Rationally designed azobenzene photoswitches for efficient two-photon neuronal excitation

Abstract

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.

Configurational Selection in Azobenzene‐Based Supramolecular Systems Through Dual‐Stimuli Processes

“…Noticeable applications range from classical supramolecular devices (recognition, self‐assembly, transport, sensing, catalysis, etc .) to photoresponsive architectures, passing through responsive materials and polymer science, energy‐related technologies, molecular machines, and biology . The preference for azobenzene with respect to other molecular switches relays on its easy preparation and functionalization, on the neat photophysical behavior that normally allows fast E / Z isomerization and selective irradiation of the two isomers, and on the large geometrical rearrangement that accompanies the isomerization process .…”

Nanoengineered Light-Harvested Proteins for Optogenetics and Photopharmacology

“…Because of its 4aminoazobenzene photochrome, MAG2P_long preserves the red-shifted absorption of MAG2P and its low cis thermal stability (τcis = 5.5 and 118 ms for MAG2P_long and MAG2P in aqueous buffer at room temperature) with respect to regular azobenzenes. In addition, it shows similar photoisomerization quantum yields, as determined in DMSO using azobenzene as a reference (trans-cis = 0.10 and cis-trans = 0.47) (Cabré et al 2019) c) Action spectra registered for HEK293 cells overexpressing GluK2-L439C-(eGFP or dsRed2) after conjugation to MAG2P_long (n=5) and MAG2P (n=3-7). Light pulse stimulation is of 1 s and ranged from 300 to 600 nm in steps of 10 nm.…”

“…In practice, only fluorescent dyes absorbing in the same region as the photoswitch or showing blue-shifted absorption will meet the latter condition, which might be considered as a limitation of photosensitization for spectrally tuning the photoactivity of optogenetic and photopharmacological systems. However, two-photon sensitization of the isomerization of visible-light absorbing azobenzenes with near-infrared radiation has already been demonstrated (Izquierdo-Serra et al 2014;Moreno et al 2016), which opens the door to exploit the lightharvesting strategy introduced herein to boost the otherwise low-to-moderate multiphoton activity of common optogenetic and photopharmacological tools (Cabré et al 2019;Carroll et al 2015;Izquierdo-Serra et al 2014;Prakash et al 2012).…”

“…light wavelength and intensity sensitivity, photoisomerization kinetics and efficiency, and thermal stability of the photoinduced state). Among them, much attention has been paid to tune the spectral response of these systems, aiming to achieve photostimulation with less phototoxic, more penetrating red and near-infrared radiation (NIR) and, even better, under multiphoton absorption to enhance excitation resolution (Cabré et al 2019;Pittolo et al 2019). In addition, broadening their action spectra will increase their sensitivity under white light illumination, a requirement for several therapeutic applications (e.g.…”

Reversible Spatiotemporal Control of Induced Protein Degradation by Bistable photoPROTACs

“…linker as shown in Figure 1D (right) which had been predicted to possess a cis-½ of about 80 days. 27 The use of Feringa's method 28 gave access to 6 from diazonium salt 4 and the organolithium derivative derived from protected 3,5-difluoroaniline 5 (Figure 2A). Subsequent palladium-catalyzed carbonylative esterification furnished aminoester 7.…”