JPH02264792A - Oligonucleotide derivative and synthetic raw material thereof - Google Patents
Oligonucleotide derivative and synthetic raw material thereofInfo
- Publication number
- JPH02264792A JPH02264792A JP8545689A JP8545689A JPH02264792A JP H02264792 A JPH02264792 A JP H02264792A JP 8545689 A JP8545689 A JP 8545689A JP 8545689 A JP8545689 A JP 8545689A JP H02264792 A JPH02264792 A JP H02264792A
- Authority
- JP
- Japan
- Prior art keywords
- group
- compound
- formula
- acid
- methyl
- 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
- 108091034117 Oligonucleotide Proteins 0.000 title claims description 6
- 239000002994 raw material Substances 0.000 title abstract description 14
- -1 (substituted) phosphoryl Chemical group 0.000 claims abstract description 25
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 6
- 239000000126 substance Substances 0.000 claims abstract description 6
- 125000002252 acyl group Chemical group 0.000 claims abstract description 4
- 125000003545 alkoxy group Chemical group 0.000 claims abstract description 3
- YCICLRBTJMLLGG-UHFFFAOYSA-N (2-chlorophenyl) dihydrogen phosphate Chemical compound OP(O)(=O)OC1=CC=CC=C1Cl YCICLRBTJMLLGG-UHFFFAOYSA-N 0.000 claims abstract 2
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 9
- 125000001424 substituent group Chemical group 0.000 claims description 7
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 5
- 125000005103 alkyl silyl group Chemical group 0.000 claims 1
- 125000000845 uracil-1-yl group Chemical group [*]N1C(=O)N([H])C(=O)C([H])=C1[H] 0.000 claims 1
- 150000001875 compounds Chemical class 0.000 abstract description 27
- 108091034057 RNA (poly(A)) Proteins 0.000 abstract description 6
- 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 abstract description 3
- 238000002955 isolation Methods 0.000 abstract description 3
- DWRXFEITVBNRMK-JXOAFFINSA-N ribothymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 DWRXFEITVBNRMK-JXOAFFINSA-N 0.000 abstract description 3
- 125000003294 thymin-1-yl group Chemical group [H]N1C(=O)N(*)C([H])=C(C1=O)C([H])([H])[H] 0.000 abstract description 3
- 125000000848 adenin-9-yl group Chemical group [H]N([H])C1=C2N=C([H])N(*)C2=NC([H])=N1 0.000 abstract 1
- 125000003738 guanin-9-yl group Chemical group O=C1N([H])C(N([H])[H])=NC2=C1N=C([H])N2[*] 0.000 abstract 1
- 239000000463 material Substances 0.000 abstract 1
- 238000002360 preparation method Methods 0.000 abstract 1
- 239000002253 acid Substances 0.000 description 33
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 27
- 239000000243 solution Substances 0.000 description 25
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 21
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 17
- 238000006243 chemical reaction Methods 0.000 description 16
- 238000000034 method Methods 0.000 description 14
- 230000015572 biosynthetic process Effects 0.000 description 13
- 238000003786 synthesis reaction Methods 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- 239000001913 cellulose Substances 0.000 description 11
- 229920002678 cellulose Polymers 0.000 description 11
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 9
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 8
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 8
- 239000002904 solvent Substances 0.000 description 8
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 7
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 7
- 229940126214 compound 3 Drugs 0.000 description 7
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 7
- 239000012071 phase Substances 0.000 description 7
- 230000008034 disappearance Effects 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 239000000741 silica gel Substances 0.000 description 6
- 229910002027 silica gel Inorganic materials 0.000 description 6
- 238000010898 silica gel chromatography Methods 0.000 description 6
- 230000000295 complement effect Effects 0.000 description 5
- 229940125904 compound 1 Drugs 0.000 description 5
- 229940125898 compound 5 Drugs 0.000 description 5
- 238000007796 conventional method Methods 0.000 description 5
- 239000010410 layer Substances 0.000 description 5
- 108020004999 messenger RNA Proteins 0.000 description 5
- 239000011347 resin Substances 0.000 description 5
- 229920005989 resin Polymers 0.000 description 5
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 4
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 4
- 229940125782 compound 2 Drugs 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 4
- 235000017557 sodium bicarbonate Nutrition 0.000 description 4
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 4
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- 239000000470 constituent Substances 0.000 description 3
- 239000006260 foam Substances 0.000 description 3
- 239000004816 latex Substances 0.000 description 3
- 229920000126 latex Polymers 0.000 description 3
- 238000010841 mRNA extraction Methods 0.000 description 3
- 239000007858 starting material Substances 0.000 description 3
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical group [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- XYFCBTPGUUZFHI-UHFFFAOYSA-N Phosphine Chemical compound P XYFCBTPGUUZFHI-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000005349 anion exchange Methods 0.000 description 2
- PASDCCFISLVPSO-UHFFFAOYSA-N benzoyl chloride Chemical compound ClC(=O)C1=CC=CC=C1 PASDCCFISLVPSO-UHFFFAOYSA-N 0.000 description 2
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000000921 elemental analysis Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- PSHKMPUSSFXUIA-UHFFFAOYSA-N n,n-dimethylpyridin-2-amine Chemical compound CN(C)C1=CC=CC=N1 PSHKMPUSSFXUIA-UHFFFAOYSA-N 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- IZUPBVBPLAPZRR-UHFFFAOYSA-N pentachlorophenol Chemical compound OC1=C(Cl)C(Cl)=C(Cl)C(Cl)=C1Cl IZUPBVBPLAPZRR-UHFFFAOYSA-N 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- NDVLTYZPCACLMA-UHFFFAOYSA-N silver oxide Chemical compound [O-2].[Ag+].[Ag+] NDVLTYZPCACLMA-UHFFFAOYSA-N 0.000 description 2
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 2
- 229940045145 uridine Drugs 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- LOSXTWDYAWERDB-UHFFFAOYSA-N 1-[chloro(diphenyl)methyl]-2,3-dimethoxybenzene Chemical compound COC1=CC=CC(C(Cl)(C=2C=CC=CC=2)C=2C=CC=CC=2)=C1OC LOSXTWDYAWERDB-UHFFFAOYSA-N 0.000 description 1
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 description 1
- 125000001731 2-cyanoethyl group Chemical group [H]C([H])(*)C([H])([H])C#N 0.000 description 1
- WDBQJSCPCGTAFG-QHCPKHFHSA-N 4,4-difluoro-N-[(1S)-3-[4-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)piperidin-1-yl]-1-pyridin-3-ylpropyl]cyclohexane-1-carboxamide Chemical compound FC1(CCC(CC1)C(=O)N[C@@H](CCN1CCC(CC1)N1C(=NN=C1C)C(C)C)C=1C=NC=CC=1)F WDBQJSCPCGTAFG-QHCPKHFHSA-N 0.000 description 1
- 108091027075 5S-rRNA precursor Proteins 0.000 description 1
- IHLOTZVBEUFDMD-UUOKFMHZSA-N 7-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-2,2-dioxo-1h-imidazo[4,5-c][1,2,6]thiadiazin-4-one Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(NS(=O)(=O)NC2=O)=C2N=C1 IHLOTZVBEUFDMD-UUOKFMHZSA-N 0.000 description 1
- OIRDTQYFTABQOQ-KQYNXXCUSA-N Adenosine Natural products C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- GSNUFIFRDBKVIE-UHFFFAOYSA-N DMF Natural products CC1=CC=C(C)O1 GSNUFIFRDBKVIE-UHFFFAOYSA-N 0.000 description 1
- 101100322735 Danio rerio aep1 gene Proteins 0.000 description 1
- 241001446459 Heia Species 0.000 description 1
- 239000007832 Na2SO4 Substances 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- DJJCXFVJDGTHFX-UHFFFAOYSA-N Uridinemonophosphate Natural products OC1C(O)C(COP(O)(O)=O)OC1N1C(=O)NC(=O)C=C1 DJJCXFVJDGTHFX-UHFFFAOYSA-N 0.000 description 1
- FOGRQMPFHUHIGU-UHFFFAOYSA-N Uridylic acid Natural products OC1C(OP(O)(O)=O)C(CO)OC1N1C(=O)NC(=O)C=C1 FOGRQMPFHUHIGU-UHFFFAOYSA-N 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- VJHCJDRQFCCTHL-UHFFFAOYSA-N acetic acid 2,3,4,5,6-pentahydroxyhexanal Chemical compound CC(O)=O.OCC(O)C(O)C(O)C(O)C=O VJHCJDRQFCCTHL-UHFFFAOYSA-N 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical group C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- ZDLBNXXKDMLZMF-UHFFFAOYSA-N cinitapride Chemical compound CCOC1=CC(N)=C([N+]([O-])=O)C=C1C(=O)NC1CCN(CC2CC=CCC2)CC1 ZDLBNXXKDMLZMF-UHFFFAOYSA-N 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
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- 238000009833 condensation Methods 0.000 description 1
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- 239000013256 coordination polymer Substances 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- WPWRNMZBLPVTAW-UHFFFAOYSA-N dichloromethane;2,2,2-trichloroacetic acid Chemical compound ClCCl.OC(=O)C(Cl)(Cl)Cl WPWRNMZBLPVTAW-UHFFFAOYSA-N 0.000 description 1
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 125000001301 ethoxy group Chemical group [H]C([H])([H])C([H])([H])O* 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- INQOMBQAUSQDDS-UHFFFAOYSA-N iodomethane Chemical compound IC INQOMBQAUSQDDS-UHFFFAOYSA-N 0.000 description 1
- 210000003127 knee Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- TXOUDPQJASQYBZ-UHFFFAOYSA-N n,n'-dihexylmethanediimine Chemical compound CCCCCCN=C=NCCCCCC TXOUDPQJASQYBZ-UHFFFAOYSA-N 0.000 description 1
- VOVWQRQDSCYAEA-UHFFFAOYSA-N n-[chloro(methoxy)phosphanyl]-n-propan-2-ylpropan-2-amine Chemical compound COP(Cl)N(C(C)C)C(C)C VOVWQRQDSCYAEA-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 239000012044 organic layer Substances 0.000 description 1
- 150000008300 phosphoramidites Chemical class 0.000 description 1
- 229910000073 phosphorus hydride Inorganic materials 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- JUJWROOIHBZHMG-UHFFFAOYSA-O pyridinium Chemical compound C1=CC=[NH+]C=C1 JUJWROOIHBZHMG-UHFFFAOYSA-O 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229910001923 silver oxide Inorganic materials 0.000 description 1
- 125000003808 silyl group Chemical group [H][Si]([H])([H])[*] 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- IHQKEDIOMGYHEB-UHFFFAOYSA-M sodium dimethylarsinate Chemical compound [Na+].C[As](C)([O-])=O IHQKEDIOMGYHEB-UHFFFAOYSA-M 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 229940014800 succinic anhydride Drugs 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- HWCKGOZZJDHMNC-UHFFFAOYSA-M tetraethylammonium bromide Chemical compound [Br-].CC[N+](CC)(CC)CC HWCKGOZZJDHMNC-UHFFFAOYSA-M 0.000 description 1
- 125000001412 tetrahydropyranyl group Chemical group 0.000 description 1
- 238000010399 three-hybrid screening Methods 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
- 238000005866 tritylation reaction Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Landscapes
- Saccharide Compounds (AREA)
Abstract
Description
【発明の詳細な説明】
ＬＬ上立且ユ±１
本発明は、有用な遺伝子のクローニングに際し、通常行
なわれているｍＲＮＡの分離操作を効率化することに用
いることができるヌクレオチドの誘導体、その合成原料
であるヌクレオチドオリゴマー及び更にその合成原料で
あるヌクレオチドに関する。これらの物質は、いずれも
、新規化合物である。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to nucleotide derivatives that can be used to improve the efficiency of mRNA isolation operations that are commonly performed in the cloning of useful genes, and their synthesis. This invention relates to a nucleotide oligomer as a raw material and a nucleotide as a raw material for its synthesis. All of these substances are new compounds.
監迷Ｊとｌ五
有用遺伝子をりｌ」−ニングする際、−殻内にはその遺
伝子が発現している生物材料より全ｍＲＮＡを分離し、
そのｍＲＮＡよりｃＤＮＡを合成し、クローニングした
侵、目的遺伝ｆをスクリーニングすることが行われてい
る。ｍＲＮＡを分離する操作においては、ｍＲＮＡがポ
リ（Ａ＞テール（ｐｏｌｙ（Ａ）−ｍＲＮＡ）を有すル
コとを利用し、ポリ（Ａ）に相補的なオリボアオキシチ
ミジル酸をセルロース等に固定化したいわゆるオリゴ（
ｄＴ）−セルロースカラムを使用して、ハイブリダイゼ
ーシジン効果を利用してポリ（Ａ）を持たないＲＮＡ
（ｐｏｌｙ（Ａ＞ −ＲＮＡ）との分離を行っている。When rinsing five useful genes, all mRNA is isolated from the biological material in which the gene is expressed in the shell,
cDNA is synthesized from the mRNA, and the cloned gene is screened for the target gene f. In the operation to separate mRNA, mRNA uses poly(A> tail (poly(A)-mRNA)), and oligoboaoxythymidylic acid complementary to poly(A) is immobilized on cellulose etc. The so-called oligo (
dT)-cellulose column to utilize the hybridization cidin effect to obtain poly(A)-free RNA.
(Separation of poly(A>-RNA) is performed.
しかし、この操作におけるｐｏｌｙ（Ａ） −ｍＲＮＡ
の単離収率が必ずしも充分でなく、クローニングの効率
を向上させる為にはポリ（Ａ）部分と更に安定なハイブ
リッドを作るオリゴマーの発見が強く望まれでいた。However, poly(A)-mRNA in this operation
The isolation yield is not necessarily sufficient, and in order to improve the efficiency of cloning, it has been strongly desired to discover an oligomer that forms a more stable hybrid with the poly(A) moiety.
発明が解決しようとする問
目的有用遺伝子をクローニングする上で、その効率に大
きくかかわるｐｏｌｙ（Ａ）−ｍＲＮＡ単離操作におい
て、オリゴ（ｄＴ）−セルロースカラムに代り収率がよ
り向上できる技術、すなわちポリ（Ａ）とより安定なハ
イブリッドを形成しうる性質を有する新規オリゴマーの
開発が￥１請されている。Problem to be Solved by the Invention In the poly(A)-mRNA isolation operation, which greatly affects the efficiency of cloning a target useful gene, there is a technology that can replace the oligo(dT)-cellulose column and improve the yield. The development of a new oligomer that has the property of forming a more stable hybrid with poly(A) has been requested for 1 yen.
の よび
本発明者は、上記問題を解決するため鋭意研究を重ねた
結果、デオキシリボヌクレオチドに比べ、オリゴ（２°
−０−メチルリボヌクレオチド）が相補鎖オリゴリボヌ
クレオチドとより安定なハイブリッドを形成することを
見い出し、ハイブリダイゼーションプローブとして有用
であることを報告した（Ｈ，Ｉｎｏｕｅら、Ｎｕｃｌｅ
ｉｃ Ａｃ１ｄｓ Ｉｔｅｓ、ｊ５（１５）６１３１頁
、 １９８／年）。更に、ハイブリッドの安定性におけ
るオリゴマーの構造上の寄与を詳細に検討した結果、２
°−０−メチルウリジル酸の代りに２゜−〇−メブ・ル
ー５−メチルウリジル酸をオリゴマーの構成ユニットに
用いることにより、相補鎖中のアデニル酸ユニットと強
固に結合しオリゴリボヌクレオチドとの熱的安定性が増
大することを見い出した。この知見に基づいて、デカリ
ボアデニル酸とその相補鎖であるデオキシリボチミジル
酸。As a result of intensive research to solve the above problems, the present inventors have discovered that oligonucleotides (2°
-0-methylribonucleotide) was found to form more stable hybrids with complementary strand oligoribonucleotides, and reported that it is useful as a hybridization probe (H, Inoue et al., Nucle.
ic Ac1ds Ites, j5(15) p. 6131, 198/). Furthermore, as a result of a detailed study of the structural contribution of oligomers to hybrid stability, we found that 2
By using 2°-0-meb-5-methyluridylic acid as the constituent unit of the oligomer instead of °-0-methyluridylic acid, it is strongly bonded to the adenylate unit in the complementary strand, and the heat exchanger between the oligoribonucleotide and found that the stability was increased. Based on this finding, decariboadenylic acid and its complementary chain, deoxyribothymidylic acid.
２°−０−メチルウリジル酸及び２°−０−メチル−５
−メチルウリジル酸のそれぞれのデカマーを合成して熱
的安定性（Ｔｍ値）をそれぞれ測定したところ、デカ２
°−０−メチル−５−メヂルウリジル酸が著しくすぐれ
た性質を有することを見出し、本発明を完成するに至っ
た。2°-0-methyluridylic acid and 2°-0-methyl-5
- When each decamer of methyluridylic acid was synthesized and the thermal stability (Tm value) was measured, it was found that deca 2
It was discovered that °-0-methyl-5-methyluridylic acid has extremely excellent properties, and the present invention was completed.
以下、本発明の詳細な説明する。The present invention will be explained in detail below.
本発明は、次の一般式（Ｉ）で表されるオリゴヌクレオ
チド誘導体に関する。The present invention relates to an oligonucleotide derivative represented by the following general formula (I).
式中、Ｒは、水素原子、０Ｍセルロース、ラテックス粒
子などのカルボニル残基等のアシル基、ホスホリル基、
又は置換基を有していてもよいホスホリル基例えばセル
ローズなどの水酸基を有する化合物とＩＦ５酸とのエス
テル結合を有するホスホリル基を表し、■は、チミン−
１−イル基を表し、Ｙｌ、Ｙ２及びＹ３は、相互に同一
であってもよく文具なっていてもよくて、水素原子、ヒ
ドロキシル基、メトキシ基、エトキシ基等の低級アルキ
ルオキシ基又は低級アルキルシリル基を表し、Ｂは、チ
ミン−１−イル基、アデニン−９−イル基。In the formula, R is a hydrogen atom, an acyl group such as a carbonyl residue such as 0M cellulose or latex particles, a phosphoryl group,
or a phosphoryl group which may have a substituent, for example, a phosphoryl group having an ester bond between a compound having a hydroxyl group such as cellulose and IF5 acid, and ■ is thymine-
It represents a 1-yl group, and Yl, Y2 and Y3 may be the same as each other or may be stationery, and represent a hydrogen atom, a lower alkyloxy group such as a hydroxyl group, a methoxy group, an ethoxy group, or a lower alkyl group. It represents a silyl group, and B is a thymin-1-yl group or an adenine-9-yl group.
グアニン−９−イル基、シトシン−９−イル基。Guanine-9-yl group, cytosine-9-yl group.
ウシシル−１−イル基又はヒボキサンチン−９−イル基
を表し、ｎは１〜１００の整数を表すが、実用的には平
均鎖長がｎ−１０〜４０のものを用いることができる。It represents a bocyl-1-yl group or a hyboxanthine-9-yl group, and n represents an integer of 1 to 100, but those having an average chain length of n-10 to 40 can be used practically.
因みに、一般式ＣＩ）の誘導体において、Ｒが上記のよ
うなアシル基（これは担体と称されることもある）又は
置換Ｕ＜これも担体と称されることがある）を有するホ
スホリル基であるものは、ｍＲＮＡの分離操作に直接使
用される化合物であり、Ｒが水素原子又は置換基を有し
ないホスホリル基である化合物（オリゴヌクレオチド）
は、前者の化合物の合成原料となりうるばかりでなく、
前者の化合物をどのような方法で合成したにしろ、１１
者の化合物は前者の化合物の構成部分としてその中心的
機能を果たすのである。Incidentally, in the derivative of the general formula CI), R is an acyl group as described above (which may also be referred to as a carrier) or a phosphoryl group having a substituted U<which may also be referred to as a carrier). Some are compounds that are directly used for mRNA separation operations, and are compounds in which R is a hydrogen atom or a phosphoryl group without a substituent (oligonucleotide).
not only can be used as a raw material for the synthesis of the former compound, but also
No matter how the former compound was synthesized, 11
The former compound fulfills its central function as a constituent part of the former compound.
本発明は、又、次の一般式（Ｉ）で表されるヌクレオチ
ド誘導体にも関する。The present invention also relates to a nucleotide derivative represented by the following general formula (I).
式中、Ｘは、モノメトキシトリプル基、ジメトキシトリ
チル基又は例えば０−クロルフェニル基等の置換基を有
していでもよいホスホリル基を表し、Ｙは、水素原子、
０−クロルフェニル燐酸、−１〕（ＯＣＩ−１３）
−Ｎ−＜ＣＦ＋ （ＣＨ３） ２ ） ２
。In the formula, X represents a monomethoxytriple group, a dimethoxytrityl group, or a phosphoryl group which may have a substituent such as an 0-chlorophenyl group, and Y represents a hydrogen atom,
0-Chlorphenyl phosphoric acid, -1] (OCI-13)
-N-<CF+ (CH3) 2) 2
.
−Ｐ （ＯＣ８２ＣＩ−１２ＯＮ）−Ｎ−（ＣＨ（ＣＨ
３）２ ’）２ 又Ｇｔ−ＧＯ−（ＣＨ，）。-P (OC82CI-12ON) -N-(CH(CH
3)2')2 Also Gt-GO-(CH,).
Ｃ０ＮＨ−（ＣＨ２’）、−（ＣＰＧＩＩ体）（コこに
、ｍ及びｎは、それぞれ、１〜１ｏの整数である）を表
わす、ただし、Ｘが置換基を有していてもよいホスホリ
ル基のときは、Ｙは水素原子である。一般式（Ｉ［）の
化合物は、一般式（１）の化合物の合成原料として使用
することができる。C0NH-(CH2'), -(CPGII form) (here, m and n are each an integer of 1 to 1o), provided that X is a phosphoryl group which may have a substituent When , Y is a hydrogen atom. The compound of general formula (I[) can be used as a raw material for the synthesis of the compound of general formula (1).
次に、これらの化合物の合成法の例を説明する。Next, examples of methods for synthesizing these compounds will be explained.
本発明のオリゴ（２°−Ｏ−メチル−５−メチルウリジ
ル酸）部分を一合成する為には、先ずその構成ヌクレオ
シドである２°−０−メチル−５−メヂルウリジン（化
合物３）を後述する様に合成し（第１図）、次に常法に
より（例えば、Ｈ，Ｙｏｓｈｉｋａｗａ ｅｔ ａｌ、
、 Ｔｅｔｒａｈｅｄｒｏｎ １ｅｔｔ、、 ５０６５
（１９６７））、５’位水酸基に直接リンＬ２基を導入
し２゛−〇−メチルー ５−・メチル−５゛−ウリジル
酸を得、それをジシク「」ヘキシルカルボジイミド（Ｄ
ＣＣ）等の縮合剤を用いて重合する方法を採用すること
ができる。または、化合物３を後述する様に３°−フォ
スホロアミデート誘導体（化合物５）とし、公知の方法
に従い（Ｓ、５ｈｉｂａｒａｈａ ｅｔ ａｌ、、 Ｈ
ｕｃｌｅｉｃＡｃｉｄｓ Ｒｃｓ、、 １５（１１）、
４４０３ （１９８７））、ＤＮＡ自動合成りｉ（ア
プライドバイオシステム社製、モデル３８ＯＡ ）を用
いて容易に合成することができる。In order to synthesize the oligo(2°-O-methyl-5-methyluridylic acid) moiety of the present invention, first, its constituent nucleoside, 2°-0-methyl-5-methyluridine (compound 3), is synthesized as described below. (Fig. 1), and then by a conventional method (for example, H. Yoshikawa et al.
, Tetrahedron 1ett,, 5065
(1967)), phosphorus L2 group was introduced directly into the 5'-position hydroxyl group to obtain 2'-0-methyl-5-methyl-5'-uridylic acid, which was then converted into di-hexylcarbodiimide (D
A method of polymerization using a condensing agent such as CC) can be adopted. Alternatively, Compound 3 is converted into a 3°-phosphoramidate derivative (Compound 5) as described below, and prepared according to a known method (S, 5hibaraha et al,, H
ucleic acids Rcs,, 15(11),
4403 (1987)) and can be easily synthesized using DNA Automation Synthesis i (Model 38OA, manufactured by Applied Biosystems).
一方、オリゴ（２°−０−メチル−５−メチルウリジル
酸）誘導体を製造する為には、先に述べた２°−〇−メ
チルー５−メチルー５°−ウリジル酸のＤＣＣによる重
合反応において、公知の方法（Ｐ、 Ｔ、 Ｇｉ Ｉｈ
ａｍ、 ＪＡＣ５，８８，４９８２（１９６４））に従
い、セルロース等を同時に反応させ、セルロース誘導体
等とすることが簡便である。また一方、先に述べた自動
合成機により合成した５°末端水Ｒｊ４を有するオリゴ
（２°−０−メチル−５−メチルウリジル酸）の５′末
端水ｗｉ基に燐ｗＩＷを導入した後に、同様にセルロー
ス等と反応させることもできる。On the other hand, in order to produce oligo(2°-0-methyl-5-methyluridylic acid) derivatives, the above-mentioned DCC polymerization reaction of 2°-〇-methyl-5-methyl-5°-uridylic acid is carried out using the known method. method (P, T, Gi Ih
Am, JAC5, 88, 4982 (1964)), it is convenient to react cellulose and the like at the same time to obtain a cellulose derivative and the like. On the other hand, after introducing phosphorus wIW into the 5' terminal water wi group of oligo (2°-0-methyl-5-methyluridylic acid) having a 5° terminal water Rj4 synthesized by the automatic synthesizer described above, It is also possible to react with cellulose etc.
また一方、５゛末端水Ｍｉｌｋを有したオリゴ（２゛−
〇−メチルー５−メチルウリジル酸）とＣＭ−セルロー
スまたはラテックス粒子等とを縮合剤（ＤＣＣ等）を用
いて結合させ、セルロースあるいはラテックス誘導体（
に、にｕｒｉｂａｙａｓｈｉ ｅｔ ａｌ、。On the other hand, the oligo with 5゛ terminal water Milk (2゛-
〇-Methyl-5-methyluridylic acid) and CM-cellulose or latex particles, etc. are bonded using a condensing agent (DCC, etc.) to form a cellulose or latex derivative (
In, Niuribayashi et al.
ＮｕＣｌｅｉＣＡｃ１ｄｓ ５ＶｌｌｐＯ３ｉｕｌ ５
ｅｒｉｅｓ、に１９．６１（１９８８）参照）等とする
ことも可能である。NuCleiCAc1ds 5VllpO3iul 5
19.61 (1988)).
化合物３の製造は、リボチミジンを出発原料として例え
ば先ず化合物１を得た後合成できる（第１図及び実施例
１〜３参照）。因みに、化合物１の合成は、リボチミジ
ンの３’、５’水酸基を公知の方法（ｔｌ、Ｉｎｏｕｃ
ｅｔ ａｌ、、Ｎｕｃｌｃｉｃ ＡｃｉｄｓＳｙｍｐ
ｏｓｉｕｎ ５ｅｒｉｅｓ、 Ｎ（１１６，１６５（
１９８５）、獣■。Compound 3 can be synthesized using ribothymidine as a starting material, for example, after first obtaining compound 1 (see FIG. 1 and Examples 1 to 3). Incidentally, compound 1 was synthesized by converting the 3' and 5' hydroxyl groups of ribothymidine to known methods (tl, Inouc).
et al., Nuclic Acids Symp.
osiun 5eries, N(116,165(
1985), Beast ■.
Ｈａｒｋｉｅｗｉｃｚ、Ｊ、Ｃｈｅｍ、Ｉｔｅ、；（Ｓ
） ２４−２５ （１９７９））に準じて、テトライ
ソプロピルジシロキサン−１，３−ジイル基で保護した
後、公知の方法（Ｈ，ｒｎｏｕｃ ｅｔａｌ、の前出の
電文（１９８７） ）に準じて、適当な溶媒中トリエチ
ルアミンの存在下塩化ベンゾイルで処理することにより
３Ｎ位を選択的に保護することにより得ることができる
。Harkiewicz, J., Chem.
) 24-25 (1979)), and then protected with a tetraisopropyldisiloxane-1,3-diyl group according to a known method (H, Rnouc et al., supra, Telegram (1987)). It can be obtained by selectively protecting the 3N position by treatment with benzoyl chloride in the presence of triethylamine in a suitable solvent.
また、２°−〇−メチルー５−メチルウリジンー３°−
フォスフォロアミダイト誘導体（化合物５）及び２°−
〇−メヂルー ５−メチルウリジン−３゛−ＣＰＧ誘導
体（化合物６）の製造は、公知の方法（Ｓ、５ｈｉｂａ
ｈａｒａ ｅｔ ａｔ、の前出の電文、Ｌ、Ｊ、ＨｃＢ
ｒｉｄ１３ ｅｔ ａｔ、、 Ｔｅｔｒａｈｅｄｒｏｎ
Ｉｅｔｔ、、２４，２４５（１９８３）参照）に従い
合成できる（第２図並びに実施例４及び５参照）。また
、ここで合成したフォスフォロアミダイト誘導体（化合
物５）の他に、亜ｇ４ｒ１１の保護基としてシアノエチ
ル基を有する化合物も、同様の方法（Ｓ、５ｈｉｂａｈ
ａｒａ ｅｔ ａｔ、の前出の電文、Ｎ、Ｄ、５ｈｉｎ
ｈａ ｅｔ ａｌ、、Ｎｕｃｌｅｉｃ Ａｃ１ｄｓ Ｒ
ＱＳ、、 １２゜４５３９（１９８４）６照）に従い
合成することができる。Also, 2°-〇-methyl-5-methyluridine-3°-
Phosphoramidite derivative (compound 5) and 2°-
〇-Mediru 5-methyluridine-3゛-CPG derivative (compound 6) can be produced by a known method (S, 5hiba
Hara et at, above mentioned telegram, L.J.HcB
rid13 et at,, Tetrahedron
Iett, 24, 245 (1983)) (see Figure 2 and Examples 4 and 5). In addition to the phosphoramidite derivative (compound 5) synthesized here, a compound having a cyanoethyl group as a protecting group for subg4r11 was also synthesized using the same method (S, 5hibah
ara et at, the aforementioned telegram, N, D, 5hin
ha et al, Nucleic Ac1ds R
QS, 12°4539 (1984) 6).
本発明に関して肝要な点は、従来より一般に使用されて
いるオリゴ（ｄＴ）−セル［］−スのオリゴ（ｄＴ）に
比べて、オリゴ（２°−〇−メチルー５−メチルウリジ
ルＭ）−がオリゴリボアデニル酸（オリゴ（ｒＡ））と
著しく安定なハイブリッドを形成する性質を有すること
を発見したことにある。The important point regarding the present invention is that oligo(2°-〇-methyl-5-methyluridyl M)- is an oligo(2°-〇-methyl-5-methyluridyl The present invention is based on the discovery that it has the property of forming an extremely stable hybrid with riboadenylic acid (oligo(rA)).
以下、この点について詳述する。すなわち、それらのハ
イブリッドの熱的安定性（Ｔｍ値）を実際に測定する為
に、デカリボアデニル酸を公知の方法（ｐ、に１ｅｒｚ
ｅｋ ｅｔ ａｌ、、Ｂｉｏｃｈｅｍｉｓｔｒｙ、 ２
５．７８４０（１９８Ｂ）参照）に従い合成しく実施例
６）、またデカ（２°−０−メチル−５−メチルウリジ
ル酸）は、実施例４及び５により製還した化合物５及び
６を用いてＤＮＡ自動合成機により常法通り（Ｓ、５ｈ
ｉｂａｈａｒａ ｅｔ ａｌ、の前出の電文参照）合成
した（実施例７）、一方、比較実験用のオリゴマーであ
るデカデオキチミジル酸は常法により市販試薬を用いて
、デカ（２°−０−メチルウリジル酸）は公知の方法（
Ｓ、５ｈｉｂａｈａｒａ ｅｔ ａｌ、の前出の電文参
照）に従い、それぞれ、ＤＮＡ自動合成機を用いて合成
した。This point will be explained in detail below. That is, in order to actually measure the thermal stability (Tm value) of those hybrids, decariboadenyl acid was purified by a known method (p,
Ek et al., Biochemistry, 2
5.7840 (198B)) and deca(2°-0-methyl-5-methyluridylic acid) was synthesized according to Example 6) using compounds 5 and 6 prepared according to Examples 4 and 5. Using an automatic synthesizer as usual (S, 5h
ibahara et al., supra)) was synthesized (Example 7). On the other hand, the oligomer for comparative experiments, decadeochitimidylic acid, was synthesized by deca(2°-0-methyluridyl) using commercially available reagents in a conventional manner. acid) by a known method (
Each of these DNAs was synthesized using an automatic DNA synthesizer according to the above-mentioned telegram of Hibahara et al.
それぞれのオリゴヌクレオチドは常法により定ゴした後
に、熱的安定性測定用試料とした。それぞれのハイブリ
ッドのＴｍ値の測定は常法に従い、ベックマンＤＵ−８
Ｂ型分光光度計を用いて測定した（第３図及び実施例８
参照）。この結果、それぞれのハイブリッドのＴｍ値は
、１８．９℃、 ２１．０”Ｃ，３１，１℃となり、デ
カ（２°−０−メチフレー５−メチルウリジル酸）とデ
カリボアデニル酸とのハイブリッドが他のハイブリッド
と比べて、Ｔｍ値が約１０℃も高く、著しく安定である
ことを発見した。After fixing each oligonucleotide by a conventional method, it was used as a sample for thermal stability measurement. The Tm value of each hybrid was measured using a Beckman DU-8 according to a conventional method.
Measured using a B-type spectrophotometer (Figure 3 and Example 8)
reference). As a result, the Tm values of each hybrid were 18.9°C, 21.0"C, and 31.1°C, and the hybrid of deca(2°-0-methifle-5-methyluridylic acid) and decariboadenyl acid It was discovered that the Tm value was about 10°C higher than that of other hybrids, and it was extremely stable.
以上のことより、オリゴ（２°−０−メチル−５−メチ
ルウリジル酸）は、そのセルロース結合体として、現在
市販されているオリゴ（（ｆＴ）−セルロースに代わる
新規ｐｏｌｙ（Ａ） −ｍＲＮＡ単離用担体として使用
できることが充分期待される。From the above, oligo(2°-0-methyl-5-methyluridylic acid), as its cellulose conjugate, is a novel poly(A)-mRNA isolation alternative to the currently commercially available oligo((fT)-cellulose. It is fully expected that it can be used as a carrier for various purposes.
以下、実施例により本発明を具体例的に説明する。Hereinafter, the present invention will be specifically explained with reference to Examples.
１塵旦 以下、実施例により本発明を更に説明する。1 dustman The present invention will be further explained below with reference to Examples.
実施例１
（Ｎ３−Ｂｅｎｚｏｙｌ−５−ｓｅｔｈｙｌ−３’ 、
５’ −０−（ｔＱｔｒａｉｓｏｌ）ｒｏｔ）ｙｌ
−ｄｉｓｉｌＯＸａｎｅ −１，３−ｄｌｌ／Ｉ）ｕｒ
ｉｄｉｎｅ（化合物１）の合成）
５−１４ｅｔｈｙｌ−１，３−Ｏ−（ｔｅｔｒａｉｓＯ
Ｉ）Ｉ’０ＤＶｌｄｉＳｉ １ＯＸａｎｅ−１，３−ｄ
ｉｙｌ） ｕｒｉｄｉｎｅ（ＩＧ、６７ ｇ 、３３．
３ｍｍｏｌ）を塩化メチレン２５０ｄに溶解し、トリエ
チルアミン（６，３ｄ、 ４５．２ｍａ＋ｏｆ）を加え
、水冷撹拌ド、塩化ベンゾイル（４，４ｄ、 ３７．９
ｎ＋ｍｏｌ）を滴下した。室温で一晩世拌１ノ、原料の
消失を確認後、０．０１ ＮＨＣｌ、水、ＮａＨＣＯ３
水溶液、水の順で反応溶液を洗浄した。有ｔＩＩＩ＠Ｎ
ａ ＳＯ４で乾燥後、溶媒を減圧トで留去し、残漬をシ
リカゲルクロマトグラフィー（Ｃ−２００、４００９、
ＣＨＣｌ３＞で精製し、化合物１を泡状物質として１３
．６９９（６７、９％）得た。Example 1 (N3-Benzoyl-5-sethyl-3',
5' -0-(tQtraisol)rot)yl
-disilOXane -1,3-dll/I)ur
Synthesis of idine (compound 1)) 5-14ethyl-1,3-O-(tetraisO
I) I'0DVldiSi 1OXane-1,3-d
iyl) uridine (IG, 67 g, 33.
3 mmol) was dissolved in 250 d of methylene chloride, triethylamine (6,3 d, 45.2 ma+of) was added, and benzoyl chloride (4,4 d, 37.9
n+mol) was added dropwise. After stirring at room temperature overnight and confirming the disappearance of the raw materials, add 0.01 NHCl, water, NaHCO3
The reaction solution was washed with an aqueous solution and then with water. tIII@N
a After drying with SO4, the solvent was distilled off under reduced pressure, and the residue was subjected to silica gel chromatography (C-200, 4009,
Compound 1 was purified as a foam with 13
．． 699 (67, 9%) was obtained.
ＩＪＶ （ＭｅＯＨ） ：λｗａｘ ２５６ｒｖ、λｓ
ｉｎ ２２５ｎｅ。IJV (MeOH): λwax 256rv, λs
in 225ne.
１Ｈ−川（ｃｏｃｔ３＞δＥ）ｌ）ｌニア、９９−７．
２９（１，６Ｈ，ｂｌＺＯＶＩ ＱｒＯＩｊｌ）、 Ｃ
６−Ｈ）。1H-kawa(coct3>δE)l)lnia, 99-7.
29 (1,6H, blZOVI QrOIjl), C
6-H).
５．７６（ｄ、 ＩＨ，Ｊ−１１１Ｚ。5.76 (d, IH, J-111Z.
Ｃ１°−Ｈ）、 １．９７（ｄ、３１１．Ｊ＝１．２Ｈ
２゜Ｃ５−ＣＨ３）。C1°-H), 1.97(d, 311.J=1.2H
2°C5-CH3).
１．１２−１．０６ （ｍ、２８Ｈ，ＴＩＰＤＳ ａ
ｒｏｕｐ）。1.12-1.06 (m, 28H, TIPDS a
roup).
実施例２
（Ｎ３−Ｂｅｎｚｏｙｌ−５−ｎｅｔｈｙｌ−２’−０
−ｍｅｔｈｙｌ−３’５’ −０−（ｔｅｔｒａｉｓｏ
ｐｒｏｐｙｌｄｉｓｉｌｏｘａｎｅ−１，３−ｄｉｙｌ
）ｕｒｉｄｉｎｅ （化合物２）の合成〉実施例１で得
た化合物１ （１３，６９７，２２，６ｍｍｏ ｌ ）
をベンゼン８０ｍに溶解し、酸化銀（１４，７ｇ、６３
．３ｕｏ ｌ ）を加え、５ＯｎｉＣａｔＯｒに約１０
分かけた。ヨウ化メチル（６５，Ｏｄ、１０４５ｍｍｏ
ｌ ）を加え、４５℃で３日間撹拌した１反応混合物を
Ｐ退し、溶媒を減圧下留去後、ＣＨＣ１３＝Ｈ２０’ｔ
’分液し、有ａｍをＮａ２ＳＯ４で乾燥後、シリカゲル
カラムクロマトグラフィー（Ｃ−２００，３００ｇ、Ｃ
ＨＣｌ３）で精製し、化合物２を泡状物質として６．７
５９（４８，２％）得た。さらに、分離できなかったフ
ラクションを染め、シリカゲル力ラムク口マトグラフィ
ー（Ｃ−２００，２Ｇ０３、ヘキサン：酢酸エチル−４
：１）でｆ６賀し、泡状物質（化合物２）を３．９６
！？ （２８，４％）得た。Example 2 (N3-Benzoyl-5-nethyl-2'-0
-methyl-3'5' -0-(tetraiso
propyldisiloxane-1,3-diyl
) uridine (Compound 2) synthesis> Compound 1 obtained in Example 1 (13,697,22,6 mmol)
was dissolved in 80ml of benzene, and silver oxide (14.7g, 63ml) was dissolved in 80ml of benzene.
．． 3 uo l) and add about 10
It took a while. Methyl iodide (65,Od, 1045mmo
1) was added and stirred at 45°C for 3 days. The reaction mixture was evaporated, and the solvent was distilled off under reduced pressure. CHC13=H20't
' Separate the liquid, dry am with Na2SO4, and then perform silica gel column chromatography (C-200, 300g, C
HCl3) to give compound 2 as a foam at 6.7
59 (48,2%) was obtained. Furthermore, the fractions that could not be separated were dyed and silica gel chromatography (C-200, 2G03, hexane:ethyl acetate-4) was performed.
:1) to f6 and foamy substance (compound 2) to 3.96
! ? (28.4%) was obtained.
ＵＶ（ＨｅＯＨ）： λｍａｘ ２５２ｔｖ、λｗｉｎ
２２５ｎｔ実施例３
（５−Ｎｅｔｈｙｌ−２’−０−ｓｅｔｈｙｌｕｒｉｄ
ｉｎｅ（化合物３）の合成）
化合物２ （６，９９ｒＪ、 １１．３ｍｍｏｌ）をジ
オキサン１００ｄ ニ溶解し、ｃＮＨ４０Ｈ（１２，２
ｍｌり ｅ加え、空温で５時間撹拌した。原料の消失を
確認し、反応溶液を濃縮し、ＣＨＣｌ３−８２０で分液
した。UV (HeOH): λmax 252tv, λwin
225 nt Example 3 (5-Nethyl-2'-0-sethylurid
Synthesis of ine (compound 3)) Compound 2 (6,99rJ, 11.3mmol) was dissolved in 100d of dioxane, and cNH40H (12,2
ml of the mixture was added, and the mixture was stirred at air temperature for 5 hours. After confirming the disappearance of the raw materials, the reaction solution was concentrated and separated with CHCl3-820.
有機層を、水、ＮａＨＣＯ３水溶液、水の順に洗浄した
後、Ｎａ ＳＯ４で乾燥し、溶媒を減圧下留去後、ヘ
キナンから結晶し、５．４６９ （９３，９％）の結晶
性化合物を得た。The organic layer was washed with water, an aqueous NaHCO3 solution, and water in this order, then dried with NaSO4, and after distilling off the solvent under reduced pressure, it was crystallized from hequinane to obtain a crystalline compound of 5.469 (93.9%). Ta.
ｍ、ｐ、：１１６．５〜１１８．０℃
ｍａｓｓ ｍ／ｅ： ４７１ （１４＋−１ｓｏｐｒｏ
ｐｙｌラジカル）元素分析： Ｃ２３＋＋４２８２
０７ Ｓ１２としての、計算値： Ｃ，５３，７０；Ｈ
，８，１７；Ｎ、５．４ｂ実験値： Ｃ，５４，５３，
Ｈ，８，１３；Ｎ、５．６８この化合物（４，００す、
７．８１１１０１）をテトラヒドロ７ラン５０−１に溶
解し、Ｔ ０ｔｒａｂｕｔＶＩａｌｌｌＯｎｉｕｌｆｌ
ｕｏｒｉｄｅ（３，２ｍ、 ３．１ｗ５ｏｌ）加え、
空温で４．５時間撹拌した。ピリジン：水： Ｍ ｅ
０１−Ｉ Ｊ３：１：１（ｖ／Ｖ）の溶液を１００ｍ！
入れて反応溶液を希釈し、これにＯｏｗｃｘ ５０
（ピリジニウム型）を３０ａｅ加え、中和した。樹脂を
濾過後、反応溶液を濃縮し、ＣＨＣｌ３−１−１２０で
分液した。水層を濃縮乾固した後、エタノールから結晶
化を行い、化合物３を１．８５９ （８７，２％）を得
た。m, p,: 116.5-118.0°C mass m/e: 471 (14+-1sopro
pyl radical) elemental analysis: C23++4282
Calculated value as 07 S12: C, 53, 70; H
, 8, 17; N, 5.4b Experimental value: C, 54, 53,
H, 8,13; N, 5.68 This compound (4,00 S,
7.811101) in Tetrahydro7ran 50-1 and
Add uoride (3.2m, 3.1w5ol),
The mixture was stirred at air temperature for 4.5 hours. Pyridine: Water: Me
01-I J3: 1:1 (v/V) solution for 100ml!
dilute the reaction solution by adding Oowcx 50
(pyridinium type) was added to neutralize the mixture. After filtering the resin, the reaction solution was concentrated and separated with CHCl3-1-120. After concentrating the aqueous layer to dryness, it was crystallized from ethanol to obtain 1.859 (87.2%) of Compound 3.
ＵＶ （Ｈｅ’ｌｌ ） ： λｗａｘ ２６６ｎｍ、
λｓｉｎ ２３３ｎｍ。UV (He'll): λwax 266nm,
λsin 233nm.
１、ｐ、 ：１９７．Ｏ〜１９８．０ ℃ｍ
ａｓｓ ｍ／ｅ： ２γ２（１４” ）元素分
析” Ｃ１１’１６ ’２ ０Ｇとしての、計算値：
Ｃ，４８，５３；Ｈ，５，８８；Ｈ，１０，２９実験
値： Ｃ，４８，３６；Ｈ，５，９６；Ｎ、１０．３７
’ＩＩ−ＮＨＲ（ＤＨ３Ｏ−ｄ６）δｐｐ１７．７８（
ｄ、１１１．Ｊ−１，２Ｈｚ、ＣＢ −Ｈ）。1, p. :197. O~198.0℃m
ass m/e: 2γ2 (14") elemental analysis"C11'16'2 Calculated value as 0G:
C, 48,53; H, 5,88; H, 10,29 Experimental value: C, 48,36; H, 5,96; N, 10.37
'II-NHR(DH3O-d6)δpp17.78(
d, 111. J-1,2Hz, CB-H).
５、８６（ｄ、 １１１． Ｊ−５，４Ｈｚ、 ＣＩ’
−Ｉｔ）。5,86(d, 111. J-5,4Hz, CI'
-It).
３．３３ （ｓ、３１１．０２’−ＣＨ３）。3.33 (s, 311.02'-CH3).
１．７８（Ｓ、３Ｎ、Ｃ５−ＣＨ３）。1.78 (S, 3N, C5-CH3).
因みに、化合物３は、・尿から単離されたとの報告があ
るが（Ｃ，Ａ、 、 Ｖｏｌ、１Ｇ１，６８６５４ａ（
１９８４）、尿からの単離では単離できる量に自ら制限
があり、上記の実施例１〜３によって製造するのが極め
て有利である。Incidentally, there is a report that Compound 3 was isolated from urine (C, A, Vol, 1G1, 68654a (
(1984), isolation from urine has its own limitations on the amount that can be isolated, and it is extremely advantageous to produce according to Examples 1 to 3 above.
実施例４
（メトキシ−５′−ジメトキシトリチル−２′−〇−メ
チルー５−メチルーウリジン−３１０−（Ｎ、Ｎ−ジイ
ソプロピルアミノ）フォスフイン（化合物５）の合成）
化合物３ （０，５４４ｔＪ、 ２．０ｍｍｏｌピリ
ジン共沸後、５−のピリジンに溶解しジメトキシトリチ
ルク１］ライド（０，８２９，２，４ｍｍｏ ｌ ）加
え、空温で４時間撹拌した。原料の消失を確認し、メタ
ノール１１１１を加え、反応を停止させた。溶媒を減圧
］；留去し、ＣＨＣ１３−Ｈ，２０で分液し、有ｔ１層
をＮａ ＳＯ４乾燥俊、シリカゲルカラムクロマトク
ラ）イー （Ｃ−２００、４０７、〜４％ＭｅＯＨ／Ｃ
ＨＣｌ３）で精製した後、ｎ・−ヘキサンから粉末化し
、化合物４を０．９２ｇ（８０％）得た。Example 4 (Synthesis of methoxy-5'-dimethoxytrityl-2'-〇-methyl-5-methyl-uridine-310-(N,N-diisopropylamino)phosphine (compound 5)) Compound 3 (0,544 tJ, 2.0 mmol After pyridine azeotropy, dimethoxytritylchloride (0,829,2,4 mmol) dissolved in 5-pyridine was added and stirred at air temperature for 4 hours. After confirming the disappearance of the raw material, methanol 1111 was added. The reaction was stopped. The solvent was removed under reduced pressure]; the layers were separated with CHC13-H, 20, and the t1 layer was purified with NaSO4, silica gel column chromatography (C-200, 407, ~4%). MeOH/C
After purification with HCl3) and powdering from n-hexane, 0.92 g (80%) of compound 4 was obtained.
この化合物４ （２８１１９１０，４９霞■０１）をピ
リジン共Ｓ侵、蒸留メチレンクロライドに溶解し、ジイ
ソプロピルエチルアミン（０，３５ａｄ！、２．０ｍｍ
ｏｌ）を加えた。密栓した侵、雰囲気をアルゴン置換し
、そこへｃｈｌｏｒｏ−Ｎ 、 Ｎ −ｄｉｉｓｏｐｒ
ｏｐｙｌａｉｉｎｏｉｅｔｈｏｘｙｐｈｏｓｐｈｉｎｅ
（０，１２ａｄ、０．６１１０１）を約１分かけて滴
下した。室温で約６０分放置し、原料の消失を確認し、
（シリカゲルＴＬＣ法：移ＦＩＪ Ｆｎ、酢酸エチル、
原料Ｒ（Ｗｉ Ｏ，４３、ＩＩ！ｉ部Ｒ，値０．７）、
酢酸エチル約１５ｍを加え、反応溶液を希釈した。This compound 4 (2811910, 49 Kasumi ■ 01) was dissolved in distilled methylene chloride with pyridine and diisopropylethylamine (0.35ad!, 2.0mm).
ol) was added. The atmosphere was replaced with argon, and then chloro-N, N-diisopr was added.
opylaiinoiethoxyphosphine
(0.12ad, 0.61101) was added dropwise over about 1 minute. Leave it at room temperature for about 60 minutes and check that the raw materials have disappeared.
(Silica gel TLC method: FIJ Fn, ethyl acetate,
Raw material R (Wi O, 43, II!i part R, value 0.7),
Approximately 15 mL of ethyl acetate was added to dilute the reaction solution.
飽和ＮａＨＣＯ３水溶液、飽和食塩水ぐ洗浄した後、無
水硫酸ナトリウムで乾燥後、濃縮し、残渣を得た。更に
、シリカゲルカラムクロマトグラフィー（Ｃ−３００，
２，５ｇ、酢酸エチル）でｍ製し、泡状物質として化合
物５を０．３４９ （９４％）を得た。After washing with a saturated aqueous NaHCO3 solution and saturated brine, the mixture was dried over anhydrous sodium sulfate and concentrated to obtain a residue. Furthermore, silica gel column chromatography (C-300,
2.5 g of ethyl acetate) to give 0.349 (94%) of compound 5 as a foam.
実施例５
（ヌクレオシド樹脂の合成）
化合物４ （０，１７９，３，０１１０１）をピリジン
共沸した侵、塩化メチレン３ｍに溶解し、無水コハク酸
（４７，８■、４．５ｕｏｌ）とジメチルアミノピリジ
ン（５５，５ｑ、 ０．４６ｉｍｏりを加え、変温で
２時間撹拌した。ＴＬＣで原料の消失を確認した後、反
応液を０．５Ｍ ＫＨＰＯ４水で洗浄した。溶媒を減
圧上留去し、残渣をシリカゲルカラムクロマトグラフィ
ー（Ｃ−３００、１０９）で精製し、ノルマルへ４−サ
ンで粉末化し、０．１８ ｇ （０，１７ｍ１ｏｌ、８
８．９％）の３’ −（２’−０−Ｈｅｔｈｌ／＋−５
’ −０−ｄｉｍｃｔｈｏｘｙｔｒｉｔｙｌ −５−１
ｃｔｈｙｌｕｒｉｄｉｎｙｌ） −ｍｏｎｏｓｕｃｃｔ
ｎａｔｅを得た。Example 5 (Synthesis of nucleoside resin) Compound 4 (0,179,3,01101) was azeotroped with pyridine, dissolved in 3 m of methylene chloride, and mixed with succinic anhydride (47.8 μl, 4.5 uol) and dimethylamino Pyridine (55.5q, 0.46imo) was added and stirred at a variable temperature for 2 hours. After confirming the disappearance of the raw materials by TLC, the reaction solution was washed with 0.5M KHPO4 water. The solvent was distilled off under reduced pressure. , the residue was purified by silica gel column chromatography (C-300, 109), powdered with normal 4-san, 0.18 g (0.17 mol, 8
8.9%) of 3'-(2'-0-Hethl/+-5
'-0-dimctoxytrityl-5-1
cthyluridinyl) -monosucct
I got nate.
全量をＤＭＦ３ａｅに溶解し、ペンタクロロフェノール
（８７，１１１Ｉ！Ｆ、約１．２当量）、ジシクロへキ
シルカルボジイミド（６６，０■、約１．２当量）を加
え、室温で一晩撹拌した。ＴＬＣで原料の消失を確認し
、生じた沈澱をＰ別した後、溶媒を減圧ド留去した。さ
らに、残渣にベンゼンを加え、析出した不溶物をＰ別し
、Ｐ液を濃縮した後、ｎ−ヘキサンから粉末化し、ｐｅ
ｎｔａｃｈ＋ｏｒｏｐｈｅｎｙ＋ −３’（２’ −
０−ｍｅｔｈｙｌ−５’ −０−ｄｉｍｅｔｈｏｘｙ
ｔｒｉｔｙｌ＝ ５−１８ｔｈｙＩＬＩｒｉｄｉｎｙ１
）−３ｕＣＣｉｎａｔｅを得た。収ｇＬ０．２３ ｇ、
０．２４９ｍｇ＋ｏ Ｉ、収率９２．３％。The entire amount was dissolved in DMF3ae, pentachlorophenol (87,111I!F, about 1.2 equivalents) and dicyclohexylcarbodiimide (66,0μ, about 1.2 equivalents) were added, and the mixture was stirred at room temperature overnight. After confirming the disappearance of the raw materials by TLC and separating the resulting precipitate with P, the solvent was distilled off under reduced pressure. Furthermore, benzene was added to the residue, the precipitated insoluble matter was separated from P, the P solution was concentrated, and then powdered from n-hexane.
ntach+oropheny+ -3'(2'-
0-methyl-5'-0-dimethoxy
trityl=5-18thyILIridiny1
)-3uCCinate was obtained. Yield gL0.23 g,
0.249 mg+o I, yield 92.3%.
コノ化合物（７５，３Ｉ！ｒｇ、８２μｍｏｌ）をＣＰ
Ｇ樹脂（１００μ１ｉｏｌ ＮＨ２／ｇ、０．２４Ｉ
Ｊ）のトリエチルアミン（１５Ｊ１１）−ＤＭＦ （３
ｍ）の懸濁液中へ加え、室温で一晩振とうした。反応液
を一過し、樹脂をＤＭＦ、ピリジン、塩化メチレンで順
次洗浄した後、減圧乾燥した。Cono compound (75,3I!rg, 82 μmol) in CP
G resin (100μ1iol NH2/g, 0.24I
J) triethylamine (15J11)-DMF (3
m) and shaken overnight at room temperature. The reaction solution was passed through, and the resin was washed successively with DMF, pyridine, and methylene chloride, and then dried under reduced pressure.
常法により定量し、ローディング量１μｍ０１／２９．
４■であることを確認した。It was determined by a conventional method, and the loading amount was 1 μm01/29.
It was confirmed that it was 4■.
０．１Ｍジメチルアミノピリジン／ピリジン４．５−と
無水酢酸０．５ｍを加えて１０分！ｆｆｉ振とうした。Add 0.1M dimethylaminopyridine/pyridine 4.5- and 0.5m acetic anhydride for 10 minutes! ffi shaken.
反応液を濾過し、樹脂をピリジン、塩化メチレンで順次
洗浄した後減圧下乾燥し、化合物６を得た。The reaction solution was filtered, and the resin was washed successively with pyridine and methylene chloride, and then dried under reduced pressure to obtain Compound 6.
実施例６
（デカリボアデニル酸（γＡ １０ｗｅｒ）の合成）
公知方法（Ｅ 、 Ｏｈｔｓｕｋａ ｅｔ ａｌ、。Example 6 (Synthesis of decariboadenylic acid (γA 10wer))
A known method (E, Ohtsuka et al.
Ｔｅｔｒａｈｅｄｒｏｎ、 ４０．４７（１９８４）参
照）に従い合成したＮ６−ペンゾイルー５′−〇−ジメ
トキシトリチルー２’−０−テトラヒドロピラニルアデ
ノシンを公知方法（Ｌ 、 Ｊ 、 Ｍ ｃ Ｂ ｒｉ
ｄｅ ｃｔａｌ、。Tetrahedron, 40.47 (1984)) N6-penzoyl-5'-〇-dimethoxytrityl-2'-0-tetrahydropyranyl adenosine was synthesized according to a known method (L, J, McBri
dectal.
Ｔｅｔｒａｈｅｄｒｏｎ Ｌｅｔｔｅｒｓ、 ２４．
２４５（１９８３）参照）従い３’ −０−（Ｎ、Ｎ−
ジイソプロピルアミノ）−メトキシホスホアミダイト誘
導体に導いた。Tetrahedron Letters, 24.
245 (1983)) Therefore, 3' -0-(N, N-
diisopropylamino)-methoxyphosphoramidite derivatives.
即ち、Ｎ６−ペンゾイルー５′−〇−ジメトキシトリチ
ルー ２’−０−テトラヒドロピラニルアデノシン（５
７６，３ｑ、 ７６６．５μｍｏｌ）をピリジン共沸
により無水にした後、ジクロルメタン１ａｉ！に溶解し
た。この溶液に窒素ガス気流「ｆジイソプロピルエチル
アミン（０，４９ｒｄ＞を加えた後、水冷下クロロ・−
メトキシ−Ｎ、Ｎ−ジイソプロピルアミノホスフィン（
０，１７ｄ）を徐々に加えた。１時間放置後、薄層りＯ
マドグラフィーにより原料の消失を確認した後、酢酸エ
チル１０ｄを加え希釈した。この希釈液を飽和炭酸水素
ナトリウム水溶液、次いで飽和食塩水で洗浄し、酢酸エ
チル層を減圧下濃縮乾固した。得られた残渣を酢酸エチ
ルに溶解し、１０ｇのシリカゲル（Ｃ−３００）を充填
したカラムクロマトグラフィー（移動相：酢酸エチル）
により精製した。溶出されたＮＧ−ペンゾイルー５′−
〇−ジメトキシトリエチルー２′−〇−テトラヒドロピ
ラニルアデノシン−３’−０−（Ｎ。That is, N6-penzoyl-5'-〇-dimethoxytrityl-2'-0-tetrahydropyranyladenosine (5
76.3q, 766.5 μmol) was made anhydrous by pyridine azeotrope, and then dichloromethane 1ai! dissolved in. After adding a nitrogen gas stream of diisopropylethylamine (0.49rd) to this solution, chloro-
Methoxy-N,N-diisopropylaminophosphine (
0.17d) was added gradually. After leaving for 1 hour, a thin layer O
After confirming the disappearance of the raw materials by mudgraphy, 10 d of ethyl acetate was added to dilute the solution. This diluted solution was washed with a saturated aqueous sodium hydrogen carbonate solution and then with saturated brine, and the ethyl acetate layer was concentrated to dryness under reduced pressure. The obtained residue was dissolved in ethyl acetate and subjected to column chromatography packed with 10 g of silica gel (C-300) (mobile phase: ethyl acetate).
It was purified by Eluted NG-penzoyl-5'-
〇-dimethoxytriethyl-2'-〇-tetrahydropyranyladenosine-3'-0-(N.
Ｎ−ジイソプロピルアミノ）メトキシホスホアミダイト
（化合物７）の純粋画分を集め濃縮乾固し、０．６７
ｇ（７３３，９μｍｏｌ ）の化合物７を９５．７％の
収率で得た。The pure fractions of N-diisopropylamino)methoxyphosphoramidite (compound 7) were collected and concentrated to dryness, giving a concentration of 0.67
g (733.9 μmol) of compound 7 was obtained in a yield of 95.7%.
次に、ＮＧ−ベンゾイル−２′−〇−テトラヒドロピラ
ニルアデノシン（１μ１０１）を結合した固相担体（Ｒ
、Ｋ １ｅｒｚｅｋ ｅｔａｌ、、 Ｂ ｉｏｃｈｅｍ
ｉｓｔｒｙ。Next, a solid phase support (R
, K. 1erzek etal, , Biochem
istry.
２５、７８４０〜７８４６ （１９８６）を出発原料に
用い、２０μｍｏｌ ／ １４０ＩＩｉの濃度になるよ
うに溶解した化合物７のアセトニトリル溶液とともにＤ
ＮＡ自動合成ｍ＜７プライドバイオシステムズ社製、モ
デル３８０Ａ ＞にて鎖長延長反応を行なった。各縮合
サイクルに先立つ脱ジメトキシトリチル化反応は、１％
ジクＬ１ル酸／メチレンクロライド溶液を用いた。反応
後の固相担体より切り出された部分的に保１ｍ３を有す
るデカアデニル酸のアンモニア水溶液は、６０℃で５時
間加温した。次いで、この溶液を濃縮し、逆相シリカゲ
ル（Ｐｒｅｌ） Ｆ’ＡＫ−５００／Ｃ−１８、ウォー
タース社製）を詰めたカラムク０マドグラフイーを行な
うことにより精製した。25, 7840-7846 (1986) as a starting material, and D together with an acetonitrile solution of compound 7 dissolved to a concentration of 20 μmol/140IIi.
The chain elongation reaction was performed using an automatic NA synthesis m<7 model 380A manufactured by Pride Biosystems. The dedimethoxytritylation reaction prior to each condensation cycle was carried out at 1%
A dichloric acid/methylene chloride solution was used. The ammonia aqueous solution of decaadenylic acid, which was cut out from the solid support after the reaction and had a partial retention capacity of 1 m3, was heated at 60° C. for 5 hours. This solution was then concentrated and purified by column chromatography packed with reverse phase silica gel (Prel F'AK-500/C-18, manufactured by Waters).
即ち、移動相として５％から３５％のアセトニトリルの
直線濃度勾配をかけた５０１Ｍ ｔ’リエチルアミンー
炭酸緩衝液（１）Ｈ７，５、以ドＴＥＡＢと略す。）を
用いて溶出し、５′末端にジメトキシトリチル基及び２
′位水ｗ１基にテトラヒドロピラニル基を有するデカア
デニル酸の純粋画分を集め、濃縮乾固した０次に、この
、残渣を０．０１ Ｎの塩酸水溶液１０ｄに溶かしてｐ
ｉ−１２，０に調整し、室温に２４時間放置した。反応
液は希釈したアンモニア水で中和後、酢酸エチルにて洗
浄したのち濃縮し、セファデックスＧ−２５を詰めたゲ
ルー過カラムクロマトグラフィー（移動相： ０．Ｉ
Ｍ ＴＥＡＢ）を行なうことにより脱塩した。That is, 501M t'-ethylamine-carbonate buffer (1) H7,5 was applied with a linear concentration gradient of 5% to 35% acetonitrile as a mobile phase, hereinafter abbreviated as TEAB. ), and dimethoxytrityl group and 2
A pure fraction of decaadenylic acid having a tetrahydropyranyl group at the water w1 group at the ' position was collected and concentrated to dryness. Next, this residue was dissolved in 10 d of a 0.01 N aqueous hydrochloric acid solution and
It was adjusted to i-12.0 and left at room temperature for 24 hours. The reaction solution was neutralized with diluted aqueous ammonia, washed with ethyl acetate, concentrated, and subjected to gel column chromatography packed with Sephadex G-25 (mobile phase: 0.I
Desalting was performed by performing MTEAB).
完全に脱保護されたデカアデニル酸は、最後に逆相シリ
カゲル（ＹＭＣＰａｃｋ ＯＤＳ、Ｃ−１８（山村科学
社製））を用いた高速液体クロマトグラフィーにより精
製し、６．５７００ユニツト、約５０ｎＩＯ＋のデカア
デニル酸を５％の通算収率で単離した。The completely deprotected decaadenylic acid was finally purified by high performance liquid chromatography using reverse phase silica gel (YMCPack ODS, C-18 (manufactured by Yamamura Kagaku)) to obtain 6.5700 units of decaadenylic acid, approximately 50 nIO+. was isolated with an overall yield of 5%.
得られたデカアデニル酸は、陰イオン交換カラム（ＤＥ
ＡＥ−２８Ｗ）を用いた高速液体クロマトグラフィーで
分析を行い、はぼ単一なピークを与えることを確認した
。The obtained decaadenylic acid was subjected to an anion exchange column (DE
Analysis was performed by high performance liquid chromatography using AE-28W), and it was confirmed that a nearly single peak was obtained.
実施例７
（デカ（２’−０−メチル−５−メチルウリジル酸）す
なわちｍ５［Ｊｍ １０ｍａｒ （Ｄ合成＞実施例５で
得た２’−０−メチル−５−メヂルウリジン（１μ５ｏ
ｌ）を結合した固相担体を出発原料にして、実施例４・
で得た化合物５のアセトニトリル溶液（２０μｍｏｆ／
１４０ ｌ１１）を用いて、常法に従い実施例６と同様
に（この場合は説ジメチルトリチル化は、３％トリクロ
ル酢酸ジクロメタン溶液を用いた）鎖長延長反応を行っ
た。反応後の固相担体より切り出された５′末端にジメ
トキシトリチル基を有するデカ（２’−０−メチル−５
−メチルウリジル酸）を実施例６と同様に逆相シリカゲ
ルカラムを用いて分ｍｍ製した。Example 7 (Deca(2'-0-methyl-5-methyluridylic acid) i.e. m5[Jm 10mar (Synthesis D>2'-0-methyl-5-methyluridine obtained in Example 5 (1 μ5o
Using the solid phase support bound with l) as a starting material, Example 4.
An acetonitrile solution (20 μmof/
140 l11), chain elongation reaction was carried out in the same manner as in Example 6 according to a conventional method (in this case, dimethyl tritylation was carried out using a 3% trichloroacetic acid dichloromethane solution). Deca(2'-0-methyl-5
-Methyl uridylic acid) was prepared in the same manner as in Example 6 using a reversed phase silica gel column.
次に、この一部をとり溶媒を減圧留去した後、８０％酢
酸水溶液１ｄを加え、室温で１０分間放直置た６反応液
を減圧留去した後、更に水と減圧共沸することにより酢
酸を除去した。残渣を水に溶解し酢酸エチルで洗浄侵減
圧留去し、実施例６で用いた逆相シリカゲルによる高速
液体クロマトグラフィーによりＩＩＩＪＬ／、７．２８
００ユニツトのデカ（２’−０−メチル−５−メチルウ
リジル酸）を得た。Next, a portion of this was taken, the solvent was distilled off under reduced pressure, 1 d of 80% acetic acid aqueous solution was added, and the reaction solution was allowed to stand for 10 minutes at room temperature. After the reaction solution was distilled off under reduced pressure, it was further azeotroped with water under reduced pressure. Acetic acid was removed by The residue was dissolved in water, washed with ethyl acetate, evaporated under reduced pressure, and subjected to high performance liquid chromatography using the same reversed-phase silica gel used in Example 6 to give IIIJL/7.28.
00 units of deca(2'-0-methyl-5-methyluridylic acid) were obtained.
得られたオリゴマーは陰イオン交換カラム（ＤＥＡＥ−
２８Ｗ）を用いた高速液体クロマトグラフィーで分析を
行い単一なピークを与え、純粋であることを確認した。The obtained oligomer was subjected to an anion exchange column (DEAE-
Analysis by high performance liquid chromatography using 28W) gave a single peak, confirming its purity.
また、同様の方法により、デカデオキシチミジル１ｌ（
ｄＴ １０ｍｅｒ）ヲ２０．８４００−Ｌニット、７
カ（２’−〇−メチルウリジル酸） （Ｕｓ １０ｍ
ａｒ ）を１５．４７００ユニツト得た。In addition, 1 liter of decadeoxythymidyl (
dT 10mer) wo20.8400-L knit, 7
Ka(2'-〇-methyluridylic acid) (Us 10m
ar) obtained 15.4700 units.
実施例８
（Ｔｉ測定実験）
実施例６及び７により得たそれぞれのデカオリゴヌクレ
オチド量は、次のε値（計算値）により定値した。Example 8 (Ti measurement experiment) The amount of each decaoligonucleotide obtained in Examples 6 and 7 was determined by the following ε value (calculated value).
ｍ−」ニー順−
γＡ １０１１１Ｏｒ （相補鎖） ： １２３
４００ｄ丁１０ｍｅｒ （対 照） ： ８１６
００ｍ Ｕｎ １０ｍｅｒ （本発明） ：
９６８００ＵｍｌＯｍｅｒ （対 照”） ：
９６８００７Ａ １０ａ＋ｅｒ（０，２０ＴＯＤ、 １
．６ｎｍｏｌ）とｄＴｌｏｍｅｒ、１５Ｕ１１１０１ｍ
＋３ｒ 、ＬＪＩ １０１１ｅｒ （７）ソれぞれ１．
６ｔｖｏｌ相当幻をエッペンドルフヂュープにとり、減
圧乾固した。それぞれバッファー（０，０１Ｍカコジル
酸ナトリウム塩、０．ＩＭ ＮａＣ１゜１１Ｍ Ｅ
ＤＴＡ、 Ｅ）Ｈ７，０） ３００ｐ１に溶解し、
ベックマン社ＩＪＤＬＩ−８８分光光度計を用いて、温
度上昇速度を２℃／ｗｉｎとし、吸光度変化を３回測定
し、それらの平均値を求め、これら３種のハイブリッド
それぞれのＴ＋ｅ値とした。m-” knee order-γA 10111Or (complementary strand): 123
400d 10mer (control): 816
00m Un 10mer (present invention):
96800UmlOmer (control):
968007A 10a+er(0,20TOD, 1
．． 6nmol) and dTlomer, 15U11101m
+3r, LJI 1011er (7) each 1.
A volume equivalent to 6 tvol was taken in an Eppendorf dupe and dried under reduced pressure. Each buffer (0.01M cacodylate sodium salt, 0.IM NaCl, 11M E
DTA, E)H7,0) dissolved in 300p1,
Using a Beckman IJDLI-88 spectrophotometer, the change in absorbance was measured three times at a temperature increase rate of 2° C./win, and the average value thereof was determined to be the T+e value for each of these three hybrids.
結果は次の第１表の通りであった（第３図）。The results are shown in Table 1 below (Figure 3).
第１表
化合物５の合成の概要を図示したものである。第３図は
、３種のハイブリッドの温度変化に対する吸光度変化を
示す（実施例８）。Table 1 shows an overview of the synthesis of Compound 5. FIG. 3 shows absorbance changes with respect to temperature changes for three types of hybrids (Example 8).
因みに、各ハイブリッドの構造式は次の通りである。Incidentally, the structural formula of each hybrid is as follows.
式中、Ｎは５− Ｍ ｅｔＭＩｕｒａｃｉｌを意味する
。In the formula, N means 5-MetMIuracil.
へイア１ノッ１：（１） ： ５’ ｒ （ＡＡＡＡ
ＡＡＡＡＡＡ）、 ３’３’ （ＴＴＴＴＴＴＴＴ
ＴＴ）ｄ ５’ハイブリッド■、 ５’ ｒ（ＡＡＡ
ＡＡＡＡＡＡＡ） ３’３’ （ＵｔｌＵＵＵＵＵ
ＵＬｔＵ）ｉ ５’ハイフリット■：５’ ｒ（ＡＡ
ＡＡＡＡＡＡＡＡ） ３’３’ （ＮＨＮＮＮＨＮ
ＮＮＮ）ｍ ５’Heia 1 no 1: (1): 5' r (AAAA
AAAAAA), 3'3' (TTTTTTTT
TT) d 5' hybrid ■, 5' r (AAA
AAAAAAAAA) 3'3' (UtlUUUUU
ULtU) i 5' high frit ■: 5' r (AA
AAAAAAAAA) 3'3' (NHNNNHN
NNN)m 5'
第１図及び第２図は、それぞれ、実施例１〜３１（埋入
弁理士 霜 越 正 夫Figures 1 and 2 show Examples 1 to 31 (embedded patent attorney Masao Shimokoshi), respectively.
Claims (1)
ド誘導体。 ▲数式、化学式、表等があります▼（ I ） 式中、Ｒは、水素原子、アシル基、又は置換基を有して
いてもよいホスホリル基を表し、Ｔは、チミン・１−イ
ル基を表し、Ｙ＿１、Ｙ＿２及びＹ＿３は、相互に同一
であってもよく又異なつていてもよくて、水素原子、ヒ
ドロキシル基、低級アルキルオキシ基又は低級アルキル
シリル基を表し、Ｂは、チミン−１−イル基、アデニン
−９−イル基、グアニン−９−イル基、シトシン−９−
イル基、ウラシル−１−イル基又はヒボキサンチン−９
−イル基を表し、ｎは１〜１００の整数を表す。 の下記一般式（II）で表されるヌクレオチド誘導体。 ▲数式、化学式、表等があります▼（II） 式中、Ｘは、モノメトキシトリチル基、ジメトキシトリ
チル基又は置換基を有していてもよいホスホリル基を表
し、Ｙは、水素原子、ｏ−クロルフエニル燐酸、−Ｐ（
ＯＣＨ＿３）−Ｎ−（ＣＨ（ＣＨ＿３）＿２）＿２、−
Ｐ（ＯＣＨ＿２ＣＨ＿２ＣＮ）−Ｎ−（ＣＨ（ＣＨ＿３
）＿２）＿２又は−ＣＯ−（ＣＨ＿２）＿ｍ−ＣＯＮＨ
−（ＣＨ＿２）＿ｎ−（ＣＰＧ誘導体）（ここに、ｍ及
びｎは、それぞれ、１〜１０の整数である）を表わす。 ただし、Ｘが置換基を有していてもよいホスホリル基の
ときは、Ｙは水素原子である。(1) An oligonucleotide derivative represented by the following general formula (I). ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (I) In the formula, R represents a hydrogen atom, an acyl group, or a phosphoryl group that may have a substituent, and T represents a thymine 1-yl group. Y_1, Y_2 and Y_3 may be the same or different and represent a hydrogen atom, a hydroxyl group, a lower alkyloxy group or a lower alkylsilyl group, and B is thymine-1 -yl group, adenine-9-yl group, guanine-9-yl group, cytosine-9-yl group
yl group, uracil-1-yl group or hypoxanthine-9
-yl group, and n represents an integer of 1 to 100. A nucleotide derivative represented by the following general formula (II). ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (II) In the formula, X represents a monomethoxytrityl group, a dimethoxytrityl group, or a phosphoryl group that may have a substituent, and Y represents a hydrogen atom, an o- Chlorphenyl phosphate, -P(
OCH_3)-N-(CH(CH_3)_2)_2,-
P(OCH_2CH_2CN)-N-(CH(CH_3
)_2)_2 or -CO-(CH_2)_m-CONH
-(CH_2)_n- (CPG derivative) (where m and n are each an integer of 1 to 10). However, when X is a phosphoryl group which may have a substituent, Y is a hydrogen atom.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8545689A JPH02264792A (en) | 1989-04-04 | 1989-04-04 | Oligonucleotide derivative and synthetic raw material thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8545689A JPH02264792A (en) | 1989-04-04 | 1989-04-04 | Oligonucleotide derivative and synthetic raw material thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH02264792A true JPH02264792A (en) | 1990-10-29 |
Family
ID=13859386
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP8545689A Pending JPH02264792A (en) | 1989-04-04 | 1989-04-04 | Oligonucleotide derivative and synthetic raw material thereof |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH02264792A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5525719A (en) * | 1991-08-30 | 1996-06-11 | Chemgenes Corporation | N-protected-2'-O-methyl-and N-protected-3'-O-methyl-ribonucleosides and their phosphoramidite derivatives |
| US5760202A (en) * | 1995-03-06 | 1998-06-02 | Isis Pharmaceuticals, Inc. | Process for the synthesis of 2'-O-substituted pyrimidines |
| US5861493A (en) * | 1995-03-06 | 1999-01-19 | Isis Pharmaceuticals, Inc. | Process for the synthesis of 2'-O-substituted pyrimidines |
| US6015886A (en) * | 1993-05-24 | 2000-01-18 | Chemgenes Corporation | Oligonucleotide phosphate esters |
| US6107094A (en) * | 1996-06-06 | 2000-08-22 | Isis Pharmaceuticals, Inc. | Oligoribonucleotides and ribonucleases for cleaving RNA |
| US7820810B2 (en) | 2007-03-19 | 2010-10-26 | Isis Pharmaceuticals, Inc. | Process for the synthesis of 2′-O-substituted purine nulceosides |
-
1989
- 1989-04-04 JP JP8545689A patent/JPH02264792A/en active Pending
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5525719A (en) * | 1991-08-30 | 1996-06-11 | Chemgenes Corporation | N-protected-2'-O-methyl-and N-protected-3'-O-methyl-ribonucleosides and their phosphoramidite derivatives |
| US6015886A (en) * | 1993-05-24 | 2000-01-18 | Chemgenes Corporation | Oligonucleotide phosphate esters |
| US6222025B1 (en) | 1995-03-06 | 2001-04-24 | Isis Pharmaceuticals, Inc. | Process for the synthesis of 2′-O-substituted pyrimidines and oligomeric compounds therefrom |
| US5861493A (en) * | 1995-03-06 | 1999-01-19 | Isis Pharmaceuticals, Inc. | Process for the synthesis of 2'-O-substituted pyrimidines |
| US6166197A (en) * | 1995-03-06 | 2000-12-26 | Isis Pharmaceuticals, Inc. | Oligomeric compounds having pyrimidine nucleotide (S) with 2'and 5 substitutions |
| US5760202A (en) * | 1995-03-06 | 1998-06-02 | Isis Pharmaceuticals, Inc. | Process for the synthesis of 2'-O-substituted pyrimidines |
| EP0813539A4 (en) * | 1995-03-06 | 2002-01-23 | Isis Pharmaceuticals Inc | Improved process for the synthesis of 2'-o-substituted pyrimidines and oligomeric compounds therefrom |
| US6642367B2 (en) | 1995-03-06 | 2003-11-04 | Isis Pharmaceuticals, Inc. | Process for the synthesis of 2′-O-substituted pyrimidines and oligomeric compounds therefrom |
| US6107094A (en) * | 1996-06-06 | 2000-08-22 | Isis Pharmaceuticals, Inc. | Oligoribonucleotides and ribonucleases for cleaving RNA |
| US7432249B2 (en) | 1996-06-06 | 2008-10-07 | Isis Pharmaceuticals, Inc. | Oligoribonucleotides and ribonucleases for cleaving RNA |
| US7432250B2 (en) | 1996-06-06 | 2008-10-07 | Isis Pharmaceuticals, Inc. | Oligoribonucleotides and ribonucleases for cleaving RNA |
| US7629321B2 (en) | 1996-06-06 | 2009-12-08 | Isis Pharmaceuticals, Inc. | Oligoribonucleotides and ribonucleases for cleaving RNA |
| US7820810B2 (en) | 2007-03-19 | 2010-10-26 | Isis Pharmaceuticals, Inc. | Process for the synthesis of 2′-O-substituted purine nulceosides |
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