A general fluorescence off/on strategy for fluorogenic probes: Steric repulsion-induced twisted intramolecular charge transfer (sr-TICT)

Abstract

Various control strategies are available for building fluorogenic probes to visualize biological events in terms of a fluorescence change. Here, we performed the time-dependent density functional theory (TD-DFT) computational analysis of the twisted intramolecular charge transfer (TICT) process in rhodamine dyes. On the basis of the results, we designed and synthesized a series of rhodamine dyes and established a fluorescence quenching strategy that we call steric repulsion-induced TICT (sr-TICT), in which the fluorescence quenching process is greatly accelerated by simple intramolecular twisting. As proof of concept of this design strategy, we used it to develop a fluorogenic probe, 2-Me PeER (pentyloxyethylrhodamine), for the N-dealkylation activity of CYP3A4. We applied 2-Me PeER for CYP3A4 activity–based fluorescence-activated cell sorting (FACS), providing access to homogeneous, highly functional human-induced pluripotent stem cell (hiPSC)–derived hepatocytes and intestinal epithelial cells. Our results suggest that sr-TICT represents a general fluorescence control method for fluorogenic probes.