Recently, a new fluorescent probeAE-Phoswas reported to detect the activity of alkaline phosphatases (ALP) in different living cell lines. Here, we present an in-depth computational analysis of the mechanism and source of the fluorescence of the AE-Phos probe. There is an intermediate product (AE-OH-Phos) in the experiment as well as a different configuration of products that may emit fluorescence. It is essential to investigate the origin of fluorescence and the detection mechanism of the probe, which could help us eliminate the interference of other substances (including an intermediate product and possible isomers) on fluorescence during the experiment. According to the change of geometric parameters and Infrared spectra, we deduce that the dual intramolecular hydrogen bonds of salicylaldehyde azine (SA) were enhanced at the excited state, while AE-OH-Phos was attenuated. Considering the complex ESIPT behavior of the dual proton-type probe, the potential energy surfaces were further discussed. It can be concluded that the single proton transfer structure of SA (SA-SPT) is the most stable form. Both the concerted double proton transfer process and stepwise single proton transfer process of SA were forbidden. The fluorescence for SA was 438 nm, while that of SA-SPT was 521 nm, which agrees with the experimentally measured fluorescence wavelength (536 nm). The conclusion that single proton transfer occurs in SA is once again verified. In addition, the distribution of electron-hole and relative index was analyzed to investigate the intrinsic mechanism for the fluorescence quenching of the probe and the intermediate product. The identification of the origin of fluorescence sheds light on the design and use of dual-proton type fluorescent probes in the future.

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To reveal the influence of different substituents on the excited-state intramolecular proton transfer (ESIPT) process and photophysical properties of 4′-N, N-dimethylamino-3-hydroxyflavone (DMA3HF), two novel molecules (DMA3HF-CN and DMA3HF-NH2) were designed by introducing the classical electron-withdrawing group cyano (-CN) and electron-donating group amino (-NH2). The three molecules in the acetonitrile phase were systematically researched by applying the density functional theory (DFT) and time-dependent DFT (TD-DFT) methods. The excited-state hydrogen bond enhancement mechanism was confirmed, and the hydrogen bond intensity followed the decreasing order of DMA3HF-NH2 > DMA3HF > DMA3HF-CN, which can be explained at the electronic level by natural bond orbital, fuzzy bond order, and frontier molecular orbital analyses. Moreover, we found from the electronic spectra that the fluorescence intensity of the three molecules in keto form is relatively strong. Moreover, the calculated absorption properties indicated that introducing the electron-withdrawing group -CN could significantly improve the absorption of DMA3HF in the ultraviolet band. In summary, the introduction of an electron-donating group -NH2 can promote the ESIPT reaction of DMA3HF, without changing the photophysical properties, while introducing the electron-withdrawing group -CN can greatly improve the absorption of DMA3HF in the ultraviolet band, but hinders the occurrence of the ESIPT reaction.

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