Photophysics and Release Kinetics of Enzyme-Activatable Optical Probes Based on H‑Dimerized Fluorophores on Self-Immolative Linkers

A series of activatable optical probes for the model enzyme penicillin G amidase based on intramolecular formation of non-fluorescent H-dimer between two identical dyes were synthesized. The probes are based on a self-immolative linker, which allows positioning both dyes in close spatial proximity to ensure efficient quenching of probes with absorption and fluorescence emission in the near-infrared (NIR) range. A detailed photophysical investigation of the novel optical probes led to a revision of a previously anticipated quenching mechanism and revealed their potential for optimizing the performance of activatable probes based on H-dimer formation. A kinetic analysis indicated that the fluorescence progress curves can be used to qualitatively extract enzyme kinetic parameters