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Fluorescent voltage indicators are an attractive alternative for studying the electrical activity of excitable cells; however, the development of indicators that are both highly sensitive and low in toxicity over long-term experiments remains a challenge. Previously, we reported a fluorene-based voltage-sensitive fluorophore that exhibits much lower phototoxicity than previous voltage indicators in cardiomyocyte monolayers, but suffers from low sensitivity to membrane potential changes. Here, we report that the addition of a single vinyl spacer in the fluorene molecular wire scaffold improves the voltage sensitivity 1.5- to 3.5-fold over fluorene-based voltage probes. Furthermore, we demonstrate the improved ability of the new vinyl-fluorene VoltageFluors (v-fVFs) to monitor action potential kinetics in both mammalian neurons and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Addition of the vinyl spacer between the aniline donor and fluorene monomer results in indicators that are significantly less phototoxic in cardiomyocyte monolayers. These results demonstrate how structural modification to the voltage sensing domain have a large effect on improving the overall properties of molecular wire-based voltage indicators.

The ability to non-invasively monitor membrane potential dynamics in excitable cells like neurons and cardiomyocytes promises to revolutionize our understanding of the physiology and pathology of the brain and heart. Here, we report the design, synthesis, and application of a new class of fluorescent voltage indicator that makes use of a fluorene-based molecular wire as a voltage sensing domain to provide fast and sensitive measurements of membrane potential in both mammalian neurons and human-derived cardiomyocytes. We show that the best of the new probes, fluorene VoltageFluor 2 (fVF 2) readily reports on action potentials in mammalian neurons, detects perturbations to cardiac action potential waveform in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, shows a substantial decrease in phototoxicity compared to existing molecular wire-based indicators, and can monitor cardiac action potentials for extended periods of time. Together, our results demonstrate the generalizability of a molecular wire approach to voltage sensing and highlights the utility of fVF 2 for interrogating membrane potential dynamics.

The direct cyanomethylation of indoles at the 2- or 3-position was achieved via photoredox catalysis. The versatile nitrile synthon is introduced as a radical generated from bromoacetonitrile, a photocatalyst, and blue LEDs as a light source. The mechanism of the reaction is explored by determination of the Stern-Volmer quenching constants. By combining photo-physical data and mass spectrometry to follow the catalyst decomposition, the catalyst ligands were tuned to enable synthetically useful yields of radical coupling products. A range of indole substrates with alkyl, aryl, halogen, ester, and ether functional groups participate in the reaction affording products in 16–90% yields. The reaction allows the rapid construction of synthetically useful cyanomethyl indoles, products that otherwise require several synthetic steps.