Structure-activity relationships of chloride-sensitive fluorescent indicators for biological application

The application of the quinoline derivative 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) to the measurement of membrane transport of chloride in biological systems was reported recently (N.P. Illsley and A.S. Verkman (1987) Biochemistry 26, 1215-1219). To understand the structure-activity relationships of compounds with chloride-sensitive fluorescence properties, 19 structural analogs of SPQ having a single quaternized nitrogen heteroatom were synthesized and characterized. The effect of variations in ring structure, length of sulfoalkyl chain, position of ring substituent, and nature of ring substituent were examined. For each compound, the water solubility, octanol:water partition coefficient, absorbance and fluorescence spectra, fluorescence lifetime, and Stern-Volmer constants (Kq) for quenching by a series of anions were measured. All compounds were quenched by chloride, bromide, iodide, and thiocyanate, but not by cations, sulfate, phosphate, nitrate, or by pH (5-8); several compounds were quenched slightly by bicarbonate (Kq = 8-12 M-1). High chloride sensitivity (Kq greater than 50 M-1) required the presence of a quinoline backbone substituted with electron-donating groups such as methyl and methoxy, but did not depend on length of the sulfoalkyl chain or on the position of ring substituents (positions 2-7). All compounds with high chloride sensitivity had fluorescence excitation spectra in the ultraviolet (excitation maximum less than 350 nm) and fluorescence lifetimes greater than 15 ns. These results establish a set of guidelines for synthesis of chloride-sensitive fluorescent indicators tailored for specific biological applications.