Reversing Intramolecular Rotation Driving Energy Enables Fluorescence Umpolung
To get a deeper understanding of this fluorescence umpolung, it’s critical to obtain the molecular geometries of these indazole-based chromophores. Fortunately, we acquired single crystals of IN-NH2, IN-Boc, IN1-NH2, DC-IN-NH2, DC-IN-Boc, and DCM-IN-NH2 (Fig. 3a and 3d, Supplementary Fig. 4, and Supplementary Tables 1–6). Specifically, the amino-substituted chromophore DC-IN-NH2 (D-indazole-π-A type dye) shows an obvious dihedral angle (around 12o) between indazole and furan (Fig. 3a). After attachment of the amino group with an electron-withdrawing Boc unit, the resulting DC-IN-Boc (EWT-indazole-π-A type dye) exhibits an exactly coplanar conformation with 0o dihedral angle (Fig. 3d). According to the single-crystal analysis, the dihedral angle between indazole and the π-bridge is distinctly different from the electron density perturbation of indazole’s C4 position, and the molecular geometries in the solution-state should be further investigated.
To support the discrete molecular geometries of D-indazole-π-A versus EWT-indazole-π-A fluorophore in solution-state, we subsequently carried out the two-dimensional 2D-NOESY NMR experiments. In 2D-NOESY NMR spectra of DC-IN-NH2, Ha shows a similar degree of couplings with Hb and Hc (Fig. 3b and 3c), which implies that the spatial distance of Ha-and-Hb is comparable with that of Ha-and-Hc. The result denotes a much more twisted conformation of DC-IN-NH2 in solution-state than that of crystal-state. Different from DC-IN-NH2, Ha of DC-IN-Boc shows only coupling with Hb rather than Hc (Fig. 3e and 3f), which further validates the planar conformation of EWT-indazole-π-A dyes in solution-state. Taken together, these single crystals and 2D-NOESY NMR analysis strongly confirm the twisted/planar conformation of D-indazole-π-A/EWT-indazole-π-A. More importantly, all the information leads us to suspect that the dark state of D-indazole-π-A dyes is due to the C-C bond rotations between indazole and the π-bridge, resulting in pronounced non-radiative decay losses (Fig. 3g).
To verify our hypothesis, we investigated the effect of viscosity on the fluorescence of these chromophores. According to a general rule, when rotation is restricted in high-viscosity environments, the non-radiative deactivation is minimized, which results in a fluorescence enhancement29–34. Indeed, the emission of DCM-IN-NH2 is partly restored in a highly viscous environment (Fig. 3h), which is typical for molecular rotors. In contrast, DCM-IN-Boc shows almost no fluorescent change with viscosity variation (Fig. 3i). Collectively, the viscosity sensitivity of DCM-IN-NH2 and the viscosity insensitivity of DCM-IN-Boc help elucidate the fluorescence umpolung of indazole-based fluorophores: (i) When modified with EDG (electron-donating group, such as amino group), the obtained fluorophores show obvious C-C bond rotations between indazole and the π-bridge, which leads to a fluorescence dark state. (ii) On the other hand, when modified with EWT (such as Boc group), the C-C bond rotation is inhibited and the fluorophores show bright emissions. Therefore, this fluorescence umpolung of indazole-based chromophores could be attributed to the effects of the electron density perturbation on intramolecular rotation (Fig. 3g).
Furthermore, quantum chemical calculations were conducted by using model molecules DM-IN-NH2 and DM-IN-Boc (Fig. 4). Their results strongly corroborate the above experimental data, and provide more details in the photoexcitation and deactivation process. Upon photoexcitation, a fluorophore could be excited from the ground state to the locally excited (LE) state, and then experience a transition from the LE state to the circa 90o twisted excited state35. As shown in Fig. 4b, we first studied the process from the ground state to the LE state. In optimized geometries, DC-IN-NH2 exhibits a twisted conformation (θ = 12o) in the ground state, whereas demonstrates a typical planar conformation (θ = 0o) in the locally excited (LE) state (Fig. 4b). This substantial rotation (Δθ = 12o) could greatly quench the emission of DM-IN-NH2. In stark contrast, we noted that DM-IN-Boc displayed a planar conformation both in the ground and excited states (Δθ = 0o), and could thus rationalize the bright emission of DM-IN-Boc (Fig. 4b). These calculation results of the process from the ground state to the LE state can in part explain the fluorescence umpolung phenomena.
Considering the rotations from the ground state to the planar LE state, we further investigated the formations of the circa 90o twisted excited state in DM-IN-NH2 and DM-IN-Boc. Calculations of the potential energy surface (PES) in the excited state (S1) show that twisted excited state formation is energetically favorable in DM-IN-NH2 (rotation driving energy ΔERDE = + 0.96 eV) (Fig. 4c). This rotation (Δθ = 90o) is accompanied by a significant reduction in oscillator strength along with enhanced charge separation in DM-IN-NH2 (Supplementary Fig. 5). In contrast, the twisted excited state is unlikely to occur in DM-IN-Boc due to a large energy barrier and the lack of driving force to enter (ΔERDE = -0.34 eV) (Fig. 4c). We attributed these different excited state tendencies to the varied electron-donating strength of the -NH2 group (strong) in DM-IN-NH2 in comparison to that of the Boc group (weak) in DM-IN-Boc (Supplementary Fig. 5). These results confirm that the electron density perturbation of indazole leads to a mutational effect on the ΔERDE, flipping between positive (enhance rotation) and negative (suppress rotation). It is thus concluded that (i) the fluorescence quenching of DM-IN-NH2 (D-indazole-π-A type dye) is largely related to the intramolecular rotation in the excited state, and (ii) the fluorescence lighting-up of DM-IN-Boc (EWT-indazole-π-A type dye) is attributed to no rotation in its’ photophysical process (Fig. 4a).
Overturning the ICT probes’ quenching mode into light-up mode for sensing EWTs
The above experiments have demonstrated that indazole-based fluorophores keep silent in the D-indazole-π-A molecules, but emit brightly when substituted with an electron-withdrawing group. We thus hypothesized that this characteristic could be used for light-up (OFF-ON) sensing of EWTs. Upon encountering the corresponding EWT analytes, the transformation from D-indazole-π-A to EWT-indazole-π-A could dramatically light-up the fluorescent signal. In this regard, various OFF-ON fluorescent probes (named Lighter EW Trackers) could be built up via using our specific indazole-based chromophores.
Detection of EWTs, especially for arylamine N-acetyltransferases (NATs) and nerve agents, has recently received growing attention in the fields of biological and environmental science. NATs are phase II metabolism enzymes that transfer an acetyl group from acetyl-CoA to aromatic amines and arylhydroxylamines36. The detection of NAT2 activity is significant for disease diagnosis and personalized therapy in a clinical setting. Unfortunately, although traditional ICT fluorophores (such as DCM-NH2) possess the large Stokes shift, they inevitably exhibit a turn-OFF response toward NAT2 (Fig. 5a-5c) owing to the EWT-induced fluorescence quenching. In contrast, our designed indazole-based probes could finely address this limitation (Fig. 5d). As shown in Fig. 5e, upon reaction with NAT2 mimic, Lighter EW Tracker (DCM-IN-NH2) shows a blue-shift (from 525 to 500 nm) in the absorption spectrum. Simultaneously, a significant fluorescence enhancement (around 56-fold) is observed at 700 nm with a large Stokes shift (200 nm, Fig. 5f). Notably, both the emission wavelength, peak shape (Supplementary Fig. 1c), and high-resolution mass spectrum (HRMS, Supplementary Fig. 6) are coincident with our synthesized DCM-IN-Ac, which further confirms the generation of acylation product upon reaction with the NAT2 mimic. Overall, we have successfully developed an OFF-ON fluorescence probe for light-up sensing of NAT2 based on our fluorescence umpolung strategy.
Encouraged by the application in biosensing, we further explored the environmental monitoring applications of Lighter EW Tracker. Nerve agents (such as sarin, soman, phosgene, and so on) have gained infamy as chemical-warfare agents (CWAs) used in wars in undeveloped countries37–40. It’s essential to analyze highly toxic CWAs and related chemicals in a rapid and precise manner. As known, the high toxicity of CWAs is due to their strong capability of nucleophilic attack. Therefore, we reasoned that the active amino group of Lighter EW Tracker could be utilized as the recognition site for light-up sensing of nerve agents.
Then, we studied the spectral response of Lighter EW Tracker toward the nerve-agent mimic diethyl chlorophosphate (DCP). As shown in Fig. 5h, the absorption band at 515 nm becomes gradually decreased and is replaced by a new peak at 470 nm. Simultaneously, the NIR emission becomes enhanced to an intensity that is around 12 times larger than that of the original solution (Fig. 5i). Moreover, the plot of the I650 nm against the concentrations of DCP ranging from 0–6 µM display a good linear relationship (R2 = 0.999, Supplementary Fig. 7). Hence, this linear curve allows for the convenient quantitative detection or tracing of DCP over this concentration range. In the HRMS of Lighter EW Tracker with DCP, the peak of DCM-IN-DCP is found at m/z 568.1739 (Supplementary Fig. 8), which strongly supports that the nucleophilic substitution causes the generation of emissive DCM-IN-DCP (EWT-indazole-π-A type dye). Consequently, Lighter EW Tracker enables the quantitative and light-up detection of nerve agents. All these results show that the indazole platform can provide a generalizable method for light-up sensing EWTs including NAT2 and nerve agents.
Light-up tracking of endogenous NAT2 in living cells and tissue homogenates
After investigating the OFF-ON response characteristics of Lighter EW Tracker (DCM-IN-NH2) as a NAT2 probe in vitro, we further explored the potential of the probe for live-cell imaging of NAT2 activation (Fig. 6). Cytotoxicity of Lighter EW Tracker was first evaluated by the widely used MTT assay. As shown in Fig. 6d, when incubated with 2, 4, 8, 16, or 32 µM Lighter EW Tracker for 24 h, the cell viabilities are close to 100%, indicating the remarkable biocompatibility of the probe. Then, fluorescence confocal microscope was used to image HepG2 and HeLa cells after incubation with Lighter EW Tracker. As well known, in humans, NAT2 is expressed only in specific cells (such as HepG2 cells)36. As expected, much stronger NIR fluorescence is detected in HepG2 cells than that of HeLa cells (Fig. 6a, 6b, and 6e). Indeed, this distinctly different fluorescent intensity (p < 0.001) in HepG2 and HeLa cells is consistent with the biodistribution of NAT2. Then, co-staining studies confirm that the probes are mainly localized in the cytoplasm, such as the cell mitochondria (Supplementary Fig. 9). Furthermore, the NIR fluorescence in cells with quercetin (inhibitor of NAT2) is much weaker (p < 0.001) than that of the cells without quercetin (Fig. 6b, 6c, and 6e). All these results strongly support that Lighter EW Tracker could be used to specifically detect endogenous NAT2 in cells with a remarkable lighting-up signal.
More convincing evidence was from the comparison of the time-dependent changes of the fluorescence signal of DCM-NH2 (turn-OFF probe) and Lighter EW Tracker (turn-ON probe). As shown in Fig. 6f and 6 g, DCM-NH2 shows a bright NIR fluorescent signal after cellular uptake within 0.5 h. Owing to the turn-OFF response toward NAT2, the fluorescence of DCM-NH2 becomes weaker with time, which inevitably results in misleading information, as the reduction in fluorescence intensities could also be attributed to photobleaching, diffusion, and so on. In contrast, Lighter EW Tracker shows a weak fluorescent signal within 0.5 h, indicating that the probe is non-fluorescent initially with low background interference. As the incubation time elapses, Lighter EW Tracker is activated by endogenous NAT2, and the fluorescence intensity increases gradually which reaches a maximum at 1.5 h (Fig. 6h and 6i). These results clearly demonstrate that Lighter EW Tracker can be used for real-time and light-up monitoring of cellular NAT2.
The cell imaging results encouraged us to further detect NAT2 in different tissue homogenates including heart, liver, spleen, lung, and kidney. Previous findings suggest that human NAT2 expression is the highest in the liver but is expressed at functional levels in other tissues41. As expected, Lighter EW Tracker exhibits the highest fluorescent intensity in livers than other tissues (Fig. 6j and 6 k). These imaging results further highlight the potential of Lighter EW Tracker for the detection of NAT2 activity in a clinical setting, which will be very useful for personalized therapy and disease diagnosis.