In Situ Ratiometric Quantitative Tracing of Intracellular Leucine Aminopeptidase Activity via an Activatable Near-Infrared Fluorescent Probe.

Leucine aminopeptidase (LAP), one of the important proteolytic enzymes, is intertwined with the progress of many pathological disorders as a well-defined biomarker. To explore fluorescent aminopeptidase probe for quantitative detection of LAP distribution and dynamic changes, herein we report a LAP-targeting near-infrared (NIR) fluorescent probe (DCM-Leu) for ratiometric quantitative trapping of LAP activity in different kinds of living cells. DCM-Leu is composed of a NIR-emitting fluorophore (DCM) as a reporter and l-leucine as a triggered moiety, which are linked together by an amide bond specific for LAP cleavage. High contrast on the ratiometric NIR fluorescence signal can be achieved in response to LAP activity, thus enabling quantification of endogenous LAP with "build-in calibration" as well as minimal background interference. Its ratiometric NIR signal can be blocked in a dose-dependent manner by bestatin, an LAP inhibitor, indicating that the alteration of endogenous LAP activity results in these obviously fluorescent signal responses. It is worth noting that DCM-Leu features striking characteristics such as a large Stokes shift (∼205 nm), superior selectivity, and strong photostability responding to LAP. Impressively, not only did we successfully exemplify DCM-Leu in situ ratiometric trapping and quantification of endogenous LAP activity in various types of living cells, but also, with the aid of three-dimensional confocal imaging, the intracellular LAP distribution is clearly observed from different perspectives for the first time, owing to the high signal-to-noise of ratiometric NIR fluorescent response. Collectively, these results demonstrate preclinical potential value of DCM-Leu serving as a useful NIR fluorescent probe for early detection of LAP-associated disease and screening inhibitor.