Synthesis of cell-impermeable Cl-sensitive fluorescent indicators with improved sensitivity and optical properties

Abstract

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. 6-methoxy-N-(3-sulfopropyl)quinolinium; dextran; fibroblast Submitted on December 20, 1990 Accepted on June 10, 1991

Measurement of rapid changes in cell volume by forward light scattering

“…The heterogeneity among ICQs is neglected and lumped to constitute a single quencher, which is denoted by subscript "P." In the absence of Cl − , Eq. 2 in terms of cell volume is (3) where V is the cell volume, M P is the mass of ICQs, and b is the cell volume that is not osmotically active water (16). Because the ICQs are entrapped in the cell, M p is a constant.…”

NBD-TMA: a novel fluorescent substrate of the peritubular organic cation transporter of renal proximal tubules

“…A number of compounds containing a quaternary nitrogen have fluorescent spectra that are sensitive to collisional quenching by halide ions [2]. In NBD-TMA the quaternary nitrogen is displaced from the light-absorbing portion of the molecule by two carbon atoms.…”

“…Due to heterogeneity in the rates of NBD-TMA uptake in tubules from different animals, or in different tubules collected from the same animal, rates of uptake were normalized to measured uptake of 10 µM NBD-TMA in each experiment. Although the saturation of a carrier-mediated process was clearly evident, the data were most effectively described if uptake is assumed to consist of a saturable process working in parallel with a process with first-order kinetics over the concentration range studied: (2) where J max is the maximal rate of NBD-TMA transport, K t the concentration of NBD-TMA that results in half- maximal transport, and D a constant reflecting the presence of a fraction of total NBD-TMA accumulation that was not saturable. From Eq.…”

“…To date, most candidate OC + s have either had comparatively poor optical properties (e.g., changes in fluorescence arising from changes in the surrounding physical environment) or have been weak bases. The former characteristic poses problems because partitioning of the transported probe into compartments of differing (and variable) physical properties can influence signal output from the probe (e.g., [2]), while transport of weak bases can be influenced profoundly by non-ionic diffusion and ion-trapping (e.g., [20]). Several studies have made use of the optical properties of the styryl dye, 4-(4-dimethylaminostyryl)-N-methylpyridinium (4-Di-1-ASP + ), to examine renal OC + transport.…”

Arylethynyl receptors for neutral molecules and anions: emerging applications in cellular imaging

“…Within these areas are several additional factors that affect utility including compartmentalization or partitioning within the cell into specific organelles,102 matching optical excitation/emission wavelengths to match common instrumentation in use103 and the use of ratiometric determination methods to help quantitate binding and ion levels 104,105. Finally, factors such as specificity versus other intracellular ions,106 water solubility,107 relative binding constants for the intracellular ion or ions108 and the effect of pH on binding99,109 and fluorescence emission101 are also important factors to be optimized for biochemical probe optimization. Of course, it is impossible to optimize all parameters, but of ultimate importance are ion selectivity and the ability to quantitate intracellular concentration.…”