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Bioorthogonal "Labeling after Recognition" Affording an FRET-based Luminescent Probe for Detecting and Imaging Caspase-3 via Photoluminescence Lifetime Imaging.

Bis-labeling with luminescent energy donor/acceptor pair onto biological substrates affords probes which give FRET readouts for detection of interaction partners. However, the covalently bound luminophores bring about steric hindrance and non-specific interaction, which probably perturb the biological recognition. Herein, we designed a highly sensitive and specific "labeling after recognition" sensing approach, where luminophore labeling occurred after the biological recognition. Taking the cutting enzyme caspase-3 as an example, we demonstrated the detection of its catalytic activity in solution and apoptotic cells using the tetrapeptide motif Asp-Glu-Val-Asp DEVD as the cleavable substrate, and an iridum(III) complex and a rhodamine derivative as the energy donor/acceptor pair. The DEVD tetrapeptide was modified with an azide and a GK-norbornylene groups at the amino and carboxyl terminuses, respectively, which allowed donor/acceptor bis-labeling via two independent catalysis-free bioorthogonal reactions. The phosphorescence lifetime of the iridium(III) complex was quenched upon bis-labeling owing to the intracellular FRET to the rhodamine derivative, and significantly elongated upon the peptide was catalytically cleaved by caspase-3. Interestingly, the sensitivity and efficiency of the lifetime response were much higher in the "labeling after recognition" sensing approach. Molecular docking analysis showed that the steric hin-drance and non-specific interactions partially inhibited the biological recognition of the DEVD substrate by caspase-3. The imaging of the catalytic activity of caspase-3 in apoptotic cells was demonstrated via photoluminescence lifetime imaging microscopy (PLIM). Lifetime analysis not only confirmed the occurrence of intracellular bioorthogonal bis-labeling and cat-alytically cleavage, but also showed the extent to which the two dynamic processes occurred.