Coumarin-coupled receptor as a membrane-permeable, Cu2+- selective fluorescent chemosensor for imaging copper(II) in HEPG-2 cell

Article abstract of DOI:10.1246/cl.2008.462

A novel fluorescent chemosensor 1 for imaging labile Cu²⁺ in living biological samples was designed and synthesized; it exhibits very strong fluorescence responses to Cu²⁺, and its LSM images strongly support the existence of Cu²⁺ in HEGP-2 cell. Copyright

Full text of DOI:10.1246/cl.2008.462

462
Chemistry Letters Vol.37, No.4 (2008)
Coumarin-coupled Receptor as a Membrane-permeable, Cu2þ-selective
Fluorescent Chemosensor for Imaging Copper(II) in HEPG-2 Cell
Mao-Xiang Wang,1 Sheng-Hai Huang,2 Xiang-Ming Meng,1 Man-Zhou Zhu,ꢀ1 and Qing-Xiang Guoꢀ1
1Department of Chemistry, University of Science and Technology of China, Hefei 230026, P. R. China
2Institute of Clinical Pharmacology, Anhui Medical University, Hefei 230026, P. R. China
(Received December 27, 2007; CL-071443; E-mail: zmz@ustc.edu.cn)
A novel fluorescent chemosensor 1 for imaging labile Cu2þ
in living biological samples was designed and synthesized; it ex-
hibits very strong fluorescence responses to Cu2þ, and its LSM
images strongly support the existence of Cu2þ in HEGP-2 cell
i
N
N
+
N
H2N
O
O
2
3
Among essential heavy metal ions in human body copper is
third in abundance.1 Cells require copper for use in a variety of
respiratory and metabolic activites, but alterations in cellular ho-
meostasis are connected to serious neuro-degenerative diseases,
including Menkes and Wilson diseases,2–4 familial amyotrophic
lateral sclerosis,5,6 Alzheimer’s disease,7 and prion diseases.8
Free copper ions are dangerous to cells owing to their oxidizing
potential. The thermodynamically estimated level of free copper
in the cytosol of bacterial model systems is less than one ion
per cell.8 So cells exert strict control over intracellular copper
distributions.8–12 Although there is compelling evidence that
the intracellular milieu does not contain any free copper ions,
the rapid kinetics of copper uptake and release suggests the pres-
ence of a labile intracellular copper pool. Yang and co-workers13
recently provided the first evidence that the labile copper pool
appears to be localized in mitochondria and the Golgi region.
MicroXANES experiments have confirmed the predominance
of low-coordinate, monovalent copper throughout the cell but
N
N
O
O
N
N
N
1
Scheme 1. Reagents and conditions: NaNO2, HCl, H2O/DMF, rt.
has advantages over previously available Cu2þ chemosensors
requiring much more cumbersome synthesis from expensive
starting materials. The final compound was characterized by
1H NMR, 13C NMR, and mass spectrometry (See Supporting
Information).18
The maximum absorption wavelength of chemosensor 1
is 435 nm, and the maximum emission wavelength is 547 nm.
Fluorescence quantum yield of free 1 is 0.00315 under physiolog-
ical conditions (pH 7.4, 0.1 M HEPES, 0.1 M KNO3). Fluores-
cence is quenched by PET reaction between the receptor and
the fluorophore.
Upon addition of Cu2þ, the fluorescence intensity of 1 in-
creased by about 7.5-fold, and the corresponding quantum yield
increased to 0.026 (see Supporting Information).18 It is impor-
tant to point out that the 7.5-fold fluorescence enhancement of
1 is significant, for most previously reported Cu2þ chemosen-
sors, the addition of Cu2þ caused fluorescence quenching of
the fluorophore.16 Furthermore, maximum fluorescence can be
obtained when the ratio of chemosensor and Cu2þ is about 1:1
did not exclude the presence of Cu2þ
.
Fluorescent chemosensors that can permeate the plasma
membrane have proven to be powerful and nondestructive tools
for the study of intracellular metal ion distributions of calcium,
magnesium, or zinc, yet rigorous analytical techniques for sensi-
tive in vivo measurements of intracellular copper levels are
lacking. Some of the currently available copper chemosensors
are fairly complex molecules.14
Here, to elucidate the presence of Cu2þ in the cytosol of the
cell, we report the development, characterization, and evaluation
of a membrane-permeable copper-selective fluorescent chemo-
sensor for imaging of kinetically labile Cu2þ. A novel chemo-
sensor 1 for Cu2þ based on photoinduced electron transfer
(PET), in which N,N-bis(pyridin-2-ylmethyl)benzenamine as a
receptor group is connected to a coumarin group via a diazo
spacer. The coumarin group is chosen as fluorophore, since it
has a strong absorption band in the visible region, emits at longer
wavelength with high quantum yield and exhibits excellent
bioactivity. By attaching an appropriate chelator group to the
diazotizated coumarin, we have obtained a novel high-quality
Cu2þ chemosensor.
8
7
6
5
4
3
2
1
Ca2+ M n2+ Fe2+ Ni2+Pb2+ Cu2+ Zn2+ Free Cd2+ Cu+ Co2+ M g2+
A=Mg2+ +Cu2+ B=Zn2+ +Cu2+ C=Cu+ +Cu2+
A
B
C
Compound 1 originated from N,N-bis(pyridine-2-ylmethyl)-
benzenamine (2) (Scheme 1). Compound 2 was coupled at the 4-
position (yield = 40%) with diazotized 7-amido-4-methylcou-
marin (3). Thus, sensor 1 was successfully synthesized via a very
short route from inexpensive starting materials. Chemosensor 1
Figure 1. The fluorescence responses of 1 to different metal cat-
ions (experimental conditions: 42 mM sensor in DMSO/H2O =
2:8(V:V), 42 mM metal cation, 100 mM HEPES buffter, 100-mM
KNO3, pH 7.4). F0 is fluorescent intensity of free 1, F is
fluorescent intensity of 1 and cations.
Copyright ꢀ 2008 The Chemical Society of Japan

Chemistry Letters Vol.37, No.4 (2008)
463
Upon addition of Cu2þ, the chemosensor exhibits a 7.5-fold
emission enhancement and excellent selectivity toward Cu2þ
.
Although the reducing environment of the cytosol is expected
to stabilize monovalent copper,17 the presence of Cu2þ in the
cytosol of living cells has been suggested. The images of
HEPG-2 provide a coherent picture with strong evidence of
the existence of the hypothesized labile Cu2þ. Because the
new chemosensor is based on fluoresence enhancement of
fluorophore, compared with the Cu2þ fluorescence quenching
effect of reported sensors, the new chemosensor offers more
practical application in celluar Cu2þ imaging and other Cu2þ
detection fields.
References and Notes
1
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Figure 2. Confocal fluorescence images of HEPG-2 cells. A:
Cells incubated in DMEM for 24 h; B: Cells supplemented with
100 mM chemosensor 1 in the growth media at 37 ꢃC for 1 h; C:
Cells supplemented with 100 mM CuCl2 in the growth media at
37 ꢃC for 24 h and stained with 100 mM chemosensor 1 for 1 h at
37 ꢃC. Upper: black-field images of live HEPG-2 cells; Lower:
black-field and bright-field images of live HEPG-2 were added
together.
2
3
4
5
6
7
(see Supporting Information).18 The result indicates that com-
pound 1 should form a 1:1 complex with Cu2þ. Dissociation
constant, Kd, between the new chemosensor and Cu2þ was
determined to be 6:6 ꢁ 10ꢂ6 M (see Supporting Information).18
Our next goal was to check the selectivity of the new chemo-
sensor, we studied the fluorescence response of 1 to other metal
cations including Cd2þ, Mg2þ, Co2þ, Ca2þ, Fe2þ, Cuþ, and
Mn2þ. As shown in Figure 1, Cu2þ is the only cation among
the tested transition elements that induces fluorescence enhance-
ment.
Emission intensities did not appear to change in the presence
of other ions (including Ca2þ, Mn2þ, Mg2þ, Cd2þ, Fe2þ, Cuþ,
and Zn2þ), except for a little enhancement on addition of Pb2þ
and Ni2þ. This is very nice because under many conditions
(e.g., physiological conditions) Fe2þ and Zn2þ may coexist at
relatively high concentrations compared to Cu2þ. Thus, our
new chemosensor can selectively detect Cu2þ under physiolog-
ical conditions.
8
9
10 D. L. Huffman, T. V. O’Halloran, Annu. Rev. Biochem.
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15 The fluorescence quantum yield was obtained by using
fluorescein (Ø = 0.95 in 0.1 mol/L NaOH) as the standard.
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We sought to evaluate the ability of 1 to operate within
living cells. HEPG-2 cells were incubated in DMEM for
20 min at 25 ꢃC (Figure 2). Supplementing cells with 100 mM
CuCl2 growth medium for 24 h at 37 ꢃC and then staining with
1 under the same loading conditions results in a marked increase
in observed intracellular fluorescence, as determined from scan-
ning confocal microscopy (LSM) on live samples. To confirm
that this fluorescence increase was due to an increase of intracel-
lular Cu2þ concentration, we further added TPEN, an intracellu-
lar Cu2þ chelator (see Supporting Information).18 This treatment
reduced the fluorescence intensity to the initial level. These
experiments show that 1 is cell-permeable and can respond to
changes in intracellular Cu2þ within living cells.
18 Supporting Information is available electronically on the
CSJ-Journal web site, http://www.csj.jp/journals/chem-lett/.
In conclusion, we have synthesized and characterized a
membrane-permeable, Cu2þ-selective fluorescent chemosensor.
Published on the web (Advance View) March 25, 2008; doi:10.1246/cl.2008.462