Balancing Brightness and Photobasicity: Modulating Excited-State Proton Transfer Pathways in Push–Pull Fluorophores for Biological Two-Photon Imaging

• Published in: The journal of physical chemistry. A, Molecules, spectroscopy, kinetics, environment, & general theory Molecules, spectroscopy, kinetics, environment, & general theory, 2024-11, Vol.128 (46), p.9904-9916
• Main Authors: McCallum, Adam M., Yu, Jiyao, Sumalekshmy, S., Hagwood, Abigail, Fahrni, Christoph J.
• Format: Article
• Language: English
• Subjects: A: Structure, Spectroscopy, and Reactivity of Molecules and Clusters
• ISSN: 1089-5639; 1520-5215; 1520-5215
• DOI: 10.1021/acs.jpca.4c05649

Push–pull fluorophores with donor–π–acceptor architectures are attractive scaffolds for the design of probes and labels for two-photon microscopy. Such fluorophores undergo a significant charge-delocalization in the excited state, which is essential for achieving a large two-photon absorption cross-section and brightness. The polarized excited state may, however, also facilitate excited-state proton transfer (ESPT) pathways that can interfere with the probe response. Herein, we employed steady-state and time-resolved spectroscopic studies to elucidate whether ESPT is responsible for the pH-dependent emission response of the Zn­(II)-selective fluorescent probe chromis-1. Composed of a push–pull architecture with a pyridine ring as the acceptor, the chromis-1 fluorophore core acts as a photobase that promotes ESPT upon acidification. Although the pK a of the pyridine acceptor increases more than six orders of magnitude upon excitation, the photobasicity is not sufficient to deprotonate solvent water molecules under neutral conditions. Rather, the pH-dependent emission response is caused by the pendant bis-isonicotinic acid chelating group which upon protonation facilitates an excited-state intramolecular proton transfer to the pyridine acceptor. A simple permutation of the core pyridine nitrogen from the para- to the ortho-position relative to the thiazole substituent was sufficient to reduce the excited-state basicity by two orders of magnitude without compromising the two-photon excited brightness. These results highlight the importance of choosing the appropriate fluorophore core and chelating moiety for minimizing pH-dependent responses in the design of fluorescent probes for biological imaging.