Targeted Covalent Photoswitch for Two-Photon Control of Endogenous Receptors

The study of intact cells and their signaling circuits with light requires a stimulation strategy that is focused, deeply penetrating, and does not damage them. Implanted optic fibers, light-emitting diodes, and luminescent materials operated externally with tissue-penetrating infrared (IR) light are invasive or limited by light attenuation around the illumination point. To overcome these barriers, two-photon pharmacology takes advantage of femtosecond-pulsed IR laser light to produce deep and spatiotemporally precise cellular stimulation using specially designed photoswitchable drugs. Compounds that can be covalently tethered to the target neuroreceptor perform particularly well. However, the tethered photoswitches reported to date require mutagenesis of the target protein, which prevents the use of photopharmacology to stimulate the nervous system in wild-type animals. Here, we report the first two-photon optimized targeted covalent photoswitch (TCP_2P) that combines the efficient two-photon isomerization of ortho-fluoro-substituted azobenzene with the ability to conjugate to nucleophilic residues of endogenous proteins (AMPA and kainate ionotropic glutamate receptors in neurons). TCP_2P is readily obtained by click coupling of two precursor compounds prior to use, and after simple incubation, it enables controlling neuronal activity at one- and two-photon excitation up to 800 nm without genetic modifications.