# Two-Photon Excitation of Azobenzene Photoswitches for Synthetic Optogenetics
## 6 Novel Photoswitches with 2 PE-Potential  
Other azobenzene-based photoswitches have also been rendered 2p-compatible by use of other strategies. Of these, the methoxy-substitutions at the _meta_ positions and C2-bridged azobenzene are particularly interesting [74]. Methoxy _meta_-substitutions are estimated to strongly shift the absorbance of the azo-core to much longer wavelengths than most reported photoswitches (i.e., >100 nm), specifically into the far-red and infra-red regimes [80] and these should likely have a high 2P-absorption cross section. However, these modifications will most likely lead to very rapid thermal relaxations. One such example is compound 28 with near-IR absorbance peaking at 620 nm (but also sufficiently activated by 730 nm), but with an ultra-rapid thermal back reaction (\(\tau_{\text{cis}}\) = 10 us) (Figure 3) [74]. The second approach, the C2-bridged azobenzene, also show a significant red-shifted absorption spectrum of the _-trans_ isomer (\(\lambda_{\text{max}}\) > 540 nm) [72; 74; 81], implying an increased 2P-absorption cross section [41], but with a much more favorable (i.e., slower) thermal relaxation rate (on the order of minutes at room temperature in aqueous solution) than methoxy _meta_-substitutions. The drawback of the molecule is that the _-cis_ state is the more thermodynamically stable form. This means that if the photoswitch is active in _-cis_, it would be active when applied onto the preparation. Thus, it is preferable should the photoswitch be active only in _-trans_. An additional drawback is that the stable _-cis_ isomer poorly absorbs red light and isomerization to _-trans_ requires UV to near UV illumination. Recently, two _trans_-active diffusible photoswitches were designed based on a C2-bridged azobenzene, denoted locked-azobenzene (LAB) [82] or Glu_brAzo [72]. Both photoswitches bear a glutamate moiety as ligand, thus intended for activation of glutamate receptors, and exhibit highly similar structural and spectral properties (Figure 3). Of note, despite their similarities, LAB is shown to selectively activate NMDARs, whereas Glu_brAzo efficiently activates both Kainate and AMPA receptors. Regardless the receptor type, Glu_brAzo shows more practical features for use in vivo, particularly increased solubility in aqueous media owing to the addition of a bulky ionic group. This is noteworthy as solubility issues are a very common limitation of most photoswitches, in particular azobenzene-based ones [9; 21]. This additional group is also suggested to improve the performance of the photoswitch by increasing the steric congestion around the glutamate moiety with the intention to lower the interaction of the glutamate moiety with the receptor when the photoswitch is found in its stable _-cis_ isomeric form. To design Glu_brAzo, the authors searched for _trans_-active photoswitches that target glutamate receptors. This was not very challenging as most diffusible GluR-photoswitches are, surprisingly, active in the extended thermally stable _-trans_ form (e.g., [83, 84]). They honed-in on a _trans_-active photoswitch denoted GluAzo [85]. For photoactivation, _cis_-Glu_brAzo (i.e., inactive) requires \(\lambda_{\text{peak}}=395\) nm to shift to _-trans_. As noted above, this is quite a limitation for two-photon activation. However, this photoswitch exhibits very desirable bi-stability, with very slow spontaneous back-isomerization from _trans_-to-_cis_ (t\({}_{1/2}\)\(\sim\)4 h, at RT in aqueous media). This step could be accelerated by green light illumination (\(\lambda_{\text{peak}}=480\) nm), suggesting that it may also be compatible with 2 PE. However, the 2P-absorption cross section was not assessed for Glu_brAzo (or LAB) and is very hard to predict based solely on 1P absorbance (seen above and e.g., [47]).