Flipping the Switch: Reverse-Demand Voltage-Sensitive Fluorophores

Fluorescence microscopy with fluorescent reporters that
respond
to environmental cues is a powerful method for interrogating biochemistry
and biophysics in living systems. Photoinduced electron transfer (PeT)
is commonly used as a trigger to modulate fluorescence in response
to changes in the biological environment. PeT-based indicators rely
on PeT either into the excited state (acceptor PeT) or out of the
excited state (donor PeT). Our group has been developing voltage-sensitive
fluorophores (VF dyes) that respond to changes in biological membrane
potential (Vm). We hypothesize that the
mechanism of voltage sensitivity arises from acceptor PeT (a-PeT)
from an electron-rich aniline-containing molecular wire into the excited-state
fluorophore, resulting in decreased fluorescence at negative Vm. In this work, we reversed the direction of
electron flow to access donor-excited PeT (d-PeT) VF dyes by introducing
electron-withdrawing rather than electron-rich molecular wires. VF
dyes containing electron-withdrawing groups show voltage-sensitive
fluorescence, but with the opposite polarity: hyperpolarizing Vm now gives fluorescence increases. We used
a combination of computation and experiment to design and synthesize
five d-PeT VF targets, two of which are voltage-sensitive.