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.
Highlights
Environment-sensitive dyes have long been popular research subjects due to their importance in chemistry and chemical biology [1]
Compounds 2 and 3 exhibit high fluorescence quantum yields (FQYs) and a lack of the aforementioned strong correlation due to an effective inhibition of twisting motions of the electron donor. It was noted for these two control compounds that weak correlations were present between log(k nr ) and ETN, which implies that some other nonradiative channels via electronic means could occur in the presence of molecular structural rigidities. These results suggest that the twisted intramolecular charge transfer (TICT) pathway is the main cause of fluorogenicity for compound 1 and higher solvent polarity favors the formation of a TICT state with a reduced excited-state twisting energy barrier
Solvent viscosity has been overlooked throughout the correlation analysis and is an important influencer for many twisting molecular systems. To take these factors into consideration for a more inclusive and comprehensive investigation, we have recently developed a multivariable linear analysis approach to identify primary contributing interactions for the observed fluorogenicity [26], though the green fluorescent protein (GFP)-derived chromophore in that work was unlocked and the fluorogenicity is readily achieved inside a fluorogen-activating protein
Summary
Environment-sensitive dyes have long been popular research subjects due to their importance in chemistry and chemical biology [1]. The development of fluorescent probes such as fluorogenic dyes to bind fluorogen-activating proteins [2,3] and fluorescent sensors to detect metal ions [4,5] has significantly facilitated the advancement of imaging and sensing toolsets for modernized life sciences. There is currently strong interest and ongoing research to discover and create more photosensitive molecules with appealing traits for the development of next-generation fluorescent probes and sensors across chemical and biological fields. To achieve this goal, a fundamental understanding of the molecular origin underlying the fluorescence–structure–environment relationships is crucial.
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