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 mechanis m without change in spectral shape. A series of "chimeric" dual-wavelength Cl- indicators were synthesized by conjugating Cl--sensitive and -insensiti ve chromophores with spacers. The SPQ chromophore (N-substituted 6-methoxyq uinolinium; MQ) was selected as the Cl--sensitive moiety [excitation wavele ngth (lambda(ex)) 350 nm, emission wavelength (lambda(em)) 450 nm]. N-subst ituted 6-amino-quinolinium (AQ) was chosen as the Cl--insensitive moiety be cause of its different spectral characteristics (lambda(ex) 380 nm, lambda( em) 546 nm), insensitivity to Cl-, positive charge (to minimize quenching b y chromophore stacking/electron transfer), and reducibility (for noninvasiv e cell loading). The dual-wavelength indicators were stable and nontoxic in cells and were distributed uniformly in cytoplasm, with occasional stainin g of the nucleus. The brightest and most Cl--sensitive indicators were alph a-MQ-alpha'-dimethyl-AQ-xylene dichloride and trans-1,2-bis(4-[1-alpha'-MQ- 1 '-alpha'-dimethyl-AQ-xylyl]-pyridinium)ethylene (bis-DMXPQ). At 365-nm ex citation, emission maxima were at 450 nm (Cl- sensitive; Stern-Volmer const ants 82 and 98 M-1) and 565 nm (Cl- insensitive). Cystic fibrosis transmemb rane conductance regulator-expressing Swiss 3T3 fibroblasts were labeled wi th bis-DMXPQ by hypotonic shock or were labeled with its uncharged reduced form (octahydro-bis-DMXPQ) by brief incubation (20 mu M, 10 min). Changes i n Cl- concentration in response to Cl-/nitrate exchange were recorded by em ission ratio imaging (450/565 nm) at 365-nm excitation wavelength. These re sults establish a first-generation set of chimeric bisquinolinium Cl- indic ators for ratiometric measurement of Cl- concentration.