Revealing the importance of the C(1) position to modulate the photophysics and Z-scan responses of o-locked GFP chromophores.

Three novel ortho-locked (o-locked) green fluorescent protein (GFP) chromophores, which are also doubly locked, with a phenyl group at the C(1) of the imidazolinone ring and substituents with varying electronic effects at the C(9) of the benzylidene moiety, have been synthesized. All the chromophores show relatively weak but much red-shifted emissions compared to their methyl counterparts at C(1), as previously reported by us [D. Paul et al., J. Phys. Chem. B 129, 692-711 (2025)]. Quantum chemical calculations indicate that the excited state dynamics of the chromophores possess initial rotation of the phenyl, followed by proton transfer and geometry twisting. Fluorescence decays confirm an early time charge transfer from the benzylidene moiety to the electron-withdrawing substituents at C(9). The relaxation processes occur within a time range of hundreds of femtoseconds to a few picoseconds, subject to respective compounds. This study supports our earlier reported findings [D. Paul et al., J. Phys. Chem. B 129, 692-711 (2025)] that the electronic effect at C(9) significantly affects the quantum yields of o-locked GFP chromophore analogs. This study also shows that solvent viscosity and temperature play dominant roles in modulating the fluorescence intensities of o-locked GFP chromophores. Furthermore, these compounds display significantly superior nonlinear optical (NLO) properties than their methyl analogs [D. Paul et al., J. Phys. Chem. B 129, 692-711 (2025)]. These findings provide valuable insight regarding the correlation between the molecular structures of o-locked GFP chromophores and their spectroscopic nature and pave the way to structurally engineering improved fluorophores. In a nutshell, these chromophores with a phenyl group at C(1), with poor but highly red-shifted emission in solution, showing viscosity dependency on emission intensity, and high NLO properties, can be very useful in bio-imaging.