Synthesis of cell-impermeable Cl-sensitive fluorescent indicators with improved sensitivity and optical properties.
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.
Department of Medicine, University of California, San Francisco 94143-0532.
J
J
Biwersi
N
N
Farah
Y X
YX
Wang
R
R
Ketcham
A S
AS
Verkman
D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D003619:Dansyl Compounds; D003911:Dextrans; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry / Q000493:pharmacokinetics*; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D011806:Quinolinium Compounds / Q000138:chemical synthesis / Q000737:chemistry / Q000493:pharmacokinetics; D012680:Sensitivity and Specificity; D013329:Structure-Activity Relationship
D002712:Chlorides; D003619:Dansyl Compounds; D003911:Dextrans; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D011806:Quinolinium Compounds; C029038:dansyl chloride