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

Abstract

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 pKa 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.