Huang_2025_ACS.Omega_10_58566

Dual-labeled, profluorescent chemical probes have been developed to quantify and visualize a specific enzyme's activity in complex biological media. The intact chemical probes often exhibit minimal fluorescence due to fluorescence quenching, but their intrinsic fluorescence can be released by a favorable enzyme-catalyzed reaction. Contact quenching represents one of several fluorescence quenching mechanisms. However, it is seldom intentionally implemented in the synthesis of dual-labeled, profluorescent chemical probes, because the structure of a contact quenching construct requires the formation of a ground-state fluorophore-quencher/-fluorophore complex. The ability of such dual-labeled molecular probes to act as intramolecular dimers cannot be predicted. We previously revealed that a mono exo-bicyclo-[6.1.0]-nonyne (exo-BCN)-derivatized cystamine framework was critical to synthesizing sensitive, dual-labeled, profluorescent chemical probes capable of contact quenching. Here, we exploited the nonsymmetrical mono-exo-BCN-cystamine backbone by sequentially coupling it with two different carboxyfluorescein (FAM) derivatives and subsequently synthesizing four nonsymmetrical bis-FAM chemical probes. The fluorescence turn-on properties of the bis-FAM chemical probe with the lowest background FAM fluorescence were characterized by kinetic studies. Moreover, the release of two FAM equivalents from the fluorescence turn-on chemical probe during reactions was utilized to develop sensitive assays for measuring the activity and inhibition of two serum biomarkers, butyrylcholinesterase (BChE) and paraoxonase 1 (PON1) lactonase. We also developed an efficient, high-throughput assay for detecting BChE activity based on the chemical probe. Finally, the fluorescence assays successfully quantified the activities of BChE and PON1 lactonase in human serum.