Heterobifunctional Dyes: Highly Fluorescent Linkers Based on Cyanine Dyes

Abstract

Herein, we present a new synthetic route to cyanine‐based heterobifunctional dyes and their application as fluorescent linkers between polymers and biomolecules. The synthesized compounds, designed in the visible spectral range, are equipped with two different reactive groups for highly selective conjugation under physiological conditions. By applying indolenine precursors with functionalized benzenes, we achieved water‐soluble asymmetric cyanine dyes bearing maleimido and N‐hydroxysuccinimidyl functionalities in a three‐step synthesis. Spectroscopic characterization revealed good molar absorption coefficients and moderate fluorescence quantum yields. Further reaction with polyethylene glycol yielded dye–polymer conjugates that were subsequently coupled to the antibody cetuximab, often applied in cancer therapy. Successful coupling was confirmed by mass shifts detected by gel electrophoresis. Receptor‐binding studies and live‐cell imaging revealed that labeling did not alter the biological function. In sum, we provided a successful synthetic pathway to rigid heterobifunctional cyanine dyes that are applicable as fluorescent linkers, for example, for connecting antibodies with macromolecules. Our approach contributes to the field of bioconjugation chemistry, such as antibody–drug conjugates by combining diagnostic and therapeutic approaches.

Synthesis of a New Amino-Furopyridine-Based Compound as a Novel Fluorescent pH Sensor in Aqueous Solution

“…Development of small fluorescent molecules, − especially pH-sensitive fluorescent dyes, has gained substantial attention owing to their promising applications in various fields, including analytical chemistry, , biomedicinal research, − clinical diagnostics, − biology, − and environmental science . They are used as strainers, biomolecular labels, , environmental indicators, etc.…”

GM1-Binding Conjugates To Improve Intestinal Permeability

“…Finally, a scrambled version of G23 was used to check the nonspecific affinity of the conjugates for GM1. , We selected a fluorophore (Cy5-NHS or FITC) as a model drug since it enables straightforward fluorescence detection and flow cytometry. The fluorophores (Cy5-NHS or FITC) were added on the amino group of a heterobifunctional linker based on a triethylene glycol unit (Scheme ), frequently used in linker design due to its flexibility and good water-solubility . The peptides (P3, G23, Scr) were condensed through their appended cysteine to the maleimide function of the linker to yield peptide conjugates FL1 or FL2 (Scheme ).…”

“…The fluorophores (Cy5-NHS or FITC) were added on the amino group of a heterobifunctional linker based on a triethylene glycol unit (Scheme 1), frequently used in linker design due to its flexibility and good water-solubility. 41 The peptides (P3, G23, Scr) were condensed through their appended cysteine to the maleimide function of the linker to yield peptide conjugates FL1 or FL2 Measured by microscale thermophoresis (MST). See Supporting Information and Figure S1.…”

Cyanine Conjugate‐Based Biomedical Imaging Probes

“…Combinatorial chemistry libraries, especially innovative dynamic combinatorial library and DNA-encoded library, commonly involved in target-based drug discovery are also applicable to ligand discovery as their underlying principles are interchangeable. [34] Additionally, biological screening platforms that rely on miscellaneous display systems, either cellular or acellular, to construct genetic mutant libraries www.advancedsciencenews.com www.advhealthmat.de (13)(14)(15) or its biomimetics (16)(17)(18)(19)(20) as targeting warheads. b) 3D image of mouse administered with conjugate 15 using fluorescence imaging tomography (FLIT) mode and confirmation of tumor cells in the primary tumor and four metastatic lymph nodes through H&E staining or cytokeratin immunostaining.…”

“…Licha's group designed (5a–d) by sequential conjugating such cyanine frameworks with PEG 5kDa and cetuximab via terminal maleimide and NHS ester groups, respectively. [ 15 ] All of the four conjugates showed characteristic cell–membrane binding and accumulation inside cellular compartments in EGFR‐positive A431 cells. They further envisioned that the heterobifunctional strategy could be extended for simultaneous targeted imaging and therapy by conjugating a targeting warhead and a therapeutic agent.…”