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.