Analytical Chemistry
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ether should be more stable. In addition, the allyl group can also
118.21, 118.06, 110.96, 101.11, 81.45, 69.41. HR-MS (ESI):
be readily removed by Pd0-mediated Tsuji–Trost reaction.28-30
Therefore, we envisaged that these allyl fluorescein ether-based
probes should have great potential for CO detection based on
the same mechanism.23, 27, 31 Indeed, our studies found that these
allyl ethers showed satisfactory stability, and more importantly,
they can be conveniently used to detect CO both in vitro and in
living cells with excellent sensing properties. As far as we know,
this is the first case of using allyl ether as reaction site for con-
struction of fluorescent CO probes. In view of the easy prepa-
ration of allyl ethers and the diversity of fluorophores, this work
provided a new way to devise fluorescent CO probes.
calcd for C26H19Cl2O5+ (M + H+): 481.0604; found 481.0605.
Optical Studies. Stock solutions of probe 1 (2 mM), probe 2
(2 mM), PdCl2 (2 mM) and CORM-3 (10 mM) were separately
prepared in HPLC grade DMSO and used freshly. Stock solu-
tions (5-10 mM) of the analytes including NaF, NaCl, NaBr,
NaI, NaN3, Na2SO4, NaHSO4, NaAcO, NaIO4, LiClO4, NaNO3,
NaSCN, KCN, NaHCO3, NaHSO3, NaHS, cysteine (Cys), ho-
mocysteine (Hcy), glutathione (GSH), alanine (Ala), leucine
(Leu), tryptophan (Trp), glycine (Gly), isoleucine (Ile), serine
(Ser), phenylalanine (Phe), glutamine (Gln), glutamic acid
(Glu), and lysine (Lys) were prepared in ultrapure water.
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−
ROS/RNS species such as ClO−, H2O2, NO2 , HNO, NO, ROO•,
EXPERIMENTAL SECTION
tBuOO• and •OH were prepared according our published proce-
dure and used freshly.32-33 For a typical optical study, a 3.0 mL
solution containing probe 1 (5 μM) and PdCl2 (5 μM) in PBS
buffer (10 mM, pH 7.4, with 0.5% DMSO, v/v) was prepared
in a quartz cuvette. The UV-Vis or fluorescent spectra were then
recorded after addition of an analyte of interest at 37 °C (con-
trolled by a temperature controller). Unless otherwise noted, the
excitation wavelength for probe 1 and 2 was set at 490 and 493
nm, respectively, and slit width was set at dex = dem = 2.5 nm.
Materials and Chemicals. All reagents and chemicals were
purchased from commercial suppliers and used without further
purification. PBS buffers were prepared with distilled water that
had been passed through a Millipore-Q ultrapurification system.
All pH measurements were made with a Sartorius basic pH-me-
ter PB-10. TLC analysis was performed using precoated silica
plates. Melting points were determined using an X-4 apparatus
and are not corrected. NMR spectra were recorded on Varian
Mercury 400 or 600 instruments. High-resolution mass spec-
trometry (HR-MS) spectra were obtained on a Bruker micro-
TOF-Q instrument. UV-Vis and fluorescence spectra were rec-
orded on an Agilent Cary-100 UV-Vis spectrophotometer and
an Agilent Cary Eclipse fluorescence spectrophotometer, re-
spectively. Cell imaging was performed in an inverted fluores-
cence microscopy with a 20× objective lens.
Cytotoxicity of the Probes. This was assessed by the stand-
ard MTT assay. In brief, HeLa cells were cultured in Dul-
becco’s Modified Eagle’s Medium (DMEM) with high glucose
medium, supplemented with 10% fetal bovine serum (FBS),
100 μg/mL penicillin and 100 μg/mL streptomycin in a 5% CO2,
water saturated incubator at 37 °C. HeLa cells were then seeded
in 96-well plates and cultured overnight. Various concentra-
tions of the probe were added into the 96-well plate. After 24 h
of treatment, 20 μL of 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphe-
nyltetrazolium bromide (MTT) solution (5 mg/mL in FBS-free
medium) were administered into the each well. After 4 h of in-
cubation at 37 °C, all media were removed from wells and 200
μL of DMSO was added into each well to dissolve the intracel-
lular formazan crystals. Absorbance of the solution was then
measured at 490 nm with a microplate reader. The cell viability
was expressed as the optical density ratio of the treatment to
control. Values were calculated according to the formula: per-
centage of cell viability = OD treatment group/OD control
group × 100%.
Synthesis of Probe 1. To a solution of fluorescein (1.00 g,
3.0 mmol) in DMF (5 mL) was added K2CO3 (1.46 g, 10.5
mmol, 3.5 eq) and the mixture was stirred at room temperature
for 15 min. Allyl bromide (3.63 g, 30 mmol, 10 eq) was then
added. After stirring at room temperature for 30 min, the result-
ing mixture was heated to 70 °C for 5 h. The mixture was di-
luted with water (50 ml) after cooled down and extracted with
ethyl acetate (50 mL × 4). The combined organic phase was
washed with saturated NaHCO3 solution and dried over sodium
sulfate. The solvent was removed under reduced pressure and
the crude solid product was purified via column chromatog-
raphy (petroleum ether:ethyl acetate =15:1, v/v) to afford probe
1 as a white crystal (236 mg, yield 19%). Mp: 120-122℃. TLC
(silica plate): Rf ~0.66 (petroleum ether:ethyl acetate 3:1, v/v).
1H NMR (400 MHz, CDCl3) δ 7.99 (d, J = 7.3 Hz, 1H), 7.61 (dt,
J = 21.6, 7.4 Hz, 2H), 7.14 (d, J = 7.6 Hz, 1H), 6.75 (d, J = 1.9
Hz, 2H), 6.65 (d, J = 8.7 Hz, 2H), 6.60 (dd, J = 9.0, 1.9 Hz, 2H),
6.02 (ddd, J = 16.2, 10.4, 5.2 Hz, 2H), 5.41 (d, J = 17.2 Hz, 2H),
5.30 (d, J = 10.4 Hz, 2H), 4.55 (d, J = 5.1 Hz, 4H). 13C NMR
Imaging of CO in Living Cells. HeLa cells were seeded in
a 24-well culture plate for one night before cell imaging exper-
iments. In a typical experiment of cell imaging, as controls, liv-
ing cells were incubated with probe 1 (1 μM) and a mixture of
probe 1 and PdCl2 (1 μM each) at 37 °C for 30 min, respectively,
and they were imaged after washing with PBS for three times.
For imaging of exogenous CO, HeLa cells were pre-treated with
CORM-3 (1, 5, and 10 μM, respectively) for 30 min at 37 °C,
and then were incubated with a mixture of probe 1 and PdCl2 (1
μM each) for 30 min at 37 °C. After washing the cells with PBS
buffer, the cells were imaged. For imaging of heme stimulation
produced CO, the procedure is almost the same to that of imag-
ing exogenous CO, but using heme (100 μM) instead of CORM-
3 in the cell pre-treatment. Similar procedures were applied for
cell imaging experiments of probe 2.
(125 MHz, CDCl3)
δ 169.01, 159.80, 152.64, 151.98, 134.58,
132.14, 129.26, 128.64, 126.37, 124.53, 123.52, 117.71, 111.69,
110.92, 101.26, 82.80, 68.60. HR-MS (ESI): calcd for
C26H21O5+ (M + H+): 413.1384; found 413.1383.
Synthesis of Probe 2. Probe 2 (120 mg, yield 10%) was ob-
tained as a white solid from the reaction of 2, 7-dichlorofluo-
rescein and allyl bromide using the same procedure for probe 1.
Mp: 182-184 °C. TLC (silica plate): Rf ~0.67 (petroleum
ether:ethyl acetate 3:1, v/v). 1H NMR (400 MHz, CDCl3) δ
8.02 (d, J = 7.2 Hz, 1H), 7.73–7.59 (m, 2H), 7.12 (d, J = 7.2 Hz,
1H), 6.76 (s, 2H), 6.71 (s, 2H), 6.09 – 5.99 (m, 2H), 5.47 (d, J
= 17.1 Hz, 2H), 5.34 (d, J = 10.6 Hz, 2H), 4.64 (d, J = 4.9 Hz,
RESULTS AND DISCUSSION
Probe Synthesis. The probe 1 and 2 were prepared directly
from the commercially available fluorescein and 2, 7-dichloro-
fluorescein with allyl bromide in DMF under basic conditions
(Scheme 2). Both probes can be isolated and purified by column
4H). 13C NMR (125 MHz, CDCl3)
150.11, 135.07, 131.29, 129.87, 128.32, 125.95, 124.96, 123.42,
δ168.49, 155.10, 151.64,
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