A new ratiometric two-photon fluorescent probe for imaging of lysosomes in living cells and tissues

Article abstract of DOI:10.1016/j.tet.2016.06.038

Intracellular pH plays a pivotal role in various biological processes. Herein, we adopted the through bond energy transfer (TBET) strategy to design a unique type of ratiometric two-photon fluorescent probe for imaging of lysosomal pH in live cells and tissues, termed Np-Rh-Lys. Specifically, a two-photon fluorophore (D-π-A-structured naphthalimide derivative) and a rhodamine B fluorophore were directly connected, and dimethylamino moiety served as a lysosomal targeting-group. The experiments demonstrate new probe Np-Rh-Lys was a reliable and specific probe for labeling lysosomes in living cells with two well-resolved emission peaks separated by 80 nm, and which showed high ratiometric imaging resolution and deep-tissue imaging depth over 180 μm, we thus may expect the new platform prompt the development of a wide variety of ratiometric two-photon fluorescent probes application in biological systems.

Full text of DOI:10.1016/j.tet.2016.06.038

Tetrahedron 72 (2016) 4637e4642
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Tetrahedron
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A new ratiometric two-photon fluorescent probe for imaging of
lysosomes in living cells and tissues
,
b
,
c
,
b
a
a
c
Liyi Zhou a
, Yongchao Liu , Shunqin Hu , Haifei Wang , Hongyan Sun ,
*
Xiaobing Zhang b
a College of Packaging and Materials Engineering, Hunan University of Technology, Hunan 412007, PR China
b Molecular Sciences and Biomedicine Laboratory, State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical
Engineering, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha 410082, PR China
c Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 April 2016
Received in revised form 19 May 2016
Accepted 12 June 2016
Available online 18 June 2016
Intracellular pH plays a pivotal role in various biological processes. Herein, we adopted the through bond
energy transfer (TBET) strategy to design a unique type of ratiometric two-photon fluorescent probe for
imaging of lysosomal pH in live cells and tissues, termed Np-Rh-Lys. Specifically, a two-photon fluo-
rophore (D-p-A-structured naphthalimide derivative) and a rhodamine B fluorophore were directly
connected, and dimethylamino moiety served as a lysosomal targeting-group. The experiments dem-
onstrate new probe Np-Rh-Lys was a reliable and specific probe for labeling lysosomes in living cells with
two well-resolved emission peaks separated by 80 nm, and which showed high ratiometric imaging
resolution and deep-tissue imaging depth over 180 mm, we thus may expect the new platform prompt
the development of a wide variety of ratiometric two-photon fluorescent probes application in biological
Keywords:
Two-photon
Ratiometric fluorescent probe
Lysosomal pH
Fluorescent imaging
systems.
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
weakly alkaline functional moieties were used for targeting
lysosome.9e12 To image and track the acidic vesicles in living cells,
In living cells, lysosome is the major subcellular organelles. Ly-
sosome contains numerous enzymes and proteins exhibiting a va-
riety of activities and functions at pH values (4.5e5.5).1 In order to
maintain the lysosomal pH values, the protons are pumped into
lysosomal interior by a membrane-bound ATPase. Importantly, the
cellular apoptosis process associates with the gradient decay of
lysosomal proton, which leads to the increase of the lysosomal pH
values.2e4 Once, the dysfunction of lysosomes are induced various
pathologies including neurodegenerative diseases,5 cancer,6 and
Alzheimer’s disease.7,8 Wherefore, it is important to monitor the
lysosomal pH values changes in living cells and tissues.
Fluorescent imaging is coming into widespread use in various
analytes in living cells, tissues and organisms by virtue of their high
sensitivity, high-speed spatial analysis, and less bio-damaging.
Traditionally, chemical probes for detecting targeting analytes in
an organelle are rationally designed by incorporating an organelle-
specific moiety as the guiding moiety. For example, positive cation
functional moieties were used for targeting mitochondria and
two main classes of pH-sensitive fluorescent probes have been
developed. The first category was turn-on probe based on a photo-
induced electron transfer (PET) system.13e15 The second category
was the ratiometric probe based on spectral shift in the emission
during proton binding.16 Of these two types of probes, the ratio-
metric probes have been mostly used for quantitative analysis,
because a turn-on response within a single detection window can
vary depending on the experimental conditions such as incident
laser power and probe distribution. Ratiometric probes, on the
other hand, can eliminate these interferences by the built-in cor-
rection of the dual emission bands, resulting in a more favorable
system for imaging living systems. However, most of these fluo-
rescent probes were used one-photon-excited and emit at a short
wavelength in the visible region, which resulted in some obvious
drawbacks for bioimaging, such as photobleaching, high auto-
fluorescence background and shallow penetration depth. There-
fore, it is of great significance and necessity to develop simple and
reliable fluorescent probe for quantitative measurement of lyso-
somal pH values changes in live cells and tissues.
To solve these problems, an effective tool is two-photon fluo-
rescence microscopy (TPFM). In this paper, based on the
* Corresponding author. Tel./fax: þ86 18711056980; e-mail address:
zhouly0817@163.com (L. Zhou).
http://dx.doi.org/10.1016/j.tet.2016.06.038
0040-4020/Ó 2016 Elsevier Ltd. All rights reserved.

4638
L. Zhou et al. / Tetrahedron 72 (2016) 4637e4642
previously works,17 we have established a new ratiometric two-
photon fluorescent probe Np-Rh-Lys, by directly connect a two-
imaging to develop a TBET-based ratiometric two-photon fluores-
cent probe. As a proof of principle, D- -A-structured naph-
p
photon D-
p
-A-structure naphthalimide derivative with a rhoda-
thalimide derivative was chosen as a donor for its excellent two-
photon properties, while rhodamine B was selected as an accep-
tor for its target-triggered ‘turn-on’ fluorescent signal that can be
easily distinguished, and alkaline dimethylamino served as a lyso-
somal targeting-group. This type of TBET-based ratiometric two-
photon fluorescent probe, termed Np-Rh-Lys (Scheme 1). The
structure of Np-Rh-Lys was characterized by 1H NMR, 13C NMR, and
MS (See the Supplementary data) (see Fig. 1).
mine B fluorophore via the through bond energy transfer (TBET)
strategy. The probe molecule exhibited a highly efficient energy
transfer with two well-resolved emission peaks, which afford
a high sensitivity for the Hþ-response, and high resolution ob-
served for Hþ ions. It showed a pronounced fluorescent response
toward Hþ ions over other common metal ions and neuter mo-
leculars. The probe was then used for TPFM imaging of pH in live
cancer cells and plant cells, showed high ratiometric imaging
resolution and large tissue-imaging depth. The synthesis processes
and the all structural characterization (using 1H NMR, 13C NMR,
and MS) are provided in the Scheme 1 and the Supplementary
data, respectively.
2.2. Optical property and selectivity of Np-Rh-Lys
The spectroscopic properties of Np-Rh-Lys was examined in
10 mM phosphate buffer solution (PBS) of different pH values. The
O
O
B
N
Br
N
Br
O
O
O
Br
O
O
O
O
O
N
O
+
Br
NH2
N
N
Br
3
O
O
4
I-1
2
1
O
Br
CHO
O
Br
O
+
O
N
OH
N
O
N
O
7
6
I-2
O
N
O
N
O
O
O
N
N
O
N
N
O
N
O
O
O
N
+
I-1 I-2
I-4
Np-Rh-Lys
N
O
I-3
N
O
O
N
O
N
N
N
O
N
O
N
I-5
Np-Rh-Lys
Scheme 1. The synthetic route of the probe Np-Rh-Lys.
2. Results and discussion
2.1. Design and synthesis of compounds Np-Rh-Lys
probe displayed different absorption (Fig. 2B) and fluorescent
(Fig. 2A) spectra with different pH values. As shown in Fig. 2B, the
probe exhibited a maximum absorption band at 400 nm at pH 7.4,
which belonged to the donor moiety. What’s more, at pH 3.0, it
showed two maximum absorption bands at 400 and 550 nm, re-
spectively, which belonged to the donor moiety and the spirocyc-
lization opened form of the xanthene unit (acceptor moiety).
Meanwhile, the probe solution’ color changed from green to red
The two-photon fluorescent activity of D-p-A-structured
naphthalimide derivatives have been reported.18,19 In order to ac-
quire the better bioimaging results, we decided to combine the
advantages of two-photon fluorescent imaging and the ratiometric

L. Zhou et al. / Tetrahedron 72 (2016) 4637e4642
4639
Fig. 1. Structure of Np-Rh-Lys and its response mechanism to pH or lysosomal pH.
8.5
8.0
0.35
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
B
pH=7.4
pH=3.0
A
2000000
1500000
1000000
500000
0
0.30
0.25
0.20
0.15
0.10
0.05
0.00
-0.05
500
550
600
650
350
400
450
500
550
600
650
Wavelength / nm
Wavelength / nm
12
10
8
pH=3.0
C
6
4
2
0
pH=7.4
-1
0
1
2
3
4
5
6
7
8
9
Cycle Index
Fig. 2. (A) Fluorescence emission spectra of Np-Rh-Lys (1
m
M) in PBS buffer (10 mM) at different pH values; (B) UV-Absorption spectra of probe Np-Rh-Lys (1
mM) under pH value
3.0 and 7.4, respectively; (C) pH reversibility study of Np-Rh-Lys between pH 3.0 and 7.4, lex¼400 nm.
was observed with pH value between 7.4 and 3.0 (see Fig. 2B). Once
upon excitation at 400 nm, the fluorescent emission intensity of
Np-Rh-Lys at 595 nm decreases significantly with the pH values
from 3.0 to 7.4, accompanying with a large increase of fluorescence
intensity at 515 nm (Fig. 2A), which provided the basis to achieve
a ratiometric (F515/F595) detection and imaging of pH. The probe
also showed a good reversibility between pH 3.0 and pH 7.4
(Fig. 2C), which was attributed to the reversible spirocyclization
opened/closed form of the probe molecule (Fig. 2). These results
indicated that the ratiometric response of Np-Rh-Lys matched well
with the physiological pH value range (pH 4.5e5.0) of lysosomes,
making it promising as a ratiometric fluorescent probe for accurate
measurement of lysosomal pH values.
Selectivity is an important parameter to evaluate the perfor-
mance of a new fluorescent probe. Particularly, for a bio-imaging
probe with potential application in complex biological samples,
a highly selective response to the target species over other poten-
tially competing kinds is necessary. As shown in Fig. S1 (see
Supplementary data), the selectivity experiments showed that var-
ious anions, cations and thiol amino acids have no interference on
the fluorescent response of Np-Rh-Lys to pH. Wherefore, Np-Rh-Lys
could be applied in living cells to track pH value of the acidic vesicles.
2.3. Colocalization and ratiometric two-photon imaging in
living cells
In order to evaluate the imaging performance of ratiometric
probe Np-Rh-Lys in vivo, the HeLa cells were chosen as the model
cell line. At first, the cytotoxic effects of Np-Rh-Lys were assessed
using the MTT assay. The results indicated that the Np-Rh-Lys was
nearly nontoxic to live cells under experimental conditions, the
IC50 was 28.2 mM (Supplementary data, Fig. S2). Following test,
the HeLa cells were co-stained with Np-Rh-Lys and LysoSensor
blue which is a commercially available lysosome-specific staining
probe. As shown in Fig. 3aed, the confocal fluorescence micros-
copy results showed that Np-Rh-Lys overlayed well with Lyso-
Sensor blue, which reveals that Np-Rh-Lys can selectively stain
lysosomes in live cells, and the ratiometric two-photon images
exhibited with high resolution between the green and the red
channels (Fig. 3eeh).
2.4. Tissues slice ratiometric imaging
At last, Np-Rh-Lys was used for onion tissue cells ratiometric
imaging. The changes of fluorescence signal intensity with scan

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L. Zhou et al. / Tetrahedron 72 (2016) 4637e4642
Fig. 3. Colocalization images and two-photon ratiometric images of HeLa cells. (A) Colocalization regent lysosome-blue (1
Rh-Lys (1
channel images. (F) Two-photon of red channel images. (G) overlay imaging of (E) and (F). (H) pseudo color imaging of (E), (F) and (G) Scale bar: 100
photon and two-photon images were acquired with a 60ꢀoil or 100ꢀoil immersion objective, respectively.
m
M, blue channel, lex¼405 nm, ¼415e455 nm). (B) Np-
l
m
M, red channel, lex¼559 nm, lem¼560e650 nm). (C) overlay imaging of (A) and (B). (D) overlay imaging of (A), (B) and bright field imaging. (E) Two-photon of green
mm, ltwo-photon ex¼780 nm, one-
depth were determined by spectral confocal multiphoton mi-
croscopy (Olympus, FV1000) in the z-scan mode. Depth scan-
ning demonstrated that the corresponding Np-Rh-Lys product
3. Conclusion
In summary, we have developed a new lysosome-targeting
ratiometric two-photon fluorescent probe (Np-Rh-Lys) by hybrid-
izing a weakly alkaline dimethylamine moiety with an xanthane
derivative and a two-photon naphthalimide fluorophore. The
xanthane derivative of the probe exhibits a pH-modulated open/
close reaction of the spirocycle, while the two-photon naph-
thalimide moiety as an internal standard, which affords the probe
a ratiometric fluorescence response towards pH. Confocal micro-
scopic images of HeLa cells labeled with Np-Rh-Lys revealed two
distinct emission ranges at 485e530 nm (green) and 560e610 nm
(red) in living cells. Colocalization experiments confirmed that the
fluorescence of probe Np-Rh-Lys overlayed with a lysosome-
specific staining dye, demonstrated its strong lysosomal localiza-
tion capability. The probe can be readily prepared and shows
lysosome-targeting ability, high resolution and low cytotoxicity.
More importantly, we successfully used the ratiometric two-
photon probe Np-Rh-Lys to monitor the pH in live animal cells
and plant cells. We believe that Np-Rh-Lys could be widely applied
in biological systems imaging.
was capable of tissue cells imaging at depths of 40e180
TPFM imaging, the results were presented in two emission
channels with different colors (see Fig. and Fig. S3 in
mm by
4
Supplementary data). These data showed that Np-Rh-Lys pos-
sessed good tissue penetration, high resolution, staining ability,
and it was also used for lysosomal pH ratiometric imaging in the
plant cells, as well as ratiometric two-color (green and red)
imaging changes.
4. Experimental
4.1. Materials and instruments
Unless otherwise specified, all chemicals were obtained from
commercial suppliers and used without further purification. Thin
layer chromatography (TLC) was carried out using silica gel F254,
and column chromatography was conducted over silica gel
(100e200 mesh), both of them were obtained from Qingdao Ocean
Chemicals (Qingdao, China). In all experiments, water used was
doubly distilled and purified by a Milli-Q system (Millipore, USA).
LC-MS analyses were performed using an Agilent 1100 HPLC/MSD
spectrometer. Mass spectra were performed using an LCQ Advan-
tage ion trap mass spectrometer (Thermo Finnigan). 1H NMR and
13C NMR spectra were obtained using a Bruker DRX-400 spec-
trometer using TMS as an internal standard. All chemical shifts are
Fig. 4. TPFM of an onion epidermal tissue slice ratiometric imaging. (A) and (B)
stained with 5 mM Np-Rh-Lys at 110 mm for 60 min. (C) The imagings overlayed (A)
and (B). (D) Ratiometric imaging of (A) and (B). The images were collected at
470e530 nm (green channel, A) and 550e650 nm (red channel, B) upon excitation at
reported in the standard
d notation of parts per million. UVevis
absorption spectra were recorded in 1.0 cm path length quartz
cuvettes on a Shimadzu 2450 UV-visible Spectrometer. HeLa cells
780 nm with femtosecond pulses. Scale bars: 100 mm.

L. Zhou et al. / Tetrahedron 72 (2016) 4637e4642
4641
and onion tissues were obtained from the College of Biological
Sciences of Hunan University, and the fluorescence images of HeLa
cells and onion tissues were obtained using Olympus FV1000-MPE
multiphoton laser scanning confocal microscope (Japan). Fluores-
cence measurements were carried out on a F4500 fluorescence
spectrometer with excitation and emission slits set at 5.0 nm and
5.0 nm, respectively. The pH was measured with a Mettler-Toledo
Delta 320 pH meter.
distilled water with final concentrations of 0.0125 mol/L, as well as,
glutathione (GSH), cysteine (Cys), and glutamate (Glu) using twice-
distilled water with final concentrations of 0.025 mol/L. Procedure
of selectivity experiments followed: for cations or anions, 20
stock solution of probe, 1948 L PBS solution (pH 7.0) and 32
m
m
L
L
m
solution of each cation or anion were combined to afford a test
solution, which contained 1ꢀ10ꢁ6 mol/L of probe and 200
mM
cation or anion; for amino acids, 20
mL stock solution of probe,
1900 mL PBS buffer solution (pH 7.4) and 80 mL solution of each
4.2. Synthesis of compounds 3, 6, 7, and Np-Rh-Lys
amino acid were combined to afford a test solution, which con-
tained 1ꢀ10ꢁ6 mol/L of probe and 1 mmol/L amino acid.
Synthesis of compound 3: compound 1 (0.01 mol, 2.57 g) and
compound 2 (0.011 mol, 1.8 g) were dissolved in glacial acetic acid
(100 mL). The mixture was heated to reflux for 4 h, then poured into
250 mL ice-water, the solid was washed with HCl(aq) (1.0 mol/L) and
NaOH(aq) (1.0 mol/L), respectively, and then the gray white solid was
obtained via the vacuum pump leak and dried under the vacuum
oven. 1H NMR (400 MHz, DMSO-d6) dH: 10.07 (s, 1H), 8.63e8.59 (t,
J¼4 Hz, 1H), 8.35e8.26 (t, J¼18 Hz, 1H), 8.03 (s, 1H), 7.76e7.74 (d,
J¼4 Hz, 1H),7.56e7.54 (d, J¼4 Hz, 1H), 7.48e7.39 (m, 4H).
4.4. Cell cytotoxic assays and imaging
The cytotoxic effects of probe were assessed using the MTT as-
say. The HeLa cells were treated with Np-Rh-Lys (0, 2.0, 4.0, 6.0, 8.0,
10.0, 20.0, 30.0, 40.0, 50.0, 60.0, 70.0, 80.0, 90.0,100.0
mM), and then
using the log([Np-Rh-Lys]/ M) as the x-axis and the cell viability as
m
the y-axis, at last via the origin fitting with a standard curve and
calculate the IC50. Before the cells imaging, the cells were washed
with PBS buffered solution, followed by incubating with probe
Synthesis of compound 4: Compound 3 (0.0046 mol, 2.0 g) and
morpholine (20 mL, excess) were added together, and then heated
to reflux for 8 h, the mixture was cooled to room temperature, and
then poured into 200 mL ice-water, and then, the yellow solid was
obtained through filtration and dried by the vacuum oven. 1H NMR
(400 MHz, DMSO-d6) dH: 8.65e8.64 (d, J¼2 Hz, 1H), 8.63e8.60 (d,
J¼6 Hz, 1H), 8.58e8.51 (d, J¼14 Hz, 1H), 7.74e7.66 (m, 3H),
7.29e7.19 (m, 3H), 4.07e4.04 (t, J¼6 Hz, 4H), 3.33e3.30 (t, J¼6 Hz,
4H). þC ESI ms¼434.3, calcd¼436.1.
(1
washing with PBS three times and imaged. Lyso-Tracher blue (1
m
M) for 30 min (in PBS containing 1% DMSO) at 37 ꢂC, then by
mM,
Invitrogen) was used for co-staining experiments. The one-photon
excitation wavelength was fixed at 405 nm and 535 nm. The fluo-
rescent emissions wavelength were recorded at (425e475) nm,
(495e540) nm and (560e650) nm, individually. The two-photon
excitation wavelength was fixed at 780 nm.
Synthesis of the I-1, I-2, and I-3 was followed the reference.17
These compounds only used the ms to be proved without further
purification, and directly to do the next steps. þC ESI ms for I-
1¼485.3, I-2¼521.3, I-3¼799.5.
4.5. Preparation and staining of plant tissue slice
The slices were cultured with 5 mM Np-Rh-Lys in an incubator at
37 ꢂC for 1 h and then washed with PBS three times for TPFM im-
aging. The TPFM images (with a magnification at 10 times) were
collected in two channels (green: 495e540 nm, and red:
560e650 nm) upon excitation at 780 nm with a pulse laser.
Synthesis of the Np-Rh-Lys: I-3 (0.63 mmol, 0.5 g), ethanol
50 mL, and 2-dimethylaminoethylamine 5 mL were added together,
to continue heating to reflux for another 4 h. The resulting mixture
solution was rotary evaporated. The yellow solid I-4/5 was obtained
through column chromatography with petroleum ether/ethyl
acetate¼5:1 (v/v). Finally, we chose a larger polarity of compound
for structural confirmation and testing. 1H NMR (400 MHz, DMSO-
d6) dH: 8.55e8.37 (m, 3H), 7.89e7.86 (t, J¼6 Hz, 2H), 7.84e7.79 (m,
1H), 7.69e7.67 (d, J¼8 Hz, 1H), 7.49e7.47 (d, J¼8 Hz, 1H), 7.39e7.34
(m, 3H), 6.41e6.39 (dd, J¼4 Hz, 6H), 3.92e3.91 (d, J¼4 Hz, 4H), 3.38
(s, 1H), 3.34e3.30 (t, J¼8 Hz, 6H), 3.23 (s, 4H), 3.10e3.03 (m, 2H),
1.90 (s, 6H), 1.08e1.05 (t, J¼6 Hz, 12H). 13C NMR (400 MHz, DMSO-
d6) dC: 169.12, 161.19, 152.72, 152.54, 152.42, 149.62, 149.46, 135.71,
128.94, 126.85, 124.79, 124.35, 112.39, 108.76, 106.99, 105.12, 102.08,
97.17, 84.28, 48.71, 47.53, 44.56, 44.11, 12.63. þC ESI ms¼869.5,
calcd¼869.4.
Acknowledgements
This work was supported by the National Key Scientific Program
of China (2011CB911000), NSFC (Grants 21325520, 21327009,
J1210040, 21177036), the Foundation for Innovative Research
Groups of NSFC (Grant 21221003), the National Natural Science
Foundation of China (21135001), the National Instrumentation
Program (2011YQ030124), State Key Laboratory of Fine Chemicals
Dalian University of Technology (KF1301) and the Hunan Provincial
Natural Science Foundation (Grant 11JJ1002).
Supplementary data
4.3. Spectrophotometric measurements
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.tet.2016.06.038.
The fluorescence measurement experiments were measured in
phosphate buffer solution (10 mM) with DMSO as co-solvent so-
lution (H2O/DMSO¼99:1, v/v). The pH value of PBS solution used
was from 3.0 to 7.4, which was achieved by adding minimal vol-
umes of HCl solution or NaOH solution. The fluorescent emission
spectra was recorded at excitation wavelength of 400 nm with
emission wavelength range from 475 to 650 nm. A 1ꢀ10ꢁ3 mol/L
stock solution of probe was prepared by dissolving probe com-
pound in DMSO. Procedure of calibration measurements with
References and notes
1. Kornfeld, S.; Mellman, I. Annu. Rev. Cell Biol. 1989, 5, 483e525.
2. Blott, E. J.; Griffiths, G. M. Nat. Rev. Mol. Cell Biol. 2002, 3, 122e131.
3. Stinchcombe, J.; Bossi, G.; Griffiths, G. M. Science 2004, 305, 55e59.
€
4. Nilsson, C.; Kagedal, K.; Johansson, U.; Ollinger, K. Methods Cell Sci. 2004, 25,
185e194.
5. Zhang, L.; Sheng, R.; Qin, Z. Acta Biochim. Biophys. Sin. 2009, 41, 437e445.
6. Schindler, M.; Grabski, S.; Hoff, E.; Simon, S. M. Biochemistry 1996, 35,
2811e2817.
probe in the buffer with different pH followed: 20 mL stock solution
7. Russell, D. A.; Pottier, R. H.; Valenzeno, D. P. Photochem. Photobiol. 1994, 59,
309e313.
8. Mogensen, H. S.; Beatty, D. M.; Morris, S. J.; Jorgensen, O. S. Neuroreport 1998, 9,
1553e1558.
of probe and 1980 mL PBS buffer solution with different pH were
combined to afford a test solution, which contained 1ꢀ10ꢁ6 mol/L
of probe. The solutions of various testing species were prepared
from NaCl, CaCl2, MgSO2, CuCl2$H2O, Zn(NO3)2$6H2O, using twice-
9. Dickinson, B. C.; Srikun, D.; Chang, C. J. Curr. Opin. Chem. Biol. 2010, 14, 50e56.

4642
L. Zhou et al. / Tetrahedron 72 (2016) 4637e4642
10. Smith, R. A.; Hartley, R. C.; Murphy, M. P. Antioxid. Redox Signal. 2011, 15,
3021e3038.
15. Yang, S.; Qi, Y.; Liu, C. H.; Wang, Y. J.; Zhao, Y. R.; Wang, L. L.; Yang, R. H. Anal.
Chem. 2014, 86, 7508e7515.
11. Fan, J. L.; Dong, H. J.; Hu, M. M.; Wang, J. Y.; Zhang, H.; Zhu, H.; Peng, X. Chem.
16. Wan, Q.; Chen, S.; Shi, W.; Li, L.; Ma, H. Angew. Chem., Int. Ed. 2014, 53,
Commun. 2014, 882e884.
10916e10920.
12. Best, Q. A.; Xu, R. S.; McCarroll, M. E.; Wang, L. C.; Dyer, D. J. Org. Lett. 2010, 12,
3219e3221.
17. Zhou, L.; Zhang, X. B.; Wang, Q.; Lv, Y.; Mao, G.; Luo, A.; Zhang, J.; Tan, W. J. Am.
Chem. Soc. 2014, 136, 9838e9841.
13. Zhu, H.; Fan, J. L.; Xu, Q. L.; Li, H. L.; Wang, J. Y.; Gao, P.; Peng, X. Chem. Commun.
2012, 11766e11768.
14. Shi, X. L.; Mao, G. J.; Zhang, X. B.; Liu, H. W.; Gong, Y. J.; Wu, Y. X.; Zhou, L.; Tan,
W. Talanta 2014, 130, 356e362.
18. Qian, Y.; Xiao, Y.; Guo, X.; Qian, J.; Zhu, W. Chem. Commun. 2010, 6418e6436.
19. Zhang, J. F.; Lim, C. S.; Bhuniya, S.; Cho, B. R.; Kim, J. S. Org. Lett. 2011, 13,
1190e1193.