Abstracts

Biothiols like Cysteine (Cys), Homocysteine (Hcy), and Glutathione (GSH) are compounds with essential physiological functions involved in redox homeostasis in living organisms. Due to their biological importance, the development of new fluorescent sensors for the detection, distinction, and quantification of these analytes has been attracting great attention. Although there are many fluorescent sensors capable of detecting biothiols, the distinction between them remains a challenge, due to their similar structures. In this work, we present the synthesis of two new seleno-BODIPYs for distinction and selective detection of biothiols, through colorimetric and fluorometric analysis, with high fluorescence quantum yields, fast response time, ratiometric response, and low detection limits, in a turn-on fluorescence process. The general procedure to obtain the new compounds started from 4-hidroxibenzaldehyde, and, after 3 reactional steps resulted in the desired BODIPY core, in reasonable overall yields (42-45%). The selenium post-functionalization of the BODIPY core was conducted through a bromination step, using N-bromosuccinimide, resulting in tetrabrominated compound in 69% yield, followed by a selenilation step, using PhSeH, generated in situ from Ph₂Se₂ and NaBH₄ (50-60% yield). The final chalcogenated compounds present a polyethylene glycol (PEG) substituent, and the main difference between these two new compounds is the chain size of this portion.The first compound obtained was successfully applied to the selective detection of Cys and GSH against 21 analytes through colorimetric and fluorometric analysis. The new probe presented low detection limits (18.4 nM for Cys and 0.42 uM for GSH), high fluorescence quantum yields_­ (31.8% for GSH and 88.1% for Cys), high sensitivity, and fast response time. Mechanistic investigation was conducted allowing to conclude that the reaction with Cys results in a monosubstituted amino-product while the reaction with GSH results in a double substituted thiol-product, by replacing the Se-containing portion. Due to the difference in the reactivity, it is possible to distinguish and detect Cys and GSH simultaneously at different wavelengths. This new compound was also applied for bioimaging in HeLa cells, acting as a fluorescent cell biomarker. The other Se-BODIPY synthesized was applied to selective detection of Cys, Hcy and GSH, in a similar way. It was possible to distinguish Cys/Hcy from GSH using different excitation wavelengths. Kinetic studies showed that the reaction with Cys is much faster than with Hcy, allowing them to be differentiated. The sensor was successfully applied to detect biothiols in urine samples, with good recovery values. Experiments using paper discs impregnated with Se-BODIPY were also conducted, making it possible to visualize a color change from blue to orange (for Cys) and from blue to pink (for GSH). This is a very advantageous and promising strategy that allows the detection of biothiols in the solid state being a practical and simple method.