A selective 'Off-On' fluorescent sensor for Zn2+ based on hydrazone-pyrene derivative and its application for imaging of intracellular Zn2+

Article abstract of DOI:10.1016/j.bmcl.2009.11.028

A simple and effective fluorescent sensor based on hydrazone-pyrene has been synthesized. This probe displays a highly selective fluorescent enhancement with Zn²⁺, and application of this probe to detect the intrinsic Zn²⁺ ions present in pancreatic β-cells was successfully demonstrated.

Full text of DOI:10.1016/j.bmcl.2009.11.028

Bioorganic & Medicinal Chemistry Letters 20 (2010) 125–128
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A selective ‘Off–On’ fluorescent sensor for Zn2+ based on hydrazone–pyrene
derivative and its application for imaging of intracellular Zn2+
Ying Zhou a, Ha Na Kim a, Juyoung Yoon a,b,
*
a Department of Chemistry and Nano Science Ewha Womans University, Seoul 120-750, Republic of Korea
b Department of Bioinspired Science, Ewha Womans University, Seoul 120-750, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A simple and effective fluorescent sensor based on hydrazone–pyrene has been synthesized. This probe
displays a highly selective fluorescent enhancement with Zn2+, and application of this probe to detect the
intrinsic Zn2+ ions present in pancreatic b-cells was successfully demonstrated.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 8 October 2009
Revised 3 November 2009
Accepted 6 November 2009
Available online 13 November 2009
Keywords:
Fluorescent chemosensor
Zinc sensor
Fluorescent probe
Pyrene
Fluorescent sensors are powerful tools to monitor ions because
of the simplicity and high sensitivity of fluorescence.1 In particular,
the development of a fluorescent probe for zinc ion in the presence
of a variety of other metal ions has received great attention.2,3 Zn2+
is involved in a variety of physiological and pathological processes,
such as Alzheimer’s disease, epilepsy, ischemic stroke, and infantile
diarrhea.4 It is also reported that zinc ion is a potent killer of neu-
rons via oxidative stress.5 In particular, compared to other tissues,
Zn2+ ions present in pancreatic b-cells and can be used as a poten-
tial real time sensor to monitor the intrinsic Zn2+ level in pancre-
atic b-cells.
As shown in Scheme 1, intermediate 4 was synthesized from 1-
bromopyrene in three steps by modifying the reported procedure9
with an improved yield of 45%. Compound 4 was then reacted with
hydrazine to give the pyrene–hydrazone derivative 110 in 46 %
yield. The detailed experimental procedures and 1H NMR, 13C
NMR spectra are explained in Supplementary data.
pancreatic islets contains relatively high concentrations of Zn2+
,
which play a critical role in insulin biosynthesis, storage and secre-
tion.6 A decrease in the concentration of Zn2+ can cause a reduction
of the ability of the islet cells to produce and secrete insulin.7
Accordingly, development of Zn2+ selective fluorescent sensors
and convenient methods to detect intracellular Zn2+ ion are cer-
tainly important issues in recent years. The total concentration of
Zn2+ in different cells varies from the nanomolar range up to about
0.3 mM,8 which means that optimized chemical probes are re-
quired to monitor zinc concentration ranging from nanomolar to
micromolar. For example, most of the fluorescence-based probes
for Zn2+ suffered limitations due to tight binding affinity or lack
of sufficient selectivity to detect intrinsic levels of Zn2+ in pancre-
atic islets.
To obtain an insight into the sensing properties of 1 towards
metal ions, the emission changes were examined with different
ions such as Ag+, Ca2+, Cd2+, Cu2+, Fe3+, Hg2+, Mg2+, Pb2+ and Zn2+
in CH3CN–HEPES buffer (0.02 M, pH 7.4) (4:6, v/v). Fluorescence
spectra were obtained by exciting probe 1 at 450 nm. As shown
in Figure 1, probe 1 exhibited a selective fluorescence enhance-
ment only with Zn2+ with maximum emission at 527 nm (Fig. 1).
In contrast, other metal ions did not induce significant fluorescence
enhancement of 1. Probe 1 displayed a remarkable fluorescence
enhancement of 12-fold upon the addition of Zn2+ (Fig. 2). A Job
plot analysis confirmed 1:1 stoichiometry between 1 and Zn2+
(Fig. 2). From the fluorescence titration experiments as shown in
Figure 2, the association constant was calculated as 2.43 ꢀ 104 M
Herein, we report a relatively simple fluorescent sensor 1,
which displayed a selective fluorescence enhancement with Zn2+
among various metal ions examined at pH 7.4. Furthermore, we
successfully demonstrated that probe 1 can detect the intrinsic
.
ꢁ1 11 The partial 1H NMR spectra of 1 upon the addition of various
amounts of Zn2+ are illustrated in the supporting information
(Fig. S1). The OH proton and NH2 protons became broad at room
temperature and imine C–H peak as well as pyrene peaks move
to upfield region as shown in Figure S1, which support two nitro-
gens on hydrazone moiety as well as phenolic oxygen may partic-
* Corresponding author. Tel.: +82 2 3277 2400; fax: +82 2 3277 3419.
E-mail address: jyoon@ewha.ac.kr (J. Yoon).
ipate in binding with Zn2+
.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.028

126
Y. Zhou et al. / Bioorg. Med. Chem. Lett. 20 (2010) 125–128
O
C
H
OCH3
Br
OCH3
i) n-butyllithium,0 ºC,
tetramethyl-
ethylenediamine, 4 h
Na, CH3OH,
CuI, reflux, 36 h
ii) DMF, 22 h
3
2
O
C
N NH2
OH
H
HC
OH
AlCl3, CH2Cl2,4 h
NH2NH2, ethanol,
reflux, 1 h
4
1
Scheme 1. Synthesis of fluorescent probe 1.
Zn2+
300
250
200
150
100
50
250
200
150
100
50
NH2
N
Ca2+, Mg2+
,
Zn2+
Hg2+, Cd2+, Ag+
1 only
Pb2+, Cu2+, Fe
3+
O
H
1 only
0
0
500
550
600
650
500
550
600
650
Wavelength (nm)
Wavelength
Figure 3. Effect of calcium ion on the binding of zinc ion to probe 1. Zinc ions (0–
50 equiv) were added to solution of 1 (20 M) containing a large excess of calcium
ions (1000 equiv) in CH3CN–HEPES buffer (0.02 M, pH 7.4) (4:6, v/v).
Figure 1. Fluorescent spectra of 1 (20
l
M) in CH3CN–HEPES buffer (0.02 M, pH 7.4)
l
(4:6, v/v) with 20 equiv of various metal ions, such as Ag+, Ca2+, Cd2+, Cu2+, Fe3+
,
Hg2+, Mg2+, Pb2+ and Zn2+. Excitation wavelength was 450 nm.
cent intensity of 1 with Zn2+ was observed in the presence of large
As shown in Figure S5, there was not any significant change in
the absorption spectra upon the addition of different amounts of
Zn2+. The weak fluorescence of probe 1 in the absence of Zn2+
and the absence of any significant change in absorption spectra
upon the addition of Zn2+ indicate that large fluorescence enhance-
ment with Zn2+ can be attributed to the blocking of photo-induced
electron transfer (PET) process from nitrogen in hydrazone moiety
to pyrene.12 As shown in Figure 3, this probe did not respond to
Ca2+, a problem associated with other Zn2+ sensors. Similar fluores-
excess Ca2+
.
In vitro studies demonstrated the ability of 1 to detect Zn2+ with
excellent selectivity. To examine whether this ability is preserved
for biological application, HaCaT cells were first used to monitor
intracellular Zn2+ ions. Human keratinocyte cell line, HaCaT were
cultured in RPMI 1640 medium supplemented with 10% fetal bo-
vine serum, 2 mM L-glutamine, 100 units/ml penicillin, 100 mg/
ml streptomycin. Cells were incubated with Zn2+ (10
lM) for
80
70
60
50
40
30
20
10
0
300
50 eq.
250
200
150
ref.
100
50
0
500
550
600
650
0.0
0.2
0.4
0.6
0.8
1.0
Zn2+/(Zn2++1):X
Wavelength (nm)
Figure 2. Left: Fluorescent emission spectra of 1 (20 l
M) in the presence of different concentrations of Zn2+ in CH3CN–HEPES buffer (0.02 M, pH 7.4) (4:6, v/v). Excitation
wavelength was 450 nm. Inset: the fluorescent intensity as a function of Zn2+ concentration. Right: the Job Plot using fluorescent intensity at 527 nm of 1 and Zn2+in CH3CN–
HEPES buffer (0.02 M, pH 7.4) (4:6, v/v). Fluorescent intensity was measured at 527 nm and total concentration of [1] and [Zn2+] was 100
lM.

Y. Zhou et al. / Bioorg. Med. Chem. Lett. 20 (2010) 125–128
127
Figure 5. (a) Fluorescence images of HaCaT cell treated with Zn2+ (10
probe 1 (20
and probe 1 (20
Fluorescence images of pancreatic b-cells with Zn2+ (5
l
M) and
l
Figure 4. (a) Bright field images of HaCaT cells treated with Zn2+ (10
1 (20 M). (b) Fluorescence images of (a). (c) Fluorescence images of pancreatic b-
cells without probe 1. (d) Fluorescence images of pancreatic b-cells with probe 1
(20 M).
lM) and probe
l
M). (b) Fluorescence images of HaCaT cell treated with Zn2+ (10
M)
M TPEN. (c)
M) and probe 1 (20 M). (d)
lM).
l
lM) and subsequent treatment of the cells with 25 l
l
l
l
Fluorescence image of (c) incubated with TPEN (50
90 min at 37 °C and washed three times with PBS to remove the re-
mained Zn2+ ion. As shown in Figure 4b, cells incubated with 5
Zn2+ and 1 (20
M) displayed clear green fluorescence.
These results encouraged us to use our probe 1 to detect biolog-
ically relevant intracellular Zn2+ ions. For the application of detect-
ing intrinsic Zn2+ ion in live cells, pancreatic b-cells (Rin-m cell
line), which contains intrinsic Zn2+ ions for storage of insulin,6
were used. Pancreatic b-cell line (Rin-m) were cultured in
Acknowledgments
lM
l
This work was supported by grants from the National Research
Foundation of Korea (NRF) funded by the Ministry of Education,
Science and Technology (20090083065, 20090063001) and WCU
program (R32-2008-000-10180-0).
Supplementary data
RPMI1640 medium (containing 2 mM L-glutamine, 10 mM HEPES,
1 mM sodium pyruvate, 4500 mg/l glucose, and 1500 mg/l sodium
bicarbonate) supplemented with 10% fetal bovine serum at 37 °C in
a humidified incubator. The detailed procedures are explained in
the supporting information. The results of fluorescence microscopy
experiments demonstrate that intracellular Zn2+ ions in pancreatic
b-cells can be fluorescently detected by using probe 1 (Fig. 4d). In
addition, even stronger green fluorescence was detected in pres-
ence of exogenous Zn2+ with 1 as shown in Figure 4c, which means
this probe can be used to detect increase or decrease of intracellu-
lar Zn2+ ion levels in the live cells.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.11.028.
References and notes
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a membrane-permeable zinc chelator (N,N,N0,N0-tetrakis(2-pyridyl-
methyl) ethylenediamine, TPEN), which is known to decrease the
intracellular level of zinc.13 As shown in Figure 5b, the TPEN trea-
ted cells displayed very weak fluorescence, indicating that green
fluorescence is caused by response of 1 to intracellular zinc ions.
Fluorescence images of pancreatic b-cells with additional exoge-
nous Zn2+ (5
The results reveal that cells treated with both 1 (20
l
M) and probe 1 (20
l
M) are explained in Figure 5c.
lM) and exter-
nal Zn2+ ions (5
lM) have a brighter fluorescence response as com-
pared to the case without adding external Zn2+ ions. The TPEN
treatment also induced the substantial decrease of green fluores-
cence (Fig. 5d).
In conclusion, we successfully demonstrated that our relatively
simple probe 1 a highly selective fluorescence enhancement with
Zn2+ at pH 7.4. In addition, this probe was successfully applied
for imaging intracellular Zn2+ ions. Most importantly, we demon-
strated this probe can monitor the level of intrinsic Zn2+ in pancre-
atic b-cells.

128
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(FAB):
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,
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(0.1 g, 0.4 mmol) and 50 of hydrazine hydrate in 25 ml ethanol was
lL
heated to reflux for 1 h under a nitrogen atmosphere. After the ethanol was
evaporated under reduced pressure, the residue was purified by silica gel
column chromatography using dichloromethane as eluent to obtain orange