A Novel Dialkylamino GFP Chromophore as an Environment-Polarity Sensor Reveals the Role of Twisted Intramolecular Charge Transfer

Abstract

We discovered a novel fluorophore by incorporating a dimethylamino group (–NMe2) into the conformationally locked green fluorescent protein (GFP) scaffold. It exhibited a marked solvent-polarity-dependent fluorogenic behavior and can potentially find broad applications as an environment-polarity sensor in vitro and in vivo. The ultrafast femtosecond transient absorption (fs-TA) spectroscopy in combination with quantum calculations revealed the presence of a twisted intramolecular charge transfer (TICT) state, which is formed by rotation of the –NMe2 group in the electronic excited state. In contrast to the bright fluorescent state (FS), the TICT state is dark and effectively quenches fluorescence upon formation. We employed a newly developed multivariable analysis approach to the FS lifetime in various solvents and showed that the FS → TICT reaction barrier is mainly modulated by H-bonding capability instead of viscosity of the solvent, accounting for the observed polarity dependence. These deep mechanistic insights are further corroborated by the dramatic loss of fluorogenicity for two similar GFP-derived chromophores in which the rotation of the –NMe2 group is inhibited by structural locking.

Excited‐State Dynamics of a meta‐Dimethylamino Locked GFP Chromophore as a Fluorescence Turn‐on Water Sensor†

“…These results between the newly synthesized contrasting chemical compounds indicate that the meta-NMe 2 substituent induces a significant effect on electronic properties of the locked GFP chromophore with a pronounced redshift (particularly for the absorption peak) due to the "double-donor" structure (38,40,41). Interestingly, when the two donor positions are switched (i.e., para-NMe 2 and meta-OH on LpHBDI, see the chemical structure in Supporting Information text), the resultant neutral chromophore shows absorption and emission peaks at 448 and 582 nm, respectively, with an FQY of 0.02 in pH = 7.4 water mainly due to facile light-induced twisting of the para-NMe 2 group enhanced with solvent polarity (38).…”

“…Therefore, we attributed this transient SE band to a dark state (i.e., no transition oscillator strength for spontaneous emission in this spectral region). As previously mentioned, the ability of -NR 2 functional groups to isomerize in the excited state and form a TICT state, whose SE band would appear at a redder position, commonly leads to nonfluorescent behavior (38,51). Our systematic DFT calculations deemed a TICT state unlikely in this molecule since no S 1 energy minimum with a twisted -NMe 2 geometry was found besides the FS, which sharply contrasts with our previously reported p-NMe 2 -m-HBDI:BF 2 (see above) wherein the dimethylamino and hydroxy groups are interchanged (38), highlighting the unusual behavior of meta effect (47,58).…”

“…As previously mentioned, the ability of -NR 2 functional groups to isomerize in the excited state and form a TICT state, whose SE band would appear at a redder position, commonly leads to nonfluorescent behavior (38,51). Our systematic DFT calculations deemed a TICT state unlikely in this molecule since no S 1 energy minimum with a twisted -NMe 2 geometry was found besides the FS, which sharply contrasts with our previously reported p-NMe 2 -m-HBDI:BF 2 (see above) wherein the dimethylamino and hydroxy groups are interchanged (38), highlighting the unusual behavior of meta effect (47,58). Knowing the limitations of current level of quantum calculations for the excited-state charge-transfer processes (59,60), we experimentally studied two other HBDI derivatives with no isomerization coordinates to evaluate whether a TICT state is a dominant factor for the fluorescence properties of m-NMe 2 -LpHBDI in solution.…”

“…Notably, the ability of -NR 2 functional groups to twist in the excited state has led many molecules to be weakly fluorescent via nonradiative relaxation pathways (38,51,52). Supported by quantum calculations in our recent work of a novel fluorogenic dye derived from GFP chromophore that incorporated a dimethylamino group at the para site and a hydroxy group at the meta site (i.e., p-NMe 2 -m-HBDI:BF 2 ) to act as a polarity sensor, an ultrafast isomerization of the p-NMe 2 group in polar solvents leads to fluorescence quenching via a dark TICT state (38). By interchanging the -NMe 2 and -OH functional groups (i.e., m-NMe 2 -LpHBDI as presented in this work), systematic quantum calculations at the same level of theory no longer support the formation of a TICT state in all solvents.…”

Editorial: Organic Fluorescent Materials as Chemical Sensors

“…The review focused on the synthetic methodologies of this group of molecules and discussed the substituent effects on the fluorescence properties as well as their use in bioimaging. C. Fang and coworkers [7] designed a dialkylamino fluorophore with imidazole moiety by engineering the green fluorescent protein (GFP) chromophore. It showed a significant polarity-dependent emission and can potentially act as an environment-polarity sensor for in vitro and in vivo applications.…”