An Excited-StateLone Pair–π* InteractionEnables Red Shift of Fluorescence of Neutral GFP-type Chromophores

Abstract Green fluorescent protein chromophores based on the 4-hydroxybenzylidene-imidazolinone (HBI) scaffold play a central role in bioimaging applications. While most structure–property studies have focused on the Z configuration, the influence of E/Z configurational isomerism on intrinsic photophysical properties remains less explored. Here, we present a systematic quantum chemical investigation of 50 HBI-type chromophore analogues to examine how E/Z configuration affects their excited-state properties. The calculations reveal that, compared to the Z isomers, E-configured chromophores exhibit a consistent red shift in fluorescence emission. Analysis of the excited-state geometries and electronic structures indicates that this behavior is associated with the formation of twisted conformations in the S1 state and the emergence of intramolecular n–π* interactions, which contribute to the reduction of the HOMO–LUMO gap. These results provide a mechanistic picture of how configurational isomerism influences emission properties at the chromophore level. The present findings offer new insight into the intrinsic electronic factors governing spectral tuning in HBI-type chromophores, and will facilitate computational design of HBI-like fluorescence probes with explicit environmental and dynamical effects included in the future.