DE19957007A1 - New cyanine dyes, used as fluorescent marking dyes, e.g. for analysis of clinical, biological, biochemical or chemical substance, have sulfoaryl and N-sulfoalkyl groups, meso-halo-methine chain and reactive binding group - Google Patents
New cyanine dyes, used as fluorescent marking dyes, e.g. for analysis of clinical, biological, biochemical or chemical substance, have sulfoaryl and N-sulfoalkyl groups, meso-halo-methine chain and reactive binding groupInfo
- Publication number
- DE19957007A1 DE19957007A1 DE1999157007 DE19957007A DE19957007A1 DE 19957007 A1 DE19957007 A1 DE 19957007A1 DE 1999157007 DE1999157007 DE 1999157007 DE 19957007 A DE19957007 A DE 19957007A DE 19957007 A1 DE19957007 A1 DE 19957007A1
- Authority
- DE
- Germany
- Prior art keywords
- dyes
- groups
- sulfoaryl
- meso
- methine chain
- 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- Withdrawn
Links
- 239000000126 substance Substances 0.000 title claims abstract description 8
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 title claims abstract 6
- 239000000975 dye Substances 0.000 title abstract description 22
- 238000004458 analytical method Methods 0.000 title description 2
- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 claims abstract description 6
- 229910052736 halogen Inorganic materials 0.000 claims abstract description 4
- 125000005843 halogen group Chemical group 0.000 claims abstract description 4
- 238000006467 substitution reaction Methods 0.000 claims abstract description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 7
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 3
- 239000003550 marker Substances 0.000 claims description 3
- 125000004964 sulfoalkyl group Chemical group 0.000 claims description 2
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical class ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims 1
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 12
- 150000003839 salts Chemical class 0.000 description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- -1 iodoacetamido group Chemical group 0.000 description 7
- 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 6
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 6
- 159000000000 sodium salts Chemical class 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical class [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 4
- 238000000354 decomposition reaction Methods 0.000 description 4
- NVRVNSHHLPQGCU-UHFFFAOYSA-N 6-bromohexanoic acid Chemical compound OC(=O)CCCCCBr NVRVNSHHLPQGCU-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 238000004220 aggregation Methods 0.000 description 3
- 230000002776 aggregation Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- NNKXEASLNXMELZ-UHFFFAOYSA-N [Br-].C(=O)(O)CCCCC[N+]1=C(C(C=2C3=C(C=CC1=2)C(=CC(=C3)S(=O)(=O)O)S(=O)(=O)O)(C)C)C Chemical compound [Br-].C(=O)(O)CCCCC[N+]1=C(C(C=2C3=C(C=CC1=2)C(=CC(=C3)S(=O)(=O)O)S(=O)(=O)O)(C)C)C NNKXEASLNXMELZ-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000008033 biological extinction Effects 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 125000002768 hydroxyalkyl group Chemical group 0.000 description 2
- 238000001499 laser induced fluorescence spectroscopy Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000008300 phosphoramidites Chemical class 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- OPSQJFXXNLXVOH-UHFFFAOYSA-N 6-(2,3,3-trimethyl-5-sulfoindol-1-ium-1-yl)hexanoic acid;bromide Chemical compound [Br-].C1=C(S(O)(=O)=O)C=C2C(C)(C)C(C)=[N+](CCCCCC(O)=O)C2=C1 OPSQJFXXNLXVOH-UHFFFAOYSA-N 0.000 description 1
- XVMSFILGAMDHEY-UHFFFAOYSA-N 6-(4-aminophenyl)sulfonylpyridin-3-amine Chemical compound C1=CC(N)=CC=C1S(=O)(=O)C1=CC=C(N)C=N1 XVMSFILGAMDHEY-UHFFFAOYSA-N 0.000 description 1
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical class S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 1
- 206010056740 Genital discharge Diseases 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- QNBRAPKFEQNQCX-UHFFFAOYSA-N [OH-].CC1=[N+](C2=CC=CC=C2C1(C)C)CCCCS(=O)(=O)O Chemical compound [OH-].CC1=[N+](C2=CC=CC=C2C1(C)C)CCCCS(=O)(=O)O QNBRAPKFEQNQCX-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 125000005189 alkyl hydroxy group Chemical group 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 238000004061 bleaching Methods 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 125000004181 carboxyalkyl group Chemical group 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 229940000425 combination drug Drugs 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- ZBKFYXZXZJPWNQ-UHFFFAOYSA-N isothiocyanate group Chemical group [N-]=C=S ZBKFYXZXZJPWNQ-UHFFFAOYSA-N 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 238000006303 photolysis reaction Methods 0.000 description 1
- 230000015843 photosynthesis, light reaction Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 229960002317 succinimide Drugs 0.000 description 1
- PXQLVRUNWNTZOS-UHFFFAOYSA-N sulfanyl Chemical class [SH] PXQLVRUNWNTZOS-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
- C09B23/0008—Methine or polymethine dyes, e.g. cyanine dyes substituted on the polymethine chain
- C09B23/0016—Methine or polymethine dyes, e.g. cyanine dyes substituted on the polymethine chain the substituent being a halogen atom
-
- 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
- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
- C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
- C09B23/08—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines
- C09B23/083—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines five >CH- groups
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Indole Compounds (AREA)
Abstract
Description
Die Erfindung betrifft Cyaninfarbstoffe, insbesondere für die Anwendung als Fluoreszenzmarker im Bereich 600 bis 700 nm, die sich gut in Wasser lösen, eine geringe Tendenz zur Aggregation aufweisen und mit Reaktivgruppen versehen sind.The invention relates to cyanine dyes, in particular for the Application as a fluorescence marker in the range 600 to 700 nm, the dissolve well in water, a small tendency for aggregation have and are provided with reactive groups.
Fluoreszenzfarbstoffe werden als Marker auf vielfältige Weise für die Analyse eines Großteils klinisch, biologisch, bioche misch oder chemisch relevanter Substanzen wie z. B. Zellen, Antikörper, Proteine, Hormone, Nucleinsäuren, Oligonucleotide, Kohlenhydrate oder Amine und Mercaptane eingesetzt. In Kombi nation mit der leicht detektierbaren, durch Laser induzierten Fluoreszenz (LIF; Laser-Induced Fluorescence), sind schnelle Analysen in den o.a. Gebieten möglich.Fluorescent dyes are used as markers in a variety of ways for the analysis of a large part of clinical, biological, bioche mixed or chemically relevant substances such. Cells, Antibodies, proteins, hormones, nucleic acids, oligonucleotides, Carbohydrates or amines and mercaptans used. In combi nation with the easily detectable, laser-induced Fluorescence (LIF, Laser-Induced Fluorescence), are fast Analyzes in the o.a. Areas possible.
Durch kovalente Bindung kann eine größere Anzahl Farbstoffmo leküle an eine Probe angeheftet werden, als es im Fall einer unspezifischen nicht-kovalenten Bindung möglich ist, so daß die Intensität des Fluoreszenzlichtsignals stärker ist. Außer dem ermöglicht eine kovalente Bindung die eindeutige Markie rung einer spezifischen Zielstruktur in einem Gemisch. Zur ko valenten Bindung des Fluoreszenzfarbstoffes an die Probe wer den die Farbstoffe deshalb mit Reaktivgruppen versehen. Ent sprechende Reaktivgruppen sind z. B. die Iodacetamidogruppe, die Isothio-cyanatgruppe, N-Succinimidester von Alkylcarbon säuren oder Phosphoramidite der Hydroxyalkylgruppen.By covalent bonding, a larger number of Farbstoffmo be attached to a sample, as in the case of a nonspecific non-covalent bond is possible, so that the intensity of the fluorescent light signal is stronger. except This allows a covalent bond the unique Markie specific target structure in a mixture. To ko valent binding of the fluorescent dye to the sample who the dyes therefore provided with reactive groups. Ent speaking reactive groups are z. The iodoacetamido group, the isothio cyanate group, N-succinimide ester of alkylcarbon acids or phosphoramidites of the hydroxyalkyl groups.
Als Reaktionspartner stehen bei Antikörpern, Proteinen, Hormo nen, Nucleinsäuren, und anderen Biomolekülen u. a. Mercapto- (-SH), Amino- (-NH2) und Hydroxygruppen (-OH) zur Verfügung. As reactants, antibodies, proteins, hormones, nucleic acids, and other biomolecules include mercapto (-SH), amino (-NH 2 ), and hydroxy groups (-OH).
Ganz wesentlich für die Verwendbarkeit der Fluoreszenzmarker ist neben der Löslichkeit in wäßrigen Systemen die Stabilität gegenüber photolytischer Zersetzung. Bisher bekannte Fluores zenzmarker neigen bei Abtasten der Proben im Laserlicht, vor allem bei Verwendung eines Fluoreszenzmikroskopes, zum Aus bleichen.Quite essential for the usability of the fluorescent markers is stability in addition to solubility in aqueous systems against photolytic decomposition. Previously known fluores Centers tend to scan the samples in laser light especially when using a fluorescence microscope, to off bleaching.
Eine Möglichkeit, die Stabilität von Cyaninfarbstoffen gegen über Photolyse zu erhöhen, besteht in der Einführung verstei fender Gruppen in die Methinkette. Bekannt ist der Einbau von partiell ungesättigten Ringen mit fünf und sechs Ringgliedern in die Methinkette. Dieses Prinzip findet Verwendung bei Heptamethin-Cyaninen.One way to counteract the stability of cyanine dyes to increase over photolysis, consists in the introduction of steal fender groups into the methine chain. Known is the installation of partially unsaturated rings with five and six ring members into the methine chain. This principle is used Heptamethine cyanines.
Es ist die Aufgabe der Erfindung, neue Farbstoffe anzugeben, die eine verbesserte Stabilität gegen photolytische Zersetzung aufweisen, in Wasser gut löslich sind, eine geringe Tendenz zur Aggregation aufweisen, mit Reaktivgruppen versehen sind, und für die Anwendung als Fluoreszenzmarker im Bereich 600 bis 700 nm geeignet sind.It is the object of the invention to provide new dyes, the improved stability against photolytic decomposition have low solubility in water, a slight tendency have aggregation, are provided with reactive groups, and for use as a fluorescence marker in the range 600 to 700 nm are suitable.
Diese Aufgabe wird durch Cyaninfarbstoffe mit den Merkmalen gemäß Anspruch 1 gelöst. Vorteilhafte Ausführungsformen und Verwendungen ergeben sich aus den abhängigen Ansprüchen.This task is accomplished by cyanine dyes with the characteristics solved according to claim 1. Advantageous embodiments and Uses result from the dependent claims.
Die Grundidee der Erfindung besteht darin, durch eine gezielte Einführung von Substituenten in die Methinkette den bekannten Nachteil der photolytischen Zersetzung offenkettiger Cyanin farbstoffe zu beseitigen. Um die Fluoreszenz-quantenausbeute nicht zu verringern und um unspezifische nicht-kovalente Bin dungen zu verhindern, kommt es auch darauf an, jede Art der Farbstoffaggregatbildung zu vermeiden. Ein erfindungsgemäßer Cyaninfarbstoff zeichnet sich durch eine Kombination von Sul foaryl- und N-Sulfoalkylgruppen, eine Halogensubstitution in meso-Position der Methinkette und eine reaktive Gruppe aus, die die Bindung an Trägersubstanzen ermöglicht.The basic idea of the invention is, by a targeted Introduction of Substituents in the Methine Chain the Known Disadvantage of the photolytic decomposition of open-chain cyanine eliminate dyes. To the fluorescence quantum yield not to decrease and unspecific non-covalent bin It also depends on preventing any kind of Avoid dye aggregation. An inventive Cyanine dye is characterized by a combination of Sul foaryl and N-sulfoalkyl groups, a halogen substitution in meso position of the methine chain and a reactive group, which allows binding to carrier substances.
Die neuen Cyaninfarbstoffe lassen sich bei Verwendung ver
schiedener Anregungslichtquellen im Absorptionsbereich zwi
schen 640 und 680 nm einsetzen und besitzen gegenüber herkömm
lichen Fluoreszenzmarkern eine erhöhte Photostabilität.
The new cyanine dyes can be used in the absorption range between 640 and 680 nm when using different excitation light sources in the absorption range and have increased photostability compared to conventional fluorescent markers.
Dabei sind n = 1, 2, R1, R2 = H, SO3M, die zur Vervollständi gung eines Benzenringes notwendigen vier CH-Gruppen bzw. die zur Vervollständigung eines Benzenringes notwendigen vier CH- Gruppen einschließlich (SO3M)n, M = H, Na, K, und X = Halogen (z. B. Cl, Br).In this case, n = 1, 2, R 1 , R 2 = H, SO 3 M, the four CH groups necessary for completing a benzene ring or the four CH groups necessary for completing a benzene ring, including (SO 3 M) n , M = H, Na, K, and X = halogen (eg Cl, Br).
In den allgemeinen Formeln 1 und 2 stehen R3 und R4 für eine Carboxyalkylgruppe oder eine Hydroxyalkylgruppe. (z. B.: R3 = Sulfoalkyl mit vier CH2-Gruppen, (CH2)mCOOH, (CH2)mCH2OH, m = 2-5, R4 = (CH2)mCOOH, (CH2)mCH2OH, m = 2-5). In den Farbstof fen mit Carboxylgruppen können diese in ihre N-Succinimid- Ester überführt werden, die dann direkt mit Aminogruppen am Biomolekül reagieren oder über einen sog. Aminolinker mit Hy droxygruppen. In den Farbstoffen mit Alkylhydroxygruppen kön nen diese in Phosphoramidite überführt werden, die direkt mit der freien 5'-OH-Gruppe von Nucleotiden zu einer Phosphitbin dung reagieren, die im Folgenden zu einer stabilen Phosphat bindung oxidiert.In the general formulas 1 and 2, R 3 and R 4 represent a carboxyalkyl group or a hydroxyalkyl group. (eg: R 3 = sulfoalkyl with four CH 2 groups, (CH 2 ) m COOH, (CH 2 ) m CH 2 OH, m = 2-5, R 4 = (CH 2 ) m COOH, ( CH 2 ) m CH 2 OH, m = 2-5). In the dyes with carboxyl groups, these can be converted into their N-succinimide esters, which then react directly with amino groups on the biomolecule or via a so-called amino linker with hydroxyl groups. In the dyes with alkyl hydroxy groups, these can be converted into phosphoramidites which react directly with the free 5'-OH group of nucleotides to form a phosphite bond, which subsequently oxidizes to a stable phosphate bond.
1-Carboxypentyl-2-[5-(1-carboxypentyl-1,3-dihydro-3,3- dimethyl-5-sulfo-2H-indol-2-yliden)-3-chlorpenta-1,3-dienyl]- 3,3-dimethyl-5-sulfo-3H-indolium-Innersalz, Natriumsalz1-carboxypentyl-2- [5- (1-carboxypentyl-1,3-dihydro-3,3- dimethyl-5-sulfo-2H-indol-2-ylidene) -3-chloro-penta-1,3-dienyl] - 3,3-dimethyl-5-sulfo-3H-indolium-internal salt, sodium salt
-
A) Natriumsalz des 1-(5-Carboxypentyl)-2,3,3-trimethyl-5-
sulfoindoliumbromids:
2,61 g Natriumsalz des 2,3,3-Trimethyl-5-sulfoindolenins und 2,1 g 6-Bromhexansäure werden in Substanz 2 h bei einer Badtemperatur von 110°C unter Rühren zur Reaktion gebracht. Nach beendeter Reaktion wird der ölige Rückstand mehrmals mit Aceton versetzt, bis ein Feststoff zurück bleibt. Ausbeute: 2,9 gA) Sodium salt of 1- (5-carboxypentyl) -2,3,3-trimethyl-5-sulfoindolium bromide:
2.61 g of sodium salt of 2,3,3-trimethyl-5-sulfoindolenine and 2.1 g of 6-bromohexanoic acid are reacted in substance for 2 h at a bath temperature of 110 ° C. with stirring. After completion of the reaction, the oily residue is added several times with acetone until a solid remains. Yield: 2.9 g -
B) 1-Carboxypentyl-2-[5-(1-carboxypentyl-1,3-dihydro-3,3-
dimethyl-5-sulfo-2H-indol-2-yliden)-3-chlorpenta-1,3-
dienyl]-3,3-dimethyl-5-sulfo-3H-indolium-Innersalz, Natrium
salz:
2,3 g des Quartärsalzes aus A) und 0,73 g des 2- Chlormalondianil-Hydrochlorids werden in 20 ml Ethanol in Ge genwart von je 1 ml Acetanhydrid und Triethylamin 25 Minuten am Rückfluß erhitzt. Nach Erkalten wird mit 2 ml 3 molarer HCl versetzt und der Farbstoff anschließend durch Etherzusatz ge fällt. (λ = 653 nm)B) 1-carboxypentyl-2- [5- (1-carboxypentyl-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene) -3-chloro-penta-1,3-dienyl ] -3,3-dimethyl-5-sulfo-3H-indolium-inner salt, sodium salt:
2.3 g of the quaternary salt of A) and 0.73 g of the 2-Chlormalondianil hydrochloride are heated in 20 ml of ethanol in Ge presence of 1 ml of acetic anhydride and triethylamine refluxed for 25 minutes. After cooling, 2 ml of 3 molar HCl are added and the dye is then added by ether addition ge. (λ = 653 nm)
3-Carboxypentyl-2-[5-(3-carboxypentyl-1,3-dihydro-1,1 dimethyl-6,8-disulfo-2H-benzo[e]indol-2-yliden)-3-chlorpenta- 1,3-dienyl]-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indolium- Innersalz, Trinatriumsalz3-carboxypentyl-2- [5- (3-carboxypentyl-1,3-dihydro-1,1 dimethyl-6,8-disulfo-2H-benzo [e] indol-2-ylidene) -3-chlorpenta- 1,3-dienyl] -1,1-dimethyl-6,8-disulfo-1H-benzo [e] indolium Inner salt, trisodium salt
-
A) Natriumsalz des 3-(5-Carboxypentyl)-1,1,2-trimethyl-6,8-
disulfobenzo[e]-indoliumbromids:
4,1 g Natriumsalz des 1,1,2-Trimethyl-6,8- disulfobenzo[e]indolenins und 2,1 g 6-Bromhexansäure werden in Substanz 2 h bei einer Badtemperatur von 110°C unter Rühren zur Reaktion gebracht. Nach beendeter Reaktion wird der ölige Rückstand mehrmals mit Aceton versetzt, bis ein Feststoff zu rück bleibt. Ausbeute: 5,3 gA) Sodium salt of 3- (5-carboxypentyl) -1,1,2-trimethyl-6,8-disulfobenzo [e] indolium bromide:
4.1 g of sodium salt of 1,1,2-trimethyl-6,8-disulfobenzo [e] indoleninsin and 2.1 g of 6-bromohexanoic acid are reacted in substance for 2 h at a bath temperature of 110 ° C with stirring. After completion of the reaction, the oily residue is added several times with acetone until a solid remains behind. Yield: 5.3 g -
B) 3-Carboxypentyl-2-[5-(3-carboxypentyl-1,3-dihydro-1,1-
dimethyl-6,8-disulfo-2H-benzo[e]indol-2-yliden)-3-chlorpenta-
1,3-dienyl]-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indolium-
Innersalz, Trinatriumsalz:
6,1 g des Quartärsalzes aus A) und 1,5 g des 2- Chlormalondia nil-Hydrochlorids werden in Methanol bei Raumtemperatur unter Zusatz von 2 ml Acetanhydrid und 3 ml Triethylamin 4,5 h ge rührt. Anschließend gibt man einen Tropfen konzentrierte HCl zu und fällt mit Ethylacetat. (λ = 692 nm)B) 3-carboxypentyl-2- [5- (3-carboxypentyl-1,3-dihydro-1,1-dimethyl-6,8-disulfo-2H-benzo [e] indol-2-ylidene) -3-chloropenta 1,3-dienyl] -1,1-dimethyl-6,8-disulfo-1H-benzo [e] indolium inner salt, trisodium salt:
6.1 g of the quaternary salt of A) and 1.5 g of the 2-Chlormalondia nil hydrochloride are stirred in methanol at room temperature with the addition of 2 ml of acetic anhydride and 3 ml of triethylamine for 4.5 h. Then add one drop of concentrated HCl and precipitate with ethyl acetate. (λ = 692 nm)
2-[5-(3-(5-Carboxypentyl)-1,3-dihydro-1,1-dimethyl-6,8- disulfo-2H-benzo[e]indol-2-yliden)-3-brompenta-1,3-dienyl]- 1,1-dimethyl-3-(4-sulfobutyl)-3H-indolium Hydroxid, Innersalz, Dinatriumsalz2- [5- (3- (5-Carboxypentyl) -1,3-dihydro-1,1-dimethyl-6,8 disulfo-2H-benzo [e] indol-2-ylidene) -3-bromopenta-1,3-dienyl] - 1,1-dimethyl-3- (4-sulfobutyl) -3H-indolium hydroxide, inner salt, disodium salt
-
A) 2-(4-Acetanilino-1,3-butadienyl)-3,3-dimethyl-1-(4-
sulfobutyl)-3H-indolium Hydroxid, Innersalz:
Ein Gemisch aus 11,8 g 1-(4-Sulfobutyl)-2,3,3-trimethyl-3H indolium Hydroxid, Innersalz und 13,55 g 2-Brommalondianil- Hydrochlorid in 50 ml Acetanhydrid werden 1 h bei einer Badtemperatur von 120°C zur Reaktion gebracht. Nach Abkühlung werden 300 ml Ethylacetat zugesetzt und der anfallende Nieder schlag abgesaugt. Ausbeute: 17,5 gA) 2- (4-Acetanilino-1,3-butadienyl) -3,3-dimethyl-1- (4-sulfobutyl) -3H-indolium hydroxide, inner salt:
A mixture of 11.8 g of 1- (4-sulfobutyl) -2,3,3-trimethyl-3H indolium hydroxide, inner salt and 13.55 g of 2-Brommalondianil hydrochloride in 50 ml of acetic anhydride are heated for 1 h at a bath temperature of 120 ° C reacted. After cooling, 300 ml of ethyl acetate are added and the resulting precipitate is filtered off with suction. Yield: 17.5 g -
B) Dinatriumsalz des 3-(5-Carboxypentyl)-6,8-disulfo-1,1,2-
trimethyl-benzo[e]-indoliumbromids:
4,13 g Dinatriumsalz des 6,8-Disulfo-1,1,2-trimethybenzo[e]- indolenins und 2,15 g 6-Bromhexansäure werden in Substanz 2 h bei einer Badtemperatur von 110°C erhitzt. Der nach beendeter Reaktion verbleibende Rückstand wird mit Aceton verrieben. Ausbeute: 5,2 gB) Disodium salt of 3- (5-carboxypentyl) -6,8-disulfo-1,1,2-trimethyl-benzo [e] -indolium bromide:
4.13 g of disodium salt of 6,8-disulfo-1,1,2-trimethylbenzo [e] -indolenine and 2.15 g of 6-bromohexanoic acid are heated in substance for 2 h at a bath temperature of 110.degree. The residue remaining after the reaction is triturated with acetone. Yield: 5.2 g -
C) 2-[5-(3-(5-Carboxypentyl)-1,3-dihydro-1,1-dimethyl-6,8-
disulfo-2H-benzo[e]indol-2-yliden)-3-brompenta-1,3-dienyl]-
1,1-dimethyl-3-(4-sulfobutyl)-3H-indolium Hydroxid, Innersalz,
Dinatriumsalz:
2,73 g der Verbindung aus A) und 3,05 g der Verbindung aus B) werden in 25 ml Pyridin in Gegenwart von 3 ml Acetanhydrid 1 h am Rückfluß erhitzt. Nach Abkühlen auf Raumtemperatur werden 80 ml Ethylacetat zugesetzt und der Niederschlag abfiltriert. Der Rückstand wird in Methanol aufgenommen, mit 4 ml Essigsäu re versetzt und durch Etherzugabe ausgefällt. Der so erhaltene Rohfarbstoff wird durch Säulenchromatographie über Kieselgel gereinigt. (λ = 674 nm)C) 2- [5- (3- (5-carboxypentyl) -1,3-dihydro-1,1-dimethyl-6,8-disulfo-2H-benzo [e] indol-2-ylidene) -3-bromopenta -1,3-dienyl] -1,1-dimethyl-3- (4-sulfobutyl) -3H-indolium hydroxide, inner salt, disodium salt:
2.73 g of the compound from A) and 3.05 g of the compound from B) are refluxed in 25 ml of pyridine in the presence of 3 ml of acetic anhydride for 1 h. After cooling to room temperature, 80 ml of ethyl acetate are added and the precipitate is filtered off. The residue is taken up in methanol, treated with 4 ml of acetic acid and precipitated by addition of ether. The crude dye thus obtained is purified by column chromatography on silica gel. (λ = 674 nm)
Zum Vergleich der photochemischen Stabilität der erfindungsge mäßen Farbstoffe 1 und 2 mit herkömmlichen Farbstoffen 4 und 5 wurden diese in eine inerte Schicht eingebracht und mit einer Lampe von 150 Watt Leistung bestrahlt. In Abhängigkeit von der Bestrahlungszeit wurde die Extinktion bestimmt. Vom Logarith mus der normalisierten Extinktion [ln (E/E0)] und der Lager zeit wurde eine Korrelation durchgeführt und die Halbwertzeit T1/2 der Zersetzung des jeweiligen Farbstoffes bestimmt. Diese Halbwertszeit dient als Maß für die photochemische Sta bilität der Farbstoffe.To compare the photochemical stability of the erfindungsge MAESSEN dyes 1 and 2 with conventional dyes 4 and 5, these were placed in an inert layer and irradiated with a lamp of 150 watts of power. Depending on the irradiation time, the extinction was determined. From the logarithm of the normalized extinction [ln (E / E 0 )] and the storage time, a correlation was carried out and the half-life T 1/2 of the decomposition of the respective dye was determined. This half-life serves as a measure of the photochemical Sta stability of the dyes.
Ermittlung der relativen photochemischen Stabilität der Farb
stoffe 1 und 2, gegen 4 und 5
Determination of the relative photochemical stability of the dyes 1 and 2, against 4 and 5
Claims (4)
mit:
n = 1, 2
R1, R2 = H, SO3M, die zur Vervollständigung eines Benzenringes notwendigen vier CH-Gruppen bzw. die zur Vervollständigung ei nes Benzenringes notwendigen vier CH-Gruppen einschließlich (SO3M)n
R3 = Sulfoalkyl mit vier CH2-Gruppen, (CH2)mCOOH, (CH2)mCH2OH, m = 2-5
R4 = (CH2)mCOOH, (CH2)mCH2OH, m = 2-5
M = H, Na, K
X = Cl, Br2. cyanine dye according to claim 1, characterized by the general structures
With:
n = 1, 2
R 1 , R 2 = H, SO 3 M, the four CH groups necessary for completing a benzene ring or the four CH groups necessary for completing a benzene ring, including (SO 3 M) n
R 3 = sulfoalkyl with four CH 2 groups, (CH 2 ) m COOH, (CH 2 ) m CH 2 OH, m = 2-5
R 4 = (CH 2 ) m COOH, (CH 2 ) m CH 2 OH, m = 2-5
M = H, Na, K
X = Cl, Br
R3, R4 = die N-Hydroxysuccinimid-Ester der Carboxylgruppe (CH2)mCOOH mit m = 2-5 und das Bis-(N,N-di isopropyl)-β-cyanethyl-phosphoramidit der Hydroxy gruppe (CH2)mCH2OH mit m = 2-5;
bedeuten.3. cyanine dye according to claim 1 or 2, characterized in that
R 3 , R 4 = the N-hydroxysuccinimide esters of the carboxyl group (CH 2 ) m COOH with m = 2-5 and the bis- (N, N-diisopropyl) -β-cyanoethyl phosphoramidite of the hydroxy group (CH 2 ) m CH 2 OH with m = 2-5;
mean.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE1999157007 DE19957007A1 (en) | 1999-11-26 | 1999-11-26 | New cyanine dyes, used as fluorescent marking dyes, e.g. for analysis of clinical, biological, biochemical or chemical substance, have sulfoaryl and N-sulfoalkyl groups, meso-halo-methine chain and reactive binding group |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE1999157007 DE19957007A1 (en) | 1999-11-26 | 1999-11-26 | New cyanine dyes, used as fluorescent marking dyes, e.g. for analysis of clinical, biological, biochemical or chemical substance, have sulfoaryl and N-sulfoalkyl groups, meso-halo-methine chain and reactive binding group |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| DE19957007A1 true DE19957007A1 (en) | 2001-05-31 |
Family
ID=7930460
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DE1999157007 Withdrawn DE19957007A1 (en) | 1999-11-26 | 1999-11-26 | New cyanine dyes, used as fluorescent marking dyes, e.g. for analysis of clinical, biological, biochemical or chemical substance, have sulfoaryl and N-sulfoalkyl groups, meso-halo-methine chain and reactive binding group |
Country Status (1)
| Country | Link |
|---|---|
| DE (1) | DE19957007A1 (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002012398A1 (en) * | 2000-08-08 | 2002-02-14 | Fuji Photo Film Co., Ltd. | Cyanine dyes |
| WO2003029356A1 (en) * | 2001-09-27 | 2003-04-10 | Fuji Photo Film Co., Ltd. | Asymmetric cyanine dye |
| WO2003074091A2 (en) * | 2002-03-07 | 2003-09-12 | Fuji Photo Film Co., Ltd. | Near infrared fluorescent contrast agent and method for fluorescence imaging |
| WO2003082988A1 (en) * | 2002-03-29 | 2003-10-09 | The General Hospital Corporation | Nir-fluorescent cyanine dyes, their synthesis and biological use |
| DE19921234B4 (en) * | 1999-05-07 | 2005-07-28 | Few Chemicals Gmbh | Cyanine dyes |
| WO2004085539A3 (en) * | 2003-03-21 | 2006-01-12 | Amersham Biosciences Corp | Cyanine dye labelling reagents with meso-substitution |
| JP2011046662A (en) * | 2009-08-28 | 2011-03-10 | Fujifilm Corp | Near infrared fluorescent imaging agent |
-
1999
- 1999-11-26 DE DE1999157007 patent/DE19957007A1/en not_active Withdrawn
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19921234B4 (en) * | 1999-05-07 | 2005-07-28 | Few Chemicals Gmbh | Cyanine dyes |
| WO2002012398A1 (en) * | 2000-08-08 | 2002-02-14 | Fuji Photo Film Co., Ltd. | Cyanine dyes |
| US6939975B2 (en) | 2000-08-08 | 2005-09-06 | Fuji Photo Film Co., Ltd. | Cyanine dyes |
| WO2003029356A1 (en) * | 2001-09-27 | 2003-04-10 | Fuji Photo Film Co., Ltd. | Asymmetric cyanine dye |
| US7473415B2 (en) | 2002-03-07 | 2009-01-06 | Fuji Photo Film Co., Ltd. | Near infrared fluorescent contrast agent and method for fluorescence imaging |
| WO2003074091A2 (en) * | 2002-03-07 | 2003-09-12 | Fuji Photo Film Co., Ltd. | Near infrared fluorescent contrast agent and method for fluorescence imaging |
| WO2003074091A3 (en) * | 2002-03-07 | 2004-03-11 | Fuji Photo Film Co Ltd | Near infrared fluorescent contrast agent and method for fluorescence imaging |
| WO2003082988A1 (en) * | 2002-03-29 | 2003-10-09 | The General Hospital Corporation | Nir-fluorescent cyanine dyes, their synthesis and biological use |
| JP2006523257A (en) * | 2003-03-21 | 2006-10-12 | ジーイー・ヘルスケア・バイオサイエンス・コーポレイション | Meso-substituted cyanine dye labeling reagent |
| US7172907B2 (en) * | 2003-03-21 | 2007-02-06 | Ge Healthcare Bio-Sciences Corp. | Cyanine dye labelling reagents with meso-substitution |
| WO2004085539A3 (en) * | 2003-03-21 | 2006-01-12 | Amersham Biosciences Corp | Cyanine dye labelling reagents with meso-substitution |
| JP4790598B2 (en) * | 2003-03-21 | 2011-10-12 | ジーイー・ヘルスケア・バイオサイエンス・コーポレイション | Meso-substituted cyanine dye labeling reagent |
| JP2011046662A (en) * | 2009-08-28 | 2011-03-10 | Fujifilm Corp | Near infrared fluorescent imaging agent |
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