Long-wavelength Fluorophores for Voltage Sensing

We present the design, synthesis, and applications of a new class of voltage-sensitive fluorescent indicators built on a modified carbofluorescein scaffold. Carbofluoresceins are an attractive target for responsive probes because they maintain oxygen substitution patterns at the 3' and 6' positions, similar to fluorescein, while simultaneously possessing excitation and emission profiles red-shifted nearly 50 nm compared to fluorescein. However, the high pK_a of carbofluorescein dyes, coupled with their tendency to cyclize to non-fluorescent configurations precludes their use in voltage-imaging applications. Here, we overcome the limitations of carbofluoresceins via chlorination to lower the pKa by 2 units to 5.2 and sulfonation to prevent cyclization to the non-absorbing form. To achieve this, we devise a synthetic route to halogenated sulfonated carbofluoresceins from readily available, inexpensive starting materials. New, chlorinated sulfone carbofluoresceins have low pK_a values (5.2) and can be incorporated into phenylenevinylene molecular wire scaffolds to create carboVoltage-sensitive Fluorophores (carboVF dyes). The best of the new carboVF dyes, carboVF2.1(OMe).Cl, possesses excitation and emission profiles >560 nm, displays high voltage sensitivity (>30% ΔF/F per 100 mV), and can be used in the presence of other blue-excited fluorophores like green fluorescent protein (GFP). Because carboVF2.1(OMe).Cl contains a phenolic oxygen, it can be incorporated into fluorogenic labeling strategies. Alkylation with a sterically bulky cyclopropylmethyl-derived acetoxymethyl ether renders carboVF weakly fluorescent; we show that fluorescence can be restored by the action of porcine liver esterase (PLE) both in vitro and on the surface of living cells and neurons. Together, these results suggest chlorinated sulfone carbofluoresceins can be promising candidates for hybrid chemical-genetic voltage imaging at wavelengths beyond typical fluorescein excitation and emission.