Exceptional Time Response, Stability and Selectivity in a Doubly-Activated Phenyl Selenium-Based Glutathione-Selective Platform
A recent paper published in Chemical Science demonstrated highly selective and extremely rapid detection of glutathione (GSH) over cysteine (Cys)/homocysteine (Hcy) without background fluorescence. The fluorescence intensity of the probe with GSH shows a ~100-fold fluorescent enhancement compared with that signal generated for other closely related amino acids including Cys and Hcy. Importantly, the substitution reaction with the sulfhydryl group of GSH occws at a particular position of the probe. This position is doubly-activated by two carbonyl groups, and reacts extremely fast, showing subsecond maximum fluorescence intensity attainment; equilibrium was reached within 100 ms (UV-vis). The probe selectivity for GSH was confirmed in Hep3B cells by confocal microscopy imaging.
Biothiols, such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), play crucial roles in biological systems as endogenous thiols. These biothiols exist in equilibrium between their oxidized disulfide and reduced free-thiol forms in biological systems. Due to the significant role of biothiols (Cys, Hcy and GSH), a number of reports have accounted for the detection of these species at the molecular level through the use of small synthetic probes. Although many probes for the detection of thiols have been reported, there is no probe for the selective detection of GSH demonstrating real-time detection as of yet. Thus, it is still challenging to design a highly selective and rapid florescent probe for a particular biothiol because of the similarity in structure and reactivity between the species.
A research team led by Professor David G. Churchill in the Department of Chemistry, KAIST and Professor Sangyong Jon in the Department of Biological Science developed a new platform for the selective and exceptional time response detection of GSH. Recently, the Churchill group has explored several fluorescent probes containing selenium as the reactive center to detect important biological analytes. Based on previous results of organoselenium probes, a new fluorescent probe has two independent potential reactive sites, aldehyde and phenylselenide, for the selective detection of GSH. A phenylselenide group was chosen to be incorporated at the coumarin 4-position; this group is able to quench fluorescence via photo-induced electron transfer (PET) and is also able to behave as a leaving-group. The proximal aldehyde group plays a dual role: it enhances the electrophilicity of the 4-position as a Michael acceptor and enables for a cyclization reaction with the sulfhydryl and primary amine groups of the biothiols.
Analyte screening was performed which includes various biothiols (Cys, Hcy, GSH) and amino acids (Pro, Thr, Val, Leu, Asn, Trp, Ile, Ala, His, Glu, Tyr, Lys, Met, Asp, Phe, Arg, Gly, Gln, Ser); the analytes were used to treat the solution (DMSO/ 10 mM PBS pH 7.4, 1 : 3, v/v) in the presence of 20 µM Conc of the probe. To help unravel the mechanism and the structure of the probe with biothiols, model compounds were synthesized and their reactivity was studied. Comparing the photophysical properties of probe, probe with GSH and model compounds, GSH undergoes nucleophilic substitution at the 4-position of the coumarin and intramolecular cyclization with the aldehyde through forming an iminium group. After understanding of the ‘turn-on’ fluorescent response with GSH, several UV-vis and fluorescent spectroscopic experiments were performed. Titration of the probe with various concentrations of GSH were studied for estimating sensitivity. Stopped-flow methods help estimate the time-response of the probe with GSH.
To evaluate the biocompatibility of the probe, cell viability testing and confocal microscopy images were used. Strong green fluorescence emissions were observed in Hep3B cells, indicating that the reaction of the probe with intracellular GSH proceeds. Additional experiments were performed to better support the conclusion that fluorescence signals were due to the interaction between the probe and GSH in the cells. A thiol-specific reagent was also used in Hep3B cells; the confocal microscopy image of NEM-treated cells showed a significant decrease in the fluorescence signal, suggesting the specific reactivity of probe with intracellular GSH.
David G. Churchill, his students Youngsam Kim, Sandip V. Mulay and Sangyong Jon, his student Minsuk Choi, Seungyoon B. Yu at KAIST were listed as the authors of this contribution published in Chemical Science Express in October, 2015 (Chemical Science, 2015, 6, 5435-5439).
* lab webpage