Balancing Brightness\nand Photobasicity: Modulating\nExcited-State Proton Transfer Pathways in Push–Pull Fluorophores\nfor Biological Two-Photon Imaging

Push–pull fluorophores with donor–π–acceptor\narchitectures are attractive scaffolds for the design of probes and\nlabels for two-photon microscopy. Such fluorophores undergo a significant\ncharge-delocalization in the excited state, which is essential for\nachieving a large two-photon absorption cross-section and brightness.\nThe polarized excited state may, however, also facilitate excited-state\nproton transfer (ESPT) pathways that can interfere with the probe\nresponse. Herein, we employed steady-state and time-resolved spectroscopic\nstudies to elucidate whether ESPT is responsible for the pH-dependent\nemission response of the Zn(II)-selective fluorescent probe chromis-1. Composed of a push–pull architecture\nwith a pyridine ring as the acceptor, the chromis-1 fluorophore core acts as a photobase that promotes ESPT upon acidification.\nAlthough the p<i>K</i><sub>a</sub> of the pyridine acceptor\nincreases more than six orders of magnitude upon excitation, the photobasicity\nis not sufficient to deprotonate solvent water molecules under neutral\nconditions. Rather, the pH-dependent emission response is caused by\nthe pendant bis-isonicotinic acid chelating group which upon protonation\nfacilitates an excited-state intramolecular proton transfer to the\npyridine acceptor. A simple permutation of the core pyridine nitrogen\nfrom the para- to the ortho-position relative to the thiazole substituent\nwas sufficient to reduce the excited-state basicity by two orders\nof magnitude without compromising the two-photon excited brightness.\nThese results highlight the importance of choosing the appropriate\nfluorophore core and chelating moiety for minimizing pH-dependent\nresponses in the design of fluorescent probes for biological imaging.