Quantitative fluorescence ratio imaging of intralysosomal chloride ions with single excitation/dual maximum emission

Article abstract of DOI:10.1002/chem.201402999

Fluorescence ratio imaging is currently being used to quantitatively detect biologically active molecules in biosystems; however, two excitations of most existing fluorescent ratiometric probes account for cumbersome operating conditions for imaging. Thus

Full text of DOI:10.1002/chem.201402999

DOI: 10.1002/chem.201402999
Full Paper
&
Imaging Agents
Quantitative Fluorescence Ratio Imaging of Intralysosomal
Chloride Ions with Single Excitation/Dual Maximum Emission
Ping Li, Shan Zhang, Nannan Fan, Haibin Xiao, Wen Zhang, Wei Zhang, Hui Wang, and
Bo Tang*[a]
Abstract: Fluorescence ratio imaging is currently being used
to quantitatively detect biologically active molecules in bio-
systems; however, two excitations of most existing fluores-
cent ratiometric probes account for cumbersome operating
conditions for imaging. Thus, a fluorescent ratiometric
probe, 6-methoxyquinolinium–dansyl (MQ-DS), for ClÀ with
single excitation/dual maximum emission has been devel-
oped. MQ-DS can preferably localize into lysosomes and dis-
play excellent photostability. Upon excitation at a single
wavelength, it responds precisely and instantaneously to
changes in ClÀ concentrations, and it can be conveniently
utilized to implement real-time fluorescence ratio imaging
to quantitatively track alterations in ClÀ levels inside cells
treated under various pH conditions, and also in zebrafish
with acute wounds. The successful application of the new
probe in bioimaging may greatly facilitate a complete un-
derstanding of the physiological functions of ClÀ.
Introduction
intensity variations that result from probe concentration, opti-
cal path length, and excitation intensity.[6] Therefore, fluores-
cent ratio imaging is an effective approach for the quantitative
detection of intracellular biologically active molecules. Howev-
er, existing fluorescent ratiometric probes, including ClÀ
probes, are mostly based on two-wavelength excitation.[7] Two
excitations require more complicated instrumental settings,
which obstruct the continuity of data collection in real time
and cause difficulties in detection in diverse biological pro-
cesses. The levels of intracellular biologically active molecules
vary continuously with time. Thus, to accurately measure their
concentrations, the development of fluorescent ratio imaging
with a single excitation wavelength has attracted interest.[8]
Nevertheless, due to electronic effects of the probe structure,
it is difficult to pursue single excitation/dual maximum emis-
sion of the probe molecule, which still remains a big challenge.
In view of the critical regulation role of chloride ions in physio-
and pathological processes, precise imaging and quantification
of ClÀ in live cells and tissues are highly desired and notewor-
thy.
Chloride ions (ClÀ), which are the most abundant physiological
anions, have a critical role in a great variety of cellular pro-
cesses, including neurotransmission, regulation of cytoplasmic
and vesicular pH, cellular volume, and charge balance.[1] As
a result, ClÀ homeostasis alterations are associated with numer-
ous diseases, such as lysosomal storage disease, cystic fibrosis,
myotonia, and osteopetrosis.[2] Previous studies revealed that
ClÀ was abundant in acidic subcellular regions, especially lyso-
somes. Lysosomes, which are membrane-bound cytoplasmic
compartments, serve as a major degradative organelle in eu-
karyotic cells.[3] Their degradative functions for internalized
macromolecules depend on maintaining an acidic environ-
ment. Indeed, there is a growing body of evidence that ClÀ
permeability of lysosomes plays an important role in lysosomal
acidification.[4] Therefore, monitoring lysosomal ClÀ has re-
ceived increasing attention, especially by means of fluores-
cence imaging approaches combined with fluorescent probes
due to high-resolution and sensitivity.[5]
Fluorescent ratiometric probes are considered to be ex-
tremely powerful bioimaging tools for biologically active mole-
cules, because such ratios effectively suppress interference of
To address this issue, we designed a series of new fluores-
cence ratiometric probes that consisted of two fluorophores
with identical excitation wavelengths, that is, a ClÀ-sensitive 6-
methoxyquinolinium (MQ) group and a ClÀ-insensitive dansyl
(DS) group (Scheme 1). Importantly, emission peaks of the two
fluorophores distinctly differ in wavelength when simultane-
ously excited at the same wavelength. Another reason for
choosing DS is a dimethylamino group in its structure, which
is comparable to commercial lysosome dyes and contributes
to probe delivery into the lysosome through binding of H+. To
screen ideal ratio imaging probes, the two moieties are linked
covalently through three different spacers, three methylenes
(1), N-methylenepiperazine (2), and benzyl (MQ-DS), to modu-
[a] Prof. P. Li, S. Zhang, N. Fan, Dr. H. Xiao, Dr. W. Zhang, W. Zhang, H. Wang,
Prof. B. Tang
College of Chemistry, Chemical Engineering and Materials Science
Collaborative Innovation Center of Functionalized Probes for
Chemical Imaging, Key Laboratory of Molecular and Nano Probes
Ministry of Education, Shandong Normal University
Jinan 250014 (P.R. China)
E-mail: tangb@sdnu.edu.cn
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201402999.
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and their fluorescence responses
to ClÀ were evaluated in detail.
Although all synthesized com-
pounds show two well-separat-
ed emission bands upon excita-
tion at a single wavelength, ex-
perimental results reveal that
compounds 1 and 2 cannot reli-
ably sense changes in ClÀ con-
centrations, whereas MQ-DS can,
as shown in Figure S1 in the
Supporting Information. Owing
to no emission cross-talk be-
tween the two fluorophores,
these two distinct measurable
signals of maximum emission
Scheme 1. Structures of the designed probes.
wavelengths
(l=440
and
560 nm) are favorable to quanti-
tative detection of ClÀ by using
fluorescence ratio changes.
As expected, the presence of
ClÀ brings about striking decline
in the fluorescence intensity of
MQ (l=440 nm) and, at the
same time, the fluorescence in-
tensity of the DS moiety (l=
560 nm) remains essentially con-
stant (Figure 1), which leads to
enhancement of the fluores-
cence ratio between DS and MQ.
The observed ratio (F560/F440
)
changes are proportional to ClÀ
concentrations, and the linear re-
gression equation was F560/F440
=
0.4801+0.0044[ClÀ] (ꢁ10À3 m)
with a correlation coefficient of
0.9937 (Figure S1c in the Sup-
porting Information); the detec-
tion limit was calculated to be
0.18 mm. These results demon-
strate that the linear response of
MQ-DS to ClÀ concentrations
within the physiological range
(0–100 mm). In contrast to two
excitations of existing fluores-
cent ratiometric probes, the
single-wavelength excitation and
Scheme 2. Synthesis of compounds 1, 2, and MQ-DS.
well-separated dual maximum
emission profiles can effectively
late electronic effects.[9] Herein, we describe the synthesis and
properties of these designed ratiometric probes.
overcome the cumbersome operation of imaging and the devi-
ation of data collection, which greatly improve the accuracy
and precision of ClÀ measurements.
To confirm whether MQ-DS could selectively detect ClÀ, we
investigated interference from various biologically relevant
anions (Figure S2 in the Supporting Information). When other
anions were present, changes in the F560/F440 ratio values were
negligible. Once ClÀ was added to the above-mentioned
Results and Discussion
The syntheses of compounds 1, 2, and MQ-DS are shown in
Scheme 2. The spectroscopic properties of three compounds
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Figure 1. Fluorescence spectroscopic responses of 12 mm MQ-DS to ClÀ in
5.0 mm phosphate/citric acid buffer (pH 4.5). The ClÀ concentrations are
from 0 to 100 mm. Fluorescence responses occur immediately upon mixing.
The excitation wavelength was l=330 nm.
system, elevation of the ratio values appeared. Therefore, we
can conclude that the ratiometric fluorescence response of
MQ-DS is ClÀ selective. Furthermore, the effect of pH on the
MQ-DS fluorescence ratio was studied. The fluorescence ratios
of MQ-DS in the reaction system remained essentially constant
from pH 4.5 to 7.5 (Figure 2). Thus, MQ-DS is believed to be pH
Figure 3. Absorption spectra of MQ-DS (48 mm) in 5.0 mm phosphate/citric
acid buffer (pH 4.5) (a) and cell extracts (b).
Figure 4E) and Lyso-Tracker DND-26 (green channel; Fig-
ure 4B), we found that distribution domains of the two com-
pounds overlapped well, which indicated that their localization
patterns were similar. The colocalization coefficient was further
calculated to be 0.95. These results suggest that MQ-DS is
readily taken up by cells and targeted to lysosomes. This evi-
dence substantiates the postulation that MQ-DS is a robust ra-
tiometric sensor to visualize changes in ClÀ within lysosomes.
Next we studied ClÀ fluctuations in HepG2 cells incubated
with Tyrode’s solution (containing 136.9 mm potassium) con-
taining various concentrations of ClÀ. Fluorescence ratio imag-
ing is readily carried out with optical windows for MQ (from
l=410 to 505 nm, blue; Figure 5a) and DS (from l=525 to
620 nm, red; Figure 5b) at a single excitation wavelength of
405 nm. The ratio of fluorescence intensity between the red
and blue channels for MQ-DS-stained cells directly reflect ClÀ
levels in HepG2 incubated with Tyrode’s solution (Figure 5c),
according to the fluorescence intensity ratio scale and graph
of output data. Moreover, there was good linearity between
the fluorescence intensity ratio and ClÀ concentrations in the
range of 0–140 mm, as depicted in Figure 5. The linear regres-
sion equation was Fred/Fblue =0.3073+0.0047[ClÀ] (ꢁ10À3 m)
with a correlation coefficient of 0.9946 (Figure 5I). Similar ex-
periments in cell extracts gave identical results (Figure S6 in
the Supporting Information). These results unequivocally prove
that MQ-DS is a reliable ratiometric sensor that can conven-
iently image changes in intracellular ClÀ by single wavelength
excitation.
Figure 2. Effect of pH on the fluorescence ratio of MQ-DS (12 mm) in the ab-
sence (a) and presence (b) of ClÀ (100 mm).
insensitive. Additionally, some other aspects of MQ-DS were
examined, and these experimental results indicated that MQ-
DS was highly photostable (Figures S3 and S4 in the Support-
ing Information) with low cytotoxicity (Figure S5 in the Sup-
porting Information).
A study of absorption spectra of MQ-DS shows that it exhib-
its absorption bands centered at l=330 nm in phosphate/
citric acid buffer (pH 4.5; Figure 3a) and cell extracts (Fig-
ure 3b), and apparently a wider band can be observed in cell
extracts. In view of the clear absorption at l=405 nm in cell
extracts, MQ-DS can be excited effectively by using a confocal
fluorescence microscope equipped with a l=405 nm laser. We
assessed the application of MQ-DS in bioimaging. First, we ex-
amined whether MQ-DS could preferably accumulate into lyso-
somes in costaining experiments with Lyso-Tracker DND-26 (a
convenient commercially available intralysosomal dye). From
analysis of merged images (Figure 4C and F) captured in
human hepatoma cells (HepG2) loaded simultaneously with
MQ-DS (MQ: blue channel; Figure 4A, and DS: red channel;
It is widely accepted that chloride ion channels in the cell
membrane play essential roles in maintaining cellular osmolali-
ty and intracellular pH stability. As a result, pH changes may
alter intracellular chloride levels because ClÀ is the main coun-
terion used to balance H+ accumulation.[10] We studied wheth-
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Figure 4. Confocal fluorescence images of live HepG2 cells costained with 100 mm MQ-DS and 5.0 mm Lyso-Tracker DND-26 for 15 min at 378C. A) Fluores-
cence image of the MQ moiety (blue). B) Fluorescence images of Lyso-Tracker DND-26 (green). C) Merged image from A) and B). D) Higher magnification of
the area in a box in C). E) Fluorescence image of the DS moiety (red). F) Merged image from E) and B). G) Higher magnification of the area in a box in F).
Upon excitation at l=405 nm, the blue and red channels were collected at l=410–505 and 525–620 nm, respectively; the green channel was collected at
l=505–520 nm provided with excitation of l=488 nm wavelength.
Figure 5. Responses of MQ-DS (100 mm) to ClÀ in HepG2 cells. Columns A to H represent ClÀ concentrations of 0, 20, 40, 60, 80, 100, 120, and 140 mm, respec-
tively, in the media. a) Fluorescence images of MQ (blue channel) and b) DS (red channel). c) Ratio images generated directly from the red and blue channels
(Fred/Fblue). d) Bright-field images of cells. Scale bars are 25 mm. I) Intracellular ClÀ calibration curve of MQ-DS in HepG2 cells from A(c)–H(c).
er the probe could visualize changes of ClÀ concentrations in
liver cancer cells (HepG2) incubated under various pH condi-
tions. Imaging experiments were performed at pH 5.0 and 6.0,
respectively (Figure 6), and displayed apparent differences in
fluorescence ratio inside the cells. By measuring the fluores-
cent intensity of cells imaged at different pH values, the corre-
sponding ClÀ concentrations were calculated to be (39.76Æ
1.54) (pH 5.0) and (23.48Æ0.87) mm (pH 6.0) respectively (Fig-
ure 5I). According to the linear regression equation obtained
in liver cancer cells, ClÀ concentrations were calculated based
on average values of fluorescence ratio in four areas (white cir-
cles in Figure 6c). These results strongly suggest that the
probe is able to quantify ClÀ fluctuations in live cells through
fluorescent ratio imaging.
It is reported that intracellular ClÀ concentrations will in-
crease when myocardial ischemia happens, and a low extracel-
lular ClÀ concentration can significantly a delay in the ische-
mia-induced increase in ClÀ concentration.[11] MQ-DS was used
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Figure 6. Pseudocolor fluorescence ratio images (Fred/Fblue) of HepG2 cells labelled with MQ-DS (100 mm) at pH 5.0 (A) and 6.0 (B). a) The blue channel was col-
lected at l=410–505 nm. b) The red channel was collected at l=525–620 nm with excitation at l=405 nm. c) Ratio images generated directly from the red
and blue channels. d) Bright-field images of cells. Scale bars are 10 mm.
to visualize fluctuations of ClÀ levels during myocardial ische-
mia, and the fluorescence ratio images are displayed in
Figure 7. Upon the treatment of ischemia in ventricular myo-
cytes, there was a gradual elevation in ClÀ levels from 0
((9.46Æ0.43) mm) to 75 min ((77.95Æ0.61) mm; Figure 7G).
These dynamic imaging results clearly demonstrate that MQ-
DS can serve for real-time quantitative tracking of the changes
in ClÀ concentrations within live cells.
We further applied MQ-DS for detecting ClÀ fluctuations in
vivo. In a previous investigation, it was found that acute
wounds were closely associated with high H+ levels.[12] Mean-
while, ClÀ is the main counterion used to balance H+ accumu-
lation, and accordingly, an increase in H+ might cause a rapid
rise in ClÀ within living systems.[10] Herein, we tested whether
MQ-DS could visualize real-time changes in ClÀ within injured
zebrafish because H+ increase induced by injury should ac-
company an increase in ClÀ levels. In zebrafish loaded with
MQ-DS, 10 min after trauma, we observed an increase in the
fluorescence intensity ratio between the red and blue channels
(Figure 8). A larger ratio means that more ClÀ is present
((43.84Æ1.23) mm, 10 min), compared with 0 min ((19.01Æ
0.61) mm), which confirms that ClÀ influx acts to control pH;
this is consistent with previous reports. These results further
demonstrate the value of this fluorescence ratiometric probe
for quantitative and dynamic tracking of ClÀ in vivo.
also displays excellent selectivity for ClÀ, less cytotoxicity, and
good membrane permeability. Furthermore, we used MQ-DS
to accurately quantify intracellular ClÀ concentrations under
various pH conditions or myocardial ischemia. In particular,
MQ-DS dynamically visualized real-time quantitative fluctua-
tions of ClÀ in zebrafish after acute injury. Altogether, these re-
sults established that the new fluorescence ratiometric probe
was a potent tool to reliably and quantitatively visualize ClÀ in
live cells and tissues; these results can contribute to complete-
ly unravel the biological functions of ClÀ. Furthermore, this
single excitation/dual maximum emission result provides
a new strategy for precise and accurate detection of other bio-
logical molecules in live cells and in vivo.
Experimental Section
Materials and reagents
Solutions of compounds 1, 2, and MQ-DS (DMSO, 0.1 mm) could
be maintained in a refrigerator at 48C. Unless stated otherwise, sol-
vents were dried by distillation. All chemicals were available com-
mercially and the solvents were purified by conventional methods
before use. MQ (ꢀ98%) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) were purchased from Sigma Aldrich.
1,3-Dibromopropane (ꢀ98%), 5-(dimethylamino)-1-naphthalene-
sulfonamide (ꢀ99%), 5-dimethylamino-1-naphthalenesulfonyl chlo-
ride (ꢀ98%), dibromomethane (ꢀ99%), piperazine (ꢀ99%), and
4-nitrobenzyl bromide (ꢀ99%) were all purchased from Aladdin
Chemical Company (Shanghai, P.R. China). Lyso-Tracker DND-26
was from a molecular probes company. All other reagents and sol-
vents were purchased from commercial sources and of analytical
reagent grade, unless indicated otherwise. HepG2 (Human hepato-
cellular liver carcinoma cell line) and H9c2 (2-1) cells (Adult rat ven-
tricular myocytes) were purchased from the Committee on Type
Culture Collection of Chinese Academy of Sciences (Shanghai, P.R.
China). Sartorius ultrapure water (18.2 MWcm) was used through-
out. Universal buffer medium (0.1m citric acid, 0.1mK2PO4, 0.1m
Conclusion
We presented the synthesis, properties, and biological applica-
tion of MQ-DS, a new fluorescence ratio imaging probe for ClÀ
in lysosomes, which allowed for single excitation/dual maxi-
mum emission ratio detection of intralysosomal ClÀ for the first
time. The single-wavelength excitation and well-separated dual
maximum emission wavelengths are beneficial for precise and
accurate monitoring of real-time changes in ClÀ levels. MQ-DS
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Figure 8. Ratiometric images of live zebrafish upon trauma: A) 0 and
B) 10 min. Zebrafish cut near the tail were pretreated with 200 mm MQ-DS in
water for 15 min. Fluorescence images of a) MQ (blue channel) and b) DS
(red channel). c) Ratio images generated directly from the red and blue
channels. Scale bars are 1 mm.
Instrumentation
NMR spectra were recorded mainly on a Bruker Avance II-400 Four-
ier transform spectrometer operating at 600 MHz for 1H and at
150 MHz for 13C. The mass spectra were obtained on a Bruker
maXis ultra-high resolution-TOF MS system and an Agilent 1200LC-
6520 Q-TOF LC/MS (US80620227) instrument. Fluorescence spectra
measurements were performed by using a Cary Eclipse fluores-
cence spectrophotometer. The slit width was 20 nm for both exci-
tation and emission. Samples were contained in 2.0 mm path
length quartz cuvettes. In experiments with cell extracts, the slit
width was 5 nm for both excitation and emission, and samples
were contained in 1.0 mm path length quartz cuvettes. Cell ex-
tracts were prepared by using a BILON92-IIL ultrasonic disintegra-
tor (Shanghai Bilon Materials Inc.).
Cell culture
HepG2 cells and H9c2 (2-1) cells were cultured in Dulbecco’s modi-
fied eagle medium (DMEM) containing 10% fetal bovine serum
(FBS), 1% penicillin, and 1% streptomycin at 378C (w/v) in a 5%
CO2/95% air incubator MCO-15AC (Sanyo, Tokyo, Japan). The con-
centrations of counted cells were adjusted to 1ꢁ106 cellsmLÀ1 for
confocal imaging in high-glucose DMEM (4.5 g of glucose/L) sup-
plemented with 10% FBS, NaHCO3 (2 ngLÀ1), and 1% antibiotics
(penicillin/streptomycin, 100 U/mL). Cultures were maintained at
378C under a humidified atmosphere containing 5% CO2.
Cell extracts
Cell concentration was adjusted to 1ꢁ106 cellsmLÀ1. HepG2 cells
were incubated with Tyrode’s solution containing ClÀ (ClÀ-treated
group) and without ClÀ (blank group) for 3 h at 378C in an incuba-
tor. The cells were suspended in a volume of phosphate/citric acid
buffer (pH 4.5) and disrupted for 6 min in an ultrasonic disintegra-
tor (<48C). The broken cell suspension was centrifuged at
12000 rpm for 30 min and the pellet was discarded to give the cell
extracts.
Figure 7. Pseudocolor ratiometric imaging of live ventricular myocytes la-
belled with MQ-DS (100 mm). Rows A to F represent simulated ischemia
groups at 0, 15, 30, 45, 60, and 75 min, respectively. Fluorescence images of
a) MQ (blue channel) and b) DS (red channel). c) Ratio images generated di-
rectly from the red and blue channels (Fred/Fblue). d) Bright-field images of
cells. Scale bars are 50 mm. G) Variation of ClÀ concentration with time; this
was calculated from output data of A(c)–F(c).
Fluorescence imaging
Fluorescent images were acquired on a Leica TCS SP5 confocal
laser scanning microscope with an objective lens (40ꢁ and 20ꢁ).
The excitation wavelengths were l=405 and 488 nm respectively.
Cell imaging was carried out after washing cells with phosphate/
citric acid buffer three times. Fluorescence images of MQ-DS were
obtained at an excitation wavelength of l=405 nm. Blue and red
channels were collected at l=410–505 and 525–620 nm, respec-
Na2B4O7, 0.1m Tris, 0.1m KCl) was adjusted to the corresponding
pH by using solutions of 98.3% sulfuric acid and saturated NaOH.
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tively, and ratio images were generated directly from the red and
blue channels.
7.56–7.58 (t, 1H), 8.00–8.02 (d, 1H), 8.06–8.07 (d, 1H), 8.14–8.16 (d,
1H), 8.31–8.32 (d, 1H), 8.48–8.50 (d, 1H), 8.77 ppm (d, 1H);
13C NMR (150 MHz, DMSO): d=151.7, 129.7, 129.4, 128.9, 128.1,
123.9, 119.9, 119.4, 115.5, 73.7, 57.6, 56.5, 45.5, 40.8, 40.5, 19.0 ppm;
HRMS: m/z: calcd for C25H28N3O3S: 450.1846 [M+H]+; found:
450.1906.
For experiments of cellular localization, ventricular myocytes were
costained with 100 mm MQ-DS and 5.0 mm Lyso-Tracker DND-26 for
15 min at 378C. For confocal fluorescence images, MQ-DS was ex-
cited at l=405 nm and Lyso-Tracker DND-26 was excited at l=
488 nm; both were collected at l=505–520 nm.
Compound 5: 1,2-Dibromomethane (1.8684 g, 10.8 mmol) and 6-
methoxyquinoline (0.8586 g, 5.4 mmol) were mixed in DMF/PhMe
(3:2, 10 mL), and heated at 1308C for 12 h. After cooling to room
temperature, the mixture was filtered. The solid was washed sever-
al times with acetone. Compound 5 was obtained by centrifuga-
tion as a white solid (ꢁ65%).
For experiments of linearity between the fluorescence intensity
ratio and ClÀ concentrations, HepG2 cells were treated with Ty-
rode’s solution[13] (containing 136.9 mm potassium) containing vari-
ous concentrations of ClÀ, nigericin (5 mm), and tributyltin (10 mm)
for 30 min at 378C, and then incubated with MQ-DS (100 mm) for
15 min.
For cell images under various pH conditions, HepG2 cells were in-
cubated with universal buffer (pH 5.0 and 6.0; CClÀ =100 mm) for
1 h at 378C, then incubated with MQ-DS (100 mm) for 15 min.
Compound 6: Piperazine (0.5618 g, 6.5 mmol) and dansyl chloride
(0.1980 g, 5.4 mmol) were added to anhydrous methanol (10 mL),
and pyridine (0.5 mL) was used as a catalyst. The mixture was
stirred at 308C for 6 h under argon. The solution was purified by
column chromatography on silica gel, eluting with hexane/ethyl
acetate (3/1, v/v), to give 6 (ꢁ60%). HRMS: m/z calcd for
C16H21N3O2S: 320.1427 [M+H]+; found: 320.1331.
For experiments of myocardial ischemia, ventricular myocytes were
incubated with MQ-DS (100 mm) for 15 min at 378C, washed twice
with ischemic solution (sodium lactate instead of glucose from Ty-
rode’s solution), then imaged by using a confocal laser scanning
microscope every 15 min after adding the ischemic solution.
Zebrafish were incubated with 200 mm MQ-DS for 15 min, then
washed three times with water. The zebrafish were anesthetized
with 5% diethyl ether. After cutting of the tail (0 and 10 min), the
zebrafish were imaged by using a confocal laser scanning micro-
scope. The zebrafish were obtained from Biology Institute of
Shangdong Academy of Sciences. The experiments were approved
by the institutional committee. All the animal experiments were
carried out in accordance with the relevant laws and guidelines
issued by the Ethical Committee of Shangdong Academy of Scien-
ces.
Compound 2: Compound 6 (1.7280, 5.4 mmol) was dissolved in
anhydrous methanol (5 mL) and triethylamine (0.5 mL) was added
as a catalyst. The mixture was heated to 1208C and 5 (1.3500 g,
5.4 mmol) was added dropwise to the solution. After 6 h, a yellow
liquid was obtained. The crude product was purified by column
chromatography on silica gel, eluting with hexane/ethyl acetate/
methanol (1/3/0.2, v/v/v), to obtain probe 2 (ꢁ47%). 1H NMR
(600 MHz, CDCl3): d=2.02–2.06 (m, 4H), 2.89–2.92 (m, 6H), 4.10 (s,
2H), 7.08 (s, 1H), 7.21–7.23 (t, 1H), 7.33–7.36 (m, 2H), 7.54–7.56 (t,
2H), 7.93–7.96 (d, 1H), 8.12–8.13 (d, 1H), 8.19–8.20 (d, 1H), 8.40–
8.41 (d, 1H), 8.60–8.61 (d, 1H), 8.73 ppm (d, 1H); 13C NMR
(150 MHz, DMSO): d=159.3, 144.0, 143.0, 136.2, 130.6, 128.9, 126.6,
124.6, 123.4, 123.0, 122.7, 107.0, 56.53, 49.0, 40.8, 40.6, 31.1 ppm;
HRMS: m/z calcd for C27H31N4O3S: 491.2111 [M+H]+; found:
491.2754.
MTT assay
Ventricular myocytes (106 cellmLÀ1) were dispersed within replicate
96-well microtiter plates to a total volume of 200 mL per well.
Plates were maintained at 378C in a 5% CO2/95% air incubator for
24 h. Then ventricular myocytes were incubated for 16 h with dif-
ferent probe concentrations of 1.0ꢁ10À6, 1.0ꢁ10À5, 1.0ꢁ10À4, and
1.0ꢁ10À3 m. MTT solution (5 mgmLÀ1, phosphate-buffered saline
(PBS)) was then added to each well. After 4 h, the remaining MTT
solution was removed, and DMSO (150 mL) was added to each well
to dissolve the formazan crystals. Absorbance was measured at l=
490 nm in a Triturus microplate reader. Calculation of IC50 values
was performed according to the Huber and Koella method.[14]
Compounds 7 and 8: 4-Nitrobenzyl bromide (2.3331 g, 10.8 mmol)
was added to 6-methoxyquinoline (1.7172 g, 10.8 mmol) in DMF/
PhMe (3/2, 10 mL). The reaction temperature was kept at 1008C
for 6 h. The orange solution was obtained and filtered. Solid 7
(yield: ꢁ75%) was washed several times with acetone. The prod-
uct was dissolved in methanol and then Pd/C was added before H2
was introduced into the mixture. The solution was stirred at 508C
for 6 h and the solvent was removed on a rotary evaporator. Com-
pound 8 was obtained as a red solid (ꢁ60%). HRMS: m/z calcd for
C17H17N2O: 265.1335 [M+H]+; found: 265.1137.
Synthesis
Compound 4: 1,3-Dibromopropane (2.1708 g, 10.8 mmol) and 6-
methoxyquinoline (0.8586 g, 5.4 mmol) were dissolved in DMF/
PhMe (3:2, 10 mL) and heated at 1008C for 4 h. After cooling to
room temperature, the mixture was filtered. The solid was washed
several times with acetone. Compound 4 was collected by centrifu-
gation as an orange solid (ꢁ75%). HRMS m/z calcd for
C13H15BrNO: 280.0331 [M+H]+; found: 280.0328.
MQ-DS: Compound 8 (0.1315 g, 0.5 mmol) and dansyl chloride
(0.135 g, 0.5 mmol) were dissolved in anhydrous methanol (5 mL)
and pyridine (0.5 mL) was added as the catalyst. The mixture was
stirred at 1208C for 6 h under argon, and purified by column chro-
matography on silica gel, eluting with hexane/ethyl acetate (3:1,
1
v/v), to give MQ-DS (ꢁ60%). H NMR (600 MHz, CDCl3): d=2.87–
Compound 1: Dansylamide sodium salt (0.1360 g, 0.5 mmol) and 4
(0.2800 g, 1.0 mmol) were dissolved in DMF (8 mL), and heated at
808C for 24 h. An orange solution was obtained, which was puri-
fied by column chromatography on silica gel by eluting with
hexane/ethyl acetate/methanol (1/2/0.5, v/v/v) to give 1 (ꢁ50%).
1H NMR (600 MHz, CDCl3): d=1.99–2.02 (d, 2H), 2.21–2.23 (m, 2H),
2.87–2.89 (m, 6H), 3.87 (t, 3H), 4.29–4.36 (m, 2H), 4.79 (s, 1H), 7.08
(s, 1H), 7.16–7.19 (d, 1H), 7.38–7.40 (m, 2H), 7.41–7.44 (t, 1H),
2.89 (m, 6H), 3.87 (t, 3H), 4.29 (s, 2H), 4.82 (s, 1H), 6.92–6.93 (d,
2H),7.08–7.09 (d, 3H), 7.16–7.18 (d, 1H), 7.35–7.40 (m, 2H), 7.41–
7.43 (t, 1H), 7.53–7.56 (t, 1H), 8.00–8.02 (d, 1H), 8.06–8.07 (d, 1H),
8.16–8.17 (d, 1H), 8.34–8.35 (d, 1H), 8.48–8.49 (d, 1H), 8.77 ppm (d,
1H); 13C NMR (150 MHz, CDCl3): d=157.7, 152.1, 147.9, 144.3,
135.9, 135.0, 134.9, 134.2, 130.8, 130.3, 129.8, 129.6, 129.3, 128.6,
128.5, 123.1, 122.3, 121.4, 121.3, 118.5, 115.2, 105.1, 77.0, 76.8, 74.0,
58.0, 55.5, 45.4, 29.7 ppm; MS: m/z: 160.1, 371.1.
Chem. Eur. J. 2014, 20, 11760 – 11767
www.chemeurj.org
11766
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
Am. Chem. Soc. 2010, 132, 1194–1195; c) X. Wang, J. Sun, W. Zhang, X.
Ma, J. Lv, B. Tang, Chem. Sci. 2013, 4, 2551–2556; d) R. G. Painter, G.
Wang, Anal. Chem. 2006, 78, 3133–3137; e) P. Li, T. Xie, N. Fan, K. Li, B.
Tang, Chem. Commun. 2012, 48, 2077–2079; f) N. D. Sonawane, F. C.
Szoka, Jr., A. S. Verkman, J. Biol. Chem. 2003, 278, 44826–44831; g) L. Al-
bertazzi, M. Brondi, G. M. Pavan, S. S. Sato, G. Signore, B. Storti, G. M.
Ratto, F. Beltram, Plos One 2011, 6, e28450; h) O. Markova, M. Mukhtar-
ov, E. Real, Y. Jacob, P. Bregestovski, J. Neurosci. Methods 2008, 170, 67–
76.
Acknowledgements
This work was supported by the 973 Program (2013CB933800),
National Natural Science Foundation of China (21227005,
21390411, 91313302, 21035003, and 21205073) and Program
for Chang jiang Scholars and Innovative Research Team in Uni-
versity.
[7] a) X. Peng, Z. Yang, J. Wang, J. Fan, Y. He, F. Song, B. Wang, S. Sun, J.
Qu, J. Qi, M. Yan, J. Am. Chem. Soc. 2011, 133, 6626–6635; b) M. Taki, M.
Desaki, A. Ojida, S. Iyoshi, T. Hirayama, I. Hamachi, Y. Yamamoto, J. Am.
Chem. Soc. 2008, 130, 12564–12565.
Keywords: anions · chlorine · fluorescence · imaging agents ·
lysosomes
[8] a) S. Bassnett, L. Reinisch, D. C. Beebe, Am. J. Physiol. 1990, 258, C171–
C178; b) C. J. Chang, J. Jaworski, E. M. Nolan, M. Sheng, S. J. Lippard,
Proc. Natl. Acad. Sci. USA 2004, 101, 1129–1134; c) E. M. Nolan, S. J. Lip-
pard, J. Am. Chem. Soc. 2007, 129, 5910–5918.
[1] a) E. K. Hoffmann, P. B. Dunham, Int. Rev. Cytol. 1995, 161, 173–262; b) P.
Bregestovski, T. Waseem, M. Mukhtarov, Front. Mol. Neurosci. 2009, 2,
108–120.
[9] S. Jayaraman, J. Biwersi, A. S. Verkman, Am. J. Physiol. Cell Physiol. 1999,
276, C747–C757.
[10] D. Arosio, F. Ricci, L. Marchetti, R. Gualdani, L. Albertazzi, F. Beltram, Nat.
Methods 2010, 7, 516–518.
[2] a) R. Planells-Cases, T. J. Jentsch, Biochim. Biophys. Acta 2009, 1792,
173–189; b) M. C. Koch, K. Steinmeyer, C. Lorenz, K. Ricker, F. Wolf, M.
Otto, B. Zoll, F. Lehmann-Horn, K. H. Grzeschik, T. J. Jentsch, Science
1992, 257, 797–800.
[11] Z. F. Lai, K. Nishi, Am. J. Physiol. Heart Circ. Physiol. 1998, 275, H1613–
H1619.
[3] a) S. Kornfeld, I. Mellman, Annu. Rev. Cell Biol. 1989, 5, 483–525; b) W.
Hunziker, H. J. Geuze, Bioessays 1996, 18, 379–389.
[12] J. Llopis, J. M. McCaffery, A. Miyawaki, M. Farquhar, R. Y. Tsien, Proc. Natl.
Acad. Sci. USA 1998, 95, 6803–6808.
[13] J. Liu, Z. F. Lai, X. D. Wang, N. Tokutomi, K. Nishi, J. Cardiovasc. Pharma-
col. 1998, 31, 558–567.
[4] a) A. R. Graves, P. K. Curran, C. L. Smith, J. A. Mindell, Nature 2008, 453,
788–792; b) S. Weinert, S. Jabs, C. Supanchart, M. Schweizer, N. Gimber,
M. Richter, J. Rademann, T. Stauber, U. Kornak, T. J. Jentsch, Science
2010, 328, 1401–1403.
[14] W. Huber, J. C. Koella, Acta Trop. 1993, 55, 257–261.
[5] a) T. Nagai, K. Ibata, E. S. Park, M. Kubota, K. Mikoshiba, A. Miyawaki,
Nat. Biotechnol. 2002, 20, 87–90; b) A. S. Verkman, Am. J. Physiol. 1990,
259, C375–C388; c) N. Marandi, A. Konnerth, O. Garaschuk, Pflꢀgers
Archiv. 2002, 445, 357–365; d) P. Li, W. Zhang, K. Li, X. Liu, H. Xiao, W.
Zhang, B. Tang, Anal. Chem. 2013, 85, 9877–9881.
Received: April 8, 2014
[6] a) S. R. Adams, A. T. Harootunian, Y. J. Buechler, S. S. Taylor, R. Y. Tsien,
Nature 1991, 349, 694–697; b) D. W. Domaille, L. Zeng, C. J. Chang, J.
Published online on August 5, 2014
Chem. Eur. J. 2014, 20, 11760 – 11767
www.chemeurj.org
11767
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim