Synthesis and spectroscopic characterisation of BODIPY® based fluorescent off–on indicators with low affinity for calcium

Abstract

Two fluorescent off–on Ca²⁺ indicators based on APTRA (o-aminophenol-N,N,O-triacetic acid) as low-affinity ligand for Ca²⁺ and BODIPY^® (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) as a fluorophore were synthesized. The new BODIPY^®–APTRA compounds absorb in the visible spectrum, with absorption maxima from 505 nm to 570 nm, and have fluorescence spectra that span the visible spectrum, with emission maxima ranging from 525 nm to 625 nm dependent on the substituents at the α-positions to the nitrogen atoms. The indicators show a large increase of the fluorescence quantum yield upon increasing Ca²⁺ concentration. The ground-state dissociation constants K_d estimated at 20 °C in 100 mM KCl aqueous buffered solution, pH 7.20, for the two complexes with Ca²⁺ were found to be around 100 µM.