WO2004092174A1 - Pyrrolopyrroles used as fluorescent labels for biomolecules and spherical particles - Google Patents

Abstract

The invention relates to pyrrolo(3,4-c)pyrroles of the typical structures (I) or (II). Said pyrroles have absorption maxima between 400 and 650 nm and are suitable in reactive form for the fluorescent dyeing of proteins, polynucleic acids and biological receptors, in addition to polymer particles. Their fluorescent quantum yields are without exception higher than 0.5. The invention also relates to corresponding fluorescently labelled proteins, oligonucleotides and particles. The materials dyed in this manner are preferably excited to fluoresce using violet, blue or blue-green light emitting diodes or diode lasers and are preferably used in analytical fluorescence determination methods, e.g. in immunoassays, in hybridisation assays, in cytometry and in pharmaceutical screening.

Description

 Pyrrolopyrrole as a fluorescent marker for biomolecules and spherical particles

description

The present invention relates to fluorescent marker dyes which are particularly suitable for labeling biomolecules and / or particles.

The fluorescence labeling of biomolecules plays an important role in bioanalytics and biological research. A distinction is made between fluorophores that are non-covalent to biomolecules such as. B. bind proteins or DNA (z. B. intercalators), and those that can be covalently (chemically) bound to biomolecules. One of the most commonly used fluorophores is that of fluorescein. It has the advantage that it can be excited very effectively with the 488 nm line of the argon ion laser, since its absorption maximum in aqueous solution is around 490 nm. Conversely, the availability of markers from the group of fluoresceins has contributed to the fact that the argon ion laser has become a standard tool in fluorescent bioanalytics.

However, fluoresceins have two disadvantages that have not yet been remedied: on the one hand, their fluorescence is strongly pH-dependent, which leads to incorrect measurement results (and thus analyzes) in the case of unexpected pH changes in a test solution, and on the other hand, fluoresceins are relatively photolabile and bleach under longer exposure, so that the fluorescence intensity slowly decreases. This also leads to incorrect results.

A large number of fluorescent markers have already been described. In general, these are fluorescent chromophores that carry a reactive group that forms a chemical (covalent) bond with another functional group of a biomolecule. The general reaction scheme of a covalent label can be shown as follows become:

F_χ + HY biomolecule ==> F— -Y biomolecule + HX

Herein Finen means fluorophore, on which there is a reactive group Jf, which enters into a fine chemical reaction with a second reactive group H1 on a biomolecule or particle. Usually an HX group is split off. Alternatively, a (fluorescence) dye can be provided with a biotin group. Biotin binds to the proteins avidin or streptavidin (SA) with high affinity (Kd approx. 10 ¹³ mol / L).

If the SA is on a biomolecule, the biotin (strept) avidin bond can be used to achieve a non-covalent attachment of dyes to biomolecules. Typical examples of the groups X and Y are given in Table 1.

Table 1. Reactive groups (X) on synthetic fluorophores, which enable their coupling to specific groups (Y) of biomolecules or of polymers. In addition, the affinity bonds between biotin and strept (avidin) listed in the last two columns are of great importance.

^*} SUI stands for the N-succinimidoyl group; NHS for the N-hydroxysuccinimide. In one of these ways, a fluorophore F can be introduced into a biomolecule and thus made accessible to a fluorescence-analytical method. These primarily include fluorescence immunoassays, ELISAs, hybridization assays, ligand-receptor interactions, pharmaceutical screening studies and enzyme inhibition tests.

Polymeric particles can also be stained via such conjugation reactions if they carry corresponding groups (Y). Alternatively, they can simply be stained with lipophilic dyes that dissolve in the corresponding polymer.

Fluorochromes are - preferably in bioanalytics - fluorescence systems that are suitable for the covalent staining of biomolecules. Numerous fluorochromes (with absorption maxima between 300 and 900 nm) are known. In the short-wave spectral range, these are primarily the coumarins, fluoresceins and Bodipy dyes. The chemical structures of each representative of the classes mentioned, namely the Alexa 430 (A), the Oregon Green (B) and a Bodipy dye (C) are shown below. The fluoresceins and many coumarins have the disadvantage of pH-dependent fluorescence. The Bodipy dyes A (described in US Pat. Nos. 6,005,113, 5,433,896, 5,338,854, 5,274,113) are photolabile in aqueous solution and are often hydrolytically unstable.

The rhodamines form another group of fluorochromes. They are largely free from the disadvantage of pH sensitivity, but they are compatible with that Argon ion lasers cannot be excited efficiently and they always carry a positive charge on the ring, which causes undesirable electrostatic effects. The reactive dyes from the group of cyanines of the following general structure D described by Waggoner and employees in US Pat. Nos. 6,048,982, 5,627,027, 5,569,766, 5,569,587, 5,486,616, 5,268,486:

where M stands for O, S, NR or C (CH ₃ ) ₂ , and n can assume the values 0 to 3, absorb at well over 500 nm, mostly even over 600 nm, and are also positively charged.

It was therefore an object of the present invention to provide new fluorescent markers for biomolecules with which the disadvantages of the prior art can be at least partially overcome. In particular, the new markers should have pH-independent fluorescence and high photostability.

This object is achieved according to the invention by pyrrolo (3,4-c) pyrroles according to the general formula I or II.

A new group of biomarkers is described here, which has the advantages of pH-independent fluorescence and great photostability. The dyes have the basic chemical structure of a pyrrolo (3,4-c) pyrrole, on which there are reactive groups that enable a reaction with biomolecules or with functional surfaces.

Compounds of the pyrrolo (3,4-c) pyrrole type have been Lichkeit and unreactivity used as color pigments for car paints, for. B. in Eur. Pat. 994,911 and 353,184; Eur. Pat Appl. 184,981 (1986); 184,982 (1986); and U.S. Patents 4,778,899 and 4,749,795 (1986). The diketopyrrolopyrroles have also been proposed as color toners (Jap. Pats. 2055-362-A, 2039-159; 3011-357) and as photoconductive substances (EPA 353.184), their insolubility and unreactivity being a basic prerequisite for their use for the above represents the purposes mentioned.

Biomarkers, on the other hand, must be readily soluble, ideally in water, but at least in water-miscible solvents, since biomarkers are almost always carried out in aqueous solution. In addition (and in contrast to color pigments) they must have a high reactivity (even at room temperature), since reactions with biomolecules can practically only be carried out in the temperature range between 0 and 50 ° C. On the one hand, the dyes according to the invention are preferred

The presence of corresponding substituents is more soluble, and is also suitable for bioconjugation due to the presence of appropriate reactive groups. The color of the new biomarkers can be adjusted so that they can be excited with the 488 nm line of the Ar ion laser. Seen overall, the pyrrolopyrroles according to the invention form a class of substances which has the following advantages:

(1) The pH sensitivity of the color often observed with other dyes is missing, at least in the physiological pH range; (2) their color can be tuned over a wide range by varying the chemical substituents;

(3) they have high quantum yields and always good photostability;

(4) they are included thanks to the lasers routinely used in clinical analysis, flow cytometry and high-throughput screening

Wavelengths between approx. 430 and 580 nm excitable, and

(5) Finally, they can be manufactured in such a way that they carry only one reactive group and thus conjugate to defined locations on a biomolecule can be; the crosslinking reactions which are often observed when several reactive groups are present are therefore avoided.

It has now been found that fluorophores of general type 1 or II are suitable as biomarkers if they are activated by introducing reactive chemical groups in such a way that they are mixed with biomocules in predominantly aqueous solution at room temperature (or only a slightly elevated temperature, for example <50 ° C) voluntarily enter into a chemical binding reaction. The compounds are preferably made more soluble by suitable chemical modification, in particular the solubility in water or aqueous solutions is increased. Solubility can e.g. can be increased by alkyl groups or hydroxyalkyl groups on nitrogen. The new biomarkers have absorption maxima between 400 nm and 650 nm, in particular between 400 and 580 nm, and can be produced and used relatively easily as monoreactive fluorochromes for staining biomolecules. Monoreactive means that the dye molecules contain exactly one group which enables a covalent bond or an affinity bond to a biomolecule or polymer particle. Compared to fluoresceins, they have significantly improved photostability and quantum yields, and their absorption and fluorescence spectra are largely pH-independent.

The reactive dyes according to the invention have the general chemical structure I,

I in what

R ¹ to R ^{4 can represent} H or any organic substituent, but in particular a linear, branched, cyclic one saturated or unsaturated alkyl radical, a (hetero) aryl radical or a heterocyclic radical, each of which individually with one or more halogen, carboxy, cyano, hydroxy, alkoxy, amino (or substituted amino), aryl, Heteroaryl, sulfo or phospho groups can be substituted, wherein

R ¹ and R ^{3 can} also represent halogen, CN, COOH, CONR ¹ , or COO-alkyl,

R ¹ and R ² or R ³ and R ⁴ can be connected to one another via aliphatic, aromatic or heterocyclic rings, X stands for O, S, C (CN) ₂ or NR ⁵ , where R ⁵ is for R ¹ - R ⁴ has the meaning indicated, and in which at least one radical R ¹ to R ⁵ represents or contains any group (including a biotin group) which enables covalent labeling or affinity labeling of a biomolecule or a polymer particle.

Further reactive dyes according to the invention have the general chemical structure II,

II worin

II in which

R ¹ to R ^{3 can represent} H or any other organic substituent, but in particular a linear, branched, cyclic, saturated or unsaturated alkyl radical, a (hetero) aryl radical, or a heterocyclic radical, each of which is may be substituted with one or more halogen, carboxy, cyano, hydroxy, alkoxy, amino (or substituted amino), aryl, heteroaryl, sulfo or phospho groups, where

R ¹ and R ² via aliphatic, aromatic or heterocyclic rings with can be linked to one another, R ¹ and R ^{3 can} also represent halogen, CN, COOH, CON (R ¹ ) ₂ or COO-alkyl, X represents O, S, C (CN) ₂ , or NR ⁵ , where R ⁵ has the meaning given for R ¹ - R ³ ,

Y stands for OR ⁶ , SR ⁶ , N (R ⁶ ) ₂ , OPOCI ₂ , where R ^{6 is} the one given for R ¹ - R ³

Has meaning, and wherein at least one radical R or Y is any group (including one

Represents or contains biotin group) which enables covalent labeling or affinity labeling of a biomolecule or a polymer particle.

The radicals R ¹ to R ⁴ in compounds of type I and the radicals R ¹ to R ³ in compounds of type II can be hydrogen or an organic substituent. The organic substituent preferably has 1 to 30 C atoms, in particular 1 to 20 C atoms. The organic substituent can furthermore have heteroatoms, in particular selected from O, S, N, P and halogens, in particular F, Cl, Br or J. The organic substituent is particularly preferably a linear branched or cyclic, saturated or unsaturated alkyl radical , in particular a Ci to C12, more preferably a C. to C ₆ and even more preferably a Ci to C ₄ alkyl radical. The organic substituent is furthermore preferably an aryl radical which can contain heteroatoms, in particular O, N, S or P or a heterocyclic radical which can contain heteroatoms, in particular selected from O, N, S and P. The organic substituent, in particular in its preferred embodiment as an alkyl radical, (hetero -) aryl radical or heterocyclic radical can be substituted one or more times, for example with halogen, carboxy, cyano, hydroxyl, alkoxy, amino, aryl, heteroaryl, sulfo - or phospho groups. The amino groups can be substituted, for example with one or two alkyl or hydroxyalkyl groups, preferably each with 1 to 6 carbon atoms. The radicals R ² and R ⁴ in compounds of type I and the radical R ² in compounds of type 2 AI are particularly preferred. alkyl radicals or hydroxyalkyl radicals, in particular with 1 to 6 carbon atoms.

R ¹ and R ² can also be connected to one another via aliphatic, aromatic or heterocyclic rings. Furthermore, R ¹ and R ^{3 can} also be halogen, in particular F, CI, Br or J, for CN, COOH; CON (R ¹ ) ₂ or COO-alkyl are available.

According to the invention, at least one of the radicals R ¹ to R ⁵ in compounds of the formula I or at least one and preferably exactly one of the radicals R ¹ to R ³ , R ⁵ to R ⁶ or Y of the compounds of the formula II contains a group which is covalent Labeling or affinity labeling of a biomolecule or a polymer particle enables. Such groups are, for example, carboxylic acid esters, in particular reactive or activated carboxylic acid ester derivatives, maleimides, haloacetyls, sulfochlorides, chlorotriazines, -SCN or -OCN groups, pyrrlium groups, phosphoramidite or biotin or streptavidin. These groups mediate the binding of the dyes according to the invention to the molecules to be labeled, that is to say in particular biomocules or polymer particles, and accordingly contain a unit which can bond with the grouping of the bio-cooler or the polymer particle.

In addition to this bond-mediated group, the marker dyes according to the invention preferably contain at least one group which increases the solubility of the dyes in aqueous media or water, in particular in media with a water content> 5% by weight, more preferably> 50% by weight , Such groups which increase solubility are preferably ionic groups, for example groups which are negatively or positively charged. The dyes preferably contain a sulfo, sulfate, phosphate, phosphonate or quaternized ammonium group.

The invention relates to pyrrolo (3,4-c) pyrroles of the typical structure I or 11. They have absorption maxima between 400 and 650 nm and are suitable in reactive form for fluorescent staining of proteins, polynucleic acids and biological receptors, but also of polymer particles. Their fluorescence quantum yields are consistently higher than 0.5. Similarly, fluorescence-labeled proteins, oligonucleotides and particles are also described. The materials stained in this way are preferably excited with violet, blue or blue-green light-emitting diodes or diode lasers for fluorescence and are preferably used in fluorescence-analytical determination methods, eg. B. in immunoassays, in hybridization, in the cytometry, and in pharmaceutical screening.

The basic bodies of the fluorochromes according to the invention can be synthesized in various ways. Corresponding general regulations can be found in the above-mentioned patents and in IP Lorenz et al. in Chemistry Eur. J. 8 (2002) 4047-4055 (where metal complexes of diketopyrrolopyrroles are described), and in CJH Morton et al. in Tetrahedron 58 (2002) 5547-5565 (where new and different heterocycles are prepared from diketo-pyrrolopyrroles). Of general importance is the reaction of succinic acid di-tert-butyl ester with a nitrile and subsequent reaction with an amine of the general structure R ⁴ -NH _2, the products of the general structure 1 are formed. The compounds of type 1 (and derivatives derived from them) serve as the starting material for further syntheses:

MethylBy reacting 1 (with R ⁴ = CH ₃ and R ³ = phenyl) with benzonitrile z. B. the N-methyl-1,4-diketo-3,6-diphenyl-pyrrolo (3,4-c) pyrrole 2a obtained, which sulfonated with fuming sulfuric acid to the sulfo compound 2b and thus can be made water-soluble. 2a: R ¹ = R ³ = phenyl; R ⁴ = methyl 2b: R ¹ = R ³ = 4-sulfophenyl; R ⁴ =

methyl

Examples of chemical structures of preferred reactive markers are given below:

6

The red (and green fluorescent) compound 3 can be activated with amino groups directly after activation to the N-hydroxy-succinimide ester (NHS ester) Response. The deep red sulfochloride 4 is an alternative in which the sulfo group causes reactivity to amino groups. The yellow (and blue-green fluorescent) quaternized compound 5 can also be conjugated to amines via an NHS ester. The red isothiocyanate 6 reacts with amines at room temperature to form a thiourea bridge in the sense of

FI-NCS + H ₂ N-Pr ====> FI-NH-CS-NH-Pr where F / stands for the pyrrolopyrrole fluorophore and Pr for protein.

The red-violet alcohol 7 shown below can, as will be shown below, be converted into a phosphoramidite ester, which can then be conjugated to the ribose residues of polynucleotides and thus enables their fluorescence labeling. Finally, with the help of the violet dicyanomethylene derivative 8, all those biomolecules which have an avididn, streptavidin or neutravidine group can be stained via its biotin group, since these have an extraordinarily high affinity for biotin.

The keto groups in the pyrrolo (3,4-c) pyrroles according to the invention can be converted with Lawesson's reagent into the corresponding thio-pyrrolo (3,4-c) pyrroles of type 9a:

Their methylation in acetone with methyl iodide in the presence of potassium carbonate leads to the thioethers 10a. Their reaction with primary alkylamines or arylamines leads to the imines 9b, with secondary amines to 10b, and the reaction with malonodinitrile in ether to the 1-dicyanomethylene-4-keto-pyrrolo (3,4-c) pyrroles from Type 9c. These dicyanomethylene derivatives have significantly longer wavelength absorption and emission wavelengths.

Virtually any of the above carboxylic acids (e.g. 3 or 5) can be activated to the reactive dye by reacting them in an aprotic organic solvent with N-hydroxysuccinimide and dicyclohexylcarbodiimide (DCC) to a reactive ester according to the following scheme:

F- (CH ₂ ) ₅ -COOH + N-hydroxysuccinimide + DCC

Here F stands for the pyrrolo (3,4-c) pyrrolo fluorophore. Compounds 3 and 5 were implemented in this way. From alcohols (eg 8), phosphoramidite II, which is suitable for labeling sugar residues of DNA or RNA, is obtained in turn by reaction with the phosphine I in a manner known per se, as will be described further below.

Alternatively, the corresponding iodoacetyl derivatives which are suitable for dyeing thiol groups can be obtained from alcohols by reaction with iodoacetyl chloride:

(1) FI-CH ₂ -CH ₂ -CH ₂ -OH (e.g. compound 7) + CI-CO-CH ₂ -l ====>

===> FI-CH2-CH2-CH2-O-CO-CH2-I

(2) FI-CH ₂ -CH ₂ -CH ₂ -O-CO-CH ₂ -l + HS-Pr ===>===> FI-CH2-CH ₂ -CH ₂ -O-CO-CH ₂ -S-Pr + Hl where F / stands for the pyrrolopyrrole fluorophore and Pr for protein.

A special form of activation consists in the reaction of pyrrolo (3,4-c) pyrroles of type 9 (if R ^{2 is} not H) with POCI ₃ , which leads to phosphoryl chloride 8c. Such phosphoric acid esters react directly with amino groups of proteins in the following sense:

FI-OPO-CI + H ₂ N-Pr ====> FI-O-PO-NH-Pr + HCl where F / stands for the pyrrolopyrrole fluorophore and Pr for protein.

The pyrrolo (3,4-c) pyrroles according to the invention have a significantly improved solubility in all solvents if there are no NH groups. The dyes described in the abovementioned patents also relate primarily to pyrrolopyrroles with free NH groups, since this is particularly advantageous because of their poor solubility with regard to the applications described there.

Solubility-improving N-alkyl groups and N-hydroxyalkyl groups can already be introduced during the synthesis of the basic structure, but subsequent alkylation can also take place, e.g. B. with the help of speaking alkyl halides or alkyl tosylates and with the addition of an acid scavenger such. B. potassium carbonate, an amine base or an alcohol. The alkylation on the one hand brings about a significant improvement in the solubility of the compounds, which makes them only suitable as a biomarker, but can also serve at the same time to introduce functional groups, e.g. B. a carboxy group, a hydroxy group, or another functional or reactive group.

The water solubility of the pyrrolo (3,4-c) pyrroles can be further improved if sulfo groups are introduced. This is done either by using sulfonated benzonitriles (or their esters) in the initial synthesis phase, or by reacting aryl-1,4-diketo-pyrrolo (3,4-c) pyrroles by sulfonation with fuming sulfuric acid.

The absorption maximum of the marker with Ri = R ₂ = alkyl is 400-430 nm, that of the marker with Ri = alkyl and R ₂ = aryl is 430-480 nm, and that the marker with R_ = R ₂ = aryl or heteroaryl at 460 - 580 nm. Specifically, the absorption maximum of dye 4 (with Ar = sulfophenyl) in aqueous solution is 478 nm, the fluorescence maximum at 525 nm (uncorrected). A significantly larger Stokes shift (~ 50 nm) is noticeable in comparison with fluorescein. The molar decadal absorption coefficient is 24,000 (M ^"1 cm ^{" 1} ). The color of the marker can be varied by other substituents in the para position of the aryl radical, or by replacing the aryl with alkyl or other aryl radicals. The absorption maximum of 1,4-diketo-3,6-bis (4-dimethylaminophenyl) pyrrolo (3,4-c) pyrrole is already at 550 nm. It therefore appears blue-violet in solution. At the same time, the molar absorbance increases to approximately 80,000 M ^"1 cm ^{" 1} .

The dyes according to the invention are outstandingly suitable for bioconjugation. Reactivated carboxylic acids (preferably their NHS esters) can be conjugated to primary amino groups at room temperature in a manner known per se: ⁺ H ₂ N biomolecule ===>

===> F - (CH ₂ ) ₅ -NH biomolecule + N-hydroxysuccinimide

Type 10a thioethers can also be reacted with primary or secondary amines and lead to type 9b products, where R stands for the protein residue.

The type II phosphoramidites described above react with a nucleoside in a manner known per se to form the primary conjugate III. After oxidation with iodine, fluorescence-labeled nucleosides of type IV are obtained. In the scheme, ß stands for a nucleobase and F for the pyrrolo (3,4-c) pyrrole fluorophore.

Examples

I. Labeling Examples 1. Labeling of Human Serum Albumin 1 mg of the NHS ester of 3 is dissolved in 100 μL of anhydrous dimethylformamide (DMF). At the same time, 5 mg human serum albumin (HSA) is dissolved in 1 mL of a 50 mM bicarbonate buffer of pH 9.0. The DMF solution is now added to the protein solution in small portions (approx. 10 μL) and with stirring. When the addition is complete, stirring is continued for 5 h.

Unconjugated dye is separated from labeled protein by gel permeation chromatography (Sephadex G25) on a 15 cm column (usually 1 cm) with 22 mM phosphate buffer (pH 7.2) as eluent. The fastest running (orange colored) band contains the labeled protein. The unconjugated excess label is in the slowest moving orange-red zone. 2. Labeling of an amino-modified nucleic acid oligomer with the marker 3c

The 15'-amino-modified 15-oligomer 3'-ATA-GTG-TCT-TAG-TAC- (CH ₂ ) 6-NH ₂ is reacted with the isothiocyanate β in the following manner: The oligomer (0.2 mg ) is dissolved in 5 mL acetonitrile and heated to 50 ° C. Then a solution of 0.1 mg of the dye in 1 mL acetonitrile is slowly added dropwise. After 1 h, the acetonitrile is evaporated off and the residue is subjected to preparative HPLC (acetonitrile as eluent). Oligomer and residual (excess) dye can be easily detected or separated using the green inherent fluorescence of the conjugate.

3. Marking of glass microparticles

Porous glass beads (1.0 g; pore size 70 nm; with aminopropyl groups on the surface (40 - 100 μmol per gram of beads; obtained from Sigma, product no. G-5019) are suspended in bicarbonate buffer solution of pH 8.0. Slowly and A solution of 3 mg of the OSl ester of 5 in DMF is added dropwise with rapid stirring at 50 ° C. After one hour, the yellow-colored glass particles are separated off, washed with copious amounts of distilled water and methanol until no dye is left in the wash water The particles are then dried and stored in a dry state, and the yellow-colored particles have a strong green fluorescence.

4. Labeling of a protein with a biotin marker The labeling takes place via the biotin-streptavidin binding reaction. Polyclonal anti-HSA (from the goat; Sigma Prod. No. A-1151) is diluted 10 times with phosphate buffer first with streptavidin-maleimide (Sigma, Prod. No. S-9415) according to the instructions from Duncan et al. in Analytical Biochemistry. 32 (1983) 68 to a thiol group of the protein. The resulting streptavidin / anti-HSA conjugate is mixed with the biotin marker 8 after electrophoretic purification in buffer solution (pH 7.0) and electrophoresed after standing for 2 h at room temperature. In this way, a red-violet colored anti body with a yellow-green fluorescence.

5. Preparation of a phosphoramidite

50 mg (145 mmol) of the alcohol 7 are dissolved in 3 ml of dry dichloromethane under an argon atmosphere. 47.5 μL (166 μmol) of 2-cyanoethyl-iS!, N, N ', N'-tetraisopropyl-phosphorodiamidite and 330 μL of a saturated tetrazole solution in acetonitrile are added. The solution is stirred for one hour at room temperature and then extracted twice with 5 ml of 5% NaHCO ₃ in water and once with 10 ml of a saturated aqueous NaCl solution. The organic phase is dried and the product is purified by means of column chromatography (reversed phase). Yield: 33.4 mg (43%) of a red-violet powder.

Claims

Expectations 1. Fluorescent maker dyes with an absorption maximum between 400 and 640 nm and the general chemical structure I

wherein R ¹ to R ^{4 can} each independently represent H or an organic substituent, in particular a linear, branched, cyclic, saturated or unsaturated alkyl radical, a (hetero) aryl radical or a heterocyclic radical, each of which individually with one or more Halogen, carboxy, cyano, hydroxy, alkoxy, Amino (or substituted amino), aryl, heteroaryl, sulfo or phospho groups can be substituted, where R ¹ and R ^{3 can} also represent halogen, CN, COOH, CONR ¹ or COO-alkyl, R ¹ and R ² or R ³ and R ⁴ can be connected to one another via aliphatic, aromatic or heterocyclic rings, X represents O, S, C (CN) ₂ or NR ⁵ , where R ^{5 has} the meaning given for R ¹ -R ⁴ , and wherein at least one of the radicals R ¹ to R ⁵ represents or contains a group which has a covalent label or an affinity label a biomolecule or a polymer particle. 2. Fluorescent marker dyes with an absorption maximum between 400 and 650 nm and the general chemical structure II

wherein R ¹ to R ^{3 can} each independently represent H or an organic substituent, in particular a linear, branched, cyclic, saturated or unsaturated alkyl radical, a (hetero) aryl radical or a heterocyclic radical, each of which individually with one or more Halogen, carboxy, cyano, hydroxy, alkoxy, amino (or substituted amino), aryl, heteroaryl, sulfo or phospho groups can be substituted, where R ¹ and R ² are linked to one another via aliphatic, aromatic or heterocyclic rings can be connected R and R ^{3 can} also represent halogen, CN, COOH, CON (R ¹ ) ₂ or COO-alkyl, X stands for O, S, C (CN) ₂ or NR ⁵ , where R ^{5 has} the meaning given for R ¹ -R ³ , Y stands for OR ⁶ , SR ⁶ , N (R ⁶ ) ₂ , OPOCI ₂ , where R ^{6 has} the meaning given for R ¹ -R ³ , and wherein at least one of the radicals R ¹ to R ³ , R ⁵ to R ⁶ or Y represents or contains a group which has a covalent label or an affinity label for a biomolecule or a polymer particle allows. 3. marker dyes according to claim 1 or 2, in which at least one of the substituents R ¹ to R ⁶ or Y is a reactive ester of a carboxylic acid, a maleimide, a haloacetyl of the type -GO-CH2-X ¹ , wherein X ^{1 is} halogen, a sulfochloride, a chlorotriazine, an -SCN or -OCN group, a pyrylium group, a phosphoramidite or a biotin or streptavidin. 4. marker dye according to one of claims 1 to 3, s thereby! characterized in that it contains an ionic group, in particular a sulfo, sulfate, phosphate, phosphonate or quaternized ammonium group. 5. Use of a marker dye according to any one of claims 1 to 4 0 for the production of fluorescently labeled biomolecules, in particular amino acids, proteins or antibodies, of DNA, RNA or PNA, of pharmaceuticals or sugars, of cells or tissue sections, of receptors and ligands or of marked spherical particles with a diameter between 10 nm and 10 μm, 5 characterized in that the marking takes place via a covalent bond between the marker and the biomolecule or particle. 6. Use of a marker dye according to any one of claims 1 to 4 0 for the production of fluorescently labeled molecules, in particular of amino acids, proteins or antibodies, of DNA, RNA or PNA, of pharmaceuticals or sugars, of cells or tissue sections, of receptors and ligands or of labeled spherical particles with a diameter between 10 nm and 10 μm, characterized in that the labeling takes place via an affinity bond, in particular between an optionally labeled biotin and an optionally labeled avidin, streptavidin or neutravidin. 7. Biomolecule, in particular selected from amino acids, proteins or antibodies, nucleosides, nucleotides and nucleic acids and their derivatives and oligomers, DNA, RNA or PNA, pharmaceuticals or sugar, cells or tissue sections, receptors or ligands, characterized in that it is marked with a fluorescent marker according to any one of claims 1 to 4. 8. Particles with a diameter between 10 nm and 10 microns, characterized in that it is marked with a fluorescent marker according to one of claims 1 to 4. 9. Particle according to claim 8, characterized in that it carries on its surface an additional carboxy group, an amino group, a biotin residue or any avidin via which the linkage to other biomolecules or cells is made possible. 10. Use of a labeled biomolecule according to claim 7 in a determination method of the type of immunoassays, hybridization assays, cellular assays, immunostaining, in-situ hybridization, the sequence analysis of proteins or polynucleotides, in affinity studies including high throughput screening or in cytometry. 11. Use of a particle according to one of claims 8 or 9 in an optical determination method including immunoassays, hybridization assays, flow cytometry, in particle sorters, in arrays and as a marker for other molecules and cells. 12. Use of a labeled biomolecule and / or particle according to one of claims 7 to 9 in a determination method in which the change in intensity, the change in the decay time, the efficiency of the resonance energy transfer of fluorescence, or the extent of Polarization of a light beam is determined. 13. Use according to one of claims 10 to 12 using of microtiter plate devices, of flow cytometers, of particle sorters, of micro-fluidic elements or other automated laboratory processes.