Graduate Thesis Or Dissertation

Dynamics and Design of Green Fluorescent Protein-Inspired Superphotoacids and Red-Emitting Fluorophores with Transient Absorption and Femtosecond Stimulated Raman Spectroscopy

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  • In the past decades, femtosecond stimulated Raman spectroscopy (FSRS) has been gaining tremendous popularity in fundamental sciences stemming from chemistry to biology. It is capable of capturing both equilibrium and non-equilibrium structural information across a broad range of timescales with simultaneously high temporal and spectral resolutions. Femtosecond transient absorption spectroscopy (fs-TA) acts as a powerful partner technique that complements the dynamics analysis and guides the resonance selection for FSRS pulses. In this dissertation, two bodies of work on the excited-state dynamics and design strategies of the green fluorescent protein (GFP) chromophore-based superphotoacids and red-emitting fluorophores studied by FSRS and TA are presented. First, FSRS and fs-TA were implemented to dissect the ultrafast excited-state proton transfer (ESPT) dynamics of superphotoacids in aqueous and nonaqueous solutions. The superphotoacids were derived from the synthetic GFP chromophore, namely, p-hydroxybenzylidiene-dimethylimidazolinone or p-HBDI. By strategic conformational locking and multi-site halogenation, p-HBDI was transformed from a dim weak photoacid to a bright superphotoacid. The locking enables a three-order increase of magnitude in fluorescence quantum yield from 10−4 to 10−1 while the halogenation remarkably elevates the photoacidity characterized by the excited-state pKa or pKa* (asterisk denotes excited state) value which is lowered from ~2.1 of p-HBDI to negative values of the derived superphotoacids. FSRS and fs-TA results showed that the ESPT dynamics of these superphotoacids in protic organic solvents such as methanol (an inert proton acceptor) are inhomogeneous. Three pathways including direct, solvent reorientation-controlled, and rotational diffusion-controlled ESPT are coexisting within the excited-state lifetime. Through a systematic comparison of different photoacids, new design principles of tuning photoacidity was proposed. Besides, a comparison between locked and unlocked halogenated superphotoacids of similar strengths was made. They exhibit drastically different ESPT dynamics and thus highlight the importance of structure-activity relationship of functional molecules. Second, ground- and excited-state FSRS in conjunction with quantum calculations allows a deep understanding of the emission mechanism in GFP chromophore and its derivatives. A unique “double-donor-one-acceptor” red-shifting strategy thereby was proposed based on the GFP chromophore and we showed that this strategy can be generalized to other fluorophore scaffolds. Excited-state intramolecular charge transfer (ICT) was extensively proven to be able to red-shift emission wavelength by stabilizing the excited state. The presence of excited-state ICT in GFP chromophore and its derivatives were both experimentally and theoretically demonstrated with site precision by FSRS and quantum calculations, respectively. Moreover, a “double-donor” structure that incorporates another electron-donating group (EDG) to the ortho site of the −OH/O− group of p-HBDI was discovered to destabilize the ground state. In synergy with the “one-acceptor” that lowers excited-state energy through ICT, the emission energy gap could be lowered to a remarkable extent. The red emission wavelength is highly desired in biological imaging due to the resultant small background noise and has drawn lots of attention in the design of imaging probes. The “double-donor-one-acceptor” strategy is advantageous in that it does not require large π-conjugation extension and hence provides space for further chemical engineering for other wanted imaging properties such as high FQY.
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