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](CO)O1 YKBGVTZYEHREMT-KVQBGUIXSA-N 0.000 description 1
- YKBGVTZYEHREMT-UHFFFAOYSA-N 2'-deoxyguanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1CC(O)C(CO)O1 YKBGVTZYEHREMT-UHFFFAOYSA-N 0.000 description 1
- IQFYYKKMVGJFEH-BIIVOSGPSA-N 2'-deoxythymidine Natural products O=C1NC(=O)C(C)=CN1[C@@H]1O[C@@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-BIIVOSGPSA-N 0.000 description 1
- CKTSBUTUHBMZGZ-SHYZEUOFSA-N 2'‐deoxycytidine Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 CKTSBUTUHBMZGZ-SHYZEUOFSA-N 0.000 description 1
- CKTSBUTUHBMZGZ-UHFFFAOYSA-N Deoxycytidine Natural products O=C1N=C(N)C=CN1C1OC(CO)C(O)C1 CKTSBUTUHBMZGZ-UHFFFAOYSA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- VECVOKKASOJPEY-SHYZEUOFSA-N [[(2r,3s,5r)-5-(4-amino-2-oxopyrimidin-1-yl)-3-aminooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](ON)C[C@@H]1N1C(=O)N=C(N)C=C1 VECVOKKASOJPEY-SHYZEUOFSA-N 0.000 description 1
- 125000005262 alkoxyamine group Chemical group 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- VGONTNSXDCQUGY-UHFFFAOYSA-N desoxyinosine Natural products C1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 VGONTNSXDCQUGY-UHFFFAOYSA-N 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 239000002920 hazardous waste Substances 0.000 description 1
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- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 239000003999 initiator Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- LVCDXCQFSONNDO-UHFFFAOYSA-N n-benzylhydroxylamine Chemical compound ONCC1=CC=CC=C1 LVCDXCQFSONNDO-UHFFFAOYSA-N 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 150000002923 oximes Chemical class 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
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- 239000011541 reaction mixture Substances 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
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- 239000000126 substance Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000000844 transformation Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/16—Purine radicals
- C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
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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
- C07H1/02—Phosphorylation
- C07H1/04—Introducing polyphosphoric acid radicals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/06—Pyrimidine radicals
- C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
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- 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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- Genetics & Genomics (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Saccharide Compounds (AREA)
- Cephalosporin Compounds (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
本发明涉及通过用芳基‑羟基胺处理将3’‑(N‑丙酮‑肟)‑2’‑脱氧核苷三磷酸转化为3’‑O‑胺‑2’‑脱氧核苷三磷酸来制备具有减少的3’‑羟基‑2’‑脱氧核糖核苷‑5’‑三磷酸污染的3’‑O‑氨基‑2’‑脱氧核糖核苷‑5’‑三磷酸的方法。
Description
近年来对多核苷酸合成的酶促方法的兴趣增加，不仅因为在许多领域例如合成生物学、基因修饰、高通量测序和分子诊断中对合成的多核苷酸的需求增加，而且因为多核苷酸合成的化学方法的限制，例如产物长度的上限以及有机溶剂的使用和需要的处置，Jensen et al,Biochemistry,57:1821-1832(2018)。酶促合成是有吸引力的，因为它具有专一性和效率，并且因为它使用温和的水相反应条件，这消除了处理有害废物的需要。
目前，大多数酶促方法采用无模板聚合酶将3’-O-封闭的核苷三磷酸重复添加到单链引发剂或与支持物连接的延长链，然后解封闭直到获得所需序列的多核苷酸，例如Jensen et al(上文引用的)。各种3’-O-封闭基团是可利用的，但特别感兴趣的是使用3’-氨基封闭基团，因为其尺寸小且易于裂解的条件，例如美国专利7544794和8212020。3’-O-氨基-2’-脱氧核糖核苷-5’-三磷酸的合成按照图1中所例示的方案，从相应的2’-脱氧核糖核苷开始。在保护5’-羟基后，通过在Mitsunobu条件下的两次随后的羟基转化，以“掩蔽的”(或“保护的”)形式作为N-羟基邻苯二甲酰亚胺部分提供O-氨基部分，其被插入在3’位置(Swamy et al,Chem.Rev.109:2551-2651(2009))。然后将核苷的3’-O-氨基和5’-羟基脱保护。然而，5’-羟基的三磷酸化需要再次保护3’-O-氨基，这是通过将其转化成肟来实现的，肟又在三磷酸化反应后通过用烷氧基胺处理而最终脱保护。然而，不幸的是，这种常规的脱保护方法进一步引起3’-O-胺的一些裂解。在多核苷酸合成中的酶促偶联反应中3’-羟基-2’-脱氧核苷三磷酸的存在允许双加成到生长的链上，以在多核苷酸产物中产生难以除去的“n+1”失效序列。
鉴于上述情况，如果可获得用于合成3’-O-氨基-2’-脱氧核苷三磷酸而不存在3’-羟基-2’-脱氧核苷三磷酸污染物的方法，则使用无模板聚合酶的多核苷酸的酶促合成将是先进的。
发明概述
本发明涉及用于制备具有减少的3’-羟基-2’-脱氧核糖核苷-5’-三磷酸污染的3’-O-氨基-2’-脱氧核糖核苷-5’-三磷酸的方法。本发明还包括通过本发明的方法制备的组合物。
在一些实施方案中，本发明的此类方法包括以下步骤：(a)保护2’-脱氧核糖核苷的5’-羟基；(b)将步骤(a)中获得的化合物的(S)-3’-羟基转化为(R)-3’-羟基；(c)使步骤(b)的产物与N-羟基邻苯二甲酰亚胺反应以产生5’-O-保护的-3’-O-邻苯二甲酰亚氨基-2’-脱氧核苷；(d)使步骤(c)的产物的5’-羟基脱保护；(e)将步骤(d)的产物转化为3’-O-(N-丙酮-肟)-2’-脱氧核苷；(f)将步骤(e)的产物的5’-羟基三磷酸化以获得3’-O-(N-丙酮-肟)-2’-脱氧核苷三磷酸；和(g)通过用芳基-羟基胺处理步骤(f)的产物，将步骤(f)的所述产物转化为3’-O-胺-2’-脱氧核苷三磷酸。。
在一些实施方案中，这样的组合物包含含有3’-O-氨基-2’-脱氧核糖核苷-5’-三磷酸和浓度小于2体积％的芳基-羟基胺的水溶液。
在许多所例示的实施方式和应用中举例说明本发明的这些上文表征的方面以及其他方面，其中一些在附图中示出并且在随后的权利要求部分中表征。然而，以上概述并非旨在描述本发明的每个所例示的实施方案或每个实施方式。
附图的简要说明
图1示例性说明了用于制备3’-O-胺-2’-脱氧核苷三磷酸单体的常规合成方案。
图2示例性说明了使用苄基羟胺将3’-丙酮-肟-核苷-三磷酸转化为或使其脱保护为3’-ONH2-核苷-三磷酸的本发明的实施方案。
图3A是实施例1中描述的粗反应产物的色谱图。
图3B是实施例1的所需的3’-O-氨基-2’-脱氧鸟苷-5’-三磷酸产物的色谱图。
图4例示出根据本发明的3’-O-氨基-2’-脱氧核糖核苷-5’-三磷酸的碱的实例，其中Z是2’-脱氧核糖-5’-三磷酸的1’碳，R是H、CH3或连接基团，以及X是N或C-R。
发明的详细说明
本发明的一般原理在本文中更详细地公开，特别是通过实例的方式，如附图中所示和详细描述的那些。然而，应当理解，意图不是将本发明限制为所描述的特定实施方案。本发明适用于各种修改和替代形式，其细节针对若干实施方案示出。意图是涵盖落入本发明的原理和范围内的所有修改、等同物和替代物。。
本发明涉及用于制备3’-O-氨基-2’-脱氧核糖核苷-5’-三磷酸的方法，该方法使用以下通用步骤：
(a)保护2’-脱氧核糖核苷的5’-羟基；
(b)将步骤(a)中获得的化合物的(S)-3’-羟基转化为(R)-3’-羟基；
(c)使步骤(b)的产物与N-羟基邻苯二甲酰亚胺反应以产生5’-O-保护的-3’-O-邻苯二甲酰亚氨基-2’-脱氧核苷；
(d)使步骤(c)的产物的5’-羟基脱保护；
(e)将步骤(d)的产物转化为3’-O-(N-丙酮-肟)-2’-脱氧核苷；
(f)将步骤(e)的产物的5’-羟基三磷酸化以获得3’-O-(N-丙酮-肟)-2’-脱氧核苷三磷酸；和
(g)将步骤(f)的所述产物转化为3’-O-胺-2’-脱氧核苷三磷酸。
在基团或部分之前的术语“(S)”或“(R)”，例如在“(R)-3’-羟基”中的(R)，是指被所述基团取代的碳的绝对构型。碳的位置也可以遵照术语“(S)”或“(R)”。在“(R)-3’-羟基”中，被羟基取代的3’位的碳具有R绝对构型。
腺嘌呤、鸟嘌呤、胞嘧啶、胸腺嘧啶和尿嘧啶的2’-脱氧核苷分别是2’-脱氧腺苷、2’-脱氧鸟苷、2’-脱氧胞苷、2’-脱氧胸苷和2’-脱氧尿苷。
在以下参考文献中公开了上述通用步骤以及许多具体实施方案：DeClercq etal,欧洲专利申请EP0381335；Kondo et al,Symp.Nucleic Acids Chem.,16:93-96(1985)；Burgess et al,J.Chem.Soc.Chem.Comm.,8:915-916(1994)；Hutter et al,Nucleosides,Nucleotides&Nucleic Acids,29(11):879-895(2010)；Sarac et al,国际专利公开WO2020/043846；和U.S.专利7544794；8034923；8212020；和10472383。
根据本发明，通过用芳基-羟基胺处理，将步骤(f)的3’-O-(N-丙酮-肟)-2’-脱氧核苷三磷酸转化为3’-O-胺-2’-脱氧核苷三磷酸，如图2所例示。在一些实施方案中，芳基-羟基胺是未取代的或取代的O-苄基羟胺或取代的或未取代的苯基羟胺。示例性的取代基包括甲氧基、硝基、卤素，其中特别地，卤素是氟、氯或溴。示例性的取代的O-苄基羟胺包括但不限于O-(2,3,4,5,6-五氟苄基)羟胺和O-(4-硝基苄基)羟胺。在一个实施方案中，用于本发明方法的芳基-羟基胺是O-苄基羟胺。在另一个实施方案中，用于本发明方法的芳基-羟基胺是取代或未取代的苯基羟胺。如上所述，示例性的取代基包括甲氧基、硝基、卤素，其中特别地卤素是氟、氯或溴。转化步骤(g)可以使用芳基-羟基胺的水溶液进行。在一些实施方案中，嘧啶3’-O-(N-丙酮-肟)-2’-脱氧核苷三磷酸在pH 6.8-pH 7.0下被转化或脱保护以形成3’-O-胺-2’-脱氧核苷三磷酸。在一些实施方案中，嘌呤3’-O-(N-丙酮-肟)-2’-脱氧核苷三磷酸在pH 6.0-pH 6.8下被转化或脱保护以形成3’-O-胺-2’-脱氧核苷三磷酸。在一些实施方案中，对于这样的转化，使用一定浓度的芳基-羟基胺，其浓度足够高以引起转化但浓度足够低以不需要在用于多核苷酸合成之前纯化3’-O-胺-2’-脱氧核苷三磷酸。在一些实施方案中，芳基-羟基胺的这种浓度为1％至100％(v/v)。在一些实施方案中，芳基-羟基胺的这种浓度为1％至2％(v/v)。在其它实施方案中，芳基-羟基胺的这种浓度小于2％(v/v)。在其它实施方案中，芳基-羟基胺的这种浓度小于1％(v/v)。
在一些实施方案中，用于本发明方法的芳基-羟基胺包括取代或未取代的O-苄基羟胺、取代或未取代的O-苯基羟胺、取代或未取代的O-苯基乙基羟胺、取代或未取代的O-苄氧基乙基羟胺和取代或未取代的O-苯氧基乙基羟胺，其中取代基选自卤素、甲氧基、硝基。在一些实施方案中，卤素是氟、氯或溴。在一些实施方案中，用于本发明方法的芳基-羟基胺包括但不限于O-苄基羟胺、O-苯基羟胺、O-[(2-甲氧基苯基)甲基]羟胺、O-[(3,5-二氯苯基)甲基]羟胺、O-(3-氯苄基)羟胺、O-(4-硝基苄基)羟胺、O-(4-甲氧基苄基)羟胺、O-[(3,4-二氯苯基)甲基]羟胺，O-(2-氯苄基)羟胺，O-[4-(三氟甲基)苄基]羟胺，O-[(2,5-二氯苯基)甲基]羟胺，O-(2-甲基苄基)羟胺，O-(2,3,4,5,6-五氟苄基)羟胺，O-(4-硝基苄基)羟胺，O-(2-(苄氧基)乙基)羟胺，O-(4-溴苯基)羟胺，O-[2-(三氟甲基)苯基]羟胺或O-(2-苯氧基乙基)羟胺。
本发明的目的是还提供组合物，其包含含有3’-O-氨基-2’-脱氧核糖核苷-5’-三磷酸和浓度小于2体积％的芳基-羟基胺的水溶液。在一个具体的实施方案中，所述芳基-羟基胺是O-苄基羟胺或O-苯基羟胺。特别地，所述3’-O-氨基-2’-脱氧核糖核苷-5’-三磷酸可以具有选自腺嘌呤、胸腺嘧啶、鸟嘌呤、胞嘧啶、尿嘧啶、肌苷、黄嘌呤或次黄嘌呤的碱。在一个实施方案中，3’-O-氨基-2’-脱氧核糖核苷-5’-三磷酸具有选自图4所例示碱的碱。有利地，连接基团是烷基接头、烯基接头、硫醚接头、氨基烷基、氨基烯丙基、叠氮基烷基、叠氮基烯丙基炔丙基胺、双炔丙基或双炔丙基-PEG。
实施例1：嘌呤核碱基(A,G)3’-O-氨基-2’-脱氧鸟苷-5’-三磷酸的脱保护
向3’-O-(N-丙酮-肟)-2’-脱氧鸟苷-5’-三磷酸(5.0mL,5.0mmol,100mM储备溶液)的溶液中加入水5.0mL和O-苄基羟胺(6.2mL,50mmol)，通过加入10％w/w的乙酸水溶液将pH调节至6.5。将反应搅拌6小时。用乙醚(3×50mL)萃取水相。将水相冷冻并冷冻干燥1小时。图3A是在2小时后上述粗反应混合物的色谱图。通过用有机溶剂液-液萃取除去过量的O-苄基羟胺。3’-ONH2-核苷三磷酸的水性组合物可能含有少量的O-苄基羟胺、乙酸钠和氯化钠。图3B是3’-O-氨基-2’-脱氧鸟苷-5’-三磷酸产物的HPLC色谱图。实施例中的液-液萃取溶剂是乙醚，但也可以是任何有机溶剂，例如甲基叔丁基醚、二氯甲烷、甲苯、戊烷等及它们的混合物。
实施例2：嘧啶核碱基(T,C)3’-O-氨基-2’-脱氧胞苷-5’-三磷酸的脱保护
向3’-O-(N-丙酮-肟)-2’-脱氧胞苷-5’-三磷酸(5.0mL,5.0mmol,100mM储备溶液)的溶液中加入水5.0mL和O-苄基羟胺(6.2mL,50mmol)，通过加入10％w/w的乙酸水溶液将pH调节至6.8。将反应搅拌6小时。用乙醚(3×50mL)萃取水相。将水相冷冻并冷冻干燥1小时。
Claims (9)
1.制备3’-O-氨基-2’-脱氧核糖核苷-5’-三磷酸的方法，包括以下步骤：
(a)保护2’-脱氧核糖核苷的5’-羟基；
(b)将步骤(a)中获得的化合物的(S)-3’-羟基转化为(R)-3’-羟基；
(c)使步骤(b)的产物与N-羟基邻苯二甲酰亚胺反应以产生5’-O-保护的-3’-O-邻苯二甲酰亚氨基-2’-脱氧核苷；
(d)使步骤(c)的产物的5’-羟基脱保护；
(e)将步骤(d)的产物转化为3’-O-(N-丙酮-肟)-2’-脱氧核苷；
(f)将步骤(e)的产物的5’-羟基三磷酸化以获得3’-O-(N-丙酮-肟)-2’-脱氧核苷三磷酸；和
(g)通过用芳基-羟基胺处理步骤(f)的产物，将步骤(f)的所述产物转化为3’-O-胺-2’-脱氧核苷三磷酸。
2.如权利要求1所述的方法，其中所述芳基-羟基胺是取代或未取代的O-苄基羟胺、取代或未取代的O-苯基羟胺、取代或未取代的O-苯基乙基羟胺、取代或未取代的O-苄氧基乙基羟胺和取代或未取代的O-苯氧基乙基羟胺，其中所述芳基-羟基胺被卤素、甲氧基或硝基取代。
3.如权利要求2所述的方法，其中所述芳基-羟基胺选自O-苄基羟胺、O-苯基羟胺、O-[(2-甲氧基苯基)甲基]羟胺、O-[(3，5-二氯苯基)甲基]羟胺、O-(3-氯苄基)羟胺、O-(4-硝基苄基)羟胺、O-(4-甲氧基苄基)羟胺、O-[(3，4-二氯苯基)甲基]羟胺、O-(2-氯苄基)羟胺、O-[4-(三氟甲基)苄基]羟胺、O-[(2，5-二氯苯基)甲基]羟胺、O-(2-甲基苄基)羟胺、O-(2，3，4，5，6-五氟苄基)羟胺、O-(4-硝基苄基)羟胺、O-(2-(苄氧基)乙基)羟胺、O-(4-溴苯基)羟胺、O-[2-(三氟甲基)苯基]羟胺和O-(2-苯氧基乙基)羟胺。
4.如权利要求3所述的方法，其中所述芳基-羟基胺是O-苄基羟胺或O-苯基羟胺。
5.组合物，其包含含有3’-O-氨基-2’-脱氧核糖核苷-5’-三磷酸和浓度小于2体积％的芳基-羟基胺的水溶液。
6.如权利要求5所述的组合物，其中所述芳基-羟基胺是O-苄基羟胺或O-苯基羟胺。
7.如权利要求5或6所述的组合物，其中所述3’-O-氨基-2’-脱氧核糖核苷-5’-三磷酸具有选自腺嘌呤、胸腺嘧啶、鸟嘌呤、胞嘧啶、尿嘧啶、肌苷、黄嘌呤或次黄嘌呤的碱。
8.如权利要求5至7中任一项所述的组合物，其中所述3’-O-氨基-2’-脱氧核糖核苷-5’-三磷酸具有选自以下的碱：
其中Z是2’-脱氧核糖-5’-三磷酸的1’碳，R是H、CH3或连接基团，以及X是N或C-R。
9.如权利要求8所述的组合物，其中所述连接基团是烷基接头、烯基接头、硫醚接头、氨基烷基、氨基烯丙基、叠氮基烷基、叠氮基烯丙基炔丙基胺、双炔丙基或双炔丙基-PEG。
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| WO2019097233A1 (en) * | 2017-11-14 | 2019-05-23 | Nuclera Nucleics Ltd | Nucleotide derivatives containing amine masked moieties and their use in a templated and non-templated enzymatic nucleic acid synthesis |
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| GB8902041D0 (en) | 1989-01-31 | 1989-03-22 | Tanabe Seiyaku Co | Antiviral agent and novel nucleoside |
| US7544794B1 (en) | 2005-03-11 | 2009-06-09 | Steven Albert Benner | Method for sequencing DNA and RNA by synthesis |
| WO2020043846A1 (en) | 2018-08-31 | 2020-03-05 | Dna Script | Method for preparing 3'-o-amino-2'-deoxyribonucleoside-5'-triphosphate |
| CN114728996B (zh) * | 2019-09-10 | 2022-11-29 | 加利福尼亚太平洋生物科学股份有限公司 | 核苷酸的可逆修饰 |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110275124A1 (en) * | 2005-03-11 | 2011-11-10 | Benner Steven A | Reagents for reversibly terminating primer extension |
| US8034923B1 (en) * | 2009-03-27 | 2011-10-11 | Steven Albert Benner | Reagents for reversibly terminating primer extension |
| US20180265537A1 (en) * | 2017-03-16 | 2018-09-20 | Steven A. Benner | Nucleoside Triphosphates with Stable Aminoxy Groups |
| WO2019097233A1 (en) * | 2017-11-14 | 2019-05-23 | Nuclera Nucleics Ltd | Nucleotide derivatives containing amine masked moieties and their use in a templated and non-templated enzymatic nucleic acid synthesis |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117143942A (zh) * | 2023-10-30 | 2023-12-01 | 泰兴合全药业有限公司 | 3’-氨基-2’,3’-双脱氧鸟苷5’-三磷酸的合成方法 |
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| IL296814A (en) | 2022-11-01 |
| US11661436B2 (en) | 2023-05-30 |
| US20210300961A1 (en) | 2021-09-30 |
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| CA3172698A1 (en) | 2021-10-07 |
| KR20220160076A (ko) | 2022-12-05 |
| AU2021246815B2 (en) | 2023-09-14 |
| JP2023519395A (ja) | 2023-05-10 |
| WO2021198040A1 (en) | 2021-10-07 |
| EP4126895A1 (en) | 2023-02-08 |
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