Thiopolyphenol-based fluorescent dyes and conjugates thereof

Abstract

A fluorescent dye according to formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X) and (XI): with R 1 selected from the group nitrile, nitro, formyl, carboxyl, substituted or unsubstituted C2-C 20 Acylalkyl, substituted or unsubstituted C5-C 20 Acylaryl, substituted or unsubstituted C2-C 20 Acyloxy, substituted or unsubstituted C2-C 20 Ester, substituted or unsubstituted C2-C 20 Amide with R 2 selected from the group hydrogen, nitrile, nitro, formyl, carboxyl, substituted or unsubstituted C2-C 20 Acylalkyl, substituted or unsubstituted C5-C 20 Acylaryl, substituted or unsubstituted C2-C 20 Acyloxy, substituted or unsubstituted C2-C 20 Ester, substituted or unsubstituted C2-C 20 Amide with R3 , R 4 , R 5 and R 6 independently selected from the group of substituted or unsubstituted C1-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkyl ethers and substituted or unsubstituted C6-C 20 Aryl; and with Z and X selected independently from a substituted or unsubstituted methylene or a substituted or unsubstituted ethane-1,2-diyl, where in formulas (I), (II), (III), (VI), (VII), (VIII), (IX) and (X) Z and X represent the substituted methylene and the substituted ethane-1,2-diyl with at least one substituted or unsubstituted C1-C 20 Alkyl and / or with a substituted or unsubstituted C2-C 20 Esters are substituted.

Description

[0001] The invention relates to novel fluorescent dyes based on thiopolyphenol derivatives having one or two thio substituents. The invention further relates to a conjugate of the aforementioned fluorescent dye and biomolecules, as well as a method for synthesizing the fluorescent dyes according to the invention. Technological background

[0002] Fluorescence is the spontaneous emission of light when an electronically excited system transitions to a lower energy state. Systems, i.e., materials, exhibiting such fluorescent properties are called fluorophores. When these are used for staining, they are referred to as fluorochromes or fluorescent dyes. Fluorescent dyes have diverse applications in biochemistry, biology, and medicine, for example, in diagnostic kits, as markers for protein structure elucidation, or in drug screening.

[0003] Fluorescent dyes are characterized by a number of parameters that allow the user to select a suitable dye depending on the intended application. The excitation wavelength λ is particularly important in this regard. exc (or λ Abs ), which corresponds to the maximum of the absorption band, the emission wavelength λ em , which corresponds to the maximum of the emission band, the Stokes shift Δλ, which is the difference between emission wavelength λ em and excitation wavelength λ exc This corresponds to the extinction coefficient ε, which represents the proportion of the excitation wavelength λ. exc reflected absorbed radiation, the fluorescence quantum yield Φ F , which corresponds to the ratio of the number of emitted to absorbed photons, and the fluorescence lifetime τ F, which corresponds to the mean time the molecule remains in its excited state before it transitions to its ground state by emitting a photon.

[0004] A large excitation wavelength λ is desirable, especially with regard to biological applications. exc , in order to penetrate biological samples as deeply as possible with the excitation radiation, a large extinction coefficient ε, so that as much of the incident light as possible is absorbed, a large Stokes shift Δλ, in order to observe as little interaction as possible between the excitation and emission radiation, a long fluorescence lifetime τ F , in order to be able to mask the short-lived natural background fluorescence of biological tissues, and ultimately achieve a large fluorescence quantum yield Φ Fto achieve the highest possible signal-to-noise ratio. Most currently known fluorescent dyes have disadvantages with regard to certain parameters, so there is always a need for new fluorescent dyes.

[0005] EP 2 399 913 B1 describes a fluorescent dye based on a 1,2,4,5-tetrahydroxybenzene derivative with a long fluorescence lifetime and large Stokes shift.

[0006] DE 10 2017 122 275 A1 describes a fluorescent dye based on a benzo[1,2-d:4,5-d']bis([1,3]dithiol) derivative with large emission and excitation wavelengths.

[0007] WO 91 / 12024 A1 describes structurally related compounds for use as NMR contrast agents. Further related derivatives were described by F. Dallacker et al. (Derivatives of methylenedioxybenzene, XV. Sulfur and selenium-containing heterocycles of pyrocatechol methylene ether. Justus Liebigs Ann. Chem., Vol. 689, 1965, No. 1, pp. 179–188. - ISSN 0075-4617), X. Zhao et al. (lodine-catalyzed thiolation of electron-rich aromatics using sulfonyl hydrazides as sulfenylation reagents. In: Org. Biomol. Chem., Vol. 14, 2016, No. 3, pp. 1131-1137. - ISSN 1477-0520) and R. Wawrzinek et al. (DBD Dyes as Fluorescence Lifetime Probes to Study Conformational Changes in Proteins. In: Chem. Eur. J., Vol. 19, 2013, No. 19, pp. 17349-17357. - ISSN 0947-6539).

[0008] There is a continuing need for new fluorescent dyes and more efficient synthesis methods. In particular, the dyes should exhibit both large emission and excitation wavelengths, as well as a long fluorescence lifetime and a large Stokes shift. Summary of the invention

[0009] A new group of fluorescent dyes is provided whose spectral properties particularly satisfy the aforementioned requirements with regard to biological applications. Fluorescent dyes according to the invention comprise derivatives of the general formulas (I), (II), (III), (IV) (hereinafter 1,2-S 2 (called DBD fluorescent dyes):

[0010] Furthermore, the fluorescent dyes according to the invention comprise derivatives of the general formulas (V), (VI) and (VII) (hereinafter 1,4-S 2 (called DBD fluorescent dyes):

[0011] Furthermore, the fluorescent dyes according to the invention comprise derivatives of the general formulas (VIII), (IX), (X) and (XI) (hereinafter S 1 (called DBD fluorescent dyes):

[0012] The rest R 1 In the aforementioned formulas, C2-C is from the group nitrile, nitro, formyl, carboxyl, substituted or unsubstituted. 20 Acylalkyl, substituted or unsubstituted C5-C 20 Acylaryl, substituted or unsubstituted C2-C 20 Acyloxy, substituted or unsubstituted C2-C 20 Ester, substituted or unsubstituted C2-C 20 Amid selected. The rest R 2 In the formulas mentioned above, C2-C is from the group hydrogen, nitrile, nitro, formyl, carboxyl, substituted or unsubstituted. 20 Acylalkyl, substituted or unsubstituted C5-C 20 Acylaryl, substituted or unsubstituted C2-C 20Acyloxy, substituted or unsubstituted C2-C 20 Ester, substituted or unsubstituted C2-C 20 Amid selected. In addition, the residues R 3 , R 4 , R 5 and R 6 independently selected from the group of substituted or unsubstituted C1-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkyl ethers and substituted or unsubstituted C6-C 20 Aryl. The (ring-forming) residues Z and X are independently selected from a substituted or unsubstituted methylene or a substituted or unsubstituted ethane-1,2-diyl, wherein in formulas (I), (II), (III), (VI), (VII), (VIII), (IX) and (X) Z and X are the substituted methylene and the substituted ethane-1,2-diyl with at least one substituted or unsubstituted C1-C6 alkyl and / or with a substituted or unsubstituted C2-C 20 Esters are substituted.

[0013] The novel fluorescent dyes have either two oxygen atoms and two sulfur atoms (hereinafter referred to as S). 2 -DBD) or three oxygen atoms and one sulfur atom (hereinafter referred to as S 1 -DBD). This novel class of fluorescent dyes offers, in comparison to the already known [1,3]-dioxolo[4.5-f]benzodioxole fluorescent dyes (DBD for short) and [1,3]-dithiolo[4.5-f]benzodithiol fluorescent dyes (S for short), 4 -DBD) exhibits, among other things, high excitation and emission wavelengths without a significant reduction in the Stokes shift. Thus, the disadvantage of a reduced Stokes shift, which results from the complete substitution of the oxygen atoms of DBD by sulfur (see p. 4 -DBD) can be resolved with virtually no loss.

[0014] The fluorescent dyes according to the invention are therefore particularly suitable for applications in the field of biology and biochemistry (e.g. for the structural elucidation of complex proteins or cells) that require a particularly clear signal and deep penetration into the biological sample, whereby the clarity of the signal is ensured by the low interaction between excitation and emission light.

[0015] Particularly preferably, Z and X in formulas (I), (II), (III), (VI), (VII), (VIII), (IX), (X) can correspond to a substituted or unsubstituted methylene. Furthermore, Z and X in formulas (I), (II), (III), (VI), (VII), (VIII), (IX), (X) can preferably be of the same type.

[0016] In a preferred embodiment, R 1 and R 2 They must be selected identically.

[0017] In another preferred embodiment, the substituted C2-C 20Acylalkyl can include a trifluoroacetyl. For example, the unsubstituted C2-C can also be used. 20 Acylalkyl comprise a residue from the group: acetyl, propionyl, butyryl and valeryl.

[0018] The following derivatives represent preferred embodiments of the fluorescent dyes according to the invention: and

[0019] Further particularly preferred embodiments are the following derivatives of the fluorescent dyes according to the invention:

[0020] Another aspect of the present disclosure relates to a conjugate comprising a fluorescent dye as previously described and a biomolecule coupled to the fluorescent dye or a cell coupled to the fluorescent dye. The biomolecule may preferably be selected from the group consisting of proteins, peptides, nucleic acids, and lipids. Brief description of the characters

[0021] The invention is explained in more detail below with reference to an exemplary embodiment and accompanying drawings. The figures show: Fig. 1 - 4 Bar charts on photophysical data of two known and three new fluorescent dyes (II-1), (S 4 -DBD-1), (DBD-1), (VI-1) and (IX-13) in acetonitrile; Fig. 5A / 5B Absorption and emission spectrum of the fluorescent dye (II-1) measured in acetonitrile; Fig. 6A / 6B Absorption and emission spectrum of the fluorescent dye (VI-1) measured in acetonitrile; and Fig. 7A / 7B Absorption and emission spectrum of the fluorescent dye (IX-13) measured in acetonitrile. Detailed description of the invention

[0022] In connection with the present invention, under “C1-C 20 “Alkyl” is understood to mean a linear or branched alkyl group that has the general formula C n H 2n+1aufweist, wobei n = 1 bis 20 bedeutet. C1-C5 Alkyl umfasst beispielsweise Methyl, Ethyl, n-Propyl, 1-Methylethyl, n-Butyl, 1-Methylpropyl, 2-Methylpropyl, 1,1-Dimethylethyl, n-Pentyl, 1-Methylbutyl, 2-Methylbutyl, 3-Methylbutyl, 1,1-Dimethylpropyl, 1,2-Dimethylpropyl, 2,2-Dimethylpropyl und 1-Ethylpropyl. Beispiele für Alkylreste mit n ≥ 6 umfassen n-Hexan, n-Heptan, n-Octan, n-Nonan, n-Decan, 1-Methylpentyl, 1-Methylhexyl, 1-Methylheptyl, 1-Methyloctyl, 1-Methylnonyl, 1-Methyldecanyl, 1-Ethylbutyl, 1-Ethylpentyl, 1-Ethylhexyl, 1-Ethylheptyl, 1-Ethyloctyl, 1-Ethylnonyl, 1-Ethyldecanyl, 2-Methylpentyl, 2-Methylhexyl, 2-Methylheptyl, 2-Methyloctyl, 2-Methylnonyl, 2-Methyldecanyl, 2-Ethylpropyl, 2-Ethylbutyl, 2-Ethylpentyl, 2-Ethylhexyl, 2-Ethylheptyl, 2-Ethyloctyl, 2-Ethylnonyl, 2-Ethyldecanyl, 1,1-Dimethylbutyl, 1,1-Dimethylpentyl, 1,1-Dimethylhexyl, 1,1-Dimethylheptyl, 1,1-Dimethyloctyl, 1,1-Dimethylnonyl, 1,1-Dimethyldecanyl, 1,2-Dimethylbutyl, 1,2-Dimethylpentyl, 1,2-Dimethylhexyl,1,2-Dimethylheptyl, 1,2-Dimethyloctyl, 1,2-Dimethylnonyl, 1,2-Dimethyldecanyl, 2-Ethyl-1-methylbutyl, 2-Ethyl-1-methylpentyl, 2-Ethyl-1-methylhexyl, 2-Ethyl-1-methylheptyl, 2-Ethyl-1-methyloctyl, 2-Ethyl-1-methylnonyl, 2-Ethyl-1-methyldecanyl, 1-Ethyl-2-methylpropyl, 1-Ethyl-2-methylbutyl, 1-Ethyl-2-methylpentyl, 1-Ethyl-2-methylhexyl, 1-Ethyl-2-methylheptyl, 1-Ethyl-2-methyloctyl, 1-Ethyl-2-methylnonyl and 1-Ethyl-2-methyldecanyl.,

[0023] In connection with the present invention, under “C2-C 20 "Acylalkyl" is understood to mean a linear or branched alkyl group of the meaning given above, which is covalently bonded to the fluorescent dye via a carbonyl group. Examples include acetyl, propionyl, butyryl, and valeryl.

[0024] In connection with the present invention, under “C6-C 20The term "aryl" refers to an aromatic residue with 6 to 20 carbon atoms. Examples include phenyl, pentalenyl, indenyl, naphthyl, biphenyl, fluorenyl, phenanthrenyl, pyrenyl, and perylenyl. Aryl residues can themselves be singly or multiply substituted with C1-C5 alkyl groups of the meaning described above. Examples of such substituted aryl residues include tolyl, xylyl, pseudocumyl, and mesityl.

[0025] In connection with the present invention, under “C5-C 20 "Acylaryl" can be understood as an aryl group of the meaning given above, which is covalently bonded to the fluorescent dye via a carbonyl group. Benzoyl is an example.

[0026] In connection with the present invention, under “C2-C 20Acyloxy is understood to be an ester covalently bonded to the fluorescent dye via the oxygen atom, whose carbonyl carbon-bonded branched or unbranched alkyl group comprises 1 to 19 carbon atoms. An example is acetyloxy.

[0027] In connection with the present invention, under “C2-C 20 An ester is understood to be an ester covalently bonded to the fluorescent dye via the carbonyl carbon. Examples include methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, and [(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl.

[0028] In connection with the present invention, under “C2-C 20An "amide" is understood to be a carboxylic acid amide covalently bonded to the fluorescent dye via the carbonyl carbon, whose nitrogen-atom-bound branched or unbranched alkyl group comprises 1 to 19 carbon atoms. Examples include methylaminocarbonyl, ethylaminocarbonyl, propylaminocarbonyl, butylaminocarbonyl, and pentylaminocarbonyl.

[0029] In connection with the present invention, under “C4-C 15 The term "alkoxy" refers to a functional group based on a linear or branched alkyl group of the meaning given above, linked to an oxygen atom. Examples include methoxy and ethoxy.

[0030] The term ‘substituted’ as used herein in reference to acylalkyl, acylaryl, acyloxy, ester, amide, alkoxy, alkyl ether and aryl refers to the fact that in these groups one, several or all of the hydrogen atoms have been replaced by fluorine, chlorine, bromine and iodine.

[0031] The term ‘substituted’ as used herein in relation to alkyl refers to the fact that in this group at least one hydrogen atom is replaced by substituents selected from the group comprising: hydroxy, tosylate (p-toluenesulfonic acid ester), azide, fluorine, chlorine, bromine and iodine.

[0032] The term “substituted”, as used herein in reference to methylene and ethane-1,2-diyl of the Z and X residues, refers to the fact that in these groups one, several or all of the hydrogen atoms are replaced by substituted or unsubstituted C1-C 20 Alkyl (as previously defined) or substituted or unsubstituted C2-C 20 Esters (as previously defined) are replaced. General synthesis procedure

[0033] A process for the synthesis of the fluorescent dyes according to the invention is described below. The process comprises step A), the reaction of a catechol derivative (e.g., veratrol or 1,2-methylenedioxybenzene) with S₂Cl₂ to form a first intermediate according to formula (A1) or (A2): and step B), the subsequent reduction of the first intermediate from step A to form a second intermediate according to formula (B1) or (B2): In the aforementioned process, the R groups 5 and R 6 from the group comprising hydrogen, substituted or unsubstituted C1-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkyl ethers and substituted or unsubstituted C6-C 20Aryl, selected independently of each other. Furthermore, X is selected from a substituted or unsubstituted methylene or a substituted or unsubstituted ethane-1,2-diyl, where for X the substituted methylene and the substituted ethane-1,2-diyl with at least one substituted or unsubstituted C1-C 20 Alkyl and / or with a substituted or unsubstituted C2-C 20 Esters are substituted. In the reduction step (reduction in step B), reducing agents including, for example, NaBH4, KBH4, NMe4BH4 and NaAlH4 can be used. Preferably, NaBH4 can be used.

[0034] Furthermore, the aforementioned procedure can include the following additional steps C) and D). Step C) corresponds to a ring-closing reaction of the second intermediate from step B to form a third intermediate according to formula (C1) or (C2):

[0035] The ring-closing reaction can be a S N-reaction or acetalization. Examples of the ring-closure reaction include the reaction with CH2BrCl or with acetone. Step D) corresponds to an (electrophilic) aromatic substitution at the third intermediate from step C:

[0036] In process steps B and D, the residues R 1 and R 2 from the group comprising hydrogen, nitrile, nitro, formyl, carboxyl, substituted or unsubstituted C2-C 20 Acylalkyl, substituted or unsubstituted C5-C 20 Acylaryl, substituted or unsubstituted C2-C 20 Acyloxy, substituted or unsubstituted C2-C 20 Ester, substituted or unsubstituted C2-C 20 Amide and substituted or unsubstituted C1-C 15 Alkoxy molecules were selected independently of each other. Furthermore, the residues R 5 and R 6Selected independently from the group comprising: hydrogen, substituted or unsubstituted C1-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkyl ethers and substituted or unsubstituted C6-C 20 Aryl. The residues Z and X are independently selected from a substituted or unsubstituted methylene or a substituted or unsubstituted ethane-1,2-diyl, wherein for Z and X the substituted methylene and the substituted ethane-1,2-diyl with at least one substituted or unsubstituted C1-C 20 Alkyl and / or with a substituted or unsubstituted C2-C 20Esters are substituted. In one embodiment, the aromatic substitution can be carried out using dimethylformamide (DMF) and an organolithium compound (e.g., n-butyllithium). Further embodiments of the (electrophilic) aromatic substitution can include Friedel-Crafts alkylations, Friedel-Crafts acylations, and nitration.

[0037] Furthermore, starting from the intermediates in step B, the procedure can include the further steps E) and F). Step E) corresponds to an S-functionalization (functionalization of the thiol groups) of the second intermediate from step B to form a fourth intermediate according to formula (D1) or (D2):

[0038] S-functionalization can, for example, involve alkylations at the sulfur atom (e.g., with methyl iodide, methyl triflate, or other alkyl reagents). Step F) corresponds to an (electrophilic) aromatic substitution at the fourth intermediate from step E:

[0039] In process steps E and F, the residues R 1 and R 2 from the group comprising hydrogen, nitrile, nitro, formyl, carboxyl, substituted or unsubstituted C2-C 20 Acylalkyl, substituted or unsubstituted C5-C 20 Acylaryl, substituted or unsubstituted C2-C 20 Acyloxy, substituted or unsubstituted C2-C 20 Ester, substituted or unsubstituted C2-C 20 Amide and substituted or unsubstituted C1-C 15 Alkoxy molecules were selected independently of each other. Furthermore, the residues R 3 , R 4 , R 5 and R 6 Selected independently from the group comprising: hydrogen, substituted or unsubstituted C1-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkyl ethers and substituted or unsubstituted C6-C 20Aryl. The residue X is selected from a substituted or unsubstituted methylene or a substituted or unsubstituted ethane-1,2-diyl, wherein for X the substituted methylene and the substituted ethane-1,2-diyl with at least one substituted or unsubstituted C1-C 20 Alkyl and / or with a substituted or unsubstituted C2-C 20 Esters are substituted.

[0040] The fluorescent dyes according to formulas (I), (II), (III), (IV), (V), (VI), (VII), (VIII) and (IX) can be prepared by the following synthesis scheme.

[0041] Exemplary reaction scheme for the synthesis of the fluorescent dyes 1,2-S 2 according to formula (I) and (II):

[0042] Exemplary reaction scheme for the synthesis of the fluorescent dyes 1,2-S 2 according to formulas (III) and (IV):

[0043] Exemplary reaction scheme for the synthesis of the fluorescent dyes 1,4-S 2according to formula (V) (VI) and (VII):

[0044] Exemplary reaction scheme for the synthesis of fluorescent dyes S 1 according to formula (VIII), (IX), (X) and (XI): Biofunctionalization

[0045] The derivatives (II-9) to (II-13) shown in the following scheme are suitable, for example, for coupling with the following functional groups found in biomolecules: Carboxylic acids (see II-9), lysine, methionine, N-heterocycles (see II-10), alkynes by click reaction (see II-11), thiols such as cysteine ​​(see II-12), and azides by click reaction (see II-13).

[0046] The functionalization of the derivatives by halogen atoms can, for example, be carried out starting from II-10 by reaction with potassium halides (Finkelstein reaction). Example 1

[0047] 1,3-Benzodioxole (2 mL, 17.60 mmol, 1.0 eq.) was dissolved in 40 mL of glacial acetic acid, sulfur monochloride (1.4 mL, 17.60 mmol, 1.0 eq.) was added, and the mixture was stirred at room temperature for 24 h. The resulting yellow solid was filtered off by filtration and washed with 50 mL of diethyl ether. After drying under vacuum, 2.69 g of compound 2 (83%, 7.30 mmol) were obtained as a yellow solid.

[0048] 50 mg (0.13 mmol, 1.0 eq.) was suspended in dry DMF (15 mL) and NaBH4 (25 mg, 0.63 mmol, 5.0 eq.) was added. The yellow mixture was stirred at room temperature for 30 min and heated to 100 °C for 1 h. After the colorless solution had cooled, H2O (20 mL) was added and the solution was extracted with DCM (20 mL). The aqueous phase was adjusted to pH 2 with 1 M HCl and extracted again with DCM (3 x 20 mL). The combined organic phases were concentrated by rotary evaporation, and the crude product was reacted twice with toluene, with the solvent removed each time, so that (I-1) was isolated as a pale green oil in 50 mg (quant., 0.25 mmol).

[0049] Dithiol 3 (50 mg, 0.22 mmol, 1.0 eq.) was dissolved in 10 mL of dry DCM, and acetone (0.03 mL, 0.37 mmol, 1.5 eq.) and boron trifluoride diethyl etherate (0.1 mL, 48%, 0.37 mmol, 1.5 eq.) were added. The greenish-clear solution was stirred for 14 h at room temperature. The reaction was stopped by adding saturated NaHCO3 solution. The phases were separated, and the aqueous phase was extracted twice with DCM (20 mL). The combined organic phases were dried over MgSO4, and the solvent was removed by rotary evaporator. After purification by flash chromatography (PE:EE 5:1), 50 mg (50%, 0.20 mmol) of 4 were obtained as a white solid.

[0050] Compound 4 (50 mg, 0.22 mmol, 1 eq.) was suspended in 3 mL of dry hexane and treated with TMEDA (0.07 mL, 0.44 mmol, 2.0 eq.). Then, at room temperature, n-BuLi (0.19 mL, 2.5 M in hexane, 0.46 mmol, 2.1 eq.) was added to the solution and stirred for 1 h. After adding dry DMF (0.42 mL, 0.55 mmol, 2.5 eq.), the mixture was stirred for another hour at room temperature. The reaction was stopped by adding 1 M HCl, the phases were separated, and the aqueous phase was extracted twice with ethyl acetate. The combined organic phases were dried with MgSO4, and the solvent was removed. After purification by flash chromatography (PE:EE 3:1) 36 mg (58%, 0.13 mmol) of compound (II-1) were obtained as a red solid. Example 2

[0051] Acetone (6.08 mL, 25.77 mmol, 1.5 eq.) and boron trifluoride diethyl etherate (6.80 mL, 48% solution, 25.77 mmol, 1.5 eq.) were added to a solution of compound 2 (3.20 g, 17.18 mmol, 1.0 eq.) in 60 mL of dry DCM and stirred for 16 h at room temperature. The mixture was washed with saturated NaHCO3 solution and the aqueous phase was extracted once with DCM. The combined organic phases were dried over MgSO4 and the solvent removed. After purification by flash chromatography (PE:EE 10:1), 3.30 g of compound 20 (55%, 9.31 mmol) were obtained as a white solid.

[0052] Compound 20 (1.71 g, 4.82 mmol, 1.0 eq.) was dissolved in 100 mL of methanol, and lithium hydroxide monohydrate (0.61 g, 14.47 mmol, 3.0 eq.) was added. The mixture was stirred at room temperature for 15 h, the solvent was removed, and the residue was dissolved in ethyl acetate. The organic phase was washed with 1 M HCl and brine and dried with MgSO4. The solvent was removed, and the residue was purified by flash chromatography (PE:EE3:1). 1.20 g of compound 21 (92%, 4.44 mmol) was obtained as a colorless oil.

[0053] A solution of 21 (100 mg, 0.37 mmol, 1.0 eq.) in 5 mL of dry hexane and TMEDA (0.12 mL, 0.77 mmol, 2.1 eq.) was cooled to 0 °C and treated with n-BuLi (0.52 mL, 2.5 M in hexane, 1.29 mmol, 3.5 eq.) and stirred for 1 h. After the addition of DMF (0.07 mL, 0.92 mmol, 2.5 eq.), the mixture was stirred again for 1 h at 0 °C, and the reaction was then stopped by the addition of 1 M HCl (15 mL). The phases were separated, and the aqueous phase was extracted twice with ethyl acetate. After drying with MgSO4, the solvent was removed, and the residue was purified by flash chromatography (PE:EE 1:1). 42 mg of compound (II-9) (35%, 0.13 mmol) were obtained as a red solid. Example 3

[0054] Benzoquinone (1.0 g, 9.25 mmol, 1.0 eq.) and ammonium thiocyanate (1.76 g, 23.1 mmol, 2.5 eq.) were dissolved in 50 mL of glacial acetic acid and stirred for 1 h at 100 °C. After cooling, water (50 mL) was added and the mixture was extracted twice with ethyl acetate. The organic phase was washed once with water and several times with saturated NaHCO3 solution, dried over MgSO4, and the solvent was removed. The brown oil was used in the next step without further purification.

[0055] Compound 12 (1.00 g, 4.46 mmol, 1.0 eq.) was dissolved in 100 mL of dry DMF and treated with NaBH4 (0.84 g, 22.30 mmol, 5.0 eq.). The mixture was stirred for 2 h at 100 °C. Under ice cooling, the solution was adjusted to pH 2 with 1 M HCl and extracted twice with ethyl acetate. The solvent was removed, and 0.10 g of the pale green residue was dissolved in 50 mL of dry DCM. After adding acetone (0.06 mL, 0.86 mmol, 1.5 eq.) and boron trifluoride diethyl etherate (0.23 mL, 48% solution, 0.86 mmol, 1.5 eq.), the mixture was stirred overnight. Saturated NaHCO3 solution. was added and the phases separated. The aqueous phase was extracted twice with DCM, and the combined organic phases were dried over MgSO4, concentrated, and purified by flash chromatography (PE:EE 10:1). 10 mg of compound 14 (1%, 0.04 mmol) was obtained as a white solid.

[0056] To a solution of 14 (10 mg, 0.04 mmol, 1.0 eq.) and TMEDA (7 µL, 0.1 mmol, 2.5 eq.) in 2 mL of dry hexane, n-BuLi (35 µL, 2.5 M in hexane, 0.08 mmol, 2.1 eq.) was added at 0 °C and stirred for 1 h. Dry DMF (8 µL, 0.1 mmol, 2.5 eq.) was then added and stirred for another hour at 0 °C. After adding 1 M HCl, the phases were separated and the aqueous phase was extracted twice with ethyl acetate. The combined organic phases were dried over MgSO4 and the solvent was removed. After purification by flash chromatography (PE:EE 10:1) 1 mg of compound VI-1 (10%, 0.004 mmol) was obtained as a red solid. Example 4

[0057] Sesamol (1.0 g, 7.24 mmol, 1.0 eq.) was placed in 10 mL of dry DMF and stirred at room temperature for 16 h after the addition of dimethylthiocarbamoyl chloride (1.34 g, 10.9 mmol, 1.5 eq.) and DABCO (1.22 g, 10.9 mmol, 1.5 eq.). Upon addition of 50 mL of water and 50 mL of ethyl acetate, the phases were separated, and the aqueous phase was extracted twice further with ethyl acetate. The combined organic phases were washed with brine and dried over MgSO4. After removal of the solvent, the residue was purified by flash chromatography (PE:EE 3:1) to yield 1.53 g of compound 16 (94%, 6.79 mmol) as a colorless oil.

[0058] Compound 16 (1.0 g, 4.44 mmol, 1.0 eq.), p-benzoquinone (0.53 g, 4.88 mmol, 1.1 eq.), p-toluenesulfonic acid monohydrate (0.08 mg, 0.44 mmol, 0.1 eq.), and palladium(II) acetate (0.05 mg, 0.22 mmol, 0.05 eq.) were added to a mixture of 4 mL glacial acetic acid and 4 mL toluene and stirred at room temperature for 10 min, followed by incubation at 120 °C for a further 14 h. All volatile compounds were removed from the black suspension, and the residue was purified by flash chromatography (PE:EE 3:1). 0.18 g of compound 17 (21%, 0.92 mmol) was isolated as a white solid.

[0059] Compound 17 (130 mg, 0.66 mmol, 1.0 eq.) was suspended in a mixture of 2 mL methanol and 2 mL water and stirred for 2 h at 60 °C after the addition of NaOH (79 mg, 1.99 mmol, 3.0 eq.). The now clear solution was adjusted to pH 2 with 1 M HCl and extracted twice with ethyl acetate. The combined organic phases were concentrated, and the residue was dissolved in 4 mL DMF. Dry K₂CO₃ (446 mg, 3.23 mmol, 5.0 eq.) and bromochloromethane were added, and the suspension was stirred for 3 h at 90 °C and then for a further 14 h at room temperature. The reaction solution was acidified with 1 M HCl (pH 2) and extracted three times with ethyl acetate. The combined organic phases were washed with brine and dried over MgSO₄. After purification of the crude product by flash chromatography (PE:EE 10:1) 52 mg of compound 19 (45%, 0.285 mmol) was obtained as a white solid.

[0060] Compound 19 (20 mg, 0.109 mmol, 1.0 eq.) was dissolved in 3 mL of dry hexane and, after the addition of TMEDA (0.03 mL, 0.219 mmol, 2.0 eq.) and n-BuLi (2.5 M in hexane, 0.09 mL, 0.230 mmol, 2.1 eq.), stirred for 1 h at 0 °C. Subsequently, dry DMF (0.02 mL, 0.275 mmol, 2.5 eq.) was added to the solution and stirred for another hour at 0 °C. The reaction was stopped by the addition of 1 M HCl, and the phases were separated. The aqueous phase was extracted once with ethyl acetate, and the combined organic phases were dried and concentrated over MgSO4. After purification by flash chromatography (PE:EE 5:1) 2 mg of compound (IX-13) (7%, 0.01 mmol) were obtained as a red solid. Comparative examples

[0061] The novel fluorescent dyes (II-1), (VI) and (IX-13) show improved fluorescence properties compared to the following structurally related compounds (S 4 -DBD-1) and (DBD-1):

[0062] Table 1 summarizes the determined fluorescence properties of the investigated fluorescent dyes and the Fig. Figures 1-4 illustrate the photophysical properties. Table 1: Photophysical properties of (II-1), (S4-DBD-1), (DBD-1), (VI-1) and (IX-13) in acetonitrile. Fluorophor l Abs [nm] l Em [nm] Δλ[nm] t F [n.s.] F F e[M -1 cm -1 ] II-1 504 637 133 9.8 0.21 4760 S 4 -DBD-1 522 643 121 3.3 0.09 5091 DBD-1 475 609 134 17.7 0.32 2950 VI-1 520 620 100 16.8 0.52 4080 IX-13 500 618 118 17.9 0.43 4220

[0063] The fluorescent dyes were measured in acetonitrile. Table 1 shows that the fluorescent dye according to the invention is particularly advantageous compared to previously known compounds, especially due to its large Stockes shift combined with large absorption and emission wavelengths.

Claims

[1] A fluorescent dye according to formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X) and (XI): with R 1 selected from the group nitrile, nitro, formyl, carboxyl, substituted or unsubstituted C2-C 20 Acylalkyl, substituted or unsubstituted C5-C 20 Acylaryl, substituted or unsubstituted C2-C 20 Acyloxy, substituted or unsubstituted C2-C 20 Ester, substituted or unsubstituted C2-C 20 Amide with R 2 selected from the group hydrogen, nitrile, nitro, formyl, carboxyl, substituted or unsubstituted C2-C 20 Acylalkyl, substituted or unsubstituted C5-C 20 Acylaryl, substituted or unsubstituted C2-C 20 Acyloxy, substituted or unsubstituted C2-C 20 Ester, substituted or unsubstituted C2-C 20 Amide with R3 , R 4 , R 5 and R 6 independently selected from the group of substituted or unsubstituted C1-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkyl ethers and substituted or unsubstituted C6-C 20 Aryl; and with Z and X selected independently from a substituted or unsubstituted methylene or a substituted or unsubstituted ethane-1,2-diyl, where in formulas (I), (II), (III), (VI), (VII), (VIII), (IX) and (X) Z and X represent the substituted methylene and the substituted ethane-1,2-diyl with at least one substituted or unsubstituted C1-C 20 Alkyl and / or with a substituted or unsubstituted C2-C 20 Esters are substituted. [2] Fluorescent dye according to claim 1, wherein Z and X in formulas (I), (II), (III), (VI), (VII), (VIII), (IX) and (X) correspond to a substituted or unsubstituted methylene. [3] Fluorescent dye according to claim 1, wherein in formulas (I), (II), (III), (VI), (VII), (VIII), (IX) and (X), Z and X are selected equally. [4] Fluorescent dye according to claim 1, wherein R 1 and R 2 are selected the same way. [5] Fluorescent dye according to claim 1, wherein the substituted C2-C 20 Acylalkyl comprises a trifluoroacetyl, and wherein the unsubstituted C2-C 20 Acylalkyl comprises a residue from the group: acetyl, propionyl, butyryl and valeryl. [6] Fluorescent dye according to claim 1 selected from the group comprising the derivatives: and [7] Fluorescent dye according to claim 1 selected from the group comprising the derivatives: [8] Conjugate comprising a fluorescent dye according to any one of claims 1 to 7 and a biomolecule coupled to the fluorescent dye or a cell coupled to the fluorescent dye. [9] The conjugate according to claim 8, wherein the biomolecule comprises from the group comprising: Proteins, peptides, nucleic acids and lipids have been selected.

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