AJP - Cell Physiology, Vol 262, Issue 1 C242-C250, Copyright © 1992 by American Physiological Society
Synthesis of cell-impermeable Cl-sensitive fluorescent indicators with improved sensitivity and optical properties
J. Biwersi, N. Farah, Y. X. Wang, R. Ketcham and A. S. Verkman
Department of Medicine, University of California, San Francisco 94143-0532.
Quinolinium compounds have been used as Cl-sensitive fluorescent indicators
in cells and cell-free membrane fractions. To improve Cl sensitivity and
for conjugation via nucleophilic reaction, the compounds
6-methoxy-N-(n-aminoalkyl)quinolinium bromide hydrochloride (AAQ) with
alkyl chain lengths (n) of 2 (AEQ), 3 (APQ), and 4 (ABQ) were synthesized.
AAQ was water soluble, fluorescent, and quenched by Cl. The Stern-Volmer
constants (KCl) for quenching of protonated AEQ, APQ and ABQ by Cl were
354, 322, and 272 M-1, respectively, higher than KCl for
6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ; 118 M-1). To eliminate
pH-dependent fluorescence,
6-methoxy-N-(3-trimethylammoniumpropyl)quinolinium dibromide (TMAPQ) was
synthesized (KCl, 310 M-1). To red shift fluorescence excitation and
emission spectra, 6-phenyl-N-(3-trimethylammoniumpropyl)quinolinium
dibromide (phenyl-TMAPQ) (emission 475 nm) and
N-(3-trimethylammoniumpropyl)phenanthridinium dibromide (TMAPP) (excitation
380 nm) were synthesized. AEQ and ABQ were conjugated with neutral dextran
activated by cyanogen bromide to give indicator-to-dextran mole ratios of 5
to 20. KCl values at pH 7.4 were 132 (AEQ-dextran) and 237 M-1
(ABQ-dextran). To construct a single molecule with Cl-sensitive and
insensitive moieties, the bichromophores 6-methoxy-N-(n-
dansylsulfonamidoalkyl)quinolinium with alkyl chains of two and four were
synthesized. The new Cl-sensitive indicators were used for measurement of
intracellular Cl activity and for the labeling of endocytic vesicles in 3T3
fibroblasts and T84 cells. Our results indicate that N-substitution of
quinoline with positively charged moieties gives increased Cl sensitivity,
and extension of ring conjugation gives indicators with red-shifted
fluorescence spectra.
