Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521
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ABSTRACT |
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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
(
ex) 350 nm, emission wavelength (
em) 450 nm]. N-substituted 6-aminoquinolinium (AQ) was
chosen as the
Cl
-insensitive moiety
because of its different spectral characteristics (
ex 380 nm,
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
-MQ-
'-dimethyl-AQ-xylene dichloride and
trans-1,2-bis(4-[1-
'-MQ-1'-
'-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
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INTRODUCTION |
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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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METHODS |
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Organic Synthesis
All chemicals were purchased from Aldrich Chemical (Milwaukee, WI). Compounds 1-MQ-n-AQ-alkane (MQanAQ) and
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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
,
'-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
-6-acetyl-AQ-
'-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:
-6-acetyl-AQ-
'-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 -(6-acetyl-AQ)-
'-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-'-MQ-1'-
'-6-acetyl-AQ-xylyl]pyridinium)ethylene
tetrachloride (bis-XPQ).
-MQ-
'-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
-MQ-
'-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.
-MQ-
'-(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-
'-MQ-1'-
'-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:
-MQ-
'-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);
-MQ-
'-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-'-1,2-dihydro-6-methoxyquinoline-1'-
'-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/FCell 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-'-MQ-1'-
'-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.
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RESULTS |
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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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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-da/
d),
where
d and
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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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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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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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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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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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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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.
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DISCUSSION |
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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
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
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.
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ACKNOWLEDGEMENTS |
---|
We thank Katherine Chen for cell cultures and Prof. J. L. Sessler for useful discussions.
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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.
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