1
13
respectively in 6-well microplate. Next, we washed the fishes
three times with E3 medium to remove the remaining drugs. The
fishes were then incubated in Cy-H O (10 µg/mL) E3 medium
characterized by H NMR, C NMR and HR-MS (Figures
ACCEPTED MANUSCRIPT
1
S10−S12). H NMR (400 MHz, CDCl , ppm) δ 8.41 (d, J = 8.9
3
Hz, 1H), 8.42-8.23 (m, 3H), 7.84 (d, J = 13.7 Hz, 2H), 7.38 (s,
1H), 7.36 (s, 1H), 7.35 (s, 1H), 7.34 (d, J = 2.6 Hz, 1H), 7.19 (d,
J = 7.4 Hz, 1H), 7.17 (s, 1H), 7.07 (s, 1H), 7.05 (s, 1H), 5.96 (s,
1H), 5.92 (s, 1H), 3.81-3.70 (m, 4H), 3.62 (s, 3H), 2.57 (t, J = 6.4
Hz, 4H), 1.93-1.82 (m, 4H), 1.75-1.66 (m, 7H), 1.25 (s, 6H), 0.88
2
2
for 1 hour. Then, we washed the larvae three times with E3
medium and imaged under an OLYMPUS IX71 fluorescence
microscope. The probe showed a response to endogenous
hydrogen peroxide with emiting fluorescence, which mainly
occurs in the abdomen of zebrafish (Figures 7 and S15). In
addition, we also studied the imaging ability of the probe for
exogenous H O in zebrafish (Figures 7 and S14). Effect of the
13
(s, 3H). C NMR (101 MHz, CDCl , ppm): δ 189.03, 170.59,
3
170.07, 164.44, 151.26, 143.11, 141.61, 140.24, 137.28, 131.43,
129.91, 128.64, 126.70, 124.26, 122.14, 109.81, 101.81, 98.38,
54.00, 53.53, 48.51, 31.99, 29.71, 28.10, 27.23, 25.57, 25.43.
2
2
probe in zebrafish was consistent with living cells.
+
+
HRMS (ESI): m/z calcd. for C H N O [M] 710.3701, found
4
4
48
5
4
3
. Experimental
7
10.3708.
3
.1. Materials and instruments
3.3. Cell cultures
All chemicals were purchased from J&K chemical reagent
The HeLa cells were provided by Chinese PLA General
Hospital (Beijing, China). All biological samples were collected
in accordance with the guidelines approved by the institutional
review board of the Chinese PLA General Hospital. The HeLa
cells were cultured on glass-bottom culture dishes (MatTek Co.)
in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented
with 10% (v/v) Fetal Bovine Serum (FBS) and 1% (v/v)
penicillin-streptomycin at 37°C in a humidified 5% CO2
incubator. Before utilized, the adherent cells were washed three
times with FBS free DMEM.
company without further purification except those with special
instructions. Fluorescent spectra were recorded with
a
PerkinElmer LS55 fluorescence spectrophotometer with a 1 cm
standard quartz cell. The fluorescence imaging of cells was
performed with laser confocal fluorescence microscopy
(
Olympus IX 71, Japan). Mass data (ESI) was measured by
quadrupole mass spectrometry. Samples for absorption and
fluorescence measurements were contained in 1 cm × 1 cm
quartz cuvettes.
3
.2. Synthesis of Cy-H O2
3.4. Zebrafish culture
2
Cy-piperazine.
Wild-type zebrafishes were purchased from Beijing
University of Chinese Medicine. The juveniles were incubated in
this laboratory. E3 culture medium contained 15 mM NaCl, 0.5
mM KCl, 1 mM MgSO , 1 mM CaCl , 0.15 mM KH PO , 0.05
Cy-piperazine was obtained as a deep-blue solid by the
previously reported methods . The total yield reached 64%
through two-step synthesis process. (Scheme 1). Cy-piperazine
26
4
2
2
4
1
13
mM Na HPO and 0.7 mM NaHCO (pH=7.5). All fishes were
2
4
3
was characterized by H NMR, C NMR and HR-MS (Figures
1
incubated at 28°C in the experiment and all animal experiments
were performed in full compliance with international ethics
guidelines.
S7−S9). H NMR (400 MHz, CDCl , ppm) δ 7.61 (d, J = 13.0
3
Hz, 2H), 7.31 (t, J = 8.1 Hz, 4H), 7.11 (t, J = 7.4 Hz, 2H), 6.98
d, J = 7.8 Hz, 2H), 5.70 (d, J = 13.0 Hz, 2H), 3.99 (s, 4H), 3.50
(
(
s, 6H), 3.25 (s, 4H), 2.48 (t, J = 6.5 Hz, 4H), 1.89 – 1.80 (m,
4
. Conclusions
13
2
2
1
H), 1.66 (s, 12H). C NMR (400 MHz, CDCl , ppm) δ 21.73,
3
4.87, 29.03, 31.16, 36.51, 47.89, 56.68, 95.16, 108.90, 122.04,
In summary, we have successfully constructed a fluorescent
23.30, 123.48, 128.38, 139.92, 140.64, 143.40, 168.94, 174.85.
probe Cy-H O , which is selective and fast responding to
2 2
+
HRMS (ESI): m/z calcd. for C H N [M - I]⁻ 533.3639, found
5
hydrogen peroxide. The probe can not only detects water medium
and living cells, but also detects endogenous and exogenous
hydrogen peroxide in zebrafish with real-time operability and
reliability. More importantly, Cy-H O displays NIR excitation
36
45
4
33.3635.
2
-(4-nitrophenyl)-2-oxoacetyl chloride (6).
2
2
4
-nitrophenylglyoxylic acid (5) was obtained as a pale
and emission spectra, which is beneficial for fluorescence
imaging in vivo. In addition, Cy-H can be used to
quantitatively detect and localize endogenous H production
27
yellowish brown solid by the previously reported methods.
Scheme 1) Then, 4-nitrophenylglyoxylic acid (39 mg, 0.2
O
2 2
(
O
2 2
mmol), oxaloyl chloride (53 µL, 0.6 mmol) and 3 mL
dichloromethane were mixed and stirred for 2 minutes, and two
drops of N, N-dimethylformamide (DMF) were added. Under the
protection of nitrogen, the temperature rose to 45°C. After one
hour, the mixture was cooled to room temperature. Then, the
solvent and excess oxaloyl chloride were evaporated, affording
the product (40.4 mg, 95%) as a light yellow transparent oil
liquid.
caused by drug oxidative damage which was mainly located at
the abdomen in zebrafish. This provides an effective method for
further monitoring of hydrogen peroxide in animals in real-time.
Conflicts of interest
There are no conflicts of interest to declare.
Acknowledgments
Cy-H O .
2
2
The authors would like to acknowledge financial support from
Beijing Key Laboratory of Environmental & Viral Oncology and
Beijing Key Laboratory for Green Catalysis and Separation.
Cy-piperazine (53 mg, 0.1 mmol), triethylamine (12 mL, 0.4
mmol) and 3 mL dichloromethane were mixed and stirred at 0°C.
Then, 6 (40.4 mg, 0.19 mmol) was dissolved in 3 mL
dichloromethane, and dripped slowly into above reaction solution.
After stirred for 3 hours, the solvent was evaporated and dried
under vacuum condition. Then, the product was purified by
References and notes
column
chromatography
over
silica
gel
using
1
H. J. Schiller, P. M. Reilly and G. B. Bulkley, Crit. Care Med., 1993,
, S92; E. W. Childs, K. F. Udobi, J. G. Wood, F. A. Hunter, D.
M. Smalley and L. Y. Cheung, Shock, 2002, 18, 423.
21
dichloromethane/MeOH (V/V=20/1) as the eluent, affording the
product (52.54 mg, 74%) as a pale blue solid. Cy-H O was
2
2