An Optimized Glutamate Receptor Photoswitch with Sensitized Azobenzene Isomerization.

A new azobenzene-based photoswitch, 2, has been designed to enable optical control of ionotropic glutamate receptors in neurons via sensitized two-photon excitation with NIR light. In order to develop an efficient and versatile synthetic route for this molecule, a modular strategy is described which relies on the use of a new linear fully protected glutamate derivative stable in basic media. The resulting compound undergoes one-photon trans-cis photoisomerization via two different mechanisms: direct excitation of its azoaromatic unit and irradiation of the pyrene sensitizer, a well-known two-photon sensitive chromophore. Moreover, 2 presents large thermal stability of its cis isomer, in contrast to other two-photon responsive switches relying on the intrinsic nonlinear optical properties of push-pull substituted azobenzenes. As a result, the molecular system developed herein is a very promising candidate for evoking large photoinduced biological responses during the multiphoton operation of neuronal glutamate receptors with NIR light, which require accumulation of the protein-bound cis state of the switch upon repeated illumination.

Two-photon neuronal and astrocytic stimulation with azobenzene-based photoswitches.

Synthetic photochromic compounds can be designed to control a variety of proteins and their biochemical functions in living cells, but the high spatiotemporal precision and tissue penetration of two-photon stimulation have never been investigated in these molecules. Here we demonstrate two-photon excitation of azobenzene-based protein switches and versatile strategies to enhance their photochemical responses. This enables new applications to control the activation of neurons and astrocytes with cellular and subcellular resolution.

Rationally designed azobenzene photoswitches for efficient two-photon neuronal excitation.

Manipulation of neuronal activity using two-photon excitation of azobenzene photoswitches with near-infrared light has been recently demonstrated, but their practical use in neuronal tissue to photostimulate individual neurons with three-dimensional precision has been hampered by firstly, the low efficacy and reliability of NIR-induced azobenzene photoisomerization compared to one-photon excitation, and secondly, the short cis state lifetime of the two-photon responsive azo switches. Here we report the rational design based on theoretical calculations and the synthesis of azobenzene photoswitches endowed with both high two-photon absorption cross section and slow thermal back-isomerization. These compounds provide optimized and sustained two-photon neuronal stimulation both in light-scattering brain tissue and in Caenorhabditis elegans nematodes, displaying photoresponse intensities that are comparable to those achieved under one-photon excitation. This finding opens the way to use both genetically targeted and pharmacologically selective azobenzene photoswitches to dissect intact neuronal circuits in three dimensions.