Two-step FRET mediated metal ion induced signalling responses in a probe appended with three fluorophores

A tri-fluorophoric molecular probe (1) with three different derivatized fluorophores, i.e. anthracene (An), 7-nitrobenz-2-oxa-1,3-diazole (NBD) and rhodamine-B (Rh) appended on to a Tren [tris-(2-aminoethyl)amine] receptor was demonstrated to exhibit metal ion induced ratiometric fluorescence signalling through the initiation of a two-step fluorescence resonance energy transfer (FRET) process owing to a compatible and substantial spectral overlap of electronic absorption and fluorescence of initial donor-intermediate donor/acceptor-final acceptor pairs. The steady-state and time-resolved fluorescence spectral investigation of probes (1a-1f) with individual excitation energy donor (D)/acceptor (A) fluorophores as well as different bi-fluorophoric D-A pairs appended to the same receptor, and comparison with those of 1 revealed a higher energy transfer efficiency between the initial donor (F_An) and the final acceptor (F_Rh) in the presence of an intermediate (F_NBD) fluorophore. The determination of FRET efficiency involving rhodamine-B derivatized systems is complicated as the dynamics and equilibria of their complexation induced spirolactam to ring-opened transformations are driven by various influential parameters; despite this, the FRET efficiencies in 1 and other intermediates were determined and compared under similar conditions for a better comprehension of individual fluorophoric contribution in two-step FRET-based sensing. The selectivity in chelation induced two-step FRET mediated fluorescence signalling in 1 is controlled by a coordination environment and solvent medium; it exhibited Hg²⁺ ion selective signalling responses in aqueous-MeCN medium while rendering discriminatory responses towards Fe³⁺/Fe²⁺ redox couples in MeCN medium.