Design, synthesis, cell imaging, kinetics and thermodynamics of reaction-based turn-on fluorescent probes for the detection of biothiols

Abstract

Two highly selective red-emitting fluorescent “OFF-ON” probes (3a and 3b) with a BODIPY core were designed and synthesized, based on the recovery of fluorescence upon the cleavage of the fluorescence quenching unit of 2, 4-dinitrobenzenesulfonyl (DNBS) by biothiols. The probes showed 43-fold (3a, λem = 610 nm) and 33-fold (3b, λem = 582 nm) fluorescence enhancement respectively in the presence of biothiols. Compared with 3b, the introduction of five strong electron-withdrawing fluorine atoms in 3a, not only caused a red shift of the emission maximum for 28 nm, but also increased the fluorescence enhancement. Since they had stable fluorescence emission within physiological pH range, the two probes can be applied in imaging of living cells. In contrast, no such fluorescence response was observed in the cells pre-treated with N-ethylmaleimide (NEM), a well-known thiol scavenger. A non-linear curve-fitting of high quality was used to fit the spectroscopic data to determine the pseudo-first-order rate constants and the chemical equilibrium constants at different given temperatures (e.g. 310 K, 298 K and 288 K). Activation energy (Ea) and thermodynamic parameters, including Gibbs free energy change (ΔG), enthalpy change (ΔH) and entropy change (ΔS), could be calculated. To the best of our knowledge, this was the first report for the chemical kinetics and thermodynamics of the reaction between the fluorescent probes and thiols. This work would greatly inspire the future design of even better fluorescent probes.