CN1158618A - Sequence-specific binding oligomers for nucleic acids and their use in antisense strategies - Google Patents
Sequence-specific binding oligomers for nucleic acids and their use in antisense strategies Download PDFInfo
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- CN1158618A CN1158618A CN 95195211 CN95195211A CN1158618A CN 1158618 A CN1158618 A CN 1158618A CN 95195211 CN95195211 CN 95195211 CN 95195211 A CN95195211 A CN 95195211A CN 1158618 A CN1158618 A CN 1158618A
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- Prior art keywords
- oligomer
- formula
- base
- heterocycle
- sequence
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- 230000000692 anti-sense effect Effects 0.000 title claims description 14
- 108020004707 nucleic acids Proteins 0.000 title claims description 6
- 150000007523 nucleic acids Chemical class 0.000 title claims description 6
- 102000039446 nucleic acids Human genes 0.000 title claims description 6
- 230000009870 specific binding Effects 0.000 title 1
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 claims abstract description 23
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 claims abstract description 18
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 claims abstract description 14
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 claims abstract description 14
- 125000000623 heterocyclic group Chemical group 0.000 claims abstract description 9
- 229940104302 cytosine Drugs 0.000 claims abstract description 6
- LRFVTYWOQMYALW-UHFFFAOYSA-N 9H-xanthine Chemical compound O=C1NC(=O)NC2=C1NC=N2 LRFVTYWOQMYALW-UHFFFAOYSA-N 0.000 claims abstract description 5
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 claims abstract description 3
- MSSXOMSJDRHRMC-UHFFFAOYSA-N 9H-purine-2,6-diamine Chemical compound NC1=NC(N)=C2NC=NC2=N1 MSSXOMSJDRHRMC-UHFFFAOYSA-N 0.000 claims abstract description 3
- 229940075420 xanthine Drugs 0.000 claims abstract 2
- 239000002777 nucleoside Substances 0.000 claims description 26
- 238000000034 method Methods 0.000 claims description 19
- 150000001875 compounds Chemical class 0.000 claims description 14
- 239000011782 vitamin Substances 0.000 claims description 14
- 229940088594 vitamin Drugs 0.000 claims description 14
- 229930003231 vitamin Natural products 0.000 claims description 14
- 235000013343 vitamin Nutrition 0.000 claims description 14
- 239000000178 monomer Substances 0.000 claims description 12
- 150000003833 nucleoside derivatives Chemical class 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 9
- 150000003839 salts Chemical group 0.000 claims description 8
- 210000001541 thymus gland Anatomy 0.000 claims description 8
- 238000005516 engineering process Methods 0.000 claims description 7
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- 230000008836 DNA modification Effects 0.000 claims description 3
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 claims description 3
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 3
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 3
- 238000003745 diagnosis Methods 0.000 claims description 3
- 238000009396 hybridization Methods 0.000 claims description 3
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical group [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims description 2
- 239000005864 Sulphur Chemical group 0.000 claims description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical group [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims description 2
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- MPCAJMNYNOGXPB-UHFFFAOYSA-N 1,5-anhydrohexitol Chemical compound OCC1OCC(O)C(O)C1O MPCAJMNYNOGXPB-UHFFFAOYSA-N 0.000 abstract description 3
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 abstract 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 abstract 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 abstract 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 abstract 1
- 229930024421 Adenine Natural products 0.000 abstract 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 abstract 1
- 229960000643 adenine Drugs 0.000 abstract 1
- 229940127073 nucleoside analogue Drugs 0.000 abstract 1
- 229940113082 thymine Drugs 0.000 abstract 1
- 229940035893 uracil Drugs 0.000 abstract 1
- 108091034117 Oligonucleotide Proteins 0.000 description 27
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 24
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Substances OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 22
- FBPFZTCFMRRESA-UHFFFAOYSA-N hexane-1,2,3,4,5,6-hexol Chemical compound OCC(O)C(O)C(O)C(O)CO FBPFZTCFMRRESA-UHFFFAOYSA-N 0.000 description 20
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 17
- 238000002844 melting Methods 0.000 description 17
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- -1 furanose compound Chemical class 0.000 description 14
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 12
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 12
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 12
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- 108020004414 DNA Proteins 0.000 description 6
- 238000004992 fast atom bombardment mass spectroscopy Methods 0.000 description 6
- 239000002773 nucleotide Substances 0.000 description 6
- 150000003214 pyranose derivatives Chemical class 0.000 description 6
- KHWCHTKSEGGWEX-RRKCRQDMSA-N 2'-deoxyadenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(O)=O)O1 KHWCHTKSEGGWEX-RRKCRQDMSA-N 0.000 description 5
- KHWCHTKSEGGWEX-UHFFFAOYSA-N deoxyadenylic acid Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(O)=O)O1 KHWCHTKSEGGWEX-UHFFFAOYSA-N 0.000 description 5
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 5
- 125000003729 nucleotide group Chemical group 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 4
- PGKUAPBTNAPDFG-UHFFFAOYSA-N dimethoxy(trityl)-$l^{3}-chlorane Chemical compound C=1C=CC=CC=1C(C=1C=CC=CC=1)(Cl(OC)OC)C1=CC=CC=C1 PGKUAPBTNAPDFG-UHFFFAOYSA-N 0.000 description 4
- 239000012044 organic layer Substances 0.000 description 4
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- 229920006395 saturated elastomer Polymers 0.000 description 4
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- 108091081021 Sense strand Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 0 *C1CC(O)I(CO)*C1 Chemical compound *C1CC(O)I(CO)*C1 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 2
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- 229910004373 HOAc Inorganic materials 0.000 description 2
- 101710163270 Nuclease Proteins 0.000 description 2
- 230000010748 Photoabsorption Effects 0.000 description 2
- 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 2
- 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 2
- 229950006790 adenosine phosphate Drugs 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 125000000649 benzylidene group Chemical group [H]C(=[*])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 2
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- 229940046166 oligodeoxynucleotide Drugs 0.000 description 2
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- RINCXYDBBGOEEQ-UHFFFAOYSA-N succinic anhydride Chemical compound O=C1CCC(=O)O1 RINCXYDBBGOEEQ-UHFFFAOYSA-N 0.000 description 2
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- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 1
- MCTWTZJPVLRJOU-UHFFFAOYSA-N 1-methyl-1H-imidazole Chemical compound CN1C=CN=C1 MCTWTZJPVLRJOU-UHFFFAOYSA-N 0.000 description 1
- OLXZPDWKRNYJJZ-RRKCRQDMSA-N 2'-deoxyadenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](CO)O1 OLXZPDWKRNYJJZ-RRKCRQDMSA-N 0.000 description 1
- 125000001731 2-cyanoethyl group Chemical group [H]C([H])(*)C([H])([H])C#N 0.000 description 1
- CFIBTBBTJWHPQV-UHFFFAOYSA-N 2-methyl-n-(6-oxo-3,7-dihydropurin-2-yl)propanamide Chemical compound N1C(NC(=O)C(C)C)=NC(=O)C2=C1N=CN2 CFIBTBBTJWHPQV-UHFFFAOYSA-N 0.000 description 1
- XVMSFILGAMDHEY-UHFFFAOYSA-N 6-(4-aminophenyl)sulfonylpyridin-3-amine Chemical compound C1=CC(N)=CC=C1S(=O)(=O)C1=CC=C(N)C=N1 XVMSFILGAMDHEY-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 229940125907 SJ995973 Drugs 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 1
- 125000003282 alkyl amino group Chemical group 0.000 description 1
- 230000029936 alkylation Effects 0.000 description 1
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- 125000003368 amide group Chemical group 0.000 description 1
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- 150000002338 glycosides Chemical class 0.000 description 1
- 125000003147 glycosyl group Chemical group 0.000 description 1
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- HZVOZRGWRWCICA-UHFFFAOYSA-N methanediyl Chemical compound [CH2] HZVOZRGWRWCICA-UHFFFAOYSA-N 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- PJUIMOJAAPLTRJ-UHFFFAOYSA-N monothioglycerol Chemical compound OCC(O)CS PJUIMOJAAPLTRJ-UHFFFAOYSA-N 0.000 description 1
- XBDUZBHKKUFFRH-UHFFFAOYSA-N n-(2-oxo-1h-pyrimidin-6-yl)benzamide Chemical compound OC1=NC=CC(NC(=O)C=2C=CC=CC=2)=N1 XBDUZBHKKUFFRH-UHFFFAOYSA-N 0.000 description 1
- XZMHJYWMCRQSSI-UHFFFAOYSA-N n-[5-[2-(3-acetylanilino)-1,3-thiazol-4-yl]-4-methyl-1,3-thiazol-2-yl]benzamide Chemical compound CC(=O)C1=CC=CC(NC=2SC=C(N=2)C2=C(N=C(NC(=O)C=3C=CC=CC=3)S2)C)=C1 XZMHJYWMCRQSSI-UHFFFAOYSA-N 0.000 description 1
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- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
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- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Substances OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
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- IGFXRKMLLMBKSA-UHFFFAOYSA-N purine Chemical compound N1=C[N]C2=NC=NC2=C1 IGFXRKMLLMBKSA-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
- C07F9/65586—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
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Abstract
The invention relates to oligomers consisting completely or partially of 1,5-anhydrohexitol nucleoside analogues represented by general formula (I), wherein B is a heterocyclic ring which is derived from a pyrimidine or purine base, such as cytosine, 5-methylcytosine, uracil and thymine, or deaza derivatives thereof, or adenine, guanine, 2,6-diaminopurine, hypoxanthine and xanthine, or deaza derivatives thereof.
Description
The present invention relates to have the oligomer of nucleic acid binding characteristic, this oligomer is completely or partially with 1, and 5-anhydrohexitol nucleoside analog is that monomeric unit constitutes.The invention still further relates to the purposes that this oligomer is used for antisense technology, and the method for preparing this oligomer.
Antisense technology can be blockaded by a complementary antisense strand based on the function of such principle: DNA or RNA molecule encoding sense strand.Antisense technology can be used for various uses, as diagnosis, treatment, dna modification with separate etc.In these technology, except the stability of antisense strand self is very important, also very important by the bonding force of the stability of described sense strand and formed duplex of antisense strand or triplex and antisense strand and sense strand.Equally, described oligomer, duplex or triplex be to degrading enzyme, also is a factor relevant with its validity as the susceptibility of nuclease.
Oligonucleotide is that monomer is the oligomer of Nucleotide.Nucleotide is the phosphoric acid ester of nucleosides, and nucleosides is by a purine or pyrimidine base and sugared a composition.The main chain of each Nucleotide is made up of alternative glycosyl and phosphate.
The stability of Nucleotide and bonding force can be subjected to the influence of base modification for instance.The research that this is carried out (1-5) shows that this modification only can cause occurring the duplex of less stable.Change main chain or structure insertion main chain that will be new can improve nuclease stability really, still, only can the bonding force of itself and complementary strand be had a negative impact.Sugar modified to improve its avidity (6-8) limitedly target molecule.
The object of the present invention is to provide new oligomer, compare with known oligomer, this oligomer has advantages of higher stability and bonding force.
Have found that, completely or partially by 1,5-dehydration-2, the 3-oligomer that two deoxidations-D-arabinose base-hexitol nucleoside analog is formed (wherein, hexitol is connected with the heterocycle of pyrimidine or purine bases by its 2-position) can be with natural oligonucleotide combination.The monomer that constitutes oligomer is at least in part represented by formula I:
Wherein, B is a heterocycle, and it is derived by pyrimidine or purine bases.Described monomer interconnects by phosphodiester bond, and the structure of this oligomer is suc as formula shown in the II:
Wherein, B is a heterocycle of being derived by pyrimidine or purine bases and coming, and l is the integer of 0-15, respectively a do for oneself integer of 1～15 of k and m, still, if k＞1, then m can be 0, and if m＞1, then k can be 0; And wherein X represents oxygen or sulphur.The present invention includes all possible salt of formula II compound.The monomer of formula I is the theme of european patent application No.92201803.1.The oligomer of formula II is a kind of new compound.It has and similar some characteristics of the oligonucleotide of being made up of natural 2 '-deoxynucleoside, but the sugar of monomer whose has been increased because the oxide compound that encircles and with carbon atom that base is connected between inserted methylene radical.
According to the present invention, have found that the oligomer of formula II and salt thereof show the sequence specificity in conjunction with natural oligonucleotide shown in the formula III:
Wherein, k is an integer, and the definition of B identical with in formula I and II.Therefore, find that hybridons that a class is new or sequence specificity are in conjunction with polymer.
To have the fact than high-bond be very surprising to the oligomer of being made up of the pyranose nucleosides of the present invention at least in part.In the past few years, the scientific research group that is made up of Eschenmoser etc. is devoted to study the oligonucleotide of being made up of monomer pyranose Nucleotide always.Eschenmoser has studied the natural selection (9) of furanose as the sugared structural unit of nucleic acid.Yet he does not point out to need suitable antisense molecule into realizing with the good combination of natural furanose-DNA.
But, the inventor confirms that class pyranose oligonucleotide can form stable duplex (10,11) with natural furanose-DNA.Theoretically, the pyranose oligonucleotide has the free energy that is better than the furanose oligomer, because its Entropy Changes in duplex forms is less.
Yet the previous class pyranose oligonucleotide of studying of the inventor can not or can not be fully in conjunction with natural furanose-DNA complementary strand.Described class pyranose oligonucleotide is respectively by 2, and the two deoxidation-β of 3--D-is red-pyranohexose nucleosides (formula V), 2, and the two deoxidation-β of 4--D-is red-pyranohexose nucleosides (formula VI) and/or 3, and the two deoxidation-β of 4--D-is red-and pyranohexose nucleosides (formula VII) forms.
More surprising is, finds to comprise pyranose and has sequence specificity binding ability as the formula II oligomer of sugared structural unit.The furan nucleus of furanose compound is zoomed into pyranoid ring, and can't produce can be with natural oligonucleotide bonded oligomer.Therefore, can not expect the furan pentose ring is zoomed into 1 5-anhydrohexitol ring.
Therefore, compound of the present invention is the oligomer of nucleoside analog, wherein, 1,5-dehydration-2, the two deoxidations of 3--D-hexitol is to connect according to the heterocycle of arabinose base-configuration by same pyrimidine in 2-position or purine bases.
Interconnect with phosphodiester or thiophosphoric acid diester form by above-mentioned nucleoside analog and to form described oligomer.This oligomer can represent that wherein, k, l, m, B and X have above definition with formula II.This oligomer can only be formed (wherein, the l in the formula II equals 0) by the hexitol nucleoside analog of formula I, or inserts natural 2 '-deoxynucleoside at interval, or connects the end (wherein, the l in the formula II is equal to or greater than 1) at this molecule.Described hexitol has (D)-configuration, and its substituent stereochemistry meets arabinose base configuration.
When group B is to be derived and when coming, it can be cytosine(Cyt), 5-methylcytosine, uridylic or thymus pyrimidine by pyrimidine base.When B is derived and when coming, it can be VITAMIN B4, guanine, 2,6-diaminopurine, xanthoglobulin or xanthine ring, or the assorted derivative of the denitrogenation of one of them by purine bases.
Can prepare monomeric unit of the present invention with different methods is nucleoside analog, and one of preparation method is disclosed among the european patent application NO.92.201803.1.Described synthetic method (12) in the article of Verheggen etc. has similarly and discloses.Be combined into oligomer by monomer and carry out, can carry out (referring to document 13) by the phosphoramidite chemical process of standard, or carry out (referring to document 14) by H-phosphoric acid chemical process according to typical method.All methods all are to carry out on the automatization dna synthesizer according to common standard oligonucleotide synthesis method.Carry out this synthetic standard conditions referring to Molecular Biology (15).
Preferred method is a phosphoramidite method, this method with the phosphoramidite of hexitol nucleoside analog as the primary formation unit, along " 6 '-direction " combination.Described phosphoramidite is suc as formula shown in the VIII, wherein B
★Be to be suitable for the protected base of oligonucleotide synthetic (for example, thymus pyrimidine, N
4-benzoyl cytosine(Cyt), N
6-benzoyl VITAMIN B4 and N
2-isobutyryl guanine is represented by formula IX, X, XI and XII respectively).
Formula VIII product can prepare according to standard method.To the protection of cytosine(Cyt), VITAMIN B4 or guanine base is (16) of realizing by to the instantaneous protection of the hydroxylic moiety of formula I compound.But, it is desirable to, is by with 4 to the protection of base, the acidylate of the nucleoside analog 1a-d of 6-benzylidene protection and realize that this analogue is the synthetic monomeric intermediate of above-mentioned formula I.
After to exocyclic amino group functionality acidylate, remove the benzylidene part with 80% acetate, obtain 3a-d.In order to obtain compound 3c, available DBU removes nitro-styroyl.
Can protect 1 with a dimethoxy trityl, the primary hydroxyl functional group of 5-anhydrohexitol analogue 3a-d is to obtain 4a-d.Can pass through 2-cyanoethyl N, N-di-isopropyl chloro phosphoramidite converts it into the phosphoramidite structural unit 5a-d that is suitable for mixing oligonucleotide chain.Contain 1; the support of 5-anhydrohexitol analogue can prepare by the succinylation to compound 4a-d; obtain 6a-d, aminofunctional polystyrene (for example, the Tentage that can utilize carbodiimide to be connected to long-chain alkylamino controlled pore glass (CCAA-CPG) or to be fit to
On-RAPPPolymere) the amido functional group, obtain 7a-d (support functionalized) referring to document 17.
Finish after the combination, the cracking from the support of the oligonucleotide that obtained got off, and by at 55 ℃ down with 16 hours deprotections of ammonia treatment.The purifying of the above-mentioned formula II oligomer of gained can be realized (18) by several method.Preferred method is to carry out anionresin FPLC under the pH value is 12 alkaline condition, to destroy all possible secondary structure (10).Can carry out lyophilize subsequently by the desalination of simple gel-filtration technology.The all available ordinary method preparation of all available salt.
The base phenyl) guanine-9-base d:B ethyl)
*=N
4-benzoyl cytosine-1-base d:B=cytosine(Cyt)-1-base CPG=controlled pore glass (solid support) is 80%HOAc (i); (ii) dimethoxy trityl chlorine, pyridine; (iii) N, N ,-diisopropylethylamine, 2-cyanogen-N, N-di-isopropyl chloro phosphoramidite, CH
2Cl
2(iv) DMAP, succinyl oxide, pyridine; (v) activate LCAA-CPG, DMAP, Et in advance
3N, 1-(3-diethyl amino propyl group)-3-ethyl carbodiimide, HCl, pyridine.
As mentioned above, described oligomer has the sequence specificity in conjunction with natural oligonucleotide.The binding ability of the sequence that the binding ability of it and the natural oligodeoxynucleotide of complementary is crossed greater than unmodified, it has higher biochemical stability.Like this, can use it for the antisense method, comprise diagnosis, hybridization, nucleic acid extraction, site-specific nature dna modification and treatment, all antisense methods are at present all at natural oligodeoxynucleotide.
Embodiment
In following examples, will the preparation of the raw material of compound of the present invention and chemosynthesis thereof be described further, but, these embodiment be not limit of the present invention.Adopted following abbreviated form: FABMS=fast atom bombardment MS Thgly=thioglycerol NBA=nitrobenzyl alcohol
Verheggen etc. (12) have disclosed 1,5-dehydration-2, and the two deoxidations of 3--2-replacement-D-arabinose base-hexitol is examined sweet analogue and 4, the synthetic method of the derivative of 6-O-benzylidene protection.The nucleoside analog 1.1.1 of example 1 base protection, 5-dehydration-2-(N
6-benzoyl VITAMIN B4-9-yl)-2,3-is two
Deoxidation-D-arabinose base hexitol (3b)
Under 0 ℃, the Benzoyl chloride of 0.9ml (7.8mmol) is added by 2.3g (6.51mmol) 1,5-dehydration-4,6-O-benzylidene-2-(VITAMIN B4-9-yl)-2, the two deoxidations of 3--D-arabinose base hexitol are dissolved in the 20ml anhydrous pyridine in the resulting solution.At room temperature stirred 4 hours, and then mixture was cooled off on ice bath, and add 2ml water.Adding the concentrated NH of 1.5ml
3Solution (33%g/v) and at room temperature after the restir 45 minutes, evaporation gained mixture.By column chromatography (CH
2Cl
2-MeOH, 98: 2) the purifying resistates, obtain 1 of 1.92g (4.19mmol, productive rate 64%), 5-dehydration-4,6-O-benzylidene-2-(N
6-benzoyl VITAMIN B4-9-yl)-2, the two deoxidations of 3--D-arabinose base hexitol.
Under 60 ℃ of temperature, further handled above-mentioned product 5 hours, to remove the benzylidene part with 100ml 80% acetate.Evaporation, with the toluene coevaporation with by column chromatography (CH
2Cl
2-MeOH, 95: 5～90: 10) purifying, obtain the title compound of 1.10g (2.98mmol, productive rate 71%) present embodiment.UV (MeOH) λ
Max282nm (∈=20200) FABMS (Thgly, NaOAc) m/e:392 (M+Na)
+.240 (B+2H)
+ 1H NMR (DMSO-d
6δ 1.94 (m, 1H, H-3 ' ax), 2.32 (m, 1H.H-3 ' eq), 3.21 (m, 1H, H-5 '), 3.42-3.76 (m, 3H, H-4 ', H-6 ', H-6 "), 3.90 (dd,
2J=13Hz, 1H, H-1 ' ax), 4.27 (dd,
2J=12.2Hz, 1H, H-1 ' eq), 4.67 (t, J=5.7Hz, 1H, 6 '=OH), 4.88-5.00 (m, 2H, H-2 ', 4 '-OH), 7.47-7.68 (m, 3H, aromatics H), (8.00-8.07 m, 2H aromatics H) 8.60 (s, 1H), 8.73 (s, 1H) (H-2, H-8) ppm.
13C NMR (DMSO-d
6) δ 35.8 (C-3 '), 50.7 (C-2 '), 60.5,60.7 (C-4 ', C-6 '), 67.9 (C-1 '), (83.1 C-5 '), 125.1 (C-5), 128.5 (Co, Cm), 132.5 (Cp), 133.6 (Cx), 143.5 (C-8), 150.3 (c-4), 151.4 (C-2), 152.4 (C-6) ppm.1.2.1,5-dehydration-2, the two deoxidation-2-(N of 3-
2-isobutyryl guanine-9
-Ji-D-arabinose base hexitol (3c))
With 1,5-dehydration-4, (1,18g is 5mmol) to N in 6-O-benzylidene 3-deoxidation-D-glucitol (glucitol)
2-isobutyryl-O
6-[2-(p-nitrophenyl) ethyl] guanine (1.85g 7.5mmol) carries out alkylation, in anhydrous pyridine, and with 1.5ml (10mmol) DBU, spend 16 hour time to remove the p-nitrophenyl ethyl, and by hurried column chromatography (CH
2Cl
2-MeOH, 99: 1～97: 3) purifying, it is rough 1 to obtain 1.35g, 5-dehydration-4,6-O-benzylidene-2, the two deoxidation-2-(N of 3-
2-isobutyryl-guanine-9-yl)-D-arabinose base hexitol.With 100ml80%HOAc benzylidene partly is hydrolyzed, through column chromatography (CH
2Cl
2-MeOH, 90: 10) after obtain required compound 3c (610mg, 1.74mmol, overall yield 34%).UV (MeOH) λ
Max273nmFABMS (Thgly, NaOAc) m/e:352 (M+H)
+ 1H NMR δ 1.11 (d, J=6.7Hz, 6H, CH
3), 1.93 (m, 1H, H-3 ' ax), 2.11-2.38 (m, 1H.H-3 ' eq), 2.80 (q, 1H, CHMe-2), 3.25 (m, 1H, H-5 '), 3.42-3.78 (m, 3H, H-4 ', H-6 ', H-6 "), 3.89 (dd,
2J=13Hz, 1H, H-1 '), 4.21 (dd,
2J=13Hz, 1H, H-1 "), 4.69
13C NMR δ 19.4 (CH
3), 34.5 (CHMe
2), 35.8, (C-3 '), (50.5 C-2 '), 60.5,60.7 (C-4 ', C-6 '), 67.9 (C-1 '), 83.1 (C-5 '), 116.7 (C-5), 141.7 (C-8), 152.0 (C-4), 153.0 (C-2), 159.8 (C-6), the dimethoxytrityl 2.1.1 of 175.2 (C=O) ppm. example, 2 nucleoside analogs, 5-dehydration-6-O-dimethoxy trityl-2-(thymus pyrimidine
-1-yl)-2, the two deoxidations of 3--D-arabinose base hexitol (4a)
With 1,5-dehydration-2-(thymus pyrimidine-2-yl)-2, (330mg 1.29mmol) is dissolved in the 20ml anhydrous pyridine, and adds 480mg (1.42mmol) dimethoxy trityl chlorine in the two deoxidations of 3--D-arabinose base-hexitol (3a).At room temperature mixture is stirred and spend the night, use 100ml CH
2Cl
2Dilution, and with the saturated NaHCO of 100ml
3Solution washing 2 times.Dry organic layer, evaporation, and with toluene-rise coevaporation.By column chromatography (employing contain 1% triethylamine by CHCl
30～3%MeOH gradient liquid of preparation) resistates that purifying obtains obtains 373mg (0.67mmol, 52%) spumescence title compound.FABMS (Thgly, NaOAc) m/e:581 (M+Na)
+.127 (B+2H)
+ 1H NMR (CDCl
3): δ 1.60-2.50 (m, 2H, H-3 ', H-3 "), 1.91 (s, 3H, CH
3), 3.12-3.62 (m, 2H, H-5 ', H-4 '), 3.77 (s, 6H, 2x OCH
3), 3.65-4.17 (m, 4H, H-6 ', H-6 ", H-1 ', H-1 "), 4.53 (s, 1H, H-2 '), 4.88 (d, 1H, J=5.1, Hz 4 '-OH), 6.81 (d, J=8.7,4H, aromatics H), (7.09-7.53 m, 9H, aromatics H), 8.09 (s, 1H, H-6), 9.10 (brs, 1H, NH) ppm
13C NMR (CDCl
3) δ 12.5 (CH3), 35.5 (C-3 '), 50.7 (C-2 '), 54.9 (OCH
3), 62.4,63.1 (C-4 ', C-6 '), 68.2 (C-1 '), 81.1 (C-5 '), 86.0 (Ph
3C) 110.0 (C-5), 138.4 (C-6), 151.0 (C-2), 163.8 (C-4), 112.9,126.6,127.5,127.8,129.7,135.6,144.6,158.3 (aromatics C) ppm.2.2.1,5-dehydration-6-O-dimethoxy trityl-2-(N
6-benzene
Formyl VITAMIN B4-9-yl)-2, the two deoxidations of 3--D-arabinose base hexitol
(4b)
370mg (1mmol) nucleosides 3b and 400mg (1.2mmol) dimethoxy trityl chlorine are dissolved in the 25ml anhydrous pyridine, at room temperature stirred 16 hours.Use 100mlCH
2Cl
2Dilution gained mixture, and with the saturated NaHCO of 100ml
3Solution washing 2 times.With organic layer drying, evaporation and with the toluene coevaporation.(use CH by column chromatography
2Cl
20～3%MeOH of preparation contains 0.2% pyridine) obtain 400mg (0.6mmol, productive rate 63%) foam-like compound 4b.FABMS(Thgly，NaOAc)m/c：694(m+Na)
+，240(B+2H)
+。2.3.1,5-dehydration-6-O-dimethoxy trityl-2-(N
2-isobutyl
Acyl guanine-9-yl)-2, the two deoxidations of 3--D-arabinose base hexitol (4c)
580mg (1.65mmol) nucleosides 3c and 670mg (2.0mmol) dimethoxy trityl chlorine are dissolved in the 25ml anhydrous pyridine, at room temperature stirred 16 hours.Use 100mlCH
2Cl
2Dilution gained mixture, and with the saturated NaHCO of 100ml
3Solution washing 2 times.With organic layer drying, evaporation and with the toluene coevaporation.By column chromatography (use contain 0.2% pyridine by CH
2Cl
20～3%MeOH gradient liquid of preparation) the purifying resistates obtains 770mg (1.18mmol, productive rate 71%) foam-like compound 4c.FABMS(NBA)m/e：654(m+H)
+。2.4. the preparation of imide (amidite) structural unit (5a-c)
6 ,-O-protection nucleosides (0.5mmol), the anhydrous N of 3 equivalents, N-di-isopropyl-ethamine and 1.5 equivalent 2-cyanoethyl-N, N-di-isopropyl chloro phosphoramidite is dissolved in the anhydrous CH of 2.5ml
2Cl
2In the mixture that obtains at room temperature stirred 3 hours.Add 0.5ml EtOH and restir after 25 minutes, use 5%NaHCO
3Solution (15ml) and saturated this mixture of NaCl solution washing, dry and evaporation.Use Et
3N carries out hurried column chromatography and obtains white foam shape imide, and it is dissolved in a small amount of anhydrous CH
2Cl
2In, and drop in 100ml freezing (50 ℃) normal hexane.Sediment separate out, with normal hexane washing, drying, and it is synthetic to use it for DNA.
Listed different imido eluting solvents and the later productive rate of precipitation in the following table:
The succinylation 3.1.1 of the nucleoside analog of example 36-O-protection, 5-dehydration-6-O-dimethoxy trityl-4-O-succinyl-
| Compound | Solvent | Solvent ratio | Productive rate | ??FABMS(NBA ??)m/e |
| ????5a | N-hexane/ethyl acetate/triethylamine | ???23∶75∶2 | ???62％ | ??759(M+H) + |
| ????5b | N-hexane/ethyl acetate/triethylamine | ???50∶48∶2 | ???65％ | ??872(M+H) + |
| ????5c | N-hexane/ethyl acetate/triethylamine | ???55∶43∶2 | ???56％ | ??854(M+H) + |
Base-2-(thymus pyrimidine-1-yl)-2, the two deoxidations of 3--D-arabinose base
Hexitol (6a)
To be dissolved in the mixture of being formed in the 5ml anhydrous pyridine by 80mg (0.14mmol) 4a, 9mg (0.07mmol) DMAP and 43mg (0.14mmol) succinyl oxide at room temperature stirred 24 hours.Before reaction finishes as yet, add 43mg (0.43mmol) again, and mixture restir 24 hours.Evaporate this solution, and with the toluene coevaporation.Resistates is dissolved in CH
2Cl
2In, with saturated NaCl solution and water washing organic layer, dry and evaporation obtains 78mg (0.12mmol, productive rate 86%) white foam shape 6a.3.2.1,5-dehydration-6-O-dimethoxy trityl-4-O-succinyl-
Base-2-(N
6-benzoyl VITAMIN B4-9-yl)-2, the two deoxidation-D of 3-
-arabinose base hexitol (6b)
The Same Way of above-mentioned synthetic 6a is used for the synthetic of 6b.With 260mg (0.39mmol) 4b is raw material, obtains 256mg (0.33mmol, productive rate 85%) spumescence title compound.The preparation of the production 4.1. solid support of example 4 oligonucleotide
Will by 80 μ mol succinates (6a, b), 400mg activates LCAA-CPG (17), 5mg (40mmol) DAMP, 35 μ l Et in advance
3N and 153mg (800 μ mol) 1-(3-dimethyl aminopropyl)-3-ethyl carbodiimide HCl was dissolved in the mixture supersound process formed in the 4ml anhydrous pyridine 5 minutes, at room temperature vibrated then 16 hours.After the vibration, the CPG solid support is leached, and use pyridine continuously, methyl alcohol and CH
2Cl
2Washing, dry under vacuum subsequently.With the unreacted position that is dissolved in 1.5ml 1-Methylimidazole (Applied Biosystems) among the THF and 1.5ml acetic anhydride-lutidine-THF (Applied Biosystems) sealing in 1: 1: 8 support surface.At room temperature vibrated 4 hours, and leached solid support then, use CH
2Cl
2Washing, and dry under vacuum.The colorimetric analysis of dimethoxy trityl shows that the load of 7a is 18.5 μ mol/g, and the load of 7b is 21.5 μ mol/g.4.2.DNA it is synthetic
Upward carrying out oligonucleotide with phosphoramidite method (removing terminal dimethoxy trityl) at an ABI 381A dna synthesizer (Applied Biosystems) synthesizes.By concentrating ammonia treatment (55 ℃, 16 hours) resulting sequence is carried out deprotection, and its cracking from the solid support is got off.At NAP-10
Post (the SephadexG25-DNA level Pharmacia) goes up purifying, with buffer A (seeing below) wash-out, and then at mono-Q
HR10/10 anion-exchange column (Pharmacia) is gone up purifying, uses following gradient system (A=10mMNaOH, pH12.0,0.1MNaCl; B=10mM NaOH, pH12.0,0.9M NaCl; Oligomer is depended in the use of gradient; Flow velocity is the 2ml/ branch).The low pressure liquid chromatographic system is by Merck-Hitachi L6200 A Intelligent Pump, a Mono Q
HR10/10 post (Pharmacia), a Uvicord SJI2138UV detector (Pharmacia-LKB) and a registering instrument are formed.At NAP-10
On the post fraction that contains product is carried out desalination, and carry out lyophilize.Example 5 melting temperature(Tm)s
Oligomer is dissolved in the following damping fluid: 0.1M NaCl, 0.02M potassiumphosphate pH=7.5,0.1mM EDTA.Determine its concentration by the photoabsorption of surveying 260nm at 80 ℃, suppose under denatured state 1,5-anhydrohexitol nucleoside analog has the optical extinction coefficient identical with natural nucleus glycoside.VITAMIN B4 monomer: ∈=15000 thymus pyrimidine monomer: ∈=8500 guanine monomers: ∈=12500 cytosine(Cyt) monomer: ∈=7500 are in all experiments, and the concentration of every chain all is approximately 4 μ M.With a Uvikon 940 spectrophotometric determination melting curves.By allowing the temperature of water stable circulation sample cell in the sample cell bearing, and measured the temperature of solution by directly immersing thermistor in the sample cell.Carry out control of automatization temperature and data gathering with an IBM/PC AT compatible.With 0.2 ℃/minute speed heating and cooling sample, do not see the difference between heating melting curve and the cooling melting curve, get photoabsorption the first order derivative of temperature curve is estimated melting curve.The example and the melting temperature(Tm) thereof of institute's synthetic oligonucleotide in table 1～4, have been provided.Table 1
By single anhydrohexitol nucleosides (A
★, T
★) mix A
13/ T
13The melting temperature(Tm) of the oligonucleotide that duplex is formed (under the NaCl of 0.1M concentration, recording).
| ????d(T) 6xd(T) 6????d(A) 6Yd(A) 6 | ||||
| ?YX | ?G | ?C | ?A | ?T |
| ?A ?A* ?XY | ?20.0 ?20.2 ?G | ?17.9 ?17.1 ?C | ?18.5 ?17.7 ?T | ?34.0 ?32.1 ?A |
| ?T ?T* | ?21.0 ?15.1 | ?20.7 ?15.2 | ?21.3 ?18.3 | ?34.0 ?28.7 |
The melting temperature(Tm) of oligonucleotide of Xiu Shiing and two terminal modified oligonucleotide records under 0.1M NaCl concentration fully.
| Tm(℃) | Hypochromicity | |
| With (dA) 13Wait molar mixture (14) | ||
| (dT) 6T*(dT) 6(8) (T*) 2(dT) 9(T*) 2(9) (T*) 13(10) | ?27.8 ?27.6 ?45.4(1) | ?33％ ?32％ ?49％ |
| With (dT) 13Wait molar mixture (15) | ||
| (dA) 6A*(dA) 6(4) (A*) 2(dA) 9(A*) 2(12) (A*) 13(13) | ?31.8 ?30.3 ?21.0 | ?31％ ?33％ ?49％ |
| (T*) 13：(A*) 13(10∶13) | ?76.3 | ?ND |
| (dT) 13∶(dA) 13(15∶14) | ?34.0 | ?35％ |
(1) measures the few A of strand at the 284nm place
★With few T
★All be ordered structure, but with under high salt concn result's opposite (result is not shown), poly T
★Do not show the tendency of similar formation homoduplex.Following result has more or less proved this-point: to few A
★With few T
★, the UV absorption value rises with the temperature rising is linear.Few T
★With the melting temperature(Tm) that waits molar mixture of few deoxyadenylic acid be 45 ℃, the hypochromicity that records at the 284nm place is 49%.Knew already, and changed salt concn, the DNA recurring structure is changed, the way it goes for the situation here.Under low salt concn, few T
★: few deoxyadenylic acid is in conjunction with more satisfactory, and under high salt concn, forms few T
★Homoduplex is comparatively desirable.Yet this complex body shows in the thermal behavior at 260nm place, few T
★: few deoxidation-adenylic acid (AMP) combination is not typical helix-coil transition.Hypochromicity at first reduces at the 260nm place, and minimum in the time of 46 ℃ (melting temperature(Tm) records at the 484nm place) increases subsequently.Measured with similar approach and to have contained VITAMIN B4 (A
★) and guanine (G
★) mixed sequence of modifying fully (two six aggressiveness and one ten dimer) of nucleoside analog.The melting temperature(Tm) of six aggressiveness that table 3 is modified fully
| Sequence (with the molar mixture that waits of complement) | Tm(℃) | |
| (16) (17) (18) (19) | 6′-A*G*G*A*G*A* 5′-AGGAGA 6′-G*A*G*A*G*A* 5′-GAGAGA | ?31.2 ?10.0 ?14.7 ?9.5 |
Condition determination: 1M NaCl, 20mM KH
2PO
4PH 7.5, and EDTA 0.1mM and complementary sequence form duplex 5 '-TCTCCT (20) and be used for 16 and 17, and 5 '-TCTCTC (21) is used for 18 and 19.
Although concerning above-mentioned some sequence, melting temperature(Tm) is six aggressiveness of surveying, and the thermally denature of these oligonucleotide is (to contain 20mM K at 1M NaCl
2HPO
4PH7.5 and 0.1mM EDTA) middle research.Most important phenomenon is, obviously formed duplex between class pyranose oligonucleotide and its natural corresponding body (counterpart).And the duplex of this modified is more stable by the duplex that Wo Sen-the Ke Like base pair is formed than absolute.
But, what form distinct contrast is sequence 16 (T
m=31.2 ℃) and 17 (T
m=14.7 ℃) and the melting temperature(Tm) of its antiparallel complementary oligonucleotide between have than big difference.And modified contain 3G
★And 3A
★Oligomer only aspect its sequence order difference is being arranged, the melting temperature(Tm) of sequence 16 is the twice of sequence 18.This sequence relies on effect and only obtain limited embodiment on control oligonucleotide 17 and 19.
Table 4
That modifies fully contains A
★And G
★Ten dimeric melting temperature(Tm)s
Under 0.1M NaCl condition, measure
| Sequence (with the molar mixture that waits of complement) | 24 Tm (℃) | |
| (22) (23) | 6′-A*G*G*G*A*G*A*G*G*A*G*A* 5′-AGG?GAG?AGG?AGA | ?64.8 ?49.0 |
(24)5′-TCT?CCT?CTC?CCT
Observe described ten dimers as can be seen, compare with its controlled sequence 23, the oligonucleotide of Xiu Shiing has advantages of higher stability fully, and these two kinds of sequences are compared with its complementary antiparallel complementary sequence 24, and melting temperature(Tm) has improved 16 ℃.
Reference
1.Beaucage, S.L. and Iyer, R.P., tetrahedron 49,6123-6194 (1993)
2.Sanghvi etc., nucleosides and Nucleotide 10,345-346 (1991)
3.Chollet etc., Chemica Scripta 26,37-40 (1986)
4.Seela, F. and Kehne, A., biological chemistry 24,7556-7561 (1985)
5.Wagner etc., science 260,1510-1513 (1993)
6.Inoue etc., nucleic acids research .15,6131-6148 (1987)
7.Perbost etc., biological chemistry and biophysical studies communication.165，742-747(1989)
8.Gagnor etc., nucleic acids research 15,10419-10436 (1987)
9.Eschenmoser, A., theoretical and applied chemistry 65,1179-1188 (1993)
10.Augustyns etc., nucleic acids research 20,4711-4716, (1992)
11.Augustyns etc., nucleic acids research 21,4670-4676, (1993)
12.Verheggen etc., journal of medicinal chemistry 36,2033-2040 (1993)
13.Matteucci en Caruthers, american Journal of the Chemical Society 103,3185-3191 (1981)
14.Froehler etc., nucleic acids research 14,5399-5407 (1986)
15. molecular biology method, Vol.20, the scheme of oligonucleotide and analogue, S.Agrawal compiles., Humana Press, Totowa, New Jersey, U.S.A.
16.Ti etc., american Journal of the Chemical Society 104,1316-1319 (1982)
17.Pon etc., biotechnology 6,768-775 (1988)
18. molecular biology method vol.26, hoofdstuk 9 " analyzes and purifying synthetic oligonucleotide with HPLC; S.Agrawel compiles., Humana Press, Totowa, NewJersey, USA.
Claims (9)
1. oligomer, wholly or in part by 1 shown in the general formula I, 5-anhydrohexitol nucleoside analog is formed
Wherein, B is derived and next heterocycle by pyrimidine or purine bases.
2. oligomer as claimed in claim 1 is characterized in that it is shown in general formula I I
Wherein, B is derived and the heterocycle that comes by pyrimidine or purine bases, and respectively do for oneself one 0～15 integer of k, l and m supposes that k and m are at least 1; But if k＞1, then m can be 0; If m＞1, then k can be 0; And X represents oxygen or sulphur, and salt.
3. as the oligomer of claim 1 or 2, base is characterised in that described heterocycle is selected from cytosine(Cyt), 5-methylcytosine, uridylic and thymus gland purine, or the assorted derivative of its denitrogenation.
4. as the oligomer of claim 1 or 2, it is characterized in that described heterocycle is selected from VITAMIN B4, guanine, 2,6-diaminopurine, xanthoglobulin and xanthine, or the assorted derivative of its denitrogenation.
5. each described oligomer in the claim as described above, wherein said formula I compound is (D)-configuration, and described substituting group is arranged in the arabinose based structures.
6. the purposes that each described oligomer in the claim 1～5 is used for antisense technology.
7. oligomer purposes as claimed in claim 6 is characterized in that, described antisense technology comprises diagnosis, hybridization, nucleic acid extraction, fixed point dna modification and treatment.
8. the method for preparation formula II oligomer comprises that the formula I monomer with suitable quantity links together.
9. the phosphoramidite shown in general formula VIII is used to prepare the purposes of the oligomer of claim 1,
Wherein, B
★It is protected base.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| NL94202342.5 | 1994-08-17 | ||
| EP94202342 | 1994-08-17 | ||
| US49515295A | 1995-06-27 | 1995-06-27 | |
| US08/495,152 | 1995-06-27 |
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| Publication Number | Publication Date |
|---|---|
| CN1158618A true CN1158618A (en) | 1997-09-03 |
Family
ID=26136490
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 95195211 Pending CN1158618A (en) | 1994-08-17 | 1995-08-14 | Sequence-specific binding oligomers for nucleic acids and their use in antisense strategies |
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|---|---|
| EP (1) | EP0777676A1 (en) |
| JP (1) | JP2000505778A (en) |
| CN (1) | CN1158618A (en) |
| AU (1) | AU3384595A (en) |
| CA (1) | CA2196306A1 (en) |
| FI (1) | FI970598A0 (en) |
| HU (1) | HUT77509A (en) |
| NO (1) | NO970716L (en) |
| NZ (1) | NZ292140A (en) |
| WO (1) | WO1996005213A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU1874397A (en) * | 1996-02-16 | 1997-09-02 | Stichting Rega Vzw | Hexitol containing oligonucleotides and their use in antisense strategies |
| US7205106B1 (en) | 2001-07-20 | 2007-04-17 | Roche Molecular Systems, Inc. | Association of polymorphisms in IL4-related genes with autoimmune disease |
| AU2003235767A1 (en) | 2002-01-08 | 2003-07-24 | Roche Diagnostics Gmbh | Use of silica material in an amplification reaction |
| ES2260569T3 (en) * | 2002-12-20 | 2006-11-01 | Roche Diagnostics Gmbh | DERIVATIVES OF MANITOL AND GLUCITOL. |
| EP1431297A1 (en) * | 2002-12-20 | 2004-06-23 | Boehringer Mannheim Gmbh | Mannitol and glucitol derivatives |
| US7560231B2 (en) | 2002-12-20 | 2009-07-14 | Roche Molecular Systems, Inc. | Mannitol and glucitol derivatives |
| EP1466919A1 (en) * | 2003-04-05 | 2004-10-13 | Roche Diagnostics GmbH | Nucleotide analogs with six membered rings |
| CA2463719A1 (en) | 2003-04-05 | 2004-10-05 | F. Hoffmann-La Roche Ag | Nucleotide analogs with six membered rings |
| US20080261905A1 (en) * | 2004-11-08 | 2008-10-23 | K.U. Leuven Research And Development | Modified Nucleosides for Rna Interference |
| US8530640B2 (en) * | 2008-02-07 | 2013-09-10 | Isis Pharmaceuticals, Inc. | Bicyclic cyclohexitol nucleic acid analogs |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NL9300058A (en) * | 1992-06-18 | 1994-01-17 | Stichting Rega V Z W | 1,5-ANHYDROHEXITOL NUCLEOSIDE ANALOGA AND PHARMACEUTICAL USE THEREOF. |
| US5314893A (en) * | 1993-01-25 | 1994-05-24 | Bristol-Myers Squibb Co. | Antiviral tetrahydropyrans |
-
1995
- 1995-08-14 JP JP08507032A patent/JP2000505778A/en active Pending
- 1995-08-14 EP EP95930468A patent/EP0777676A1/en not_active Withdrawn
- 1995-08-14 AU AU33845/95A patent/AU3384595A/en not_active Abandoned
- 1995-08-14 NZ NZ292140A patent/NZ292140A/en unknown
- 1995-08-14 CA CA 2196306 patent/CA2196306A1/en not_active Abandoned
- 1995-08-14 CN CN 95195211 patent/CN1158618A/en active Pending
- 1995-08-14 HU HU9800097A patent/HUT77509A/en unknown
- 1995-08-14 WO PCT/EP1995/003248 patent/WO1996005213A1/en not_active Application Discontinuation
-
1997
- 1997-02-12 FI FI970598A patent/FI970598A0/en not_active Application Discontinuation
- 1997-02-17 NO NO970716A patent/NO970716L/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| AU3384595A (en) | 1996-03-07 |
| HUT77509A (en) | 1998-05-28 |
| WO1996005213A1 (en) | 1996-02-22 |
| EP0777676A1 (en) | 1997-06-11 |
| FI970598A (en) | 1997-02-12 |
| JP2000505778A (en) | 2000-05-16 |
| NZ292140A (en) | 1998-02-26 |
| MX9701111A (en) | 1998-03-31 |
| CA2196306A1 (en) | 1996-02-22 |
| NO970716L (en) | 1997-02-17 |
| FI970598A0 (en) | 1997-02-12 |
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