A fluorogenic probe for in vitro and in vivo detection of biothiols and vitamin-C with an in-depth mechanistic understanding

Abstract

Thiol-containing biomolecules like Cysteine (Cys), Homocysteine (Hcy), and reduced Glutathione (rGSH) are involved in various biological processes. In addition, vitamin-C (ascorbic acid, AA), being a common antioxidant, plays a significant role in various biological reactions. In vivo detection of these biologically relevant small molecules by using a fluorogenic probe provides a pivotal tool to unravel real-time biomolecular dynamics. The development of a cell-permeable, water-soluble and non-toxic fluorogenic molecules are always advantageous for their spatiotemporal sensitivity and accuracy. Although the application of fluorogenic probes is widely studied, the deeper investigation of underlying mechanisms has remained curtained. In this report, a fluorogenic probe, 4-azido-7-nitrobenzo-2-oxa-1,3-diazole (NBD-N3), has been developed for detection of AA, Cys, Hcy among a range of biologically relevant molecules including rGSH with an extensive mechanistic investigation. The weakly fluorescent NBD-N3 transforms into a highly fluorescent NBD-NH2 with the action of AA by redox mechanism. However, Cys, and Hcy convert NBD-N3 into highly fluorescent NBD-NHR (R=Cys/Hcy) via aromatic nucleophilic substitution (SNAr) followed by S-N rearrangement. The reaction mechanisms for the fluorogenic response have also been authenticated by UV–vis., fluorescence, NMR spectroscopy and mass spectrometry. Furthermore, the probe has also been employed to validate in vitro and in vivo monitoring of biothiols and AA in mammalian cancer cells and in multicellular living Caenorhabditis elegans.