Reversing the Solvent Polarity Effect on Fluorescence Quantum Yields of a GFP Chromophore Analogue by Inhibiting the Polarity-Promoted Hula Twist

Flexible solvatochromic fluorescent dyes (SFD) typically exhibit a decrease in fluorescence quantum yield (Phi(F)) and a red shift in fluorescence with increasing solvent polarity. In this study, we report a new E,Z-isomerization-active green fluorescence protein chromophore (GFPc) analogue (4), which uniquely demonstrates a reversed solvent polarity effect on Phi(F) and a record-high Phi(F) of 0.43 in the red (>600 nm) region. This is achieved by ring-bridging both the exocyclic C-C bond (the phi-bond) and the phenyl-amino C-N bond (the omega-bond) of a known meta-amino-substituted GFPc (1), which shows typical SFD behavior in aprotic solvents but fluorescence quenching in protic solvents. Based on the reference compounds 2 (omega-bridged) and 3 (phi-bridged), the reversed solvent effect is attributed to enhanced one-bond-flip (OBF) E,Z-isomerization in nonpolar solvents and the inhibition of hula twist (HT) pathway, which is accelerated in polar solvents. The drastic difference in fluorescence characteristics between 1 and 4 is further underscored by the superior performance of 4 in cell imaging. This work demonstrates the versatility of the GFP chromophore, whose properties could be drastically reshaped through specific structural editing at key positions.