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 < 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.