SPECIAL COMMUNICATION
Synthesis and characterization of dual-wavelength Clminus -sensitive fluorescent indicators for ratio imaging

Sujatha Jayaraman, Joachim Biwersi, and A. S. Verkman

Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521


    ABSTRACT
Top
Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

The fluorescence of quinolinium-based Cl- indicators such as 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) is quenched by Cl- by a collisional mechanism without change in spectral shape. A series of "chimeric" dual-wavelength Cl- indicators were synthesized by conjugating Cl--sensitive and -insensitive chromophores with spacers. The SPQ chromophore (N-substituted 6-methoxyquinolinium; MQ) was selected as the Cl--sensitive moiety [excitation wavelength (lambda ex) 350 nm, emission wavelength (lambda em) 450 nm]. N-substituted 6-aminoquinolinium (AQ) was chosen as the Cl--insensitive moiety because of its different spectral characteristics (lambda ex 380 nm, lambda em 546 nm), insensitivity to Cl-, positive charge (to minimize quenching by chromophore stacking/electron transfer), and reducibility (for noninvasive cell loading). The dual-wavelength indicators were stable and nontoxic in cells and were distributed uniformly in cytoplasm, with occasional staining of the nucleus. The brightest and most Cl--sensitive indicators were alpha -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 excitation, emission maxima were at 450 nm (Cl- sensitive; Stern-Volmer constants 82 and 98 M-1) and 565 nm (Cl- insensitive). Cystic fibrosis transmembrane conductance regulator-expressing Swiss 3T3 fibroblasts were labeled with bis-DMXPQ by hypotonic shock or were labeled with its uncharged reduced form (octahydro-bis-DMXPQ) by brief incubation (20 µM, 10 min). Changes in Cl- concentration in response to Cl-/nitrate exchange were recorded by emission ratio imaging (450/565 nm) at 365-nm excitation wavelength. These results establish a first-generation set of chimeric bisquinolinium Cl- indicators for ratiometric measurement of Cl- concentration.

6-methoxy-N-3-(sulfopropyl)quinolinium; fluorescence; chloride transport; membrane permeability; organic synthesis


    INTRODUCTION
Top
Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

THE AVAILABLE CHLORIDE-SENSITIVE fluorescent indicators contain heterocyclic structures containing a positively charged quaternary nitrogen (23, 24). The first such compound described for use in biological systems is 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) (11). SPQ absorbs ultraviolet light (320-365 nm) to yield blue fluorescence (440-460 nm). SPQ fluorescence is quenched by Cl- and several other anions (I-, Br-, and SCN-, but not by NO-3) by a collisional mechanism without change in spectral shape. SPQ and related compounds have been used to measure Cl- concentration and transport properties in biomembrane vesicles (1), liposomes (27), cells (5, 7, 9, 25), intact epithelia (13), and extracellular fluid spaces in complex tissues (21, 28). In addition, a fiber-optic halide sensor was developed by indicator immobilization on porous glass beads (12). Recently, SPQ has been used to assess the efficacy of cystic fibrosis transmembrane conductance regulator (CFTR) gene delivery in human gene therapy trials for the disease cystic fibrosis (8, 10, 17, 19).

A series of structure-activity studies was done to develop heterocyclic Cl- indicators with improved optical and physical properties, and anion sensitivity and selectivity profiles. It was found that the Cl- sensitivity and optical properties of quinolinium-based indicators could be modified substantially by the location and nature of ring substitutions (14, 26), yielding indicators with threefold-greater Cl- sensitivities (2). Because SPQ is a polar compound with low membrane permeability, prolonged incubation or invasive procedures (such as hypotonic shock) are required to stain cell cytoplasm. For rapid noninvasive loading, the uncharged compound 6-methoxy-N-ethyl-1,2-dihydroquinoline (diH-MEQ) was synthesized (4). DiH-MEQ is membrane permeable and is oxidized in the cell to the charged and membrane-impermeable Cl- indicator 6-methoxy-N-ethylquinolinium (MEQ). A class of long-wavelength Cl- indicators for extracellular use that contained the acridinium chromophore was introduced (3); however, the Cl--sensitive acridiniums synthesized to date are unstable in cytoplasm and chemically modified to Cl--insensitive chromophores.

A significant limitation in the use of heterocyclic Cl- indicators for some applications has been the lack of a Cl--dependent change in spectral shape, which precludes ratiometric measurements. This limitation is of particular concern in the analysis of the efficacy of CFTR gene delivery by imaging or cell sorting methods, which involve cells that are very heterogeneous in appearance and properties. We previously attempted to construct dual-wavelength Cl- indicators by conjugating quinolinium-based Cl--sensitive chromophores with Cl--insensitive chromophores having different optical properties (such as dansyl chromophore; Refs. 2, 23). Not unexpectedly, the products were nonfluorescent as a result of ring-ring interactions and dark-complex formation. Dual-wavelength dextrans have been synthesized (24) but are not suitable for cytoplasmic loading. There is a possibility of de novo design of multiwavelength Cl- binding indicators based on calix[4]pyrrole (18) or other structures (20), but this is a challenging endeavor that has not been successful to date.

We report here the synthesis and properties of a series of fluorescent dual-wavelength Cl- indicators, including their characterization in cells. Spacer groups and Cl--sensitive and -insensitive chromophores were screened to yield conjugates that were 1) fluorescent, 2) suitable for ratio imaging, 3) nontoxic and not metabolized in cells, and 4) loadable into cells. As shown in Fig. 1, 6-methoxyquinolinium (MQ) was used as the Cl--sensitive chromophore and 6-aminoquinolinium (AQ) was used as the Cl--insensitive chromophore. Nonrigid alkyl (Fig. 1A) and rigid xylyl (Fig. 1B) and trans-1,2-bis(4-[1-xylyl]-pyridinium)ethylene (Fig. 1C) spacers were used. AQ was used as the Cl--insensitive chromophore because of its similar excitation but red-shifted emission spectra compared with those of Cl--sensitive chromophore MQ, its positively charged heterocyclic nitrogen which prevented ring-ring interactions, and its reversible reducibility to a cell-permeable dihydroquinoline (Fig. 1D). In addition, alkylation of the amino group in AQ was found to modify indicator optical properties. The utility of the synthesized indicators with the best optical properties and Cl- sensitivities in cells was demonstrated.


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Fig. 1.   Chemical structures of the synthesized dual-wavelength Cl- indicators. 6-Methoxyquinolinium (MQ; Cl- sensitive, blue fluorescence) and 6-aminoquinolinium (AQ; Cl- insensitive, green fluorescence) were linked by various spacer groups: flexible alkyl chain spacers (A), a rigid xylyl spacer (B), or trans-1,2-bis-(4-[1-xylyl]-pyridinium)ethylene (C). MQanAQ, 1-MQ-n-AQ-alkane; DMAQ, 6-(dimethylamino)quinolinium; MQanDMAQ, 1-MQ-n-DMAQ-alkane; MQxyDMAQ, alpha -MQ-alpha '-DMAQ-xylene; bis-XPQ, trans-1,2-bis[4-(1-alpha '-MQ-1'-alpha '-AQ-xylyl)pyridinium]ethylene tetrachloride; bis-DMXPQ, trans-1,2-bis[4-(1-alpha '-MQ-1'-alpha '-DMAQ-xylyl)-pyridinium] ethylene. D: reduced, cell-permeable form of MQa4AQ, 1-(1,2-dihydro-6-methoxyquinoline)-4-(1,2-dihydro-6-aminoquinoline) butane (tetrahydro-MQa4AQ), diffuses across the cell membrane and is reoxidized in cytoplasm to charged cell-impermeable MQa4AQ.


    METHODS
Top
Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

Organic Synthesis

All chemicals were purchased from Aldrich Chemical (Milwaukee, WI). Compounds 1-MQ-n-AQ-alkane (MQanAQ) and alpha -MQ-alpha '-xylene dichloride (MQxyAQ) were synthesized in four steps as shown by the synthesis scheme in Fig. 2.


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Fig. 2.   Synthesis of dual-wavelength indicators with different spacer groups. See organic synthesis section of METHODS for details.

Step I. 6-Aminoquinoline (1 g, 6.9 mmol) was converted to 6-(acetylamino)quinoline (step I product) by refluxing with a twofold molar excess of acetic anhydride (1.41 g, 13.8 mmol) for 15 min. The precipitated product (yield 1.2 g, 94%) was filtered, washed with water, and recrystallized from methanol-water (1:3).

Step IIa. The step I product (1 g, 5.4 mmol) was mixed with a twofold excess of 1,n-dibromoalkane (n = 2, 4, 6) in a dimethylformamide (DMF):toluene (3:2) solvent mixture (5 ml) and heated to 100°C for 4 h to give a light brownish-yellow (n = 2, 4) or light yellow (n = 6) precipitate of 1-(n-bromoalkyl)-6-acetyl-AQ bromide (step IIa product) (yield 65-75%). The solid was filtered and washed with acetone until pure as judged by TLC (ethyl acetate). The following NMR results were obtained: 1-(2-bromoethyl)-6-acetyl-AQ bromide 1H-NMR (D2O; 300 MHz), 8.0-9.5 (m, 6H), 5.4 (t, 2H), 4.1 (t, 2H), 2.3 (s, 3H); 1-(4-bromobutyl)-6-acetyl-AQ bromide 1H-NMR (D2O; 300 MHz), 8.0-9.5 (m, 6H), 5.0 (t, 2H), 3.8 (t, 2H), 2.4 (s, 3H), 2.2 (m, 4H); 1-(6-bromohexyl)-6-acetyl-AQ bromide 1H-NMR (D2O; 300 MHz), 8.0-9.5 (m, 6H), 5.0 (t, 2H), 3.5 (t, 2H), 2.3 (s, 3H), 2.1 (m, 2H), 1.7 (m, 2H), 1.4 (m, 4H).

Step IIb. The step I product (1 g, 5.4 mmol) was added to a twofold excess of alpha ,alpha '-dibromo-p-xylene (2.84 g, 10.8 mmol) in DMF:toluene (3:2) (5 ml) at 70°C. The mixture was heated to 100°C for 3 h to give a light yellow precipitate of alpha -6-acetyl-AQ-alpha '-bromoxylene (step IIb product). The product was filtered and washed three times with chloroform and then three or four times with acetone (yield 2 g, 83%). The following NMR results were obtained: alpha -6-acetyl-AQ-alpha '-bromoxylene 1H-NMR (D2O; 300 MHz), 7.0-9.5 (m, 10H), 6.25 (s, 2H), 4.6 (s, 2H), 2.3 (s, 3H).

Step III. The product of step IIa or IIb (0.5 g) was mixed with a fourfold excess of 6-methoxyquinoline (0.64-1.28 g), and the mixture was heated to 100°C for 4 h to give 1-(6-acetyl-AQ)-n-MQ-alkane dibromide (n = 2, 4, 6) (step IIIa product) or alpha -(6-acetyl-AQ)-alpha '-MQ-xylene dibromide (step IIIb product), respectively. The resulting product was triturated with acetone, filtered, and stirred with acetone overnight to remove unreacted 6-methoxyquinoline (yield 95-98%). Product purity was confirmed by reverse-phase TLC (1:1 ethanol:water, 1% trichloroacetic acid).

Step IV. The product of step III (0.5 g) was refluxed with 3 ml of 2 N HCl overnight to deprotect the amino group to give MQanAQ (n = 2, 4, 6) (step IVa product) and MQxyAQ (step IVb product). These products were obtained as bright yellow solids after removal of water by evaporation. The product obtained in quantitative yield was washed with acetone and recrystallized from 50% methanol-water. The following NMR results were obtained: MQa2AQ 1H-NMR (DMSO; 300 MHz), 7.2-9.1 (m, 12H), 6.6 (s, 2H), 4.8 (t, 2H), 4.9 (t, 2H), 4.0 (s, 3H); MQa4AQ 1H-NMR (D2O; 300 MHz), 7.0-9.0 (m, 12H), 5.1 (t, 2H), 4.9 (t, 2H), 4.0 (s, 3H), 2.0 (m, 4H); MQa6AQ 1H-NMR (DMSO; 300 MHz), 7.0-9.5 (m, 12H), 6.6 (s, 2H), 5.1(t, 2H), 4.9 (t, 2H), 4.0 (s, 3H), 2.0 (m, 4H), 1.4 (m, 4H); MQxyAQ 1H-NMR (D2O; 300 MHz), 7.0-9.3 (m, 16H), 6.25 (s, 2H), 6.15 (s, 2H), 4.0 (s, 3H).

Step IVc. The following steps describe the synthesis of trans-1,2-bis(4-[1-alpha '-MQ-1'-alpha '-6-acetyl-AQ-xylyl]pyridinium)ethylene tetrachloride (bis-XPQ). alpha -MQ-alpha '-bromoxylene was synthesized as described in step IIb and was reacted (0.5 g, 1.11 mmol) with trans-1,2-bis(4-pyridyl)ethylene (0.26 g, 1.42 mmol) in DMF (3 ml) for 6 h. The precipitated alpha -MQ-alpha '-trans-1,2-bis[4-pyridyl]ethylene-xylene dibromide (yield 0.65 g, 90%) was filtered and washed twice with ethanol and two or three times with acetone. alpha -MQ-alpha '-(trans-1,2-bis[4-pyridyl]ethylene)-xylene dibromide (0.10 g, 0.23 mmol) was mixed with the product of step IIb (0.14 g, 0.3 mmol) in DMF (3 ml) and heated at 110°C for 48 h. The precipitated trans-1,2-bis(4-[1-alpha '-MQ-1'-alpha '-6-acetyl-AQ-xylyl]-pyridinium)ethylene tetrabromide was filtered, washed with acetone, and refluxed with 2 N HCl overnight to deprotect the amino group. Bis-XPQ was obtained as a red-brown solid after removal of water by evaporation. (yield 0.36 g, 90%). The following NMR results were obtained: alpha -MQ-alpha '-trans-1,2-bis[4-pyridyl]ethylene-xylene dichloride 1H-NMR (D2O; 300 MHz), 7.2-9.4 (m, 20H), 6.25 (s, 2H), 5.8 (s, 2H), 4.0 (s, 3H); bis-XPQ 1H-NMR (D2O; 300 MHz), 7.2-9.5 (m, 30H), 6.25 (s, 2H), 6.15 (s, 2H), 5.8 (s, 4H), 4.0 (s, 3H).

Methylation of the amino group on AQ. The product of step IV (MQa4AQ, MQxyAQ, or bis-XPQ) (0.25 g) was dissolved in 10 ml of 0.1 M K2CO3 and heated to 90°C, and a 10-fold excess of dimethyl sulfate was added. The reaction mixture was refluxed overnight. The resultant solution was extracted with ethyl acetate, and the aqueous phase was treated with 0.1 N NaOH to dissociate any residual dimethyl sulfate. The aqueous phase was then concentrated, and the pH was adjusted to 4-5 with 0.1 N HCl. The product was dialyzed (molecular weight cutoff 100) to remove the salts and then lyophilized (yield >95%). The following NMR results were obtained: 1-MQ-4-6-(dimethylamino)quinolinium-alkane (MQa4DMAQ) 1H-NMR (D2O; 300 MHz), 7.0-9.0 (m, 12H), 5.1 (t, 2H), 4.9 (t, 2H), 4.0 (s, 3H), 3.3 (s, 6H), 2.0 (m, 4H); alpha -MQ-alpha '-DMAQ-xylene (MQxyDMAQ) 1H-NMR (D2O; 300 MHz), 7.0-9.3 (m, 16H), 6.25 (s, 2H), 6.15 (s, 2H), 4.0 (s, 3H), 3.3 (s, 6H).

Reduction of MQa4AQ and bis-XPQ. The reduction of MQa4AQ to 1-(1,2-dihydro-6-methoxyquinoline)-4-(1,2-dihydro-6-aminoquinoline) butane (tetrahydro-MQa4AQ) and of bis-XPQ to trans-1,2-bis(4-[1-alpha '-1,2-dihydro-6-methoxyquinoline-1'-alpha '-1,2-dihydro-6-aminoquinoline-xylyl]-1,2-dihydropyridyl)ethylene (octahydro-bis-XPQ) was done with NaBH4 as reported previously (4).

Spectroscopic Measurements

Absorption spectra were recorded with a Hewlett-Packard photodiode array spectrophotometer (HP 8452A). Fluorescence spectra were measured with an SLM 8000c fluorometer (SLM Instruments, Urbana, IL). Quantum yields were calculated from integrated emission spectra by using reference compounds SPQ (quantum yield 0.69) and 6-amino-N-(3-sulfopropyl)quinolinium (ASPQ; quantum yield 0.065). Fluorescence lifetimes were measured by the frequency domain method at 40 modulation frequencies of 4-160 MHz. Fluorescence was excited at 322 nm by a He-Cd laser and detected at 450 nm with an interference filter (450 ± 25 nm) and at 565 nm with a filter with a >530-nm cutoff. The reference fluorophore for lifetime measurements was dimethyl-1,4-bis(4-methyl-5-phenyloxazol-2-yl)benzene in ethanol (lifetime, 1.45 ns) for 450-nm emission and fluorescein in NaOH (lifetime, 4 ns) for 550-nm emission.

Fluorescence Quenching

Fluorescence quenching measurements were carried out at peak excitation and emission wavelengths. Microliter aliquots of the sodium salt of the quenching anions (1 M stock) were added to 3 ml of each compound (10 µM in 5 mM Na2HPO4-NaH2PO4) at pH 7.2. Stern-Volmer constants (Ksv) were calculated from the slope of a Stern-Volmer plot of Fo/F - 1 vs. quencher concentration: Fo/F - 1 = Ksv[Q], where Fo is compound fluorescence in the absence of quencher Q, and F is the fluorescence in the presence of the quencher.

Cell Fluorescence Measurements

Swiss 3T3 fibroblasts (ATCC CCL 92) expressing CFTR were provided by Dr. Michael Welsh. CHO cells (ATCC CRL 9606) and Swiss 3T3 fibroblasts were maintained at 37°C in a 95% air-5% CO2 incubator in H21 DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were grown on 18-mm-diameter round glass coverslips until nearly confluent for use in a perfusion chamber as described previously (5). Cells were loaded with the dyes by either hypotonic shock or by incubation with the cell-permeable reduced compounds. For loading by hypotonic shock, cells were incubated with 2-5 mM dye in 1:1 PBS:water for 5 min at room temperature and allowed to recover in PBS for 10 min at 37°C. Cells were loaded with the reduced derivatives (20-50 µM) by incubation in PBS at 37°C for 10 min followed by a 15-min incubation at 37°C in growth medium to permit complete reoxidation to the parent quinolinium dyes.

Cells were imaged with a Leitz epifluorescence microscope equipped with a Nipkow wheel coaxial-confocal attachment (Technical Instruments, San Francisco, CA). Cells were mounted in a perfusion chamber and viewed with a Nikon ×60 oil-immersion objective [numerical aperture (NA) 1.4]. Confocal images were detected by a cooled charged-coupled device camera (AT 200; Photometrics, Tucson, AZ). Continuous integrated cell fluorescence was measured after mounting the cells in a 200-µl perfusion chamber in a Nikon inverted epifluorescence microscope with an immersion objective (Nikon ×40, oil-immersion, NA 1.3) by using a photomultiplier (5).

For ratio imaging of CHO cells loaded with MQxyDMAQ or trans-1,2-bis(4-[1-alpha '-MQ-1'-alpha '-DMAQ-xylyl]pyridinium)ethylene (bis-DMXPQ), MQ fluorescence was imaged at 450 nm with 365 ± 20-nm excitation filters and 450 ± 25-nm emission filters. DMAQ fluorescence was imaged at 565 nm with 450 ± 25-nm excitation filters and >530-nm emission filters.

Image pairs were acquired (exposure time, 2 s) for the same field containing one or more cells. Ratio images were computed by pixel-by-pixel division of the 450-nm image by the 565-nm image after background subtraction.


    RESULTS
Top
Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

Chloride indicators of potential suitability for ratio imaging were synthesized by covalently linking the Cl--sensitive chromophore MQ (excitation 350-365 nm, peak emission 450 nm) and the Cl--insensitive chromophore AQ (excitation 380-410 nm, peak emission 546 nm) by a spacer. Indicators with different spacers and functional groups were synthesized to examine structure-function relationships. As shown in Fig. 1, the spacers chosen were flexible n-alkyl chains with 2, 4, and 6 carbon atoms, and rigid xylyl and trans-1,2-bis(4-[1-xylyl]-pyridinium)ethylene.

The absorption and fluorescence excitation and emission spectra of MQxyAQ, MQxyDMAQ, and bis-XPQ are shown in Fig. 3 and spectroscopic data for all compounds are summarized in Table 1. The absorption maxima for MQanAQ and MQxyAQ (Fig. 3A) (318, 350, and 396 nm) and MQanDMAQ and MQxyDMAQ (Fig. 3B) (318, 350, and 440 nm) corresponded to the maxima of free MQ and AQ, suggesting the absence of intramolecular ring stacking. We believe that the repulsion afforded by the positively charged chromophores prevented the stacking (which would give nonfluorescent conjugates) observed in previous failed attempts to generate dual-wavelength Cl- indicators. Although free and conjugated MQ and AQ had similar absorbance maxima, there were considerable differences in their molar extinction coefficients and fluorescence properties (Table 1). For bis-XPQ, absorption maxima at 324, 342, and 410 nm were seen (Fig. 3C). The shift in the absorption maxima is due to the overlap with the strong absorption of the trans-1,2-bis(4-[1-xylyl]-pyridinium)ethylene spacer (absorption maxima at 322 and 342 nm).


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Fig. 3.   Absorption, excitation, and emission spectra of 20 µM MQxyAQ (A), MQxyDMAQ (B), and bis-XPQ (C) in 5 mM sodium phosphate (pH 7.2). lambda em, emission wavelength; lambda ex, excitation wavelength.

                              
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Table 1.   Spectroscopic properties of indicators

For MQanAQ, MQxyAQ, and bis-XPQ, excitation at 350 nm produced fluorescence emission maxima at 450 and 550 nm (Fig. 3, A and C, and Table 1), corresponding to the Cl--sensitive MQ and the Cl--insensitive AQ chromophores, respectively. As expected, direct excitation of AQ at 410 nm gave a single emission maximum at 550 nm. Similarly, MQanDMAQ, MQxyDMAQ, and bis-DMXPQ emit at 450 and 565 nm for 350-nm excitation, whereas excitation at 440 nm gave emission only at 565 nm (Fig. 3B). The nonzero emission from both the Cl--sensitive and -insensitive chromophores indicates that a dark complex is not formed. Note that spectral overlap of AQ absorption and MQ fluorescence emission exists, suggesting the possibility of intramolecular resonance energy transfer. Energy transfer can have both favorable and unfavorable consequences: quenching of MQ donor fluorescence results in decreased blue fluorescence and Cl- sensitivity of the MQ moiety, whereas measurement of sensitized emission might be exploited to improve Cl- sensitivity or to measure Cl- by single-emission (565 nm), dual-excitation ratio imaging. In addition, there may be interactions between the chromophores and the spacers, resulting in altered spectral and/or anion sensitivity properties.

Energy transfer between chromophores was examined by measurement of quantum yields and nanosecond fluorescence lifetimes (Table 1). Compared with that of SPQ, there was an approximately fivefold reduction in the quantum yields of the MQ chromophore in MQa2AQ, MQa4AQ, and MQxyAQ and a two- to threefold decrease in the quantum yields of bis-XPQ, MQxyDMAQ, and bis-DMXPQ. The quantum yields of AQ in all of the bichromophoric indicators were similar to that of free ASPQ. Lifetime analysis was done to determine if the decreased MQ quantum yield was due to an intramolecular energy transfer mechanism. Compared with that of the free MQ chromophore (lifetime 26 ns), the fluorescence lifetime of MQ was reduced nearly threefold in MQanAQ, MQxyAQ, and bis-XPQ. A greater lifetime of 23.1 ns was obtained for MQxyDMAQ. From the lifetime of MQ in the presence and absence of AQ, the energy transfer efficiency (E) was calculated: E = (1-tau da/tau d), where tau d and tau da are the lifetimes of MQ in the absence and presence of the acceptor, respectively (Table 1). The increased quantum yield of MQ corresponding to the decreased energy transfer efficiency indicates an intramolecular energy transfer mechanism.

The quenching of MQxyAQ fluorescence by Cl- is shown in Fig. 4. At a 365-nm excitation wavelength, the fluorescence emission at 450 nm was quenched by 50% at 18 mM Cl-. Fluorescence emission at 546 nm was not quenched; the small amount of apparent quenching observed for 365 nm excitation was due to a decrease in the broad MQ emission. When only AQ was excited at 410 nm (Fig. 4), no decrease in the fluorescence intensity was observed on the addition of 150 mM Cl-, confirming that AQ fluorescence is insensitive to Cl-. Qualitatively similar results were obtained for bichromophoric indicators with different spacer groups.


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Fig. 4.   Fluorescence emission of MQxyAQ (20 µM) for 365-nm (solid lines) and 410-nm (dashed line) excitation in the presence of specified NaCl concentrations in 5 mM sodium phosphate at pH 7.2.

Stern-Volmer plots for Cl- quenching of MQ fluorescence in the dual-wavelength indicators are shown in Fig. 5. The Cl- sensitivity of MQ (slope) was dependent on spacer group identity and optical properties of the second chromophore. Compounds MQxyDMAQ and bis-DMXPQ had the highest Cl- sensitivities with KCl values of 98 and 82 M-1, respectively, which were slightly lower than the KCl of 118 M-1 for SPQ.


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Fig. 5.   Stern-Volmer plots for the fluorescence quenching of indicated compounds (20 µM) by Cl-. Stern-Volmer constants are reported in Table 2. SPQ, 6-methoxy-N-(3-sulfopropyl)quinolinium; Fo, compound fluorescence in absence of quencher; F, fluorescence in presence of quencher.

Table 2 summarizes the Stern-Volmer constants for the quenching of MQ and AQ chromophores by various anions. Cl- sensitivity was reduced for all synthesized dual-wavelength indicators (MQ-spacer-AQ) compared with that for SPQ, whereas conjugation of the spacer only to MQ (MQ-spacer; see MQa4 and MQxy in Table 1) had no influence on the Cl- sensitivity. This suggests that the reduced Cl- sensitivity in the dual-wavelength indicators is due to energy transfer between MQ and AQ. Dimethylation of the amino group in AQ to DMAQ produced an increase in Cl- sensitivity for all spacer groups. The AQ chromophore was quenched mildly by I- and SCN-, but not by Cl- and Br-. In general, the pattern of sensitivity of MQ quenching to halides was Cl- < Br- < I-. MQ and AQ fluorescence was not quenched significantly by nitrate, phosphate, or sulfate or by changes in pH in the range of 4-8.

                              
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Table 2.   Stern-Volmer quenching constants

The utility of the dual-wavelength indicators for measurement of Cl- in living cells was investigated. For use in cells, the indicators should be nontoxic and distributed uniformly in the cytoplasm. The dual-wavelength indicators did not affect viability or growth of CHO cells and 3T3 fibroblasts when the cells were incubated overnight with growth medium containing 5 mM indicator. Figure 6 shows confocal fluorescence micrographs of CHO cells labeled with several of the compounds. SPQ (Fig. 6A), MQxyDMAQ (Fig. 6B), and bis-DMXPQ (Fig. 6C) were loaded by a transient permeabilization procedure in which cells were incubated for 5 min with a 2-5 mM concentration of each indicator in hypotonic buffer. Figure 6D shows cells loaded by a 10-min incubation with 25-50 µM of the cell-permeable tetrahydro-MQa4AQ derivative, followed by a 15-min incubation to allow for reoxidation to the parent quinolinium indicator. Hypotonic shock loading gave fairly uniform staining of cytoplasm for indicators with the rigid xylyl (Fig. 6B) and trans-1,2-bis(4-[1-xylyl]-pyridinium)ethylene (Fig. 6C) spacers. For MQanAQ, the hypotonic shock loading procedure resulted in compartmentalization in cells (not shown), whereas loading by the reduction/reoxidation procedure gave fairly uniform staining of cytoplasm (Fig. 6D). In contrast to SPQ (Fig. 6A), the dual-wavelength indicators were found to occasionally accumulate in subnuclear compartments.


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Fig. 6.   Fluorescence confocal micrographs of CHO cells labeled with SPQ (A), MQxyDMAQ (B), or bis-DMXPQ (C) by being incubated for 5 min with a 2-5 mM concentration of each indicator in hypotonic buffer. D: MQa4AQ was loaded by a 10-min incubation of cells with 25-50 µM cell-permeable tetrahydro-MQa4AQ derivative, followed by 15-min incubation to allow for reoxidation to parent cell-impermeable quinolinium indicator. Bar = 10 µm.

The sensitivity of MQ fluorescence in MQxyDMAQ (Fig. 7B) and bis-DMXPQ (Fig. 7A) to changes in external Cl- concentration was measured in CFTR-expressing Swiss 3T3 fibroblasts. Cl- efflux and influx were induced by the exchange of extracellular Cl- with NO-3 in the presence of forskolin to allow for fast Cl-/NO-3 exchange. At a 365-nm excitation wavelength, reversible changes in fluorescence at 450 nm were observed upon removal and addition of solution Cl-, while emission at 565 nm did not change. In cells loaded with bis-DMXPQ, an 18% change in the fluorescence intensity ratio was observed in response to Cl-/NO-3 exchange, whereas a 12% change was obtained with MQxyDMAQ. There was no significant photobleaching or leakage of the indicators (<2%) from the cells during the course of the experiments.


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Fig. 7.   Intracellular Cl- activity measurement by using entrapped bis-DMXPQ (A) and MQxyDMAQ (B) in Swiss 3T3 fibroblasts expressing cystic fibrosis transmembrane conductance regulator (CFTR). Where indicated Cl- was replaced by NO-3. CFTR Cl- channels were stimulated by addition of forskolin (20 µM) to increase membrane Cl- permeability. Change in fluorescence at 450 and 565 nm and the fluorescence ratio (450/565 nm) are shown.

Figure 8A shows ratio imaging of intracellular Cl- activity by using the dual-wavelength indicators bis-DMXPQ (top) and MQxyDMAQ (bottom). The fluorescence micrographs of the blue and yellow emission showed mildly heterogeneous staining with microcompartmentalization in the nucleus. By ratio imaging of the blue-to-yellow fluorescence, the microheterogeneity in staining was reduced. The fluorescence intensity ratio was quite uniform in the cytoplasm and nucleus and, interestingly, slightly lower in the nucleus. The possibility of a higher Cl- concentration in the nucleus requires further investigation.


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Fig. 8.   A: fluorescence micrographs of CHO cells loaded with bis-DMXPQ (top) and MQxyDMAQ (bottom) for MQ (left) and DMAQ (middle) emission at 450 and 565 nm, respectively. Bar = 8 µm. Right: corresponding ratio image of 450/565 nm emission. Ratio images were obtained by pixel-by-pixel division of the 450-nm image by the 565-nm image after background subtraction. B: Calibration of bis-DMXPQ fluorescence vs. Cl- in Swiss 3T3 fibroblasts expressing CFTR. Cells were perfused with buffers containing 100 mM K+, 38 mM Na+, indicated Cl- concentrations (remaining anion NO-3), 5 µM valinomycin, 5 µM nigericin, and 20 µM forskolin.

Calibration of dual-wavelength indicator fluorescence against intracellular Cl- activity was done with Swiss 3T3 fibroblasts expressing CFTR. Cells were perfused with buffer having high K+ and containing forskolin (for stimulation of CFTR Cl- channels) and the K+ ionophores valinomycin and nigericin to facilitate fast equilibration of intracellular and outside Cl-. The Cl- ionophore tributyltin was not used because of its high cell toxicity. Upon perfusion with the high-K+ calibration solutions, the fluorescence at 450 nm was quenched progressively and reversibly with increasing Cl- concentration, whereas the 565-nm emission remained unchanged (Fig. 8B). There was little photobleaching or dye leakage. As has been observed for SPQ (5), the sensitivity of dual-wavelength indicators to Cl- is lower in cells than in vitro.


    DISCUSSION
Top
Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

The objective of this study was to design, synthesize, and characterize dual-wavelength Cl- indicators for ratio imaging. The design strategy was to link Cl--sensitive and -insensitive chromophores by optically inert spacers. The chromophores and spacers were selected to fulfill specific optical and cellular requirements as described in the introduction. Methoxy- and amino-substituted quinolinium chromophores were used for the synthesis of fluorescent single-excitation, dual-emission indicators. The prior problem of ring-ring interactions resulting in dark complex formation was solved by using charge repulsion and appropriate spacers to prevent chromophore interactions. The favorable redox properties of the quinoliniums were exploited to design uncharged, cell-permeable compounds that are oxidized in cytoplasm to polar, multiply charged indicators. The indicators remained trapped in the aqueous cytoplasm with a fairly uniform distribution. Like the previous quinolinium Cl- indicators, the hybrid indicators were chemically inert and nontoxic in cells.

The quantum yield and Cl- sensitivity of MQ in the dual-wavelength indicators were reduced compared with those of free MQ (not linked to AQ). A major cause of these effects was fluorescence resonance energy transfer between the MQ and AQ chromophores. The MQ fluorescence emission spectrum partially overlaps with the absorption spectrum of AQ, giving an experimentally determined Förster distance, Ro (at which 50% of donor fluorescence is quenched), of 9.2 Å. An energy transfer mechanism was confirmed by the decrease in MQ fluorescence lifetime in the presence of conjugated AQ. The decreased MQ lifetime would account for the decreased MQ quantum yield (even in the absence of Cl-), as well as the decreased Stern-Volmer constant for quenching of MQ fluorescence by Cl-. Energy transfer produces an apparent decrease in Cl- sensitivity because the fluorescence decay rate of the excited MQ chromophore is the sum of an intrinsic radiative decay rate (reciprocal fluorescence lifetime in the absence of Cl- or energy transfer), a nonradiative Cl- quenching rate, and an additional nonradiative energy transfer rate. If energy transfer produces 50% quenching of MQ fluorescence in the absence of Cl-, then the apparent Stern-Volmer constant for MQ quenching by Cl- will decrease by a factor of 2. Based on calculated energy transfer efficiencies (Table 1), a 2- to 4.5-fold decrease (compared with that for SPQ) in KCl was expected for all the dual-wavelength indicators. Although the measured Stern-Volmer constants were reduced compared with that for SPQ, they were greater for all indicators than those that would be predicted on the basis of the energy transfer efficiency alone. The greater Stern-Volmer constants were probably due to the additional positive charge of the AQ chromophore. It has been shown that a second positive charge near the Cl--sensitive MQ chromophore increases Cl- sensitivity, probably because of increased coulombic interactions between the fluorophore and negatively charged chloride (4, 16).

MQ fluorescence and Cl- sensitivity can be increased by maneuvers designed to decrease Ro and/or increase MQ-AQ separation. Ro can be decreased by the selection of chromophores with less spectral overlap or by fixing chromophore orientation so that donor and acceptor dipoles are nearly perpendicular (decreasing kappa  orientation factor) (6). The latter strategy was not practical. Given the restrictions imposed on the chromophores (bright, dual-wavelength fluorescence; effective cell loading and trapping), we were unable to identify a suitable second chromophore with low spectral overlap to minimize energy transfer. As an alternative strategy, a Cl--sensitive donor-Cl--insensitive acceptor pair with very high energy transfer efficiency can be used to generate a dual-excitation, single-emission indicator. Upon excitation of the donor, the high energy transfer efficiency leads to Cl--sensitive emission of the acceptor, whereas direct excitation of the acceptor is Cl- insensitive. To increase the spectral overlap between MQ emission and AQ absorbance, the 6-amino group of AQ was methylated, yielding DMAQ. The absorbance spectrum of DMAQ was red-shifted by 40 nm compared with that of AQ (see Fig. 3B), resulting in a greater spectral overlap with the MQ emission. The calculated Ro increased to 22 Å for MQ-DMAQ, compared with 9.2 Å for MQ-AQ. Surprisingly, energy transfer was reduced in the indicators for which the MQ and DMAQ were separated by rigid spacers. A possible explanation for this finding is a more fixed conformation of MQ and DMAQ so that the transition dipoles are oriented almost perpendicular to each other, reducing kappa  and thus energy transfer efficiency. Because energy transfer can also be reduced by increasing MQ-AQ separation, indicators containing a long rigid spacer consisting of bis-pyridinium ethylene flanked by xylyl groups (~23 Å) were synthesized. Because methylation of the amino group was found to decrease energy transfer, bis-DMXPQ, which combined the addition of the long rigid spacer with methylation of AQ, was synthesized. Bis-DMXPQ had low energy transfer efficiency, giving a Stern-Volmer Cl- quenching constant reduced by only 30% compared with that for SPQ.

The mechanism(s) by which quinolinium indicators are sensitive to Cl- has not been elucidated, although photophysical studies of related compounds suggest a collisional quenching mechanism involving the transient formation of a charge transfer complex and subsequent electron transfer. Fluorescence quenching of aromatic molecules, such as naphthalene and its substituents (carbocyclic analogs of quinoline), by halides was found to involve electron transfer from the halide anion to the excited aromatic molecule (15, 22). This mechanism was established from the dependence of the fluorescence quenching rate on the free energy of electron transfer given by the Rehm-Weller equation and from the observation of intermediate free-radical species (15). Also, pyridinium compounds (analogs of quinolinium) are known to quench the fluorescence of metal complexes by an electron transfer mechanism (16). On the basis of these findings, it is likely that the mechanism of the quenching of quinolinium compounds by Cl- involves electron transfer quenching.

In summary, the results in this paper document the ability to generate dual-wavelength Cl- indicators for ratiometric measurement of Cl- concentration. The structure-activity correlations with different spacers and aminoquinoline substituents defined the principal photophysical issues in the design of hybrid Cl- indicators. Although several of the compounds synthesized had good optical properties in vitro and could be used to monitor Cl- transport in cells, there remain significant limitations. The quantum yield and Cl- sensitivity of the MQ chromophore are lower than those of SPQ, even with the extended trans-1,2-bis(4-[1-xylyl]-pyridinium)ethylene spacer and the spectral shift produced by amino group alkylation in AQ. An intrinsic problem with the use of independent chromophores is the possibility of differential photobleaching, resulting in a change in fluorescence ratio that is unrelated to a change in Cl- concentration. Although quinolinium chromophores are relatively resistant to photobleaching compared with fluorescein, differential photobleaching is a concern in measurements involving continuous illumination; photobleaching is less of a concern in measurements involving intermittent illumination or single-pulse illumination, such as those used in cell cytometry applications. Last, it is noted that the fluorescence of the MQ chromophore is weakly quenched by cytoplasmic proteins so that the determination of absolute intracellular Cl- concentration, even by a ratiometric approach, requires an ionophore calibration procedure for each cell type studied. The compounds reported here should thus be considered the first-generation ratiometric Cl- indicators with the possibility of substantial future improvements.


    ACKNOWLEDGEMENTS

We thank Katherine Chen for cell cultures and Prof. J. L. Sessler for useful discussions.


    FOOTNOTES

This work was supported by National Institutes of Health Grants HL-60288 and DK-43840, Gene Therapy Core Center Grant DK-47766, and Research Development Grant R613 from the National Cystic Fibrosis Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: Alan S. Verkman, 1246 Health Sciences East Tower, Cardiovascular Research Institute, Univ. of California, San Francisco, CA 94143-0521. (E-mail: verkman{at}itsa.ucsf.edu; http://www.ucsf.edu/verklab).

Received 19 June 1998; accepted in final form 5 November 1998.


    REFERENCES
Top
Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

1.   Bae, H. R., and A. S. Verkman. Protein kinase A regulates chloride conductance in endocytic vesicles from proximal tubule. Nature 348: 635-637, 1990.

2.   Biwersi, J., N. Farah, Y.-X. Wang, R. Ketchum, and A. S. Verkman. Synthesis of cell-impermeable Cl-sensitive fluorescent indicators with improved sensitivity and optical properties. Am. J. Physiol. 262 (Cell Physiol. 31): C243-C250, 1992.

3.   Biwersi, J., B. Tulk, and A. S. Verkman. Long wavelength chloride-sensitive fluorescent indicators. Anal. Biochem. 219: 139-143, 1994[Medline].

4.   Biwersi, J., and A. S. Verkman. Cell permeable fluorescent indicator for cytosolic chloride. Biochemistry 30: 7879-7883, 1991[Medline].

5.   Chao, A. C., J. A. Dix, M. Sellers, and A. S. Verkman. Fluorescence measurement of chloride transport in monolayer cultured cells: mechanisms of chloride transport in fibroblasts. Biophys. J. 56: 1071-1081, 1989[Abstract].

6.   Chattoraj, M., B. Bal, G. L. Gloss, and D. H. Levy. Conformation-dependent intramolecular electronic energy transfer in a molecular beam. J. Phys. Chem. 95: 9666-9672, 1991.

7.   Dechecchi, M. C., A. Tamanini, G. Berton, and G. Cabrini. Protein kinase C activates chloride conductance in C127 cells stably expressing the cystic fibrosis gene. J. Biol. Chem. 268: 11321-11325, 1993[Abstract/Free Full Text].

8.   Florence, D., T. Chinet, J. M. Zahm, D. Pierrot, J. Hinnrasky, H. Kaplan, N. Bonnet, and E. Puchelle. Introduction of a cAMP-stimulated chloride secretion in regenerating poorly differentiated airway epithelial cells by adenovirus-mediated CFTR gene transfer. Hum. Gene Ther. 8: 1439-1450, 1997[Medline].

9.   Foskett, J. K. [Ca2+]i modulation of Cl- content controls cell volume in single salivary acinar cells during fluid secretion. Am. J. Physiol. 259 (Cell Physiol. 28): C998-C1004, 1990[Abstract/Free Full Text].

10.   Gill, D. R., K. W. Southern, K. A. Mofford, T. Seddon, L. Huang, F. Sorgi, A. Thomson, L. J. MacVinish, R. Ratcliff, D. Bilton, D. J. Lane, J. M. Littlewood, A. K. Webb, P. G. Middleton, W. H. Colledge, A. W. Cuthbert, M. J. Evans, C. F. Higgins, and S. C. Hyde. A placebo-controlled study of liposome-mediated gene transfer to the nasal epithelium of patients with cystic fibrosis. Gene Ther. 4: 199-209, 1997[Medline].

11.   Illsley, N. P., and A. S. Verkman. Membrane chloride transport measured using a chloride-sensitive fluorescent indicator. Biochemistry 26: 1215-1219, 1987[Medline].

12.   Kao, H. P., J. Biwersi, and A. S. Verkman. Fiber optic halide sensor based on fluorescence quenching. Proc. SPIE 1648: 194-201, 1992.

13.   Krapf, R., C. A. Berry, and A. S. Verkman. Estimation of intracellular chloride activity in isolated perfused rabbit proximal tubules using a fluorescent probe. Biophys. J. 53: 955-962, 1988[Abstract].

14.   Krapf, R., N. P. Illsley, H. C. Tseng, and A. S. Verkman. Structure-activity relationships of chloride-sensitive fluorescent indicators for biological application. Anal. Biochem. 169: 142-150, 1988[Medline].

15.   Mac, M., and J. Wirz. Deriving intrinsic electron-transfer rates from nonlinear Stern-Volmer dependencies for fluorescence quenching of aromatic molecules by inorganic anions in acetonitrile. Chem. Phys. Lett. 211: 20-26, 1993.

16.   Mark, D. N., I. C. Robert, and G. N. Daniel. Contribution of long-range Coulomb interactions to biomolecular luminescence quenching rates. J. Phys. Chem. 95: 9660-9666, 1991.

17.   McLachlan, G., L.-P. Ho, H. Davidson-Smith, J. Samways, H. Davidson, B. J. Stevenson, A. D. Carothers, E. W. F. W. Alton, P. G. Middleton, S. N. Smith, G. Kallmeyer, U. Michalis, S. Seeber, K. Naujoks, A. P. Greening, J. A. Innes, J. R. Dorin, and D. J. Porteous. Laboratory and clinical studies in support of cystic fibrosis gene therapy using pCMV-CFTR-DOTAP. Gene Ther. 3: 1113-1123, 1996[Medline].

18.   Philip, A. G., J. L. Sessler, V. Kral, and V. Lynch. Calix[4]pyrroles: old yet new anion-binding agents. J. Am. Chem. Soc. 118: 5140-5141, 1997.

19.   Porteous, D. J., J. R. Dorin, G. McLachlan, H. Davidson-Smith, H. Davidson, B. J. Stevenson, A. D. Carothers, W. A. H. Wallace, S. Moralee, C. Hoenes, G. Kallmeyer, U. Michaelis, K. Naujoks, L.-P. Ho, J. M. Samways, M. Imrie, A. P. Greening, and J. A. Innes. Evidence for safety and efficacy of DOTAP cationic liposome mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis. Gene Ther. 4: 210-218, 1997[Medline].

20.   Sessler, J. L., A. Andrievsky, and J. W. Genge. Anion binding by sapphyrins. Adv. Supramol. Chem. 4: 97-142, 1997.

21.   Shi, L. B., K. Fushimi, and A. S. Verkman. Solvent drag measurement of transcellular and basolateral membrane NaCl reflection coefficient in mammalian proximal tubule. J. Gen. Physiol. 98: 379-398, 1991[Abstract].

22.   Shizuka, H., M. Nakamoto, and T. Morita. Anion induced fluorescence quenching of aromatic molecules. J. Phys. Chem. 84: 989-994, 1980.

23.   Verkman, A. S. Development and biological applications of chloride-sensitive fluorescent indicators. Am. J. Physiol. 259 (Cell Physiol. 28): C375-C388, 1990[Abstract/Free Full Text].

24.   Verkman, A. S., and J. Biwersi. Chloride-sensitive fluorescent indicators. In: Methods in Neurosciences, edited by J. Kraicer, and S. J. Dixon. Academic, 1995, vol. 27, p. 328-339.

25.   Verkman, A. S., A. C. Chao, and T. Hartmann. Hormonal regulation of chloride conductance in cultured polar airway cells measured by a fluorescent indicator. Am. J. Physiol. 262 (Cell Physiol. 31): C23-C31, 1992[Abstract/Free Full Text].

26.   Verkman, A. S., M. Sellers, A. C. Chao, T. Leung, and R. Ketcham. Synthesis and characterization of improved chloride-sensitive fluorescent indicators for biological applications. Anal. Biochem. 178: 355-361, 1989[Medline].

27.   Verkman, A. S., R. Takla, B. Sefton, C. Basbaum, and J. H. Widdicombe. Quantitative fluorescence measurement of chloride transport in phospholipid vesicles. Biochemistry 28: 4240-4244, 1989[Medline].

28.   Xia, P., B. E. Persson, and K. R. Spring. The chloride concentration in the lateral intercellular spaces of MDCK cell monolayers. J. Membr. Biol. 144: 21-30, 1995[Medline].


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