DE102006057345A1 - New hydrophilic marker based on cyanine diastereomer useful e.g. for marking biomolecule and as a cyanine-conjugate in e.g. optical procedure, immune tests, hybridization procedure and fluorescence resonance energy transfer system - Google Patents

Abstract

Hydrophilic marker based on cyanine diastereomer (I) and/or (II) is new. Hydrophilic marker based on cyanine diastereomer of formula (I) and/or (II) is new. R 1, R 2aliphatic (preferably methyl) or heteroaliphatic group; L, L 1-L 4divalent linear -(CH 2) o-, branched or cyclic alkylene (optionally interrupted by one or more O, substituted N and/or S atom); o : 1-15; X 1-OH, -SH, NH 2, -NH-NH 2, -F, -Cl, -Br, -I, -O-succinimidyl, O-sulfosuccinimidyl, -O-(4-sulfo-2,3,5,6-tetrafluorophenoxy), -O-(2,3,5,6-tetrafluorophenoxy), benzotriazole, -NR-L-OH, -NR-L-O-phosphoramidite, -NR-L-SH, -NR-L-CO-NH 2, -NR-L-CO-NH-NH 2, -NR-L-CHO, -NR-L-COOH, -NR-L-COO-succinimidyl, NR-L-COO-sulfosuccinimidyl, -NR-L-CO-O-4-sulfo-2,3,5,6-tetrafluorophenoxy, -NR-L-CO-O-2,3,5,6-tetrafluorophenoxy, -NR-L-NH-CO-CH 2-I or -NR-L-maleimide; R : H, R 1or R 2; X 2, X 3H, alkyl, tert.alkyl, aryl, carboxyaryl, dicarboxyaryl, heteroaryl, cycloalkyl, heterocycloalkyl, alkoxy, alkylmercapto, aryloxy, arylmercapto, OH, amino, NO 2, CN or solubilizing and/or ionizable and/or ionized group e.g. SO 3->, PO 32->, CO 2->, tert-ammonium, cyclodextrin or sugar; Y 1halo (F, Cl or Br), substituted phenoxy or substituted arylmercapto (phenylsulfanyl); Cation : Na +>, K +>, Ca 2+>, ammonium or other cation to balance negative charge in (I) and/or (II); n : 0-3; R 3, R 4aliphatic (preferably methyl) or heteroaliphatic group; or R 3+R 4-(CH 2) m-, -(CH 2) mO(CH 2) m-, -(CH 2) mS(CH 2) m-, -(CH 2) mCH=CH- or -OCH=CH-; and m : 2-6. [Image] [Image].

Description

The The invention relates to markers suitable, highly hydrophilic compounds based on diastereomeric cyanines and their use in optical, in particular fluorescence-optical determination and detection methods.

typical Process applications are based on the reaction of dye-labeled Biomolecules, like for example, antigens, antibodies or DNA segments with the respective complementary species. This will be under other measurements of enzyme kinetics, receptor-ligand interactions and nucleic acid hybridization kinetics in vitro as well as in vivo. Furthermore, the claimed markers for the pharmacological characterization of receptors or drugs interesting.

applications result, for example, in medicine and pharmacy, in biology and materials science, in environmental control and the proof of occurring in nature and technology organic and inorganic micro samples as well as others.

For these applications, xanthylium salts ( US 5,846,737 ; Molecular Probes) or cyanines, as claimed, for example, in US Pat. No. 5,627,027.

These Markers have the disadvantage of being due to the planarity of the π-electron system for aggregation and dimer formation, especially in aqueous Tend to systems. Furthermore, insufficiently hydrophilic markers do not work specific interactions with different surfaces, causing problems in the purification of the corresponding conjugates and leads to an unsatisfactory signal-to-noise ratio.

In the younger ones There has been no lack of attempts in the past, through the introduction of additional, Hydrophilicity of the marker-enhancing substituents the known disadvantages to reduce the previously known cyanines on. This happened mainly through the introduction of additional Sulfonic acid functions in the Cyanine chromophore.

In US 6,083,485 (Licha) or US 09 / 969,853 and US 09 / 968,401 (Molecular Probes) describe cyanines in which one of the methyl substituents customary at the terminal indole heterocycle in the 3-position by an ω-carboxyalkyl function is substituted and the previous (eg in Cy3 or Cy5) N-alkyl or N-ω-carboxyalkyl functions have been replaced by N-ω-sulfonic acid alkyl functions.

In WO 05/044923 A1 (Amersham) describes cyanines in which the usual at the terminal indole heterocycle in 3-position methyl substituent by an ω-sulfonic acid alkyl function is substituted.

The cyanines having more than two sulfonic acid functions described in these patents have opposite to those in US 5,627,027 described cyanines (Cy3, Cy5) an increased solubility and associated therewith a lower tendency for dimer formation.

We now found that it is of particular advantage in their substituent pattern hitherto unknown cyanins to use in which the one terminal indole heterocycle in 3-position with a solubilizing or ionizing group (eg ω-sulfonic acid alkyl function) and the another terminal heterocycle having a coupling function biomolecules (eg, an ω-carboxyalkyl function) is.

By the derivatization of the two terminal indole heterocycles in 3-position, once with a ω-sulfonic acid alkyl function and once with an ω-carboxyalkyl function the cyanines described herein each have 3-position chiral carbon atoms which means that there is a mixture of diastereomers here.

The Advantages of the diastereomers first described by us at this point Cyanine lies in the fact that due to the different spatial Arrangement of the substituents on the cyanine chromophore further minimization the aggregation between the dye molecules is observed. conditioned through this particular substituent pattern, coupled with the strong Hydrophilicity of the new cyanines (at least three sulfonic acid residues) will fail of cyanine-protein conjugates high cyanine-protein ratio from aqueous solution prevents and unwanted unspecific interactions with those in the prior art known cyanines are significantly reduced.

wobei es auch möglich ist, die Diastereomeren-Gemische, wie am Beispiel der allgemeinen Formel I gezeigt, zu trennen und dann die Enantiomeren-Gemische für optische Detektionsmethoden zu verwenden. Diastereomer 1 (Enantiomeren-Gemisch)

Diastereomer 2 (Enantiomeren-Gemisch)

wobei

• ⧫ R₁ und R₂ gleich oder unterschiedlich, jeweils eine aliphatisch- (vorzugsweise CH₃-) oder heteroaliphatische Gruppe sein können,
• ⧫ L₁–L₄ gleich oder unterschiedlich, jeweils eine divalente lineare (-(CH₂)_o- mit o = 1–15), gekreuzte oder cyclische Alkylen-Gruppe, die durch eine oder mehrere Heteroatome wie Sauerstoff, substituierter Stickstoff oder Schwefel substituiert sein kann, darstellen,
• ⧫ X₁ gleich -OH, -SH, NH₂, -NH-NH₂, -F, -Cl, -Br, -I, -O-NHS (Succinimidyl/Sulfo-Succinimidyl), -O-STP (4-Sulfonsäure-2,3,5,6-tetrafluorobenzen), Benzotriazol bzw. -L-OH, -L-O-Phosphoramidit, -L-SH, -L-NH₂, -L-NH-NH₂, -L-N=OH, -L-CHO und -L-Maleimid, wobei L eine divalente lineare (-(CH₂)_o'- mit o' = 1–15), gekreuzte oder cyclische Alkylen-Gruppe darstellt, die durch ein oder mehrere Heteroatome wie Sauerstoff, substituierter Stickstoff oder Schwefel substituiert sein kann,
• ⧫ X₂ und X₃ gleich oder unterschiedlich Wasserstoff, Alkyl-, tert-Alkyl-, Aryl-, Carboxyaryl-, Dicarboxyaryl-, Heteroaryl-, Cycloalkyl-, Heterocycloalkyl-, Alkyloxy-, Alkylmercapto-, Aryloxy-, Arylmercapto-, Hydroxy-, Amino-, Nitro-, Cyano-Reste sein können bzw. einen solubilisierenden bzw. ionisierbaren bzw. ionisierten Substituenten wie -SO₃ ^–, -PO₃ ^2–, -CO₂ ^–, tert-Ammonium, Cyclodextrin oder Zucker darstellen,
• ⧫ Y für ein Halogenatom wie Fluor, Chlor oder Brom, eine substituierte Phenoxy- oder eine substituierte Arylmercapto-(Phenyl-sulfanyl-)Funktion steht,
• ⧫ Kat für eine bestimmte Anzahl von Na⁺, K⁺, Ca²⁺, Ammonium oder andere Kationen steht, die zum Ausgleich der durch das Cyanin I bzw. II vorgegebenen negativen Ladung notwendig sind,
• ⧫ für n im Cyanin I der Wert von 0–3 stehen kann und
• ⧫ R₃ und R₄ in II gleich oder unterschiedlich, jeweils eine aliphatisch- (vorzugsweise CH₃-) oder heteroaliphatische Gruppe sein kann bzw. zusammen das divalente Strukturelement -(CH₂)_m-, -(CH₂)_mO(CH₂)_m'-, -(CH₂)_mS(CH₂)_m'-, -(CH₂)_mCH=CH- oder -OCH=CH- bilden und m bzw. m' gleich oder unterschiedlich die Werte von 2–6 darstellen können,

The invention now relates to novel, especially substituted and strongly hydrophilic markers based on diastereomeric cyanines of the general formulas I and II,

wherein it is also possible to separate the diastereomeric mixtures as shown by the example of general formula I, and then to use the enantiomeric mixtures for optical detection methods. Diastereomer 1 (enantiomeric mixture)

Diastereomer 2 (enantiomeric mixture)

in which

• ⧫ R ₁ and R _{2 may be} identical or different and each may be an aliphatic (preferably CH ₃ ) or heteroaliphatic group,
• ⧫ L ₁ -L _{4 are the} same or different, each one divalent linear (- (CH ₂ ) _o - with o = 1-15), crossed or cyclic alkylene group represented by one or more heteroatoms such as oxygen, substituted nitrogen or sulfur may be substituted,
• X ₁ is -OH, -SH, NH ₂ , -NH-NH ₂ , -F, -Cl, -Br, -I, -O-NHS (succinimidyl / sulfo-succinimidyl), -O-STP (4- Sulfonic acid 2,3,5,6-tetrafluorobenzene), benzotriazole or -L-OH, -LO-phosphoramidite, -L-SH, -L-NH ₂ , -L-NH-NH ₂ , -LN = OH, -L-CHO and -L-maleimide, wherein L represents a divalent linear (- (CH ₂ ) _{o '} - with o' = 1-15), a crossed or cyclic alkylene group represented by one or more heteroatoms, such as oxygen, substituted nitrogen or sulfur may be substituted,
• ⧫ X ₂ and X _{3 are} identical or different and are hydrogen, alkyl, tert-alkyl, aryl, carboxyaryl, dicarboxyaryl, heteroaryl, cycloalkyl, heterocycloalkyl, alkyloxy, alkylmercapto, aryloxy, arylmercapto, hydroxy -, amino, nitro, cyano radicals may be or represent a solubilizing or ionizable or ionized substituents such as -SO ₃ ^- , -PO ₃ ^2- , -CO ₂ ^- , tert-ammonium, cyclodextrin or sugar,
• ⧫ Y is a halogen atom such as fluorine, chlorine or bromine, a substituted phenoxy or a substituted arylmercapto (phenylsulfanyl) function,
• ⧫ cat represents a certain number of Na ⁺ , K ⁺ , Ca ²⁺ , ammonium or other cations necessary to balance the negative charge given by the cyanine I or II,
• ⧫ for n in cyanine I the value of 0-3 can be and
• ⧫ R ₃ and R ₄ in II may be identical or different, each may be an aliphatic (preferably CH ₃ ) or heteroaliphatic group or together the divalent structural element - (CH ₂ ) _m -, - (CH ₂ ) _m O (CH ₂ ) _{m '} -, - (CH ₂ ) _m S (CH ₂ ) _m' -, - (CH ₂ ) _m CH = CH- or -OCH = CH- form and m or m 'the same or different values of 2-6,

The cyanines according to the invention can as chromophores / fluorophores for optical labeling and thus the qualitative and / or quantitative detection of proteins, nucleic acids, oligomers, DNA, RNA, biological cells, lipids, mono-, oligo- and polysaccharides, Ligands, receptors, polymers, pharmaceuticals or polymer particles be used.

The Covalent coupling of the new cyanines takes place via the in the 3-position on the indole ring standing ω-carboxyalkyl function, the for example, according to known methods of protein chemistry as NHS ester, acid fluoride or STP ester can be activated and with the amine function of the biomolecule under formation of an acid amide function is implemented.

These Coupling reaction is advantageously carried out in organic or aqueous solutions. From the cyanines according to the invention and biomolecules Existing conjugates have fluorescent properties.

The cyanines according to the invention find in optical, especially fluorescent optical qualitative and quantitative determination methods including immunoassays, hybridization methods, chromatographic or electrophoretic methods, FRET systems and high-throughput screening or analysis of receptor-ligand interactions on a microarray use.

The Cyanines of the general formulas I and / or II can be used as dyes for optical Marking of organic or inorganic recognition units, for example, of amino acids, Peptides, proteins, antigens, haptens, enzyme substrates, enzyme cofactors, Biotin, carotenoids, hormones, neurohormones, neurotransmitters, Growth factors, lectins, toxins, carbohydrates, oligosaccharides, Polysaccharides, dextrans, nucleic acids, oligonucleotides, DNA, RNA, biological cells, lipids, receptor binding drugs or organic or inorganic polymeric support materials are used.

The Marking the recognition units can also by training of ionic interactions between the cyanines of the general Formulas I and / or II and the materials to be marked are made.

Farther there is also the possibility the recognition unit or the support material covalently with the Cyanine connect. This coupling reaction can be in aqueous or predominantly aqueous solution and preferably carried out at room temperature become. This results in a conjugate to the qualitative or quantitative Determination of different biomaterials or other organic and inorganic materials using optical methods.

Either the cyanines of general formulas I and / or II and derived therefrom Systems can in optical, in particular fluorescence optical, qualitative and quantitative determination method for the diagnosis of cell properties (Molecular imaging), in biosensors (point of care measurements), for the study of the genome and in miniaturization technologies be used. Typical applications are in cytometry and cell sorting, fluorescence correlation spectroscopy (FCS), in the Ultra High Throughput Screening (UHTS), in multicolor fluorescence in situ hybridization (FISH), in FRET systems and in microarrays (DNA and protein chips).

there a microarray is a grid-like arrangement of at least a surface immobilized molecules, which can be used to study receptor-ligand interactions. A grid-like arrangement means more than two different ones molecules which is within a surface and there in different, previously defined regions are immobilized with a known position.

One Receptor is a molecule that's an affinity to a given ligand. Receptors can be naturally occurring or artificial produced molecules be. Receptors can used in pure form or bound to other species. Receptors can covalent or noncovalent either directly or by particular Coupling agents are linked to a binding partner.

Examples for receptors, which can be detected by this invention include agonists and Antagonists for Cell membrane receptors, toxins and other toxins, viral epitopes, Hormones such as opiates and steroids, hormone receptors, peptides, enzymes, Enzyme substrates, cofactors, lectins, sugars, Oligonucleotides, nucleic acids, Oligosaccharides, cells, cell fragments, tissue fragments, proteins and antibodies but are not limited to the listed substances.

One Ligand is a molecule that is recognized by a particular receptor. Examples of ligands that by the compounds according to the invention can be detected shut down Agonists and antagonists for Cell membrane receptors, Toxins and other toxins, viral epitopes, hormones like opiates and steroids, hormone receptors, peptides, enzymes, enzyme substrates, cofactors, lectins, sugars, oligonucleotides, nucleic acids, Oligosaccharides, proteins and antibodies, but are not on the cited Limited substances.

The cyanines according to the invention are relatively easy due to the condensation of the two substituted CH-acidic indole heterocycles and a C-1, C-3 or C-5 building blocks.

Further Methods for their preparation are that in a 1. Reaction stage of one of the CH-acidic indole heterocycles with the C-1, C-3 or C-5 building block is condensed and after isolation of the 1: 1 condensation product in a subsequent condensation with the second CH-acidic indole heterocycle is converted to cyanine. Here is the order of use the CH-acidic indole heterocycles irrelevant. This is in a few Reaction steps a variety of different functionalized, strong hydrophilic, diastereomeric cyanines, which vary in overall charge and the specificity / reactivity of the Immobilization used activated groups differ simple way to produce.

The Invention will be explained in more detail below with reference to the embodiments 1 and 2.

embodiment 1 (compounds of general formula I)

1. Synthesis of 1-ethyl-3-methyl-2 - ((E) -2-phenylamino-vinyl) -5-sulfo-3- (3-sulfopropyl) -3H-indolium sodium salt

0,794 g (2 mmol) of 3- (3-sulfopropyl) -2,3-dimethyl-5-sulfonato-1-ethyl-3H-indolium Sodium salt and 0.49 g (2.5 mmol) of N, N'-diphenyl-formamidine are dissolved in 20 ml Methanol was added and stirred for about 4 h at reflux. After cooling to RT becomes the solvent mixture distilled off in vacuo. The residue is washed well with ethyl acetate. A dark yellow solid is obtained which can be further reacted without further purification.

2. 2 - {(E) -3- [3- (3-carboxy-propyl) -3-methyl-5-sulfo-1- (3-sulfo-propyl) -1,3-dihydro-indole (2E) - ylidene] -propenyl} -1-ethyl-3-methyl-5-sulfo-3- (3-sulfo-propyl) -3H-indolium trisodium salt

500 mg (1 mmol) of 1-ethyl-3-methyl-2 - ((E) -2-phenylamino-vinyl) -5-sulfo-3- (3-sulfopropyl) -3H-indolium sodium salt and 483 mg of 3- (3-carboxypropyl) -2,3-dimethyl-5-sulfonato-1- (3-sulfopropyl) -3H-indolium Sodium salt in a mixture of 10 ml of acetic acid and 10 ml of acetic anhydride dissolved. Now 5 ml of pyridine are added. The solution is stirred for about 15 min at reflux.

After this cooled to RT was added, are added to about 20 ml of ether. The resulting precipitate is filtered off with suction, washed with ether and dried.

The residue is purified by column chromatography: RP-18: acetonitrile / water, 2/8 + 1% conc. HCl; Here, the diastereomers are separated from each other. The good fractions are, separated according to the diastereomers, combined, neutralized with NaHCO ₃ and evaporated. The separation is repeated on an RP-18 column - acetonitrile / water, 2/8 - without acid.

The corresponding fractions are combined and the solvent is distilled off. The two products (diastereomers) will be dried in a high vacuum.

The Yield is about 15% per diastereomer.

Diastereomer 1:

• UV vis (ethanol): λ _max = 560 nm
• MS (ES, -) [M / z]: 271.8 [M] ^3-

Diastereomer 2:

• UV-vis (ethanol): λ _max = 562 nm
• MS (ES, -) [M / z]: 419.1 [M + Na] ^2- ; 271.8 [M] ^3-

3. Synthesis of 1-ethyl-3-methyl-2 - ((1E, 3E) -4-phenylamino-buta-1,3-dienyl) -5-sulfo-3- (3-sulfo-propyl) -3H- indolium sodium salt

0,794 g (2 mmol) of 3- (3-sulfopropyl) -2,3-dimethyl-5-sulfonato-1-ethyl-3H-indolium Sodium salt and 0.65 g (2.5 mmol) of malonaldehyde bisphenylimine hydrochloride are dissolved in a mixture of 10 ml of acetic acid and 10 ml of acetic anhydride solved and about 4 h at 120 ° C touched. After cooling on RT, the solvent mixture distilled off in vacuo. The residue is washed with ethyl acetate. This gives a dark brown solid, which can be reacted without further purification.

4. Synthesis of 2 - {(1E, 3E) -5- [3- (3-carboxy-propyl) -3-methyl-5-sulfo-1- (3-sulfopropyl) -1,3-dihydro-indole (2E) -ylidene] penta-1,3-dienyl} -1-ethyl-3-methyl-5-sulfo-3- (3-sulfo-propyl) -3H-indolium trisodium salt

516 mg (1 mmol) of 1-ethyl-3-methyl-2 - ((1E, 3E) -4-phenylamino-buta-1,3-dienyl) -5-sulfo-3- (3-sulfopropyl) -3H -indolium Sodium salt and 483 mg of 3- (3-carboxypropyl) -2,3-dimethyl-5-sulfonato-1- (3-sulfopropyl) -3H-indolium Sodium salt in a mixture of 10 ml of acetic acid and 10 ml of acetic anhydride dissolved. Now 5 ml of pyridine are added. The solution is stirred for about 15 min at reflux.

After this cooled to RT was added, are added to about 20 ml of ether. The resulting precipitate is filtered off with suction, washed with ether and dried.

Of the Residue becomes column chromatographic Purified: RP-18 acetonitrile / water, 2/8 + 1% conc. HCl; in this connection the diastereomers are separated from each other. The good factions are separated after the diastereomers, combined, neutralized and evaporated. The separation is carried out on an RP-18 column - acetonitrile / water, 2/8 - without Acid repeated.

The corresponding fractions are combined and the solvent is distilled off. The two products (diastereomers) will be dried in a high vacuum.

The Yield is about 15% per diastereomer.

Diastereomer 1:

• UV-vis (ethanol): λ _max = 655 nm
• MS (ES, -) [M / z]: 432.2 [M + Na] ^2- ; 280.5 [M] ^3-

Diastereomer 2:

• UV-vis (ethanol): λ _max = 654 nm
• MS (ES, -) [M / z]: 432.1 [M + Na] ^2- ; 280.5 [M] ^3-

5. Synthesis of 3-methyl-2 - ((1E, 3E) -4-phenylamino-buta-1,3-dienyl) -5-sulfo-1,3-bis (3-sulfo-propyl) -3H- indolium Bisnatriumsalz

1.03 g (2 mmol) 1,3-bis (3-sulfopropyl) -2,3-dimethyl-5-sulfonato-3H-indolium Bis sodium salt and 0.65 g (2.5 mmol) of malonaldehyde bisphenylimine hydrochloride are dissolved in a mixture of 10 ml of acetic acid and 10 ml of acetic anhydride solved and about 4 h at 120 ° C touched. After cooling on RT, the solvent mixture distilled off in vacuo. The residue is washed with ethyl acetate. This gives a dark brown solid, which can be reacted without further purification.

6. Synthesis of 2 - {(1E, 3E) -5- [3- (3-carboxy-propyl) -3-methyl-5-sulfo-1- (3-sulfopropyl) -1,3-dihydro-indole (2E) -ylidene] penta-1,3-dienyl} -3-methyl-5-sulfo-1,3-bis- (3-sulfo-propyl) -3H-indolium Tetrasodium salt

642 mg (1 mmol) of 3-methyl-2 - ((1E, 3E) -4-phenylamino-buta-1,3-dienyl) -5-sulfo-1,3-bis (3-sulfo-pro pyl) -3H-indolium bis-sodium salt and 483 mg (1 mmol) of 3- (3-carboxypropyl) -2,3-dimethyl-5-sulfonato-1- (3-sulfopropyl) -3H-indolium sodium salt are dissolved in a mixture of 10 Dissolved acetic acid and 10 ml of acetic anhydride. Now 5 ml of pyridine are added. The solution is stirred for about 15 min at reflux. After cooling to RT, about 20 ml of ether are added. The resulting precipitate is filtered off with suction, washed with ether and dried.

The residue is purified by column chromatography: RP-18 acetonitrile / water, 2/8 + 1% conc. HCl. Here, the diastereomers are separated from each other. The good fractions are combined separately according to the diastereomers, neutralized with NaHCO ₃ and evaporated: The separation is repeated on an RP-18 column - acetonitrile / water, 2/8 - without acid.

The corresponding fractions are combined and the solvent is distilled off. The two products (diastereomers) will be dried in a high vacuum.

The Yield is about 10% per diastereomer.

Diastereomer 1:

• UV-vis (ethanol): λ _max = 654 nm
• MS (ES, -) [M / z]: 490.2 [M + 2Na] ^2- ; 319.4 [M + Na] ^3- ; 233.6 [M] ^4-

Diastereomer 2:

• UV-vis (ethanol): λ _max = 655 nm
• MS (ES, -) [M / z]: 490.2 [M + 2Na] ^2- ; 319.3 [M + Na] ^3-; 233.5 [M] ^4-

7. Synthesis of 2 - {(1E, 3E, 5E) -7- [3- (3-carboxy-propyl) -3-methyl-5-sulfo-1- (3-sulfopropyl) -1,3-dihydro- -ylidene indole (2E)] hepta-1,3,5-trienyl} -1-ethyl-3-methyl-5-sulfo-3- (3-sulfo-propyl) -3H-indolium trisodium salt

397 mg (1 mmol) of 3- (3-sulfopropyl) -2,3-dimethyl-5-sulfonato-1-ethyl-3H-indolium Sodium salt and 284 mg (1 mmol) of phenyl - [(2E, 4E) -5-phenylamino-penta-2,4-dien- (E) -ylidene] -ammonium chloride in 10 ml of acetic acid and 10 ml of acetic anhydride dissolved and 1 h at 120 ° C touched. Now, 483 mg (1 mmol) of 3- (3-carboxypropyl) -2,3-dimethyl-5-sulfonato-1- (3-sulfopropyl) -3H-indolium Sodium salt and 5 ml of pyridine. The mixture is now at 170 ° C approx. Stirred for 20 min. After cooling RT is added to about 20 ml of ether. The resulting precipitate is filtered off with suction, washed with ether and dried.

The residue is purified by column chromatography: RP-18 acetonitrile / water, 2/8 + 1% conc. HCl. Here, the diastereomers are separated from each other. The good fractions are combined separately according to the diastereomers, neutralized with NaHCO ₃ and evaporated. The separation is repeated on an RP-18 column - acetonitrile / water, 2/8 - without acid.

The corresponding fractions are combined and the solvent is distilled off. The two products (diastereomers) will be dried in a high vacuum.

The Yield is about 2% per diastereomer.

Diastereomer 1:

• UV-vis (ethanol): λ _max = 752 nm
• MS (ES, -) [M / z]: 445.1 [M + Na] ^2- ; 289.2 [M] ^3-

Diastereomer 2:

• UV vis (ethanol): λ _max = 751 nm
• MS (ES, -) [M / z]: 445.1 [M + Na] ^2- ; 289.2 [M] ^3-

Embodiment 2 - (Compounds of the general formula II)

8. 2 - ((E) -2- {3- [2- [3- (3-carboxy-propyl) -3-methyl-5-sulfo-1- (3-sulfo-propyl) -1,3- dihydro-indol- (2E) -ylidene] -eth- (e) -ylidene] -2-chloro-cyclohexyl} -vinyl) -1-ethyl-3-methyl-5-sulfo-3- (3-sulfo-propyl ) -3-indolium trisodium salt

397 mg (1 mmol) of 3- (3-sulfopropyl) -2,3-dimethyl-5-sulfonato-1-ethyl-3H-indolium Sodium salt and 358 mg (1 mmol) of [1- {2-chloro-3- [1-phenylamino-meth (E) -ylidene] -cyclohex-1-enyl} -meth- (E) -ylidene] -phenyl- ammonium chloride in 10 ml of acetic acid and 10 ml of acetic anhydride dissolved and 1 h at 120 ° C touched. Now, 483 mg (1 mmol) of 3- (3-carboxypropyl) -2,3-dimethyl-5-sulfonato-1- (3-sulfopropyl) -3H-indolium sodium salt and 5 ml of pyridine added. The mixture is now at 170 ° C about 20 stirred for a few minutes.

To cooling RT is added to about 20 ml of ether. The resulting precipitate is filtered off with suction, washed with ether and dried.

The residue is purified by column chromatography: RP-18 acetonitrile / water, 2/8 + 1% conc. HCl. Here, the diastereomers are separated from each other. The good fractions are combined separately according to the diastereomers, neutralized with NaHCO ₃ and evaporated. The separation is repeated on an RP-18 column - acetonitrile / water, 2/8 - without acid.

The corresponding fractions are combined and the solvent is distilled off. The two products (diastereomers) will be dried in a high vacuum.

The Yield is about 1% per diastereomer.

Diastereomer 1:

• UV-vis (ethanol): λ _max = 784 nm
• MS (ES, -) [M / z]: 482.1 [M + Na] ^2- ; 313.7 [M] ^3-

Diastereomer 2:

• UV-vis (ethanol): λ _max = 784 nm
• MS (ES -) [M / z]: 482.2 (M + Na] ^2-; 313.7 [M] ^3-

General Method for the synthesis of N-hydroxysuccinimide esters

20 μmol Type I or II dye with X ₁ = OH, 8 mg (40 μmol) dicyclohexylcarbodiimide and 5 mg (40 μmol) of N-hydroxysuccinimide are dissolved in 1 ml of dry DMF. Then 6 .mu.l (40 .mu.mol) of triethylamine are added. The reaction mixture is stirred for 24 hours at RT and then filtered. Following this, the solvent is stripped off and the residue is washed with diethyl ether. This reaction is quantitative.

Claims (7)

Hydrophilic markers based on diastereomeric cyanines of the general structure I or II

wherein R ₁ and R _{2 may be the} same or different, each an aliphatic (preferably CH ₃ ) or heteroaliphatic group, L ₁ -L _{4 are the} same or different, each having a divalent linear (- (CH ₂ ) _o - with o = 1-15), crossed or cycli alkylene group which may be substituted by one or more heteroatoms such as oxygen, substituted nitrogen or sulfur, X ₁ is -OH, -SH, NH ₂ , -NH-NH ₂ , -F, -Cl, -Br , -I, -O-NHS (succinimidyl / sulfo-succinimidyl), -O-STP (4-sulfonic acid-2,3,5,6-tetrafluorobenzene), benzotriazole or -L-OH, -LO-phosphoramidite, - L-SH, -L-NH ₂ , -L-NH-NH ₂ , -LN = OH, -L-CHO and -L-maleimide, where L is a divalent linear (- (CH ₂ ) _{o '} - with o' = 1-15), a crossed or cyclic alkylene group which may be substituted by one or more heteroatoms such as oxygen, substituted nitrogen or sulfur, X ₂ and X _{3 are the} same or different hydrogen, alkyl, tertiary alkyl, aryl -, carboxyaryl, dicarboxyaryl, heteroaryl, cycloalkl, heterocycloalkyl, alkyloxy, alkylmercapto, aryloxy, arylmercapto, hydroxy, amino, nitro, cyano radicals or a solubilizing or ionizable or ionized substituents such as -SO ₃ ^- , -P O _{3 is} ^2- , -CO ₂ ^- , tert-ammonium, cyclodextrin or sugar, Y is a halogen atom such as fluorine, chlorine or bromine, a substituted phenoxy or a substituted arylmercapto (phenyl-sulfanyl) function, cat is a certain number of Na ⁺ , K ⁺ , Ca ²⁺ , ammonium or other cations necessary to balance the negative charge given by cyanine I or II, for which n in cyanine I can be 0-3 and R ₃ and R ₄ in II may be identical or different, may each be an aliphatic (preferably CH ₃ ) or heteroaliphatic group or, together, the divalent structural element - (CH ₂ ) _m -, - (CH ₂ ) _m O (CH ₂ ) _{m '} -, - (CH ₂ ) _m S (CH ₂ ) _m' -, - (CH ₂ ) _m CH = CH- or -OCH = CH- form and m or m 'the same or different values of 2-6. Hydrophilic marker according to claim 1, characterized in that that in 3 position ω-carboxyalkyl function on the indole ring, for example as NHS ester, acid fluoride or STP ester activated. Hydrophilic marker according to claim 1, characterized in that that in 3 position on the indole ring standing ω-carboxyalkyl function is used to over an acid amide function the respective cyanines as maleimides, haloacetamides, hydrazides, Activate amines or phosphoramidites. Hydrophilic markers according to claims 1-3, characterized that these are used to label biomolecules. Hydrophilic markers according to claims 1 and 3, characterized that instead of the diastereomeric mixtures, the corresponding enantiomers for labeling biomolecules be used. Hydropile marker according to claims 1-3, characterized in that that instead of the enantiomers, the corresponding isolated stereoisomers for labeling biomolecules be used. Use of the hydrophilic markers according to claims 1-6 as cyanines for the covalent attachment of amino acids, proteins, antibodies, nucleic acids, oligomers, DNA, RNA, biological cells, lipids, haptens, polymers, pharmaceuticals or polymer particles and application of the cyanine conjugates in optical, especially fluorescent optical qualitative and quantitative Including determination Immunoassays, hybridization methods, FRET systems, chromatographic or electrophoretic methods and high-throughput screening.