DE19915867A1 - New nucleoside derivatives with photolabile protecting groups, useful in oligonucleotide synthesis, particularly on solid phases, e.g. for hybridization testing - Google Patents
New nucleoside derivatives with photolabile protecting groups, useful in oligonucleotide synthesis, particularly on solid phases, e.g. for hybridization testingInfo
- Publication number
- DE19915867A1 DE19915867A1 DE19915867A DE19915867A DE19915867A1 DE 19915867 A1 DE19915867 A1 DE 19915867A1 DE 19915867 A DE19915867 A DE 19915867A DE 19915867 A DE19915867 A DE 19915867A DE 19915867 A1 DE19915867 A1 DE 19915867A1
- Authority
- DE
- Germany
- Prior art keywords
- group
- nucleoside
- radical
- alkyl
- opn
- 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.)
- Pending
Links
- 150000003833 nucleoside derivatives Chemical class 0.000 title claims abstract description 38
- 125000006239 protecting group Chemical group 0.000 title claims abstract description 25
- 239000007790 solid phase Substances 0.000 title claims abstract description 7
- 238000009396 hybridization Methods 0.000 title description 3
- 238000002515 oligonucleotide synthesis Methods 0.000 title 1
- -1 dimethoxytrityl Chemical group 0.000 claims abstract description 55
- 238000000034 method Methods 0.000 claims abstract description 19
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 17
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 16
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 claims abstract description 14
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 claims abstract description 14
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 14
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 14
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 14
- 125000000217 alkyl group Chemical group 0.000 claims abstract description 12
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000002360 preparation method Methods 0.000 claims abstract description 11
- 125000003545 alkoxy group Chemical group 0.000 claims abstract description 10
- 108091034117 Oligonucleotide Proteins 0.000 claims abstract description 8
- 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 claims abstract description 8
- 125000004183 alkoxy alkyl group Chemical group 0.000 claims abstract description 8
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims abstract description 7
- 229930024421 Adenine Natural products 0.000 claims abstract description 7
- 229960000643 adenine Drugs 0.000 claims abstract description 7
- 229940104302 cytosine Drugs 0.000 claims abstract description 7
- 229940035893 uracil Drugs 0.000 claims abstract description 7
- 229940113082 thymine Drugs 0.000 claims abstract description 6
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 5
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 4
- MSSXOMSJDRHRMC-UHFFFAOYSA-N 9H-purine-2,6-diamine Chemical group NC1=NC(N)=C2NC=NC2=N1 MSSXOMSJDRHRMC-UHFFFAOYSA-N 0.000 claims abstract description 3
- 125000000524 functional group Chemical group 0.000 claims abstract description 3
- 229910052717 sulfur Inorganic materials 0.000 claims abstract description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 54
- 239000003153 chemical reaction reagent Substances 0.000 claims description 20
- 238000005917 acylation reaction Methods 0.000 claims description 19
- 230000010933 acylation Effects 0.000 claims description 17
- 125000004432 carbon atom Chemical group C* 0.000 claims description 17
- 239000002777 nucleoside Substances 0.000 claims description 17
- 229910052736 halogen Inorganic materials 0.000 claims description 15
- 150000002367 halogens Chemical class 0.000 claims description 15
- 150000001875 compounds Chemical class 0.000 claims description 13
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 10
- MCTWTZJPVLRJOU-UHFFFAOYSA-N 1-methyl-1H-imidazole Chemical compound CN1C=CN=C1 MCTWTZJPVLRJOU-UHFFFAOYSA-N 0.000 claims description 9
- 150000005840 aryl radicals Chemical class 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 7
- 150000003254 radicals Chemical class 0.000 claims description 6
- 229910052739 hydrogen Inorganic materials 0.000 claims description 5
- 239000002773 nucleotide Substances 0.000 claims description 5
- 125000003729 nucleotide group Chemical group 0.000 claims description 5
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- 125000002777 acetyl group Chemical class [H]C([H])([H])C(*)=O 0.000 claims description 3
- 229910052801 chlorine Inorganic materials 0.000 claims description 3
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 3
- 229910052731 fluorine Inorganic materials 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 150000003536 tetrazoles Chemical class 0.000 claims description 3
- DHKHKXVYLBGOIT-UHFFFAOYSA-N acetaldehyde Diethyl Acetal Natural products CCOC(C)OCC DHKHKXVYLBGOIT-UHFFFAOYSA-N 0.000 claims description 2
- 229910052794 bromium Inorganic materials 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 239000012022 methylating agents Substances 0.000 claims description 2
- PSHKMPUSSFXUIA-UHFFFAOYSA-N n,n-dimethylpyridin-2-amine Chemical compound CN(C)C1=CC=CC=N1 PSHKMPUSSFXUIA-UHFFFAOYSA-N 0.000 claims description 2
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 2
- 150000003852 triazoles Chemical class 0.000 claims description 2
- 229910052799 carbon Inorganic materials 0.000 claims 2
- 125000002947 alkylene group Chemical group 0.000 claims 1
- 238000010276 construction Methods 0.000 claims 1
- KPUWHANPEXNPJT-UHFFFAOYSA-N disiloxane Chemical group [SiH3]O[SiH3] KPUWHANPEXNPJT-UHFFFAOYSA-N 0.000 claims 1
- 239000003960 organic solvent Substances 0.000 claims 1
- 239000002798 polar solvent Substances 0.000 claims 1
- 125000003107 substituted aryl group Chemical group 0.000 abstract 2
- PWCLNQLPYQVWAB-UHFFFAOYSA-N 5-aminoimidazole-4-carboxylic acid Chemical compound NC=1NC=NC=1C(O)=O PWCLNQLPYQVWAB-UHFFFAOYSA-N 0.000 abstract 1
- 125000005083 alkoxyalkoxy group Chemical group 0.000 abstract 1
- 125000001475 halogen functional group Chemical group 0.000 abstract 1
- 125000000472 sulfonyl group Chemical group *S(*)(=O)=O 0.000 abstract 1
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 24
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 18
- 238000006243 chemical reaction Methods 0.000 description 18
- 238000000018 DNA microarray Methods 0.000 description 13
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 description 9
- 239000002808 molecular sieve Substances 0.000 description 8
- 125000003835 nucleoside group Chemical group 0.000 description 8
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 8
- LRWLGGPRIFYAPO-UHFFFAOYSA-N 2-(2-nitrophenyl)propyl carbonochloridate Chemical compound ClC(=O)OCC(C)C1=CC=CC=C1[N+]([O-])=O LRWLGGPRIFYAPO-UHFFFAOYSA-N 0.000 description 7
- 239000012074 organic phase Substances 0.000 description 7
- LCNXGABVMBGKSJ-UHFFFAOYSA-M 2-(2-nitrophenyl)propyl 3-methylimidazol-3-ium-1-carboxylate;trifluoromethanesulfonate Chemical compound [O-]S(=O)(=O)C(F)(F)F.C=1C=CC=C([N+]([O-])=O)C=1C(C)COC(=O)N1C=C[N+](C)=C1 LCNXGABVMBGKSJ-UHFFFAOYSA-M 0.000 description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 6
- 230000011987 methylation Effects 0.000 description 6
- 238000007069 methylation reaction Methods 0.000 description 6
- OISVCGZHLKNMSJ-UHFFFAOYSA-N 2,6-dimethylpyridine Chemical compound CC1=CC=CC(C)=N1 OISVCGZHLKNMSJ-UHFFFAOYSA-N 0.000 description 5
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 5
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 5
- 238000006911 enzymatic reaction Methods 0.000 description 5
- 239000012299 nitrogen atmosphere Substances 0.000 description 5
- 239000003495 polar organic solvent Substances 0.000 description 5
- 229910052938 sodium sulfate Inorganic materials 0.000 description 5
- 235000011152 sodium sulphate Nutrition 0.000 description 5
- INMIVEZLEVVJNJ-UHFFFAOYSA-M 2-(2-nitrophenyl)propyl 3-methylimidazol-3-ium-1-carboxylate;chloride Chemical compound [Cl-].C=1C=CC=C([N+]([O-])=O)C=1C(C)COC(=O)N1C=C[N+](C)=C1 INMIVEZLEVVJNJ-UHFFFAOYSA-M 0.000 description 4
- 125000001731 2-cyanoethyl group Chemical group [H]C([H])(*)C([H])([H])C#N 0.000 description 4
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 4
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 4
- ANVKJBSWPJROCX-GUPZPADCSA-N [(2r,3s,5r)-2-(hydroxymethyl)-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-3-yl] 2-(2-nitrophenyl)propyl carbonate Chemical compound N1([C@H]2C[C@@H]([C@H](O2)CO)OC(=O)OCC(C)C=2C(=CC=CC=2)[N+]([O-])=O)C=C(C)C(=O)NC1=O ANVKJBSWPJROCX-GUPZPADCSA-N 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- LYGJENNIWJXYER-UHFFFAOYSA-N nitromethane Chemical compound C[N+]([O-])=O LYGJENNIWJXYER-UHFFFAOYSA-N 0.000 description 4
- 150000008300 phosphoramidites Chemical class 0.000 description 4
- 125000004742 propyloxycarbonyl group Chemical group 0.000 description 4
- GQHTUMJGOHRCHB-UHFFFAOYSA-N 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine Chemical compound C1CCCCN2CCCN=C21 GQHTUMJGOHRCHB-UHFFFAOYSA-N 0.000 description 3
- 229910004298 SiO 2 Inorganic materials 0.000 description 3
- RXXXJYQORYSSDI-IIEJJZGLSA-N [(2r,3s,5r)-5-[6-[[2-(4-tert-butylphenoxy)acetyl]amino]purin-9-yl]-2-(hydroxymethyl)oxolan-3-yl] 2-(2-nitrophenyl)propyl carbonate Chemical compound N1=CN=C2N([C@H]3C[C@@H]([C@H](O3)CO)OC(=O)OCC(C)C=3C(=CC=CC=3)[N+]([O-])=O)C=NC2=C1NC(=O)COC1=CC=C(C(C)(C)C)C=C1 RXXXJYQORYSSDI-IIEJJZGLSA-N 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- UAOMVDZJSHZZME-UHFFFAOYSA-N diisopropylamine Chemical compound CC(C)NC(C)C UAOMVDZJSHZZME-UHFFFAOYSA-N 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000006260 foam Substances 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 230000005855 radiation Effects 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- SCYULBFZEHDVBN-UHFFFAOYSA-N 1,1-Dichloroethane Chemical compound CC(Cl)Cl SCYULBFZEHDVBN-UHFFFAOYSA-N 0.000 description 2
- CIVPZPJJPWVVIP-UHFFFAOYSA-N 2-(2-nitrophenyl)propan-1-ol Chemical compound OCC(C)C1=CC=CC=C1[N+]([O-])=O CIVPZPJJPWVVIP-UHFFFAOYSA-N 0.000 description 2
- LBLYYCQCTBFVLH-UHFFFAOYSA-N 2-Methylbenzenesulfonic acid Chemical compound CC1=CC=CC=C1S(O)(=O)=O LBLYYCQCTBFVLH-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 2
- HOPRXXXSABQWAV-UHFFFAOYSA-N anhydrous collidine Natural products CC1=CC=NC(C)=C1C HOPRXXXSABQWAV-UHFFFAOYSA-N 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 238000010804 cDNA synthesis Methods 0.000 description 2
- PFKFTWBEEFSNDU-UHFFFAOYSA-N carbonyldiimidazole Chemical compound C1=CN=CN1C(=O)N1C=CN=C1 PFKFTWBEEFSNDU-UHFFFAOYSA-N 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 239000000460 chlorine Substances 0.000 description 2
- UTBIMNXEDGNJFE-UHFFFAOYSA-N collidine Natural products CC1=CC=C(C)C(C)=N1 UTBIMNXEDGNJFE-UHFFFAOYSA-N 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- JXTHNDFMNIQAHM-UHFFFAOYSA-N dichloroacetic acid Chemical compound OC(=O)C(Cl)Cl JXTHNDFMNIQAHM-UHFFFAOYSA-N 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 238000007169 ligase reaction Methods 0.000 description 2
- 238000007403 mPCR Methods 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- AOJFQRQNPXYVLM-UHFFFAOYSA-N pyridin-1-ium;chloride Chemical compound [Cl-].C1=CC=[NH+]C=C1 AOJFQRQNPXYVLM-UHFFFAOYSA-N 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 239000011877 solvent mixture Substances 0.000 description 2
- GFYHSKONPJXCDE-UHFFFAOYSA-N sym-collidine Natural products CC1=CN=C(C)C(C)=C1 GFYHSKONPJXCDE-UHFFFAOYSA-N 0.000 description 2
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 2
- 229940086542 triethylamine Drugs 0.000 description 2
- ITMCEJHCFYSIIV-UHFFFAOYSA-M triflate Chemical compound [O-]S(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-M 0.000 description 2
- MIOPJNTWMNEORI-GMSGAONNSA-N (S)-camphorsulfonic acid Chemical compound C1C[C@@]2(CS(O)(=O)=O)C(=O)C[C@@H]1C2(C)C MIOPJNTWMNEORI-GMSGAONNSA-N 0.000 description 1
- SGUVLZREKBPKCE-UHFFFAOYSA-N 1,5-diazabicyclo[4.3.0]-non-5-ene Chemical compound C1CCN=C2CCCN21 SGUVLZREKBPKCE-UHFFFAOYSA-N 0.000 description 1
- UBTJZUKVKGZHAD-UHFFFAOYSA-N 1-[5-[[bis(4-methoxyphenyl)-phenylmethoxy]methyl]-4-hydroxyoxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound C1=CC(OC)=CC=C1C(C=1C=CC(OC)=CC=1)(C=1C=CC=CC=1)OCC1C(O)CC(N2C(NC(=O)C(C)=C2)=O)O1 UBTJZUKVKGZHAD-UHFFFAOYSA-N 0.000 description 1
- JDIIGWSSTNUWGK-UHFFFAOYSA-N 1h-imidazol-3-ium;chloride Chemical compound [Cl-].[NH2+]1C=CN=C1 JDIIGWSSTNUWGK-UHFFFAOYSA-N 0.000 description 1
- IHCCLXNEEPMSIO-UHFFFAOYSA-N 2-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperidin-1-yl]-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C1CCN(CC1)CC(=O)N1CC2=C(CC1)NN=N2 IHCCLXNEEPMSIO-UHFFFAOYSA-N 0.000 description 1
- XWKFPIODWVPXLX-UHFFFAOYSA-N 2-methyl-5-methylpyridine Natural products CC1=CC=C(C)N=C1 XWKFPIODWVPXLX-UHFFFAOYSA-N 0.000 description 1
- 125000003903 2-propenyl group Chemical group [H]C([*])([H])C([H])=C([H])[H] 0.000 description 1
- RKVHNYJPIXOHRW-UHFFFAOYSA-N 3-bis[di(propan-2-yl)amino]phosphanyloxypropanenitrile Chemical compound CC(C)N(C(C)C)P(N(C(C)C)C(C)C)OCCC#N RKVHNYJPIXOHRW-UHFFFAOYSA-N 0.000 description 1
- DFGKGUXTPFWHIX-UHFFFAOYSA-N 6-[2-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]acetyl]-3H-1,3-benzoxazol-2-one Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)CC(=O)C1=CC2=C(NC(O2)=O)C=C1 DFGKGUXTPFWHIX-UHFFFAOYSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 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 1
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical class [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 1
- JAKNTMZCBVZZMJ-UHFFFAOYSA-N C(C)(C)N(C(C)C)POCCC#N.[Cl] Chemical compound C(C)(C)N(C(C)C)POCCC#N.[Cl] JAKNTMZCBVZZMJ-UHFFFAOYSA-N 0.000 description 1
- KZBUYRJDOAKODT-UHFFFAOYSA-N Chlorine Chemical compound ClCl KZBUYRJDOAKODT-UHFFFAOYSA-N 0.000 description 1
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 1
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Abstract
Description
Gegenstand der vorliegenden Erfindung sind Nucleosid-Derivate mit photolabilen Schutzgruppen, Verfahren zu deren Herstellung, deren Verwendung sowie daraus aufgebaute Nucleinsäure-Chips.The present invention relates to nucleoside derivatives with photolabile Protecting groups, processes for their production, their use and nucleic acid chips built from it.
Photolabile Schutzgruppen für die Hydroxy- und Phosphatfunktionen in Nucleosi den bzw. Nucleotiden sind von Bedeutung, da sie sich für die lichtgesteuerte Parallel-Synthese von Oligonukleotiden auf einer soliden Trägeroberfläche eignen (Fodor et al., Science 1991, 251, S. 767 ff.). Hiermit können Oligonukleotide oder Nukleinsäure-Chips aufgebaut werden, die für eine effiziente Sequenzierung von Nukleinsäuren eingesetzt werden können.Photolabile protecting groups for the hydroxy and phosphate functions in nucleosi The or nucleotides are important because they are for the light-controlled Parallel synthesis of oligonucleotides on a solid support surface are suitable (Fodor et al., Science 1991, 251, pp. 767 ff.). This allows oligonucleotides or nucleic acid chips are built up for efficient sequencing of nucleic acids can be used.
Bislang sind einzig photolithografische Herstellungsverfahren von DNA-Chips unter Verwendung von 3'-O-Phosphitamiden, die entsprechend die temporäre photolabile Schutzgruppe an der 5'-O-Position aufweisen, bekannt (WO-A- 96/18634). Unter Verwendung dieser Nucleinsäurebausteine lassen sich DNA- Chips herstellen, wobei der Aufbau des Oligomers vom 3'- zum 5'-Ende erfolgt. Das fertiggestellte Oligomer ist somit über das 3'-O-Ende an der festen Phase verankert, das 5'-OH-Ende ist frei zugänglich. DNA-Chips, die mit dieser Metho de erzeugt wurden, lassen sich für Hybridisierungsexperimente verwenden, aber nicht für bestimmte Enzymreaktionen (z. B. mit der der DNA-Polymerase oder Ligase), die ein freies 3'-OH erfordern.So far, only photolithographic manufacturing processes of DNA chips are using 3'-O-phosphitamides, which correspond to the temporary have photolabile protecting group at the 5'-O position, known (WO-A- 96/18634). Using these nucleic acid building blocks, DNA Prepare chips, building the oligomer from the 3 'to the 5' end. The completed oligomer is thus at the 3'-O end on the solid phase anchored, the 5'-OH end is freely accessible. DNA chips using this metho de, can be used for hybridization experiments, but not for certain enzyme reactions (e.g. with that of DNA polymerase or Ligase), which require a free 3'-OH.
Die Aufgabe der vorliegenden Erfindung besteht deshalb darin, 3'-photolabile Nucleoside und deren Derivate bereitzustellen und mit den daraus gewonnenen 3'-photolabilen Nucleosiden Nukleinsäure-Chips zu generieren, bei denen die über die lichtgesteuerte Synthese aufgebauten Oligomeren über das 5'-Ende an die feste Phase gekoppelt sind und somit Enzymreaktionen am 3'-Ende möglich machen. The object of the present invention is therefore 3'-photolabile To provide nucleosides and their derivatives and with those obtained from them 3'-photolabile nucleosides to generate nucleic acid chips in which the oligomers built up via the light-controlled synthesis via the 5 'end the solid phase is coupled and enzyme reactions at the 3 'end are possible do.
Diese Aufgabe wird erfindungsgemäß durch die Nucleosid-Derivate der allgemei nen Formel (I) entsprechend Patentanspruch 1 gelöst. Vorteilhafte Ausgestaltun gen ergeben sich aus den Unteransprüchen.According to the invention, this object is achieved in general by the nucleoside derivatives NEN formula (I) according to claim 1 solved. Advantageous design conditions result from the subclaims.
Erfindungsgemäße Nucleosid-Derivate haben folgende Formel:
Nucleoside derivatives according to the invention have the following formula:
mit
R1 = H, NO2, CN, OCH3, Halogen, Alkyl-, Alkoxy- oder Alkoxyalkylrest
mit 1 bis 4 C-Atomen
R2 = H, OCH3, NO2, CN, Halogen, Allkoxy-, Alkoxyalkyl- oder Alkylrest
mit 1 bis 4 C-Atomen oder ein Arylrest
R3 = H, Halogen, NO2, CN, Alkylrest mit 1 bis 4 C-Atomen oder Arylrest
oder aliphatischer Acylrest mit 2 bis 5 C-Atomen
R4 = H, Halogen, OCH3, CN, NO2, Alkoxy-, Alkoxyalkyl- oder Alkylrest
mit 1 bis 4 C-Atomen oder Arylrest
R5 = H oder eine bei der Herstellung von Oligonukleotiden übliche Sch
utzgruppe
R6 = H, OH, Halogen, Alkoxy- oder Alkoxyalkylrest mit 1 bis 4 C-Ato
men oder XR8, wobei X = O oder S und R8 = eine in der Nukleo
tidchemie übliche Schutzgruppe
R7 = H, NO2, CN, OCH3, Halogen, Alkyl- oder Alkoxyalkylgruppe mit 1
bis 4 C-Atomen oder ein Arylrest
X = SO2, OCO, OCS
B = H, Adenin, Cytosin, Guanin, Thyimin, Uracil, 2,6-Diaminopurin-9-yl,
Hypoxanthin-9-yl, 5-Methylcytosin-1-yl, 5-Amino-4-Imidazolcarbon
säure-1-yl oder 5-Amino-4-Imidazolcarbonsäureamid-3-yl, wobei im
Falle von B = Adenin, Cytosin oder Guanin die primäre Aminofunk
tion ggf. eine permanente Schutzgruppe aufweist bzw. Thymin
oder Uracil an der O4-Position ggf. eine permanente Schutzgruppe
aufweist.With
R 1 = H, NO 2 , CN, OCH 3 , halogen, alkyl, alkoxy or alkoxyalkyl radical having 1 to 4 carbon atoms
R 2 = H, OCH 3 , NO 2 , CN, halogen, alkoxy, alkoxyalkyl or alkyl radical having 1 to 4 carbon atoms or an aryl radical
R 3 = H, halogen, NO 2 , CN, alkyl radical with 1 to 4 carbon atoms or aryl radical or aliphatic acyl radical with 2 to 5 carbon atoms
R 4 = H, halogen, OCH 3 , CN, NO 2 , alkoxy, alkoxyalkyl or alkyl radical having 1 to 4 carbon atoms or aryl radical
R 5 = H or a protective group customary in the production of oligonucleotides
R 6 = H, OH, halogen, alkoxy or alkoxyalkyl radical with 1 to 4 C atoms or XR 8 , where X = O or S and R 8 = a protective group customary in nucleotide chemistry
R 7 = H, NO 2 , CN, OCH 3 , halogen, alkyl or alkoxyalkyl group with 1 to 4 carbon atoms or an aryl radical
X = SO 2 , OCO, OCS
B = H, adenine, cytosine, guanine, thyimin, uracil, 2,6-diaminopurin-9-yl, hypoxanthine-9-yl, 5-methylcytosin-1-yl, 5-amino-4-imidazole carbonate 1-yl or 5-amino-4-imidazolecarboxamide-3-yl, where in the case of B = adenine, cytosine or guanine the primary amino function may have a permanent protective group or thymine or uracil at the O 4 position may have a permanent protective group having.
Die Alkyl-, Alkoxy- oder Alkoxyalkylgruppe der Reste R1, R2, R3, R4 und R7 kann linear oder verzweigt sein, substituiert (insbesondere mit einem oder mehreren Halogenatomen) oder unsubstituiert sowie gesättigt oder ungesättigt sein. Analoges gilt für die Arylgruppe der Reste R2, R3, R4 oder R7 bzw. den Acylrest des Rests R3.The alkyl, alkoxy or alkoxyalkyl group of the radicals R 1 , R 2 , R 3 , R 4 and R 7 can be linear or branched, substituted (in particular with one or more halogen atoms) or unsubstituted and saturated or unsaturated. The same applies analogously to the aryl group of the radicals R 2 , R 3 , R 4 or R 7 or the acyl radical of the radical R 3 .
Vorzugsweise stellt R4 H oder einen Methylrest dar. Im Falle von R4 ≠ H sind die Substituenten R1-R3 am Phenylring vorzugsweise Wasserstoffreste. Außerdem stellt im Falle von R2 = OCH3 R3 vorzugsweise einen Wasserstoffrest dar.R 4 preferably represents H or a methyl radical. In the case of R 4 ≠ H, the substituents R 1 -R 3 on the phenyl ring are preferably hydrogen radicals. In addition, in the case of R 2 = OCH 3 R 3, it is preferably a hydrogen radical.
In der Position R5 bedeutet "eine bei der Herstellung von Oligonukleotiden übliche Schutzgruppe" beispielsweise eine Phosphitamid-Gruppe, wie p-NC-CH2- CH2-O-P-N(Q)2, p-NC-C6H4-CH2-CH2-O-P-N(Q)2, p-NO2-C6H4-CH2-CH2-O-P-N(Q)2 oder CH2 = CH-CH2-O-P-N(Q)2, wobei die Q-Gruppen gleich oder verschieden sein können und lineare oder verzweigte Alkylreste mit 1 bis 4 C-Atomen, vorzugs weise Ethyl- oder Isopropylreste, bedeuten.In position R 5 is a 'common in the preparation of oligonucleotides protecting group ", for example, a phosphite group such as p-NC-CH 2 - CH 2 -OPN (Q) 2, p-NC-C 6 H 4 -CH 2 -CH 2 -OPN (Q) 2 , p-NO 2 -C 6 H 4 -CH 2 -CH 2 -OPN (Q) 2 or CH 2 = CH-CH 2 -OPN (Q) 2 , where the Q- Groups can be the same or different and mean linear or branched alkyl radicals with 1 to 4 carbon atoms, preferably as ethyl or isopropyl radicals.
In der Position R6 bedeutet "eine in der Nukleotidchemie übliche Schutzgruppe" (= R8) insbesondere H sowie die üblichen Alkyl-, Alkenyl-, Acetal- oder Silylet her-Schutzgruppen. Bevorzugte Schutzgruppen sind Methyl- oder Ethylreste, Allylreste, Tetrahydropyranyl- bzw. Methoxytetrahydropyranyl-Reste sowie t- Butyldimethylsilyl-Reste.In position R 6 , “a protective group customary in nucleotide chemistry” (= R 8 ) means in particular H and the customary alkyl, alkenyl, acetal or silylet protective groups. Preferred protective groups are methyl or ethyl radicals, allyl radicals, tetrahydropyranyl or methoxytetrahydropyranyl radicals and t-butyldimethylsilyl radicals.
Die an den Basen B ggf. permanent vorkommenden Schutzgruppen basieren vorzugsweise auf Acyl-Schutzgruppen. Bevorzugt sind vor allem Phenoxyacetyl-, tert-Butylphenoxyacetyl-, Isobutyryl-, Acetyl-, Benzoyl-, Allyl-, Phthaloyl-, Dansylethyloxycarbonyl-, 2-(4-Nitrophenyl)ethoxycarbonyl-oder Dimethylforma midino-Reste. Im Falle von Adenin, Cytosin und Guanin handelt es sich vorzugs weise um Phenoxyacetyl-, tert-Butylphenoxyacetyl, Acetyl- oder 2-(4-Nitrophe nyl)ethoxycarbonyl-Gruppen zum Schutz der exocyclischen Aminofunktionen. Die O6-Position von Guanin kann ggf. durch eine Schutzgruppe wie 2-(4-Nitro phenylsulfonyl)ethyl- oder 2-(4-Nitrophenyl)ethyl- geschützt sein. Ebenso kann die O4-Position von Thymin oder Uracil eine Schutzgruppe wie 2-(4-Nitrophenyl sulfonyl)ethyl- oder 2-(4-Nitrophenyl)ethyl- aufweisen.The protective groups which may occur permanently at the bases B are preferably based on acyl protective groups. Phenoxyacetyl, tert-butylphenoxyacetyl, isobutyryl, acetyl, benzoyl, allyl, phthaloyl, dansylethyloxycarbonyl, 2- (4-nitrophenyl) ethoxycarbonyl or dimethylforma midino radicals are particularly preferred. In the case of adenine, cytosine and guanine, preference is given to phenoxyacetyl, tert-butylphenoxyacetyl, acetyl or 2- (4-nitrophenyl) ethoxycarbonyl groups to protect the exocyclic amino functions. The O 6 position of guanine can optionally be protected by a protective group such as 2- (4-nitrophenylsulfonyl) ethyl or 2- (4-nitrophenyl) ethyl. Likewise, the O 4 position of thymine or uracil can have a protective group such as 2- (4-nitrophenyl sulfonyl) ethyl or 2- (4-nitrophenyl) ethyl.
Halogen bedeutet erfindungsgemäß F, Cl, Br, I, wobei die drei erstgenannten bevorzugt sind.Halogen according to the invention means F, Cl, Br, I, the first three mentioned are preferred.
Die Herstellung der erfindungsgemäßen Nucleosid-Derivate ist beispielhaft in Fig. 2 gezeigt, worauf nachfolgend Bezug genommen wird. Die Erwähnung von be stimmten Halogen- und Alkylsubstitutionen schließt immer gleichwirkende Äquivalente ein, z. B. "Chlor-" schließt nicht aus, daß auch die entsprechenden Fluor- oder Bromverbindungen einsetzbar sind. Ebensolches gilt für "Methyl-", das auch die entsprechenden anderen Niederalkylverbindungen, wie Ethyl-, Propyl- oder Butyl mit einschließt. Die Reste R1, R2, R3, R4, R6, R7 und X haben die oben genannten Bedeutungen.The preparation of the nucleoside derivatives according to the invention is shown by way of example in FIG. 2, to which reference is made below. The mention of certain halogen and alkyl substitutions always includes equivalent equivalents, e.g. B. "Chlorine" does not exclude that the corresponding fluorine or bromine compounds can also be used. The same applies to "methyl", which also includes the corresponding other lower alkyl compounds, such as ethyl, propyl or butyl. The radicals R 1 , R 2 , R 3 , R 4 , R 6 , R 7 and X have the meanings given above.
Die Herstellung beginnt mit der Präparation eines Acylierungsreagenzes. Hierzu wird auf Fig. 1 verwiesen. Ausgegangen wird hierfür bevorzugt von einem Chlorkohlensäureester (II, mit X = OCO), der sich beispielsweise gemäß der Vorschrift in WO-A-96/18634 oder gemäß nachfolgendem Beispiel 1 erhalten läßt. Analog ist ein gewünschter Chlorthiokohlensäureester (II, mit X = OCS) über die analoge Umsetzung mit Thiophosgen zugänglich. Das Acylierungs reagenz (IV) wird dann durch Umsetzung des Chlorkohlensäureesters (II, mit X = OCO) oder des Chlorthiokohlensäureesters (II, mit X = OCS) oder ein entsprechendes Sulfonylchlorid-Derivat (II, X = SO2) mit einer Verbindung (III), vorzugsweise N-Methylimidazol, generiert. Diese Reaktionen werden in einem polaren organischen Lösungsmittel, vorzugsweise Dichlormethan, bei Temperatu ren zwischen -10°C und +10°C, vorzugsweise bei 0°C, durchgeführt. Vor zugsweise wird der Reaktion Molekularsieb zugesetzt und mit einem Überschuß an Verbindung (III), bevorzugt N-Methylimidazol, in bezug auf die eingesetzte Verbindung (II) gearbeitet: 1-10 Äquivalente, bevorzugt 2-5 Äquivalente. Alter nativ zu N-Methylimidazol kann die Generation des Acylierungsreagenzes auch mittels anderer heterocyclischer Verbindungen (III), wie Pyridin, 4-N,N-Dimethyl aminopyridin (DMAP), Triazol, Tetrazol oder Imidazol verlaufen.Production begins with the preparation of an acylation reagent. For this purpose, reference is made to FIG. 1. The starting point for this is preferably a chlorocarbonic acid ester (II, with X = OCO), which can be obtained, for example, according to the specification in WO-A-96/18634 or according to Example 1 below. A desired chlorothiocarbonic acid ester (II, with X = OCS) is accessible analogously via the analogous reaction with thiophosgene. The acylation reagent (IV) is then reacted with the chlorocarbonic acid ester (II, with X = OCO) or the chlorothiocarbonic acid ester (II, with X = OCS) or a corresponding sulfonyl chloride derivative (II, X = SO 2 ) with a compound (III ), preferably N-methylimidazole. These reactions are carried out in a polar organic solvent, preferably dichloromethane, at temperatures between -10 ° C. and + 10 ° C., preferably at 0 ° C. Molecular sieve is preferably added to the reaction and an excess of compound (III), preferably N-methylimidazole, is used in relation to the compound (II) used: 1-10 equivalents, preferably 2-5 equivalents. The generation of the acylation reagent using other heterocyclic compounds (III), such as pyridine, 4-N, N-dimethylaminopyridine (DMAP), triazole, tetrazole or imidazole, can also proceed in an alternative way to N-methylimidazole.
Alternativ ist das Acylierungsreagenz (IV, Z = triflat) ausgehend von N,N-Carbo nyldiimidazol (V, Y = CO) oder N,N-Thiocarbonyldiimidazol (V, Y = CS) nach Methylierung mit einem Methylierungsreagenz (VI), vorzugsweise Trifluorme thansulfonsäuremethylester, und Umsetzung mit dem entsprechenden Alkohol (VIII) zugänglich. Hierbei wird die Reaktion bevorzugt in einem polaren organi schen Lösungsmittel, vorzugsweise in Nitromethan oder einem Gemisch von Nitromethan und Dichlormethan, bei Temperaturen zwischen -10 und +10°C, vorzugsweise 0°C, durchgeführt. Die Methylierung von (V) erfolgt beispiels weise gemäß Rapoport et al., J. Am. Chem. Soc. 1989, 111, S. 4856-4859. Nach erfolgter Methylierung wird der entsprechende Alkohol (VIII) zugesetzt und somit das Acylierungsmittel vom Imidazoliumtyp (IV, Z = triflat) in Form eines Triflatsalzes generiert. Werden bei der Methylierung von (V) als Methylierungs reagenz (VI) Methyljodid oder Meerweinsalze eingesetzt, können die Acylierungs reagenzien (IV) entsprechend in Form ihrer Iodid oder Tetrafluoroborat-Salze erzeugt werden. Die Umsetzung von Verbindung (V) mit dem entsprechenden Methylierungsmittel erfolgt vorzugsweise im Verhältnis 1 : 1 bis 1 : 10, bevorzugt im Verhältnis 1 : 2. Die Umsetzung der methylierten Form (VI) mit dem entspre chenden Alkohol (VIII) erfolgt vorzugsweise im Verhältnis 1 : 1 bis 1 : 10, ganz bevorzugt im Verhältnis 1 : 1 bis 1 : 2.Alternatively, the acylation reagent (IV, Z = triflate) is based on N, N-carbo nyldiimidazole (V, Y = CO) or N, N-thiocarbonyldiimidazole (V, Y = CS) Methylation with a methylation reagent (VI), preferably trifluorine methyl thanesulfonic acid, and reaction with the corresponding alcohol (VIII) accessible. The reaction is preferably carried out in a polar organic rule solvent, preferably in nitromethane or a mixture of Nitromethane and dichloromethane, at temperatures between -10 and + 10 ° C, preferably 0 ° C, carried out. The methylation of (V) takes place, for example wise according to Rapoport et al., J. Am. Chem. Soc. 1989, 111, pp. 4856-4859. After methylation, the corresponding alcohol (VIII) is added and thus the imidazolium type acylating agent (IV, Z = triflate) in the form of a Triflatsalzes generated. Are in the methylation of (V) as methylation reagent (VI) methyl iodide or sea wine salts used, the acylation reagents (IV) in the form of their iodide or tetrafluoroborate salts be generated. The reaction of compound (V) with the corresponding one Methylating agent is preferably in a ratio of 1: 1 to 1:10, preferably in a ratio of 1: 2. The reaction of the methylated form (VI) with the corresponding The alcohol (VIII) is preferably in a ratio of 1: 1 to 1:10, whole preferably in a ratio of 1: 1 to 1: 2.
Das Acylierungsreagenz (IV) wird weiter mit einem ggf. geschützten Nucleosid (IX) umgesetzt. 5'-DMTr-geschützte Nucleoside der allgemeinen Formel (IX) sind beispielsweise käuflich erhältlich von den Firmen Proligo, Fluka, Sigma oder Aldrich. The acylation reagent (IV) is further protected with an optionally protected nucleoside (IX) implemented. 5'-DMTr-protected nucleosides of the general formula (IX) for example commercially available from the companies Proligo, Fluka, Sigma or Aldrich.
Die Umsetzung des Acylierungsreagenz (IV) mit den geschützen Nukleosiden (IX) erfolgt vorzugsweise in Dichlormethan oder einem Lösungsmittelgemisch aus Dichlormethan und einem polaren organischen Lösungsmittel ggf. in Gegenwart einer Base, wie Pyridin, N-Methylimidazol, 4,N,N-Dimethylaminopyridin, Ethyldii sopropylamin (EtN(i-pr)2) oder Triethylamin, bei Temperaturen zwischen -60 und +25°C, bevorzugt 0°C. Als polares organisches Lösungsmittel wird vorzugs weise Dichlorethan, Nitromethan, DMF oder Pyridin eingesetzt. Das Mischungs verhältnis von Dichlormethan zu dem polaren organischen Lösungsmittel unter liegt keiner Beschränkung. Vorzugsweise werden jedoch 1 bis 3 Vol.-Teile Dichlormethan pro Vol.-Teil polarem organischem Lösungsmittel eingesetzt. Bevorzugt wird eine Lösung des Acylierungsreagenz (IV) in Dichlormethan vor gelegt und das Nucleosid (IX), welches ebenso in Dichlormethan gelöst wurde, zugetropft. Das Molverhältnis von Acylierungsreagenz zu Nucleosid kann vor zugsweise zwischen 1 : 1 bis 5 : 1, bevorzugt bei 3 : 1, ganz bevorzugt bei 2 : 1 liegen, d. h. das Acylierungsreagenz wird bevorzugt im Überschuß verwendet. Die Konzentration des Nucleosids im Lösungsmittelgemisch unterliegt keiner Be schränkung. Sie liegt jedoch bevorzugt im Bereich von 0,1 bis 3,0 mmol pro 10 ml Lösungsmittel.The reaction of the acylation reagent (IV) with the protected nucleosides (IX) is preferably carried out in dichloromethane or a solvent mixture of dichloromethane and a polar organic solvent, optionally in the presence of a base such as pyridine, N-methylimidazole, 4, N, N-dimethylaminopyridine, Ethyldiisopropylamine (EtN (i-pr) 2 ) or triethylamine, at temperatures between -60 and + 25 ° C, preferably 0 ° C. As a polar organic solvent, dichloroethane, nitromethane, DMF or pyridine is preferably used. The mixing ratio of dichloromethane to the polar organic solvent is not limited. However, 1 to 3 parts by volume of dichloromethane are preferably used per part by volume of polar organic solvent. A solution of the acylation reagent (IV) in dichloromethane is preferably placed in front and the nucleoside (IX), which was also dissolved in dichloromethane, is added dropwise. The molar ratio of acylating reagent to nucleoside can preferably be between 1: 1 to 5: 1, preferably 3: 1, most preferably 2: 1, ie the acylating reagent is preferably used in excess. The concentration of the nucleoside in the solvent mixture is not subject to any restrictions. However, it is preferably in the range from 0.1 to 3.0 mmol per 10 ml of solvent.
Nach erfolgter Umsetzung (bevorzugte Reaktionszeit: 1-12 Std.) kann das erhaltene Nucleosid-Derivat (X) isoliert werden. Danach erfolgt das Abspalten der 5'-Schutzgruppe am Nucleosidbestandteil durch Umsetzen mit bevorzugt Trichloressigsäure oder Toluolsulfonsäure, ggf. mit Camphersulfonsäure oder Dichloressigsäure in Dichlormethan. Es wird das Nucleosid-Derivat (XI) erhalten, das Formel (I) gehorcht.After the reaction has taken place (preferred reaction time: 1-12 hours), this can be done obtained nucleoside derivative (X) can be isolated. This is followed by splitting off the 5'-protecting group on the nucleoside component by reaction with preferred Trichloroacetic acid or toluenesulfonic acid, optionally with camphorsulfonic acid or Dichloroacetic acid in dichloromethane. The nucleoside derivative (XI) is obtained the formula (I) obeys.
Falls es gewünscht ist, kann an der 5'-Position des Nucleosid-Derivats (XI) eine Phosphitamid-Gruppe eingeführt werden. Dies geschieht beispielsweise durch die Umsetzung des Nucleosid-Derivats (XI) mit Bis(diisopropylamino)(β-cyanoethoxy) phosphin unter Zusatz eines leicht aciden Katalysators (beispielsweise Tetrazol, Pyridin-Hydrochlorid) oder durch Umsetzung des Nucleosidderivats mit Chlor- Diisopropylamino-2-cyanoethoxylphosphin unter Zusatz einer Base (z. B. Dii sopropylethylamin, N-Methylmorphin, Lutidin oder Collidin) und einem Lösungs mittel (z. B. THF). Dabei entsteht Verbindung (XII).If desired, one can be located at the 5 'position of the nucleoside derivative (XI) Phosphitamide group are introduced. This happens for example through the Reaction of the nucleoside derivative (XI) with bis (diisopropylamino) (β-cyanoethoxy) phosphine with the addition of a slightly acidic catalyst (for example tetrazole, Pyridine hydrochloride) or by reacting the nucleoside derivative with chlorine Diisopropylamino-2-cyanoethoxylphosphine with the addition of a base (e.g. Dii sopropylethylamine, N-methylmorphine, lutidine or collidine) and a solution medium (e.g. THF). This creates compound (XII).
Der Vorteil die Umsetzung des geschützten Nucleosids mit einem milden Acylie rungsreagenz durchzuführen, liegt in der Selektivität der Reaktion. Es werden quantitative Acylierungen der 3'-O-Position des Nucleosidbausteins ohne nach teilige Nebenproduktformation erhalten. Werden hierzu reaktivere Acylierungs reagenzien, wie z. B. der Chlorkohlensäureester selbst, verwendet, tritt eine unkontrollierte Reaktion ein. Es werden eine große Anzahl von Nebenprodukten gebildet, d. h. es besteht hierbei keinerlei Selektivität für das gewünschte 3- monoacylierte Produkt. In einer ganz besonders bevorzugten Ausführungsform wird das geschützte Nucleosid mit dem Acylierungsreagenz unter Zusatz von Molekularsieb durchgeführt. Mit Molekularsieb ist eine Steigerung der Selektivi tät für das gewünschte 3'-monoacylierte Produkt zu beobachten.The advantage of implementing the protected nucleoside with a mild acyly Carrying out reagent lies in the selectivity of the reaction. It will quantitative acylations of the 3'-O-position of the nucleoside building block without after partial by-product formation obtained. Do this with more reactive acylation reagents such as B. the chlorocarbonic acid itself used, occurs uncontrolled reaction. There are a large number of by-products formed, d. H. there is no selectivity for the desired 3- monoacylated product. In a very particularly preferred embodiment the protected nucleoside with the acylation reagent with the addition of Molecular sieve carried out. With molecular sieve there is an increase in selectivity for the desired 3'-monoacylated product.
Die erfindungsgemäßen 3'-photolabilen Nucleoside können bei der photolithogra fischen Nukleinsäurechip-Synthese eingesetzt werden. Verfahren hierzu sind dem Fachmann ausreichend bekannt (z. B. Fodor et al., s. o.). Ein geeignetes Verfahren hierzu ist beispielsweise auch in der deutschen Anmeldung DE 198 58 440.7 gezeigt. Dort wird zwar ein Verfahren gezeigt, das von 5'-photolabilen Nucleosiden ausgeht, aber die dort gezeigte Methodik läßt sich auf die Ver wendung von 3'-photolabilen 5'-Phosphitamiden analog übertragen. Bei diesem Verfahren wird der bei der Chip-Synthese übliche Bestrahlungsschritt in Anwe senheit einer Base durchgeführt wird. Dieses Verfahren zur photolithographischen Biochip-Synthese bietet den Vorteil, daß eine effiziente Abspaltung von photola bilen Schutzgruppen stattfindet.The 3'-photolabile nucleosides according to the invention can be used in photolithography fish nucleic acid chip synthesis can be used. Procedures for this are sufficiently known to the person skilled in the art (e.g. Fodor et al., see above). A suitable one The procedure for this is also described, for example, in German application DE 198 58 440.7 shown. A process is shown there, that of 5'-photolabile Nucleosides, but the methodology shown there can be applied to Ver analogous use of 3'-photolabile 5'-phosphitamides. With this The procedure is the irradiation step usual in chip synthesis in Anwe base is carried out. This process for photolithographic Biochip synthesis has the advantage that an efficient cleavage of photola protection groups.
Unter einem Nucleinsäurechip sollen erfindungsgemäß auf einem Träger aufge baute Biomoleküle, wie DNA oder RNA, sowie Nucleinsäureanaloga, wie PNA, LNA oder Chimären von diesen mit DNA, RNA oder untereinander verstanden werden. According to the invention, a nucleic acid chip is intended to be applied to a support built biomolecules, such as DNA or RNA, and nucleic acid analogues, such as PNA, LNA or chimeras of these understood with DNA, RNA or with each other become.
Erfindungsgemäß ist jegliche(r) auf diesem Gebiet übliche Träger bzw. Matrix bei der Nucleinsäurechip-Herstellung einsetzbar. Dies sind insbesondere Glas, Folien bzw. Membranen aus Polypropylen, Nylon, Cellulose, Cellulosederivate (z. B. Celluloseacetat, Cellulose-Mischester), Polyethersulfonen, Polyamiden, Polyvinyl chlorid, Polyvinylidenfluorid, Polyester, Teflon oder Polyethylen. Die Trägerober flächen können auch mit freien oder geschützten funktionellen Gruppen versehen sein, z. B. eine Amino-Gruppe, Hydroxyl-Gruppe, Carboxyl-Gruppe, Carbonyl- Gruppe, Thiol-, Amid- oder Phosphat-Gruppe tragen. In einer bevorzugten Ausführungsform weisen die Trägeroberflächen eine Derivatisierung gemäß der deutschen Patentanmeldung 198 53 242.3 auf.According to the invention, any carrier or matrix customary in this field is included of nucleic acid chip production can be used. These are especially glass, foils or membranes made of polypropylene, nylon, cellulose, cellulose derivatives (e.g. Cellulose acetate, mixed cellulose esters), polyether sulfones, polyamides, polyvinyl chloride, polyvinylidene fluoride, polyester, Teflon or polyethylene. The porters Areas can also be provided with free or protected functional groups be, e.g. B. an amino group, hydroxyl group, carboxyl group, carbonyl Wear group, thiol, amide or phosphate group. In a preferred one The carrier surfaces have a derivatization according to the embodiment German patent application 198 53 242.3.
Bei dem oben genannten bevorzugten Verfahren zur photolithographischen Bio chip-Synthese gemäß der deutschen Anmeldung DE 198 58 440.7 werden die Schritte Kondensation, Oxidation und Capping wie üblich (Fodor et al., Science 1991, 251, S. 767 ff.) durchgeführt. Allerdings findet der erste Schritt der Syn these, nämlich die Bestrahlung, unter Zusatz von Basen, bevorzugt starken Basen, insbesondere nicht-nukleophilen Basen, statt, was in Zusammenwirken mit dem bei der Bestrahlung angewendeten Licht zu einer überraschend effekti ven Abspaltung der Schutzgruppen führt. Als Basen eignen sich die dem Fach mann bekannten Basen, wie z. B. DBU (1,8-Diazabicyclo[5.4.0]undec-7-en, DBN (1,5-Diazabicyclo[4.3.0]non-5-en, Diiisopropylethylamin, Pyridin, Piperidin, Triethyl amin, Diisopropylamin, N-Methylmorpholin, 2,6-Lutidin, Collidin, N-Methylimi dazol, Dabco, N,N,-Dimethylaminopyridin. Die Bestrahlung kann unter den üblichen Bedingungen stattfinden. Die Wellenlänge der Bestrahlung ist von der verwendeten Schutzgruppe abhängig. Die geeigneten Wellenlängen sind dem Fachmann bekannt. Die Menge an während der Bestrahlung anwesender Base variiert zwischen 0,01 M und 1,0 M und ist natürlich von der Basenstärke abhängig. So hat sich es sich bewährt 0,03 bis 1 M (bevorzugt 0,05 bis 0,5 M) DBU in Acetonitril, 0,03 bis 0,8 M (bevorzugt 0,05 M) Diisoproylethylamin in Acetronitril oder 0,03 bis 1 M (bevorzugt 0,05 M) Piperidin in Acetonitril zu verwenden. In the above preferred method for photolithographic bio Chip synthesis according to the German application DE 198 58 440.7 Condensation, oxidation and capping steps as usual (Fodor et al., Science 1991, 251, p. 767 ff.). However, the first step of the syn thesis, namely the radiation, with the addition of bases, preferably strong Bases, especially non-nucleophilic bases, instead of what works together with the light used in the irradiation to a surprisingly effective ven deprotection leads. The bases are suitable for the subject man-known bases, such as. B. DBU (1,8-diazabicyclo [5.4.0] undec-7-en, DBN (1,5-diazabicyclo [4.3.0] non-5-ene, diiisopropylethylamine, pyridine, piperidine, triethyl amine, diisopropylamine, N-methylmorpholine, 2,6-lutidine, collidine, N-methylimi dazol, Dabco, N, N, -dimethylaminopyridine. The radiation can be among the usual conditions take place. The wavelength of the radiation is of the protective group used. The suitable wavelengths are Known specialist. The amount of base present during the irradiation varies between 0.01 M and 1.0 M and is of course based on base strength dependent. So it has proven itself 0.03 to 1 M (preferably 0.05 to 0.5 M) DBU in acetonitrile, 0.03 to 0.8 M (preferably 0.05 M) diisoproylethylamine in Acetronitrile or 0.03 to 1 M (preferably 0.05 M) piperidine in acetonitrile use.
Nucleinsäure-Chips, die unter Verwendung erfindungsgemäßer Nucleoside herge stellt worden sind, sind dadurch gekennzeichnet, daß das fertiggestellte Oligo mer mit der 5'-Position mit der festen Phase verbunden ist, das 3'-OH aber frei zugänglich ist (vgl. Fig. 3). DNA-Chips, die mit dieser Methode erzeugt wurden, lassen sich sowohl für Hybridisierungsexperimente als auch für bestimmte Enzymreaktionen (z. B. DNA-Polymerase), die ein freies 3'-OH erfordern, ver wenden. Somit haben Nucleinsäure-Chips (bevorzugt DNA-Chips), die mit dieser Strategie erzeugt wurden, einen weit größeren Anwendungsbereich, da mit diesen sowohl alle Experimente durchgeführt werden können wie mit den "üblichen" DNA-Chips, aber darüberhinaus noch hochparallel festphasengestütz te Enzymreaktionen (z. B. cDNA-Synthese, Ligase-Reaktionen, reverse Trans kription, PCR, multiplex-PCR) durchgeführt werden können. Damit erschließen sich neue Anwendungsgebiete (z. B. DNA-Computing, festphasengestützte Sequenzierung).Are nucleic acid chips, according to the invention using nucleosides Herge is, are characterized in that the finished oligo is mer connected to the 5 'position with the solid phase, but the 3'-OH is freely accessible (see. Fig. 3). DNA chips generated with this method can be used for hybridization experiments as well as for certain enzyme reactions (e.g. DNA polymerase) that require a free 3'-OH. Thus, nucleic acid chips (preferably DNA chips), which were generated with this strategy, have a much wider field of application, since with them all experiments can be carried out as with the "usual" DNA chips, but also highly parallel-based enzyme reactions (e.g. cDNA synthesis, ligase reactions, reverse transcription, PCR, multiplex-PCR) can be carried out. This opens up new areas of application (e.g. DNA computing, solid phase-based sequencing).
Die Erfindung wird weiter anhand der nachfolgenden Figuren beschrieben.The invention is further described with reference to the following figures.
Fig. 1 Herstellung eines Acylierungsreagenzes (allgemein)
(a) Herstellung des Acylierungsreagenzes für X = OCO
(b) Herstellung des Acylierungsreagenzes für X = OCS
(c) Herstellung des Acylierungsreagenzes für X = SO2 Fig. 1 Preparation of an acylating reagent (general)
(a) Preparation of the acylation reagent for X = OCO
(b) Preparation of the acylation reagent for X = OCS
(c) Preparation of the acylation reagent for X = SO 2
Fig. 2 Allgemeiner Syntheseplan erfindungsgemäßer 3'-photolabiler Nu cleosid-Derivate Fig. 2 General synthesis plan of 3'-photolabile nucloside derivatives according to the invention
Fig. 3-6 Synthesepläne der Verbindungen gemäß der Beispiele 1-6 Fig. 3-6 synthesis plans of the compounds according to Examples 1-6
Fig. 7 Aufbau eines Oligonukleotids unter Verwendung 3'-photolabiler 5'- Phosphitamide Fig. 7 Structure of an oligonucleotide using 3'-photolabile 5'-phosphitamides
Fig. 8 Bio-Chip mit d(T9), hergestellt unter Verwendung von 3'-O-[2-(2- Nitrophenyl)-propoxycarbonyl]thymidin-5'-O-[2-cyanoethyl]-N,N- diisopropylphosphoramidit] Fig. 8 bio-chip with d (T 9), prepared using 3'-O- [2- (2-nitrophenyl) propoxycarbonyl] thymidine-5'-O- [2-cyanoethyl] -N, N- diisopropyl phosphoramidite]
Fig. 9 DNA-Chip-Syntheseprinzip Fig. 9 DNA chip synthesis principle
Die Erfindung wird weiter anhand der nachfolgenden Beispiele beschrieben. Die Reaktionsschemata zur Herstellung der nachfolgenden Verbindungen sind in den Fig. 3-6 gezeigt, worauf nachfolgend Bezug genommen wird.The invention is further described on the basis of the following examples. The reaction schemes for the preparation of the following compounds are shown in FIGS. 3-6, to which reference is made below.
Zu 5 ml Diphosgen (41,4 mmol) in 10 ml absolutem THF werden unter Stick stoffatmosphäre bei 0°C über eine Kanüle eine Lösung bestehend aus 7,2 g 2- (2-Nitrophenyl)propanol (39,9 mmol) und 4,4 ml N-Methylmorpholin (39,7 mmol) in 15 ml absolutem THF langsam zugegeben. Nach 1 Std. Rühren bei 0°C wird vom gebildeten Niederschlag abgesaugt und das Filtrat am Hochvakuum abgezogen. Man erhält 6,91 g von (2) in Form eines braunes Öls (71%).5 ml of diphosgene (41.4 mmol) in 10 ml of absolute THF are added under stick atmosphere at 0 ° C via a cannula a solution consisting of 7.2 g 2- (2-nitrophenyl) propanol (39.9 mmol) and 4.4 ml of N-methylmorpholine (39.7 mmol) slowly added in 15 ml of absolute THF. After stirring for 1 hour at 0 ° C is suctioned off from the precipitate formed and the filtrate in a high vacuum deducted. 6.91 g of (2) are obtained in the form of a brown oil (71%).
Unter Stickstoff und unter Ausschluß von Licht werden bei 0°C zu 0,8 ml N- Methylimidazol (12,66 mol) und Molekularsieb 4 Å in 20 ml absolutem Dichlor methan 0,55 ml 2-(2-Nitrophenyl)propoxycarbonylchlorid (2) (5 mmol) zugege ben. Die Reaktionslösung wird 15 Minuten bei 0°C gerührt. Diese Reaktions lösung wird direkt für Acylierungen eingesetzt.Under nitrogen and in the absence of light, 0.8 ml of N- Methylimidazole (12.66 mol) and molecular sieve 4 Å in 20 ml absolute dichlor methane 0.55 ml of 2- (2-nitrophenyl) propoxycarbonyl chloride (2) (5 mmol) was added ben. The reaction solution is stirred at 0 ° C for 15 minutes. This reaction solution is used directly for acylations.
Unter Stickstoffatmosphäre werden 2,19 g N,N-Carbonyldiimidazol (13,5 mmol) in 40 ml absolutem Dichlormethan und 10 ml absolutem Nitromethan gelöst und auf 0°C gekühlt. Es werden 3 ml Trifluormethansulfonsäuremethylester (27 mmol) zugegeben und bei 0°C gerührt. Nach 30 Min. wird eine Lösung, beste hend aus 1,22 g 2-(2-Nitrophenyl)propanol (6,75 mmol) in 10 ml absolutem Dichlormethan zugegeben. Die Reaktionslösung kann nach 1 Std. Reaktionszeit direkt für Acylierungen eingesetzt werden.Under a nitrogen atmosphere, 2.19 g of N, N-carbonyldiimidazole (13.5 mmol) dissolved in 40 ml of absolute dichloromethane and 10 ml of absolute nitromethane and Chilled to 0 ° C. 3 ml of methyl trifluoromethanesulfonate (27th mmol) added and stirred at 0 ° C. After 30 minutes a solution will be the best Starting from 1.22 g of 2- (2-nitrophenyl) propanol (6.75 mmol) in 10 ml of absolute Dichloromethane added. The reaction solution can after 1 hour reaction time can be used directly for acylations.
1,088 g 5'-O-Dimethoxytrityl-thymidin ((5), 2 mmol; Fa. Proligo, Hamburg) werden unter Stickstoffatmosphäre und unter Ausschluß von Licht zu einer 1- Methyl-3-[2-(2-nitrophenyl)propoxycarbonyl]imidazoliumchlorid-Acylierungs reaktion (3) (hergestellt aus 0,55 ml 2-[2-Nitrophenyl)propoxycarbonylchlorid (4,9 mmol), 0,8 ml N-Methylimidazol (10 mmol) und Molekularsieb 4 Å) zugefügt. Das Reaktionsgemisch wird abgedunkelt über Nacht bei 4°C gelagert. Das Molekularsieb wird abgetrennt und die organische Phase gegen 50 ml gesättigte Natriumbicarbonatlösung extrahiert. Nach Trocknen der organischen Phase über Natriumsulfat wird 100 ml einer 3% Trichloressigsäurelösung in Dichlorethan zugefügt. Die stark rot gefärbte Lösung wird mit 100 ml gesättigter Natriumbi carbonatlösung extrahiert, die organische Phase über Natriumsulfat getrocknet und evaporiert. Die Reinigung erfolgt über Säulenchromatografie [30 g SiO2; Elution mit Toluol/Ethylacetat (5 : 4, 250 ml), dann Toluol/Ethylacetat/Methanol (5 : 4 : 0,5, 300 ml)]. Die Ausbeute an Verbindung (7) beträgt 781 mg in Form eines weißen Schaumes (87% Ausbeute).1.088 g of 5'-O-dimethoxytrityl-thymidine ((5), 2 mmol; from Proligo, Hamburg) are converted to a 1-methyl-3- [2- (2-nitrophenyl) propoxycarbonyl] under a nitrogen atmosphere and in the absence of light. imidazolium chloride acylation reaction (3) (made from 0.55 ml of 2- [2-nitrophenyl) propoxycarbonyl chloride (4.9 mmol), 0.8 ml of N-methylimidazole (10 mmol) and molecular sieve 4 Å) were added. The reaction mixture is stored darkened at 4 ° C. overnight. The molecular sieve is separated off and the organic phase is extracted against 50 ml of saturated sodium bicarbonate solution. After drying the organic phase over sodium sulfate, 100 ml of a 3% trichloroacetic acid solution in dichloroethane is added. The strongly red colored solution is extracted with 100 ml of saturated sodium bicarbonate solution, the organic phase is dried over sodium sulfate and evaporated. The purification is carried out by column chromatography [30 g SiO 2 ; Elution with toluene / ethyl acetate (5: 4, 250 ml), then toluene / ethyl acetate / methanol (5: 4: 0.5, 300 ml)]. The yield of compound (7) is 781 mg in the form of a white foam (87% yield).
Unter Stickstoffatmosphäre werden 449 mg 3'-O-[2-(2-Nitrophenyl)propoxy carbonyl]thymidin (7) (1 mmol) in 20 ml absolutem Dichlormethan gelöst. Es werden unter Stickstoffatmosphäre und unter Ausschluß von Licht 0,22 ml N- Methylmorpholin (2 mmol) und 0,24 ml (2-Cyanoethyl)-N,N-diisopropylphos phorimidochloridit (8) (1,1 mmol) zugefügt und 30 Minuten gerührt. Es wird gegen 50 ml gesättigte Natriumbicarbonatlösung extrahiert, die organische Phase über Natriumsulfat getrocknet und evaporiert. Die Reinigung erfolgt über Säulenchromatografie [10 g SiO2; Elution mit Toluol (50 ml), Toluol/Ethylacetat (4 : 1; 50 ml), Toluol/Ethylacetat (3 : 1, 40 ml), Toluol/Ethylacetat (2 : 1, 120 ml)]. Die Ausbeute beträgt 300 mg Verbindung (9) in Form eines weißen Schaumes (46% Ausbeute). 449 mg of 3'-O- [2- (2-nitrophenyl) propoxycarbonyl] thymidine (7) (1 mmol) are dissolved in 20 ml of absolute dichloromethane under a nitrogen atmosphere. 0.22 ml of N-methylmorpholine (2 mmol) and 0.24 ml (2-cyanoethyl) -N, N-diisopropylphosphorimidochloridite (8) (1.1 mmol) are added under a nitrogen atmosphere and in the absence of light and 30 minutes touched. It is extracted against 50 ml of saturated sodium bicarbonate solution, the organic phase is dried over sodium sulfate and evaporated. The purification is carried out by column chromatography [10 g SiO 2 ; Elution with toluene (50 ml), toluene / ethyl acetate (4: 1; 50 ml), toluene / ethyl acetate (3: 1, 40 ml), toluene / ethyl acetate (2: 1, 120 ml)]. The yield is 300 mg of compound (9) in the form of a white foam (46% yield).
1,0 g 5'-O-Dimethoxytrityl-N6-[(4-(tertbutyl)phenoxy)acetyl]-2'-desoxyadenosin (1,34 mmol) in 20 ml absolutem Dichlormethan werden durch eine Kanüle unter Stickstoffatmosphäre und unter Ausschluß von Licht zu einer 1-Methyl-3-[2-(2- nitrophenyl)propoxycarbonyl]imidazoliumchlorid-Acylierungsreaktion (3) (her gestellt aus 0,30 ml 2-[2-Nitrophenyl)propoxycarbonylchlorid (2,68 mmol), 0,534 ml N-Methylimidazol (6,7 mmol) und Molekularsieb 4 Å) zugefügt. Das Reaktionsgemisch wird abgedunkelt über Nacht bei 4°C gelagert. Das Moleku larsieb wird abgetrennt und die organische Phase gegen 50 ml gesättigte Natri umbicarbonatlösung extrahiert. Nach Trocknen der organischen Phase über Natriumsulfat wird diese 30 Min. in einer 1% Toluolsulfonsäurelösung in Di chlormethan/Methanol (4 : 1) gerührt. Die stark rot gefärbte Lösung wird mit 100 ml gesättigter Natriumbicarbonatlösung extrahiert, die organische Phase über Natriumsulfat getrocknet und evaporiert. Die Reinigung erfolgt über Säulen chromatografie [30 g SiO2; Elution mit Toluol, dann Toluol/Ethylacetat (4 : 1, 100 ml), (7 : 3, 100 ml), (3.2, 100 ml), (1 : 1, 200 ml), (2 : 3, 200 ml), (3 : 7, 200 ml). Die Ausbeute an Verbindung (12) beträgt 324 mg in Form eines weißen Schau mes (37% Ausbeute).1.0 g of 5'-O-dimethoxytrityl-N 6 - [(4- ( tert- butyl) phenoxy) acetyl] -2'-deoxyadenosine (1.34 mmol) in 20 ml of absolute dichloromethane are passed through a cannula under a nitrogen atmosphere and under Exclusion of light to a 1-methyl-3- [2- (2-nitrophenyl) propoxycarbonyl] imidazolium chloride acylation reaction (3) (prepared from 0.30 ml of 2- [2-nitrophenyl) propoxycarbonyl chloride (2.68 mmol), 0.534 ml of N-methylimidazole (6.7 mmol) and molecular sieve 4 Å) were added. The reaction mixture is stored darkened at 4 ° C. overnight. The molecular sieve is separated off and the organic phase is extracted against 50 ml of saturated sodium bicarbonate solution. After drying the organic phase over sodium sulfate, it is stirred for 30 minutes in a 1% toluenesulfonic acid solution in dichloromethane / methanol (4: 1). The strongly red colored solution is extracted with 100 ml of saturated sodium bicarbonate solution, the organic phase is dried over sodium sulfate and evaporated. The purification is carried out using column chromatography [30 g SiO 2 ; Elution with toluene, then toluene / ethyl acetate (4: 1, 100 ml), (7: 3, 100 ml), (3.2, 100 ml), (1: 1, 200 ml), (2: 3, 200 ml) , (3: 7, 200 ml). The yield of compound (12) is 324 mg in the form of a white foam (37% yield).
Die DNA-Chip-Synthese wurde analog dem bekannten Verfahren von Fodor et al. (Science 1991, 251, S. 767 ff) auf einer Glasoberfläche als Träger durchgeführt. Hinsichtlich des Bestrahlungsschritts wurde jedoch gemäß der deutschen Paten tanmeldung DE 198 58 440.7 verfahren. Der Reaktionsablauf ist schematisch in Fig. 9 gezeigt. Als Nucleosid wurde die erfindungsgemäße Verbindung (9) (3'- O-[2-(2-Nitrophenyl)-propoxycarbonyl]thymidin-5'-O-[2-cyanoethyl]-(N,N-di isopropylamino)phosphoramidit]; s. Beispiel 5) eingesetzt.The DNA chip synthesis was carried out analogously to the known method by Fodor et al. (Science 1991, 251, p. 767 ff) carried out on a glass surface as a carrier. With regard to the irradiation step, however, the German patent application DE 198 58 440.7 was used. The course of the reaction is shown schematically in FIG. 9. Compound (9) (3'-O- [2- (2-nitrophenyl) propoxycarbonyl] thymidine-5'-O- [2-cyanoethyl] - (N, N-diisopropylamino) phosphoramidite]; see Example 5) used.
Der angewendete Zyklus und die speziellen Synthesebedingungen sind in der deutschen Patentanmeldung DE 198 58 440.7 gezeigt, worauf hier Bezug genommen wird. Das allgemeine Syntheseprinzip ist auch in Fig. 7 gezeigt.The cycle used and the special synthesis conditions are shown in German patent application DE 198 58 440.7, to which reference is made here. The general principle of synthesis is also shown in FIG. 7.
In Fig. 8 ist eine Fluoreszenzanalyse des hergestellten Chips mit d(T9) zu sehen. Das Muster entspricht der angewendeten Maske II, d. h. es war eine erfolgreiche DNA-Chip-Synthese möglich.In FIG. 8 is a fluorescence analysis is shown of the chips produced with d (T 9). The pattern corresponds to the mask II used, ie a successful DNA chip synthesis was possible.
Dieser Chip hat den Vorteil, daß die Ankopplung des Nucleotidstrangs über das 5'-Ende erfolgt und somit das 3'-Ende frei verfügbar ist. Dadurch können Enzym reaktionen, die ein freies 3'-Ende verlangen (z. B. PCR, cDNA-Synthese, Ligase- Reaktionen, reverse Transkription, Sequenzierungen, multiplex-PCR usw.) an diesem Bio-Chip durchgeführt werden.This chip has the advantage that the coupling of the nucleotide strand via the 5 'end occurs and thus the 3' end is freely available. This allows enzyme reactions that require a free 3 'end (e.g. PCR, cDNA synthesis, ligase Reactions, reverse transcription, sequencing, multiplex-PCR etc.) this bio-chip.
Claims (19)
mit
R1 = H, NO2, CN, OCH3, Halogen, Alkyl-, Alkoxy- oder Alkoxyalkylrest mit 1 bis 4 C-Atomen
R2 = H, OCH3, NO2, CN, Halogen, Alkoxy-, Alkoxyalkyl- oder Alkylrest mit 1 bis 4 C-Atomen oder ein Arylrest
R3 = H, Halogen, NO2, CN, Alkylrest mit 1 bis 4 C-Atomen oder Arylrest oder aliphatischer Acylrest mit 2 bis 5 C-Atomen
R4 = H, Halogen, OCH3, CN, NO2, Alkoxy-, Alkoxyalkyl- oder Alkylrest mit 1 bis 4 C-Atomen oder Arylrest
R5 = H oder eine bei der Herstellung von Oligonukleotiden übliche Schutzgruppe
R6 = H, OH, Halogen, Alkoxy- oder Alkoxyalkylrest mit 1 bis 4 C-Ato men oder XR8, wobei X = O oder S und R8 = eine in der Nukleo tidchemie übliche Schutzgruppe
R7 = H, NO2, CN, OCH3, Halogen, Alkyl- oder Alkoxyalkylgruppe mit 1 bis 4 C-Atomen oder ein Arylrest
X = SO2, OCO, OCS
B = H, Adenin, Cytosin, Guanin, Thymin, Uracil, 2,6-Diaminopurin-9-yl, Hypoxanthin-9-yl, 5-Methylcytosin-1-yl, 5-Amino-4-Imidazolcarbon säure-1-yl oder 5-Amino-4-Imidazolcarbonsäureamid-3-yl, wobei im Falle von B = Adenin, Cytosin oder Guanin die primäre Aminofunk tion ggf. eine permanente Schutzgruppe aufweist bzw. Thymin oder Uracil an der O4-Position ggf. eine permanente Schutzgruppe aufweist.1. Nucleoside derivatives with photolabile protecting groups of the general formula (I)
With
R 1 = H, NO 2 , CN, OCH 3 , halogen, alkyl, alkoxy or alkoxyalkyl radical having 1 to 4 carbon atoms
R 2 = H, OCH 3 , NO 2 , CN, halogen, alkoxy, alkoxyalkyl or alkyl radical having 1 to 4 carbon atoms or an aryl radical
R 3 = H, halogen, NO 2 , CN, alkyl radical with 1 to 4 carbon atoms or aryl radical or aliphatic acyl radical with 2 to 5 carbon atoms
R 4 = H, halogen, OCH 3 , CN, NO 2 , alkoxy, alkoxyalkyl or alkyl radical having 1 to 4 carbon atoms or aryl radical
R 5 = H or a protective group customary in the production of oligonucleotides
R 6 = H, OH, halogen, alkoxy or alkoxyalkyl radical with 1 to 4 C atoms or XR 8 , where X = O or S and R 8 = a protective group customary in nucleotide chemistry
R 7 = H, NO 2 , CN, OCH 3 , halogen, alkyl or alkoxyalkyl group with 1 to 4 carbon atoms or an aryl radical
X = SO 2 , OCO, OCS
B = H, adenine, cytosine, guanine, thymine, uracil, 2,6-diaminopurin-9-yl, hypoxanthine-9-yl, 5-methylcytosin-1-yl, 5-amino-4-imidazole carbonate 1-yl or 5-amino-4-imidazolecarboxamide-3-yl, where in the case of B = adenine, cytosine or guanine the primary amino function may have a permanent protective group or thymine or uracil at the O 4 position may have a permanent protective group having.
p-NC-CH2-CH2-O-P-N(Q)2, p-NC-C6H4-CH2-CH2-O-P-N(Q)2, p-NO2- C6H4-CH2-CH2-O-P-N(Q)2 oder OH2=CH-CH2-O-P-N(Q)2 ist,
wobei die Q-Gruppen gleich oder verschieden sein können und lineare oder verzweigte Alkylreste mit 1 bis 4 C-Atomen bedeuten.5. Nucleoside derivative according to any one of claims 1 to 4, wherein the usual functional group for the preparation of oligonucleotides at position R 5 is a phosphitamide group of the formula
p-NC-CH 2 -CH 2 -OPN (Q) 2 , p-NC-C 6 H 4 -CH 2 -CH 2 -OPN (Q) 2 , p-NO 2 - C 6 H 4 -CH 2 - Is CH 2 -OPN (Q) 2 or OH 2 = CH-CH 2 -OPN (Q) 2 ,
where the Q groups can be the same or different and mean linear or branched alkyl radicals having 1 to 4 carbon atoms.
- 1. eine Verbindung der allgemeinen Formel (II)
mit N-Methylimidazol, Pyridin, N,N-Dimethylaminopyridin, Triazol oder Tetrazol umsetzt, oder9. A process for the preparation of nucleoside derivatives according to any one of claims 1-8, wherein
- 1. a compound of the general formula (II)
with N-methylimidazole, pyridine, N, N-dimethylaminopyridine, triazole or tetrazole, or
Y = C = O oder C = S
mit einem Methylierungsmittel umsetzt sowie das erhaltene Pro dukt mit einem Alkohol reagieren läßt und anschließend(a2) a compound of the general formula (V)
Y = C = O or C = S
reacted with a methylating agent and the product obtained can react with an alcohol and then
Y = C = O oder C = S
mit einem Nucleosid der allgemeinen Formel (IX)
in der R5, R6 und B die oben angegebene Bedeutung haben, reagieren läßt.(b) the acylation reagent (IV) formed in step (a1) or (a2)
Y = C = O or C = S
with a nucleoside of the general formula (IX)
in which R 5 , R 6 and B have the meaning given above, can react.
p-NC-CH2-CH2-O-P-N(Q)2, p-NC-C6H4-CH2-CH2-O-P-N(Q)2, p-NO2- C6H4-CH2-CH2-O-P-N(Q)2 oder CH2=CH-CH2-O-P-N(Q)2 ist,
wobei die Q-Gruppen gleich oder verschieden sein können und lineare oder verzweigte Alkylreste mit 1 bis 4 C-Atomen bedeuten, einführt.10. The method according to claim 9, wherein in the 5'-position of the resulting nucleoside derivatives, a phosphitamide group of the formula
p-NC-CH 2 -CH 2 -OPN (Q) 2 , p-NC-C 6 H 4 -CH 2 -CH 2 -OPN (Q) 2 , p-NO 2 - C 6 H 4 -CH 2 - Is CH 2 -OPN (Q) 2 or CH 2 = CH-CH 2 -OPN (Q) 2 ,
where the Q groups may be the same or different and represent linear or branched alkyl radicals having 1 to 4 carbon atoms.
Priority Applications (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19915867A DE19915867A1 (en) | 1999-04-08 | 1999-04-08 | New nucleoside derivatives with photolabile protecting groups, useful in oligonucleotide synthesis, particularly on solid phases, e.g. for hybridization testing |
| AU50598/00A AU5059800A (en) | 1999-04-08 | 2000-04-07 | Nucleoside derivatives with photo-unstable protective groups |
| US09/958,610 US6756492B1 (en) | 1999-04-08 | 2000-04-07 | Nucleoside derivatives with photo-unstable protective groups |
| PCT/DE2000/001148 WO2000061594A2 (en) | 1999-04-08 | 2000-04-07 | Nucleoside derivatives with photo-unstable protective groups |
| EP00934905A EP1212338B1 (en) | 1999-04-08 | 2000-04-07 | Nucleoside derivatives with photolabile protective groups |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19915867A DE19915867A1 (en) | 1999-04-08 | 1999-04-08 | New nucleoside derivatives with photolabile protecting groups, useful in oligonucleotide synthesis, particularly on solid phases, e.g. for hybridization testing |
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| Publication Number | Publication Date |
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| DE19915867A1 true DE19915867A1 (en) | 2000-10-19 |
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| DE19915867A Pending DE19915867A1 (en) | 1999-04-08 | 1999-04-08 | New nucleoside derivatives with photolabile protecting groups, useful in oligonucleotide synthesis, particularly on solid phases, e.g. for hybridization testing |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000061594A2 (en) | 1999-04-08 | 2000-10-19 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | Nucleoside derivatives with photo-unstable protective groups |
| DE10105079A1 (en) * | 2001-02-05 | 2002-08-08 | Febit Ferrarius Biotech Gmbh | Photolabile protecting groups for the synthesis of biopolymers |
| DE10105077A1 (en) * | 2001-02-05 | 2002-08-08 | Febit Ferrarius Biotech Gmbh | Hybrid-protecting group |
| WO2003006476A1 (en) * | 2001-07-09 | 2003-01-23 | Chemogenix Gmbh | Multimer polynucleotide synthesis |
| WO2003035664A3 (en) * | 2001-10-25 | 2003-10-09 | Chemogenix Gmbh | Method for covalently attaching nucleosides and/or nucleotides on surfaces and method for determining coupling yields in the synthesis of nucleotides |
| DE102004019098A1 (en) * | 2004-04-20 | 2005-11-10 | Chemogenix Gmbh | Photolabile protecting groups |
| US7687618B2 (en) | 2002-10-14 | 2010-03-30 | Nimblegen Systems Gmbh | Method of manufacturing labelled oligonucleotide conjugates |
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| DE3606394A1 (en) * | 1986-02-27 | 1987-09-03 | Wolfgang Prof Dr Dr Pfleiderer | Ribonucleosides, process for their preparation and their use |
| US5623068A (en) * | 1994-03-07 | 1997-04-22 | Beckman Instruments, Inc. | Synthesis of DNA using substituted phenylacetyl-protected nucleotides |
| US5843655A (en) * | 1995-09-18 | 1998-12-01 | Affymetrix, Inc. | Methods for testing oligonucleotide arrays |
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| US5623068A (en) * | 1994-03-07 | 1997-04-22 | Beckman Instruments, Inc. | Synthesis of DNA using substituted phenylacetyl-protected nucleotides |
| US5843655A (en) * | 1995-09-18 | 1998-12-01 | Affymetrix, Inc. | Methods for testing oligonucleotide arrays |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000061594A2 (en) | 1999-04-08 | 2000-10-19 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | Nucleoside derivatives with photo-unstable protective groups |
| US6756492B1 (en) | 1999-04-08 | 2004-06-29 | Deutsches Krebsforschungszentrum Stiftund Des Offentlichen Rechts | Nucleoside derivatives with photo-unstable protective groups |
| DE10105079A1 (en) * | 2001-02-05 | 2002-08-08 | Febit Ferrarius Biotech Gmbh | Photolabile protecting groups for the synthesis of biopolymers |
| DE10105077A1 (en) * | 2001-02-05 | 2002-08-08 | Febit Ferrarius Biotech Gmbh | Hybrid-protecting group |
| WO2003006476A1 (en) * | 2001-07-09 | 2003-01-23 | Chemogenix Gmbh | Multimer polynucleotide synthesis |
| WO2003035664A3 (en) * | 2001-10-25 | 2003-10-09 | Chemogenix Gmbh | Method for covalently attaching nucleosides and/or nucleotides on surfaces and method for determining coupling yields in the synthesis of nucleotides |
| US7687618B2 (en) | 2002-10-14 | 2010-03-30 | Nimblegen Systems Gmbh | Method of manufacturing labelled oligonucleotide conjugates |
| DE102004019098A1 (en) * | 2004-04-20 | 2005-11-10 | Chemogenix Gmbh | Photolabile protecting groups |
| US7432368B2 (en) | 2004-04-20 | 2008-10-07 | Roche Nimblegen, Inc. | Photolabile protecting groups |
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