High Stokes shift fluorescent dyes based on cross-linked benzopyrylene salts

Abstract

This invention provides a novel, water-soluble fluorescent dye compound that exhibits a high fluorescence quantum yield based on an oxygen-containing heterocycle. [Solution] General formula 1 or 4 JPEG2026143433000063.jpg30161 Compounds thereof, as well as their salts and solvates, are provided.

Description

[Technical Field]

[0001] Fluorescent markers have been used for decades in biological, biotechnological, and medical research and medical diagnostics [Brinkley M., A Brief Survey of Methods for Preparing Protein Conjugates with Dyes, Haptens and Cross-Linking Reagents, Bioconjugate Chem, 3 (1992) 2-12; Waggoner A., ​​Covalent Labeling of Proteins and Nucleic Acids with Fluorophores, Meth. Enzymol., 246 (1995) 362-373; Hermanson GT, Bioconjugate Techniques, Academic Press 2013; Springer Series on Fluorescence 18, 2019 (Pedras B. Editor): Fluorescence in Industry]. Chromophores, originally known in the fields of textiles and photosensitive dyes, have been derivatized to optimize them for application in aqueous physiological environments. A specific focus of the development of this invention was water solubility and high quantum yield in aqueous solutions.

[0002] In biological analysis, efforts to obtain more information are reflected in multicolor analyses, which are now routine experimental procedures, such as flow cytometry [Lee LG, NEAR-IR Dyes in Three Color Volumetric Capillary Cytometry: Cell Analysis With 633- and 785-nm Laser Excitation, Cytometry, 21 (1995) 120-128], DNA sequencing, and various PCR methods (Roche's LightCycler).

[0003] Furthermore, dyes with large Stokes shifts are used in high-resolution optical microscopy, particularly in STED microscopy [Sednev MV, Belov VN, Hell SW, Fluorescent dyes with large Stokes shifts for super-resolution optical microscopy of biological objects: a review, MethodsAppl.Fluoresc. 3 (2015) 042004]. Initially, combinations of dyes were used that provided spectrally distinguishable multiple signals by transferring energy from one donor to multiple different acceptors when excited by an identical (monochromatic) light source. Examples of this include DNA sequencers from Amersham (now GE Healthcare) and ABI (now Life Technologies) that appeared on the market in the late 1990s.

[0004] Another approach for composite applications is the use of dyes that enable spectral discrimination without energy transfer between donor and acceptor. An example of this can be seen in the MegaStokes dyes (European Patent No. 1318177B1, European Patent No. 1535969B1), which have been known since around 2002. These are preferably tuned to excitation wavelengths of 470 nm (blue-green LED) to 500 nm (488 nm Ar ion laser), which were best suited to the state of excitation light sources at the time.

[0005] Combinations of several excitation light sources, each having more than one chromophore, are also a cutting-edge technology today (Solexa, International Publication No. 2007 / 135368A2). A typical application is next-generation sequencing (NGS).

[0006] The availability of high-intensity, short-wavelength excitation sources such as UV-LEDs or violet laser diodes, along with the expansion of phosphor bases suitable for bioanalytical applications, are becoming more widespread. Examples include Molecular Probes' Pacific Orange (U.S. Patent No. 8,158,801) and a series of benzoxazole dyes from Dyomics (U.S. Patent No. 9,453,010B2, European Patent No. 2,886,542B1), which are part of MegaStokes. TM This enabled multicolor analysis similar to that of dyes, but the excitation wavelength was around 400 nm.

[0007] The present invention relates to the production of a fluorescent marker based on an available crosslinked benzopyrillium compound, wherein the labeled molecule K may be linked via a linker L and a reactive group A, and the fluorescent marker has as many of the following properties as possible: high Stokes shift, high photostability and storage stability, solubility in aqueous media and high fluorescence quantum yield.

[0008] The present invention relates to General Formula 1 [ka] [During the ceremony, R11 and R12 are each independently hydrogen or alkyl, preferably C1-C4 alkyl, and particularly preferably methyl. R2 is hydrogen, an alkyl group, preferably a C1-C4-alkyl group or an alkenyl group, and in some embodiments, hydrogen is preferred. R3 is hydrogen, alkyl, preferably C1-C4-alkyl, particularly preferably methyl, aryl, hydroxy or oxo, alkoxy, preferably methoxy or ethoxy, particularly preferably ethoxy, aryloxy, NR18R19 or group Q. Here, R18 and R19 are each independently (i) hydrogen, (ii) benzyl, (iii) aryl, (iv) heteroaryl, (v) a reactive group A linked via linker L, (vi) alkyl, preferably C1-C4 alkyl, more preferably ethyl, and (vii) alkyl ω-sulfonate (-(CH2)) where x is preferably 1 to 5, particularly preferably 3. x -SO3 - ), (viii)y is preferably 1 to 8, particularly preferably 6, ω-carboxylate alkyl (-(CH2) y Selected from the ethyl esters of (-CO2H) and (ix)(viii), Furthermore, the NR18R19 is particularly preferably selected from 3-aminopropanesulfonate, N-methylaniline, 4-(methylamino)benzenesulfonate, aniline, 5-carboxypentylamine, and 3-carboxypropyl(methyl)amine. Q is given by equation 2 or 3 [ka] (In the formula, n=1, 2 or 3; each R20 is independently an alkyl ω-sulfonate (-(CH2)) where x is preferably 1-5, particularly preferably 3, and is an alkyl, preferably C1-C8 alkyl, and x is preferably 1-5, particularly preferably 3. x -SO3 - ) or a reactive group A bonded via linker L, where R21, R22, R23, and R24 are each independently hydrogen, sulfonic acid, or a sulfonic acid derivative, and R25 is hydrogen, alkyl, preferably C1-C8 alkyl, x is preferably 1 to 5, and particularly preferably 3, ω-sulfonic acid alkyl (-(CH2) x -SO3 - ) or reactive group A bonded via linker L) It is a heterocyclic structure selected from the structure of, Alternatively, R2 and R3 may bridge together with the carbon atoms to which they are bonded to form a saturated ring, a partially unsaturated ring, an aromatic ring, or a heteroaromatic ring, which may contain further substituents. R4 is hydrogen, bromine, chlorine, sulfonic acid or a sulfonic acid derivative, alkyl, aryl or heteroaryl, with hydrogen, sulfonic acid and pyridine (4-pyridyl) being preferred, or R3 and R4, together with the carbon atom to which they are bonded, are crosslinked to form a saturated ring, partially unsaturated ring, aromatic ring or heteroaromatic ring which may comprise a further substituent, in particular sulfonic acid or a sulfonic acid derivative, R5 is hydrogen, sulfonic acid or a sulfonic acid derivative, preferably hydrogen, R6 is hydrogen, bromine, chlorine, hydroxy, alkoxy (for example 6-oxy-hexanoic acid), aryloxy or NR29R30, wherein R29 and R30 are each independently hydrogen, alkyl, aryl or a reactive group A bonded via a linker L, wherein R29 and R30 are each independently preferably (i) C1-C4 alkyl, preferably ethyl, (ii) ω-sulfonic acid alkyl wherein x is preferably 1 to 5, particularly preferably 3 (-(CH2) x -SO3 - ), (iii) ω-carboxylic acid alkyl wherein y is preferably 1 to 8, particularly preferably 6 (-(CH2) y -CO2H) and (iv) ethyl esters of (iii), or R5 and R6, together with the carbon atom to which they are bonded, are crosslinked to form a saturated ring, partially unsaturated ring, aromatic ring or heteroaromatic ring which may comprise a further substituent, in particular sulfonic acid or a sulfonic acid derivative, R7 is hydrogen, bromine, chlorine, hydroxy, alkoxy, aryloxy, NR31R32, sulfonic acid or a sulfonic acid derivative, with hydrogen, sulfonic acid or a sulfonic acid derivative being particularly preferred, wherein said R31 and R32 are each independently (i) hydrogen, (ii) benzyl, (iii) aryl, (iv) heteroaryl, (v) a reactive group A bonded via a linker L, (vi) alkyl, preferably C1-C4 alkyl, more preferably ethyl, (vii) ω-sulfonic acid alkyl wherein x is preferably 1 to 5, particularly preferably 3 (-(CH2) x -SO3 -), (viii)y is preferably 1 to 8, particularly preferably 6, ω-carboxylate alkyl (-(CH2) y Selected from the ethyl esters of (-CO2H) and (ix)(viii), Alternatively, R6 and R7, together with the carbon atoms to which they are bonded, may bridge to form a saturated ring, a partially unsaturated ring, an aromatic ring, or a heteroaromatic ring that may contain further substituents, particularly sulfonic acid or sulfonic acid derivatives. R8 is hydrogen, methyl, or ethyl, preferably hydrogen. Alternatively, R7 and R8, together with the carbon atoms to which they are bonded, may bridge to form a saturated ring, a partially unsaturated ring, an aromatic ring, or a heteroaromatic ring that may contain further substituents, particularly sulfonic acid or sulfonic acid derivatives. R9 is hydrogen, alkyl, preferably C1-C6 alkyl or 2-carboxyphenyl, preferably hydrogen. L is -(CH2) s -and-[(CH2) m -O] p -(CH2) m - is a linker selected from, where m is an integer between 2 and 5, and p and s are independently integers between 1 and 10, where, Each compound either does not contain linker L, or contains linker L having a reactive group A bonded to L for covalent bonding to the labeled molecule K, where, A is an amine (-NH2), hydroxy (-OH), or phosphoramidite (-OP-[O-CH2-CH2-CN]-N[(CH(CH3)2]2) functional group, carboxylic acid (-COOH), alkyl ester or active ester derived therefrom (NHS ester, sulfo-NHS ester, tetrafluorophenyl ester, p-sulfo-tetrafluorophenyl ester), carboxylic acid hydrazide (-CONHNH2), or -(CH2) t -It is a carboxylic acid amide (-CONHR28) having R28 equivalent to -Y, Here, Y is -OH, -NH2, -NH3 +, maleimide (-N[CO-CH]2), -NCS, -NCO, -NH-CO-CH2-I, -NH-CO-CH2-Br, -azide (-N3), -alkyne (-CCH), or -phosphoramidite (-OP-[O-CH2-CH2-CN]-N-[CH-(CH3)2]2), where t is an integer from 1 to 10. K represents haptens (molecules that represent incomplete antigens and exhibit antigenic effects only when bound to a protein or cell structure), proteins, antibodies (proteins formed in response to antigens), low molecular weight drugs (for example, active components in drugs that can enter cells due to their relatively small molecular weight of up to approximately 800 g / mol, in contrast to proteins as extremely large molecules), peptides (small molecules or short-chain proteins composed of up to approximately 100 amino acids), nucleotides (composed of a phosphate group, a monosaccharide group, and a nucleic acid base group, e.g., adenine, guanine, cytosine, thymine, or uracil, DNA or The component is selected from the group consisting of (base components of nucleic acids such as RNA), nucleosides (base components of nucleic acids such as DNA or RNA that lack a phosphate group and consist only of a monosaccharide group and a nucleic acid base group), DNA oligomers (deoxyribonucleic acid molecules that are relatively small molecules, in contrast to DNA as a macromolecule, but have a number of nucleotides that is not precisely defined), and polymers (synthetic or natural, chain-like or branched chemical compounds consisting of repeating units and monomers; polymers can also exist as copolymers consisting of at least two different monomers with different proportions and arrangements). This invention relates to compounds (particularly cross-linked benzopyrylene salts), their salts, and solvates.

[0009] Preferably, the aryl substituent and / or heteroaryl substituent (as described in R3, R4, R6, R7, R18, R19, R29, R30, R31, R32) includes further substituents such as sulfonic acid or sulfonic acid derivatives and / or alkoxy groups and / or substituted amino groups.

[0010] Preferably, the compound according to the present invention comprises at least one group selected from a sulfonic acid group, a sulfonic acid derivative, an alkoxy group, and an amino group, preferably a sulfonic acid group.

[0011] The term sulfonic acid also includes the term sulfonate, and the term sulfonate also includes the term sulfonic acid. The term carboxylic acid also includes the term carboxylate, and the term carboxylate also includes the term carboxylic acid.

[0012] The verbs "comprise" and "contain," as well as their conjugations, also include the verb "consist of" and its conjugations.

[0013] Preferred embodiments are also shown in the dependent claims.

[0014] Preferably, the compounds according to the present invention exhibit fluorescence.

[0015] Preferably, the compound according to the present invention is a fluorescent dye. In other words, preferably, the compound is suitable as a fluorescent dye.

[0016] In one embodiment, R3 = hydroxyl.

[0017] When R3 = hydroxyl, the crosslinked compound is a 3-hydroxyxanthenium salt at low pH values, depending on the pH. 1 3-oxo-2H-xanthene exists as a compound that exhibits neutral to basic properties at high pH levels. 4 It exists as such. In one embodiment, R3 is preferably hydroxyl, and the compound is 3-oxo-2H-xanthene, which exhibits neutral to basic properties depending on the pH. 4 It exists as such. [ka]

[0018] In the compounds according to the present invention, one or more combinations selected from R2-R3, R3-R4, R5-R6, R6-R7, and R7-R8 can be crosslinked to independently form saturated rings, partially unsaturated rings, aromatic rings, or heteroaromatic rings, each containing further substituents, particularly sulfonic acid or sulfonic acid derivatives.

[0019] In one embodiment, at least one pair selected from R3 and R4, R5 and R6, R6 and R7, and R7 and R8 bridge together with the carbon atoms to which they are bonded to form a saturated ring, a partially unsaturated ring, an aromatic ring, or a heteroaromatic ring, where each ring independently may contain further substituents, particularly sulfonic acid or sulfonic acid derivatives.

[0020] In one embodiment, R3 and R4 are not bridging together with the carbon atoms to which they are bonded to form any ring selected from saturated rings, partially unsaturated rings, aromatic rings, or heteroaromatic rings.

[0021] In one embodiment, R3 and R4 are not crosslinked so as to form an aromatic ring together with the carbon atoms to which they are bonded.

[0022] Furthermore, the compounds according to the present invention are represented, for example, by formula 5 (chromeno-xanthenium compound) formed by R2-R3 crosslinking. [ka]

[0023] In addition to the general explanation, the following applies to the further substituents R13-R17: R13 is hydrogen, alkyl, preferably C1-C4 alkyl or 2-carboxyphenyl, particularly preferably hydrogen. R14 is hydrogen or an alkyl group, preferably a C1-C4-alkyl group, and particularly preferably hydrogen. R15 is hydrogen, bromine, chlorine, hydroxyl, alkoxy, aryloxy, NR33R34, sulfonic acid or sulfonic acid derivative, or crosslinked with R16, and is particularly preferably hydrogen. Here, R33 and R34 are each independently (i) hydrogen, (ii) benzyl, (iii) aryl, (iv) heteroaryl, (v) reactive group A linked via linker L, (vi) alkyl, preferably C1-C4 alkyl, more preferably ethyl, and (vii) alkyl ω-sulfonate (-(CH2)x-SO3) where x is preferably 1 to 5, particularly preferably 3. - ), (viii)y is preferably 1 to 8, particularly preferably 6, ω-carboxylate alkyl (-(CH2) y Selected from the ethyl esters of (-CO2H) and (ix)(viii), Alternatively, R14 and R15 may bridge together with the carbon atoms to which they are bonded to form a saturated ring, a partially unsaturated ring, an aromatic ring, or a heteroaromatic ring that may contain further substituents, particularly sulfonic acid or sulfonic acid derivatives. R16 is hydrogen, bromine, chlorine, hydroxy, alkoxy, aryloxy or NR35R36, with NR35R36 being particularly preferred. R35 and R36 are, respectively, (i) hydrogen, (ii) benzyl, (iii) aryl, (iv) heteroaryl, (v) reactive group A linked via linker L, (vi) alkyl, preferably C1-C4 alkyl, more preferably ethyl, and (vii) alkyl ω-sulfonate (-(CH2)x-SO3) where x is preferably 1 to 5, particularly preferably 3. - ), (viii)y is preferably 1 to 8, particularly preferably 6, ω-carboxylate alkyl (-(CH2) y It is preferable to select from the ethyl esters of (-CO2H) and (ix)(viii), Particularly preferably, R35 and R36 are each independently preferably C1-C4 alkyl, preferably ethyl, and ω-carboxylate alkyl (-(CH2)) where y is preferably 1-8, particularly preferably 6. y Selected from -CO2H, Alternatively, R15 and R16, together with the carbon atoms to which they are bonded, may bridge to form a saturated ring, a partially unsaturated ring, an aromatic ring, or a heteroaromatic ring that may contain further substituents, particularly sulfonic acid or sulfonic acid derivatives. R17 is hydrogen, sulfonic acid, or sulfonic acid derivative, particularly preferably hydrogen. Alternatively, R16 and R17, together with the carbon atoms to which they are bonded, may bridge to form a saturated ring, a partially unsaturated ring, an aromatic ring, or a heteroaromatic ring that may contain further substituents, particularly sulfonic acid or sulfonic acid derivatives.

[0024] Preferably, the aryl substituent and / or heteroaryl substituent (as indicated by R4, R6, R7, R15, R16, R18, R19, R33, R34, R35, R36) includes further substituents such as sulfonic acid or sulfonic acid derivatives and / or alkoxy groups and / or substituted amino groups.

[0025] Several examples of this type of simple, unfunctionalized compound are known. German Patent No. 2942931A1; BASF AG; 07.05.1981; Schmidt R., Koch V.[ 1 In the formula 5 The basic structure and its application to staining of anionically modified fibers were described for the first time. Detailed information regarding fluorescence was not provided.

[0026] In the Russian Journal of Organic Chemistry, 37(4), 2001, 527-538; Olekhnovich EP, Boroshko SL, Korobka IV, Metelitsa AV, Olekhnovich LP [2], several compounds of this type with simple, non-functionalized substituents in limited combinations were prepared and their absorption and emission properties were tested in more detail. In these, different synthetic approaches were used, thereby formula 1 and 4These compounds also became available. Specific applications of these compounds were not described. Their basic structures are soluble only in organic solvents and do not contain any functional groups that would provide solubility in aqueous solutions, or enable, for example, covalent bonding of biomolecules.

[0027] The following advantages are achieved by the production of the functionalized crosslinked benzopyrillium compounds that we request: The introduction of linkers and reactive groups into these types of cross-linked benzopyrillium salts enables the formation of covalent bonds with suitable biomolecules, which is a prerequisite for their use as fluorescent markers. The wide selection of substituents, particularly the diverse substituted alkoxy or amino groups, allows for tuning of a broad range of absorption or emission wavelengths. Surprisingly, derivatives with R3=NR18R19 are extremely chemically stable, and especially photostable. Due to its basic structure and substituents, the maximum absorption is present across the entire visible light spectrum, concentrated in the 500-530 nm range. The compound exhibits a high Stokes shift in the 80 nm range in aqueous or ethanolic solution at 25°C with an absorbance of 1.0, for example, using Analytik Jena's Specord 205, where the aqueous solution is preferably a phosphate buffer solution (PBS buffer; approximately 100 mM sodium chloride and 100 mM total phosphate) with a pH of 7.5. For example, luminescence was measured at 25°C in a diluted aqueous or ethanolic solution with an absorbance of 0.1, using Jasco's FP-6600 spectrofluorometer, where the diluted aqueous solution is preferably a phosphate buffer solution (PBS buffer; approximately 100 mM sodium chloride and 100 mM total phosphate) with a pH of 7.5.

[0028] Preferably, the compounds according to the present invention are characterized in that the compound is at least a fluorescent compound.

[0029] In one embodiment, the compound according to the present invention has maximum absorption in the wavelength range of 400 nm to 650 nm, preferably 500 nm to 550 nm.

[0030] In one embodiment, the compound according to the present invention exhibits maximum absorption in the wavelength range of 630 nm to 700 nm, preferably 650 nm to 690 nm.

[0031] Preferably, the Stokes shift is at least 40 nm, preferably 50 nm to 120 nm, and more preferably 70 nm to 90 nm.

[0032] Furthermore, for high stability, in one embodiment, R3 is NR18R19, where R18 and R19 are independently (i) hydrogen, (ii) benzyl, (iii) aryl, (iv) heteroaryl, (v) reactive group A linked via linker L, (vi) alkyl, preferably C1-C4 alkyl, more preferably ethyl, and (vii) alkyl ω-sulfonate (-(CH2)) where x is preferably 1 to 5, particularly preferably 3. x -SO3 - ), (viii)y is preferably 1 to 8, particularly preferably 6, ω-carboxylate alkyl (-(CH2) y It is preferable to select from the ethyl esters of (-CO2H) and (ix)(viii), Furthermore, NR18R19 is preferably selected from 3-aminopropanesulfonate, N-methylaniline, 4-(methylamino)benzenesulfonate, aniline, 5-carboxypentylamine, and 3-carboxypropyl(methyl)amine.

[0033] The addition of substituents with radicals that enable solubility in water (e.g., sulfonic acids) allows these dyes to be used for analytical or diagnostic purposes in protic solvents. The hydrophilic properties of various compounds can be adjusted to a specific range by the number of water-soluble groups. Therefore, the compounds according to the present invention are preferably soluble in water. Particularly preferably, the compounds according to the present invention are characterized by the fact that at least 1 mg, preferably 2 mg to 4 mg of the compound is soluble in 1,000 g (1,000 mg) of water at 25°C.

[0034] Furthermore, sulfonic acid groups influence aggregation behavior, reducing non-covalent dimer formation and non-covalent bonding to biomolecules and surfaces. In particular, sulfonic acid substituents directly bonded to the basic structure of the dye also affect the physicochemical properties of the dye, shifting absorption and emission wavelengths, and, in principle, achieving a significant increase in quantum yield.

[0035] For these reasons, in certain embodiments, the compound preferably contains at least one sulfonic acid group.

[0036] Preferably, the compound according to the present invention comprises at least one group selected from a sulfonic acid group, a sulfonic acid derivative, a hydroxyl group, an amino group, a carboxylic acid, and a carboxylic acid derivative.

[0037] Preferably, compounds in which R7 is methoxy and R3 is an amino group, particularly NR18R19, especially NH(4-HOOCC6H4), NH(4-C2H5COOC6H4), NH(2-C2H5COOC6H4), NH(C6H5), N(C2H5)2, or N(CH2)2(CH2)2O are not included in the present invention.

[0038] Compounds in which R4 is bromine and R6 and R16 are N(CH3)2 are preferably not included in the present invention.

[0039] Preferably, compounds in which R6 and R16 are present, and one of R6 and R16 is a hydroxyl group, are not included in the present invention.

[0040] Crosslinking allows for the stable acquisition of benzopyrillium compounds, achieving remarkably high fluorescence quantum yields in aqueous solutions depending on the substituents. Preferably, the fluorescence quantum yield of the compounds according to the present invention is 0.1 to 0.95, more preferably 0.5 to 0.9. The fluorescence quantum yield is preferably determined at 25°C in a diluted aqueous or ethanolic solution with an absorbance of 0.1 using, for example, a Hamamatsu Absolute Photoluminescence Quantum Yield Measurement System C-9920, where the diluted aqueous solution is preferably a phosphate buffer solution (PBS buffer; about 100 mM sodium chloride and 100 mM total phosphate) with a pH of 7.5. Thus, the compounds complement or are superior fluorescent alternatives to already commercially available dyes with high Stokes shifts.

[0041] Derivatives with R3=OH exist as hydroxybenzopyrylene salts in strongly acidic solutions and are deprotonated at higher pH values. Subsequently, they exist as 3-oxo-2H-xanthenes with a neutral basic structure. These exhibit high fluorescence quantum yield and are stable within the physiological pH range.

[0042] The compound exhibits extremely high photostability. Its photostability is significantly higher than that of comparable coumarin dyes, such as DY-510XL (see Figure 1).

[0043] The compounds according to the present invention are preferably selected from the following compounds and their salts and solvates (in the case of ions, the counterions, which may be selected from tetrafluoroborates, chlorides, and sodium): 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]aminohexanoic acid, 3-[(6-ethoxy-8,8-dimethyl-7H-xanthene-10-ium-3-yl)-(6-ethoxy-6-oxohexyl)amino]propane-1-sulfonate, 3-[[6-[5-carboxypentyl(3-sulfonatopropyl)amino]-1,1-dimethyl-2H-xanthene-10-ium-3-yl]amino]propane-1-sulfonate, 3-[[6-[[6-(2,5-dioxopyrrolidine-1-yl)oxy-6-oxohexyl]-(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]hexanoate 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]benzene sulfonate, 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]oxyhexanoic 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]quinoline-11-ium-1-yl)propane-1-sulfonate, 6-(9-ethoxy-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinoline-11-ium-1-yl) ethyl hexanoate, 3-[9-(5-carboxypentylamino)-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinoline-11-ium-1-yl]propane-1-sulfonate, 3-[9-[3-carboxypropyl(methyl)amino]-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinoline-11-ium-1-yl]propane-1-sulfonate, 6-[2,2,4,7,7-pentamethyl-9-(N-methylanilino)-8H-chromeno[3,2-g]quinoline-11-ium-1-yl]hexanoic acid, 4-[[1-(5-carboxypentyl)-2,2,7,7-tetramethyl-4-(sulfonatomethyl)-8H-chromeno[3,2-g]quinoline-11-ium-9-yl]-methyl-amino]benzene sulfonate, 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-enilidene e]-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-1-sulfonate, 6-[5-carboxypentyl(3-sulfonatopropyl)amino]-1,1-dimethyl-3-oxo-2H-xanthene-4-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]quinoline-1-yl)hexanoic acid, 1-(5-carboxypentyl)-2,2,4,7,7-pentamethyl-9-oxo-8H-chromeno[3,2-g]quinoline-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.

[0044] The compounds according to the present invention are particularly preferably selected from the following: 6-Ethoxy-N,N-diethyl-8,8-dimethyl-7H-xanthene-10-ium-3-amine tetrafluoroborate, 6-[[6-(diethylamino)-1,1-dimethyl-2H-xanthene-10-ium-3-yl]aminohexanoic acid chloride salt, 3-[(6-ethoxy-8,8-dimethyl-7H-xanthene-10-ium-3-yl)-(6-ethoxy-6-oxohexyl)amino]propane-1-sulfonate, 3-[[6-[5-carboxypentyl(3-sulfonatopropyl)amino]-1,1-dimethyl-2H-xanthene-10-ium-3-yl]amino]propane-1-sulfonate sodium salt, 3-[[6-[[6-(2,5-dioxopyrrolidine-1-yl)oxy-6-oxohexyl]-(3-sulfonatopropyl)amino]-1,1-dimethyl-2H-xanthene-10-ium-3-yl]amino]propane-1-sulfonate sodium salt, 6-[(6-ethoxy-8,8-dimethyl-7H-xanthene-10-ium-3-yl)-ethyl-amino]hexanoate ethyl ester tetrafluoroborate, 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 sodium salt, 6-[[8,8-dimethyl-6-(N-methylanilino)-7H-xanthene-10-ium-3-yl]-ethyl-amino]hexanoic acid chloride salt, 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 chloride salt, 6-[5-carboxypentyl(ethyl)amino]-1,1-dimethyl-3-(4-sulfonatoanilino)-2H-xanthene-10-ium-4-sulfonate sodium salt, 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 tetrafluoroborate, 8,8-dimethyl-6-(N-methylanilino)-7H-xanthen-10-ium-3-ol tetrafluoroborate, 6-[[8,8-dimethyl-6-(N-methylanilino)-7H-xanthene-10-ium-3-yl]oxy]hexanoic acid chloride salt, 3-(5-carboxypentoxy)-8,8-dimethyl-6-(N-methyl-4-sulfonato-anilino)-7H-xanthene-10-ium-2-sulfonate sodium salt, 3-(9-ethoxy-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinoline-11-ium-1-yl)propane-1-sulfonate, 6-(9-ethoxy-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinoline-11-ium-1-yl) ethyl hexanoate tetrafluoroborate, 3-[9-(5-carboxypentylamino)-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinoline-11-ium-1-yl]propane-1-sulfonate, 3-[9-[3-carboxypropyl(methyl)amino]-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinoline-11-ium-1-yl]propane-1-sulfonate, 6-[2,2,4,7,7-pentamethyl-9-(N-methylanilino)-8H-chromeno[3,2-g]quinoline-11-ium-1-yl]hexanoic acid chloride salt, 4-[[1-(5-carboxypentyl)-2,2,7,7-tetramethyl-4-(sulfonatomethyl)-8H-chromeno[3,2-g]quinoline-11-ium-9-yl]-methyl-amino]benzenesulfonate sodium salt, 6-[[6-[3-(dimethylamino)anilino]-8,8-dimethyl-7H-xanthene-10-ium-3-yl]-ethyl-amino]hexanoic acid chloride salt, 6-[[6-(4-aminoanilino)-8,8-dimethyl-7H-xanthene-10-ium-3-yl]-ethyl-amino]hexanoic acid chloride salt, 6-[[6-[bis(2-pyridylmethyl)amino]-8,8-dimethyl-7H-xanthene-10-ium-3-yl]-ethyl-amino]hexanoic acid chloride salt, 6-[[8,8-dimethyl-6-[(E)-1H-pyridine-2-ylidenemethyl]-7H-xanthene-10-ium-3-yl]-ethyl-amino]hexanoic acid chloride salt, 6-[[6-[4-(dimethylamino)phenyl]-8,8-dimethyl-7H-xanthene-10-ium-3-yl]-ethyl-amino]hexanoic acid chloride salt, 6-[ethyl-(6,8,8-trimethyl-7H-xanthene-10-ium-3-yl)amino]hexanoic acid chloride salt, (2E)-1-(5-carboxypentyl)-2-[(E)-3-[6-(diethylamino)-1,1-dimethyl-2H-xanthene-10-ium-3-yl]prop-2-enilidene]-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 sodium salt, 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 sodium salt, 3-[5-carboxypentyl(8,8-dimethyl-6-oxo-7H-xanthene-3-yl)amino]propane-1-sulfonate sodium salt, 6-[5-carboxypentyl(3-sulfonatopropyl)amino]-1,1-dimethyl-3-oxo-2H-xanthene-4-sulfonate disodium salt, 6-hydroxy-1,1-dimethyl-3-oxo-2H-xanthene-4-sulfonate sodium salt, 6-(5-carboxypentoxy)-1,1-dimethyl-3-oxo-2H-xanthene-4-sulfonate sodium salt, 6-(2,2,4,7,7-pentamethyl-9-oxo-8H-chromeno[3,2-g]quinoline-1-yl)hexanoic acid, 1-(5-carboxypentyl)-2,2,4,7,7-pentamethyl-9-oxo-8H-chromeno[3,2-g]quinoline-10-sulfonate sodium salt, 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 sodium salt, 2-[3,9-bis(diethylamino)-13,13-dimethyl-chromeno[3,2-b]xanthene-5-ium-14-yl]benzoic acid chloride salt, 6-[[9-(diethylamino)-13,13-dimethyl-chromeno[3,2-b]xanthene-7-ium-3-yl]ethyl-amino]hexanoic acid chloride salt, and 6-[ethyl-(3-methoxy-13,13-dimethyl-chromeno[3,2-b]xanthene-5-ium-9-yl)amino]hexanoic acid chloride salt.

[0045] The compounds according to the present invention are particularly preferably selected from the following compounds and their salts and solvates: 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-4-sulfonate, and 6-(5-carboxypentoxy)-1,1-dimethyl-3-oxo-2H-xanthene-4-sulfonate.

[0046] The compounds according to the present invention are particularly preferably selected from the following: 3-[[6-[5-carboxypentyl(3-sulfonatopropyl)amino]-1,1-dimethyl-2H-xanthene-10-ium-3-yl]amino]propane-1-sulfonate sodium salt, 4-[[6-[5-carboxypentyl(3-sulfonatopropyl)amino]-1,1-dimethyl-2H-xanthene-10-ium-3-yl]-methyl-amino]benzenesulfonate sodium salt, 6-[5-carboxypentyl(ethyl)amino]-1,1-dimethyl-3-oxo-2H-xanthene-4-sulfonate sodium salt, 6-[5-carboxypentyl(3-sulfonatopropyl)amino]-1,1-dimethyl-3-oxo-2H-xanthene-4-sulfonate disodium salt, and 6-(5-carboxypentoxy)-1,1-dimethyl-3-oxo-2H-xanthene-4-sulfonate sodium salt.

[0047] The compounds according to the present invention can be used as dyes for the optical labeling of organic or inorganic recognition units, such as amino acids, peptides, proteins, antibodies, antigens, haptens, enzyme substrates, enzyme cofactors, biotin, carotenoids, hormones, neurohormones, neurotransmitters, growth factors, lectins, toxins, carbohydrates, oligosaccharides, polysaccharides, dextran, nucleic acids, oligonucleotides, DNA, RNA, biological cells, lipids, receptor-binding pharmaceuticals, or organic or inorganic polymer carriers.

[0048] Labeling of the recognition unit can be achieved by the formation of ionic or van der Waals interactions between the marker (compound of the present invention) and the substance to be labeled.

[0049] Furthermore, it is also possible to conjugate recognition units or carrier materials to fluorescent dyes. This coupling reaction can be carried out in an aqueous solution or a solution mainly composed of an aqueous solution, preferably at room temperature. This generates fluorescent probes (conjugates) for the qualitative or quantitative determination of various biomaterials or other organic and inorganic substances.

[0050] Both the compounds according to the present invention and compound systems derived therefrom can be used in optical, particularly fluorescent, qualitative and quantitative determination methods for the diagnosis of cellular characteristics, in biosensors (point-of-care measurements), in genome research (DNA sequencing), and in miniaturization technologies. Typical applications include cytometry and cell sorting, fluorescence correlation spectroscopy (FCS), ultra-high-throughput screening (UHTS), multicolor fluorescence in situ hybridization (FISH), and microarrays (DNA and protein chips).

[0051] A receptor is a molecule that has affinity for a particular ligand. Receptors can be naturally occurring molecules or synthetic molecules. Receptors can be used in their pure form or in combination with other species. Receptors can bind to their binding partners covalently or noncovalently, directly or via a coupling medium.

[0052] A ligand is a molecule recognized by a specific receptor. Examples of ligands that can be detected by the present invention include, but are not limited to, cell membrane receptor agonists and antagonists, toxins and other toxins, viral epitopes, hormones such as opioates and steroids, hormone receptors, peptides, enzymes, enzyme substrates, substances that act as cofactors, lectins, sugars, oligonucleotides, nucleic acids, oligosaccharides, proteins, and antibodies.

[0053] The use of any of the compounds described herein, either as a fluorescent dye and / or in a fluorescent probe, or as a fluorescent probe, for the purposes and / or methods described herein is also part of the present invention.

[0054] The use of one of the compounds described herein as a fluorescent dye and / or in a fluorescent probe, or 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, dextran, nucleic acids, oligonucleotides, DNA, RNA, lipids, receptor-binding pharmaceuticals, and one of the compounds described herein as a fluorescent probe for labeling cells, is also part of the present invention.

[0055] The compounds according to the present invention may have at least one reactive group A in the form of an active ester, and the active ester is preferably the same as the NHS ester (N-hydroxysuccinimidyl ester), sulfo-NHS ester (sulfo-hydroxysuccinimidyl ester), TFP ester (tetrafluorophenyl ester), or STP ester (p-sulfo-tetrafluorophenyl ester) shown in the table below. [Table 1]

[0056] In further embodiments, the compounds of the present invention may contain A in the form of a carboxylic acid derivative, the carboxylic acid derivative being preferably a hydrazide, amine, iodoacetamide, maleimide, alkyne, or azide as shown in the table below. [Table 2]

[0057] A is an equation [ka] Compounds in the form of phosphoramidites, which are the basis of the compound, are also described.

[0058] Furthermore, the subject of the present invention is a method for producing a compound of formula 1, preferably a method for producing a compound of formula 1 according to the present invention.

[0059] The method comprises the reaction of (E)-(3-ethoxy-5,5-dimethyl-cyclohexy-2-ene-1-ylidene)-ethyl-oxonium or (E)-[3-ethoxy-5,5-dimethyl-2-(4-pyridyl)cyclohexy-2-ene-1-ylidene]-ethyl-oxonium with a benzaldehyde compound, wherein the benzaldehyde compound has a benzaldehyde group and a hydroxyl group at the ortho position of the benzaldehyde group.

[0060] Preferably, the reaction is carried out in an organic solvent, which is preferably triethyl orthoformate. Preferably, the reaction is carried out at 70°C to 200°C, more preferably at 90°C to 130°C. Preferably, (E)-(3-ethoxy-5,5-dimethyl-cyclohexy-2-en-1-ylidene)-ethyl-oxonium tetrafluoroborate or (E)-[3-ethoxy-5,5-dimethyl-2-(4-pyridyl)cyclohexy-2-en-1-ylidene]-ethyl-oxonium tetrafluoroborate is used. The benzaldehyde compound is preferably selected from 2-hydroxy-4-diethylaminobenzaldehyde, 3-(N-(6-ethoxy-6-oxohexyl)-4-formyl-3-hydroxyanilino)propane-1-sulfonate, ethyl 6-(N-ethyl-4-formyl-3-hydroxyanilino)hexanoic acid, 2,4-dihydroxybenzaldehyde, 3-(6-formyl-7-hydroxy-2,2,4-trimethyl-1-quinolyl)propane-1-sulfonate, and 6-(6-formyl-7-hydroxy-2,2,4-trimethyl-1-quinolyl)hexanoic acid.

[0061] The method may involve, in an upstream reaction, the conversion of the compound 5,5-dimethylcyclohexane-1,3-dione or 3-hydroxy-5,5-dimethyl-2-(4-pyridyl)cyclohexy-2-en-1-one to the compound (E)-(3-ethoxy-5,5-dimethyl-cyclohexy-2-en-1-ylidene)-ethyl-oxonium or (E)-[3-ethoxy-5,5-dimethyl-2-(4-pyridyl)cyclohexy-2-en-1-ylidene]-ethyl-oxonium. Preferably, this is carried out at a temperature of 5 to 50°C.

[0062] The subject of the present invention is also a method for producing a compound of formula 5, preferably a method for producing a compound of formula 5 according to the present invention.

[0063] The method involves 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]hexanoate ethyl ester with a benzaldehyde compound, wherein the benzaldehyde compound has a benzaldehyde group and a hydroxyl group at the ortho position of the benzaldehyde group.

[0064] Preferably, the reaction is carried out in an organic solvent, the organic solvent being preferably glacial acetic acid. Preferably, the reaction is carried out at 70°C to 200°C, more preferably 90°C to 110°C. Preferably, 6-ethoxy-N,N-diethyl-8,8-dimethyl-7H-xanthene-10-ium-3-amine tetrafluoroborate or 6-[(6-ethoxy-8,8-dimethyl-7H-xanthene-10-ium-3-yl)-ethyl-amino]hexanoate ethyl ester tetrafluoroborate is used. Preferably, the benzaldehyde compound is selected from 2-[4-(diethylamino)-2-hydroxy-benzoyl]benzoic acid, 4-(diethylamino)-2-hydroxy-benzaldehyde and 2-hydroxy-4-methoxy-benzaldehyde.

[0065] Preferably, the present invention's method for producing compounds of formula 1 or formula 5 involves introducing at least one group selected from a sulfonic acid group, a sulfonic acid derivative, an alkoxy group, and an amino group, and particularly preferably a sulfonic acid group.

[0066] The present invention is described in more detail by typical embodiments and figures below. [Brief explanation of the drawing]

[0067] [Figure 1] The photostability of compounds 5, 8, 11, and 38 compared to MegaStokes dye DY-510XL is shown. [Figure 2]The emission spectra of selected compounds according to the present invention in PBS are shown. [Modes for carrying out the invention]

[0068] Examples of Embodiments Compound 1: (E)-(3-ethoxy-5,5-dimethylcyclohexy-2-ene-1-ylidene)-ethyl-oxonium tetrafluoroborate [ka] 5 mmol of 5,5-dimethylcyclohexane-1,3-dione is suspended in 10 ml of triethyl orthoformate, and 1.5 ml of 48% tetrafluoroboric acid is added at room temperature. After stirring at room temperature for 30 minutes, 50 ml of dry diethyl ether is added, and the mixture is allowed to stand at room temperature for several hours. The resulting colorless precipitate is filtered, washed with a small amount of dry ether, and dried in a vacuum. Yield: 1.1g (78%) (C 12 H 21 BF4O2; 284.10 g / mol) MS ESI+(m / z):197.2[M + ]

[0069] Compound 2: 6-Ethoxy-N,N-diethyl-8,8-dimethyl-7H-xanthene-10-ium-3-amine tetrafluoroborate [ka] Prepare a 15 ml solution of 3 mmol of compound 1 in triethyl orthoformate, and add a 10 ml solution of 3 mmol of 2-hydroxy-4-diethylaminobenzaldehyde in triethyl orthoformate. Stir the mixture at 130°C for 30 minutes. After cooling, precipitate with diethyl ether, and recrystallize the precipitate from glacial acetic acid. Yield: 980 mg (80%) (C 21 H 28BF4NO2; 413.26 g / mol) MS ESI +(m / z):326.1([M] + ) UV-Vis:λ in ethanol max :531nm;λem:605nm

[0070] Compound 3: 6-[[6-(diethylamino)-1,1-dimethyl-2H-xanthene-10-ium-3-yl]aminohexanoic acid chloride salt [ka] 485 μmol of compound 2 and 1.5 mmol of sodium 6-aminohexanoate are suspended in 10 ml of DMF and stirred at room temperature for 8 hours. The solvent is removed by distillation, and the residue is purified by RP chromatography. Yield: 100 mg (46%) (C 25 H 35 ClN2O3; 447.01 g / mol) MS ESI-(m / z):411.2([M] + ) UV-Vis:λ in PBS max :505nm;λ em :570nm;ε=39,200l / mol*cm;QY:0.58 UV-Vis:λ in ethanol max :510nm;λ em :575nm;ε=32,500l / mol*cm;QY:0.82

[0071] Compound 4: 3-[(6-ethoxy-8,8-dimethyl-7H-xanthene-10-ium-3-yl)-(6-ethoxy-6-oxohexyl)amino]propane-1-sulfonate [ka] Prepare a 5 ml solution of 1 mmol of compound 1 with triethyl orthoformate, and add 5 ml of glacial acetic acid solution of 1 mmol of aldehyde 3-(N-(6-ethoxy-6-oxo-hexyl)-4-formyl-3-hydroxy-anilino)propane-1-sulfonate sodium salt. Stir the mixture at 130°C for 30 minutes. After cooling, precipitate with diethyl ether, and recrystallize the precipitate from glacial acetic acid. Yield: 385 mg (72%) (C 28 H 39 NO7S; 533.68 g / mol) MS ESI+(m / z):534.3([M+H + ] + )

[0072] Compound 5: 3-[[6-[5-carboxypentyl(3-sulfonatopropyl)amino]-1,1-dimethyl-2H-xanthene-10-ium-3-yl]amino]propane-1-sulfonate sodium salt [ka] 375 μmol of compound 4 and 1.5 mmol of sodium 3-aminopropanesulfonic acid salt are stirred in 5 ml of DMF at 40°C for 2 hours. The solvent is removed by distillation, and 10 ml of 3 M HCl is added to the residue for ester cleavage. The mixture is boiled at reflux temperature for 1 hour. After neutralization with sodium bicarbonate, the mixture is purified by RP chromatography. Yield: 98 mg (42%) (C 27 H 37 N2NaO9S2; 620.71 g / mol) MS ESI-(m / z): 597.2 (base, [M] - );297.9(25%,[MH + ] 2- ) UV-Vis:λ in PBS max :505nm;λ em :572nm;ε=45,000l / mol*cm;QY:0.72 UV-Vis:λ in ethanol max:512nm;λ em :570nm;ε=49,800l / mol*cm;QY:0.87 1 H NMR(400MHz D2O):δ(ppm)=1.10(S, 6H, CH3); 1.24(M, 2H, CH2); 1.40(M, 2H, CH2); 1.67(M, 2H, CH2 );1.89(M, 2H, CH2);2.04(M, 2H, CH2);2.32(T, 2H, CH2);2.45(S, 2H, CH2);2.87(T, 2 H, CH2);2.92(T,2H,CH2);3.14(T,2H,CH2);3.35(T,2H,CH2);3.46(T,2H,CH2);5.6 4(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) 13 C-NMR(400MHz D2O):δ(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;4 9.08;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

[0073] Compound 6: 3-[[6-[[6-(2,5-dioxopyrrolidine-1-yl)oxy-6-oxohexyl]-(3-sulfonatopropyl)amino]-1,1-dimethyl-2H-xanthene-10-ium-3-yl]amino]propane-1-sulfonate sodium salt [ka] Dissolve 125 µmol of Compound 5 in 3 ml of DMF. Add 45 mg of TSTU (N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate) and 26 µl of DIPEA (diisopropylethylamine) at 0°C, and stir the mixture at room temperature for 20 minutes. Distill off the solvent in vacuo and purify the residue by reversed-phase chromatography. Yield: 80 mg (90%) (C 31 H 40 N3NaO 11 S2; 717.78 g / mol) MS ESI- (m / z): 694.2 (base, [M - ; 346.5 (25%, [M-H + 2- ) UV-Vis in ethanol: λ max : 512 nm; λ em : 570 nm; ε=44,000 l / mol·cm

[0074] Compound 7: Ethyl 6-[(6-ethoxy-8,8-dimethyl-7H-xanthen-10-ium-3-yl)-ethyl-amino]hexanoate tetrafluoroborate

Chemical Structure

[0075] Compound 8: 3-[[6-[5-carboxypentyl(ethyl)amino]-1,1-dimethyl-2H-xanthen-10-ium-3-yl]amino]propane-1-sulfonate ​[ka] 375 μmol of compound 7 and sodium 3-aminopropanesulfonic acid salt are reacted according to the synthesis method described for compound 5. Yield: 110 mg (58%) (C 26 H 36 N2O6S; 504.63 g / mol) MS ESI-(m / z):503.2([MH + ] - );MS ESI+(m / z):505.3(base,M+H + ] + );527.3(15%, [M+Na + ] + ) UV-Vis:λ in PBS max :509nm;λ em :578nm;ε=38,500l / mol*cm;QY:0.58 UV-Vis:λ in ethanol max :511nm;λ em :571nm;ε=41,600l / mol*cm;QY 0.84

[0076] Compound 9: 3-[5-carboxypentyl-[8,8-dimethyl-6-(N-methylanilino)-7H-xanthene-10-ium-3-yl]amino]propane-1-sulfonate [ka] 375 μmol of compound 4 and 750 μmol of N-methylaniline are stirred in 5 ml of DMF at 150°C for 2 hours. The solvent is removed by distillation, and 10 ml of 3 M HCl is added to the residue for ester cleavage. The mixture is boiled at reflux temperature for 1 hour. After neutralization with sodium bicarbonate, the mixture is purified by RP chromatography. Yield: 80 mg (38%) (C 31 H 38 N2O6S; 566.71 g / mol) MS ESI-(m / z):565.2([MH + ]- ); MS ESI+(m / z): 567.3 (80%, [M+H + + ); 589.3 (base, [M+Na + + ); 605.3 (70%, [M+Ka + + ) UV-Vis in PBS: λ max : 523 nm; λ em : 600 nm; ε=39,000 l / mol*cm; QY: 0.24 UV-Vis in ethanol: λ max : 531 nm; λ em : 601 nm; ε=41,000 l / mol*cm; QY 0.47

[0077] Compound 10: Sodium 4-[[6-[5-carboxypentyl(3-sulfonatopropyl)amino]-1,1-dimethyl-2H-xanthen-10-ium-3-yl]-methyl-amino]benzenesulfonate

Chemical Formula

[0078] Compound 11: 6-[[8,8-dimethyl-6-(N-methylanilino)-7H-xanthen-10-ium-3-yl]-ethyl-amino]hexanoic acid chloride salt

Chemical

[0079] Compound 12: 4-[[6-[5-carboxypentyl(ethyl)amino]-1,1-dimethyl-2H-xanthen-10-ium-3-yl]-methyl-amino]benzenesulfonate

Chemical

[0080] Compound 13: 6-[(6-anilino-8,8-dimethyl-7H-xanthene-10-ium-3-yl)-ethyl-amino]hexanoic acid chloride salt [ka] 375 μmol of compound 7 and 750 μmol of dry aniline are stirred in 5 ml of DMF at 150°C for 2 hours. The solvent is removed by distillation, and 10 ml of 3 M HCl is added to the residue for ester cleavage. The mixture is boiled at reflux temperature for 1 hour. After neutralization with sodium bicarbonate, the mixture is purified by RP chromatography. Yield: 50 mg (27%) (C 29 H 35 ClN2O3; 495.05 g / mol) MS ESI+(m / z):459.3([M] + );MS ESI-(m / z):457.2([M-2H + ] - ) UV-Vis:λ in PBS max :526nm;λ em :609nm;ε=38,000l / mol*cm UV-Vis:λ in ethanol max :532nm;λ em :603nm;ε=44,000l / mol*cm

[0081] Compound 14: 6-[5-carboxypentyl(ethyl)amino]-1,1-dimethyl-3-(4-sulfonatoanilino)-2H-xanthene-10-ium-4-sulfonate sodium salt [ka] Dissolve 177 μmol of compound 13 in 2 ml of fuming sulfuric acid (20% SO3) and stir at room temperature for 2 hours. Pour the mixture onto ice and stir at room temperature for a further 1 hour. After slowing the reaction with sodium carbonate, purify by RP chromatography. Yield: 30 mg (26%) (C 29 H 33 N2O9S2Na; 640.70 g / mol) MS ESI-(m / z):617.3([M] - ) UV-Vis:λ in PBS max :545nm;λ em :625nm;ε=40,100l / mol*cm;QY:0.11 UV-Vis:λ in ethanol max :542nm;λ em :616nm;ε=45,300l / mol*cm;QY 0.61

[0082] Compound 15: 6-[[6-[5-carboxypentyl(ethyl)amino]-1,1-dimethyl-2H-xanthene-10-ium-3-yl]amino]naphthalene-2-sulfonate [ka] 375 μmol of compound 7 and 420 μmol of 6-amino-2-naphthalenesulfonic acid hydrate are stirred in 5 ml of glacial acetic acid at 120°C for 8 hours. The solvent is removed by distillation, and 10 ml of 3 M HCl is added to the residue to cleave the ester, and the mixture is heated under reflux for 1 hour. After neutralization with sodium bicarbonate, the mixture is purified by RP chromatography. Yield: 18 mg (8%) (C 33 H 36 N2O6S; 588.71 g / mol) MS ESI-(m / z):587.3([MH + ] - ) MS ESI+(m / z):589.3([M+H + ] + ) UV-Vis:λ in ethanol max :546nm;λ em :619nm;ε=32,000l / mol*cm

[0083] Compound 16: 6-Ethoxy-8,8-dimethyl-7H-xanthene-10-ium-3-ol tetrafluoroborate [ka] Prepare a 25 ml solution of 5 mmol of compound 1 with triethyl orthoformate, and add 5 mmol of 2,4-dihydroxybenzaldehyde. Heat the mixture at 130°C for 30 minutes. After cooling, precipitate with diethyl ether, and recrystallize the precipitate from glacial acetic acid. Yield: 500 mg (28%) (C 17 H 19 BF4O3; 358.14 g / mol) MS ESI+(m / z):271.2([M] + )

[0084] Compound 17: 8,8-dimethyl-6-(N-methylanilino)-7H-xanthen-10-ium-3-ol tetrafluoroborate [ka] 750 μmol of compound 16 and 1.5 mmol of N-methylaniline are stirred in 10 ml of DMF at 140°C for 2 hours. After cooling, the mixture is precipitated using diethyl ether, and the precipitate is recrystallized from glacial acetic acid. Yield: 125 mg (40%) (C 22 H 22 BF4NO2; 419.22 g / mol) MS ESI+(m / z):332.3([M] + )

[0085] Compound 18: 6-[[8,8-dimethyl-6-(N-methylanilino)-7H-xanthene-10-ium-3-yl]oxy]hexanoic acid chloride salt [ka] 286 μmol of compound 17 is stirred at 120°C for 1 hour in 5 ml of DMF containing 80 mg of K2CO3 and 100 mg of ethyl 6-bromohexanoate. The solvent is removed by distillation, and 10 ml of 3 M HCl is added to the residue for ester cleavage. The mixture is heated under reflux for 1 hour. After neutralization with sodium bicarbonate, the mixture is purified by RP chromatography. Yield: 35 mg (24%) (C 28 H 32 ClNO4; 482.01 g / mol) MS ESI+(m / z):446.4([M] + ) UV-Vis:λ in ethanol max :452+469nm;λ em :520nm

[0086] Compound 19: 3-(5-carboxypentoxy)-8,8-dimethyl-6-(N-methyl-4-sulfonatoanilino)-7H-xanthene-10-ium-2-sulfonate sodium salt [ka] Dissolve 35 mg of compound 18 in 1 ml of fuming sulfuric acid (20% SO3) and stir at room temperature for 2 hours. Pour the mixture onto ice and stir at room temperature for a further 1 hour. After slowing the reaction with sodium carbonate, purify by RP chromatography. Yield: 27 mg (59%) (C28 H 30 ClNNaO 10 S2; 627.66 g / mol) MS ESI-(m / z):604.2([M] - ) UV-Vis:λ in ethanol max :465nm;λ em :522nm;ε=18,000l / mol*cm. UV-Vis:λ in PBS max :460nm;λ em :509nm;ε=16.500l / mol*cm

[0087] Compound 20: 3-(9-ethoxy-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinoline-11-ium-1-yl)propane-1-sulfonate [ka] Prepare a 5 ml solution of 1 mmol of compound 1 with triethyl orthoformate, and add 5 ml of glacial acetic acid solution of 1 mmol of aldehyde 3-(6-formyl-7-hydroxy-2,2,4-trimethyl-1-quinolyl)propane-1-sulfonate sodium salt. Stir the mixture at 130°C for 30 minutes. After cooling, precipitate with diethyl ether, and recrystallize the precipitate from glacial acetic acid. Yield: 320 mg (68%) (C 26 H 33 NO5S; 471.61 g / mol) MS ESI+(m / z):472.2(base,M+H) + ] + );494.3(60%, [M+Na + ] + );510.3(20%, [M+K + ] + )

[0088] Compound 21: 6-(9-ethoxy-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinoline-11-ium-1-yl) ethyl hexanoate tetrafluoroborate [ka] Prepare a 5 ml solution of 1 mmol of compound 1 with triethyl orthoformate, and add 5 ml of glacial acetic acid solution of 1 mmol of aldehyde 6-(6-formyl-7-hydroxy-2,2,4-trimethyl-1-quinolyl)hexanoic acid. Stir the mixture at 130°C for 30 minutes. After cooling, precipitate with diethyl ether, and recrystallize the precipitate from glacial acetic acid. Yield: 245 mg (42%) (C 31 H 42 BF4NO4; 579.47 g / mol) MS ESI+(m / z):492.3([M] + )

[0089] Compound 22: 3-[9-(5-carboxypentylamino)-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinoline-11-ium-1-yl]propane-1-sulfonate [ka] 375 μmol of compound 20 and 1.5 mmol of sodium 6-aminohexanoate are stirred in 5 ml of DMF at 40°C for 2 hours. The solvent is removed by distillation, and the mixture is purified by RP chromatography. Yield: 25 mg (12%) (C 30 H 40 N2NaO6S; 556.71 g / mol) MS ESI-(m / z): 555.2([MH + ] - ) MS ESI+(m / z):557.2(50%, [M+H + ] + ); 579.5 (base, [M+Na + ] +);595.3(20%, [M+K + ] + UV-Vis:λ in PBS max :516nm;λ em :590nm;ε=37.500l / mol*cm UV-Vis:λ in ethanol max :531nm;λ em :589nm;ε=39,000l / mol*cm

[0090] Compound 23: 3-[9-[3-carboxypropyl(methyl)amino]-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinoline-11-ium-1-yl]propane-1-sulfonate [ka] 375 μmol of compound 20 and 1.5 mmol of sodium N-methylbutyrate are stirred in 5 ml of DMF at 40°C for 2 hours. The solvent is removed by distillation, and the mixture is purified by RP chromatography. Yield: 30 mg (15%) (C 29 H 38 N2O6S; 542.69 g / mol) MS ESI-(m / z):541.2([MH + ] - ) MS ESI+(m / z): 553.3 (base, M+H + ] + );565.5(20%, [M+Na + ] + );581.3(15%, [M+K + ] + ) UV-Vis:λ in PBS max :527nm;λ em :600nm;ε=45,400l / mol*cm;QY:0.58 UV-Vis:λ in ethanol max :542nm;λ em :600nm;ε=55,000l / mol*cm;QY:0.88

[0091] Compound 24: 6-[2,2,4,7,7-pentamethyl-9-(N-methylanilino)-8H-chromeno[3,2-g]quinoline-11-ium-1-yl]hexanoic acid chloride salt [ka] 750 μmol of compound 21 and 1.5 mmol of N-methylaniline are stirred in 5 ml of DMF at 150°C for 2 hours. The solvent is removed by distillation, and 10 ml of 3 M HCl is added to the residue for ester cleavage. The mixture is boiled at reflux temperature for 1 hour. After neutralization with sodium bicarbonate, the mixture is purified by RP chromatography. Yield: 95 mg (22%) (C 34 H 41 NClN2O3; 561.15 g / mol) MS ESI-(m / z):341.3 (base, M - -H + ] 2- );683.3(15%, [M] - UV-Vis:λ in PBS max :548nm;λ em :632nm;ε=39.000l / mol*cm UV-Vis:λ in ethanol max :555nm;λ em :630nm;ε=41,000l / mol*cm

[0092] Compound 25: 4-[[1-(5-carboxypentyl)-2,2,7,7-tetramethyl-4-(sulfonatomethyl)-8H-chromeno[3,2-g]quinoline-11-ium-9-yl]-methyl-amino]benzenesulfonate sodium salt [ka] Dissolve 150 μmol of compound 24 in 2 ml of fuming sulfuric acid (20% SO3) and stir at 50°C for 2 hours. Pour the mixture onto ice and stir at room temperature for a further 1 hour. After slowing the reaction with sodium carbonate, purify by RP chromatography. Yield: 70 mg (67%) (C 34 H 39 NaN2O9S2; 706.80 g / mol) MS ESI+(m / z):525.3([M] + UV-Vis:λ in PBS max :540nm;λ em :620nm;ε=42,000l / mol*cm UV-Vis:λ in ethanol max :545nm;λ em :615nm;ε=43,000l / mol*cm

[0093] Compound 26: 6-[[6-[3-(dimethylamino)anilino]-8,8-dimethyl-7H-xanthene-10-ium-3-yl]-ethyl-amino]hexanoic acid chloride salt [ka] 375 μmol of compound 7, 420 μmol of 4-amino-N,N-dimethylaniline dihydrochloride, and 700 μmol of diisopropylethylamine are stirred in 5 ml of glacial acetic acid at 120°C for 2 hours. The solvent is removed by distillation, and 10 ml of 3 M HCl is added to the residue for ester cleavage, and the mixture is stirred at reflux temperature for 1 hour. After neutralization with sodium bicarbonate, the mixture is purified by RP chromatography. Yield: 44 mg (22%) (C 31 H 40 ClN3O3; 538.12 g / mol) MS ESI+(m / z):502.4([M] + ) UV-Vis:λ in ethanol max :531nm;λ em :573nm;ε=34,000l / mol*cm

[0094] Compound 27: 6-[[6-(4-aminoanilino)-8,8-dimethyl-7H-xanthene-10-ium-3-yl]-ethyl-amino]hexanoic acid chloride salt [ka] 375 μmol of compound 7 and 420 μmol of p-phenylenediamine are stirred in 5 ml of DMF at 140°C for 2 hours. The solvent is removed by distillation, and 10 ml of 3 M HCl is added to the residue for ester cleavage. The mixture is boiled at reflux temperature for 1 hour. After neutralization with sodium bicarbonate, the mixture is purified by RP chromatography. Yield: 63 mg (33%) (C 29 H 36 ClN3O3; 510.07 g / mol) MS ESI+(m / z):474.3([M] + ) UV-Vis:λ in ethanol max :530nm;λ em :612nm;ε=35,000l / mol*cm

[0095] Compound 28: 6-[[6-[bis(2-pyridylmethyl)amino]-8,8-dimethyl-7H-xanthene-10-ium-3-yl]-ethyl-amino]hexanoic acid chloride salt [ka] 375 μmol of compound 7 and 420 μmol of di-(2-picolyl)amine are stirred in 5 ml of DMF at 140°C for 2 hours. The solvent is removed by distillation, and 10 ml of 3 M HCl is added to the residue for ester cleavage. The mixture is heated under reflux for 1 hour. After neutralization with sodium bicarbonate, the mixture is purified by RP chromatography. Yield: 180 mg (80%) (C 35 H 41 ClN4O3; 601.18 g / mol) MS ESI+(m / z):565.4([M]+ ) UV-Vis:λ in ethanol max :531nm;λ em :593nm;ε=28,000l / mol*cm

[0096] Compound 29: 6-[[8,8-dimethyl-6-[(E)-1H-pyridine-2-ylidenemethyl]-7H-xanthene-10-ium-3-yl]-ethyl-amino]hexanoic acid chloride salt [ka] Dissolve 1.2 ml of 1.0 M THF / hexane solution of lithium diisopropylamide in 10 ml of dry THF and 1 mmol, and add dry 2-methylpyridine to the mixture at -10°C. Raise the temperature of this solution to 0°C and stir at 0°C for 30 minutes. Slowly add 336 μmol of compound 35 in 10 ml of dry THF solution to this. Once the addition is complete, raise the temperature to room temperature and stir for a further 1 hour at room temperature. After hydrolysis with dilute HCl, remove the organic solvent by distillation and isolate the product from the aqueous layer by RP chromatography. Yield: 85 mg (51%) (C 29 H 35 ClN2O3; 495.05 g / mol) MS ESI+(m / z):459.3([M] + ) UV-Vis:λ in ethanol max :434nm;λ em :541nm;ε=24,000l / mol*cm

[0097] Compound 30: 6-[[6-[4-(dimethylamino)phenyl]-8,8-dimethyl-7H-xanthene-10-ium-3-yl]-ethyl-amino]hexanoic acid chloride salt [ka] Dissolve 1.0 mol of compound 35 in 10 ml of dried pyridine, and slowly add 5 ml of 0.5 M THF solution of 4-(N,N-dimethylaniline)-magnesium bromide at -40°C. After the addition is complete, raise the temperature of the solution to room temperature and stir at room temperature for 3 hours. After hydrolysis with HCl, remove the organic solvent by distillation, and isolate the product from the aqueous phase by RP chromatography. Yield: 100 mg (18%) (C 31 H 39 ClN2O3; 523.11 g / mol) MS ESI+(m / z):487.5([M] + ) UV-Vis:λ in ethanol max :664nm;λ em :713nm;ε=30,000l / mol*cm

[0098] Compound 31: 6-[ethyl-(6,8,8-trimethyl-7H-xanthene-10-ium-3-yl)amino]hexanoic acid chloride salt [ka] Dissolve 1.0 mol of compound 35 in 10 ml of dry THF, and slowly add 3 ml of 1.4 M THF / toluene solution of methylmagnesium bromide while stirring at -20°C. After the addition is complete, raise the temperature of the solution to room temperature and stir at room temperature for 1 hour. After hydrolysis with dilute HCl, remove the organic solvent by distillation, and isolate the product from the aqueous phase by RP chromatography. Yield: 125 mg (30%) (C 20 H 26 ClNO; 331.88 g / mol) MS ESI+(m / z):296.1([M] + ) UV-Vis:λ in ethanol max :538nm;λ em :628nm;ε=9,800l / mol*cm

[0099] Compound 32: (2E)-1-(5-carboxypentyl)-2-[(E)-3-[6-(diethylamino)-1,1-dimethyl-2H-xanthene-10-ium-3-yl]prop-2-enilidene]-3,3-dimethyl-indoline-5-sulfonate [ka] Dissolve 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 in 4 ml of acetic anhydride and 4 ml of glacial acetic acid, add 250 mg of sodium acetate, and heat under reflux temperature for 15 minutes. After cooling, precipitate with diethyl ether to obtain an oily precipitate, which is then analyzed by RP chromatography. Yield: 92 mg (14%) (C 38 H 46 N2O6S; 658.85 g / mol) MS ESI+(m / z):659.4(bass, M+H + ] + );681.6(30%, [M+Na + ] + );697.6(20%, [M+K + ] + MS ESI-(m / z):657.3([MH + ] - ) UV-Vis:λ in PBS max :755nm;λ em :788nm;ε=75.000l / mol*cm UV-Vis:λ in ethanol max :770nm;λ em :795nm;ε=143,600l / mol*cm

[0100] Compound 33: 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 sodium salt [ka] 1.0 mol of compound 31 and 1 mmol of 3-[5-formyl-3-(6-methoxy-6-oxohexyl)-2,4,6-trioxo-hexahydropyrimidine-1-yl]propane-1-sulfonate sodium salt were dissolved in 5 ml of acetic anhydride and 5 ml of glacial acetic acid, 250 mg of sodium acetate was added, and the mixture was heated under reflux for 15 minutes. After cooling, the precipitate was obtained by precipitation with diethyl ether. For ester cleavage, 10 ml of 3 M HCl and 10 ml of acetone were added to the precipitate and the mixture was heated under reflux for 1 hour. After neutralization with sodium bicarbonate, the mixture was purified by RP chromatography. Yield: 150 mg (22%) (C 34 H 42 NaN3O9S; 691.77 g / mol) MS ESI-(m / z):668.1([M] - ) UV-Vis:λ in ethanol max :683nm;λ em :719nm;ε=67,000l / mol*cm

[0101] Compound 34: 6-(diethylamino)-1,1-dimethyl-2H-xanthene-3-one [ka] 500 μmol of compound 2 is stirred at 50°C for 1 hour in 50 ml of acetone and 10 ml of 0.5 M pH 9 buffer. The solvent is removed by distillation, and the compound is purified by RP chromatography. Yield: 80 mg (54%) reddish-brown oily substance (C 19 H 23NO2; 297.39 g / mol) MS ESI-(m / z):298.1 (base, M+H + ] + );617.4(90%, [2M+Na + ] + ) UV-Vis:λ in PBS max :450nm;λ em :558nm;ε=23.00l / mol*cm pH3 underwater UV-Vis:λ max :xnm;λ em :xnm (exists as hydroxy-BPS) UV-Vis:λ in ethanol max :440nm;λ em :540nm;ε=24,800l / mol*cm

[0102] Compound 35: 6-[(8,8-dimethyl-6-oxo-7H-xanthene-3-yl)-ethyl-amino]hexanoic acid [ka] 500 μmol of compound 7 is stirred at 50°C for 1 hour in 50 ml of acetone and 10 ml of 0.5 M pH 9 buffer. The solvent is removed by distillation, and the compound is purified by RP chromatography. Yield: 150 mg (78%) (C 23 H 29 NO4; 383.48 g / mol) MS ESI-(m / z):382.2([MH + ] - );MS ESI+(m / z):384.3([M+H + ] + ) UV-Vis:λ in PBS max :457nm;λ em :559nm;ε=24,200l / mol*cm UV-Vis:λ in ethanol max :441nm;λ em :540nm;ε=25,300l / mol*cm

[0103] Compound 36: 6-[5-carboxypentyl(ethyl)amino]-1,1-dimethyl-3-oxo-2H-xanthene-4-sulfonate sodium salt [ka] Dissolve 177 μmol of compound 35 in 2 ml of fuming sulfuric acid (20% SO3) and stir at room temperature for 2 hours. Pour the mixture onto ice and stir at room temperature for a further 1 hour. After slowing the reaction with sodium carbonate, purify by RP chromatography. Yield: 17 mg (20%) (C 23 H 28 NO7SNa; 485.53 g / mol) MS ESI-(m / z):462.0(60%, [M] - );230.4(base, [MH + ] 2- ) UV-Vis:λ in PBS max :482nm;λ em :565nm;ε=30,100l / mol*cm;QY:0.66 UV-Vis:λ in ethanol max :446nm;λ em :542nm;ε=25,000l / mol*cm;QY 0.90

[0104] Compound 37: 3-[5-carboxypentyl-(8,8-dimethyl-6-oxo-7H-xanthene-3-yl)amino]propane-1-sulfonate sodium salt [ka] 375 μmol of compound 4 is stirred at 50°C for 1 hour in 50 ml of acetone and 10 ml of 0.5 M pH 9 buffer. The solvent is removed by distillation, and the residue is purified by RP chromatography using an acetonitrile-water gradient. Yield: 105 mg (56%) (C 24 H 30NO7S Na; 499.55 g / mol) MS ESI-(m / z):476.0 (base, [M] - );237.5(15%,[M - - H + ] 2- ) UV-Vis:λ in PBS max :454nm;λ em :553nm;ε=26,000l / mol*cm UV-Vis:λ in ethanol max :439nm;λ em :538nm;ε=27,000l / mol*cm

[0105] Compound 38: 6-[5-carboxypentyl(3-sulfonatopropyl)amino]-1,1-dimethyl-3-oxo-2H-xanthene-4-sulfonate disodium salt [ka] Dissolve 177 μmol of compound 37 in 2 ml of fuming sulfuric acid (20% SO3) and stir at room temperature for 2 hours. Pour the mixture onto ice and stir at room temperature for a further 1 hour. After slowing the reaction with sodium carbonate, purify by RP chromatography. Yield: 90 mg (85%) (C 24 H 29 NO 10 SNa; 601.60 g / mol) MS ESI-(m / z):556.2(15%,[M 2- +H + ] - );277.6(base, [M] 2- ) UV-Vis:λ in PBS max :479nm;λ em :557nm;ε=28,000l / mol*cm;QY:0.78 UV-Vis:λ in ethanol max :463nm;λ em :544nm;ε=31,000l / mol*cm;QY 0.91 1 H NMR(400MHz D2O):δ(ppm)=1.26(S, 6H, CH3);1.32(M, 2H, CH2);1.51(M, 2H, CH2);1.54(M, 2H, CH2);1.94(M, 2H, CH2);2.30(T, 2H, CH2);2.31(S, 2 H, CH2); 2.89 (T, 2H, CH2); 3.27 (T, 2H, CH2); 3.43 (T, 2H, CH2); 6.71 (D, 1H, 7-H); 6.86 (S, 1H, 5-H); 7.34 (D, 1H, 8-H); 7.52 (S, 1H, 9-H) 13 C-NMR(400MHz D2O):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

[0106] Compound 39: 6-Hydroxy-1,1-dimethyl-3-oxo-2H-xanthene-4-sulfonate sodium salt [ka] 750 μmol of compound 16 is stirred at 50°C for 1 hour in 50 ml of acetone and 10 ml of 0.5 M pH 9 buffer. The solvent is removed by distillation, and the compound is purified by RP chromatography. Yield: 115 mg (45%) (C 15 H 13 NaO6S; 344.31 g / mol) MS ESI-(m / z):321.3 (base, [M] - ) UV-Vis:λ in PBS max :409nm;λ em :485nm UV-Vis:λ in pH 9 buffer max :462nm;λ em :536nm

[0107] Compound 40: 6-(5-carboxypentoxy)-1,1-dimethyl-3-oxo-2H-xanthene-4-sulfonate sodium salt [ka] In 5 ml of DMF, 290 μmol of compound 39 was mixed with 80 mg of K2CO3 and 100 mg of ethyl 6-bromohexanoate and stirred at 120°C for 1 hour. The solvent was removed by distillation, and 10 ml of 3 M HCl was added to the residue for ester cleavage. The mixture was heated under reflux for 1 hour. After neutralization with sodium bicarbonate, the mixture was purified by RP chromatography. Yield: 33 mg (25%) (C 21 H 23 NaO8SNa; 458.46 g / mol) MS ESI-(m / z):435.2 (base, [M] - );217.0(30%,[M - -H + ] 2- ) UV-Vis:λ in PBS max :407nm;λ em :483nm;ε=21,000l / mol*cm UV-Vis:λ in ethanol max :403nm;λ em :487nm;ε=19,500l / mol*cm

[0108] Compound 41: 6-(2,2,4,7,7-pentamethyl-9-oxo-8H-chromeno[3,2-g]quinoline-1-yl)hexanoic acid [ka] 375 μmol of compound 21 is stirred at 50°C for 1 hour in 50 ml of acetone and 10 ml of 0.5 M pH 9 buffer. The solvent is removed by distillation. To cleave the ester, 10 ml of 3 M HCl is added to the residue and heated at reflux temperature for 1 hour. After neutralization with sodium bicarbonate, the compound is purified by RP chromatography. Purification is performed by RP chromatography using an acetonitrile-water gradient. Yield: 60 mg (38%) (C 27 H 33 NO4; 435.56 g / mol) MS ESI+(m / z):436.2([M+H + ] + ) UV-Vis:λ in PBS max :460nm;λ em :575nm;ε=23,000l / mol*cm UV-Vis:λ in ethanol max :463nm;λ em :557nm;ε=24,000l / mol*cm

[0109] Compound 42: 1-(5-carboxypentyl)-2,2,4,7,7-pentamethyl-9-oxo-8H-chromeno[3,2-g]quinoline-10-sulfonate sodium salt [ka] Dissolve 177 μmol of compound 41 in 2 ml of fuming sulfuric acid (20% SO3) and stir at room temperature for 2 hours. Pour the mixture onto ice and stir at room temperature for a further 1 hour. After slowing the reaction with sodium carbonate, purify by RP chromatography. Yield: 45 mg (48%) (C 27 H 32 NO7SNa; 537.60 g / mol) MS ESI-(m / z):514.2([M] - ) UV-Vis:λ in PBS max :487nm;λ em :578nm;ε=28,000l / mol*cm UV-Vis:λ in ethanol max :473nm;λ em :559nm;ε=30,000l / mol*cm

[0110] Compound 43: (E)-[3-ethoxy-5,5-dimethyl-2-(4-pyridyl)cyclohexy-2-ene-1-ylidene]-ethyl-oxonium tetrafluoroborate [ka] 4 mmol of 3-hydroxy-5,5-dimethyl-2-(4-pyridyl)cyclohexy-2-en-1-one is suspended in 10 ml of triethyl orthoformate, and 1.2 ml of 48% tetrafluoroboric acid is added at room temperature. After stirring for 30 minutes at room temperature, 50 ml of dry diethyl ether is added, and the mixture is allowed to stand at room temperature for several hours. The resulting pale yellow precipitate is filtered, washed with a small amount of dry ether, and dried in a vacuum. Yield: 970 mg (67%) (C 17 H 24 BF4NO2; 361.18 g / mol) MS ESI+(m / z):274.2[M + ]

[0111] Compound 44: 6-[[8,8-dimethyl-6-oxo-5-(4-pyridyl)-7H-xanthene-3-yl]-ethyl-amino]hexanoic acid [ka] 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 synthesis method of compound 2. The solvent is removed by distillation, and 10 ml of 3 M HCl is added to the residue for ester cleavage. The mixture is boiled at reflux temperature for 1 hour. After neutralization with sodium bicarbonate, the mixture is purified by RP chromatography. Yield: 97 mg (21%) (C 28 H 32 N2O4; 460.56 g / mol) MS ESI+(m / z):460.3([M+H + ] + ) UV-Vis:λ in PBS max :472nm;λem :573nm;ε=13,000l / mol*cm UV-Vis:λ in pH5 buffer max :523nm;λ em :600nm;ε=13.300l / mol*cm

[0112] Compound 45: 3-[5-carboxypentyl-[8,8-dimethyl-6-oxo-5-(4-pyridyl)-7H-xanthene-3-yl]amino]propane-1-sulfonate sodium salt [ka] 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 synthesis method of compound 2. The solvent is removed by distillation, and 10 ml of 3 M HCl is added to the residue for ester cleavage. The mixture is boiled at reflux temperature for 1 hour. After neutralization with sodium bicarbonate, the mixture is purified by RP chromatography. Yield: 97 mg (21%) (C 29 H 34 N2O4S; 554.65 g / mol) MS ESI-(m / z):553.2([MH + ] - ) MS ESI+(m / z):555.4([M+H + ] + ) UV-Vis:λ in PBS max :470nm;λ em :570nm;ε=15.000l / mol*cm UV-Vis:λ in pH5 buffer max :524nm;λ em :594nm;ε=15.400l / mol*cm

[0113] Compound 46: 2-[3,9-bis(diethylamino)-13,13-dimethyl-chromeno[3,2-b]xanthene-5-ium-14-yl]benzoic acid chloride salt [ka] 1 mmol of compound 2 and 1 mmol of 2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid are stirred in 5 ml of glacial acetic acid at 110°C for 16 hours. After cooling, the mixture is precipitated using diethyl ether, and the precipitate is purified by RP chromatography. Yield: 214 mg (34%) (C 37 H 39 ClN2O4; 611.17 g / mol) MS ESI+(m / z):575.4([M] + ) UV-Vis:λ in ethanol max :664nm;λ em :710nm;ε=60,000l / mol*cm UV-Vis:λ in PBS max :663nm;λ em :711nm;ε=42.000l / mol*cm

[0114] Compound 47: 6-[[9-(diethylamino)-13,13-dimethyl-chromeno[3,2-b]xanthene-7-ium-3-yl]-ethyl-amino]hexanoic acid chloride salt [ka] 500 μmol of compound 7 and 500 μmol of 4-(diethylamino)-2-hydroxybenzaldehyde are stirred in 5 ml of glacial acetic acid at 110°C for 16 hours. After cooling, the mixture is precipitated with diethyl ether, and the precipitate is purified by RP chromatography. Yield: 70 mg (24%) (C 34 H 41 ClN2O4; 577.15 g / mol) MS ESI+(m / z):541.3([M] + ) UV-Vis:λ in ethanol max :679nm;λ em:713nm;ε=80,300l / mol*cm UV-Vis:λ in PBS max :670nm;λ em :710nm;ε=37.000l / mol*cm

[0115] Compound 48: 6-[ethyl-(3-methoxy-13,13-dimethyl-chromeno[3,2-b]xanthene-5-ium-9-yl)amino]hexanoic acid chloride salt [ka] 1 mmol of compound 7 and 1 mmol of 2-hydroxy-4-methoxy-benzaldehyde are stirred in 5 ml of glacial acetic acid at 110°C for 16 hours. After cooling, the mixture is precipitated with diethyl ether, and the precipitate is purified by RP chromatography. Yield: 43 mg (8%) (C 31 H 34 ClNO5; 536.06 g / mol) MS ESI+(m / z):500.2([M] + ) UV-Vis:λ in ethanol max :654nm;λ em :712nm;ε=53,000l / mol*cm.

[0116] Photostability of the compound according to the present invention Figure 1 shows the irradiation results of aqueous solutions of compounds 5, 8, 11, and 38 (PBS pH 7.5, 100 mM and 100 mM NaCl and 5 mM NaN3) compared to MegaStokes dye DY-510XL. The solutions were adjusted so that the absorbance at the maximum absorption of a 1 cm layer thickness was "1". White light was irradiated from a 150 W Xe lamp on a fluorescence spectrometer (JASCO FP-6600, monochromator, 0 nm, slit position L: 10 nm), and the absorption was monitored at 5-minute intervals at the maximum value for 1 hour.

[0117] Maximum fluorescence of selected compounds according to the present invention Figure 2 shows the maximum fluorescence of the selected compound.

[0118] This invention relates to novel water-soluble fluorescent dyes having high fluorescence quantum yield based on oxygen-containing heterocycles, their reactive derivatives and dye conjugates, and their use for sample labeling and specimen analysis. The novel class of dye compounds are compatible with commercial excitation light sources and are characterized by a Stokes shift greater than 50 nm.

[0119] Further aspects of the present invention Further preferred embodiments of the present invention are shown below: Appearance 1 General formula 1 [ka] [During the ceremony, R11 and R12 are independently hydrogen or alkyl. R2 is hydrogen, alkyl, or alkenyl. R3 is hydrogen, alkyl, aryl, hydroxy, alkoxy, aryloxy, NR18R19, or group Q. Here, R18 and R19 are each independently (i) hydrogen, (ii) benzyl, (iii) aryl, (iv) heteroaryl, (v) reactive group A linked via linker L, (vi) alkyl, preferably C1-C4 alkyl, more preferably ethyl, and (vii) alkyl ω-sulfonate (-CH2) with x = 1 to 5. x -SO3 - ), (viii) ω-alkyl carboxylate (-(CH2) where y is 1-8 y Selected from the ethyl esters of (-CO2H) and (ix)(viii), Q is the structure of equation 2 or 3. [ka] (In the formula, n=1, 2, or 3; each R20 independently is alkyl, ω-sulfonic acid alkyl (-(CH2) x -SO3 -) or a reactive group A bonded to linker L, where each R21, R22, R23, R24 is independently hydrogen, sulfonic acid, or a sulfonic acid derivative, and R25 is hydrogen, alkyl, ω-alkyl sulfonate (-(CH2) x -SO3 - ) or a reactive group A bonded by a linker L, where x is an integer between 1 and 5. A heterocyclic structure selected from, R4 is hydrogen, bromine, chlorine, sulfonic acid or sulfonic acid derivative, alkyl, aryl or heteroaryl, R5 is hydrogen, sulfonic acid, or a sulfonic acid derivative. R6 is hydrogen, bromine, chlorine, hydroxy, alkoxy, aryloxy or NR29R30, where R29 and R30 are each independently a reactive group A bonded via hydrogen, alkyl, aryl or linker L, and R29 and R30 are each independently preferably (i) C1-C4 alkyl, (ii) ω-sulfonic acid alkyl (-(CH2)) where x is 1-5 x -SO3 - ), (iii) ω-alkyl carboxylate (-(CH2) where y is 1 to 8 y Selected from the ethyl esters of (-CO2H) and (iv)(iii), R7 is hydrogen, bromine, chlorine, hydroxyl, alkoxy, aryloxy, NR31R32, sulfonic acid, or sulfonic acid derivative, where R31 and R32 are independently (i) hydrogen, (ii) benzyl, (iii) aryl, (iv) heteroaryl, (v) reactive group A linked via linker L, (vi) alkyl, preferably C1-C4 alkyl, (vii) ω-alkyl sulfonate (-(CH2)) where x is 1 to 5 x -SO3 - ), (viii) ω-alkyl carboxylate (-(CH2) where y is 1-8 y Selected from the ethyl esters of (-CO2H) and (ix)(viii), R8 is hydrogen, methyl, or ethyl. R9 is hydrogen, alkyl, or 2-carboxyphenyl. L is -(CH2) s -and-[(CH2) m -O] p -(CH2) m - A linker selected from, where m is an integer between 2 and 5, and p and s are independently integers between 1 and 10. Each compound either does not contain linker L, or contains one linker L having a reactive group A bonded to the labeled molecule K, where A is an amine (-NH2), hydroxy (-OH), or phosphoramidite (-OP-[O-CH2-CH2-CN]-N[(CH(CH3)2]2) functional group, carboxylic acid (-COOH), alkyl ester or active ester derived therefrom (NHS ester, sulfo-NHS ester, tetrafluorophenyl ester, p-sulfo-tetrafluorophenyl ester), carboxylic acid hydrazide (-CONHNH2), or -(CH2) t -A carboxylic acid amide (-CONHR28) having R28 equivalent to -Y, where, Y is -OH, -NH2, -NH3 + , maleimide (-N[CO-CH]2), -NCS, -NCO, -NH-CO-CH2-I, -NH-CO-CH2-Br, -azide (-N3), -alkyne (-CCH), or -phosphoramidite (-OP-[O-CH2-CH2-CN]-N-[CH-(CH3)2]2), where t is an integer from 1 to 10. K is a hapten (a molecule representing an incomplete antigen that exhibits antigenic effects only when bound to a protein or cell structure), protein, antibody (a protein formed in response to an antigen), low molecular weight drugs (for example, the active component in drugs that can enter cells due to their relatively small molecular weight of up to approximately 800 g / mol, in contrast to proteins as extremely large molecules), peptides (small molecules or short-chain proteins with up to approximately 100 amino acids), nucleotides (consisting of a phosphate moiety, a monosaccharide moiety, and a nucleic acid base moiety, e.g., adenine, guanine, cytosine, thymine, or uracil), D [The component is selected from the group consisting of: nucleotide base components (such as NA or RNA), nucleosides (nucleotide base components such as DNA or RNA that lack a phosphate group and consist only of a monosaccharide group and a nucleic acid base group), DNA oligomers (deoxyribonucleic acid molecules that are relatively small molecules, in contrast to DNA as a macromolecule, but have a number of nucleotides that is not precisely defined), and polymers (synthetic or natural, chain-like or branched chemical compounds consisting of repeating units and monomers; polymers can also exist as copolymers consisting of at least two different monomers with different proportions and arrangements). Compounds of the same and their salts and solvates.

[0120] Appearance 2 R3 = hydroxyl, and the compound has a neutral to basic structure depending on the pH, such as 3-oxo-2H-xanthene. 4 [ka] A compound of embodiment 1, characterized by its existence as such.

[0121] Appearance 3 A compound according to embodiment 1 or 2, wherein at least one selected from R2-R3, R3-R4, R5-R6, R6-R7, and R7-R8 is crosslinked by forming a saturated ring, a partially unsaturated ring, an aromatic ring, or a heteroaromatic ring, each of which is independently characterized by comprising further substituents, particularly a sulfonic acid or a sulfonic acid derivative.

[0122] Pattern 4 formula 5 Structure [ka] [During the ceremony, R13 is hydrogen, alkyl, or 2-carboxyphenyl. R14 is hydrogen, alkyl, or 2-carboxyphenyl. R15 is hydrogen, bromine, chlorine, hydroxyl, alkoxy, aryloxy, NR33R34, sulfonic acid or sulfonic acid derivative, or crosslinked with R16, where R33 and R34 are independently (i) hydrogen, (ii) benzyl, (iii) aryl, (iv) heteroaryl, (v) reactive group A linked via linker L, (vi) alkyl, preferably C1-C4 alkyl, (vii) ω-alkyl sulfonate (-(CH2)) where x is 1 to 5 x -SO3 - ), (viii) ω-alkyl carboxylate (-(CH2) where y is 1-8 y Selected from the ethyl esters of (-CO2H) and (ix)(viii), R16 is hydrogen, bromine, chlorine, hydroxy, alkoxy, aryloxy or NR35R36, where R35 and R36 are independently (i) hydrogen, (ii) benzyl, (iii) aryl, (iv) heteroaryl, (v) reactive group A linked via linker L, (vi) alkyl, preferably C1-C4 alkyl, (vii) alkyl ω-sulfonate (-(CH2)) where x is 1-5 x -SO3 - ), (viii) ω-alkyl carboxylate (-(CH2) where y is 1-8 y Selected from the ethyl esters of (-CO2H) and (ix)(viii), R17 is hydrogen, sulfonic acid, or a sulfonic acid derivative. One or more elements selected from R5-R6, R6-R7, R7-R8, R14-R15, R15-R16, and R16-R17 can be crosslinked by forming a saturated ring, a partially unsaturated ring, an aromatic ring, or a heteroaromatic ring, each independently having further substituents, particularly sulfonic acid or sulfonic acid derivatives. Preferably, the aryl substituent and / or heteroaryl substituent (in R4, R6, R7, R15, R16, R18, R19, R33, R34, R35, R36) includes further substituents such as sulfonic acid or sulfonic acid derivatives and / or alkoxy groups and / or substituted amino groups. A compound according to any of embodiments 1 to 3, characterized by the above.

[0123] Appearance 5 The compound according to any one of embodiments 1 to 4, wherein the compound has at least one sulfonic acid group.

[0124] Appearance 6 The compound according to any one of embodiments 1 to 5, wherein R3 is NR18R19, where R18 and R19 are each independently reactive groups A bonded via hydrogen, alkyl, benzyl, aryl, heteroaryl, or linker L, and where NR18R19 is preferably characterized by being selected from 3-aminopropanesulfonate, N-methylaniline, 4-(methylamino)benzenesulfonate, aniline, 5-carboxypentylamine, and 3-carboxypropyl(methyl)amine.

[0125] Appearance 7 The compound according to any one of embodiments 1 to 6, characterized in that at least 1 mg of the compound is soluble in 1,000 g of water at 25°C.

[0126] Appearance 8 The compound according to any one of embodiments 1 to 7, wherein the compound is a fluorescent compound having maximum absorption at a wavelength in the range of 400 nm to 650 nm, preferably in the range of 500 nm to 550 nm.

[0127] Appearance 9 The compound according to embodiment 8, characterized by having a Stokes shift of at least 40 nm, preferably 50 nm to 120 nm, and more preferably 70 nm to 90 nm.

[0128] Appearance 10 A compound according to any one of embodiments 1 to 9, characterized by being selected from the following: 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]aminohexanoic acid, 3-[(6-ethoxy-8,8-dimethyl-7H-xanthene-10-ium-3-yl)-(6-ethoxy-6-oxohexyl)amino]propane-1-sulfonate, 3-[[6-[5-carboxypentyl(3-sulfonatopropyl)amino]-1,1-dimethyl-2H-xanthene-10-ium-3-yl]amino]propane-1-sulfonate, 3-[[6-[[6-(2,5-dioxopyrrolidine-1-yl)oxy-6-oxohexyl]-(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]hexanoate 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]oxyhexanoic 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]quinoline-11-ium-1-yl)propane-1-sulfonate, 6-(9-ethoxy-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinoline-11-ium-1-yl) ethyl hexanoate, 3-[9-(5-carboxypentylamino)-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinoline-11-ium-1-yl]propane-1-sulfonate, 3-[9-[3-carboxypropyl(methyl)amino]-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinoline-11-ium-1-yl]propane-1-sulfonate, 6-[2,2,4,7,7-pentamethyl-9-(N-methylanilino)-8H-chromeno[3,2-g]quinoline-11-ium-1-yl]hexanoic acid, 4-[[1-(5-carboxypentyl)-2,2,7,7-tetramethyl-4-(sulfonatomethyl)-8H-chromeno[3,2-g]quinoline-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-enilidene e]-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-1-sulfonate, 6-[5-carboxypentyl(3-sulfonatopropyl)amino]-1,1-dimethyl-3-oxo-2H-xanthene-4-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]quinoline-1-yl)hexanoic acid, 1-(5-carboxypentyl)-2,2,4,7,7-pentamethyl-9-oxo-8H-chromeno[3,2-g]quinoline-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.

[0129] Appearance 11 A method for producing a compound of formula 1, comprising reacting (E)-(3-ethoxy-5,5-dimethylcyclohexy-2-ene-1-ylidene)ethyl-oxonium or (E)-[3-ethoxy-5,5-dimethyl-2-(4-pyridyl)cyclohexy-2-ene-1-ylidene]-ethyl-oxonium with a benzaldehyde compound, wherein the benzaldehyde compound has a benzaldehyde group and the benzaldehyde compound has a hydroxyl group at the ortho position of the benzaldehyde group.

[0130] Appearance 12 A method for producing a compound of formula 5, comprising reacting 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]hexanoate ethyl ester with a benzaldehyde compound, wherein the benzaldehyde compound has a benzaldehyde group and the benzaldehyde compound has a hydroxyl group at the ortho position of the benzaldehyde group.

[0131] Appearance 13 Use of any compound of Embodiments 1 to 10 as a fluorescent dye and / or in a fluorescent probe, or as a fluorescent probe.

[0132] Appearance 14 The use according to embodiment 13 for labeling 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, dextran, nucleic acids, oligonucleotides, DNA, RNA, cells, lipids, and receptor-binding pharmaceuticals.

[0133] Appearance 15 Uses according to aspect 13 or 14 in optical, particularly fluorescent, qualitative and / or quantitative determination methods for diagnosing cellular characteristics, for studies in biosensors (point-of-care measurements), genome (DNA sequencing), cytometry and cell sorting, fluorescence correlation spectroscopy (FCS), ultra-high-throughput screening (UHTS), multi-color fluorescence in situ hybridization (FISH), and microarrays (DNA and protein chips).

Claims

1. General formula 1 or 4 【Chemistry 1】 [During the ceremony, R11 and R12 are independently hydrogen or alkyl. R2 is hydrogen, alkyl, or alkenyl. R3 is hydrogen, alkyl, aryl, hydroxy, alkoxy, aryloxy, NR18R19, or group Q. Wherein R¹⁸ and R¹⁹ are each independently (i) hydrogen, (ii) benzyl, (iii) aryl, (iv) heteroaryl, (v) a reactive group A bonded via a linker L, (vi) alkyl, preferably C １ -C ４ alkyl, more preferably ethyl, (vii) ω-sulfoalkyl wherein x is 1 to 5 (-(CH ２ ) ｘ -SO ３ － ), (viii) ω-carboxyalkyl wherein y is 1 to 8 (-(CH ２ ) ｙ -CO ２ H), and (ix) ethyl ester of (viii). Q is the structure of formula 2 or 3. 【Chemistry 2】 (In the formula, n = 1, 2, or 3; each R20 independently is alkyl, ω-alkyl sulfonate (-(CH ２ ) ｘ -SO ３ － ) or a reactive group A bonded to linker L; each R21, R22, R23, R24 is independently hydrogen, sulfonic acid, or a sulfonic acid derivative; R25 is hydrogen, alkyl, ω-alkyl sulfonate (-(CH ２ ) ｘ -SO ３ － ) or a reactive group A bonded by a linker L, where x is an integer from 1 to 5. A heterocyclic structure selected from, Alternatively, R2 and R3, together with the carbon atoms to which they are bonded, may bridge to form a saturated ring, a partially unsaturated ring, an aromatic ring, or a heteroaromatic ring that may contain further substituents. R4 is hydrogen, bromine, chlorine, sulfonic acid or sulfonic acid derivative, alkyl, aryl or heteroaryl, R3 and R4, together with the carbon atoms to which they are bonded, bridge to form a saturated ring, a partially unsaturated ring, an aromatic ring, or a heteroaromatic ring that may contain further substituents, particularly sulfonic acid or sulfonic acid derivatives. R5 is hydrogen, sulfonic acid, or a sulfonic acid derivative. R6 is hydrogen, bromine, chlorine, hydroxy, alkoxy, aryloxy, or NR29R30, where R29 and R30 are each independently a reactive group A bonded via hydrogen, alkyl, aryl, or linker L, and R29 and R30 are each independently preferably (i)C １ -C ４ Alkyl, (ii) x is 1 to 5 ω-sulfonic acid alkyl (-(CH ２ ) ｘ -SO ３ － ), (iii) ω-alkyl carboxylate (-(CH) where y is 1 to 8 ２ ) ｙ -CO ２ Selected from the ethyl esters of H) and (iv)(iii), Alternatively, R5 and R6, together with the carbon atoms to which they are bonded, bridge to form a saturated ring, a partially unsaturated ring, an aromatic ring, or a heteroaromatic ring that may contain further substituents, particularly sulfonic acid or sulfonic acid derivatives. R7 is hydrogen, bromine, chlorine, hydroxyl, alkoxy, aryloxy, NR31R32, sulfonic acid, or a sulfonic acid derivative, where R31 and R32 are independently (i) hydrogen, (ii) benzyl, (iii) aryl, (iv) heteroaryl, (v) a reactive group A linked via a linker L, (vi) alkyl, preferably C １ -C ４ Alkyl, (vii)x is 1 to 5 ω-sulfonate alkyl (-(CH ２ ) ｘ -SO ３ － ), (viii) ω-alkyl carboxylate (-(CH) where y is 1 to 8 ２ ) ｙ -CO ２ Selected from the ethyl esters of H, (ix) and (viii), Alternatively, R6 and R7 may bridge together with the carbon atoms to which they are bonded to form a saturated ring, a partially unsaturated ring, an aromatic ring, or a heteroaromatic ring that may contain further substituents, particularly sulfonic acid or sulfonic acid derivatives. R8 is hydrogen, methyl, or ethyl. Alternatively, R7 and R8 may bridge together with the carbon atoms to which they are bonded to form a saturated ring, a partially unsaturated ring, an aromatic ring, or a heteroaromatic ring that may contain further substituents, particularly sulfonic acid or sulfonic acid derivatives. R9 is hydrogen, alkyl, or 2-carboxyphenyl. L is -(CH ２ ) ｓ - and - [(CH ２ ) ｍ -O] ｐ -(CH ２ ) ｍ - A linker selected from, where m is an integer from 2 to 5, and p and s are each an integer from 1 to 10 independently. Each compound either does not contain linker L, or contains one linker L having a reactive group A bonded to the labeled molecule K, where A is an amine (-NH ２ ), hydroxy(-OH) or phosphoramidite (-O-P-[O-CH ２ -CH ２ -CN]-N[(CH(CH ３ ) ２ ] ２ ) Functional groups, carboxylic acids (-COOH), alkyl esters or active esters derived therefrom (NHS esters, sulfo-NHS esters, tetrafluorophenyl esters, p-sulfo-tetrafluorophenyl esters), carboxylic acid hydrazides (-CONHNH ２ ), or -(CH ２ ) ｔ -A carboxylic acid amide having R28 equivalent to -Y (-CONHR28), where, Y is -OH, -NH ２ , -NH ３ ＋ Maleimide (-N[CO-CH] ２ ), -NCS, -NCO, -NH-CO-CH ２ -I, -NH-CO-CH ２ -Br, -Azide(-N ３ ), -alkynes (-CCH) or -phosphoramidites (-O-P-[O-CH) ２ -CH ２ -CN]-N-[CH-(CH ３ ) ２ ] ２ ) where t is an integer from 1 to 10, K is a component selected from the group consisting of haptens, proteins, antibodies, low molecular weight drugs, peptides, nucleotides, nucleosides, DNA oligomers, and polymers. Compounds of the same and their salts and solvates.

2. The compound according to claim 1, characterized in that R3 = hydroxyl.

3. The compound according to claim 1 or 2, characterized in that R3 and R4 do not crosslink together with the carbon atoms to which they are bonded to form an aromatic ring.

4. Structure of Equation 5 【Transformation 3】 [During the ceremony, R13 is hydrogen, alkyl, or 2-carboxyphenyl. R14 is hydrogen, alkyl, or 2-carboxyphenyl. R15 is hydrogen, bromine, chlorine, hydroxyl, alkoxy, aryloxy, NR33R34, sulfonic acid or sulfonic acid derivative, or crosslinked with R16, where R33 and R34 are independently (i) hydrogen, (ii) benzyl, (iii) aryl, (iv) heteroaryl, (v) reactive group A linked via linker L, (vi) alkyl, preferably C １ -C ４ Alkyl, (vii)x is 1 to 5 ω-sulfonate alkyl (-(CH ２ ) ｘ -SO ３ － ), (viii) ω-alkyl carboxylate (-(CH) where y is 1 to 8 ２ ) ｙ -CO ２ Selected from the ethyl esters of H, (ix) and (viii), Alternatively, R14 and R15, together with the carbon atoms to which they are bonded, bridge to form a saturated ring, a partially unsaturated ring, an aromatic ring, or a heteroaromatic ring that may contain further substituents, particularly sulfonic acid or sulfonic acid derivatives. R16 is hydrogen, bromine, chlorine, hydroxy, alkoxy, aryloxy or NR35R36, where R35 and R36 are independently (i) hydrogen, (ii) benzyl, (iii) aryl, (iv) heteroaryl, (v) a reactive group A linked via linker L, (vi) alkyl, preferably C １ -C ４ Alkyl, (vii)x is 1 to 5 ω-sulfonate alkyl (-(CH ２ ) ｘ -SO ３ － ), (viii) ω-alkyl carboxylate (-(CH) where y is 1 to 8 ２ ) ｙ -CO ２ Selected from the ethyl esters of H, (ix) and (viii), Alternatively, R15 and R16, together with the carbon atoms to which they are bonded, bridge to form a saturated ring, a partially unsaturated ring, an aromatic ring, or a heteroaromatic ring that may contain further substituents, particularly sulfonic acid or sulfonic acid derivatives. R17 is hydrogen, sulfonic acid, or a sulfonic acid derivative. Alternatively, R16 and R17, together with the carbon atoms to which they are bonded, may bridge to form a saturated ring, a partially unsaturated ring, an aromatic ring, or a heteroaromatic ring that may contain further substituents, particularly sulfonic acid or sulfonic acid derivatives. The compound according to claim 1 or 3, characterized by the above.

5. The compound comprises at least one group selected from a sulfonic acid group, a sulfonic acid derivative, a hydroxyl group, an amino group, a carboxylic acid, and a carboxylic acid derivative, wherein preferably the compound is a) R7 is methoxy, R3 is an amino group, particularly NR18R19, particularly NH(4-HOOCC ６ H ４ ), NH(4-C ２ H ５ COOC ６ H ４ ), NH(2-C ２ H ５ COOC ６ H ４ ), NH(C ６ H ５ ), N(C ２ H ５ ) ２ or N(CH ２ ) ２ (CH ２ ) ２ O; b) R4 is bromine, and R6 and R16 are N(CH ３ ) ２ And; c) R6 and R16 are present, and one of R6 or R16 is a hydroxyl group. A compound according to any one of claims 1 to 4, characterized in that it is not a compound.

6. The compound according to any one of claims 1 to 5, wherein the compound comprises at least one sulfonic acid group.

7. The compound according to any one of claims 1 to 6, wherein R3 is NR18R19, where R18 and R19 are each independently reactive groups A bonded via hydrogen, alkyl, benzyl, aryl, heteroaryl, or linker L, and where NR18R19 is preferably selected from 3-aminopropanesulfonate, N-methylaniline, 4-(methylamino)benzenesulfonate, aniline, 5-carboxypentylamine, and 3-carboxypropyl(methyl)amine.

8. The compound according to any one of claims 1 to 7, characterized in that the compound is soluble in water, and at least 1 mg of the compound is soluble in 1,000 g of water at 25°C.

9. The compound according to any one of claims 1 to 8, wherein the compound is a fluorescent compound having maximum absorption at a wavelength in the range of 400 nm to 650 nm, preferably in the range of 500 nm to 550 nm.

10. The compound according to claim 8, characterized in that the Stokes shift is at least 40 nm, preferably 50 nm to 120 nm, and more preferably 70 nm to 90 nm.

11. A compound according to any one of claims 1 to 10, characterized in that the compound is selected from the following: 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]aminohexanoic acid, 3-[(6-ethoxy-8,8-dimethyl-7H-xanthene-10-ium-3-yl)-(6-ethoxy-6-oxohexyl)amino]propane-1-sulfonate, 3-[[6-[5-carboxypentyl(3-sulfonatopropyl)amino]-1,1-dimethyl-2H-xanthene-10-ium-3-yl]amino]propane-1-sulfonate, 3-[[6-[[6-(2,5-dioxopyrrolidine-1-yl)oxy-6-oxohexyl]-(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]hexanoate 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-xanthen-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-sulfonatoanilino)-7H-xanthene-10-ium-2-sulfonate, 3-(9-ethoxy-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinoline-11-ium-1-yl)propane-1-sulfonate, 6-(9-ethoxy-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinoline-11-ium-1-yl) ethyl hexanoate, 3-[9-(5-carboxypentylamino)-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinoline-11-ium-1-yl]propane-1-sulfonate, 3-[9-[3-carboxypropyl(methyl)amino]-2,2,4,7,7-pentamethyl-8H-chromeno[3,2-g]quinoline-11-ium-1-yl]propane-1-sulfonate, 6-[2,2,4,7,7-pentamethyl-9-(N-methylanilino)-8H-chromeno[3,2-g]quinoline-11-ium-1-yl]hexanoic acid, 4-[[1-(5-carboxypentyl)-2,2,7,7-tetramethyl-4-(sulfonatomethyl)-8H-chromeno[3,2-g]quinoline-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-enilidene e]-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-1-sulfonate, 6-[5-carboxypentyl(3-sulfonatopropyl)amino]-1,1-dimethyl-3-oxo-2H-xanthene-4-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]quinoline-1-yl)hexanoic acid, 1-(5-carboxypentyl)-2,2,4,7,7-pentamethyl-9-oxo-8H-chromeno[3,2-g]quinoline-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 any one of claims 1 to 11, characterized in that the compound is selected from the following: 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-4-sulfonate, and 6-(5-carboxypentoxy)-1,1-dimethyl-3-oxo-2H-xanthene-4-sulfonate.

13. A method for producing a compound of formula 1 according to any one of claims 1 to 12, comprising reacting (E)-(3-ethoxy-5,5-dimethyl-cyclohexy-2-ene-1-ylidene)-ethyl-oxonium or (E)-[3-ethoxy-5,5-dimethyl-2-(4-pyridyl)cyclohexy-2-ene-1-ylidene]-ethyl-oxonium with a benzaldehyde compound, wherein the benzaldehyde compound has a benzaldehyde group and the benzaldehyde compound has a hydroxyl group at the ortho position of the benzaldehyde group.

14. A method for producing a compound of formula 5, comprising reacting 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]hexanoate ethyl ester with a benzaldehyde compound, wherein the benzaldehyde compound has a benzaldehyde group and the benzaldehyde compound has a hydroxyl group at the ortho position of the benzaldehyde group.

15. Use of the compound according to any one of claims 1 to 12 as a fluorescent dye and / or in a fluorescent probe, or as a fluorescent probe.

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