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DE-102020114139-B4 - Thiopolyphenol-based fluorescent dyes and conjugates thereof

DE102020114139B4DE 102020114139 B4DE102020114139 B4DE 102020114139B4DE-102020114139-B4

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 consisting of nitrile, nitro, formyl, carboxyl, substituted or unsubstituted C 2 -C 20 acylalkyl, substituted or unsubstituted C 5 -C 20 acylaryl, substituted or unsubstituted C2 - C20 acyloxy, substituted or unsubstituted C2 - C20 ester, substituted or unsubstituted C2 - C20 amide with R 2 selected from the group 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 C2 - C20 ester, substituted or unsubstituted C2 - C20 amide with R 3 , R 4 , R 5 and R 6 independently selected from the group of substituted or unsubstituted C 1 -C 20 alkyl, substituted or unsubstituted C 2 -C 20 Alkyl ethers and substituted or unsubstituted C6 - C20 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 C 1 -C 20 alkyl and/or are substituted with a substituted or unsubstituted C 2 -C 20 ester.

Inventors

  • Pablo Wessig
  • Leonard John

Assignees

  • UNIVERSITÄT POTSDAM

Dates

Publication Date
20260513
Application Date
20200527

Claims (9)

  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 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 with R 2 selected from the group 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 with R 3 , R 4 , R 5 and R 6 independently selected from the group of substituted or unsubstituted C 1 -C 20 alkyl, substituted or unsubstituted C 2 -C 20 alkyl ether 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, wherein in formulas (I), (II), (III), (VI), (VII), (VIII), (IX) and (X) Z and X are substituted methylene and substituted ethane-1,2-diyl respectively, with at least one substituted or unsubstituted C 1 -C 20 alkyl and/or with a substituted or unsubstituted C 2 -C 20 ester.
  2. Fluorescent dye according to Claim 1 , where 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 , where in formulas (I), (II), (III), (VI), (VII), (VIII), (IX) and (X), Z and X are selected as the same.
  4. Fluorescent dye according to Claim 1 , where R1 and R2 are selected equally.
  5. 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 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 one of the Claims 1 until 7 and a biomolecule coupled to the fluorescent dye or a cell coupled to the fluorescent dye.
  9. The conjugate after Claim 8 , where the biomolecule is selected from the group comprising: proteins, peptides, nucleic acids and lipids.

Description

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 Fluorescence is the spontaneous emission of light during the transition of an electronically excited system 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. Fluorescent dyes are characterized by a number of parameters that allow the user to select a suitable dye depending on the intended use. The most important parameters to mention here are the excitation wavelength λ <sub>exc</sub> (or λ <sub>Abs</sub> ), which corresponds to the maximum of the absorption band; the emission wavelength λ <sub>em </sub>, which corresponds to the maximum of the emission band; the Stokes shift Δλ, which corresponds to the difference between the emission wavelength λ <sub>em</sub> and the excitation wavelength λ <sub>exc</sub> ; the extinction coefficient ε, which reflects the proportion of radiation absorbed at the excitation wavelength λ <sub>exc</sub> ; the fluorescence quantum yield Φ<sub> F </sub>, which corresponds to the ratio of the number of emitted to absorbed photons; and the fluorescence lifetime τ<sub> F </sub>, which corresponds to the average time the molecule remains in its excited state before transitioning to its ground state through the emission of a photon. Desirable properties, especially for biological applications, include a long excitation wavelength λ <sub>exc</sub> to allow the excitation radiation to penetrate biological samples as deeply as possible, a large extinction coefficient ε to maximize the absorption of the incident light, a large Stokes shift Δλ to minimize interactions between the excitation and emission radiation, a long fluorescence lifetime τ<sub> F </sub> to mask the short-lived natural background fluorescence of biological tissues, and finally, a high fluorescence quantum yield Φ<sub> F </sub> to achieve the highest possible signal-to-noise ratio. Most currently known fluorescent dyes have drawbacks with respect to certain parameters, so there is a constant 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 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. 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 catechol 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 ). 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 A new group of fluorescent dyes is provided whose spectral properties particularly meet the aforementioned requirements with regard to biological applications. Suitable fluorescent dyes include derivatives of the general formulas (I), (II), (III), (IV) (hereinafter referred to as 1,2-S 2 -DBD fluorescent dyes): Furthermore, 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): Furthermore, the fluorescent dyes according to the invention comprise derivatives of the general formulas (VIII), (IX), (X) and (XI) (hereinafter referred to as S 1 -DBD fluorescent dyes): The residue R 1 in the aforementioned formulas is selected from the group consisting of 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. The R