Designer Aromatic Cations for Photoinduced Protein Ligation, Imaging, and Intracellular Labeling at Extended Wavelengths

Photoinduced protein labeling strategies have become essential tools in chemical biology, but most strategies require high-energy wavelengths of light as input to drive reactivity. Recently, we reported a biocompatible method for engaging photoinduced electron transfer to drive protein labeling using biarylpyridinium salts, and here, we report the design of a series of aromatic cation salts that trigger this process using longer wavelengths of light while maintaining a sterically minimal profile. We achieved this through the systematic study of structure–reactivity relationships of various donor–acceptor pyridinium salts possessing extended conjugation, and these studies revealed the need of a constrained trans-stilbene relationship between the probe’s donor and acceptor substituents in order to achieve protein labeling. Probes with chromene-based donor groups in particular showed either robust protein labeling, significant fluorescence quantum yields, or state-dependent photophysical properties, in turn enabling the same probes to be used for both photoinduced protein labeling and wash-free, live cell imaging. These qualities were then harnessed for live cell labeling using green light photoactivation with two different aromatic cation probes that show complementary photophysical properties and microenvironment responsiveness. Mass spectrometry-based analysis of labeled proteins revealed that each probe enriched highly distinct (<10% protein enrichment overlap) proteomic subsections from primarily mitochondria and the endoplasmic reticulum. This series of experiments not only demonstrates the ability of this latest generation of probes to engage in photoinduced labeling using lower-energy light in complex proteomes but also reveals new capabilities for photophysical state-dependent reactivity and measurements.