WO2021193954A1 - 核酸オリゴマーの製造方法 - Google Patents
核酸オリゴマーの製造方法 Download PDFInfo
- Publication number
- WO2021193954A1 WO2021193954A1 PCT/JP2021/013023 JP2021013023W WO2021193954A1 WO 2021193954 A1 WO2021193954 A1 WO 2021193954A1 JP 2021013023 W JP2021013023 W JP 2021013023W WO 2021193954 A1 WO2021193954 A1 WO 2021193954A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- group
- nucleic acid
- formaldehyde
- dichloroacetic acid
- formula
- Prior art date
Links
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 148
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 148
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 135
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 57
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims abstract description 217
- JXTHNDFMNIQAHM-UHFFFAOYSA-N dichloroacetic acid Chemical compound OC(=O)C(Cl)Cl JXTHNDFMNIQAHM-UHFFFAOYSA-N 0.000 claims abstract description 210
- 229960005215 dichloroacetic acid Drugs 0.000 claims abstract description 105
- 238000000034 method Methods 0.000 claims abstract description 71
- -1 hydroxyalkyl ammonium ion Chemical compound 0.000 claims description 78
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 62
- 125000006239 protecting group Chemical group 0.000 claims description 52
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 42
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 29
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 27
- 239000007790 solid phase Substances 0.000 claims description 23
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 claims description 21
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 claims description 21
- 239000002904 solvent Substances 0.000 claims description 19
- 150000001875 compounds Chemical class 0.000 claims description 18
- 125000005647 linker group Chemical group 0.000 claims description 18
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Natural products CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 16
- 239000002585 base Substances 0.000 claims description 16
- 229920002477 rna polymer Polymers 0.000 claims description 16
- 239000002773 nucleotide Substances 0.000 claims description 15
- 125000000548 ribosyl group Chemical group C1([C@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims description 14
- 239000002635 aromatic organic solvent Substances 0.000 claims description 13
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 12
- 125000004432 carbon atom Chemical group C* 0.000 claims description 11
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 10
- 125000003545 alkoxy group Chemical group 0.000 claims description 9
- 238000009835 boiling Methods 0.000 claims description 9
- 229910052799 carbon Inorganic materials 0.000 claims description 9
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 8
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 claims description 7
- 125000001153 fluoro group Chemical group F* 0.000 claims description 7
- 125000003729 nucleotide group Chemical group 0.000 claims description 7
- 125000004200 2-methoxyethyl group Chemical group [H]C([H])([H])OC([H])([H])C([H])([H])* 0.000 claims description 6
- 229910001413 alkali metal ion Inorganic materials 0.000 claims description 6
- 125000004093 cyano group Chemical group *C#N 0.000 claims description 6
- 229910052731 fluorine Inorganic materials 0.000 claims description 6
- 125000004430 oxygen atom Chemical group O* 0.000 claims description 6
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims description 5
- 125000005210 alkyl ammonium group Chemical group 0.000 claims description 4
- 125000006575 electron-withdrawing group Chemical group 0.000 claims description 3
- 125000000816 ethylene group Chemical group [H]C([H])([*:1])C([H])([H])[*:2] 0.000 claims description 3
- 125000000219 ethylidene group Chemical group [H]C(=[*])C([H])([H])[H] 0.000 claims description 3
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 claims description 3
- 229910052717 sulfur Inorganic materials 0.000 claims description 3
- 125000004434 sulfur atom Chemical group 0.000 claims description 2
- 125000003944 tolyl group Chemical group 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 2
- 108091034117 Oligonucleotide Proteins 0.000 description 37
- 238000006243 chemical reaction Methods 0.000 description 28
- 238000000691 measurement method Methods 0.000 description 18
- 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 17
- FPGGTKZVZWFYPV-UHFFFAOYSA-M tetrabutylammonium fluoride Chemical compound [F-].CCCC[N+](CCCC)(CCCC)CCCC FPGGTKZVZWFYPV-UHFFFAOYSA-M 0.000 description 15
- 125000000217 alkyl group Chemical group 0.000 description 14
- 239000000047 product Substances 0.000 description 13
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 12
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 12
- 239000002777 nucleoside Substances 0.000 description 11
- 150000003839 salts Chemical class 0.000 description 11
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- 238000005859 coupling reaction Methods 0.000 description 9
- 239000011630 iodine Substances 0.000 description 9
- 229910052740 iodine Inorganic materials 0.000 description 9
- MCTWTZJPVLRJOU-UHFFFAOYSA-N 1-methyl-1H-imidazole Chemical compound CN1C=CN=C1 MCTWTZJPVLRJOU-UHFFFAOYSA-N 0.000 description 8
- 125000002103 4,4'-dimethoxytriphenylmethyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)(C1=C([H])C([H])=C(OC([H])([H])[H])C([H])=C1[H])C1=C([H])C([H])=C(OC([H])([H])[H])C([H])=C1[H] 0.000 description 8
- 239000002253 acid Substances 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 8
- 238000010511 deprotection reaction Methods 0.000 description 8
- 238000003786 synthesis reaction Methods 0.000 description 8
- RHFKLGNCRGPQAE-UHFFFAOYSA-N 2,2-dichloroacetic acid;toluene Chemical compound OC(=O)C(Cl)Cl.CC1=CC=CC=C1 RHFKLGNCRGPQAE-UHFFFAOYSA-N 0.000 description 7
- 239000003795 chemical substances by application Substances 0.000 description 7
- 239000007800 oxidant agent Substances 0.000 description 7
- 125000001424 substituent group Chemical group 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 0 CC*C1OC2(CO*)[C@](*CCI*3IC3)C1OC2 Chemical compound CC*C1OC2(CO*)[C@](*CCI*3IC3)C1OC2 0.000 description 6
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 6
- XMGZWGBXVLJOKE-UHFFFAOYSA-N acetic acid;toluene Chemical compound CC(O)=O.CC1=CC=CC=C1 XMGZWGBXVLJOKE-UHFFFAOYSA-N 0.000 description 6
- 150000001413 amino acids Chemical group 0.000 description 6
- 125000003963 dichloro group Chemical group Cl* 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 150000008300 phosphoramidites Chemical class 0.000 description 6
- OJMIONKXNSYLSR-UHFFFAOYSA-N phosphorous acid Chemical group OP(O)O OJMIONKXNSYLSR-UHFFFAOYSA-N 0.000 description 6
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 6
- 210000002700 urine Anatomy 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- XSWODFNOUCZADO-UHFFFAOYSA-N 5-benzyl-1-sulfanyltetrazole Chemical compound SN1N=NN=C1CC1=CC=CC=C1 XSWODFNOUCZADO-UHFFFAOYSA-N 0.000 description 5
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 5
- 230000008878 coupling Effects 0.000 description 5
- 238000010168 coupling process Methods 0.000 description 5
- 239000012046 mixed solvent Substances 0.000 description 5
- 230000003647 oxidation Effects 0.000 description 5
- 238000007254 oxidation reaction Methods 0.000 description 5
- 230000001590 oxidative effect Effects 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 238000010532 solid phase synthesis reaction Methods 0.000 description 5
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 4
- QUSNBJAOOMFDIB-UHFFFAOYSA-N Ethylamine Chemical compound CCN QUSNBJAOOMFDIB-UHFFFAOYSA-N 0.000 description 4
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 4
- BAVYZALUXZFZLV-UHFFFAOYSA-N Methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 description 4
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 4
- YRKCREAYFQTBPV-UHFFFAOYSA-N acetylacetone Chemical compound CC(=O)CC(C)=O YRKCREAYFQTBPV-UHFFFAOYSA-N 0.000 description 4
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound 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 4
- 235000011114 ammonium hydroxide Nutrition 0.000 description 4
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 description 4
- 238000009833 condensation Methods 0.000 description 4
- 230000005494 condensation Effects 0.000 description 4
- 239000007789 gas Substances 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 150000003833 nucleoside derivatives Chemical class 0.000 description 4
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 4
- KJUGUADJHNHALS-UHFFFAOYSA-N 1H-tetrazole Chemical compound C=1N=NNN=1 KJUGUADJHNHALS-UHFFFAOYSA-N 0.000 description 3
- SXUXMRMBWZCMEN-UHFFFAOYSA-N 2'-O-methyl uridine Natural products COC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 SXUXMRMBWZCMEN-UHFFFAOYSA-N 0.000 description 3
- OFOBLEOULBTSOW-UHFFFAOYSA-N Malonic acid Chemical compound OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- WMZXICYWVDCASN-UHFFFAOYSA-N acetonitrile;2,6-dimethylpyridine Chemical compound CC#N.CC1=CC=CC(C)=N1 WMZXICYWVDCASN-UHFFFAOYSA-N 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 125000002768 hydroxyalkyl group Chemical group 0.000 description 3
- 239000002808 molecular sieve Substances 0.000 description 3
- LYGJENNIWJXYER-UHFFFAOYSA-N nitromethane Chemical compound C[N+]([O-])=O LYGJENNIWJXYER-UHFFFAOYSA-N 0.000 description 3
- 125000003835 nucleoside group Chemical group 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 230000002194 synthesizing effect Effects 0.000 description 3
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 3
- 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 3
- CCSBNBKMACZDGN-UHFFFAOYSA-N (2-phenoxyacetyl) 2-phenoxyacetate Chemical compound C=1C=CC=CC=1OCC(=O)OC(=O)COC1=CC=CC=C1 CCSBNBKMACZDGN-UHFFFAOYSA-N 0.000 description 2
- NAWXUBYGYWOOIX-SFHVURJKSA-N (2s)-2-[[4-[2-(2,4-diaminoquinazolin-6-yl)ethyl]benzoyl]amino]-4-methylidenepentanedioic acid Chemical compound C1=CC2=NC(N)=NC(N)=C2C=C1CCC1=CC=C(C(=O)N[C@@H](CC(=C)C(O)=O)C(O)=O)C=C1 NAWXUBYGYWOOIX-SFHVURJKSA-N 0.000 description 2
- JUDOLRSMWHVKGX-UHFFFAOYSA-N 1,1-dioxo-1$l^{6},2-benzodithiol-3-one Chemical compound C1=CC=C2C(=O)SS(=O)(=O)C2=C1 JUDOLRSMWHVKGX-UHFFFAOYSA-N 0.000 description 2
- SLZAMKNXRDJWLA-UHFFFAOYSA-N 1-(5-acetyl-2,6-dimethyl-1,4-dihydropyridin-3-yl)ethanone Chemical compound CC(=O)C1=C(C)NC(C)=C(C(C)=O)C1 SLZAMKNXRDJWLA-UHFFFAOYSA-N 0.000 description 2
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 description 2
- XBCXJKGHPABGSD-UHFFFAOYSA-N 1-methyluracil Chemical compound CN1C=CC(=O)NC1=O XBCXJKGHPABGSD-UHFFFAOYSA-N 0.000 description 2
- SXUXMRMBWZCMEN-ZOQUXTDFSA-N 2'-O-methyluridine Chemical compound CO[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 SXUXMRMBWZCMEN-ZOQUXTDFSA-N 0.000 description 2
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- 125000001731 2-cyanoethyl group Chemical group [H]C([H])(*)C([H])([H])C#N 0.000 description 2
- 125000001999 4-Methoxybenzoyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1OC([H])([H])[H])C(*)=O 0.000 description 2
- OLXZPDWKRNYJJZ-UHFFFAOYSA-N 5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-ol Chemical compound C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(CO)O1 OLXZPDWKRNYJJZ-UHFFFAOYSA-N 0.000 description 2
- YWZHEXZIISFIDA-UHFFFAOYSA-N 5-amino-1,2,4-dithiazole-3-thione Chemical compound NC1=NC(=S)SS1 YWZHEXZIISFIDA-UHFFFAOYSA-N 0.000 description 2
- GXGKKIPUFAHZIZ-UHFFFAOYSA-N 5-benzylsulfanyl-2h-tetrazole Chemical compound C=1C=CC=CC=1CSC=1N=NNN=1 GXGKKIPUFAHZIZ-UHFFFAOYSA-N 0.000 description 2
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 2
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 description 2
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 description 2
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 description 2
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 235000011054 acetic acid Nutrition 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 229960005305 adenosine Drugs 0.000 description 2
- 125000004183 alkoxy alkyl group Chemical group 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 125000003710 aryl alkyl group Chemical group 0.000 description 2
- 125000003118 aryl group Chemical group 0.000 description 2
- 239000012298 atmosphere Substances 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical group [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 2
- 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
- FPUGCISOLXNPPC-IOSLPCCCSA-N cordysinin B Chemical compound CO[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(N)=C2N=C1 FPUGCISOLXNPPC-IOSLPCCCSA-N 0.000 description 2
- 125000000753 cycloalkyl group Chemical group 0.000 description 2
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- HPNMFZURTQLUMO-UHFFFAOYSA-N diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- 230000009368 gene silencing by RNA Effects 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 229940029575 guanosine Drugs 0.000 description 2
- 125000005843 halogen group Chemical group 0.000 description 2
- 125000004415 heterocyclylalkyl group Chemical group 0.000 description 2
- 125000004051 hexyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 2
- 239000011261 inert gas Substances 0.000 description 2
- 125000000959 isobutyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])* 0.000 description 2
- 125000001972 isopentyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])C([H])([H])* 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 229940098779 methanesulfonic acid Drugs 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 125000004108 n-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 2
- 125000000740 n-pentyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 2
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- IXGZXXBJSZISOO-UHFFFAOYSA-N s-(2-phenylacetyl)sulfanyl 2-phenylethanethioate Chemical compound C=1C=CC=CC=1CC(=O)SSC(=O)CC1=CC=CC=C1 IXGZXXBJSZISOO-UHFFFAOYSA-N 0.000 description 2
- 125000006850 spacer group Chemical group 0.000 description 2
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 229940104230 thymidine Drugs 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 2
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 2
- ITMCEJHCFYSIIV-UHFFFAOYSA-N triflic acid Chemical compound OS(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-N 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
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 1
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 1
- DZCRSVQAXDNLEY-UHFFFAOYSA-N 1-ethylsulfanyltetrazole Chemical compound CCSN1C=NN=N1 DZCRSVQAXDNLEY-UHFFFAOYSA-N 0.000 description 1
- XGDRLCRGKUCBQL-UHFFFAOYSA-N 1h-imidazole-4,5-dicarbonitrile Chemical compound N#CC=1N=CNC=1C#N XGDRLCRGKUCBQL-UHFFFAOYSA-N 0.000 description 1
- FPUGCISOLXNPPC-UHFFFAOYSA-N 2'-O-Methyladenosine Natural products COC1C(O)C(CO)OC1N1C2=NC=NC(N)=C2N=C1 FPUGCISOLXNPPC-UHFFFAOYSA-N 0.000 description 1
- RFCQJGFZUQFYRF-UHFFFAOYSA-N 2'-O-Methylcytidine Natural products COC1C(O)C(CO)OC1N1C(=O)N=C(N)C=C1 RFCQJGFZUQFYRF-UHFFFAOYSA-N 0.000 description 1
- OVYNGSFVYRPRCG-UHFFFAOYSA-N 2'-O-Methylguanosine Natural products COC1C(O)C(CO)OC1N1C(NC(N)=NC2=O)=C2N=C1 OVYNGSFVYRPRCG-UHFFFAOYSA-N 0.000 description 1
- RFCQJGFZUQFYRF-ZOQUXTDFSA-N 2'-O-methylcytidine Chemical compound CO[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)N=C(N)C=C1 RFCQJGFZUQFYRF-ZOQUXTDFSA-N 0.000 description 1
- OVYNGSFVYRPRCG-KQYNXXCUSA-N 2'-O-methylguanosine Chemical compound CO[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(N=C(N)NC2=O)=C2N=C1 OVYNGSFVYRPRCG-KQYNXXCUSA-N 0.000 description 1
- YKBGVTZYEHREMT-KVQBGUIXSA-N 2'-deoxyguanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](CO)O1 YKBGVTZYEHREMT-KVQBGUIXSA-N 0.000 description 1
- YKBGVTZYEHREMT-UHFFFAOYSA-N 2'-deoxyguanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1CC(O)C(CO)O1 YKBGVTZYEHREMT-UHFFFAOYSA-N 0.000 description 1
- CKTSBUTUHBMZGZ-SHYZEUOFSA-N 2'‐deoxycytidine Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 CKTSBUTUHBMZGZ-SHYZEUOFSA-N 0.000 description 1
- ASJSAQIRZKANQN-CRCLSJGQSA-N 2-deoxy-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 description 1
- BMYNFMYTOJXKLE-UHFFFAOYSA-N 3-azaniumyl-2-hydroxypropanoate Chemical compound NCC(O)C(O)=O BMYNFMYTOJXKLE-UHFFFAOYSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- GONFBOIJNUKKST-UHFFFAOYSA-N 5-ethylsulfanyl-2h-tetrazole Chemical compound CCSC=1N=NNN=1 GONFBOIJNUKKST-UHFFFAOYSA-N 0.000 description 1
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 description 1
- LKDWQHQNDDNHSC-UHFFFAOYSA-N 5-phenyl-1,2,4-dithiazol-3-one Chemical compound S1SC(=O)N=C1C1=CC=CC=C1 LKDWQHQNDDNHSC-UHFFFAOYSA-N 0.000 description 1
- IELBPRXADXDMJU-UHFFFAOYSA-N 6-nitro-1h-benzimidazol-3-ium;trifluoromethanesulfonate Chemical compound [O-]S(=O)(=O)C(F)(F)F.[O-][N+](=O)C1=CC=C2[NH2+]C=NC2=C1 IELBPRXADXDMJU-UHFFFAOYSA-N 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 108091023037 Aptamer Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- GAWIXWVDTYZWAW-UHFFFAOYSA-N C[CH]O Chemical group C[CH]O GAWIXWVDTYZWAW-UHFFFAOYSA-N 0.000 description 1
- 102000053642 Catalytic RNA Human genes 0.000 description 1
- 108090000994 Catalytic RNA Proteins 0.000 description 1
- 229920001393 Crofelemer Polymers 0.000 description 1
- CKTSBUTUHBMZGZ-UHFFFAOYSA-N Deoxycytidine Natural products O=C1N=C(N)C=CN1C1OC(CO)C(O)C1 CKTSBUTUHBMZGZ-UHFFFAOYSA-N 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- HBBGRARXTFLTSG-UHFFFAOYSA-N Lithium ion Chemical compound [Li+] HBBGRARXTFLTSG-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 1
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- FKNQFGJONOIPTF-UHFFFAOYSA-N Sodium cation Chemical compound [Na+] FKNQFGJONOIPTF-UHFFFAOYSA-N 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical group OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- FLQHMGIPWICELL-UHFFFAOYSA-N acetyl acetate;oxolane Chemical compound C1CCOC1.CC(=O)OC(C)=O FLQHMGIPWICELL-UHFFFAOYSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 125000005042 acyloxymethyl group Chemical group 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 125000003342 alkenyl group Chemical group 0.000 description 1
- 125000004390 alkyl sulfonyl group Chemical group 0.000 description 1
- 125000000304 alkynyl group Chemical group 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- AZDRQVAHHNSJOQ-UHFFFAOYSA-N alumane Chemical class [AlH3] AZDRQVAHHNSJOQ-UHFFFAOYSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 125000004103 aminoalkyl group Chemical group 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- 125000004391 aryl sulfonyl group Chemical group 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 239000005667 attractant Substances 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 125000001246 bromo group Chemical group Br* 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 125000004063 butyryl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 125000001309 chloro group Chemical group Cl* 0.000 description 1
- 229960004106 citric acid Drugs 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 125000004966 cyanoalkyl group Chemical group 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 125000001316 cycloalkyl alkyl group Chemical group 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- VGONTNSXDCQUGY-UHFFFAOYSA-N desoxyinosine Natural products C1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 VGONTNSXDCQUGY-UHFFFAOYSA-N 0.000 description 1
- 125000004663 dialkyl amino group Chemical group 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- CCIVGXIOQKPBKL-UHFFFAOYSA-M ethanesulfonate Chemical compound CCS([O-])(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-M 0.000 description 1
- 125000001301 ethoxy group Chemical group [H]C([H])([H])C([H])([H])O* 0.000 description 1
- 239000008098 formaldehyde solution Substances 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 239000001530 fumaric acid Substances 0.000 description 1
- 229960002598 fumaric acid Drugs 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 229940093915 gynecological organic acid Drugs 0.000 description 1
- 125000001188 haloalkyl group Chemical group 0.000 description 1
- 229910000856 hastalloy Inorganic materials 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 125000001072 heteroaryl group Chemical group 0.000 description 1
- 125000004446 heteroarylalkyl group Chemical group 0.000 description 1
- 150000004677 hydrates Chemical class 0.000 description 1
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 description 1
- 229940071870 hydroiodic acid Drugs 0.000 description 1
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 1
- 125000004029 hydroxymethyl group Chemical group [H]OC([H])([H])* 0.000 description 1
- 150000007529 inorganic bases Chemical class 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 125000002346 iodo group Chemical group I* 0.000 description 1
- JJWLVOIRVHMVIS-UHFFFAOYSA-N isopropylamine Chemical compound CC(C)N JJWLVOIRVHMVIS-UHFFFAOYSA-N 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 229960000448 lactic acid Drugs 0.000 description 1
- 229910001416 lithium ion Inorganic materials 0.000 description 1
- 159000000003 magnesium salts Chemical class 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- 239000011976 maleic acid Substances 0.000 description 1
- 229940098895 maleic acid Drugs 0.000 description 1
- 239000001630 malic acid Substances 0.000 description 1
- 235000011090 malic acid Nutrition 0.000 description 1
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 125000004123 n-propyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 229960003104 ornithine Drugs 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 125000006245 phosphate protecting group Chemical group 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- 125000001501 propionyl group Chemical group O=C([*])C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 125000003808 silyl group Chemical group [H][Si]([H])([H])[*] 0.000 description 1
- 229910001415 sodium ion Inorganic materials 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000012453 solvate Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 229940095064 tartrate Drugs 0.000 description 1
- DZLFLBLQUQXARW-UHFFFAOYSA-N tetrabutylammonium Chemical compound CCCC[N+](CCCC)(CCCC)CCCC DZLFLBLQUQXARW-UHFFFAOYSA-N 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 125000004954 trialkylamino group Chemical group 0.000 description 1
- 125000004665 trialkylsilyl group Chemical group 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 150000005691 triesters Chemical class 0.000 description 1
- ZMANZCXQSJIPKH-UHFFFAOYSA-O triethylammonium ion Chemical compound CC[NH+](CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-O 0.000 description 1
- 125000004953 trihalomethyl group Chemical group 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
- C07C51/43—Separation; Purification; Stabilisation; Use of additives by change of the physical state, e.g. crystallisation
- C07C51/44—Separation; Purification; Stabilisation; Use of additives by change of the physical state, e.g. crystallisation by distillation
- C07C51/46—Separation; Purification; Stabilisation; Use of additives by change of the physical state, e.g. crystallisation by distillation by azeotropic distillation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Definitions
- the present invention relates to a method for producing a nucleic acid oligomer.
- nucleic acid oligomers In recent years, there has been increasing interest in the application of nucleic acid oligomers to the medical field. For example, antisense nucleic acids, aptamers, ribozymes, and nucleic acids that induce RNA interference (RNAi) such as siRNA are mentioned, and these are called nucleic acid drugs.
- RNAi RNA interference
- Nucleic acid oligomers can be synthesized by the solid-phase synthesis method, and the nucleic acid oligomer synthesized by extending the nucleic acid on the solid-phase carrier is cut out from the solid-phase carrier, and then the nucleic acid oligomer containing ribose is 2'of ribose.
- the desired nucleic acid oligomer is produced by deprotecting and removing the protecting group of the hydroxyl group at the position.
- phosphoramidite of nucleoside (hereinafter referred to as "amidite”) is used as a raw material, and it is known that the protecting group of the hydroxyl group at the 5'position is deprotected by using a dichloroacetic acid solution.
- a dichloroacetic acid solution the yield of the nucleic acid oligomer synthesized using the conventional dichloroacetic acid solution was not always satisfactory, and the synthesis was not efficient (Patent Document 1).
- An object of the present invention is to provide an efficient method for producing a nucleic acid oligomer.
- the present inventors used a dichloroacetic acid solution having a formaldehyde concentration below a certain level or improved the quality of dichloroacetic acid when synthesizing a nucleic acid oligomer.
- a dichloroacetic acid solution having a formaldehyde concentration below a certain level or improved the quality of dichloroacetic acid when synthesizing a nucleic acid oligomer.
- an efficient method for producing a nucleic acid oligomer which is characterized by being used later.
- Equation (1) (During the ceremony, G 2 represents a hydroxyl-protecting group B a is independently, the same or different and each represents a nucleic acid base which may be protected by a protecting group, R 1 , R 2 and R 3 independently represent the same or different hydrogen atoms or alkoxy groups. R independently represents the same or differently protected hydroxyl group, hydrogen atom, fluorine atom, methoxy group, 2-methoxyethyl group, or OQ'group.
- Q' is independently the same or different from the methylene group bonded to the carbon atom at the 4'position of ribose, the ethylene group bonded to the carbon atom at the 4'position of ribose, or the ribose.
- Y represents an oxygen atom or a sulfur atom independently, the same or differently, respectively.
- n represents an integer of 1 to 200 and represents W 1 represents an OZ group and X 1 represents an R group, or W 1 represents an OV group and X 1 represents an OZ group.
- V represents a hydroxyl-protecting group Z is a group having a structure consisting of a solid phase carrier and a linking group.
- the formula (2) includes a step of reacting the nucleic acid oligomer represented by (1) with a dichloroacetic acid solution in which the molar ratio of formaldehyde to dichloroacetic acid (formaldehyde mol / dichloroacetic acid mol) is 90 ⁇ 10-6 or less.
- B c represents a nucleobase independently, identically or differently.
- G 4 are each, independently, the same or different and each represents a hydrogen atom, an alkali metal ion, an ammonium ion, an alkyl ammonium ion, or a hydroxyalkyl ammonium ion, R'independently represents a hydroxyl group, a hydrogen atom, a fluorine atom, a methoxy group, a 2-methoxyethyl group, or an OQ'group, respectively.
- Q' is as described above, X 3 and W 3 each independently represent a hydroxyl group, or X 3 represents an R'group and W 3 represents a hydroxyl group.
- the nucleic acid oligomer represented by the formula (2) is arbitrarily extended in chain length by the amidite method (3): (During the ceremony, G 2 , Ba , R, Y, X 1 and W 1 are as described above. G 5 is Indicates a hydroxyl-protecting group or a hydrogen atom indicated by R 1, R 2 and R 3 are as defined above, m is an integer satisfying m ⁇ n. ) The step of obtaining the nucleic acid compound represented by the formula (3), and the formula (4): from the compound represented by the formula (3). (During the ceremony, G 5 , R, Y and m are as described above.
- G 4 are each, independently, the same or different and each represents a hydrogen atom, an alkali metal ion, an ammonium ion, an alkyl ammonium ion, or a hydroxyalkyl ammonium ion
- B C are each independently the same or different, it represents a nucleobase
- X 2 represents a hydroxyl group and W 2 represents an OV group
- X 2 represents an R group and W 2 represents a hydroxyl group.
- V represents a hydroxyl-protecting group.
- R'independently represents a hydroxyl group, a hydrogen atom, a fluorine atom, a methoxy group, a 2-methoxyethyl group, or an OQ'group, respectively.
- the linker consisting of an amino acid skeleton is a linker having a structure selected from the group consisting of the following formulas (A14-1), (A14-2) and (A14-3).
- Y is as described above.
- the dichloroacetic acid solution contains at least one solvent selected from the group consisting of dichloromethane, acetonitrile, and aromatic organic solvents. 7.
- the molar ratio of formaldehyde to dichloroacetic acid (formaldehyde mol / dichloroacetic acid mol) in the dichloroacetic acid solution is 22 ⁇ 10-6 or less.
- the nucleic acid oligomer is ribonucleic acid (RNA).
- RNA ribonucleic acid
- the protecting group for the hydroxyl group at the 2'position of ribose is the protecting group represented by the formula (6).
- R a and R b are hydrogen atoms at the same time, and, E W is a cyano group, a manufacturing method according to item 10.
- nucleic acid oligomer is an oligomer having a length of 80 chains or more.
- nucleic acid oligomer is an oligomer having a chain length of 100 or more.
- a dichloroacetic acid solution having a molar ratio of formaldehyde to dichloroacetic acid (formaldehyde mol / dichloroacetic acid mol) of 90 ⁇ 10-6 or less. 18.
- a method for producing a nucleic acid oligomer which comprises the step of purifying the dichloroacetic acid solution according to the preceding item 20 and the step according to any one of the preceding items 1 to 3 using the purified dichloroacetic acid solution obtained in the step.
- the present invention provides an efficient method for producing a nucleic acid oligomer.
- the production method of the present invention can be expected to improve the yield of the produced nucleic acid oligomer.
- FIG. 1 is a scheme A showing a typical example of producing the nucleic acid oligomer represented by the formula (5) from the nucleic acid oligomer represented by the formula (1).
- the G 1 can be used without particular limitation so long as it can function as a protecting group of a hydroxyl group, can be widely used known protecting groups used in amidite compound.
- G 3 may independently represent an alkyl group, which is the same or different from each other, or two G 3s may be bonded to each other to form a cyclic structure.
- the G 3 each independently, the same or different from each other, alkyl groups such as methyl group, ethyl group, preferably a propyl group or an isopropyl group, and more preferably both are isopropyl.
- alkyl groups such as methyl group, ethyl group, preferably a propyl group or an isopropyl group, and more preferably both are isopropyl.
- Other symbols are as described above.
- a method of reacting the nucleic acid oligomer represented by the formula (1) with a dichloroacetic acid solution having a formaldehyde concentration of a certain level or less to obtain the nucleic acid oligomer represented by the formula (2) will be described.
- the molar ratio of formaldehyde to dichloroacetic acid (mol of formaldehyde / mol of dichloroacetic acid) in the dichloroacetic acid solution of the present invention is usually 90 ⁇ 10-6 or less, preferably 43 ⁇ 10-6 or less, more preferably 22 ⁇ . It is 10-6 or less.
- Methods for measuring the formaldehyde concentration in a dichloroacetic acid solution include a gas chromatograph method and a high performance liquid chromatograph method. In the gas chromatograph method, formaldehyde is directly analyzed and the concentration is calculated.
- the concentration of dichloroacetic acid in the dichloroacetic acid solution is usually 0.1 to 2.4 M, preferably 0.1 to 1.2 M, more preferably 0.1 to 0.6 M, still more preferably 0. It is 2 to 0.4 M.
- the diluting solvent for dichloroacetic acid is not particularly limited as long as it is not involved in the reaction, and examples thereof include dichloromethane, acetonitrile, aromatic organic solvent, water or any mixed solvent, preferably dichloromethane, acetonitrile, and aromatic organic. At least one solvent selected from the group consisting of solvents can be mentioned, and more preferably an aromatic organic solvent can be mentioned. Examples of the aromatic organic solvent include toluene.
- the reaction temperature in the above reaction is preferably 0 to 40 ° C, more preferably 10 to 30 ° C.
- Formaldehyde in the dichloroacetic acid solution can be removed by co-boiling with an arbitrary solvent or an arbitrary mixed solvent, and the co-boiling solvent is not particularly limited as long as it has a lower boiling point than dichloroacetic acid.
- Dichloromethane, acetonitrile, aromatic organic solvents or any mixed solvent can be mentioned, preferably dichloromethane, acetonitrile, or aromatic organic solvents can be mentioned, and more preferably aromatic organic solvents can be mentioned.
- the aromatic organic solvent include toluene.
- the boiling point of the azeotropic solvent is preferably 200 ° C. or lower, more preferably 194 ° C. or lower.
- a glass container, a plastic container, or a metal container can be used for storing the dichloroacetic acid solution.
- a container made of polyethylene or polypropylene can be used
- a container made of SUS or Hastelloy can be used as the metal container.
- the oxidizing solution can be stored in an air atmosphere or an inert gas atmosphere, and as the inert gas, argon, nitrogen, carbon dioxide, helium or the like can be used.
- nucleic acid compound having a protecting group at the hydroxyl group at the 5'position examples include the nucleic acid compound of the above formula (1).
- nucleic acid compound produced by reacting the dichloroacetic acid solution examples include the nucleic acid compound represented by the above formula (2).
- the methylene group represented by Q' which is independently the same or different from the carbon atom at the 4'position of ribose, and the 4'position of ribose.
- LNA-1, LNA-2, or LNA-2 of the following formula (7) examples thereof include the structure represented by LNA-3.
- B a represents a nucleic acid base which may be protected.
- the group As a group having a structure consisting of a solid-phase carrier represented by Z and a linking group connecting the solid-phase carrier and the oxygen atom of the hydroxyl group at the 2'- or 3'-position of ribose at the 3'end of the nucleic acid oligomer, the group is more suitable.
- a structure represented by the following formula (8) can be mentioned.
- Sp represents a spacer.
- Examples of the spacer (Sp) include those having the structural formula shown in the following formula (9).
- the Linker may have, for example, a structure represented by the following formula (10), or a structure having no hexamethyleneamino group moiety in the structure of the formula (10) and having an aminopropyl group bonded to Si. ..
- the Linker may have a structure represented by the following formula (11).
- A may be either a hydroxyl group, an alkoxy group, or an alkyl group.
- the alkoxy group include a methoxy group and an ethoxy group.
- Examples of the alkyl group include a methyl group, an ethyl group, an isopropyl group and an n-propyl group.
- Si indicates that it is bonded to oxygen of the hydroxyl group on the surface of the carrier.
- the solid support include an inorganic porous carrier and an organic resin carrier.
- the inorganic porous carrier include Controlled Pole Glass (CPG).
- the organic resin carrier include a carrier made of polystyrene.
- 2'-F RNA and the above-mentioned LNA are exemplified, but the above-mentioned nucleoside is not limited thereto.
- the method for synthesizing a nucleic acid oligomer by a solid phase synthesis method typically includes the following steps. (1) A step of deprotecting the 5'-position hydroxyl group of a nucleoside in which the hydroxyl group bonded to the solid phase carrier via a linker is protected. (2) A step of coupling the 5'-position hydroxyl group generated in the above step with a phosphoramidite compound to obtain a phosphorous acid triester compound.
- the above steps (1) to (3) that is, a deprotection step of the 5'-position hydroxyl group of the produced nucleic acid molecule, a coupling step of the 5'-position hydroxyl group and the amidite compound, and the produced subphosphorus.
- a series of reaction cycles consisting of an acid triester oxidation step is repeated an arbitrary number of times to synthesize a nucleic acid molecule on the solid phase carrier, and (5) on the solid phase carrier produced in step (4).
- the method for synthesizing the nucleic acid oligomer includes a step of capping the hydroxyl group at the 5'position in which the coupling reaction with the phosphoramidite compound has not proceeded, following the step (2) or (3).
- a capping step may be added between any steps of the series of reaction cycles constituting step (4).
- the nucleic acid molecules on the solid-phase carrier produced in the step (4) are carried out in the order of the following steps (5-1) and (5-2). Then, it is carried out by subjecting it to the reaction of step (5-3).
- the reaction of the step (5-1) may be carried out arbitrarily, and the reaction of the step (5-2) may be carried out by the method described in Japanese Patent No. 4705716.
- step (1) or step (4) shown in FIG. 1 The schemes of the steps (1) to (5) are shown in FIG.
- the deprotection reaction in step (1) or step (4) shown in FIG. 1 is carried out using the above-mentioned dichloroacetic acid solution.
- the definition of the substituent in the chemical formula in Scheme A is as described above.
- the nucleic acid compound of the formula (1) can be further extended by an arbitrary chain length using a nucleotide-type or non-nucleotide-type linker by the amidite method, and used for producing the nucleic acid compound represented by the formula (3). can. Only the nucleic acid compound is cut out from the nucleic acid compound bound to the solid phase carrier of the formula (3) to obtain the nucleic acid oligomer represented by the formula (4), and then further deprotected and represented by the formula (5). Nucleic acid oligomers can also be obtained.
- the substituents in each formula will be described in more detail.
- B nucleobase represented by protected nucleic acid base which may be protected by a group and B c represented by a is not particularly limited.
- the nucleobase include adenine, cytosine, guanine, uracil, thymine, 5-methylcytosine, pseudouracil, 1-methyl uracil and the like.
- the nucleobase may be substituted with a substituent.
- substituents include, for example, halogen atoms such as fluoro groups, chloro groups, bromo groups and iodo groups, acyl groups such as acetyl groups, alkyl groups such as methyl and ethyl groups, and benzyl groups.
- Arylalkyl group alkoxy group such as methoxy group, alkoxyalkyl group such as methoxyethyl group, cyanoalkyl group such as cyanoethyl group, hydroxy group, hydroxyalkyl group, acyloxymethyl group, amino group, monoalkylamino group , Dialkylamino groups, carboxy groups, cyano groups, nitro groups and the like, as well as combinations of two or more substituents thereof.
- protecting group of the nucleic acid base which may be protected by a protecting group represented by B a
- B a can be used protecting groups used in the known nucleic acid chemistry, as such protecting groups
- protecting groups For example, benzoyl group, 4-methoxybenzoyl group, 4-methylbenzoyl group, acetyl group, propionyl group, butyryl group, isobutyryl group, phenylacetyl group, phenoxyacetyl group, 4-tert-butylphenoxyacetyl group, 4-isopropyl
- protecting groups thereof include a phenoxyacetyl group, a (dimethylamino) methylene group, and a combination of two or more protecting groups thereof.
- R 4 represents a hydrogen atom, a methyl group, phenoxyacetyl group, 4-tert-butyl-phenoxyacetyl, 4-isopropyl phenoxyacetyl, phenylacetyl group, an acetyl group or a benzoyl group
- R 5 represents a hydrogen atom, an acetyl group, an isobutyryl group or a benzoyl group.
- R 6 represents a hydrogen atom, a phenoxyacetyl group, a 4-tert-butylphenoxyacetyl group, a 4-isopropylphenoxyacetyl group, a phenylacetyl group, an acetyl group or an isobutylyl group.
- R 7 represents a 2-cyanoethyl group
- R 8 represents a hydrogen atom, a methyl group, a benzoyl group, a 4-methoxybenzoyl group or a 4-methylbenzoyl group
- R 9 represents a dimethylaminomethylene group.
- examples of B c include a group obtained by removing a protecting group from the specific example of B a.
- G 5 is preferably the following groups. (During the ceremony, R 1 , R 2 and R 3 independently represent a hydrogen atom or an alkoxy group, which are the same or different from each other. )
- R 1 , R 2 and R 3 is a hydrogen atom, and the other two are preferably the same or different (preferably the same) alkoxy groups, and the methoxy group is particularly preferable as the alkoxy group. More preferably, G 5 is a 4,4'-dimethoxytrityl group (DMTr group).
- DMTr group 4,4'-dimethoxytrityl group
- G 2 any one that can function as a protecting group for a hydroxyl group can be used without particular limitation, and a known protecting group used in an amidite compound can be widely used.
- Examples of G 2 include an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a haloalkyl group, an aryl group, a heteroaryl group, an arylalkyl group, a cycloalkyl group, a cycloalkylalkyl group, a cyclylalkyl group, and a hydroxyalkyl.
- aminoalkyl group alkoxyalkyl group, heterocyclylalkyl group, heterocyclylalkyl group, heteroarylalkyl group, silyl group, silyloxyalkyl group, mono, di or trialkylsilyl group, mono, di or trialkylsilyloxyalkyl group Etc., which may be substituted with one or more electron attractants.
- G 2 is preferably an alkyl group substituted with an electron attracting group.
- the electron attracting group include a cyano group, a nitro group, an alkylsulfonyl group, a halogen atom, an arylsulfonyl group, a trihalomethyl group, a trialkylamino group and the like, and a cyano group is preferable.
- the alkyl group in the definition of R 1 , R 2 , R 3 and G 2 may be linear or branched, preferably an alkyl group having 1 to 12 carbon atoms, and more preferably 1 to 6 carbon atoms.
- Alkyl group of. Specific examples of alkyl groups include methyl group, ethyl group, n-provyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, n-pentyl group, isopentyl group, and hexyl group. Can be mentioned.
- the alkyl group moiety constituting the alkoxy group in the definition of the substituent has the same definition as the definition of the alkyl group here.
- the amidite compound can be used in a free state or in a salt state.
- the salt of the amidite compound include base addition salts and acid addition salts, but are not particularly limited.
- Specific examples of the base addition salt include salts with inorganic bases such as sodium salt, magnesium salt, potassium salt, calcium salt and aluminum salt; salts with organic bases such as methylamine, ethylamine and ethanolamine; lysine, Salts with basic amino acids such as ornithine, arginine; and ammonium salts.
- the acid addition salt include mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid; formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, and malic acid.
- Organic acids such as tartrate, fumaric acid, succinic acid, lactic acid, maleic acid, citric acid, methanesulfonic acid, trifluoromethanesulfonic acid, ethanesulfonic acid; and acid addition salts with acidic amino acids such as aspartic acid and glutamic acid. Be done.
- Amidite compounds also include forms such as salts, hydrates, solvates, and polymorphs.
- R preferably represents a protected hydroxyl group.
- the protecting group when R indicates a protected hydroxyl group or the protecting group of the hydroxyl group represented by V may be any one that can be used in the amidite method, for example, a 2'-tert-butyldimethylsilyl (TBS) group, 2'-bis (2-acetoxy) methyl (ACE) group, 2'-(triisopropylsilyloxy) methyl (TOM) group, 2'-(2-cyanoethoxy) ethyl (CEE) group, 2'-(2) -Cyanoethoxy) methyl (CEM) group, 2'-para-toluylsulfonylethoxymethyl (TEM) group, 2'-EMM group (International Publication No.
- V is preferably a 2'-tert-butyldimethylsilyl (TBS) group.
- TBS 2'-tert-butyldimethylsilyl
- ribose is contained in the nucleic acid oligomer, such as when the nucleic acid oligomer produced by the method of the present invention is ribonucleic acid (RNA), the above formula (6) is used as a protecting group for the hydroxyl group at the 2'position of the ribose.
- RNA ribonucleic acid
- the protecting group represented by the formula (12) having a cyano group are exemplified as the electron withdrawing group represented by E W.
- E W electron withdrawing group represented by E W.
- q, Ra and R b are synonymous with the definitions in the above formula (6).
- a group in which q is 1 and Ra and R b are hydrogen atoms at the same time is exemplified.
- the protecting group represented by the formula (12) can be synthesized, for example, as described in WO 2013/027843 and WO 2019/208571, and an amidite compound having such a protecting group can be used to prepare a nucleic acid compound. Can be used for.
- the amidite compound of the formula (13) shown in Scheme A of FIG. 1 is used.
- non-nucleotide linker examples include a linker having an amino acid skeleton (for example, a linker having an amino acid skeleton described in Japanese Patent No. 5157168 or Japanese Patent No. 5554881). Specifically, as a non-limiting example, it is represented by, for example, the formula (A14-1) or (A14-2) or (A14-3) (for example, described in International Publication No. 2019/0741010). A linker is exemplified. In addition to these linkers, the linkers described in International Publication No. 2012/005368, International Publication No. 2018/182008, or International Publication No. 2019/0741010 are exemplified. (In the formula, Y is as described above.)
- Nucleotides and amidites in which the R group in the formula (13) and the R'group in the formula (5) are substituents other than hydroxyl groups are known methods described in Japanese Patent No. 3745226 and the like, International Publication No. 2001/053528. It can also be produced from JP-A-2014-221817 and nucleosides synthesized by known methods cited therein, and further, commercially available products will be used in Examples described later. It can be produced according to the described methods or by a method obtained by appropriately modifying these methods.
- G 4 represents a hydrogen atom, an alkali metal ion, an ammonium ion, an alkylammonium ion, or a hydroxyalkylammonium ion.
- the alkali metal ion include sodium ion and lithium ion.
- the alkylammonium ion specific examples of the alkyl group include a methyl group, an ethyl group, an n-provyl group, an isopropyl group, an n-butyl group, an isobutyl group, a tert-butyl group and an n-pentyl group. , Isopentyl group, and hexyl group.
- examples thereof include diethylammonium ion, triethylammonium ion, tetrabutylammonium ion, hexylammonium ion, and dibutylammonium ion.
- examples of a specific hydroxyalkyl moiety include, for example, hydroxymethyl, hydroxyethyl, hydroxy-n-provir, hydroxyisopropyl, hydroxy-n-butyl, and trishydroxymethyl.
- More specific examples of hydroxyalkylammonium ions include trishydroxymethylammonium ions.
- G 4 preferably represents a hydrogen atom.
- G 5 represents a protecting group of a hydrogen atom or the hydroxyl group, and when representing a protecting group, G 1 also represents the same protecting group.
- G 5 is a hydrogen atom when deprotected, but the nucleotide compound in that case is also subjected to a series of nucleic acid extension reaction steps.
- Y is preferably an oxygen atom.
- W 1 and X 1 preferably W 1 represents an OZ group and X 1 represents an R group.
- W 2 and X 2 preferably W 2 represents a hydroxyl group and X 2 represents an R group.
- W 3 and X 3 preferably each independently represent a hydroxyl group.
- R' is preferably a hydroxyl group.
- nucleic acid extension reaction means a reaction in which an oligonucleotide is extended by sequentially binding nucleotides via a phosphodiester bond.
- the nucleic acid extension reaction can be carried out according to the procedure of a general phosphoramidite method.
- the nucleic acid extension reaction may be carried out using an automatic nucleic acid synthesizer or the like that employs the phosphoramidite method.
- the chain length of the nucleic acid oligomer is, for example, 20 mer or more (that is, n ⁇ 19), 40 mer or more (that is, n ⁇ 39), 50 mer or more (that is, n ⁇ 49), 60 mer or more (that is, n ⁇ 59), 80 mer or more.
- n ⁇ 79 100 mer or more (ie, n ⁇ 99), 2 to 200 mer (ie, 1 ⁇ n ⁇ 199), 10 to 150 mer (ie, 9 ⁇ n ⁇ 149), 15 to 110 mer (ie, 1 ⁇ n ⁇ 149). , 14 ⁇ n ⁇ 109).
- the deprotection step of the step (1) is a step of deprotecting the protecting group of the 5'hydroxyl group at the terminal of the oligonucleotide chain supported on the solid phase carrier.
- a general protecting group a 4,4'-dimethoxytrityl group (DMTr group), a 4-monomethoxytrityl group, and a 4,4', 4 "-trimethoxytrityl group are used.
- DMTr group 4,4'-dimethoxytrityl group
- 4-monomethoxytrityl group 4-monomethoxytrityl group
- a 4,4', 4 "-trimethoxytrityl group are used for deprotection.
- an acid is used for deprotection.
- deprotecting acid examples include trifluoroacetic acid, dichloroacetic acid, trifluoromethanesulfonic acid, trichloroacetic acid, methanesulfonic acid, hydrochloric acid, acetic acid, p-toluenesulfonic acid and the like. ..
- the nucleoside phosphoramidite represented by the following formula (13) shown in the scheme A of FIG. 1 is applied to the 5'hydroxyl group at the terminal of the oligonucleotide chain deprotected by the deprotection step. It is a reaction to bind.
- amidite compounds represented by the formulas (13) or (A9) to (A12) are used.
- a protecting group eg, DMTr group
- the condensation step can be carried out using an activator or a condensing agent that activates the nucleoside phosphoramidite.
- Examples of the activator or condensing agent include 5-benzylthio-1H-tetrazole (BTT) (also referred to as 5-benzyl mercapto-1H-tetrazole), 1H-tetrazole, 4,5-dicyanoimidazole (DCI), 5- Ethylthio-1H-tetrazole (ETT), N-methylbenzimidazolium trifurate (N-MeBIT), benzimidazolium trifurate (BIT), N-phenylimidazolium trifurate (N-PhIMT), imidazolium trifurate (IMT), 5 -Nitrobenzimidazolium triflate (NBT), 1-hydroxybenzotriazole (HOBT) or 5- (bis-3,5-trifluoromethylphenyl) -1H-tetrazole and the like can be mentioned.
- BTT 5-benzylthio-1H-tetrazole
- DCI 5-benzylthio-1H-
- amidite The nucleoside phosphoramidite (hereinafter referred to as amidite) represented by the formula (13) described in Scheme A of FIG. 1 is as follows. formula: (During the ceremony, G 1 , G 2 , G 3 , Ba , and R are as described above. ).
- Capping can be performed using a known capping solution such as acetic anhydride-tetrahydrofuran solution or phenoxyacetic acid anhydride / N-methylimidazole solution.
- the oxidation step of the step (3) is a step of converting the phosphorous acid group formed by the condensation step into a phosphorous acid group or a thiophosphate group.
- This step is a reaction for converting trivalent phosphorus to pentavalent phosphorus using an oxidizing agent, and can be carried out by reacting an oligonucleic acid derivative supported on a solid phase carrier with an oxidizing agent. ..
- iodine can be used as the "oxidizing agent”.
- the oxidizing agent can be prepared and used so as to have a concentration of 0.005 to 2M.
- Water can be used as the oxygen source for oxidation, and pyridine, N-methylimidazole (NMI), N-methylmorpholin, triethylamine, or the like can be used as the base for advancing the reaction.
- the solvent is not particularly limited as long as it does not participate in the reaction, and examples thereof include acetonitrile, tetrahydrofuran (THF), and a mixed solvent of any ratio thereof.
- acetonitrile acetonitrile
- THF tetrahydrofuran
- iodine / water / pyridine / acetonitrile, iodine / water / pyridine, iodine / water / pyridine / NMI, or iodine / water / pyridine / THF can be used.
- the reaction temperature is preferably 5 ° C to 50 ° C.
- the reaction time is usually 1 to 30 minutes.
- the amount of the reagent used is preferably 1 to 100 mol, more preferably 1 to 10 mol, based on 1 mol of the compound supported on the solid phase carrier.
- an "oxidizing agent" for example, sulfur, 3H-1,2-benzodithiol-3-one-1,1-dioxide (Beaucage reagent) ), 3-Amino-1,2,4-dithiazole-5-thione (ADTT), 5-phenyl-3H-1,2,4-dithiazole-3-one (POS), [(N, N-dimethylamino) Methylidene) amino] -3H-1,2,4-dithiazolin-3-thione (DDTT), and phenylacetyldisulfide (PADS) can be used.
- an "oxidizing agent” for example, sulfur, 3H-1,2-benzodithiol-3-one-1,1-dioxide (Beaucage reagent) ), 3-Amino-1,2,4-dithiazole-5-thione (ADTT), 5-phenyl-3H-1,2,4-dithiazole-3-one (POS), [(N, N-dimethyl
- the oxidizing agent can be used by diluting it with a suitable solvent so as to have a concentration of 0.001 to 2M.
- the solvent used in the reaction is not particularly limited as long as it is not involved in the reaction, and examples thereof include dichloromethane, acetonitrile, pyridine, and a mixed solvent of any ratio thereof.
- the oxidation step may be performed after the capping operation, or conversely, the capping operation may be performed after the oxidation step, and the order is not limited.
- step (5-1) the protecting group for the 5'-position hydroxyl group of the nucleotide introduced at the end of extension is protected from the 5'-position hydroxyl group after excision from the solid phase carrier and deprotection of the protecting group, which will be described later. It may be used for column purification using a group as a tag, or the protecting group of the hydroxyl group at the 5'position may be deprotected after column purification.
- step (5-2) the step of deprotecting a phosphate protecting group is to allow an amine compound to act to deprotect the protecting group of the phosphate moiety after the synthesis of the nucleic acid having the desired sequence is completed.
- the amine compound include diethylamine described in Japanese Patent No. 4705716.
- the nucleic acid oligomer extended to a desired chain length on the solid phase carrier is usually cut out from the solid phase carrier using concentrated aqueous ammonia as a cutout agent.
- the oligonucleotide chain is cleaved from the solid phase carrier and recovered.
- the amine compound include methylamine, ethylamine, isopropylamine, ethylenediamine, diethylamine and the like.
- step (5-3) the protecting group for the hydroxyl group at the 2'- or 3'-position of ribose of the nucleic acid compound (4) excised from the solid-phase carrier in step (5-2) was published in International Publication No. 2006/02223.
- the deprotected nucleic acid oligomer (5) can be obtained by removing the nucleic acid oligomer (5) according to the method described in JP-A), WO2013 / 027443, or WO2019 / 208571.
- the nucleoside contained in the nucleic acid oligomer has RNA, DNA, and 2'-O-MOE, 2'-O-Me, and 2'-F.
- examples include, but are not limited to, RNA and nucleic acid oligomers that are LNAs.
- RNA and nucleic acid oligomers that are LNAs.
- the nucleic acid oligomer produced by the method of the invention is RNA.
- nucleic acid oligomers that can be used in the production method of the present invention are shown below in addition to the examples described in Examples, but the present invention is not limited thereto.
- U stands for uridine
- C stands for cytidine
- A stands for adenosine
- G stands for guanosine.
- Nucleic acid oligomers having the following sequences (A) and (B), which are described in WO 2019/060442, can be mentioned.
- a typical example is a nucleic acid oligomer having the following sequence (C). Sequence (C): 5'-AGAGCCAGCCUUCUUAUUGUUUUAGAGCUAUGCUGU-3' (SEQ ID NO: 3) 36mer Examples thereof include nucleic acid oligomers described in Japanese Patent No. 4965745. A typical example is a nucleic acid oligomer having the following sequence (D). Sequence (D): 5'-CCAUGAGAAGUAUGACAACAGCC-P-GGCUGUUGUCAUACUUCUCAUGGUU-3' (SEQ ID NO: 4, 5) 49mer In the sequence (D), "P” is represented by a partial structure separated by wavy lines in the following formula (A5).
- SEQ ID NO: 4 indicates the base sequence of the following sequence (D1) from the 5'end of the sequence (D) to the front of "P", and the description of SEQ ID NO: 5 is the sequence ( The base sequence of the following sequence (D2) from after the "P” in D) to the 3'end is shown.
- Examples thereof include nucleic acid oligomers having the following sequence (E) described in Nucleic Acids Research, 2019, Vol. 47, No. 2: 547.
- nucleic acid oligomers having the following sequence (F) which are described in JP-A-2015-523856, page 173.
- Examples thereof include nucleic acid oligomers described in JP-A-2017-537626.
- Typical examples include nucleic acid oligomers having the following sequences (G), (H), (I), and (J).
- U indicates 2'-O-methyluridine
- Am indicates 2'-O-methyladenosine
- Gm indicates 2'-O-methylguanosine
- s indicates phosphorothioate modification.
- Oligonucleotide purity was measured using HPLC.
- the HPLC measurement conditions are shown in Table 1 below. (Measurement method 1: Measurement of oligonucleotide purity)
- Methods for measuring the formaldehyde concentration in a dichloroacetic acid solution include a gas chromatograph method and a high performance liquid chromatograph method.
- gas chromatograph method formaldehyde is directly analyzed and the concentration is calculated.
- high performance liquid chromatography method formaldehyde is reacted with acetylacetone, the amount of 3,5-diacetyl-1,4-dihydrolutidine obtained is measured, and the concentration of formaldehyde is calculated.
- dichloroacetic acid solutions having different formaldehyde concentrations used in the following tests were prepared by preparing a dichloroacetic acid solution having a low formaldehyde concentration in advance and adding an aqueous formaldehyde solution to the obtained dichloroacetic acid solution.
- C is represented by a partial structure separated by wavy lines in the following equation (A2).
- G is represented by a partial structure separated by wavy lines in the following equation (A3).
- U is represented by a partial structure separated by wavy lines in the following equation (A4).
- the "U” at the 3'end is represented by a partial structure separated by a wavy line in the following formula (A8).
- the "G” at the 5'end is represented by a partial structure separated by a wavy line in the following formula (A6), and the "A” at the 5'end in the sequences (II) and (III). Is represented by a partial structure separated by wavy lines in the following equation (A7).
- CPG Controlled Pole Glass
- NTS M-4MX-E manufactured by Nippon Techno Service Co., Ltd.
- the above sequence (I) was performed by a phosphoramidite solid-phase synthesis method.
- (II) and (III) were synthesized from the 3'side to the 5'side. The synthesis was carried out on a scale of about 1 ⁇ mol.
- uridine EMM amidite (A11) according to Example 2 of US2012 / 0035246, cytidine EMM amidite (A9) according to Example 3, adenosine EMM amidite (A12) according to Example 4, and Using the guanosine EMM amidite (A10) described in Example 5, a 3% toluene dichloroacetate solution as a deblocking solution, 5-benzyl mercapto-1H-tetrazole as a condensing agent, and an iodine solution as an oxidizing agent. And a phenoxyacetic acid anhydride solution and an N-methylimidazole solution were used as capping solutions.
- the oligonucleotide produced by the production method of the present invention in the following examples is an oligonucleotide having the sequences (I), (II) and (III).
- the uridine derivative described in the following Examples and Comparative Examples means a compound represented by the following structural formula.
- the circles illustrated in the following structural formulas schematically represent CPG.
- Example 1 It is shown in the sequence (I) using the Controlled Pole Glass (CPG) carrying a 0.98 ⁇ mol uridine derivative and the amidite represented by the formula (A9), the formula (A10), the formula (A11), or the formula (A12).
- Nucleic acid oligomers were automatically synthesized from the 3'side to the 5'side by NTS M-4MX-E (manufactured by Nippon Techno Service Co., Ltd.). The procedure for automatic synthesis was as follows: First, a 3% toluene dichloroacetate solution was sent to CPG to deprotect the trityl protecting group at the 5'position.
- the concentration of formaldehyde in the dichloroacetic acid solution used can be measured by the measuring method 3, and the molar ratio of formaldehyde to dichloroacetic acid (mol formaldehyde mol / mol dichloroacetic acid) in the dichloroacetic acid solution is 43 ⁇ 10 ⁇ . It was 7. Subsequently, 5-benzyl mercapto-1H-tetrazole was sent to CPG as a condensing agent with various amidites, and the coupling reaction was allowed to proceed to the hydroxyl group at the 5'position. Subsequently, an oxidizing solution containing 50 mM iodine was sent to convert the phosphite group into a phosphate group.
- 1M tetra-n-butylammonium fluoride (1M tetra-n-butylammonium fluoride) was prepared by dissolving the free oligonucleotide in 1.5 mL of dimethyl sulfoxide, adding 1.0 mL of acetonitrile, 20 ⁇ L of nitromethane and a stirrer, and then dehydrating with molecular sieve 4A. 2.08 mL of a dimethyl sulfoxide solution of TBAF) was flowed in at room temperature under stirring with a stirrer, and the mixture was kept warm at 33 ° C. for 4 hours to deprotect the 2'-EMM protecting group.
- the product of the nucleic acid oligomer was obtained by a precipitation operation.
- the purity was 76.2%.
- the yield of the oligonucleotide was measured using the method described in the measurement method 2
- the yield was 4032 ⁇ g, which was 4114 ⁇ g in terms of the yield per CPG carrying a 1.00 ⁇ mol uridine derivative. The results are shown in Table 2.
- Example 2 In the experiment of Example 1, 3% dichloro having a molar ratio of formaldehyde to dichloroacetic acid (formaldehyde mol / dichloroacetic acid mol) of 90 ⁇ 10-6 with Controlled Pole Glass (CPG) carrying a 1.02 ⁇ mol urine derivative.
- the nucleic acid oligomer of sequence (I) was obtained in the same manner except that a toluene acetate solution was used.
- the purity of the product was 75.0%.
- Example 3 It is shown in the sequence (II) using the Controlled Pole Glass (CPG) carrying a 1.03 ⁇ mol uridine derivative and the amidite represented by the formula (A9), the formula (A10), the formula (A11), or the formula (A12).
- Nucleic acid oligomers were automatically synthesized from the 3'side to the 5'side by NTS M-4MX-E (manufactured by Nippon Techno Service Co., Ltd.). The procedure for automatic synthesis was as follows: First, a 3% toluene dichloroacetate solution was sent to CPG to deprotect the trityl protecting group at the 5'position.
- the concentration of formaldehyde in the dichloroacetic acid solution used can be measured by the measuring method 3, and the molar ratio of formaldehyde to dichloroacetic acid (mol formaldehyde mol / mol dichloroacetic acid) in the dichloroacetic acid solution is 43 ⁇ 10 ⁇ . It was 7. Subsequently, 5-benzyl mercapto-1H-tetrazole was sent to CPG as a condensing agent with various amidites, and the coupling reaction was allowed to proceed to the hydroxyl group at the 5'position. Subsequently, an oxidizing solution containing 50 mM iodine was sent to convert the phosphite group into a phosphate group.
- Example 4 In the experiment of Example 3, Controlled Pole Glass (CPG) carrying a 1.05 ⁇ mol urine derivative and 3% dichloro having a molar ratio of formaldehyde to dichloroacetic acid (formaldehyde mol / dichloroacetic acid mol) of 90 ⁇ 10-6.
- the nucleic acid oligomer of sequence (II) was obtained in the same manner except that a toluene acetate solution was used.
- the purity of the product was 47.8%.
- Example 5 It is shown in the sequence (III) using the Controlled Pole Glass (CPG) carrying a 0.99 ⁇ mol uridine derivative and the amidite represented by the formula (A9), the formula (A10), the formula (A11), or the formula (A12).
- Nucleic acid oligomers were automatically synthesized from the 3'side to the 5'side by NTS M-4MX-E (manufactured by Nippon Techno Service Co., Ltd.). The procedure for automatic synthesis was as follows: First, a 3% toluene dichloroacetate solution was sent to CPG to deprotect the trityl protecting group at the 5'position.
- the concentration of formaldehyde in the dichloroacetic acid solution used can be measured by the measuring method 3, and the molar ratio of formaldehyde to dichloroacetic acid (mol formaldehyde mol / mol dichloroacetic acid) in the dichloroacetic acid solution is 43 ⁇ 10 ⁇ . It was 7. Subsequently, 5-benzyl mercapto-1H-tetrazole was sent to CPG as a condensing agent with various amidites, and the coupling reaction was allowed to proceed to the hydroxyl group at the 5'position. Subsequently, an oxidizing solution containing 50 mM iodine was sent to convert the phosphite group into a phosphate group.
- 1M tetra-n-butylammonium fluoride (1M tetra-n-butylammonium fluoride) was prepared by dissolving the free oligonucleotide in 1.5 mL of dimethyl sulfoxide, adding 1.0 mL of acetonitrile, 20 ⁇ L of nitromethane and a stirrer, and then dehydrating with molecular sieve 4A. 2.08 mL of a dimethyl sulfoxide solution of TBAF) was flowed in at room temperature under stirring with a stirrer, and the mixture was kept warm at 33 ° C. for 4 hours to deprotect the 2'-EMM protecting group.
- the product of the nucleic acid oligomer was obtained by a precipitation operation.
- the purity was 33.1%.
- the yield of the oligonucleotide was measured using the method described in the measurement method 2
- the yield was 12889 ⁇ g, which was 13019 ⁇ g in terms of the yield per CPG carrying a 1.00 ⁇ mol uridine derivative. The results are shown in Table 2.
- Example 6 Controlled Pole Glass (CPG) carrying a 1.04 ⁇ mol urine derivative and 3% dichloro having a molar ratio of formaldehyde to dichloroacetic acid (formaldehyde mol / dichloroacetic acid mol) of 90 ⁇ 10-6.
- the nucleic acid oligomer of sequence (III) was obtained in the same manner except that a toluene acetate solution was used.
- the purity of the product was 29.7%.
- Example 7 To 30 g of a dichloroacetic acid solution having a molar ratio of formaldehyde to dichloroacetic acid (mol formaldehyde mol / mol dichloroacetic acid) of 25 ⁇ 10-5 , 300 mL of toluene was added, and toluene and formaldehyde were co-boiling distilled at 40 ° C. using an evaporator. Then, 34 g of a slightly yellow oily dichloroacetic acid solution was obtained. When formaldehyde contained in the obtained dichloroacetic acid solution was analyzed by the method described in Measurement Method 3, the molar ratio of formaldehyde to dichloroacetic acid was 43 ⁇ 10-7 .
- the present invention provides an efficient method for producing a nucleic acid oligomer. Further, the yield of the nucleic acid oligomer produced according to the method for producing the nucleic acid oligomer can be expected to be improved.
- SEQ ID NOs: 1 to 14 in the sequence listing represent the base sequences of oligonucleotides produced according to the production method of the present invention.
Abstract
Description
本発明は、核酸オリゴマーの製造方法に関する。
1.式(1):
G2は、水酸基の保護基を示し、
Baは、それぞれ独立して、同一又は相異なって、保護基で保護されていてもよい核酸塩基を表し、
R1、R2及びR3は、それぞれ独立して、同一又は相異なって、水素原子又はアルコキシ基を表し、
Rは、それぞれ独立して、同一又は相異なって、保護された水酸基、水素原子、フッ素原子、メトキシ基、2-メトキシエチル基、またはOQ’基を表し、
Q’は、それぞれ独立して、同一又は相異なって、リボースの4’位の炭素原子と結合しているメチレン基、リボースの4’位の炭素原子と結合しているエチレン基、またはリボースの4’位の炭素原子と結合しているエチリデン基を表し、
Yは、それぞれ独立して、同一又は相異なって、酸素原子または硫黄原子を表し、
nは、1~200の何れかの整数を表し、
W1は、OZ基を表し、かつ、X1は、R基を表すか、あるいは
W1は、OV基を表し、かつ、X1は、OZ基を表し、
Vは、水酸基の保護基を表し、
Zは、固相担体および連結基からなる構造を有する基である。
そして、nが2以上の整数のとき、式(1)で示される核酸オリゴマーは、それぞれのヌクレオチドの間に、非ヌクレオチドリンカーが組み込まれていてもよい。)
で示される核酸オリゴマーと、ホルムアルデヒドとジクロロ酢酸のモル比(ホルムアルデヒドmol/ジクロロ酢酸mol)が、90×10-6以下であるジクロロ酢酸溶液を反応させる工程を含む、式(2):
G2、Ba、R、Y、X1、W1およびnは、前記のとおりであり、そして、
式(1)において定義されたとおり、ヌクレオチドの間に、非ヌクレオチドリンカーが組み込まれていてもよい。)
で示される核酸オリゴマーの製造方法。
2.前項1に記載の工程と、さらに、当該工程で生成する式(2)で示される核酸オリゴマーからZで表される基を除く工程、ならびに水酸基および核酸塩基の保護基を除く工程を含む、式(2’):
Yおよびnは、前記のとおりであり、
Bcは、それぞれ独立して、同一又は相異なって、核酸塩基を表し、
G4は、それぞれ独立して、同一又は相異なって、水素原子、アルカリ金属イオン、アンモニウムイオン、アルキルアンモニウムイオン、またはヒドロキシアルキルアンモニウムイオンを表し、
R’は、それぞれ独立して、同一又は相異なって、水酸基、水素原子、フッ素原子、メトキシ基、2-メトキシエチル基、またはOQ’基を表し、
Q’は、前記のとおりであり、
X3およびW3は各々、それぞれ独立して、水酸基を表すか、あるいは
X3は、R’基を表し、かつ、W3は、水酸基を表す。)
で示される核酸オリゴマーの製造方法。
3.式(2)で示される核酸オリゴマーを、アミダイト法で任意に鎖長を伸長した式(3):
G2、Ba、R、Y、X1およびW1は、前記のとおりであり、
G5は、
R1、R2及びR3は、前記のとおりであり、
mは、m≧nを満たす整数である。)
で示される核酸化合物を得る工程、ならびに
式(3)で示される化合物から式(4):
G5、R、Yおよびmは、前記のとおりであり、
G4は、それぞれ独立して、同一又は相異なって、水素原子、アルカリ金属イオン、アンモニウムイオン、アルキルアンモニウムイオン、またはヒドロキシアルキルアンモニウムイオンを表し、
BCは、それぞれ独立して、同一又は相異なって、核酸塩基を表し、
X2は、水酸基を表し、かつ、W2は、OV基を表すか、あるいは
X2は、R基を表し、かつ、W2は、水酸基を表し、
Vは、水酸基の保護基を表す。)
で示される化合物を切り出し、
さらに式(4)で示される化合物を脱保護して、式(5):
G4、Bc、Yおよびmは、前記のとおりであり、
R’は、それぞれ独立して、同一又は相異なって、水酸基、水素原子、フッ素原子、メトキシ基、2-メトキシエチル基、またはOQ’基を表し、
Q’は、前記のとおりであり、
X3およびW3は各々、それぞれ独立して、水酸基を表すか、あるいは
X3は、R’基を表し、かつ、W3は、水酸基を表す。)
で示される核酸オリゴマーを製造する工程をさらに含む、前項1に記載の製造方法。
4.非ヌクレオチドリンカーが、アミノ酸骨格からなるリンカーである、前項1~3の何れか一項に記載の製造方法。
5.アミノ酸骨格からなるリンカーが、下記式(A14-1)、(A14-2)および(A14-3)からなる群から選ばれる構造を有するリンカーである、前項4に記載の製造方法。
6.ジクロロ酢酸溶液が、ジクロロメタン、アセトニトリル、および芳香族有機溶媒からなる群から選ばれる少なくとも1つの溶媒を含む、前項1~5の何れか一項に記載の製造方法。
7.ジクロロ酢酸溶液中のホルムアルデヒドとジクロロ酢酸のモル比(ホルムアルデヒドmol/ジクロロ酢酸mol)が、43×10-6以下である、前項1~6の何れか一項に記載の製造方法。
8.ジクロロ酢酸溶液中のホルムアルデヒドとジクロロ酢酸のモル比(ホルムアルデヒドmol/ジクロロ酢酸mol)が、22×10-6以下である、前項1~6の何れか一項に記載の製造方法。
9.核酸オリゴマーが、リボ核酸(RNA)である、前項1~8の何れか一項に記載の製造方法。
10.核酸オリゴマーが、リボ核酸(RNA)であり、そのリボースの2’位の水酸基の保護基が、式(6)で示される保護基である、前項1~8の何れか一項に記載の製造方法。
式(6):
qは、1~5の何れかの整数を表し、
RaおよびRbは、それぞれ独立して、同一又は相異なって、メチル基、エチル基または水素原子を表し、
*印は、リボースの2’位の水酸基由来の酸素原子との結合点を表し、そして、
EWは、電子求引基を表す。)
11.RaおよびRbが同時に水素原子であり、かつ、EWがシアノ基である、前項10に記載の製造方法。
12.核酸オリゴマーが、40鎖長以上のオリゴマーである、前項1~11の何れか一項に記載の製造方法。
13.核酸オリゴマーが、50鎖長以上のオリゴマーである、前項1~11の何れか一項に記載の製造方法。
14.核酸オリゴマーが、60鎖長以上のオリゴマーである、前項1~11の何れか一項に記載の製造方法。
15.核酸オリゴマーが、80鎖長以上のオリゴマーである、前項1~11の何れか一項に記載の製造方法。
16.核酸オリゴマーが、100鎖長以上のオリゴマーである、前項1~11の何れか一項に記載の製造方法。
17.ホルムアルデヒドとジクロロ酢酸のモル比(ホルムアルデヒドmol/ジクロロ酢酸mol)が、90×10-6以下である、ジクロロ酢酸溶液。
18.ホルムアルデヒドとジクロロ酢酸のモル比(ホルムアルデヒドmol/ジクロロ酢酸mol)が、43×10-6以下である、前項17に記載のジクロロ酢酸溶液。
19.ホルムアルデヒドとジクロロ酢酸のモル比(ホルムアルデヒドmol/ジクロロ酢酸mol)が、22×10-6以下である、前項17に記載のジクロロ酢酸溶液。
20.ホルムアルデヒドを含む未精製ジクロロ酢酸溶液とホルムアルデヒドと共沸する溶媒を含む溶液からホルムアルデヒドを共沸させて留去することにより精製ジクロロ酢酸溶液を得る工程を含む、前項17~19の何れか一項に記載のジクロロ酢酸溶液の製造方法。
21.共沸溶媒の沸点が、194℃以下である、前項20に記載の製造方法。
22.共沸溶媒が、ジクロロメタン、アセトニトリルまたは芳香族有機溶媒である、前項20または21に記載の製造方法。
23.芳香族有機溶媒がトルエンである、前項22に記載の製造方法。
24.前項20に記載のジクロロ酢酸溶液の精製工程および当該工程で得られた精製ジクロロ酢酸溶液を使用する前項1~3の何れか一項に記載の工程を含む、核酸オリゴマーの製造方法。
空気雰囲気下または不活性ガス雰囲気下で酸化溶液を保管することができ、不活性ガスとしては、アルゴン、窒素、二酸化炭素またはヘリウム等を使用することができる。
前記式(1)および(2)において、Q’で表される、それぞれ独立して同一又は相異なって、リボースの4’位の炭素原子と結合しているメチレン基、リボースの4’位の炭素原子と結合しているエチレン基、またはリボースの4’位の炭素原子と結合しているエチリデン基を表す化合物として、具体的には下記式(7)のLNA-1、LNA-2、またはLNA-3で示される構造が挙げられる。
スペーサー(Sp)としては、例えば、下記式(9)に示す構造式を有するものが例示される。
Aは、水酸基、アルコキシ基、またはアルキル基のいずれかであってもよい。アルコキシ基としては、例えばメトキシ基およびエトキシ基が挙げられる。アルキル基としては、例えばメチル基、エチル基、イソプロピル基、n-プロピル基が挙げられる。Siは、担体表面の水酸基の酸素と結合していることを示す。)
Solid supportとしては、無機多孔質担体や有機系樹脂担体などが挙げられる。無機多孔質担体には、例えば、Controlled Pore Glass(CPG)が挙げられる。有機系樹脂担体には、例えば、ポリスチレンからなる担体が挙げられる。
(1)固相担体にリンカーを介して結合している水酸基が保護されたヌクレオシドの5’位の水酸基を脱保護する工程、
(2)前記工程で生成した5’位の水酸基をホスホロアミダイト化合物とカップリング反応させて亜リン酸トリエステル化合物を得る工程、
(3)前記工程で生成した亜リン酸トリエステルを酸化してリン酸トリエステルに変換して伸長した核酸分子を製造する工程、あるいは、チオリン酸トリエステルに変換する任意の工程、
(4)前記工程(1)~(3)、すなわち、生成した核酸分子の5’位の水酸基の脱保護工程、5’位の水酸基とアミダイト化合物とのカップリング工程、および、生成した亜リン酸トリエステルの酸化工程、から構成される一連の反応のサイクルを、任意の回数繰り返し、固相担体上に核酸分子を合成する工程、および
(5)工程(4)で生成した固相担体上の核酸分子を、切り出しおよび脱保護する工程に供し、固相担体から遊離させて、保護基が除かれた核酸オリゴマーを製造する工程。
ただし、前記核酸オリゴマーの合成方法においては、工程(2)または(3)に続けて、ホスホロアミダイト化合物とのカップリング反応が進行しなかった5’位の水酸基をキャッピングする工程を含んでいてもよく、工程(4)を構成する一連の反応のサイクルの何れかの工程の間にキャッピング工程が付加されていてもよい。
(5-1)核酸分子の5’末端の水酸基の保護基を脱保護する反応、
(5-2)核酸分子を固相担体から切りだして遊離させる反応、および、
(5-3)核酸分子を構成するリボースの2’位もしくは3’末端の3’位の水酸基の保護基を脱保護する反応。
R4は、水素原子、メチル基、フェノキシアセチル基、4-tert-ブチルフェノキシアセチル基、4-イソプロピルフェノキシアセチル基、フェニルアセチル基、アセチル基又はベンゾイル基を表し、
R5は、水素原子、アセチル基、イソブチリル基又はベンゾイル基を表し、
R6は、水素原子、フェノキシアセチル基、4-tert-ブチルフェノキシアセチル基、4-イソプロピルフェノキシアセチル基、フェニルアセチル基、アセチル基又はイソブチリル基を表し、
R7は、2-シアノエチル基を表し、
R8は、水素原子、メチル基、ベンゾイル基、4-メトキシベンゾイル基又は4-メチルベンゾイル基を表し、そして、
R9は、ジメチルアミノメチレン基を表す。)
のいずれかで表される基を表す。
q、RaおよびRbは、前記式(6)における定義と同義である。)
さらに好ましくは、式(12)で示される基において、qが1であり、RaおよびRbが同時に水素原子である基が例示される。
核酸の伸長反応には、図1のスキームAに記載の式(13)のアミダイト化合物が使用される。
本明細書において、「核酸伸長反応」とは、ホスホジエステル結合を介して、ヌクレオチドを順次結合させることにより、オリゴヌクレオチドを伸長させる反応を意味する。核酸伸長反応は、一般的なホスホロアミダイト法の手順に従い行うことができる。核酸伸長反応は、ホスホロアミダイト法を採用する核酸自動合成装置等を用いて行ってもよい。
式:
G1、G2、G3、Ba、およびRは、前記の通りである。)で示される化合物。
亜リン酸基をリン酸基に変換する場合には、「酸化剤」として、例えば、ヨウ素を使用することができる。該酸化剤は、0.005~2Mの濃度になるように調製して使用することができる。酸化の酸素源としては水を用いることができ、反応を進行させる塩基としてはピリジン、N-メチルイミダゾール(NMI)、N-メチルモルフォリン、またはトリエチルアミンなどを用いることができる。また、溶媒としては、反応に関与しなければ特に限定されないが、アセトニトリル、テトラヒドロフラン(THF)又はこれらの任意の割合の混合溶媒が挙げられる。例えば、ヨウ素/水/ピリジン/アセトニトリル、あるいはヨウ素/水/ピリジンあるいはヨウ素/水/ピリジン/NMI、あるいはヨウ素/水/ピリジン/THFを用いることができる。反応温度は、5℃~50℃が好ましい。反応時間は、通常1分~30分が適当である。使用する試薬の量は固相担体に担持されている化合物1molに対して1~100molが好ましく、より好ましくは1~10molである。
以下、配列の説明中、Uはウリジンを、Cはシチジンを、Aはアデノシンを、またGはグアノシンを示す。
国際公開第2019/060442号公報に記載されている、下記の配列(A)および(B)を有する核酸オリゴマーが挙げられる。
配列(A):5’-AUGGAAUmACUCUUGGUUmACdTdT-3’(Antisense)(配列番号1)21mer
配列(B):5’-GUmAACmCmAAGAGUmAUmUmCmCmAUmdTdT-3’(Sense)(配列番号2)21mer
配列(A)および(B)中、Umは2'-O-メチルウリジンを、Cmは2'-O-メチルシチジンを、またdTはチミジンを示す。
Daniel O'Reillyら著、Nucleic Acids Research, 2019, Vol. 47, No.2, 546-558に記載されている核酸オリゴマー(553頁参照)が挙げられる。典型例として、下記の配列(C)を有する核酸オリゴマーが挙げられる。
配列(C):5’-AGAGCCAGCCUUCUUAUUGUUUUAGAGCUAUGCUGU-3’(配列番号3)36mer
特許第4965745号公報に記載されている核酸オリゴマーが挙げられる。典型例として、下記の配列(D)を有する核酸オリゴマーが挙げられる。
配列(D):5’-CCAUGAGAAGUAUGACAACAGCC-P-GGCUGUUGUCAUACUUCUCAUGGUU-3’(配列番号4,5)49mer
配列(D)中、“P”は、以下の式(A5)において波線で区切られる部分構造で示される。
なお、配列表中の配列番号4の記載は、配列(D)の5’末端から「P」の前までの下記の配列(D1)の塩基配列を示し、配列番号5の記載は、配列(D)の「P」の後から3’末端までの下記の配列(D2)の塩基配列を示す。
配列(D1):5’-CCAUGAGAAGUAUGACAACAGCC-3’(配列番号4)23mer
配列(D2):5’-GGCUGUUGUCAUACUUCUCAUGGUU-3’(配列番号5)25mer
Nucleic Acids Research, 2019, Vol. 47, No. 2: 547に記載されている、下記の配列(E)を有する核酸オリゴマーが挙げられる。
配列(E):5’-ACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU-3’(配列番号6)67mer
特表2015-523856号公報、173頁に記載されている、下記の配列(F)を有する核酸オリゴマーが挙げられる。
配列(F):5’-GUUUUCCCUUUUCAAAGAAAUCUCCUGGGCACCUAUCUUCUUAGGUGCCCUCCCUUGUUUAAACCUGACCAGUUAACCGGCUGGUUAGGUUUUU-3’(配列番号7)94mer
特表2017-537626号公報に記載されている核酸オリゴマーが挙げられる。典型例として、下記の配列(G)、(H)、(I)、および(J)を有する核酸オリゴマーが挙げられる。
配列(G):5’-AGUCCUCAUCUCCCUCAAGCGUUUUAGAGCUAGUAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3’(配列番号8)100mer
配列(H):5’-GCAGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU-3’(配列番号9)113mer
配列(I):5’-dAdGdTdCdCdTdCdAdTdCdTdCdCdCdTdCdAdAdGdCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU-3’(配列番号10)113mer
配列(I)中、dTはチミジンを、dCは2'-デオキシシチジンを、dAは2'-デオキシアデノシンを、またdGは2'-デオキシグアノシンを示す。
配列(J):5’-AmsGmsUmsCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUmsUmsUmsU-3’(配列番号11)113mer
配列(J)中、Umは2'-O-メチルウリジンを、Amは2'-O-メチルアデノシンを、Gmは2'-O-メチルグアノシンを、またsはホスホロチオエート修飾を示す。
<測定方法>
まず、以下の試験で用いた各種測定方法を以下に示す。
粗生成物のOD260を測定した。OD260とは1mL溶液(pH=7.5)における10mm光路長あたりのUV260nmの吸光度を表す。一般的にRNAでは1OD=40μgであることが知られていることから、前記OD260の測定値に基づき、収量を算出した。
ジクロロ酢酸溶液中のホルムアルデヒド濃度測定方法には、ガスクロマトグラフ法または高速液体クロマトグラフ法がある。ガスクロマトグラフ法ではホルムアルデヒドを直接分析し濃度を算出する。高速液体クロマトグラフ法では、ホルムアルデヒドとアセチルアセトンを反応させ、得られた3,5―ジアセチル―1,4―ジヒドロルチジンの量を測定し、ホルムアルデヒドの濃度を算出する。
以下の試験で用いたホルムアルデヒド濃度の異なるジクロロ酢酸溶液は、予めホルムアルデヒド濃度の低いジクロロ酢酸溶液を調製し、得られたジクロロ酢酸溶液にホルムアルデヒド水溶液を添加して調製した。
配列(I):5’-GGCACCGAGUCGGUGCUUUU-3’(配列番号12)20mer
配列(II):5’-AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3’(配列番号13)50mer
配列(III):5’-AUAACUCAAUUUGUAAAAAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3’(配列番号14)100mer
前記配列(I)、(II)および(III)中、“A”は、以下の式(A1)において波線で区切られる部分構造で示される。“C”は、以下の式(A2)において波線で区切られる部分構造で示される。“G”は、以下の式(A3)において波線で区切られる部分構造で示される。Uは、以下の式(A4)において波線で区切られる部分構造で示される。なお、3’末端の“U”は、以下の式(A8)において波線で区切られる部分構造で示される。また、配列(I)中、5’末端の“G”は、以下の式(A6)において波線で区切られる部分構造で示され、配列(II)および(III)中、5’末端の“A”は、以下の式(A7)において波線で区切られる部分構造で示される。
また、以下の実施例および比較例中に記載するウリジン誘導体とは、下記の構造式で示される化合物を意味する。下記構造式において図示されたサークルは、CPGを模式的に示すものである。
0.98μmolのウリジン誘導体を担持したControlled Pore Glass(CPG)と、式(A9)、式(A10)、式(A11)、または式(A12)に示すアミダイトを用いて、配列(I)に示す核酸オリゴマーをNTS M-4MX-E(日本テクノサービス社製)により、3’側から5’側に向かって自動合成した。自動合成の手順は、まず、3%ジクロロ酢酸トルエン溶液をCPGに送液し、5’位のトリチル保護基を脱保護した。この際、使用したジクロロ酢酸溶液中のホルムアルデヒド濃度は、測定方法3により測定することができ、ジクロロ酢酸溶液中のホルムアルデヒドとジクロロ酢酸のモル比(ホルムアルデヒドmol/ジクロロ酢酸mol)は、43×10-7であった。続いて、各種アミダイトと縮合剤として5-ベンジルメルカプト-1H-テトラゾールをCPGに送液し、5’位の水酸基にカップリング反応を進行させた。続いて、50mMヨウ素を含む酸化溶液を送液し、亜リン酸基をリン酸基に変換した。続いて、キャッピング溶液として0.1Mフェノキシ酢酸無水物アセトニトリル溶液と10%N-メチルイミダゾール/10%2,6-ルチジンアセトニトリル溶液を使用し、カップリングが進行しなかった反応点にキャッピングを施した。更にこれらの工程を合計19回繰り返した後、5’末端の塩基における保護基(DMTr基)を3%ジクロロ酢酸トルエン溶液で脱保護し、配列(I)に示される配列の核酸オリゴヌクレオチドをCPG担体上に合成した。その後、0.98μmol分のオリゴヌクレオチドを担持したCPG担体に対して、28%アンモニア水1.5mLとエタノール0.5mLを流入し、混合物を40℃で4時間保温することで核酸オリゴマーを固相担体から遊離させた後、濃縮により溶媒を除去した。次いで遊離オリゴヌクレオチドをジメチルスルホキシド1.5mLに溶解後、アセトニトリル1.0mL、ニトロメタン20μLと撹子を入れた後、モレキュラーシーブ4Aにて脱水処理を施した1Mのフッ化テトラ-n-ブチルアンモニウム(TBAF)のジメチルスルホキシド溶液2.08mLをスターラーによる攪拌下室温で流入し、混合物を33℃で4時間保温することで2’-EMM保護基の脱保護を行った。その後、核酸オリゴマーの生成物を沈殿操作により得た。得られた生成物について、前記測定方法1に記載の方法を用いて、オリゴヌクレオチドの純度を測定した結果、純度は76.2%であった。また、前記測定方法2に記載の方法を用いて、オリゴヌクレオチドの収量を測定したところ、収量は4032μgであり、1.00μmolのウリジン誘導体を担持したCPGあたりの収量に換算すると4114μgであった。結果を表2に示す。
実施例1の実験において、1.02μmolのウリジン誘導体を担持したControlled Pore Glass(CPG)と、ホルムアルデヒドとジクロロ酢酸のモル比(ホルムアルデヒドmol/ジクロロ酢酸mol)が90×10-6である3%ジクロロ酢酸トルエン溶液を用いる以外は、同様の方法で配列(I)の核酸オリゴマーを得た。前記測定方法1に記載の方法を用いて、オリゴヌクレオチドの純度を測定した結果、生成物の純度は75.0%であった。また、前記測定方法2に記載の方法を用いて、オリゴヌクレオチドの収量を測定したところ、収量は4147μgであり、1.00μmolのウリジン誘導体を担持したCPGあたりの収量に換算すると4066μgであった。結果を表2に示す。
実施例1の実験において、1.03μmolのウリジン誘導体を担持したControlled Pore Glass(CPG)と、ホルムアルデヒドとジクロロ酢酸のモル比(ホルムアルデヒドmol/ジクロロ酢酸mol)が20×10-5である3%ジクロロ酢酸トルエン溶液を用いる以外は、同様の方法で配列(I)の核酸オリゴマーを得た。前記測定方法1に記載の方法を用いて、オリゴヌクレオチドの純度を測定した結果、生成物の純度は73.8%であった。また、前記測定方法2に記載の方法を用いて、オリゴヌクレオチドの収量を測定したところ、収量は3979μgであり、1.00μmolのウリジン誘導体を担持したCPGあたりの収量に換算すると3863μgであった。結果を表2に示す。
1.03μmolのウリジン誘導体を担持したControlled Pore Glass(CPG)と、式(A9)、式(A10)、式(A11)、または式(A12)に示すアミダイトを用いて、配列(II)に示す核酸オリゴマーをNTS M-4MX-E(日本テクノサービス社製)により、3’側から5’側に向かって自動合成した。自動合成の手順は、まず、3%ジクロロ酢酸トルエン溶液をCPGに送液し、5’位のトリチル保護基を脱保護した。この際、使用したジクロロ酢酸溶液中のホルムアルデヒド濃度は、測定方法3により測定することができ、ジクロロ酢酸溶液中のホルムアルデヒドとジクロロ酢酸のモル比(ホルムアルデヒドmol/ジクロロ酢酸mol)は、43×10-7であった。続いて、各種アミダイトと縮合剤として5-ベンジルメルカプト-1H-テトラゾールをCPGに送液し、5’位の水酸基にカップリング反応を進行させた。続いて、50mMヨウ素を含む酸化溶液を送液し、亜リン酸基をリン酸基に変換した。続いて、キャッピング溶液として0.1Mフェノキシ酢酸無水物アセトニトリル溶液と10%N-メチルイミダゾール/10%2,6-ルチジンアセトニトリル溶液を使用し、カップリングが進行しなかった反応点にキャッピングを施した。更にこれらの工程を合計49回繰り返した後、5’末端の塩基における保護基(DMTr基)を3%ジクロロ酢酸トルエン溶液で脱保護し、配列(II)に示される配列の核酸オリゴヌクレオチドをCPG担体上に合成した。その後、1.03μmol分のオリゴヌクレオチドを担持したCPG担体に対して、28%アンモニア水1.5mLとエタノール0.5mLを流入し、混合物を40℃で4時間保温することで核酸オリゴマーを固相担体から遊離させた後、濃縮により溶媒を除去した。次いで遊離オリゴヌクレオチドをジメチルスルホキシド1.5mLに溶解後、アセトニトリル1.0mL、ニトロメタン20μLと撹子を入れた後、モレキュラーシーブ4Aにて脱水処理を施した1Mのフッ化テトラ-n-ブチルアンモニウム(TBAF)のジメチルスルホキシド溶液2.08mLをスターラーによる攪拌下室温で流入し、混合物を33℃で4時間保温することで2’-EMM保護基の脱保護を行った。その後、核酸オリゴマーの生成物を沈殿操作により得た。得られた生成物について、前記測定方法1に記載の方法を用いて、オリゴヌクレオチドの純度を測定した結果、純度は50.8%であった。また、前記測定方法2に記載の方法を用いて、オリゴヌクレオチドの収量を測定したところ、収量は9156μgであり、1.00μmolのウリジン誘導体を担持したCPGあたりの収量に換算すると8889μgであった。結果を表2に示す。
実施例3の実験において、1.05μmolのウリジン誘導体を担持したControlled Pore Glass(CPG)と、ホルムアルデヒドとジクロロ酢酸のモル比(ホルムアルデヒドmol/ジクロロ酢酸mol)が90×10-6である3%ジクロロ酢酸トルエン溶液を用いる以外は、同様の方法で配列(II)の核酸オリゴマーを得た。前記測定方法1に記載の方法を用いて、オリゴヌクレオチドの純度を測定した結果、生成物の純度は47.8%であった。また、前記測定方法2に記載の方法を用いて、オリゴヌクレオチドの収量を測定したところ、収量は9378μgであり、1.00μmolのウリジン誘導体を担持したCPGあたりの収量に換算すると8931μgであった。結果を表2に示す。
実施例3の実験において、1.05μmolのウリジン誘導体を担持したControlled Pore Glass(CPG)と、ホルムアルデヒドとジクロロ酢酸のモル比(ホルムアルデヒドmol/ジクロロ酢酸mol)が20×10-5である3%ジクロロ酢酸トルエン溶液を用いる以外は、同様の方法で配列(II)の核酸オリゴマーを得た。前記測定方法1に記載の方法を用いて、オリゴヌクレオチドの純度を測定した結果、生成物の純度は42.8%であった。また、前記測定方法2に記載の方法を用いて、オリゴヌクレオチドの収量を測定したところ、収量は9307μgであり、1.00μmolのウリジン誘導体を担持したCPGあたりの収量に換算すると8864μgであった。結果を表2に示す。
0.99μmolのウリジン誘導体を担持したControlled Pore Glass(CPG)と、式(A9)、式(A10)、式(A11)、または式(A12)に示すアミダイトを用いて、配列(III)に示す核酸オリゴマーをNTS M-4MX-E(日本テクノサービス社製)により、3’側から5’側に向かって自動合成した。自動合成の手順は、まず、3%ジクロロ酢酸トルエン溶液をCPGに送液し、5’位のトリチル保護基を脱保護した。この際、使用したジクロロ酢酸溶液中のホルムアルデヒド濃度は、測定方法3により測定することができ、ジクロロ酢酸溶液中のホルムアルデヒドとジクロロ酢酸のモル比(ホルムアルデヒドmol/ジクロロ酢酸mol)は、43×10-7であった。続いて、各種アミダイトと縮合剤として5-ベンジルメルカプト-1H-テトラゾールをCPGに送液し、5’位の水酸基にカップリング反応を進行させた。続いて、50mMヨウ素を含む酸化溶液を送液し、亜リン酸基をリン酸基に変換した。続いて、キャッピング溶液として0.1Mフェノキシ酢酸無水物アセトニトリル溶液と10%N-メチルイミダゾール/10%2,6-ルチジンアセトニトリル溶液を使用し、カップリングが進行しなかった反応点にキャッピングを施した。更にこれらの工程を合計99回繰り返した後、5’末端の塩基における保護基(DMTr基)を3%ジクロロ酢酸トルエン溶液で脱保護し、配列(III)に示される配列の核酸オリゴヌクレオチドをCPG担体上に合成した。その後、1.03μmol分のオリゴヌクレオチドを担持したCPG担体に対して、28%アンモニア水1.5mLとエタノール0.5mLを流入し、混合物を40℃で4時間保温することで核酸オリゴマーを固相担体から遊離させた後、濃縮により溶媒を除去した。次いで遊離オリゴヌクレオチドをジメチルスルホキシド1.5mLに溶解後、アセトニトリル1.0mL、ニトロメタン20μLと撹子を入れた後、モレキュラーシーブ4Aにて脱水処理を施した1Mのフッ化テトラ-n-ブチルアンモニウム(TBAF)のジメチルスルホキシド溶液2.08mLをスターラーによる攪拌下室温で流入し、混合物を33℃で4時間保温することで2’-EMM保護基の脱保護を行った。その後、核酸オリゴマーの生成物を沈殿操作により得た。得られた生成物について、前記測定方法1に記載の方法を用いて、オリゴヌクレオチドの純度を測定した結果、純度は33.1%であった。また、前記測定方法2に記載の方法を用いて、オリゴヌクレオチドの収量を測定したところ、収量は12889μgであり、1.00μmolのウリジン誘導体を担持したCPGあたりの収量に換算すると13019μgであった。結果を表2に示す。
実施例5の実験において、1.04μmolのウリジン誘導体を担持したControlled Pore Glass(CPG)と、ホルムアルデヒドとジクロロ酢酸のモル比(ホルムアルデヒドmol/ジクロロ酢酸mol)が90×10-6である3%ジクロロ酢酸トルエン溶液を用いる以外は、同様の方法で配列(III)の核酸オリゴマーを得た。前記測定方法1に記載の方法を用いて、オリゴヌクレオチドの純度を測定した結果、生成物の純度は29.7%であった。また、前記測定方法2に記載の方法を用いて、オリゴヌクレオチドの収量を測定したところ、収量は13375μgであり、1.00μmolのウリジン誘導体を担持したCPGあたりの収量に換算すると12861μgであった。結果を表2に示す。
実施例5の実験において、0.99μmolのウリジン誘導体を担持したControlled Pore Glass(CPG)と、ホルムアルデヒドとジクロロ酢酸のモル比(ホルムアルデヒドmol/ジクロロ酢酸mol)が20×10-5である3%ジクロロ酢酸トルエン溶液を用いる以外は、同様の方法で配列(III)の核酸オリゴマーを得た。前記測定方法1に記載の方法を用いて、オリゴヌクレオチドの純度を測定した結果、生成物の純度は26.4%であった。また、前記測定方法2に記載の方法を用いて、オリゴヌクレオチドの収量を測定したところ、収量は12675μgであり、1.00μmolのウリジン誘導体を担持したCPGあたりの収量に換算すると12803μgであった。結果を表2に示す。
ホルムアルデヒドとジクロロ酢酸のモル比(ホルムアルデヒドmol/ジクロロ酢酸mol)が25×10-5であるジクロロ酢酸溶液30gに、トルエン300mLを加え、エバポレーターを用いて、40℃でトルエンとホルムアルデヒドを共沸留去し、微黄色油状のジクロロ酢酸溶液34gを得た。得られたジクロロ酢酸溶液に含まれるホルムアルデヒドを測定方法3に記載の方法で分析したところ、ホルムアルデヒドとジクロロ酢酸のモル比は、43×10-7であった。
Claims (24)
- 式(1):
G2は、水酸基の保護基を示し、
Baは、それぞれ独立して、同一又は相異なって、保護基で保護されていてもよい核酸塩基を表し、
R1、R2及びR3は、それぞれ独立して、同一又は相異なって、水素原子又はアルコキシ基を表し、
Rは、それぞれ独立して、同一又は相異なって、保護された水酸基、水素原子、フッ素原子、メトキシ基、2-メトキシエチル基、またはOQ’基を表し、
Q’は、それぞれ独立して、同一又は相異なって、リボースの4’位の炭素原子と結合しているメチレン基、リボースの4’位の炭素原子と結合しているエチレン基、またはリボースの4’位の炭素原子と結合しているエチリデン基を表し、
Yは、それぞれ独立して、同一又は相異なって、酸素原子または硫黄原子を表し、
nは、1~200の何れかの整数を表し、
W1は、OZ基を表し、かつ、X1は、R基を表すか、あるいは
W1は、OV基を表し、かつ、X1は、OZ基を表し、
Vは、水酸基の保護基を表し、
Zは、固相担体および連結基からなる構造を有する基である。
そして、nが2以上の整数のとき、式(1)で示される核酸オリゴマーは、それぞれのヌクレオチドの間に、非ヌクレオチドリンカーが組み込まれていてもよい。)
で示される核酸オリゴマーと、ホルムアルデヒドとジクロロ酢酸のモル比(ホルムアルデヒドmol/ジクロロ酢酸mol)が、90×10-6以下であるジクロロ酢酸溶液を反応させる工程を含む、式(2):
G2、Ba、R、Y、X1、W1およびnは、前記のとおりであり、そして、
式(1)において定義されたとおり、ヌクレオチドの間に、非ヌクレオチドリンカーが組み込まれていてもよい。)
で示される核酸オリゴマーの製造方法。 - 請求項1に記載の工程と、さらに、当該工程で生成する式(2)で示される核酸オリゴマーからZで表される基を除く工程、ならびに水酸基および核酸塩基の保護基を除く工程を含む、式(2’):
Yおよびnは、前記のとおりであり、
Bcは、それぞれ独立して、同一又は相異なって、核酸塩基を表し、
G4は、それぞれ独立して、同一又は相異なって、水素原子、アルカリ金属イオン、アンモニウムイオン、アルキルアンモニウムイオン、またはヒドロキシアルキルアンモニウムイオンを表し、
R’は、それぞれ独立して、同一又は相異なって、水酸基、水素原子、フッ素原子、メトキシ基、2-メトキシエチル基、またはOQ’基を表し、
Q’は、前記のとおりであり、
X3およびW3は各々、それぞれ独立して、水酸基を表すか、あるいは
X3は、R’基を表し、かつ、W3は、水酸基を表す。)
で示される核酸オリゴマーの製造方法。 - 式(2)で示される核酸オリゴマーを、アミダイト法で任意に鎖長を伸長した式(3):
G2、Ba、R、Y、X1およびW1は、前記のとおりであり、
G5は、
R1、R2及びR3は、前記のとおりであり、
mは、m≧nを満たす整数である。)
で示される核酸化合物を得る工程、ならびに
式(3)で示される化合物から式(4):
G5、R、Yおよびmは、前記のとおりであり、
G4は、それぞれ独立して、同一又は相異なって、水素原子、アルカリ金属イオン、アンモニウムイオン、アルキルアンモニウムイオン、またはヒドロキシアルキルアンモニウムイオンを表し、
BCは、それぞれ独立して、同一又は相異なって、核酸塩基を表し、
X2は、水酸基を表し、かつ、W2は、OV基を表すか、あるいは
X2は、R基を表し、かつ、W2は、水酸基を表し、
Vは、水酸基の保護基を表す。)
で示される化合物を切り出し、
さらに式(4)で示される化合物を脱保護して、式(5):
G4、Bc、Yおよびmは、前記のとおりであり、
R’は、それぞれ独立して、同一又は相異なって、水酸基、水素原子、フッ素原子、メトキシ基、2-メトキシエチル基、またはOQ’基を表し、
Q’は、前記のとおりであり、
X3およびW3は各々、それぞれ独立して、水酸基を表すか、あるいは
X3は、R’基を表し、かつ、W3は、水酸基を表す。)
で示される核酸オリゴマーを製造する工程をさらに含む、請求項1に記載の製造方法。 - 非ヌクレオチドリンカーが、アミノ酸骨格からなるリンカーである、請求項1~3の何れか一項に記載の製造方法。
- ジクロロ酢酸溶液が、ジクロロメタン、アセトニトリル、および芳香族有機溶媒からなる群から選ばれる少なくとも1つの溶媒を含む、請求項1~5の何れか一項に記載の製造方法。
- ジクロロ酢酸溶液中のホルムアルデヒドとジクロロ酢酸のモル比(ホルムアルデヒドmol/ジクロロ酢酸mol)が、43×10-6以下である、請求項1~6の何れか一項に記載の製造方法。
- ジクロロ酢酸溶液中のホルムアルデヒドとジクロロ酢酸のモル比(ホルムアルデヒドmol/ジクロロ酢酸mol)が、22×10-6以下である、請求項1~6の何れか一項に記載の製造方法。
- 核酸オリゴマーが、リボ核酸(RNA)である、請求項1~8の何れか一項に記載の製造方法。
- RaおよびRbが同時に水素原子であり、かつ、EWがシアノ基である、請求項10に記載の製造方法。
- 核酸オリゴマーが、40鎖長以上のオリゴマーである、請求項1~11の何れか一項に記載の製造方法。
- 核酸オリゴマーが、50鎖長以上のオリゴマーである、請求項1~11の何れか一項に記載の製造方法。
- 核酸オリゴマーが、60鎖長以上のオリゴマーである、請求項1~11の何れか一項に記載の製造方法。
- 核酸オリゴマーが、80鎖長以上のオリゴマーである、請求項1~11の何れか一項に記載の製造方法。
- 核酸オリゴマーが、100鎖長以上のオリゴマーである、請求項1~11の何れか一項に記載の製造方法。
- ホルムアルデヒドとジクロロ酢酸のモル比(ホルムアルデヒドmol/ジクロロ酢酸mol)が、90×10-6以下である、ジクロロ酢酸溶液。
- ホルムアルデヒドとジクロロ酢酸のモル比(ホルムアルデヒドmol/ジクロロ酢酸mol)が、43×10-6以下である、請求項17に記載のジクロロ酢酸溶液。
- ホルムアルデヒドとジクロロ酢酸のモル比(ホルムアルデヒドmol/ジクロロ酢酸mol)が、22×10-6以下である、請求項17に記載のジクロロ酢酸溶液。
- ホルムアルデヒドを含む未精製ジクロロ酢酸溶液とホルムアルデヒドと共沸する溶媒を含む溶液からホルムアルデヒドを共沸させて留去することにより精製ジクロロ酢酸溶液を得る工程を含む、請求項17~19の何れか一項に記載のジクロロ酢酸溶液の製造方法。
- 共沸溶媒の沸点が、194℃以下である、請求項20に記載の製造方法。
- 共沸溶媒が、ジクロロメタン、アセトニトリルまたは芳香族有機溶媒である、請求項20または21に記載の製造方法。
- 芳香族有機溶媒がトルエンである、請求項22に記載の製造方法。
- 請求項20に記載のジクロロ酢酸溶液の精製工程および当該工程で得られた精製ジクロロ酢酸溶液を使用する請求項1~3の何れか一項に記載の工程を含む、核酸オリゴマーの製造方法。
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17/906,820 US20230167152A1 (en) | 2020-03-27 | 2021-03-26 | Method for producing nucleic acid oligomer |
CN202180023282.5A CN115335387A (zh) | 2020-03-27 | 2021-03-26 | 核酸寡聚物的制造方法 |
JP2022510762A JPWO2021193954A1 (ja) | 2020-03-27 | 2021-03-26 | |
KR1020227035118A KR20220160010A (ko) | 2020-03-27 | 2021-03-26 | 핵산 올리고머의 제조 방법 |
EP21774023.2A EP4130018A4 (en) | 2020-03-27 | 2021-03-26 | PROCESS FOR THE PRODUCTION OF NUCLEIC ACID OLIGOMERS |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2020-058880 | 2020-03-27 | ||
JP2020058880 | 2020-03-27 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2021193954A1 true WO2021193954A1 (ja) | 2021-09-30 |
Family
ID=77892293
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2021/013023 WO2021193954A1 (ja) | 2020-03-27 | 2021-03-26 | 核酸オリゴマーの製造方法 |
Country Status (6)
Country | Link |
---|---|
US (1) | US20230167152A1 (ja) |
EP (1) | EP4130018A4 (ja) |
JP (1) | JPWO2021193954A1 (ja) |
KR (1) | KR20220160010A (ja) |
CN (1) | CN115335387A (ja) |
WO (1) | WO2021193954A1 (ja) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2023054350A1 (ja) * | 2021-09-28 | 2023-04-06 | 住友化学株式会社 | 精製ジクロロ酢酸の製造方法 |
Citations (22)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999043694A1 (en) | 1998-02-26 | 1999-09-02 | Isis Pharmaceuticals, Inc. | Improved methods for synthesis of oligonucleotides |
WO2001053528A1 (en) | 2000-01-18 | 2001-07-26 | Isis Pharmaceuticals, Inc. | Antisense inhibition of ptp1b expression |
JP3745226B2 (ja) | 1998-09-29 | 2006-02-15 | アイシス・ファーマシューティカルス・インコーポレーテッド | サービビン発現のアンチセンス・モジュレーション |
WO2006022323A1 (ja) | 2004-08-26 | 2006-03-02 | Nippon Shinyaku Co., Ltd. | ホスホロアミダイト化合物及びオリゴrnaの製法 |
JP4705716B2 (ja) | 1999-02-05 | 2011-06-22 | ジーイー・ヘルスケア・バイオサイエンス・コーポレイション | オリゴヌクレオチドの脱保護法 |
WO2012005368A1 (ja) | 2010-07-08 | 2012-01-12 | 株式会社ボナック | 遺伝子発現制御のための一本鎖核酸分子 |
WO2012017919A1 (ja) * | 2010-08-03 | 2012-02-09 | 株式会社ボナック | 含窒素脂環式骨格を有する一本鎖核酸分子 |
US20120035246A1 (en) | 2010-08-03 | 2012-02-09 | Bonac Corporation | Single-stranded nucleic acid molecule having nitrogen-containing alicyclic skeleton |
WO2013027843A1 (ja) | 2011-08-25 | 2013-02-28 | 株式会社ボナック | 配糖体化合物、チオエーテルの製造方法、エーテル、エーテルの製造方法、配糖体化合物の製造方法、核酸の製造方法 |
WO2013103146A1 (ja) * | 2012-01-07 | 2013-07-11 | 株式会社ボナック | アミノ酸骨格を有する一本鎖核酸分子 |
JP2014221817A (ja) | 2006-10-18 | 2014-11-27 | アイシス ファーマシューティカルズ, インコーポレーテッド | アンチセンス化合物 |
JP2015523856A (ja) | 2012-05-25 | 2015-08-20 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフ | Rna依存性標的dna修飾およびrna依存性転写調節のための方法および組成物 |
WO2016158809A1 (ja) * | 2015-03-27 | 2016-10-06 | 株式会社ボナック | デリバリー機能と遺伝子発現制御能を有する一本鎖核酸分子 |
WO2017131237A1 (ja) * | 2016-01-30 | 2017-08-03 | 株式会社ボナック | 人工単一ガイドrna及びその用途 |
JP2017537626A (ja) | 2014-12-03 | 2017-12-21 | アジレント・テクノロジーズ・インクAgilent Technologies, Inc. | 化学修飾を有するガイドrna |
WO2018182008A1 (ja) | 2017-03-31 | 2018-10-04 | 株式会社ボナック | 遺伝子発現制御機能を有する環状型核酸分子 |
WO2019060442A1 (en) | 2017-09-19 | 2019-03-28 | Alnylam Pharmaceuticals, Inc. | COMPOSITIONS AND METHODS FOR TREATMENT OF TRANSTHYRETIN MEDIATED AMYLOSIS (TTR) |
WO2019074110A1 (ja) | 2017-10-13 | 2019-04-18 | 株式会社ボナック | 一本鎖核酸分子およびその製造方法 |
WO2019189722A1 (ja) * | 2018-03-30 | 2019-10-03 | 東レ株式会社 | ヘアピン型一本鎖rna分子の製造方法 |
WO2019208571A1 (ja) | 2018-04-24 | 2019-10-31 | 住友化学株式会社 | アミダイト化合物及び該化合物を用いたポリヌクレオチドの製造方法 |
JP2020058880A (ja) | 2015-07-31 | 2020-04-16 | ザ プロクター アンド ギャンブル カンパニーThe Procter & Gamble Company | 成形された不織布を用いた吸収性物品のパッケージ |
WO2021024465A1 (ja) * | 2019-08-08 | 2021-02-11 | 住友化学株式会社 | 核酸分子の製造方法 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011005595A2 (en) * | 2009-06-24 | 2011-01-13 | Alios Biopharma, Inc. | 2-5a analogs and their methods of use |
JP6728075B2 (ja) * | 2014-06-24 | 2020-07-22 | ヤンセン バイオファーマ インク. | 置換ヌクレオシド、ヌクレオチドおよびその類似体 |
-
2021
- 2021-03-26 US US17/906,820 patent/US20230167152A1/en active Pending
- 2021-03-26 WO PCT/JP2021/013023 patent/WO2021193954A1/ja active Application Filing
- 2021-03-26 JP JP2022510762A patent/JPWO2021193954A1/ja active Pending
- 2021-03-26 CN CN202180023282.5A patent/CN115335387A/zh active Pending
- 2021-03-26 KR KR1020227035118A patent/KR20220160010A/ko unknown
- 2021-03-26 EP EP21774023.2A patent/EP4130018A4/en active Pending
Patent Citations (25)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999043694A1 (en) | 1998-02-26 | 1999-09-02 | Isis Pharmaceuticals, Inc. | Improved methods for synthesis of oligonucleotides |
JP3745226B2 (ja) | 1998-09-29 | 2006-02-15 | アイシス・ファーマシューティカルス・インコーポレーテッド | サービビン発現のアンチセンス・モジュレーション |
JP4705716B2 (ja) | 1999-02-05 | 2011-06-22 | ジーイー・ヘルスケア・バイオサイエンス・コーポレイション | オリゴヌクレオチドの脱保護法 |
WO2001053528A1 (en) | 2000-01-18 | 2001-07-26 | Isis Pharmaceuticals, Inc. | Antisense inhibition of ptp1b expression |
WO2006022323A1 (ja) | 2004-08-26 | 2006-03-02 | Nippon Shinyaku Co., Ltd. | ホスホロアミダイト化合物及びオリゴrnaの製法 |
JP5157168B2 (ja) | 2004-08-26 | 2013-03-06 | 日本新薬株式会社 | ホスホロアミダイト化合物及びオリゴrnaの製法 |
JP2014221817A (ja) | 2006-10-18 | 2014-11-27 | アイシス ファーマシューティカルズ, インコーポレーテッド | アンチセンス化合物 |
WO2012005368A1 (ja) | 2010-07-08 | 2012-01-12 | 株式会社ボナック | 遺伝子発現制御のための一本鎖核酸分子 |
WO2012017919A1 (ja) * | 2010-08-03 | 2012-02-09 | 株式会社ボナック | 含窒素脂環式骨格を有する一本鎖核酸分子 |
US20120035246A1 (en) | 2010-08-03 | 2012-02-09 | Bonac Corporation | Single-stranded nucleic acid molecule having nitrogen-containing alicyclic skeleton |
JP4965745B2 (ja) | 2010-08-03 | 2012-07-04 | 株式会社ボナック | 含窒素脂環式骨格を有する一本鎖核酸分子 |
JP5554881B2 (ja) | 2011-08-25 | 2014-07-23 | 株式会社ボナック | 配糖体化合物、チオエーテルの製造方法、エーテル、エーテルの製造方法、配糖体化合物の製造方法、核酸の製造方法 |
WO2013027843A1 (ja) | 2011-08-25 | 2013-02-28 | 株式会社ボナック | 配糖体化合物、チオエーテルの製造方法、エーテル、エーテルの製造方法、配糖体化合物の製造方法、核酸の製造方法 |
WO2013103146A1 (ja) * | 2012-01-07 | 2013-07-11 | 株式会社ボナック | アミノ酸骨格を有する一本鎖核酸分子 |
JP2015523856A (ja) | 2012-05-25 | 2015-08-20 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフ | Rna依存性標的dna修飾およびrna依存性転写調節のための方法および組成物 |
JP2017537626A (ja) | 2014-12-03 | 2017-12-21 | アジレント・テクノロジーズ・インクAgilent Technologies, Inc. | 化学修飾を有するガイドrna |
WO2016158809A1 (ja) * | 2015-03-27 | 2016-10-06 | 株式会社ボナック | デリバリー機能と遺伝子発現制御能を有する一本鎖核酸分子 |
JP2020058880A (ja) | 2015-07-31 | 2020-04-16 | ザ プロクター アンド ギャンブル カンパニーThe Procter & Gamble Company | 成形された不織布を用いた吸収性物品のパッケージ |
WO2017131237A1 (ja) * | 2016-01-30 | 2017-08-03 | 株式会社ボナック | 人工単一ガイドrna及びその用途 |
WO2018182008A1 (ja) | 2017-03-31 | 2018-10-04 | 株式会社ボナック | 遺伝子発現制御機能を有する環状型核酸分子 |
WO2019060442A1 (en) | 2017-09-19 | 2019-03-28 | Alnylam Pharmaceuticals, Inc. | COMPOSITIONS AND METHODS FOR TREATMENT OF TRANSTHYRETIN MEDIATED AMYLOSIS (TTR) |
WO2019074110A1 (ja) | 2017-10-13 | 2019-04-18 | 株式会社ボナック | 一本鎖核酸分子およびその製造方法 |
WO2019189722A1 (ja) * | 2018-03-30 | 2019-10-03 | 東レ株式会社 | ヘアピン型一本鎖rna分子の製造方法 |
WO2019208571A1 (ja) | 2018-04-24 | 2019-10-31 | 住友化学株式会社 | アミダイト化合物及び該化合物を用いたポリヌクレオチドの製造方法 |
WO2021024465A1 (ja) * | 2019-08-08 | 2021-02-11 | 住友化学株式会社 | 核酸分子の製造方法 |
Non-Patent Citations (6)
Title |
---|
DANIEL O'REILLY ET AL., NUCLEIC ACIDS RESEARCH, vol. 47, no. 2, 2019, pages 547 - 558 |
LONNBERG,H.: "Synthesis of oligonucleotides on a soluble support", BEILSTEIN JOURNAL OF ORGANIC CHEMISTRY, vol. 13, 2017, pages 1368 - 1387, XP055798685, ISSN: 1860-5397, DOI: 10.3762/bjoc.13.134 * |
PAUL,C.H. ET AL.: "Acid binding and detritylation during oligonucleotide synthesis", NUCLEIC ACIDS RESEARCH, vol. 24, no. 15, pages 3048 - 3052, XP055787716, ISSN: 0305-1048, DOI: 10.1093/nar/24.15.3048 * |
See also references of EP4130018A4 |
SEPTAK,M.: "Kinetic studies on depurination and detritylation of CPG-bound intermediates during oligonucleotide synthesis", NUCLEIC ACIDS RESEARCH, vol. 24, no. 15, 1996, pages 3053 - 3058, XP055784527, ISSN: 0305-1048, DOI: 10.1093/nar/24.15.3053 * |
XIULONG, SHEN ET AL., NUCLEIC ACIDS RESEARCH, vol. 46, no. 46, 2018, pages 1584 - 1600 |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2023054350A1 (ja) * | 2021-09-28 | 2023-04-06 | 住友化学株式会社 | 精製ジクロロ酢酸の製造方法 |
Also Published As
Publication number | Publication date |
---|---|
EP4130018A4 (en) | 2024-04-17 |
CN115335387A (zh) | 2022-11-11 |
US20230167152A1 (en) | 2023-06-01 |
JPWO2021193954A1 (ja) | 2021-09-30 |
KR20220160010A (ko) | 2022-12-05 |
EP4130018A1 (en) | 2023-02-08 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP2019514999A (ja) | ヌクレオシドまたはオリゴヌクレオチドへの、立体的に規定されたオキサザホスホリジンホスホルアミダイト単量体のカップリングの増大法 | |
WO2022009959A1 (ja) | 核酸オリゴマーの製造方法 | |
JP7164540B2 (ja) | 立体的に規定されたホスホロチオエートオリゴヌクレオチドの調製のための直交保護基 | |
AU716391B2 (en) | Solid phase synthesis of oligonucleotides | |
JPH09510206A (ja) | オリゴヌクレオチドの合成に用いる組成物および方法 | |
WO2021193954A1 (ja) | 核酸オリゴマーの製造方法 | |
WO2021153047A1 (ja) | 核酸オリゴマーの製造方法 | |
WO2021153770A1 (en) | Process of preparing nucleic acid oligomer | |
WO2002018406A1 (en) | Alkylated hexitol nucleoside analogues and oligomers thereof | |
WO2023054350A1 (ja) | 精製ジクロロ酢酸の製造方法 | |
WO2024024873A1 (ja) | チオ化溶液 | |
WO2024019137A1 (ja) | オリゴヌクレオチドの製造方法 | |
WO2024089953A1 (ja) | オリゴヌクレオチドの製造方法 | |
CN117980285A (zh) | 纯化二氯乙酸的制造方法 | |
WO2022064908A1 (ja) | 核酸オリゴマーの製造方法 | |
WO2021210408A1 (ja) | 核酸オリゴマーの製造方法 | |
WO2021070494A1 (ja) | 核酸オリゴマーの製造方法 | |
EP1828218B1 (en) | Synthesis of phosphitylated compounds using a quaternary heterocyclic activator | |
WO2021075423A1 (ja) | ホスホロアミダイト活性化剤 | |
CN115315430A (zh) | 使用了链段型亚磷酰胺化合物的核酸合成法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 21774023 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2022510762 Country of ref document: JP Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 20227035118 Country of ref document: KR Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2021774023 Country of ref document: EP |
|
ENP | Entry into the national phase |
Ref document number: 2021774023 Country of ref document: EP Effective date: 20221027 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |