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The hydroxyl radical (•OH), one of the reactive oxygen species (ROS) in biosystems, is found to be involved in many physiological and pathological processes. However, specifically detecting endogenous •OH remains an outstanding challenge owing to the high reactivity and short lifetime of this radical. Herein, inspired by the scavenging mechanism of a neuroprotective drug edaravone toward •OH, we developed a new •OH-specific fluorescent probe RH-EDA. RH-EDA is a hybrid of rhodamine and edaravone and exploits a •OH-specific 3-methyl-pyrazolone moiety to control its fluorescence behavior. RH-EDA itself is almost nonfluorescent in physiological conditions, which was attributed to the formation of a twisted intramolecular charge transfer (TICT) state upon photoexcitation and the acylation of its rhodamine nitrogen at the 3′ position. However, upon a treatment with •OH, its edaravone subunit was converted to the corresponding 2-oxo-3-(phenylhydrazono)-butanoic acid (OPB) derivative (to afford RH-OPB), thus leading to a significant fluorescence increase (ca. 195-fold). RH-EDA shows a high sensitivity and selectivity to •OH without interference from other ROS. RH-EDA has been utilized for imaging endogenous •OH production in living cells and zebrafishes under different stimuli. Moreover, RH-EDA allows a high-contrast discrimination of cancer cells from normal ones by monitoring their different •OH levels upon stimulation with β-Lapachone (β-Lap), an effective ROSgenerating anticancer therapeutic agent. The present study provides a promising methodology for the construction of probes through a drug-guided approach.

Selenocysteine (Sec) is a primary kind of reactive selenium species in cells, and its vital roles in physiological processes have been featured. Thus, the development of highly sensitive and selective methods for the sensing of Sec is of great significance. This work reports a turn-on fluorescent probe for selenol based on the unique fluorescence OFF-ON switching between the Schiff base (SB) and its complementary protonated Schiff base (PSB) form of merocyanine dyes. The probe consists of a merocyanine Schiff base fluorophore and a 2,4-dinitrobenzenesulfonamide moiety that reacts especially with selenol. The fluorescence turn-on response of MC-Sec is realized via the selective removal of the strongly electron withdrawing 2,4-dinitrobenzenesulfonyl group by Sec, leading to a shift in the p K of the imine nitrogen of the probe from 6.40 to 9.04 and thus significantly increasing the population of the fluorescent PSB form of the dye at physiological pH. MC-Sec shows good selectivity and sensitivity for Sec and has been applied in the imaging of exogenous and endogenous selenol in living cells by confocal fluorescence microscopy. The proposed mechanism should be useful for developing future probes directed to other target molecules by employing this simple but effective p K shift strategy.

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Vicinal dithiol-containing proteins (VDPs) play fundamental roles in intracellular redox homeostasis and are responsible for many diseases. In this work, we report a far-red fluorescence turn-on probe MCAs for VDPs exploiting the pK shift of the imine functionality of the probe. MCAs is composed of a merocyanine Schiff base as the fluorescent reporter and a cyclic 1,3,2-dithiarsenolane as the specific ligand for VDPs. The imine pK of MCAs is 4.8, and it exists predominantly in the Schiff base (SB) form at physiological pH. Due to the absence of a resonating positive charge, it absorbs at a relatively short wavelength and is essentially nonfluorescent. Upon selective binding to reduced bovine serum albumin (rBSA, selected as the model protein), MCAs was brought from aqueous media to the binding pockets of the protein, causing a large increase in pK value of MCAs (pK = 7.1). As a result, an increase in the protonated Schiff base (PSB) form of MCAs was observed at the physiological pH conditions, which in turn leads to a bathochromically shifted chromophore (λ = 634 nm) and a significant increase in fluorescence intensity (λ = 657 nm) simultaneously. Furthermore, molecular dynamics simulations indicate that the salt bridges formed between the iminium in MCAs and the residues D72 and D517 in rBSA resist the dissociation of proton from the probe, thus inducing an increase of the pK value. The proposed probe shows excellent sensitivity and specificity toward VDPs over other proteins and biologically relevant species and has been successfully applied for imaging of VDPs in living cells. We believe that the present pK shift switching strategy may facilitate the development of new fluorescent probes that are useful for a wide range of applications.

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