Comprehensive Studies on an Overall Proton Transfer Cycle of the ortho-Green Fluorescent Protein Chromophore

Abstract

Initiated by excited-state intramolecular proton transfer (ESIPT) reaction, an overall reaction cycle of 4-(2-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one (o-HBDI), an analogue of the core chromophore of the green fluorescent protein (GFP), has been investigated. In contrast to the native GFP core, 4-(4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one (p-HBDI), which requires hydrogen-bonding relay to accomplish proton transfer in vivo, o-HBDI possesses a seven-membered-ring intramolecular hydrogen bond and thus provides an ideal system for mimicking an intrinsic proton-transfer reaction. Upon excitation, ESIPT takes place in o-HBDI, resulting in a ∼600 nm proton-transfer tautomer emission. The o-HBDI tautomer emission, resolved by fluorescence upconversion, is comprised of an instantaneous rise to a few hundred femtosecond oscillation in the early relaxation stage. Frequency analysis derived from ultrashort pulse gives two low-frequency vibrations at 115 and 236 cm(-1), corresponding to skeletal deformation motions associated with the hydrogen bond. The results further conclude that ESIPT in o-HBDI is essentially triggered by low-frequency motions and may be barrierless along the reaction coordinate. Femtosecond UV/vis transient absorption spectra also provide supplementary evidence for the structural evolution during the reaction. In CH(3)CN, an instant rise of a 530 nm transient is resolved, which then undergoes 7.8 ps decay, accompanied by the growth of a rather long-lived 580 nm transient species. It is thus concluded that following ESIPT the cis-proton transfer isomer undergoes cis-trans-isomerization. The results of viscosity-dependent dynamics are in favor of the one-bond-flip mechanism, which is in contrast to the volume-conserving isomerization behavior for cis-stilbene and p-HBDI. Further confirmation is given by the picosecond-femtosecond transient IR absorption spectra, where several new and long-lived IR bands in the range of 1400-1500 cm(-1) are assigned to the phenyl in-plane breathing motions of the trans-proton transfer tautomer. Monitored by the nanosecond transient absorption, the 580 nm transient undergoes a ∼7.7 μs decay constant, accompanied by the growth of a new ∼500 nm band. The latter is assigned to a deprotonated tautomer species, which then undergoes the ground-state reverse proton recombination to the original o-HBDI in ∼50 μs, achieving an overall, reversible proton transfer cycle. This assignment is unambiguously supported by pump-probe laser induced fluorescence studies. On these standpoints, a comparison of photophysical properties among o-HBDI, p-HBDI, and wild-type GFP is discussed in detail.

A cascade synthetic route to new bioactive spiroindolinepyrido[1,2-a]indolediones from indirubin

“…29,30 However, the opposite is observed with the calculated values (2 to 9). This bathochromic shift can be explained by referring to the 'cross-conjugated' system ('H-chromophore'), which was found to be an important feature to explain (although other explanations have been proposed 31 ), the colour of indigoid molecules.…”

Photoacid behaviour in a fluorinated green fluorescent protein chromophore: ultrafast formation of anion and zwitterion states

“…By thus extending the excited state lifetime, ESPT was observed to occur two to three orders of magnitude slower than in the protein 14. Solntsev et al also showed that the meta hydroxy derivative, m -HBDI, does exhibit picosecond ESPT in aqueous solution,15 while Hsieh et al showed that o -HBDI in aprotic solvents undergoes efficient intramolecular ESPT, protonating the imidazole nitrogen with which the o -OH group forms an intramolecular H-bond 16. However, neither the o -HBDI nor the m -HBDI isomers of the chromophore occur naturally in the FP family.…”

Excited‐State Intramolecular Proton Transfer in a Blue Fluorescence Chromophore Induces Dual Emission

“…More interestingly,c ompared with GFP analogues with ESIPT,t his BFP chromophore exhibits ag reatlye nhanced fluorescence quantum yield and am uch slowerE SIPT rate of about 18 ps, which is significantly longert han 25 fs for 4-(2-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one (o-HBDI). [47][48][49] Even Compared with green fluorescencep rotein (GFP) chromophores,t he recently synthesized blue fluorescencep rotein (BFP) chromophore variant presentsi ntriguing photochemical properties, for example, dual fluorescencee mission, enhanced fluorescenceq uantumy ield, and ultra-slow excited-state intramolecular proton transfer (ESIPT; J. Phys. Chem.…”