A Molecular Hybrid for Double Stepwise Nitric Oxide Photorelease with Double Fluorescence Readout in Living Cells

The strict dependence of the multifaceted biological effects of nitric oxide (NO) on the generation site and dose demands precise spatiotemporal control of its delivery. Light-activatable NO precursors enable this requirement by allowing for the appropriate localization of the excitation light source on the area of interest and regulation of the light dose. However, monitoring the NO released in living cells with the aid of noninvasive fluorescence techniques, without the use of additional NO probes, is very challenging for the bioapplications of this inorganic free radical. We report herein the design, synthesis, characterization, and biological validation of a molecular hybrid consisting of two chromogenic nitroso-derivatives covalently joined through a flexible alkyl spacer, which can release two NO molecules exclusively under visible light input through a stepwise mechanism accompanied by a double fluorescence readout. Specifically, the two distinct NO release processes are paralleled by a turn-on and turn-off of the green fluorescence of the corresponding stable coproducts, respectively, which act as optical NO reporters. This feature permits monitoring of the two individual steps of NO photouncaging in real time by fluorescence spectroscopy. Proof-of-principle fluorescence microscopy experiments demonstrate that, not trivially, the photochemical properties of the hybrid are well-preserved in living cancer cells, where the stepwise release of NO can be imaged in real time without the use of additional chemical probes. This makes the present compound an intriguing candidate for fundamental and potential applicative research studies, where the short- and long-term release of spatiotemporally controlled NO delivery, associated with its direct optical detection, is required.