Ultrafast Excited-state Dynamics of GFP Chromophores and Pyrene Derivatives

Abstract

Photophysical processes are energy transitions that occur as a molecule absorbs light. Among these processes, excited-state proton transfer (ESPT), where protons are transferred in the excited state, and excited- state intramolecular charge transfer (ICT), where charges are redistributed within a molecule, are particularly noteworthy. These proton transfer (PT) and charge transfer (CT) processes can alter a molecule's electronic structure and energy states. Femtosecond transient absorption spectroscopy (TA) is an effective tool for observing such processes, enabling detailed investigations of ultrafast dynamics and energy relaxation pathways in the excited state. Studying photophysical processes is essential in understanding the energy transition mechanisms of molecules and exploring pathways to control these processes. This study elucidates the ESPT dynamics of green fluorescent protein (GFP) chromophores and the ICT dynamics of pyrene derivatives using TA. This study aims to identify design directions for improving fluorescence quantum yields or fluorescence lifetimes by observing the ESPT dynamics of two basic forms of GFP chromophores, 4-(4-hydroxybenzylidene)-1,2-dimethyl-1H- imidazol-5(4H)-one (p-HBDI) and 4-(2-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one (o-HBDI) and discussing the impact of electron acceptors on the ICT dynamics of pyrene derivatives and the solvent effects on the excited-state dynamics. GFP, derived from the jellyfish aequorea victoria, emits strong green fluorescence through intermolecular proton transfer with water. GFP exhibits a high quantum efficiency of 80% and a long fluorescence lifetime of 3 ns, making it the subject of extensive research for the emission mechanism. One of the widely used model chromophores for GFP is p-HBDI. p-HBDI is virtually non-fluorescent and undergoes energy dissipation through ultrafast non-radiative pathways in the excited state. In contrast, o-HBDI emits strong fluorescence through intramolecular ESPT, which is strongly solvent-dependent. The ESPT process slows down in water due to the intermolecular hydrogen bonding with water. This study would contribute to a deeper understanding of the photophysical processes of GFP chromophores and their analogs, aiding in developing new fluorescent probes and potential applications. The photophysical properties and ICT dynamics of two pyrene derivatives, (E)-2-cyano-3-(pyren-1- yl)acrylamide (pyr-ca) and (Z)-3-(pyren-1-yl)-2-(pyridin-3-yl)acrylonitrile (pyr-pn) will be discussed. pyr-ca exhibits the increase of Stokes shifts with the solvent polarity increase: 3515 cm⁻¹ (toluene) to 5344 cm⁻¹ (acetonitrile). In contrast, pyr-pn shows smaller Stokes shift increase with the solvent polarity increase: 4342 cm⁻¹ (toluene) to 5103 cm⁻¹ (acetonitrile). The ICT dynamics of pyr-ca is determined as the ultrafast time constant of 430 fs in acetonitrile, which is followed by vibrational relaxation (3.7 ps) and population decay (36 ps) of the ICT state. In contrast, the ultrafast ICT process is not observed for pyr-pn regardless of the solvent polarity.