Fluorescence Enhancement of Unconstrained GFP Chromophore Analogues Based on the Push–Pull Substituent Effect

Unlike the high fluorescence quantum yield of the naturally occurring green fluorescence protein (GFP, Φf ∼ 0.8), the GFP chromophore, a benzylidenedimethylimidazolinone (BDI) dye, is nearly nonfluorescent (Φf ortho-CN and meta-dimethylamino substituent electronic effect that largely suppresses the Z → E photoisomerization (the τ torsion) reaction, which is the major nonradiative decay channel of uGFPc. The structural design relied on the assumptions that the τ torsion of the meta-amino-substituted BDI systems leads to a zwitterionic twisted intermediate state (1p*) and that destabilizing the 1p* state by an electron-withdrawing CN substituent at the ortho or para position could slow down the τ torsion. The observed CN position effect conforms to the design concept. The push–pull substitution of BDI also leads to sensitive fluorescence-quenching responses to electron donors such as trimethylamine and to H-bond donors such as methanol.