New insights into the detection mechanism of β-galactosidase in living cells with fluorescent probes

Highlights: • Using DFT and TDDFT to investigate the ESIPT mechanism of ESIPT-GAL and ESIPT-OH. • Explaining the reason for the single fluorescence of ESIPT-OH. • Investigating the charge transfer mechanism in ESIPT-GAL and ESIPT-OH in S₁ state. • Exploring the reasons for the weak fluorescence of ESIPT-GAL. The mechanism of β-galactosidase detection probes ESIPT-GAL and ESIPT-OH have been investigated. The torsional potential energy curve proves the planar characteristics of the system. Combined with frontier molecular orbital and "hole-electron" analysis, there are apparent planar intramolecular charge transfer (PICT) characteristics in ESIPT-GAL and ESIPT-OH. The barrier-free intramolecular proton transfer explained the single fluorescence. Furthermore, the mechanism of ESIPT-OH fluorescent probe is the cooperation of excited-state intramolecular proton transfer (ESIPT) and PICT. The relationship between the singlet and triplet states combined with the spin coupling constant reveals that the weak fluorescence characteristic of ESIPT-GAL is caused by the intersystem crossing.