NJC
Paper
were recorded on an Agilent 8453 UV/Vis spectrometer. NMR 4.4 Cell imaging
spectra were obtained on a Bruker DRX-300 NMR and Varian Unity
The cell line HeLa was provided by the Food Industry Research
and Development Institute (Taiwan). HeLa cells were cultured
in Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% fetal bovine serum (FBS) at 37 1C under an atmosphere
Inova 500 NMR spectrometer. IR data were obtained on Bomem
DA8.3 Fourier-transform infrared spectrometer. Fluorescence
images were obtained on a Leica TCS SP5 X AOBS confocal
fluorescence microscope.
2
of 5% CO . Cells were plated on 18 mm glass coverslips and
allowed to adhere for 24 h.
Experiments to assess the Cu uptake were performed in
phosphate-buffered saline (PBS) with 20 mM Cu(BF ) . The cells
cultured in DMEM were treated with 20 mM solutions of
chemosensor 1 (2 mL; final concentration: 20 mM) dissolved in
DMSO and incubated at 37 1C for 30 min. The treated cells were
washed with PBS (3 Â 2 mL) to remove any remaining sensor.
DMEM (2 mL) was added to the cell culture, which was then treated
with a 10 mM solution of Cu(BF4)2 (2 mL; final concentration:
4
.2 Synthesis of chemosensors 1
2
+
2
9
2-(Hydrazonomethyl)phenol (136 mg, 1.00 mmol) and 7-diethyl-
4
2
30
aminocoumarin-3-aldehyde (245 mg, 1.00 mmol) were dissolved
in 10 mL of methanol, and stirred overnight at room temperature.
A red precipitate was formed, and the crude product was filtered,
thoroughly washed with methanol to give 1. Yield: 254 mg (70%).
Melting point: 196–197 1C; H-NMR (300 MHz, DMSO-d ): d 11.23
1
6
(s, 1H), 8.90 (s, 1H), 8.65 (s, 1H), 8.54 (s, 1H), 7.67 (t, J = 7.5 Hz,
2
9
4
H), 7.39 (t, J = 6.9 Hz, 1H), 6.96 (t, J = 8.1 Hz, 2H), 6.80 (dd, J =
.0 Hz, J = 2.1 Hz, 1H), 6.64 (d, J = 1.8 Hz, 1H), 3.48 (q, J = 6.6 Hz,
H), 1.15 (t, J = 6.6 Hz, 6H); C-NMR (125 MHz, DMSO-d ):
6
20 mM) dissolved in sterilized PBS (pH = 7.4). The samples were
incubated at 37 1C for 30 min. The culture medium was removed,
and the treated cells were washed with PBS (3 Â 2 mL) before
observation. Confocal fluorescence imaging of cells was performed
using a Leica TCS SP5 X AOBS confocal fluorescence microscope
13
d 162.1, 160.4, 158.6, 157.2, 156.8, 152.1, 141.4, 132.9, 131.5, 131.0,
119.5, 118.3, 116.4, 111.1, 110.0, 108.0, 96.4, 44.3, 12.3; IR (KBr):
À1
3417, 2974, 2933, 1709, 1620, 1576 cm ; MS(EI): m/z (%) = 363
(
Germany), and a 63Â oil-immersion objective lens was used. The
(100), 348 (26.6), 346 (37.8), 243 (38.4), 229 (59.5), 173 (87.4);
cells were excited with a white light laser at 467 nm, and emission
was collected at 517–557 nm.
+
21 3 3
HRMS (EI): m/z, calcd for C21H N O (M ): 363.1583; found:
363.1588.
4
.5 Quantum chemical calculation
4
.3 Metal ion binding study by fluorescence spectroscopy
Quantum chemical calculations based on density functional
theory (DFT) were carried out using a Gaussian 09 program.
The ground-state structures of chemosensor 1 and the Cu –1
Chemosensor 1 (10 mM) was added with different metal ions
20 mM). All spectra were measured in 1.0 mL methanol–water
(
2+
2
solution (v/v = 1 : 1, 10 mM HEPES, pH 7.0). The light path
length of the cuvette was 1.0 cm.
For pH dependence experiments, the buffers were: pH 3–4:
PBS; pH 4.5–6: MES; pH 6.5–8.5: HEPES; pH 9–10: Tris-HCl. The
binding stoichiometry of 1–Cu complexes was determined by
Job plot experiments. The fluorescence intensity at 537 nm was
plotted against molar fraction of 1 at a constant total concen-
complexes were computed using the density functional theory
DFT) method with the hybrid-generalized gradient approximation
HGGA) functional B3LYP. The 6-31G basis set was assigned to
(
(
2+
nonmetal elements (C, H, N and O). For the Cu –1 complexes, the
LANL2DZ basis set was used for Cu , whereas the 6-31G basis set
was used for other atoms.
2
+
2
2+
2
+
tration (20 mM) of 1 and Cu . The fluorescence approached a
minimum intensity when the molar fraction was 0.67. These Acknowledgements
results indicate that chemosensor 1 forms a 2 : 1 complex with
2
+
2+
We gratefully acknowledge the financial support of Ministry
of Science and Technology (Taiwan) and National Chiao Tung
University.
Cu . The stability constants K of 2 : 1 1–Cu complexes were
a
3
1
determined by the equation:
2
a /(1 À a) = 1/(2K
a
C
F
[M])
(1)
where C
system and a is defined as the ratio between the free chemosensor
and the total concentration of chemosensor 1. The value "a" was
obtained using eqn (2)
F
is the total concentration of chemosensor 1 in the
Notes and references
1
1 A. P. de Silva, H. Q. N. Gunaratne, T. Gunnlaugsson, A. J. M.
Huxley, C. P. McCoy, J. T. Rademacher and T. E. Rice, Chem.
Rev., 1997, 97, 1515–1566.
a = [F À F
0
]/[F
1
À F
0
]
(2)
2
E. M. Nolan and S. J. Lippard, Chem. Rev., 2008, 108,
where F is the fluorescence intensity at 537 nm at any given
3443–3480.
2
+
Cu concentration, F
in the absence of Cu , F is the maxima fluorescence intensity
at 537 nm in the presence of Cu . The association constant K
1
is the fluorescence intensity at 537 nm
3 N. Kaur and S. Kumar, Tetrahedron, 2011, 67, 9233–9264.
4 M. Dutta and D. Das, TrAC, Trends Anal. Chem., 2012, 32,
113–132.
5 H. N. Kim, W. X. Ren, J. S. Kim and J. Yoon, Chem. Soc. Rev.,
2012, 41, 3210–3244.
2
+
0
2
+
a
2
2+
was evaluated graphically by plotting a /(1 À a) against 1/[Cu ].
2
2+
The plot a /(1 À a) vs. 1/[Cu ] is shown in Fig. 6. Data were
linearly fitted according to eqn (1) and the K
a
value was
6 J. A. Cowan, Inorganic Biochemistry: An Introduction, Wiley-
VCH, New York, 1997, pp. 133-134.
obtained from the slope of the line.
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