Aiming to extend the scope of indigoid photoswitches to polar protic environments, we have synthesized a sulfonated thioindigo derivative highly soluble in water. Studies by UV/Vis absorption, fluorescence, and NMR spectroscopy indicate that, despite aggregation effects at micromolar concentrations, the novel dye offers satisfactory performance in aqueous solution in the absence of solvation aides. Enrichment of the metastable cis isomer by irradiation may exceed 65 % and its half-life at room temperature may exceed several hours. Degradation after 20 yellow and blue light irradiation cycles within 2 hours is less than 2 %. Performance can be expected to further improve in future molecular designs if the tendency towards self-association is further reduced. Crucially, photoisomerization of indigoids is not necessarily inhibited by water and, thus, the superior spatial control offered by this class of molecular switches may be of great benefit also in biological systems.

We have synthesized and characterized a thioindigo photoswitch featuring phenolic residues forming an intramolecular hydrogen bond exclusively in the cis isomer. In acetone–water mixtures, wavelength‐dependent photostationary states reached their maximum and minimum values of up to 95 % and down to 11 % cis isomers at circa λ=580 and 480 nm, respectively. Within a few hours, room‐temperature thermal isomerization is negligible in neutral and mildly acidic solutions. Protic media decrease trans→cis but increase cis→trans quantum yields. In CD3OD solution, in which light of any wavelength absorbed triggers the latter process almost exclusively, a 19F NMR titration is used to calculate pKa values of 7.2 (trans) and 5.0 (cis; both rescaled to water) corresponding to a change in dissociation constant of 2.2 orders of magnitude. Future derivatives featuring solubility and reversible photochromism in water may provide pH modulation of similar amplitude in a range adjustable by the choice of phenolic substituents.

Potent and selective small-molecule inhibitors are valuable tools to elucidate the functions of protein kinases within complex signaling networks. Incorporation of a photoswitchable moiety into the inhibitor scaffold offers the opportunity to steer inhibitor potency with temporal precision, while the challenge of selective inhibition can often be addressed by employing a chemical genetic approach, termed the analog-sensitive method. Here, we combine the perks of these two approaches and report photoswitchable azopyrazoles to target calcium-dependent protein kinase 1 (CDPK1) from Toxoplasma gondii, a kinase naturally susceptible to analog-sensitive kinase inhibitors due to its glycine gatekeeper residue. The most promising azopyrazoles display favorable photochemical properties, thermal stability, and a substantial difference in IC 50 values between both photostationary states. Consequently, the CDPK1 kinase reaction can be controlled dynamically and reversibly by applying light of different wavelengths. Inhibition of CDPK1 by the azopyrazoles drastically relies on the nature of the gatekeeper residue as a successive increase in gatekeeper size causes a concurrent loss of inhibitory activity. Furthermore, two photoswitchable inhibitors exhibit activity against T. gondii and Cryptosporidium parvum infection in a cell culture model, making them a promising addition to the toolbox for dissecting the role of CDPK1 in the infectious cycle with high temporal control. Overall, this work merges the benefits of the analog-sensitive approach and photopharmacology without compromising inhibitory potency and thus holds great promise for application to other protein kinases in the future.