EP0229053A1 - Method for synthesizing deoxyoligonucleotides - Google Patents
Method for synthesizing deoxyoligonucleotidesInfo
- Publication number
- EP0229053A1 EP0229053A1 EP19850903503 EP85903503A EP0229053A1 EP 0229053 A1 EP0229053 A1 EP 0229053A1 EP 19850903503 EP19850903503 EP 19850903503 EP 85903503 A EP85903503 A EP 85903503A EP 0229053 A1 EP0229053 A1 EP 0229053A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- compound
- formula
- phosphoramidites
- morpholino
- bis
- 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.)
- Withdrawn
Links
- 238000000034 method Methods 0.000 title claims description 27
- 230000002194 synthesizing effect Effects 0.000 title description 3
- 238000006243 chemical reaction Methods 0.000 claims abstract description 14
- -1 1-thyminyl Chemical group 0.000 claims description 27
- 150000001875 compounds Chemical class 0.000 claims description 26
- 238000002360 preparation method Methods 0.000 claims description 16
- 125000004573 morpholin-4-yl group Chemical group N1(CCOCC1)* 0.000 claims description 9
- 239000011541 reaction mixture Substances 0.000 claims description 7
- 230000003197 catalytic effect Effects 0.000 claims description 5
- 239000000725 suspension Substances 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 4
- 125000000217 alkyl group Chemical group 0.000 claims 2
- 241000907681 Morpho Species 0.000 claims 1
- BQJCRHHNABKAKU-KBQPJGBKSA-N morphine Chemical compound O([C@H]1[C@H](C=C[C@H]23)O)C4=C5[C@@]12CCN(C)[C@@H]3CC5=CC=C4O BQJCRHHNABKAKU-KBQPJGBKSA-N 0.000 claims 1
- 230000015572 biosynthetic process Effects 0.000 abstract description 25
- 238000003786 synthesis reaction Methods 0.000 abstract description 22
- 150000008300 phosphoramidites Chemical class 0.000 abstract description 21
- 238000011065 in-situ storage Methods 0.000 abstract description 9
- 229920000642 polymer Polymers 0.000 abstract description 8
- 150000003536 tetrazoles Chemical class 0.000 abstract description 5
- 239000003054 catalyst Substances 0.000 abstract description 3
- 125000005265 dialkylamine group Chemical group 0.000 abstract 1
- XYFCBTPGUUZFHI-UHFFFAOYSA-N Phosphine Chemical compound P XYFCBTPGUUZFHI-UHFFFAOYSA-N 0.000 description 12
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 9
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 7
- 239000002777 nucleoside Substances 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- YNAVUWVOSKDBBP-UHFFFAOYSA-N Morpholine Chemical compound C1COCCN1 YNAVUWVOSKDBBP-UHFFFAOYSA-N 0.000 description 6
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 6
- UAOMVDZJSHZZME-UHFFFAOYSA-N diisopropylamine Chemical compound CC(C)NC(C)C UAOMVDZJSHZZME-UHFFFAOYSA-N 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical class CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 6
- 229910000073 phosphorus hydride Inorganic materials 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 230000002255 enzymatic effect Effects 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- YFYBXOIQXOOUCI-UHFFFAOYSA-N n-[[di(propan-2-yl)amino]-methoxyphosphanyl]-n-propan-2-ylpropan-2-amine Chemical compound CC(C)N(C(C)C)P(OC)N(C(C)C)C(C)C YFYBXOIQXOOUCI-UHFFFAOYSA-N 0.000 description 5
- 108091033319 polynucleotide Proteins 0.000 description 5
- 102000040430 polynucleotide Human genes 0.000 description 5
- 239000002157 polynucleotide Substances 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 108091034117 Oligonucleotide Proteins 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- 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 4
- 150000001412 amines Chemical class 0.000 description 4
- 238000001704 evaporation Methods 0.000 description 4
- 230000008020 evaporation Effects 0.000 description 4
- 239000012299 nitrogen atmosphere Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 230000003595 spectral effect Effects 0.000 description 4
- CSDQQAQKBAQLLE-UHFFFAOYSA-N 4-(4-chlorophenyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine Chemical compound C1=CC(Cl)=CC=C1C1C(C=CS2)=C2CCN1 CSDQQAQKBAQLLE-UHFFFAOYSA-N 0.000 description 3
- 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 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- USJRLGNYCQWLPF-UHFFFAOYSA-N chlorophosphane Chemical class ClP USJRLGNYCQWLPF-UHFFFAOYSA-N 0.000 description 3
- HCSDJECSMANTCX-UHFFFAOYSA-N dichloro(methoxy)phosphane Chemical compound COP(Cl)Cl HCSDJECSMANTCX-UHFFFAOYSA-N 0.000 description 3
- 229960004132 diethyl ether Drugs 0.000 description 3
- 150000003833 nucleoside derivatives Chemical class 0.000 description 3
- 125000003835 nucleoside group Chemical group 0.000 description 3
- 229920006395 saturated elastomer Polymers 0.000 description 3
- 239000000377 silicon dioxide Substances 0.000 description 3
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- OXJVPEPMGZHRJB-UHFFFAOYSA-N aminophosphinoamine Chemical compound NPN OXJVPEPMGZHRJB-UHFFFAOYSA-N 0.000 description 2
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 2
- 239000012267 brine Substances 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 239000013058 crude material Substances 0.000 description 2
- 229940043279 diisopropylamine Drugs 0.000 description 2
- WBMWCLOACCAWIJ-UHFFFAOYSA-N dimorpholin-4-ylmethoxyphosphane Chemical compound C1COCCN1C(OP)N1CCOCC1 WBMWCLOACCAWIJ-UHFFFAOYSA-N 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000004508 fractional distillation Methods 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000012044 organic layer Substances 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 238000001394 phosphorus-31 nuclear magnetic resonance spectrum Methods 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 2
- 229940104230 thymidine Drugs 0.000 description 2
- PPKPKFIWDXDAGC-IHWYPQMZSA-N (z)-1,2-dichloroprop-1-ene Chemical compound C\C(Cl)=C\Cl PPKPKFIWDXDAGC-IHWYPQMZSA-N 0.000 description 1
- 238000004679 31P NMR spectroscopy Methods 0.000 description 1
- QAJJXHRQPLATMK-UHFFFAOYSA-N 4,5-dichloro-1h-imidazole Chemical compound ClC=1N=CNC=1Cl QAJJXHRQPLATMK-UHFFFAOYSA-N 0.000 description 1
- QCQCHGYLTSGIGX-GHXANHINSA-N 4-[[(3ar,5ar,5br,7ar,9s,11ar,11br,13as)-5a,5b,8,8,11a-pentamethyl-3a-[(5-methylpyridine-3-carbonyl)amino]-2-oxo-1-propan-2-yl-4,5,6,7,7a,9,10,11,11b,12,13,13a-dodecahydro-3h-cyclopenta[a]chrysen-9-yl]oxy]-2,2-dimethyl-4-oxobutanoic acid Chemical compound N([C@@]12CC[C@@]3(C)[C@]4(C)CC[C@H]5C(C)(C)[C@@H](OC(=O)CC(C)(C)C(O)=O)CC[C@]5(C)[C@H]4CC[C@@H]3C1=C(C(C2)=O)C(C)C)C(=O)C1=CN=CC(C)=C1 QCQCHGYLTSGIGX-GHXANHINSA-N 0.000 description 1
- GIKHXAUMMAGJBH-UHFFFAOYSA-N 4-methoxyphosphanyl-n,n-di(propan-2-yl)morpholin-2-amine Chemical compound COPN1CCOC(N(C(C)C)C(C)C)C1 GIKHXAUMMAGJBH-UHFFFAOYSA-N 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 230000006820 DNA synthesis Effects 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
- 229910019142 PO4 Inorganic materials 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- YQVISGXICTVSDQ-UHFFFAOYSA-O [c-]1nn[nH]n1.CC(C)[NH2+]C(C)C Chemical class [c-]1nn[nH]n1.CC(C)[NH2+]C(C)C YQVISGXICTVSDQ-UHFFFAOYSA-O 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 239000003377 acid catalyst Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000010420 art technique Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 description 1
- IWFOEMYKRGNBNE-UHFFFAOYSA-N bis(morpholin-4-ylmethoxy)phosphane Chemical compound C1COCCN1COPOCN1CCOCC1 IWFOEMYKRGNBNE-UHFFFAOYSA-N 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- HSHIMRFRQDTJJF-UHFFFAOYSA-N chloro(methoxy)phosphane Chemical compound COPCl HSHIMRFRQDTJJF-UHFFFAOYSA-N 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000006642 detritylation reaction Methods 0.000 description 1
- WDIIYWASEVHBBT-UHFFFAOYSA-N di(propan-2-yl)phosphane Chemical compound CC(C)PC(C)C WDIIYWASEVHBBT-UHFFFAOYSA-N 0.000 description 1
- ZDTQJPVEXIUREJ-UHFFFAOYSA-N dichlorophosphinous acid Chemical compound OP(Cl)Cl ZDTQJPVEXIUREJ-UHFFFAOYSA-N 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- NDNQLUHJMUHTSD-UHFFFAOYSA-N dipyrrolidin-1-ylmethoxyphosphane Chemical compound C1CCCN1C(OP)N1CCCC1 NDNQLUHJMUHTSD-UHFFFAOYSA-N 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 229910052736 halogen Inorganic materials 0.000 description 1
- 150000002367 halogens Chemical class 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010952 in-situ formation Methods 0.000 description 1
- 229920000592 inorganic polymer Polymers 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 125000005647 linker group Chemical group 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 230000003340 mental effect Effects 0.000 description 1
- JVRGLGIDPIOAFN-UHFFFAOYSA-N methoxyphosphane Chemical compound COP JVRGLGIDPIOAFN-UHFFFAOYSA-N 0.000 description 1
- KQCKTXVUXOQWDN-UHFFFAOYSA-N n,n-di(propan-2-yl)-2h-tetrazol-5-amine Chemical compound CC(C)N(C(C)C)C=1N=NNN=1 KQCKTXVUXOQWDN-UHFFFAOYSA-N 0.000 description 1
- ITZFQBIVZJBQOZ-UHFFFAOYSA-N n-$l^{1}-phosphanyl-n-propan-2-ylpropan-2-amine Chemical compound CC(C)N([P])C(C)C ITZFQBIVZJBQOZ-UHFFFAOYSA-N 0.000 description 1
- APVPOHHVBBYQAV-UHFFFAOYSA-N n-(4-aminophenyl)sulfonyloctadecanamide Chemical compound CCCCCCCCCCCCCCCCCC(=O)NS(=O)(=O)C1=CC=C(N)C=C1 APVPOHHVBBYQAV-UHFFFAOYSA-N 0.000 description 1
- FVGJFFQRXUXGOM-UHFFFAOYSA-N n-(chloromethoxyphosphanyl)-n-propan-2-ylpropan-2-amine Chemical compound CC(C)N(C(C)C)POCCl FVGJFFQRXUXGOM-UHFFFAOYSA-N 0.000 description 1
- ZYQPNKHEEHSDCO-UHFFFAOYSA-N n-[[di(propan-2-yl)amino]-ethoxyphosphanyl]-n-propan-2-ylpropan-2-amine Chemical compound CCOP(N(C(C)C)C(C)C)N(C(C)C)C(C)C ZYQPNKHEEHSDCO-UHFFFAOYSA-N 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
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000005580 one pot reaction Methods 0.000 description 1
- 229920000620 organic polymer Polymers 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- OJMIONKXNSYLSR-UHFFFAOYSA-N phosphorous acid Chemical compound OP(O)O OJMIONKXNSYLSR-UHFFFAOYSA-N 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002212 purine nucleoside Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- IOVGROKTTNBUGK-SJCJKPOMSA-N ritodrine Chemical compound N([C@@H](C)[C@H](O)C=1C=CC(O)=CC=1)CCC1=CC=C(O)C=C1 IOVGROKTTNBUGK-SJCJKPOMSA-N 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 150000003335 secondary amines Chemical class 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000010626 work up procedure Methods 0.000 description 1
Classifications
-
- 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
-
- 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
- C07F9/2408—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic of hydroxyalkyl compounds
-
- 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
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6527—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having nitrogen and oxygen atoms as the only ring hetero atoms
- C07F9/6533—Six-membered rings
-
- 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
-
- 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
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
Definitions
- the present invention relates to a new and novel method for the preparation of deoxynucleoside phos ⁇ phoramidites in situ from deoxynucleosides, bis- dial ylaminophosphines, and the corresponding dialkyl ⁇ amine hydrotetrazolide or tetrazole as catalyst for the reaction.
- These phosphoramidites lead to the direct synthesis of deoxyoligonucleotides on various polymer supports.
- Modified inorganic polymers are known in the prior art, primarily for use as absorption materials, for example, in liquid chromatography.
- the attach ⁇ ment of nucleosidephosphates to silica gel using a trityl linking group is described in the prior art but the method is apparently applicable only to pyri idine nucleosides.
- the cleavage of the nucleo- side from the silica support can only be accomplished with acid to which the purine nucleosides are sensi ⁇ tive.
- phosphotriester derivatives of oligothymidylates is described in the prior art by reaction of a phosphorodichloridite with a 5'-0 blocked thymidine and subsequent reaction of the product with a 3'-0 blocked ' thymidine. This is followed by oxidation of the resulting phosphite to a phosphate and removal of blocking groups to obtain the phosphotriesters.
- the process requires separation and purification of products at each stage to ensure proper sequencing of the added nucleosides. Separation techniques including precipi ⁇ tation and washing of precipitates are necessary to implement each successive stage reaction.
- U.S. Patent No. 4,415,732 describes a new class of nucleoside phosphoramidites derived from saturated secondary amines, which are relatively stable. This permits isolation and storage of the compounds, at room temperature. These phosphoramidite compounds have been found to have excellent use in the forma ⁇ tion of the internucleotide bonds described in the earlier mentioned U.S. Patent. The disclosure of this patent is incorporated in toto herein.
- B may be 1-thyminyl; l-(N-4 benzoyl cyto- sinyl) ; 9-(N-2-isobutyrylquaninyl) ; or 9-(N-6-benzoyl- adeninyl) , wherein Z may be morpholino or N(lower alkyl)-, preferably (i-propyl)_; and wherein DMT is di-p-anisylphenyl methyl.
- phosphoramidites are activated by tetra ⁇ zole, with the 5'-hydroxyl group of a deoxynucleoside or deoxyoligonucleotide attached covalently to a polymer support (see, for example, Caruthers, M. H. , Beaucage, S. L., Becker, C. , Efcavitch, J. . , Fisher, E. F. , Galluppi, G. , Goldman, R. A., de- Haseth, P. L., Martin, F. , Matteucci, M. D. and Stabinsky, Y. ,(1982) in Genetic Engineering, Setlow, J. and Hollaender, A. Eds., Vol.
- phosphoramidites can be prepared by existing methods (see, for example, Beaucage, S. L. and Caruthers, M. H. (1981) Tetrahedron Lett. 22, 1859-1862; and McBride, L. J. and Caruthers, M. H. (1983) ibid. 24, 245-248), from the appropriately protected deoxynucleosides of the following struc ⁇ tural formula:
- DMT and B are as defined for the compounds of Formula I above, and chlorphosphines of the following structural formula:
- chlorophosphines used in the preparation of the compounds of Formula I, are difficult to prepare and easily react with trace amounts of water.
- the high reactivity of the chlorophosphines and the concomitant production- of insoluble amine hydrochloride salts preclude their use for any strategy involving the in situ generation of deoxynu ⁇ cleoside phosphoramidites for deoxyoligonucleotide synthesis on solid supports, (see, for example Fourrey, J. L. and Shire, D. (1981) Tetrahedron Lett. 22, 729-732) .
- the reaction is furthermore found to be catalytic in either tetrazole or the salt, as well as a greater selectivity of activation.
- This preparation has been successfully applied to the synthesis of deoxyoligo- nucleotides directly on a solid support via an in situ approach.
- Example IV shows a typical experi ⁇ mental procedure for the preparation of phosphor ⁇ amidites according to the present invention. Al ⁇ though the description is specific for the formation of the preparation of the compound of general Formula I wherein B is 1-thyminyl and Z is diisopropyl amino, the remaining compounds of general Formula I may be prepared along similar protocols by those skilled in the synthesis art.
- the 31P NMR spectral data for phosphoramidites prepared following the typical procedure given in Example IV are contained in Table
- the bis-morpholinomethoxyphosphine was prepared in a similar manner (80% yield) and had stability comparable to that obtained for the bis-(diisopropylamino)methoxy ⁇ phosphine.
- the mixed (diisopropylaminomorpholino) methoxyphosphine could not be obtained cleanly in the same one pot procedure as above; however, treatment of the mixed (diisopropylaminochloro)methoxyphosphine with one equivalent of morpholine and triethylamine gave a 77% yield of methoxyphosphine contaminated with about 2% of the thermodynamically more stable bis-(diisopropylamino) methoxyphosphine.
- the typical preparation of phosphoramidites depicted in Example IV required the addition of 1.1 equivalents of the diisopropyl phosphine to a mixture of the nucleoside and 0.5 equivalents of the catalyst (general Formula V) in dichloromethane.
- the TLC showed the reaction to be complete in about 20 min. After an aqueous work-up and precipitation of the material in cold hexanes, the product was obtained in 87% yield as an amorphous solid.
- the 31P NMR spectrum showed two signals corresponding to a 1:1 diastereo eric mixture of phosphoramidites.
- Phosphoramidites prepared using the procedure according to the present invention were tested as syn- thons by constructing d(GGGAATTCCC) , a self-complementary segment containing the EcoRI recognition sequence.
- the deoxyoligonucleotide was synthesized and deprotected using standard procedures according to Caruthers in Chemical and Enzymatic Synthesis of Gene Fragments, A Laboratory Manual, and isolated by gel electrophoresis in a 57% yield.
- the average coupling yield (measured spectrophotometrically from the dimethoxytrityl cation,
- Bis-(diisopropylamino) ethoxyphosphine was also tested as part of an _in situ synthesis protocol.
- Phos ⁇ phoramidites according to general Formula I were each prepared as 0.1M solutions in dry acetonitrile containing bis-(diisopropylamino)methoxyphosphine (1.0 eq) and the diisopropylamine tetrazolides of general Formula V (0.5 eq) .
- the segment d(GGGAATTCCC) was then prepared in 50% isolated yield (average coupling yield was 94%) using the following procedure.
- the present invention demonstrates that very stable dialkylamino phosphines according to general Formula IV can be used as phosphitylating reagents to form deoxynucleoside phosphoramidites cleanly and in good yields.
- the reactions are catalytic in either tetrazole or the corresponding amine hydrotetrazolides. These reactions have furthermore been found to be selective to the formation of only 3'-deoxynucleoside phosphoramidites without concurrent hydrolysis to phosphonus acid or synthesis of the 3'-3 * dinucleoside phosphit-3. This selectivity renders these reagents attractive for the in situ generation of phosphoramidites useful ' for DNA synthesis on solid supports.
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Abstract
Des phosphoramidites de désoxynucléosides sont préparés avec un rendement élevé à partir de désoxynucléosides, de bis-dialkylamino-phosphines, avec l'hydrotétrazolure de dialkylamine ou le tétrazole correspondant comme catalyseur pour la réaction. Ces phosphoramidites produits in situ conduisent à la synthèse directe de désoxyoligonucléotides sur des supports polymères.Deoxynucleoside phosphoramidites are prepared in high yield from deoxynucleosides, bis-dialkylamino-phosphines, with the dialkylamine hydrotetrazolide or the corresponding tetrazole as a catalyst for the reaction. These phosphoramidites produced in situ lead to the direct synthesis of deoxyoligonucleotides on polymer supports.
Description
Claims
Applications Claiming Priority (1)
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PCT/US1985/001148 WO1986007362A1 (en) | 1985-06-14 | 1985-06-14 | Method for synthesizing deoxyoligonucleotides |
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EP0229053A1 true EP0229053A1 (en) | 1987-07-22 |
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EP19850903503 Withdrawn EP0229053A1 (en) | 1985-06-14 | 1985-06-14 | Method for synthesizing deoxyoligonucleotides |
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WO (1) | WO1986007362A1 (en) |
Families Citing this family (9)
Publication number | Priority date | Publication date | Assignee | Title |
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US5071974A (en) * | 1986-10-31 | 1991-12-10 | Amoco Corporation | Compositions and methods for the synthesis of oligonucleotides having 5'-phosphorylated termini |
US5256775A (en) * | 1989-06-05 | 1993-10-26 | Gilead Sciences, Inc. | Exonuclease-resistant oligonucleotides |
EP0476071A4 (en) * | 1989-06-05 | 1992-11-04 | Gilead Sciences, Inc. | Exonuclease-resistant oligonucleotides and methods for preparing the same |
US5281701A (en) * | 1991-07-12 | 1994-01-25 | Applied Biosystems, Inc. | Process and compounds for RNA synthesis |
US5623068A (en) * | 1994-03-07 | 1997-04-22 | Beckman Instruments, Inc. | Synthesis of DNA using substituted phenylacetyl-protected nucleotides |
US5705621A (en) * | 1995-11-17 | 1998-01-06 | Isis Pharmaceuticals, Inc. | Oligomeric phosphite, phosphodiester, Phosphorothioate and phosphorodithioate compounds and intermediates for preparing same |
ES2212097T3 (en) * | 1996-05-03 | 2004-07-16 | Avecia Biotechnology Inc | IN SITU PREPARATION OF NUCLEOSID PHOSPHORAMIDITES AND ITS USE IN OLIGONUCLEOTIDOS SYNTHESIS. |
US6248877B1 (en) | 1998-04-07 | 2001-06-19 | Biolink Partners | Solid phase synthesis of organic compounds via phosphitylating reagents |
US6451998B1 (en) | 1999-10-18 | 2002-09-17 | Agilent Technologies, Inc. | Capping and de-capping during oligonucleotide synthesis |
Family Cites Families (6)
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US4500707A (en) * | 1980-02-29 | 1985-02-19 | University Patents, Inc. | Nucleosides useful in the preparation of polynucleotides |
EP0173356B1 (en) * | 1980-02-29 | 1990-09-19 | University Patents, Inc. | Process for preparing modified inorganic polymers |
US4458066A (en) * | 1980-02-29 | 1984-07-03 | University Patents, Inc. | Process for preparing polynucleotides |
US4415732A (en) * | 1981-03-27 | 1983-11-15 | University Patents, Inc. | Phosphoramidite compounds and processes |
DE3239888A1 (en) * | 1982-10-28 | 1984-05-03 | Hubert Prof. Dr. 2000 Hamburg Köster | METHOD FOR PRODUCING OLIGONUCLEOSIDE PHOSPHONATES |
DE3247923A1 (en) * | 1982-12-24 | 1984-06-28 | Dr. Karl Thomae Gmbh, 7950 Biberach | Oligonucleotides and process for their preparation |
-
1985
- 1985-06-14 EP EP19850903503 patent/EP0229053A1/en not_active Withdrawn
- 1985-06-14 WO PCT/US1985/001148 patent/WO1986007362A1/en unknown
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