A fluorophore's electron-deficiency does matter in designing high-performance near-infrared fluorescent probes

The applications of most fluorescent probes available for Glutathione S -Transferases (GSTs), including NI3 which we developed recently based on 1,8-naphthalimide ( NI ), are limited by their short emission wavelengths due to insufficient penetration. To realize imaging at a deeper depth, near-infrared (NIR) fluorescent probes are required. Here we report for the first time the designing of NIR fluorescent probes for GSTs by employing the NIR fluorophore HCy which possesses a higher brightness, hydrophilicity and electron-deficiency relative to NI . Intriguingly, with the same receptor unit, the HCy -based probe is always more reactive towards glutathione than the NI -based one, regardless of the specific chemical structure of the receptor unit. This was proved to result from the higher electron-deficiency of HCy instead of its higher hydrophilicity based on a comprehensive analysis. Further, with caging of the autofluorescence being crucial and more difficult to achieve via photoinduced electron transfer (PET) for a NIR probe, the quenching mechanism of HCy -based probes was proved to be PET for the first time with femtosecond transient absorption and theoretical calculations. Thus, HCy2 and HCy9 , which employ receptor units less reactive than the one adopted in NI3 , turned out to be the most appropriate NIR probes with high-sensitivity and little nonenzymatic background noise. They were then successfully applied to detecting GST in cells, tissues and tumor xenografts in vivo . Additionally, unlike HCy2 with a broad isoenzyme selectivity, HCy9 is specific for GSTA1-1, which is attributed to its lower reactivity and the higher effectiveness of GSTA1-1 in stabilizing the active intermediate via H-bonds based on docking simulations. An abnormal and intriguing phenomenon that the fluorophore's electron-deficiency could affect a probe's performance is now revealed for the first time.