Design and synthesis of a ratiometric fluorescent chemosensor for Cu(II) with a fluorophore hybridization approach

Article abstract of DOI:10.1021/ol301849z

A new ratiometric fluorescent sensor for Cu²⁺, WLN, has been developed via integrating a 1,8-naphthalimide fluorophore with 8-aminoquinoline. WLN exhibits a highly selective ratiometric response to Cu²⁺ over other transition metal ions in aqueous media. Moreover, its practical ratiometric imaging ability for intracellular Cu²⁺ has been confirmed in human breast adenocarcinoma cells (MCF-7 cells) using a confocal microscope.

Full text of DOI:10.1021/ol301849z

ORGANIC
LETTERS
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012
Vol. 14, No. 17
378–4381
Design and Synthesis of a Ratiometric
Fluorescent Chemosensor for Cu(II) with a
Fluorophore Hybridization Approach
4
,
†,‡
§
†
Zhipeng Liu,* Changli Zhang, Xiaoqing Wang, Weijiang He,* and Zijian Guo
,‡
‡
School of Chemistry and Chemical Engineering and School of Material Science and
Engineering, Liaocheng University, Liaocheng, P. R. China, State Key Laboratory of
Coordination Chemistry, Coordination Chemistry Institute, School of Chemistry and
Chemical Engineering, Nanjing University, Nanjing, P. R. China, and School of
Biochemical and Environmental Engineering, Nanjing Xiaozhuang College,
Nanjing 210017, P. R. China
chliuzp@163.com; heweij69@nju.edu.cn.
Received July 6, 2012
ABSTRACT
2þ
A new ratiometric fluorescent sensor for Cu , WLN, has been developed via integrating a 1,8-naphthalimide fluorophore with 8-aminoquinoline.
þ
2
WLN exhibits a highly selective ratiometric response to Cu over other transition metal ions in aqueous media. Moreover, its practical ratiometric
2
imaging ability for intracellular Cu has been confirmed in human breast adenocarcinoma cells (MCF-7 cells) using a confocal microscope.
þ
Development of fluorescent sensors for chemical species
ofbiological and environmental significanceiscurrentlyan
and trafficking was proposed to be associated with certain
3
diseases suchas Menkes syndrome, Wilson’s disease, and
4
1
attractive field for scientists. As the third most abundant
5
Alzheimer’s disease owing to the aberrant oxidative and
2þ
2þ
transition metal ions in the human body, Cu plays vital
roles in various biological processes, and its homeostasis is
critical for the metabolism and development of living
nitrosative stress induced by Cu . Moreover, long-term
2þ
exposure tohigh levels of Cu has been reported to induce
6
liver and kindey damage. According to the U.S. Environ-
2
2þ
organisms. The Cu disorder in its uptake, storage,
mental Protection Agency (EPA), the maximum accepta-
7
2þ
ble level of Cu in drinking water is ∼20 μM. Therefore,
†
there is considerable interest in developing specific fluores-
Liaocheng University.
Nanjing University
Nanjing Xiaozhuang College
2þ
‡
cent sensors for sensitive Cu detection, especially in
environmental and physiological conditions.
§
(
1) (a) Thomas, S. W., III; Joly, G. D.; Swager, T. M. Chem. Rev.
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1
0.1021/ol301849z r 2012 American Chemical Society
Published on Web 08/10/2012

2
þ
A number of fluorescent Cu sensors have been re-
ported, and some of them have been successfully applied
fluorophore 8-aminoquinoline (AQ) was incoporated into
2þ
ANP as a Cu ionophore via a piperazine linker. Since
there is a large overlap between the excitation spectra of
2
d,8
both in biological and in environmental samples. Most
of these reported sensors sense Cu via the mechanism of
Cu induced chemical reactions or Cu coordination.
Due to the paramagnetic nature of Cu , Cu coordina-
tion often results in fluorescence quenching, and very
few systems have been found to exhibit fluorescence
To overcome the quenching nature of
Cu , several turn-on chemodosimeters have been devel-
2
þ
10
ANP (340ꢀ420 nm) and AQ (300ꢀ410 nm), the same
2þ
2þ
excitation wavelength would excitate the emission of ANP
and AQ fluorophores simutaneously. It was envisioned that
2þ
2þ
2þ
Cu coordination to AQ might alter the emission ratio of
2þ
ANP and AQ, offering the Cu ratiometric sensing ability.
The synthesis of WLN is depicted in Scheme 1. WLN was
synthesized through a three-step procedure from the start-
ing materials 4-bromo-1,8-naphthalimide and AQ. WLN
8
a,b
enhancement.
2þ
2þ
oped via the specific Cu -induced reaction to form a
8
cꢀh
2þ
1
13
fluorophore.
For example, Cu -induced opening of
was well characterized by H, C NMR, ESI-MS, and
elemental analysis (see Supporting Information).
As shown in Figure 1, free WLN in HEPES buffer
the spirolactam of xanthenes and related derivatives have
beenwell developed for the construction ofturn-on sensors
for Cu . However, most of these fluoresent che-
modosimeters displayed a slower and irreversible response to
Cu when compared with those from the Cu coordina-
tion mechanism. This drawback limited the application of
these chemodosimeters especially in the real-time detection
2
þ 8h
(50 mM HEPES; ethanol/H O = 35:65, v/v; pH 7.2)
2
shows two absorption bands centered at 252 nm (band
4
2þ
2þ
ꢀ1
ꢀ1
A, ε = 2.3 ꢁ 10 M cm ) and 403nm (band B, ε= 1.0 ꢁ
4
10 M cm ), which can be assigned respectively as a
ꢀ1 ꢀ1
2þ
πꢀπ* transition and an ICT band. Upon Cu addition
(0ꢀ1.5 equiv), band A increased gradually, while broad
band B underwent a slight decrease and minor hyposo-
chromic shift, which can be ascribed to the decrease of
electron-donating ability induced by Cu coordination.
The linear increase of absorbance at 252 nm with
2þ
of Cu flunctuation in biological samples, and reversible
2þ
2
þ
fluorescent Cu sensor functioning directly via Cu
coordiantion is more appealing. However, quantitative
2þ
2þ
Cu detection in living systems requires a sensor display-
2þ
ing a ratiometric response for Cu to reduce the inter-
ference ascribed to deviations in detecting parameters and
microenvironments. Different approaches have been pro-
2þ
2þ
[Cu ]total up to a molar ratio ([Cu ]total/[WLN]) of 1:1
2þ
and the stable spectrum at even higher [Cu ]total implied a
2þ
2þ
posed to realize ratiometric Cu sensing and overcome
1:1 Cu binding stoichiometry of WLN.
2þ
the emission quenching nature of Cu , and a few ratio-
8iꢀp
2þ
metric Cu sensors have been reported.
diversified and complicated biological systems demand
However,
2þ
ratiometric Cu sensors of differnt natures to satisfy
Cu imaging in differnt microenvironments, and explor-
ing a new rationale to construct ratiometric Cu fluor-
escent sensors of different properties for quantitative Cu
detection is of great significance and challenging. In this
communication, we describe a new ratiometric fluorescent
Cu sensor, WLN, which was constructed by integrating
a 4-amino-1,8-naphthalimide (ANP) fluorophore with an
2þ
2þ
2þ
2þ
8
-aminoquinoline (AQ) fluorophore.
Scheme 1. Synthesis of WLN
Figure 1. Absorption spectra of WLN (10 μM) in HEPES buffer
(
50 mM, pH 7.2) obtained by adding aliquots of 25 μL of CuCl
1.2 mM) solution. Inset, the titration profile based on the
2
(
absorbance at 252 nm.
When excited at its excitation maximum of 395 nm,
WLN showed two characteristic fluorescence bands cen-
tered at 435 and 526 nm, which can be assigned to the
2þ
emission band of AQ and ANP, respectively. Cu titra-
tion demonstrated a distinct emission decrease of the band
centered at 526 nm, while the emission band of 435 nm
remained almost intact. The emission ratio at 435 and
In WLN, ANP was adopted as a fluorophore due to its
long emission wavelength, large Stokes shift, and pH
9
innertness under near-neutral conditions. The second
2þ
526 nm (F435/F526) increased linearly with [Cu ]total from
2þ
0.15 to 0.41 until the [Cu
]
total/[WLN] ratio reached 1:1.
Org. Lett., Vol. 14, No. 17, 2012
4379

After that, the emission spectrum of WLN became stable.
2þ
The Cu titration profile according to F435/F526 is con-
2þ
sistent with a 1:1 Cu binding stoichiometry disclosed by
UVꢀvis titration (Figure 2). The association constant was
4
ꢀ1
calculated to be 2.9 ꢁ 10 M according to the fluores-
cence titration profile (Figure S4).
The sensing selectivity of WLN toward Cu was eval-
2þ
uated by adding 1 equiv of various metal ions including
2þ
2
þ
2þ
2þ
2þ
2þ
2þ
þ
2þ
2þ
Hg , Cd , Pd , Cu , Co , Ag , Ni , Mn , Fe ,
þ
þ
2þ
Na , K , Ca , and Mg , respectively. As shown in
Figure 3, the addition of other metal cations did not
distinctly alter the emission ratio (F435/F526) of WLN
2
þ
except for the addition of Cu . Moreover, the ratiometric
sensing behavior of WLN to Cu experienced no inter-
ference by the presence of other metal ions.
2þ
Figure 3. Emission ratio at 435 and 526 nm (F435/F526) of WLN
10 μM) in HEPES buffer (50 mM, pH 7.2) induced by different
(
metal cations. Black bars represent the F435/F526 ratio of free
2
þ
þ
2þ
2þ
2þ
þ
2þ
2þ
sensor or in the presence of 1 equiv of Cu , Hg , Zn , Fe
Co , Ag , Ni , Pb , Cd , Mn , Na , Mg , K , Ca
,
.
2
þ
þ
2þ
2þ
2þ
2þ
Red bars, the F435/F526 ratio of WLN determined after the
addition of 1 equiv of indicated metal ions followed by addition
2þ
of 1 equiv of Cu . λ , 395 nm.
ex
and ratiometric images were obtained by mediating the
image from the green channel with the corresponding
one from the red channel. The bright fluorescence inside
the cells shown in both green and red channel images
indicated that WLN can be loaded into cells in 1 h,
displaying the fine membrane permeability of WLN. The
ratiometric imaging of cells loaded with WLN showed
very low levels of the background intracellular emission
Figure 2. Emission spectra of WLN (10 μM) in HEPES buffer
(
50 mM, pH 7.2) obtained by adding aliquots of 25 μL of CuCl
1.2 mM) solution. Inset, the titration profile based on the emission
2
2
þ
ratio, indicating the low Cu level inside MCF-7 cells
(
incubation with CuCl solution, an intensive blue to
(
ratio at 435 and 526 nm, F435/F526. Excitation was at 395 nm.
2
Figure 4a). When exogenous Cu was introduced via
þ
2
green color change was observed inside the cell, display-
2þ
The practical ratiometric imaging application of WLN
ing an enhanced intracellular Cu level (Figure 4b).
Treatment with the metal ion chelator TPEN for 1 min at
2þ
to track Cu levels was investigated in MCF-7 cells
stained by WLN via a dual emission imaging mode. There-
fore, two series of confocal fluorescence images were
obtained respectively from the green channel of band path
2
5 °C reduced the emission ratio enhancement distinctly
Figure 4d), implying WLN can monitor intracelluar
(
Cu flunctuation reversibly.
In summary, we have developed a new ratiometric
2þ
4
20ꢀ470 nm and red channel of band path 480ꢀ580 nm,
2
Cu sensor (WLN) via a fluorophore hybridization
þ
2
approach. WLN showed a specific Cu -induced devia-
þ
(
8) (a) Jung, H. S.; Park, M.; Han, D. Y.; Kim, E.; Lee, C.; Ham, S.;
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tion in the ratio of its two emission bands due to the
2þ
´
´
different quenching effects of Cu on its two consti-
tuent fluorophores. The specific ratiometric sensing
2
2
ability for Cu implied WLN as a potential imaging
þ
Mo, T.; Li, K.; Liu, F. Anal. Chem. 2005, 77, 7294. (e) Kim, M. H.; Jang,
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2
þ
candidate for intracelluar Cu ratiometric imaging.
Indeed, the imaging experiment clearly confirmed the
(
1
2
2þ
ratiometric imaging ability of WLN to monitor Cu
levels in living cells.
3029. (l) Yang, H.; Liu, Z.; Zhou, Z.; Shi, E.; Li, F. Tetrahedron Lett.
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Org. Lett., Vol. 14, No. 17, 2012

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Figure 4. Confocal fluorescence images of intracellular Cu in MCF-7 cells with WLN-staining. MCF-7 cells incubated with WLN
(
10 μM) at 25 °C for 1 h (a, b, c, d). The stained cells were exposed to 200 μM CuCl solution at 37 °C for 20 h, followed by washing with
2
WLN solution (e, f, g, h); the cells in (e) were further treated by TPEN solution (100 μM, 30 min, i, j, k, l). (a, e, i) Bright-field
transmission images. (b, f, j) Fluorescence images obtained according to the emission collected by the green channel (band path
4
20ꢀ470 nm). (c, g, k) Fluorescence images obtained from the red channel (band path 480ꢀ580 nm). (d, h, l) Ratiometric images
generated from (b, f, j) and (c, g, k). λex, 405 nm; bar = 20 μM.
Acknowledgment. We thank the Natural Science Foun-
dation of Shandong Province (ZR2011BQ010) and the
Liaocheng University Funds for Young Scientists (31805)
for financial support. W.H. is thankful for support from
the National Basic Research Program of China (No.
Supporting Information Available. Synthesis of WLN;
13
H, C NMR and ESI-MS spectra of WLN. This
material is available free of charge via the Internet at
http://pubs.acs.org.
1
2
011CB935800).
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 17, 2012
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