New photochemical tools based on thioketal

Photoactivable fluorescent probe, also known as photocage, is an important class of optical probes used in biological imaging. These probes typically exhibit a unique feature: the fluorescence of the probe is quenched when bound to a “cage”, but upon irradiation of light at specific wavelength, the “cage” is cleaved, releasing the fluorophore and restoring its fluorescence. This transition from a non-fluorescent to a fluorescent state is entirely dependent on an external light signal, allowing for precise control over the fluorescence signal emitted by the photocage. Since the external light signal is highly adjustable, photocages offer superior controllability and higher spatiotemporal selectivity compared to traditional always-on or reactive fluorescent probes. While various photocages have been developed, there are few universal strategies for designing photocages. To meet the increasingly complex demands of molecular design and research, the development of novel universal photocage design strategies is a key focus in the field.

Recently, Professor Kun Li's group from Sichuan University published an article titled “Thioketal-photocage: universal modified strategy to construct new photochemical tools for real-time imaging in living cells”. They report a new universal photocage modification strategy based on thioketal, which can be applied for live cell subcellular imaging, and investigate the mechanism of photocage photoresponse in this system. This class of molecules can be directly synthesized by condensation of a carbonyl-containing fluorophore with a dithiol, where the thioketal moiety serves as the photoresponsive component. It completely quenches the fluorescence, while being capable of light-induced deprotection under UV-visible light irradiation, thereby restoring the fluorescence of the original fluorophore. Using the thioketal-based fluorescence probe SiR-EDT as an example, we characterized the products, conditions, conversion rates, and limits during photoreaction using techniques such as UV-visible spectroscopy, fluorescence spectroscopy, Nuclear Magnetic Resonance Spectroscopy (NMR), high-resolution mass spectra (HRMS), and high-performance liquid chromatography (HPLC). Based on experimental results and theoretical calculations, we propose a mechanism for the photoreaction of these thioketal probes. The results reveal that the photolysis mechanism of this probe is distinct from previously reported thioketal deprotection mechanisms. This process requires only UV-visible light and oxygen to proceed, demonstrating non-dependence on external photosensitizers or exogenous oxidants. The photocage SiR-EDT exhibits up to a 68-fold fluorescence activation upon light irradiation in vitro and shows good dark stability in the presence of various analytes (reactive oxygen species (ROS), metal ions, reducing agents). In live cells, SiR-EDT maintains good dark stability, can be specifically activated by UV-visible light, and can be further modified to target specific organelles or label particular proteins. This enables selective activation and imaging of subcellular structures and specific proteins.

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