CN108610360A - Phosphoramidite and its preparation method and application - Google Patents
Phosphoramidite and its preparation method and application Download PDFInfo
- Publication number
- CN108610360A CN108610360A CN201810363792.7A CN201810363792A CN108610360A CN 108610360 A CN108610360 A CN 108610360A CN 201810363792 A CN201810363792 A CN 201810363792A CN 108610360 A CN108610360 A CN 108610360A
- Authority
- CN
- China
- Prior art keywords
- phosphoramidite
- ether
- reaction
- hydroxyl
- dimethoxytrityls
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 150000008300 phosphoramidites Chemical class 0.000 title claims abstract description 40
- 238000002360 preparation method Methods 0.000 title claims abstract description 34
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 51
- 238000006243 chemical reaction Methods 0.000 claims abstract description 42
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 23
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 19
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 19
- -1 cyanoethyls N, N diisopropylphosphoramidite ester Chemical class 0.000 claims abstract description 17
- LQNUZADURLCDLV-UHFFFAOYSA-N nitrobenzene Substances [O-][N+](=O)C1=CC=CC=C1 LQNUZADURLCDLV-UHFFFAOYSA-N 0.000 claims abstract description 15
- 230000032050 esterification Effects 0.000 claims abstract description 10
- 238000005886 esterification reaction Methods 0.000 claims abstract description 10
- 239000007858 starting material Substances 0.000 claims abstract description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 28
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 22
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 21
- WVDDGKGOMKODPV-UHFFFAOYSA-N hydroxymethyl benzene Natural products OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 claims description 15
- 238000003756 stirring Methods 0.000 claims description 15
- 229910052757 nitrogen Inorganic materials 0.000 claims description 14
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 14
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical class OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 claims description 12
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 11
- 239000012043 crude product Substances 0.000 claims description 11
- YDNNTXXWYHETTD-UHFFFAOYSA-N 1-methoxy-4-[methoxy-(4-methoxyphenyl)-phenylmethyl]benzene Chemical compound C1=CC(OC)=CC=C1C(OC)(C=1C=CC(OC)=CC=1)C1=CC=CC=C1 YDNNTXXWYHETTD-UHFFFAOYSA-N 0.000 claims description 8
- JHSWKMHTUVSEOQ-UHFFFAOYSA-N 1-[2-chloro-1-(4-methoxyphenyl)-1-phenylethyl]-4-methoxybenzene Chemical class C1=CC(OC)=CC=C1C(CCl)(C=1C=CC(OC)=CC=1)C1=CC=CC=C1 JHSWKMHTUVSEOQ-UHFFFAOYSA-N 0.000 claims description 7
- 150000001875 compounds Chemical class 0.000 claims description 7
- 238000006266 etherification reaction Methods 0.000 claims description 7
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 7
- 235000019445 benzyl alcohol Nutrition 0.000 claims description 6
- 239000012074 organic phase Substances 0.000 claims description 6
- 239000000047 product Substances 0.000 claims description 6
- 238000000746 purification Methods 0.000 claims description 6
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- 238000002390 rotary evaporation Methods 0.000 claims description 5
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 5
- 125000002103 4,4'-dimethoxytriphenylmethyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)(C1=C([H])C([H])=C(OC([H])([H])[H])C([H])=C1[H])C1=C([H])C([H])=C(OC([H])([H])[H])C([H])=C1[H] 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 4
- 239000003960 organic solvent Substances 0.000 claims description 4
- FYBOFPSQYFXGLL-UHFFFAOYSA-N C1=CC(OC)=CC=C1C(C=1C=CC(OC)=CC=1)(C=1C=CC=CC=1)OCC1=CC=CC=C1 Chemical compound C1=CC(OC)=CC=C1C(C=1C=CC(OC)=CC=1)(C=1C=CC=CC=1)OCC1=CC=CC=C1 FYBOFPSQYFXGLL-UHFFFAOYSA-N 0.000 claims description 3
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 238000001704 evaporation Methods 0.000 claims description 3
- 239000002994 raw material Substances 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- KCXMKQUNVWSEMD-UHFFFAOYSA-N benzyl chloride Chemical class ClCC1=CC=CC=C1 KCXMKQUNVWSEMD-UHFFFAOYSA-N 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims description 2
- QUSNBJAOOMFDIB-UHFFFAOYSA-N Ethylamine Chemical compound CCN QUSNBJAOOMFDIB-UHFFFAOYSA-N 0.000 claims 1
- 150000002240 furans Chemical class 0.000 claims 1
- JBWKIWSBJXDJDT-UHFFFAOYSA-N triphenylmethyl chloride Chemical compound C=1C=CC=CC=1C(C=1C=CC=CC=1)(Cl)C1=CC=CC=C1 JBWKIWSBJXDJDT-UHFFFAOYSA-N 0.000 claims 1
- 238000003786 synthesis reaction Methods 0.000 abstract description 10
- 230000015572 biosynthetic process Effects 0.000 abstract description 9
- 230000004048 modification Effects 0.000 abstract description 6
- 238000012986 modification Methods 0.000 abstract description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract description 5
- 125000002887 hydroxy group Chemical group [H]O* 0.000 abstract description 5
- 238000007306 functionalization reaction Methods 0.000 abstract description 3
- 238000001668 nucleic acid synthesis Methods 0.000 abstract description 2
- 239000007787 solid Substances 0.000 abstract description 2
- BWRBVBFLFQKBPT-UHFFFAOYSA-N (2-nitrophenyl)methanol Chemical compound OCC1=CC=CC=C1[N+]([O-])=O BWRBVBFLFQKBPT-UHFFFAOYSA-N 0.000 abstract 1
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 3
- 238000005481 NMR spectroscopy Methods 0.000 description 3
- 150000001412 amines Chemical class 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
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- 210000003705 ribosome Anatomy 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 239000000908 ammonium hydroxide Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000012620 biological material Substances 0.000 description 2
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N dimethyl sulfoxide Natural products CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
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- 238000001415 gene therapy Methods 0.000 description 2
- JMANVNJQNLATNU-UHFFFAOYSA-N glycolonitrile Natural products N#CC#N JMANVNJQNLATNU-UHFFFAOYSA-N 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
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- 238000004519 manufacturing process Methods 0.000 description 2
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- 102000004169 proteins and genes Human genes 0.000 description 2
- 150000003222 pyridines Chemical class 0.000 description 2
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- 239000007790 solid phase Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- YJTKZCDBKVTVBY-UHFFFAOYSA-N 1,3-Diphenylbenzene Chemical group C1=CC=CC=C1C1=CC=CC(C=2C=CC=CC=2)=C1 YJTKZCDBKVTVBY-UHFFFAOYSA-N 0.000 description 1
- ASJSAQIRZKANQN-CRCLSJGQSA-N 2-deoxy-D-ribose Chemical group OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 description 1
- 241000023308 Acca Species 0.000 description 1
- 108091023037 Aptamer Proteins 0.000 description 1
- DRSHXJFUUPIBHX-UHFFFAOYSA-N COc1ccc(cc1)N1N=CC2C=NC(Nc3cc(OC)c(OC)c(OCCCN4CCN(C)CC4)c3)=NC12 Chemical compound COc1ccc(cc1)N1N=CC2C=NC(Nc3cc(OC)c(OC)c(OCCCN4CCN(C)CC4)c3)=NC12 DRSHXJFUUPIBHX-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- FKLJPTJMIBLJAV-UHFFFAOYSA-N Compound IV Chemical compound O1N=C(C)C=C1CCCCCCCOC1=CC=C(C=2OCCN=2)C=C1 FKLJPTJMIBLJAV-UHFFFAOYSA-N 0.000 description 1
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- FIWILGQIZHDAQG-UHFFFAOYSA-N NC1=C(C(=O)NCC2=CC=C(C=C2)OCC(F)(F)F)C=C(C(=N1)N)N1N=C(N=C1)C1(CC1)C(F)(F)F Chemical compound NC1=C(C(=O)NCC2=CC=C(C=C2)OCC(F)(F)F)C=C(C(=N1)N)N1N=C(N=C1)C1(CC1)C(F)(F)F FIWILGQIZHDAQG-UHFFFAOYSA-N 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 206010034960 Photophobia Diseases 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
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- NEHMKBQYUWJMIP-UHFFFAOYSA-N chloromethane Chemical class ClC NEHMKBQYUWJMIP-UHFFFAOYSA-N 0.000 description 1
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- NLFBCYMMUAKCPC-KQQUZDAGSA-N ethyl (e)-3-[3-amino-2-cyano-1-[(e)-3-ethoxy-3-oxoprop-1-enyl]sulfanyl-3-oxoprop-1-enyl]sulfanylprop-2-enoate Chemical compound CCOC(=O)\C=C\SC(=C(C#N)C(N)=O)S\C=C\C(=O)OCC NLFBCYMMUAKCPC-KQQUZDAGSA-N 0.000 description 1
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- 238000001254 matrix assisted laser desorption--ionisation time-of-flight mass spectrum Methods 0.000 description 1
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 1
- 229910000474 mercury oxide Inorganic materials 0.000 description 1
- UKWHYYKOEPRTIC-UHFFFAOYSA-N mercury(ii) oxide Chemical compound [Hg]=O UKWHYYKOEPRTIC-UHFFFAOYSA-N 0.000 description 1
- GRVDJDISBSALJP-UHFFFAOYSA-N methyloxidanyl Chemical compound [O]C GRVDJDISBSALJP-UHFFFAOYSA-N 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- XUZLXCQFXTZASF-UHFFFAOYSA-N nitro(phenyl)methanol Chemical compound [O-][N+](=O)C(O)C1=CC=CC=C1 XUZLXCQFXTZASF-UHFFFAOYSA-N 0.000 description 1
- 230000005311 nuclear magnetism Effects 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 239000003504 photosensitizing agent Substances 0.000 description 1
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- 239000000843 powder Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000011403 purification operation Methods 0.000 description 1
- RDRCCJPEJDWSRJ-UHFFFAOYSA-N pyridine;1h-pyrrole Chemical compound C=1C=CNC=1.C1=CC=NC=C1 RDRCCJPEJDWSRJ-UHFFFAOYSA-N 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
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- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
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- 239000003643 water by type Substances 0.000 description 1
- 229910052724 xenon Inorganic materials 0.000 description 1
- FHNFHKCVQCLJFQ-UHFFFAOYSA-N xenon atom Chemical compound [Xe] FHNFHKCVQCLJFQ-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/22—Amides of acids of phosphorus
- C07F9/24—Esteramides
- C07F9/2404—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic
- C07F9/242—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic of hydroxyaryl compounds
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Saccharide Compounds (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
A kind of phosphoramidite of present invention proposition and its preparation method and application, the phosphoramidite includes using 5 hydroxyl, 2 nitrobenzyl alcohol as starting material, it is etherified that 5 hydroxyl, 2 nitrobenzene methyl (4 is obtained by the reaction, 4' dimethoxytrityls) after ether, 2 nitrobenzene methyl (4 are prepared through esterification again, 4' dimethoxytrityls) ether 5 (2 O cyanoethyls N, N diisopropylphosphoramidite ester).Wherein phosphoramidite can be used for preparing the nucleic acid containing photosensitive unit.The phosphoramidite of the present invention shows good stability in room temperature and be protected from light under the conditions of, delicate structure, synthesis are simple, photoresponse is efficient, and it can be directly used for the functional modification in nucleic acid synthesis in solid state, nucleic acid sequence arbitrary site introduce photosensitive unit, for nucleic acid functionalization provide one kind being more concisely and efficiently selection.
Description
Technical field
The invention belongs to chemosynthesis technical fields more particularly to a kind of phosphoramidite and its preparation method and application.
Background technology
With the development of biotechnology, it is bio-based materials design, gene inspection to be functionalized to natural biological macromolecular
The important means in the fields such as survey and gene therapy.It is led in the research of poly-nuclear ribosomal ribonucleic acid (RNA) and poly- DNA (DNA)
In domain, the introducing of non-enzymatic cut-out connection unit is to carry out aptamers screening, gene therapy, protein engineering and biomaterial to set
The Basic Design of meter.The controllable of nucleic acid sequence is breaking at molecular biology, highly sensitive detection technique, protein engineering and novel
The fields such as biomaterial structure have important theory significance and application value.In recent years it has been developed that a variety of can be in physics
Or the connection reagent of degradation is generated under chemical stimulation.
Biologist and material scholar have had devised a variety of non-digestion connection units at present, and achieve preferable application
Effect.Wherein the degradable connection unit of light (photolabile linker) is a kind of degradation unit received significant attention, and
Traditional chemical degradation unit is different, and light degradable unit does not introduce other chemical compositions when degrading, and reduces and draws in system
Enter the possibility of unpredictable factor；Wavelength, intensity of illumination and the time of light source are controllable simultaneously, have in practical operation
Great flexibility, is a kind of connection reagent with larger market value, such reagent has as follows compared with other reagents
Advantage：Pollution is hardly introduced using process, operating flexibility is higher, and it is larger that space may be selected in light source.
Since last century is over 90 years, the artificial synthesized technology of nucleic acid is ripe and market-oriented, can be used for functional nucleic acid
The degradable unit of light of preparation is also constantly reported.People can carry out photodegradative 2- nitrobenzyls in ultraviolet and visible region light
Base is chemically modified in its foundation structure as the degradable unit of light so that it being automatically synthesized for nucleic acid.But mesh
The degradable reagent of light that uses has that synthesis step is cumbersome, isolates and purifies difficult and valence in preceding commercialization and document patent
The problems such as lattice are high limits its further application.
Invention content
The present invention for it is above-mentioned the technical issues of, propose a kind of phosphoramidite and its preparation method and application.
In order to achieve the above object, the technical solution adopted by the present invention is：
A kind of phosphoramidite, shown in the structural formula as I of the compound：
A kind of preparation method of above-mentioned phosphoramidite, including using 5- hydroxyl -2- nitrobenzyl alcohols as starting material, by it
Benzyl alcohol hydroxyl is etherified that 5- hydroxyl -2- nitros are obtained by the reaction by 4,4 '-dimethoxytrityl chloromethanes selective protections
After benzyl-(4,4'- dimethoxytrityl) ether, 2- nitrobenzenes methyl-(4,4'- dimethoxies are prepared through esterification
Base triphenyl) ether -5- (2-O- cyanoethyls-N, N- diisopropylphosphoramidite ester).
As preferred：The etherification reaction specifically includes following steps：5- hydroxyl -2- nitrobenzyl alcohols are taken to be dissolved in anhydrous pyrrole
Pyridine, the anhydrous pyridine being added dropwise under ice bath stirring dissolved with 4,4'- dimethoxytrityl chloromethanes obtains reaction solution, to the end of reaction
Evaporation organic phase obtains crude product afterwards, and the purified processing of crude product obtains 5- hydroxyl -2- nitrobenzenes methyl-(4,4'- diformazans
Oxygroup triphenyl) ether.
As preferred：The reaction condition of the etherification reaction is：The reaction solution stirs 1 hour at 0 DEG C, is restored to room
Temperature is stirred overnight；The mass ratio 1.7 of the 5- hydroxyls -2- nitrobenzyl alcohols and the 4,4'- dimethoxytrityls chloromethanes：
4；It is dissolved in a concentration of 0.017g/mL of the 5- hydroxyl -2- nitrobenzyl alcohols in the anhydrous pyridine；Dissolved with 5- hydroxyl -2- nitros
The volume ratio of the anhydrous pyridine of benzyl alcohol and the anhydrous pyridine dissolved with 4,4'- dimethoxytrityl chloromethanes is 10:3.
As preferred：The purification processes specifically include following steps：The crude product is taken to be dissolved in ethyl acetate, through water
It washes, dry, filtering, after rotary evaporation removing organic solvent, product is dissolved in ethyl acetate, cooling n-hexane is added dropwise
In, it filters, be dried to obtain 5- hydroxyl -2- nitrobenzenes methyl-(4,4'- dimethoxytrityl) ether.
As preferred：The esterification includes with 5- hydroxyl -2- nitrobenzenes methyl-(4,4'- dimethoxytrityls)
Ether is that raw material prepares 2- nitrobenzenes methyl-(4,4'- diformazans with the reaction of 2-O- cyanoethyls-N, N- diisopropyl chloro phosphoramidite
Oxygroup triphenyl) ether -5- (2-O- cyanoethyls-N, N- diisopropylphosphoramidite ester) the step of.
As preferred：Take 5- hydroxyls -2- nitrobenzenes methyl-(4,4'- dimethoxytrityls) ether and anhydrous three second
Amine is dissolved in anhydrous tetrahydro furan, and 2-O- cyanoethyls-N, N- diisopropyl chloro phosphoramidite, reaction knot are added dropwise under ice bath stirring
Be filtered, washed after beam, dry after obtain 2- nitrobenzenes methyl-(4,4'- dimethoxytrityls) ether -5- (2-O- cyanoethyl-N,
N- diisopropylphosphoramidites ester).
As preferred：The reaction condition of the esterification is：It stirs 2 hours at room temperature；5- hydroxyls -2- the nitros
Benzyl-(4,4'- dimethoxytrityls) ether, anhydrous triethylamine and 2-O- cyanoethyls-N, N- diisopropyl chloro phosphorous acyl
The mass ratio of amine is 2.3:1.2:1.27, a concentration of 0.024g/ml of anhydrous triethylamine described in the anhydrous tetrahydro furan.
As preferred：It is described to filter insoluble matter after reaction, it takes filtrate to be washed using saturated sodium bicarbonate solution, is added
Anhydrous sodium sulfate is dried to obtain 2- nitrobenzenes methyl-(4,4'- dimethoxytrityls) ether -5-, and (2-O- cyanoethyls-N, N- bis- is different
Phosphoramidite ester).
A kind of application of above-mentioned phosphoramidite in preparing the nucleic acid containing photosensitive unit.
Compared with prior art, the advantages and positive effects of the present invention are：
1, phosphoramidite of the invention shows good stability in room temperature and be protected from light under the conditions of, delicate structure,
Synthesis is simple, and photoresponse is efficient, and can be directly used for the functional modification in nucleic acid synthesis in solid state, in the arbitrary of nucleic acid sequence
Site introduce photosensitive unit, for nucleic acid functionalization provide one kind being more concisely and efficiently selection；
2, the preparation method of phosphoramidite provided by the invention is simple to operation, is greatly reducing the same of synthesis cost
When remain the degradable high efficiency of light, prepare 2- nitros by the organism unit reaction of standards such as being simply etherified and being esterified
Benzyl-(4,4'- dimethoxytrityl) ether -5- (2-O- cyanoethyls-N, N- diisopropylphosphoramidite ester), in having simplified
Mesosome isolates and purifies, and simplifies the operating procedure of experiment, improves the yield of final product, final product is made to can be directly used for
On nucleic acid automatic synthesizer, there is preferable substrate adaptability, greatly simplify modification step；Show good light simultaneously
Response.This design provides a kind of design, preparation and the application processes of new photoresponse connection unit, are greatlying simplify
While organic synthesis operates, enrich the type of the connection reagent of photoresponse in nucleic acid and nucleic acid sill, be nucleic acid end group and
Photosensitizer modification provides a kind of simple and direct efficient method in sequence, has preferable market value.
3, phosphoramidite of the invention can be used for preparing the DNA containing photosensitive unit, experiment show its
With very high reactivity, good substrate adaptability, enough chemical stabilities and good optical Response when preparation,
A kind of method for being more concisely and efficiently selection, simplifying nucleic acid of the preparation containing photosensitive unit is provided for the functionalization of nucleic acid,
Its manufacturing cost is reduced, the market application for the nucleic acid for preparing photosensitive unit is expanded.
Description of the drawings
Fig. 1 is the hydrogen nuclear magnetic resonance spectrogram of phosphoramidite of the present invention；
Fig. 2 is the carbon-13 nmr spectra figure of phosphoramidite of the present invention；
Fig. 3 is the mass spectrogram of phosphoramidite of the present invention；
Fig. 4 is the mass spectrogram of the DNA of the invention containing photosensitive unit；
Fig. 5 is the mass spectrogram of the DNA after obtained light degradation after illumination of the present invention；
Fig. 6 is the mass spectrogram of DNA of the present invention without photosensitive unit；
Fig. 7 is the HPLC spectrograms of the DNA of the invention containing photosensitive unit；
Fig. 8 is the HPLC spectrograms of the DNA after obtained light degradation after illumination of the present invention；
Fig. 9 is the HPLC spectrograms of the control DNA after illumination of the present invention.
Specific implementation mode
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
The every other embodiment that technical staff is obtained without making creative work belongs to the model that the present invention protects
It encloses.
An embodiment of the present invention provides a kind of phosphoramidites, shown in the structural formula as I of the compound：
The present embodiment also provides a kind of preparation method of above-mentioned phosphoramidite, including with 5- hydroxyl -2- nitrobenzyl alcohols
It is etherified to react by its benzyl alcohol hydroxyl by 4,4 '-dimethoxytrityl chloromethanes selective protections for starting material
To after 5- hydroxyl -2- nitrobenzenes methyl-(4,4'- dimethoxytrityl) ether, 2- nitrobenzoyls are prepared through esterification
Base-(4,4'- dimethoxytrityls) ether -5- (2-O- cyanoethyls-N, N- diisopropylphosphoramidite ester).
In an alternative embodiment：The etherification reaction specifically includes following steps：Take 5- hydroxyl -2- nitrobenzyl alcohols molten
The anhydrous pyridine being added dropwise under anhydrous pyridine, ice bath stirring dissolved with 4,4'- dimethoxytrityl chloromethanes obtains reaction solution, waits for
Evaporation organic phase obtains crude product after reaction, and the purified processing of crude product obtains 5- hydroxyl -2- nitrobenzenes methyl -
(4,4'- dimethoxytrityls) ether.
Wherein selection under ice bath stirring using being added dropwise, to reduce by 4,4'- dimethoxytrityls chloromethanes and 5- hydroxyls -2-
The reaction probabilities of phenolic hydroxyl group in nitrobenzyl alcohol simplify post-processing.
In an alternative embodiment：The reaction condition of the etherification reaction is：The reaction solution stirs 1 hour at 0 DEG C,
It is restored to and is stirred overnight at room temperature；The matter of the 5- hydroxyls -2- nitrobenzyl alcohols and the 4,4'- dimethoxytrityls chloromethanes
Measure ratio 1.7：4；It is dissolved in a concentration of 0.017g/mL of the 5- hydroxyl -2- nitrobenzyl alcohols in the anhydrous pyridine；Dissolved with 5- hydroxyls
The anhydrous pyridine of base -2- nitrobenzyl alcohols and the volume ratio of the anhydrous pyridine dissolved with 4,4'- dimethoxytrityl chloromethanes are
10:3。
Etherification reaction in preparation method wherein of the invention under the above-described reaction conditions so that 4,4'- dimethoxys three
Phenyl chloromethanes while can avoid on remaining phenyl ring preferentially with hydroxyl reaction in the benzyl alcohol in 5- hydroxyl -2- nitrobenzyl alcohols
Phenolic hydroxyl group be etherified.This operates at 0 DEG C and carries out, and also can avoid 4,4'- dimethoxytrityls and at relatively high temperatures may be used
It caused can decompose, help to simplify subsequent purification step, reduce operating cost.
In an alternative embodiment：The purification processes specifically include following steps：The crude product is taken to be dissolved in acetic acid second
Product is dissolved in ethyl acetate after washing, drying, filtering, rotary evaporation remove organic solvent, cooling is added dropwise by ester
N-hexane in, filter, be dried to obtain 5- hydroxyl -2- nitrobenzenes methyl-(4,4'- dimethoxytrityl) ether.
The wherein present invention further uses above-mentioned purification processes step, purification operations simple on the basis of its preparation principle
It is easy to operate, improve preparation efficiency.
In an alternative embodiment：The esterification includes with 5- hydroxyl -2- nitrobenzenes methyl-(4,4'- dimethoxys
Triphenyl) ether be the reaction of raw material and 2-O- cyanoethyls-N, N- diisopropyl chloro phosphoramidite prepare 2- nitrobenzenes methyl-(4,
4'- dimethoxytrityls) ether -5- (2-O- cyanoethyls-N, N- diisopropylphosphoramidite ester) the step of.
In an alternative embodiment：Take 5- hydroxyls -2- nitrobenzenes methyl-(4,4'- dimethoxytrityls) ether and
Anhydrous triethylamine is dissolved in anhydrous tetrahydro furan, and 2-O- cyanoethyls-N, N- diisopropyl chloro phosphorous acyl is added dropwise under ice bath stirring
Amine, be filtered, washed after reaction, dry after obtain 2- nitrobenzenes methyl-(4,4'- dimethoxytrityl) ether -5- (2-O-
Cyanoethyl-N, N- diisopropylphosphoramidite ester).
In an alternative embodiment：The reaction condition of the esterification is：It stirs 2 hours at room temperature；The 5- hydroxyls
Base -2- nitrobenzenes methyl-(4,4'- dimethoxytrityls) ether, anhydrous triethylamine and 2-O- cyanoethyls-N, N- diisopropyl chlorine
Mass ratio for phosphoramidite is 2.3:1.2:1.27, anhydrous triethylamine is a concentration of described in the anhydrous tetrahydro furan
0.024g/ml。
By above-mentioned reaction condition it is found that this method operating condition designed by the present invention is mild, easy, reduction is produced into
This, suitable for promoting on the market.
In an alternative embodiment：It is described to filter insoluble matter after reaction, take filtrate to use saturated sodium bicarbonate solution
Washing is added anhydrous sodium sulfate and is dried to obtain 2- nitrobenzenes methyl-(4,4'- dimethoxytrityl) ether -5- (2-O- cyanogen second
Base-N, N- diisopropylphosphoramidite ester).
Above-mentioned steps are directly over for product after reaction to be filtered, washed, is dried and can wait to obtain, into one
Step illustrates that preparation method of the present invention has simplified isolating and purifying for intermediate, simplifies the operating procedure of experiment, improves final production
The yield of object.
For the preparation method of the phosphoramidite of above-mentioned offer, reaction process is as shown in reaction equation I：
Wherein compound III be 5- hydroxyl -2- nitrobenzyl alcohols, compound IV be 5- hydroxyl -2- nitrobenzenes methyl-(4,
4'- dimethoxytrityls) ether, compound I is 2- nitrobenzenes methyl-(4,4'- dimethoxytrityl) ether -5- (2-O- cyanogen
Ethyl-N, N- diisopropylphosphoramidite ester).
A kind of application of above-mentioned phosphoramidite in preparing the DNA containing photosensitive unit.
It is wherein directed to the preparation method of the DNA containing photosensitive unit, is included the following steps：Take the phosphorous
Carboxylic acid amide esters, which are dissolved in anhydrous acetonitrile, is used as end group modification position reagent and Synthesis of DNA, and the product after synthesis is taken to be dispersed through
In ammonium hydroxide, and after airtight heating, it is centrifuged off insoluble matter, the concentrated processing of solution is taken to obtain the deoxidation core containing photosensitive unit
Ribosomal ribonucleic acid.
For the preparation method of the DNA containing photosensitive unit of above-mentioned offer, reaction process such as reaction equation
Shown in II：
For wherein compound V to be immobilized in the 5' terminal hydroxy group DNA on carrier, compound VI is immobilized in load
The ends 5' on body are functionalized DNA, and compound VII is to be supported on the DNA not cut off on solid phase carrier,
Compound II is the DNA containing photosensitive unit.
In order to become apparent from introduce in detail a kind of phosphoramidite that the embodiment of the present invention is provided and preparation method thereof and
Purposes is described below in conjunction with specific embodiment.
Embodiment 1：
A kind of preparation method of phosphoramidite, includes the following steps：
Step 1：Prepare 5- hydroxyl -2- nitrobenzenes methyl-(4,4'- dimethoxytrityls) ether：Take 1.7 grams of 5- hydroxyls-
2- nitrobenzyl alcohols are dissolved in 100 milliliters of anhydrous pyridines, under ice bath stirring, are added dropwise dissolved with 4.0 grams of 4,4'- dimethoxy triphens
In 30 milliliters of anhydrous pyridines of base chloromethanes, 0 DEG C is placed reaction liquid into after being added dropwise and is stirred lower 1 hour, is restored to room temperature
It is stirred overnight；It waits for rotary evaporation organic phase after reaction, crude product is dissolved in 30 milliliters of ethyl acetate, washed using 50 milliliters
After twice, organic phase is dried using anhydrous sodium sulfate；Rotary evaporation removes organic solvent, obtained buff oily after filtering
Liquid is dissolved in 5 milliliters of ethyl acetate, is added dropwise 100 milliliters and is cooled in 0 DEG C of n-hexane, obtains yellow mercury oxide,
Vacuum drying obtains 2.5 grams of yellow powders, yield 53% after filtering；
Step 2：Prepare phosphoramidite：Take 2.3 grams of 5- hydroxyl -2- nitrobenzenes methyl-(4,4'- dimethoxy triphens
Base) ether and 1.2 grams of anhydrous triethylamines are dissolved in 50 milliliters of anhydrous tetrahydro furans, 1.27 grams of 2-O- cyanogen second are added dropwise under ice bath stirring
Base-N, N- diisopropyl chloro phosphoramidite；Reaction is stirred 2 hours at room temperature, filters out insoluble matter, filtrate is with unsaturated carbonate hydrogen
Sodium solution washs；Organic phase is dried using anhydrous sodium sulfate；Obtain 2- nitrobenzenes methyl-(4,4'- dimethoxytrityls) ether-
5- (2-O- cyanoethyls-N, N- diisopropylphosphoramidite ester) (yellow viscous liquid), 2.0 grams of yields 60%.
The nuclear magnetic resonance spectroscopy testing result of compound I in the phosphoramidite of above-mentioned preparation, that is, above-mentioned reaction equation I is such as
Shown in Fig. 1：
1H-NMR(d6-DMSO),400MHz:7.79-7.80 (d, J=4.0Hz, ArH, 1H), 7.43-7.44 (d, J=
4.0Hz, ArH, 1H), 7.32-7.33 (d, J=4.0Hz, ArH, 1H), 7.24-7.26 (t, J=4.0Hz, ArH, 2H), 7.21-
7.23 (d, J=8.0Hz, ArH, 4H), 7.12-7.21 (m, ArH, 2H), 7.02-7.03 (d, J=4.0Hz, ArH, 1H),
6.89-6.91 (d, J=8.0Hz, ArH, 4H), 6.84-6.85 (d, J=4.0Hz, ArH, 1H), 4.47 (s, CH2O,2H),
3.89-4.03(m,CH2OP,2H),3.73(s,CH3O, 6H), 3.33-3.49 (m, NCH, 2H), 2.80-2.87 (t, J=
4.0Hz,CH2CN,2H),1.17-1.29(d,CH3,12H)。
The carbon-13 nmr spectra testing result of the phosphoramidite of above-mentioned preparation is as shown in Figure 2：
13C-NMR(d6-DMSO),100MHz:163.8,158.7,145.3,139.3,135.9,130.1,129.4,
128.6,128.1,127.4,119.1,114.8,114.5,113.9,113.3,87.0,63.3,58.7,55.6,45.1,
23.2,19.3。
The results are shown in Figure 3 for the Mass Spectrometer Method of the phosphoramidite of above-mentioned preparation：
ESI-MS:C37H42N3O7P,calc 671.2760,found 687.2714(M++O),710.2607(M++O+Na+),726.2347(M++O+K+)。
Embodiment 2
A kind of preparation method of the DNA containing photosensitive unit, includes the following steps：
Step 1：2- nitrobenzenes methyl-(4,4'- dimethoxytrityls) ether -5- (2- for taking 600mg embodiments 1 to prepare
O- cyanoethyls-N, N- diisopropylphosphoramidite ester) it is dissolved in 10 milliliters of anhydrous acetonitriles, it is transferred to ABI under nitrogen protection
In reagent bottle of the 3400DNA synthesizers marked as " 5 ", it is defined as X, inputs DNA sequence dna, the synthesis model retained using DMT is held
The synthesis of 1 μm of ol magnitude of row；
Step 2：Solid phase carrier is taken out after synthesis, is scattered in 1 milliliter of 23% ammonium hydroxide, airtight heating 2 at 55 DEG C
Hour, centrifugation removal insoluble matter, solution concentration obtains crude product；The nucleic acid that target product contains photosensitive unit is passed through efficient
Liquid phase separation, and structural information characterization is carried out by mass spectrum and high performance liquid chromatography, result as shown in figs. 4 and 7, such as Fig. 7
Shown in the nucleic acid appearance time containing photosensitive unit be 19.233 minutes.
The light sensitivity detection experiment of nucleic acid containing photosensitive unit：
Test method：The sources Zhong Jiaojin company is used by the nucleic acid containing photosensitive unit is got according to the method for embodiment 2
CEL-HXF300 type xenon lamps install 350-380nm optical filters, optical power density 20mW/cm additional2, irradiation time is after 2 minutes, point
The mass spectrum and HPLC spectrograms of DNA after other detection light pre-irradiation.Simultaneously not carry out the deoxyribose core of end group modification
Sour DNA as a control group, sequence is as shown in SEQ ID No.1, accordingly in the nucleic acid containing photosensitive unit
DNA sequence dna is also as shown in SEQ ID No.1.Mass spectrum and HPLC spectrograms are detected after carrying out illumination according to the method described above.
Test result：
Before illumination the DNA containing photosensitive unit mass spectrum as shown in Figure 4, HPLC spectrograms as shown in fig. 7, its
Appearance time is 19.233 minutes；
DNA mass spectrogram after the light degradation obtained after illumination as shown in Figure 5, HPLC spectrograms as shown in figure 8,
Its appearance time is 23.401 and 7.214 minutes；
Shown in mass spectrogram 6 after control group DNA illumination, HPLC spectrograms are as shown in figure 9, appearance time is
19.105 minutes.
Above-mentioned three groups of middle-molecular-weihydroxyethyls are calculated, data result is as shown in table 1：
1 molecular weight data result of table
By above-mentioned profiling results and test data it is found that the deoxidation core containing photosensitive unit prepared by the embodiment of the present invention 2
Ribosomal ribonucleic acid is degraded under conditions of illumination, and specific degradation is as shown in reaction equation III：
In the above, the structure and purity of organic molecule by nuclear magnetic resonance spectroscopy (1H-NMR), carbon spectrum (13C-NMR)
It is determined with electron spray ionisation (ESI-MS), the structural information and light degradation process of DNA are by ground substance assistant laser electricity
Analysis-flight time mass spectrum (MALDI-TOF) is dissociated to determine, reaction efficiency is determined by high performance liquid chromatography (HPLC).
400 Spectrometer (400MHz) of nuclear-magnetism model Bruker AMX, solvent for use is deuterated dimethyl sulfoxide
(d6- DMSO), band TMS internal standards；ESI mass spectrum model Agilent 6510Q-TOF, detection pattern is ion mode；
MALDI-TOF mass spectrum model Shimadazu Biotech Axima Performance, detection pattern is ion mode；
High performance liquid chromatography model Waters 2695, detection column type number are XBriage Oligonucleotides BEH C18
(2.1mm×50mm；Column2.5 μm), elution condition is as shown in table 2：
Table 2 elutes condition
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, any made by repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Sequence table
<110>China University Of Petroleum Beijing（East China）
<120>Phosphoramidite and its preparation method and application
<141> 2018-03-30
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
ccaacatcta ttcaacttga acca 24
Claims (10)
1. a kind of phosphoramidite, it is characterised in that：Shown in the structural formula as I of the compound：
2. a kind of preparation method of phosphoramidite described in claim 1, it is characterised in that：Include the following steps：With 5- hydroxyls
Base -2- nitrobenzyl alcohols are starting material, its benzyl alcohol hydroxyl is selectively protected by 4,4 '-dimethoxytrityl chloromethanes
Shield, it is etherified 5- hydroxyl -2- nitrobenzenes methyl-(4,4'- dimethoxytrityl) ether is obtained by the reaction after, then through esterification system
It is standby to obtain 2- nitrobenzenes methyl-(4,4'- dimethoxytrityls) ether -5- (2-O- cyanoethyls-N, N- diisopropylphosphoramidites
Ester).
3. the preparation method of phosphoramidite according to claim 2, it is characterised in that：The etherification reaction specifically includes
Following steps：It takes 5- hydroxyl -2- nitrobenzyl alcohols to be dissolved in anhydrous pyridine, is added dropwise dissolved with 4,4'- dimethoxys three under ice bath stirring
The anhydrous pyridine of phenyl chloromethanes obtains reaction solution, waits for that evaporating organic phase after reaction obtains crude product, the crude product warp
Purification processes obtain 5- hydroxyl -2- nitrobenzenes methyl-(4,4'- dimethoxytrityls) ether.
4. the preparation method of phosphoramidite according to claim 3, it is characterised in that：The reaction item of the etherification reaction
Part is：The reaction solution stirs 1 hour at 0 DEG C, is restored to and is stirred overnight at room temperature；5- hydroxyls -2- the nitrobenzyl alcohols with
The mass ratio 1.7 of the 4,4'- dimethoxytrityls chloromethanes：4；5- hydroxyl -2- the nitros being dissolved in the anhydrous pyridine
A concentration of 0.017g/mL of benzyl alcohol；Dissolved with 5- hydroxyl -2- nitrobenzyl alcohols anhydrous pyridine with dissolved with 4,4'- dimethoxys
The volume ratio of the anhydrous pyridine of triphenylchloromethane is 10:3.
5. the preparation method of phosphoramidite according to claim 3, it is characterised in that：The purification processes specifically include
Following steps：The crude product is taken to be dissolved in ethyl acetate, it, will after washing, drying, filtering, rotary evaporation remove organic solvent
Product is dissolved in ethyl acetate, is added dropwise in cooling n-hexane, is filtered, is dried to obtain 5- hydroxyl -2- nitrobenzenes methyl -
(4,4'- dimethoxytrityls) ether.
6. the preparation method of phosphoramidite according to claim 2, it is characterised in that：The esterification includes with 5-
Hydroxyl -2- nitrobenzenes methyl-(4,4'- dimethoxytrityls) ether is raw material and 2-O- cyanoethyls-N, N- diisopropyl chloro
Phosphoramidite reaction prepares 2- nitrobenzenes methyl-(4,4'- dimethoxytrityls) ether -5- (2-O- cyanoethyls-N, N- diisopropyls
Base phosphoramidite) the step of.
7. the preparation method of phosphoramidite according to claim 6, it is characterised in that：Take the 5- hydroxyls -2- nitros
Benzyl-(4,4'- dimethoxytrityl) ether and anhydrous triethylamine are dissolved in anhydrous tetrahydro furan, and 2- is added dropwise under ice bath stirring
O- cyanoethyls-N, N- diisopropyl chloro phosphoramidite, be filtered, washed after reaction, dry after obtain 2- nitrobenzenes methyl-
(4,4'- dimethoxytrityls) ether -5- (2-O- cyanoethyls-N, N- diisopropylphosphoramidite ester).
8. the preparation method of phosphoramidite according to claim 7, it is characterised in that：The reaction item of the esterification
Part is：It stirs 2 hours at room temperature；5- hydroxyls -2- nitrobenzenes the methyl-(4,4'- dimethoxytrityls) ether, anhydrous three
The mass ratio of ethamine and 2-O- cyanoethyls-N, N- diisopropyl chloro phosphoramidite is 2.3:1.2:1.27, the anhydrous tetrahydrochysene
A concentration of 0.024g/ml of anhydrous triethylamine described in furans.
9. the preparation method of phosphoramidite according to claim 7, it is characterised in that：It is described to filter after reaction not
Molten object takes filtrate to be washed using saturated sodium bicarbonate solution, and anhydrous sodium sulfate is added and is dried to obtain 2- nitrobenzenes methyl-(4,4'-
Dimethoxytrityl) ether -5- (2-O- cyanoethyls-N, N- diisopropylphosphoramidite ester).
10. a kind of application of phosphoramidite described in claim 1 in preparing the nucleic acid containing photosensitive unit.
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Cited By (2)
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| CN109810141A (en) * | 2019-01-30 | 2019-05-28 | 中国石油大学(华东) | Sulfur-bearing phosphorous imide ester, preparation method and its application |
| CN114685560A (en) * | 2020-12-31 | 2022-07-01 | 沈阳药科大学 | Synthesis and application of phosphoramidite monomer containing piperidine skeleton and oligonucleotide |
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| CN102439025A (en) * | 2008-09-06 | 2012-05-02 | 坎姆根公司 | Rna synthesis - phosphoramidites for synthetic rna in the reverse direction, and application in convenient introduction of ligands, chromophores and modifications of synthetic rna at the 3' - end |
| CN103819501A (en) * | 2014-03-19 | 2014-05-28 | 厦门大学 | Methyl propenyl phosphoramidite monomer and synthetic method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109810141A (en) * | 2019-01-30 | 2019-05-28 | 中国石油大学(华东) | Sulfur-bearing phosphorous imide ester, preparation method and its application |
| CN109810141B (en) * | 2019-01-30 | 2021-08-24 | 中国石油大学(华东) | Sulfur-containing phosphoramidite ester, preparation method and application thereof |
| CN114685560A (en) * | 2020-12-31 | 2022-07-01 | 沈阳药科大学 | Synthesis and application of phosphoramidite monomer containing piperidine skeleton and oligonucleotide |
| CN114685560B (en) * | 2020-12-31 | 2024-05-14 | 沈阳药科大学 | Synthesis and application of phosphoramidite monomer containing piperidine skeleton and oligonucleotide |
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