FLUORESCENT DYES HAVING HIGH STOKES SHIFT, ON THE BASIS OF BRIDGED BENZOPYRYLIUM SALTS

Abstract

The invention relates to novel, water-soluble fluorescent dyes with high fluorescence quantum yield based on oxygen-containing heterocycles, their reactive derivatives and dye conjugates, and their use for labelling samples and detecting analytes. The compounds of the new dye class are compatible with commercial excitation light sources and are characterised by Stokes shifts of more than 50 nm.

Claims

1. A compound of the general formula 1 or 4 ##STR00068## and salts as well as solvates thereof, wherein R11 and R12 are each independently of the other hydrogen or alkyl, R2 is hydrogen, alkyl or alkenyl, R3 is hydrogen, alkyl, aryl, hydroxy, alkoxy, aryloxy, NR18R19 or a group Q, wherein R18 and R19 are each independently of the other selected from (i) hydrogen, (ii) benzyl, (iii) aryl, (iv) heteroaryl, (v) a reactive group A bound via a linker L, (vi) alkyl, preferably C.sub.1-C.sub.4 alkyl, more preferably ethyl, (vii) ω-sulfonic acid alkyl (—(CH.sub.2).sub.x—SO.sub.3.sup.−) where x is 1-5, (viii) ω-carboxylic acid alkyl (—(CH.sub.2).sub.y—CO.sub.2H) where y is 1-8, and (ix) ethyl esters of (viii), and Q is a heterocyclic structure selected from a structure of formula 2 or 3 ##STR00069## where n=1, 2 or 3; wherein each R20 is independently of the other alkyl, ω-sulfonic acid alkyl (—(CH.sub.2).sub.x—SO.sub.3.sup.−) or a reactive group A bound through a linker L; R21, R22, R23, R24 are independently of the other each hydrogen, a sulfonic acid or a sulfonic acid derivative; R25 is hydrogen, alkyl, ω-sulfonic acid alkyl (—(CH.sub.2).sub.x—SO.sub.3.sup.−) or a reactive group A bound through a linker L, wherein each x is an integer from 1-5, or R2 and R3 are bridged to form a saturated ring, partially unsaturated ring, aromatic ring or heteroaromatic ring together with the carbon atoms to which they are bound, which ring may contain further substituents, R4 is hydrogen, bromine, chlorine, sulfonic acid or a sulfonic acid derivative, alkyl, aryl or heteroaryl or R3 and R4 are bridged to form a saturated ring, partially unsaturated ring, aromatic ring or heteroaromatic ring together with the carbon atoms to which they are bound, which ring may contain further substituents, in particular a sulfonic acid or a sulfonic acid derivative, R5 is hydrogen, sulfonic acid or a sulfonic acid derivative, R6 is hydrogen, bromine, chlorine, hydroxy, alkoxy, aryloxy or NR29R30, wherein R29 and R30 are each independently of the other hydrogen, alkyl, aryl or a reactive group A bound via a linker L wherein R29 and R30 are each independently of the other preferably selected from (i) C.sub.1-C.sub.4 alkyl, (ii) ω-sulfonic acid alkyl (—(CH.sub.2).sub.x—SO.sub.3.sup.−) wherein x is 1-5, (iii) ω-carboxylic acid alkyl (—(CH.sub.2).sub.y—CO.sub.2H) wherein y is 1-8, and (iv) ethyl esters of (iii), or R5 and R6 are bridged to form a saturated ring, partially unsaturated ring, aromatic ring or heteroaromatic ring together with the carbon atoms to which they are bound, which ring may contain further substituents, in particular a sulfonic acid or sulfonic acid derivative, R7 is hydrogen, bromine, chlorine, hydroxy, alkoxy, aryloxy, NR31R32, sulfonic acid or a sulfonic acid derivative, wherein R31 and R32 are each independently of the other selected from (i) hydrogen, (ii) benzyl, (iii) aryl, (iv) heteroaryl, (v) a reactive group A bound via a linker L, (vi) alkyl, preferably C.sub.1-C.sub.4 alkyl, (vii) ω-sulfonic acid alkyl (—(CH.sub.2).sub.x—SO.sub.3.sup.−) where x is 1-5, (viii) ω-carboxylic acid alkyl (—(CH.sub.2).sub.y—CO.sub.2H) where y is 1-8, and (ix) ethyl esters of (viii), or R6 and R7 are bridged to form a saturated ring, partially unsaturated ring, aromatic ring or heteroaromatic ring together with the carbon atoms to which they are bound, which ring may contain further substituents, in particular a sulfonic acid or sulfonic acid derivative, R8 is hydrogen, methyl or ethyl, or R7 and R8 are bridged to form a saturated ring, partially unsaturated ring, aromatic ring or heteroaromatic ring together with the carbon atoms to which they are bound, which ring may contain further substituents, in particular a sulfonic acid or a sulfonic acid derivative, R9 is hydrogen, alkyl or 2-carboxyphenyl, L is a linker selected from —(CH.sub.2).sub.s— and —[(CH.sub.2).sub.m—O].sub.p—(CH.sub.2).sub.m—, where m is an integer from 2-5 and p and s are each independently of the other an integer from 1-10, wherein the compound contains a linker L having a reactive group A bonded to L for covalently bonding to a molecule K to be labelled, wherein A is an amine (—NH.sub.2), hydroxy (—OH) or phosphoramidite (—O—P—[O—CH.sub.2—CH.sub.2—CN]—N[(CH(CH.sub.3).sub.2].sub.2) function, a carboxylic acid (—COOH), an alkyl ester or active ester derived therefrom (NHS ester, sulfo-NHS ester, tetrafluoro-phenyl ester, p-sulfo-tetrafluoro-phenyl ester), a carboxylic acid hydrazide (—CONHNH.sub.2) or a carboxylic acid amide (—CONHR28) with R28 equal to —(CH.sub.2).sub.t—Y, where Y is —OH, —NH.sub.2, —NH.sub.3.sup.+, maleimide (—N[CO—CH].sub.2), —NCS, —NCO, —NH—CO—CH.sub.2—I, —NH—CO—CH.sub.2—Br, -azide (—N.sub.3), -alkyne (—CCH) or -phosphoramidite (—O—P—[O—CH.sub.2—CH.sub.2—CN]—N—[CH—(CH.sub.3).sub.2].sub.2) and t is an integer from 1-10, K is a component selected from the group consisting of haptens, proteins, antibodies, low-molecular weight drugs, peptides, nucleotides, nucleosides, DNA oligomers, polymers.

2. A compound according to claim 1, wherein R3=hydroxy.

3. A compound according to claim 1, wherein R3 and R4 are not bridged together in such a way that they form an aromatic ring together with the carbon atoms to which they are bound.

4. A compound according to claim 1, comprising: a structure of formula 5, ##STR00070## wherein R13 is hydrogen, alkyl or 2-carboxyphenyl, R14 is hydrogen, alkyl or 2-carboxyphenyl, R15 is hydrogen, bromine chlorine, hydroxy, alkoxy, aryloxy, NR33R34, sulfonic acid or a sulfonic acid derivative or bridged to R16, wherein R33 and R34 are each independently of the other selected from (i) hydrogen, (ii) benzyl, (iii) aryl, (iv) heteroaryl, (v) a reactive group A bound via a linker L, (vi) alkyl, preferably C.sub.1-C.sub.4 alkyl, (vii) ω-sulfonic acid alkyl (—(CH.sub.2).sub.x—SO.sub.3.sup.−) where x is 1-5, (viii) ω-carboxylic acid alkyl (—(CH.sub.2).sub.y—CO.sub.2H) where y is 1-8, and (ix) ethyl esters of (viii), or R14 and R15 are bridged to form a saturated ring, partially unsaturated ring, aromatic ring or heteroaromatic ring together with the carbon atoms to which they are bound, which ring may contain further substituents, in particular a sulfonic acid or sulfonic acid derivative, R16 is hydrogen, bromine, chlorine, hydroxy, alkoxy, aryloxy or NR35R36, where R35 and R36 are each independently of the other selected from (i) hydrogen, (ii) benzyl, (iii) aryl, (iv) heteroaryl, (v) a reactive group A bound via a linker L, (vi) alkyl, preferably C.sub.1-C.sub.4 alkyl, (vii) ω-sulfonic acid alkyl (—(CH.sub.2).sub.x—SO.sub.3.sup.−), where x is 1-5, (viii) ω-carboxylic acid alkyl (—(CH.sub.2).sub.y—CO.sub.2H), where y is 1-8, and (ix) ethyl esters of (viii), or R15 and R16 are bridged to form a saturated ring, partially unsaturated ring, aromatic ring or heteroaromatic ring together with the carbon atoms to which they are bound, which ring may contain further substituents, in particular a sulfonic acid or sulfonic acid derivative, R17 is hydrogen, sulfonic acid or a sulfonic acid derivative, or R16 and R17 are bridged to form a saturated ring, partially unsaturated ring, aromatic ring or heteroaromatic ring together with the carbon atoms to which they are bound, which ring may contain further substituents, in particular a sulfonic acid or a sulfonic acid derivative.

5. A compound according to claim 1, wherein the compound contains at least one group selected from a sulfonic acid group, a sulfonic acid derivative, a hydroxy group, an amino group, a carboxylic acid and a carboxylic acid derivative, wherein preferably compounds are excluded in which a) R7 is methoxy and R3 is an amino group, in particular NR18R19, in particular NH(4-HOOCC.sub.6H.sub.4), NH(4-C.sub.2H.sub.5COOC.sub.6H.sub.4), NH(2-C.sub.2H.sub.5COOC.sub.6H.sub.4), NH(C.sub.6H.sub.5), N(C.sub.2H.sub.5).sub.2 or N(CH.sub.2).sub.2(CH.sub.2).sub.2O; b) R4 is bromine and R6 and R16 are N(CH.sub.3).sub.2; c) R6 and R16 are present and one of R6 and R16 is a hydroxy group.

6. A compound according to claim 1, wherein the compound contains at least one sulfonic acid group.

7. A compound according to claim 1, wherein R3 is NR18R19, wherein R18 and R19 are each independently of the other hydrogen, alkyl, benzyl, aryl, heteroaryl or a reactive group A bound via a linker L, wherein NR18R19 is preferably selected from 3-aminopropanesulfonate, N-methylaniline, 4-(methylamino)benzenesulfonate, aniline, 5-carboxypentylamine and 3-carboxypropyl(methyl)amine.

8. A compound according to claim 1, wherein the compound is water soluble, preferably that at least 1 mg of the compound is soluble in 1000 mg of water at 25° C.

9. A compound according to claim 1, wherein the compound is a fluorescent compound having an absorption maximum in the wavelength range from 400 nm to 650 nm.

10. A compound according to claim 8, wherein the Stokes shift is at least 40 nm.

11. A compound according to claim 1, wherein the compound is selected from 6-ethoxy-N,N-diethyl-8,8-dimethyl-7H-xanthene-10-ium-3-amine, 6-[[6-(diethylamino)-1,1-dimethyl-2H-xanthene-10-ium-3-yl]amino]hexanoic acid, 3-[(6-ethoxy-8,8-dimethyl-7H-xanthene-10-ium-3-yl)-(6-ethoxy-6-oxo-hexyl)amino]propane-1-sulfonate, 3-[[6-[5-carboxypentyl(3-sulfonatopropyl)amino]-1,1-dimethyl-2H-xanthene ium-3-yl]amino]propane-1-sulfonate, 3-[[6-[[6-(2,5-dioxopyrrolidine-1-yl)oxy-6-oxo-hexyl]-(3-sulfonatopropyl)amino]-1,1-dimethyl-2H-xanthene-10-ium-3-yl]amino]propane-1-sulfonate, 6-[(6-ethoxy-8,8-dimethyl-7H-xanthene-10-ium-3-yl)-ethyl-amino]hexanoic acid ethyl ester, 3-[[6-[5-carboxypentyl(ethyl)amino]-1,1-dimethyl-2H-xanthene-10-ium-3-yl]amino]propane-1-sulfonate, 3-[5-carboxypentyl-[8,8-dimethyl-6-(N-methylanilino)-7H-xanthene-10-ium-3-yl]amino]propane-1-sulfonate, 4-[[6-[5-carboxypentyl(3-sulfonatopropyl)amino]-1,1-dimethyl-2H-xanthene-10-ium-3-yl]-methyl-amino]benzenesulfonate, 6-[[8,8-dimethyl-6-(N-methylanilino)-7H-xanthene-10-ium-3-yl]-ethyl-amino]hexanoic acid, 4-[[6-[5-carboxypentyl(ethyl)amino]-1,1-dimethyl-2H-xanthene-10-ium-3-yl]-methyl-amino]benzenesulfonate, 6-[(6-anilino-8,8-dimethyl-7H-xanthene-10-ium-3-yl)-ethyl-amino]hexanoic acid, 6-[5-carboxypentyl(ethyl)amino]-1,1-dimethyl-3-(4-sulfonatoanilino)-2H-xanthene-10-ium-4-sulfonate, 6-[[6-[5-carboxypentyl(ethyl)amino]-1,1-dimethyl-2H-xanthene-10-ium-3-yl]amino]naphthalene-2-sulfonate, 6-ethoxy-8,8-dimethyl-7H-xanthene-10-ium-3-ol, 8,8-dimethyl-6-(N-methylanilino)-7H-xanthene-10-ium-3-ol, 6-[[8,8-dimethyl-6-(N-methylanilino)-7H-xanthene-10-ium-3-yl]oxy]hexanoic acid, 3-(5-carboxypentoxy)-8,8-dimethyl-6-(N-methyl-4-sulfonato-anilino)-7H-xanthene-10-ium-2-sulfonate, 3-(9-ethoxy-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinolin-11-ium-1-yl)propane-1-sulfonate, 6-(9-ethoxy-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinolin-11-ium-1-yl)hexanoic acid ethyl ester, 3-[9-(5-carboxypentylamino)-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinolin-11-ium-1-yl]propane-1-sulfonate, 3-[9-[3-carboxypropyl(methyl)amino]-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinolin-11-ium-1-yl]propane-1-sulfonate, 6-[2,2,4,7,7-pentamethyl-9-(N-methylanilino)-8H-chromeno[3,2-g]quinolin-11-ium-1-yl]hexanoic acid, 4-[[1-(5-carboxypentyl)-2,2,7,7-tetramethyl-4-(sulfonatomethyl)-8H-chromeno[3,2-g]quinolin-11-ium-9-yl]-methyl-amino]benzenesulfonate, 6-[[6-[3-(dimethylamino)anilino]-8,8-dimethyl-7H-xanthene-10-ium-3-yl]-ethyl-amino]hexanoic acid, 6-[[6-(4-aminoanilino)-8,8-dimethyl-7H-xanthene-10-ium-3-yl]-ethyl-amino]hexanoic acid, 6-[[6-[bis(2-pyridylmethyl)amino]-8,8-dimethyl-7H-xanthene-10-ium-3-yl]-ethyl-amino]hexanoic acid, 6-[[8,8-dimethyl-6-[(E)-1H-pyridine-2-ylidenemethyl]-7H-xanthene-10-ium-3-yl]-ethyl-amino]hexanoic acid, 6-[[6-[4-(dimethylamino)phenyl]-8,8-dimethyl-7H-xanthene-10-ium-3-yl]-ethyl-amino]hexanoic acid, 6-[ethyl-(6,8,8-trimethyl-7H-xanthene-10-ium-3-yl)amino]hexanoic acid, (2E)-1-(5-carboxypentyl)-2-[(E)-3-[6-(diethylamino)-1,1-dimethyl-2H-xanthene-10-ium-3-yl]prop-2-enylidene]-3,3-dimethyl-indoline-5-sulfonate, 3-[(5Z)-3-(5-carboxypentyl)-5-[(2E)-2-[6-(diethylamino)-1,1-dimethyl-2H-xanthene-3-ylidene]ethylidene]-2,4,6-trioxo-hexahydropyrimidine-1-yl]propane-1-sulfonate, 6-(diethylamino)-1,1-dimethyl-2H-xanthene-3-one, 6-[(8,8-dimethyl-6-oxo-7H-xanthene-3-yl)-ethyl-amino]hexanoic acid, 6-[5-carboxypentyl(ethyl)amino]-1,1-dimethyl-3-oxo-2H-xanthene-4-sulfonate, 3-[5-carboxypentyl(8,8-dimethyl-6-oxo-7H-xanthene-3-yl)amino]propane sulfonate, 6-[5-carboxypentyl(3-sulfonatopropyl)amino]-1,1-dimethyl-3-oxo-2H-xanthene sulfonate, 6-hydroxy-1,1-dimethyl-3-oxo-2H-xanthene-4-sulfonate, 6-(5-carboxypentoxy)-1,1-dimethyl-3-oxo-2H-xanthene-4-sulfonate, 6-(2,2,4,7,7-pentamethyl-9-oxo-8H-chromeno[3,2-g]quinolin-1-yl)hexanoic acid, 1-(5-carboxypentyl)-2,2,4,7,7-pentamethyl-9-oxo-8H-chromeno[3,2-g]quinolin-10-sulfonate, 6-[[8,8-dimethyl-6-oxo-5-(4-pyridyl)-7H-xanthene-3-yl]-ethyl-amino]hexanoic acid, 3-[5-carboxypentyl-[8,8-dimethyl-6-oxo-5-(4-pyridyl)-7H-xanthene-3-yl]amino]propane-1-sulfonate, 2-[3,9-bis(diethylamino)-13,13-dimethyl-chromeno[3,2-b]xanthene-5-ium-14-yl]benzoic acid, 6-[[9-(diethylamino)-13,13-dimethyl-chromeno[3,2-b]xanthene-7-ium-3-yl]-ethyl-amino]hexanoic acid, and 6-[ethyl-(3-methoxy-13,13-dimethyl-chromeno[3,2-b]xanthene-5-ium-9-yl)amino]hexanoic acid.

12. A compound according to claim 1, wherein the compound is selected from 3-[[6-[5-carboxypentyl(3-sulfonatopropyl)amino]-1,1-dimethyl-2H-xanthene-10-ium-3-yl]amino]propane-1-sulfonate, 4-[[6-[5-carboxypentyl(3-sulfonatopropyl)amino]-1,1-dimethyl-2H-xanthene-10-ium-3-yl]-methyl-amino]benzenesulfonate, 6-[5-carboxypentyl(ethyl)amino]-1,1-dimethyl-3-oxo-2H-xanthene-4-sulfonate, 6-[5-carboxypentyl(3-sulfonatopropyl)amino]-1,1-dimethyl-3-oxo-2H-xanthene sulfonate, and 6-(5-carboxypentoxy)-1,1-dimethyl-3-oxo-2H-xanthene-4-sulfonate.

13. A method for preparing a compound of formula 1 according to claim 1, which method comprises the reaction of (E)-(3-ethoxy-5,5-dimethyl-cyclohex-2-en-1-ylidene)-ethyl-oxonium or (E)-[3-ethoxy-5,5-dimethyl-2-(4-pyridyl)cyclohex-2-en-1-ylidene]-ethyl-oxonium with a benzaldehyde compound, wherein the benzaldehyde compound has a benzaldehyde group and the benzaldehyde compound has a hydroxy group in ortho-position to the benzaldehyde group.

14. A method for preparing a compound of formula 5 according to claim 1, which method comprises the reaction of a compound of formula 1, preferably of 6-ethoxy-N,N-diethyl-8,8-dimethyl-7H-xanthene-10-ium-3-amine or 6-[(6-ethoxy-8,8-dimethyl-7H-xanthene-10-ium-3-yl)-ethyl-amino]hexanoic acid ethyl ester, with a benzaldehyde compound, wherein the benzaldehyde compound has a benzaldehyde group and the benzaldehyde compound has a hydroxy group in ortho-position to the benzaldehyde group.

15. Use of a compound according to claim 1 as a fluorescent dye and/or in a fluorescent probe or as a fluorescent probe.

Description

[0171] The invention is described in more detail below with reference to exemplary embodiments and figures.

[0172] They show

[0173] FIG. 1: Photostability of compounds 5, 8, 11 and 38 compared to the MegaStokes dye DY-510XL.

[0174] FIG. 2: Emission spectra of selected compounds according to the invention in PBS.

EXEMPLARY EMBODIMENTS

Compound 1

(E)-(3-ethoxy-5,5-dimethyl-cyclohex-2-en-1-ylidene)-ethyl-oxonium tetrafluoroborate

[0175] ##STR00016##

[0176] 5 mmol of 5,5-dimethylcyclohexane-1,3-dione are suspended in 10 ml of orthoformic acid triethyl ester and 1.5 ml of 48% tetrafluoroboric acid are added at room temperature. After stirring for 30 minutes at RT, 50 ml of dry diethyl ether is added and the mixture is left at RT for several hours. The resulting colourless precipitate is filtered, washed with a little dry ether and dried in vacuo.

[0177] Yield: 1.1 g (78%) (C.sub.12H.sub.21BF.sub.4O.sub.2; 284.10 g/mol)

[0178] MS ESI+ (m/z): 197.2 [M.sup.+]

Compound 2

6-ethoxy-N,N-diethyl-8,8-dimethyl-7H-xanthene-10-ium-3-amine tetrafluoroborate

[0179] ##STR00017##

[0180] 3 mmol of compound 1 in 15 ml of orthoformic acid triethyl ester are provided and a solution of 3 mmol of 2-hydroxy-4-diethylaminobenzaldehyde in 10 ml of orthoformic acid triethyl ester are added. The mixture is stirred at 130° C. for 30 minutes. After cooling, it is precipitated with diethyl ether and the precipitate is recrystallized from glacial acetic acid.

[0181] Yield: 980 mg (80%) (C.sub.21H.sub.28BF.sub.4NO.sub.2; 413.26 g/mol)

[0182] MS ESI+ (m/z): 326.1 ([M].sup.+)

[0183] UV-Vis in ethanol: λ.sub.max: 531 nm; λ.sub.em: 605 nm

Compound 3

6-[[6-(diethylamino)-1,1-dimethyl-2H-xanthene-10-ium-3-yl]amino]hexanoic Acid Chloride Salt

[0184] ##STR00018##

[0185] 485 μmol compound 2 and 1.5 mmol 6-aminohexanoic acid sodium salt are suspended in 10 ml DMF and stirred at RT for 8 hours. The solvent is distilled off and the residue is purified by RP chromatography.

[0186] Yield: 100 mg (46%) (C.sub.25H.sub.35ClN.sub.2O.sub.3; 447.01 g/mol)

[0187] MS ESI− (m/z): 411.2 ([M].sup.+)

[0188] UV-Vis in PBS: λ.sub.max: 505 nm; λ.sub.em: 570 nm; ε=39,200 l/mol*cm; QY: 0.58

[0189] UV-Vis in ethanol: λ.sub.max: 510 nm; λ.sub.em: 575 nm; ε=32,500 l/mol*cm; QY: 0.82

Compound 4

3-[(6-ethoxy-8,8-dimethyl-7H-xanthene-10-ium-3-yl)-(6-ethoxy-6-oxo-hexyl)amino]propane-1-sulfonate

[0190] ##STR00019##

[0191] 1 mmol of compound 1 in 5 ml of orthoformic acid triethyl ester are provided and a solution of 1 mmol of the aldehyde 3-(N-(6-ethoxy-6-oxo-hexyl)-4-formyl-3-hydroxy-anilino)propane-1-sulfonate sodium salt in 5 ml of glacial acetic acid are added. The mixture is stirred at 130° C. for 30 minutes. After cooling, it is precipitated with diethyl ether and the precipitate recrystallized from glacial acetic acid.

[0192] Yield: 385 mg (72%) (C.sub.28H.sub.39NO.sub.7S; 533.68 g/mol)

[0193] MS ESI+ (m/z): 534.3 ([M+H.sup.+].sup.+)

Compound 5

3-[[6-[5-carboxypentyl(3-sulfonatopropyl)amino]-1,1-dimethyl-2H-xanthene-10-ium-3-yl]amino]propane-1-sulfonate Sodium Salt

[0194] ##STR00020##

[0195] 375 μmol compound 4 and 1.5 mmol 3-aminopropane sulfonic acid sodium salt are stirred in 5 ml DMF at 40° C. for 2 hours. The solvent is distilled off and 10 ml of 3 M HCl are added to the residue for ester cleavage and boiled at reflux for 1 hour. After neutralisation with sodium hydrogen carbonate, purification is carried out by RP chromatography.

[0196] Yield: 98 mg (42%) (C.sub.27H.sub.37N.sub.2NaO.sub.9S.sub.2; 620.71 g/mol)

[0197] MS ESI− (m/z): 597.2 (base, [M].sup.−); 297.9 (25%, [M−H.sup.+].sup.2−)

[0198] UV-Vis in PBS: λ.sub.max: 505 nm; λ.sub.em: 572 nm; ε=45,000 l/mol*cm; QY: 0.72

[0199] UV-Vis in ethanol: λ.sub.max: 512 nm; λ.sub.em: 570 nm; ε=49,800 l/mol*cm; QY: 0.87

[0200] .sup.1H NMR (400 MHz D.sub.2O): δ (ppm)=1.10 (S, 6H, CH.sub.3); 1.24 (M, 2H, CH.sub.2); 1.40 (M, 2H, CH.sub.2); 1.67 (M, 2H, CH.sub.2); 1.89 (M, 2H, CH.sub.2); 2.04 (M, 2H, CH.sub.2); 2.32 (T, 2H, CH.sub.2); 2.45 (S, 2H, CH.sub.2); 2.87 (T, 2H, CH.sub.2); 2.92 (T, 2H, CH.sub.2); 3.14 (T, 2H, CH.sub.2); 3.35 (T, 2H, CH.sub.2); 3.46 (T, 2H, CH.sub.2); 5.64 (S, 1H, 4-H); 6.16 (S, 1H, 5-H); 6.47 (D, 1H, 7-H); 6.95 (D, 1H, 8-H); 7.26 (S, 1H, 9-H)

[0201] .sup.13C-NMR (400 MHz D.sub.2O): 3 (ppm)=22.27; 23.43; 24.31; 25.71; 26.17; 26.31, 26.86, 33.78; 34.20; 42.14; 42.76; 48.15; 48.29; 49.08; 50.29; 89.31; 95.96; 111.06; 111.34; 126.90; 129.46; 134.58; 151.33; 153.71; 168.62; 169.42; 178.23

Compound 6

3-[[6-[[6-(2,5-dioxopyrrolidine-1-yl)oxy-6-oxo-hexyl]-(3-sulfonatopropyl)amino]-1,1-dimethyl-2H-xanthene-10-ium-3-yl]amino]propane-1-sulfonate Sodium Salt

[0202] ##STR00021##

[0203] 125 μmol compound 5 are dissolved in 3 ml DMF. At 0° C., 45 mg of TSTU (N,N,N′,N′-tetramethyl-O—(N-succinimidyl)uronium tetrafluoroborate) and 26 μl of DIPEA (diisopropyl-ethylamine) are added and stirred at RT for 20 minutes. The solvent is distilled off in vacuo and the residue is purified by RP chromatography.

[0204] Yield: 80 mg (90%) (C.sub.31H.sub.40N.sub.3NaO.sub.11S.sub.2; 717.78 g/mol)

[0205] MS ESI− (m/z): 694.2 (base, [M].sup.−); 346.5 (25%, [M−H].sup.2−)

[0206] UV-Vis in ethanol: λ.sub.max: 512 nm; λ.sub.em: 570 nm; ε=44,000 l/mol*cm

Compound 7

6-[(6-ethoxy-8,8-dimethyl-7H-xanthene-10-ium-3-yl)-ethyl-amino]hexanoic acid ethyl ester tetrafluoroborate

[0207] ##STR00022##

[0208] 1 mmol compound 1 and ethyl 6-(N-ethyl-4-formyl-3-hydroxy-anilino)hexanoic acid are reacted according to the synthetic method for compound 2.

[0209] Yield: 420 mg (80%) (C.sub.27H.sub.38BF.sub.4NO.sub.4; 527.40 g/mol)

[0210] MS ESI+ (m/z): 440.3 ([M].sup.+)

Compound 8

3-[[6-[5-Carboxypentyl(ethyl)amino]-1,1-dimethyl-2H-xanthene-10-ium-3-yl]amino]propane-1-sulfonate

[0211] ##STR00023##

[0212] 375 μmol compound 7 and 3-aminopropane sulfonic acid sodium salt are reacted according to the synthesis method for compound 5.

[0213] Yield: 110 mg (58%) (C.sub.26H.sub.36N.sub.2O.sub.6S; 504.63 g/mol)

[0214] MS ESI− (m/z): 503.2 ([M−H.sup.+].sup.−); MS ESI+ (m/z): 505.3 (base, [M+H].sup.+); 527.3 (15%, [M+Na].sup.+).sup.+)

[0215] UV-Vis in PBS: λ.sub.max: 509 nm; λ.sub.em: 578 nm; ε=38,500 l/mol*cm; QY: 0.58

[0216] UV-Vis in ethanol: λ.sub.max: 511 nm; λ.sub.em: 571 nm; ε=41,600 l/mol*cm; QY 0.84

Compound 9

3-[5-carboxypentyl-[8,8-dimethyl-6-(N-methylanilino)-7H-xanthene-10-ium-3-yl]amino]propane-1-sulfonate

[0217] ##STR00024##

[0218] 375 μmol compound 4 and 750 μmol N-methylaniline are stirred in 5 ml DMF at 150° C. for 2 hours. The solvent is distilled off and 10 ml of 3 M HCl are added to the residue for ester cleavage and boiled at reflux for 1 hour. After neutralisation with sodium hydrogen carbonate, purification is carried out by RP chromatography.

[0219] Yield: 80 mg (38%) (C.sub.31H.sub.38N.sub.2O.sub.6S; 566.71 g/mol)

[0220] MS ESI− (m/z): 565.2 ([M−H.sup.+].sup.−); MS ESI+ (m/z): 567.3 (80%, [M+H.sup.+].sup.+); 589.3 (base, [M+Na.sup.+].sup.+); 605.3 (70%, [M+Ka.sup.+].sup.+)

[0221] UV-Vis in PBS: λ.sub.max: 523 nm; λ.sub.em: 600 nm; ε=39,000 l/mol*cm; QY: 0,24

[0222] UV-Vis in ethanol: λ.sub.max: 531 nm; λ.sub.em: 601 nm; ε=41,000 l/mol*cm; QY 0.47

Compound 10

4-[[6-[5-carboxypentyl(3-sulfonatopropyl)amino]-1,1-dimethyl-2H-xanthene-10-ium-3-yl]-methyl-amino]benzenesulfonate sodium Salt

[0223] ##STR00025##

[0224] 177 μmol of compound 9 in 2 ml of oleum (20% SO.sub.3) are dissolved and stirred at RT for 2 hours. The mixture is poured onto ice and stirred for 1 h more at RT. After blunting with sodium carbonate, purification is carried out by RP chromatography.

[0225] Yield: 75 mg (64%) (C.sub.31H.sub.38N.sub.2O.sub.6S; 668.75 g/mol)

[0226] MS ESI− (m/z): 645.5 (base, [M].sup.−); 322.1 (95%, [M.sup.−−H].sup.2−)

[0227] UV-Vis in PBS: λ.sub.max: 527 nm; λ.sub.em: 612 nm; ε=44,000 l/mol*cm; QY: 0.35

[0228] UV-Vis in ethanol: λ.sub.max: 533 nm; λ.sub.em: 609 nm; ε=48,000 l/mol*cm; QY 0.57

Compound 11

6-[[8,8-dimethyl-6-(N-methylanilino)-7H-xanthene-10-ium-3-yl]-ethyl-amino]hexanoic Acid Chloride Salt

[0229] ##STR00026##

[0230] 375 μmol compound 7 and N-methylaniline are reacted according to the synthesis method for compound 9.

[0231] Yield: 100 mg (53%) (C.sub.30H.sub.37ClN.sub.2O.sub.3; 509.08 g/mol)

[0232] MS ESI+ (m/z): 473.3 ([M].sup.+); MS ESI− (m/z): 471.2 ([M.sup.+−2H.sup.+].sup.−)

[0233] UV-Vis in PBS: λ.sub.max: 526 nm; λ.sub.em: 606 nm; ε=39,000 l/mol*cm; QY: 0.27

[0234] UV-Vis in ethanol: λ.sub.max: 531 nm; λ.sub.em: 601 nm; ε=44,000 l/mol*cm; QY 0.42

Compound 12

4-[[6-[5-carboxypentyl(ethyl)amino]-1,1-dimethyl-2H-xanthene-10-ium-3-yl]-methyl-amino]benzenesulfonate

[0235] ##STR00027##

[0236] 177 μmol of compound 11 are dissolved in 2 ml of oleum (20% SO.sub.3) and stirred at RT for 2 hours. The mixture is poured onto ice and stirred for 1 h more at RT. After blunting with sodium carbonate, purification is carried out by RP chromatography.

[0237] Yield: 67 mg (69%) (C.sub.30H.sub.36N.sub.2O.sub.6S; 552.68 g/mol)

[0238] MS ESI− (m/z): 551.2 ([M−H.sup.−].sup.−); MS ESI+ (m/z): 553.4 ([M+H.sup.+].sup.+)

[0239] UV-Vis in PBS: λ.sub.max: 532 nm; λ.sub.em: 619 nm; ε=45,000 l/mol*cm; QY: 0.26

[0240] UV-Vis in ethanol: λ.sub.max: 533 nm; λ.sub.em: 610 nm; ε=50,000 l/mol*cm; QY 0.51

Compound 13

6-[(6-anilino-8,8-dimethyl-7H-xanthene-10-ium-3-yl)-ethyl-amino]hexanoic Acid Chloride Salt

[0241] ##STR00028##

[0242] 375 μmol compound 7 and 750 μmol dry aniline are stirred in 5 ml DMF at 150° C. for 2 hours. The solvent is distilled off and 10 ml of 3 M HCl are added to the residue for ester cleavage and boiled at reflux for 1 hour. After neutralisation with sodium hydrogen carbonate, purification is carried out by RP chromatography.

[0243] Yield: 50 mg (27%) (C.sub.29H.sub.35ClN.sub.2O.sub.3; 495.05 g/mol)

[0244] MS ESI+ (m/z): 459.3 ([M].sup.+); MS ESI− (m/z): 457.2 ([M−2H.sup.+].sup.−)

[0245] UV-Vis in PBS: λ.sub.max: 526 nm; λ.sub.em: 609 nm; ε=38,000 l/mol*cm

[0246] UV-Vis in ethanol: λ.sub.max: 532 nm; λ.sub.em: 603 nm; ε=44,000 l/mol*cm

Compound 14

6-[5-carboxypentyl(ethyl)amino]-1,1-dimethyl-3-(4-sulfonatoanilino)-2H-xanthene-10-ium-4-sulfonate Sodium Salt

[0247] ##STR00029##

[0248] 177 μmol of compound 13 are dissolved in 2 ml of oleum (20% SO.sub.3) and stirred at RT for 2 hours. The mixture is poured onto ice and stirred for 1 h more at RT. After blunting with sodium carbonate, purification is carried out by RP chromatography.

[0249] Yield: 30 mg (26%) (C.sub.29H.sub.33N.sub.2O.sub.9S.sub.2 Na; 640.70 g/mol)

[0250] MS ESI− (m/z): 617.3 ([M].sup.−)

[0251] UV-Vis in PBS: λ.sub.max: 545 nm; λ.sub.em: 625 nm; ε=40,100 l/mol*cm; QY: 0.11

[0252] UV-Vis in ethanol: λ.sub.max: 542 nm; λ.sub.em: 616 nm; ε=45,300 l/mol*cm; QY 0.61

Compound 15

6-[[6-[5-carboxypentyl(ethyl)amino]-1,1-dimethyl-2H-xanthene-10-ium-3-yl]amino]naphthalene-2-sulfonate

[0253] ##STR00030##

[0254] 375 μmol compound 7 and 420 μmol 6-amino-2-naphthalenesulfonic acid hydrate are stirred in 5 ml glacial acetic acid at 120° C. for 8 hours. The solvent is distilled off and 10 ml of 3 M HCl are added to the residue for easter cleavage and boiled at reflux for 1 hour. After neutralisation with sodium hydrogen carbonate, purification is carried out by RP chromatography.

[0255] Yield: 18 mg (8%) (C.sub.33H.sub.36N.sub.2O.sub.6S; 588.71 g/mol)

[0256] MS ESI− (m/z): 587.3 ([M−H.sup.+].sup.−)

[0257] MS ESI+ (m/z): 589.3 ([M+H.sup.+].sup.+)

[0258] UV-Vis in ethanol: λ.sub.max: 546 nm; λ.sub.em: 619 nm; ε=32,000 l/mol*cm

Compound 16

6-ethoxy-8,8-dimethyl-7H-xanthene-10-ium-3-ol tetrafluoroborate

[0259] ##STR00031##

[0260] 5 mmol compound 1 are provided in 25 ml orthoformic acid triethyl ester and 5 mmol 2,4-dihydroxybenzaldehyde are added. The mixture is stirred at 130° C. for 30 minutes. After cooling, it is precipitated with diethyl ether and the precipitate recrystallized from glacial acetic acid.

[0261] Yield: 500 mg (28%) (C.sub.17H.sub.19BF.sub.4O.sub.3; 358.14 g/mol)

[0262] MS ESI+ (m/z): 271.2 ([M].sup.+)

Compound 17

8,8-dimethyl-6-(N-methylanilino)-7H-xanthene-10-ium-3-ol tetrafluoroborate

[0263] ##STR00032##

[0264] 750 μmol compound 16 and 1.5 mmol N-methylaniline are stirred in 10 ml DMF at 140° C. for 2 hours. After cooling, it is precipitated with diethyl ether and the precipitate recrystallized from glacial acetic acid.

[0265] Yield: 125 mg (40%) (C.sub.22H.sub.22BF.sub.4NO.sub.2; 419.22 g/mol)

[0266] MS ESI+ (m/z): 332.3 ([M].sup.+)

Compound 18

6-[[8,8-dimethyl-6-(N-methylanilino)-7H-xanthene-10-ium-3-yl]oxy]hexanoic Acid Chloride Salt

[0267] ##STR00033##

[0268] 286 μmol of compound 17 are stirred in 5 ml DMF containing 80 mg K.sub.2CO.sub.3 and 100 mg 6-bromohexanoic acid ethyl ester at 120° C. for 1 hour. The solvent is distilled off and 10 ml of 3 M HCl are added to the residue for ester cleavage and boiled at reflux for 1 hour. After neutralisation with sodium hydrogen carbonate, purification is carried out by RP chromatography.

[0269] Yield: 35 mg (24%) (C.sub.28H.sub.32ClNO.sub.4; 482.01 g/mol)

[0270] MS ESI+ (m/z): 446.4 ([M].sup.+)

[0271] UV-Vis in ethanol: λ.sub.max: 452+469 nm; λ.sub.em: 520 nm

Compound 19

3-(5-carboxypentoxy)-8,8-dimethyl-6-(N-methyl-4-sulfonato-anilino)-7H-xanthene-10-ium-2-sulfonate Sodium Salt

[0272] ##STR00034##

[0273] 35 mg of compound 18 are dissolved in 1 ml of oleum (20% SO.sub.3) and stirred at RT for 2 hours. The mixture is poured onto ice and stirred for 1 h more at RT. After blunting with sodium carbonate, purification is carried out by RP chromatography.

[0274] Yield: 27 mg (59%) (C.sub.28H.sub.30ClNNaO.sub.10S.sub.2; 627.66 g/mol)

[0275] MS ESI− (m/z): 604.2 ([M].sup.−)

[0276] UV-Vis in ethanol: λ.sub.max: 465 nm; λ.sub.em: 522 nm; ε=18,000 l/mol*cm.

[0277] UV-Vis in PBS: λ.sub.max: 460 nm; λ.sub.em: 509 nm; ε=16.500 l/mol*cm

Compound 20

3-(9-ethoxy-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinolin-11-ium-1-yl)propane-1-sulfonate

[0278] ##STR00035##

[0279] 1 mmol of compound 1 is provided in 5 ml of orthoformic acid triethyl ester and a solution of 1 mmol of the aldehyde 3-(6-formyl-7-hydroxy-2,2,4-trimethyl-1-quinolyl) propane-1-sulfonate sodium salt in 5 ml of glacial acetic acid are added. The mixture is stirred at 130° C. for 30 minutes. After cooling, it is precipitated with diethyl ether and the precipitate recrystallized from glacial acetic acid.

[0280] Yield: 320 mg (68%) (C.sub.26H.sub.33NO.sub.5S; 471.61 g/mol)

[0281] MS ESI+ (m/z): 472.2 (base, [M+H.sup.+].sup.+); 494.3 (60%, [M+Na.sup.+].sup.+); 510.3 (20%, [M+K.sup.+].sup.+)

Compound 21

6-(9-ethoxy-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinolin-11-ium yl)hexanoic Acid Ethyl Ester Tetrafluoroborate

[0282] ##STR00036##

[0283] 1 mmol of compound 1 is provided in 5 ml of orthoformic acid triethyl ester and a solution of 1 mmol of the aldehyde 6-(6-formyl-7-hydroxy-2,2,4-trimethyl-1-quinolyl)hexanoic acid in 5 ml of glacial acetic acid are added. The mixture is stirred at 130° C. for 30 minutes. After cooling, it is precipitated with diethyl ether and the precipitate recrystallized from glacial acetic acid.

[0284] Yield: 245 mg (42%) (C.sub.31H.sub.42BF.sub.4NO.sub.4; 579.47 g/mol)

[0285] MS ESI+ (m/z): 492.3 ([M].sup.+)

Compound 22

3-[9-(5-carboxypentylamino)-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinolin-11-ium-1-yl]propane-1-sulfonate

[0286] ##STR00037##

[0287] 375 μmol compound 20 and 1.5 mmol 6-aminohexanoic acid sodium salt are stirred in 5 ml DMF at 40° C. for 2 hours. The solvent is distilled off and purification is by RP chromatography.

[0288] Yield: 25 mg (12%) (C.sub.30H.sub.40N.sub.2NaO.sub.6S; 556.71 g/mol)

[0289] MS ESI− (m/z): 555.2 ([M−H.sup.+].sup.−)

[0290] MS ESI+ (m/z): 557.2 (50%, [M+H.sup.+].sup.+); 579.5 (base, [M+Na.sup.+].sup.+); 595.3 (20%, [M+K.sup.+].sup.+

[0291] UV-Vis in PBS: λ.sub.max: 516 nm; λ.sub.em: 590 nm; ε=37.500 l/mol*cm

[0292] UV-Vis in ethanol: λ.sub.max: 531 nm; λ.sub.em: 589 nm; ε=39,000 l/mol*cm

Compound 23

3-[9-[3-carboxypropyl(methyl)amino]-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinolin-11-ium-1-yl]propane-1-sulfonate

[0293] ##STR00038##

[0294] 375 μmol compound 20 and 1.5 mmol N-methylbutyric acid sodium salt are stirred in 5 ml DMF at 40° C. for 2 hours. The solvent is distilled off and purification is by RP chromatography.

[0295] Yield: 30 mg (15%) (C.sub.29H.sub.38N.sub.2O.sub.6S; 542.69 g/mol)

[0296] MS ESI− (m/z): 541.2 ([M−H.sup.+].sup.−)

[0297] MS ESI+ (m/z): 553.3 (base, [M+H.sup.+].sup.+); 565.5 (20%, [M+Na.sup.+].sup.+); 581.3 (15%, [M+K.sup.+].sup.+

[0298] UV-Vis in PBS: λ.sub.max: 527 nm; λ.sub.em: 600 nm; ε=45,400 l/mol*cm; QY: 0.58

[0299] UV-Vis in ethanol: λ.sub.max: 542 nm; λ.sub.em: 600 nm; ε=55,000 l/mol*cm; QY: 0.88

Compound 24

6-[2,2,4,7,7-pentamethyl-9-(N-methylanilino)-8H-chromeno[3,2-g]quinolin-11-ium-1-yl]hexanoic Acid Chloride Salt

[0300] ##STR00039##

[0301] 750 μmol compound 21 and 1.5 mmol N-methylaniline are stirred in 5 ml DMF at 150° C. for 2 hours. The solvent is distilled off and 10 ml of 3 M HCl are added to the residue for ester cleavage and boiled at reflux for 1 hour. After neutralisation with sodium hydrogen carbonate, purification is carried out by RP chromatography.

[0302] Yield: 95 mg (22%) (C.sub.34H.sub.41NClN.sub.2O.sub.3; 561.15 g/mol)

[0303] MS ESI− (m/z): 341.3 (base, [M.sup.−−H.sup.+].sup.2−); 683.3 (15%, [M].sup.−

[0304] UV-Vis in PBS: λ.sub.max: 548 nm; λ.sub.em: 632 nm; ε=39.000 l/mol*cm

[0305] UV-Vis in ethanol: λ.sub.max: 555 nm; λ.sub.em: 630 nm; ε=41,000 l/mol*cm

Compound 25

4-[[1-(5-carboxypentyl)-2,2,7,7-tetramethyl-4-(sulfonatomethyl)-8H-chromeno[3,2-g]quinolin-11-ium-9-yl]-methyl-amino]benzenesulfonate Sodium Salt

[0306] ##STR00040##

[0307] 150 μmol of compound 24 are dissolved in 2 ml of oleum (20% SO.sub.3) and stirred at 50° C. for 2 hours. The mixture is poured onto ice and stirred for 1 h more at RT. After blunting with sodium carbonate, purification is carried out by RP chromatography.

[0308] Yield: 70 mg (67%) (C.sub.34H.sub.39NaN.sub.2O.sub.9S.sub.2; 706.80 g/mol)

[0309] MS ESI+ (m/z): 525.3 ([M].sup.+

[0310] UV-Vis in PBS: λ.sub.max: 540 nm; λ.sub.em: 620 nm; ε=42,000 l/mol*cm

[0311] UV-Vis in ethanol: λ.sub.max: 545 nm; λ.sub.em: 615 nm; ε=43,000 l/mol*cm

Compound 26

6-[[6-[3-(dimethylamino)anilino]-8,8-dimethyl-7H-xanthene-10-ium-3-yl]-ethyl-amino]hexanoic Acid Chloride Salt

[0312] ##STR00041##

[0313] 375 μmol compound 7, 420 μmol 4-amino-N,N-dimethylaniline dihydrochloride and 700 μmol diisopropylethylamine are stirred in 5 ml glacial acetic acid at 120° C. for 2 hours. The solvent is distilled off and 10 ml of 3 M HCl are added to the residue for ester cleavage and boiled at reflux for 1 hour. After neutralisation with sodium hydrogen carbonate, purification is carried out by RP chromatography.

[0314] Yield: 44 mg (22%) (C.sub.31H.sub.40ClN.sub.3O.sub.3; 538.12 g/mol)

[0315] MS ESI+ (m/z): 502.4 ([M].sup.+)

[0316] UV-Vis in ethanol: λ.sub.max: 531 nm; λ.sub.em: 573 nm; ε=34,000 l/mol*cm

Compound 27

6-[[6-(4-aminoanilino)-8,8-dimethyl-7H-xanthene-10-ium-3-yl]-ethyl-amino]hexanoic Acid Chloride Salt

[0317] ##STR00042##

[0318] 375 μmol compound 7 and 420 μmol p-phenylenediamine are stirred in 5 ml DMF at 140° C. for 2 hours. The solvent is distilled off and 10 ml of 3 M HCl are added to the residue for ester cleavage and boiled at reflux for 1 hour. After neutralisation with sodium hydrogen carbonate, purification is carried out by RP chromatography.

[0319] Yield: 63 mg (33%) (C.sub.29H.sub.36ClN.sub.3O.sub.3; 510.07 g/mol)

[0320] MS ESI+ (m/z): 474.3 ([M].sup.+)

[0321] UV-Vis in ethanol: λ.sub.max: 530 nm; λ.sub.em: 612 nm; ε=35,000 l/mol*cm

Compound 28

6-[[6-[bis(2-pyridylmethyl)amino]-8,8-dimethyl-7H-xanthene-10-ium-3-yl]-ethyl-amino]hexanoic Acid Chloride Salt

[0322] ##STR00043##

[0323] 375 μmol compound 7 and 420 μmol di-(2-picolyl)amine are stirred in 5 ml DMF at 140° C. for 2 hours. The solvent is distilled off and 10 ml of 3 M HCl are added to the residue for ester cleavage and boiled at reflux for 1 hour. After neutralisation with sodium hydrogen carbonate, purification is carried out by RP chromatography.

[0324] Yield: 180 mg (80%) (C.sub.35H.sub.41ClN.sub.4O.sub.3; 601.18 g/mol)

[0325] MS ESI+ (m/z): 565.4 ([M].sup.+)

[0326] UV-Vis in ethanol: λ.sub.max: 531 nm; λ.sub.em: 593 nm; ε=28,000 l/mol*cm

Compound 29

6-[[8,8-dimethyl-6-[(E)-1H-pyridine-2-ylidenemethyl]-7H-xanthene-10-ium-3-yl]-ethyl-amino]hexanoic Acid Chloride Salt

[0327] ##STR00044##

[0328] 1.2 ml of a 1.0 M solution of lithium diisopropylamide in THF/hexane are solved in 10 ml dry THF and 1 mmol dry 2-methylpyridine are added to the mixture at −10° C. This solution is warmed to 0° C. and stirred at 0° C. for 30 minutes. To this is added slowly a solution of 336 μmol of compound 35 in 10 ml of dry THF. When the addition is complete, it is warmed to RT and stirred at RT for 1 hour more. After hydrolysis with dilute HCl, the organic solvents are distilled off and the product is isolated from the aqueous phase by RP chromatography.

[0329] Yield: 85 mg (51%) (C.sub.29H.sub.35ClN.sub.2O.sub.3; 495.05 g/mol)

[0330] MS ESI+ (m/z): 459.3 ([M].sup.+)

[0331] UV-Vis in ethanol: λ.sub.max: 434 nm; λ.sub.em: 541 nm; ε=24,000 l/mol*cm

Compound 30

6-[[6-[4-(dimethylamino)phenyl]-8,8-dimethyl-7H-xanthene-10-ium-3-yl]-ethyl-amino]hexanoic Acid Chloride Salt

[0332] ##STR00045##

[0333] 1.0 mol of compound 35 are dissolved in 10 ml of dry pyridine and at −40° C. 5 ml of a 0.5 M solution of 4-(N,N-dimethylaniline)-magnesium bromide in THF are slowly added. After completion of the addition, this solution is warmed to RT and stirred at RT for 3 hours. After hydrolysis with dilute HCl, the organic solvents are distilled off and the product is isolated from the aqueous phase by RP chromatography.

[0334] Yield: 100 mg (18%) (C.sub.31H.sub.39ClN.sub.2O.sub.3; 523.11 g/mol)

[0335] MS ESI+ (m/z): 487.5 ([M].sup.+)

[0336] UV-Vis in ethanol: λ.sub.max: 664 nm; λ.sub.em: 713 nm; ε=30,000 l/mol*cm

Compound 31

6-[ethyl-(6,8,8-trimethyl-7H-xanthene-10-ium-3-yl)amino]hexanoic Acid Chloride Salt

[0337] ##STR00046##

[0338] 1.0 mol of compound 35 are dissolved in 10 ml of dry THF and slowly 3 ml of a 1.4 M solution of methyl magnesium bromide in THF/toluene are added while stirring at −20° C. After completion of the addition, this solution is warmed to RT and stirred at RT for 1 hour. After hydrolysis with diluted HCl, the organic solvents are distilled off and the product is isolated from the aqueous phase by RP chromatography.

[0339] Yield: 125 mg (30%) (C.sub.20H.sub.26ClNO; 331.88 g/mol)

[0340] MS ESI+ (m/z): 296.1 ([M].sup.+)

[0341] UV-Vis in ethanol: λ.sub.max: 538 nm; λ.sub.em: 628 nm; ε=9,800 l/mol*cm

Compound 32

(2E)-1-(5-sarboxypentyl)-2-[(E)-3-[6-(diethylamino)-1,1-dimethyl-2H-xanthene ium-3-yl]prop-2-enyliden]-3,3-dimethyl-indoline-5-sulfonate

[0342] ##STR00047##

[0343] 1.0 mol of compound 31 and 1.1 mmol of 2-[(E)-2-anilinovinyl]-1-(5-carboxypentyl)-3,3-dimethyl-indole-1-ium-5-sulfonate are dissolved in 4 ml of acetic anhydride and 4 ml of glacial acetic acid and boiled at reflux for 15 minutes with the addition of 250 mg of sodium acetate. After cooling, an oily precipitate is obtained by precipitation with diethyl ether, which precipitate is purified by RP chromatography.

[0344] Yield: 92 mg (14%) (C.sub.38H.sub.46N.sub.2O.sub.6S; 658.85 g/mol)

[0345] MS ESI+ (m/z): 659.4 (base, [M+H.sup.+].sup.+); 681.6 (30%, [M+Na.sup.+].sup.+); 697.6 (20%, [M+K.sup.+].sup.+

[0346] MS ESI− (m/z): 657.3 ([M−H.sup.+].sup.−)

[0347] UV-Vis in PBS: λ.sub.max: 755 nm; λ.sub.em: 788 nm; ε=75.000 l/mol*cm

[0348] UV-Vis in ethanol: λ.sub.max: 770 nm; λ.sub.em: 795 nm; ε=143,600 l/mol*cm

Compound 33

3-[(5Z)-3-(5-carboxypentyl)-5-[(2E)-2-[6-(diethylamino)-1,1-dimethyl-2H-xanthene-3-ylidene]ethylidene]-2,4,6-trioxo-hexahydropyrimidin-1-yl]propane-1-sulfonate Sodium Salt

[0349] ##STR00048##

[0350] 1.0 mol of compound 31 and 1 mmol of 3-[5-formyl-3-(6-methoxy-6-oxo-hexyl)-2,4,6-trioxo-hexahydropyrimidine-1-yl]propane-1-sulfonate sodium salt are dissolved in 5 ml of acetic anhydride and 5 ml of glacial acetic acid and boiled at reflux for 15 minutes with the addition of 250 mg of sodium acetate. After cooling, a precipitate is obtained by precipitation with diethyl ether. 10 ml 3 M HCl and 10 ml acetone are added to the precipitate for ester cleavage and boiled at reflux for 1 hour. After neutralisation with sodium hydrogen carbonate, purification is carried out by RP chromatography.

[0351] Yield: 150 mg (22%) (C.sub.34H.sub.42NaN.sub.3O.sub.9S; 691.77 g/mol)

[0352] MS ESI− (m/z): 668.1 ([M].sup.−)

[0353] UV-Vis in ethanol: λ.sub.max: 683 nm; λ.sub.em: 719 nm; ε=67,000 l/mol*cm

Compound 34

6-(diethylamino)-1,1-dimethyl-2H-xanthene-3-one

[0354] ##STR00049##

[0355] 500 μmol compound 2 are stirred in 50 ml acetone and 10 ml 0.5 M pH9 buffer at 50° C. for 1 hour. The solvent is distilled off and purification is carried out by RP chromatography.

[0356] Yield: 80 mg (54%) red-brown oil (C.sub.19H.sub.23NO.sub.2; 297.39 g/mol)

[0357] MS ESI− (m/z): 298.1 (base, [M+H].sup.+); 617.4 (90%, [2M+Na]).sup.+

[0358] UV-Vis in PBS: λ.sub.max: 450 nm; λ.sub.em: 558 nm; ε=23.00 l/mol*cm

[0359] UV-Vis in water pH 3: λ.sub.max: x nm; λ.sub.em: x nm (present as hydroxy-BPS)

[0360] UV-Vis in ethanol: λ.sub.max: 440 nm; λ.sub.em: 540 nm; ε=24,800 l/mol*cm

Compound 35

6-[(8,8-dimethyl-6-oxo-7H-xanthene-3-yl)-ethyl-amino]hexanoic Acid

[0361] ##STR00050##

[0362] 500 μmol compound 7 are stirred in 50 ml acetone and 10 ml 0.5 M pH9 buffer at 50° C. for 1 hour. The solvent is distilled off and purification is carried out by RP chromatography.

[0363] Yield: 150 mg (78%) (C.sub.23H.sub.29NO.sub.4; 383.48 g/mol)

[0364] MS ESI− (m/z): 382.2 ([M−H.sup.+].sup.−); MS ESI+ (m/z): 384.3 ([M+H.sup.+].sup.+)

[0365] UV-Vis in PBS: λ.sub.max: 457 nm; λ.sub.em: 559 nm; ε=24,200 l/mol*cm

[0366] UV-Vis in ethanol: λ.sub.max: 441 nm; λ.sub.em: 540 nm; ε=25,300 l/mol*cm

Compound 36

6-[5-carboxypentyl(ethyl)amino]-1,1-dimethyl-3-oxo-2H-xanthene-4-sulfonate Sodium Salt

[0367] ##STR00051##

[0368] 177 μmol of compound 35 are dissolved in 2 ml of oleum (20% SO.sub.3) and stirred at RT for 2 hours. The mixture is poured onto ice and stirred for 1 h more at RT. After blunting with sodium carbonate, purification is carried out by RP chromatography.

[0369] Yield: 17 mg (20%) (C.sub.23H.sub.28NO.sub.7S Na; 485.53 g/mol)

[0370] MS ESI− (m/z): 462.0 (60%, [M].sup.−); 230.4 (base, [M−H.sup.+].sup.2−)

[0371] UV-Vis in PBS: λ.sub.max: 482 nm; λ.sub.em: 565 nm; ε=30,100 l/mol*cm; QY: 0.66

[0372] UV-Vis in ethanol: λ.sub.max: 446 nm; λ.sub.em: 542 nm; ε=25,000 l/mol*cm; QY 0.90

Compound 37

3-[5-carboxypentyl-(8,8-dimethyl-6-oxo-7H-xanthene-3-yl)amino]propane-1-sulfonate Sodium Salt

[0373] ##STR00052##

[0374] 375 μmol compound 4 are stirred in 50 ml acetone and 10 ml 0.5 M pH9 buffer at 50° C. for 1 hour. The solvent is distilled off and the residue is purified by RP chromatography with acetonitrile-water gradient.

[0375] Yield: 105 mg (56%) (C.sub.24H.sub.30NO.sub.7S Na; 499.55 g/mol)

[0376] MS ESI− (m/z): 476.0 (base, [M].sup.−); 237.5 (15%, [M.sup.−−H.sup.+].sup.2−)

[0377] UV-Vis in PBS: λ.sub.max: 454 nm; λ.sub.em: 553 nm; ε=26,000 l/mol*cm

[0378] UV-Vis in ethanol: λ.sub.max: 439 nm; λ.sub.em: 538 nm; ε=27,000 l/mol*cm

Compound 38

6-[5-carboxypentyl(3-sulfonatopropyl)amino]-1,1-dimethyl-3-oxo-2H-xanthene-4-sulfonate di-sodium SALT

[0379] ##STR00053##

[0380] 177 μmol of compound 37 are dissolved in 2 ml of oleum (20% SO.sub.3) and stirred at RT for 2 hours. The mixture is poured onto ice and stirred for 1 h more at RT. After blunting with sodium carbonate, purification is carried out by RP chromatography.

[0381] Yield: 90 mg (85%) (C.sub.24H.sub.29NO.sub.10S Na; 601.60 g/mol)

[0382] MS ESI− (m/z): 556.2 (15%, [M.sup.2−+H.sup.+].sup.−); 277.6 (base, [M].sup.2−)

[0383] UV-Vis in PBS: λ.sub.max: 479 nm; λ.sub.em: 557 nm; ε=28,000 l/mol*cm; QY: 0.78

[0384] UV-Vis in ethanol: λ.sub.max: 463 nm; λ.sub.em: 544 nm; ε=31,000 l/mol*cm; QY 0.91

[0385] .sup.1H NMR (400 MHz D.sub.2O): δ (ppm)=1.26 (S, 6H, CH.sub.3); 1.32 (M, 2H, CH.sub.2); 1.51 (M, 2H, CH.sub.2); 1.54 (M, 2H, CH.sub.2); 1.94 (M, 2H, CH.sub.2); 2.30 (T, 2H, CH.sub.2); 2.31 (S, 2H, CH.sub.2); 2.89 (T, 2H, CH.sub.2); 3.27 (T, 2H, CH.sub.2); 3.43 (T, 2H, CH.sub.2); 6.71 (D, 1H, 7-H); 6.86 (S, 1H, 5-H); 7.34 (D, 1H, 8-H); 7.52 (S, 1H, 9-H)

[0386] .sup.13C-NMR (400 MHz D.sub.2O): δ (ppm)=22.02; 24.23; 25.61; 25.98; 26.32; 33.34; 33.91; 48.47; 49.29; 50.23; 50.57; 97.71; 110.71; 111.46; 112.52; 128.02; 129.20; 133.93; 150.95; 153.95; 165.49; 179.17; 193.27

Compound 39

6-hydroxy-1,1-dimethyl-3-oxo-2H-xanthene-4-sulfonate Sodium Salt

[0387] ##STR00054##

[0388] 750 μmol of compound 16 are stirred in 50 ml acetone and 10 ml 0.5 M pH9 buffer at 50° C. for 1 hour. The solvent is distilled off and purification is carried out by RP chromatography.

[0389] Yield: 115 mg (45%) (C.sub.15H.sub.13NaO.sub.6S; 344.31 g/mol)

[0390] MS ESI− (m/z): 321.3 (base, [M].sup.−)

[0391] UV-Vis in PBS: λ.sub.max: 409 nm; λ.sub.em: 485 nm

[0392] UV-Vis in buffer pH 9: λ.sub.max: 462 nm; λ.sub.em: 536 nm

Compound 40

6-(5-carboxypentoxy)-1,1-dimethyl-3-oxo-2H-xanthene-4-sulfonate Sodium Salt

[0393] ##STR00055##

[0394] 290 μmol of compound 39 are stirred in 5 ml DMF with 80 mg K.sub.2CO.sub.3 and 100 mg 6-bromohexanoic acid ethyl ester at 120° C. for 1 hour. The solvent is distilled off and 10 ml of 3 M HCl are added to the residue for ester cleavage and boiled at reflux for 1 hour. After neutralisation with sodium hydrogen carbonate, purification is carried out by RP chromatography.

[0395] Yield: 33 mg (25%) (C.sub.21H.sub.23NaO.sub.8S Na; 458.46 g/mol)

[0396] MS ESI− (m/z): 435.2 (base, [M].sup.−); 217.0 (30%, [M.sup.−−H.sup.+].sup.2−)

[0397] UV-Vis in PBS: λ.sub.max: 407 nm; λ.sub.em: 483 nm; ε=21,000 l/mol*cm

[0398] UV-Vis in ethanol: λ.sub.max: 403 nm; λ.sub.em: 487 nm; ε=19,500 l/mol*cm

Compound 41

6-(2,2,4,7,7-pentamethyl-9-oxo-8H-chromeno[3,2-g]quinolin-1-yl)hexanoic Acid

[0399] ##STR00056##

[0400] 375 μmol compound 21 are stirred in 50 ml acetone and 10 ml 0.5 M pH9 buffer at 50° C. for 1 hour. The solvent is distilled off. To the residue 10 ml 3 M HCl is added for ester cleavage and boiled at reflux for 1 hour. After neutralisation with sodium hydrogen carbonate, purification is carried out by RP chromatography.

purified by RP chromatography with acetonitrile-water gradient.

[0401] Yield: 60 mg (38%) (C.sub.27H.sub.33NO.sub.4; 435.56 g/mol)

[0402] MS ESI+ (m/z): 436.2 ([M+H.sup.+].sup.+)

[0403] UV-Vis in PBS: λ.sub.max: 460 nm; λ.sub.em: 575 nm; ε=23,000 l/mol*cm

[0404] UV-Vis in ethanol: λ.sub.max: 463 nm; λ.sub.em: 557 nm; ε=24,000 l/mol*cm

Compound 42

1-(5-carboxypentyl)-2,2,4,7,7-pentamethyl-9-oxo-8H-chromeno[3,2-g]quinoline-10-sulfonate Sodium Salt

[0405] ##STR00057##

[0406] 177 μmol of compound 41 are dissolved in 2 ml of oleum (20% SO.sub.3) and stirred at RT for 2 hours. The mixture is poured onto ice and stirred for 1 h more at RT. After blunting with sodium carbonate, purification is carried out by RP chromatography.

[0407] Yield: 45 mg (48%) (C.sub.27H.sub.32NO.sub.7S Na; 537.60 g/mol)

[0408] MS ESI− (m/z): 514.2 ([M].sup.−)

[0409] UV-Vis in PBS: λ.sub.max: 487 nm; λ.sub.em: 578 nm; ε=28,000 l/mol*cm

[0410] UV-Vis in ethanol: λ.sub.max: 473 nm; λ.sub.em: 559 nm; ε=30,000 l/mol*cm

Compound 43

(E)-[3-ethoxy-5,5-dimethyl-2-(4-pyridyl)cyclohex-2-en-1-ylidene]-ethyl-oxonium tetrafluoroborate

[0411] ##STR00058##

[0412] 4 mmol of 3-hydroxy-5,5-dimethyl-2-(4-pyridyl)cyclohex-2-en-1-one are suspended in 10 ml of orthoformic acid triethyl ester and 1.2 ml of 48% tetrafluoroboric acid are added at room temperature. After stirring for 30 minutes at RT, 50 ml of dry diethyl ether are added and the mixture is left at RT for several hours. The resulting slightly yellowish precipitate is filtered, washed with a little dry ether and dried in vacuo.

[0413] Yield: 970 mg (67%) (C.sub.17H.sub.24BF.sub.4NO.sub.2; 361.18 g/mol)

[0414] MS ESI+ (m/z): 274.2 [M.sup.+]

Compound 44

6-[[8,8-dimethyl-6-oxo-5-(4-pyridyl)-7H-xanthene-3-yl]-ethyl-amino]hexanoic Acid

[0415] ##STR00059##

[0416] 1 mmol of compound 43 and 1 mmol of ethyl 6-(N-ethyl-4-formyl-3-hydroxy-anilino)hexanoic acid are reacted according to the synthetic method for compound 2. The solvent is distilled off and 10 ml of 3 M HCl are added to the residue for ester cleavage and boiled at reflux for 1 hour. After neutralisation with sodium hydrogen carbonate, purification is carried out by RP chromatography.

[0417] Yield: 97 mg (21%) (C.sub.28H.sub.32N.sub.2O.sub.4; 460.56 g/mol)

[0418] MS ESI+ (m/z): 460.3 ([M+H.sup.+].sup.+)

[0419] UV-Vis in PBS: λ.sub.max: 472 nm; λ.sub.em: 573 nm; ε=13,000 l/mol*cm

[0420] UV-Vis in buffer pH 5: λ.sub.max: 523 nm; λ.sub.em: 600 nm; ε=13.300 l/mol*cm

Compound 45

3-[5-carboxypentyl-[8,8-dimethyl-6-oxo-5-(4-pyridyl)-7H-xanthene-3-yl]amino]propane-1-sulfonate Sodium Salt

[0421] ##STR00060##

[0422] 1 mmol of compound 43 and 1 mmol of ethyl 6-(N-ethyl-4-formyl-3-hydroxy-anilino)hexanoic acid are reacted according to the synthetic method for compound 2. The solvent is distilled off and 10 ml of 3 M HCl are added to the residue for ester cleavage and boiled at reflux for 1 hour. After neutralisation with sodium hydrogen carbonate, purification is carried out by RP chromatography.

[0423] Yield: 97 mg (21%) (C.sub.29H.sub.34N.sub.2O.sub.4S; 554.65 g/mol)

[0424] MS ESI− (m/z): 553.2 ([M−H.sup.+].sup.−)

[0425] MS ESI+ (m/z): 555.4 ([M+H.sup.+].sup.+)

[0426] UV-Vis in PBS: λ.sub.max: 470 nm; λ.sub.em: 570 nm; ε=15.000 l/mol*cm

[0427] UV-Vis in buffer pH 5: λ.sub.max: 524 nm; λ.sub.em: 594 nm; ε=15.400 l/mol*cm

Compound 46

2-[3,9-bis(diethylamino)-13,13-dimethyl-chromeno[3,2-b]xanthene-5-ium-14-yl]benzoic Acid Chloride Salt

[0428] ##STR00061##

[0429] 1 mmol of compound 2 and 1 mmol of 2-[4-(diethylamino)-2-hydroxy-benzoyl] benzoic acid are stirred in 5 ml of glacial acetic acid at 110° C. for 16 hours. After cooling, it is precipitated with diethyl ether and the precipitate purified by RP chromatography.

[0430] Yield: 214 mg (34%) (C.sub.37H.sub.39ClN.sub.2O.sub.4; 611.17 g/mol)

[0431] MS ESI+ (m/z): 575.4 ([M].sup.+)

[0432] UV-Vis in ethanol: λ.sub.max: 664 nm; λ.sub.em: 710 nm; ε=60,000 l/mol*cm

[0433] UV-Vis in PBS: λ.sub.max: 663 nm; λ.sub.em: 711 nm; ε=42.000 l/mol*cm

Compound 47

6-[[9-(diethylamino)-13,13-dimethyl-chromeno[3,2-b]xanthene-7-ium-3-yl]-ethyl-amino]hexanoic Acid Chloride Salt

[0434] ##STR00062##

[0435] 500 μmol compound 7 and 500 μmol 4-(diethylamino)-2-hydroxy-benzaldehyde are stirred in 5 ml glacial acetic acid at 110° C. for 16 hours. After cooling, it is precipitated with diethyl ether and the precipitate purified by RP chromatography.

[0436] Yield: 70 mg (24%) (C.sub.34H.sub.41ClN.sub.2O.sub.4; 577.15 g/mol)

[0437] MS ESI+ (m/z): 541.3 ([M].sup.+)

[0438] UV-Vis in ethanol: λ.sub.max: 679 nm; 713 nm; ε=80,300 l/mol*cm

[0439] UV-Vis in PBS: λ.sub.max: 670 nm; 710 nm; ε=37.000 l/mol*cm

Compound 48

6-[ethyl-(3-methoxy-13,13-dimethyl-chromeno[3,2-b]xanthene-5-ium-9-yl)amino]hexanoic Acid Chloride Salt

[0440] ##STR00063##

[0441] 1 mmol compound 7 and 1 mmol 2-hydroxy-4-methoxy-benzaldehyde are stirred in 5 ml glacial acetic acid at 110° C. for 16 hours. After cooling, it is precipitated with diethyl ether and the precipitate purified by RP chromatography.

[0442] Yield: 43 mg (8%) (C.sub.31H.sub.34ClNO.sub.5; 536.06 g/mol)

[0443] MS ESI+ (m/z): 500.2 ([M].sup.+)

[0444] UV-Vis in ethanol: λ.sub.max: 654 nm; 712 nm; ε=53,000 l/mol*cm.

[0445] Photostability of Selected Compounds According to the Invention

[0446] FIG. 1 shows the results of the irradiation of aqueous solutions (PBS pH 7.5, 100 mM & 100 mM NaCl plus 5 mM NaN.sub.3) of compounds 5, 8, 11 and 38 in comparison to the MegaStokes dye DY-510XL. The solutions were adjusted to an extinction of “1” in the absorption maximum at a layer thickness of 1 cm and irradiated with white light from the 150 W Xe lamp of a fluorescence spectrometer (JASCO FP-6600, monochromator at 0 nm, slit position L: 10 nm) and the absorption in the maximum was monitored in 5 min intervals over one hour.

[0447] Fluorescence Maxima of Selected Compounds According to the Invention

[0448] FIG. 2 shows the fluorescence maxima of selected compounds.

[0449] The invention relates to novel, water-soluble fluorescent dyes with high fluorescence quantum yields based on oxygen-containing heterocycles, their reactive derivatives and dye conjugates, and their use for labelling samples and detecting analytes. The compounds of the new dye class are compatible with commercial excitation light sources and are characterised by Stokes shifts of more than 50 nm.

FURTHER ASPECTS OF THE INVENTION

[0450] Further preferred aspects of the invention are set out below:

1. Aspect

[0451] A compound of the general formula 1

##STR00064##

and salts and solvates thereof, wherein
R11 and R12 are each independently of the other hydrogen or alkyl,
R2 is hydrogen, alkyl or alkenyl,
R3 is hydrogen, alkyl, aryl, hydroxy, alkoxy, aryloxy, NR18R19 or a group Q,
wherein R18 and R19 are each independently of the other selected from (i) hydrogen, (ii) benzyl, (iii) aryl, (iv) heteroaryl, (v) a reactive group A bound via a linker L, (vi) alkyl, preferably C.sub.1-C.sub.4 alkyl, more preferably ethyl, (vii) ω-sulfonic acid alkyl (—CH.sub.2).sub.x—SO.sub.3.sup.−) where x is 1-5, (viii) ω-carboxylic acid alkyl (—(CH.sub.2).sub.y—CO.sub.2H) where y is 1-8, and (ix) ethyl esters of (viii),
and Q is a heterocyclic structure selected from a structure of formula 2 or 3

##STR00065##

where n=1, 2 or 3; wherein each R20 is independently of the other alkyl, ω-sulfonic acid alkyl (—(CH.sub.2).sub.x—SO.sub.3.sup.−) or a reactive group A bound through a linker L, each R21, R22, R23, R24 is independently of the other hydrogen, a sulfonic acid or a sulfonic acid derivative, R25 is hydrogen, alkyl, ω-sulfonic acid alkyl (—(CH.sub.2).sub.x—SO.sub.3.sup.−) or a reactive group A bound through a linker L, wherein each x is an integer from 1-5, and
R4 is hydrogen, bromine, chlorine, sulfonic acid or a sulfonic acid derivative, alkyl, aryl or heteroaryl, and
R5 is hydrogen, sulfonic acid or a sulfonic acid derivative,
R6 is hydrogen, bromine, chlorine, hydroxy, alkoxy, aryloxy or NR29R30 wherein R29 and R30 are each independently of the other hydrogen, alkyl, aryl or a reactive group A bound via a linker L, wherein R29 and R30 are each independently of the other preferably selected from (i) C.sub.1-C.sub.4 alkyl, (ii) ω-sulfonic acid alkyl (—(CH.sub.2).sub.x—SO.sub.3.sup.−) wherein x is 1-5, (iii) ω-carboxylic acid alkyl (—(CH.sub.2).sub.y—CO.sub.2H) wherein y is 1-8, and (iv) ethyl esters of (iii),
R7 is hydrogen, bromine, chlorine, hydroxy, alkoxy, aryloxy, NR31R32, sulfonic acid or a sulfonic acid derivative, wherein R31 and R32 are each independently of the other selected from (i) hydrogen, (ii) benzyl, (iii) aryl, (iv) heteroaryl, (v) a reactive group A bound via a linker L, (vi) alkyl, preferably C.sub.1-C.sub.4 alkyl, (vii) ω-sulfonic acid alkyl (—(CH.sub.2).sub.x—SO.sub.3.sup.−) where x is 1-5, (viii) ω-carboxylic acid alkyl (—(CH.sub.2).sub.y—CO.sub.2H) where y is 1-8, and (ix) ethyl esters of (viii),
R8 is hydrogen, methyl or ethyl,
R9 is hydrogen, alkyl or 2-carboxyphenyl, and
L is a linker selected from —(CH.sub.2).sub.s— and —[(CH.sub.2).sub.m—O].sub.p—(CH.sub.2).sub.m—, where m is an integer from 2-5 and p and s are each independently of the other an integer from 1-10, wherein
each compound contains no or one linker L having a reactive group A bound to L for covalently bonding to a molecule K to be labelled, wherein
A is an amine (—NH.sub.2), hydroxy (—OH) or phosphoramidite (—O—P—[O—CH.sub.2—CH.sub.2—CN]—N[(CH(CH.sub.3).sub.2].sub.2) function, a carboxylic acid (—COOH), an alkyl ester or active ester derived therefrom (NHS ester, sulfo-NHS ester, tetrafluoro-phenyl ester, p-sulfo-tetrafluoro-phenyl ester), a carboxylic acid hydrazide (—CONHNH.sub.2) or a carboxylic acid amide (—CONHR28) with R28 equal to —(CH.sub.2).sub.t—Y, where
Y is —OH, —NH.sub.2, —NH.sub.3.sup.+, maleimide (—N[CO—CH].sub.2), —NCS, —NCO, —NH—CO—CH.sub.2—I, —NH—CO—CH.sub.2—Br, -azide (—N.sub.3), -alkyne (—CCH) or -phosphoramidite (—O—P—[O—CH.sub.2—CH.sub.2—CN]—N—[CH—(CH.sub.3).sub.2].sub.2) and t is an integer from 1-10,
K is a component selected from the group haptens (molecules which represent an incomplete antigen and exhibit the effect of an antigen only when bound to proteins or cell structures), proteins, antibodies (proteins which are formed in response to antigens), low-molecular-weight drugs (active constituents in drugs which, because of their relatively low molar mass of up to about 800 g/mol, in contrast to, for example, proteins as very large molecules, are able to penetrate into cells), peptides (small or short-chain proteins up to about 100 linked amino acids), nucleotides (basic building blocks of nucleic acids such as DNA or RNA, which consist of a phosphate part, a monosaccharide part and a nucleobase part such as adenine, guanine, cytosine, thymine or uracil), nucleosides (basic building blocks of nucleic acids such as DNA or RNA, which do not have a phosphate part, but consist only of a monosaccharide part and a nucleobase part), DNA oligomers (in contrast to DNA as a macromolecule, molecules of deoxyribonucleic acid with a relatively small, not exactly defined number of nucleotides), polymers (synthetic or natural, chain-like or branched chemical compound consisting of repeating units, the monomers; polymers may also as copolymers consist of at least two different monomers in different proportions and arrangements).

2. Aspect

[0452] Compound according to aspect 1, characterized by

R3=hydroxy, wherein the compound, depending on the pH, is present as the neutral base structure 3-oxo-2H-xanthene 4

##STR00066##

3. Aspect

[0453] Compound according to aspect 1 or 2, characterized in that

at least one selected from R2-R3, R3-R4, R5-R6, R6-R7 and R7-R8 is bridged by forming saturated rings, partially unsaturated rings, aromatic rings or heteroaromatic rings which independently of the other contain further substituents, in particular sulfonic acids or sulfonic acid derivatives.

4. Aspect

[0454] Compound according to any one of the preceding aspects, characterised by a structure of formula 5,

##STR00067##

wherein
R13 is hydrogen, alkyl or 2-carboxyphenyl,
R14 is hydrogen, alkyl or 2-carboxyphenyl,
R15 is hydrogen, bromine, chlorine, hydroxy, alkoxy, aryloxy, NR33R34, sulfonic acid or a sulfonic acid derivative or bridged to R16, wherein R33 and R34 are each independently of the other selected from (i) hydrogen, (ii) benzyl, (iii) aryl, (iv) heteroaryl, (v) a reactive group A bound via a linker L, (vi) alkyl, preferably C.sub.1-C.sub.4 alkyl, (vii) ω-sulfonic acid alkyl (—(CH.sub.2).sub.x—SO.sub.3.sup.−) where x is 1-5, (viii) ω-carboxylic acid alkyl (—(CH.sub.2).sub.y—CO.sub.2H) where y is 1-8, and (ix) ethyl esters of (viii),
R16 is hydrogen, bromine, chlorine, hydroxy, alkoxy, aryloxy or NR35R36, wherein R35 and R36 are each independently of the other selected from (i) hydrogen, (ii) benzyl, (iii) aryl, (iv) heteroaryl, (v) a reactive group A bound via a linker L, (vi) alkyl, preferably C.sub.1-C.sub.4 alkyl, (vii) ω-sulfonic acid alkyl (—(CH.sub.2).sub.x—SO.sub.3.sup.−) where x is 1-5, (viii) ω-carboxylic acid alkyl (—(CH.sub.2).sub.y—CO.sub.2H) where y is 1-8, and (ix) ethyl esters of (viii),
R17 is hydrogen, sulfonic acid or a sulfonic acid derivative, and
one or more selected from R5-R6, R6-R7, R7-R8, R14-R15, R15-R16 and R16-R17 may be bridged by forming saturated rings, partially unsaturated rings, aromatic rings or heteroaromatic rings which may independently of the other contain further substituents, in particular sulfonic acids or sulfonic acid derivatives, and
preferably aryl substituents and/or heteroaryl substituents (as in R4, R6, R7, R15, R16, R18, R19, R33, R34, R35, R36) contain further substituents such as sulfonic acids or sulfonic acid derivatives and/or alkoxy groups and/or substituted amino groups.

5. Aspect

[0455] Compound according to any one of the preceding aspects, characterized in that the compound contains at least one sulfonic acid group.

6. Aspect

[0456] Compound according to any one of the preceding aspects, characterized in that R3 is NR18R19, wherein R18 and R19 are each independently of the other hydrogen, alkyl, benzyl, aryl, heteroaryl or a reactive group A bound via a linker L, wherein NR18R19 is preferably selected from 3-aminopropane sulfonate, N-methylaniline, 4-(methylamino)benzenesulfonate, aniline, 5-carboxypentylamine and 3-carboxypropyl(methyl)amine.

7. Aspect

[0457] Compound according to any one of the preceding aspects, characterised in that at least 1 mg of the compound is soluble in 1,000 g of water at 25° C.

8. Aspect

[0458] Compound according to any one of the preceding aspects, characterized in that the compound is a fluorescent compound having an absorption maximum in the wavelength range from 400 nm to 650 nm, preferably from 500 nm to 550 nm.

9. Aspect

[0459] Compound according to aspect 8, characterized in that the Stokes shift is at least 40 nm, preferably from 50 nm to 120 nm, more preferably from 70 nm to 90 nm.

10. Aspect

[0460] Compound according to any one of the preceding aspects, characterized in that the compound is selected from [0461] 6-ethoxy-N,N-diethyl-8,8-dimethyl-7H-xanthene-10-ium-3-amin, [0462] 6-[[6-(diethylamino)-1,1-dimethyl-2H-xanthene-10-ium-3-yl]amino]hexanoic acid, [0463] 3-[(6-ethoxy-8,8-dimethyl-7H-xanthene-10-ium-3-yl)-(6-ethoxy-6-oxo-hexyl)amino]propane-1-sulfonate, [0464] 3-[[6-[5-carboxypentyl(3-sulfonatopropyl)amino]-1,1-dimethyl-2H-xanthene-10-ium-3-yl]amino]propane-1-sulfonate, [0465] 3-[[6-[[6-(2,5-dioxopyrrolidine-1-yl)oxy-6-oxo-hexyl]-(3-sulfonatopropyl)amino]-1,1-dimethyl-2H-xanthene-10-ium-3-yl]amino]propane-1-sulfonate, [0466] 6-[(6-ethoxy-8,8-dimethyl-7H-xanthene-10-ium-3-yl)-ethyl-amino]hexanoic acid ethyl ester, [0467] 3-[[6-[5-carboxypentyl(ethyl)amino]-1,1-dimethyl-2H-xanthene-10-ium-3-yl]amino]propane-1-sulfonate, [0468] 3-[5-carboxypentyl-[8,8-dimethyl-6-(N-methylanilino)-7H-xanthene-10-ium-3-yl]amino]propane-1-sulfonate, [0469] 4-[[6-[5-carboxypentyl(3-sulfonatopropyl)amino]-1,1-dimethyl-2H-xanthene-10-ium-3-yl]-methyl-amino]benzenesulfonate, [0470] 6-[[8,8-dimethyl-6-(N-methylanilino)-7H-xanthene-10-ium-3-yl]-ethyl-amino]hexanoic acid, [0471] 4-[[6-[5-carboxypentyl(ethyl)amino]-1,1-dimethyl-2H-xanthene-10-ium-3-yl]-methyl-amino]benzenesulfonate, [0472] 6-[(6-anilino-8,8-dimethyl-7H-xanthene-10-ium-3-yl)-ethyl-amino]hexanoic acid, [0473] 6-[5-carboxypentyl(ethyl)amino]-1,1-dimethyl-3-(4-sulfonatoanilino)-2H-xanthene-10-ium-4-sulfonate, [0474] 6-[[6-[5-carboxypentyl(ethyl)amino]-1,1-dimethyl-2H-xanthene-10-ium-3-yl]amino]naphthalene-2-sulfonate, [0475] 6-ethoxy-8,8-dimethyl-7H-xanthene-10-ium-3-ol, [0476] 8,8-dimethyl-6-(N-methylanilino)-7H-xanthene-10-ium-3-ol, [0477] 6-[[8,8-dimethyl-6-(N-methylanilino)-7H-xanthene-10-ium-3-yl]oxy]hexanoic acid, [0478] 3-(5-carboxypentoxy)-8,8-dimethyl-6-(N-methyl-4-sulfonato-anilino)-7H-xanthene-10-ium-2-sulfonate, [0479] 3-(9-ethoxy-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinolin-11-ium yl)propane-1-sulfonate, [0480] 6-(9-ethoxy-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinolin-11-ium-1-yl)hexanoic acid ethyl ester, [0481] 3-[9-(5-carboxypentylamino)-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinolin-11-ium-1-yl]propane-1-sulfonate, [0482] 3-[9-[3-carboxypropyl(methyl)amino]-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinolin-11-ium-1-yl]propane-1-sulfonate, [0483] 6-[2,2,4,7,7-pentamethyl-9-(N-methylanilino)-8H-chromeno[3,2-g]quinolin-11-ium-1-yl]hexanoic acid, [0484] 4-[[1-(5-carboxypentyl)-2,2,7,7-tetramethyl-4-(sulfonatomethyl)-8H-chromeno[3,2-g]quinolin-11-ium-9-yl]-methyl-amino]benzenesulfonate, [0485] 6-[[6-[3-(dimethylamino)anilino]-8,8-dimethyl-7H-xanthene-10-ium-3-yl]-ethyl-amino]hexanoic acid, [0486] 6-[[6-(4-aminoanilino)-8,8-dimethyl-7H-xanthene-10-ium-3-yl]-ethyl-amino]hexanoic acid, [0487] 6-[[6-[bis(2-pyridylmethyl)amino]-8,8-dimethyl-7H-xanthene-10-ium-3-yl]-ethyl-amino]hexanoic acid, [0488] 6-[[8,8-dimethyl-6-[(E)-1H-pyridine-2-ylidenemethyl]-7H-xanthene-10-ium-3-yl]-ethyl-amino]hexanoic acid, [0489] 6-[[6-[4-(dimethylamino)phenyl]-8,8-dimethyl-7H-xanthene-10-ium-3-yl]-ethyl-amino]hexanoic acid, [0490] 6-[ethyl-(6,8,8-trimethyl-7H-xanthene-10-ium-3-yl)amino]hexanoic acid, [0491] (2E)-1-(5-carboxypentyl)-2-[(E)-3-[6-(diethylamino)-1,1-dimethyl-2H-xanthene-10-ium-3-yl]prop-2-enylidene]-3,3-dimethyl-indoline-5-sulfonate, [0492] 3-[(5Z)-3-(5-carboxypentyl)-5-[(2E)-2-[6-(diethylamino)-1,1-dimethyl-2H-xanthene-3-ylidene]ethylidene]-2,4,6-trioxo-hexahydropyrimidine-1-yl]propane-1-sulfonate, [0493] 6-(diethylamino)-1,1-dimethyl-2H-xanthene-3-one, [0494] 6-[(8,8-dimethyl-6-oxo-7H-xanthene-3-yl)-ethyl-amino]hexanoic acid, [0495] 6-[5-carboxypentyl(ethyl)amino]-1,1-dimethyl-3-oxo-2H-xanthene-4-sulfonate, [0496] 3-[5-carboxypentyl(8,8-dimethyl-6-oxo-7H-xanthene-3-yl)amino]propane-1-sulfonate, [0497] 6-[5-carboxypentyl(3-sulfonatopropyl)amino]-1,1-dimethyl-3-oxo-2H-xanthene-4-sulfonate, [0498] 6-hydroxy-1,1-dimethyl-3-oxo-2H-xanthene-4-sulfonate, [0499] 6-(5-carboxypentoxy)-1,1-dimethyl-3-oxo-2H-xanthene-4-sulfonate, [0500] 6-(2,2,4,7,7-pentamethyl-9-oxo-8H-chromeno[3,2-g]quinolin-1-yl)hexanoic acid, [0501] 1-(5-carboxypentyl)-2,2,4,7,7-pentamethyl-9-oxo-8H-chromeno[3,2-g]quinoline sulfonate, [0502] 6-[[8,8-dimethyl-6-oxo-5-(4-pyridyl)-7H-xanthene-3-yl]-ethyl-amino]hexanoic acid, [0503] 3-[5-carboxypentyl-[8,8-dimethyl-6-oxo-5-(4-pyridyl)-7H-xanthene-3-yl]amino]propane-1-sulfonate, [0504] 2-[3,9-bis(diethylamino)-13,13-dimethyl-chromeno[3,2-b]xanthene-5-ium-14-yl]benzoic acid, [0505] 6-[[9-(diethylamino)-13,13-dimethyl-chromeno[3,2-b]xanthene-7-ium-3-yl]-ethyl-amino]hexanoic acid,
und [0506] 6-[ethyl-(3-methoxy-13,13-dimethyl-chromeno[3,2-b]xanthene-5-ium-9-yl)amino]hexanoic acid.

11. Aspect

[0507] Method for preparing a compound of Formula 1, wherein the method comprises the reaction of (E)-(3-ethoxy-5,5-dimethyl-cyclohex-2-en-1-ylidene)ethyl-oxonium or (E)-[3-ethoxy-5,5-dimethyl-2-(4-pyridyl)cyclohex-2-en-1-ylidene]-ethyl-oxonium with a benzaldehyde compound, wherein the benzaldehyde compound has a benzaldehyde group and the benzaldehyde compound has a hydroxy group in ortho-position to the benzaldehyde group.

12. Aspect

[0508] Method for preparing a compound of Formula 5, wherein the method comprises the reaction of a compound of Formula 1, preferably 6-ethoxy-N,N-diethyl-8,8-dimethyl-7H-xanthene-10-ium-3-amine or 6-[(6-ethoxy-8,8-dimethyl-7H-xanthene-10-ium-3-yl)-ethyl-amino]hexanoic acid ethyl ester, with a benzaldehyde compound, wherein the benzaldehyde compound has a benzaldehyde group and the benzaldehyde compound has a hydroxy group in ortho-position to the benzaldehyde group.

13. Aspect

[0509] Use of a compound according to any one of aspects 1 to 10 as a fluorescent dye and/or in a fluorescent probe or as a fluorescent probe.

14. Aspect

[0510] Use according to aspect 13 for labelling one or more compounds selected from amino acids, peptides, proteins, antibodies, antigens, haptens, enzyme substrates, enzyme cofactors, biotin, carotenoids, hormones, neurohormones, neurotransmitters, growth factors, lectins, toxins, carbohydrates, oligosaccharides, polysaccharides, dextrans, nucleic acids, oligonucleotides, DNA, RNA, cells, lipids, receptor-binding pharmaceuticals.

15. Aspect

[0511] Use according to aspect 13 or 14 in optical, in particular fluorescence-optical, qualitative and/or quantitative determination methods for the diagnosis of cell properties, in biosensors (point of care measurements), for researching a genome (DNA sequencing), in cytometry and cell sorting, fluorescence correlation spectroscopy (FCS), ultra-high throughput screening (UHTS), multicolour fluorescence in situ hybridisation (FISH) and in microarrays (DNA and protein chips).