DE102005050834A1 - Use of long chain azo dye as blotter for fluorescent based detection reactions and as dye for optical detection e.g. in qualitative and quantitative determination procedure e.g. immune tests and hybridization procedure - Google Patents
Use of long chain azo dye as blotter for fluorescent based detection reactions and as dye for optical detection e.g. in qualitative and quantitative determination procedure e.g. immune tests and hybridization procedure Download PDFInfo
- Publication number
- DE102005050834A1 DE102005050834A1 DE200510050834 DE102005050834A DE102005050834A1 DE 102005050834 A1 DE102005050834 A1 DE 102005050834A1 DE 200510050834 DE200510050834 DE 200510050834 DE 102005050834 A DE102005050834 A DE 102005050834A DE 102005050834 A1 DE102005050834 A1 DE 102005050834A1
- Authority
- DE
- Germany
- Prior art keywords
- aliphatic
- substituents
- dye
- azo dyes
- hetero
- 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.)
- Withdrawn
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- 239000000975 dye Substances 0.000 title claims abstract description 47
- 239000000987 azo dye Substances 0.000 title claims abstract description 30
- 238000001514 detection method Methods 0.000 title claims abstract description 13
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 11
- 230000003287 optical effect Effects 0.000 title claims description 7
- 238000000034 method Methods 0.000 title claims description 5
- 238000009396 hybridization Methods 0.000 title claims 2
- -1 tricyanovinyl Chemical group 0.000 claims abstract description 43
- 125000001424 substituent group Chemical group 0.000 claims abstract description 34
- 125000001931 aliphatic group Chemical group 0.000 claims abstract description 32
- 125000003118 aryl group Chemical group 0.000 claims abstract description 24
- 125000006850 spacer group Chemical group 0.000 claims abstract description 15
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical group NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 claims abstract description 14
- LEQAOMBKQFMDFZ-UHFFFAOYSA-N glyoxal Chemical compound O=CC=O LEQAOMBKQFMDFZ-UHFFFAOYSA-N 0.000 claims abstract description 14
- 150000001732 carboxylic acid derivatives Chemical class 0.000 claims abstract description 13
- 125000004122 cyclic group Chemical group 0.000 claims abstract description 12
- 235000000346 sugar Nutrition 0.000 claims abstract description 9
- 229910052739 hydrogen Inorganic materials 0.000 claims abstract description 8
- DHQPQGBAUAMAEK-UHFFFAOYSA-N 4,6-dibromotriazine Chemical compound BrC1=CC(Br)=NN=N1 DHQPQGBAUAMAEK-UHFFFAOYSA-N 0.000 claims abstract description 7
- NIXOWILDQLNWCW-UHFFFAOYSA-M Acrylate Chemical compound [O-]C(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 claims abstract description 7
- NOWKCMXCCJGMRR-UHFFFAOYSA-N Aziridine Chemical compound C1CN1 NOWKCMXCCJGMRR-UHFFFAOYSA-N 0.000 claims abstract description 7
- 229920000858 Cyclodextrin Polymers 0.000 claims abstract description 7
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 claims abstract description 7
- 150000001299 aldehydes Chemical class 0.000 claims abstract description 7
- 125000003545 alkoxy group Chemical group 0.000 claims abstract description 7
- 125000000217 alkyl group Chemical group 0.000 claims abstract description 7
- 125000004414 alkyl thio group Chemical group 0.000 claims abstract description 7
- 150000001412 amines Chemical group 0.000 claims abstract description 7
- 125000004104 aryloxy group Chemical group 0.000 claims abstract description 7
- 229910052794 bromium Inorganic materials 0.000 claims abstract description 7
- 150000001733 carboxylic acid esters Chemical class 0.000 claims abstract description 7
- 229910052801 chlorine Inorganic materials 0.000 claims abstract description 7
- 125000000753 cycloalkyl group Chemical group 0.000 claims abstract description 7
- 229940015043 glyoxal Drugs 0.000 claims abstract description 7
- 150000002463 imidates Chemical class 0.000 claims abstract description 7
- 239000012948 isocyanate Substances 0.000 claims abstract description 7
- 150000002513 isocyanates Chemical group 0.000 claims abstract description 7
- 150000002540 isothiocyanates Chemical group 0.000 claims abstract description 7
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 claims abstract description 7
- 230000003381 solubilizing effect Effects 0.000 claims abstract description 7
- 125000004044 trifluoroacetyl group Chemical group FC(C(=O)*)(F)F 0.000 claims abstract description 7
- 125000002023 trifluoromethyl group Chemical group FC(F)(F)* 0.000 claims abstract description 7
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 claims abstract description 7
- 229920002554 vinyl polymer Polymers 0.000 claims abstract description 7
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 3
- 229910052711 selenium Inorganic materials 0.000 claims abstract description 3
- 229910052717 sulfur Inorganic materials 0.000 claims abstract description 3
- 229910052714 tellurium Inorganic materials 0.000 claims abstract description 3
- 150000003254 radicals Chemical group 0.000 claims description 11
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- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical group [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims description 6
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 claims description 6
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims description 6
- CERQOIWHTDAKMF-UHFFFAOYSA-M Methacrylate Chemical compound CC(=C)C([O-])=O CERQOIWHTDAKMF-UHFFFAOYSA-M 0.000 claims description 6
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 claims description 6
- 239000000460 chlorine Substances 0.000 claims description 6
- 125000005553 heteroaryloxy group Chemical group 0.000 claims description 6
- 125000000592 heterocycloalkyl group Chemical group 0.000 claims description 6
- 239000001257 hydrogen Substances 0.000 claims description 6
- 125000004435 hydrogen atom Chemical class [H]* 0.000 claims description 6
- PGLTVOMIXTUURA-UHFFFAOYSA-N iodoacetamide Chemical compound NC(=O)CI PGLTVOMIXTUURA-UHFFFAOYSA-N 0.000 claims description 6
- NMHMNPHRMNGLLB-UHFFFAOYSA-N phloretic acid Chemical compound OC(=O)CCC1=CC=C(O)C=C1 NMHMNPHRMNGLLB-UHFFFAOYSA-N 0.000 claims description 6
- 150000008300 phosphoramidites Chemical class 0.000 claims description 6
- 150000003461 sulfonyl halides Chemical class 0.000 claims description 6
- 239000003814 drug Substances 0.000 claims description 5
- 230000003993 interaction Effects 0.000 claims description 5
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- 239000003446 ligand Substances 0.000 claims description 4
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- 238000005516 engineering process Methods 0.000 claims description 2
- 125000001072 heteroaryl group Chemical group 0.000 claims description 2
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- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 claims 3
- 239000005556 hormone Substances 0.000 claims 3
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- 125000004356 hydroxy functional group Chemical group O* 0.000 claims 3
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- 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 claims 2
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- 150000001720 carbohydrates Chemical class 0.000 claims 1
- 235000014633 carbohydrates Nutrition 0.000 claims 1
- 235000021466 carotenoid Nutrition 0.000 claims 1
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- 125000006852 aliphatic spacer Chemical group 0.000 abstract 1
- DMSZORWOGDLWGN-UHFFFAOYSA-N ctk1a3526 Chemical compound NP(N)(N)=O DMSZORWOGDLWGN-UHFFFAOYSA-N 0.000 abstract 1
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- QGKMIGUHVLGJBR-UHFFFAOYSA-M (4z)-1-(3-methylbutyl)-4-[[1-(3-methylbutyl)quinolin-1-ium-4-yl]methylidene]quinoline;iodide Chemical compound [I-].C12=CC=CC=C2N(CCC(C)C)C=CC1=CC1=CC=[N+](CCC(C)C)C2=CC=CC=C12 QGKMIGUHVLGJBR-UHFFFAOYSA-M 0.000 description 1
- FBMQNRKSAWNXBT-UHFFFAOYSA-N 1,4-diaminoanthracene-9,10-dione Chemical compound O=C1C2=CC=CC=C2C(=O)C2=C1C(N)=CC=C2N FBMQNRKSAWNXBT-UHFFFAOYSA-N 0.000 description 1
- RZVHIXYEVGDQDX-UHFFFAOYSA-N 9,10-anthraquinone Chemical group C1=CC=C2C(=O)C3=CC=CC=C3C(=O)C2=C1 RZVHIXYEVGDQDX-UHFFFAOYSA-N 0.000 description 1
- DRSHXJFUUPIBHX-UHFFFAOYSA-N COc1ccc(cc1)N1N=CC2C=NC(Nc3cc(OC)c(OC)c(OCCCN4CCN(C)CC4)c3)=NC12 Chemical compound COc1ccc(cc1)N1N=CC2C=NC(Nc3cc(OC)c(OC)c(OCCCN4CCN(C)CC4)c3)=NC12 DRSHXJFUUPIBHX-UHFFFAOYSA-N 0.000 description 1
- AYRHWIPMJUEWMD-ZQHSETAFSA-N C[S](CCCN(CCC[S](C)(O)(=O)=O)c(cc1)ccc1/N=N/C1Sc(cccc2)c2N1CCCCCC(O)=O)(O)(=O)=O Chemical compound C[S](CCCN(CCC[S](C)(O)(=O)=O)c(cc1)ccc1/N=N/C1Sc(cccc2)c2N1CCCCCC(O)=O)(O)(=O)=O AYRHWIPMJUEWMD-ZQHSETAFSA-N 0.000 description 1
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- NLFBCYMMUAKCPC-KQQUZDAGSA-N ethyl (e)-3-[3-amino-2-cyano-1-[(e)-3-ethoxy-3-oxoprop-1-enyl]sulfanyl-3-oxoprop-1-enyl]sulfanylprop-2-enoate Chemical compound CCOC(=O)\C=C\SC(=C(C#N)C(N)=O)S\C=C\C(=O)OCC NLFBCYMMUAKCPC-KQQUZDAGSA-N 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B29/00—Monoazo dyes prepared by diazotising and coupling
- C09B29/0025—Monoazo dyes prepared by diazotising and coupling from diazotized amino heterocyclic compounds
- C09B29/0074—Monoazo dyes prepared by diazotising and coupling from diazotized amino heterocyclic compounds the heterocyclic ring containing nitrogen and sulfur as heteroatoms
- C09B29/0077—Monoazo dyes prepared by diazotising and coupling from diazotized amino heterocyclic compounds the heterocyclic ring containing nitrogen and sulfur as heteroatoms containing a five-membered heterocyclic ring with one nitrogen and one sulfur as heteroatoms
- C09B29/0085—Thiazoles or condensed thiazoles
- C09B29/0088—Benzothiazoles
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B29/00—Monoazo dyes prepared by diazotising and coupling
- C09B29/06—Monoazo dyes prepared by diazotising and coupling from coupling components containing amino as the only directing group
- C09B29/08—Amino benzenes
- C09B29/0801—Amino benzenes containing acid groups, e.g. COOH, SO3H, PO3H2, OSO3H, OPO3H2; SO2NHSO2R or salts thereof, R being hydrocarbonyls
- C09B29/0802—Amino benzenes containing acid groups, e.g. COOH, SO3H, PO3H2, OSO3H, OPO3H2; SO2NHSO2R or salts thereof, R being hydrocarbonyls containing COOH
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B29/00—Monoazo dyes prepared by diazotising and coupling
- C09B29/06—Monoazo dyes prepared by diazotising and coupling from coupling components containing amino as the only directing group
- C09B29/08—Amino benzenes
- C09B29/0801—Amino benzenes containing acid groups, e.g. COOH, SO3H, PO3H2, OSO3H, OPO3H2; SO2NHSO2R or salts thereof, R being hydrocarbonyls
- C09B29/0803—Amino benzenes containing acid groups, e.g. COOH, SO3H, PO3H2, OSO3H, OPO3H2; SO2NHSO2R or salts thereof, R being hydrocarbonyls containing SO3H, OSO3H
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B29/00—Monoazo dyes prepared by diazotising and coupling
- C09B29/06—Monoazo dyes prepared by diazotising and coupling from coupling components containing amino as the only directing group
- C09B29/095—Amino naphthalenes
- C09B29/0955—Amino naphthalenes containing water solubilizing groups
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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Abstract
Description
Die Erfindung betrifft die Verwendung von langwelligen Azofarbstoffen als Löscher für fluoreszenzbasierende Nachweisreaktionen. Typische Verfahrensanwendungen beruhen auf der Reaktion von farbstoffmarkierten Antigenen und Antikörpern oder DNA-Segmenten mit der jeweils komplementären Spezies. Damit werden unter anderem Messungen von Enzymkinetiken, Rezeptor-Ligand-Interaktionen und Nukleinsäurehybridisierungskinetiken ermöglicht. Des Weiteren sind die beanspruchten Löscher für die pharmakologische Charakterisierung von Rezeptoren oder Wirkstoffen interessant.The This invention relates to the use of long wavelength azo dyes as a extinguisher for fluorescence-based Detection reactions. Typical process applications are based on the Reaction of dye-labeled antigens and antibodies or With DNA segments each complementary Species. This will include measurements of enzyme kinetics, Receptor-ligand interactions and nucleic acid hybridization kinetics allows. Furthermore, the claimed quenchers are for pharmacological characterization of receptors or drugs interesting.
Einsatzmöglichkeiten ergeben sich beispielsweise in der Medizin und Pharmazie, in der Bio- und Materialwissenschaft, bei der Umweltkontrolle sowie für den Nachweis von in Natur und Technik vorkommenden organischen und anorganischen Mikroproben.applications arise, for example, in medicine and pharmacy, in the Biology and materials science, environmental control and detection from occurring in nature and technology organic and inorganic Micro samples.
Es sind bereits Farbstoffe als Löscher für Fluoreszenz bekannt. So wurde in der Arbeit von Jonathan P. May ein 1,4-Diaminoanthraquinonfarbstoff für die Anwendung in Molecular Beacons beschrieben (Chem. Comm., 2003, 970-971). Dieser Farbstoff ist aber auf Grund fehlender hydrophiler Funktionen (wie z. B. Carbonsäure- oder Sulfonsäuregruppen) wasserunlöslich und daher nur schlecht zum Labeln von Biomolekülen einsetzbar. Darüber hinaus führt die sperrige Anthraquinonstruktur zur Farbstoffaggregation und damit zu instabilen spektralen Eigenschaften.It are already dyes as extinguishers for fluorescence known. Thus, in the work of Jonathan P. May, a 1,4-diaminoanthraquinone dye for the Application in Molecular Beacons (Chem. Comm., 2003, 970-971). However, this dye is due to lack of hydrophilic functions (such as carboxylic acid or sulfonic acid groups) insoluble in water and therefore only badly used for labeling biomolecules. Furthermore leads the bulky anthraquinone structure for dye aggregation and thus to unstable spectral properties.
Mary K. Johannson beschreibt ebenfalls Farbstoffe (J. Am. Chem. Soc. 126, 2004, 16451), welche auf Grund fehlender hydrophiler Funktionen wasserunlöslich sind und durch ihre voluminöse Bisazostruktur zur Aggregation neigen. Darüber hinaus sind diese Farbstoffe über ihre Hydroxyethylfunktionen nur eingeschränkt an Biomoleküle immobilisierbar.Mary K. Johannson also describes dyes (J. Am. Chem. Soc. 126, 2004, 16451), which due to the lack of hydrophilic functions insoluble in water are and through their voluminous Bisazostruktur tend to aggregate. In addition, these dyes are about theirs Hydroxyethyl functions immobilized only limited to biomolecules.
Dies
gilt auch für
die von Andrei Laikhter in der
In
Ferner sind langwellige Azofarbstoffe an sich bekannt, die beispielsweise zur Herstellung flüssigkristalliner Materialien diskutiert wurden (S. Yasui, M. Matsuoka, M. Takao, T. Kitao, The effect of annelation on the absorption spectra of polyazo dyes, J. Soc. Dyers Colour. 104 (1988) 284-288).Further Long-wave azo dyes are known per se, for example for the production of liquid crystalline Materials were discussed (S. Yasui, M. Matsuoka, M. Takao, T. Kitao, The effect of annealing on the absorption spectra of polyazo dyes, J. Soc. Dyer's Color. 104 (1988) 284-288).
Des weiteren wurde die Verwendung von langwelligen Azofarbstoffen für die Herstellung von Materialien mit nichtlinearen optischen Eigenschaften vorgeschlagen (H. E. Katz, K. D. Singer, J. E. Sohn, C. W. Dirk, L. A. King, H. M. Gordon: Greatly enhanced secondorder nonlinear optical susceptibilities in donor-acceptor organic molecules, J. Am. Chem. Soc. 109, 1987, 6561-6563). Über Verwendungen als Löscher für fluoreszenzbasierende Nachweisreaktionen ist in der Fachwelt nichts bekannt geworden.Of Another was the use of long-wave azo dyes for the production proposed materials with nonlinear optical properties (H.E. Katz, K.D. Singer, J.E. Sohn, C.W. Dirk, L.A. King, H. M. Gordon: Greatly second-order nonlinear optical susceptibilities in donor-acceptor organic molecules, J. Am. Chem. Soc. 109, 1987, 6561-6563). about Uses as extinguisher for fluorescence-based Detection reactions have not become known in the art.
Der Erfindung liegt die Aufgabe zu Grunde, wasserlösliche Löscherfarbstoffe hoher Extinktion und guter Photostabilität zu schaffen, welche möglichst kovalent an Biomoleküle angebunden werden können und auf möglichst einfache Weise zuverlässige fluoreszenzbasierende Nachweisreaktionen ermöglichen.Of the The invention is based on the object, water-soluble extinguisher high extinction and good photostability to create which possible covalently attached to biomolecules can be connected and on as possible simple way reliable enable fluorescence-based detection reactions.
Überraschend
hat sich gezeigt, dass langwellige Azofarbstoffe der allgemeinen
Formel I wobei:
R1-R5
gleich oder unterschiedlich sind und Wasserstoff, ein oder mehrere
Chlor-, Brom- und/oder Iodatome, Alkyl-, Aryl-, Hereroaryl-, Cycloalkyl-,
Heterocycloalkyl-, Alkyloxy-, Alkylmercapto-, Aryloxy-, Arylmercapto-, Heteroaryloxy-,
Heteroarylmercapto-, Nitro-Reste, Carbonsäure-, Carbonsäureester-,
Carbonsäureamid-, Trifluormethyl,
Trifluoracetyl, Tricyanovinyl- oder Cyano-Reste, eine oder mehrere
Hydroxy-, eine oder mehrere alkylsubstituierte oder cyclische Aminofunktionen
sein können
und R2 und R3 und/oder R4 und R5 weitere aliphatische oder aromatische
Ringe bzw. zwei benachbarte Reste zusammen einen oder mehrere aliphatische
oder aromatische Ringe bilden können,
X ein Element aus der Gruppe O, S, Se, Te oder das Strukturelement
(CR2)n mit n = 1 – 4, NR
oder SO2 sein und für die Strukturelemente -CR2-O-, -O-CR2, -CO-O-
oder -O-CO- stehen kann, R6 ein substituiertes aromatisches oder
heteroaromatisches System ist, welches an die in (I) angegebene
Grundstruktur gebunden ist, einer oder mehrere der Substituenten
R1-R5 solubilisierende bzw. ionisierbare bzw. ionisierte Substituenten
wie SO3-, PO3 2–,
CO2 –, O–,
NR3 + oder Cyclodextrin
oder Zucker darstellen, welche die hydrophilen Eigenschaften der
Farbstoffe bestimmen, wobei diese Substituenten auch über eine
aliphatische oder heteroaliphatische, beispielsweise zyklische,
Spacergruppe am Farbstoff angebunden sein können, mindestens einer der
Substituenten R1-R5 für
eine reaktive Gruppe vom Typ Isocyanat, Isothiocyanat, Hydrazin,
Amin, Mono- und Dichlor- bzw. Mono- und Dibromtriazin, Aziridin, Methacrylat, Acrylat,
Vinyl, Vinylsulfonyl, Sulfonylhalogenid, N-Hydroxysuccinimidester,
Imido-Ester, Glyoxal oder Aldehyd bzw. Maleimid oder Iodacetamid
sowie Phosphoramidit steht, wobei der jeweilige Substituent über eine
aliphatische oder heteroaliphatische, beispielsweise zyklische,
Spacergruppe am Farbstoff angebunden sein kann und die aliphatische
oder hereroaliphatische Spacergruppe aus einem Strukturelement -[(CH2)a-Y-(CH2)b]c besteht,
worin Y gleich oder verschieden eine CR2-,
O-, S-, SO2, SO2NH-,
NR-, COO- oder CONR-Funktion sein kann, wobei R gleich oder unterschiedlich
die Funktionen von R1-R5 einnehmen kann und a und b gleich oder verschieden
die Werte von 0-18 und c die Werte von 1-18 darstellen.
hervorragend
als wasserlösliche
Löscher
mit hoher Extinktion und stabilen spektralen Eigenschaften für fluoreszenzbasierende
Nachweisreaktionen verwendet werden können. Die neuen Löscherfarbstoffe,
deren Absorptionsmaxima üblicherweise
größer als
560 nm liegen, weisen eine gute Photostabilität auf und können in der Regel kovalent
an Biomoleküle
angebunden werden. Durch diese zweckmäßige Anbindung der besagten Azofarbstoff-Derivate
an die Biomoleküle
entstehende Konjugate besitzen sehr gute lichtabsorbierende sowie lumineszenzlöschende
Eigenschaften und können
damit auf verfahrenstechnisch einfache Weise effiziente fluoreszenzbasierende
Nachweisreaktionen ermöglichen.
Die breiten Absorptionsbanden der Azofarbstoffe überlappen mit nahezu allen
kommerziell erhältlichen
Fluoreszenzfarbstoffen.Surprisingly, it has been found that long-wave azo dyes of the general formula I in which:
R 1 and R 5 are identical or different and are hydrogen, one or more chlorine, bromine and / or iodine atoms, alkyl, aryl, hereroaryl, cycloalkyl, heterocycloalkyl, alkyloxy, alkylmercapto, aryloxy, arylmercapto, Heteroaryloxy, heteroarylmercapto, nitro radicals, carboxylic acid, carboxylic acid ester, carboxylic acid amide, trifluoromethyl, trifluoroacetyl, tricyanovinyl or cyano radicals, one or more hydroxy, one or more alkyl-substituted or cyclic amino functions and R2 and R3 and or R 4 and R 5 further aliphatic or aromatic rings or two adjacent radicals together may form one or more aliphatic or aromatic rings, X is an element from the group O, S, Se, Te or the structural element (CR 2 ) n with n = 1 - 4, NR or SO 2 and for the structural elements -CR 2 -O-, -O-CR 2 , -CO-O- or -O-CO-, R6 is a substituted aromatic or heteroaromatic system which bonded to the basic structure given in (I) en, one or more of the substituents R 1 -R 5 solubilizing or ionizable or ionized substituents such as SO 3 -, PO 3 2- , CO 2 - , O - , NR 3 + or cyclodextrin or sugar representing the hydrophilic properties of the Determine dyes, these substituents may also be attached to the dye via an aliphatic or heteroaliphatic, such as cyclic, spacer group, at least one of the substituents R 1 -R 5 for a reactive group of the isocyanate, isothiocyanate, hydrazine, amine, mono- and dichloro- or Mono- and dibromotriazine, aziridine, methacrylate, acrylate, vinyl, vinylsulfonyl, sulfonyl halide, N-hydroxysuccinimide ester, imido-ester, glyoxal or aldehyde or maleimide or iodoacetamide and phosphoramidite, wherein the respective substituent via an aliphatic or heteroaliphatic, for example cyclic , Spacergruppe can be attached to the dye and the aliphatic or hereroaliphatic spacer group au s is a structural element - [(CH 2 ) a -Y- (CH 2 ) b ] c wherein Y, identically or differently, is a CR 2 -, O-, S-, SO 2 , SO 2 NH-, NR-, COO or CONR function, where R may be the same or different from the functions of R1-R5, and a and b, identically or differently, represent the values of 0-18 and c represent the values of 1-18.
can be used excellently as water-soluble quenchers with high extinction and stable spectral properties for fluorescence-based detection reactions. The new quencher dyes whose absorption maxima are usually greater than 560 nm have good photostability and can usually be covalently attached to biomolecules. By this convenient attachment of said azo dye derivatives to the biomolecules resulting conjugates have very good light-absorbing and luminescence-quenching properties and can thus in a technically simple manner efficient fluorescence-based detection reactions allow. The broad absorption bands of the azo dyes overlap with almost all commercially available fluorescent dyes.
Für spezielle Markierungen (z. B. der Markierung von negativ geladener DNA über elektrostatische Wechselwirkung mit positiv geladenen Farbstoffen, oder geladenen Proteinen mit ionischen Farbstoffen) kann es hingegen auch zweckmäßig sein, wenn die Markierung statt unter kovalenter Anbindung durch Ausbildung von ionischen Wechselwirkungen zwischen den Azofarbstoffen und den zu markierenden Materialien erfolgt. Auch bei solchen Verwendungen der langwelligen Azofarbstoffe sind die besagten Vorzüge für fluoreszenzbasierende Nachweisreaktionen gegeben.For special Labeling (eg labeling of negatively charged DNA via electrostatic interaction with positively charged dyes, or charged proteins with ionic dyes), it may also be appropriate if the labeling takes place under covalent bonding through the formation of ionic interactions between the azo dyes and the marking materials takes place. Even with such uses The long-wavelength azo dyes are the said advantages for fluorescence-based Given evidence reactions.
Durch die Wahl geeigneter Substituenten R6 der allgemeinen Formel I (beispielsweise aromatischer oder heteroaromatischer Rest gemäß den Verbindungen II und III) können die spektralen Eigenschaften zusätzlich modifiziert werden, um die Löscherfarbstoffe an spezielle Fluoreszenzfarbstoffe anzupassen und noch effizientere Löschung zu erreichen.By the choice of suitable substituents R6 of the general formula I (for example aromatic or heteroaromatic radical according to compounds II and III) can the spectral properties in addition modified to the quencher dyes to adapt to specific fluorescent dyes and even more efficient deletion to reach.
Die Photostabilität der neuartigen Azofarbstoffe ist vielen bereits bekannten Farbstoffen wie z. B. Cyanin-, Hemicyanin-, Merocyanin- Oxonol und Stilbenfarbstoffen deutlich überlegen.The photostability The novel azo dyes are many already known dyes such as As cyanine, hemicyanine, merocyanine oxonol and stilbene dyes clearly superior.
Die Azofarbstoffe sind durch einfache Syntheseschritte beispielsweise in wässrigem Medium ökonomisch leicht zugänglich. Sie können mittels in Biochemie und Biologie etablierter Methoden einfach und effizient an Biomoleküle gekoppelt werden. Durch spezielle Substitution der Reste R1-R5 in Verbindung I, R7-R12 in Verbindung II und R7-R15 in Verbindung III können sowohl kastionische, elektrisch neutrale sowie auch anionische Farbstoffe erhalten werden, um so unspezifische Wechselwirkungen mit Biomolekülen sowie Aggregation vermeiden zu können. Dabei kann der hydrophile Charakter der jeweils unterschiedlich absorbierenden Farbstoffe jedoch nahezu identisch gestaltet werden.The azo dyes are easily accessible by simple synthetic steps, for example, in an aqueous medium. They can be easily and efficiently coupled to biomolecules using methods established in biochemistry and biology. By special substitution of the radicals R1-R5 in compound I, R7-R12 In compound II and R7-R15 in compound III, both cationic, electrically neutral and also anionic dyes can be obtained in order to be able to avoid unspecific interactions with biomolecules and aggregation. However, the hydrophilic character of each differently absorbing dyes can be made almost identical.
In den Unteransprüchen sind vorteilhafte Verwendungen der langwelligen Azofarbstoffe nach allgemeiner Formel I angeführt. So kann als Rest R6 beispielsweise eine aromatische Verbindung der allgemeinen Formel II In the subclaims advantageous uses of the long-wave azo dyes are given by general formula I. Thus, as the radical R6, for example, an aromatic compound of general formula II
Verwendung
finden,
wobei:
R7-R12 gleich oder unterschiedlich sind
und Wasserstoff, ein oder mehrere Chlor-, Brom- und/oder Iodatome, Alkyl-,
Aryl-, Hereroaryl-, Cycloalkyl-, Heterocycloalkyl-, Alkyloxy-, Alkylmercapto-,
Aryloxy-, Arylmercapto-, Heteroaryloxy-, Heteroarylmercapto-, Nitro-Reste,
Carbonsäure-,
Carbonsäureester-,
Carbonsäureamid-, Trifluormethyl,
Trifluoracetyl, Tricyanovinyl- oder Cyano-Reste, eine oder mehrere
Hydroxy-, eine oder mehrere alkylsubstituierte oder cyclische Aminofunktionen
sein können
und R7 und R8 und/oder R11 und R12 weitere aliphatische oder aromatische
Ringe bzw. zwei benachbarte Reste zusammen einen oder mehrere aliphatische
oder aromatische Ringe bilden können,
einer oder mehrere der Substituenten R7-R12 solubilisierende bzw. ionisierbare
bzw. ionisierte Substituenten wie SO3 –,
PO3 2–, CO2 –,
O-, NR3 + oder Cyclodextrin
oder Zucker darstellen, welche die hydrophilen Eigenschaften der
Farbstoffe bestimmen, wobei diese Substituenten auch über eine
aliphatische oder heteroaliphatische, beispielsweise zyklische,
Spacergruppe am Farbstoff angebunden sein können, mindestens einer der
Substituenten R7-R12 für
eine reaktive Gruppe vom Typ Isocyanat, Isothiocyanat, Hydrazin,
Amin, Mono- und Dichlor- bzw. Mono- und Dibromtriazin, Aziridin, Methacrylat, Acrylat,
Vinyl, Vinylsulfonyl, Sulfonylhalogenid, N-Hydroxysuccinimidester,
Imido-Ester, Glyoxal oder Aldehyd bzw. Maleimid oder Iodacetamid
sowie Phosphoramidit stehen kann, wobei der jeweilige Substituent über eine aliphatische
oder heteroaliphatische, beispielsweise zyklische, Spacergruppe
am Farbstoff angebunden sein kann und die aliphatische oder hereroaliphatische
Spacergruppe aus einem Strukturelement -[(CH2)a-Y-(CH2)b]c besteht, worin
Y gleich oder verschieden eine CR2-, O-,
S-, SO2, SO2NH-,
NR-, COO- oder CONR- Funktion sein kann, wobei R gleich oder unterschiedlich
die Funktionen von R1 bis R5 und R7 bis R12 einnehmen kann und a
und b gleich oder verschieden die Werte von 0-18 und c die Werte
von 1-18 darstellen.Find use
in which:
R 7 -R 12 are identical or different and are hydrogen, one or more chlorine, bromine and / or iodine atoms, alkyl, aryl, hereroaryl, cycloalkyl, heterocycloalkyl, alkyloxy, alkylmercapto, aryloxy, arylmercapto, Heteroaryloxy, heteroarylmercapto, nitro radicals, carboxylic acid, carboxylic acid ester, carboxylic acid amide, trifluoromethyl, trifluoroacetyl, tricyanovinyl or cyano radicals, one or more hydroxy, one or more alkyl-substituted or cyclic amino functions and R7 and R8 and or R 11 and R 12 further aliphatic or aromatic rings or two adjacent radicals together may form one or more aliphatic or aromatic rings, one or more of the substituents R 7 -R 12 solubilizing or ionizable or ionized substituents such as SO 3 - , PO 3 2 - , CO 2 - , O-, NR 3 + or cyclodextrin or sugar, which determine the hydrophilic properties of the dyes, these substituents also via an aliphatic e or heteroaliphatic, for example cyclic, spacer group may be attached to the dye, at least one of the substituents R7-R12 for a reactive group of the type isocyanate, isothiocyanate, hydrazine, amine, mono- and dichloro- or mono- and dibromotriazine, aziridine, methacrylate , Acrylate, vinyl, vinylsulfonyl, sulfonyl halide, N-hydroxysuccinimide ester, imido-ester, glyoxal or aldehyde or maleimide or iodoacetamide and phosphoramidite, wherein the respective substituent can be attached via an aliphatic or heteroaliphatic, for example cyclic, spacer group on the dye and the aliphatic or hereroaliphatic spacer group consists of a structural element - [(CH 2 ) a -Y- (CH 2 ) b ] c , in which Y is, identically or differently, a CR 2 -, O-, S-, SO 2 , SO 2 NH-- , NR, COO or CONR function, where R may be the same or different from R1 to R5 and R7 to R12 and a and b may be the same or different from 0-18 and c represent the values of 1-18.
Vorteilhaft
wäre als
Rest R6 auch eine aromatische Verbindung der allgemeinen Formel
III wobei:
R7-R15
gleich oder unterschiedlich sind und Wasserstoff, ein oder mehrere
Chlor-, Brom- und/oder Iodatome, Alkyl-, Aryl-, Hereroaryl-, Cycloalkyl-,
Heterocycloalkyl-, Alkyloxy-, Alkylmercapto-, Aryloxy-, Arylmercapto-, Heteroaryloxy-,
Heteroarylmercapto-, Nitro-Reste, Carbonsäure-, Carbonsäureester-,
Carbonsäureamid-, Trifluormethyl,
Trifluoracetyl, Tricyanovinyl- oder Cyano-Reste, eine oder mehrere
Hydroxy-, eine oder mehrere alkylsubstituierte oder cyclische Aminofunktionen
sein können
und R7 und R8 und/oder R11 und R12 weitere aliphatische oder aromatische
Ringe bzw. zwei benachbarte Reste zusammen einen oder mehrere aliphatische
oder aromatische Ringe bilden können,
einer oder mehrere der Substituenten R7-R15 solubilisierende bzw. ionisierbare
bzw. ionisierte Substituenten wie SO3 –,
PO3 2–, CO2 –,
O-, NR3 + oder Cyclodextrin
oder Zucker darstellen, welche die hydrophilen Eigenschaften der
Farbstoffe bestimmen, wobei diese Substituenten auch über eine
aliphatische oder heteroaliphatische, beispielsweise zyklische,
Spacergruppe am Farbstoff angebunden sein können, mindestens einer der
Substituenten R7-R15 für
eine reaktive Gruppe vom Typ Isocyanat, Isothiocyanat, Hydrazin,
Amin, Mono- und Dichlor- bzw. Mono- und Dibromtriazin, Aziridin, Methacrylat, Acrylat,
Vinyl, Vinylsulfonyl, Sulfonylhalogenid, N-Hydroxysuccinimidester,
Imido-Ester, Glyoxal oder Aldehyd bzw. Maleimid oder Iodacetamid
sowie Phosphoramidit stehen kann, wobei der jeweilige Substituent über eine aliphatische
oder heteroaliphatische, beispielsweise zyklische, Spacergruppe
am Farbstoff angebunden sein kann und die aliphatische oder hereroaliphatische
Spacergruppe aus einem Strukturelement -[(CH2)a-Y-(CH2)b]c besteht, worin
Y gleich oder verschieden eine CR2-, O-,
S-, SO2, SO2NH-,
NR-, COO- oder CONR-Funktion sein kann, wobei R gleich oder unterschiedlich
die Funktionen von R1 bis R5 und R7 bis R15 einnehmen kann und a
und b gleich oder verschieden die Werte von 0-18 und c die Werte
von 1-18 darstellen.An advantageous radical R6 would also be an aromatic compound of general formula III in which:
R 7 -R 15 are identical or different and are hydrogen, one or more chlorine, bromine and / or iodine atoms, alkyl, aryl, hereroaryl, cycloalkyl, heterocycloalkyl, alkyloxy, alkylmercapto, aryloxy, arylmercapto, Heteroaryloxy, heteroarylmercapto, nitro radicals, carboxylic acid, carboxylic acid ester, carboxylic acid amide, trifluoromethyl, trifluoroacetyl, tricyanovinyl or cyano radicals, one or more hydroxy, one or more alkyl-substituted or cyclic amino functions and R7 and R8 and or R 11 and R 12 further aliphatic or aromatic rings or two adjacent radicals together may form one or more aliphatic or aromatic rings, one or more of the substituents R 7 -R 15 solubilizing or ionizable or ionized substituents such as SO 3 - , PO 3 2 - , CO 2 - , O-, NR 3 + or cyclodextrin or sugar, which determine the hydrophilic properties of the dyes, these substituents also via an aliphatic or heteroaliphatic, for example cyclic, spacer group may be attached to the dye, at least one of the substituents R7-R15 for a reactive group of the isocyanate, isothiocyanate, hydrazine, amine, mono- and dichloro- or mono- and dibromotriazine, aziridine , Methacrylate, acrylate, vinyl, vinylsulfonyl, sulfonyl halide, N-hydroxysuccinimide ester, imido-ester, glyoxal or aldehyde or maleimide or iodoacetamide and phosphoramidite, the respective substituent being attached to the dye via an aliphatic or heteroaliphatic, for example cyclic, spacer group and the aliphatic or heteroaliphatic spacer group consists of a structural element - [(CH 2 ) a -Y- (CH 2 ) b ] c , wherein Y, identically or differently, is a CR 2 -, O-, S-, SO 2 , SO 2 NH, NR, COO or CONR function, where R may be the same or different from R1 to R5 and R7 to R15 and a and b are the same or different the values of 0-18 and c represent the values of 1-18.
Nachstehend sollen beispielhaft vier spezielle Strukturen für langwellige Azofarbstoffe angegeben werden:below For example, four specific structures for long-wavelength azo dyes be specified:
Beispiel 1: Example 1:
Beispiel 2: Example 2:
Beispiel 3: Example 3:
Beispiel 4: Example 4:
Claims (13)
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DE200510050834 DE102005050834A1 (en) | 2005-10-20 | 2005-10-20 | Use of long chain azo dye as blotter for fluorescent based detection reactions and as dye for optical detection e.g. in qualitative and quantitative determination procedure e.g. immune tests and hybridization procedure |
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DE200510050834 DE102005050834A1 (en) | 2005-10-20 | 2005-10-20 | Use of long chain azo dye as blotter for fluorescent based detection reactions and as dye for optical detection e.g. in qualitative and quantitative determination procedure e.g. immune tests and hybridization procedure |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016160051A1 (en) | 2015-03-30 | 2016-10-06 | Enzo Biochem, Inc. | Monoazo dyes with cyclic amine as fluorescence quenchers |
US9925278B2 (en) | 2008-10-23 | 2018-03-27 | Koninklijke Philips N.V. | Molecular imaging |
-
2005
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9925278B2 (en) | 2008-10-23 | 2018-03-27 | Koninklijke Philips N.V. | Molecular imaging |
WO2016160051A1 (en) | 2015-03-30 | 2016-10-06 | Enzo Biochem, Inc. | Monoazo dyes with cyclic amine as fluorescence quenchers |
US9951227B2 (en) | 2015-03-30 | 2018-04-24 | Enzo Biochem, Inc. | Monoazo dyes with cyclic amine as fluorescence quenchers |
US9957393B2 (en) | 2015-03-30 | 2018-05-01 | Enzo Biochem, Inc. | Monoazo dyes with cyclic amine as fluorescence quenchers |
US10131789B2 (en) | 2015-03-30 | 2018-11-20 | Enzo Biochem, Inc. | Monoazo dyes with cyclic amine as fluorescence quencers |
US10865309B2 (en) | 2015-03-30 | 2020-12-15 | Enzo Biochem, Inc. | Monoazo dyes with cyclic amine as fluorescence quenchers |
EP4008790A1 (en) | 2015-03-30 | 2022-06-08 | Enzo Biochem, Inc. | Monoazo dyes with cyclic amine as fluorescence quenchers |
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