A ratiometric near-infrared fluorescent probe based on a novel reactive cyanine platform for mitochondrial pH detection

A near-infrared reactive cyanine platform (probe A) was prepared by condensation of 9-chloro-1,2,3,4-tetrahydro-10-methyl-acridinium iodide with Fisher's aldehyde. A near-infrared fluorescent probe (probe B) was prepared by modifying a reactive chlorine atom of probe A with tert-butyl(2-aminoethyl)carbamate through a substitution reaction. The deprotection of the Boc group of probe B was achieved under an acidic condition, affording an amine-functionalized cyanine dye (probe C). A near-infrared ratiometric fluorescent probe (probe D) for mitochondrial pH detection was synthesized by conjugating a FRET coumarin donor to a FRET cyanine acceptor (probe C) through an amide bond connection. Probe A shows low fluorescence of 2% due to an electron-withdrawing chlorine atom, while probes B-D display high fluorescence quantum yields of 60%, 32%, and 35% in aqueous solutions containing 10% ethanol, respectively. Probes B-D show strong fluorescence with push-pull molecular structures in neutral and basic pH conditions. However, protonation of the probe's second amine at the 9-position under acidic condition disrupts the push-pull feature of the probes, resulting in fluorescence quenching of the new cyanine fluorophores. The probes can selectively stain mitochondria, while probe D was employed to detect pH changes in HeLa cells and Drosophila melanogaster first-instar larvae.

Automated summary

In this study, four fluorescent probes with different structures were synthesized for the detection and study of mitochondrial pH. These probes exhibited varying fluorescence properties under different conditions, with probe D showing potential applications through the detection of pH changes in HeLa cells and Drosophila melanogaster first-instar larvae.