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

Abstract Flexible solvatochromic fluorescent dyes (SFD) typically exhibit a decrease in fluorescence quantum yield (Φ 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 Φ F and a record‐high Φ F of 0.43 in the red (>600 nm) region. This is achieved by ring‐bridging both the exocyclic C−C bond (the ϕ‐bond) and the phenyl‐amino C−N bond (the ω‐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 (ω‐bridged) and 3 (ϕ‐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.