A general strategy to increase emission shift of two-photon ratiometric pH probes using a reversible intramolecular reaction of spiro-oxazolidine.

Fluorescent pH probes have been served as powerful tools in biological and pathological studies in recent years due to the important roles of pH values in various physiological processes. Although plenty of pH probes have been delivered, development of two-photon ratiometric pH probes with large emission shift for detecting the variation of intracellular pH values is still a greatly challenging task. To address this concern, in this work, we have discovered a general strategy designing pH probes by means of a pH-dependent reversible intramolecular reaction of spiro-oxazolidine which can efficiently change their conjugation length and the electronic effect concurrently. To display the generality of the strategy, we have synthesized six pH probes, and all these probes exhibit short emission in basic conditions and dramatically red-shifted emission in acid environments. The emission shift of the six probes is more than 150 nm and even up to 210 nm, much larger than shift of all commercial and reported pH probes. The chemical sensing mechanism of intramolecular ring opening/closing reaction of spiro-oxazolidine has been confirmed with 1H NMR spectra and density functional theory (DFT) calculations. Finally, we have used one of six with one- and two-photon properties to successfully image lysosomal pH changes under confocal and two-photon microscopes in a ratiometric manner. We believed that this spiro-oxazolidine strategy can serve as a general and powerful platform for the design of ideal pH probes.