USRE39324E1 - Pyrimidine derivatives and oligonucleotides containing same - Google Patents
Pyrimidine derivatives and oligonucleotides containing same Download PDFInfo
- Publication number
- USRE39324E1 USRE39324E1 US10/080,074 US8007402A USRE39324E US RE39324 E1 USRE39324 E1 US RE39324E1 US 8007402 A US8007402 A US 8007402A US RE39324 E USRE39324 E US RE39324E
- Authority
- US
- United States
- Prior art keywords
- compound
- independently
- protecting group
- oligonucleotide
- alkyl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 108091034117 Oligonucleotide Proteins 0.000 title claims description 293
- 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 title description 147
- 150000003230 pyrimidines Chemical class 0.000 title description 8
- 229940083082 pyrimidine derivative acting on arteriolar smooth muscle Drugs 0.000 title 1
- 150000001875 compounds Chemical class 0.000 claims abstract description 239
- 125000006239 protecting group Chemical group 0.000 claims abstract description 108
- -1 —N3 Chemical group 0.000 claims description 73
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 54
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 48
- 125000004432 carbon atom Chemical group C* 0.000 claims description 46
- 150000007523 nucleic acids Chemical class 0.000 claims description 37
- 230000008878 coupling Effects 0.000 claims description 35
- 238000010168 coupling process Methods 0.000 claims description 35
- 238000005859 coupling reaction Methods 0.000 claims description 35
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 34
- 108020004707 nucleic acids Proteins 0.000 claims description 34
- 102000039446 nucleic acids Human genes 0.000 claims description 34
- 125000000217 alkyl group Chemical group 0.000 claims description 32
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 claims description 32
- 150000003839 salts Chemical class 0.000 claims description 32
- 150000004713 phosphodiesters Chemical group 0.000 claims description 31
- 150000002772 monosaccharides Chemical class 0.000 claims description 30
- 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 29
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 claims description 29
- 229910052739 hydrogen Inorganic materials 0.000 claims description 29
- 239000001257 hydrogen Substances 0.000 claims description 28
- 125000005647 linker group Chemical group 0.000 claims description 25
- 229910052736 halogen Inorganic materials 0.000 claims description 22
- 150000002367 halogens Chemical class 0.000 claims description 22
- 229910052799 carbon Inorganic materials 0.000 claims description 20
- 125000006850 spacer group Chemical group 0.000 claims description 20
- 125000004093 cyano group Chemical group *C#N 0.000 claims description 19
- 239000007864 aqueous solution Substances 0.000 claims description 16
- 150000001412 amines Chemical group 0.000 claims description 15
- 125000004433 nitrogen atom Chemical group N* 0.000 claims description 14
- 229910052760 oxygen Inorganic materials 0.000 claims description 14
- 150000003290 ribose derivatives Chemical class 0.000 claims description 14
- 239000007787 solid Substances 0.000 claims description 14
- 125000004209 (C1-C8) alkyl group Chemical group 0.000 claims description 13
- 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 13
- 150000001413 amino acids Chemical class 0.000 claims description 13
- 229940104302 cytosine Drugs 0.000 claims description 13
- 101710163270 Nuclease Proteins 0.000 claims description 12
- 229940024606 amino acid Drugs 0.000 claims description 12
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims description 12
- 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 claims description 11
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 claims description 10
- KDCGOANMDULRCW-UHFFFAOYSA-N Purine Natural products N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 claims description 10
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 claims description 10
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 claims description 9
- QNNARSZPGNJZIX-UHFFFAOYSA-N 6-amino-5-prop-1-ynyl-1h-pyrimidin-2-one Chemical compound CC#CC1=CNC(=O)N=C1N QNNARSZPGNJZIX-UHFFFAOYSA-N 0.000 claims description 9
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 claims description 9
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 claims description 9
- 229910052757 nitrogen Inorganic materials 0.000 claims description 9
- 150000008300 phosphoramidites Chemical class 0.000 claims description 9
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 8
- 125000005842 heteroatom Chemical group 0.000 claims description 8
- 229910019142 PO4 Inorganic materials 0.000 claims description 7
- 125000005843 halogen group Chemical group 0.000 claims description 7
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 claims description 7
- 125000004430 oxygen atom Chemical group O* 0.000 claims description 7
- 239000010452 phosphate Substances 0.000 claims description 7
- KMEMIMRPZGDOMG-UHFFFAOYSA-N 2-cyanoethoxyphosphonamidous acid Chemical compound NP(O)OCCC#N KMEMIMRPZGDOMG-UHFFFAOYSA-N 0.000 claims description 6
- 229930024421 Adenine Natural products 0.000 claims description 6
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims description 6
- UEZVMMHDMIWARA-UHFFFAOYSA-N Metaphosphoric acid Chemical compound OP(=O)=O UEZVMMHDMIWARA-UHFFFAOYSA-N 0.000 claims description 6
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 claims description 6
- 229960000643 adenine Drugs 0.000 claims description 6
- 125000004123 n-propyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])* 0.000 claims description 6
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 6
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 5
- UJBCLAXPPIDQEE-UHFFFAOYSA-N 5-prop-1-ynyl-1h-pyrimidine-2,4-dione Chemical compound CC#CC1=CNC(=O)NC1=O UJBCLAXPPIDQEE-UHFFFAOYSA-N 0.000 claims description 5
- 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 claims description 5
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 claims description 5
- 238000003776 cleavage reaction Methods 0.000 claims description 5
- 229940029575 guanosine Drugs 0.000 claims description 5
- 150000002431 hydrogen Chemical group 0.000 claims description 5
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 5
- 230000007017 scission Effects 0.000 claims description 5
- 239000012453 solvate Substances 0.000 claims description 5
- 125000004434 sulfur atom Chemical group 0.000 claims description 5
- 229940113082 thymine Drugs 0.000 claims description 5
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 claims description 4
- LEQAOMBKQFMDFZ-UHFFFAOYSA-N glyoxal Chemical compound O=CC=O LEQAOMBKQFMDFZ-UHFFFAOYSA-N 0.000 claims description 4
- UBQKCCHYAOITMY-UHFFFAOYSA-N pyridin-2-ol Chemical compound OC1=CC=CC=N1 UBQKCCHYAOITMY-UHFFFAOYSA-N 0.000 claims description 4
- 125000004178 (C1-C4) alkyl group Chemical group 0.000 claims description 3
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 3
- DTQVDTLACAAQTR-UHFFFAOYSA-M Trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-M 0.000 claims description 3
- 150000004676 glycans Chemical class 0.000 claims description 3
- 125000001475 halogen functional group Chemical group 0.000 claims description 3
- 150000003573 thiols Chemical class 0.000 claims description 3
- 125000006832 (C1-C10) alkylene group Chemical group 0.000 claims description 2
- JQPFYXFVUKHERX-UHFFFAOYSA-N 2-hydroxy-2-cyclohexen-1-one Natural products OC1=CCCCC1=O JQPFYXFVUKHERX-UHFFFAOYSA-N 0.000 claims description 2
- WMLUAVXIVYIPKK-UHFFFAOYSA-N 5-but-1-ynyl-1h-pyrimidine-2,4-dione Chemical compound CCC#CC1=CNC(=O)NC1=O WMLUAVXIVYIPKK-UHFFFAOYSA-N 0.000 claims description 2
- DOMUCQWDJSGOOU-UHFFFAOYSA-N 6-amino-5-but-1-ynyl-1h-pyrimidin-2-one Chemical compound CCC#CC1=CNC(=O)N=C1N DOMUCQWDJSGOOU-UHFFFAOYSA-N 0.000 claims description 2
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 claims description 2
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 claims description 2
- OILAIQUEIWYQPH-UHFFFAOYSA-N cyclohexane-1,2-dione Chemical compound O=C1CCCCC1=O OILAIQUEIWYQPH-UHFFFAOYSA-N 0.000 claims description 2
- 229940015043 glyoxal Drugs 0.000 claims description 2
- 150000002466 imines Chemical class 0.000 claims description 2
- 150000002500 ions Chemical class 0.000 claims description 2
- MZDMEAKVGTZHES-UHFFFAOYSA-N methylphosphonamidous acid Chemical compound CP(N)O MZDMEAKVGTZHES-UHFFFAOYSA-N 0.000 claims description 2
- 125000000636 p-nitrophenyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1*)[N+]([O-])=O 0.000 claims description 2
- 229920001282 polysaccharide Polymers 0.000 claims description 2
- 239000005017 polysaccharide Substances 0.000 claims description 2
- IGFXRKMLLMBKSA-UHFFFAOYSA-N purine Chemical compound N1=C[N]C2=NC=NC2=C1 IGFXRKMLLMBKSA-UHFFFAOYSA-N 0.000 claims description 2
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 claims description 2
- 229940035893 uracil Drugs 0.000 claims description 2
- 125000002947 alkylene group Chemical group 0.000 claims 2
- MDFFNEOEWAXZRQ-UHFFFAOYSA-N aminyl Chemical compound [NH2] MDFFNEOEWAXZRQ-UHFFFAOYSA-N 0.000 claims 2
- 125000004400 (C1-C12) alkyl group Chemical group 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 74
- 230000027455 binding Effects 0.000 abstract description 60
- 230000000295 complement effect Effects 0.000 abstract description 33
- 239000000543 intermediate Substances 0.000 abstract description 22
- 238000003556 assay Methods 0.000 abstract description 12
- 239000002585 base Substances 0.000 description 202
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 108
- 239000000178 monomer Substances 0.000 description 75
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 51
- 229920000642 polymer Polymers 0.000 description 51
- 239000011541 reaction mixture Substances 0.000 description 42
- 239000000243 solution Substances 0.000 description 42
- 239000000203 mixture Substances 0.000 description 41
- 238000006243 chemical reaction Methods 0.000 description 40
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 39
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 37
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 36
- 0 [1*]N1C=C2[34*]c3cccc([2*])c3NC2=NC1=O Chemical compound [1*]N1C=C2[34*]c3cccc([2*])c3NC2=NC1=O 0.000 description 33
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 31
- 238000003786 synthesis reaction Methods 0.000 description 31
- 230000015572 biosynthetic process Effects 0.000 description 30
- 235000000346 sugar Nutrition 0.000 description 30
- MALIONKMKPITBV-UHFFFAOYSA-N 2-(3-chloro-4-hydroxyphenyl)-n-[2-(4-sulfamoylphenyl)ethyl]acetamide Chemical group C1=CC(S(=O)(=O)N)=CC=C1CCNC(=O)CC1=CC=C(O)C(Cl)=C1 MALIONKMKPITBV-UHFFFAOYSA-N 0.000 description 29
- 238000005160 1H NMR spectroscopy Methods 0.000 description 28
- 125000003729 nucleotide group Chemical group 0.000 description 27
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 23
- 125000004429 atom Chemical group 0.000 description 23
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 23
- 239000000047 product Substances 0.000 description 22
- 210000004027 cell Anatomy 0.000 description 21
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 21
- 239000000741 silica gel Substances 0.000 description 21
- 229910002027 silica gel Inorganic materials 0.000 description 21
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 20
- 125000003367 polycyclic group Chemical group 0.000 description 20
- GQHTUMJGOHRCHB-UHFFFAOYSA-N 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine Chemical compound C1CCCCN2CCCN=C21 GQHTUMJGOHRCHB-UHFFFAOYSA-N 0.000 description 18
- 239000002773 nucleotide Substances 0.000 description 18
- 238000001890 transfection Methods 0.000 description 17
- 102000004190 Enzymes Human genes 0.000 description 16
- 108090000790 Enzymes Proteins 0.000 description 16
- 238000003818 flash chromatography Methods 0.000 description 16
- 125000000547 substituted alkyl group Chemical group 0.000 description 16
- 108020004414 DNA Proteins 0.000 description 15
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 14
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 14
- 238000002515 oligonucleotide synthesis Methods 0.000 description 14
- 238000002360 preparation method Methods 0.000 description 14
- 125000001424 substituent group Chemical group 0.000 description 14
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 14
- 230000014509 gene expression Effects 0.000 description 13
- 238000002844 melting Methods 0.000 description 13
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 12
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 12
- 239000002253 acid Substances 0.000 description 12
- 125000003277 amino group Chemical group 0.000 description 12
- 238000009396 hybridization Methods 0.000 description 12
- 230000001965 increasing effect Effects 0.000 description 12
- 230000008018 melting Effects 0.000 description 12
- 238000006467 substitution reaction Methods 0.000 description 12
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium on carbon Substances [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 11
- 150000001721 carbon Chemical group 0.000 description 11
- 239000000706 filtrate Substances 0.000 description 11
- RIOQSEWOXXDEQQ-UHFFFAOYSA-N triphenylphosphine Chemical compound C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-N 0.000 description 11
- JBWYRBLDOOOJEU-UHFFFAOYSA-N 1-[chloro-(4-methoxyphenyl)-phenylmethyl]-4-methoxybenzene Chemical compound C1=CC(OC)=CC=C1C(Cl)(C=1C=CC(OC)=CC=1)C1=CC=CC=C1 JBWYRBLDOOOJEU-UHFFFAOYSA-N 0.000 description 10
- OKKJLVBELUTLKV-MZCSYVLQSA-N Deuterated methanol Chemical compound [2H]OC([2H])([2H])[2H] OKKJLVBELUTLKV-MZCSYVLQSA-N 0.000 description 10
- 235000001014 amino acid Nutrition 0.000 description 10
- 230000000692 anti-sense effect Effects 0.000 description 10
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 10
- 239000003054 catalyst Substances 0.000 description 10
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 10
- 238000003756 stirring Methods 0.000 description 10
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 9
- 239000003153 chemical reaction reagent Substances 0.000 description 9
- 238000000338 in vitro Methods 0.000 description 9
- 230000004048 modification Effects 0.000 description 9
- 238000012986 modification Methods 0.000 description 9
- 238000000746 purification Methods 0.000 description 9
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 8
- 230000003321 amplification Effects 0.000 description 8
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 8
- 230000005764 inhibitory process Effects 0.000 description 8
- 238000003199 nucleic acid amplification method Methods 0.000 description 8
- 229940046166 oligodeoxynucleotide Drugs 0.000 description 8
- 238000003752 polymerase chain reaction Methods 0.000 description 8
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 238000013518 transcription Methods 0.000 description 8
- 230000035897 transcription Effects 0.000 description 8
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 7
- 239000003795 chemical substances by application Substances 0.000 description 7
- 239000001301 oxygen Substances 0.000 description 7
- 229920006395 saturated elastomer Polymers 0.000 description 7
- 239000007858 starting material Substances 0.000 description 7
- 230000000707 stereoselective effect Effects 0.000 description 7
- 229910052717 sulfur Inorganic materials 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 230000001225 therapeutic effect Effects 0.000 description 7
- PYOKUURKVVELLB-UHFFFAOYSA-N trimethyl orthoformate Chemical compound COC(OC)OC PYOKUURKVVELLB-UHFFFAOYSA-N 0.000 description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 101500027983 Rattus norvegicus Octadecaneuropeptide Proteins 0.000 description 6
- 150000007513 acids Chemical class 0.000 description 6
- 150000001299 aldehydes Chemical class 0.000 description 6
- 239000003431 cross linking reagent Substances 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 238000004896 high resolution mass spectrometry Methods 0.000 description 6
- 150000002632 lipids Chemical class 0.000 description 6
- LHSQKTSXCBNOMJ-UHFFFAOYSA-N n-(phosphanyloxymethyl)-n-propan-2-ylpropan-2-amine Chemical compound CC(C)N(C(C)C)COP LHSQKTSXCBNOMJ-UHFFFAOYSA-N 0.000 description 6
- 239000002777 nucleoside Substances 0.000 description 6
- 239000003961 penetration enhancing agent Substances 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 125000000548 ribosyl group Chemical group C1([C@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- 150000008163 sugars Chemical class 0.000 description 6
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 description 6
- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical class C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 description 5
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 5
- 102100034343 Integrase Human genes 0.000 description 5
- 102000001788 Proto-Oncogene Proteins c-raf Human genes 0.000 description 5
- 108010029869 Proto-Oncogene Proteins c-raf Proteins 0.000 description 5
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 5
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 5
- 150000001298 alcohols Chemical class 0.000 description 5
- 150000001408 amides Chemical class 0.000 description 5
- 229920001222 biopolymer Polymers 0.000 description 5
- 238000004132 cross linking Methods 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 125000000524 functional group Chemical group 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 238000010348 incorporation Methods 0.000 description 5
- 229910052751 metal Inorganic materials 0.000 description 5
- 239000002184 metal Substances 0.000 description 5
- 239000012074 organic phase Substances 0.000 description 5
- 230000003389 potentiating effect Effects 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- 238000010992 reflux Methods 0.000 description 5
- 238000010898 silica gel chromatography Methods 0.000 description 5
- 239000011593 sulfur Substances 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 4
- 125000006273 (C1-C3) alkyl group Chemical group 0.000 description 4
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 4
- KBPLFHHGFOOTCA-UHFFFAOYSA-N 1-Octanol Chemical compound CCCCCCCCO KBPLFHHGFOOTCA-UHFFFAOYSA-N 0.000 description 4
- JEPCLNGRAIMPQV-UHFFFAOYSA-N 2-aminobenzene-1,3-diol Chemical compound NC1=C(O)C=CC=C1O JEPCLNGRAIMPQV-UHFFFAOYSA-N 0.000 description 4
- KUZSBKJSGSKPJH-VXGBXAGGSA-N 5-[(9R)-6-[(3R)-3-methylmorpholin-4-yl]-11-oxa-1,3,5-triazatricyclo[7.4.0.02,7]trideca-2,4,6-trien-4-yl]pyrazin-2-amine Chemical compound C[C@@H]1COCCN1c1nc(nc2N3CCOC[C@H]3Cc12)-c1cnc(N)cn1 KUZSBKJSGSKPJH-VXGBXAGGSA-N 0.000 description 4
- BRLVENRVLQFFHX-UHFFFAOYSA-N CC1=C(C)[Ra]=[Ra][Ra]=[Ra]1 Chemical compound CC1=C(C)[Ra]=[Ra][Ra]=[Ra]1 BRLVENRVLQFFHX-UHFFFAOYSA-N 0.000 description 4
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 4
- 238000001712 DNA sequencing Methods 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-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
- 238000007334 copolymerization reaction Methods 0.000 description 4
- 210000000805 cytoplasm Anatomy 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 230000002255 enzymatic effect Effects 0.000 description 4
- 229910052731 fluorine Inorganic materials 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 230000004060 metabolic process Effects 0.000 description 4
- 229940098779 methanesulfonic acid Drugs 0.000 description 4
- 125000003835 nucleoside group Chemical group 0.000 description 4
- 150000007530 organic bases Chemical class 0.000 description 4
- 239000003960 organic solvent Substances 0.000 description 4
- 229910000027 potassium carbonate Inorganic materials 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 150000003212 purines Chemical class 0.000 description 4
- 238000006798 ring closing metathesis reaction Methods 0.000 description 4
- 238000007363 ring formation reaction Methods 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 238000010561 standard procedure Methods 0.000 description 4
- 238000005987 sulfurization reaction Methods 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- TZMSYXZUNZXBOL-UHFFFAOYSA-N 10H-phenoxazine Chemical compound C1=CC=C2NC3=CC=CC=C3OC2=C1 TZMSYXZUNZXBOL-UHFFFAOYSA-N 0.000 description 3
- ASJSAQIRZKANQN-CRCLSJGQSA-N 2-deoxy-D-ribose Chemical group OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 description 3
- BUYUFJZVMVFQLQ-UHFFFAOYSA-N 4-[2-(5,5,8,8-tetramethyl-6,7-dihydroquinoxalin-2-yl)ethynyl]benzoic acid Chemical compound CC1(C=2N=CC(=NC=2C(CC1)(C)C)C#CC1=CC=C(C(=O)O)C=C1)C BUYUFJZVMVFQLQ-UHFFFAOYSA-N 0.000 description 3
- WCDLCPLAAKUJNY-UHFFFAOYSA-N 4-[4-[3-(1h-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidin-6-yl]phenyl]morpholine Chemical compound C1COCCN1C1=CC=C(C2=CN3N=CC(=C3N=C2)C2=CNN=C2)C=C1 WCDLCPLAAKUJNY-UHFFFAOYSA-N 0.000 description 3
- MITGKKFYIJJQGL-UHFFFAOYSA-N 9-(4-chlorobenzoyl)-6-methylsulfonyl-2,3-dihydro-1H-carbazol-4-one Chemical compound ClC1=CC=C(C(=O)N2C3=CC=C(C=C3C=3C(CCCC2=3)=O)S(=O)(=O)C)C=C1 MITGKKFYIJJQGL-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 3
- 108020004394 Complementary RNA Proteins 0.000 description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 108090000371 Esterases Proteins 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 101710203526 Integrase Proteins 0.000 description 3
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 3
- 239000004472 Lysine Substances 0.000 description 3
- BAVYZALUXZFZLV-UHFFFAOYSA-N Methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 3
- 229910006069 SO3H Inorganic materials 0.000 description 3
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 3
- NELWQUQCCZMRPB-UBPLGANQSA-N [(2r,3r,4r,5r)-4-acetyloxy-5-(4-amino-5-ethenyl-2-oxopyrimidin-1-yl)-2-methyloxolan-3-yl] acetate Chemical compound CC(=O)O[C@@H]1[C@H](OC(C)=O)[C@@H](C)O[C@H]1N1C(=O)N=C(N)C(C=C)=C1 NELWQUQCCZMRPB-UBPLGANQSA-N 0.000 description 3
- 230000002378 acidificating effect Effects 0.000 description 3
- 238000000137 annealing Methods 0.000 description 3
- 150000001479 arabinose derivatives Chemical class 0.000 description 3
- HSDAJNMJOMSNEV-UHFFFAOYSA-N benzyl chloroformate Chemical compound ClC(=O)OCC1=CC=CC=C1 HSDAJNMJOMSNEV-UHFFFAOYSA-N 0.000 description 3
- 230000001588 bifunctional effect Effects 0.000 description 3
- 150000001720 carbohydrates Chemical class 0.000 description 3
- 239000003184 complementary RNA Substances 0.000 description 3
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 description 3
- FAMRKDQNMBBFBR-BQYQJAHWSA-N diethyl azodicarboxylate Substances CCOC(=O)\N=N\C(=O)OCC FAMRKDQNMBBFBR-BQYQJAHWSA-N 0.000 description 3
- KPUWHANPEXNPJT-UHFFFAOYSA-N disiloxane Chemical compound [SiH3]O[SiH3] KPUWHANPEXNPJT-UHFFFAOYSA-N 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical group O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 3
- 239000011737 fluorine Substances 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 125000000623 heterocyclic group Chemical group 0.000 description 3
- 238000003018 immunoassay Methods 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 150000002576 ketones Chemical class 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 235000018977 lysine Nutrition 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000036961 partial effect Effects 0.000 description 3
- 238000005897 peptide coupling reaction Methods 0.000 description 3
- PTMHPRAIXMAOOB-UHFFFAOYSA-L phosphoramidate Chemical compound NP([O-])([O-])=O PTMHPRAIXMAOOB-UHFFFAOYSA-L 0.000 description 3
- 229910052698 phosphorus Inorganic materials 0.000 description 3
- 238000005932 reductive alkylation reaction Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 235000004400 serine Nutrition 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 238000001308 synthesis method Methods 0.000 description 3
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 239000001226 triphosphate Substances 0.000 description 3
- 235000011178 triphosphate Nutrition 0.000 description 3
- DIOHEXPTUTVCNX-UHFFFAOYSA-N 1,1,1-trifluoro-n-phenyl-n-(trifluoromethylsulfonyl)methanesulfonamide Chemical compound FC(F)(F)S(=O)(=O)N(S(=O)(=O)C(F)(F)F)C1=CC=CC=C1 DIOHEXPTUTVCNX-UHFFFAOYSA-N 0.000 description 2
- FJZNNKJZHQFMCK-LRDDRELGSA-N 1-[(3S,4R)-4-(2,6-difluoro-4-methoxyphenyl)-2-oxopyrrolidin-3-yl]-3-phenylurea Chemical compound C1(=CC(=CC(=C1[C@H]1[C@@H](C(=O)NC1)NC(=O)NC1=CC=CC=C1)F)OC)F FJZNNKJZHQFMCK-LRDDRELGSA-N 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 2
- HPZMWTNATZPBIH-UHFFFAOYSA-N 1-methyladenine Chemical compound CN1C=NC2=NC=NC2=C1N HPZMWTNATZPBIH-UHFFFAOYSA-N 0.000 description 2
- PVJZBZSCGJAWNG-UHFFFAOYSA-N 2,4,6-trimethylbenzenesulfonyl chloride Chemical compound CC1=CC(C)=C(S(Cl)(=O)=O)C(C)=C1 PVJZBZSCGJAWNG-UHFFFAOYSA-N 0.000 description 2
- LMRDBJZQDUVCQH-UHFFFAOYSA-N 2-(1,3-dioxoisoindol-2-yl)acetaldehyde Chemical compound C1=CC=C2C(=O)N(CC=O)C(=O)C2=C1 LMRDBJZQDUVCQH-UHFFFAOYSA-N 0.000 description 2
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- FZWGECJQACGGTI-UHFFFAOYSA-N 2-amino-7-methyl-1,7-dihydro-6H-purin-6-one Chemical compound NC1=NC(O)=C2N(C)C=NC2=N1 FZWGECJQACGGTI-UHFFFAOYSA-N 0.000 description 2
- LQLQRFGHAALLLE-UHFFFAOYSA-N 5-bromouracil Chemical compound BrC1=CNC(=O)NC1=O LQLQRFGHAALLLE-UHFFFAOYSA-N 0.000 description 2
- 239000004475 Arginine Substances 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
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- XTEGARKTQYYJKE-UHFFFAOYSA-M Chlorate Chemical compound [O-]Cl(=O)=O XTEGARKTQYYJKE-UHFFFAOYSA-M 0.000 description 2
- 108020004635 Complementary DNA Proteins 0.000 description 2
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Chemical class CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 2
- 125000000824 D-ribofuranosyl group Chemical group [H]OC([H])([H])[C@@]1([H])OC([H])(*)[C@]([H])(O[H])[C@]1([H])O[H] 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 230000004543 DNA replication Effects 0.000 description 2
- 230000005526 G1 to G0 transition Effects 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 2
- XDMCWZFLLGVIID-SXPRBRBTSA-N O-(3-O-D-galactosyl-N-acetyl-beta-D-galactosaminyl)-L-serine Chemical compound CC(=O)N[C@H]1[C@H](OC[C@H]([NH3+])C([O-])=O)O[C@H](CO)[C@H](O)[C@@H]1OC1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 XDMCWZFLLGVIID-SXPRBRBTSA-N 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 2
- 108010083644 Ribonucleases Proteins 0.000 description 2
- 102000006382 Ribonucleases Human genes 0.000 description 2
- 108020004682 Single-Stranded DNA Proteins 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- STSCVKRWJPWALQ-UHFFFAOYSA-N TRIFLUOROACETIC ACID ETHYL ESTER Chemical compound CCOC(=O)C(F)(F)F STSCVKRWJPWALQ-UHFFFAOYSA-N 0.000 description 2
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical class OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 2
- 239000004473 Threonine Substances 0.000 description 2
- 229910052770 Uranium Inorganic materials 0.000 description 2
- 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 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
- 235000004279 alanine Nutrition 0.000 description 2
- 230000029936 alkylation Effects 0.000 description 2
- 238000005804 alkylation reaction Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 150000001450 anions Chemical group 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- WGQKYBSKWIADBV-UHFFFAOYSA-N benzylamine Chemical compound NCC1=CC=CC=C1 WGQKYBSKWIADBV-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical class OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-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
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 150000001735 carboxylic acids Chemical class 0.000 description 2
- 150000001768 cations Chemical class 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000002425 crystallisation Methods 0.000 description 2
- 230000008025 crystallization Effects 0.000 description 2
- 125000000753 cycloalkyl group Chemical group 0.000 description 2
- 238000010511 deprotection reaction Methods 0.000 description 2
- 238000002405 diagnostic procedure Methods 0.000 description 2
- JXTHNDFMNIQAHM-UHFFFAOYSA-N dichloroacetic acid Chemical compound OC(=O)C(Cl)Cl JXTHNDFMNIQAHM-UHFFFAOYSA-N 0.000 description 2
- WKHJZIRDAONVML-UHFFFAOYSA-N dichloromethane;tetrachloromethane Chemical compound ClCCl.ClC(Cl)(Cl)Cl WKHJZIRDAONVML-UHFFFAOYSA-N 0.000 description 2
- RXKJFZQQPQGTFL-UHFFFAOYSA-N dihydroxyacetone Chemical class OCC(=O)CO RXKJFZQQPQGTFL-UHFFFAOYSA-N 0.000 description 2
- 239000000539 dimer Substances 0.000 description 2
- AUZONCFQVSMFAP-UHFFFAOYSA-N disulfiram Chemical compound CCN(CC)C(=S)SSC(=S)N(CC)CC AUZONCFQVSMFAP-UHFFFAOYSA-N 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 229940093499 ethyl acetate Drugs 0.000 description 2
- 235000019439 ethyl acetate Nutrition 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 238000005755 formation reaction Methods 0.000 description 2
- 239000012458 free base Substances 0.000 description 2
- 150000002243 furanoses Chemical class 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- 150000002402 hexoses Chemical class 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 150000002430 hydrocarbons Chemical class 0.000 description 2
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 2
- 238000005984 hydrogenation reaction Methods 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- WQYVRQLZKVEZGA-UHFFFAOYSA-N hypochlorite Chemical compound Cl[O-] WQYVRQLZKVEZGA-UHFFFAOYSA-N 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 230000003834 intracellular effect Effects 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
- 239000003446 ligand Substances 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 231100000682 maximum tolerated dose Toxicity 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 150000007522 mineralic acids Chemical class 0.000 description 2
- 238000002156 mixing Methods 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
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 150000007524 organic acids Chemical class 0.000 description 2
- 235000005985 organic acids Nutrition 0.000 description 2
- NFHFRUOZVGFOOS-UHFFFAOYSA-N palladium;triphenylphosphane Chemical compound [Pd].C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 NFHFRUOZVGFOOS-UHFFFAOYSA-N 0.000 description 2
- 238000005192 partition Methods 0.000 description 2
- 125000006340 pentafluoro ethyl group Chemical group FC(F)(F)C(F)(F)* 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- UEZVMMHDMIWARA-UHFFFAOYSA-M phosphonate Chemical compound [O-]P(=O)=O UEZVMMHDMIWARA-UHFFFAOYSA-M 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 229910000073 phosphorus hydride Inorganic materials 0.000 description 2
- 230000026731 phosphorylation Effects 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- NROKBHXJSPEDAR-UHFFFAOYSA-M potassium fluoride Chemical compound [F-].[K+] NROKBHXJSPEDAR-UHFFFAOYSA-M 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 125000001501 propionyl group Chemical group O=C([*])C([H])([H])C([H])([H])[H] 0.000 description 2
- 230000006340 racemization Effects 0.000 description 2
- 239000000376 reactant Substances 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 2
- 150000003335 secondary amines Chemical class 0.000 description 2
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- 230000000087 stabilizing effect Effects 0.000 description 2
- 150000003462 sulfoxides Chemical class 0.000 description 2
- FRACPXUHUTXLCX-BELIEFIBSA-N tert-butyl N-{1-[(1S)-1-{[(1R,2S)-1-(benzylcarbamoyl)-1-hydroxy-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl]carbamoyl}-2-cyclopropylethyl]-2-oxopyridin-3-yl}carbamate Chemical compound CC(C)(C)OC(=O)NC1=CC=CN(C1=O)[C@@H](CC2CC2)C(=O)N[C@@H](C[C@@H]3CCNC3=O)[C@H](C(=O)NCC4=CC=CC=C4)O FRACPXUHUTXLCX-BELIEFIBSA-N 0.000 description 2
- HWCKGOZZJDHMNC-UHFFFAOYSA-M tetraethylammonium bromide Chemical compound [Br-].CC[N+](CC)(CC)CC HWCKGOZZJDHMNC-UHFFFAOYSA-M 0.000 description 2
- DHCDFWKWKRSZHF-UHFFFAOYSA-L thiosulfate(2-) Chemical compound [O-]S([S-])(=O)=O DHCDFWKWKRSZHF-UHFFFAOYSA-L 0.000 description 2
- 229940104230 thymidine Drugs 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 150000005691 triesters Chemical class 0.000 description 2
- 239000013638 trimer Substances 0.000 description 2
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- SZUVGFMDDVSKSI-WIFOCOSTSA-N (1s,2s,3s,5r)-1-(carboxymethyl)-3,5-bis[(4-phenoxyphenyl)methyl-propylcarbamoyl]cyclopentane-1,2-dicarboxylic acid Chemical compound O=C([C@@H]1[C@@H]([C@](CC(O)=O)([C@H](C(=O)N(CCC)CC=2C=CC(OC=3C=CC=CC=3)=CC=2)C1)C(O)=O)C(O)=O)N(CCC)CC(C=C1)=CC=C1OC1=CC=CC=C1 SZUVGFMDDVSKSI-WIFOCOSTSA-N 0.000 description 1
- YPYSAKSNRPFOET-UHFFFAOYSA-N (2,4-dibromophenyl) dihydrogen phosphate Chemical compound OP(O)(=O)OC1=CC=C(Br)C=C1Br YPYSAKSNRPFOET-UHFFFAOYSA-N 0.000 description 1
- IIMQBSMLPGKJKO-UHFFFAOYSA-N (2,4-dichlorophenyl) dihydrogen phosphate Chemical compound OP(O)(=O)OC1=CC=C(Cl)C=C1Cl IIMQBSMLPGKJKO-UHFFFAOYSA-N 0.000 description 1
- YMXHPSHLTSZXKH-RVBZMBCESA-N (2,5-dioxopyrrolidin-1-yl) 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoate Chemical compound C([C@H]1[C@H]2NC(=O)N[C@H]2CS1)CCCC(=O)ON1C(=O)CCC1=O YMXHPSHLTSZXKH-RVBZMBCESA-N 0.000 description 1
- WMSUFWLPZLCIHP-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 9h-fluoren-9-ylmethyl carbonate Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)ON1C(=O)CCC1=O WMSUFWLPZLCIHP-UHFFFAOYSA-N 0.000 description 1
- APPOKADJQUIAHP-GGWOSOGESA-N (2e,4e)-hexa-2,4-diene Chemical compound C\C=C\C=C\C APPOKADJQUIAHP-GGWOSOGESA-N 0.000 description 1
- NJYXSKVOTDPOAT-LMVFSUKVSA-N (2r,3r,4r)-2-fluoro-3,4,5-trihydroxypentanal Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](F)C=O NJYXSKVOTDPOAT-LMVFSUKVSA-N 0.000 description 1
- BBAMTDMNXVSCRU-UHFFFAOYSA-N (4-chlorophenyl) dihydrogen phosphate Chemical compound OP(O)(=O)OC1=CC=C(Cl)C=C1 BBAMTDMNXVSCRU-UHFFFAOYSA-N 0.000 description 1
- WSWCOQWTEOXDQX-MQQKCMAXSA-M (E,E)-sorbate Chemical compound C\C=C\C=C\C([O-])=O WSWCOQWTEOXDQX-MQQKCMAXSA-M 0.000 description 1
- MIOPJNTWMNEORI-XVKPBYJWSA-N (R)-camphorsulfonic acid Chemical compound C1C[C@]2(CS(O)(=O)=O)C(=O)C[C@H]1C2(C)C MIOPJNTWMNEORI-XVKPBYJWSA-N 0.000 description 1
- 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 1
- BINWKEIURMBREF-UHFFFAOYSA-N 1,3-dihydroimidazo[4,5-b]quinolin-2-one Chemical class C1=CC=C2N=C(NC(=O)N3)C3=CC2=C1 BINWKEIURMBREF-UHFFFAOYSA-N 0.000 description 1
- QLOCVMVCRJOTTM-TURQNECASA-N 1-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-prop-1-ynylpyrimidine-2,4-dione Chemical group O=C1NC(=O)C(C#CC)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 QLOCVMVCRJOTTM-TURQNECASA-N 0.000 description 1
- BDFUGVNODPORPL-JPVQNKNJSA-N 1-[(2r,4s,5r)-4-acetyl-4-hydroxy-5-(1-hydroxy-2-oxopropyl)oxolan-2-yl]-5-bromopyrimidine-2,4-dione Chemical compound C1[C@](C(C)=O)(O)[C@@H](C(O)C(=O)C)O[C@H]1N1C(=O)NC(=O)C(Br)=C1 BDFUGVNODPORPL-JPVQNKNJSA-N 0.000 description 1
- YLYUOOLFHOEBNQ-APTVDZGQSA-N 1-[(2r,4s,5r)-4-acetyl-4-hydroxy-5-(1-hydroxy-2-oxopropyl)oxolan-2-yl]pyrimidine-2,4-dione Chemical compound C1[C@](C(C)=O)(O)[C@@H](C(O)C(=O)C)O[C@H]1N1C(=O)NC(=O)C=C1 YLYUOOLFHOEBNQ-APTVDZGQSA-N 0.000 description 1
- PISWNSOQFZRVJK-XLPZGREQSA-N 1-[(2r,4s,5r)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-methyl-2-sulfanylidenepyrimidin-4-one Chemical compound S=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 PISWNSOQFZRVJK-XLPZGREQSA-N 0.000 description 1
- 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 1
- SATCOUWSAZBIJO-UHFFFAOYSA-N 1-methyladenine Natural products N=C1N(C)C=NC2=C1NC=N2 SATCOUWSAZBIJO-UHFFFAOYSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- UPQQXPKAYZYUKO-UHFFFAOYSA-N 2,2,2-trichloroacetamide Chemical compound OC(=N)C(Cl)(Cl)Cl UPQQXPKAYZYUKO-UHFFFAOYSA-N 0.000 description 1
- HVHZEKKZMFRULH-UHFFFAOYSA-N 2,6-ditert-butyl-4-methylpyridine Chemical compound CC1=CC(C(C)(C)C)=NC(C(C)(C)C)=C1 HVHZEKKZMFRULH-UHFFFAOYSA-N 0.000 description 1
- HLYBTPMYFWWNJN-UHFFFAOYSA-N 2-(2,4-dioxo-1h-pyrimidin-5-yl)-2-hydroxyacetic acid Chemical compound OC(=O)C(O)C1=CNC(=O)NC1=O HLYBTPMYFWWNJN-UHFFFAOYSA-N 0.000 description 1
- OZAIFHULBGXAKX-UHFFFAOYSA-N 2-(2-cyanopropan-2-yldiazenyl)-2-methylpropanenitrile Chemical compound N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 description 1
- MWFLUYFYHANMCM-UHFFFAOYSA-N 2-(2-hydroxyethyl)isoindole-1,3-dione Chemical compound C1=CC=C2C(=O)N(CCO)C(=O)C2=C1 MWFLUYFYHANMCM-UHFFFAOYSA-N 0.000 description 1
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 description 1
- BLDFSDCBQJUWFG-UHFFFAOYSA-N 2-(methylamino)-1,2-diphenylethanol Chemical compound C=1C=CC=CC=1C(NC)C(O)C1=CC=CC=C1 BLDFSDCBQJUWFG-UHFFFAOYSA-N 0.000 description 1
- SGAKLDIYNFXTCK-UHFFFAOYSA-N 2-[(2,4-dioxo-1h-pyrimidin-5-yl)methylamino]acetic acid Chemical compound OC(=O)CNCC1=CNC(=O)NC1=O SGAKLDIYNFXTCK-UHFFFAOYSA-N 0.000 description 1
- SVBOROZXXYRWJL-UHFFFAOYSA-N 2-[(4-oxo-2-sulfanylidene-1h-pyrimidin-5-yl)methylamino]acetic acid Chemical compound OC(=O)CNCC1=CNC(=S)NC1=O SVBOROZXXYRWJL-UHFFFAOYSA-N 0.000 description 1
- FEDLEBCVFZMHBP-UHFFFAOYSA-N 2-amino-3-methylphenol Chemical compound CC1=CC=CC(O)=C1N FEDLEBCVFZMHBP-UHFFFAOYSA-N 0.000 description 1
- KUCWUAFNGCMZDB-UHFFFAOYSA-N 2-amino-3-nitrophenol Chemical compound NC1=C(O)C=CC=C1[N+]([O-])=O KUCWUAFNGCMZDB-UHFFFAOYSA-N 0.000 description 1
- JRYMOPZHXMVHTA-DAGMQNCNSA-N 2-amino-7-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1h-pyrrolo[2,3-d]pyrimidin-4-one Chemical compound C1=CC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O JRYMOPZHXMVHTA-DAGMQNCNSA-N 0.000 description 1
- 125000004182 2-chlorophenyl group Chemical group [H]C1=C([H])C(Cl)=C(*)C([H])=C1[H] 0.000 description 1
- 150000005749 2-halopyridines Chemical class 0.000 description 1
- 125000004200 2-methoxyethyl group Chemical group [H]C([H])([H])OC([H])([H])C([H])([H])* 0.000 description 1
- GDTSJMKGXGJFGQ-UHFFFAOYSA-N 3,7-dioxido-2,4,6,8,9-pentaoxa-1,3,5,7-tetraborabicyclo[3.3.1]nonane Chemical compound O1B([O-])OB2OB([O-])OB1O2 GDTSJMKGXGJFGQ-UHFFFAOYSA-N 0.000 description 1
- GJAKJCICANKRFD-UHFFFAOYSA-N 4-acetyl-4-amino-1,3-dihydropyrimidin-2-one Chemical compound CC(=O)C1(N)NC(=O)NC=C1 GJAKJCICANKRFD-UHFFFAOYSA-N 0.000 description 1
- JVVRCYWZTJLJSG-UHFFFAOYSA-N 4-dimethylaminophenol Chemical compound CN(C)C1=CC=C(O)C=C1 JVVRCYWZTJLJSG-UHFFFAOYSA-N 0.000 description 1
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 1
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-dimethylaminopyridine Substances CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 1
- OVONXEQGWXGFJD-UHFFFAOYSA-N 4-sulfanylidene-1h-pyrimidin-2-one Chemical compound SC=1C=CNC(=O)N=1 OVONXEQGWXGFJD-UHFFFAOYSA-N 0.000 description 1
- AVVWPBAENSWJCB-UHFFFAOYSA-N 5-(1,2-dihydroxyethyl)oxolane-2,3,4-triol Chemical compound OCC(O)C1OC(O)C(O)C1O AVVWPBAENSWJCB-UHFFFAOYSA-N 0.000 description 1
- MQJSSLBGAQJNER-UHFFFAOYSA-N 5-(methylaminomethyl)-1h-pyrimidine-2,4-dione Chemical compound CNCC1=CNC(=O)NC1=O MQJSSLBGAQJNER-UHFFFAOYSA-N 0.000 description 1
- LMNPKIOZMGYQIU-UHFFFAOYSA-N 5-(trifluoromethyl)-1h-pyrimidine-2,4-dione Chemical compound FC(F)(F)C1=CNC(=O)NC1=O LMNPKIOZMGYQIU-UHFFFAOYSA-N 0.000 description 1
- SVXNJCYYMRMXNM-UHFFFAOYSA-N 5-amino-2h-1,2,4-triazin-3-one Chemical compound NC=1C=NNC(=O)N=1 SVXNJCYYMRMXNM-UHFFFAOYSA-N 0.000 description 1
- KSNXJLQDQOIRIP-UHFFFAOYSA-N 5-iodouracil Chemical compound IC1=CNC(=O)NC1=O KSNXJLQDQOIRIP-UHFFFAOYSA-N 0.000 description 1
- KELXHQACBIUYSE-UHFFFAOYSA-N 5-methoxy-1h-pyrimidine-2,4-dione Chemical compound COC1=CNC(=O)NC1=O KELXHQACBIUYSE-UHFFFAOYSA-N 0.000 description 1
- ZLAQATDNGLKIEV-UHFFFAOYSA-N 5-methyl-2-sulfanylidene-1h-pyrimidin-4-one Chemical compound CC1=CNC(=S)NC1=O ZLAQATDNGLKIEV-UHFFFAOYSA-N 0.000 description 1
- VYBOLMKVLWOAIV-UHFFFAOYSA-N 5-prop-1-ynyl-2-sulfanylidene-1h-pyrimidin-4-one Chemical compound CC#CC1=CNC(=S)NC1=O VYBOLMKVLWOAIV-UHFFFAOYSA-N 0.000 description 1
- RIFSDIDBSTTXND-UHFFFAOYSA-N 5-prop-1-ynyl-4-sulfanylidene-1h-pyrimidin-2-one Chemical compound CC#CC1=CNC(=O)NC1=S RIFSDIDBSTTXND-UHFFFAOYSA-N 0.000 description 1
- UDZRZGNQQSUDNP-UHFFFAOYSA-N 6-(aminomethyl)-5-methoxy-2-sulfanylidene-1H-pyrimidin-4-one Chemical compound COC=1C(NC(NC=1CN)=S)=O UDZRZGNQQSUDNP-UHFFFAOYSA-N 0.000 description 1
- PLUDYDNNASPOEE-UHFFFAOYSA-N 6-(aziridin-1-yl)-1h-pyrimidin-2-one Chemical compound C1=CNC(=O)N=C1N1CC1 PLUDYDNNASPOEE-UHFFFAOYSA-N 0.000 description 1
- SXQMWXNOYLLRBY-UHFFFAOYSA-N 6-(methylamino)purin-8-one Chemical compound CNC1=NC=NC2=NC(=O)N=C12 SXQMWXNOYLLRBY-UHFFFAOYSA-N 0.000 description 1
- YNBNVGKHAZGDOS-UHFFFAOYSA-N 6-amino-5-prop-1-ynyl-1h-pyrimidine-2-thione Chemical compound CC#CC1=CNC(=S)N=C1N YNBNVGKHAZGDOS-UHFFFAOYSA-N 0.000 description 1
- LXHOFEUVCQUXRZ-RRKCRQDMSA-N 6-azathymidine Chemical compound O=C1NC(=O)C(C)=NN1[C@@H]1O[C@H](CO)[C@@H](O)C1 LXHOFEUVCQUXRZ-RRKCRQDMSA-N 0.000 description 1
- CKOMXBHMKXXTNW-UHFFFAOYSA-N 6-methyladenine Chemical compound CNC1=NC=NC2=C1N=CN2 CKOMXBHMKXXTNW-UHFFFAOYSA-N 0.000 description 1
- LOSIULRWFAEMFL-UHFFFAOYSA-N 7-deazaguanine Chemical compound O=C1NC(N)=NC2=C1CC=N2 LOSIULRWFAEMFL-UHFFFAOYSA-N 0.000 description 1
- MSSXOMSJDRHRMC-UHFFFAOYSA-N 9H-purine-2,6-diamine Chemical compound NC1=NC(N)=C2NC=NC2=N1 MSSXOMSJDRHRMC-UHFFFAOYSA-N 0.000 description 1
- OZAIFHULBGXAKX-VAWYXSNFSA-N AIBN Substances N#CC(C)(C)\N=N\C(C)(C)C#N OZAIFHULBGXAKX-VAWYXSNFSA-N 0.000 description 1
- 208000035657 Abasia Diseases 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=C[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-KKGHZKTASA-N 0.000 description 1
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 1
- 108091023037 Aptamer Proteins 0.000 description 1
- DJHGAFSJWGLOIV-UHFFFAOYSA-K Arsenate3- Chemical compound [O-][As]([O-])([O-])=O DJHGAFSJWGLOIV-UHFFFAOYSA-K 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- IWTZBJFLLKUMEB-UHFFFAOYSA-N BrCC1=CC=CC2=C1/N=C\O2.CC1=C(N)C(O)=CC=C1.CC1=CC=CC2=C1/N=C\O2.OCCOCC1=CC=CC2=C1/N=C\O2.[N-]=[N+]=NCCOCC1=CC=CC(O)=C1N.[N-]=[N+]=NCCOCC1=CC=CC2=C1/N=C\O2 Chemical compound BrCC1=CC=CC2=C1/N=C\O2.CC1=C(N)C(O)=CC=C1.CC1=CC=CC2=C1/N=C\O2.OCCOCC1=CC=CC2=C1/N=C\O2.[N-]=[N+]=NCCOCC1=CC=CC(O)=C1N.[N-]=[N+]=NCCOCC1=CC=CC2=C1/N=C\O2 IWTZBJFLLKUMEB-UHFFFAOYSA-N 0.000 description 1
- FERIUCNNQQJTOY-UHFFFAOYSA-M Butyrate Chemical compound CCCC([O-])=O FERIUCNNQQJTOY-UHFFFAOYSA-M 0.000 description 1
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Natural products CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- DEPUQAANYJDLOH-UHFFFAOYSA-N CC(=O)OCC1OC(N2=C(=O)N=C(NC3=CC=CC=C3C)C(Br)=C2)CC1OC(C)=O.CC(=O)OCC1OC(N2C=C(Br)C(OS(=O)(=O)C3=CC(C)=CC(C)=C3)=CC2=O)CC1OC(C)=O.CC1=CC=CC(O)=C1N Chemical compound CC(=O)OCC1OC(N2=C(=O)N=C(NC3=CC=CC=C3C)C(Br)=C2)CC1OC(C)=O.CC(=O)OCC1OC(N2C=C(Br)C(OS(=O)(=O)C3=CC(C)=CC(C)=C3)=CC2=O)CC1OC(C)=O.CC1=CC=CC(O)=C1N DEPUQAANYJDLOH-UHFFFAOYSA-N 0.000 description 1
- VWKJQDBUHQZFEK-CNXRQGPKSA-N CC1=CC=CC(O)=C1N.CC1=CC=CC2=C1/N=C\O2.O=C(NC/C=C\C1=CC=CC2=C1/N=C\O2)C(F)(F)F.O=C(NCCCC1=CC=CC2=C1/N=C\O2)C(F)(F)F.O=S(=O)(OC1=CC=CC2=C1/N=C\O2)C(F)(F)F.O=[N+]([O-])C1=C(O)C=CC=C1O.OC1=CC=CC2=C1/N=C\O2 Chemical compound CC1=CC=CC(O)=C1N.CC1=CC=CC2=C1/N=C\O2.O=C(NC/C=C\C1=CC=CC2=C1/N=C\O2)C(F)(F)F.O=C(NCCCC1=CC=CC2=C1/N=C\O2)C(F)(F)F.O=S(=O)(OC1=CC=CC2=C1/N=C\O2)C(F)(F)F.O=[N+]([O-])C1=C(O)C=CC=C1O.OC1=CC=CC2=C1/N=C\O2 VWKJQDBUHQZFEK-CNXRQGPKSA-N 0.000 description 1
- HEXHUSWVFVCSKN-UHFFFAOYSA-N CN(C)CCOc1cccc2c1NC1=NC(=O)N(C)C=C1O2.CN1C=C2Oc3cccc(CCCN)c3NC2=NC1=O.CN1C=C2Oc3cccc(NCCN)c3NC2=NC1=O.CN1C=C2Oc3cccc(OCCCN)c3NC2=NC1=O.CN1C=C2Oc3cccc(OCCN)c3NC2=NC1=O Chemical compound CN(C)CCOc1cccc2c1NC1=NC(=O)N(C)C=C1O2.CN1C=C2Oc3cccc(CCCN)c3NC2=NC1=O.CN1C=C2Oc3cccc(NCCN)c3NC2=NC1=O.CN1C=C2Oc3cccc(OCCCN)c3NC2=NC1=O.CN1C=C2Oc3cccc(OCCN)c3NC2=NC1=O HEXHUSWVFVCSKN-UHFFFAOYSA-N 0.000 description 1
- JPUZQJSYPZTYBV-UHFFFAOYSA-N CN1C=C2Oc3cccc(COCCN)c3NC2=NC1=O.CN1C=C2Oc3cccc(OCCNC4=NC(=O)N(C5CC(O)C(CO)O5)C=C4)c3NC2=NC1=O.CN1C=C2Oc3ccccc3NC2=NC1=O Chemical compound CN1C=C2Oc3cccc(COCCN)c3NC2=NC1=O.CN1C=C2Oc3cccc(OCCNC4=NC(=O)N(C5CC(O)C(CO)O5)C=C4)c3NC2=NC1=O.CN1C=C2Oc3ccccc3NC2=NC1=O JPUZQJSYPZTYBV-UHFFFAOYSA-N 0.000 description 1
- DCERHCFNWRGHLK-UHFFFAOYSA-N C[Si](C)C Chemical compound C[Si](C)C DCERHCFNWRGHLK-UHFFFAOYSA-N 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- 229920002284 Cellulose triacetate Polymers 0.000 description 1
- 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 1
- PCDQPRRSZKQHHS-UHFFFAOYSA-N Cytidine 5'-triphosphate Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 PCDQPRRSZKQHHS-UHFFFAOYSA-N 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- YTBSYETUWUMLBZ-UHFFFAOYSA-N D-Erythrose Chemical class OCC(O)C(O)C=O YTBSYETUWUMLBZ-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-CBPJZXOFSA-N D-Gulose Chemical class OC[C@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O WQZGKKKJIJFFOK-CBPJZXOFSA-N 0.000 description 1
- WQZGKKKJIJFFOK-WHZQZERISA-N D-aldose Chemical class OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-WHZQZERISA-N 0.000 description 1
- WQZGKKKJIJFFOK-IVMDWMLBSA-N D-allopyranose Chemical class OC[C@H]1OC(O)[C@H](O)[C@H](O)[C@@H]1O WQZGKKKJIJFFOK-IVMDWMLBSA-N 0.000 description 1
- LKDRXBCSQODPBY-JDJSBBGDSA-N D-allulose Chemical class OCC1(O)OC[C@@H](O)[C@@H](O)[C@H]1O LKDRXBCSQODPBY-JDJSBBGDSA-N 0.000 description 1
- ZGUNAGUHMKGQNY-SSDOTTSWSA-N D-alpha-phenylglycine Chemical compound OC(=O)[C@H](N)C1=CC=CC=C1 ZGUNAGUHMKGQNY-SSDOTTSWSA-N 0.000 description 1
- 150000008574 D-amino acids Chemical class 0.000 description 1
- YTBSYETUWUMLBZ-IUYQGCFVSA-N D-erythrose Chemical class OC[C@@H](O)[C@@H](O)C=O YTBSYETUWUMLBZ-IUYQGCFVSA-N 0.000 description 1
- RGHNJXZEOKUKBD-SQOUGZDYSA-M D-gluconate Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O RGHNJXZEOKUKBD-SQOUGZDYSA-M 0.000 description 1
- MNQZXJOMYWMBOU-VKHMYHEASA-N D-glyceraldehyde Chemical class OC[C@@H](O)C=O MNQZXJOMYWMBOU-VKHMYHEASA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical class OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- ZAQJHHRNXZUBTE-NQXXGFSBSA-N D-ribulose Chemical class OC[C@@H](O)[C@@H](O)C(=O)CO ZAQJHHRNXZUBTE-NQXXGFSBSA-N 0.000 description 1
- ZAQJHHRNXZUBTE-UHFFFAOYSA-N D-threo-2-Pentulose Chemical class OCC(O)C(O)C(=O)CO ZAQJHHRNXZUBTE-UHFFFAOYSA-N 0.000 description 1
- YTBSYETUWUMLBZ-QWWZWVQMSA-N D-threose Chemical class OC[C@@H](O)[C@H](O)C=O YTBSYETUWUMLBZ-QWWZWVQMSA-N 0.000 description 1
- ZAQJHHRNXZUBTE-WUJLRWPWSA-N D-xylulose Chemical class OC[C@@H](O)[C@H](O)C(=O)CO ZAQJHHRNXZUBTE-WUJLRWPWSA-N 0.000 description 1
- 239000003298 DNA probe Substances 0.000 description 1
- 102000052510 DNA-Binding Proteins Human genes 0.000 description 1
- 108700020911 DNA-Binding Proteins Proteins 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- BWLUMTFWVZZZND-UHFFFAOYSA-N Dibenzylamine Chemical compound C=1C=CC=CC=1CNCC1=CC=CC=C1 BWLUMTFWVZZZND-UHFFFAOYSA-N 0.000 description 1
- XBPCUCUWBYBCDP-UHFFFAOYSA-N Dicyclohexylamine Chemical compound C1CCCCC1NC1CCCCC1 XBPCUCUWBYBCDP-UHFFFAOYSA-N 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 206010056474 Erythrosis Diseases 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 1
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 1
- 229930091371 Fructose Chemical class 0.000 description 1
- 239000005715 Fructose Chemical class 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical class OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Chemical class C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 108010035713 Glycodeoxycholic Acid Proteins 0.000 description 1
- 229930186217 Glycolipid Natural products 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 1
- 229930010555 Inosine Natural products 0.000 description 1
- LKDRXBCSQODPBY-AMVSKUEXSA-N L-(-)-Sorbose Chemical class OCC1(O)OC[C@H](O)[C@@H](O)[C@@H]1O LKDRXBCSQODPBY-AMVSKUEXSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VSOAQEOCSA-N L-altropyranose Chemical class OC[C@@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-VSOAQEOCSA-N 0.000 description 1
- 150000008575 L-amino acids Chemical class 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical class C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 1
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 101710128836 Large T antigen Proteins 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 239000012359 Methanesulfonyl chloride Substances 0.000 description 1
- 241001072332 Monia Species 0.000 description 1
- SGSSKEDGVONRGC-UHFFFAOYSA-N N(2)-methylguanine Chemical compound O=C1NC(NC)=NC2=C1N=CN2 SGSSKEDGVONRGC-UHFFFAOYSA-N 0.000 description 1
- HYVABZIGRDEKCD-UHFFFAOYSA-N N(6)-dimethylallyladenine Chemical compound CC(C)=CCNC1=NC=NC2=C1N=CN2 HYVABZIGRDEKCD-UHFFFAOYSA-N 0.000 description 1
- QIAFMBKCNZACKA-UHFFFAOYSA-N N-benzoylglycine Chemical compound OC(=O)CNC(=O)C1=CC=CC=C1 QIAFMBKCNZACKA-UHFFFAOYSA-N 0.000 description 1
- PCLIMKBDDGJMGD-UHFFFAOYSA-N N-bromosuccinimide Chemical compound BrN1C(=O)CCC1=O PCLIMKBDDGJMGD-UHFFFAOYSA-N 0.000 description 1
- UEEJHVSXFDXPFK-UHFFFAOYSA-N N-dimethylaminoethanol Chemical compound CN(C)CCO UEEJHVSXFDXPFK-UHFFFAOYSA-N 0.000 description 1
- HTLZVHNRZJPSMI-UHFFFAOYSA-N N-ethylpiperidine Chemical compound CCN1CCCCC1 HTLZVHNRZJPSMI-UHFFFAOYSA-N 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
- 102000011931 Nucleoproteins Human genes 0.000 description 1
- 108010061100 Nucleoproteins Proteins 0.000 description 1
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- BPQQTUXANYXVAA-UHFFFAOYSA-N Orthosilicate Chemical compound [O-][Si]([O-])([O-])[O-] BPQQTUXANYXVAA-UHFFFAOYSA-N 0.000 description 1
- 108010010677 Phosphodiesterase I Proteins 0.000 description 1
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 description 1
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 description 1
- LGRFSURHDFAFJT-UHFFFAOYSA-N Phthalic anhydride Natural products C1=CC=C2C(=O)OC(=O)C2=C1 LGRFSURHDFAFJT-UHFFFAOYSA-N 0.000 description 1
- 108010039918 Polylysine Proteins 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- WUGQZFFCHPXWKQ-UHFFFAOYSA-N Propanolamine Chemical compound NCCCO WUGQZFFCHPXWKQ-UHFFFAOYSA-N 0.000 description 1
- 108020004518 RNA Probes Proteins 0.000 description 1
- 239000003391 RNA probe Substances 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-N Sulfurous acid Chemical compound OS(O)=O LSNNMFCWUKXFEE-UHFFFAOYSA-N 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 101710136739 Teichoic acid poly(glycerol phosphate) polymerase Proteins 0.000 description 1
- ZMZDMBWJUHKJPS-UHFFFAOYSA-M Thiocyanate anion Chemical compound [S-]C#N ZMZDMBWJUHKJPS-UHFFFAOYSA-M 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 102100040396 Transcobalamin-1 Human genes 0.000 description 1
- 101710124861 Transcobalamin-1 Proteins 0.000 description 1
- 102000004357 Transferases Human genes 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 229910021536 Zeolite Inorganic materials 0.000 description 1
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 1
- NNLVGZFZQQXQNW-ADJNRHBOSA-N [(2r,3r,4s,5r,6s)-4,5-diacetyloxy-3-[(2s,3r,4s,5r,6r)-3,4,5-triacetyloxy-6-(acetyloxymethyl)oxan-2-yl]oxy-6-[(2r,3r,4s,5r,6s)-4,5,6-triacetyloxy-2-(acetyloxymethyl)oxan-3-yl]oxyoxan-2-yl]methyl acetate Chemical compound O([C@@H]1O[C@@H]([C@H]([C@H](OC(C)=O)[C@H]1OC(C)=O)O[C@H]1[C@@H]([C@@H](OC(C)=O)[C@H](OC(C)=O)[C@@H](COC(C)=O)O1)OC(C)=O)COC(=O)C)[C@@H]1[C@@H](COC(C)=O)O[C@@H](OC(C)=O)[C@H](OC(C)=O)[C@H]1OC(C)=O NNLVGZFZQQXQNW-ADJNRHBOSA-N 0.000 description 1
- TUQDFJQANVWYLL-UHFFFAOYSA-N [H]N1C2=NC(=O)N(C3CC(C)C(CC)O3)C=C2OC2=CC=CC(CCCNC(=O)OCC3C4=C(C=CC=C4)C4=C3C=CC=C4)=C21.[H]N1C2=NC(=O)N(C3CC(O)C(CC)O3)C=C2OC2=CC=CC(CCCNC(=O)OCC3C4=C(C=CC=C4)C4=C3C=CC=C4)=C21.[H]N1C2=NC(=O)N(C3CC(O)C(CO)O3)C=C2OC2=CC=CC(C)=C21.[H]N1C2=NC(=O)N(C3CC(O)C(CO)O3)C=C2OC2=CC=CC(CCCN)=C21 Chemical compound [H]N1C2=NC(=O)N(C3CC(C)C(CC)O3)C=C2OC2=CC=CC(CCCNC(=O)OCC3C4=C(C=CC=C4)C4=C3C=CC=C4)=C21.[H]N1C2=NC(=O)N(C3CC(O)C(CC)O3)C=C2OC2=CC=CC(CCCNC(=O)OCC3C4=C(C=CC=C4)C4=C3C=CC=C4)=C21.[H]N1C2=NC(=O)N(C3CC(O)C(CO)O3)C=C2OC2=CC=CC(C)=C21.[H]N1C2=NC(=O)N(C3CC(O)C(CO)O3)C=C2OC2=CC=CC(CCCN)=C21 TUQDFJQANVWYLL-UHFFFAOYSA-N 0.000 description 1
- LXLXFUHGWSSOKB-UHFFFAOYSA-N [H]N1C2=NC(=O)N(C3CC(O)C(CC)O3)C=C2OC2=C1C(NCCCN1C(=O)C3=C(C=CC=C3)C1=O)=CC=C2.[H]N1C2=NC(=O)N(C3CC(O)C(CO)O3)C=C2OC2=C1C(NCCCN1C(=O)C3=C(C=CC=C3)C1=O)=CC=C2 Chemical compound [H]N1C2=NC(=O)N(C3CC(O)C(CC)O3)C=C2OC2=C1C(NCCCN1C(=O)C3=C(C=CC=C3)C1=O)=CC=C2.[H]N1C2=NC(=O)N(C3CC(O)C(CO)O3)C=C2OC2=C1C(NCCCN1C(=O)C3=C(C=CC=C3)C1=O)=CC=C2 LXLXFUHGWSSOKB-UHFFFAOYSA-N 0.000 description 1
- 239000000370 acceptor Substances 0.000 description 1
- DZBUGLKDJFMEHC-UHFFFAOYSA-N acridine Chemical class C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000010933 acylation Effects 0.000 description 1
- 238000005917 acylation reaction Methods 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 229910001413 alkali metal ion Inorganic materials 0.000 description 1
- 229910001420 alkaline earth metal ion Inorganic materials 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 150000001345 alkine derivatives Chemical class 0.000 description 1
- 125000004183 alkoxy alkyl group Chemical group 0.000 description 1
- 125000003545 alkoxy group Chemical group 0.000 description 1
- 150000003973 alkyl amines Chemical class 0.000 description 1
- 125000005599 alkyl carboxylate group Chemical group 0.000 description 1
- 125000005907 alkyl ester group Chemical group 0.000 description 1
- 125000005600 alkyl phosphonate group Chemical group 0.000 description 1
- 125000006350 alkyl thio alkyl group Chemical group 0.000 description 1
- 239000002168 alkylating agent Substances 0.000 description 1
- 229940100198 alkylating agent Drugs 0.000 description 1
- 125000000304 alkynyl group Chemical group 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical class OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- SRBFZHDQGSBBOR-STGXQOJASA-N alpha-D-lyxopyranose Chemical class O[C@@H]1CO[C@H](O)[C@@H](O)[C@H]1O SRBFZHDQGSBBOR-STGXQOJASA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 229940059260 amidate Drugs 0.000 description 1
- 150000001414 amino alcohols Chemical class 0.000 description 1
- 238000007098 aminolysis reaction Methods 0.000 description 1
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 description 1
- 229960003942 amphotericin b Drugs 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 210000001557 animal structure Anatomy 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 239000000074 antisense oligonucleotide Substances 0.000 description 1
- 238000012230 antisense oligonucleotides Methods 0.000 description 1
- 239000002152 aqueous-organic solution Substances 0.000 description 1
- 229940000489 arsenate Drugs 0.000 description 1
- 229910052785 arsenic Inorganic materials 0.000 description 1
- RQNWIZPPADIBDY-UHFFFAOYSA-N arsenic atom Chemical compound [As] RQNWIZPPADIBDY-UHFFFAOYSA-N 0.000 description 1
- OMLQKKZJMXIQIH-UHFFFAOYSA-N arsorous acid;formic acid Chemical compound OC=O.O[As](O)O OMLQKKZJMXIQIH-UHFFFAOYSA-N 0.000 description 1
- 125000005228 aryl sulfonate group Chemical group 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 238000011948 assay development Methods 0.000 description 1
- 238000011914 asymmetric synthesis Methods 0.000 description 1
- 229910052788 barium Inorganic materials 0.000 description 1
- DSAJWYNOEDNPEQ-UHFFFAOYSA-N barium atom Chemical compound [Ba] DSAJWYNOEDNPEQ-UHFFFAOYSA-N 0.000 description 1
- 230000037429 base substitution Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- UPABQMWFWCMOFV-UHFFFAOYSA-N benethamine Chemical compound C=1C=CC=CC=1CNCCC1=CC=CC=C1 UPABQMWFWCMOFV-UHFFFAOYSA-N 0.000 description 1
- JUHORIMYRDESRB-UHFFFAOYSA-N benzathine Chemical compound C=1C=CC=CC=1CNCCNCC1=CC=CC=C1 JUHORIMYRDESRB-UHFFFAOYSA-N 0.000 description 1
- SRSXLGNVWSONIS-UHFFFAOYSA-N benzenesulfonic acid Chemical compound OS(=O)(=O)C1=CC=CC=C1 SRSXLGNVWSONIS-UHFFFAOYSA-N 0.000 description 1
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid group Chemical group C(C1=CC=CC=C1)(=O)O WPYMKLBDIGXBTP-UHFFFAOYSA-N 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
- SAGINAGERRNGGV-UHFFFAOYSA-N benzyl n-(2-hydroxyethyl)carbamate Chemical compound OCCNC(=O)OCC1=CC=CC=C1 SAGINAGERRNGGV-UHFFFAOYSA-N 0.000 description 1
- WXQCFKYWSKKNKY-UHFFFAOYSA-N benzyl n-(3-hydroxypropyl)carbamate Chemical compound OCCCNC(=O)OCC1=CC=CC=C1 WXQCFKYWSKKNKY-UHFFFAOYSA-N 0.000 description 1
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 description 1
- 102000005936 beta-Galactosidase Human genes 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 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 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 229910052797 bismuth Inorganic materials 0.000 description 1
- JCXGWMGPZLAOME-UHFFFAOYSA-N bismuth atom Chemical compound [Bi] JCXGWMGPZLAOME-UHFFFAOYSA-N 0.000 description 1
- 229910052794 bromium Inorganic materials 0.000 description 1
- JHIWVOJDXOSYLW-UHFFFAOYSA-N butyl 2,2-difluorocyclopropane-1-carboxylate Chemical compound CCCCOC(=O)C1CC1(F)F JHIWVOJDXOSYLW-UHFFFAOYSA-N 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 229910052793 cadmium Inorganic materials 0.000 description 1
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 150000004657 carbamic acid derivatives Chemical class 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 125000002837 carbocyclic group Chemical group 0.000 description 1
- 150000004649 carbonic acid derivatives Chemical class 0.000 description 1
- JLQUFIHWVLZVTJ-UHFFFAOYSA-N carbosulfan Chemical compound CCCCN(CCCC)SN(C)C(=O)OC1=CC=CC2=C1OC(C)(C)C2 JLQUFIHWVLZVTJ-UHFFFAOYSA-N 0.000 description 1
- 150000001734 carboxylic acid salts Chemical class 0.000 description 1
- 229940082638 cardiac stimulant phosphodiesterase inhibitors Drugs 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 229940099352 cholate Drugs 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000012539 chromatography resin Substances 0.000 description 1
- 229940126543 compound 14 Drugs 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000005289 controlled pore glass Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 125000001559 cyclopropyl group Chemical group [H]C1([H])C([H])([H])C1([H])* 0.000 description 1
- 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 1
- PCDQPRRSZKQHHS-ZAKLUEHWSA-N cytidine-5'-triphosphate Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO[P@](O)(=O)O[P@@](O)(=O)OP(O)(O)=O)O1 PCDQPRRSZKQHHS-ZAKLUEHWSA-N 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 229940009976 deoxycholate Drugs 0.000 description 1
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 description 1
- 229960005215 dichloroacetic acid Drugs 0.000 description 1
- SOCTUWSJJQCPFX-UHFFFAOYSA-N dichromate(2-) Chemical compound [O-][Cr](=O)(=O)O[Cr]([O-])(=O)=O SOCTUWSJJQCPFX-UHFFFAOYSA-N 0.000 description 1
- 239000005546 dideoxynucleotide Substances 0.000 description 1
- 150000001993 dienes Chemical class 0.000 description 1
- 150000005690 diesters Chemical class 0.000 description 1
- 125000004177 diethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 229940120503 dihydroxyacetone Drugs 0.000 description 1
- 230000000447 dimerizing effect Effects 0.000 description 1
- 150000002009 diols Chemical class 0.000 description 1
- NAGJZTKCGNOGPW-UHFFFAOYSA-K dioxido-sulfanylidene-sulfido-$l^{5}-phosphane Chemical compound [O-]P([O-])([S-])=S NAGJZTKCGNOGPW-UHFFFAOYSA-K 0.000 description 1
- HNPSIPDUKPIQMN-UHFFFAOYSA-N dioxosilane;oxo(oxoalumanyloxy)alumane Chemical compound O=[Si]=O.O=[Al]O[Al]=O HNPSIPDUKPIQMN-UHFFFAOYSA-N 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- WBZKQQHYRPRKNJ-UHFFFAOYSA-L disulfite Chemical compound [O-]S(=O)S([O-])(=O)=O WBZKQQHYRPRKNJ-UHFFFAOYSA-L 0.000 description 1
- GRWZHXKQBITJKP-UHFFFAOYSA-L dithionite(2-) Chemical compound [O-]S(=O)S([O-])=O GRWZHXKQBITJKP-UHFFFAOYSA-L 0.000 description 1
- NAGJZTKCGNOGPW-UHFFFAOYSA-N dithiophosphoric acid Chemical class OP(O)(S)=S NAGJZTKCGNOGPW-UHFFFAOYSA-N 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000003821 enantio-separation Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 150000002118 epoxides Chemical class 0.000 description 1
- UQPHVQVXLPRNCX-UHFFFAOYSA-N erythrulose Chemical class OCC(O)C(=O)CO UQPHVQVXLPRNCX-UHFFFAOYSA-N 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 125000005448 ethoxyethyl group Chemical group [H]C([H])([H])C([H])([H])OC([H])([H])C([H])([H])* 0.000 description 1
- FAMRKDQNMBBFBR-UHFFFAOYSA-N ethyl n-ethoxycarbonyliminocarbamate Chemical compound CCOC(=O)N=NC(=O)OCC FAMRKDQNMBBFBR-UHFFFAOYSA-N 0.000 description 1
- NPUKDXXFDDZOKR-LLVKDONJSA-N etomidate Chemical compound CCOC(=O)C1=CN=CN1[C@H](C)C1=CC=CC=C1 NPUKDXXFDDZOKR-LLVKDONJSA-N 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 125000004030 farnesyl group Chemical group [H]C([*])([H])C([H])=C(C([H])([H])[H])C([H])([H])C([H])([H])C([H])=C(C([H])([H])[H])C([H])([H])C([H])([H])C([H])=C(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 238000002875 fluorescence polarization Methods 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 125000004216 fluoromethyl group Chemical group [H]C([H])(F)* 0.000 description 1
- 229960002949 fluorouracil Drugs 0.000 description 1
- 230000005021 gait Effects 0.000 description 1
- 229930182830 galactose Chemical class 0.000 description 1
- 229940050410 gluconate Drugs 0.000 description 1
- 150000002303 glucose derivatives Chemical group 0.000 description 1
- WVULKSPCQVQLCU-BUXLTGKBSA-N glycodeoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 WVULKSPCQVQLCU-BUXLTGKBSA-N 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 125000001072 heteroaryl group Chemical group 0.000 description 1
- KUQWZSZYIQGTHT-UHFFFAOYSA-N hexa-1,5-diene-3,4-diol Chemical compound C=CC(O)C(O)C=C KUQWZSZYIQGTHT-UHFFFAOYSA-N 0.000 description 1
- FYAQQULBLMNGAH-UHFFFAOYSA-N hexane-1-sulfonic acid Chemical class CCCCCCS(O)(=O)=O FYAQQULBLMNGAH-UHFFFAOYSA-N 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 150000004677 hydrates Chemical class 0.000 description 1
- 125000000717 hydrazino group Chemical group [H]N([*])N([H])[H] 0.000 description 1
- BHEPBYXIRTUNPN-UHFFFAOYSA-N hydridophosphorus(.) (triplet) Chemical compound [PH] BHEPBYXIRTUNPN-UHFFFAOYSA-N 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000003840 hydrochlorides Chemical class 0.000 description 1
- 239000012433 hydrogen halide Chemical class 0.000 description 1
- 229910000039 hydrogen halide Chemical class 0.000 description 1
- ZMZDMBWJUHKJPS-UHFFFAOYSA-N hydrogen thiocyanate Natural products SC#N ZMZDMBWJUHKJPS-UHFFFAOYSA-N 0.000 description 1
- 150000002443 hydroxylamines Chemical class 0.000 description 1
- UWYVPFMHMJIBHE-OWOJBTEDSA-N hydroxymaleic acid group Chemical group O/C(/C(=O)O)=C/C(=O)O UWYVPFMHMJIBHE-OWOJBTEDSA-N 0.000 description 1
- CBOIHMRHGLHBPB-UHFFFAOYSA-N hydroxymethyl Chemical compound O[CH2] CBOIHMRHGLHBPB-UHFFFAOYSA-N 0.000 description 1
- 150000002454 idoses Chemical class 0.000 description 1
- 150000002463 imidates Chemical class 0.000 description 1
- 125000002883 imidazolyl group Chemical group 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 150000002484 inorganic compounds Chemical class 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
- 229910052809 inorganic oxide Inorganic materials 0.000 description 1
- 229960003786 inosine Drugs 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 150000002540 isothiocyanates Chemical class 0.000 description 1
- GCHPUFAZSONQIV-UHFFFAOYSA-N isovaline Chemical compound CCC(C)(N)C(O)=O GCHPUFAZSONQIV-UHFFFAOYSA-N 0.000 description 1
- BJHIKXHVCXFQLS-PQLUHFTBSA-N keto-D-tagatose Chemical class OC[C@@H](O)[C@H](O)[C@H](O)C(=O)CO BJHIKXHVCXFQLS-PQLUHFTBSA-N 0.000 description 1
- 238000007834 ligase chain reaction Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 150000002634 lipophilic molecules Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 201000005296 lung carcinoma Diseases 0.000 description 1
- 239000001095 magnesium carbonate Substances 0.000 description 1
- ZLNQQNXFFQJAID-UHFFFAOYSA-L magnesium carbonate Chemical compound [Mg+2].[O-]C([O-])=O ZLNQQNXFFQJAID-UHFFFAOYSA-L 0.000 description 1
- 229910000021 magnesium carbonate Inorganic materials 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 150000002736 metal compounds Chemical class 0.000 description 1
- 229910000000 metal hydroxide Inorganic materials 0.000 description 1
- 150000004692 metal hydroxides Chemical class 0.000 description 1
- QARBMVPHQWIHKH-UHFFFAOYSA-N methanesulfonyl chloride Chemical compound CS(Cl)(=O)=O QARBMVPHQWIHKH-UHFFFAOYSA-N 0.000 description 1
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 1
- YACKEPLHDIMKIO-UHFFFAOYSA-N methylphosphonic acid Chemical compound CP(O)(O)=O YACKEPLHDIMKIO-UHFFFAOYSA-N 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- IFNALFAUNCRFBS-UHFFFAOYSA-N morpholin-2-ylcarbamic acid Chemical class OC(=O)NC1CNCCO1 IFNALFAUNCRFBS-UHFFFAOYSA-N 0.000 description 1
- 125000004573 morpholin-4-yl group Chemical group N1(CCOCC1)* 0.000 description 1
- IJCJJNXCMVURNM-UHFFFAOYSA-N n-[(2,4-dichlorophenyl)methylsulfanylphosphanyl]-n-methylmethanamine Chemical compound CN(C)PSCC1=CC=C(Cl)C=C1Cl IJCJJNXCMVURNM-UHFFFAOYSA-N 0.000 description 1
- SKUSIJOMOKTHIX-UHFFFAOYSA-N n-[di(propan-2-yl)amino]phosphanyl-n-propan-2-ylpropan-2-amine Chemical compound CC(C)N(C(C)C)PN(C(C)C)C(C)C SKUSIJOMOKTHIX-UHFFFAOYSA-N 0.000 description 1
- MBHMVABTPGEBDY-UHFFFAOYSA-N n-ethyl-n-methoxyphosphanylethanamine Chemical compound CCN(CC)POC MBHMVABTPGEBDY-UHFFFAOYSA-N 0.000 description 1
- GHRFGOHSYPDQTL-UHFFFAOYSA-N n-methoxyphosphanyl-n-propan-2-ylpropan-2-amine Chemical compound COPN(C(C)C)C(C)C GHRFGOHSYPDQTL-UHFFFAOYSA-N 0.000 description 1
- KVBGVZZKJNLNJU-UHFFFAOYSA-N naphthalene-2-sulfonic acid Chemical compound C1=CC=CC2=CC(S(=O)(=O)O)=CC=C21 KVBGVZZKJNLNJU-UHFFFAOYSA-N 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- YCWSUKQGVSGXJO-NTUHNPAUSA-N nifuroxazide Chemical group C1=CC(O)=CC=C1C(=O)N\N=C\C1=CC=C([N+]([O-])=O)O1 YCWSUKQGVSGXJO-NTUHNPAUSA-N 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- GHLZUHZBBNDWHW-UHFFFAOYSA-N nonanamide Chemical compound CCCCCCCCC(N)=O GHLZUHZBBNDWHW-UHFFFAOYSA-N 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- 229920000620 organic polymer Polymers 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 101150115162 p27 gene Proteins 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 125000001147 pentyl group Chemical group C(CCCC)* 0.000 description 1
- VLTRZXGMWDSKGL-UHFFFAOYSA-M perchlorate Inorganic materials [O-]Cl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-M 0.000 description 1
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 1
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 150000002991 phenoxazines Chemical class 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- WLJVXDMOQOGPHL-UHFFFAOYSA-N phenylacetic acid Chemical compound OC(=O)CC1=CC=CC=C1 WLJVXDMOQOGPHL-UHFFFAOYSA-N 0.000 description 1
- UYWQUFXKFGHYNT-UHFFFAOYSA-N phenylmethyl ester of formic acid Natural products O=COCC1=CC=CC=C1 UYWQUFXKFGHYNT-UHFFFAOYSA-N 0.000 description 1
- 239000002571 phosphodiesterase inhibitor Substances 0.000 description 1
- ABLZXFCXXLZCGV-UHFFFAOYSA-N phosphonic acid group Chemical group P(O)(O)=O ABLZXFCXXLZCGV-UHFFFAOYSA-N 0.000 description 1
- 150000008298 phosphoramidates Chemical class 0.000 description 1
- PTMHPRAIXMAOOB-UHFFFAOYSA-N phosphoramidic acid Chemical class NP(O)(O)=O PTMHPRAIXMAOOB-UHFFFAOYSA-N 0.000 description 1
- SXADIBFZNXBEGI-UHFFFAOYSA-N phosphoramidous acid Chemical group NP(O)O SXADIBFZNXBEGI-UHFFFAOYSA-N 0.000 description 1
- 125000004437 phosphorous atom Chemical group 0.000 description 1
- 230000000865 phosphorylative effect Effects 0.000 description 1
- NAYYNDKKHOIIOD-UHFFFAOYSA-N phthalamide Chemical group NC(=O)C1=CC=CC=C1C(N)=O NAYYNDKKHOIIOD-UHFFFAOYSA-N 0.000 description 1
- 229940075930 picrate Drugs 0.000 description 1
- OXNIZHLAWKMVMX-UHFFFAOYSA-M picrate anion Chemical compound [O-]C1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O OXNIZHLAWKMVMX-UHFFFAOYSA-M 0.000 description 1
- 210000002706 plastid Anatomy 0.000 description 1
- 239000003495 polar organic solvent Substances 0.000 description 1
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 1
- 229920000656 polylysine Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000011698 potassium fluoride Substances 0.000 description 1
- 235000003270 potassium fluoride Nutrition 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 150000003141 primary amines Chemical class 0.000 description 1
- 230000037452 priming Effects 0.000 description 1
- MFDFERRIHVXMIY-UHFFFAOYSA-N procaine Chemical compound CCN(CC)CCOC(=O)C1=CC=C(N)C=C1 MFDFERRIHVXMIY-UHFFFAOYSA-N 0.000 description 1
- 229960004919 procaine Drugs 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- JKANAVGODYYCQF-UHFFFAOYSA-N prop-2-yn-1-amine Chemical compound NCC#C JKANAVGODYYCQF-UHFFFAOYSA-N 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 239000002719 pyrimidine nucleotide Substances 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 230000011514 reflex Effects 0.000 description 1
- HSSLDCABUXLXKM-UHFFFAOYSA-N resorufin Chemical compound C1=CC(=O)C=C2OC3=CC(O)=CC=C3N=C21 HSSLDCABUXLXKM-UHFFFAOYSA-N 0.000 description 1
- 150000008223 ribosides Chemical class 0.000 description 1
- 125000006413 ring segment Chemical group 0.000 description 1
- XTDHBAVVPOKCKF-UHFFFAOYSA-N s-(benzoyltrisulfanyl) benzenecarbothioate Chemical compound C=1C=CC=CC=1C(=O)SSSSC(=O)C1=CC=CC=C1 XTDHBAVVPOKCKF-UHFFFAOYSA-N 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000007423 screening assay Methods 0.000 description 1
- 125000002914 sec-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- QRUBYZBWAOOHSV-UHFFFAOYSA-M silver trifluoromethanesulfonate Chemical compound [Ag+].[O-]S(=O)(=O)C(F)(F)F QRUBYZBWAOOHSV-UHFFFAOYSA-M 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 229940075554 sorbate Drugs 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 108010068698 spleen exonuclease Proteins 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 229910052712 strontium Inorganic materials 0.000 description 1
- CIOAGBVUUVVLOB-UHFFFAOYSA-N strontium atom Chemical compound [Sr] CIOAGBVUUVVLOB-UHFFFAOYSA-N 0.000 description 1
- 229940014800 succinic anhydride Drugs 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- IIACRCGMVDHOTQ-UHFFFAOYSA-M sulfamate Chemical compound NS([O-])(=O)=O IIACRCGMVDHOTQ-UHFFFAOYSA-M 0.000 description 1
- IIACRCGMVDHOTQ-UHFFFAOYSA-N sulfamic acid Chemical class NS(O)(=O)=O IIACRCGMVDHOTQ-UHFFFAOYSA-N 0.000 description 1
- 229940124530 sulfonamide Drugs 0.000 description 1
- 150000003456 sulfonamides Chemical class 0.000 description 1
- 150000003871 sulfonates Chemical class 0.000 description 1
- 150000003457 sulfones Chemical class 0.000 description 1
- 150000003460 sulfonic acids Chemical class 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- WBWWGRHZICKQGZ-HZAMXZRMSA-N taurocholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCS(O)(=O)=O)C)[C@@]2(C)[C@@H](O)C1 WBWWGRHZICKQGZ-HZAMXZRMSA-N 0.000 description 1
- AWDRATDZQPNJFN-VAYUFCLWSA-N taurodeoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCS(O)(=O)=O)C)[C@@]2(C)[C@@H](O)C1 AWDRATDZQPNJFN-VAYUFCLWSA-N 0.000 description 1
- QBYUNVOYXHFVKC-GBURMNQMSA-N taurolithocholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCS(O)(=O)=O)C)[C@@]2(C)CC1 QBYUNVOYXHFVKC-GBURMNQMSA-N 0.000 description 1
- 125000001981 tert-butyldimethylsilyl group Chemical group [H]C([H])([H])[Si]([H])(C([H])([H])[H])[*]C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 150000003512 tertiary amines Chemical class 0.000 description 1
- FPGGTKZVZWFYPV-UHFFFAOYSA-M tetrabutylammonium fluoride Chemical compound [F-].CCCC[N+](CCCC)(CCCC)CCCC FPGGTKZVZWFYPV-UHFFFAOYSA-M 0.000 description 1
- MPLHNVLQVRSVEE-UHFFFAOYSA-N texas red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 description 1
- 150000003568 thioethers Chemical class 0.000 description 1
- ZEMGGZBWXRYJHK-UHFFFAOYSA-N thiouracil Chemical compound O=C1C=CNC(=S)N1 ZEMGGZBWXRYJHK-UHFFFAOYSA-N 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000041 toxicology testing Toxicity 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- 238000000844 transformation Methods 0.000 description 1
- LGSAOJLQTXCYHF-UHFFFAOYSA-N tri(propan-2-yl)-tri(propan-2-yl)silyloxysilane Chemical compound CC(C)[Si](C(C)C)(C(C)C)O[Si](C(C)C)(C(C)C)C(C)C LGSAOJLQTXCYHF-UHFFFAOYSA-N 0.000 description 1
- 125000005270 trialkylamine group Chemical group 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 125000000026 trimethylsilyl group Chemical group [H]C([H])([H])[Si]([*])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000002264 triphosphate group Chemical group [H]OP(=O)(O[H])OP(=O)(O[H])OP(=O)(O[H])O* 0.000 description 1
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 1
- YFTHZRPMJXBUME-UHFFFAOYSA-N tripropylamine Chemical compound CCCN(CCC)CCC YFTHZRPMJXBUME-UHFFFAOYSA-N 0.000 description 1
- PBYZMCDFOULPGH-UHFFFAOYSA-N tungstate Chemical compound [O-][W]([O-])(=O)=O PBYZMCDFOULPGH-UHFFFAOYSA-N 0.000 description 1
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 1
- 229940045145 uridine Drugs 0.000 description 1
- 210000003934 vacuole Anatomy 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 229910052727 yttrium Inorganic materials 0.000 description 1
- 239000010457 zeolite Substances 0.000 description 1
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
- C07H19/06—Pyrimidine radicals
-
- 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
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
- C12Q2525/10—Modifications characterised by
- C12Q2525/101—Modifications characterised by incorporating non-naturally occurring nucleotides, e.g. inosine
-
- 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
- This invention relates to the field of labels, particularly labels for diagnostic or analytical use.
- it relates to oligonucleotides that are modified to enhance the binding affinity or the binding specificity of the oligonucleotides for complementary sequences and that in addition optionally bear a readily detectable characteristic.
- U.S. Pat. No. 5,502,177 discloses phenoxazine polycycle-containing oligonucleotides and monomers for preparing the oligonucleotides.
- compositions or methods accomplish one or more of the following objects.
- An object of this invention is to increase the affinity of oligonucleotides for their complementary sequences.
- An object of this invention is to increase the specificity of oligonucleotides for their complementary sequences.
- Another object of this invention is to provide detectable labels for use in diagnostic assays.
- Another object is to enhance diagnostic assays that use oligonucleotides.
- Another object is to improve the therapeutic efficacy of oligonucleotides.
- Another object is to improve the potency of oligonucleotides as antisense reagents that affect gene expression by altering intracellular metabolism of complementary RNA sequences encoding a target gene(s).
- Another object is to provide chemical intermediates and synthesis methods to prepare the invention compositions.
- the invention provides compounds having the structure (1)
- embodiments of the compounds of this invention include oligonucleotides of structure (2), (2A), (2B), or (2C) wherein
- Embodiments include compositions useful as intermediates in making the structure (1) compounds, including intermediates having structure (4) and tautomers, solvates and salts thereof wherein,
- the invention includes contacting a structure (2), (2A), (2B), or (2C) oligonucleotide, wherein n is at least about 7, with a sample suspected to contain a nucleic acid having a base sequence that is at least substantially complementary to the structure (2), (2A), (2B), or (2C) oligonucleotide.
- the invention includes detecting the presence, absence or amount of a complex comprising a structure (2), (2A), (2B), or (2C) oligonucleotide, wherein n is at least about 7, and a nucleic acid having a base sequence that is at least substantially complementary to the structure (2), (2A), (2B), or (2C) oligonucleotide.
- the invention includes converting a structure (4) compound to a compound of structure (1) where R 34 is —O— or —S— and the R 2 atom or moiety alpha to the ring containing R 27 is —O—, —S— or —CH 2 —, by displacing R 24 .
- the invention includes converting a structure (4A) compound (a structure (1) compound where R 2 is replaced with —NH 2 ); to a compound of structure (1), by reductive alkylation of the —NH 2 group to yield a structure (1) compound where the R 2 moiety alpha to the ring containing R 27 is —NH—.
- the invention comprises all combinations formed by independently selecting individual Markush group members and assembling them in accordance with the teachings herein.
- the invention optionally excludes any feature or substance found in, or obvious over, the prior art.
- Structural formulas are designated as parenthetical numerals. We intend that designation of aromaticity with respect to carbocycles and heterocycles herein includes any highly resonant unsaturated ring structure. Alternatively, placement of double bonds, where indicated, represents one potential structure for the depicted compound but we intend this depiction to include other resonant states of the compound as well as protonated and charged species, only one of which a structure may show.
- the invention includes invention compounds in unpurified, substantially purified and purified forms. It includes invention compounds that are present with any additional component(s) such as a solvent, reactant or by-product that is present during invention compound synthesis or purification, and any additional component(s) that is present during the use or manufacture of an invention compound.
- additional component(s) such as a solvent, reactant or by-product that is present during invention compound synthesis or purification, and any additional component(s) that is present during the use or manufacture of an invention compound.
- Halo and halogen mean F, Cl, Br or I.
- Alkyl means unbranched, branched or cyclic hydrocarbons that are saturated or unsaturated, or combinations thereof. Alkyl includes all isomers, e.g., stereoisomers, positional isomers, diastereomers and regioisomers. Alkyl moieties that are unsaturated will typically contain 1, 2, 3 or more —CH ⁇ CH— or —C ⁇ C— groups, usually one such group.
- Substituted alkyl means unbranched, branched or cyclic hydrocarbons that are saturated or unsaturated, or combinations thereof, where the hydrocarbon contains a heteroatom linked to a carbon or a heteroatom that replaces a carbon atom.
- Substituted alkyl includes all isomers, e.g., stereoisomers, positional isomers, diastereomers and regioisomers.
- Substituted alkyl moieties that are unsaturated will typically contain 1, 2, 3 or more —CH ⁇ CH— or —C ⁇ —C— groups, usually one such group.
- Substituted alkyl includes alkyl groups having substituents linked to a carbon atom or substituents that interrupt a carbon atom chain, and unless otherwise defined, substituents include ethers (—O—), ketones (—C(O)—), —OR 5 , —C(O)OR 5 , —C(O)O—, —OC(O)—, —C(O)H, —OCH 2 —, —OCH 2 CH 2 —, —OCH 2 O—, —OCH 2 CH 2 O—, —NR 5 —, —NHR 5 , —NHC(O)—, —C(O)NH—, C(O)NHR 5 , —OC(O)NR 5 —, —OC(O)NHR 5 , —NR 5 C(O)NR 5 —, —NR 5 C(O)NHR 5 , —NR 5 CH 2 —, —NR 5 CH 2 CH 2 —, —S
- the invention compounds herein do not include obviously unstable structures, e.g., —O—O—, —O—S— or unsaturated cyclopropyl, unless they are useful as transitory intermediates in the preparation of more stable compounds.
- “monosaccharide” means a polyhydroxy aldehyde or ketone having the empirical formula (CH 2 O) n where n is 3, 4, 5, 6 or 7. Monosaccharide includes open-chain and closed-chain forms, but will usually be closed chain forms. Monosaccharide includes hexofuranose and pentofuranose sugars such as 2′-deoxyribose, ribose, arabinose, xylose, their 2′-deoxy and 3′-deoxy derivatives, their 2′,3′-dideoxy derivatives, and their derivatives containing R 21 linked to the 2′ or 3′ position, usually the 2′ position.
- hexofuranose and pentofuranose sugars such as 2′-deoxyribose, ribose, arabinose, xylose, their 2′-deoxy and 3′-deoxy derivatives, their 2′,3′-dideoxy derivatives, and their derivatives containing R 21 linked to the 2′ or 3′ position,
- Monosaccharide also includes the 2′,3′ dideoxydidehydro derivative of ribose.
- Monosaccharides include the D- and L-isomers of glucose, fructose, mannose, idose, galactose, allose, gulose, altrose, talose, fucose, erythrose, threose, lyxose, erythrulose, ribulose, xylulose, ribose, arabinose, xylose, psicose, sorbose, tagatose, glyceraldehyde, dihydroxyacetone and their monodeoxy derivatives such as rhamnose. Monosaccharides are optionally protected or partially protected.
- a “protecting group” means a moiety that prevents the atom to which it is linked from participating in unwanted reactions.
- R 5 is a protecting group for the oxygen atom found in a hydroxyl or carboxyl group
- R 5 is a protecting group for sulfur in thiols for instance
- R 5 is a nitrogen atom protecting group for primary or secondary amines. Hydroxyl, amine and other reactive groups are found in invention compounds at, e.g., R 2 , R 21 or oligonucleotide linkages or oligonucleotide bases.
- the protecting groups for oxygen, sulfur or nitrogen atoms are usually used to prevent unwanted reactions with electrophilic compounds, such as acylating or phosphorylating agents used, e.g., in nucleoside, nucleotide or oligonucleotide chemistry.
- Protecting groups are intended to be removed by known procedures, although it will be understood that the protected intermediates fall within the scope of this invention. The removal of the protecting group may be arduous or straightforward, depending upon the economics and nature of the conversions involved. In general, one will use a protecting group with exocyclic amines in the B groups of the compounds of this invention. For oligonucleotide containing such B groups to be fully binding competent, exocyclic amines must be deprotected because the amine groups participate in hydrogen bonding with complementary bases.
- reversible protecting groups for the 5′ and 3′ hydroxyl groups of pentofuranose sugars in nucleotides intended for use as monomers in synthesis of oligonucleotides containing 3,5′ linkages.
- Protecting groups commonly are employed to protect against covalent modification of a sensitive group in reactions such as phosphorylation, alkylation or acylation. Ordinarily, protecting groups are removed by, e.g. hydrolysis, elimination or aminolysis.
- Suitable protecting groups and criteria for their selection are described in T. W. Greene and P. G. M. Wuts, Eds. “Protective Groups in Organic Synthesis” 2nd edition, Wiley Press, at pps. 10-142, 143-174, 175-223, 224-276, 277-308, 309-405 and 406-454.
- Embodiments include salts and complexes of invention compounds, including pharmaceutically acceptable or salts that are relatively non-toxic.
- the invention compounds may have one or more moieties that carry at least a partial positive or negative charge in aqueous solutions, typically at a pH of about 4-10, that can participate in forming a salt, a complex, a composition with partial salt and partial complex properties or other noncovalent interactions, all of which we refer to as a “salt(s)”.
- Salts are usually biologically compatible or pharmaceutically acceptable or non-toxic, particularly for mammalian cells. Salts that are biologically toxic are optionally used with synthetic intermediates of invention compounds. When a water soluble composition is desired, monovalent salts are usually preferred.
- Metal salts typically are prepared by reacting the metal hydroxide with a compound of this invention.
- metal salts which are optionally prepared in this way are salts containing Li + , Na + , and K + .
- a less soluble metal salt can be precipitated from the solution of a more soluble salt by adding a suitable metal compound.
- Invention salts may be formed from acid addition of certain organic acids, such as organic carboxylic acids, and inorganic acids, such as alkylsulfonic acids or hydrogen halide acids, to acidic or basic centers on invention compounds, such as basic centers on the invention pyrimidine base analogs.
- Metal salts include ones containing Na + , Li + , K + , Ca ++ or Mg ++ .
- Other metal salts may contain aluminum, barium, strontium, cadmium, bismuth, arsenic or zinc ion.
- Salt(s) of invention compounds may comprise a combination of appropriate cations such as alkali and alkaline earth metal ions or ammonium and quaternary ammonium ions with the acid anion moiety of the phosphoric acid or phosphonic acid group, which may be present in invention polymers or monomers.
- Salts are produced by standard methods, including dissolving free base in an aqueous, aqueous-alcohol or aqueous-organic solution containing the selected acid, optionally followed by evaporating the solution.
- the free base is reacted in an organic solution containing the acid, in which case the salt usually separates directly or one can concentrate the solution.
- Suitable amine salts include amines having sufficient basicity to form a stable salt, preferably amines of low toxicity including trialkyl amines (tripropylamine, triethylamine, trimethylamine), procaine, dibenzylamine, N-benzyl-betaphenethylamine, ephenamine, N,N′-dibenzylethylenediamine, N-ethylpiperidine, benzylamine and dicyclohexylamine.
- trialkyl amines tripropylamine, triethylamine, trimethylamine
- procaine dibenzylamine
- dibenzylamine N-benzyl-betaphenethylamine
- ephenamine N,N′-dibenzylethylenediamine
- N-ethylpiperidine benzylamine and dicyclohexylamine.
- Salts include organic sulfonic acid or organic carboxylic acid salts, made for example by addition of the acids to basic centers, typically amines.
- Exemplary sulfonic acids include C 6-16 aryl sulfonic acids, C 6-16 heteroaryl sulfonic acids and C 1-16 alkyl sulfonic acids such as phenyl sulfonic acid, a-naphthalene sulfonic acid, ⁇ -naphthalene sulfonic acid, (S)-camphorsulfonic acid, methyl (CH 3 SO 3 H), ethyl (C 2 H 5 SO 3 H), n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, pentyl and hexyl sulfonic acids.
- Exemplary organic carboxylic acids include C 1-16 alkyl, C 6-16 aryl carboxylic acids and C 4-16 heteroaryl carboxylic acids such as acetic, glycolic, lactic, pyruvic, malonic, glutaric, tartaric, citric, fumaric, succinic, malic, maleic, oxalic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic, salicylic, nicotinic and 2-phenoxybenzoic.
- Invention salts include those made from inorganic acids, e.g., HF, HCl, HBr, HI, H 2 SO 4 , H 3 PO 4 , Na 2 CO 3 , K 2 CO 3 , CaCO 3 , MgCO 3 and NaClO 3 .
- Suitable anions which are optionally present with a cation such a Ca ++ , Mg ++ , Li + , Na + or K + , include arsenate, arsenite formate, sorbate, chlorate, perchlorate, periodate, dichromate, glycodeoxycholate, cholate, deoxycholate, desoxycholate, taurocholate, taurodeoxycholate, taurolithocholate, tetraborate, nitrate, nitrite, sulfite, sulfamate, hyposulfite, bisulfite, metabisulfite, thiosulfate, thiocyanate, silicate, metasilicate, CN ⁇ , gluconate, gulcuronate, hippurate, picrate, hydrosulfite, hexafluorophosphate, hypochlorite, hypochlorate, borate, metaborate, tungstate and urate.
- Salts also include the invention compound salts with one or more amino acids.
- Many amino acids are suitable, especially the naturally-occurring amino acids found as protein components, although the amino acid typically is one bearing a side chain with a basic or acidic group, e.g., lysine, arginine, histidine or glutamic acid, or a neutral group such as glycine, serine, threonine, alanine, isoleucine, or leucine.
- compositions include compounds in their un-ionized, as well as zwitterionic form, and combinations with stoichiometric amounts of water as in hydrates.
- the compounds of the invention include enriched or resolved optical isomers at any or all asymmetric atoms as are apparent from the depictions. Both racemic and diasteromeric mixtures, as well as the individual optical isomers isolated or synthesized so as to be substantially free of their enantiomeric or diastereomeric partners, are all within the scope of the invention. Chiral centers may be found in invention compounds at, for example, R 1 , R 2 and R 21 .
- One or more of the following enumerated methods are used to prepare the enantiomerically enriched or pure isomers herein.
- the methods are listed in approximately their order of preference, i.e., one ordinarily should employ stereospecific synthesis from chiral precursors before chromatographic resolution before spontaneous crystallization.
- Stereospecific synthesis is described in the examples. Methods of this type conveniently are used when the appropriate chiral starting material is available and reaction steps are chosen do not result in undesired racemization at chiral sites.
- One advantage of stereospecific synthesis is that it does not produce undesired enantiomers that must be removed from the final product, thereby lowering overall synthetic yield.
- those skilled in the art would understand what starting materials and reaction conditions should be used to obtain the desired enantiomerically enriched or pure isomers by stereospecific synthesis. If an unexpected racemization occurs in a method thought to be stereospecific then one needs only to use one of the following separation methods to obtain the desired product.
- Another method entails converting the enantiomers in the mixture to diasteriomers with chiral auxiliaries and then separating the conjugates by ordinary column chromatography.
- This is a very suitable method, particularly when the embodiment contains free carboxyl, amino or hydroxyl that will form a salt or covalent bond to a chiral auxiliary.
- Chirally pure amino acids, organic acids or organosulfonic acids are all worthwhile exploring as chiral auxiliaries, all of which are well known in the art. Salts with such auxiliaries can be formed, or they can be covalently (but reversibly) bonded to the functional group.
- pure D or L amino acids can be used to amidate the carboxyl group of embodiments of this invention and then separated by chromatography.
- Enzymatic resolution is another method of potential value.
- one prepares covalent derivatives of the enantiomers in the racemic mixture, generally lower alkyl esters (for example of carboxyl), and then exposes the derivative to enzymatic cleavage, generally hydrolysis.
- an enzyme must be chosen that is capable of stereospecific cleavage, so it is frequently necessary to routinely screen several enzymes. If esters are to be cleaved, then one selects a group of esterases, phosphatases, and lipases and determines their activity on the derivative. Typical esterases are from liver, pancreas or other animal organs, and include porcine liver esterase.
- the enantiomeric mixture separates from solution or a melt as a conglomerate, i.e., a mixture of enantiomerically-pure crystals, then the crystals can be mechanically separated, thereby producing the enantiomerically enriched preparation.
- This method is not practical for large scale preparations and is of no value for true racemic compounds.
- Asymmetric synthesis is another technique for achieving enantiomeric enrichment. For example, a chiral protecting group is reacted with the group to be protected and the reaction mixture allowed to equilibrate. If the reaction is enantiomerically specific then the product will be enriched in that enantiomer.
- R 2 is a key functionality. It is substituted on the polycycle depicted in structure (1), less R 2 .
- the combination of the polycycle and R 2 is termed the polycyclic substructure.
- R 2 consists of two principal structural features denominated —A(Z) X1 .
- Group A is a spacer that is used to position the Z group(s) and attach it to the remainder of the polycycle.
- the Z group(s) serve as a site for attachment of a detectable label or to enhance the hydrogen bonding of the polycycle to the complementary guanine base. In some embodiments, Z is capable of performing both functions.
- Z groups capable of hydrogen bonding are useful as sites for covalent bonding to detectable labels, but not all Z groups that are useful as label-bonding sites are capable of hydrogen bonding to guanine.
- Z contains at least one atom other than carbon, typically O, N or S.
- the R 2 groups possess at least one of these practical utilities. It would be routine to make and test them to determine the best use of any one embodiment.
- Z groups capable of base-pairing are believed to hydrogen bond with N 7 of guanine in a complementary nucleic acid sequence when incorporated into a polycyclic substructure-substituted oligonucleotide.
- the resulting duplex has greater stability than one containing a native GC pair because the R 2 group provides an additional point for hydrogen bonding to the complementary guanine base.
- these embodiments serve as cytosine surrogates for supplemented Watson-Crick base-pairing.
- a base-pairing substituent Z is defined functionally as any group that, when taken together with the remainder of R 2 , is capable of increasing the temperature of melting of any of the oligonucleotides 3-9 in Table 1 by at least about 2 degrees Centigrade when substituted as shown in Table 1.
- R 2 does not contain a substituent that is capable of contributing to base pairing or hydrogen bonding then R 2 is useful at least as a point of attachment for a detectable label.
- Z groups need only be reactive with a bifunctional cross-linking agent or with the label directly.
- the polycyclic substructure is itself fluorescent, and in these cases it is not necessary to link the Z group to a detectable label.
- the polycyclic substructure is detectable by fluorescent emissions, or by adsorption and energy transfer to an emitting (second) label present on a binding partner in the same fashion as is used in EMIT technologies well-known in the diagnostics field.
- Spacer A is substituted with from 1 to 3 Z groups.
- Z is a base-pairing hydrogen bonding group then “X1” is preferably 1 or 2, ordinarily 1. Similarly, for reasons of steric access it is preferred that only 1 or 2 Z groups are present on spacer A when R 2 is intended to function as a label bonding site.
- the spacer group A and the Z substituent(s) will interact functionally, i.e., changes in group A may have an impact on the physical or chemical properties of Z, and vice-versa.
- changes in group A may have an impact on the physical or chemical properties of Z, and vice-versa.
- changes in spacer A that would reduce or increase the ability of Z to hydrogen bond or to react with a label or cross-linking agent.
- a readily apparent instance of this would be substitution of A with electron donors or acceptors proximal to a Z group, which may affect hydrogen bonding between Z and guanine.
- Spacer A typically contains a backbone chain of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 carbon atoms, any 1, 2 or 3 of which are optionally replaced with N, O or S atoms, usually 1 N, O or S atom.
- the backbone chain refers to the atoms that connect the Z group(s) to the ring carbon atom at the R 2 binding site on the polycycle.
- the number of spacer backbone atoms does not include terminal Z group atoms.
- R 2 does not include protected amine as described in U.S. Pat. No. 5,502,177.
- the spacer A backbone is linear or one or more backbone atoms are substituted, which results in branching. Ordinarily, when 1 Z group is present then A will contain a linear backbone of 2 to 8, usually 2 to 4 atoms.
- the backbone generally is carbon only, bonded by saturated or unsaturated bonds. If unsaturated bonds are present, the backbone generally will contain 1 to 2 double or triple bonds. Preferably, the backbone is saturated. If a heteroatom is present in the backbone it typically will be O or S. Preferably the heteroatom is O, and preferably only 1 O is present in the backbone chain.
- Heteroatoms are used to replace any of the backbone carbon atoms, but preferably are used to replace the carbon atom alpha (adjacent) to the polycyclic ring.
- the atom in the spacer chain that is bonded to the polycyclic substructure is unsubstituted, e.g., —O—, —S—, —NH— or —CH 2 —, and, in general, the next 1, 2 or 3 atoms in the spacer are unsubstituted carbon.
- the spacer A backbone is optionally substituted independently with 1, 2 or 3 of the following: C 1 -C 8 alkyl, —OR 5 , ⁇ O, —NO 2 , —N 3 , —COOR 5 , —N(R 5 ) 2 , or —CN groups, C 1 -C 8 alkyl substituted with —OH, ⁇ O, —NO 2 , —N 3 , —COOR 5 , —N(R 5 ) 2 , or —CN groups, or any of the foregoing in which —CH 2 — is replaced with —O—, —NH— or —N(C 1 -C 8 alkyl), wherein R 5 is H or a protecting group. Certain of these groups may function as Z sites for linking to detectable labels, but need not be used for that purpose unless desired. In some embodiments these substituents are useful in increasing the lipophilicity of the compounds of this invention.
- Group Z detectable labels include all of the conventional assayable substances used heretofore in labeling oligonucleotides or proteins. Examples are well known and include fluorescent moieties such as fluorescein, chemiluminescent substances, radioisotopes, chromogens, or enzymes such as horseradish peroxidase.
- fluorescent moieties such as fluorescein, chemiluminescent substances, radioisotopes, chromogens, or enzymes such as horseradish peroxidase.
- the residue of any bifunctional or multifunctional agent used to crosslink the Z group(s) to the A backbone is defined to be part of the Z group, and the residue of the detectable label is considered also to represent part of Z.
- Group Z also encompasses substituents that are not detectable by conventional diagnostic means used in clinical chemistry settings (e.g., UV or visible light absorption or emission, scintillation or gamma counting, or the like) but which are nonetheless capable of reacting with a crosslinking agent or a detectable label to form a covalent bond.
- the Z groups function as intermediates in the synthesis of the labelled reagent.
- Typical Z groups useful for this purpose include —NH 2 , —CHO, —SH, —CO 2 Y or OY, where Y is H, 2-hydroxypyridine, N-hydroxysuccinimide, p-nitrophenyl, acylimidazole, maleimide, trifluoroacetate, an imido, a sulfonate, an imine 1,2-cyclohexanedione, glyoxal or an alpha-halo ketone.
- Suitable spacers, reactive groups and detectable labels have been described, e.g., U.S. Pat. Nos.
- Z also is a hydrogen bond donor moiety or a moiety, when taken together with the influence of spacer A, has a net positive charge of at least about +0.5 at pH 6-8 in aqueous solutions.
- Z groups are designated R 2D .
- R 2D is covalently linked to a short spacer A having a backbone (otherwise described above) of 2, 3, 4, 5 or 6 atoms, designated R 2C .
- R 2C short spacer chain backbone atoms are C atoms and optionally one or two atoms independently selected from the group consisting of O, N or S atoms.
- R 2C short spacer chain backbones include unbranched and branched alkyl that optionally contain one or two independently selected O, N or S atoms.
- R 2C is unbranched, i.e. the backbone has no hydrocarbon substituents.
- Any branching if present, will usually consist of a C 1 -C 3 alkyl group, usually a methyl or ethyl group, or C 1 -C 3 alkyl substituted with —OH, ⁇ O, —O(C 1 -C 3 alkyl), —CN, N 3 or 1, 2, 3 or 4 halogen atoms.
- R 36 is —H, —CH 3 , —CH 2 CH 3 , a protecting group, a monosaccharide, where the monosaccharide is usually linked at the monosaccharide's 1′ position and where any monosaccharide hydroxyl groups are optionally protected, typically an R 36 monosaccharide is 2′-deoxyribose, a 2′-deoxy-2′-R 21 -substituted ribose or arabinose such as 2′-deoxy-2′-fluororibose or 2′-deoxy-2′-fluoroarabinose, or the monosaccharide is ribose or arabinose;
- R 2 moieties having the structure —R 59 —NH 2 where R 59 has the structure —R 6 —R 60 —, including —R 6 —(CH 2 ) t —N(R 3 ) 2 , where R 6 is usually —O—, —S—, —NH— or —CH 2 —, R 60 is —CHR 51 —(CHR 51 ) Z3 —(R 61 ) Z1 —(CHR 51 ) Z2 —CHR 51 — where R 61 is —O—, —S—, C(O), —CHR 5 or —NR 5 — and usually 0, 1 or 2 R 51 are methyl or ethyl; R 51 independently is —H, methyl or ethyl; Z3 is 1, 2 or 3, usually 1; Z1 is 0 or 1, usually 0; and Z2 is 1, 2 or 3, usually 1.
- any functional groups e.g., —OH, —NH 2 , —COOH, or —SH, that are optionally present at R 21 are usually protected.
- These embodiments are useful as intermediates useful to make monomers for oligonucleotide synthesis.
- R 2 moieties having the structure where R 61 is —H, alkyl having 1, 2, 3 or 4 carbon atoms or optionally protected substituted alkyl having 1, 2, 3, 4, 5 or 6 carbon atoms including —CH 3 and —CH 2 CH 3 , and R 62 is —H, —NH 2 or —NH(CH 3 ).
- R 62 is —H, —NH 2 or —NH(CH 3 ).
- Other embodiments are —R 6 —(CH 2 ) 2-8 —NH 2 , —R 6 —(CH 2 ) 2-8 —OR 5 or —R 6 —(CH 2 ) 2-8 —CO 2 R 5A .
- R 1 linker groups to covalently bond the invention base to the selected binding partner, although it will be understood that this need not be the sole use for the linker functionality.
- a group present in R 1 linkers principally serves as the site for covalently bonding the invention base to a binding partner, typically by incorporating the invention base via the linker residue into a polymeric binding partner by grafting or copolymerization.
- R 1 linkers also optionally are substituted with groups that ordinarily will not participate in binding to the binding partner, e.g., halo, azido and protected hydroxyl. Generally, such linker groups will contain from 2 to about 50 atoms. If it contains a cycle the cyclic functionality typically will be an oxygen, sulfur or phosphorus-containing saturated or unsaturated heterocycle having a total of about from 5 to 7 ring atoms and 1 to 3 heteroatoms.
- the cycle will be a monosaccharide, typically (i) a hexose, (ii) a hexose such as glucose substituted with phosphate, protected phosphate, hydrogen phosphonate, a phosphoramidate, hydroxyl or protected hydroxyl, (iii) a furanose or (iv) a furanose substituted with phosphate, protected phosphate, hydrogen phosphonate, a phosphoramidate, hydroxyl or protected hydroxyl.
- Typical furanose sugars include ribose, 2′-deoxyribose, 2′-deoxy-2′-R 21 -substituted ribose and their 2′ ara isomers.
- R 1 is an abasic nucleotide residue or such a residue derivatized so as to be capable of incorporation into an oligonucleotide.
- the R 1 linker frequently comprises an activated group or other group which can react with a polymer or other binding partner to be labeled with the polycyclic substructure.
- groups described below that are compatible with commonly available oligonucleotide synthetic chemistries are useful.
- Other examples of reactant groups for covalent labeling are well-known from the diagnostic fields and have heretofore been used commonly to label proteins and oligonucleotide probes, as is more fully discussed below.
- R 1 is a bifunctional or multifunctional organic linker group such as alkyl, alkene, alkyne, alkoxyalkyl, alkylthioalkyl, alkoxy, saturated or unsaturated heterocycle that is substituted with at least one group capable of being crosslinked with or incorporated into a polymer, e.g., such groups as hydroxy, amino, carboxyl, vinyl, phosphate or phosphonate.
- a bifunctional or multifunctional organic linker group such as alkyl, alkene, alkyne, alkoxyalkyl, alkylthioalkyl, alkoxy, saturated or unsaturated heterocycle that is substituted with at least one group capable of being crosslinked with or incorporated into a polymer, e.g., such groups as hydroxy, amino, carboxyl, vinyl, phosphate or phosphonate.
- U.S. Pat. No. 5,502,177 describes suitable linker groups.
- R 1 linker suitable for oligonucleotide synthesis is protected monosaccharides, such as ribofuranose and deoxyribofuranose sugars of structure (5) where an invention base is linked to the open valence at the 1′ position.
- D is hydroxyl, protected hydroxyl or is an oligonucleotide coupling group and D 1 is independently R 21 or an oligonucleotide coupling group, but both D 1 are not coupling groups.
- R 1 is typically structure (5) where one D 1 is an oligonucleotide coupling group and D is —OH or protected hydroxyl.
- Coupling group as used herein means any group suitable for generating a phosphodiester linkage or phosphodiester substitute linkage between nucleotide bases or their analogs. These coupling groups are conventional and well-known for the preparation of oligonucleotides, and are prepared and used in the same fashion here. They are usually configured as the b anomers as denoted in structure (5) or optionally as the alpha anomers. In general, each compound comprising structure (5) will contain two coupling groups: D or D 1 , but with only one D 1 being a coupling group. The coupling groups are used as intermediates in the preparation of 3′,5′ 5′,3′, 5′,2′ and 2′,5′ internucleotide linkages in accord with known methods.
- Suitable coupling groups for phosphodiester linkages or phosphodiester substitute linkages containing phosphorus include OH, H-phosphonate; (for amidite chemistries) alkylphosphonamidites or phosphoramidites such as ⁇ -cyanoethylphosphoramidite, N,N-diisopropylamino- ⁇ -cyanoethoxyphosphine, N,N-diisopropylamino-methoxyphosphine, N,N-diethylamino-methoxyphosphine, N,N-diethylamino- ⁇ -cyanoethoxyphosphine, N-morpholino- ⁇ -cyanoethoxyphosphine, N-morpholino methoxyphosphine, bismorpholino-phosphine, N,N-dimethylamino- ⁇ -cyanoethylmercapto-phosphine, N,N-dimethylamino-2,4-dichlorobenz
- D 1 is a coupling group
- D typically will be hydroxyl protected with a group suitable for ensuring that the monomer is added to the oligonucleotide rather than dimerizing.
- groups are well known and include DMT, MMT, FMOC (9-fluorenylmethoxycarbonyl), PAC (phenoxyacetyl), a trialkyl (C 1 -C 6 alkyl, each alkyl group is independently chosen) silyl ether or an alkyl (C 1 -C 6 alkyl) diaryl (e.g., phenyl) silyl ether such as TBDMS (t-butyldiphenylsilyl) and TMS (trimethylsilyl).
- TBDMS t-butyldiphenylsilyl
- TMS trimethylsilyl
- D is DMT
- D 1 is located on the 3′ carbon
- R 21 is H
- the D 1 and R 21 groups are in the alpha anomer conformation.
- R 1 includes an optionally protected monosaccharides of structure (4) and (4A).
- monosaccharides in structure (4) and (4A) compounds are 2′-deoxyribose, 2′-deoxy-2′-R 21 -substituted ribose, 2′-deoxy-2′-R 21 -substituted arabinose, ribose or arabinose, any of which are optionally protected at sugar all or some hydroxyls or at optionally present R 21 functional groups such as —OH —SH or —NH 2 groups.
- Invention embodiments include compositions of the formula where B is a structure (3), (4) or (4A) base, R 56 is a diene or a dieneophile, both as defined in WO 97/14706, R 57 is —OR 5 , a coupling group including —OH, H-phosphonate, a phosphoramidite or an optionally protected oligonucleotide having a 3′-terminal group selected from a coupling group and —OR 5 and any reactive moiety in R 21 is optionally protected.
- R 56 dienes are independently chosen and include 2,4-hexadiene and 3,5-hexadiene.
- R 56 are linked to a solid support such as a crosslinked organic polymer, polystyrene, TentagelTM, polyethylene glycol or an inorganic oxide such as silica gel, alumina, controlled pore glass or a zeolite. These compositions are useful for making oligonucleotides containing one or more invention bases.
- a solid support such as a crosslinked organic polymer, polystyrene, TentagelTM, polyethylene glycol or an inorganic oxide such as silica gel, alumina, controlled pore glass or a zeolite.
- Invention embodiments include compositions and their isomers of the formula where R 57 is independently —H, a protecting group or both R 57 together are a dihydroxy protecting group, R 58 is —H or alkyl containing 1, 2, 3 or 4 carbon atoms and B is a structure (3), (4) or (4A) base.
- Suitable R 5 at the nitrogen atom include —H, FMOC and tBOC (t-butyloxycarbonyl) and suitable R 5 at the carboxyl group include —H, t-butyl and benzyl, see, e.g., WO 97/14709.
- These compositions are useful for making oligonucleotides containing one or more invention bases.
- Protecting groups suitable for use with amine groups that may be present at R 2 include FMOC and trichloroacetamide. Monomers and polymers may contain such protecting groups at R 2 .
- R 1 when functioning as a binding partner, is a substance that non-covalently binds to a target compound.
- the target compound is an analyte whose presence is desired to be detected.
- Binding partners are well-known from the immunoassay art and include hapten-antibody pairs such as those used in drug immunoassays using EMIT or ELISA technologies. Binding partners are employed analytically in enzymology, where the substrate or the enzyme is labeled.
- Binding partners also are known from the oligonucleotide hybridization art, including oligonucleotide-nucleic acid binding partners (as in diagnostic probes or therapeutic antisense oligonucleotides) or oligonucleotide-protein binding partners (aptamers).
- an invention base is substituted at R 1 by any binding partner. While the binding partner may be a small molecule such as a drug, hapten, substrate or the like, ordinarily it is a polymer.
- R 1 is a polymer
- an R 1 linker group has been subsumed into the polymer structure, either as a monomer unit or by grafting onto pre-existing polymer. Therefore, when R 1 is a polymer, the polymer may comprise the residue of a linking group derived from a monomer or where the linking group differs from the polymer's monomeric subunits.
- the invention base must be covalently linked to the polymer.
- Typical R 1 polymers include a biopolymer such as an oligonucleotide, a protein (including antibodies, enzymes, cell membrane proteins, glycoproteins, glycolipids, lipoproteins, and nucleoproteins), a peptide, a nucleic acid, or a glycan or other polysaccharide or carbohydrate.
- a biopolymer such as an oligonucleotide, a protein (including antibodies, enzymes, cell membrane proteins, glycoproteins, glycolipids, lipoproteins, and nucleoproteins), a peptide, a nucleic acid, or a glycan or other polysaccharide or carbohydrate.
- the polymer is an oligonucleotide in which either or both of the sugar or phosphodiester monomer subunits are substituted by groups that continue to permit base pairing by the invention base analogs but which have other desirable characteristics that are not shared with native substituents, e.g., those which mask the negative charges of the phosphodiester linkages or replace the phosphodiester linkage with another group.
- the site at which one links the invention base analogs to a polymer is typically not critical. In general, any reactive group on the polymer is satisfactory when one wants to graft the polycycle-R 2 substructure onto a pre-existing polymer. Obviously, the site of the substitution should not be in a location in which the polycycle-R 2 substructure will interfere with the intended function for the polymer, e.g. enzyme active site, antibody CDR, and the like as will be understood by the artisan.
- amino acid side chain such as that of lysine, glutamic acid, serine, asparagine and the like will be satisfactory for grafting to protein R 1 , as will alpha amino groups, provided that the amino acids in question do not participate in the binding partner or ligand/substrate interaction involved in the assay in which the labeled protein is to be used.
- the 1′ position of ribose or deoxyribose is satisfactory as the site of substitution of an oligonucleotide by the invention base analogs. Suitable sites will be known to the artisan, particularly in those instances where the one intends to substitute an invention base analog for purine or pyrimidine bases, usually for cytosine, or for fluorescent labels.
- the degree of polymer substitution by the invention base analogs is not critical.
- One skilled in the art will choose the reaction conditions such that the resulting labeled polymer will be substituted with sufficient molar proportion of base analog to facilitate its use in the desired analytical, therapeutic or preparative procedure. This is accomplished by preparing the labeled polymers under a variety of conventional conditions, e.g., the time, temperature or duration of the labeling reaction, to yield a matrix of multiply-labeled polymers. These then are screened for suitability in the intended application. Molar ratios of about from 1:1 to 10:1 invention base to polymer generally are suitable. Where the labeled polymer is prepared by monomer incorporation, the resulting polymer may contain about from 1% to 100% invention base analog substitution. In this embodiment each invention base is considered a monomer unit (even though the polymer may have been assembled from intermediate synthons containing 2 or more invention bases per synthon).
- Oligonucleotides are polymers containing at least 2 covalently linked nucleotides or nucleotide analogs (collectively monomers), at least one of which comprises an invention base.
- at least one invention base is covalently linked to a nucleotide sugar
- typical invention oligonucleotides will contain about 2-75% of the bases as invention base analogs, usually about 5-25%.
- Small oligonucleotides, e.g., 2-6 mers, that serve as synthetic intermediates for larger oligonucleotides will optionally contain the higher proportions of invention base analogs, e.g., about 50-75%.
- oligonucleotides e.g., about 7-21 mers, will generally contain 1, 2, 3, or 4 invention bases, occasionally 5 and usually not more than about 5 invention bases, unless the oligonucleotide is relatively long, e.g., about 22-50-mer.
- invention embodiments include polymers and oligonucleotides where the invention bases are located on 2, 3 or more adjacent monomers or nucleotide residues, or the invention bases may be located on monomers or nucleotide residues that are separated from each other by about 1, 2, 3, 4, 6, 8, 10, 12, 15, 18 or more monomers or nucleotide residues that do not contain these bases.
- the oligonucleotide may contain 1, 2 or 3 of these labeled monomers, usually 1 or 2.
- Such labelled monomers are often located at the 3′ or 5′ terminus, but they may reside at an internal position such as one, two or more monomer residues from either terminus.
- invention oligonucleotides which contain 1, 2, 3 or more invention bases, will typically have sufficient binding affinity for complementary nucleic acid sequences to allow facile detection of the duplex or triplex resulting from the base sequence-specific binding interaction.
- an invention oligonucleotide will have a Tm of at least about 15° C., usually at least about 20° C., when tested under typical in vitro binding conditions, such as those described herein and elsewhere, (Jones, “J Org Chem” [hereafter “JOC”] 58:2983-91 1993, Froehler, “Tet. Lett.” 34:1003-06 1993).
- Complementary nucleic acid means a natural or synthetic compound that is capable of forming a hydrogen bonded complex in a sequence-specific manner with an invention oligonucleotide such as a structure (2) oligonucleotide.
- Complementary nucleic acid base sequences contain no mismatches, while “substantially complementary” base sequences contain only a limited number of mismatches, e.g., at most about 1 mismatch per about 15-20 bases, relative to an invention oligonucleotide.
- embodiments include short oligonucleotides or oligonucleotide domains, e.g., having about 2, 3, 4, 5 or 6 linked monomers, where the domain may have low binding affinity, but even in this case are useful as intermediates to make longer oligonucleotides so as to increase affinity sufficiently to confer a Tm of at least about 15° C.
- invention oligonucleotide analogs will contain about 40% or less, usually about 25% or less, of monomers that significantly reduce binding affinity, i.e., monomers that decrease the Tm more than about 2° C. per monomer, compared to a corresponding unmodified oligonucleotide.
- Invention embodiments include protected, partially protected and deprotected monomers and polymers including oligonucleotides.
- Partially protected compounds arise during the course of deprotection and they are thus intermediates in the process of preparing deprotected compounds. Typically, one would not recover partially deprotected compounds.
- Deprotected compounds have been subject to a treatment that removes the protecting group(s), although the preparation may contain some compounds with unremoved protecting groups. Typically, any remaining protecting groups that remain after deprotection are present in small amounts that may be removed by suitable purification methods if desired.
- Invention embodiments include oligonucleotides of structure (2) where R 37 is oxygen.
- Invention oligonucleotides including those where R 37 is oxygen, typically contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 linked monomers usually about 5-21.
- Such oligonucleotides optionally contain about 30-100%, typically about 60-100%, of the linkages as phosphodiester or phosphorothioate linkages, or other linkages of similar binding affinity.
- Invention oligonucleotides include support-bound oligonucleotides, which are typically used in solid phase synthesis and separation applications. Support-bound oligonucleotides are typically protected during synthesis, e.g., bases, sugar hydroxyls, linkages and functional groups optionally present are protected as needed, e.g., a sugar hydroxyl group present at R 1 , an amine group at R 2 or a hydroxyl or amine group at R 21 .
- R 1 is covalently linked to a solid support or R 1 is an oligonucleotide linked to a solid support. When one removes invention oligonucleotides from a support, the protecting groups are generally removed at the same time or shortly thereafter.
- Invention embodiments include highly lipophilic polymers and oligonucleotides that comprise (i) one or more structure (3) bases, usually about 1, 2, 3 or 4, and (ii) lipophilic modifications such that the polymer or oligonucleotide has an octanol:water partition coefficient of about ⁇ 0.5 to about 2.5, typically about 0.0-2.0, usually about 0.2-1.5, and a solubility in water of at least 0.001 ⁇ g/mL, usually at least 0.1 ⁇ g/mL.
- These highly lipophilic polymers and oligonucleotides need not be binding competent for cell staining, detecting or visualizing applications and they are optionally labeled using standard labels, e.g., radiolabels ( 32 p, 35 S, 131 I, 14 C, 3 H), fluorescent labels such as fluorescein, Texas Red, Rhodamine, BODIPY, resorufin or arylsulfonate cyanines and chemiluminescent labels, e.g., acridinium esters.
- radiolabels 32 p, 35 S, 131 I, 14 C, 3 H
- fluorescent labels such as fluorescein, Texas Red, Rhodamine, BODIPY, resorufin or arylsulfonate cyanines and chemiluminescent labels, e.g., acridinium esters.
- Embodiments of such optionally labeled highly lipophilic invention oligonucleotides include species where (i) at least about 30%, typically at least about 40%, usually at least about 60% (often at least about 80%), of the internucleotide linkages are non-ionic internucleotide linkages (typically containing a lipophilic moiety at each non-ionic linkage), or (ii) at least about 30%, typically at least about 40% usually at least about 60%, of the bases included in said oligonucleotide contain a lipophilic substitution, (iii) at least about 30%, typically at least about 40% usually at least about 60%, of the sugars, usually at the 2′ position, included in said oligonucleotide contain a lipophilic substitution or (iv) the percent non-ionic nucleotide linkage and the percent lipophilic bases and the percent lipophilic sugars sum to at least about 30%, typically at least about 40% usually at least about 60% (or at least about 80%).
- the invention base analogs and other noninvention bases that are present in binding-competent oligonucleotides are linked together by an organic moiety that is sufficiently flexible to permit the invention base analog(s) to hybridize to complementary bases.
- the linkage may be a conventional phosphodiester linkage in which a nucleotide analog containing a structure (1) compound, where R 1 is deoxyribosyl, ribosyl or an analog thereof, which is incorporated into an oligonucleotide by conventional methods.
- R 1 is deoxyribosyl, ribosyl or an analog thereof, which is incorporated into an oligonucleotide by conventional methods.
- other groups are used to replace the phosphodiester linkage or, in some instances, both of the phosphodiester linkage and the sugar group. These replacement groups are termed “phosphodiester substitute linkages” for the purposes herein.
- Phosphodiester substitute linkages are well-known from the prior literature. They include for example phosphorodithioates (Marshal, “Science” 259:1564, 1993), phosphorothioates and alkylphosphonates (U.S. Pat. No. 5,212,295, Kibler-Herzog, “Nucleic Acids Research” [hereafter “NAR”] 19:2979, 1991; PCT 92/01020; EP 288,163; FIG.
- Phosphodiester substitute linkages per se also are known for the replacement of the entire phosphoribosyl linkage of conventional oligonucleotides. These include for example morpholino-carbamates (Stirchak, “NAR” 17:6129, 1989), peptides (Nielsen et al., “Science” 254:1497, 1991; U.S. Ser. Nos. 07/892,902 and 07/894,397), riboacetal linkages (PCT 92/10793) and morpholino-based linkages disclosed in U.S. Pat. Nos. 5,521,063 and 5,185,144.
- Invention embodiments include oligonucleotides having 1, 2, 3 or more optionally protected invention bases, 0 to about 30 other optionally protected bases, usually guanosine, adenine, thymine, cytosine or 5-methylcytosine, and at least one modified linkage, e.g., 3′—N(R 11 )—O—5′, where R 11 is hydrogen, C 1-6 alkyl, usually —CH 3 or —C 2 H 5 and the terminal atoms are linked to the 3′ and 5′ carbons of adjacent ribose or 2′-deoxy-2′-R 21 substituted ribose sugars, where R 38 independently is O, CH 2 or NH; R 40 independently is hydrogen, C 1-8 alkyl (methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, etc.), a protecting group or both R 40 together with the nitrogen atom to which they are attached
- R 40 pairs include ones where one R 40 is hydrogen and the other R 40 is alkyl containing 1, 2, 3, 4, 5 or 6 carbon atoms or substituted alkyl containing 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms, including methyl, ethyl, methoxyethyl and ethoxyethyl.
- R 40 When R 40 is substituted alkyl, it will usually contain 1, 2, 3 or 4 non-carbon atoms, but may contain additional non-carbon atoms, particularly when the non-carbon atoms are halogens or when a group is present as a protecting group, e.g., R 39 , R 40 , R 41 or R 42 .
- oligonucleotides include species where one or more R 21 is —F, —O(CH 2 ) 2 NHR 5 , —O(CH 2 ) 3 NHR 5 , —O(CH 2 ) 4 NHR 5 , —O(CH 2 ) 2 OCH 3 , —O(CH 2 ) 3 OCH 3 , —O(CH 2 ) 2 OR 5 , —O(CH 2 ) 2 F, —O(CH 2 ) 3 OR 5 , or —O(CH 2 ) 3 F (see, e.g., Griffey “J Med Chem” 39:5100-5109 1996, Schultze “Cell” 24:2966-2973 1996).
- Such oligonucleotides include oligonucleotides where 1, 2, 3, 4, 5, 6, 7, 8 or more monomers are substituted with R 21 , which will optionally comprise one of these substituents and the remaining R 21 are all hydrogen.
- Embodiments also include optionally protected monomers containing an optionally protected invention base for synthesis of phosphoramidate-linked oligonucleotides. Oligonucleotides containing one or more of these linkages are optionally prepared as highly lipophilic oligonucleotides and they are suitable for cell staining uses, diagnostic uses and for antisense applications that optionally rely at least in part on an RNase H mechanism.
- Invention embodiments include oligonucleotides or monomers described in U.S. Pat. Nos. 5,670,489, 5,667,976, 5,652,355, 5,652,356 and 5,212,295 where one or more optionally protected invention bases is present, usually 1, 2, 3 or 4.
- Invention embodiments include oligonucleotides having 1, 2, 3 or more optionally protected invention bases, 0 to about 30 other bases (optionally protected) and at least one amide linkage, e.g., a compound of structure (2B) where n is 0 to about 50, usually about 5-21.
- amide linkages have been described, e.g., (Haaima “Agnew Chem Int Ed Engl” 35:1939-1942 1996; Nielsen “Bioconjugate Chem” 5:3-7 1994).
- Other amide linkages that are suitable have been described, e.g., WO 92/20702 and WO 93/24507.
- Embodiments also include optionally protected monomers containing an optionally protected invention base for synthesis of amide-linked oligonucleotides.
- structure (2B) oligonucleotides will contain only amide linkages, but they may also comprise a domain of monomers linked by non-amide linkages.
- Suitable D 2 and D 3 have been described, e.g., WO 86/05518, WO 92/20702, WO 93/24507.
- D 2 optionally comprises a peptide coupling group, a protecting group, or a solid support.
- D 3 optionally comprises —H, a peptide coupling group, a protecting group, or a solid support, but D 2 and D 3 are not both a peptide coupling group or a solid support.
- WO 92/20702 described activated derivatives of —CO 2 H and —SO 3 H.
- the phosphodiester or phosphodiester substitute linkages herein are used to bond the 2′ or 3′ carbon atoms of ribose or ribose analogs to the 5′ carbon atoms of the adjacent ribose or ribose analog.
- the linkages in oligonucleotides are used to bond the 3′ atom of the 5′ terminal oligonucleotide to the 5′ carbon atom of the next 3′-adjacent nucleotide or its analog.
- linkages that contain a phosphorus atom will be 3′,5′ linkages and not 2′,5′ linkages because such linkages usually confer reduced binding affinity on the oligonucleotide in which they are present.
- Table 1 of U.S. Pat. No. 5,502,177 describes examples of suitable phosphodiester substitute linkages for use with the invention base analogs.
- the starting materials in Table 1, or those used to prepare the starting materials of Table 1, generally possess structure (1) in which R 1 is ribose, 2′-deoxyribose, a ribose analog or a 2′-deoxyribose analog comprising a 5′ hydroxyl group and a 3′ or 2′ hydroxyl group, prepared as described herein or in the citations, with an invention base analog(s) being substituted for the bases used in the citations.
- the reactions are repeated or ganged with phosphodiester or other linkages in order to produce trimers, tetramers, pentamers or larger oligonucleotides, including ones up to about 100 bases.
- oligonucleotides of this invention contain naturally occurring nucleotides or derivatives thereof.
- the companion nucleotide residues contain pyrimidine nucleotides substituted at the 5 position with a carbon atom which is distally Pi bonded to another atom as for instance 1-alkenyl, 1-alkynyl, heteroaromatic and 1-alkynyl-heteroaromatic groups such as 5-(1-propynyl)-cytosine and -uridine nucleotides (see PCT Publication No. WO 93/10820 and U.S. Pat. No. 5,594,121).
- analogs of native bases for use herein include alkylated purines or pyrimidines, acylated purines or pyrimidines, or other analogs of purine or pyrimidine bases and their aza and deaza analogs. These include, for example N 4 ,N 4 -ethanocytosine, 7-deazaxanthosine, 7-deazaguanosine, 8-oxo-N 6 -methyladenine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyl uracil, inosine, N 6 -isopentenyl-adenine, 1-methyladenine, 2-methylguanine, 5-methylcytosine, N 6 -methyladenine, 7-methylguanine, 5-methylaminomethyl uracil, 5-methoxy aminomethyl-2-thiouraci
- oligonucleotides in addition to these base analogs, one can conveniently incorporate into the invention oligonucleotides other base analogs, including pyrimidine analogs including 6-azacytosine, 6-azathymidine, 5-trifluoromethyluracil or other bases previously described, see, e.g., bases, monomers or oligonucleotides described in WO 92/02258, WO 97/32888 and U.S. Pat. No. 5,614,617.
- bases monomers or oligonucleotides described in WO 92/02258, WO 97/32888 and U.S. Pat. No. 5,614,617.
- Preferred bases include adenine, guanine, thymine, uracil, cytosine, 5-methylcytosine, 5-(1-propynyl)uracil, 5-(1-propynyl)cytosine and 5-(1-butynyl)uracil, 5-(1-butynyl)cytosine.
- Invention embodiments include the protected derivatives of native bases, their analogs and optionally protected monomer synthons containing such bases, which one would typically use as intermediates to prepare invention oligonucleotides (see, e.g., Internal Publication No. WO 96/37504, U.S. Pat. No. 5,614,622 Iyer et al., “Nucleosides & Nucleotides”, 14:1349-57, 1995, Uhlmann et al., “Chem Revs”, 90:543-587, 1990, S. Agrawal, Ed. Methods in Molecular Biology, Vol. 20, Protocols for Oligonucleotides and Analogs, pp. 165-189, Humana Press, 1993).
- Embodiments of the oligonucleotides of the invention comprise a moiety which is capable of effecting at least one covalent bond between the oligonucleotide and a nucleic acid duplex or strand. Multiple covalent bonds can also be formed by providing a multiplicity of such crosslinking moieties.
- the covalent bond is preferably to a base residue in the target strand, but can also be made with other portions of the target, including the saccharide or phosphodiester.
- Preferred crosslinking moieties include acylating and alkylating agents, and, in particular, those positioned relative to the sequence specificity-conferring portion so as to permit reaction with the target location in the strand.
- crosslinking moieties are disclosed and claimed in PCT 91/03680. See also Praseuth (“Proc Natl Acad Sci” 8:1349, 1988), Fedorova (“FEBS” 228:273, 1988), Meyer (“JACS” 111:8517, 1989), Lee (“Biochemistry” 27:3197, 1988), Home (“JACS” 112:2435, 1990), Shaw (“JACS” 113:7765, 1991).
- Invention embodiments include monomers and oligonucleotides containing 1, 2, 3 or more invention bases and a 5′ hydroxyl group protected with a base labile protecting group, including a dansylethoxycarbonyl group, which has been described, e.g., U.S. Pat. No. 5,631,362. Such embodiments optionally include additional protecting groups.
- Invention embodiments include monomers and oligonucleotides containing 1, 2, 3 or more invention bases and an invention base or a non-invention base having an exocyclic nitrogen atom, where the nitrogen atom is protected with a protecting group as previously described, e.g., specification and claims 1, 2, 3, 4, 5, 6, 7 and 8 of U.S. Pat. No. 5,623,068. Such embodiments optionally include additional protecting groups.
- Invention embodiments include optionally protected monomers and optionally protected oligonucleotides containing 1, 2, 3 or more invention bases wherein the compositions possess N-branching, which has been described, e.g., U.S. Pat. No. 5,623,049.
- Invention embodiments include “hybrid” oligonucleotides, which contain 1, 2, 3 or more invention bases and 2′ modifications in one or two regions or domains that comprise adjacent linked monomers, typically about 2-8 linked monomers, usually about 2-3. One domain contains 2′ modifications, while hydrogen is linked to the remaining monomers, typically about 4-10 adjacent linked monomers, usually about 6-8.
- Such oligonucleotides contain at least one domain that is competent to serve as a RNase H substrate and comprises hydrogen at each 2′ position and phosphodiester, phosphorothioate or phosphorodithioate 3′,5′ linkages.
- the other domain(s) contain a 2′ modification(s), such as —O—(CH 2 ) 2 F or —O—(CH 2 ) 2 —O—CH 3 , that enhances binding affinity or nuclease stability.
- the 2′-modified domain(s) is usually not an efficient RNase H substrate.
- the bases in hybrid oligonucleotides are the typical purines and pyrimidines found in nucleic acids (G, A, T, C or U) or their analogs, which one finds in some oligonucleotide analogs (e.g., U.S. Pat. Nos. 5,484,908, 5,594,121 and 5,502,177, International Publication No. WO 93/10820). Intermediates used to prepare hybrid oligonucleotides will typically contain appropriately protected derivatives of any bases.
- Oligonucleotides of inverted polarity also fall within the scope of this invention.
- “Inverted polarity” means that the oligonucleotide contains tandem sequences which have opposite polarity, i.e., one having polarity 5′ ⁇ 3′ followed by another with polarity 3′ ⁇ 5′, or vice versa. These sequences thus are joined by linkages which can be thought of as effectively a 3′—3′ internucleoside junction (however the linkage is accomplished), or effectively a 5′—5′ internucleoside junction.
- suitable methods for making such oligonucleotides see, e.g., WO 93/10820.
- compositions of “parallel-stranded DNA” designed to form hairpins secured with AT linkages using either a 3′-3′ inversion or a 5′-5′ inversion have been synthesized by Van de Sande, “Science” 241:551, 1988.
- oligonucleotides which contain 3′-3′ linkages have been described (Horne, op cit; and Froehler, “Biochemistry” 31:1603, 1992). These oligonucleotides are useful as binding partners for double stranded nucleic acids to form triple helix (or triplex) complexes as a means for detecting complementary sequences and inhibiting of target gene expression (PCT 89/05769 and 91/09321).
- Invention embodiments include polymers such as oligonucleotides containing 1, 2, 3 or more invention bases, where the polymer is a component of a complex or composition useful for transfecting the polymer into a cell in vitro or in vivo.
- transfection complexes optionally comprise one or more lipids, e.g., cationic or anionic lipids, as well as other lipophilic compounds such as cholesterol or colipids such as DOPE.
- the complexes optionally comprise an uncharged or a charged polymer such as polyethylene glycol, polybrene or a peptide, e.g., polylysine.
- the complexes optionally comprise unilamellar or multilamellar liposomes or vesicles.
- any compound(s), reagent(s) or treatment that enhances delivery of an invention oligonucleotide into a cell or tissue is a “permeation enhancing agent.”
- Permeation enhancing agents are well known and are usually present as transfection complexes containing oligonucleotides, e.g., unilamellar or multilamellar liposomes or vesicles.
- the permeation enhancing agent are used in essentially the same manner as is used to prepared transfection complexes containing nucleic acids, non-invention oligonucleotides or polymers into cells or tissues.
- Invention transfection complexes optionally comprise an additional non-invention polymer(s), e.g., a nucleic acid expression vector(s), a therapeutic agent(s) (e.g., amphotericin B) or a peptide(s).
- an additional non-invention polymer(s) e.g., a nucleic acid expression vector(s), a therapeutic agent(s) (e.g., amphotericin B) or a peptide(s).
- Invention transfection complexes comprising a lipid may be, as defined herein, “large”, i.e., complexes having a maximum average dimension of at least about 200 nm in length or diameter, typically having an average length or diameter of about 200-400 nm, occasionally having an average length or diameter of about 400-800 nm.
- Transfection complexes comprising a lipid may be “small”, i.e., complexes having a maximum average dimension of about 15-200 nm in length or diameter, e.g., an average dimension of about 60-120 nm.
- Transfection complexes may comprise a mixture of large and small complexes in about equal proportions or they may comprise a preponderance of small or large transfection complexes, e.g., at least about 55%, or at least about 60-80% of the complexes in a given preparation are large or small.
- Transfection complexes comprising a lipid optionally include a stabilizing compound(s), e.g., a monosaccharide or a disaccharide such as glucose, trehalose, maltose or sucrose, that is present at the outer surface or at the inner surface or at both surfaces of transfection complexes.
- a stabilizing compound(s) e.g., a monosaccharide or a disaccharide such as glucose, trehalose, maltose or sucrose, that is present at the outer surface or at the inner surface or at both surfaces of transfection complexes.
- suitable compounds such as lipids, colipids and stabilizing compounds for making transfection complexes, methods to size the complexes and methods to use the complexes to deliver a polymer or monomer into the cytoplasm of a cell in vitro or in vivo, e.g., U.S. Pat. Nos.
- Invention transfection complexes useful for delivering the invention oligonucleotides into cell cytoplasm also include complexes comprising inorganic compounds, e.g., calcium phosphate.
- the R 21 moiety is linked to invention oligonucleotides or monomers useful for oligonucleotide synthesis.
- R 21 is usually linked to the 2′ or the 3′ position of furanose sugars.
- R 21 is a nuclease stability enhancing moiety, a broad range of structures may be used to increase stability of oligodeoxynucleotides or oligoribonucleotides containing phosphodiester linkages.
- Oligonucleotides having moieties other than hydrogen or hydroxyl at the 2′ position usually confer increased nuclease stability or increased binding affinity on the oligonucleotide relative to hydrogen or hydroxyl.
- Enhanced nuclease stability is conveniently measured using dimers or short oligonucleotides as essentially described, e.g., WO 92/05186.
- One or two R 21 moieties at the 3′ and 5′ terminal monomers in an oligonucleotide will increase stability of the oligonucleotide to 3′- and 5′-exonucleases.
- R 21 moieties enhance binding affinity of the oligonucleotide to which they are linked.
- These moieties include fluorine and short unbranched optionally substituted O-alkyl groups containing about 2-8 carbon atoms, where the alkyl group is optionally substituted at the distal carbon atom with, e.g. —F, —OH or —NH 2 , and optionally substituted with an ether at an internal carbon, e.g., —O—(CH 2 ) 2 —O—(CH 2 ) 2 F, —O—(CH 2 ) 2 OCH 3 or —O—(CH 2 ) 2 —O—(CH 2 ) 2 CH 3 .
- R 21 When R 21 is a nuclease stability enhancing moiety, it will typically comprise —CH 3 , ⁇ O—NHR 5 or a chain having a backbone containing 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 linked atoms, wherein the chain usually comprises carbon (C) atoms and optionally 1, 2, 3 or 4 atoms independently selected from the group consisting of oxygen (O), nitrogen (N) and sulfur (S) atoms.
- the chain is usually linked to the sugar carbon atom through —O—, —S—, —S(O)—, S(O)(O)—, —CH 2 —, ⁇ CH— or —NH—.
- the chain is branched or unbranched, often it is unbranched or has only limited branching, e.g., —CH 3 , —CH 2 OH, —C 2 H 5 or —C 2 H 4 OH.
- the R 21 chain may comprise a C 2-12 alkyl group or a C 2-20 substituted alkyl group. If R 21 is a substituted alkyl group, usually only 1, 2 or 3 carbon atoms are substituted. Suitable substituents include those described above for substituted alkyl groups, e.g., halogen (usually fluorine), —O— or —OR 5 .
- Invention embodiments include oligonucleotides and monomers where one or more monomers comprise 2′-deoxyribose, ribose or arabinose sugars, or their carbocyclic analogs, having one or more 2′ R 21 modification such as, —O—alkyl, —NH—alkyl, —S—alkyl, —OR 5 , —NHR 5 , —SR 5 , -halo (usually —F), —R 44 -alkyl or —R44-substituted alkyl wherein the alkyl or substituted alkyl group usually comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms, usually about 2-6 carbon atoms, where R 44 is independently —O—, —S—, —NH— or —CH 2 —, usually —O—.
- R 21 modification such as, —O—alkyl, —NH—alkyl, —S—alkyl, —OR 5
- the oligonucleotide linkages connecting such monomers are 3′,5′ linkages.
- the alkyl groups at the 2′ position typically have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms which are optionally present as methylene groups (—CH 2 ) and optionally have 1, 2, 3 or 4 ether (—O—) or other substitutions, e.g., O-alkoxyalkyl (C 2 -C 12 alkyl), —O—(CH 2 ) 2-8 —CH 2 CO 2 H, —O—(CH 2 ) 2-8 —CH 2 N(R 5 ) 2 , —(—O—(CH 2 ) 2-4 —O—(CH 2 ) 2-4 —O—(CH 2 ) 2-4 —R 65 ), —O(CH 2 CH 2 O) r CH 2 —R 65 , —O—CH 2 CH 2 —R 65 , —O(CH 2 CH 2 )O(CH 2 CH 2 )R
- the alkyl groups at the 2′ position also include substituted alkyl, e.g., —O— alkylamino, —S-alkylamino , —NH-alkylamino, or their protected derivatives, wherein the alkyl group contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms, which are all optionally present as methylene carbons (—CH 2 —).
- substituted alkyl e.g., —O— alkylamino, —S-alkylamino , —NH-alkylamino, or their protected derivatives, wherein the alkyl group contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms, which are all optionally present as methylene carbons (—CH 2 —).
- Usually the alkyl group or substituted alkyl group at R 2 ′ will contain 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms.
- Such groups include —O—methyl, —O—ethyl, —O—n-propyl, —O-ally, —O—(CH 2 ) 2-6 OH including —O—(CH 2 ) 2 OH, —O—(CH 2 ) 2 F, —O—CH 2 CHF 2 , —O—CH 2 CF 3 , —O—(CH 2 ) 2-6 OCH 3 , including —O—(CH 2 ) 2 OCH 3 , —O—(CH 2 ) 2 OCH 2 CH 3 , —O—(CH 2 ) 2 OCH 2 CH 2 OH, —O—(CH 2 ) 2 OCH 2 CH 2 F, —O—(CH 2 ) 2 NHR 5 , —O—(CH 2 ) 3 NHR 5 , —O—(CH 2 ) 4 NHR 5 , —O—(CH 2 ) 2 F, —O—(CH 2 ) 3 F, —O—(CH
- R 21 at the 2′ or 3′ position of optionally protected invention monomers or optionally protected invention oligonucleotides include — 2 H, — 3 H, —NHOR 55 , ⁇ NH, —N 3 , —CN, —CH 2 CN, —CHCl 2 , —CFH 2 , —CT 2 H, ⁇ CH 2 , ⁇ CF 2 , —CH 2 CH ⁇ CH 2 , ⁇ O, ⁇ CHC(O))OR 55 (including ⁇ CHC(O)OCH 3 and ⁇ CHC(O)OCH 2 CH 3 ), —OC(S)OC 6 H 5 , 1-butyldimethylsilyl ether, triisopropylsilyl ether, a 2′ amino group protected by an N-phthaloyl protecting group or a fluorescent label, R 55 is independently R 5 , alkyl having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms of substituted alkyl having 1, 2, 3, 4, 5, 6,
- R 1 is a linker or H an prepared by methods known in the art per se and as more fully described below.
- such compounds are prepared from a 5-bromouracil, 5-bromouridin-1-yl, 5-iodouracil, 5-iodouridin-1-yl substituted derivative as shown in the synthetic schemes below and subsequent reactions close the polycyclic ring, In these embodiments the hydroxyl, amino and any other labile groups of R 1 are protected as required.
- R 1 of the starting material is H or a protecting group and one adds the linker after the ring closure steps set forth in the schemes, in the same fashion as has heretofore been employed in the alkylation of pyrimidine bases intended for use as antiviral compounds.
- R 1 is a binding partner such as a polymer
- the compounds of this invention are synthesized by covalently crosslinking the linker modified polycyclic base of this invention to the binding partner, or (where the binding partner is a polymer) by incorporating into the polymer a monomer unit which is substituted by an invention polycyclic base.
- a R 1 -substituted polycyclic substructure is covalently bonded via any conventional cross-linking agent to the polymer.
- structure (1) compounds in which R 1 is hydroxyl- or amino-substituted alkyl are readily cross-linked to reactive groups present in the molecule to be labeled as noted above.
- cross-linking agents include succinic anhydride, N-hydroxysuccinimide esters (biotin NHS ester), epoxides, isothiocyanates, imidates, DCC (dicclohexylcarbodiimide), EDC (1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide), BOP, and glutaraldehyde, see, e.g., EP 0 063 879, Ruth “J Org Chem” 43:2870, 1978, Bergstrom, JACS 100:8106, 1978, Bigge, JACS 102:2033,1980. Cyanogen bromide activated carbohydrates also are used.
- the cross-linking agents are used to bond the R 2 -substituted polycycle to the polymer in the same fashion as polymers heretofore have been cross-linked to ligands, e.g., to hydroxyl or amino-bearing moieties.
- ligands e.g., to hydroxyl or amino-bearing moieties.
- An example of a suitable method is described per se in Cook et al., U.S. Pat. No. 5,218,105. This method is readily applied to covalently bond an amino-substituted R 1 linker to the 5′ terminus of an oligonucleotide.
- R 1 or R 2 are amino substituted, the following exemplary synthetic approaches are suitable for crosslinking amines with other moieties:
- the R 1 linker is capable of functioning as a monomer for copolymerization with other monomer units that may or may not be substituted with the polycyclic substructure of structure (1).
- the R 1 linker is an alkyl carboxylate, an alkyl amine or an amino acid for incorporation into peptides by in vitro methods.
- the R 1 polymeric binding partner is an oligonucleotide as depicted in structure (2), and these conveniently are made by copolymerization with a nucleotide analog substituted with the polycyclic substructure.
- the starting materials for the synthesis of structure (2) generally are compounds of structure (1) in which R 1 is ribose or deoxyribose substituted with appropriate protecting and coupling groups further described above.
- Suitable starting monomers for oligonucleotides having phosphodiester substitute linkages are set forth in Table 1, and they an prepared in the same fashion as other nucleotide analog bases described in the literature.
- conventional phosphodiester or phosphorothioate linkages are prepared from nucleotide analogs containing coupling groups D and D 1 described above.
- the compounds of this invention then are incorporated into the desired oligonucleotide by known methods of in vitro synthesis described in the referenced methods.
- polycylic substructure-substituted nucleotide triphosphates may be incorporated iota oligonucleotides as cytosine analogs by DNA polymerase or reverse transcriptase in vivo or in vitro (see Ward, U.S. Pat. No. 4,711,955).
- R 1 is ribosyl or deoxribosyl triphosphate, or a triphosphorylated analog thereof recognized by DNA polymerase or reverse transcriptase which is then incorporated into an oligonucleotide by template-directed transcription.
- Synthesis of oligonucleotides containing 3 or more nucleotide residues is optionally accomplished using synthons such as dimers (which contain substitute or diester linkages) or trimers, each carrying a terminal coupling group suitable for use with amidite, H-phosphonate or triester chemistries.
- the synthon is then linked to the oligonucleotide or another synthon via a phosphodiester or phosphorous-containing phosphodiester substitute linkage.
- Oligonucleotides containing phosphorothioate, methylphosphonate and phosphodiester linkages are readily prepared by solid-phase oligonucleotide synthesis techniques. A description of modifications useful in the synthesis of phosphorothioate linked oligonucleotides are found, for example, in EP 288,163, wherein the oxidation step in solid phase automated synthesis using amidite chemistry can be independently adjusted at any step to obtain the phosphorothioate. An alternate method for synthesis of oligonucleotides with phosphorothioate linkages, via hydrogen phosphonate chemistry, has also been described (Froehler “NAR” 14:5399, 1986).
- Sulfurization is accomplished using reagents such as tetraethylthiuram disulfide, dibenzoyl tetrasulfide, thiophosphoric acid disulfide, 3H-1,2-benzodithiole-3-one 1,1-dioxide and the like as described (Vu, “Tel Lett” 26:3005, 1991; Rao, “Tel Lett” 33:4539, 1992; U.S. Pat. No. 5,515,510; Iyer, “JOC” 55:4693, 1990; Dahl, “Sulfur Reports” 11:167, 1991). These sulfurization reagents are used with either phosphoramidite or hydrogen-phosphonate chemistries.
- Synthesis of phosphorothioate oligonucleotides having controlled stereochemistry is used to generate stereoregular invention oligonucleotides as described (EP 506,242).
- Thionomethyl phosphonate is prepared with methylphosphonamidite followed by sulfurization as described (Roelen, “Tel Lett” 33:2357,1992) or, with the sulfurization reagents described above.
- Scheme A depicts preparation of structure (1) compounds where R 47 is —O— or —S—; and R 24 —OH is R 2 which has a free hydroxyl.
- Step one is conducted by bearing the reaction mixture containing (100) in an organic solvent to at least about 50° C., generally for about 3-4 hours.
- Step two is performed by reacting (102) in an organic solvent for about 6-48 hours, generally for about 10-20 hours at about 15° C. to reflex temperature, generally at about 18-25° C.
- the R 2 moiety is linked under Mitsunobu conditions to (103) in step 3 by reacting about 1-1.5 equivalents of the alcohol, i.e., R 24 —OH, using an activating agent as a leaving group, such as triphenylphosphine (Ph 3 P) and diethyl diazocarboxylate (DEAD) to obtain (104).
- an activating agent as a leaving group, such as triphenylphosphine (Ph 3 P) and diethyl diazocarboxylate (DEAD) to obtain (104).
- step 4 (105) is prepared by forming the ring containing R 47 by (1) incubating (104) in a polar organic solvent, typically an alkanol containing 1, 2, 3, 4, 5 or 6 carbon atoms, such as methanol or ethanol, containing a mild base such as NH 3 , TEA (triethylamine), DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) or (2) refluxing in ethanol in the presence of potassium fluoride.
- a polar organic solvent typically an alkanol containing 1, 2, 3, 4, 5 or 6 carbon atoms, such as methanol or ethanol
- a mild base such as NH 3 , TEA (triethylamine), DBU (1,8-diazabicyclo[5.4.0]undec-7-ene
- a mild base such as NH 3 , TEA (triethylamine), DBU (1,8-diazabicyclo[5.4.0]undec-7-ene
- R 1 is a monosaccharide, e.g., 2′-deoxyribose, 2′-deoxy-2′-R 21 -substituted ribose or arabinose
- the sugar hydroxyls in (100) are usually protected, generally using base-labile protecting groups, e.g., acetate, proprionate, butyrate, phenoxyacetyl.
- base-labile protecting groups e.g., acetate, proprionate, butyrate, phenoxyacetyl.
- the step 4 reaction under basic conditions removes the protecting groups, which facilitates the ring formation reaction resulting in (105).
- R 3 is not a monosaccharide, it is generally a protecting group or an optionally protected linker, e.g., —(R 48 ) X —R 49 , where R 48 is independently —CH 2 —, —O—, —S—, —NH— or —C(O)—, X is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and R49 usually is a functional group suitable for linking to a solid support, monomer or polymer, including —OR 5 , —SR 5 , —N(R 5 ) 2 , —C(O)N(R 5 ) 2 , —NR 5 C(O)H.
- R 48 is independently —CH 2 —, —O—, —S—, —NH— or —C(O)—
- X is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
- R49 usually is a functional group suitable for linking to a solid support, monomer or polymer, including —OR 5 ,
- R 5 is independently —H, a protecting group, or both R 5 together are a protecting group.
- R 48 group that is adjacent to R 49 is ethylene (—CH 2 —CH 2 —) and generally only 0, 1 or 2 R 48 are moieties other than —CH 2 —, e.g., one R 48 is —O—or —C(O)— and the remaining R 47 are all —CH 2 —.
- Exemplary compounds where R 1 is a monosaccharide have the structures (130)-(134), which correspond to compounds (100)-(101) and (103)-(105), respectively. For compounds (130)-(132).
- R 5 at sugar hydroxyls is typically a base labile protecting group such as acetyl and R 37 is typically —O—.
- R 5 protecting group is removed from the sugar under ring closure reaction conditions, which facilitates ring closure.
- Structure (134) compounds may be converted to monomers suitable for oligonucleotide synthesis.
- Such monomers typically have a coupling group at the 3′ position, e.g., H-phosphonate, or a phosphoramidite such as a ⁇ -cyanoethylphosphoramidite, N,N-diisopropylamino- ⁇ -cyanoethoxyphosphine or N,N-diisopropylaminomethoxyphosphine.
- the 5′ position will contain a DMT-O— or other protecting group suitable for oligonucleotide synthesis.
- the monomers may alternatively have a coupling group et the 5′ position and a protecting group at the 3′ position.
- Scheme B shows synthesis of structure (1) compounds where R 6 in R 2 is —CH 2 —.
- Y is 1, 2, 3 or 4;
- R 50 is independently —CH 2 —, —C(O)—, —(CH 2 ) 2 —O—(CH 2 ) 2 —(CH 2 ) 2 —NR 5 —(CH 2 ) 2 —, —(CH 2 ) 2 —S—(CH 2 ) 2 —, —CH(N(R 5 ) 2 )—, —CH(COOR 5 )— or —C(CH 3 )—, —C(C 2 H 5 )— but adjacent moieties are not C(O), usually R 50 is —CH 2 —; TFA is trifluoroacetate; and CBZ is carboxybenzoyl; and (96) is HC ⁇ C(R 50 ) y —NH-TFA.
- Protecting groups present in R 50 are stable to the reaction conditions
- Compound (97) is converted to (98) by hydrogenation reaction is alcohol, usually methanol or ethanol, at about 15-25° C. for about 10-24 hours, usually about 12-18 hours.
- the catalyst is removed and the filtrate is concentrated.
- Compound (98) in trimethyl orthoformate is converted to (99) in the presence of acid, e.g., methanesulfonic acid at about 15-25° C., usually about 18-22° C., for about 20-120 minutes.
- acid e.g., methanesulfonic acid
- the reaction is cooled and quenched with a base, e.g., an organic base such as TFA
- a base e.g., an organic base such as TFA
- the reaction mixture is concentrated and purified, e.g., by flash column chromatography on silica gel.
- Compound (106) is prepared by reacting (99) in organic solution such as DMF, CH 2 Cl 2 or CH 2 Cl 2 /DMF (about 2:1 v/v) with K 2 CO 3 at about 15-25° C. for about 30 minutes, followed by adding phenyltrifluoromethanesufonimide and stirring the mixture for about 10-24 hours, usually about 12-16 hours. The reaction mixture is then diluted with CH 2 Cl 2 , washed with water owe or twice and concentrated and purified.
- organic solution such as DMF, CH 2 Cl 2 or CH 2 Cl 2 /DMF (about 2:1 v/v)
- K 2 CO 3 about 15-25° C. for about 30 minutes
- Compound (96) is prepared by reacting HC ⁇ —(R 50 ) Y NH 2 with ethyl trifluoroacetate at about 15-25° C. for about 10-24 hours, washing with saturated aqueous NaHCO 3 and concentrated. Compound (96) is purified by distillation.
- Compound (107) is prepared by stirring an organic solvent such as DMF containing about 2 equivalents of (96), about 2 equivalents of an organic base such as TEA, and Pd((PPh) 3 ) 4 , Cul and about 1 equivalent of (106) at about 15-25° C. for about 18-36 hours.
- the organic phase is washed with water, dried and purified by silica gel chromatography, to obtain (107).
- Compound (110) is prepared by treating (109) in ethanol with aqueous HCl (about 3 N) at about 15-45° C., usually about 40° C., for about 30-120 minutes, usually about 60 minutes.
- the product is dried and optionally azeotroped using e.g., CH 3 CN, several times.
- Compound (111) is prepared by reaction of (100) with mesitylenesulfonyl chloride in the presence of a tertiary amine such as TEA.
- Compound (112) is prepared by stirring a mixture of (110) and (111) in organic solution containing about 2 equivalents of an organic base such as DBU or TEA at about 15-25° C. for about 10-24 hours. The reaction mixture is washed with an aqueous 10% citric acid solution, dried and purified by silica gel chromatography.
- organic base such as DBU or TEA
- Compound (113) is prepared by treating (112) with saturated NH 3 in methanol at about 15-25° C. for about 3-4 days. The reaction mixture is dried, concentrated and purified by silica gel chromatography.
- Compound (114) is prepared by hydrogenation of (113) in the presence of 10% Pd/C at about 15-25° C. for about 3-6 hours. Catalyst is removed, washed, and the filtrate is concentrated to dryness. The amino group in (114) is protected with FMOC to afford (115).
- R 1 in scheme B is an optionally protected monosaccharide such as 2′-deoxyribose, 2′-deoxy-2′—R 21 -substituted ribose, 2′-deoxy-2′-R 21 -substituted arabinose, ribose or arabinose
- the sugar's hydroxyl groups in compounds (111) and (112) are usually protected with a base-labile protecting group such as acetyl, propionyl and phenoxyacetyl. These protecting groups are removed by treatment with base during synthesis of (113).
- R 1 is a monosaccharide
- R 5 at sugar hydroxyls is typically a base labile protecting group such as acetyl.
- R 37 is typically —O—.
- Structure (139) compounds may be converted to monomers suitable for oligonucleotide synthesis.
- Such monomers typically have a coupling group at the 3′ position, e.g., H-phosphonate, or a phosphoramidite such as a ⁇ -cyanoethylphosphoramidite, N,N-diisopropyl-amino- ⁇ -cyanoethoxyphosphine or N,N-diisopropylaminomethoxyphosphine.
- the 5′ position will contain a DMT-O- or other protecting group suitable for oligonucleotide synthesis.
- the monomers may alternatively have a coupling group at the 5′ position and a protecting group at the 3′ position. The protecting and coupling groups are added sequentially.
- Scheme C shows synthesis of structure (1) compounds where R 6 in R 2 is —NH—.
- R 52 is —(CHR 52A )—(R 52B )—CHR 52A —, where R 52A is —H or C 1 -C 6 alkyl (typically C 1 -C 3 ), usually —H, and R 52B is a bond, —CHR 52A —O—CHR 52A —, —CHR 52A —S—CHR 2A —, —CHR 52A —NR 5 —CHR 52A —, C 1 -C 10 alkylene (typically C 3 -C 6 ) optionally substituted with 1 or 2 moieties selected from the group consisting of C 1 -C 6 alkyl (typically C 1 -C 3 alkyl), —OR 5 , ⁇ O, —NO 2 , —N 3 , —CN, —COOR 5 , or —N(R 5 ) 2 .
- R 52A is —H or C 1 -C 6 alkyl (typically C 1 -
- any heteroatom in the spacer chain will be separated from the nitrogen atoms that R 52 is linked to by one methylene and one or more —CHR 52A —moieties.
- adjacent carbon atoms in R 52B are not substituted with —OR 5 , ⁇ O, —NO 2 , —N 3 , —CH, —COOR 5 , or —N(R 5 ) 2 .
- Protecting groups present on (119A) are usually stable to oxidizing conditions and labile to basic conditions.
- the protected intermediate (119A) is prepared by reacting phthalic anhydride with the appropriate HOCH—R 52 —NH 2 moiety to yield a phthalimide compound or by reacting a phthalimide compound with HOCH-R 52 —Br.
- Compound (117) is prepared by reacting (101) or (111) with about 1.0-1.5 equivalents, usually about 1.1 equivalents of (116) and about 1-2 equivalents, usually about 2 equivalents of organic base such as DBU or TEA in an organic solvent such as CH 2 Cl 2 /CCl 4 (about 1:1) or CH 2 Cl 2 by reaction at about 10-30° C., usually at about 18-25° C. for about 16-48 hours, usually about 20-28 bouts.
- organic base such as DBU or TEA
- organic solvent such as CH 2 Cl 2 /CCl 4 (about 1:1) or CH 2 Cl 2
- R 1 in scheme C is an optionally protected monosaccharide such as 2′-deoxyribose, 2′-deoxy-2′-R 23 -substituted ribose, 2′deoxy-2′-R 21 ′-substituted arabinose, ribose or arabinose
- the sugar's hydroxyl groups in compounds (101), (111) and (117) are usually protected with a base-labile protecting group such as acetyl, propionyl and phenoxyacetyl. These protecting groups an removed by treatment with base during synthesis of (118).
- Compound (118) was hydrogenated in ethanol and acid to obtain (119).
- Compound (120) was obtained by reductive alkylation of (119) with aldehydes.
- Compound (120) is optionally converted, without removing the nitrogen protecting group linked to the exocyclic amine, to a monomer suitable for use in oligonucleotide synthesis, e.g., (121) (not shown), by protecting the 5′ hydroxyl group by reaction with a protecting group reagent such as DMT-Cl (4,4′-dimethoxytrityl chloride) to obtain the 5′-protected derivative (not shown).
- a protecting group reagent such as DMT-Cl (4,4′-dimethoxytrityl chloride
- Compound (121) is then converted to a derivative (121A) (not shown) suitable for coupling the 3′ hydroxyl group with another 5′ hydroxyl group, i.e., a coupling group such as H-phosphonate or a phosphoramidite, e.g., N,N-diisopropylamino- ⁇ -cyanoethoxyphosphine or N,N-diisopropylaminomethoxyphosphine, is linked to the 3′ position.
- a coupling group such as H-phosphonate or a phosphoramidite, e.g., N,N-diisopropylamino- ⁇ -cyanoethoxyphosphine or N,N-diisopropylaminomethoxyphosphine
- R 1 is a monosaccharide
- R 5 at sugar hydroxyls is typically a base labile protecting group such as acetyl and R 37 is typically —O—.
- R 5′ or 3′ oxygen is linked to —H or a coupling group such as H-phosphonate, or a phosphoramidite such as ⁇ -cyanoethylphosphoramidite, N,N-diisopropylamino- ⁇ -cyanoethoxyphosphine or N, N-diisopropylaminomethoxyphosphine.
- the R 5 protecting group is removed from the sugar under ring closure reaction conditions, which facilitates ring closure.
- Structure (143) compounds may be converted to monomers suitable for oligonucleotide synthesis.
- Such monomers typically have a coupling group at the 3′ position, e.g., H-phosphonate, or a phosphoramidite such as a ⁇ -cyanoethylphosphoramidite, N,N-diisopropylamino- ⁇ -cyanoethoxyphosphine or N,N-diisopropylaminomethoxyphosphine.
- the 5′ position will contain a DMT, MMT or other protecting group suitable for oligonucleotide synthesis.
- the monomers may alternatively have a coupling group at the 5′ position and a protecting group at the 3′ position.
- (143) is incorporated into an oligonucleotide such, as one of structure (2), by standard methods and the phthalamide protecting group, along with other base labile protecting groups at R 52 are removed using basic conditions, e.g., NH 4 OH or NH 2 CH 3 , to yield deprotected or partially deprotected —NH—R 52 —NH 2 as R 2 .
- basic conditions e.g., NH 4 OH or NH 2 CH 3
- scheme D depicts a method to prepare structure (1) compounds where R 2 comprises a cytosine derivative.
- iPr is isopropyl
- R 59 is a portion of an R 2 moiety having the structure —R 6 —R 60 —, where R 6 is usually —Q—, —S—, —NH— or —CH 2 —
- R 60 is —(CH 2 ) Z3 —(R 61 ) Z1 —(CH 2 ) 22 —
- R 61 is —O—, —S—, —NR 5 —, —C(O)—, —CH 2 —O—CH 2 —, —CH 2 —NR 5 —CH 2 — or —CH 2 —S—CH 2 —
- Z3 is 1, 2 or 3, usually 1
- Z1 is 0 or 1, usually 0
- Z2 is 1, 2 or 3, usually 1; any functional groups, e
- Compound (126) of scheme D is converted to a monomer suitable for oligonucleotide synthesis using standard methods, e.g., treatment of (126) with a protecting group such as DMT C1 protects the 5′ oxygen atom and yields compound (127) (not shown).
- a protecting group such as DMT C1 protects the 5′ oxygen atom and yields compound (127) (not shown).
- R 2 is where R 61 is —H, alkyl having 1, 2, 3 or 4 carbon atoms or optionally protected substituted alkyl having 1, 2, 3, 4, 5 or 6 carbon atoms including —CH 3 and —CH 2 CH 3 , and R 6 is —O— or —S— are synthesized using scheme A, while scheme B is used when R 6 is —CH 2 — and scheme C is used when R 6 is —NH—.
- Schemes E and F illustrate the synthesis of alcohols containing an imidazole moiety (see e.g., Munk “J. Med. Chem.” 40:181997), to scheme E, R 62 is R 5 or alkyl having 1, 2, 3, or 4 carbon atoms and R 63 is —OR 5 , —(CH 2 ) Z3 OR 5 or —(CH 2 ) 1-2 —R 47 —(CH 2 ) 1-2 —ORS and Z4 is 0, 1, 2, 3, 4 or 5.
- Z5 is 1, 3, 3, 4 or 5.
- Scheme D is also used to obtain compounds of structure (1) where R 2 comprises, for example, a moiety such as —(CH 2 ) 2-4 —NH((CH 2 ) 2-4 NH) 0-4 —(CH 2 ) 2-4 NH 2 , —(CH 2 ) 2-4 NH(CH 2 ) 2-4 N((CH 2 ) 0-4 —NH 2 ), or when R 2 is —R 6 (CH 2 ) 1 N(R 3 ) 2 and one R 4 is H, CH 3 , CH 2 CH, a protecting group or —(CH 2 ) v —N(R 33 ) 2 and the other R 4 is —(CH 2 ) v —N(R 33 ) 2 , —CH(N[R 33 ] 2 )—N(R 33 ) 2 , (48), (49) or (50).
- R 2 comprises, for example, a moiety such as —(CH 2 ) 2-4 —NH((CH 2 ) 2-4 NH
- Converting a compound of structure (4) to a compound of structure (1) is accomplished by a method comprising displacing R 24 .
- R 24 usually is —Br and R 27 is usually —CH—.
- R 2 groups containing a diol e.g. (CH 2 OH) 2 —CH—(CH 2 ) n —O— where n is 1-8, are converted by cleavage to the aldehyde CHO—CH 2 —(CH 2 ) n —O— using sodium periodate.
- Reductive alkylation of the aldehyde to a primary or secondary amine is accomplished using N(R 3A ) 2 where R 3A independently is —H, C 1-6 alkyl, including —CH 3 , —CH 2 CH 3 or a protecting group, usually —H or —OH 3 .
- the compounds of this invention find uses in the diagnostic, analytic and therapeutic fields, or as intermediates in the preparation of compounds useful in such fields.
- the R 2 -substituted compounds of structure (1) are useful as intermediates in the preparation of the labeled biopolymers of structure (1), wherein a biopolymer is rendered fluorescent or otherwise delectably labeled by linkage to the polycyclic substructure. It is most convenient, however, to use the appropriate structure (1) compounds as monomers in the preparation of nucleic acids or oligonucleotides.
- the labeled biopolymers are employed in diagnostic assays or preparative procedures in the same fashion as other fluorophor-labeled biopolymers, e.g. in fluorescence polarization methods, fluorescence activated cell sorting, competitive-type EMIT immunoassays and the like.
- the linker- and hydrogen-substituted compounds of structure (1) are useful as intermediates to prepare materials suitable for ruse in affinity purification of guanine or guanine-containing compounds, e.g., nucleosides.
- the structure (1) compounds form hydrogen bonds with guanine and one can thus link the structure (1) base structure to an appropriate support material or polymer to prepare an affinity resin suitable for binding to guanine-containing compounds, e.g., nucleosides, nucleotides and oligonucleotides or similar compounds containing guanine analogs, e.g., 7-deazaguanine.
- Typical linker-derivatized structure (1) compounds optionally contain a linker of structure —(R 48 ) X —R 49 , defined above in the discussion under Scheme A.
- the monomers are of particular use in preparing oligonucleotides for diagnostic or therapeutic use. Since oligonucleotides having 2 or more nucleotides or nucleotide analogs bearing the polycyclic substructure will usually exhibit greatly increased Tm, such oligonucleotides are particularly useful in therapeutic or diagnostic utilities where highly stable duplex hybridization structures are desired. Since these oligonucleotides frequently are fluorescent, changes in the oligonucleotide fluorescence can be followed as the oligonucleotide binds to complementary nucleic acid or oligonucleotide sequences. These changes are detectable as modifications in energy transfer, e.g., fluorescence quenching or shifts in activation or emission wavelength(s).
- the polycyclic substructure labeled oligonucleotides are employed in diagnostic or analytic methods in the same fashion as other labeled oligonucleotides.
- the oligonucleotides are used in hybridization methods in which an antibody capable of binding base-paired structure (1) is used to detect binding of the oligonucleotide to a target nucleic acid, sequence.
- changes in fluorescent character can be assayed as described above.
- 2, 3 or more polycyclic substructure labeled oligonucleotides are used in a hybridization assay.
- One oligonucleotide is labeled at its 3′ end with a polycyclic substructure containing nucleotide while the other nucleotide is labeled at its 5′ end with the same or another polycyclic substructure or with a different fluorophor such as fluorescein or rhodamine capable of energy transfer.
- the two oligonucleotides recognize a complementary sequence in which the 3′ end of the target sequence binds the oligonucleotide bearing the 3′-terminal fluorophor and the adjacent 5′ sequence of the target binds to the oligonucleotide bearing the 5′ terminal fluorophor, Binding is assayed by measuring a change in fluorescence of either or both of the oligonucleotides when they bind in tandem according to the illustrated model. In other embodiments only a single labeled oligonucleotide is employed in the hybridization method.
- the oligonucleotides of this invention thus are useful in solution phase hybridization diagnostics, i.e., it is not necessary to perform a phase separation in order to detect labeled oligonucleotide binding.
- Detecting a target base sequence in a nucleic acid or an oligonucleotide using an invention oligonucleotide is accomplished by a method comprising (i) mixing or contacting a sample suspected of containing a nucleic acid with an optionally labeled invention oligonucleotide comprising at least about 7 bases, usually about 12-30 bases, where the protecting groups have been removed, (ii) allowing time sufficient for the invention oligonucleotide to bind to the target base sequence, (iii) separating unbound invention oligonucleotides from bound invention oligonucleotides and (iii) detecting the presence, absence or amount of bound invention oligonucleotide. Aspects of the invention include conducting any one of these steps individually, which are each part of the complete method. The use of known hybridization methods and conditions are applied to accomplish the method.
- target base sequences may optionally use invention oligonucleotide and target base sequences that are substantially complementary to each other, i.e., sequences having 1 or no mismatches per about every 15-30 bases.
- the target base: sequences to be detected are generally present in a all, in a cell or tissue extract or, usually, in a purified nucleic acid or oligonucleotide preparation, e.g., a sequence encoding a portion of a cytokine, cell surface molecule, an enzyme such as farnesyl protein transferase or an oncogene such as neu, myc, raf, ras or c-ras.
- the target base sequences to be detected are generally present in RNA or single stranded DNA, although the invention oligonucleotides are also useful to detect base sequences in duplex nucleic acids.
- Another invention embodiment is a method comprising incubating a cell with a deprotected invention oligonucleotide containing at least about 7 bases, usually about 12-30 bases, wherein the invention oligonucleotide is optionally present in a transfection complex comprising the invention oligonucleotide and a permeation enhancing agent. This method is used to introduce optionally labeled invention oligonucleotides into cell cytoplasm or vacuoles.
- no permeation enhancing agent is usually needed to introduce the compound into the cells.
- the invention compounds as described is step (a) or step (b) or step (c) (or, in sequence, steps a, b, or steps b, c or steps a, b, c) of claim 1 of U.S. Pat. No. 5,589 29 to practice that claimed method (or to practice necessary steps in the claim 1 method, e.g., contacting an RNA probe containing an invention base with a single stranded nucleic acid to form a duplex).
- oligonucleotides containing an invention base(s) can similarly use screening assay methods described in U.S. Pat. No. 5,589,330.
- the invention compounds as described in the first part of the second step or the second part of the second step or the third part of the second step or the fourth part of the second step or (or, in sequence, in the first step and in the first part of the second step, etc.) of claim 1 of 5,593,839 to practice that claimed method (or to practice necessary steps in the claim 1 method).
- invention monomer compositions containing a 5′a- 35 S-thiotriphosphate group or a 5′ triphosphate group linked to 2′,3′-dideoxyribose to perform dideoxy DNA sequencing methods.
- the invention monomers may be advantageously used in enzymatic DNA sequencing protocols because the invention monomers, which act as cytosine surrogates, have a high affinity for guanosine and may perform better than cytidine 5′ triphosphate in sequencing reactions, particularly where the DNA to be sequenced contains a high proportion of guanosine residues, which can cause sequencing problems.
- Oligonucleotide analogs containing 1, 2, 3 or more invention bases are also suitable for binding to open complexes in cells or cell lysates of eukaryotic or prokaryotic cells.
- Open complexes may also arise is systems comprising at least partially petrified cell components, e.g., RNA polymerase, nucleotide triphosphates, suitable transcription cofactors, DNA binding proteins and duplex DNA capable of transcription.
- Open complexes am regions of single stranded DNA or RNA that occur at least transiently during duplex nucleic acid metabolism, e.g., during DNA replication or RNA transcription in the nucleus, cytoplasm, plastids or mitochondria.
- Such DNA replication or RNA transcription may involve metabolism of cellular, viral or other nucleic acids.
- Such oligonucleotides will typically comprise about 8 to 30 monomers, usually about 12-21 monomers, baying a sequence complementary to a single stranded open complex region(s) involved in the initiation of a nucleic acid metabolic event, e.g., initiation of RNA transcription in a promoter or transcription initiation region.
- an invention oligonucleotide in one or more of the steps needed to practice the methods described in these publications.
- an invention oligonucleotide containing (i) about 7-50, usually about 8-30 linked monomers, usually where the oligonucleotide has a uniform polarity, (ii) 1, 2 or 3 invention bases, (iii) purine and pyrimidine bases having a base sequence complementary or substantially complementary to a target sequence, i.e., a defined base sequence having no more than about 1, 2, 3 or, for relatively long oligonucleotides (about 35-50-mers), 4 base mismatches, and, optionally, (iv) other moieties or features, which are readily apparent to the skilled artisan who has read this disclosure, that facilitate using the oligonucleotide in the intended application, e.g., (i) a free hydroxyl group at the 3′ terminus for applications where the one wishes
- one will use known hybridization conditions, enzyme (such as polymerase, RNase or DNase) reaction conditions or detection technologies to design the invention oligonucleotide in a way that does not interfere with the intended use, or in a way that improves the intended use.
- enzyme such as polymerase, RNase or DNase
- detection technologies to design the invention oligonucleotide in a way that does not interfere with the intended use, or in a way that improves the intended use.
- a previously described assay or diagnostic method calls for conducting an oligonucleotide hybridization or enzyme amplification protocol at a specified temperature
- one will have the option of increasing the hybridization or enzyme amplification temperature due to the presence of an invention base(s) in the oligonucleotide.
- one can use the enhanced binding affinity or enhanced binding specificity property of the invention oligonucleotides to increase hybridization stringency.
- an invention oligonucleotide in a polymerase chain reaction (PCR) amplification method
- PCR polymerase chain reaction
- Skilled artisans routinely design diagnostic or assay protocols by testing varying temperature, salt composition and concentration, pH, oligonucleotide concentration, enzyme concentration, enzyme reaction buffer, act oligonucleotide charge, or oligonucleotide base, sugar or linkage structure during assay development.
- Embodiments include a method comprising preparing a series of oligonucleotides, each having the same base sequence, which sequence contains 2 or more cytosine bases, where each member of the series contains an invention base of structure (3) in place of one or more of the cytosine residues.
- Such oligonucleotides typically comprise 2 to about 6 cytosine residues.
- oligonucleotide compounds such as those of structure (2) are capable of participating in Watson-Crick base pairing they will hind to nucleic acids and therefore are useful in detecting the presence of nucleic acids.
- Bases of structure (1) in such oligonucleotides will recognize guanosine as its complementary base in natural nucleic acids.
- oligonucleotides including many structure (2), (2A), (2B) and (2C) oligonucleotides capable of forming high melting duplexes with complementary sequences, are useful in numerous applications, including antisense or codeblocking utilities in vivo or in vitro as well as diagnostics and probe uses.
- High melting duplexes are those having melting temperatures substantially above the melting temperatures of oligonucleotide or nucleic acid duplexes of the same sequence that contain the ordinary, naturally occurring bases, e.g., adenosine, cytidine, uridine, guanosine, thymidine and the like.
- “Substantially above” means that the derivative oligonucleotide, when hybridized with its complementary sequence, will not dissociate from the duplex until the temperature is raised from about 2 to 40° C., ordinarily about 8 to 40° C., above the dissociation temperature of the same oligonucleotide having the analogous normal A, C, U, G or T bases, but to no greater temperature than about 95° C. This is known as the D Tm.
- D Tm is measured by comparing control oligonucleotide binding to complementary RNA or DNA with the binding of test oligonucleotide to the same RNA or DNA, following, e.g., the method described in Jones et al., “JOC” 58:2983 (1993).
- riboside and deoxyriboside compounds are fluorescent.
- the compounds remain fluorescent upon incorporation, into oligonucleotides and are visible intracellularly, including when bound to target sequences after direct injection or after transfection into cells in accord with known methods.
- tandem arrangements will contain from 2 to about 10 invention polycyclic bases, usually 2, 3 or 4, which can be the same or different polycycles but generally are the same invention polycycle. They also optionally are copolymerized with purine or pyrimidine bases containing known alkynyl substitutions (e.g., U.S. Pat. Nos. 5,645,985 and 5,594,121), in particular pyrimidine bases substituted at the 5 position with a carbon atom which is bonded to another atom by a Pi bond, or the fluorescent cytosine derivatives of Inoue et al. (op cit).
- the compounds of this invention are useful in improved methods for polymerase chain reaction (“PCR”) or ligase chain reaction (“LCR”) amplification and detection of nucleic acids.
- PCR polymerase chain reaction
- LCR ligase chain reaction
- the high melting oligonucleotides are used as one or both primers in classical PCR or as probes in LCR.
- the elevated melting temperature of duplexes with high melting primers avoids the need to thermally cycle the reaction because at these elevated temperatures (about 68 to 95° C., preferably greater than about 75° C.; the derivative primer will continue in at least some proportion to anneal to the target but extension product will not.
- primers will not hybridize and the polymerase will not initiate transcription until the reaction mixture is cooled to a level at which the primer will anneal to the target sequence (usually, about 55° C.).
- the elevated temperature that is chosen for use with the high-melting derivative oligonucleotides is one at which a substantial proportion of the extended primer population (about 10 to 90 mole %) is found dissociated from the target, but sufficient unextended primer is bound to permit extension. Optimally, this is about from 85 to 95° C., ordinarily 92 to 95° C.
- the optimal temperature is determined empirically by simply selecting a range of temperatures within the melting range of the extended sequence, but within the annealing range of the derivative primers, and measuring the amount of amplification product to achieve satisfactory levels for the diagnostic or preparative processes at hand.
- Amplification methods have been described, e.g., U.S. Pat. No. 5,667,974.
- An exemplary method to use an invention oligonucleotide to amplify a nucleic acid base sequence comprises, providing an invention oligonucleotide and target nucleic acid sequence that forms a complex having a Tm of about 85 to 95° C., optionally heating the complex to about 85 to 95° C. (e.g., to a temperature within about 5° C. of the Tm) to provide a brand complex, and optionally mixing the heated complex with a DNA polymerase such as Taq polymerase or other suitable heat stable enzyme.
- the complex and the heated complex is typically a duplex.
- the polymerase reaction will contain cofactors and buffer conditions suitable for amplification purposes.
- the optimal temperature will vary considerably depending upon the derivative bases chosen, whether they are adjacent or separated by other bases, the number of bases in the primers (toe highest annealing temperatures are found with primers having greater than about 18 bases or base analogs), the proportions of pyrimidines and purines and the like.
- the heat stable polymerase useful in this system is for example Tag polymerase or other suitable bear stable enzyme.
- the fluorescent properties of the primers also facilitate detection of the extension products.
- the extension products are readily separated from the unextended primers, e.g. on the basis of molecular weight, and detected by their fluorescence, thereby avoiding staining with agents such as ethidium bromide.
- the fluorescence is enhanced by using NTP's comprising the fluorescent substructures of this invention in primer extension so that the fluorescent NTPs are incorporated into the extension products as well.
- the polycyclic substructure used in the NTP's may be the same or different than the one incorporated into the primers.
- 2-Aminoresorcinol (#1019-43): A ethanol solution (500 mL) of 3-nitroresorinol (20 g 0.129 mmole) was hydrogenated (H 2 in balloon) in the presence of 10% Pd/C (1 g) at room temperature overnight. The catalyst was filtered off through a celite pad. The filtrate was concentrated and purified by flash column chromatography, affording 15.58, 96% of 2-aminoresorcinol. 1 H NMR (DMSO-d 6 ): ⁇ 8.80 (bs, 2H), 6.15-6.30 (m, 3H), 3.85 (bs, 2H).
- N-TFA-propargylamine (#1019-44): Propargylamine (25 g; 0.45 mmole) was dissolved in CH 3 OH (500 mL), followed by addition of ethyl trifluoroacetate (84 g; 0.59 mole). The resulting mixture was stirred at room temperature overnight, then concentrated to dryness. The residue was redissolved in CH 2 Cl 2 (200 mL), washed with saturated NaHCO 3 aqueous solution. The organic phase was isolated, dried, concentrated to a brown residue (liquid). The product #1019-44, 54.0 g was distilled off, 78.8%.
- Compound #1090-18 Compound #1090-15 has been described (Lira, “JACS” 117:3873-3874, 1995).
- a mixture of Compound #1090-15 (15 g; 26 mmole), #1090-05 (2.6 mmole) and DBU (0.8 g; 5.2 mmole) in CH 2 Cl 2 (30 mL) was stirred at room temperature overnight.
- the reaction mixture was washed with 10% citric acid aqueous solution, dried and purified on silica gel column chromatography, affording 1.21 g, 69% of product.
- Compound #1090-25 Compound #1090-22 (0.6 g) was dissolved in ethanol (10 mL) and was hydrogenated (H 2 in a balloon) in the presence of 10% Pd/C (50 mg) at room temperature for 4 hrs. The catalyst was filtered off, washed with CH 3 OH. The filtrate was concentrated and dried to afford 0.3 g 68% of product.
- Compound #1090-31 Compound #1090-26 (150 mg; 0.167 mmole) in CH 2 Cl 2 (2 mL) was added to a 0° C. cold CH 2 Cl 2 solution (1 mL) of PA (0.25 mL of 1M CH 2 Cl 2 solution, 0.25 mmole) and pyridine (66 mg, 0.83 mmole). The resulting mixture was then gradually warmed to room temperature. After 30 minutes, the reaction mixture was diluted with CH 2 Cl 2 , washed with 1 M TEAB aqueous solution, dried and purified on silica gel, eluted with 5% CH 3 OH/CH 2 Cl 2 , the 15%, H 2 O in CH 3 CN. The combined fractions of product were concentrated and then partitioned between CH 2 Cl 2 and 1M TEAB aqueous solution dried, yielding 180 mg of H-phosphonate derivative, quantitatively.
- the isolated product contained Ph 3 P ⁇ O, was treated with saturated NH 3 in CH 3 OH for 5 days at room temperature. After removal of all solvent, the residue (crude #1090-51) was redissolved in CH 3 OH (100 mL), and hydrogenated with H 2 in a balloon in the presence of 10% Pd/C and 4 N HCl in dioxane (200 mL, 0.8 mmole). After 5 hrs, the catalyst was filtered off, washed with CH 3 OH. The filtrate was concentrated and purified by flash column chromatography to afford 0.83 g of #1090-68, in 50% yield for 3 steps.
- Phthalimidoacetaldehyde #1090-70A A mixture of N-(2-hydroxyethyl)phthalimide (90 mg; 0.47 mmole), DCC (0.15 g, 0.7 mmole), DMSO (1 mL) and dichloroacetic acid (20 mL) was stirred at room temperature for 2 hrs. The reaction mixture was diluted with CH 2 Cl 2 , washed with H 2 O twice, dried and concentrated. The crude product #1090-70A contained DCC-urea by-product, was used for the next reaction without further purification.
- Compound #1090-89 Compound #1090-70 (35 mg; 69 mmole) was dissolved in CH 2 Cl 2 (2 mL) and pyridine (0.5 mL), then reacted with DMT-Cl (28 mg, 83 mmole) at room temperature for 3 hrs. The reaction mixture was worked up and purified by flash column chromatography, yielding 44 mg, 78.6% of corresponding 5′-O-DMT-derivatives.
- Compound #1090-91 Compound #1090-89 (44 mg) was converted into 3′-H-phosphonate in normal fashion, 52 mg, in 77% yield.
- N-CBZ-3-aminopropanol (Compound #1154-74, CBZ; benzyloxycarbonyl): A CH 2 Cl 2 solution (300 mL) of 3-aminopropanol (10 g, 0.133 mole) and TEA (20 g, 0.2 mole) was cooled to 0° C., followed by slow addition of CH 2 Cl 2 solution (25 mL) of benzyl chloroformate (25 g, 0.147 mole). The resulting solution was gradually warmed to room temperature. The stirring was continued at room temperature overnight.
- N-CBZ-2-aminoethanol (Compound #1154-104): To 0° C. cold of CH 2 Cl 2 solution (300 mL) of 2-aminoethanol (10.8 g, 0.177 mole) and TEA (26.8 g; 0.265 mole) was added slowly a CH 2 Cl 2 solution (50 mL) of benzyl chloroformate (33.2 g, 0.194 mole). After complete addition, the resulting solution was gradually warmed to room temperature and stirring was continued at room temperature overnight. The reaction was worked up and purified by flash column chromatography on silica gel, affording 22.8 g in 66% yield of title compound.
- Compound 12a (1154-106, 1154-111): Compound 12a (2.5 g, 40.8%, 2 steps) was prepared from 1154-093 (6.0 g, 12 mmole) and 1154-104 (3.0 g, 15.3 mole).
- Compound 12b Compound 12b (0.30 g; 57.6%) was prepared from 1154-93 (0.5 g; 1.0 mmole) and compound 1154-074 (0.31 g; 1.5 mmole).
- Compound 11c Compound 11c (86 mg; 37.7%) was prepared from Compound #1154-093 (200 mg, 0.4 mmole) and N,N-dimethylaminoethanol (43 mg, 0.48 mmole).
- reaction mixture was concentrated, partitioned between CH 2 Cl 2 and saturated NaHCO 3 aqueous solution, dried and purified on silica gel, eluted with 5% CH 3 OH/CH 2 Cl 2 , then 10% CH 3 OH/CH 2 Cl 2 to yield compound 13c, 60 mg, 34% yield.
- Compound #1154-125 A CH 2 Cl 2 solution (20 mL) of Compound #1154-112 (0.28 g, 1.3 mmole), ethylene glycol (0.82 g, 13.2 mmole), silver triflate (0.5 g, 1.95 mmole) and 2,6-di-t-butyl-4-methylpyridine (0.52 g, 2.6 mmole) was stirred at room temperature for 3 hrs. The precipitate was filtered off, washed with CH 2 Cl 2 . The filtrate was washed with H 2 O, dried, concentrated and purified on silica gel, eluted with 3% CH 3 OH/CH 2 Cl 2 , affording 0.17 g, 68% of product #1154-125.
- Compound #1154-126 Compound #1154-125 (170 mg, 0.88 mmole) was dissolved in CH 2 Cl 2 (5 mL) containing TEA (0.27 g, 2.6 mmole) and treated with CH 3 SO 2 Cl (0.15 g, 1.32 mmole). After 30 min at room temperature, the reaction mixture was washed with H 2 O, dried and concentrated. The residue was dissolved in DMF (2 mL) followed by addition of sodium azide (86 mg, 1.3 mmole). The resulting solution was heated at reflux for 2 hrs. The reaction mixture was then partitioned between CH 2 Cl 2 and water.
- Compound #1154-132 Compound #1154-126 (1.1 g, 5.0 mmole) was treated with 3N HCl aqueous solution (10 mL) and ethanol (10 mL) at 50° C. for 1 hr. The reaction mixture was concentrated to dryness, azeotroped with CH 3 CN three times, used for next reaction without further purification.
- Compound #1154-138 The crude #1154-133 (Theoretically 2.5 mmole) was treated with saturated NH 3 in CH 3 OH at room temperature for 3 days, concentrated to dryness, and purified in silica gel, yield 0.28 g of 28% yield of product (2 steps).
- Compound #1154-148 A CH 3 OH solution (25 mL) of Compound #1154-138 (160 mg, 0.38 mmole) was hydrogenated (H 2 in a balloon) in the presence of 10% Pd/C at room temperature for 2 hrs. The catalyst was filtered off, washed with CH 3 OH. The filtrate was concentrated to dryness to afford #1154-148.
- 3′-5′-Diacetyl-O 4 -sulfonyl-2′-deoxyuridine (#1154-136): A CH 2 Cl 2 solution (10 mL) of 3′,5′-diacetyl-2′-deoxyuridine (0.76 g, 2.4 mmole), 2-mesitylenesulfonyl chloride (1.0 g, 4.8 mmole), TEA (1.23 g, 12.1 mmole) and catalytic amount of DMAP (0.1 g) was stirred at room temperature overnight.
- reaction mixture was diluted with CH 2 Cl 2 , washed with 5% citric acid aqueous solution, dried and purified on silica gel, eluted with CH 2 Cl 2 , 35% ethyl acetate in CH 2 Cl 2 , to give the title compound, 0.76 g in 63% yield.
- Compound #1154-137 A CH 2 Cl 2 solution (10 mL) of compound #1154-136 (0.3 g, 0.61 mmole), compound #1154-135 (crude, 0.61 mmole), and DBU (0.46 g, 3.0 mmole) was stirred at room temperature overnight. The reaction mixture was washed with 5% citric acid aqueous solution, dried, and purified by flash column chromatography (silica gel, CH 3 OH/CH 2 Cl 2 ), to give 0.21 g, 38% of product #1154-137. FAB LRMS calculated for C 42 H 61 N 6 O 13 Si 2 (M+H + ) 912, found 913.
- the tested DNA oligonucleotide analogs had the base sequence shown in Table I below. This sequence is complementary to a the 12 base RNA sequence expressed by the large SV40 virus large T antigen gene, 5′ GTA GTG AGG AGG 3′ (SEQ ID NO. 1). In the tested oligonucleotides, which are shown in Table I, each linkage was a phosphorothioate linkage and all sugars were 2′-deoxyribose.
- the tested oligonucleotides had the base sequences shown in Table I.
- bases were designated as follows.
- T m values obtained from the RNA hybrids and the intracellular IC 50 values for T-antigen inhibition derived from microinjection analysis are shown below. In all cases, ⁇ -galactosidase inhibition was concurrently tested as an internal control and we saw no inhibition at 5 ⁇ M, the highest concentration tested.
- ⁇ -galactosidase inhibition was concurrently tested as an internal control and we saw no inhibition at 5 ⁇ M, the highest concentration tested.
- Oligonucleotides containing a base of structure (3) at an internal position in the oligonucleotide where R 2 was (a) the (S) isomer of —O—CH 2 —C*H(CH 3 )—NH 2 or (b) the (R) isomer of —O—CH 2 —C*H(CH 3 )—NH 2 were prepared and tested for binding affinity in a similar manner. Both oligonucleotides had an increased binding affinity compared to the control oligonucleotide.
- ODN13 Antisense inhibition of p27 kip1 expression.
- the sequence of ODN13 was 5′ UGGCUCUCCUGCGCC 3′ (SEQ ID NO. 18) and it contained phosphorothioate linkages, all sugars were 2′-deoxyribose, all G positions contained guanine, all U positions contained 5-(1-propynyl)uracil and all C positions contained 5-(1-propynyl)-cytosine.
- ODN14 had the same sequence and base composition as ODN13, except that all C positions contained 5-methylcytosine instead of 5-(1-propynyl)cytosine and thymidine instead of 5-(1-propynyl)uracil.
- ODN15 had the same sequence and base composition as ODN14, except that the 9th C position from the 5′ end contained a (57) base instead of 5-methylcytosine.
- Each ODN was tested for its capacity to inhibit p27 gene expression in CV-1 cells in cell culture using cationic lipid to deliver the oligonucleotides into the cells, essentially as described (Coats “Science” 272:877-880, 1996).
- ODN IC 50 (nM) 13 10 14 >20 15 ⁇ 5 The results showed that ODN15 containing one (57) base is more potent than the all-propyne derivative ODN13.
- phosphorothioate-linked oligonucleotides containing 5-1(propynyl)-modified pyrimidine bases were the among the most potent reported class of antisense agents.
- ODN13 was tested in rats and, in a 10 day toxicological evaluation, it was found that the MTD (maximum tolerated dose) was 0.6 mg/kg/d i.v. ODN15 was tested in the same manner with no toxicity observed at a dose of 6.0 mg/kg/d i.v.
- ODN22 having the base sequence, 5′ TCTCCCTCTC 3′ (SEQ ID NO. 19).
- ODN22 contained only phosphorothioate linkages, all sugars were 2′-deoxyribose, bases designated T were thymine, bases designated C were 5-methylcytosine.
- ODN23 was the same as ODN22, except that the 5th base position from the 5′ end contained 5-(1-propynyl)cytosine (base designated “D” in Table IV).
- ODN24 was the same as ODN22, except that the 5th base position from the 5′ end contained a structure (60) base.
- ODN25 was the same as ODN22, except that the 5th base position from the 5′ end contained a structure (55) base.
- ODN26 was the same as ODN22, except that the 5th base position from the 5′ end contained a structure (57) base.
- ODN27 was the same as ODN22, except that the 5th base position from the 5′ end contained a structure (59) base.
- ODN28 was the same as ODN22, except that the 5th base position from the 5′ end contained a structure (58) base.
- ODN29 was the same as ODN22, except that the 5th base position from the 5′ end contained a structure (61) base.
- ODN30 was the same as ODN22, except that the 5th base position from the 5′ end contained a structure (65) base.
- T m (° C.) of ODN22-ODN30 using 4 different oligonucleotides: A complementary RNA oligonucleotide (ODN31), an RNA oligonucleotide (ODN32) having adenine at the 11th position from its 5′ end, i.e., a single A: test base mismatch, a complementary DNA oligonucleotide (ODN33), and a DNA oligonucleotide (ODN34) having adenine at the 11th position from its 5′ end.
- ODN31 complementary RNA oligonucleotide
- ODN32 RNA oligonucleotide having adenine at the 11th position from its 5′ end
- a single A test base mismatch
- ODN33 complementary DNA oligonucleotide
- ODN34 DNA oligonucleotide
- Target DNA 33)/RNA (31): 5′-AAA-AAG-AGA-GGG-AGA (SEQ ID NO. 21, 22)
- Target DNA 34)/RNA (32): 5′-AAA-AAG-AGA-GAG-AGA (SEQ ID NO.
- oligonucleotide 5′ TCC-CGC- X TG-TGA-CAT-CGA-TT 3′ (SEQ ID NO. 25), was X was a structure (57) base.
- the oligonucleotide was complementary to the 3′ untranslated region of the c-raf mRNA.
- a control oligonucleotide had the same sequence except that the X base was replaced with cytosine.
- Each oligonucleotide was tested to determine its potency at inhibiting expression c-raf gene expression essentially as described (Monia “Nature Med” 2:668-675 1966, WO 97/32604). Briefly, a range of concentrations of each oligonucleotide was transfected into A549 small lung carcinoma cells on two consecutive days, followed by preparing cell extracts 48 hours after the first transfection. Immunoblot assay for c-raf protein expression showed the control oligonucleotide reduced c-raf protein expression with an IC 50 of about 20 nM. The test oligonucleotide containing the structure (57) base in place of cytosine was at least 20-fold more potent and had an IC 50 of less than 1 nM.
- oligonucleotide containing about 8-18 bases that are complementary to raf or c-raf e.g., the oligonucleotide sequence used in this example or a shortened version thereof, is accomplished in a similar manner using invention oligonucleotides containing 1, 2 or 3 invention bases having an R 2 moiety that increases binding affinity compared to a control oligonucleotide containing cytosine.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Saccharide Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Compounds having structure (1)
wherein R1 is —H a protecting group, a linker or a binding partner; and R2 and R34 are as defined in the specification. The invention also provides intermediates and methods make the structure (1) compounds, as well as methods to use the compounds as labels in diagnostic assays and to enhance binding to complementary bases.
wherein R1 is —H a protecting group, a linker or a binding partner; and R2 and R34 are as defined in the specification. The invention also provides intermediates and methods make the structure (1) compounds, as well as methods to use the compounds as labels in diagnostic assays and to enhance binding to complementary bases.
Description
This invention relates to the field of labels, particularly labels for diagnostic or analytical use. In particular, it relates to oligonucleotides that are modified to enhance the binding affinity or the binding specificity of the oligonucleotides for complementary sequences and that in addition optionally bear a readily detectable characteristic.
Sequence specific binding of oligonucleotides both to single stranded RNA and DNA and to duplex DNA is widely known. This phenomenon has been harnessed for a great variety of diagnostic, therapeutic and analytical, e.g., sequence determination or gene mapping, purposes. Previously, one objective of research in this field has been to increase the affinity of such oligonucleotides for their complementary sequences. For example, workers have described oligonucleotides containing 5-substituted pyrimidine bases that substantially increase the Tm for oligonucleotide binding to complementary bases (International Publication No. WO 93/10820).
Publications have described the use of fluorescent cytosine derivatives to prepare labeled DNA probes. See Inoue et al., Jpn. Kokai JP 62059293, (1987). In addition, fluorescent labeled nucleotides have been employed in DNA sequencing. See Prober et al., “Science” 238:336-341 (1987).
1,3-Dihydro-2H-imidazo[4,5-b]-quinolin-2-one derivatives as phosphodiesterase inhibitors are disclosed by Raeymackers et al. (EP 541,153).
U.S. Pat. No. 5,502,177, discloses phenoxazine polycycle-containing oligonucleotides and monomers for preparing the oligonucleotides.
The invention compositions or methods accomplish one or more of the following objects.
An object of this invention is to increase the affinity of oligonucleotides for their complementary sequences.
An object of this invention is to increase the specificity of oligonucleotides for their complementary sequences.
Another object of this invention is to provide detectable labels for use in diagnostic assays.
Another object is to enhance diagnostic assays that use oligonucleotides.
Another object is to improve the therapeutic efficacy of oligonucleotides.
Another object is to improve the potency of oligonucleotides as antisense reagents that affect gene expression by altering intracellular metabolism of complementary RNA sequences encoding a target gene(s).
Another object is to provide chemical intermediates and synthesis methods to prepare the invention compositions.
These and other objects of the invention will be apparent when one considers the disclosure as a whole.
-
- and tautomers, solvates and salts thereof, wherein
- R1 is a binding partner, a protecting group, a linker or —H;
- R2 is A(Z)X1, wherein A is a spacer and Z independently is a label bonding group optionally bonded to a detectable label, but R2 is not amine, protected amine, nitro or cyano;
- R27 is independently —CH═, —N═, —C(C1-8 alkyl)═ or —C(halogen)═, but no adjacent R27 are both —N═, or two adjacent R27 are taken together to form a ring having the structure,
where Ra is independently —CH═, —N═, —C(C1-8 alkyl)═ or —C(halogen)═, but no adjacent Ra are both —N═; - R34 is —O—, —S— or —N(CH3)—; and
- X1 is 1, 2 or 3.
When the binding partner R1 is an oligonucleotide, embodiments of the compounds of this invention include oligonucleotides of structure (2), (2A), (2B), or (2C)
wherein
wherein
-
- D is —OH, protected —OH, an oligonucleotide coupling group or a solid support;
- D1 is an oligonucleotide coupling group, —OH, protected —OH or a solid support, wherein D1 is bonded to one 2′ or 3′ position in the oligonucleotide of structure (2) and the adjacent 2′ or 3′ position in structure (2) is substituted with R21, provided that D and D1 are not both an oligonucleotide coupling group or they are not both a solid support;
- D2 is —CO2R5, —C(O)N(R5)2, —SO3R5, —SO2N(R5)2 or an activated derivative of —CO2H or —SO3H;
- D3 is a protecting group, —H or —(CH2)2-6—N(R5)2;
- R4 is independently a phosphodiester linkage or a phosphodiester substitute linkage, wherein R4 is bonded to one 2′ or 3′ position in the structure (2) oligonucleotide and the adjacent 2′ or 3′ position in structure (2) is substituted with R21;
- R5 is independently —H or a protecting group;
- R21 is independently —H, —OH, halogen or a moiety that enhances the oligonucleotide against nuclease cleavage;
- R37 is independently —O—, —CH2— or —CF2—;
- n is an integer from 0 to 98; and
- B independently is a purine or pyrimidine base or a protected derivative thereof, provided that at least one B is a base of structure (3)
Embodiments include compositions useful as intermediates in making the structure (1) compounds, including intermediates having structure (4)
and tautomers, solvates and salts thereof wherein,
and tautomers, solvates and salts thereof wherein,
-
- R24 is a halogen; and
- R25 is —SH, —OH, ═S or ═O.
In a further embodiment, the invention includes contacting a structure (2), (2A), (2B), or (2C) oligonucleotide, wherein n is at least about 7, with a sample suspected to contain a nucleic acid having a base sequence that is at least substantially complementary to the structure (2), (2A), (2B), or (2C) oligonucleotide.
In a further embodiment, the invention includes detecting the presence, absence or amount of a complex comprising a structure (2), (2A), (2B), or (2C) oligonucleotide, wherein n is at least about 7, and a nucleic acid having a base sequence that is at least substantially complementary to the structure (2), (2A), (2B), or (2C) oligonucleotide.
In a further embodiment, the invention includes converting a structure (4) compound to a compound of structure (1) where R34 is —O— or —S— and the R2 atom or moiety alpha to the ring containing R27 is —O—, —S— or —CH2—, by displacing R24.
In a further embodiment, the invention includes converting a structure (4A) compound (a structure (1) compound where R2 is replaced with —NH2); to a compound of structure (1), by reductive alkylation of the —NH2 group to yield a structure (1) compound where the R2 moiety alpha to the ring containing R27 is —NH—.
The invention comprises all combinations formed by independently selecting individual Markush group members and assembling them in accordance with the teachings herein. The invention optionally excludes any feature or substance found in, or obvious over, the prior art.
Structural formulas are designated as parenthetical numerals. We intend that designation of aromaticity with respect to carbocycles and heterocycles herein includes any highly resonant unsaturated ring structure. Alternatively, placement of double bonds, where indicated, represents one potential structure for the depicted compound but we intend this depiction to include other resonant states of the compound as well as protonated and charged species, only one of which a structure may show.
The invention includes invention compounds in unpurified, substantially purified and purified forms. It includes invention compounds that are present with any additional component(s) such as a solvent, reactant or by-product that is present during invention compound synthesis or purification, and any additional component(s) that is present during the use or manufacture of an invention compound.
Halo and halogen mean F, Cl, Br or I.
Alkyl means unbranched, branched or cyclic hydrocarbons that are saturated or unsaturated, or combinations thereof. Alkyl includes all isomers, e.g., stereoisomers, positional isomers, diastereomers and regioisomers. Alkyl moieties that are unsaturated will typically contain 1, 2, 3 or more —CH═CH— or —C≡C— groups, usually one such group.
Substituted alkyl means unbranched, branched or cyclic hydrocarbons that are saturated or unsaturated, or combinations thereof, where the hydrocarbon contains a heteroatom linked to a carbon or a heteroatom that replaces a carbon atom. Substituted alkyl includes all isomers, e.g., stereoisomers, positional isomers, diastereomers and regioisomers. Substituted alkyl moieties that are unsaturated will typically contain 1, 2, 3 or more —CH═CH— or —C≡—C— groups, usually one such group. Substituted alkyl includes alkyl groups having substituents linked to a carbon atom or substituents that interrupt a carbon atom chain, and unless otherwise defined, substituents include ethers (—O—), ketones (—C(O)—), —OR5, —C(O)OR5, —C(O)O—, —OC(O)—, —C(O)H, —OCH2—, —OCH2CH2—, —OCH2O—, —OCH2CH2O—, —NR5—, —NHR5, —NHC(O)—, —C(O)NH—, C(O)NHR5, —OC(O)NR5—, —OC(O)NHR5, —NR5C(O)NR5—, —NR5C(O)NHR5, —NR5CH2—, —NR5CH2CH2—, —S—, —SR5, —S(O)—, —S(O)(O)—, —S(O)OR5, —S(O)H, halogen, CN, NO2, and combinations of these substituents where R5 is hydrogen or a protecting group.
The invention compounds herein do not include obviously unstable structures, e.g., —O—O—, —O—S— or unsaturated cyclopropyl, unless they are useful as transitory intermediates in the preparation of more stable compounds.
As used herein, “monosaccharide” means a polyhydroxy aldehyde or ketone having the empirical formula (CH2O)n where n is 3, 4, 5, 6 or 7. Monosaccharide includes open-chain and closed-chain forms, but will usually be closed chain forms. Monosaccharide includes hexofuranose and pentofuranose sugars such as 2′-deoxyribose, ribose, arabinose, xylose, their 2′-deoxy and 3′-deoxy derivatives, their 2′,3′-dideoxy derivatives, and their derivatives containing R21 linked to the 2′ or 3′ position, usually the 2′ position. Monosaccharide also includes the 2′,3′ dideoxydidehydro derivative of ribose. Monosaccharides include the D- and L-isomers of glucose, fructose, mannose, idose, galactose, allose, gulose, altrose, talose, fucose, erythrose, threose, lyxose, erythrulose, ribulose, xylulose, ribose, arabinose, xylose, psicose, sorbose, tagatose, glyceraldehyde, dihydroxyacetone and their monodeoxy derivatives such as rhamnose. Monosaccharides are optionally protected or partially protected.
As used herein, a “protecting group” means a moiety that prevents the atom to which it is linked from participating in unwanted reactions. For example, for —OR5, R5 is a protecting group for the oxygen atom found in a hydroxyl or carboxyl group, for —SR5, R5 is a protecting group for sulfur in thiols for instance, and for —NHR5 or —N(R5)—, R5 is a nitrogen atom protecting group for primary or secondary amines. Hydroxyl, amine and other reactive groups are found in invention compounds at, e.g., R2, R21 or oligonucleotide linkages or oligonucleotide bases. These groups may require protection against reactions taking place elsewhere in the molecule. The protecting groups for oxygen, sulfur or nitrogen atoms are usually used to prevent unwanted reactions with electrophilic compounds, such as acylating or phosphorylating agents used, e.g., in nucleoside, nucleotide or oligonucleotide chemistry.
Protecting groups are intended to be removed by known procedures, although it will be understood that the protected intermediates fall within the scope of this invention. The removal of the protecting group may be arduous or straightforward, depending upon the economics and nature of the conversions involved. In general, one will use a protecting group with exocyclic amines in the B groups of the compounds of this invention. For oligonucleotide containing such B groups to be fully binding competent, exocyclic amines must be deprotected because the amine groups participate in hydrogen bonding with complementary bases. Similarly, one will typically use reversible protecting groups for the 5′ and 3′ hydroxyl groups of pentofuranose sugars in nucleotides intended for use as monomers in synthesis of oligonucleotides containing 3,5′ linkages. Protecting groups commonly are employed to protect against covalent modification of a sensitive group in reactions such as phosphorylation, alkylation or acylation. Ordinarily, protecting groups are removed by, e.g. hydrolysis, elimination or aminolysis. Thus, simple functional considerations will suffice to guide the selection of a reversible or an irreversible protecting group at a given locus on the invention compounds. Suitable protecting groups and criteria for their selection are described in T. W. Greene and P. G. M. Wuts, Eds. “Protective Groups in Organic Synthesis” 2nd edition, Wiley Press, at pps. 10-142, 143-174, 175-223, 224-276, 277-308, 309-405 and 406-454.
Embodiments include salts and complexes of invention compounds, including pharmaceutically acceptable or salts that are relatively non-toxic. The invention compounds may have one or more moieties that carry at least a partial positive or negative charge in aqueous solutions, typically at a pH of about 4-10, that can participate in forming a salt, a complex, a composition with partial salt and partial complex properties or other noncovalent interactions, all of which we refer to as a “salt(s)”. Salts are usually biologically compatible or pharmaceutically acceptable or non-toxic, particularly for mammalian cells. Salts that are biologically toxic are optionally used with synthetic intermediates of invention compounds. When a water soluble composition is desired, monovalent salts are usually preferred.
Metal salts typically are prepared by reacting the metal hydroxide with a compound of this invention. Examples of metal salts which are optionally prepared in this way are salts containing Li+, Na+, and K+. A less soluble metal salt can be precipitated from the solution of a more soluble salt by adding a suitable metal compound. Invention salts may be formed from acid addition of certain organic acids, such as organic carboxylic acids, and inorganic acids, such as alkylsulfonic acids or hydrogen halide acids, to acidic or basic centers on invention compounds, such as basic centers on the invention pyrimidine base analogs. Metal salts include ones containing Na+, Li+, K+, Ca++ or Mg++. Other metal salts may contain aluminum, barium, strontium, cadmium, bismuth, arsenic or zinc ion.
Salt(s) of invention compounds may comprise a combination of appropriate cations such as alkali and alkaline earth metal ions or ammonium and quaternary ammonium ions with the acid anion moiety of the phosphoric acid or phosphonic acid group, which may be present in invention polymers or monomers.
Salts are produced by standard methods, including dissolving free base in an aqueous, aqueous-alcohol or aqueous-organic solution containing the selected acid, optionally followed by evaporating the solution. The free base is reacted in an organic solution containing the acid, in which case the salt usually separates directly or one can concentrate the solution.
Suitable amine salts include amines having sufficient basicity to form a stable salt, preferably amines of low toxicity including trialkyl amines (tripropylamine, triethylamine, trimethylamine), procaine, dibenzylamine, N-benzyl-betaphenethylamine, ephenamine, N,N′-dibenzylethylenediamine, N-ethylpiperidine, benzylamine and dicyclohexylamine.
Salts include organic sulfonic acid or organic carboxylic acid salts, made for example by addition of the acids to basic centers, typically amines. Exemplary sulfonic acids include C6-16 aryl sulfonic acids, C6-16 heteroaryl sulfonic acids and C1-16 alkyl sulfonic acids such as phenyl sulfonic acid, a-naphthalene sulfonic acid, β-naphthalene sulfonic acid, (S)-camphorsulfonic acid, methyl (CH3SO3H), ethyl (C2H5SO3H), n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, pentyl and hexyl sulfonic acids. Exemplary organic carboxylic acids include C1-16 alkyl, C6-16 aryl carboxylic acids and C4-16 heteroaryl carboxylic acids such as acetic, glycolic, lactic, pyruvic, malonic, glutaric, tartaric, citric, fumaric, succinic, malic, maleic, oxalic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic, salicylic, nicotinic and 2-phenoxybenzoic.
Invention salts include those made from inorganic acids, e.g., HF, HCl, HBr, HI, H2SO4, H3PO4, Na2CO3, K2CO3, CaCO3, MgCO3 and NaClO3. Suitable anions, which are optionally present with a cation such a Ca++, Mg++, Li+, Na+ or K+, include arsenate, arsenite formate, sorbate, chlorate, perchlorate, periodate, dichromate, glycodeoxycholate, cholate, deoxycholate, desoxycholate, taurocholate, taurodeoxycholate, taurolithocholate, tetraborate, nitrate, nitrite, sulfite, sulfamate, hyposulfite, bisulfite, metabisulfite, thiosulfate, thiocyanate, silicate, metasilicate, CN−, gluconate, gulcuronate, hippurate, picrate, hydrosulfite, hexafluorophosphate, hypochlorite, hypochlorate, borate, metaborate, tungstate and urate.
Salts also include the invention compound salts with one or more amino acids. Many amino acids are suitable, especially the naturally-occurring amino acids found as protein components, although the amino acid typically is one bearing a side chain with a basic or acidic group, e.g., lysine, arginine, histidine or glutamic acid, or a neutral group such as glycine, serine, threonine, alanine, isoleucine, or leucine.
The invention compositions include compounds in their un-ionized, as well as zwitterionic form, and combinations with stoichiometric amounts of water as in hydrates.
The compounds of the invention include enriched or resolved optical isomers at any or all asymmetric atoms as are apparent from the depictions. Both racemic and diasteromeric mixtures, as well as the individual optical isomers isolated or synthesized so as to be substantially free of their enantiomeric or diastereomeric partners, are all within the scope of the invention. Chiral centers may be found in invention compounds at, for example, R1, R2 and R21.
One or more of the following enumerated methods are used to prepare the enantiomerically enriched or pure isomers herein. The methods are listed in approximately their order of preference, i.e., one ordinarily should employ stereospecific synthesis from chiral precursors before chromatographic resolution before spontaneous crystallization.
Stereospecific synthesis is described in the examples. Methods of this type conveniently are used when the appropriate chiral starting material is available and reaction steps are chosen do not result in undesired racemization at chiral sites. One advantage of stereospecific synthesis is that it does not produce undesired enantiomers that must be removed from the final product, thereby lowering overall synthetic yield. In general, those skilled in the art would understand what starting materials and reaction conditions should be used to obtain the desired enantiomerically enriched or pure isomers by stereospecific synthesis. If an unexpected racemization occurs in a method thought to be stereospecific then one needs only to use one of the following separation methods to obtain the desired product.
If a suitable stereospecific synthesis cannot be empirically designed or determined with routine experimentation then those skilled in the art would turn to other methods. One method of general utility is chromatographic resolution of enantiomers on chiral chromatography resins. These resins are packed in columns, commonly called Pirkle columns, and are commercially available. The columns contain a chiral stationary phase. The racemate is placed in solution and loaded onto the column, and thereafter separated by HPLC. See for example, Proceedings Chromatographic Society—International Symposium on Chiral Separations, Sep. 3-4, 1987. Examples of chiral columns that could be used to screen for the optimal separation technique would include Diacel Chriacel OD, Regis Pirkle Covalent D-phenylglycine, Regis Pirkle Type 1A, Astec Cyclobond II, Astec Cyclobond III, Serva Chiral D-DL=Daltosil 100, Bakerbond DNBLeu, Sumipax OA-1000, Merck Cellulose Triacetate column, Astec Cyclobond I-Beta, or Regis Pirkle Covalent D-Naphthylalanine. Not all of these columns are likely to be effective with every racemic mixture. However, those skilled in the art understand that a certain amount of routine screening may be required to identify the most effective stationary phase. When using such columns it is desirable to employ embodiments of the compounds of this invention in which the charges are not neutralized, e.g., where acidic functionalities such as carboxyl are not esterified or amidated.
Another method entails converting the enantiomers in the mixture to diasteriomers with chiral auxiliaries and then separating the conjugates by ordinary column chromatography. This is a very suitable method, particularly when the embodiment contains free carboxyl, amino or hydroxyl that will form a salt or covalent bond to a chiral auxiliary. Chirally pure amino acids, organic acids or organosulfonic acids are all worthwhile exploring as chiral auxiliaries, all of which are well known in the art. Salts with such auxiliaries can be formed, or they can be covalently (but reversibly) bonded to the functional group. For example, pure D or L amino acids can be used to amidate the carboxyl group of embodiments of this invention and then separated by chromatography. Enzymatic resolution is another method of potential value. In such methods one prepares covalent derivatives of the enantiomers in the racemic mixture, generally lower alkyl esters (for example of carboxyl), and then exposes the derivative to enzymatic cleavage, generally hydrolysis. For this method to be successful an enzyme must be chosen that is capable of stereospecific cleavage, so it is frequently necessary to routinely screen several enzymes. If esters are to be cleaved, then one selects a group of esterases, phosphatases, and lipases and determines their activity on the derivative. Typical esterases are from liver, pancreas or other animal organs, and include porcine liver esterase.
If the enantiomeric mixture separates from solution or a melt as a conglomerate, i.e., a mixture of enantiomerically-pure crystals, then the crystals can be mechanically separated, thereby producing the enantiomerically enriched preparation. This method, however, is not practical for large scale preparations and is of no value for true racemic compounds.
Asymmetric synthesis is another technique for achieving enantiomeric enrichment. For example, a chiral protecting group is reacted with the group to be protected and the reaction mixture allowed to equilibrate. If the reaction is enantiomerically specific then the product will be enriched in that enantiomer.
Further guidance in the separation of enantiomeric mixtures can be found, by way of example and not limitation, in “Enantiomers, Racemates, and resolutions”, Jean Jacques, Andre Collet, and Samuel H. Wilen (Krieger Publishing Company, Malabar, Fla., 1991, ISBN 0-89464-618-4): Part 2, Resolution of Enantiomer Mixture, pages 217-435; more particularly, section 4, Resolution by Direct Crystallization, pages 217-251, section 5, Formation and Separation of Diastereomers, pages 251-369, section 6, Crystallization-Induced Asymmetric Transformations, pages 369-378; and section 7, Experimental Aspects and Art of Resolutions, pages 378-435; still more particularly, section 5.1.4, Resolution of Alcohols, Transformation of Alcohols into Salt-Forming Derivatives, pages 263-266, section 5.2.3, Covalent Derivatives of Alcohols, Thiols, and Phenols, pages 332-335, section 5.1.1, Resolution of Acids, pages 257-259, section 5.1.2, Resolution of Bases, pages 259-260, Section 5.1.3, Resolution of Amino Acids, page 261-263, section 5.2.1, Covalent Derivatives of Acids, page 329, section 5.2.2, Covalent derivatives of Amines, pages 330-331, section 5.2.4, Covalent Derivatives of Aldehydes, Ketones, and Sulfoxides, pages 335-339, and section 5.2.7, Chromatographic Behavior of Covalent Diastereomers, pages 348-354.
R2 is a key functionality. It is substituted on the polycycle depicted in structure (1), less R2. The combination of the polycycle and R2 is termed the polycyclic substructure. R2 consists of two principal structural features denominated —A(Z)X1. Group A is a spacer that is used to position the Z group(s) and attach it to the remainder of the polycycle. The Z group(s) serve as a site for attachment of a detectable label or to enhance the hydrogen bonding of the polycycle to the complementary guanine base. In some embodiments, Z is capable of performing both functions. For the most part, Z groups capable of hydrogen bonding are useful as sites for covalent bonding to detectable labels, but not all Z groups that are useful as label-bonding sites are capable of hydrogen bonding to guanine. Z contains at least one atom other than carbon, typically O, N or S. In any case, the R2 groups possess at least one of these practical utilities. It would be routine to make and test them to determine the best use of any one embodiment.
Z groups capable of base-pairing are believed to hydrogen bond with N7 of guanine in a complementary nucleic acid sequence when incorporated into a polycyclic substructure-substituted oligonucleotide. The resulting duplex has greater stability than one containing a native GC pair because the R2 group provides an additional point for hydrogen bonding to the complementary guanine base. Thus, these embodiments serve as cytosine surrogates for supplemented Watson-Crick base-pairing. In general, a base-pairing substituent Z is defined functionally as any group that, when taken together with the remainder of R2, is capable of increasing the temperature of melting of any of the oligonucleotides 3-9 in Table 1 by at least about 2 degrees Centigrade when substituted as shown in Table 1.
If R2 does not contain a substituent that is capable of contributing to base pairing or hydrogen bonding then R2 is useful at least as a point of attachment for a detectable label. Such Z groups need only be reactive with a bifunctional cross-linking agent or with the label directly. In some embodiments, the polycyclic substructure is itself fluorescent, and in these cases it is not necessary to link the Z group to a detectable label. In these embodiments the polycyclic substructure is detectable by fluorescent emissions, or by adsorption and energy transfer to an emitting (second) label present on a binding partner in the same fashion as is used in EMIT technologies well-known in the diagnostics field.
Spacer A is substituted with from 1 to 3 Z groups. When Z is a base-pairing hydrogen bonding group then “X1” is preferably 1 or 2, ordinarily 1. Similarly, for reasons of steric access it is preferred that only 1 or 2 Z groups are present on spacer A when R2 is intended to function as a label bonding site.
In some embodiments the spacer group A and the Z substituent(s) will interact functionally, i.e., changes in group A may have an impact on the physical or chemical properties of Z, and vice-versa. For example, it will be understood by those skilled in the art that changes can be introduced in spacer A that would reduce or increase the ability of Z to hydrogen bond or to react with a label or cross-linking agent. A readily apparent instance of this would be substitution of A with electron donors or acceptors proximal to a Z group, which may affect hydrogen bonding between Z and guanine. However, it is conceptually useful to consider these domains to be functionally and structurally discrete taking into account interdomain interactions that would be apparent to the ordinary artisan.
Spacer A typically contains a backbone chain of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 carbon atoms, any 1, 2 or 3 of which are optionally replaced with N, O or S atoms, usually 1 N, O or S atom. The backbone chain refers to the atoms that connect the Z group(s) to the ring carbon atom at the R2 binding site on the polycycle. The number of spacer backbone atoms does not include terminal Z group atoms. R2 does not include protected amine as described in U.S. Pat. No. 5,502,177.
The spacer A backbone is linear or one or more backbone atoms are substituted, which results in branching. Ordinarily, when 1 Z group is present then A will contain a linear backbone of 2 to 8, usually 2 to 4 atoms. The backbone generally is carbon only, bonded by saturated or unsaturated bonds. If unsaturated bonds are present, the backbone generally will contain 1 to 2 double or triple bonds. Preferably, the backbone is saturated. If a heteroatom is present in the backbone it typically will be O or S. Preferably the heteroatom is O, and preferably only 1 O is present in the backbone chain. Heteroatoms are used to replace any of the backbone carbon atoms, but preferably are used to replace the carbon atom alpha (adjacent) to the polycyclic ring. Usually the atom in the spacer chain that is bonded to the polycyclic substructure is unsubstituted, e.g., —O—, —S—, —NH— or —CH2—, and, in general, the next 1, 2 or 3 atoms in the spacer are unsubstituted carbon.
The spacer A backbone is optionally substituted independently with 1, 2 or 3 of the following: C1-C8 alkyl, —OR5, ═O, —NO2, —N3, —COOR5, —N(R5)2, or —CN groups, C1-C8 alkyl substituted with —OH, ═O, —NO2, —N3, —COOR5, —N(R5)2, or —CN groups, or any of the foregoing in which —CH2— is replaced with —O—, —NH— or —N(C1-C8 alkyl), wherein R5 is H or a protecting group. Certain of these groups may function as Z sites for linking to detectable labels, but need not be used for that purpose unless desired. In some embodiments these substituents are useful in increasing the lipophilicity of the compounds of this invention.
Group Z detectable labels include all of the conventional assayable substances used heretofore in labeling oligonucleotides or proteins. Examples are well known and include fluorescent moieties such as fluorescein, chemiluminescent substances, radioisotopes, chromogens, or enzymes such as horseradish peroxidase. For the purposes herein, the residue of any bifunctional or multifunctional agent used to crosslink the Z group(s) to the A backbone is defined to be part of the Z group, and the residue of the detectable label is considered also to represent part of Z.
Group Z also encompasses substituents that are not detectable by conventional diagnostic means used in clinical chemistry settings (e.g., UV or visible light absorption or emission, scintillation or gamma counting, or the like) but which are nonetheless capable of reacting with a crosslinking agent or a detectable label to form a covalent bond. In this regard, the Z groups, function as intermediates in the synthesis of the labelled reagent. Typical Z groups useful for this purpose include —NH2, —CHO, —SH, —CO2Y or OY, where Y is H, 2-hydroxypyridine, N-hydroxysuccinimide, p-nitrophenyl, acylimidazole, maleimide, trifluoroacetate, an imido, a sulfonate, an imine 1,2-cyclohexanedione, glyoxal or an alpha-halo ketone. Suitable spacers, reactive groups and detectable labels have been described, e.g., U.S. Pat. Nos. 5,668,266, 5,659,022, 5,646,261, 5,629,153, 5,525,465 and 5,260,433, WO 88/10264, WO 97/31008, EP 063 879 B1, Urdea “NAR” 16:4937-4956 (1988), Prober “Science” 238:336-341 (1987).
Z also is a hydrogen bond donor moiety or a moiety, when taken together with the influence of spacer A, has a net positive charge of at least about +0.5 at pH 6-8 in aqueous solutions. Such Z groups are designated R2D. In these embodiments, R2D is covalently linked to a short spacer A having a backbone (otherwise described above) of 2, 3, 4, 5 or 6 atoms, designated R2C.
The R2C short spacer chain backbone atoms are C atoms and optionally one or two atoms independently selected from the group consisting of O, N or S atoms. R2C short spacer chain backbones include unbranched and branched alkyl that optionally contain one or two independently selected O, N or S atoms. Usually R2C is unbranched, i.e. the backbone has no hydrocarbon substituents. Any branching, if present, will usually consist of a C1-C3 alkyl group, usually a methyl or ethyl group, or C1-C3 alkyl substituted with —OH, ═O, —O(C1-C3 alkyl), —CN, N3 or 1, 2, 3 or 4 halogen atoms.
Exemplary —R2C—R2D and related structures are
-
- (a) —R6—(CH2)t—NR5C(NR5)(NR3)2,including —O—(CH2)t—NR5C(NR5)(NR3)2, —NH—(CH2)t—NR5C(NR5)(NR3)2 and —(CH2)2-5NR5C(NR5)(NR3)2, —R6 —(CH 2)t NR 5C(NR 5)N(R 3)2 , including —O—(CH 2)t NR 5C(NR 5)N(R 3)2 , —N—(CH 2)t NR 5 C(NR 5)N(R 3)2 , and —(CH 2)2-5 NR 5 C(NR 5)N(R 3)2 ,
- (b) —R6—CH2—CNR31—N(R3)2, —R6—(R7)y—N(R3)2, —R6—(CH2)tN(R3)2, —(CH2)tN(R3)2, —CH2—O—(CH2)t—N(R3)2,
where R6 is usually —O—,
where R6 is usually —O— and R8 is usually —CH2— or —CH2CH2— in structures (42)-(46) and adjacent R6 and R8 are not —O—O—, —O—S— or —S—S—; - R3 is independently —H, —CH3, —CH2CH3, —(CH2)w—N(R33)2 or a protecting group, usually —H or —CH3,
- or, both R3 together are joined to form a protecting group,
- or, when R2 is —R6(CH2)tN(R3)2, one R3 is H, CH3, CH2CH3, a protecting group or —(CH2)w—N(R33)2 and the other R3 is —H, —CH3, —CH2CH3, —(CH2)w—N(R33)2, —CH(N[R33]2)—N(R33)2,
usually when one R3 is —H, —CH3, —CH2CH3, or —(CH2)v—N(R33)2, the other R3 is —H or a protecting group; - R5 is independently —H or a protecting group;
- R6 is independently —S—, —NR5—, —O— or —CH2—;
- R7 is independently linear alkyl having 1, 2, 3 or 4 carbon atoms, linear alkyl having 2, 3 or 4 carbon atoms and containing one —CH═CH—, —C≡C— or —CH2—O—CH2— moiety, or R7 is cyclic alkyl having 3, 4 or 5 carbon atoms, wherein one of the linear alkyl carbon atoms is optionally substituted with a single —CH3, —CN, ═O, —OH or protected hydroxyl, provided that the carbon atoms in any —CH═CH— or —CH2—O—CH2— moiety are not substituted with ═O, —OH or protected hydroxyl, and usually R7 is —CH2—, —CH2—CH2—, —CH2—CH2—CH2— or —CH2—CH2—CH2—CH2—;
- R8 is linear alkyl having 1 or 2 carbon atoms wherein one of the linear alkylene carbon atoms is optionally substituted with a single —CH3, —CN, ═O, —OH or protected hydroxyl, or R8 is absent and R6 is linked directly to the ring in R2 structures (42)-(46), usually R8 is —CH2— or —CH2—CH2—;
- R28 is independently —CH2—, —CH(CH3)—, —CH(OCH3)—, —CH(OR5)— or —O—, but both are not —O—;
- R29 is independently —N—, —N(CH3)—, —CH—, —C(CH3)—, but both are not —N(CH3)—;
- R30 is —H or —N(R3)2, usually —H or —NH2;
- R31 is the side chain of an amino acid, usually the side chain of a naturally occurring amino acid, e.g. glycine, alanine, valine, isovaline, leucine, threonine, serine, lysine or arginine;
- R33 is independently —H, —CH3, —CH2CH3 or a protecting group;
- R35 is H, C1-C4 alkyl (including —CH3, —CH2CH3) or a protecting group, usually —H or a protecting group;
R36 is —H, —CH3, —CH2CH3, a protecting group, a monosaccharide, where the monosaccharide is usually linked at the monosaccharide's 1′ position and where any monosaccharide hydroxyl groups are optionally protected, typically an R36 monosaccharide is 2′-deoxyribose, a 2′-deoxy-2′-R21-substituted ribose or arabinose such as 2′-deoxy-2′-fluororibose or 2′-deoxy-2′-fluoroarabinose, or the monosaccharide is ribose or arabinose;
-
- t is 1, 2, 3 or 4, but when R6 is —O—, —S— or —NR5—, t is 2,3 or 4;
- v is independently 0, 1 or 2; and
- w is 1 or 2.
Invention embodiments include R2 moieties having the structure —R59—NH2 where R59 has the structure —R6—R60—, including —R6—(CH2)t—N(R3)2, where R6 is usually —O—, —S—, —NH— or —CH2—, R60 is —CHR51—(CHR51)Z3—(R61)Z1—(CHR51)Z2—CHR51— where R61 is —O—, —S—, C(O), —CHR5 or —NR5— and usually 0, 1 or 2 R51 are methyl or ethyl; R51 independently is —H, methyl or ethyl; Z3 is 1, 2 or 3, usually 1; Z1 is 0 or 1, usually 0; and Z2 is 1, 2 or 3, usually 1. In these embodiments, any functional groups, e.g., —OH, —NH2, —COOH, or —SH, that are optionally present at R21 are usually protected. These embodiments are useful as intermediates useful to make monomers for oligonucleotide synthesis.
Other invention embodiments include R2 moieties having the structure
where R61 is —H, alkyl having 1, 2, 3 or 4 carbon atoms or optionally protected substituted alkyl having 1, 2, 3, 4, 5 or 6 carbon atoms including —CH3 and —CH2CH3, and R62 is —H, —NH2 or —NH(CH3). Other embodiments are —R6—(CH2)2-8—NH2, —R6—(CH2)2-8—OR5 or —R6—(CH2)2-8—CO2R5A.
where R61 is —H, alkyl having 1, 2, 3 or 4 carbon atoms or optionally protected substituted alkyl having 1, 2, 3, 4, 5 or 6 carbon atoms including —CH3 and —CH2CH3, and R62 is —H, —NH2 or —NH(CH3). Other embodiments are —R6—(CH2)2-8—NH2, —R6—(CH2)2-8—OR5 or —R6—(CH2)2-8—CO2R5A.
One uses R1 linker groups to covalently bond the invention base to the selected binding partner, although it will be understood that this need not be the sole use for the linker functionality. Thus, a group present in R1 linkers principally serves as the site for covalently bonding the invention base to a binding partner, typically by incorporating the invention base via the linker residue into a polymeric binding partner by grafting or copolymerization.
R1 linkers also optionally are substituted with groups that ordinarily will not participate in binding to the binding partner, e.g., halo, azido and protected hydroxyl. Generally, such linker groups will contain from 2 to about 50 atoms. If it contains a cycle the cyclic functionality typically will be an oxygen, sulfur or phosphorus-containing saturated or unsaturated heterocycle having a total of about from 5 to 7 ring atoms and 1 to 3 heteroatoms. For the most part, the cycle will be a monosaccharide, typically (i) a hexose, (ii) a hexose such as glucose substituted with phosphate, protected phosphate, hydrogen phosphonate, a phosphoramidate, hydroxyl or protected hydroxyl, (iii) a furanose or (iv) a furanose substituted with phosphate, protected phosphate, hydrogen phosphonate, a phosphoramidate, hydroxyl or protected hydroxyl. Typical furanose sugars include ribose, 2′-deoxyribose, 2′-deoxy-2′-R21-substituted ribose and their 2′ ara isomers. Ordinarily, R1 is an abasic nucleotide residue or such a residue derivatized so as to be capable of incorporation into an oligonucleotide.
Thus, the R1 linker frequently comprises an activated group or other group which can react with a polymer or other binding partner to be labeled with the polycyclic substructure. For example, groups described below that are compatible with commonly available oligonucleotide synthetic chemistries are useful. Other examples of reactant groups for covalent labeling are well-known from the diagnostic fields and have heretofore been used commonly to label proteins and oligonucleotide probes, as is more fully discussed below.
In one embodiment, R1 is a bifunctional or multifunctional organic linker group such as alkyl, alkene, alkyne, alkoxyalkyl, alkylthioalkyl, alkoxy, saturated or unsaturated heterocycle that is substituted with at least one group capable of being crosslinked with or incorporated into a polymer, e.g., such groups as hydroxy, amino, carboxyl, vinyl, phosphate or phosphonate. U.S. Pat. No. 5,502,177 describes suitable linker groups. An example of such an R1 linker suitable for oligonucleotide synthesis is protected monosaccharides, such as ribofuranose and deoxyribofuranose sugars of structure (5)
where an invention base is linked to the open valence at the 1′ position. D is hydroxyl, protected hydroxyl or is an oligonucleotide coupling group and D1 is independently R21 or an oligonucleotide coupling group, but both D1 are not coupling groups.
where an invention base is linked to the open valence at the 1′ position. D is hydroxyl, protected hydroxyl or is an oligonucleotide coupling group and D1 is independently R21 or an oligonucleotide coupling group, but both D1 are not coupling groups.
In embodiments of the invention where the compound of structure (1) is to be used as a monomer in the preparation of oligonucleotides, R1 is typically structure (5) where one D1 is an oligonucleotide coupling group and D is —OH or protected hydroxyl.
“Coupling group” as used herein means any group suitable for generating a phosphodiester linkage or phosphodiester substitute linkage between nucleotide bases or their analogs. These coupling groups are conventional and well-known for the preparation of oligonucleotides, and are prepared and used in the same fashion here. They are usually configured as the b anomers as denoted in structure (5) or optionally as the alpha anomers. In general, each compound comprising structure (5) will contain two coupling groups: D or D1, but with only one D1 being a coupling group. The coupling groups are used as intermediates in the preparation of 3′,5′ 5′,3′, 5′,2′ and 2′,5′ internucleotide linkages in accord with known methods.
Suitable coupling groups for phosphodiester linkages or phosphodiester substitute linkages containing phosphorus include OH, H-phosphonate; (for amidite chemistries) alkylphosphonamidites or phosphoramidites such as β-cyanoethylphosphoramidite, N,N-diisopropylamino-β-cyanoethoxyphosphine, N,N-diisopropylamino-methoxyphosphine, N,N-diethylamino-methoxyphosphine, N,N-diethylamino-β-cyanoethoxyphosphine, N-morpholino-β-cyanoethoxyphosphine, N-morpholino methoxyphosphine, bismorpholino-phosphine, N,N-dimethylamino-β-cyanoethylmercapto-phosphine, N,N-dimethylamino-2,4-dichlorobenzylmercaptophosphine, and bis(N,N-diisopropylamino)-phosphine; and (for triester chemistries) 2-, or 4-chlorophenyl phosphate, 2,4-dichlorophenyl phosphate, or 2,4-dibromophenyl phosphate. See for example U.S. Pat. Nos. 4,725,677; 4,973,679; 4,997,927; 4,415,732; 4,458,066; 5,047,524; 4,959,463; 5,624,621; and International Publication Nos. WO 97/14706 and WO 92/07864.
For structure (2) embodiments, if D1 is a coupling group then D typically will be hydroxyl protected with a group suitable for ensuring that the monomer is added to the oligonucleotide rather than dimerizing. Such groups are well known and include DMT, MMT, FMOC (9-fluorenylmethoxycarbonyl), PAC (phenoxyacetyl), a trialkyl (C1-C6 alkyl, each alkyl group is independently chosen) silyl ether or an alkyl (C1-C6 alkyl) diaryl (e.g., phenyl) silyl ether such as TBDMS (t-butyldiphenylsilyl) and TMS (trimethylsilyl). The opposite will apply when one desires to synthesize an oligonucleotide in the opposite direction (5′→3′). Ordinarily in structure (5) compounds, D is DMT, D1 is located on the 3′ carbon, R21 is H and the D1 and R21 groups are in the alpha anomer conformation.
As noted above, R1 includes an optionally protected monosaccharides of structure (4) and (4A). Usually the monosaccharides in structure (4) and (4A) compounds are 2′-deoxyribose, 2′-deoxy-2′-R21-substituted ribose, 2′-deoxy-2′-R21-substituted arabinose, ribose or arabinose, any of which are optionally protected at sugar all or some hydroxyls or at optionally present R21 functional groups such as —OH —SH or —NH2 groups.
Invention embodiments include compositions of the formula
where B is a structure (3), (4) or (4A) base, R56 is a diene or a dieneophile, both as defined in WO 97/14706, R57 is —OR5, a coupling group including —OH, H-phosphonate, a phosphoramidite or an optionally protected oligonucleotide having a 3′-terminal group selected from a coupling group and —OR5 and any reactive moiety in R21 is optionally protected. R56 dienes are independently chosen and include 2,4-hexadiene and 3,5-hexadiene. One or both R56 are linked to a solid support such as a crosslinked organic polymer, polystyrene, Tentagel™, polyethylene glycol or an inorganic oxide such as silica gel, alumina, controlled pore glass or a zeolite. These compositions are useful for making oligonucleotides containing one or more invention bases.
where B is a structure (3), (4) or (4A) base, R56 is a diene or a dieneophile, both as defined in WO 97/14706, R57 is —OR5, a coupling group including —OH, H-phosphonate, a phosphoramidite or an optionally protected oligonucleotide having a 3′-terminal group selected from a coupling group and —OR5 and any reactive moiety in R21 is optionally protected. R56 dienes are independently chosen and include 2,4-hexadiene and 3,5-hexadiene. One or both R56 are linked to a solid support such as a crosslinked organic polymer, polystyrene, Tentagel™, polyethylene glycol or an inorganic oxide such as silica gel, alumina, controlled pore glass or a zeolite. These compositions are useful for making oligonucleotides containing one or more invention bases.
Invention embodiments include compositions and their isomers of the formula
where R57 is independently —H, a protecting group or both R57 together are a dihydroxy protecting group, R58 is —H or alkyl containing 1, 2, 3 or 4 carbon atoms and B is a structure (3), (4) or (4A) base. Suitable R5 at the nitrogen atom include —H, FMOC and tBOC (t-butyloxycarbonyl) and suitable R5 at the carboxyl group include —H, t-butyl and benzyl, see, e.g., WO 97/14709. These compositions are useful for making oligonucleotides containing one or more invention bases.
where R57 is independently —H, a protecting group or both R57 together are a dihydroxy protecting group, R58 is —H or alkyl containing 1, 2, 3 or 4 carbon atoms and B is a structure (3), (4) or (4A) base. Suitable R5 at the nitrogen atom include —H, FMOC and tBOC (t-butyloxycarbonyl) and suitable R5 at the carboxyl group include —H, t-butyl and benzyl, see, e.g., WO 97/14709. These compositions are useful for making oligonucleotides containing one or more invention bases.
Protecting groups suitable for use with amine groups that may be present at R2 include FMOC and trichloroacetamide. Monomers and polymers may contain such protecting groups at R2.
R1, when functioning as a binding partner, is a substance that non-covalently binds to a target compound. Generally, the target compound is an analyte whose presence is desired to be detected. Binding partners are well-known from the immunoassay art and include hapten-antibody pairs such as those used in drug immunoassays using EMIT or ELISA technologies. Binding partners are employed analytically in enzymology, where the substrate or the enzyme is labeled. Binding partners also are known from the oligonucleotide hybridization art, including oligonucleotide-nucleic acid binding partners (as in diagnostic probes or therapeutic antisense oligonucleotides) or oligonucleotide-protein binding partners (aptamers). In accordance with this invention, an invention base is substituted at R1 by any binding partner. While the binding partner may be a small molecule such as a drug, hapten, substrate or the like, ordinarily it is a polymer.
Compounds of structure (1) wherein R1 is a polymer are an important feature of this invention. For the most part, when R1 is a polymer an R1 linker group has been subsumed into the polymer structure, either as a monomer unit or by grafting onto pre-existing polymer. Therefore, when R1 is a polymer, the polymer may comprise the residue of a linking group derived from a monomer or where the linking group differs from the polymer's monomeric subunits. The invention base must be covalently linked to the polymer.
The nature of the polymer is not critical. Typical R1 polymers include a biopolymer such as an oligonucleotide, a protein (including antibodies, enzymes, cell membrane proteins, glycoproteins, glycolipids, lipoproteins, and nucleoproteins), a peptide, a nucleic acid, or a glycan or other polysaccharide or carbohydrate. In certain embodiments the polymer is an oligonucleotide in which either or both of the sugar or phosphodiester monomer subunits are substituted by groups that continue to permit base pairing by the invention base analogs but which have other desirable characteristics that are not shared with native substituents, e.g., those which mask the negative charges of the phosphodiester linkages or replace the phosphodiester linkage with another group.
The site at which one links the invention base analogs to a polymer is typically not critical. In general, any reactive group on the polymer is satisfactory when one wants to graft the polycycle-R2 substructure onto a pre-existing polymer. Obviously, the site of the substitution should not be in a location in which the polycycle-R2 substructure will interfere with the intended function for the polymer, e.g. enzyme active site, antibody CDR, and the like as will be understood by the artisan. An amino acid side chain such as that of lysine, glutamic acid, serine, asparagine and the like will be satisfactory for grafting to protein R1, as will alpha amino groups, provided that the amino acids in question do not participate in the binding partner or ligand/substrate interaction involved in the assay in which the labeled protein is to be used. One applies the same reasoning to select a binding site or sites on other analytes such as sugars, glycans, lipids, and the like. For example, the 1′ position of ribose or deoxyribose is satisfactory as the site of substitution of an oligonucleotide by the invention base analogs. Suitable sites will be known to the artisan, particularly in those instances where the one intends to substitute an invention base analog for purine or pyrimidine bases, usually for cytosine, or for fluorescent labels.
The degree of polymer substitution by the invention base analogs is not critical. One skilled in the art will choose the reaction conditions such that the resulting labeled polymer will be substituted with sufficient molar proportion of base analog to facilitate its use in the desired analytical, therapeutic or preparative procedure. This is accomplished by preparing the labeled polymers under a variety of conventional conditions, e.g., the time, temperature or duration of the labeling reaction, to yield a matrix of multiply-labeled polymers. These then are screened for suitability in the intended application. Molar ratios of about from 1:1 to 10:1 invention base to polymer generally are suitable. Where the labeled polymer is prepared by monomer incorporation, the resulting polymer may contain about from 1% to 100% invention base analog substitution. In this embodiment each invention base is considered a monomer unit (even though the polymer may have been assembled from intermediate synthons containing 2 or more invention bases per synthon).
Oligonucleotides are polymers containing at least 2 covalently linked nucleotides or nucleotide analogs (collectively monomers), at least one of which comprises an invention base. In oligonucleotide invention embodiments at least one invention base is covalently linked to a nucleotide sugar, and typical invention oligonucleotides will contain about 2-75% of the bases as invention base analogs, usually about 5-25%. Small oligonucleotides, e.g., 2-6 mers, that serve as synthetic intermediates for larger oligonucleotides will optionally contain the higher proportions of invention base analogs, e.g., about 50-75%. Larger oligonucleotides, e.g., about 7-21 mers, will generally contain 1, 2, 3, or 4 invention bases, occasionally 5 and usually not more than about 5 invention bases, unless the oligonucleotide is relatively long, e.g., about 22-50-mer.
Invention embodiments include polymers and oligonucleotides where the invention bases are located on 2, 3 or more adjacent monomers or nucleotide residues, or the invention bases may be located on monomers or nucleotide residues that are separated from each other by about 1, 2, 3, 4, 6, 8, 10, 12, 15, 18 or more monomers or nucleotide residues that do not contain these bases. When a detectable label is linked to an invention base at R2, the oligonucleotide may contain 1, 2 or 3 of these labeled monomers, usually 1 or 2. Such labelled monomers are often located at the 3′ or 5′ terminus, but they may reside at an internal position such as one, two or more monomer residues from either terminus.
Invention oligonucleotides, which contain 1, 2, 3 or more invention bases, will typically have sufficient binding affinity for complementary nucleic acid sequences to allow facile detection of the duplex or triplex resulting from the base sequence-specific binding interaction. Typically, an invention oligonucleotide will have a Tm of at least about 15° C., usually at least about 20° C., when tested under typical in vitro binding conditions, such as those described herein and elsewhere, (Jones, “J Org Chem” [hereafter “JOC”] 58:2983-91 1993, Froehler, “Tet. Lett.” 34:1003-06 1993). Complementary nucleic acid means a natural or synthetic compound that is capable of forming a hydrogen bonded complex in a sequence-specific manner with an invention oligonucleotide such as a structure (2) oligonucleotide. Complementary nucleic acid base sequences contain no mismatches, while “substantially complementary” base sequences contain only a limited number of mismatches, e.g., at most about 1 mismatch per about 15-20 bases, relative to an invention oligonucleotide.
One optionally measures the binding of an invention oligonucleotide to a complementary nucleic acid by detecting or measuring a Tm, by detecting the presence of a label present on the invention oligonucleotide or on the complementary nucleic acid (after separating bound invention oligonucleotide from unbound invention oligonucleotide), by amplifying nucleic acids containing a region(s) complementary to an invention oligonucleotide and so forth. Because of this, invention oligonucleotides optionally include species containing one or more modifications that decrease binding affinity, while the oligonucleotide still retains sufficient binding affinity for a given application. In addition, embodiments include short oligonucleotides or oligonucleotide domains, e.g., having about 2, 3, 4, 5 or 6 linked monomers, where the domain may have low binding affinity, but even in this case are useful as intermediates to make longer oligonucleotides so as to increase affinity sufficiently to confer a Tm of at least about 15° C. Generally, invention oligonucleotide analogs will contain about 40% or less, usually about 25% or less, of monomers that significantly reduce binding affinity, i.e., monomers that decrease the Tm more than about 2° C. per monomer, compared to a corresponding unmodified oligonucleotide.
Invention embodiments include protected, partially protected and deprotected monomers and polymers including oligonucleotides. Partially protected compounds arise during the course of deprotection and they are thus intermediates in the process of preparing deprotected compounds. Typically, one would not recover partially deprotected compounds. Deprotected compounds have been subject to a treatment that removes the protecting group(s), although the preparation may contain some compounds with unremoved protecting groups. Typically, any remaining protecting groups that remain after deprotection are present in small amounts that may be removed by suitable purification methods if desired.
Invention embodiments include oligonucleotides of structure (2) where R37 is oxygen. Invention oligonucleotides, including those where R37 is oxygen, typically contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 linked monomers usually about 5-21. Such oligonucleotides optionally contain about 30-100%, typically about 60-100%, of the linkages as phosphodiester or phosphorothioate linkages, or other linkages of similar binding affinity.
Invention oligonucleotides include support-bound oligonucleotides, which are typically used in solid phase synthesis and separation applications. Support-bound oligonucleotides are typically protected during synthesis, e.g., bases, sugar hydroxyls, linkages and functional groups optionally present are protected as needed, e.g., a sugar hydroxyl group present at R1, an amine group at R2 or a hydroxyl or amine group at R21. In these embodiments, R1 is covalently linked to a solid support or R1 is an oligonucleotide linked to a solid support. When one removes invention oligonucleotides from a support, the protecting groups are generally removed at the same time or shortly thereafter.
Invention embodiments include highly lipophilic polymers and oligonucleotides that comprise (i) one or more structure (3) bases, usually about 1, 2, 3 or 4, and (ii) lipophilic modifications such that the polymer or oligonucleotide has an octanol:water partition coefficient of about −0.5 to about 2.5, typically about 0.0-2.0, usually about 0.2-1.5, and a solubility in water of at least 0.001 μg/mL, usually at least 0.1 μg/mL.
One can use such highly lipophilic polymers and oligonucleotides as reagents to stain, detect or visualize living cells in vitro or in vivo, as described in U.S. Pat. No. 5,633,360 and in Application No. PCT US 96/12530. These highly lipophilic polymers and oligonucleotides need not be binding competent for cell staining, detecting or visualizing applications and they are optionally labeled using standard labels, e.g., radiolabels (32p, 35S, 131I, 14C, 3H), fluorescent labels such as fluorescein, Texas Red, Rhodamine, BODIPY, resorufin or arylsulfonate cyanines and chemiluminescent labels, e.g., acridinium esters.
Embodiments of such optionally labeled highly lipophilic invention oligonucleotides include species where (i) at least about 30%, typically at least about 40%, usually at least about 60% (often at least about 80%), of the internucleotide linkages are non-ionic internucleotide linkages (typically containing a lipophilic moiety at each non-ionic linkage), or (ii) at least about 30%, typically at least about 40% usually at least about 60%, of the bases included in said oligonucleotide contain a lipophilic substitution, (iii) at least about 30%, typically at least about 40% usually at least about 60%, of the sugars, usually at the 2′ position, included in said oligonucleotide contain a lipophilic substitution or (iv) the percent non-ionic nucleotide linkage and the percent lipophilic bases and the percent lipophilic sugars sum to at least about 30%, typically at least about 40% usually at least about 60% (or at least about 80%).
Usually, the invention base analogs and other noninvention bases that are present in binding-competent oligonucleotides are linked together by an organic moiety that is sufficiently flexible to permit the invention base analog(s) to hybridize to complementary bases. The linkage may be a conventional phosphodiester linkage in which a nucleotide analog containing a structure (1) compound, where R1 is deoxyribosyl, ribosyl or an analog thereof, which is incorporated into an oligonucleotide by conventional methods. Alternatively, other groups are used to replace the phosphodiester linkage or, in some instances, both of the phosphodiester linkage and the sugar group. These replacement groups are termed “phosphodiester substitute linkages” for the purposes herein.
Phosphodiester substitute linkages are well-known from the prior literature. They include for example phosphorodithioates (Marshal, “Science” 259:1564, 1993), phosphorothioates and alkylphosphonates (U.S. Pat. No. 5,212,295, Kibler-Herzog, “Nucleic Acids Research” [hereafter “NAR”] 19:2979, 1991; PCT 92/01020; EP 288,163; FIG. 12-1), phosphoroamidates (Froehler, “NAR” 16:4831, 1988), 3′-NH phosphoramidates (Schultz, “NAR” 24:2966, 1996; Gryaznov, “J Am Chem Soc” [hereafter “JACS”] 116:3143, 1994; Chen, “NAR” 22:2661, 1995; Gryaznov, “Proc Natl Acad Sci” USA 92:5798, 1995), phosphotriesters (Marcus-Sekura, “NAR” 15:5749, 1987), boranophosphates (Sood, “JACS” 112:9000, 1991), 3′-O-5′-S-phosphorothioates (Mag, “NAR” 19:1437, 1991), 3-S-5′-O-phosphorothioates (Kyle, Biochemistry 31:3012, 1992), 3′-CH2-5′-O-phosphonates (Heinemann, “NAR” 19:427, 1991), 3′-NH-5′-O-phosphonates (Mag, “Tet. Lett.” 33:7323, 1992), sulfonates and sulfonamides (Reynolds, “JOC” 57:2983, 1992), sulfones (Huie, “JOC” 57:4519, 1992), sulfoxides (Huang, “JOC” 56:3869, 1991), sulfides (Schneider, “Tet Lett.” 30:335, 1989), sulfamates, ketals and formacetals (Matteucci, “JACS” 113:7767, 1991, PCT 92/03385 and PCT 90/06110), 3′-thioformacetals (Jones, “JOC” 58:2983, 1993), 5′-S-thioethers (Kawai, “Nucleosides Nucleotides” 10:1485, 1991), carbonates (Gait, “J Chem Soc Perkin Trans 1” 1389, 1979), carbamates (Stirchak “JOC” 52:4202, 1987), hydroxylamines (Vasseur, “JACS” 114:4006, 1992), methylamine (methylimines) and methyleneoxy (methylimino) (Debart, “Bioorg Med Chem Lett” 2:1479, 1992) and amino (PCT 91/06855). Also of interest are hydrazino and siloxane (U.S. Pat. No. 5,214,134) linkages, thionotriester linkages (WO 96/29337) and related synthesis methods (WO 97/31009).
Phosphodiester substitute linkages per se also are known for the replacement of the entire phosphoribosyl linkage of conventional oligonucleotides. These include for example morpholino-carbamates (Stirchak, “NAR” 17:6129, 1989), peptides (Nielsen et al., “Science” 254:1497, 1991; U.S. Ser. Nos. 07/892,902 and 07/894,397), riboacetal linkages (PCT 92/10793) and morpholino-based linkages disclosed in U.S. Pat. Nos. 5,521,063 and 5,185,144.
Additional disclosure of phosphodiester substitute linkages is found in U.S. Pat. No. 5,386,023, U.S. Pat. No. 5,489,677, WO 95/18623, WO 94/00467, WO 93/08296, WO 92/20822, WO 92/20823, PCT 91/08213, 90/15065, 91/15500, 92/20702, 92/20822, 92/20823, 89/12060 and 91/03680; Mertes, “J Med Chem” 12:154, 1969; Mungall, “JOC” 42:703, 1977; Wang, “Tet Lett” 32:7385, 1991; Stirchak, “NAR” 17:6129, 1989; Hewitt, “Nucleosides and Nucleotides” 11:1661, 1992; Van Aerschot, “Agnew Chem Int Ed Engl” 34:1338, 1995; and U.S. Pat. Nos. 5,034,506 and 5,142,047.
Invention embodiments include oligonucleotides having 1, 2, 3 or more optionally protected invention bases, 0 to about 30 other optionally protected bases, usually guanosine, adenine, thymine, cytosine or 5-methylcytosine, and at least one modified linkage, e.g., 3′—N(R11)—O—5′, where R11 is hydrogen, C1-6 alkyl, usually —CH3 or —C2H5 and the terminal atoms are linked to the 3′ and 5′ carbons of adjacent ribose or 2′-deoxy-2′-R21 substituted ribose sugars,
where R38 independently is O, CH2 or NH; R40 independently is hydrogen, C1-8 alkyl (methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, etc.), a protecting group or both R40 together with the nitrogen atom to which they are attached form
or both R40 together are a protecting group, or R40 is alkyl (C1-C12), usually unbranched or branched once containing 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms, including methyl, ethyl, n-propyl and isopropyl) or R40 is substituted alkyl (C1-C12, usually unbranched or branched once containing 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms, with substituents including one, two or more —O—, —C(O)—, —OC(O)—, —C(O)O—, —OR42, —SR43, —C(O)NR39—, —C(O)N(R41)2, —NR41—, —N(R41)2, halo (e.g., —F, —Cl), —CN, —NO2 moieties); R41 independently is hydrogen, a protecting group (or both R41 together are a protecting group), alkyl (C1-C4 including methyl, ethyl and n-propyl); R42 is hydrogen or a protecting group; R43 is C1-6 alkyl or a protecting group; and R45 is —H, a counter ion or a hydrolyzable moiety such as
R46 is alkyl containing 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms. R40 pairs include ones where one R40 is hydrogen and the other R40 is alkyl containing 1, 2, 3, 4, 5 or 6 carbon atoms or substituted alkyl containing 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms, including methyl, ethyl, methoxyethyl and ethoxyethyl. When R40 is substituted alkyl, it will usually contain 1, 2, 3 or 4 non-carbon atoms, but may contain additional non-carbon atoms, particularly when the non-carbon atoms are halogens or when a group is present as a protecting group, e.g., R39, R40, R41 or R42.
where R38 independently is O, CH2 or NH; R40 independently is hydrogen, C1-8 alkyl (methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, etc.), a protecting group or both R40 together with the nitrogen atom to which they are attached form
or both R40 together are a protecting group, or R40 is alkyl (C1-C12), usually unbranched or branched once containing 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms, including methyl, ethyl, n-propyl and isopropyl) or R40 is substituted alkyl (C1-C12, usually unbranched or branched once containing 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms, with substituents including one, two or more —O—, —C(O)—, —OC(O)—, —C(O)O—, —OR42, —SR43, —C(O)NR39—, —C(O)N(R41)2, —NR41—, —N(R41)2, halo (e.g., —F, —Cl), —CN, —NO2 moieties); R41 independently is hydrogen, a protecting group (or both R41 together are a protecting group), alkyl (C1-C4 including methyl, ethyl and n-propyl); R42 is hydrogen or a protecting group; R43 is C1-6 alkyl or a protecting group; and R45 is —H, a counter ion or a hydrolyzable moiety such as
R46 is alkyl containing 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms. R40 pairs include ones where one R40 is hydrogen and the other R40 is alkyl containing 1, 2, 3, 4, 5 or 6 carbon atoms or substituted alkyl containing 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms, including methyl, ethyl, methoxyethyl and ethoxyethyl. When R40 is substituted alkyl, it will usually contain 1, 2, 3 or 4 non-carbon atoms, but may contain additional non-carbon atoms, particularly when the non-carbon atoms are halogens or when a group is present as a protecting group, e.g., R39, R40, R41 or R42.
Structure (2) and (52) oligonucleotides include species where one or more R21 is —F, —O(CH2)2NHR5, —O(CH2)3NHR5, —O(CH2)4NHR5, —O(CH2)2OCH3, —O(CH2)3OCH3, —O(CH2)2OR5, —O(CH2)2F, —O(CH2)3OR5, or —O(CH2)3F (see, e.g., Griffey “J Med Chem” 39:5100-5109 1996, Schultze “Cell” 24:2966-2973 1996). Such oligonucleotides include oligonucleotides where 1, 2, 3, 4, 5, 6, 7, 8 or more monomers are substituted with R21, which will optionally comprise one of these substituents and the remaining R21 are all hydrogen. Embodiments also include optionally protected monomers containing an optionally protected invention base for synthesis of phosphoramidate-linked oligonucleotides. Oligonucleotides containing one or more of these linkages are optionally prepared as highly lipophilic oligonucleotides and they are suitable for cell staining uses, diagnostic uses and for antisense applications that optionally rely at least in part on an RNase H mechanism.
Invention embodiments include oligonucleotides or monomers described in U.S. Pat. Nos. 5,670,489, 5,667,976, 5,652,355, 5,652,356 and 5,212,295 where one or more optionally protected invention bases is present, usually 1, 2, 3 or 4.
Invention embodiments include oligonucleotides having 1, 2, 3 or more optionally protected invention bases, 0 to about 30 other bases (optionally protected) and at least one amide linkage, e.g., a compound of structure (2B) where n is 0 to about 50, usually about 5-21. Such amide linkages have been described, e.g., (Haaima “Agnew Chem Int Ed Engl” 35:1939-1942 1996; Nielsen “Bioconjugate Chem” 5:3-7 1994). Other amide linkages that are suitable have been described, e.g., WO 92/20702 and WO 93/24507. Embodiments also include optionally protected monomers containing an optionally protected invention base for synthesis of amide-linked oligonucleotides. In general, structure (2B) oligonucleotides will contain only amide linkages, but they may also comprise a domain of monomers linked by non-amide linkages. Suitable D2 and D3 have been described, e.g., WO 86/05518, WO 92/20702, WO 93/24507. D2 optionally comprises a peptide coupling group, a protecting group, or a solid support. D3 optionally comprises —H, a peptide coupling group, a protecting group, or a solid support, but D2 and D3 are not both a peptide coupling group or a solid support. WO 92/20702 described activated derivatives of —CO2H and —SO3H.
The phosphodiester or phosphodiester substitute linkages herein are used to bond the 2′ or 3′ carbon atoms of ribose or ribose analogs to the 5′ carbon atoms of the adjacent ribose or ribose analog. Ordinarily, the linkages in oligonucleotides are used to bond the 3′ atom of the 5′ terminal oligonucleotide to the 5′ carbon atom of the next 3′-adjacent nucleotide or its analog. In general, linkages that contain a phosphorus atom will be 3′,5′ linkages and not 2′,5′ linkages because such linkages usually confer reduced binding affinity on the oligonucleotide in which they are present.
Table 1 of U.S. Pat. No. 5,502,177 describes examples of suitable phosphodiester substitute linkages for use with the invention base analogs. The starting materials in Table 1, or those used to prepare the starting materials of Table 1, generally possess structure (1) in which R1 is ribose, 2′-deoxyribose, a ribose analog or a 2′-deoxyribose analog comprising a 5′ hydroxyl group and a 3′ or 2′ hydroxyl group, prepared as described herein or in the citations, with an invention base analog(s) being substituted for the bases used in the citations. The reactions are repeated or ganged with phosphodiester or other linkages in order to produce trimers, tetramers, pentamers or larger oligonucleotides, including ones up to about 100 bases.
The oligonucleotides of this invention contain naturally occurring nucleotides or derivatives thereof. In some oligonucleotide embodiments the companion nucleotide residues contain pyrimidine nucleotides substituted at the 5 position with a carbon atom which is distally Pi bonded to another atom as for instance 1-alkenyl, 1-alkynyl, heteroaromatic and 1-alkynyl-heteroaromatic groups such as 5-(1-propynyl)-cytosine and -uridine nucleotides (see PCT Publication No. WO 93/10820 and U.S. Pat. No. 5,594,121). Other analogs of native bases for use herein include alkylated purines or pyrimidines, acylated purines or pyrimidines, or other analogs of purine or pyrimidine bases and their aza and deaza analogs. These include, for example N4,N4-ethanocytosine, 7-deazaxanthosine, 7-deazaguanosine, 8-oxo-N6-methyladenine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyl uracil, inosine, N6-isopentenyl-adenine, 1-methyladenine, 2-methylguanine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyl uracil, 5-methoxy aminomethyl-2-thiouracil, 5-methoxyuracil, pseudouracil, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-(1-propynyl)-4-thiouracil, 5-(1-propynyl)-2-thiouracil, 5-(1-propynyl)-2-thiocytosine, 2-thiothymidine, and 2,6-diaminopurine. In addition to these base analogs, one can conveniently incorporate into the invention oligonucleotides other base analogs, including pyrimidine analogs including 6-azacytosine, 6-azathymidine, 5-trifluoromethyluracil or other bases previously described, see, e.g., bases, monomers or oligonucleotides described in WO 92/02258, WO 97/32888 and U.S. Pat. No. 5,614,617.
Preferred bases include adenine, guanine, thymine, uracil, cytosine, 5-methylcytosine, 5-(1-propynyl)uracil, 5-(1-propynyl)cytosine and 5-(1-butynyl)uracil, 5-(1-butynyl)cytosine.
Invention embodiments include the protected derivatives of native bases, their analogs and optionally protected monomer synthons containing such bases, which one would typically use as intermediates to prepare invention oligonucleotides (see, e.g., Internal Publication No. WO 96/37504, U.S. Pat. No. 5,614,622 Iyer et al., “Nucleosides & Nucleotides”, 14:1349-57, 1995, Uhlmann et al., “Chem Revs”, 90:543-587, 1990, S. Agrawal, Ed. Methods in Molecular Biology, Vol. 20, Protocols for Oligonucleotides and Analogs, pp. 165-189, Humana Press, 1993).
Embodiments of the oligonucleotides of the invention comprise a moiety which is capable of effecting at least one covalent bond between the oligonucleotide and a nucleic acid duplex or strand. Multiple covalent bonds can also be formed by providing a multiplicity of such crosslinking moieties. The covalent bond is preferably to a base residue in the target strand, but can also be made with other portions of the target, including the saccharide or phosphodiester. Preferred crosslinking moieties include acylating and alkylating agents, and, in particular, those positioned relative to the sequence specificity-conferring portion so as to permit reaction with the target location in the strand. Exemplary crosslinking moieties are disclosed and claimed in PCT 91/03680. See also Praseuth (“Proc Natl Acad Sci” 8:1349, 1988), Fedorova (“FEBS” 228:273, 1988), Meyer (“JACS” 111:8517, 1989), Lee (“Biochemistry” 27:3197, 1988), Home (“JACS” 112:2435, 1990), Shaw (“JACS” 113:7765, 1991).
Invention embodiments include monomers and oligonucleotides containing 1, 2, 3 or more invention bases and a 5′ hydroxyl group protected with a base labile protecting group, including a dansylethoxycarbonyl group, which has been described, e.g., U.S. Pat. No. 5,631,362. Such embodiments optionally include additional protecting groups.
Invention embodiments include monomers and oligonucleotides containing 1, 2, 3 or more invention bases and an invention base or a non-invention base having an exocyclic nitrogen atom, where the nitrogen atom is protected with a protecting group as previously described, e.g., specification and claims 1, 2, 3, 4, 5, 6, 7 and 8 of U.S. Pat. No. 5,623,068. Such embodiments optionally include additional protecting groups.
Invention embodiments include optionally protected monomers and optionally protected oligonucleotides containing 1, 2, 3 or more invention bases wherein the compositions possess N-branching, which has been described, e.g., U.S. Pat. No. 5,623,049.
Invention embodiments include “hybrid” oligonucleotides, which contain 1, 2, 3 or more invention bases and 2′ modifications in one or two regions or domains that comprise adjacent linked monomers, typically about 2-8 linked monomers, usually about 2-3. One domain contains 2′ modifications, while hydrogen is linked to the remaining monomers, typically about 4-10 adjacent linked monomers, usually about 6-8. Such oligonucleotides contain at least one domain that is competent to serve as a RNase H substrate and comprises hydrogen at each 2′ position and phosphodiester, phosphorothioate or phosphorodithioate 3′,5′ linkages. The other domain(s) contain a 2′ modification(s), such as —O—(CH2)2F or —O—(CH2)2—O—CH3, that enhances binding affinity or nuclease stability. The 2′-modified domain(s) is usually not an efficient RNase H substrate. The bases in hybrid oligonucleotides are the typical purines and pyrimidines found in nucleic acids (G, A, T, C or U) or their analogs, which one finds in some oligonucleotide analogs (e.g., U.S. Pat. Nos. 5,484,908, 5,594,121 and 5,502,177, International Publication No. WO 93/10820). Intermediates used to prepare hybrid oligonucleotides will typically contain appropriately protected derivatives of any bases.
Oligonucleotides of inverted polarity also fall within the scope of this invention. “Inverted polarity” means that the oligonucleotide contains tandem sequences which have opposite polarity, i.e., one having polarity 5′→3′ followed by another with polarity 3′→5′, or vice versa. These sequences thus are joined by linkages which can be thought of as effectively a 3′—3′ internucleoside junction (however the linkage is accomplished), or effectively a 5′—5′ internucleoside junction. For a further description of suitable methods for making such oligonucleotides see, e.g., WO 93/10820. Compositions of “parallel-stranded DNA” designed to form hairpins secured with AT linkages using either a 3′-3′ inversion or a 5′-5′ inversion have been synthesized by Van de Sande, “Science” 241:551, 1988. In addition, oligonucleotides which contain 3′-3′ linkages have been described (Horne, op cit; and Froehler, “Biochemistry” 31:1603, 1992). These oligonucleotides are useful as binding partners for double stranded nucleic acids to form triple helix (or triplex) complexes as a means for detecting complementary sequences and inhibiting of target gene expression (PCT 89/05769 and 91/09321).
Invention embodiments include polymers such as oligonucleotides containing 1, 2, 3 or more invention bases, where the polymer is a component of a complex or composition useful for transfecting the polymer into a cell in vitro or in vivo. These complexes or compositions are referred to herein as “transfection complexes”. Such transfection complexes optionally comprise one or more lipids, e.g., cationic or anionic lipids, as well as other lipophilic compounds such as cholesterol or colipids such as DOPE. The complexes optionally comprise an uncharged or a charged polymer such as polyethylene glycol, polybrene or a peptide, e.g., polylysine. The complexes optionally comprise unilamellar or multilamellar liposomes or vesicles.
As used herein, any compound(s), reagent(s) or treatment that enhances delivery of an invention oligonucleotide into a cell or tissue is a “permeation enhancing agent.” Permeation enhancing agents are well known and are usually present as transfection complexes containing oligonucleotides, e.g., unilamellar or multilamellar liposomes or vesicles. One uses permeation enhancing agents to prepare transfection complexes containing invention oligonucleotides. The permeation enhancing agent are used in essentially the same manner as is used to prepared transfection complexes containing nucleic acids, non-invention oligonucleotides or polymers into cells or tissues.
Invention transfection complexes optionally comprise an additional non-invention polymer(s), e.g., a nucleic acid expression vector(s), a therapeutic agent(s) (e.g., amphotericin B) or a peptide(s).
Invention transfection complexes comprising a lipid may be, as defined herein, “large”, i.e., complexes having a maximum average dimension of at least about 200 nm in length or diameter, typically having an average length or diameter of about 200-400 nm, occasionally having an average length or diameter of about 400-800 nm. Transfection complexes comprising a lipid may be “small”, i.e., complexes having a maximum average dimension of about 15-200 nm in length or diameter, e.g., an average dimension of about 60-120 nm. Transfection complexes may comprise a mixture of large and small complexes in about equal proportions or they may comprise a preponderance of small or large transfection complexes, e.g., at least about 55%, or at least about 60-80% of the complexes in a given preparation are large or small.
Transfection complexes comprising a lipid optionally include a stabilizing compound(s), e.g., a monosaccharide or a disaccharide such as glucose, trehalose, maltose or sucrose, that is present at the outer surface or at the inner surface or at both surfaces of transfection complexes. Workers have described suitable compounds such as lipids, colipids and stabilizing compounds for making transfection complexes, methods to size the complexes and methods to use the complexes to deliver a polymer or monomer into the cytoplasm of a cell in vitro or in vivo, e.g., U.S. Pat. Nos. 5,635,491, 5,633,156, 5,631,018, 5,629,184, 5,627,159, 5,626,867, 5,620,589, 5,595,756, 5,543,152, 5,478,860, 5,459,127, 5,264,618, 5,223,263, 5,194,654, 4,981,692, 5,077,056, 4,522,803, 4,588,578, 4,885,172, 4,975,282, 5,059,421, 5,000,958, 5,030,453 and 5,047,245, WO 96/01840, WO 96/01841, WO 97/30969, WO 97/30732, Lewis, “Proc Natl Acad Sci” 93:3176-3181, 1996, U.S. patent application Ser. No. 08/672,206.
Invention transfection complexes useful for delivering the invention oligonucleotides into cell cytoplasm also include complexes comprising inorganic compounds, e.g., calcium phosphate.
The R21 moiety is linked to invention oligonucleotides or monomers useful for oligonucleotide synthesis. R21 is usually linked to the 2′ or the 3′ position of furanose sugars. When R21 is a nuclease stability enhancing moiety, a broad range of structures may be used to increase stability of oligodeoxynucleotides or oligoribonucleotides containing phosphodiester linkages. Oligonucleotides having moieties other than hydrogen or hydroxyl at the 2′ position usually confer increased nuclease stability or increased binding affinity on the oligonucleotide relative to hydrogen or hydroxyl. Enhanced nuclease stability is conveniently measured using dimers or short oligonucleotides as essentially described, e.g., WO 92/05186. One or two R21 moieties at the 3′ and 5′ terminal monomers in an oligonucleotide will increase stability of the oligonucleotide to 3′- and 5′-exonucleases. One may increase an oligonucleotide's stability to endonucleases by incorporating R23 moieties that increase nuclease stability at internal monomer positions.
In addition to increasing nuclease stability, some R21 moieties enhance binding affinity of the oligonucleotide to which they are linked. These moieties include fluorine and short unbranched optionally substituted O-alkyl groups containing about 2-8 carbon atoms, where the alkyl group is optionally substituted at the distal carbon atom with, e.g. —F, —OH or —NH2, and optionally substituted with an ether at an internal carbon, e.g., —O—(CH2)2—O—(CH2)2F, —O—(CH2)2OCH3 or —O—(CH2)2—O—(CH2)2CH3.
When R21 is a nuclease stability enhancing moiety, it will typically comprise —CH3, ═O—NHR5 or a chain having a backbone containing 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 linked atoms, wherein the chain usually comprises carbon (C) atoms and optionally 1, 2, 3 or 4 atoms independently selected from the group consisting of oxygen (O), nitrogen (N) and sulfur (S) atoms. The chain is usually linked to the sugar carbon atom through —O—, —S—, —S(O)—, S(O)(O)—, —CH2—, ═CH— or —NH—. The chain is branched or unbranched, often it is unbranched or has only limited branching, e.g., —CH3, —CH2OH, —C2H5 or —C2H4OH. The R21 chain may comprise a C2-12 alkyl group or a C2-20 substituted alkyl group. If R21 is a substituted alkyl group, usually only 1, 2 or 3 carbon atoms are substituted. Suitable substituents include those described above for substituted alkyl groups, e.g., halogen (usually fluorine), —O— or —OR5.
Invention embodiments include oligonucleotides and monomers where one or more monomers comprise 2′-deoxyribose, ribose or arabinose sugars, or their carbocyclic analogs, having one or more 2′ R21 modification such as, —O—alkyl, —NH—alkyl, —S—alkyl, —OR5, —NHR5, —SR5, -halo (usually —F), —R44-alkyl or —R44-substituted alkyl wherein the alkyl or substituted alkyl group usually comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms, usually about 2-6 carbon atoms, where R44 is independently —O—, —S—, —NH— or —CH2—, usually —O—. The oligonucleotide linkages connecting such monomers are 3′,5′ linkages. The alkyl groups at the 2′ position typically have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms which are optionally present as methylene groups (—CH2) and optionally have 1, 2, 3 or 4 ether (—O—) or other substitutions, e.g., O-alkoxyalkyl (C2-C12 alkyl), —O—(CH2)2-8—CH2CO2H, —O—(CH2)2-8—CH2N(R5)2, —(—O—(CH2)2-4—O—(CH2)2-4—O—(CH2)2-4—R65), —O(CH2CH2O)rCH2—R65, —O—CH2CH2—R65, —O(CH2CH2)O(CH2CH2)R65, —OCH2CF2CF3, when R65 is —H, halo (usually fluorine), —OR5, —OCH3, —NNR5, —SR5, and r is 1, 2, 3 or 4, usually 1 or 2.
The alkyl groups at the 2′ position also include substituted alkyl, e.g., —O— alkylamino, —S-alkylamino , —NH-alkylamino, or their protected derivatives, wherein the alkyl group contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms, which are all optionally present as methylene carbons (—CH2—). Usually the alkyl group or substituted alkyl group at R2′ will contain 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms. Such groups include —O—methyl, —O—ethyl, —O—n-propyl, —O-ally, —O—(CH2)2-6OH including —O—(CH2)2OH, —O—(CH2)2F, —O—CH2CHF2, —O—CH2CF3, —O—(CH2)2-6OCH3, including —O—(CH2)2OCH3, —O—(CH2)2OCH2CH3, —O—(CH2)2OCH2CH2OH, —O—(CH2)2OCH2CH2F, —O—(CH2)2NHR5, —O—(CH2)3NHR5, —O—(CH2)4NHR5, —O—(CH2)2F, —O—(CH2)3F, —O—(CH2)4F, —O—CH2—CF2CF3, —O—(CH2)sR65, —O—(CH2)2—[O—(CH2)2]rR65, —NH-methyl, —NH-ethyl, —NH-n-propyl, —NH—(CH2)2OH, —NH—(CH2)3OH, —NH—(CH2)2F, —NH—CH2—CF2CF3, —NH—(CH2)5R65, —S-methyl, —S-ethyl, —S-n-propyl, —S-allyl, —S—(CH2)sOH, —S—(CH2)sOH —S—(CH2)2F and —S—(CH2)s—[O—2)2]rR65, where s is 2, 3, 4, 5, 6, 7 or 8. R21 moieties do not include unstable species at the 2′ position, e.g., —O—O— or —S—O—.
Other suitable R21 at the 2′ or 3′ position of optionally protected invention monomers or optionally protected invention oligonucleotides include —2H, —3H, —NHOR55, ═NH, —N3, —CN, —CH2CN, —CHCl2, —CFH2, —CT2H, ═CH2, ═CF2, —CH2CH═CH2, ═O, ═CHC(O))OR55 (including ═CHC(O)OCH3 and ═CHC(O)OCH2CH3), —OC(S)OC6H5, 1-butyldimethylsilyl ether, triisopropylsilyl ether, a 2′ amino group protected by an N-phthaloyl protecting group or a fluorescent label, R55 is independently R5, alkyl having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms of substituted alkyl having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms where the carbon atoms in R55 are optionally all present as methylene (—CH2—) or substituted methylene (—CH(substitution)-) moieties. Workers have described suitable 2′ modified monomers and oligonucleotides e.g., WO 97/14706, WO 96/05298, WO 93/13121, and WO 91/06556 and U.S. Pat. Nos. 5,631,360, 5,627,053, 5,623,065, 5,576,302 and 5,578,718.
The compounds of structure (1) where R1 is a linker or H an prepared by methods known in the art per se and as more fully described below. Typically, such compounds are prepared from a 5-bromouracil, 5-bromouridin-1-yl, 5-iodouracil, 5-iodouridin-1-yl substituted derivative as shown in the synthetic schemes below and subsequent reactions close the polycyclic ring, In these embodiments the hydroxyl, amino and any other labile groups of R1 are protected as required. )n another approach, R1 of the starting material is H or a protecting group and one adds the linker after the ring closure steps set forth in the schemes, in the same fashion as has heretofore been employed in the alkylation of pyrimidine bases intended for use as antiviral compounds.
In those embodiments in which R1 is a binding partner such as a polymer the compounds of this invention are synthesized by covalently crosslinking the linker modified polycyclic base of this invention to the binding partner, or (where the binding partner is a polymer) by incorporating into the polymer a monomer unit which is substituted by an invention polycyclic base.
In the first embodiment (polymer grafting) a R1-substituted polycyclic substructure is covalently bonded via any conventional cross-linking agent to the polymer. Most conveniently, structure (1) compounds in which R1 is hydroxyl- or amino-substituted alkyl are readily cross-linked to reactive groups present in the molecule to be labeled as noted above. Exemplary cross-linking agents include succinic anhydride, N-hydroxysuccinimide esters (biotin NHS ester), epoxides, isothiocyanates, imidates, DCC (dicclohexylcarbodiimide), EDC (1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide), BOP, and glutaraldehyde, see, e.g., EP 0 063 879, Ruth “J Org Chem” 43:2870, 1978, Bergstrom, JACS 100:8106, 1978, Bigge, JACS 102:2033,1980. Cyanogen bromide activated carbohydrates also are used. The cross-linking agents are used to bond the R2-substituted polycycle to the polymer in the same fashion as polymers heretofore have been cross-linked to ligands, e.g., to hydroxyl or amino-bearing moieties. An example of a suitable method is described per se in Cook et al., U.S. Pat. No. 5,218,105. This method is readily applied to covalently bond an amino-substituted R1 linker to the 5′ terminus of an oligonucleotide.
When R1 or R2 are amino substituted, the following exemplary synthetic approaches are suitable for crosslinking amines with other moieties:
-
- —CH2NH+R10—C(N+H2)—OR10→—CH2NHC(N+H2)—R10
- —CH2NH2+R10—N═C—S→—CH2NHC(S)NH—R10
- —CH2NH2+R10-epoxide→—CH2NHCH2CH(OH)—R10
where R10 is an organic moiety optionally containing a hapten or other detectable moiety such as biotin or avadin.
In the second embodiment (copolymerization) the R1 linker is capable of functioning as a monomer for copolymerization with other monomer units that may or may not be substituted with the polycyclic substructure of structure (1). In some embodiments, the R1 linker is an alkyl carboxylate, an alkyl amine or an amino acid for incorporation into peptides by in vitro methods. However, in the typical embodiment the R1 polymeric binding partner is an oligonucleotide as depicted in structure (2), and these conveniently are made by copolymerization with a nucleotide analog substituted with the polycyclic substructure. The starting materials for the synthesis of structure (2) generally are compounds of structure (1) in which R1 is ribose or deoxyribose substituted with appropriate protecting and coupling groups further described above. Suitable starting monomers for oligonucleotides having phosphodiester substitute linkages are set forth in Table 1, and they an prepared in the same fashion as other nucleotide analog bases described in the literature. Similarly, conventional phosphodiester or phosphorothioate linkages are prepared from nucleotide analogs containing coupling groups D and D1 described above. The compounds of this invention then are incorporated into the desired oligonucleotide by known methods of in vitro synthesis described in the referenced methods. Alternatively polycylic substructure-substituted nucleotide triphosphates may be incorporated iota oligonucleotides as cytosine analogs by DNA polymerase or reverse transcriptase in vivo or in vitro (see Ward, U.S. Pat. No. 4,711,955). In this R1 is ribosyl or deoxribosyl triphosphate, or a triphosphorylated analog thereof recognized by DNA polymerase or reverse transcriptase which is then incorporated into an oligonucleotide by template-directed transcription.
Synthesis of oligonucleotides containing 3 or more nucleotide residues is optionally accomplished using synthons such as dimers (which contain substitute or diester linkages) or trimers, each carrying a terminal coupling group suitable for use with amidite, H-phosphonate or triester chemistries. The synthon is then linked to the oligonucleotide or another synthon via a phosphodiester or phosphorous-containing phosphodiester substitute linkage.
Oligonucleotides containing phosphorothioate, methylphosphonate and phosphodiester linkages are readily prepared by solid-phase oligonucleotide synthesis techniques. A description of modifications useful in the synthesis of phosphorothioate linked oligonucleotides are found, for example, in EP 288,163, wherein the oxidation step in solid phase automated synthesis using amidite chemistry can be independently adjusted at any step to obtain the phosphorothioate. An alternate method for synthesis of oligonucleotides with phosphorothioate linkages, via hydrogen phosphonate chemistry, has also been described (Froehler “NAR” 14:5399, 1986). Sulfurization is accomplished using reagents such as tetraethylthiuram disulfide, dibenzoyl tetrasulfide, thiophosphoric acid disulfide, 3H-1,2-benzodithiole-3-one 1,1-dioxide and the like as described (Vu, “Tel Lett” 26:3005, 1991; Rao, “Tel Lett” 33:4539, 1992; U.S. Pat. No. 5,515,510; Iyer, “JOC” 55:4693, 1990; Dahl, “Sulfur Reports” 11:167, 1991). These sulfurization reagents are used with either phosphoramidite or hydrogen-phosphonate chemistries. Synthesis of phosphorothioate oligonucleotides having controlled stereochemistry is used to generate stereoregular invention oligonucleotides as described (EP 506,242). Thionomethyl phosphonate is prepared with methylphosphonamidite followed by sulfurization as described (Roelen, “Tel Lett” 33:2357,1992) or, with the sulfurization reagents described above.
Scheme A depicts preparation of structure (1) compounds where R47 is —O— or —S—; and R24—OH is R2 which has a free hydroxyl.
Step one is conducted by bearing the reaction mixture containing (100) in an organic solvent to at least about 50° C., generally for about 3-4 hours. Step two is performed by reacting (102) in an organic solvent for about 6-48 hours, generally for about 10-20 hours at about 15° C. to reflex temperature, generally at about 18-25° C. The R2 moiety is linked under Mitsunobu conditions to (103) in step 3 by reacting about 1-1.5 equivalents of the alcohol, i.e., R24—OH, using an activating agent as a leaving group, such as triphenylphosphine (Ph3P) and diethyl diazocarboxylate (DEAD) to obtain (104). In step 4, (105) is prepared by forming the ring containing R47 by (1) incubating (104) in a polar organic solvent, typically an alkanol containing 1, 2, 3, 4, 5 or 6 carbon atoms, such as methanol or ethanol, containing a mild base such as NH3, TEA (triethylamine), DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) or (2) refluxing in ethanol in the presence of potassium fluoride. Generally (104) is incubated in saturated NH3 in methanol for about 2-3 days to afford (105).
The use of (102) in which one R47 is —O— and the other is —S— will produce a mixture. One optionally isolates each (104) component or one optionally converts the (104) mixture to a (105), Mixture. One optionally separates the mixtures at any convenient point by standard methods, e.g., silica gel chromatography, or HPLC.
When one prepares compounds according to scheme A and R1 is a monosaccharide, e.g., 2′-deoxyribose, 2′-deoxy-2′-R21-substituted ribose or arabinose, the sugar hydroxyls in (100) are usually protected, generally using base-labile protecting groups, e.g., acetate, proprionate, butyrate, phenoxyacetyl. The step 4 reaction under basic conditions removes the protecting groups, which facilitates the ring formation reaction resulting in (105). When R3 is not a monosaccharide, it is generally a protecting group or an optionally protected linker, e.g., —(R48)X—R49, where R48 is independently —CH2—, —O—, —S—, —NH— or —C(O)—, X is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and R49 usually is a functional group suitable for linking to a solid support, monomer or polymer, including —OR5, —SR5, —N(R5)2, —C(O)N(R5)2, —NR5C(O)H. —C(O)H and —C(O)OR5, R5 is independently —H, a protecting group, or both R5 together are a protecting group. Generally the R48 group that is adjacent to R49 is ethylene (—CH2—CH2—) and generally only 0, 1 or 2 R48 are moieties other than —CH2—, e.g., one R48 is —O—or —C(O)— and the remaining R47 are all —CH2—. Exemplary compounds where R1 is a monosaccharide have the structures (130)-(134), which correspond to compounds (100)-(101) and (103)-(105), respectively. For compounds (130)-(132). R5 at sugar hydroxyls is typically a base labile protecting group such as acetyl and R37 is typically —O—. During conversion of (133) to (234), the R5 protecting group is removed from the sugar under ring closure reaction conditions, which facilitates ring closure.
Structure (134) compounds may be converted to monomers suitable for oligonucleotide synthesis. Such monomers typically have a coupling group at the 3′ position, e.g., H-phosphonate, or a phosphoramidite such as a β-cyanoethylphosphoramidite, N,N-diisopropylamino-β-cyanoethoxyphosphine or N,N-diisopropylaminomethoxyphosphine. The 5′ position will contain a DMT-O— or other protecting group suitable for oligonucleotide synthesis. The monomers may alternatively have a coupling group et the 5′ position and a protecting group at the 3′ position. The protecting and coupling groups are added sequentially. Scheme B shows synthesis of structure (1) compounds where R6 in R2 is —CH2—. In scheme B, Y is 1, 2, 3 or 4; R50 is independently —CH2—, —C(O)—, —(CH2)2—O—(CH2)2—(CH2)2—NR5—(CH2)2—, —(CH2)2—S—(CH2)2—, —CH(N(R5)2)—, —CH(COOR5)— or —C(CH3)—, —C(C2H5)— but adjacent moieties are not C(O), usually R50 is —CH2—; TFA is trifluoroacetate; and CBZ is carboxybenzoyl; and (96) is HC═C(R50)y—NH-TFA. Protecting groups present in R50 are stable to the reaction conditions shown.
Compound (97) is converted to (98) by hydrogenation reaction is alcohol, usually methanol or ethanol, at about 15-25° C. for about 10-24 hours, usually about 12-18 hours. The catalyst is removed and the filtrate is concentrated.
Compound (98) in trimethyl orthoformate is converted to (99) in the presence of acid, e.g., methanesulfonic acid at about 15-25° C., usually about 18-22° C., for about 20-120 minutes. The reaction is cooled and quenched with a base, e.g., an organic base such as TFA The reaction mixture is concentrated and purified, e.g., by flash column chromatography on silica gel.
Compound (106) is prepared by reacting (99) in organic solution such as DMF, CH2Cl2 or CH2Cl2/DMF (about 2:1 v/v) with K2CO3 at about 15-25° C. for about 30 minutes, followed by adding phenyltrifluoromethanesufonimide and stirring the mixture for about 10-24 hours, usually about 12-16 hours. The reaction mixture is then diluted with CH2Cl2, washed with water owe or twice and concentrated and purified.
Compound (96) is prepared by reacting HC═—(R50)YNH2 with ethyl trifluoroacetate at about 15-25° C. for about 10-24 hours, washing with saturated aqueous NaHCO3 and concentrated. Compound (96) is purified by distillation.
Compound (107) is prepared by stirring an organic solvent such as DMF containing about 2 equivalents of (96), about 2 equivalents of an organic base such as TEA, and Pd((PPh)3)4, Cul and about 1 equivalent of (106) at about 15-25° C. for about 18-36 hours. The organic phase is washed with water, dried and purified by silica gel chromatography, to obtain (107).
Compound (107) is hydrogenated in ethanol in the presence of 10% Pd/C at room temperature. The catalyst is filtered off, the filtrate is concentrated and then treated with cone. NH4OH:dioxane (1:1) to afford (108). The amino group in (108) was protected with CBZ to afford (109).
Compound (110) is prepared by treating (109) in ethanol with aqueous HCl (about 3 N) at about 15-45° C., usually about 40° C., for about 30-120 minutes, usually about 60 minutes. The product is dried and optionally azeotroped using e.g., CH3CN, several times.
Compound (111) is prepared by reaction of (100) with mesitylenesulfonyl chloride in the presence of a tertiary amine such as TEA.
Compound (112) is prepared by stirring a mixture of (110) and (111) in organic solution containing about 2 equivalents of an organic base such as DBU or TEA at about 15-25° C. for about 10-24 hours. The reaction mixture is washed with an aqueous 10% citric acid solution, dried and purified by silica gel chromatography.
Compound (113) is prepared by treating (112) with saturated NH3 in methanol at about 15-25° C. for about 3-4 days. The reaction mixture is dried, concentrated and purified by silica gel chromatography.
Compound (114) is prepared by hydrogenation of (113) in the presence of 10% Pd/C at about 15-25° C. for about 3-6 hours. Catalyst is removed, washed, and the filtrate is concentrated to dryness. The amino group in (114) is protected with FMOC to afford (115).
Where R1 in scheme B is an optionally protected monosaccharide such as 2′-deoxyribose, 2′-deoxy-2′—R21-substituted ribose, 2′-deoxy-2′-R21-substituted arabinose, ribose or arabinose, the sugar's hydroxyl groups in compounds (111) and (112) are usually protected with a base-labile protecting group such as acetyl, propionyl and phenoxyacetyl. These protecting groups are removed by treatment with base during synthesis of (113).
Exemplary compounds where R1 is a monosaccharide have the structures (135)-(139), which correspond to compounds (111)-(115) respectively. For compounds (135)-(136), R5 at sugar hydroxyls is typically a base labile protecting group such as acetyl. R37 is typically —O—. During conversion of (136) to (137), the R5 protecting group is removed from the sugar under ring closure reaction conditions, which facilitates ring closure.
Structure (139) compounds may be converted to monomers suitable for oligonucleotide synthesis. Such monomers typically have a coupling group at the 3′ position, e.g., H-phosphonate, or a phosphoramidite such as a β-cyanoethylphosphoramidite, N,N-diisopropyl-amino-β-cyanoethoxyphosphine or N,N-diisopropylaminomethoxyphosphine. The 5′ position will contain a DMT-O- or other protecting group suitable for oligonucleotide synthesis. The monomers may alternatively have a coupling group at the 5′ position and a protecting group at the 3′ position. The protecting and coupling groups are added sequentially.
In scheme C, R52 is —(CHR52A)—(R52B)—CHR52A—, where R52A is —H or C1-C6 alkyl (typically C1-C3), usually —H, and R52B is a bond, —CHR52A—O—CHR52A—, —CHR52A—S—CHR2A—, —CHR52A—NR5—CHR52A—, C1-C10 alkylene (typically C3-C6) optionally substituted with 1 or 2 moieties selected from the group consisting of C1-C6 alkyl (typically C1-C3 alkyl), —OR5, ═O, —NO2, —N3, —CN, —COOR5, or —N(R5)2. In R52, any heteroatom in the spacer chain will be separated from the nitrogen atoms that R52 is linked to by one methylene and one or more —CHR52A—moieties. Typically, adjacent carbon atoms in R52B are not substituted with —OR5, ═O, —NO2, —N3, —CH, —COOR5, or —N(R5)2. Protecting groups present on (119A) are usually stable to oxidizing conditions and labile to basic conditions. The protected intermediate (119A) is prepared by reacting phthalic anhydride with the appropriate HOCH—R52—NH2 moiety to yield a phthalimide compound or by reacting a phthalimide compound with HOCH-R52—Br.
Compound (117) is prepared by reacting (101) or (111) with about 1.0-1.5 equivalents, usually about 1.1 equivalents of (116) and about 1-2 equivalents, usually about 2 equivalents of organic base such as DBU or TEA in an organic solvent such as CH2Cl2/CCl4 (about 1:1) or CH2Cl2 by reaction at about 10-30° C., usually at about 18-25° C. for about 16-48 hours, usually about 20-28 bouts.
Where R1 in scheme C is an optionally protected monosaccharide such as 2′-deoxyribose, 2′-deoxy-2′-R23-substituted ribose, 2′deoxy-2′-R21 ′-substituted arabinose, ribose or arabinose, the sugar's hydroxyl groups in compounds (101), (111) and (117) are usually protected with a base-labile protecting group such as acetyl, propionyl and phenoxyacetyl. These protecting groups an removed by treatment with base during synthesis of (118). Compound (118) was hydrogenated in ethanol and acid to obtain (119). Compound (120) was obtained by reductive alkylation of (119) with aldehydes.
Compound (120) is optionally converted, without removing the nitrogen protecting group linked to the exocyclic amine, to a monomer suitable for use in oligonucleotide synthesis, e.g., (121) (not shown), by protecting the 5′ hydroxyl group by reaction with a protecting group reagent such as DMT-Cl (4,4′-dimethoxytrityl chloride) to obtain the 5′-protected derivative (not shown). Compound (121) is then converted to a derivative (121A) (not shown) suitable for coupling the 3′ hydroxyl group with another 5′ hydroxyl group, i.e., a coupling group such as H-phosphonate or a phosphoramidite, e.g., N,N-diisopropylamino-β-cyanoethoxyphosphine or N,N-diisopropylaminomethoxyphosphine, is linked to the 3′ position. Exemplary compounds where R1 is a monosaccharide have the structures (140)-(143), which correspond to compounds (117)-(120) respectively For compounds (140)-(141), R5 at sugar hydroxyls is typically a base labile protecting group such as acetyl and R37 is typically —O—. In structure (143) compounds, the 5′ or 3′ oxygen, usually the 3′ oxygen, is linked to —H or a coupling group such as H-phosphonate, or a phosphoramidite such as β-cyanoethylphosphoramidite, N,N-diisopropylamino-β-cyanoethoxyphosphine or N, N-diisopropylaminomethoxyphosphine. During conversion of compound (140) to (141), the R5 protecting group is removed from the sugar under ring closure reaction conditions, which facilitates ring closure.
Structure (143) compounds may be converted to monomers suitable for oligonucleotide synthesis. Such monomers typically have a coupling group at the 3′ position, e.g., H-phosphonate, or a phosphoramidite such as a β-cyanoethylphosphoramidite, N,N-diisopropylamino-β-cyanoethoxyphosphine or N,N-diisopropylaminomethoxyphosphine. The 5′ position will contain a DMT, MMT or other protecting group suitable for oligonucleotide synthesis. The monomers may alternatively have a coupling group at the 5′ position and a protecting group at the 3′ position. The protecting and coupling groups are added sequentially During oligonucleotide synthesis, (143) is incorporated into an oligonucleotide such, as one of structure (2), by standard methods and the phthalamide protecting group, along with other base labile protecting groups at R52 are removed using basic conditions, e.g., NH4OH or NH2CH3, to yield deprotected or partially deprotected —NH—R52—NH2 as R2.
Straightforward variations of schemes A-C can be used to prepare other structure (1) compounds. For example, scheme D depicts a method to prepare structure (1) compounds where R2 comprises a cytosine derivative. In scheme D, iPr is isopropyl; R59 is a portion of an R2 moiety having the structure —R6—R60—, where R6 is usually —Q—, —S—, —NH— or —CH2—; R60 is —(CH2)Z3—(R61)Z1—(CH2)22—; R61 is —O—, —S—, —NR5—, —C(O)—, —CH2—O—CH2—, —CH2—NR5—CH2— or —CH2—S—CH2—; Z3 is 1, 2 or 3, usually 1; Z1 is 0 or 1, usually 0; and Z2 is 1, 2 or 3, usually 1; any functional groups, e.g., —OH, —NH2, —COOH, —SH, that are optionally present at R21 are protected. Compound(124) is 3′,5′-diacetyl-O4-sulfonyl-2′-deoxyuridine, the synthesis of which is described in the examples.
Compound (126) of scheme D is converted to a monomer suitable for oligonucleotide synthesis using standard methods, e.g., treatment of (126) with a protecting group such as DMT C1 protects the 5′ oxygen atom and yields compound (127) (not shown). One then links a coupling group such as H-phosphonate or a phosphoramidite group such as N,N-diisopropylamino-β-cyanoethoxyphosphine or N,N-diisopropylaminomethoxyphosphine to the 3′ hydroxyl group, of (127) to obtain (128) (not shown), which is suitable for oligonucleotide synthesis. Variation of the synthesis shown in scheme D is used to prepare compounds of structure (129) or (129A).
Similarly, structure (1) compounds where R2 is
are synthesized using variations of scheme D and the corresponding intermediates, e.g., reaction of (123) with 2-halopyridine (Bernatowicz “JOC” 57:2497 1992).
Similarly, structure (1) compounds where R2 is
are synthesized using variations of scheme D and the corresponding intermediates, e.g., reaction of (123) with 2-halopyridine (Bernatowicz “JOC” 57:2497 1992).
Compounds of structure (1) where R2 is
where R29 is —N—, —CH— or —C(CH3)—, R62 is —H, —NH2 or —NH(CH3) and R6 is —O— or —S— are synthesized using scheme A, while scheme B is used when R6 is —CH2— and scheme C is used when R6 is —NH—.
where R29 is —N—, —CH— or —C(CH3)—, R62 is —H, —NH2 or —NH(CH3) and R6 is —O— or —S— are synthesized using scheme A, while scheme B is used when R6 is —CH2— and scheme C is used when R6 is —NH—.
Compounds of structure (1) where R2 is
where R61 is —H, alkyl having 1, 2, 3 or 4 carbon atoms or optionally protected substituted alkyl having 1, 2, 3, 4, 5 or 6 carbon atoms including —CH3 and —CH2CH3, and R6 is —O— or —S— are synthesized using scheme A, while scheme B is used when R6 is —CH2 — and scheme C is used when R6 is —NH—.
where R61 is —H, alkyl having 1, 2, 3 or 4 carbon atoms or optionally protected substituted alkyl having 1, 2, 3, 4, 5 or 6 carbon atoms including —CH3 and —CH2CH3, and R6 is —O— or —S— are synthesized using scheme A, while scheme B is used when R6 is —CH2 — and scheme C is used when R6 is —NH—.
Compounds of structure (1) where R2 is —R6—R60—N(R3)2, including —R6—(CH 2)1—N(R3)2, and R6 is —O— or —S— are synthesized using scheme A, while scheme B is used when R6 is —CH2— and scheme C is used when R6 is —NH—.
Compounds of structure (1) where R2 is
and R6 is —O— or —S— are synthesized using scheme A, while scheme B is used when R5 is —CH2— and scheme C is used when R6 is —NH—. Compounds containing these structures where R6 is —S— or —O—, in general are synthesized using Scheme A and compound (103), e.g., using #1154-093 described in the examples below, and the corresponding protected alcohols. Compounds where R6 is —CH2— are obtained by following Scheme B. Compounds where R6 is—NH— can be obtained using compound (119), e.g., using #1090-68 and protected aldehydes described in the examples below, by following Scheme C.
and R6 is —O— or —S— are synthesized using scheme A, while scheme B is used when R5 is —CH2— and scheme C is used when R6 is —NH—. Compounds containing these structures where R6 is —S— or —O—, in general are synthesized using Scheme A and compound (103), e.g., using #1154-093 described in the examples below, and the corresponding protected alcohols. Compounds where R6 is —CH2— are obtained by following Scheme B. Compounds where R6 is—NH— can be obtained using compound (119), e.g., using #1090-68 and protected aldehydes described in the examples below, by following Scheme C.
Schemes E and F illustrate the synthesis of alcohols containing an imidazole moiety (see e.g., Munk “J. Med. Chem.” 40:181997), to scheme E, R62 is R5 or alkyl having 1, 2, 3, or 4 carbon atoms and R63 is —OR5, —(CH2)Z3OR5 or —(CH2)1-2—R47—(CH2)1-2—ORS and Z4 is 0, 1, 2, 3, 4 or 5. In scheme F, Z5 is 1, 3, 3, 4 or 5.
Scheme D is also used to obtain compounds of structure (1) where R2 comprises, for example, a moiety such as —(CH2)2-4—NH((CH2)2-4NH)0-4—(CH2)2-4NH2, —(CH2)2-4NH(CH2)2-4N((CH2)0-4—NH2), or when R2 is —R6(CH2)1N(R3)2 and one R4 is H, CH3, CH2CH, a protecting group or —(CH2)v—N(R33)2 and the other R4 is —(CH2)v—N(R33)2, —CH(N[R33]2)—N(R33)2, (48), (49) or (50).
Converting a compound of structure (4) to a compound of structure (1) is accomplished by a method comprising displacing R24. In this method, R24 usually is —Br and R27 is usually —CH—.
R2 groups containing a diol, e.g. (CH2OH)2—CH—(CH2)n—O— where n is 1-8, are converted by cleavage to the aldehyde CHO—CH2—(CH2)n—O— using sodium periodate. Reductive alkylation of the aldehyde to a primary or secondary amine is accomplished using N(R3A)2 where R3A independently is —H, C1-6 alkyl, including —CH3, —CH2CH3 or a protecting group, usually —H or —OH3.
To the extent that a compound within the claims' scope can not be directly synthesized using the schemes above or the examples below, the artisan will employ straightforward methods known for preparing such compounds, see e.g., B. M. Trost & I. Fleming, eds. “Comprehensive Organic Synthesis”, volumes 1-8, Pergamon Press; M. Fieser, ed. “Fieser and Fieser's Reagents for Organic Synthesis”, volumes 1-17, John Wiley & Sons; and A. F. Finch Ed. “Theilbeimer's Synthetic Methods of Organic Cbemistry”, volumes 1-49, latest editions, S. Karger AG.
The compounds of this invention find uses in the diagnostic, analytic and therapeutic fields, or as intermediates in the preparation of compounds useful in such fields.
The R2-substituted compounds of structure (1) are useful as intermediates in the preparation of the labeled biopolymers of structure (1), wherein a biopolymer is rendered fluorescent or otherwise delectably labeled by linkage to the polycyclic substructure. It is most convenient, however, to use the appropriate structure (1) compounds as monomers in the preparation of nucleic acids or oligonucleotides. The labeled biopolymers are employed in diagnostic assays or preparative procedures in the same fashion as other fluorophor-labeled biopolymers, e.g. in fluorescence polarization methods, fluorescence activated cell sorting, competitive-type EMIT immunoassays and the like.
The linker- and hydrogen-substituted compounds of structure (1) are useful as intermediates to prepare materials suitable for ruse in affinity purification of guanine or guanine-containing compounds, e.g., nucleosides. The structure (1) compounds form hydrogen bonds with guanine and one can thus link the structure (1) base structure to an appropriate support material or polymer to prepare an affinity resin suitable for binding to guanine-containing compounds, e.g., nucleosides, nucleotides and oligonucleotides or similar compounds containing guanine analogs, e.g., 7-deazaguanine. Typical linker-derivatized structure (1) compounds optionally contain a linker of structure —(R48)X—R49, defined above in the discussion under Scheme A.
The monomers are of particular use in preparing oligonucleotides for diagnostic or therapeutic use. Since oligonucleotides having 2 or more nucleotides or nucleotide analogs bearing the polycyclic substructure will usually exhibit greatly increased Tm, such oligonucleotides are particularly useful in therapeutic or diagnostic utilities where highly stable duplex hybridization structures are desired. Since these oligonucleotides frequently are fluorescent, changes in the oligonucleotide fluorescence can be followed as the oligonucleotide binds to complementary nucleic acid or oligonucleotide sequences. These changes are detectable as modifications in energy transfer, e.g., fluorescence quenching or shifts in activation or emission wavelength(s).
The polycyclic substructure labeled oligonucleotides are employed in diagnostic or analytic methods in the same fashion as other labeled oligonucleotides. For example, the oligonucleotides are used in hybridization methods in which an antibody capable of binding base-paired structure (1) is used to detect binding of the oligonucleotide to a target nucleic acid, sequence. In addition, changes in fluorescent character can be assayed as described above. Typically, 2, 3 or more polycyclic substructure labeled oligonucleotides are used in a hybridization assay. One oligonucleotide is labeled at its 3′ end with a polycyclic substructure containing nucleotide while the other nucleotide is labeled at its 5′ end with the same or another polycyclic substructure or with a different fluorophor such as fluorescein or rhodamine capable of energy transfer. The two oligonucleotides recognize a complementary sequence in which the 3′ end of the target sequence binds the oligonucleotide bearing the 3′-terminal fluorophor and the adjacent 5′ sequence of the target binds to the oligonucleotide bearing the 5′ terminal fluorophor, Binding is assayed by measuring a change in fluorescence of either or both of the oligonucleotides when they bind in tandem according to the illustrated model. In other embodiments only a single labeled oligonucleotide is employed in the hybridization method. The oligonucleotides of this invention thus are useful in solution phase hybridization diagnostics, i.e., it is not necessary to perform a phase separation in order to detect labeled oligonucleotide binding.
Detecting a target base sequence in a nucleic acid or an oligonucleotide using an invention oligonucleotide is accomplished by a method comprising (i) mixing or contacting a sample suspected of containing a nucleic acid with an optionally labeled invention oligonucleotide comprising at least about 7 bases, usually about 12-30 bases, where the protecting groups have been removed, (ii) allowing time sufficient for the invention oligonucleotide to bind to the target base sequence, (iii) separating unbound invention oligonucleotides from bound invention oligonucleotides and (iii) detecting the presence, absence or amount of bound invention oligonucleotide. Aspects of the invention include conducting any one of these steps individually, which are each part of the complete method. The use of known hybridization methods and conditions are applied to accomplish the method.
In this method to detect target base sequences, one may optionally use invention oligonucleotide and target base sequences that are substantially complementary to each other, i.e., sequences having 1 or no mismatches per about every 15-30 bases. The target base: sequences to be detected are generally present in a all, in a cell or tissue extract or, usually, in a purified nucleic acid or oligonucleotide preparation, e.g., a sequence encoding a portion of a cytokine, cell surface molecule, an enzyme such as farnesyl protein transferase or an oncogene such as neu, myc, raf, ras or c-ras. The target base sequences to be detected are generally present in RNA or single stranded DNA, although the invention oligonucleotides are also useful to detect base sequences in duplex nucleic acids.
Another invention embodiment is a method comprising incubating a cell with a deprotected invention oligonucleotide containing at least about 7 bases, usually about 12-30 bases, wherein the invention oligonucleotide is optionally present in a transfection complex comprising the invention oligonucleotide and a permeation enhancing agent. This method is used to introduce optionally labeled invention oligonucleotides into cell cytoplasm or vacuoles.
One optionally conducts this method using invention oligonucleotides having a octanol:water partition coefficient of about −0.5 to about 2.5, typically about 0.0 to about 2.0, usually about 0.5-1.5, and a solubility in water of at least 0.001 μg/mL. However, in these embodiments, no permeation enhancing agent is usually needed to introduce the compound into the cells.
One can use oligonucleotides containing 1, 2, 3 or more invention base(s) to detect a base pair mismatch in a nucleic acid sample using ribonuclease protection assay methods described in U.S. Pat. No. 5,589,329. Thus one can use the invention compounds as described is step (a) or step (b) or step (c) (or, in sequence, steps a, b, or steps b, c or steps a, b, c) of claim 1 of U.S. Pat. No. 5,589 29 to practice that claimed method (or to practice necessary steps in the claim 1 method, e.g., contacting an RNA probe containing an invention base with a single stranded nucleic acid to form a duplex). One can similarly use oligonucleotides containing an invention base(s) to screen mammalian genomic DNA samples for insertions, deletions or substitutions using screening assay methods described in U.S. Pat. No. 5,589,330. Thus, one can use the invention compounds as described in step (ii) or step (iii) or step (iv) or step (v) (or, in sequence, steps i, ii, or steps i, ii, iii or steps iii, iv, or steps i, ii, iii, iv, etc.) of claim 1 of 5,589,330 to practice that claimed method (or to practice necessary steps in the claim 1 method, e.g., contacting an oligonucleotide containing an invention base with an immobilized genomic DNA sample to form a triplex or duplex). One can similarly use oligonucleotides containing an invention base(s) to design a synthesis method for an array of materials to be synthesized on a substrate using methods described in U.S. Pat. No. 5,93,839. Thus, one can use the invention compounds as described in the first part of the second step or the second part of the second step or the third part of the second step or the fourth part of the second step or (or, in sequence, in the first step and in the first part of the second step, etc.) of claim 1 of 5,593,839 to practice that claimed method (or to practice necessary steps in the claim 1 method).
One can also use the invention monomer compositions containing a 5′a-35S-thiotriphosphate group or a 5′ triphosphate group linked to 2′,3′-dideoxyribose to perform dideoxy DNA sequencing methods. One may use invention monomers in kits that optionally contain buffers or enzymes suitable for DNA sequencing. The invention monomers may be advantageously used in enzymatic DNA sequencing protocols because the invention monomers, which act as cytosine surrogates, have a high affinity for guanosine and may perform better than cytidine 5′ triphosphate in sequencing reactions, particularly where the DNA to be sequenced contains a high proportion of guanosine residues, which can cause sequencing problems.
Oligonucleotide analogs containing 1, 2, 3 or more invention bases are also suitable for binding to open complexes in cells or cell lysates of eukaryotic or prokaryotic cells. Open complexes may also arise is systems comprising at least partially petrified cell components, e.g., RNA polymerase, nucleotide triphosphates, suitable transcription cofactors, DNA binding proteins and duplex DNA capable of transcription. Open complexes am regions of single stranded DNA or RNA that occur at least transiently during duplex nucleic acid metabolism, e.g., during DNA replication or RNA transcription in the nucleus, cytoplasm, plastids or mitochondria. Such DNA replication or RNA transcription may involve metabolism of cellular, viral or other nucleic acids. One can use binding of invention oligonucleotides to single stranded open complex sequences to affect nucleic acid metabolism, e.g., one may inhibit RNA transcription or one may use the oligonucleotides, which are optionally labeled, to detect the presence of open complexes. Such oligonucleotides will typically comprise about 8 to 30 monomers, usually about 12-21 monomers, baying a sequence complementary to a single stranded open complex region(s) involved in the initiation of a nucleic acid metabolic event, e.g., initiation of RNA transcription in a promoter or transcription initiation region. Workers have described open complexes and their formation in vitro and in cells, e.g., Burns, “Biochem. J.” 317:305-11 1996, Smith, “Proc. Natl. Acad. Sci. USA” 93:8868-72 1996, Jiang, “J Biol Chem” 268:6535-40 1993.
Workers have described other applications that one can practice in a similar straightforward manner using optionally labeled oligonucleotides containing 1, 2, 3 or more invention bases, see e.g., U.S. Pat. Nos. 4,910,300, 5,093,232, 5,124,246, 5,202,231, 5,258,506, 5,202,231, 5,525,464, 5,578,717, 5,578,715, 5,578,467, 5,591,584, 5,599,932, 5,599,668, 5,593,841, 5,578,458, 5,589,332, 5,589,333, 5,589,339, 5,589,342, 5,593,830, 5,593,831, 5,593,832, 5,593,836, 5,593,840, 5,593,841, 5,593,863, 5,604,097, 5,604,099, 5,605,793, 5,605,794, 5,605,796, 5,605,798, 5,605,824, 5,606,047, 5,608,063, 5,614,617, 5,594,117, 5,633,364, 5,639,612, 5,639,611, 5,639,608, 5,639,647, 5,639,736, 5,639,626, 5,641,631International Publication Nos. WO 97/07246, WO 97/06252, WO 97/06183, WO 97/04787, WO 97/05280, WO 95/41017, WO 96/41012, WO 96/40994, WO 96/40996, WO 96/40991, WO 96/40992, WO 96/41016, WO 97/04126, WO 97/04129, WO 96/06950 and European Publication No. 761 822. For each of these applications, one would use an invention oligonucleotide in place of one or more of the described oligonucleotides. To practice these and other typical applications, one will typically use an invention oligonucleotide in one or more of the steps needed to practice the methods described in these publications. In many of these applications, one will use an invention oligonucleotide containing (i) about 7-50, usually about 8-30 linked monomers, usually where the oligonucleotide has a uniform polarity, (ii) 1, 2 or 3 invention bases, (iii) purine and pyrimidine bases having a base sequence complementary or substantially complementary to a target sequence, i.e., a defined base sequence having no more than about 1, 2, 3 or, for relatively long oligonucleotides (about 35-50-mers), 4 base mismatches, and, optionally, (iv) other moieties or features, which are readily apparent to the skilled artisan who has read this disclosure, that facilitate using the oligonucleotide in the intended application, e.g., (i) a free hydroxyl group at the 3′ terminus for applications where the one wishes to use the invention oligonucleotide as a primer in enzyme-mediated chain elongation applications, (ii) a fluorescent label, enzyme label or radiolabel to facilitate oligonucleotide detection in a particular assay, (iii) biotin linked to a convenient location such as the 5′ or 3′ terminus, or (iv) a free 5′ hydroxyl group for enzymatic phosphorylation.
When one adapts the presently claimed compounds to uses known in the an, one will use known hybridization conditions, enzyme (such as polymerase, RNase or DNase) reaction conditions or detection technologies to design the invention oligonucleotide in a way that does not interfere with the intended use, or in a way that improves the intended use. For example, when a previously described assay or diagnostic method calls for conducting an oligonucleotide hybridization or enzyme amplification protocol at a specified temperature, one will have the option of increasing the hybridization or enzyme amplification temperature due to the presence of an invention base(s) in the oligonucleotide. Thus, one can use the enhanced binding affinity or enhanced binding specificity property of the invention oligonucleotides to increase hybridization stringency. Similarly, when one intends to use an invention oligonucleotide in a polymerase chain reaction (PCR) amplification method, one would initially test to see if the presence of an invention base at the 3′ terminus improves the oligonucleotide's primer function and then adjust the reaction conditions accordingly, e.g., by altering the primer to target sequence ratio, primer concentration or by altering the temperature at which one denatures and amplifies the PCR reaction. If the presence of an invention base significantly affects polymerase-mediated primer elongation, then one could choose to design invention oligonucleotides for this use without an invention base(s) at the 3′ terminal 1, 2 or 3 monomer positions. Skilled artisans routinely design diagnostic or assay protocols by testing varying temperature, salt composition and concentration, pH, oligonucleotide concentration, enzyme concentration, enzyme reaction buffer, act oligonucleotide charge, or oligonucleotide base, sugar or linkage structure during assay development.
Embodiments include a method comprising preparing a series of oligonucleotides, each having the same base sequence, which sequence contains 2 or more cytosine bases, where each member of the series contains an invention base of structure (3) in place of one or more of the cytosine residues. Such oligonucleotides typically comprise 2 to about 6 cytosine residues. One prepares oligonucleotides containing an invention base at each cytosine position and optionally one prepares oligonucleotides containing an invention base at each of two cytosine positions, at each of three cytosine positions and so forth. One uses this method to determine which oligonucleotide(s), compared to a control containing no invention base(s), has optimal properties for a desired application, e.g., hybridization affinity for use as a probe or optimal antisense activity for inhibiting target gene expression in a cell.
Structure (5) monomers, when triphosphorylated and containing R1 ribose or deoxyribose derivatives that are chain terminating (e.g. where the 3′ position is not hydroxyl), are useful in methods for fluorescent chain-terminating dideoxynucleotide sequencing in the same general fashion as ddNTPs having other linker-attached fluorophores.
Since oligonucleotide compounds such as those of structure (2) are capable of participating in Watson-Crick base pairing they will hind to nucleic acids and therefore are useful in detecting the presence of nucleic acids. Bases of structure (1) in such oligonucleotides will recognize guanosine as its complementary base in natural nucleic acids.
Invention oligonucleotides, including many structure (2), (2A), (2B) and (2C) oligonucleotides capable of forming high melting duplexes with complementary sequences, are useful in numerous applications, including antisense or codeblocking utilities in vivo or in vitro as well as diagnostics and probe uses. High melting duplexes are those having melting temperatures substantially above the melting temperatures of oligonucleotide or nucleic acid duplexes of the same sequence that contain the ordinary, naturally occurring bases, e.g., adenosine, cytidine, uridine, guanosine, thymidine and the like. “Substantially above” means that the derivative oligonucleotide, when hybridized with its complementary sequence, will not dissociate from the duplex until the temperature is raised from about 2 to 40° C., ordinarily about 8 to 40° C., above the dissociation temperature of the same oligonucleotide having the analogous normal A, C, U, G or T bases, but to no greater temperature than about 95° C. This is known as the D Tm. Ordinarily, D Tm is measured by comparing control oligonucleotide binding to complementary RNA or DNA with the binding of test oligonucleotide to the same RNA or DNA, following, e.g., the method described in Jones et al., “JOC” 58:2983 (1993).
Some of the invention riboside and deoxyriboside compounds are fluorescent. The compounds remain fluorescent upon incorporation, into oligonucleotides and are visible intracellularly, including when bound to target sequences after direct injection or after transfection into cells in accord with known methods.
One can optionally prepare oligonucleotides having tandem arrangements of the novel bases. In general, such tandem arrangements will contain from 2 to about 10 invention polycyclic bases, usually 2, 3 or 4, which can be the same or different polycycles but generally are the same invention polycycle. They also optionally are copolymerized with purine or pyrimidine bases containing known alkynyl substitutions (e.g., U.S. Pat. Nos. 5,645,985 and 5,594,121), in particular pyrimidine bases substituted at the 5 position with a carbon atom which is bonded to another atom by a Pi bond, or the fluorescent cytosine derivatives of Inoue et al. (op cit).
The compounds of this invention, or other oligonucleotides capable of forming high melting duplexes (e.g. the Pi bonded bases discussed above), are useful in improved methods for polymerase chain reaction (“PCR”) or ligase chain reaction (“LCR”) amplification and detection of nucleic acids. In one embodiment, the high melting oligonucleotides are used as one or both primers in classical PCR or as probes in LCR. Particularly in PCR processes, the elevated melting temperature of duplexes with high melting primers avoids the need to thermally cycle the reaction because at these elevated temperatures (about 68 to 95° C., preferably greater than about 75° C.; the derivative primer will continue in at least some proportion to anneal to the target but extension product will not. Ordinary primers will not hybridize and the polymerase will not initiate transcription until the reaction mixture is cooled to a level at which the primer will anneal to the target sequence (usually, about 55° C.). The elevated temperature that is chosen for use with the high-melting derivative oligonucleotides (a temperature suitable for all of annealing, extension and melting) is one at which a substantial proportion of the extended primer population (about 10 to 90 mole %) is found dissociated from the target, but sufficient unextended primer is bound to permit extension. Optimally, this is about from 85 to 95° C., ordinarily 92 to 95° C. Alternatively, the optimal temperature is determined empirically by simply selecting a range of temperatures within the melting range of the extended sequence, but within the annealing range of the derivative primers, and measuring the amount of amplification product to achieve satisfactory levels for the diagnostic or preparative processes at hand. Amplification methods have been described, e.g., U.S. Pat. No. 5,667,974.
An exemplary method to use an invention oligonucleotide to amplify a nucleic acid base sequence comprises, providing an invention oligonucleotide and target nucleic acid sequence that forms a complex having a Tm of about 85 to 95° C., optionally heating the complex to about 85 to 95° C. (e.g., to a temperature within about 5° C. of the Tm) to provide a brand complex, and optionally mixing the heated complex with a DNA polymerase such as Taq polymerase or other suitable heat stable enzyme. In this method, the complex and the heated complex is typically a duplex. The polymerase reaction will contain cofactors and buffer conditions suitable for amplification purposes.
It will be understood that the optimal temperature will vary considerably depending upon the derivative bases chosen, whether they are adjacent or separated by other bases, the number of bases in the primers (toe highest annealing temperatures are found with primers having greater than about 18 bases or base analogs), the proportions of pyrimidines and purines and the like. The heat stable polymerase useful in this system is for example Tag polymerase or other suitable bear stable enzyme. Thus, whatever the optimum temperature chosen, the amplification and priming reactions are conducted conventionally but at a substantially constant temperature.
Not only do the oligonucleotides of this invention facilitate PCR or LCR processes, the fluorescent properties of the primers also facilitate detection of the extension products. The extension products are readily separated from the unextended primers, e.g. on the basis of molecular weight, and detected by their fluorescence, thereby avoiding staining with agents such as ethidium bromide. In some embodiments, the fluorescence is enhanced by using NTP's comprising the fluorescent substructures of this invention in primer extension so that the fluorescent NTPs are incorporated into the extension products as well. The polycyclic substructure used in the NTP's may be the same or different than the one incorporated into the primers.
We incorporate herein all citations by reference with specificity.
The following examples further illustrate and do not limit the invention.
The following example shows synthesis of representative starting materials and intermediates for making invention compounds.
Conditions: a: H2,10% Pd/C, CH3OH, RT; b: HC(OCH3)3, methanesulfonic acid, 47.8%; c: Tf2NPh, CH2Cl2/DMF, K2CO3, RT, quantitative; d: HC═CCH2NHTFA, Pd(Ph3P)4 Cul, TEA, DMF, 60%; e: H2, 10% Pd/C, CH3COOEt; f: con. NH4OH/Dioxane (1/1), RT; g: PhCH2OC(O)Cl, pyridine; h: 3N HCl/EtOH (1/1), RT, 1 hr or 40° C., 30 min.
9-(3′-Aminopropyl phenoxazine
Conditions: a: H2,10% Pd/C, CH3OH, RT; b: HC(OCH3)3, methanesulfonic acid, 47.8%; c: Tf2NPh, CH2Cl2/DMF, K2CO3, RT, quantitative; d: HC═CCH2NHTFA, Pd(Ph3P)4 Cul, TEA, DMF, 60%; e: H2, 10% Pd/C, CH3COOEt; f: con. NH4OH/Dioxane (1/1), RT; g: PhCH2OC(O)Cl, pyridine; h: 3N HCl/EtOH (1/1), RT, 1 hr or 40° C., 30 min.
9-(3′-Aminopropyl phenoxazine
2-Aminoresorcinol (#1019-43): A ethanol solution (500 mL) of 3-nitroresorinol (20 g 0.129 mmole) was hydrogenated (H2 in balloon) in the presence of 10% Pd/C (1 g) at room temperature overnight. The catalyst was filtered off through a celite pad. The filtrate was concentrated and purified by flash column chromatography, affording 15.58, 96% of 2-aminoresorcinol. 1H NMR (DMSO-d6): δ8.80 (bs, 2H), 6.15-6.30 (m, 3H), 3.85 (bs, 2H).
Compound #1019-45: A trimethyl orthoformate solution (100 mL) of 2-aminoresorcinol (6.0 g; 48 mmole) was treated with methanesulfonic acid (4.6 g; 48 mmole). After 30 min stirring at room temperature, the reaction was cooled to 0° C. and was quenched with TEA (4.8 g; 48 mmole). The reaction mixture was concentrated and purified by column chromatography on silica gel, yielding 3.10 g of product, 47.8%. 1H NMR (DMSO-d6): δ 10.3 (bs, 1H), 8.53 (s, 1H), 7.18 (dd, 1H, J=8.0 Hz, J=7.8 Hz), 7.12 (d. 1H, J=8.0 Hz), 6.73 (d, 1H, J=7.8 Hz).
Compound #1019-46: A CH2Cl2/DMF (N,N-dimethylformamide) solution (20 mL/10 mL) of #1019-45 (2.90 g; 21.4 mmole) was stirred with solid K2CO3 (14.8 g; 107 mmole) at room temperature for 30 min, followed by addition of N-phenyltrifluromethanesulfonimide (850 g; 23.6 mmole). The resulting mixture was stirred at room temperature overnight, then diluted with CH2Cl2, washed with water twice, dried, concentrated, and purified, affording 5.76 g, quantitatively, of #1019-46. 1H NMR (CDCl3): δ 8.19 (s, 1H), 7.66 (d,1H, J=8.2 Hz), 7.48 (t, 1H, J=8.2 Hz), 7.33 (d, 1H, J=8.2 Hz).
N-TFA-propargylamine (#1019-44): Propargylamine (25 g; 0.45 mmole) was dissolved in CH3OH (500 mL), followed by addition of ethyl trifluoroacetate (84 g; 0.59 mole). The resulting mixture was stirred at room temperature overnight, then concentrated to dryness. The residue was redissolved in CH2Cl2 (200 mL), washed with saturated NaHCO3 aqueous solution. The organic phase was isolated, dried, concentrated to a brown residue (liquid). The product #1019-44, 54.0 g was distilled off, 78.8%. 1H NMR (CDCl3): δ 6.60 (bs, 1H), 4.18 (m, 2H), 2.38 (s, 1H).
Compound #1019-97: A DMF solution (12 mL) of compound #1019-46 (4.9 g; 18.3 mmole), N-TFA-propargylamine (5.5 g; 36.7 mmole), Pd(PPh3)4 (4.3 g; 3.7 mmole), Cul (1.78 g; 9.3 mmole) and TEA (3.7 g; 36.7 mmole) was stirred at room temperature for 24 hours. The reaction mixture was diluted with CH2Cl2 (100 mL), stirred with Dowex 1×800 (HCO3—form). The organic phase was washed with water, dried, and purified, yielding 2.94 g, 60%, of product #1019-97. 1H NMR (CDCl3): δ 8.18 (s, 1N), 7.62 (d, 1N, J=9 Hz), 7.48 (d, 1H, J=9 Hz), 7.36 (t, 1H, J=9Hz), 6.85 (bs, 1H), 4.51 (d, 2H, J=3 Hz).
Compound #1019-102: A ethylacetate solution (60 mL) of #1019-97 (1.7 g; 6.3 mmole) was hydrogenated in the presence of 10% Pd/C (200 mg) at room temperature. The catalyst was filtered off. The filtrate was concentrated to dryness, used for next reaction without further purification. 1H NMR (CDCl3): δ 8.25 (bs, 1H8.12 (s, 1H), 7.49 (d, 1H, J=82 Hz), 7.36 (dd, 1H, J=7.6 & 8.1 Hz), 7.20 (d, 1H, J=7.5 Hz), 3.25 (q, 2H) 3.07 (t, 2H, J=6.6 Hz), 2.01 (p, 2H).
Compound #1090-05A: A 1.4-dioxane solution (10 mL) of compound #1019-102 (1.0 g; 3.6 mmole) was treated with concentrated NHOH (15 mL) at room temperature for 16 hrs. The reaction mixture was concentrated to dryness. The residue was redissolved in CH2Cl2(30 mL), containing TEA (0.74 g; 735 mmole), cooled to 0° C., followed by addition of benzyl chloroformate (0.75 g, 4.4 mmole). The resulting solution was stirred at room temperature for 4 hrs, washed with H2O, dried and purified by flash column chromatography to give 0.81 g, 71% of #1090-05A.
Compound #1090-05: A ethanol solution (10 mL) of compound #1090-05A(0.81 g, 2.6 mmole) was treated with 3 NHCl aqueous solution (10 mL) at 40° C. for 1 hr. The reaction mixture was concentrated to dryness, azeotroped with CH3CN three times. The crude product was used for next reaction, without further purification, 1H NMR (CDCl3): δ 7.25-7.40 (m, 5H), 7.20 (t, 1H), 6.78-6.90 (m, 2H), 5.07 (s, 2H), 3.17 (t, 2H, J=6.8 Hz), 2.67 (t, 2H, J=7.6 Hz), 1.79 (p, 2H).
Compound #1090-18: Compound #1090-15 has been described (Lira, “JACS” 117:3873-3874, 1995). A mixture of Compound #1090-15 (15 g; 26 mmole), #1090-05 (2.6 mmole) and DBU (0.8 g; 5.2 mmole) in CH2Cl2(30 mL) was stirred at room temperature overnight. The reaction mixture was washed with 10% citric acid aqueous solution, dried and purified on silica gel column chromatography, affording 1.21 g, 69% of product. 1H NMR (CDCl3): δ8.86 (s, 1H), 7.99 (s, 1H), 7.29-7.40 (m, 5H), 7.15 (t, 1H), 6.98 (d, 1H, J=8 Hz), 7.78 (d, 1H, J=7 Hz), 6.27 (t, 1H), 5.20-5.23 (m, 1H), 5.09 (s, 2H), 4.78 (bs, 1H, 4.30-4.52 (m, 3H), 3.20-3.30 (q, 2H), 2.65-2.80 (t+m, 3H), 2.10-2.20 (2 s+m, 7H), 1.80 (p, 2H).
Compound #1090-22: Compound #1019-18 (1.20 g; 1.78 mmole) was treated with saturated NH3 in CH3OH (200 mL) at room temperature for 5 days. The reaction mixture was concentrated to dryness and purified by flash column chromatography affording 0.63 g of product #1090-22, 70% 1H NMR (CDCl3+10% CD3OD): δ 7.30-7.38 (m, 5H), 6.81 (t, 1H, J=7.3 Hz), 6.73 (d, 1H, J=7.9 Hz), 6.60 (d, 1H), 6.20 (t, 1H), 5.10 (s, 2H), 4.36-4.40 (m, 1H), 3.94-4.0 (m, 1H), 3.70 3.90 (m, 2H), 3.23 (t, 2H), 2.58 (t, 2H), 2.30-2.40 (m, 1H), 2.10-2.24 (m, 1H), 1.70-1.82 (m, 2H).
Compound #1090-25: Compound #1090-22 (0.6 g) was dissolved in ethanol (10 mL) and was hydrogenated (H2 in a balloon) in the presence of 10% Pd/C (50 mg) at room temperature for 4 hrs. The catalyst was filtered off, washed with CH3OH. The filtrate was concentrated and dried to afford 0.3 g 68% of product. 1H NMR (CD3OD: δ 7.38 (s, 1H), 6.7-6.82 (m, 2H), 6.57 (d, 1H, J=6.3 Hz), 6.23 (dd, 1H, J=6.5 Hz, J=6.7 Hz), 4.35-4.38 (m, 1H), 3.80-3.90 (m, 1H), 3.70-3.80 (m, 1H), 2.60-2.80 (m, 4H), 2.10-2.30 (m, 2H), 1.75-1.86 (m, 2H).
Compound #1090-26: Compound #1090-25 (0.13 g, 0.34 mmole) was dissolved in DMF/CH2Cl2 (1 mL/3 mL), followed by addition of 9-fluorenylmethyl-N-succinimidylcarbonate (FMOC-NHS, 0.14 g; 0.41 mmole). After 1 hr of stirring at room temperature, to the reaction mixture pyridine (140 mg, 1.7 mmole), and DMT-Cl (4,4′-dimethoxytrityl chloride; 0.14 g; 0.41 mmole) were added. The resulting mixture was stirred at room temperature for 2 hrs and then diluted with CH2Cl2, washed with water twice. The organic phase was isolated, dried and purified by flash column chromatography on silica gel, to give 153 mg, 49%, FAB HRMS (high resolution mass spectroscopy) calculated for M+H+ 899.366.
Compound #1090-31: Compound #1090-26 (150 mg; 0.167 mmole) in CH2Cl2 (2 mL) was added to a 0° C. cold CH2Cl2 solution (1 mL) of PA (0.25 mL of 1M CH2Cl2 solution, 0.25 mmole) and pyridine (66 mg, 0.83 mmole). The resulting mixture was then gradually warmed to room temperature. After 30 minutes, the reaction mixture was diluted with CH2Cl2, washed with 1 M TEAB aqueous solution, dried and purified on silica gel, eluted with 5% CH3OH/CH2Cl2, the 15%, H2O in CH3CN. The combined fractions of product were concentrated and then partitioned between CH2Cl2 and 1M TEAB aqueous solution dried, yielding 180 mg of H-phosphonate derivative, quantitatively.
Compound #1090-68: A CH2Cl2—CCl4 (40 mL/40 mL) solution of 5-bromo-3′-5′-diacetyl-2′-deoxyuridine (2.0 g, 5.1 mmole) and triphenyl phosphine (2.0 g, 7.6 mmole) was heated at reflux for 3 hrs. The reaction mixture was cooled to room temperature, followed by addition of 2-amino-3-nitrophenol (0.79 g, 5.1 mmole) and DBU (1.6 g, 10 mmole). After stirring at room temperature overnight, the reaction mixture was washed with 10% citric acid aqueous solution, dried, concentrated and purified on silica gel. The isolated product contained Ph3P═O, was treated with saturated NH3 in CH3OH for 5 days at room temperature. After removal of all solvent, the residue (crude #1090-51) was redissolved in CH3OH (100 mL), and hydrogenated with H2 in a balloon in the presence of 10% Pd/C and 4 N HCl in dioxane (200 mL, 0.8 mmole). After 5 hrs, the catalyst was filtered off, washed with CH3OH. The filtrate was concentrated and purified by flash column chromatography to afford 0.83 g of #1090-68, in 50% yield for 3 steps. 1H NMR (DMSO-Cl6): δ 9.6 (b, 1H), 6.56 (t, 1H, J=8.0 Hz), 6.22 (d, 1H, J=7.8 Hz), 6.10 (t, 1H, J=6.8 Hz), 5.96 (d, 1H, J=7.1 Hz), 5.17 (d, 1H), 4.92-5.08 (m, 3H), 4.16-4.20 (m, 1H), 3.70-3.80 (m, 1H), 3.50-3.60 (m, 2H), 2.0 (m, 2H).
Phthalimidoacetaldehyde #1090-70A: A mixture of N-(2-hydroxyethyl)phthalimide (90 mg; 0.47 mmole), DCC (0.15 g, 0.7 mmole), DMSO (1 mL) and dichloroacetic acid (20 mL) was stirred at room temperature for 2 hrs. The reaction mixture was diluted with CH2Cl2, washed with H2O twice, dried and concentrated. The crude product #1090-70A contained DCC-urea by-product, was used for the next reaction without further purification.
Compound #1090-70: A DMF/CH3OH (0.5 mL/2 mL) solution of #1090-68 (0.2 g, 0.42 mmole), phthalimidoacetaldehyde #1090-70A (0.47 mmole) in CH3OH (2 mL) and acetic acid (85 mg, 1.4 mmole) was reacted with sodium cyanoborohydride (87 mg, 1.4 mmole) at room temperature for 4 hrs. The reaction mixture was concentrated, and purified on silica gel, yielding 243 mg, 78% yield of compound #1154-70. 1H NMR (CDCl3+10% DMSO-d6): δ 9.45 (s, 1H), 7.60-7.68 (m, 2H), 7.50-7.58 (m, 2H), 6.51 (t, 1H, J=8.2 Hz), 6.12 (d, 1H, J=8.2 Hz), 6.06 (t, 1H, J=6.7 Hz), 5.78 (d, 1H, J=8.0 Hz), 4.94 (t, 1H), 4.49-4.52 (m, 1H), 4.18-4.25 (m, 2H), 3.50-3.72 (m, 4H), 3.19-3.30 (m, 2H), 1.85-2.05 (m, 2H).
Compound #1090-89: Compound #1090-70 (35 mg; 69 mmole) was dissolved in CH2Cl2 (2 mL) and pyridine (0.5 mL), then reacted with DMT-Cl (28 mg, 83 mmole) at room temperature for 3 hrs. The reaction mixture was worked up and purified by flash column chromatography, yielding 44 mg, 78.6% of corresponding 5′-O-DMT-derivatives. 1H NMR (CDCl3): δ 7.80-7.90 (m, 2H), 7.68-7.75 (m, 2H), 7.18-7.50 (m, 10H), 7.0 (b, 1H), 6.80-6.90 (m, 4H), 6.77 (t, 1H), 6.30-6.42 (m, 2H), 5.94 (d, 1H, J=8.0 Hz), 5.50 (bs, 1H), 4.50-4.60 (m, 1H), 4.10-4.18 (m, 1H), 3.86-4.0 (m, 2H), 3.25-3.30 (2s, 6H), 3.28-3.52 (m, 4H), 2.58-2.68 (m, 1H), 2.20-2.32 (m, 1H).
Compound #1090-91: Compound #1090-89 (44 mg) was converted into 3′-H-phosphonate in normal fashion, 52 mg, in 77% yield.
N-CBZ-3-aminopropanol (Compound #1154-74, CBZ; benzyloxycarbonyl): A CH2Cl2 solution (300 mL) of 3-aminopropanol (10 g, 0.133 mole) and TEA (20 g, 0.2 mole) was cooled to 0° C., followed by slow addition of CH2Cl2 solution (25 mL) of benzyl chloroformate (25 g, 0.147 mole). The resulting solution was gradually warmed to room temperature. The stirring was continued at room temperature overnight. The reaction mixture was washed with H2O, dried and purified by flash column chromatography (on silica gel, CH3OH—CH2Cl2) to yield 16.4 g, 58.9% of title compound. 1H NMR (CDCl3): δ 7.26-7.40 (m, 5H), 5.10 & 5.0-5.10 (s+m, 3H), 3.68 (q, 2H), 3.36 (q, 2H), 2.58 (t, 1H), 1.70 (p, 2H).
N-CBZ-2-aminoethanol (Compound #1154-104): To 0° C. cold of CH2Cl2 solution (300 mL) of 2-aminoethanol (10.8 g, 0.177 mole) and TEA (26.8 g; 0.265 mole) was added slowly a CH2Cl2 solution (50 mL) of benzyl chloroformate (33.2 g, 0.194 mole). After complete addition, the resulting solution was gradually warmed to room temperature and stirring was continued at room temperature overnight. The reaction was worked up and purified by flash column chromatography on silica gel, affording 22.8 g in 66% yield of title compound. 1H NMR (CDCl3): δ 7.34-7.40 (m, 5H), 5.20-5.32 (m, 1H), 5.14 (s, 2H), 3.74 (q, 2H), 3.37 (q, 2H), 2.38 (bs, 1H).
3′-5′-Diacetyl-N4-(2″,6″-dihydroxyphenyl)-2′-deoxy-5-bromo-cytidine (Compound #1154-093): A CCl4—CH2Cl2 solution (150 mL-150 mL) of 5-bromo-3′-5′-diacetyl-2′-deoxyuridine (15 g, 38.3 mmole) and Ph3P (15 g, 57.5 mmole) was heated at reflux under N2 for 3 hrs. The reaction mixture was cooled to room temperature, followed by addition of 2-amino-resorcinol (5.2 g, 42 mmole) and DBU (8.7 g, 57.5 mmole). The resulting solution was stirred at room temperature overnight. The reaction mixture was concentrated to about ½ volume, then poured into citric acid aqueous solution (7.5 g in 300 mL H2O) with vigorously stirring. The precipitate was filtered off, washed with H2O, CH2Cl2 then CH3CN, dried in a vacuum oven overnight, weighed 12.9 g, 67.8% yield of title compound. 1H NMR (DMSO-d6): δ 9.63 (s, 2H), 8.21 (s, 1H), 8.0 (s, 1H), 6.89 (t, 1H, J=8.1 Hz), 6.33 (d, 2H, J=8.1 Hz), 6.10 (t, 1H, J=7.4 Hz), 5.10-5.17 (m, 1H), 4.12-4.30 (m, 3H), 2.30-2.40 (m, 2H), 2.06 &2.03 (2s, 6H).
General procedure for synthesis of compounds 11a-11c and 12a-12c: Compounds 11a-11c: To a CH2Cl2 solution of proper protected amino alcohol, Ph3P (1.5 eq) and diethyl azodicarboxylate (DEAD, 1.5 eq), was added compound #1154-093 (1 eq). The resulting mixture was stirred at room temperature overnight. The reaction mixture was washed with H2O, dried and purified by a silica gel (eluted with CH3OH—CH2Cl2) to yield compound 11a11c, normally they contaminated with Ph3P═O. However, the crude compound 11a-11c was directly used for compounds 12a-12c without further purification.
Compound 12a-12c: Crude compound 11a-11c was treated with saturated NH3 in CH3OH at room temperature for 1-3 days. After removal of solvent, the reaction mixture was purified by flash column chromatography on silica gel (eluted with CH3OH—CH2Cl2), to yield compounds 12a-12c.
Compound 12a (1154-106, 1154-111): Compound 12a (2.5 g, 40.8%, 2 steps) was prepared from 1154-093 (6.0 g, 12 mmole) and 1154-104 (3.0 g, 15.3 mole). 1H NMR (DMSO-d6): δ 9.82 (bs, 1H), 7.81 (bs, 1H), 7.69 (bs, 1H), 7.25-7.36 (m, 5H), 6.82 (t, 1H, J=8.2 Hz), 6.60-6.80 (m, 2H), 6.46 (d, 1H, J=8.2 Hz), 6.14 (t, 1H, J=6.7 Hz), 5.22 (d, 1H), 5.06-5.13 (s+s, 3H), 4.20-4.25 (m, 1H), 3.94-4.0 (m, 2H), 3.79-3.81 (m, 1H), 3.58-3.65 (m, 2H), 3.40-3.50 (m, 2H), 1.98-2.20 (m, 2H).
Compound 12b: Compound 12b (0.30 g; 57.6%) was prepared from 1154-93 (0.5 g; 1.0 mmole) and compound 1154-074 (0.31 g; 1.5 mmole). 1H NMR (CD3OD): δ 7.65 (s, 1H), 7.24-7.36 (m, 5H), 6.79 (t, 1H, J=8.6 Hz), 6.54 (d, 1H, J=8.2 Hz), 6.36 (d, 1H, J=8.2 Hz), 6.23 (t, 1H), 5.06 (s, 2H), 4.38-4.45 (m, 1H), 3.92-4.11 (m, 3H), 3.81 (q, 2H), 3.38 (t, 2H), 2.30-2.40 (m, 1H), 2.10-2.22 (m, 1H).
Compound 11c: Compound 11c (86 mg; 37.7%) was prepared from Compound #1154-093 (200 mg, 0.4 mmole) and N,N-dimethylaminoethanol (43 mg, 0.48 mmole). 1H NMR (CDCl3): δ 10.9 (s, 1H), 8.49 (s, 1H), 7.97 (s, 1H), 7.11 (t, 1H, J=8.2 Hz), 6.77 (d, 1H, J=8.2 Hz), 6.55 (d, 1H, J=8.0 Hz), 6.32 (dd, 1H), 5.23-5.27 (m, 1H), 4.40-4.46 (m, 2H), 4.33-4.37 (m, 1H), 4.20 (t, 2H, J=5.5 Hz), 2.70-2.80 (m+t, 3H), 2.30 (s, 6H), 2.20 (s, 3H), 2.10-2.20 (m+s, 3H).
Compound 13c: Compound 11c (130 mg, 228 mmole) was treated with saturated NH3 in CH3OH (30 mL). After 2 days at room temperature the reaction mixture was concentrated to dryness to give crude 12c. The crude 12c was then dissolved in pyridine (2 mL) containing TEA (89 mg, 1.2 mmole), followed by addition of DMT-Cl (115 mg, 340 mmole). After 2 hrs at room temperature, the reaction mixture was concentrated, partitioned between CH2Cl2 and saturated NaHCO3 aqueous solution, dried and purified on silica gel, eluted with 5% CH3OH/CH2Cl2, then 10% CH3OH/CH2Cl2 to yield compound 13c, 60 mg, 34% yield. 1H NMR (CDCl3): δ 7.20-7.50 (m, 10H), 6.86 (dd, 4H), 6.75 (t, 1H, J=8.2 Hz), 6.55 (d, 1H, J=8.3 Hz), 6.33 (t, 1H, J=6.1 Hz), 6.28 (d, 1H, J=7.9 Hz), 4.52-4.60 (m, 1H), 4.14 (q, 1H), 4.0-4.10 (m, 2H), 3.77 & 3.79 (2s, 6H), 3.32-3.45 (m, 2H) 2.62-2.74 (m, 3H), 2.39 (s, 6H), 2.20-2.34 (m, 1H).
Compound 13a (1154-175): A methanol solution (150 mL) of compound 12a (1154-111, 2.2 g, 4.3 mmole) was hydrogenated (H2 in a balloon) in the presence of 10% Pd/C at room temperature overnight. The catalyst was filtered off, washed with CH3OH. The filtrate was concentrated to dryness. The crude unprotected 12a derivative was dissolved in DMF/CH2Cl2 (10 mL/5 mL), and reacted with FMOC-NHS (1.73 g, 5.1 mmole) at room temperature for 1 hr. The reaction mixture was diluted with CH2Cl2 (50 mL), containing pyridine (1.0 g, 12.7 mmole), followed by addition of DMT-Cl (1.8 g, 5.3 mmole). After 2 hrs the reaction mixture was worked up and purified by flash column chromatography, affording 2.87 g, 73% of compound 13a. FAB HRMS calculated for C53H49N4O10 (M+H+) 901.345, found 901.333.
Compound 13b: A ethanol solution (15 mL) of compound 12b (1154-077) was hydrogenated (H2 in a balloon) in the presence of 10% Pd/C at room temperature for 4 hrs. Catalyst was filtered off. The filtrate was concentrated to yield unprotected 12b derivative. Unprotected 12b derivative (125 mg, 0.32 mmole) was dissolved in DMF (2 mL) and reacted with FMOC-NHS (130 mg, 0.38 mmole) at room temperature for 1 hr. The reaction mixture was diluted with CH2Cl2 (5 mL) containing pyridine (132 mg, 1.7 mmole), followed by addition of DMT-Cl (135 mg, 0.38 mmole). After 2 hrs, the reactionmixture was diluted with CH2Cl2 (10 mL), washed with H2O, dried and purified by flash column chromatography to give compound 13b, 86 mg, 29% yield. FAB HRMS calculated for C54H51N4O10 (M+H+) 915.360, found 915.361.
Compound #1154-108: 2-Amino-m-cresol (2.1 g, 17.6 mmole) was dissolved in trimethylorthoformate (15 mL), followed by addition of methanesulfonic acid (0.32 g, 3.4 mmole, 20 molar %). The resulting solution was stirred at room temperature for 3 hrs. The reaction mixture was neutralized with TEA (0.4 g, 4.1 mmole), concentrated and purified on silica gel, eluted with 2% CH3OH/CH2Cl2, affording 1.6 g, 72% yield. 1H NMR (CDCl3): δ 8.10 (s, 1H), 7.43 (d, 1H, J=8.2 Hz), 7.31 (dd, 1H, J=7.3 Hz, J=8.2 Hz), 7.20 (d, 1H, J=7.3 Hz), 2.68 (s, 3H).
Compound #1154-112: Compound #1154-108 (4.3 g, 32 mmole) was dissolved in CCl4 (50 mL), followed by addition of N-bromosuccimide (6.3 g, 35 mmole) and AIBN (240 mg). The resulting solution was refluxed for 2 hrs under N2. The reaction mixture was washed with H2O, dried, concentrated and purified by flash column chromatography on silica gel, eluted with 2% CH3OH/CH2Cl2, affording 5.67 g, of product, 82.7%. 1H NMR (CDCl3): δ 8.16 (s, 1H), 7.55 (d, 1H), 7.40-7.50 (m, 2H), 4.90 (s, 2H).
Compound #1154-125: A CH2Cl2 solution (20 mL) of Compound #1154-112 (0.28 g, 1.3 mmole), ethylene glycol (0.82 g, 13.2 mmole), silver triflate (0.5 g, 1.95 mmole) and 2,6-di-t-butyl-4-methylpyridine (0.52 g, 2.6 mmole) was stirred at room temperature for 3 hrs. The precipitate was filtered off, washed with CH2Cl2. The filtrate was washed with H2O, dried, concentrated and purified on silica gel, eluted with 3% CH3OH/CH2Cl2, affording 0.17 g, 68% of product #1154-125. 1H NMR (CDCl3): δ 8.14 (s, 1H), 7.55 (d, 1H), 7.36-7.40 (m, 2H), 4.97 (s, 2H), 3.70-3.85 (m, 4H),
Compound #1154-126: Compound #1154-125 (170 mg, 0.88 mmole) was dissolved in CH2Cl2 (5 mL) containing TEA (0.27 g, 2.6 mmole) and treated with CH3SO2Cl (0.15 g, 1.32 mmole). After 30 min at room temperature, the reaction mixture was washed with H2O, dried and concentrated. The residue was dissolved in DMF (2 mL) followed by addition of sodium azide (86 mg, 1.3 mmole). The resulting solution was heated at reflux for 2 hrs. The reaction mixture was then partitioned between CH2Cl2 and water. The organic phase was isolated, dried and purified on silica gel, eluted with 1% CH3OH/CH2Cl2, to afford #1154-126, 162 gm, 84%. 1H NMR (CDCl3): δ 8.10 (s, 1H), 4.99 (s, 2H), 3.76 (t, 2H, J=4.8 Hz), 3.45 (t, 2H, J=4.6 Hz).
Compound #1154-132: Compound #1154-126 (1.1 g, 5.0 mmole) was treated with 3N HCl aqueous solution (10 mL) and ethanol (10 mL) at 50° C. for 1 hr. The reaction mixture was concentrated to dryness, azeotroped with CH3CN three times, used for next reaction without further purification. 1H NMR (DMSO-d6): δ 10.6 (bs), 6.85-7.20 (m, 3H), 4.60-4.70 (m, 2H), 3.60-3.72 (m, 2H), 3.50-3.60 (m, 2H).
Compound #1154-133: 5-Bromo-3′,5′-diacetyl-2′-deoxyuridine (0.98 g, 2.5 mmole) and Ph3P (0.8 g, 3.0 mmole) were dissolved in CCl4/CH2Cl2 (10 mL/10 mL), and were heated at reflux for 3 hrs. The reaction mixture was cooled to room temperature, followed by addition of #1154-132 (0.62 g, HCl salt, 2.5 mmole) and DBU (0.78 g, 5.1 mmole). The resulting mixture was then stirred at room temperature for 2 days, washed with 5%, citric acid aqueous solution, dried, concentrated and purified on silica gel. The isolated product contained Ph3P═O, without further purification and used for next reaction.
Compound #1154-138: The crude #1154-133 (Theoretically 2.5 mmole) was treated with saturated NH3 in CH3OH at room temperature for 3 days, concentrated to dryness, and purified in silica gel, yield 0.28 g of 28% yield of product (2 steps). 1H NMR (CD3OD): δ 7.56 (bs, 1H), 6.78-6.85 (m, 2H), 6.64-6.70 (m, 1H), 6.19 (t, 1H, J=6.4 Hz), 4.54 (s, 2H), 4.35-4.40 (m, 1H), 3.88-3.94 (m, 1H), 3.60-3.85 (m, 4H), 3.62 (t, 2H), 2.22-2.35 (m, 1H), 2.10-2.20 (m, 1H).
Compound #1154-148: A CH3OH solution (25 mL) of Compound #1154-138 (160 mg, 0.38 mmole) was hydrogenated (H2 in a balloon) in the presence of 10% Pd/C at room temperature for 2 hrs. The catalyst was filtered off, washed with CH3OH. The filtrate was concentrated to dryness to afford #1154-148. 1H NMR (CD3OD): δ 7.63 (s, 1H), 6.81-6.86 (m, 2H), 6.70-6.74 (m, 1H), 6.22 (t, 1H), 4.65 (s, 2H), 4.35-4.40 (m, 1H), 3.89-3.93 (m, 1H), 3.68-3.84 (m, 2H), 3.55 (t, 2H), 2.80-2.90 (m, 2H), 2.25-2.36 (m, 1H), 2.10-2.21 (m, 1H).
Compound #1154-149: A DMF solution (2 mL) of compound #1154-148 (170 mg, 0.43 mmole) was treated with FMOC-NHS (150 mg, 0.52 mmole). After 2 hr reaction at room temperature the reaction mixture was diluted with CH2Cl2 (10 mL) containing pyridine (0.7 g, 8.7 mmole), followed by addition of DMT-Cl (0.22 g, 0.65 mmole). After 1 hr, the reaction was worked up and purified by flash column chromatography, to yield 232 mg, 58.2% of 5′-O-DMT-N-FMOC derivative #1154-149. FAB HRMS calculated for C54H51N4O10 (M+H+) 915.360, found 915.359.
3′-5′-Diacetyl-O4-sulfonyl-2′-deoxyuridine (#1154-136): A CH2Cl2 solution (10 mL) of 3′,5′-diacetyl-2′-deoxyuridine (0.76 g, 2.4 mmole), 2-mesitylenesulfonyl chloride (1.0 g, 4.8 mmole), TEA (1.23 g, 12.1 mmole) and catalytic amount of DMAP (0.1 g) was stirred at room temperature overnight. The reaction mixture was diluted with CH2Cl2, washed with 5% citric acid aqueous solution, dried and purified on silica gel, eluted with CH2Cl2, 35% ethyl acetate in CH2Cl2, to give the title compound, 0.76 g in 63% yield. 1H NMR (CDCl3): δ 8.0 (d, 1H, J=9 Hz), 6.99 (s, 10 2H), 6.13 (d, 1H, J=9 Hz), 6.07 (t, 1H, J=6.0 Hz), 5.15-5.20 (m, 1H), 4.33 (s, 3H), 2.70-2.85 (m+s, 7H), 2.73 (s, 3H), 2.09 (s, 3H), 1.98-2.07 (m+s, 4H).
3′-5′-O-(1,1,3,3-Tetraisopropyl-1,3-disiloxyanediyl)-9″-(aminoethoxy)phenoxazine (#1154-135): Compound 14 (see preparation of 12a and 13a) was dissolved in DMF/pyridine (2 mL/2 mL) followed by addition of 1,1,3,3-tetraisopropyl-dichlorodisilane (0.28 g, 0.91 mmole). The stirring was continued at room temperature for 3 hr. The reaction mixture was concentrated to dryness. The residue was used for next reaction without further purification.
Compound #1154-137: A CH2Cl2 solution (10 mL) of compound #1154-136 (0.3 g, 0.61 mmole), compound #1154-135 (crude, 0.61 mmole), and DBU (0.46 g, 3.0 mmole) was stirred at room temperature overnight. The reaction mixture was washed with 5% citric acid aqueous solution, dried, and purified by flash column chromatography (silica gel, CH3OH/CH2Cl2), to give 0.21 g, 38% of product #1154-137. FAB LRMS calculated for C42H61N6O13Si2 (M+H+) 912, found 913.
Compound #1154-144. Compound #1154-137 (0.20 g, 0.22 mmole) in THF 1 mL, was treated with 1 M Bu4NF in CH2Cl2 solution (0.87 mL, 0.87 mmole) at room temperature for 30 min, then concentrated to dryness. The residue was redissolved in CH2Cl2 (5 mL) containing pyridine (173 mg; 2.2 mmole), followed by addition of DMT-Cl (330 mg, 1 mmole). After 2 hrs, the reaction mixture was washed with saturated NaHCO3 aqueous solution, dried, and purified by flash column chromatography (silica gel, CH3OH/CH2Cl2) to yield 0.14 g, 65% of product #1154-144. FAB HRMS calculated for C51H53N6O14 (M+H+) 973.362, found 973.364.
Antisense inhibition of T-antigen expression. The tested DNA oligonucleotide analogs, had the base sequence shown in Table I below. This sequence is complementary to a the 12 base RNA sequence expressed by the large SV40 virus large T antigen gene, 5′ GTA GTG AGG AGG 3′ (SEQ ID NO. 1). In the tested oligonucleotides, which are shown in Table I, each linkage was a phosphorothioate linkage and all sugars were 2′-deoxyribose.
The tested oligonucleotides had the base sequences shown in Table I. In the Table I oligonucleotides, bases were designated as follows.
Base | |
abbreviation | base |
C | 5-methylcytnxine |
U | 5-(1-propynyl)uracil |
A | adenine |
T | thymine |
D | 5-(1-propynyl)cytosine |
Z | structure (58) |
V | structure (61) |
X | structure (57) |
Y | structure (60) |
The Tm values obtained from the RNA hybrids and the intracellular IC50 values for T-antigen inhibition derived from microinjection analysis are shown below. In all cases, β-galactosidase inhibition was concurrently tested as an internal control and we saw no inhibition at 5 μM, the highest concentration tested. We measured inhibition of T antigen expression in CV-1 cells in tissue culture essentially as described (Wagner et al., “Science” 260:1510-1513 1993). We measured the ΔTm (° C.) values relative to control ODN1 essentially as described (Jones et al. “JOC” 58:2983 1993).
TABLE I | ||||
ODN | ΔTm | IC50 | ||
# | BASE | ODN sequence 5′ to 3′ | (° C.)* | (μM) |
1 | C control | CCU-CCU-CAC-UAC | SEQ ID NO. 2 | (65.0) | 0.5-1.0 |
2 | D control | DDU-DDU-DAD-UAD | SEQ ID NO. 3 | +13.0 | 0.25 |
3 | (57) | XCU-CCU-CAC-UAC | SEQ ID NO. 4 | +2.5 | 1.0 |
4 | (57) | CXU-CCU-CAC-UAC | SEQ ID NO. 5 | +9.5 | 0.05 |
5 | (57) | CCU-XCU-CAC-UAC | SEQ ID NO. 6 | +6.5 | 0.1 |
6 | (57) | CCU-CXU-CAC-UAC | SEQ ID NO. 7 | +9.0 | 0.05 |
7 | (57) | CCU-CCU-XAC-UAC | SEQ ID NO. 8 | +6.5 | 0.05-0.1 |
8 | (57) | CCU-CCU-CAX-UAC | SEQ ID NO. 9 | +5.0 | 0.1 |
9 | (57) | CCU-CCU-CAC-UAX | SEQ ID NO. 10 | +5.5 | 2.0 |
10 | mismatch | CCX-CCU-UAC-UAC | SEQ ID NO. 11 | −12.5 | >0.5 |
11 | T control | CCT-CCT-CAC-TAC | SEQ ID NO. 12 | −11.0 | >1.0 |
12 | T-(57) | CCT-CCT-XAC-TAC | SEQ ID NO. 13 | −2.0 | 0.25 |
16 | D | CCU-CCU-DAC-UAC | SEQ ID NO. 14 | 0 | 0.5-1 |
17 | (60) | CCU-CCU-YAC-UAC | SEQ ID NO. 15 | +0.5 | >0.5 |
18 | (58) | CCU-CCU-ZAC-UAC | SEQ ID NO. 16 | +5.0 | 0.3 |
19 | (61) | CCU-CCU-VAC-UAC | SEQ ID NO. 17 | +1.0 | 0.35 |
*ΔTm Relative to control ODN1 |
The results above show that the presence of an invention base elicits potent and specific antisense inhibition of target gene expression (compare ODN1 vs. ODN4 and ODN6). Incorporation of one invention base at an internal C position resulted in a potency enhancement which exceeds that obtained from the substitution of seven 5-(1-propynyl)cytosine bases for 5-methylcytosine (compare ODN2 and ODN4). The ODN12 results showed that oligonucleotides containing limited base substitutions and only a single invention base may have significant antisense potency. Phosphorothioate linked oligodeoxynucleotides containing propynyl-substituted pyrimidine bases were previously the most potent class of antisense agents that workers had described.
Oligonucleotides containing a base of structure (3) at an internal position in the oligonucleotide where R2 was (a) the (S) isomer of —O—CH2—C*H(CH3)—NH2 or (b) the (R) isomer of —O—CH2—C*H(CH3)—NH2 were prepared and tested for binding affinity in a similar manner. Both oligonucleotides had an increased binding affinity compared to the control oligonucleotide.
Antisense inhibition of p27kip1 expression. The sequence of ODN13 was 5′ UGGCUCUCCUGCGCC 3′ (SEQ ID NO. 18) and it contained phosphorothioate linkages, all sugars were 2′-deoxyribose, all G positions contained guanine, all U positions contained 5-(1-propynyl)uracil and all C positions contained 5-(1-propynyl)-cytosine. ODN14 had the same sequence and base composition as ODN13, except that all C positions contained 5-methylcytosine instead of 5-(1-propynyl)cytosine and thymidine instead of 5-(1-propynyl)uracil. ODN15 had the same sequence and base composition as ODN14, except that the 9th C position from the 5′ end contained a (57) base instead of 5-methylcytosine. Each ODN was tested for its capacity to inhibit p27 gene expression in CV-1 cells in cell culture using cationic lipid to deliver the oligonucleotides into the cells, essentially as described (Coats “Science” 272:877-880, 1996).
ODN | IC50(nM) | ||
13 | 10 | ||
14 | >20 | ||
15 | <5 | ||
The results showed that ODN15 containing one (57) base is more potent than the all-propyne derivative ODN13. Previously, phosphorothioate-linked oligonucleotides containing 5-1(propynyl)-modified pyrimidine bases were the among the most potent reported class of antisense agents.
ODN13 was tested in rats and, in a 10 day toxicological evaluation, it was found that the MTD (maximum tolerated dose) was 0.6 mg/kg/d i.v. ODN15 was tested in the same manner with no toxicity observed at a dose of 6.0 mg/kg/d i.v.
Increased oligonucleotide binding affinity and specificity. We made a 10-mer oligonucleotide, ODN22, having the base sequence, 5′ TCTCCCTCTC 3′ (SEQ ID NO. 19). ODN22 contained only phosphorothioate linkages, all sugars were 2′-deoxyribose, bases designated T were thymine, bases designated C were 5-methylcytosine. ODN23 was the same as ODN22, except that the 5th base position from the 5′ end contained 5-(1-propynyl)cytosine (base designated “D” in Table IV). ODN24 was the same as ODN22, except that the 5th base position from the 5′ end contained a structure (60) base. ODN25 was the same as ODN22, except that the 5th base position from the 5′ end contained a structure (55) base. ODN26 was the same as ODN22, except that the 5th base position from the 5′ end contained a structure (57) base. ODN27 was the same as ODN22, except that the 5th base position from the 5′ end contained a structure (59) base. ODN28 was the same as ODN22, except that the 5th base position from the 5′ end contained a structure (58) base. ODN29 was the same as ODN22, except that the 5th base position from the 5′ end contained a structure (61) base. ODN30 was the same as ODN22, except that the 5th base position from the 5′ end contained a structure (65) base.
We measured the Tm (° C.) of ODN22-ODN30 using 4 different oligonucleotides: A complementary RNA oligonucleotide (ODN31), an RNA oligonucleotide (ODN32) having adenine at the 11th position from its 5′ end, i.e., a single A: test base mismatch, a complementary DNA oligonucleotide (ODN33), and a DNA oligonucleotide (ODN34) having adenine at the 11th position from its 5′ end. We measured the ΔTm (° C.) values are relative to control ODN22 essentially as described (Jones “JOC” 58-2983 1993). The results are shown in Table II.
TABLE II |
Thermal Denaturation Data for 9-Modified Phenoxazine ODNs |
Target DNA (33)/RNA (31): 5′-AAA-AAG-AGA-GGG-AGA (SEQ ID NO. 21, 22) |
Target DNA (34)/RNA (32): 5′-AAA-AAG-AGA-GAG-AGA (SEQ ID NO. 23, 24) |
ΔTm | ΔTm | ||||||||
ODN | test base | 31 | ΔTm* | 32 | (31 − 32) | 33 | ΔTm* | 34 | (33 − 34) |
22 | C control | 61.5 | — | 42.5 | 19.0 | 50.5 | — | 32.0 | 18.5 |
23 | D (control) | 65.0 | 3.5 | 44.5 | 20.5 | 54.0 | 3.5 | 33.0 | 21.0 |
24 | (60) (control) | 66.5 | 5.0 | 50.0 | 16.5 | 57.0 | 6.5 | 44.5 | 12.5 |
25 | (55) | 73.5 | 12.0 | 56.0 | 17.5 | 63.5 | 13.0 | 44.0 | 19.0 |
26 | (57) | 77.5 | 16.0 | 52.0 | 25.5 | 68.5 | 18.5 | 43.0 | 25.5 |
27 | (59) | 74.0 | 12.5 | 51.0 | 23.0 | — | — | — | — |
28 | (58) | 73.5 | 12.0 | 52.5 | 21.0 | — | — | — | — |
29 | (61) | 70.5 | 9.0 | 55.0 | 15.5 | — | — | — | — |
30 | (65) | 61.5 | 0 | 44.0 | 17.5 | — | — | — | — |
ΔTm relative to ODN22. |
This data demonstrates the enhancement in melting point afforded by oligonucleotides containing invention bases. The increased ΔTm(31-32) and ΔTm(33-34) values obtained with invention bases (57), (58) and (59) indicate that these invention bases have an increased binding specificity compared to 5-methylcytosine or 5-(1-propynyl)cytosine.
Increased potency of gene expression inhibition. We made a 20-mer phosphorothioate-linked DNA oligonucleotide, 5′ TCC-CGC-XTG-TGA-CAT-CGA-TT 3′ (SEQ ID NO. 25), was X was a structure (57) base. The oligonucleotide was complementary to the 3′ untranslated region of the c-raf mRNA. A control oligonucleotide had the same sequence except that the X base was replaced with cytosine. Each oligonucleotide was tested to determine its potency at inhibiting expression c-raf gene expression essentially as described (Monia “Nature Med” 2:668-675 1966, WO 97/32604). Briefly, a range of concentrations of each oligonucleotide was transfected into A549 small lung carcinoma cells on two consecutive days, followed by preparing cell extracts 48 hours after the first transfection. Immunoblot assay for c-raf protein expression showed the control oligonucleotide reduced c-raf protein expression with an IC50 of about 20 nM. The test oligonucleotide containing the structure (57) base in place of cytosine was at least 20-fold more potent and had an IC50 of less than 1 nM.
Similar assays using an oligonucleotide containing about 8-18 bases that are complementary to raf or c-raf, e.g., the oligonucleotide sequence used in this example or a shortened version thereof, is accomplished in a similar manner using invention oligonucleotides containing 1, 2 or 3 invention bases having an R2 moiety that increases binding affinity compared to a control oligonucleotide containing cytosine.
Claims (30)
and tautomers, solvates and salts thereof, wherein
R1 is an oligonucleotide, a protecting group, a linker or —H;
R2 is A(Z)X1, wherein A is a spacer and Z independently is a label bonding group optionally bonded to a detectable label, but R2 is not amine NH2, protected amine NH2, nitro or cyano;
wherein Ra is independently —CH═, —N═, —C(C1-8alkyl)═ or —C(halogen)═, but no adjacent Ra are both —N═;
R34 is —O—, —S— or —N(CH3)—; and
X1 is 1, 2 or 3.
2. The compound of claim 1 wherein R2 is —R2C—R2D, wherein R2C is a short spacer chain and R2D is a hydrogen bond donor moiety or a moiety having a net positive charge of at least about +0.5 at pH 6-8 in aqueous solutions.
3. The compound of claim 1 wherein R2 is —R6—(CH2)tNR5C(NR5)(NR3)2, —R6 —(CH 2)t NR 5 C(NR 5)N(R 3)2, —R6—CH2—CHR31—N(R3)2, —R6—(R7)v—N(R3)2, —R6—(CH2)t—N(R3)2, —(CH2)1-2—O—(CH2)t—N(R3)2,
R3 is independently —H, —CH3, —CH2CH3, —(CH2)w—N(R33)2 or a protecting group, or both R3 together are a protecting group, or when R2 is —R6—(CH2)t—N(R3)2, one R3 is —H, —CH3, —CH2CH3, a protecting group or —(CH2)w—N(R33)2 and the other R3 is —H, —CH3, —CH2CH3, —(CH2)w—N(R33)2, —CH(N(R33)2)—N(R33)2,
R5 is independently H or a protecting group;
R6 is independently —S—, —NR5—, —O— or —CH2—;
R7 is independently linear alkyl having 1, 2, 3 or 4 carbon atoms optionally substituted with one —CH≡CH—, —C═C— or —CH2—O—CH2— moiety, or R7 is cyclic alkyl having 3, 4 or 5 carbon atoms, wherein one of the linear alkyl carbon atoms is optionally substituted with a single —CH3, —CN, ═O, —OH or protected hydroxyl, provided that the carbon atoms in any —CH═CH— or —CH2—O—CH2— moiety are not substituted with ═O, —OH or protected hydroxyl;
R8 is linear alkylene having 1 or 2 carbon atoms wherein one alkylene carbon atom is optionally substituted with a single —CH3, —CN, ═O, —OH or protected hydroxyl, or R8 is absent;
R28 is independently —CH2—, —CH(CH3)—, —CH)-(OCH3)—, —CH(OR5)— or —O—, but both are not —O—;
R29 is independently —N—, —N(CH3)—, —CH—, —C(CH3)—, but both are not —N(CH3)—;
R30 is —H or —N(R3)2;
R31 is the side chain of an amino acid;
R33 is independently —H, —CH3, —CH2CH3 or a protecting group;
R35 is H, C1-C4 alkyl or a protecting group;
R36 is H, —CH3, —CH2CH3, a protecting group or an optionally protected monosaccharide;
t is 1, 2, 3 or 4, but when R6 is —O—, —S— or —NR5—, t is 2, 3 or 4;
v is independently 0, 1 or 2; and
w is independently 1 or 2.
4. The compound of claim 3 wherein R2 is —CH2—(CH2)tN(R3)2, —NR5—(CH2)tN(R3)2, —S—(CH2)tN(R3)2, —O—(CH2)tN(R3)2, —O—(CH2)tNR5C(NR5)(NR3)2, —O—(CH 2)t NR 5C(NR5)N(R3)2, —(CH2)1-2—O—(CH2)tN(R3)2, —R6—CH2—CHR31—N(R3)2, —R6—(R7)v—N(R3)2, —R6—(CH2)t—NR5C(NR5)(NR3)2, —R6 —(CH 2)t—NR 5 C(NR5)N(R3)2, or —CH2(CH2)tNR5C(NR5)(NR3)2. —CH2—(CH 2)tNR 5C(NR5)N(R3)2.
5. The compound of claim 4 wherein t is 2.
6. The compound of claim 5 wherein R3 independently is —H, —CH3—C2H5 or a protecting group.
7. The compound of claim 6 wherein R2 is —O—(CH2)2—NH2, —O—(CH2)3—NH2, —O—(CH2)2—N(CH3)2, —O—(CH2)3—N(CH3)2, —O—(CH2)2—NHCH3, —O—(CH2)3—NHCH3, —O—CH2—CH(CH3)—NH2, —CH2—O—(CH2)2—NH2, —CH2—O—(CH2)3—NH2 or —(CH2)2—O—(CH2)2—NH2.
8. The compound of claim 3 wherein t is 2 or 3.
9. The compound of claim 1 wherein R1 comprises —H, an optionally protected monosaccharide, hydroxyl, phosphate or hydrogen phosphonate.
10. The compound of claim 1 wherein R1 is optionally protected 2′-deoxy-R21-substituted ribose, 2′-deoxyribose or ribose, wherein R21 is H, —OH, halogen or a moiety that enhances the nuclease stability of an oligonucleotide containing the optionally protected 2′-deoxy-R21-substituted ribose, 2′-deoxyribose or ribose.
11. The compound of claim 1 having the structure designated by the numbers selected from the group consisting of (104), (105), (133), (134), (111), (112), (113), (115) ( 114 ), (135), (136), (137), (138), (139), (120), (121), (121A), (143), (122), (123), (125), or (126):
wherein
R1 is an optionally protected monosaccharide;
R2A is —OH;
R5 is independently —H or a protecting group;
R6 is —O—, —S—, —NH— or —CH2—,
R21 is H, —OH, halogen or a moiety that enhances the nuclease stability of an oligonucleotide;
R24 is a halogen;
where Ra is independently —CH═, —N═, —C(C1-8alkyl)═ or —C(halogen)═, but no adjacent Ra are both —N═;
R34 is —O—, —S— or —N(CH3)—;
R37 is —O—, —CH2— or —CF2—;
R47 is —O— or —S—;
R50 is —CH2—, —C(O)—, —(CH2)—O—(CH2)2— —(CH2)2—O—(CH2)2—, —(CH2)2—NR5—(CH2)2—, —(CH2)2—S—(CH2)2—, —CH(N(R5)2)—, —CH(COOR5)— or —C(CH3)—, —C(C2C5)— but adjacent moieties are not C(O);
R52 is —(CHR52A)—(R52B)—CHR52A—, —CHR52A—O—CHR52A—, —CHR52A—S—CHR52A—, —CHR52A—NR5—CHR52A—, C1-C10 alkylene optionally substituted with 1 or 2 moieties selected from the group consisting of C1-C6 alkyl, —OR5, ═O, —NO2, —N3, —CN, —COOR5, or —N(R5)2, wherein any heteroatom is separated from the nitrogen atoms that R52 is linked to by one methylene and one or more —CHR52A—;
R52A is —H or C1-C6 alkyl;
R52B is a bond;
R59 is —R6—R60—;
R60 is —(CH2)Z3—(R61)Z1—(CH2)Z2—;
R61 is —O—, —S—, —NR5—, —C(O)—, —CH2—O—CH2—, —CH2—NR5—CH2— or CH2—S—CH2—;
Z1 is 0 or 1;
Z2 is 1, 2 or 3;
Z3 is 1, 2 or 3;
Y is 1, 2, 3 or 4;
CBZ is carboxybenzoyl;
Fmoc is 9-fluorenylmethoxycarbonyl;
iPr is isopropyl; and
Ac is acetyl.
wherein
D is —OH, protected —OH, an oligonucleotide coupling group or a solid support;
D1 is an oligonucleotide coupling group, —OH, protected —OH or a solid support, wherein D1 is bonded to one 2′ or 3′ position in the oligonucleotide of structure (2) and the adjacent 2′ or 3′ position in structure (2) is substituted with R21, provided that D and D1 are not both an oligonucleotide coupling group or they are not both a solid support;
R4 is independently a phosphodiester linkage or a phosphodiester substitute linkage, wherein R4 is bonded to one 2′ or 3′ position in the structure (2) oligonucleotide and the adjacent 2′ or 3′ position in structure (2) is substituted with R21;
R21 is independently —H, —OH, halogen or a moiety that enhances the oligonucleotide against nuclease cleavage;
R37 is independently —O—, —CH2—, —CF2—;
n is an integer from 0 to 98; and
13. The compound of claim 12 wherein R4 is independently 3′-O—P(S)(S)—O-5′, 3′-O—P(S)(O)—O-5′, 3′-O— P(O)(O)—O-5′, 3′-O—P(Me)(O)—O-5′, 3′-NH—P(O)(O)—O-5′, 3′-S—CH2—O-5′, 2′-S—CH2—O-5′, 3′-O—CH2—O-5′, 2′-O—CH2—O-5′, 3′O—P(Me)(S)—O-5′, 3′-CH2—N(CH3)—O-5′, 2′-CH2—N(CH3)—O-5′, or 3′-R38—P(N2)(O)—O-5′, wherein
R38 independently is —O—, —CH2— or —NH—;
R39 is a protecting group; .
or both R40 together are a protecting group;
R41 independently is hydrogen, a protecting group, alkyl (C1-C4 or both R41 together are a protecting group;
R42 is hydrogen or a protecting group;
R46 is alkyl containing 1-8 carbon atoms.
14. The compound of claim 12 wherein R2 is —R6—(CH2)tNR5C(NR5)(NR3)2, —R6 —(CH2)tNR 5 C(NR5)N(R3)2, —R6—CH2—CHR31—N(R3)2, —R6—(R7)v—N(R3)2, —R6—(CH2)t—N(R3)2, —(CH2)1-2—O—(CH2)t—N(R3)2,
R3 is independently —H, —CH3, —CH2CH3, —(CH2)w—N(R33)2 or a protecting group, or both R3 together are a protecting group, or when R2 is —R6—(CH2)t—N(R3)2 —R6—(CH2)t—N(R33)2, one R3 is —H, —CH2CH3, a protecting group or —(CH2)w—N(R33)2 and the other R3 is —H, —CH3, —CH2CH3, —(CH2)w—N(R33)2, —CH(N[R33]2)—N(R33)2,
R5 is independently H or a protecting group;
R6 is independently —S—, —NR5—, —O— or —CH2—;
R7 is independently linear alkyl having 1, 2, 3 or 4 carbon atoms optionally substituted with one —CH═CH—, —C≡C— or —CH2—O—CH2— moiety, or R7 is cyclic alkyl having 3, 4 or 5 carbon atoms, wherein one of the linear alkyl carbon atoms is optionally substituted with a single —CH3, —CN, ═O, —OH or protected hydroxyl, provided that the carbon atoms in any —CH═CH— or —CH2—O—CH2— moiety are not substituted with ═O, —OH or protected hydroxy;
R8 is linear alkylene having 1 or 2 carbon atoms wherein one alkylene carbon atom is optionally substituted with a single —CH3, —CN, ═O, —OH or protected hydroxyl, or R8 is absent;
R28 is independently —CH2—, —CH(CH3)—, —CH(OCH3)—, —CH(OR5)— or —O—, but both are not —O—;
R29 is independently —N—, —N(CH3)—, —CH—, —C(CH3)—, but both are not —N(CH3)—;
R30 is —H or —N(R3)2;
R31 is the side chain of an amino acid;
R33 is independently —H, —CH3, —CH2CH3 or a protecting group;
R35 is H, C1-C4 alkyl or a protecting group;
R36 is —H, —CH3, —CH2CH3, a protecting group or an optionally protected monosaccharide;
t is 1, 2, 3 or 4, but when R6 is —O—, —S— or —NR5—, t is 2, 3 or 4;
v is independently 0, 1 or 2; and
w is independently 1 or 2.
15. The compound of claim 14 wherein R2 is —CH2—(CH2)tN(R3)2, —NR5—(CH2)tN(R3)2, —S—(CH2)tN(R3)2, —O—(CH2)tN(R3)2, —O—(CH2)tNR5C(NR5)(NR3)2, —O—(CH 2)tNR 5C(NR5)N(R3)2, —(CH2)1-2—O—(CH2)tN(R3)2, —R6—CH2—CHR31—N(R3)2, —R6—(R7)v—N(R3)2, —R6—(CH2)t—NR5C(NR5)(NR3)2, —R6—(CH2)tNR 5C(NR5)N(R3)2, or —CH2—(CH2)tNR5C(NR5)(NR3)2. —CH2—(CH2)tNR 5C(NR5)N(R3)2.
16. The compound of claim 15 wherein t is 2 or 3.
17. The compound of claim 16 wherein R3 independently is —H, —CH3, —C2H5 or a protecting group.
18. The compound of claim 17 wherein R2 is —O—(CH2)2—NH2, —O—(CH2)3—NH2, —O—(CH2)2—N(CH3)2, —O—(CH2)3—N(CH3)2, —O—(CH2)2—NHCH3, —O—(CH2)3—NHCH3, —O—CH2—CH(CH3)—NH2, —CH2—O—(CH2)2—NH2, —CH2—O—(CH2)3—NH2 or —(CH2)2—O—(CH2)2—NH2.
19. The compound of claim 12 wherein R21 is independently —H, —OH, halogen, protected hydroxyl, —O-methyl, —O-ethyl, —O-n-propyl, —O-allyl, —O—(CH2)2OH, —O—(CH2)3OH, —O—(CH2)2F, —O—(CH2)sR65, —O—(CH2)2—[O—(CH2)2]r—R65, —O—(CH2)r—O—(CH2)r—O—(CH2)r—R65) —O—(CH2)t—O—(CH2)r—O—(CH2)r—R 65, —NH-methyl, —NH-ethyl, —NH-n-propyl, —NH—(CH2)2OH, —NH—(CH2)3OH, —NH—(CH2)sR65, —S-methyl, —S-ethyl, —S-n-propyl, —S-allyl, —S—(CH2)2OH, —S—(CH2)3OH —S—(CH2)2—F, —S—(CH2)s—R65, or —S—(CH2)2—[O—(CH2)2]rR65, wherein
R65 is —H, —F, —OH, —OCH3, —NH2, —SH, protected hydroxyl, protected amino or protected thiol;
r is 1, 2, 3 or 4; and
s is 2, 3, 4, 5, 6, 7 or 8.
20. The compound of claim 19 wherein R21 is independently —H, —OH, —F, protected hydroxyl, —OCH3, —O—CH2CH3, —O—CH2CH2OH, —O—CH2CH2F, —O—CH2CH2CH3, —O—CH2CH2CH2OH, —O—CH2CH2CH2F, —O—CH2CF2H, —O—CH2CF3 or —O—CH2CH2—O—CH3.
21. The compound of claim 12 wherein B independently are selected from the group consisting of a base of structure (3), guanosine, adenine, thymine, uracil, cytosine, 5-methylcytosine, 5-(1-propynyl)uracil, 5-(1-propynyl)cytosine, 5-(1-butynyl)uracil therefore 5-(1-butynyl)cytosine.
22. The compound of claim 12 wherein D1 is H-phosphonate, a methylphosphonamidite, a β-cyanoethylphosphoramidite or a phosphoramidite.
24. The compound of claim 23 wherein R1 is —H or an optionally protected monosaccharide.
25. The compound of claim 24 wherein the optionally protected monosaccharide is 2′-deoxy-R21-substituted ribose, wherein R21 is H, —OH, halogen or a moiety that enhances the nuclease stability of an oligonucleotide containing the optionally protected 2′-deoxy-R21-substituted ribose, 2′-deoxyribose or ribose.
26. The compound of claim 25 wherein R21 is —H, —OH, —F, protected hydroxyl, —OCH3, —O—CH2CH3, —O—CH2CH2OH, —O—CH2CH2F, —O—CH2CH2CH3, —O—CH2CH2CH2OH, —O—CH2CH2CH2F, —O—CH2CF2H, —O—CH2CF3 or —O—CH2CH2—O—CH3.
and tautomers, solvates and salts thereof, wherein
R1 is a protecting group, an oligonucleotide, a nucleic acid, a polysaccharide, an optionally protected monosaccharide, hydroxyl, phosphate, hydrogen phosphonate, halo, azido, protected hydroxyl, or —H;
R2 is A(Z)X1, but R2 is not amine, protected amine, nitro or cyano;
R5 is H or a protecting group;
R27 is indepedently —CH═, —N═, —C(C1-C8alkyl)═ or —C(halogen)═, but no adjacent R27 are both —N═, or two adjacent R27 are taken together to form a ring having the structure,
R34 is —O—, —S— or —N(CH3)—;
Ra is independently —CH═, —N═, —C(C1-8 alkyl)═ or —C(halogen)═, but no adjacent Ra are both —N═;
A is a backbone chain of 2-16 carbon atoms, any 1, 2 or 3 of which are optionally replaced with N, O or S atoms, wherein the backbone chain is optionally substituted independently with 1, 2 or 3 of the following: C1-C8 alkyl, —OR5, ═O, —NO2, —N3, —COOR5, —N(R5)2, or —CN groups, C1-C8 alkyl substituted with —OH, ═O, —NO2, —N3, —COOR5, —N(R5)2, or —CN groups, or any of the foregoing in which —CH2— is replaced with —O—, —NH— or —N(C1-C8 alkyl);
X1 is 1, 2 or 3;
Y is H, 2-hydroxypyridine, N-hydroxysuccinimide, p-nitrophenyl, acylimidazole, maleimide, trifluoroacetate, an imido, a sulfonate, an imine 1,2-cyclohexanedione, glyoxal or an alpha-halo ketone; and
Z independently is —NH2, —CHO, —SH, —CO2Y, OY.
28. The compound of claim 27 wherein Z is bonded to a detectable label.
29. The compound of claim 27 wherein R1 is an oligonucleotide.
30. The compound of claim 27 wherein R1 is an optionally protected monosaccharide.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/080,074 USRE39324E1 (en) | 1997-11-07 | 2002-02-21 | Pyrimidine derivatives and oligonucleotides containing same |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/966,392 US6028183A (en) | 1997-11-07 | 1997-11-07 | Pyrimidine derivatives and oligonucleotides containing same |
US10/080,074 USRE39324E1 (en) | 1997-11-07 | 2002-02-21 | Pyrimidine derivatives and oligonucleotides containing same |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US08/966,392 Reissue US6028183A (en) | 1997-11-07 | 1997-11-07 | Pyrimidine derivatives and oligonucleotides containing same |
Publications (1)
Publication Number | Publication Date |
---|---|
USRE39324E1 true USRE39324E1 (en) | 2006-10-03 |
Family
ID=25511327
Family Applications (6)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US08/966,392 Ceased US6028183A (en) | 1997-11-07 | 1997-11-07 | Pyrimidine derivatives and oligonucleotides containing same |
US09/400,502 Expired - Lifetime US6414127B1 (en) | 1997-11-07 | 1999-09-21 | Pyrimidine derivatives for labeled binding partners |
US10/080,074 Expired - Lifetime USRE39324E1 (en) | 1997-11-07 | 2002-02-21 | Pyrimidine derivatives and oligonucleotides containing same |
US10/152,123 Expired - Fee Related US6800743B2 (en) | 1997-11-07 | 2002-05-21 | Pyrimidine derivatives for labeled binding partners |
US10/294,181 Expired - Lifetime US6951931B2 (en) | 1997-11-07 | 2002-11-14 | Pyrimidine derivatives for labeled binding partners |
US10/921,734 Expired - Fee Related US7511125B2 (en) | 1997-11-07 | 2004-08-19 | Pyrimidine derivatives for labeled binding partners |
Family Applications Before (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US08/966,392 Ceased US6028183A (en) | 1997-11-07 | 1997-11-07 | Pyrimidine derivatives and oligonucleotides containing same |
US09/400,502 Expired - Lifetime US6414127B1 (en) | 1997-11-07 | 1999-09-21 | Pyrimidine derivatives for labeled binding partners |
Family Applications After (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/152,123 Expired - Fee Related US6800743B2 (en) | 1997-11-07 | 2002-05-21 | Pyrimidine derivatives for labeled binding partners |
US10/294,181 Expired - Lifetime US6951931B2 (en) | 1997-11-07 | 2002-11-14 | Pyrimidine derivatives for labeled binding partners |
US10/921,734 Expired - Fee Related US7511125B2 (en) | 1997-11-07 | 2004-08-19 | Pyrimidine derivatives for labeled binding partners |
Country Status (1)
Country | Link |
---|---|
US (6) | US6028183A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013041194A1 (en) | 2011-09-23 | 2013-03-28 | Roche Diagnostics Gmbh | Use of g-clamp for improved allele-specific pcr |
Families Citing this family (148)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7812149B2 (en) * | 1996-06-06 | 2010-10-12 | Isis Pharmaceuticals, Inc. | 2′-Fluoro substituted oligomeric compounds and compositions for use in gene modulations |
US20050053976A1 (en) * | 1996-06-06 | 2005-03-10 | Baker Brenda F. | Chimeric oligomeric compounds and their use in gene modulation |
US5898031A (en) * | 1996-06-06 | 1999-04-27 | Isis Pharmaceuticals, Inc. | Oligoribonucleotides for cleaving RNA |
US20050119470A1 (en) * | 1996-06-06 | 2005-06-02 | Muthiah Manoharan | Conjugated oligomeric compounds and their use in gene modulation |
US20040171031A1 (en) * | 1996-06-06 | 2004-09-02 | Baker Brenda F. | Sugar surrogate-containing oligomeric compounds and compositions for use in gene modulation |
US20040033973A1 (en) * | 2002-08-16 | 2004-02-19 | Muthiah Manoharan | Compounds and oligomeric compounds comprising novel nucleobases |
US20040203024A1 (en) * | 1996-06-06 | 2004-10-14 | Baker Brenda F. | Modified oligonucleotides for use in RNA interference |
US9096636B2 (en) | 1996-06-06 | 2015-08-04 | Isis Pharmaceuticals, Inc. | Chimeric oligomeric compounds and their use in gene modulation |
US20040147022A1 (en) * | 1996-06-06 | 2004-07-29 | Baker Brenda F. | 2'-methoxy substituted oligomeric compounds and compositions for use in gene modulations |
US6028183A (en) * | 1997-11-07 | 2000-02-22 | Gilead Sciences, Inc. | Pyrimidine derivatives and oligonucleotides containing same |
US7098192B2 (en) | 1999-04-08 | 2006-08-29 | Isis Pharmaceuticals, Inc. | Antisense oligonucleotide modulation of STAT3 expression |
US7286724B2 (en) * | 1999-12-06 | 2007-10-23 | Hyundai Curitel, Inc. | Method and apparatus for searching, browsing and summarizing moving image data using fidelity for tree-structure moving image hierarchy |
US20040081959A9 (en) * | 1999-12-08 | 2004-04-29 | Epoch Biosciences, Inc. | Fluorescent quenching detection reagents and methods |
US6727356B1 (en) * | 1999-12-08 | 2004-04-27 | Epoch Pharmaceuticals, Inc. | Fluorescent quenching detection reagents and methods |
DE10010959A1 (en) * | 2000-03-06 | 2001-09-20 | Eppendorf Geraetebau Netheler | Transferring material through cell membranes, useful e.g. in genetic engineering or hydride production, using aqueous solution containing trehalose, increases cell survival |
ATE384072T1 (en) * | 2000-09-26 | 2008-02-15 | Idera Pharmaceuticals Inc | MODULATION OF THE IMMUNOSTIMULATIVE ACTIVITY OF IMMUNOSTIMULATIVE OLIGONUCLEOTIDE ANALOGS THROUGH POSITIONAL CHEMICAL CHANGES |
US20040121310A1 (en) * | 2002-12-18 | 2004-06-24 | Ecker David J. | Methods for rapid detection and identification of bioagents in forensic studies |
US20040121309A1 (en) * | 2002-12-06 | 2004-06-24 | Ecker David J. | Methods for rapid detection and identification of bioagents in blood, bodily fluids, and bodily tissues |
US20030027135A1 (en) * | 2001-03-02 | 2003-02-06 | Ecker David J. | Method for rapid detection and identification of bioagents |
US7666588B2 (en) * | 2001-03-02 | 2010-02-23 | Ibis Biosciences, Inc. | Methods for rapid forensic analysis of mitochondrial DNA and characterization of mitochondrial DNA heteroplasmy |
US20040121314A1 (en) * | 2002-12-06 | 2004-06-24 | Ecker David J. | Methods for rapid detection and identification of bioagents in containers |
US7226739B2 (en) | 2001-03-02 | 2007-06-05 | Isis Pharmaceuticals, Inc | Methods for rapid detection and identification of bioagents in epidemiological and forensic investigations |
US7718354B2 (en) * | 2001-03-02 | 2010-05-18 | Ibis Biosciences, Inc. | Methods for rapid identification of pathogens in humans and animals |
US7217510B2 (en) * | 2001-06-26 | 2007-05-15 | Isis Pharmaceuticals, Inc. | Methods for providing bacterial bioagent characterizing information |
US8073627B2 (en) | 2001-06-26 | 2011-12-06 | Ibis Biosciences, Inc. | System for indentification of pathogens |
US7070933B2 (en) * | 2001-09-28 | 2006-07-04 | Gen-Probe Incorporated | Inversion probes |
US7169916B2 (en) * | 2002-04-01 | 2007-01-30 | Isis Pharmaceuticals, Inc. | Chloral-free DCA in oligonucleotide synthesis |
AU2003269809A1 (en) * | 2002-04-01 | 2003-12-12 | Isis Pharmaceuticals, Inc. | Method for rapid detection and identification of viral bioagents |
WO2004026804A1 (en) * | 2002-09-20 | 2004-04-01 | Integrated Dna Technologies, Inc. | Anthraquinone quencher dyes, their methods of preparation and use |
AU2003291755A1 (en) * | 2002-11-05 | 2004-06-07 | Isis Pharmaceuticals, Inc. | Oligomers comprising modified bases for binding cytosine and uracil or thymine and their use |
AU2003287505A1 (en) | 2002-11-05 | 2004-06-03 | Isis Pharmaceuticals, Inc. | Chimeric oligomeric compounds and their use in gene modulation |
WO2004043367A2 (en) * | 2002-11-06 | 2004-05-27 | Bristol-Myers Squibb Company | Fused heterocyclic compounds and use thereof |
EP1578399A4 (en) | 2002-12-06 | 2007-11-28 | Isis Pharmaceuticals Inc | Methods for rapid identification of pathogens in humans and animals |
US20040121312A1 (en) * | 2002-12-18 | 2004-06-24 | Ecker David J. | Methods for rapid detection and identification of the absence of bioagents |
US20040122857A1 (en) * | 2002-12-18 | 2004-06-24 | Ecker David J. | Secondary structure defining database and methods for determining identity and geographic origin of an unknown bioagent in forensic studies thereby |
US20040121315A1 (en) * | 2002-12-18 | 2004-06-24 | Ecker David J. | Secondary structure defining database and methods for determining identity and geographic origin of an unknown bioagent in containers thereby |
US20040198640A1 (en) * | 2003-04-02 | 2004-10-07 | Dharmacon, Inc. | Stabilized polynucleotides for use in RNA interference |
US8046171B2 (en) * | 2003-04-18 | 2011-10-25 | Ibis Biosciences, Inc. | Methods and apparatus for genetic evaluation |
US8057993B2 (en) | 2003-04-26 | 2011-11-15 | Ibis Biosciences, Inc. | Methods for identification of coronaviruses |
US7964343B2 (en) * | 2003-05-13 | 2011-06-21 | Ibis Biosciences, Inc. | Method for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture |
US8158354B2 (en) | 2003-05-13 | 2012-04-17 | Ibis Biosciences, Inc. | Methods for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture |
US7276599B2 (en) * | 2003-06-02 | 2007-10-02 | Isis Pharmaceuticals, Inc. | Oligonucleotide synthesis with alternative solvents |
CA2524495A1 (en) | 2003-06-03 | 2005-01-13 | Eli Lilly And Company | Modulation of survivin expression |
US20060241072A1 (en) * | 2003-06-20 | 2006-10-26 | Isis Pharmaceuticals, Inc. | Oligomeric compounds for use in gene modulation |
EP1648914A4 (en) | 2003-07-31 | 2009-12-16 | Regulus Therapeutics Inc | Oligomeric compounds and compositions for use in modulation of small non-coding rnas |
US20050053981A1 (en) * | 2003-09-09 | 2005-03-10 | Swayze Eric E. | Gapped oligomeric compounds having linked bicyclic sugar moieties at the termini |
US20100035239A1 (en) * | 2003-09-11 | 2010-02-11 | Isis Pharmaceuticals, Inc. | Compositions for use in identification of bacteria |
US8546082B2 (en) | 2003-09-11 | 2013-10-01 | Ibis Biosciences, Inc. | Methods for identification of sepsis-causing bacteria |
US20120122101A1 (en) * | 2003-09-11 | 2012-05-17 | Rangarajan Sampath | Compositions for use in identification of bacteria |
US20080138808A1 (en) * | 2003-09-11 | 2008-06-12 | Hall Thomas A | Methods for identification of sepsis-causing bacteria |
US8097416B2 (en) | 2003-09-11 | 2012-01-17 | Ibis Biosciences, Inc. | Methods for identification of sepsis-causing bacteria |
US20060240412A1 (en) * | 2003-09-11 | 2006-10-26 | Hall Thomas A | Compositions for use in identification of adenoviruses |
AU2004274021B2 (en) * | 2003-09-18 | 2009-08-13 | Isis Pharmaceuticals, Inc. | 4'-thionucleosides and oligomeric compounds |
PE20050469A1 (en) | 2003-09-18 | 2005-09-10 | Lilly Co Eli | MODULATION OF THE EXPRESSION OF EIF4E |
US7439341B2 (en) | 2003-11-14 | 2008-10-21 | Integrated Dna Technologies, Inc. | Fluorescence quenching azo dyes, their methods of preparation and use |
US8163895B2 (en) | 2003-12-05 | 2012-04-24 | Ibis Biosciences, Inc. | Compositions for use in identification of orthopoxviruses |
US20090280567A1 (en) * | 2004-02-06 | 2009-11-12 | Dharmacon, Inc. | Stabilized sirnas as transfection controls and silencing reagents |
EP2123759B1 (en) * | 2004-02-06 | 2014-01-15 | Thermo Fisher Scientific Biosciences Inc. | Stabilized RNAS as transfection controls and silencing reagents |
US7666592B2 (en) | 2004-02-18 | 2010-02-23 | Ibis Biosciences, Inc. | Methods for concurrent identification and quantification of an unknown bioagent |
US8119336B2 (en) * | 2004-03-03 | 2012-02-21 | Ibis Biosciences, Inc. | Compositions for use in identification of alphaviruses |
US8569474B2 (en) * | 2004-03-09 | 2013-10-29 | Isis Pharmaceuticals, Inc. | Double stranded constructs comprising one or more short strands hybridized to a longer strand |
EP2700720A3 (en) | 2004-03-15 | 2015-01-28 | Isis Pharmaceuticals, Inc. | Compositions and methods for optimizing cleavage of RNA by RNASE H |
KR101147147B1 (en) * | 2004-04-01 | 2012-05-25 | 머크 샤프 앤드 돔 코포레이션 | Modified polynucleotides for reducing off-target effects in rna interference |
WO2005117270A2 (en) | 2004-05-24 | 2005-12-08 | Isis Pharmaceuticals, Inc. | Mass spectrometry with selective ion filtration by digital thresholding |
US20050266411A1 (en) * | 2004-05-25 | 2005-12-01 | Hofstadler Steven A | Methods for rapid forensic analysis of mitochondrial DNA |
EP1766052A4 (en) * | 2004-06-03 | 2009-12-16 | Isis Pharmaceuticals Inc | Chimeric gapped oligomeric compositions |
US8394947B2 (en) * | 2004-06-03 | 2013-03-12 | Isis Pharmaceuticals, Inc. | Positionally modified siRNA constructs |
JP2008501693A (en) * | 2004-06-03 | 2008-01-24 | アイシス ファーマシューティカルズ、インク. | Double-stranded composition with individually regulated strands for use in gene regulation |
US7811753B2 (en) | 2004-07-14 | 2010-10-12 | Ibis Biosciences, Inc. | Methods for repairing degraded DNA |
US7884086B2 (en) | 2004-09-08 | 2011-02-08 | Isis Pharmaceuticals, Inc. | Conjugates for use in hepatocyte free uptake assays |
US7923206B2 (en) * | 2004-11-22 | 2011-04-12 | Dharmacon, Inc. | Method of determining a cellular response to a biological agent |
US7923207B2 (en) | 2004-11-22 | 2011-04-12 | Dharmacon, Inc. | Apparatus and system having dry gene silencing pools |
US7935811B2 (en) * | 2004-11-22 | 2011-05-03 | Dharmacon, Inc. | Apparatus and system having dry gene silencing compositions |
EP1859040A2 (en) * | 2005-02-25 | 2007-11-28 | Isis Pharmaceuticals, Inc. | Compositions and their uses directed to il-4r alpha |
US8084207B2 (en) * | 2005-03-03 | 2011-12-27 | Ibis Bioscience, Inc. | Compositions for use in identification of papillomavirus |
CA2600184A1 (en) * | 2005-03-03 | 2006-09-08 | Isis Pharmaceuticals, Inc. | Compositions for use in identification of adventitious viruses |
US20060223777A1 (en) * | 2005-03-29 | 2006-10-05 | Dharmacon, Inc. | Highly functional short hairpin RNA |
JP2008545659A (en) | 2005-05-20 | 2008-12-18 | インテグレイテッド ディーエヌエイ テクノロジーズ インコーポレイテッド | Compounds and methods for labeling oligonucleotides |
AU2006272776B2 (en) * | 2005-07-21 | 2012-01-19 | Ibis Biosciences, Inc. | Methods for rapid identification and quantitation of nucleic acid variants |
JP5523705B2 (en) | 2005-08-29 | 2014-06-18 | レグルス・セラピューティクス・インコーポレイテッド | Method of using to modulate MIR-122A |
CN101321869A (en) | 2005-09-19 | 2008-12-10 | 强生医药研究及开发有限责任公司 | Modulation of glucagon receptor expression |
JP2009508527A (en) | 2005-09-19 | 2009-03-05 | ジヨンソン・アンド・ジヨンソン・フアーマシユーチカル・リサーチ・アンド・デベロツプメント・エルエルシー | Regulation of glucocorticoid receptor expression |
EP1969143A4 (en) * | 2005-12-20 | 2009-07-22 | Isis Pharmaceuticals Inc | Double stranded nucleic acid molecules targeted to il-4 receptor alpha |
EP2388328A1 (en) | 2006-01-27 | 2011-11-23 | Isis Pharmaceuticals, Inc. | Oligomeric compounds and compositions for the use in modulation of micrornas |
EP2023938A4 (en) * | 2006-05-23 | 2010-11-10 | Isis Pharmaceuticals Inc | Modulation of chrebp expression |
WO2008011473A2 (en) | 2006-07-19 | 2008-01-24 | Isis Pharmaceuticals, Inc. | Compositions and their uses directed to hbxip |
US9149473B2 (en) * | 2006-09-14 | 2015-10-06 | Ibis Biosciences, Inc. | Targeted whole genome amplification method for identification of pathogens |
US8252755B2 (en) * | 2006-09-22 | 2012-08-28 | Dharmacon, Inc. | Duplex oligonucleotide complexes and methods for gene silencing by RNA interference |
EP2126132B1 (en) * | 2007-02-23 | 2013-03-20 | Ibis Biosciences, Inc. | Methods for rapid foresnsic dna analysis |
WO2008118809A1 (en) * | 2007-03-23 | 2008-10-02 | Ibis Biosciences, Inc. | Compositions for use in identification of mixed populations of bioagents |
US9598724B2 (en) | 2007-06-01 | 2017-03-21 | Ibis Biosciences, Inc. | Methods and compositions for multiple displacement amplification of nucleic acids |
US7845686B2 (en) * | 2007-12-17 | 2010-12-07 | S & B Technical Products, Inc. | Restrained pipe joining system for plastic pipe |
US8188060B2 (en) | 2008-02-11 | 2012-05-29 | Dharmacon, Inc. | Duplex oligonucleotides with enhanced functionality in gene regulation |
CN104975020B (en) * | 2008-02-11 | 2020-01-17 | 菲奥医药公司 | Modified RNAi polynucleotides and uses thereof |
WO2009124295A2 (en) * | 2008-04-04 | 2009-10-08 | Isis Pharmaceuticals, Inc. | Oligomeric compounds comprising bicyclic nucleosides and having reduced toxicity |
US8341614B2 (en) * | 2008-06-06 | 2012-12-25 | Apple Inc. | Memory management for closures |
WO2010008582A2 (en) | 2008-07-18 | 2010-01-21 | Rxi Pharmaceuticals Corporation | Phagocytic cell drug delivery system |
WO2010019967A1 (en) * | 2008-08-15 | 2010-02-18 | Georgetown University | Fluorescent cdk inhibitors for treatment of cancer |
EP2347254A2 (en) | 2008-09-16 | 2011-07-27 | Ibis Biosciences, Inc. | Sample processing units, systems, and related methods |
EP2349549B1 (en) | 2008-09-16 | 2012-07-18 | Ibis Biosciences, Inc. | Mixing cartridges, mixing stations, and related kits, and system |
EP2344893B1 (en) | 2008-09-16 | 2014-10-15 | Ibis Biosciences, Inc. | Microplate handling systems and methods |
US10138485B2 (en) | 2008-09-22 | 2018-11-27 | Rxi Pharmaceuticals Corporation | Neutral nanotransporters |
EP2358398A2 (en) | 2008-10-24 | 2011-08-24 | Isis Pharmaceuticals, Inc. | Oligomeric compounds and methods |
US9074211B2 (en) | 2008-11-19 | 2015-07-07 | Rxi Pharmaceuticals Corporation | Inhibition of MAP4K4 through RNAI |
WO2010080616A1 (en) | 2008-12-19 | 2010-07-15 | Abbott Laboratories | Molecular assay for diagnosis of malaria |
JP2012512665A (en) * | 2008-12-19 | 2012-06-07 | アボット・ラボラトリーズ | Diagnostic test for mutations in codons 12-13 of human K-RAS |
WO2010078536A1 (en) | 2009-01-05 | 2010-07-08 | Rxi Pharmaceuticals Corporation | Inhibition of pcsk9 through rnai |
WO2010090762A1 (en) | 2009-02-04 | 2010-08-12 | Rxi Pharmaceuticals Corporation | Rna duplexes with single stranded phosphorothioate nucleotide regions for additional functionality |
US20120021515A1 (en) | 2009-02-06 | 2012-01-26 | Swayze Eric E | Oligomeric compounds and methods |
EP2396803A4 (en) * | 2009-02-12 | 2016-10-26 | Ibis Biosciences Inc | Ionization probe assemblies |
WO2010114842A1 (en) | 2009-03-30 | 2010-10-07 | Ibis Biosciences, Inc. | Bioagent detection systems, devices, and methods |
WO2011008971A1 (en) * | 2009-07-17 | 2011-01-20 | Ibis Biosciences, Inc. | Lift and mount apparatus |
WO2011008972A1 (en) | 2009-07-17 | 2011-01-20 | Ibis Biosciences, Inc. | Systems for bioagent identification |
WO2011014811A1 (en) | 2009-07-31 | 2011-02-03 | Ibis Biosciences, Inc. | Capture primers and capture sequence linked solid supports for molecular diagnostic tests |
WO2011041695A1 (en) * | 2009-10-02 | 2011-04-07 | Ibis Biosciences, Inc. | Determination of methylation status of polynucleotides |
ES2628739T3 (en) * | 2009-10-15 | 2017-08-03 | Ibis Biosciences, Inc. | Multiple displacement amplification |
ES2683311T3 (en) | 2009-10-16 | 2018-09-26 | Melinta Therapeutics, Inc. | Antimicrobial compounds and methods for manufacturing and using them |
EA023350B1 (en) | 2009-10-16 | 2016-05-31 | Мелинта Терапьютикс, Инк. | Antimicrobial compounds, methods of making and using the same |
AU2011232365A1 (en) | 2010-03-24 | 2012-10-25 | Rxi Pharmaceuticals Corporation | RNA interference in dermal and fibrotic indications |
US9095504B2 (en) | 2010-03-24 | 2015-08-04 | Rxi Pharmaceuticals Corporation | RNA interference in ocular indications |
RU2615143C2 (en) | 2010-03-24 | 2017-04-04 | Адвирна | Self-delivered rnai compounds of reduced size |
US8916345B2 (en) * | 2010-03-26 | 2014-12-23 | Integrated Dna Technologies, Inc. | Methods for enhancing nucleic acid hybridization |
US9506057B2 (en) | 2010-03-26 | 2016-11-29 | Integrated Dna Technologies, Inc. | Modifications for antisense compounds |
CN106434648A (en) | 2010-07-19 | 2017-02-22 | F·C·贝内特 | Modulation of dystrophia myotonica-protein kinase (dmpk) expression |
WO2012033848A1 (en) | 2010-09-07 | 2012-03-15 | Integrated Dna Technologies, Inc. | Modifications for antisense compounds |
DK2790736T3 (en) | 2011-12-12 | 2018-05-07 | Oncoimmunin Inc | In vivo delivery of oligonucleotides |
US9970061B2 (en) | 2011-12-27 | 2018-05-15 | Ibis Biosciences, Inc. | Bioagent detection oligonucleotides |
EP3434789A1 (en) | 2012-01-13 | 2019-01-30 | Data2Bio | Genotyping by next-generation sequencing |
TW201536329A (en) | 2013-08-09 | 2015-10-01 | Isis Pharmaceuticals Inc | Compounds and methods for modulation of dystrophia myotonica-protein kinase (DMPK) expression |
US10106543B2 (en) | 2013-09-09 | 2018-10-23 | Melinta Therapeutics, Inc. | Antimicrobial compounds and methods of making and using the same |
JP2016529325A (en) | 2013-09-09 | 2016-09-23 | メリンタ セラピューティクス,インコーポレイテッド | Antimicrobial compounds and methods for their production and use |
WO2015084897A2 (en) | 2013-12-02 | 2015-06-11 | Mirimmune, Llc | Immunotherapy of cancer |
WO2015168108A2 (en) | 2014-04-28 | 2015-11-05 | Rxi Pharmaceuticals Corporation | Methods for treating cancer using nucleic targeting mdm2 or mycn |
HRP20220379T1 (en) | 2014-06-10 | 2022-05-27 | Erasmus University Medical Center Rotterdam | Antisense oligonucleotides useful in treatment of pompe disease |
WO2015191303A2 (en) * | 2014-06-10 | 2015-12-17 | Agilent Technologies, Inc. | Protecting groups for "z nucleotide" and methods thereof |
JP2017522015A (en) | 2014-06-24 | 2017-08-10 | アボツト・モレキユラー・インコーポレイテツド | Detection of single nucleotide polymorphisms in human KRAS |
WO2016037071A2 (en) | 2014-09-05 | 2016-03-10 | Rxi Pharmaceuticals Corporation | Methods for treating aging and skin disorders using nucleic acids targeting tyr or mmp1 |
MX2017011616A (en) | 2015-03-11 | 2018-04-11 | Melinta Therapeutics Inc | Antimicrobial compounds and methods of making and using the same. |
WO2017007813A1 (en) | 2015-07-06 | 2017-01-12 | Rxi Pharmaceuticals Corporation | Nucleic acid molecules targeting superoxide dismutase 1 (sod1) |
US10808247B2 (en) | 2015-07-06 | 2020-10-20 | Phio Pharmaceuticals Corp. | Methods for treating neurological disorders using a synergistic small molecule and nucleic acids therapeutic approach |
WO2017070151A1 (en) | 2015-10-19 | 2017-04-27 | Rxi Pharmaceuticals Corporation | Reduced size self-delivering nucleic acid compounds targeting long non-coding rna |
CA3007152A1 (en) | 2015-12-07 | 2017-06-15 | Erasmus University Medical Center Rotterdam | Enzymatic replacement therapy and antisense therapy for pompe disease |
JP2019515041A (en) | 2016-05-06 | 2019-06-06 | メリンタ セラピューティクス、 インコーポレイテッドMelinta Therapeutics, Inc. | Antimicrobial agents and methods of making and using the same |
NL2017294B1 (en) | 2016-08-05 | 2018-02-14 | Univ Erasmus Med Ct Rotterdam | Natural cryptic exon removal by pairs of antisense oligonucleotides. |
NL2017295B1 (en) | 2016-08-05 | 2018-02-14 | Univ Erasmus Med Ct Rotterdam | Antisense oligomeric compound for Pompe disease |
US11447519B2 (en) | 2016-11-10 | 2022-09-20 | San Diego State University Research Foundation | Compounds for fluorescence sensing of duplex formation |
GB2600372A (en) * | 2020-06-25 | 2022-05-04 | Chalmers Ventures Ab | Fluorescent Cytosine analogues and their application in transcription and translation |
EP4396352A2 (en) | 2021-09-01 | 2024-07-10 | Ionis Pharmaceuticals, Inc. | Compounds and methods for reducing dmpk expression |
Citations (26)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4233402A (en) * | 1978-04-05 | 1980-11-11 | Syva Company | Reagents and method employing channeling |
JPS6259293A (en) * | 1985-09-09 | 1987-03-14 | Teijin Ltd | Fluorescent nucleoside or nucleotide |
WO1988010264A1 (en) * | 1987-06-24 | 1988-12-29 | Howard Florey Institute Of Experimental Physiology | Nucleoside derivatives |
WO1990015065A1 (en) * | 1989-06-05 | 1990-12-13 | Gilead Sciences, Inc. | Exonuclease-resistant oligonucleotides and methods for preparing the same |
WO1991006626A2 (en) * | 1989-10-23 | 1991-05-16 | Gilead Sciences, Inc. | Triple helix formation in oligonucleotide therapy |
WO1991006629A1 (en) * | 1989-10-24 | 1991-05-16 | Gilead Sciences, Inc. | Oligonucleotide analogs with novel linkages |
WO1992002258A1 (en) * | 1990-07-27 | 1992-02-20 | Isis Pharmaceuticals, Inc. | Nuclease resistant, pyrimidine modified oligonucleotides that detect and modulate gene expression |
EP0487289A2 (en) * | 1990-11-20 | 1992-05-27 | BEHRINGWERKE Aktiengesellschaft | Cyclosporin immunoassay |
WO1992020702A1 (en) * | 1991-05-24 | 1992-11-26 | Ole Buchardt | Peptide nucleic acids |
EP0541153A1 (en) * | 1991-10-30 | 1993-05-12 | Janssen Pharmaceutica N.V. | 1,3-Dihydro-2H-imidazo[4,5-b]-quinolin-2-one derivatives as phosphodiesterase inhibitors |
WO1993010820A1 (en) * | 1991-11-26 | 1993-06-10 | Gilead Sciences, Inc. | Enhanced triple-helix and double-helix formation with oligomers containing modified pyrimidines |
WO1993013121A1 (en) * | 1991-12-24 | 1993-07-08 | Isis Pharmaceuticals, Inc. | Gapped 2' modified oligonucleotides |
WO1993024507A1 (en) * | 1992-05-28 | 1993-12-09 | Gilead Sciences, Inc. | Conformationally restrained oligomers containing amide or carbamate linkages for sequence-specific binding |
US5286717A (en) * | 1987-03-25 | 1994-02-15 | The United States Of America As Represented By The Department Of Health And Human Services | Inhibitors for replication of retroviruses and for the expression of oncogene products |
WO1996005298A1 (en) * | 1994-08-09 | 1996-02-22 | Ciba-Geigy Ag | Antitumor antisense oligonucleotides |
US5502177A (en) * | 1993-09-17 | 1996-03-26 | Gilead Sciences, Inc. | Pyrimidine derivatives for labeled binding partners |
WO1996037504A1 (en) * | 1995-05-23 | 1996-11-28 | Hybridon, Inc. | Novel synthons for stereoselective oligonucleotide synthesis |
US5594121A (en) * | 1991-11-07 | 1997-01-14 | Gilead Sciences, Inc. | Enhanced triple-helix and double-helix formation with oligomers containing modified purines |
US5614622A (en) * | 1995-06-01 | 1997-03-25 | Hybridon, Inc. | 5-pentenoyl moiety as a nucleoside-amino protecting group, 4-pentenoyl-protected nucleotide synthons, and related oligonucleotide syntheses |
US5623068A (en) * | 1994-03-07 | 1997-04-22 | Beckman Instruments, Inc. | Synthesis of DNA using substituted phenylacetyl-protected nucleotides |
WO1997014706A1 (en) * | 1995-10-19 | 1997-04-24 | Nexstar Pharmaceuticals, Inc. | Method for solution phase synthesis of oligonucleotides |
WO1997028176A1 (en) * | 1996-02-01 | 1997-08-07 | Amersham Pharmacia Biotech Uk Limited | Nucleoside analogues |
WO1997031008A1 (en) * | 1996-02-23 | 1997-08-28 | Industrial Research Limited | Enzyme detection/assay method and substrates |
WO1997032888A1 (en) * | 1996-03-08 | 1997-09-12 | Isis Pharmaceuticals, Inc. | Substituted nucleic acid mimics |
US5668272A (en) * | 1995-06-30 | 1997-09-16 | National Research Council Of Canada | Method for producing synthetic N-linked glycoconjugates |
US5728528A (en) * | 1995-09-20 | 1998-03-17 | The Regents Of The University Of California | Universal spacer/energy transfer dyes |
Family Cites Families (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4182914A (en) | 1974-01-22 | 1980-01-08 | Nippon Oil Company Limited | Process for continuously producing diisopropyl ether |
US4857664A (en) | 1987-12-30 | 1989-08-15 | Mobil Oil Corporation | Process for the production of ether and alcohol |
US4906787A (en) | 1987-12-30 | 1990-03-06 | Mobil Oil Corporation | Process for the production of ethers |
US5057629A (en) | 1989-05-01 | 1991-10-15 | Shell Oil Company | Process for reducing isopropyl alcohol in diisopropyl ether |
US5154801A (en) | 1990-05-24 | 1992-10-13 | Mobil Oil Corporation | Advances in product separation in dipe process |
US5113024A (en) | 1991-06-07 | 1992-05-12 | Mobil Oil Corporation | Process for product separation in the production of di-isopropyl ether |
US5208387A (en) | 1991-12-27 | 1993-05-04 | Mobil Oil Corporation | Two stage process for production of diisopropyl ether |
US5324866A (en) | 1993-03-23 | 1994-06-28 | Uop | Integrated process for producing diisopropyl ether from isopropyl alcohol |
US5371301A (en) | 1993-06-21 | 1994-12-06 | Uop | Single stage diisopropyl ether process using aqueous extraction and ion exchange treating for SO3 removal |
US5600023A (en) | 1993-06-21 | 1997-02-04 | Uop | Single stage diisopropyl ether process using organic solvent aqueous extraction and ion exchange treating for SO3 removal |
US5504257A (en) | 1994-09-26 | 1996-04-02 | Uop | Process for producing diisopropyl ether with removal of acid material |
DE19608588A1 (en) | 1996-03-06 | 1997-09-11 | Thomae Gmbh Dr K | Pyrimido [5,4-d] pyrimidines, medicaments containing these compounds, their use and processes for their preparation |
US6028183A (en) * | 1997-11-07 | 2000-02-22 | Gilead Sciences, Inc. | Pyrimidine derivatives and oligonucleotides containing same |
US6007992A (en) * | 1997-11-10 | 1999-12-28 | Gilead Sciences, Inc. | Pyrimidine derivatives for labeled binding partners |
-
1997
- 1997-11-07 US US08/966,392 patent/US6028183A/en not_active Ceased
-
1999
- 1999-09-21 US US09/400,502 patent/US6414127B1/en not_active Expired - Lifetime
-
2002
- 2002-02-21 US US10/080,074 patent/USRE39324E1/en not_active Expired - Lifetime
- 2002-05-21 US US10/152,123 patent/US6800743B2/en not_active Expired - Fee Related
- 2002-11-14 US US10/294,181 patent/US6951931B2/en not_active Expired - Lifetime
-
2004
- 2004-08-19 US US10/921,734 patent/US7511125B2/en not_active Expired - Fee Related
Patent Citations (28)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4233402A (en) * | 1978-04-05 | 1980-11-11 | Syva Company | Reagents and method employing channeling |
JPS6259293A (en) * | 1985-09-09 | 1987-03-14 | Teijin Ltd | Fluorescent nucleoside or nucleotide |
US5286717A (en) * | 1987-03-25 | 1994-02-15 | The United States Of America As Represented By The Department Of Health And Human Services | Inhibitors for replication of retroviruses and for the expression of oncogene products |
WO1988010264A1 (en) * | 1987-06-24 | 1988-12-29 | Howard Florey Institute Of Experimental Physiology | Nucleoside derivatives |
WO1990015065A1 (en) * | 1989-06-05 | 1990-12-13 | Gilead Sciences, Inc. | Exonuclease-resistant oligonucleotides and methods for preparing the same |
WO1991006626A2 (en) * | 1989-10-23 | 1991-05-16 | Gilead Sciences, Inc. | Triple helix formation in oligonucleotide therapy |
WO1991006629A1 (en) * | 1989-10-24 | 1991-05-16 | Gilead Sciences, Inc. | Oligonucleotide analogs with novel linkages |
WO1992002258A1 (en) * | 1990-07-27 | 1992-02-20 | Isis Pharmaceuticals, Inc. | Nuclease resistant, pyrimidine modified oligonucleotides that detect and modulate gene expression |
US5614617A (en) * | 1990-07-27 | 1997-03-25 | Isis Pharmaceuticals, Inc. | Nuclease resistant, pyrimidine modified oligonucleotides that detect and modulate gene expression |
EP0487289A2 (en) * | 1990-11-20 | 1992-05-27 | BEHRINGWERKE Aktiengesellschaft | Cyclosporin immunoassay |
WO1992020702A1 (en) * | 1991-05-24 | 1992-11-26 | Ole Buchardt | Peptide nucleic acids |
EP0541153A1 (en) * | 1991-10-30 | 1993-05-12 | Janssen Pharmaceutica N.V. | 1,3-Dihydro-2H-imidazo[4,5-b]-quinolin-2-one derivatives as phosphodiesterase inhibitors |
US5594121A (en) * | 1991-11-07 | 1997-01-14 | Gilead Sciences, Inc. | Enhanced triple-helix and double-helix formation with oligomers containing modified purines |
WO1993010820A1 (en) * | 1991-11-26 | 1993-06-10 | Gilead Sciences, Inc. | Enhanced triple-helix and double-helix formation with oligomers containing modified pyrimidines |
US5645985A (en) * | 1991-11-26 | 1997-07-08 | Gilead Sciences, Inc. | Enhanced triple-helix and double-helix formation with oligomers containing modified pyrimidines |
WO1993013121A1 (en) * | 1991-12-24 | 1993-07-08 | Isis Pharmaceuticals, Inc. | Gapped 2' modified oligonucleotides |
WO1993024507A1 (en) * | 1992-05-28 | 1993-12-09 | Gilead Sciences, Inc. | Conformationally restrained oligomers containing amide or carbamate linkages for sequence-specific binding |
US5502177A (en) * | 1993-09-17 | 1996-03-26 | Gilead Sciences, Inc. | Pyrimidine derivatives for labeled binding partners |
US5623068A (en) * | 1994-03-07 | 1997-04-22 | Beckman Instruments, Inc. | Synthesis of DNA using substituted phenylacetyl-protected nucleotides |
WO1996005298A1 (en) * | 1994-08-09 | 1996-02-22 | Ciba-Geigy Ag | Antitumor antisense oligonucleotides |
WO1996037504A1 (en) * | 1995-05-23 | 1996-11-28 | Hybridon, Inc. | Novel synthons for stereoselective oligonucleotide synthesis |
US5614622A (en) * | 1995-06-01 | 1997-03-25 | Hybridon, Inc. | 5-pentenoyl moiety as a nucleoside-amino protecting group, 4-pentenoyl-protected nucleotide synthons, and related oligonucleotide syntheses |
US5668272A (en) * | 1995-06-30 | 1997-09-16 | National Research Council Of Canada | Method for producing synthetic N-linked glycoconjugates |
US5728528A (en) * | 1995-09-20 | 1998-03-17 | The Regents Of The University Of California | Universal spacer/energy transfer dyes |
WO1997014706A1 (en) * | 1995-10-19 | 1997-04-24 | Nexstar Pharmaceuticals, Inc. | Method for solution phase synthesis of oligonucleotides |
WO1997028176A1 (en) * | 1996-02-01 | 1997-08-07 | Amersham Pharmacia Biotech Uk Limited | Nucleoside analogues |
WO1997031008A1 (en) * | 1996-02-23 | 1997-08-28 | Industrial Research Limited | Enzyme detection/assay method and substrates |
WO1997032888A1 (en) * | 1996-03-08 | 1997-09-12 | Isis Pharmaceuticals, Inc. | Substituted nucleic acid mimics |
Non-Patent Citations (8)
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013041194A1 (en) | 2011-09-23 | 2013-03-28 | Roche Diagnostics Gmbh | Use of g-clamp for improved allele-specific pcr |
Also Published As
Publication number | Publication date |
---|---|
US6800743B2 (en) | 2004-10-05 |
US6951931B2 (en) | 2005-10-04 |
US20030176677A1 (en) | 2003-09-18 |
US6028183A (en) | 2000-02-22 |
US7511125B2 (en) | 2009-03-31 |
US6414127B1 (en) | 2002-07-02 |
US20030072712A1 (en) | 2003-04-17 |
US20050020826A1 (en) | 2005-01-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
USRE39324E1 (en) | Pyrimidine derivatives and oligonucleotides containing same | |
US6007992A (en) | Pyrimidine derivatives for labeled binding partners | |
EP1027364B1 (en) | Pyrimidine derivatives for labeled binding partners | |
EP0719272B1 (en) | Pyrimidine derivatives for labeled binding partners | |
KR0155574B1 (en) | Back bone modified oligonucleotide analogs | |
RU2145964C1 (en) | Nucleosides with modified sugars and oligonucleotides | |
US7399845B2 (en) | 6-modified bicyclic nucleic acid analogs | |
EP1015469B2 (en) | Bi- and tri-cyclic nucleoside, nucleotide and oligonucleoide analogues | |
US7034133B2 (en) | Oligonucleotide analogues | |
US8153365B2 (en) | Oligonucleotide analogues | |
CA2057475A1 (en) | 3'-(2')-amino- or thiol-modified, fluorescent dye-coupled nucleosides, nucleotides and oligonucleotides, and a process for the preparation and the use thereof | |
JP4887500B2 (en) | Nucleoside analogue or salt thereof | |
EP2006293B1 (en) | 2'-hydroxyl-modified ribonucleoside derivative | |
WO1994022886A1 (en) | Heteroatomic oligonucleoside linkages | |
Pankiewicz et al. | Nucleosides. 164. Studies directed toward the synthesis of 2'-deoxy-2'-substituted arabino nucleosides. 10. Synthesis of 2'-. beta.-fluoro-and 3'-. alpha.-fluoro-substituted guanine nucleosides. Effect of sugar conformational shifts on nucleophilic displacement of the 2'-hydroxy and 3'-hydroxy group with DAST |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
CC | Certificate of correction | ||
FPAY | Fee payment |
Year of fee payment: 8 |
|
FPAY | Fee payment |
Year of fee payment: 12 |
|
AS | Assignment |
Owner name: IONIS PHARMACEUTICALS, INC., CALIFORNIA Free format text: CHANGE OF NAME;ASSIGNOR:ISIS PHARMACEUTICALS, INC.;REEL/FRAME:043341/0571 Effective date: 20151218 |