DE102012205807A1 - New cyanine dye comprising e.g. sulfo-aryl group, and methine chain, which is bridged with five or six-membered ring structure, and comprises differently substituted pyrimidinetrione in meso-position, useful e.g. in assay for detecting cell - Google Patents

Abstract

Cyanine dye comprising sulfo-aryl- and N-sulfo-alkyl groups or trialkylammonium-alkyl groups, where the alkyl group exhibits 1-4 carbon atoms, and a methine chain, which is bridged with a five- or six-membered ring structure, and comprises a differently substituted pyrimidinetrione in its meso-position comprising a reactive group, which allows binding to carrier substances, where at least one oxygen atom, preferably oxygen atom in para position to the bond to the methine chain, in the pyrimidinetrione, is optionally substituted by a sulfur atom, is new. An independent claim is also included for preparing the cyanine dye.

Description

The invention relates to cyanine dyes for use as fluorescent markers in the range of 700 to 800 nm, which dissolve well in water, are provided with reactive groups and in comparison to conventional fluorescent markers do not tend to form aggregates in aqueous solution and in buffer systems, and Process for their preparation and their use.

State of the art

Fluorescent dyes are used as markers in a variety of ways for the analysis of a large part of clinically, biologically, biochemically or chemically relevant substances such. As cells, antibodies, proteins, hormones, nucleic acids, oligonucleotides, carbohydrates or amines and mercaptans used. In combination with the easily detectable, laser-induced fluorescence (LIF), rapid analyzes in the above-mentioned. Areas possible.

By covalent bonding, a larger number of dye molecules can be attached to a sample than is possible in the case of non-specific non-covalent binding, so that the intensity of the fluorescent light signal is stronger. In addition, covalent bonding allows unique labeling of a specific target in a mixture. For covalent bonding of the fluorescent dye to the sample, the dyes are therefore provided with reactive groups. Corresponding reactive groups are z. The iodoacetamido group, the isothiocyanato group, N-succinimide esters of alkylcarboxylic acids or phosphoramidites of the hydroxyalkyl groups.

Reactants for antibodies, proteins, hormones, nucleic acids, and other biomolecules include mercapto - (- SH), amino - (- NH ₂ ), and hydroxy groups (-OH).

An essential goal is to reduce the detection limit ever further. The prerequisite for this is a low level of background fluorescence (autofluorescence of accompanying substances, solvents), light scattering (Rayleigh, Tyndall and Raman scattering) and fluorescence quenching. Especially in clinically and biologically significant applications, these effects, due to the colloidal nature of many biomolecules, increasingly occur. Compared to the analytics in the visible spectral range, the background fluorescence and the light scattering in the near infrared (NIR) do not occur or only in a small form. With the development of low-cost and long-wave laser diodes (780 nm), the prerequisites required by the technical side for the detection technology in the NIR were in principle present. Dyes which absorb in the NIR range were available, inter alia, from silver halide photography. One of the earliest works in which an NIR dye was used for bioanalytical applications has returned Sauda et al. (Anal Chem 58 (1986) 2649) That indocyanine green (ICG) 1 (λ _max = 785 nm; MeOH) were used for protein analysis in human serum and thus increase the detection sensitivity about two orders of magnitude.

However, this dye has various handling issues. So he is z. B. bound to the sample only by physical effects (non-covalent bond), which leads to a partial separation of the dye during the chromatographic separation of the sample. Despite this serious problem, it was possible to impressively demonstrate the advantages of NIR fluorescent markers and to stimulate the work of synthesizing NIR fluorescent markers with reactive groups for the range around 780 nm.

Quite essential for the usability of the fluorescent markers is, in addition to the stability to photolytic decomposition, the solubility in aqueous systems. Previously known fluorescence markers tend to form aggregates when introduced into water, saline buffer solutions or at the target molecule.

One way to reduce the aggregation tendency of cyanine dyes is to introduce functional groups that increase water solubility, as well as bulky space-filling substituents that inhibit intermolecular interactions between the dye molecules. The incorporation of alkyl and arylsulfonic acid groups is known.

NIR absorbers with phenoxy and thiophenoxy substituents in the meso position were first described in 1992 ( Synth. Commun. 1992, 22, 2593 ). Based on these principles, for example, the IRD 40 ^TM colorant developed by LI-COR Inc. has been developed (http://www.licor.com/bio). It is known that ether and thioether bridges tend to base-catalyzed hydrolysis in the alkaline range. This finding turns out to be a drawback of such NIR absorbers that the compounds with phenyl or barbituric acid substituents do not have in the meso position.

The problem of ether cleavage is also not solved by the dyes Ba and Bb offered under IRD 41 ^™ and IRD 800 ^™ , respectively (http://www.licor.com/bio).

In order to overcome this disadvantage, the phenoxy or thiophenoxy group was replaced by the phenyl group, which led to the dyes having the structure C ( WO2010121163A2 . J. Org. Chem. 2006, 71, 7862-7865 ,).

In addition to the pH stability, the tendency for dimerization / aggregation in aqueous buffer solution is an important criterion. In particular, the heptamethine cyanines show due to the high polarizability of their π-electron system and low water solubility an extremely strong tendency for dimerization / aggregation. This problem also occurs with the dyes having the structure C.

The previously known dyes, when introduced into water, saline buffer solutions or the target molecule still tend to form aggregates and are not sufficiently soluble in water.

Summary of the invention

It is the object of the invention to provide novel dyes which are readily soluble in water, have a greatly reduced tendency to aggregate in water and buffer systems, are provided with reactive groups and are suitable for use as fluorescence markers in the range between 700 to 800 nm.

This object is achieved by cyanine dyes having the features of claim 1. Advantageous embodiments and uses emerge from the dependent claims.

Also disclosed is a process for the preparation of the dyes of the invention and the use of the dyes in arrays (assays) and assays (assays), in particular in arrays and assays for the detection of cells, antibodies, proteins, hormones, nucleic acids, oligonucleotides, carbohydrates, amines and / or mercaptans.

Description of the pictures

The to show absorption spectra of various dyes from the reference example to illustrate the suppression of aggregation in aqueous media by the dyes of the invention.

Detailed description of the invention

The basic idea of the invention is to eliminate the known disadvantage of aggregation and twisting of open-chain cyanine dyes by a targeted introduction of substituents into the methine chain. In order not to reduce the fluorescence quantum yield too much and to prevent unspecific non-covalent interactions, it is important to avoid any kind of dye aggregation by introducing sterically demanding substituents.

A cyanine dye according to the invention is characterized by a combination of sulfoaryl and N-sulfoalkyl groups or trialkylammoniumalkyl groups, wherein the alkyl comprises in each case 1 to 4 C atoms, a methine chain which is bridged with five- or six-membered ring structures, and a pyrimidinetrione of various substituents in the meso position of the methine chain and a reactive group on the latter which allows binding to carrier substances, wherein in the pyrimidinetrione at least one oxygen atom, in particular the oxygen atom in the para position for attachment to the methine chain, may be substituted by a sulfur atom.

Preferred substituents on Pyrimidintrion are alkyl and sulfoalkyl groups having one to four CH ₂ groups, wherein the alkyl comprises 1 to 4 carbon atoms, and carboxyalkyl groups and hydroxyalkyl groups having 1 to 5 carbon atoms, preferably carboxyalkyl groups having 1 to 5 carbon atoms and hydroxyalkyl groups having 2 to 5 C atoms.

Preferably, the dye according to the invention has a methine chain which is bridged with five- or six-membered structures, and at both ends of idol or indoline derived groups are present, which characterized by a combination of sulfoaryl and N-sulfoalkyl or Trialkylammoniumalkylgruppen as substituents are. In this case, the idol or indoline group may be condensed with another benzene ring. The sulfoaryl and N-sulfoalkyl groups may also be present as the alkali metal derivative of the group.

The bridging of the methine chain is preferably carried out with five or six-ring structures around the meso position.

The new cyanine dyes can be used when using different excitation light sources in the absorption range between 700 and 800 nm, in particular between 740 and 790 nm.

mit:
Y = O, S;
Z = (CH₂)_n und n = 0, 1;
R₁, R₂, R₃, R₄ = H, SO₃M, die zur Vervollständigung eines Benzenringes notwendigen vier CH-Gruppen bzw. die zur Vervollständigung eines Benzenringes notwendigen vier CH-Gruppen einschließlich (SO₃M)_n, wobei mindestens einer der Reste SO₃M darstellt oder mindestens eine SO₃M-Gruppe an einem der durch R₁ und R₂ und/oder R₃ und R₄ gebildeten Benzenringe vorhanden ist, und wobei bevorzugt R₁ und/oder R₃ SO₃M;
M = ein Alkalimetall, bevorzugt Na und K, insbesondere Na;
R₅, R₆ = Sulfoalkyl mit ein bis vier CH₂-Gruppen und/oder Trialkylammoniumalkyl, wobei das Alkyl jeweils 1 bis 4 C-Atome umfasst, insbesondere Trimethylammoniumpropyl
R₇ = Alkyl, Sulfoalkyl mit ein bis vier CH₂-Gruppen, wobei das Alkyl 1 bis 4 C-Atome umfasst;
R₈ = (CH₂)_nCOOH mit n = 1–5, (CH₂)_mCH₂OH mit m = 2–5.Preferred cyanine dyes are those of the following formula A:

With:
Y = O, S;
Z = (CH ₂ ) _n and n = 0, 1;
R ₁ , R ₂ , R ₃ , R ₄ = H, SO ₃ M, the necessary for completing a Benzenringes four CH groups or necessary to complete a Benzenringes four CH groups including (SO ₃ M) _n , wherein at least one of the radicals SO ₃ M or at least one SO ₃ M group is present on one of the benzene rings formed by R ₁ and R ₂ and / or R ₃ and R ₄ , and wherein preferably R ₁ and / or R ₃ SO ₃ M ;
M = an alkali metal, preferably Na and K, in particular Na;
R ₅ , R ₆ = sulfoalkyl having one to four CH ₂ groups and / or trialkylammoniumalkyl, wherein the alkyl in each case comprises 1 to 4 C-atoms, in particular trimethylammoniumpropyl
R ₇ = alkyl, sulfoalkyl having one to four CH ₂ groups, wherein the alkyl comprises 1 to 4 C atoms;
R ₈ = (CH ₂ ) _n COOH with n = 1-5, (CH ₂ ) _m CH ₂ OH with m = 2-5.

More preferably, R ₁ and R _{3 are} SO ₃ M, and in particular M is Na.

In the general formula A, R _{8 is} preferably a carboxyalkyl group or a hydroxyalkyl group as defined above. 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. Aminolinker with hydroxy groups. Furthermore, in the dyes with carboxyl groups, these can be converted into the maleimides which react with mercapto groups. In the dyes with alkylhydroxy groups, these can be converted to 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. Thus, preferred dyes of the invention are also characterized in that R _{8 is} the N-hydroxysuccinimide ester of the carboxy group (CH ₂ ) _n COOH with n = 1-5 or the maleimide of the carboxy group (CH ₂ ) _n COOH with n = 1-5 or the bis- (N, N-di-isopropyl) -cyanethyl-phosphoramidite of the hydroxy group (CH ₂ ) _m CH ₂ OH with m = 2-5.

The cyanine dyes according to the invention are suitable for use in assays, in particular immunoassays.

For bio / medical applications, immunoassays have become the most valuable analytical instrument for the detection of pico- to femtomolar substances. The high affinity and specificity to the analyte is achieved by the antibody / antigen reaction. For sensitive detection, specific antibodies are chemically linked to a fluorescent dye via a reactive group.

The use of fluorescent labels for these purposes has increased rapidly in recent years. However, especially in the NIR range, the fluorescence markers used so far are not optimal. Obstacles are the comparatively low water solubility, the low pH stability and the tendency to dimerization / aggregation. The last property concerns not only the self-aggregation of the dye molecules with each other but also the nonspecific, disruptive application of the dyes to other components of the sample to be examined (eg blood, urine). The cyanine dyes according to the invention provide fluorescence in the NIR range, are readily water-soluble, pH-stable and, above all, do not aggregate in aqueous or saline solution or attract unspecifically to biomolecules and are therefore particularly suitable for use in immunoassays. In particular, they are also suitable for the detection of cells, antibodies, proteins, hormones, nucleic acids, oligonucleotides, carbohydrates, amines and / or mercaptans.

Further disclosed is a process for the preparation of the cyanine dyes of the invention.

The process for the preparation of a cyanine dye according to the invention is characterized in that a compound comprising a methine chain which is bridged with five- or six-membered ring structures and which is substituted by sulfoaryl and N-sulfoalkyl groups or trialkylaminoalkyl groups, with a differently substituted Pyrimidintrion in meso Position of the methine chain is reacted, wherein the Pyrimidintrion has a reactive group on this, which allows binding to carrier substances, wherein in Pyrimidintrion at least one oxygen atom, in particular the oxygen atom in the para position for attachment to the methine chain, may be substituted by a sulfur atom ,

Preferred substituents on Pyrimidintrion are alkyl and sulfoalkyl groups having one to four CH ₂ groups, wherein the alkyl comprises 1 to 4 carbon atoms, and carboxyalkyl groups and hydroxyalkyl groups having 1 to 5 carbon atoms, preferably carboxyalkyl groups having 1 to 5 carbon atoms and hydroxyalkyl groups having 2 to 5 C atoms.

Preferably, the dye according to the invention has a methine chain which is bridged with five- or six-membered structures and at both ends of which indole or indoline-derived groups are present, which are characterized by a combination of sulfoaryl and N-sulfoalkyl groups or trialkylaminoalkyl groups as substituents are. In this case, the idol or indoline group may be condensed with another benzene ring. The sulfoaryl and N-sulfoalkyl groups may also be present as the alkali metal derivative of the group.

The bridging of the methine chain is preferably carried out with five or six-ring structures around the meso position.

mit:
Y = O, S;
Z = (CH₂)_n und n = 0, 1;
R₁, R₂, R₃, R₄ = H, SO₃M, die zur Vervollständigung eines Benzenringes notwendigen vier CH-Gruppen bzw. die zur Vervollständigung eines Benzenringes notwendigen vier CH-Gruppen einschließlich (SO₃M)_n, wobei mindestens einer der Reste SO₃M darstellt oder mindestens eine SO₃M-Gruppe an einem der durch R₁ und R₂ und/oder R₃ und R₄ gebildeten Benzenringe vorhanden ist, und wobei bevorzugt R₁ und/oder R₃ SO₃M;
M = ein Alkalimetall, bevorzugt Na und K, insbesondere Na;
R₅, R₆ = Sulfoalkyl mit ein bis vier CH₂-Gruppen und/oder Trialkylammoniumalkyl, wobei das Alkyl jeweils 1 bis 4 C-Atome umfasst, insbesondere Trimethylammoniumpropyl
R₇ = Alkyl, Sulfoalkyl mit ein bis vier CH₂-Gruppen, wobei das Alkyl 1 bis 4 C-Atome umfasst;
R₈ = (CH₂)_nCOOH mit n = 1–5, (CH₂)_mCH₂OH mit m = 2–5.A preferred process is characterized by the following reaction scheme:

With:
Y = O, S;
Z = (CH ₂ ) _n and n = 0, 1;
R ₁ , R ₂ , R ₃ , R ₄ = H, SO ₃ M, the necessary for completing a Benzenringes four CH groups or necessary to complete a Benzenringes four CH groups including (SO ₃ M) _n , wherein at least one of the radicals SO ₃ M or at least one SO ₃ M group is present on one of the benzene rings formed by R ₁ and R ₂ and / or R ₃ and R ₄ , and wherein preferably R ₁ and / or R ₃ SO ₃ M ;
M = an alkali metal, preferably Na and K, in particular Na;
R ₅ , R ₆ = sulfoalkyl having one to four CH ₂ groups and / or trialkylammoniumalkyl, wherein the alkyl in each case comprises 1 to 4 C-atoms, in particular trimethylammoniumpropyl
R ₇ = alkyl, sulfoalkyl having one to four CH ₂ groups, wherein the alkyl comprises 1 to 4 C atoms;
R ₈ = (CH ₂ ) _n COOH with n = 1-5, (CH ₂ ) _m CH ₂ OH with m = 2-5.

Furthermore, in a method according to the invention in the compound

R _{8 is} further reacted to give an N-hydroxysuccinimide ester of the carboxy group (CH ₂ ) _n COOH with n = 1-5 or to a maleimide of the carboxy group (CH ₂ ) _n COOH with n = 1-5, when R ₈ = ( CH ₂ ) _n COOH with n = 1-5, or to a bis- (N, N-di-isopropyl) -cyanethyl-phosphoramidite of the hydroxy group (CH ₂ ) _m CH ₂ OH with m = 2-5 R ₈ = (CH ₂ ) _m CH ₂ OH with m = 2-5.

The invention will now be further clarified by means of exemplary embodiments.

embodiments

The following embodiments illustrate the invention and are not intended to be limiting thereof.

reference example

Absorption spectra of various dyes found in to are shown, measured.

The dyes were present in PBS buffer as 0.01 M phosphate buffered saline (NaCl = 0.138 M, KCl = 0.0027 M, pH = 7.4 at 25 ° C).

In the influence on the aggregation of water-soluble dyes are phenyl ( ), Thiophenoxy- ( ), Phenoxy- ( ) and barbituric acid substituents ( ) in meso position of methine chain similar in their effect. When examined in PBS buffer in particular, the dye with meso-phenyl group tends strongly to the formation of aggregate bands. It is clear when comparing the absorption spectra of the dyes in water and in the PBS buffer in the - 3 no aggregate band is found in the spectra of the dyes with pyrimidine trio substituent in the meso position of the methine chain ( ).

There is no noticeable difference to the spectrum in pure water; so that this modification effectively prevented dimerization / aggregation. Example 1: Preparation of 3-butyl-1- (5-carboxypentyl) -5- (2- (2- (3,3-dimethyl-5-sulfo-1- (4-sulfobutyl) -3H-indolium-2-one yl) vinyl) -6- (2- (3,3-dimethyl-5-sulfo-1- (4-sulfobutyl) indolin-2-ylidene) ethylidene) cyclohex-1-en-1-yl) -2,6 dioxo-1,2,3,6-tetrahydropyrimidin-4-olate, tetrasodium salt

A) 3,3-Dimethyl-2- [2- [2-chloro-3- [2- [1,3-dihydro-3,3-dimethyl-5-sulfo-1- (4-sulfobutyl) -2H- indol-2-ylidene] ethylidene] -1-cyclohexen-1-yl] -ethenyl] -5-sulfo-1- (4-sulfobutyl) -3H-indolium hydroxide, inner salt, trisodium salt:

3.97 g of the sodium salt of 1-sulfobutyl-2,3,3-trimethyl-5-sulfoindolenine and 0.86 g of 2-chloro-3-hydroxymethylenecyclohex-1-en-carbaldehyde are dissolved in 40 ml of n-propanol in the presence of 2 ml of acetic anhydride and 0.82 g of sodium acetate was reacted for 1 h at a bath temperature of 12 ° C with stirring. After completion of the reaction, the precipitated dye is filtered off and rinsed with n-propanol.
Yield: 3.4 g
λ = 790 nm, ε = 2.88 × 10 ⁵ l / mol · cm

B) 3-Butyl-1- (5-ethoxycarbonylpentyl) -5- (2- (2- (3,3-dimethyl-5-sulfo-1- (4-sulfobutyl) -3H-indolium-2-yl) -vinyl ) -6- (2- (3,3-dimethyl-5-sulfo-1- (4-sulfobutyl) indolin-2-ylidene) ethylidene) cyclohex-1-en-1-yl) -2,6-dioxo- 1,2,3,6-tetrahydropyrimidin-4-olate, tetrasodium salt:

0.95 g of the dye from A) are dissolved in 8 ml of water and heated to 70 ° C. To this is added slowly a solution of 0.33 g of 3-butyl-1- (5-ethoxycarbonylpentyl) hexahydropyrimidine-2,4,6-trione and 0.08 g of sodium acetate in 3 ml of water. After 90 minutes, the reaction is complete and the mixture is concentrated on a rotary evaporator. The target dye is obtained by column chromatography on silica gel from the crude product.

C) 3-Butyl-1- (5-carboxypentyl) -5- (2- (2- (3,3-dimethyl-5-sulfo-1- (4-sulfobutyl) -3H-indolium-2-yl) -vinyl ) -6- (2- (3,3-dimethyl-5-sulfo-1- (4-sulfobutyl) indolin-2-ylidene) ethylidene) cyclohex-1-en-1-yl) -2,6-dioxo- 1,2,3,6-tetrahydropyrimidin-4-olate, tetrasodium salt:

1.26 g of the dye from B) are dissolved in 30 ml of a 1: 1 mixture of water and ethanol and treated at room temperature with 2 ml of half-concentrated hydrochloric acid. After a two-hour reaction at room temperature, the target dye is separated by successive addition of acetone by fractional precipitation. The purification is carried out by column chromatography on silica gel.
λ = 765 nm, ε = 2.41 × 10 ⁵ l / mol · cm

Examples 2 to 7:

λ = 759 nm, ε = 2,37·10⁵ l/mol·cm Beispiel 3: 1-(2-Carboxyethyl)-5-(2-(2-(3,3-dimethyl-5-sulfo-1-(4-sulfobutyl)-3H-indolium-2-yl)vinyl)-5-(2-(3,3-dimethyl-5-sulfo-1-(4-sulfobutyl)indolin-2-ylidene)ethyliden)cyclopent-1-en-1-yl)-2,6-dioxo-3-(4-sulfobutyl)-1,2,3,6-tetrahydropyrimidin-4-olat, Pentanatriumsalz

λ = 789 nm, ε = 2,39·10⁵ l/mol·cm Beispiel 4: 1-(3-Carboxypropyl)-5-(2-(2-(3,3-dimethyl-5-sulfo-1-(3-(trimethylammonio)propyl)-3H-indolium-2-yl)vinyl)-5-(2-(3,3-dimethyl-5-sulfo-1-(3-(trimethylammonio)propyl)indolin-2-ylidene)ethyliden)cyclopent-1-en-1-yl)-3-ethyl-6-oxo-2-thioxo-1,2,3,6-tetrahydropyrimidin-4-olat, Dibromid, Dinatriumsalz

λ = 784 nm, ε = 2,33·10⁵ l/mol·cm Beispiel 5: 5-(2-(2-(3,3-Dimethyl-5-sulfo-1-(4-sulfobutyl)-3H-indolium-2-yl)vinyl)-6-(2-(3,3-dimethyl-5-sulfo-1-(4-sulfobutyl)indolin-2-yliden)ethyliden)cyclohex-1-en-1-yl)-1-(6-hydroxyhexyl)-2,6-dioxo-3-propyl-1,2,3,6-tetrahydropyrimidin-4-olat, Tetranatriumsalz

λ = 647 nm, ε = 2,29·10⁵ l/mol·cm Beispiel 6: 5-(2-(2-(3,3-Dimethyl-5-sulfo-1-(3-(trimethylammonio)propyl)-3H-indolium-2-yl)vinyl)-6-(2-(3,3-dimethyl-5-sulfo-1-(3-(trimethylammonio)propyl)indolin-2-yliden)ethyliden)cyclohex-1-en-1-yl)-1-(4-((2,5-dioxopyrrolidin-1-yl)oxy)-4-oxobutyl)-3-methyl-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-olat, Dibromid, Dinatriumsalz N-Hydroxysuccinimidester aus Beispielfarbstoff 2 hergestellt nach literaturbekannten Methoden

λ = 759 nm, ε = 2,21·10⁵ l/mol·cm Beispiel 7: 5-(2-(2-(3,3-Dimethyl-5-sulfo-1-(4-sulfobutyl)-3H-indolium-2-yl)vinyl)-5-(2-(3,3-dimethyl-5-sulfo-1-(4-sulfobutyl)indolin-2-yliden)ethyliden)cyclopent-1-en-1-yl)-1-(3-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-3-oxopropyl)-2,6-dioxo-3-(4-sulfobutyl)-1,2,3,6-tetrahydropyrimidin-4-olat, Pentanatriumsalz Maleinimid aus Beispielfarbstoff 3, hergestellt nach literaturbekannten Methoden

λ = 789 nm, ε = 2,19·10⁵ l/mol·cmThe following dyes were prepared according to the synthesis method given in Example 1 using the appropriate precursors and intermediates or chain building blocks. Example 2: 1- (3-Carboxypropyl) -5- (2- (2- (3,3-dimethyl-5-sulfo-1- (3- (trimethylammonio) propyl) -3H-indolium-2-yl) vinyl ) -6- (2- (3,3-dimethyl-5-sulfo-1- (3- (trimethylammonio) propyl) indolin-2-ylidene) ethylidene) cyclohex-1-en-1-yl) -3-methyl -2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-olate, dibromide, disodium salt

λ = 759 nm, ε = 2.37 × 10 ⁵ l / mol · cm Example 3: 1- (2-Carboxyethyl) -5- (2- (2,3-dimethyl-5-sulfo-1- (4-sulfobutyl) -3H-indolium-2-yl) vinyl) -5 - (2- (3,3-dimethyl-5-sulfo-1- (4-sulfobutyl) indolin-2-ylidene) ethylidene) cyclopent-1-en-1-yl) -2,6-dioxo-3- ( 4-sulfobutyl) -1,2,3,6-tetrahydropyrimidin-4-olate, pentasodium salt

λ = 789 nm, ε = 2.39 × 10 ⁵ l / mol · cm Example 4: 1- (3-Carboxypropyl) -5- (2- (2,3-dimethyl-5-sulfo-1) (3- (trimethylammonio) propyl) -3H-indolium-2-yl) vinyl) -5- (2- (3,3-dimethyl-5-sulfo-1- (3- (trimethylammonio) propyl) indolin-2- ylidenes) ethylidene) cyclopent-1-en-1-yl) -3-ethyl-6-oxo-2-thioxo-1,2,3,6-tetrahydropyrimidin-4-olate, dibromide, disodium salt

λ = 784 nm, ε = 2.33 × 10 ⁵ l / mol · cm Example 5: 5- (2- (2- (3,3-Dimethyl-5-sulfo-1- (4-sulfobutyl) -3H-indolium-2-yl) vinyl) -6- (2- (3,3 dimethyl-5-sulfo-1- (4-sulfobutyl) indolin-2-ylidene) ethylidene) cyclohex-1-en-1-yl) -1- (6-hydroxyhexyl) -2,6-dioxo-3-propyl 1,2,3,6-tetrahydropyrimidine-4-olate, tetrasodium salt

λ = 647 nm, ε = 2.29 × 10 ⁵ l / mol · cm Example 6: 5- (2- (3,3-Dimethyl-5-sulfo-1- (3- (trimethylammonio) propyl) -3H-indolium-2-yl) vinyl) -6- (2- (3,3-dimethyl-5-sulfo-1- (3- (trimethylammonio) propyl) indolin-2-ylidene) ethylidene) cyclohex-1- en-1-yl) -1- (4 - ((2,5-dioxo-pyrrolidin-1-yl) oxy) -4-oxobutyl) -3-methyl-2,6-dioxo-1,2,3,6- tetrahydropyrimidin-4-olate, dibromide, disodium salt N-hydroxysuccinimide ester from example dye 2 prepared by methods known from the literature

λ = 759 nm, ε = 2.21 × 10 ⁵ l / mol · cm Example 7: 5- (2- (2- (3,3-Dimethyl-5-sulfo-1- (4-sulfobutyl) -3H-indolium-2-yl) vinyl) -5- (2- (3,3 dimethyl-5-sulfo-1- (4-sulfobutyl) indolin-2-ylidene) ethylidene) cyclopent-1-en-1-yl) -1- (3 - ((2- (2,5-dioxo-2 , 5-dihydro-1H-pyrrol-1-yl) ethyl) amino) -3-oxopropyl) -2,6-dioxo-3- (4-sulfobutyl) -1,2,3,6-tetrahydropyrimidin-4-olate , Pentasodium salt maleimide from Example dye 3, prepared by literature methods

λ = 789 nm, ε = 2.19 × 10 ⁵ l / mol · cm

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• WO 2010121163 A2 [0011]

Cited non-patent literature

• Sauda et al. (Anal Chem 58 (1986) 2649) [0005]
• Synth. Commun. 1992, 22, 2593 [0009]
• J. Org. Chem. 2006, 71, 7862-7865 [0011]

Claims (9)

Cyanine dye, characterized by a combination of sulfoaryl and N-sulfoalkyl groups or trialkylammoniumalkyl groups, wherein the alkyl comprises in each case 1 to 4 C atoms, a methine chain which is bridged with five- or six-membered ring structures, a differently substituted pyrimidinetrione in meso Position of the methine chain and a reactive group on this, which allows binding to carrier substances, wherein in Pyrimidintrion at least one oxygen atom, in particular the oxygen atom in the para position for attachment to the methine chain, may be substituted by a sulfur atom. Cyanine dye according to claim 1, characterized by the general structures

with: Y = O, S; Z = (CH ₂ ) _n and n = 0, 1; R ₁ , R ₂ , R ₃ , R ₄ = H, SO ₃ M, the necessary for completing a Benzenringes four CH groups or necessary to complete a Benzenringes four CH groups including (SO ₃ M) _n , wherein at least one of the radicals SO ₃ M or at least one SO ₃ M group is present on one of the benzene rings formed by R ₁ and R ₂ and / or R ₃ and R ₄ , and wherein preferably R ₁ and / or R ₃ SO ₃ M ; M = an alkali metal, preferably Na and K, in particular Na; R ₅ , R ₆ = sulfoalkyl having one to four CH ₂ groups and / or trialkylammoniumalkyl, wherein the alkyl in each case comprises 1 to 4 C-atoms, in particular trimethylaminopropyl R ₇ = alkyl, sulfoalkyl having one to four CH ₂ groups, wherein the alkyl comprises 1 to 4 C atoms; R ₈ = (CH ₂ ) _n COOH with n = 1-5, (CH ₂ ) _m CH ₂ OH with m = 2-5. Cyanine dye according to claim 2, characterized in that R _{8 is} the N-hydroxysuccinimide ester of the carboxy group (CH ₂ ) _n COOH with n = 1-5 or the maleimide of the carboxy group (CH ₂ ) _n COOH with n = 1-5 or the bis- (N, N-di-isopropyl) -cyanethyl-phosphoramidite of the hydroxy group (CH ₂ ) _m CH ₂ OH with m = 2-5. Cyanine dye according to Claim 2, characterized in that R ₈ is an N-hydroxysuccinimide ester of the carboxy group (CH ₂ ) _n COOH with n = 1-5 or to a maleimide of the carboxy group (CH ₂ ) _n COOH with n = 1 -5 or to a bis (N, N-di-isopropyl) -cyanethyl-phosphoramidite of the hydroxy group (CH ₂ ) _m CH ₂ OH where m = 2-5. Use of a cyanine dye according to any one of claims 1 to 4 in an array or assay. Use of a cyanine dye according to any one of claims 1 to 4 for the detection of cells, antibodies, proteins, hormones, nucleic acids, oligonucleotides, carbohydrates, amines and / or mercaptans. A process for producing a cyanine dye according to any one of claims 1 to 4, characterized in that a compound comprising a methine chain which is bridged with five or six-membered ring structure and which is substituted by sulfoaryl and N-sulfoalkyl or Trialkylaminoalkylgruppen, with a Substituted substituted Pyrimidintrionen is substituted in the meso position of the methine chain, wherein the Pyrimidintrion additionally has a reactive group which allows binding to carrier substances and wherein in Pyrimidintrion at least one oxygen atom, in particular the oxygen atom in the para position for binding to the methine chain, by a Sulfur atom may be substituted. Process according to claim 7, characterized by the following reaction scheme:

with: Y = O, S; Z = (CH ₂ ) _n and n = 0, 1; R ₁ , R ₂ , R ₃ , R ₄ = H, SO ₃ M, the four CH groups necessary to complete a benzene ring, or the four CH groups necessary to complete a benzene ring, including (SO ₃ M), wherein at least one the radicals SO ₃ M or at least one SO ₃ M group is present on one of the benzene rings formed by R ₁ and R ₂ and / or R ₃ and R ₄ , and wherein preferably R ₁ and / or R ₃ SO ₃ M; M = an alkali metal, preferably Na and K, in particular Na; R ₅ , R ₆ = sulfoalkyl having one to four CH ₂ groups and / or trialkylammoniumalkyl, wherein the alkyl in each case comprises 1 to 4 C-atoms, in particular trimethylammoniumpropyl R ₇ = alkyl, sulfoalkyl having one to four CH ₂ groups, wherein the alkyl comprises 1 to 4 C atoms; R ₈ = (CH ₂ ) _n COOH with n = 1-5, (CH ₂ ) _m CH ₂ OH with m = 2-5. The method of claim 8, wherein R ₈ in the compound

is further converted to an N-hydroxysuccinimide ester of the carboxy group (CH ₂ ) _n COOH with n = 1-5 or to a maleimide of the carboxy group (CH ₂ ) _n COOH with n = 1-5, when R ₈ = (CH ₂ ) _n COOH with n = 1-5, or to a bis (N, N-di-isopropyl) -cyanethyl-phosphoramidite of the hydroxy group (CH ₂ ) _m CH ₂ OH with m = 2-5 is reacted when R ₈ = (CH ₂ ) _m CH ₂ OH with m = 2-5.

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