Linker Molecules Convert Commercial Fluorophores into Tailored Functional Probes during Biolabelling

• Published in: Angewandte Chemie International Edition Vol. 61; no. 19; pp. e202112959 - n/a
• Main Authors: Zhang, Lei, Isselstein, Michael, Köhler, Jens, Eleftheriadis, Nikolaos, Huisjes, Nadia M., Guirao‐Ortiz, Miguel, Narducci, Alessandra, Smit, Jochem H., Stoffels, Janko, Harz, Hartmann, Leonhardt, Heinrich, Herrmann, Andreas, Cordes, Thorben
• Format: Journal Article
• Language: English
• Published: WEINHEIM Wiley 02.05.2022; Wiley Subscription Services, Inc; John Wiley and Sons Inc
• Edition: International ed. in English
• Subjects: Biolabelling; Biomedical materials; Chemical compounds; Chemical synthesis; Chemistry; Chemistry, Multidisciplinary; DNA probes; DNA sequencing; Dyes; Fluorescence; Fluorescent Dyes - chemistry; Fluorophores; Healing; Ionophores; Labeling; Labels; Metal–Chelating Fluorophores; Modular design; Physical Sciences; Rhodamines - chemistry; Science & Technology; Self-Healing Dyes; Single Molecule Imaging; Spectroscopy; Super-Resolution Microscopy; Metal-Chelating Fluorophores; Self-Healing Dyes; Biolabelling; SINGLE; PROTEIN; SUPERRESOLUTION MICROSCOPY; CONFORMATIONAL DYNAMICS; ORGANIC FLUOROPHORES; INTRAMOLECULAR PHOTOSTABILIZATION; BIOCONJUGATION; SPECTROSCOPY; LIVE-CELL; Fluorophores; FLUORESCENT-PROBES; Super-Resolution Microscopy
• ISSN: 1433-7851; 1521-3773
• DOI: 10.1002/anie.202112959

Many life‐science techniques and assays rely on selective labeling of biological target structures with commercial fluorophores that have specific yet invariant properties. Consequently, a fluorophore (or dye) is only useful for a limited range of applications, e.g., as a label for cellular compartments, super‐resolution imaging, DNA sequencing or for a specific biomedical assay. Modifications of fluorophores with the goal to alter their bioconjugation chemistry, photophysical or functional properties typically require complex synthesis schemes. We here introduce a general strategy that allows to customize these properties during biolabelling with the goal to introduce the fluorophore in the last step of biolabelling. For this, we present the design and synthesis of ‘linker’ compounds, that bridge biotarget, fluorophore and a functional moiety via well‐established labeling protocols. Linker molecules were synthesized via the Ugi four‐component reaction (Ugi‐4CR) which facilitates a modular design of linkers with diverse functional properties and bioconjugation‐ and fluorophore attachment moieties. To demonstrate the possibilities of different linkers experimentally, we characterized the ability of commercial fluorophores from the classes of cyanines, rhodamines, carbopyronines and silicon‐rhodamines to become functional labels on different biological targets in vitro and in vivo via thiol‐maleimide chemistry. With our strategy, we showed that the same commercial dye can become a photostable self‐healing dye or a sensor for bivalent ions subject to the linker used. Finally, we quantified the photophysical performance of different self‐healing linker–fluorophore conjugates and demonstrated their applications in super‐resolution imaging and single‐molecule spectroscopy. Fluorophore functionality with a click: A new biolabelling strategy using bespoke linker structures is introduced that allows to convert commercially available fluorophores into functional probes with high photostability (self‐healing dyes) or dyes with the ability to sense bivalent metal ions resulting in blinking emission.