Synthesis and characterization of dual-wavelength Cl−-sensitive fluorescent indicators for ratio imaging

The fluorescence of quinolinium-based Cl− indicators such as 6-methoxy- N-(3-sulfopropyl)quinolinium (SPQ) is quenched by Cl− by a collisional mechanism without change in spectral shape. A series of “chimeric” dual-wavelength Cl− indicators were synthesized by conjugating Cl−-sensitive and -insensitive chromophores with spacers. The SPQ chromophore (N-substituted 6-methoxyquinolinium; MQ) was selected as the Cl−-sensitive moiety [excitation wavelength (λex) 350 nm, emission wavelength (λem) 450 nm]. N-substituted 6-aminoquinolinium (AQ) was chosen as the Cl−-insensitive moiety because of its different spectral characteristics (λex 380 nm, λem 546 nm), insensitivity to Cl−, positive charge (to minimize quenching by chromophore stacking/electron transfer), and reducibility (for noninvasive cell loading). The dual-wavelength indicators were stable and nontoxic in cells and were distributed uniformly in cytoplasm, with occasional staining of the nucleus. The brightest and most Cl−-sensitive indicators were α-MQ-α′-dimethyl-AQ-xylene dichloride and trans-1,2-bis(4-[1-α′-MQ-1′-α′-dimethyl-AQ-xylyl]-pyridinium)ethylene (bis-DMXPQ). At 365-nm excitation, emission maxima were at 450 nm (Cl− sensitive; Stern-Volmer constants 82 and 98 M−1) and 565 nm (Cl−insensitive). Cystic fibrosis transmembrane conductance regulator-expressing Swiss 3T3 fibroblasts were labeled with bis-DMXPQ by hypotonic shock or were labeled with its uncharged reduced form (octahydro-bis-DMXPQ) by brief incubation (20 μM, 10 min). Changes in Cl− concentration in response to Cl−/nitrate exchange were recorded by emission ratio imaging (450/565 nm) at 365-nm excitation wavelength. These results establish a first-generation set of chimeric bisquinolinium Cl− indicators for ratiometric measurement of Cl− concentration.