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

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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 < 0.001) in common solutions at room temperature. While many efforts have been devoted into the BDI chromophore engineering for fluorescence recovery, limited success has been achieved for structurally unconstrained GFP chromophore analogues (uGFPc). Herein we report a rational design of uGFPc toward an unprecedentedly high fluorescence quantum efficiency of 0.60 in hexane. This is achieved by a combined 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.