DE102020114139A1 - Thiopolyphenol-based fluorescent dyes - Google Patents

Abstract

The invention relates to novel fluorescent dyes based on oxygen- and sulfur-containing heterocycles. The invention also relates to a conjugate of said fluorescent dye and biomolecules and a method for synthesizing the fluorescent dyes according to the invention.

Description

The invention relates to novel fluorescent dyes based on thiopolyphenol derivatives which have one or two thio substituents. The invention also relates to a conjugate of said fluorescent dye and biomolecules and a method for synthesizing the fluorescent dyes according to the invention.

Technological background

Fluorescence is the spontaneous emission of light when an electronically excited system changes into a state of lower energy. Systems, i.e. materials that have such fluorescent properties, are called fluorophores. If these are used for coloring, they are called fluorochromes or fluorescent dyes. Fluorescent dyes have a wide range of uses in biochemistry, biology and medicine, e.g. in diagnostic kits, as markers in the structure determination of proteins or in drug screening.

Fluorescent dyes are characterized by a number of parameters that allow the user to select a suitable dye depending on the intended use. Above all, the excitation wavelength λ _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 (Stokes shift) Δλ, which is the difference The emission wavelength λ _em and the excitation wavelength λ _exc , the extinction coefficient ε, which _{reflects the proportion of the radiation absorbed at the excitation wavelength λ exc} , the fluorescence quantum yield Φ _F , which corresponds to the quotient of the number of emitted photons to be absorbed, and the fluorescence lifetime τ _F , which corresponds to the mean time in which the molecule remains in its excited state before it passes into its ground state through the emission of a light quantum.

_{A large excitation wavelength λ exc is} desirable, especially with regard to biological applications, in order to be able to penetrate as deep as possible into biological samples 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 the least possible interactions between the excitation and emission _{radiation, a long fluorescence lifetime τ F} in order to be able to mask out the short-lived natural background _{fluorescence of biological tissue, and finally a large fluorescence quantum yield Φ F} in order to achieve the highest possible signal-to-noise ratio . Most of the previously known fluorescent dyes have disadvantages in certain parameters, so that there is always a need for new fluorescent dyes.

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

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.

There is a continuing need for new fluorescent dyes and more efficient synthesis methods. In particular, the dyes should have long emission and excitation wavelengths as well as a long fluorescence lifetime and a large Stokes shift.

Summary of the invention

A new group of fluorescent dyes is provided, the spectral properties of which in particular meet the above-mentioned requirements with regard to biological applications. Fluorescent dyes according to the invention include derivatives of the general formulas (I), (II), (III), (IV) (hereinafter referred to as 1,2-S ² -DBD fluorescent dyes):

In addition, the fluorescent dyes according to the invention comprise derivatives of the general formulas (V), (VI) and (VII) (hereinafter referred to as 1,4-S ² -DBD fluorescent dyes):

The fluorescent dyes according to the invention also include derivatives of the general formulas (VIII), (IX), (X) and (XI) (hereinafter referred to as S ¹ -DBD fluorescent dyes):

The radicals R ¹ and R ² in the aforementioned formulas are from the group comprising: hydrogen, nitrile, nitro, formyl, carboxyl, substituted or unsubstituted C ₂ -C ₂₀ acylalkyl, substituted or unsubstituted C ₅ -C ₂₀ acylaryl, substituted or unsubstituted C ₂ -C ₂₀ acyloxy, substituted or unsubstituted C ₂ -C ₂₀ ester, substituted or unsubstituted C ₂ -C ₂₀ amide and substituted or unsubstituted C ₁ -C ₂₀ alkoxy are selected independently of one another. In addition, the radicals R ³ , R ⁴ , R ⁵ and R ^{6 are} independently selected from the group comprising: hydrogen, substituted or unsubstituted C ₁ -C ₂₀ alkyl, substituted or unsubstituted C ₂ -C ₂₀ alkyl ethers and substituted or unsubstituted C ₆ -C ₂₀ aryl. The (ring-forming) radicals Z and X are independently selected from a substituted or unsubstituted methylene or a substituted or unsubstituted ethane-1,2-diyl, where in the formulas (I), (II), (III), (VI) , (VII), (VIII), (IX) and (X) for Z and X, the substituted methylene and the substituted ethane-1,2-diyl with at least one substituted or unsubstituted C ₁ -C ₆ alkyl and / or with one substituted or unsubstituted C ₂ -C ₂₀ esters are substituted.

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). The new class of fluorescent dyes offers compared 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 (p ⁴ -DBD), among other things, high excitation and emission wavelengths without any significant reduction in the Stokes shift. Thus, the disadvantage of a reduced Stokes shift, which was caused by the complete substitution of the oxygen atoms of the DBD by sulfur (see S ⁴ -DBD), could be eliminated with almost no loss.

The fluorescent dyes according to the invention are therefore particularly suitable for applications in the field of biology and biochemistry (for example for the structure elucidation of complex proteins or cells), which have a require a particularly clear signal and deep penetration into the biological sample, the clarity of the signal being ensured by the low interaction between excitation and emission light.

Z and X in the formulas (I), (II), (III), (VI), (VII), (VIII), (IX), (X) can particularly preferably correspond to a substituted or unsubstituted methylene. In addition, Z and X in formulas (I), (II), (III), (VI), (VII), (VIII), (IX), (X) can preferably be selected to be the same.

In a preferred embodiment, R ¹ and R ² can be selected to be the same (identical).

In a further preferred embodiment, the substituted C ₂ -C ₂₀ acylalkyl can comprise a trifluoroacetyl. _{The unsubstituted C 2} -C ₂₀ acylalkyl, for example, can also comprise a radical from the group: acetyl, propionyl, butyryl and valeryl.

und

The following derivatives represent preferred embodiments of the fluorescent dyes according to the invention:

and

The following derivatives of the fluorescent dyes according to the invention are further particularly preferred embodiments:

Another aspect of the present disclosure relates to a conjugate comprising a fluorescent dye as described above and a biomolecule coupled to the fluorescent dye or a cell coupled to the fluorescent dye. Here, the biomolecule can preferably be selected from the group of proteins, peptides, nucleic acids and lipids.

Figure list

• 1 - 4 Balkendiagramme zu photophysikalischen Daten von zwei bekannten und drei neuen Fluoreszenzfarbstoffe (II-1), (S⁴-DBD-1), (DBD-1), (VI-1) und (IX-13) in Acetonitril;
• 5A/5B Absorptions- und Emissionsspektrum des Fluoreszenzfarbstoffs (II-1) gemessen in Acetonitril;
• 6A/6B Absorptions- und Emissionsspektrum des Fluoreszenzfarbstoffs (VI-1) gemessen in Acetonitril; und
• 7A/7B Absorptions- und Emissionsspektrum des Fluoreszenzfarbstoffs (IX-13) gemessen in Acetonitril.

The invention is explained in more detail below using an exemplary embodiment and the associated drawings. The figures show:

• 1 - 4th Bar graphs of 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;
• 5A / 5B Absorption and emission spectrum of the fluorescent dye (II-1) measured in acetonitrile;
• 6A / 6B Absorption and emission spectrum of the fluorescent dye (VI-1) measured in acetonitrile; and
• 7A / 7B Absorption and emission spectrum of the fluorescent dye (IX-13) measured in acetonitrile.

Detailed description of the invention

In connection with the present invention, “C ₁ -C ₂₀ alkyl” is understood to mean a linear or branched alkyl radical which has the general formula C _n H _{2n + 1} , where n = 1 to 20. C ₁ -C ₅ alkyl includes, for example, 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 and 1-ethylpropyl. Examples of alkyl radicals with n ≥ 6 include n-hexane, n-heptane, n-octane, n-nonane, n-decane, 1-methylpentyl, 1-methylhexyl, 1-methylheptyl, 1-methyloctyl, 1-methylnonyl, 1- Methyldecanyl, 1-ethylbutyl, 1-ethylpentyl, 1-ethylhexyl, 1-ethylheptyl, 1-ethyl octyl, 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-ethyl octyl, 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-methylp entyl, 1-ethyl-2-methylhexyl, 1-ethyl-2-methylheptyl, 1-ethyl-2-methyloctyl, 1-ethyl-2-methylnonyl and 1-ethyl-2-methyldecanyl.

In connection with the present invention, “C ₂ -C ₂₀ acylalkyl” is understood to mean a linear or branched alkyl radical of the meaning given above, which is covalently bonded to the fluorescent dye via a carbonyl group. Examples include acetyl, propionyl, butyryl, and valeryl.

In connection with the present invention, “C ₆ -C ₂₀ aryl” is understood to mean an aromatic radical having 6 to 20 carbon atoms. Examples include phenyl, pentalenyl, indenyl, naphthyl, biphenyl, fluorenyl, phenanthrenyl, pyrenyl and perylenyl. The aryl radicals can for their part be _{substituted one or more times by C 1} -C ₅ alkyl radicals of the meaning given above. Examples of such substituted aryl radicals include tolyl, xylyl, pseudocumyl and mesityl.

In connection with the present invention, “C ₅ -C ₂₀ acylaryl” is understood to mean an aryl radical of the meaning given above, which is covalently bonded to the fluorescent dye via a carbonyl group. One example is benzoyl.

In connection with the present invention, “C ₂ -C ₂₀ acyloxy” is understood to mean an ester which is covalently bonded to the fluorescent dye via the oxygen atom and whose carbonyl carbon-bonded branched or unbranched alkyl radical comprises 1 to 19 carbon atoms. An example is acetyloxy.

In connection with the present invention, “C ₂ -C ₂₀ ester” is understood to mean 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.

In connection with the present invention, “C ₂ -C ₂₀ amide” is understood to mean a carboxamide which is covalently bonded to the fluorescent dye via the carbonyl carbon and whose nitrogen atom-bonded branched or unbranched alkyl radical comprises 1 to 19 carbon atoms. Examples include methylaminocarbonyl, ethylaminocarbonyl, propylaminocarbonyl, butylaminocarbonyl, and pentylaminocarbonyl.

In connection with the present invention, “C ₁ -C ₁₅ alkoxy” is understood to mean a functional group which is based on a linear or branched alkyl group of the meaning given above, linked to an oxygen atom. Examples include methoxy and ethoxy.

The term “substituted”, as used herein in relation to acylalkyl, acylaryl, acyloxy, ester, amide, alkoxy, alkylether and aryl, refers to the fact that in these groups one, more or all of the hydrogen atoms are replaced by fluorine, chlorine, bromine and iodine are replaced.

Fluor, Chlor, Brom und lod, ersetzt ist.The term “substituted”, as it is used 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.

The term "substituted", as used herein in relation to methylene and ethane-1, 2-diyl of the radicals Z and X, refers to the fact that in these groups one, more or all of the hydrogen atoms are replaced by substituted or unsubstituted C ₁ - C ₂₀ alkyl (as defined above) or substituted or unsubstituted C ₂ -C ₂₀ esters (as defined above).

General synthesis instructions

und den Schritt B), der anschließenden Reduktion des ersten Intermediates aus Schritt A zur Bildung eines zweiten Intermediates gemäß Formel (B1) oder (B2):

A method for synthesizing the fluorescent dyes according to the invention is described below. The process comprises step A), the reaction of a catechol derivative (e.g. veratrole or 1,2-methylenedioxybenzene) with S ₂ Cl ₂ 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 process mentioned, the radicals R ⁵ and R ^{6 are} from the group comprising hydrogen, substituted or unsubstituted C ₁ -C ₂₀ alkyl, substituted or unsubstituted C ₂ -C ₂₀ alkyl ethers and substituted or unsubstituted C ₆ -C ₂₀ aryl, independently of one another selected. In addition, 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 C ₁ -C ₂₀ Alkyl and / or substituted by a substituted or unsubstituted C ₂ -C ₂₀ ester. In the reduction step (reduction in step B), for example, reducing agents comprising NaBH ₄ , KBH ₄ , NMe ₄ BH ₄ and NaAlH ₄ can be used. Preferably NaBH ₄ can be used.

Furthermore, the aforementioned method can comprise the following further steps C) and D). Step C) corresponds to a ring closure reaction of the second intermediate from step B to form a third intermediate according to formula (C1) or (C2):

The ring closure reaction can _{comprise an S N} reaction or acetalization. Examples of the ring closure reaction can include reaction with CH ₂ BrCl or with acetone.

Step D) corresponds to an (electrophilic) aromatic substitution on the third intermediate from step C:

In process steps B and D, the radicals R ¹ and R ^{2 are selected} from the group comprising hydrogen, nitrile, nitro, formyl, carboxyl, substituted or unsubstituted C ₂ -C ₂₀ acylalkyl, substituted or unsubstituted C ₅ -C ₂₀ acylaryl, substituted or unsubstituted C ₂ -C ₂₀ acyloxy, substituted or unsubstituted C ₂ -C ₂₀ ester, substituted or unsubstituted C ₂ -C ₂₀ amide and substituted or unsubstituted C ₁ -C ₁₅ alkoxy are selected independently of one another. In addition, the radicals R ⁵ and R ^{6 are} independently selected from the group comprising: hydrogen, substituted or unsubstituted C ₁ -C ₂₀ alkyl, substituted or unsubstituted C ₂ -C ₂₀ alkyl ethers and substituted or unsubstituted C ₆ -C ₂₀ aryl. The radicals Z and X are independently selected from a substituted or unsubstituted methylene or a substituted or unsubstituted ethane-1,2-diyl, where for Z and X the substituted methylene and the substituted ethane-1,2-diyl with at least one substituted one or unsubstituted C ₁ -C ₂₀ alkyl and / or substituted by a substituted or unsubstituted C ₂ -C ₂₀ ester. In one embodiment, the aromatic substitution can take place by means of dimethylformamide (DMF) and an organyl lithium compound (for example n-butyllithium). Further embodiments of the (electrophilic) aromatic substitution can include Friedel-Crafts alkylations, Friedel-Crafts acylations and nitration.

Furthermore, based on the intermediates from step B, the method can comprise 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):

The S-functionalization can include, for example, alkylations on the sulfur atom (for example with methyl iodide, methyl triflate or other alkyl reagents). Step F) corresponds to an (electrophilic) aromatic substitution on the fourth intermediate from step E:

In process steps E and F, the radicals R ¹ and R ^{2 are selected} from the group comprising hydrogen, nitrile, nitro, formyl, carboxyl, substituted or unsubstituted C ₂ -C ₂₀ acylalkyl, substituted or unsubstituted C ₅ -C ₂₀ acylaryl, substituted or unsubstituted C ₂ -C ₂₀ acyloxy, substituted or unsubstituted C ₂ -C ₂₀ ester, substituted or unsubstituted C ₂ -C ₂₀ amide and substituted or unsubstituted C ₁ -C ₁₅ alkoxy are selected independently of one another. In addition, the radicals R ³ , R ⁴ , R ⁵ and R ^{6 are} independently selected from the group comprising: hydrogen, substituted or unsubstituted C ₁ -C ₂₀ alkyl, substituted or unsubstituted C ₂ -C ₂₀ alkyl ethers and substituted or unsubstituted C ₆ -C ₂₀ aryl. The radical 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 C ₁ -C _{20 are} alkyl and / or substituted by a substituted or unsubstituted C ₂ -C ₂₀ ester.

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

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

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

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

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

Biofunctionalization

• Carbonsäuren (vgl. II-9), Lysin, Methionin, N-Heterozyklen (vgl. 11-10), Alkine durch Click-Reaktion (vgl. II-11), Thiole wie Cystein (vgl. II-12), und Azide durch Click-Reaktion (vgl. II-13).

The derivatives (II-9) to (11-13) shown in the following scheme are suitable, for example, for coupling with the following functional groups occurring in biomolecules:

• Carboxylic acids (cf. II-9), lysine, methionine, N-heterocycles (cf. 11-10), alkynes by the click reaction (cf. II-11), thiols such as cysteine (cf. II-12), and azides by the click reaction (cf. II-13).

The derivatives can be functionalized by halogen atoms, for example, starting from 11-10 by reacting them with potassium halides (Finkelstein reaction).

example 1

Benzo [2,3,8,9] dioxole-1,2,5,6-tetrathiocine

1,3-Benzodioxol (2 mL, 17.60 mmol, 1.0 eq.) wurden in 40 mL Eisessig vorgelegt, mit Schwefelmonochlorid versetzt (1.4 mL, 17.60 mmol, 1.0 eq.) und für 24 h bei Raumtemperatur gerührt. Der entstandene gelbe Feststoff wurde abgesaugt und mit 50 mL Diethylether gewaschen. Nach dem Trocknen im Vakuum wurden 2.69 g von Verbindung 2 (83%,7.30 mmol) als gelber Feststoff erhalten.

1,3-Benzodioxole (2 mL, 17.60 mmol, 1.0 eq.) Was placed in 40 mL of glacial acetic acid, mixed with sulfur monochloride (1.4 mL, 17.60 mmol, 1.0 eq.) And stirred for 24 h at room temperature. The yellow solid formed was filtered off with suction and washed with 50 mL diethyl ether. After drying in vacuo, 2.69 g of compound 2 (83%, 7.30 mmol) were obtained as a yellow solid.

Benzo [d] [1,3] dioxole-5,6-dithiol

(50 mg, 0.13 mmol, 1.0 eq.) Were suspended in dry DMF (15 ml) and NaBH ₄ (25 mg, 0.63 mmol, 5.0 eq.) Was added. The yellow mixture was stirred for 30 min at room temperature and heated to 100 ° C. for 1 h. After the colorless solution had cooled, H ₂ O (20 mL) was added to the solution and the solution was extracted with DCM (20 mL). The aqueous phase was adjusted to pH = 2 with 1M HCl and extracted again with DCM (3x 20 mL). The combined organic phases were concentrated on a rotary evaporator, toluene was added twice to the crude product and the solvent was removed in each case, so that (I-1) was isolated as a pale green oil in 50 mg (quant., 0.25 mmol).

6,6-dimethyl- [1,3] dithiolo [4 ', 5': 4.5] benzo [1,2-d] [1,3] dioxole

The dithiol 3 (50 mg, 0.22 mmol, 1.0 eq.) Was dissolved in 10 mL 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.) admitted. The greenish-clear solution was stirred for 14 h at room temperature. The reaction was ended by adding saturated NaHCO _{3 solution.} The phases were separated and the aqueous phase was extracted twice with DCM (20 mL). The combined organic phases were _{dried over MgSO 4} and the solvent was removed on a rotary evaporator. After purification by means of flash chromatography (PE: EE 5: 1), 50 mg (50%, 0.20 mmol) of 4 were obtained as a white solid.

6,6-Dimethyl- [1,3] dithiolo [4 ', 5': 4.5] benzo [1,2-d] [1,3] dioxole-4,8-dicarbaldehyde

Compound 4 (50 mg, 0.22 mmol, 1 eq.) Was suspended in 3 mL dry hexane and treated with TMEDA (0.07 mL, 0.44 mmol, 2.0 eq.). Then 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 a further hour at room temperature. The reaction was terminated by adding 1M HCl, the phases were separated and the aqueous phase was extracted twice with ethyl acetate. The combined organic phases were _{dried with MgSO 4} and the solvent was removed. After purification by means of flash chromatography (PE: EE 3: 1), 36 mg (58%, 0.13 mmol) of compound (II-1) were obtained as a red solid.

Example 2

3- (6-Methyl- [1,3] dithiolo [4 ', 5': 4.5] benzo [1,2-d] [1,3] dioxol-6-yl) propyl pivalate

To a solution of compound 2 (3.20 g, 17.18 mmol, 1.0 eq.) In 60 mL dry DCM, 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.) And stirred for 16 h at room temperature. The mixture was _{washed with saturated NaHCO 3} solution and the aqueous phase was extracted once with DCM. The combined organic phases were _{dried over MgSO 4} and the solvent was removed. After purification by means of flash chromatography (PE: EE 10: 1), 3.30 g of compound 20 (55%, 9.31 mmol) were obtained as a white solid.

3- (6-Methyl- [1,3] dithiolo [4 ', 5': 4.5] benzo [1,2-d] [1,3] dioxol-6-yl) propan-1-ol

Compound 20 (1.71 g, 4.82 mmol, 1.0 eq.) Were 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 for 15 h at room temperature, the solvent was removed and the residue was taken up in ethyl acetate. The organic phase was washed with 1MHCl and Brine and dried _{with MgSO 4.} The solvent was removed and the residue was purified by means of flash chromatography (PE: EE3: 1). 1.20 g of compound 21 (92%, 4.44 mmol) were obtained as a colorless oil.

6- (3-hydroxypropyl) -6-methyl- [1,3] dithiolo [4 ', 5': 4.5] benzo [1,2-d] [1,3] dioxole-4,8-dicarbaldehyde

A solution of 21 (100 mg, 0.37 mmol, 1.0 eq.) In 5 mL 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 addition of DMF (0.07 mL, 0.92 mmol, 2.5 eq.), The mixture was again stirred for 1 h at 0 ° C. and then the reaction was ended by adding 1M HCl (15 mL). The phases were separated and the aqueous phase was extracted twice with ethyl acetate. After drying with MgSO ₄ , the solvent was removed and the residue was purified by means of flash chromatography (PE: EE 1: 1). 42 mg of compound (II-9) (35%, 0.13 mmol) were obtained as a red solid.

Example 3

Polythiocyanate hydroquinone

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 glacial acetic acid and stirred at 100 ° C. for 1 h. After cooling, water (50 mL) was added and the mixture was extracted twice with ethyl acetate. The organic phase was once with water and several times with saturated NaHCO ₃ solution. washed, _{dried over MgSO 4} and the solvent removed. The brown oil was used in the next step without further purification.

2,2,6,6-Tetramethylbenzo [1,2-d: 4,5-d '] to [1,3] oxathiol

Compound 12 (1.00 g, 4.46 mmol, 1.0 eq.) Was dissolved in 100 mL dry DMF, and NaBH ₄ (0.84 g, 22.30 mmol, 5.0 eq.) Was added. The mixture was stirred at 100 ° C. for 2 h. While cooling with ice, the solution was adjusted to pH = 2 with 1M 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 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 NaHCO ₃ solution. was added and the phases separated. The aqueous phase was extracted twice with DCM and the combined organic phases were _{dried over MgSO 4} , concentrated and purified by means of flash chromatography (PE: EE 10: 1). 10 mg of compound 14 (1%, 0.04 mmol) were obtained as a white solid.

2,2,6,6-tetramethylbenzo [1,2-d: 4,5-d '] bis ([1,3] oxathiols) -4,8-dicarbaldehyde

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 dry hexane was added n-BuLi (35 µL, 2.5 M in hexane) at 0 ° C , 0.08 mmol, 2.1 eq.) And stirred for 1 h. Then dry DMF (8 μL, 0.1 mmol, 2.5 eq.) Was added and the mixture was stirred at 0 ° C. for a further hour. After 1M HCl had been added, the phases were separated and the aqueous phase was extracted twice with ethyl acetate. The combined organic phases were _{dried over MgSO 4} and the solvent was removed. After purification by means of flash chromatography (PE: EE 10: 1), 1 mg of compound VI-1 (10%, 0.004 mmol) was obtained as a red solid.

Example 4

O- (Benzo [d] [1,3] dioxol-5-yl) dimethyl carbamothioate

Sesamol (1.0 g, 7.24 mmol, 1.0 eq.) Was placed in 10 mL dry DMF and after addition of dimethylthiocarbamoyl chloride (1.34 g, 10.9 mmol, 1.5 eq.) And DABCO (1.22 g, 10.9 mmol, 1.5 eq.) For 16 stirred at room temperature for h. After adding 50 ml of water and 50 ml of ethyl acetate, the phases were separated and the aqueous phase was extracted twice more with ethyl acetate. The combined organic phases were washed with brine and dried _{over MgSO 4.} After removing the solvent, the residue was purified by means of flash chromatography (PE: EE 3: 1) and 1.53 g of compound 16 (94%, 6.79 mmol) was obtained as a colorless oil.

[1,3] Dioxolo [4 ', 5': 4.5] benzo [1,2-d] [1,3] oxathiol-6-one

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.) and stirred at room temperature for 10 min and a further 14 h at 120 ° C. All volatile compounds were removed from the black suspension and the residue was purified by means of flash chromatography (PE: EE 3: 1). 0.18 g of compound 17 (21%, 0.92 mmol) were isolated as a white solid.

[1,3] Dioxolo [4 ', 5': 4.5] benzo [1,2-d] [1,3] oxathiol

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

[1,3] Dioxolo [4 ', 5': 4.5] benzo [1,2-d] [1,3] oxathiols-4,8-dicarbaldehyde

Compound 19 (20 mg, 0.109 mmol, 1.0 eq.) Was dissolved in 3 mL dry hexane and, after 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.) for 1 h at 0 ° C. Then dry DMF (0.02 mL, 0.275 mmol, 2.5 eq.) Was added to the solution and the mixture was stirred at 0 ° C. for a further hour. The reaction was terminated by adding 1M HCl and the phases were separated. The aqueous phase was extracted once with ethyl acetate and the combined organic phases were _{dried over MgSO 4} and concentrated. After purification by means of flash chromatography (PE: EE 5: 1), 2 mg of compound (IX-13) (7%, 0.01 mmol) were obtained as a red solid.

Comparative examples

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

In Table 1, the determined fluorescence properties of the fluorescent dyes examined are summarized and the 1 - 4th illustrate the photophysical properties.

Table 1: Photophysical properties of (II-1), (S ⁴ -DBD-1), (DBD-1), (VI-1) and (IX-13) in acetonitrile. Fluorophore λ _Abs [nm] λ _Em [nm] Δλ [nm] τ _F [ns] Φ _F ε [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

The fluorescent dyes were measured in acetonitrile. From Table 1 it can be deduced that the fluorescent dye according to the invention is particularly advantageous, in particular due to the large Stockes shift with simultaneously large absorption and emission wavelengths, compared to the compounds already known.

QUOTES INCLUDED IN THE DESCRIPTION

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Patent literature cited

• EP 2399913 B1 [0005]
• DE 102017122275 A1 [0006]

Claims (9)

A fluorescent dye according to the formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X) and (XI):

with R ¹ and R ² independently selected from the group comprising: hydrogen, nitrile, nitro, formyl, carboxyl, substituted or unsubstituted C ₂ -C ₂₀ acylalkyl, substituted or unsubstituted C ₅ -C ₂₀ acylaryl, substituted or unsubstituted C ₂ - C ₂₀ acyloxy, substituted or unsubstituted C ₂ -C ₂₀ ester, substituted or unsubstituted C ₂ -C ₂₀ amide and substituted or unsubstituted C ₁ -C ₁₅ alkoxy; with R ³ , R ⁴ , R ⁵ and R ⁶ independently selected from the group comprising: hydrogen, substituted or unsubstituted C ₁ -C ₂₀ alkyl, substituted or unsubstituted C ₂ -C ₂₀ alkyl ethers and substituted or unsubstituted C ₆ -C ₂₀ Aryl; and with Z and X independently selected from a substituted or unsubstituted methylene or a substituted or unsubstituted ethane-1,2-diyl, where in the formulas (I), (II), (III), (VI), (VII) , (VIII), (IX) and (X) for Z and X the substituted methylene and the substituted ethane-1,2-diyl with at least one substituted or unsubstituted C ₁ -C ₂₀ alkyl and / or with a substituted or unsubstituted C. ₂ -C ₂₀ esters are substituted. Fluorescent dye according to Claim 1 , where Z and X in the formulas (I), (II), (III), (VI), (VII), (VIII), (IX) and (X) correspond to a substituted or unsubstituted methylene. Fluorescent dye according to Claim 1 , wherein in the formulas (I), (II), (III), (VI), (VII), (VIII), (IX) and (X), Z and X are selected to be the same. Fluorescent dye according to Claim 1 where R ¹ and R ² are selected to be the same. Fluorescent dye according to Claim 1 wherein the substituted C ₂ -C ₂₀ acylalkyl comprises a trifluoroacetyl, and wherein the unsubstituted C ₂ -C ₂₀ acylalkyl comprises a radical from the group: acetyl, propionyl, butyryl and valeryl. Fluorescent dye according to Claim 1 selected from the group comprising the derivatives:

and

Fluorescent dye according to Claim 1 selected from the group comprising the derivatives:

A conjugate comprising a fluorescent dye according to one of Claims 1 until 7th and a biomolecule coupled to the fluorescent dye or a cell coupled to the fluorescent dye. The conjugate after Claim 8 , wherein the biomolecule is selected from the group comprising: proteins, peptides, nucleic acids and lipids.

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