Evolution of Fluorescein as a Platform for Finely Tunable Fluorescence Probes

TLDR

Fluorescence imaging is the premier method for continuous observation of dynamic intracellular processes, yet probe design remains largely empirical and limited to a few biomolecules. The study seeks to establish a rational, photochemically grounded design strategy that departs from traditional fluorescein structures. This strategy replaces the essential carboxylic group of fluorescein with alternative substituents guided by strict photochemical principles, generating diverse new dyes. The method produces novel fluorescents with substituted carboxyl groups and demonstrates its value by creating a highly sensitive, membrane‑permeable β‑galactosidase probe.

Abstract

Fluorescence imaging is the most powerful technique currently available for continuous observation of dynamic intracellular processes in living cells. Suitable fluorescence probes are naturally of critical importance for fluorescence imaging, but only a very limited range of biomolecules can currently be visualized because of the lack of flexible design strategies for fluorescence probes. At present, design is largely empirical. Here we show that the carboxylic group of traditional fluorescein dyes, formerly considered indispensable, has been replaced with other substituents, affording various kinds of new fluoresceins. Further, by breaking out of the traditional structure of fluorescein, we developed the first and totally rational design strategy for novel fluorescence probes based on a strict photochemical basis. The value of this approach is exemplified by its application to develop a novel, highly sensitive, and membrane-permeable fluorescence probe for β-galactosidase, which is the most widely used reporter enzyme.