A selective and sensitive fluorescence probe for imaging endogenous zinc in living cells

Article abstract of DOI:10.1016/j.tet.2012.11.007

A carboxamidoquinoline-based fluorescent Zn²⁺ probe 1 containing N/S/S heteroatoms as a receptor was designed and readily synthesized, which was featured by the Zn²⁺-induced red-shift of emission (45 nm) based on internal charge transfer (ICT) in an aqueous HEPES buffer (pH=7.4). Moreover, spectroscopic studies indicated that the composition of the complex 1-Zn ²⁺ was 1:1, which was also confirmed by X-ray crystallography. In addition, ¹H NMR titration experiment suggested that probe 1 transformed from the amide tautomer to imidic acid tautomer after binding with Zn²⁺. The binding of zinc with 1 was easily reversed by addition of N,N,N′,N′-tetrakis(2-picolyl)ethylenediamine (TPEN) or EDTA. Furthermore, probe 1 was successfully applied to image both exogenous and endogenous zinc in living HeLa cells.

Full text of DOI:10.1016/j.tet.2012.11.007

Accepted Manuscript
A selective and sensitive fluorescence probe for imaging endogenous zinc in living
cells
Liangwei Zhang, Dongzhu Duan, Xuemei Cui, Jinyu Sun, Jianguo Fang
PII:
S0040-4020(12)01667-5
10.1016/j.tet.2012.11.007
TET 23689
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 14 August 2012
Revised Date: 17 October 2012
Accepted Date: 2 November 2012
Please cite this article as: Zhang L, Duan D, Cui X, Sun J, Fang J, A selective and sensitive
fluorescence probe for imaging endogenous zinc in living cells, Tetrahedron (2012), doi: 10.1016/
j.tet.2012.11.007.
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ACCEPTED MANUSCRIPT
A selective and sensitive fluorescence probe for imaging endogenous
zinc in living cells
Liangwei Zhang, Dongzhu Duan, Xuemei Cui, Jinyu Sun and Jianguo Fang*
State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou
University, Lanzhou 730000, P. R. China.
Fax: (+86) 931-8915557; Tel: (+86) 931-8912500; E-mail: fangjg@lzu.edu.cn.
Abstract A carboxamidoquinoline-based fluorescent Zn2+ probe 1 containing N/S/S heteroatoms as a receptor
was designed and readily synthesized, which was featured by the Zn2+-induced red-shift of emission (45 nm)
based on internal charge transfer (ICT) in an aqueous HEPES buffer (pH=7.4). Moreover, spectroscopic studies
indicated that the composition of the complex 1-Zn2+ was 1:1, which was also confirmed by X-ray
1
crystallography. In addition, H NMR titration experiment suggested that probe 1 transformed from the amide
tautomer to imidic acid tautomer after binding with Zn2+. The binding of zinc with 1 was easily reversed by
addition of N, N, N’, N’-tetrakis(2-picolyl)ethylenediamine (TPEN) or EDTA. Furthermore, probe 1 was
successfully applied to image both exogenous and endogenous zinc in living HeLa cells.
Keywords: Zinc; Fluorescent probe; Internal charge transfer; Living cell imaging
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Graphic abstract
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Introduction
Zinc is the second most abundant d-block metal ion in human body,1 and plays a vital role in various
biological processes such as brain function, gene expression, and mammalian reproduction.2 Disturbance of the
zinc homeostasis in living organisms is critically related to a series of disease.3 Therefore, the detection of zinc
has attracted increasing interests. Numerous preceding works have demonstrated that fluorescent sensor is a
powerful tool to detect metal ions,4 anions and small molecules5 in vitro and in vivo due to the simplicity and
high sensitivity. Fluorescence imaging of zinc is especially suitable because this ion has no facile spectroscopic
or magnetic signature.6a The past years have witnessed intense research devoted to the development of novel
fluorescent probes for recognizing zinc6 and biological applications.7 Among the reported zinc sensors,
quinoline and its derivatives, due to their unique chemical and physical properties, have aroused enormous
attentions.8, 13b However, most of them suffer from multiple drawbacks, such as selectivity, sensitivity, water
solubility and cell membrane permeability,8b, c, f, g, 10b which prevent them to satisfy various pratical needs. And
thus, it still remains a challenge to develop novel fluorescent probes to overcome those disadvantages.
8-Aminoquinoline was commonly employed as fluorophore for the metal ion sensors based on internal
charge transfer (ICT), which is one of most important signaling mechanism in designing metal ion sensors. In
these sense, the fluorescent probe 1 based on carboxamidoquinoline was designed, which bears N/S/S
heteroatoms as a receptor. The spectroscopic studies demonstrated that it is a reversible probe with broad pH
stability, highly sensitivity and selectivity for Zn2+. The binding mode was determined by X-ray
1
crystallography and H NMR titration experiments. Binding of Zn2+ with probe 1 induces deprotonation of
carboxamido group and the breakage of intramolecular hydrogen bond of 8-aminoquinoline, which in turn
enhance the ICT process as described in the previous report.8a As a consequence, the red-shift and enhancement
of fluorescence emission were observed. The probe was also successfully used in imaging both exogenous and
endogenous zinc in living HeLa cells, demonstrating its pratical applications.
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Scheme 1 Synthesis of 1
Results and discussion
Synthesis
The synthesis of sensor 1 is depicted in Scheme 1 according to the reported procedures with some
modifications.8a, 9 The probe 1, obtained in 2 steps with 62 % overall yield starting from readily available
8-aminoquinoline, was fully characterized by 1H NMR, 13C NMR and MS spectra (Fig. S1- 10).
UV-Vis absorption and emission spectra of probe 1
The spectral properties of probe 1 were examined by UV-Vis absorbance and fluorescence titrations. Two
bands were both red-shifted with addition of of Zn2+ (0-1.0 equiv) in the absorption spectra, accompanying
three isosbestic points at 245, 279 and 327 nm (Fig. 1). Fluorescence spectra of Zn2+ titration were shown in
Fig. 2. Probe 1 has a very weak fluorescence emission at about 445 nm with excitation at 345 nm. Upon
addition of Zn2+, it displays a 45 nm red-shift of emission to 490 nm and a remarkable enhancement of
fluorescence intensity, which is saturated by 1.5 eq. of Zn2+with a ~ 21-fold increase. It is worth noting that this
fluorescence titration experiment was performed in a pure aqueous solution, which is different from other
probes determined in organic solvent mixed solutions,8 and has potential application in biological imaging (vide
infra). The solubility of the probe 1 in HEPES buffer was estimated to be more than 500 µM (containing 0.5 %
organic solvent) by serial dilution from a concentrated stock solution (100 mM in acetonitrile). The quick
changes of the emission wavelength as well as intensity upon znic addition can be explained by
carboxam-idoquinoline’s deprotonation process and enhancement of internal charge transfer.
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Fig. 1 UV-Vis absorption spectra of probe 1 in HEPES buffer (10 mM, pH= 7.4) in the presence of different
concentrations of Zn2+. [1] = 10 ꢀM, [Zn2+] = 0-10 ꢀM.
Fig. 2 Fluorescence spectra of 1 (10 ꢀM) in the presence of an increasing Zn2+ concentration (0-15ꢀM) in
HEPES buffer (10 mM, pH=7.4) excited at 345 nm. Inset: fluorescence changes of 1 (10 ꢀM) in absence and
presence of Zn2+ (10 ꢀM).
Job’s plot and 1H NMR titration
To determin the binding stoichiometry of 1-Zn2+ complex, we adapted the Job’s plot based on the
fluorescence titration. The maximal fluorescence intensity appears at about 0.5 mole fraction (Fig. 3), indicating
that 1-Zn2+ complex has 1:1 stoichiometry. The most convincing evidence was provided by the crystal structure
of 1 complexed with zinc (Fig. 4a), which can better explain the fluorescence sensing mechanism. The crystal
structure unambiguously demonstrates that the probe 1, acting as a quinquidentate ligand via its N and S atoms,
forms a 1:1 complex with Zn2+. The stronger coordination of Cu2+ by 1 (Fig. 4b), reflected by the shorter bond
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lengths in the complex (Tables S1 and S2), supported that Cu2+ is a potent competitive quencher against Zn2+
1
(vide infra). The bond formation between 1 and Zn2+ is further supported by H NMR titration. In Fig. 5, as
addition of 1 eq. Zn(ClO4)2, the NH proton shifted to upfield from 11.34 ppm and diminished gradually, and a
new peak formed at 10.01 ppm, which results from the existence of the OH proton. This suggested that probe 1
complexed with Zn2+ in the imidic acid tautomer in CH3CN predominantly (scheme S1). The probe 1 is also a
senetive sensor for other zinc metal sources demonstrated in Fig. 6.
Fig. 3 Job’s plot of 1-Zn2+ in 10 mM HEPES buffer (pH=7.4). The total molar concentration of 1 and Zn2+ is
10µM.
-
Fig. 4 (a) The structure of 1-Zn2+ complex, showing thermal ellipsoid at the 30% probability level. ClO4 and H
atoms are omitted for clarity. (b) The crystal structure of 1-Cu2+ complex.
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Fig. 5 1H NMR spectra of probe 1 in the presence of a different amount of Zn2+ in CD3CN-CDCl3 (3: 2, v/v).
Fig. 6 Fluorescence responses of probe 1 (10 µM) to zinc metal sources (10 µM) in HEPES buffer (10 mM,
pH=7.4) at 490 nm. The response is normalized with respect to the background fluorescence of the free probe 1
(F0).
Determination of dissociation constant and the detection limit
Determination of the dissociation constant, Kd, of 1-zinc complex was performed by the equation (1)
proposed by Kowalczy et al.10a from direct fluorometric titration (Fig. 7a). In the Equation (1), F stands for the
fluorescence intensities at [X] (X = Zn2+), Fmin and Fmax are the emission intensities in the absence of, presence
of saturated X at 490 nm, respectively. n is the number of X bound per ligand. Value of Kd between the probe 1
(1)
(2)
and Zn2+ was obtained by nonlinear fitting of the titration curve assuming 1 : 1 stoichiometry (Fig. 7b). The
detection limit was calculated with the equation (2).10b In the equation (2), σ is the standard deviation of blank
measurement, which can be acquired by ten times measurement of emission spectrum of probe 1. k is the slope
from plotting the fluorescence intensity versus Zn2+ concentration (Fig. 7C). In Fig. 7C, it shows a good linear
relationship between the fluorescence intensity and the Zn2+ concentration in the range of 0 to 4 µM (R2=
0.9944). The dissociation constants Kd and the detection limit, calculated from the fluorescence titration curves,
are 6.54 × 10-7 M and 8.14 ×10-8 M, respectively, which indicated that probe 1 has moderate affinity and high
sensitivity for Zn2+.
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Fig. 7 (a) Emission spectrum of 5 µM probe 1 in presence of various concentrations of Zn2+ from 0 to 5 µM; (b)
Nonlinear fitting curve and (c) linear fitting curve of the fluorescence intensity versus [Zn2+] at 490 nm. λex=
345 nm.
Fig. 8 Reversibility of probe 1 binding to Zn2+ by addition of TPEN and EDTA in 10 mM HEPES buffer
(pH=7.40). λex= 345 nm.
Reversibility of probe 1
The response of probe 1 to Zn2+ can be reversed. N,N,N’,N’-tetrakis(2-picolyl)ethylenediamine) (TPEN)
and EDTA are well-known metal ion chelator used in reversibility studies.11 The fluorescence emission restored
to initial intensity of probe 1 (10 ꢀM) by adding either TPEN (10 ꢀM) or EDTA (40 ꢀM) after probe 1 was
saturated with Zn2+ (Fig. 8).
Metal ion selectivity and competition experiments
The selectivity of probe 1 toward different metal ions was investigated as shown in Fig. 9a. There is no
significant influence by addition of 10 eq. of KClO4, NaCl, Al(ClO4)3, CaCl2, MgCl2 or 1 eq. of Zn(ClO4)2,
Cd(ClO4)2, Fe(ClO4)3, Ni(ClO4)2, Co(ClO4)2, Mn(ClO4)2, FeSO4, CrCl3, Cu(ClO4)2, HgCl2, AgClO4. However,
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addition of Cd(ClO4)2 caused a red-shift of emission from 445 to 500 nm and slight enhancement of the
intensity, as zinc and cadmium are in the same group of the periodic table and have similar properties. Probe 1
can distinguish Zn2+ from Cd2+ more than ~ 4.3-fold (Fig. 9a). Next, we determined if probe 1 can recognize
zinc when it coexists with other metal ions. As shown in Fig. 9b, Cr3+, Cu2+, Hg2+, and Ag+ quench the
fluorescene signal dose-dependently, while other ions have little interference (Fig. 9c). This is probably due to
the displacement of Zn2+ by Cr3+, Cu2+, Hg2+, Ag+ since sulfur atom has also high affinity for them. The further
evidence was provided by comparison the bond lengths in the crystal structure 1-Cu2+ and 1-Zn2+ complex.
(Fig. 4b, Tables S1 and S2). Cd2+ and Cr3+ are ultratrace element, and have extremely low concentration. Hg2+
and Ag+ are not essential element, and rarely found in human body. Although copper belongs to essential
element, its concentration usually more than 10 times lower than that of zinc in vivo,12 and its influence could
be neglected (Fig. S11). Taken together, the interference caused by these ions in vitro should not prevent the
application of probe 1 for imaging zinc in vivo.
Fig. 9 (a) Fluorescence emission spectra of probe 1 in the presence of different metal ion. The following metal
ions (100 ꢀM) (3-7) were added: KCl (3), NaCl (4), Al(ClO4)3 (5), CaCl2 (6), and MgCl2 (7). Other metal ions
(2, 8-17) were added at 10 ꢀM: Zn(ClO4)2 (2), Fe(ClO4)3 (8), FeSO4 (9), Cd(ClO4)2 (10), Ni(ClO4)2 (11),
Co(ClO4)2 (12), Mn(ClO4)2 (13), CrCl3 (14), Cu(ClO4)2 (15), HgCl2 (16), and AgClO4 (17). (b) The fluorescence
spectra of probe 1 (10 µM) saturated with Zn2+ in presence of increasing concentrations of Cr3+, Cu2+, Hg2+, or
Ag+. (c) The fluorescence intensity of probe 1 at 490 nm with Mn+ (From 3 to 13: 100 ꢀM for KClO4 (3), NaCl
(4), Al(ClO4)3 (5), CaCl2 (6), MgCl2 (7), 10 ꢀM for Cd(ClO4)2 (8), Fe(ClO4)3 (9), Ni(ClO4)2 (10), Co(ClO4)2 (11),
Mn(ClO4)2 (12), and FeSO4 (13) followed by 10 ꢀM Zn2+. 1 and 2 represented free probe 1 and direct added
Zn(ClO4)2,. The response is normalized with respect to the background fluorescence of the free probe 1 (F0).
The effect of pH
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The effect of pH on the fluorescence signal of probe 1 in the absence or presence of Zn2+ was measured.
As shown in Fig. 10, the free ligand has no fluorescence signal within the pH range of 2-13. After addition of
Zn2+, the fluorescence signal increased gradually when pH > 4, and is stable from pH 6.8 to pH 11.0, which
covers the physiological pH range, indicating the potential application of the probe in biological systems.
Fig. 10 Emission of probe 1 and 1-Zn2+ complex (10 ꢀM) in aqueous solutions at different pH at 490 nm. λex=
345 nm
Fig. 11 Fluorescence images of HeLa cells. (a) Normal HeLa cells. (b) HeLa cells incubated with probe 1 (20
ꢀM) for 1 h. (c) HeLa cells incubated with probe 1 for 1 h, and then ZnCl2 (25 ꢀM) and pyrithione (5 ꢀM) for
0.5 h. (d) HeLa cells incubated with probe 1 for 1 h, and then ZnCl2 (25 ꢀM) and pyrithione (5 ꢀM) for 1 h. (e)
HeLa cells incubated with NEM (200 ꢀM). (f) HeLa cells incubated with probe 1 (20 ꢀM) and NEM (200 ꢀM)
for 1h.
Living cell imaging
The exciting in vitro results eventually prompt us to determine if probe 1 can be applied in imaging zinc in
living cells. The Zn2+ sensing and imaging were carried out in HeLa cells.13 HeLa cells showed no fluorescence,
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and very weak fluorescence treated with probe 1 only, probably caused by the endogenous zinc (Fig. 11a and
11b). As Zn2+ accumulation by treatment of ZnCl2 and sodium pyrithione, a zinc ionorphore,13b a significant
increasing green fluorescence in cells was observed (Fig. 11c and 11d). If the HeLa cells were simultaneously
treated with probe 1 and N-ethylmaleimide (NEM), a known alkylating reagent to release zinc in cells,13c
a
strong green fluorescence was also observed (Fig. 11e and 11f). The latter experiments reveal that probe 1 is
sensitive enough to view intracellular endogenous Zn2+. Bright field fluorescence images of HeLa cells were
illustrated in Fig. S12. These experiments unambiguously demonstrated that probe 1 is an effective probe in
imaging both exogenous and endogenous Zn2+ in living cells.
Zn2+ is an indispensable metal ion in cells, and plays multiple vital roles in various
physiological/pathophysiological processes. Although tremendous improvements have been achieved by
different groups,6, 7, 8 specific and sensitive detection of endogenous Zn2+ still remains a challenge. The probe 1
described in this paper shows improved water solubility, high sensitivity, good selectivity over a panel of
potential interfering ions, and broad pH stability. Furthermore, the moderate affinity and reversibility of 1 to
zinc indicates that the probe will not perturb the homeostasis of Zn2+ in normal cells when applied for in vivo
imaging. Indeed, the probe 1 appears no cytotoxicity when HeLa cells were treated with 20 µM of 1 for 24h
(data not shown). ZS5, a precedent zinc probe reported by Lippard et al.,8d was applied to detect endogenous
Zn2+ in living cells. However, the dissociation constant (Kd) of ZS5 is 3-times greater than that of 1 (1.5 µM vs
0.6 µM), indicating that the higher sensitivity of 1 could be obtained. Using thiol-alkylating agent NEM, we
successfully imaged the endogenous zinc in living HeLa cells, demonstrating the practical application of 1.
Conclusions
In conclusion, we have developed a reversible, highly sensitive, and readily synthesized fluorescent probe
1 with broad pH stability, water solubility, and cell membrane permeability. The probe displays good selectivity
and sensitivity for Zn2+ with a 45 nm red-shift and about ~ 21-fold enhancement of emission based on
carboxamidoquinoline’s deprotonation process and ICT mechanism. We also successfully applied the probe 1
in imaging intracellularly endogenous zinc in living HeLa cells.
Experimental section
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Materials and general methods
All reagents and other chemicals were obtained from commercial supplies and used without further
purification. Nuclear magnetic resonance spectra were recorded on Bruker 400 MHz or Varian 300M
instruments with TMS as an internal standard. Mass spectra were determined using Bruker Daltonics esquire
6000 mass spectrometer. Fluorescence spectra were performed with a Hitachi F-4500 fluorescence
spectrofluorometer at room temperature, except the influence of pH on the fluorescence of probe 1 were
obtained on a Shimadzu RF-5301 spectrophotometer. Absorption spectra were carried out on a Thermo
Scientific Multiskan GO. The pH measurement was made with a Mettler Toledo EL-20 meter.
The HEPES buffer (10mM, pH=7.4) were prepared using deionized water. Stock solutions (1 mM and 10
mM) of NaCl, CaCl2, KClO4, ZnSO4, Zn(NO3)2, Zn(CHCOO)2 in deionized water and the others MgCl2，
Al(ClO4)3, Zn(ClO4)2, Cd(ClO4)2, Fe(ClO4)3, Ni(ClO4)2, Co(ClO4)2, Mn(ClO4)2, FeSO4, CrCl3, Cu(ClO4)2,
HgCl2, AgClO4 in methanol were prepared. A stock solution of 1 (1 mM) was prepared in acetonitrile. For all
measurements, the zinc metal sources using Zn(ClO4)2, unless otherwise noted. The fluorescence spectra were
obtained by excitation at 345 nm in HEPES buffer (10mM, pH=7.4). Both the excitation and emission slit
widths were 5 nm, respectively.
Imaging in HeLa Cells
HeLa cells were cultured in Dulbecco’s Modified Eagle Medium (Sigma) supplemented with 10 % fetal
bovine serum (Sijiqing Biological Engineering Materials Co., Ltd., Hangzhou, China), 2 mM glutamine
(Beyotime, Haimen, China), penicillin (100 units/ml), streptomycin (100 mg/ml) (Beyotime ) and 5% CO2 at 37
°C. When cells reached ~50% confluence, probe 1 (20 ꢀM) was added, and the cells were incubated for another
1 h. The cells were rinsed with the medium for three times to remove the remaining probe 1. Then the cells
were further treated with ZnCl2 (25 ꢀM) plus sodium pyrithione (5 ꢀM), or N-ethylmaleimide (NEM) (200
ꢀM). The cells were washed three times with PBS and imaged by inverted fluorescent microscope (Leica DMI
4000 B).
Synthesis of 2
Compound 2 (2-chloro-N-(quinol-8-yl)acetamide) was synthesized according to the reported procedures.7a
Chloroacetyl chloride (461 mg, 2.4 mmol) was dissolved in chloroform (5 ml), then added dropwise to a cooled,
stirred solution of 8-aminoquinoline (288 mg, 2.0 mmol) and pyridine (222 mg, 2.8 mmol) in chloroform (10
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ml) within 1 h, after stirred 2 h at room temperature, the mixture was removed under reduced pressure to obtain
a white solid, which was purified by silica gel column chromatography using dichloromethane as eluent to
afford compound 2 (78%). 1H NMR (400 MHz, CDCl3) δ(ppm): 10.90 (s, 1H), 8.84 (t, J = 4 Hz, 1H), 8.75 (dd,
J = 5.6, 3.2 Hz, 1H), 8.16 (d, J = 8.2 Hz, 1H), 7.54 (dd, J = 8.3, 5.8 Hz, 2H), 7.46 (dd, J = 8.2, 4.2 Hz, 1H), 4.32
(s, 2H). 13C-NMR (CDCl3, 400 MHz) δ (ppm): 164.28, 148.59, 138.63, 136.18, 133.49, 127.07, 122.45, 121.72,
116.53, 43.31. ESI–MS: (m/z) 221.2 [M+H]+, calcd. for C19H27N3OS2=220.04.
Synthesis of 3
Compound 3 (bis[2-(ethylthio)ethyl]amine) was synthesized according to the reported procedures.9
Diethanolamine (20 g, 190 mmol)) were added dropwise to a stirred solution of thionyl chloride (55.6 ml, 768
mmol) in chloroform (45 ml) in 2.5 h. After addition, the reaction mixture was kept at room temperature for 1 h
and then heated under reflux for 1 h. After cooling the reaction mixture to room temperature, the precipitate was
filtered, washed with chloroform and dried to give bis(2-chloroethyl)amine hydrochloride. The hydrochloride
was used without any further treatment. Under an argon atmosphere, sodium (4.6 g, 200 mmol) was dissolved
in ethanol (200 ml), and ethanethiol (7.44 g, 120 mmol) was added to the solution. An ethanol solution (100 ml)
of bis(2-chloroethyl)amine hydrochloride (7.14 g, 40 mmol) was added dropwise with refluxing, and the
reaction mixture was refluxed for 2 h under nitrogen atmosphere. The residue obtained by solvent evaporation
was poured into water, and the product was extracted with chloroform. The solvent was evaporated result in the
crude product. 1H NMR (CDCl3, 400 MHz): δ (ppm): 2.80 (m, 4H), 2.66 (m, 4H), 2.50 (m, 4H), 2.10 (s, 1H),
1.22 (m, 6H). 13C-NMR (CDCl3, 400 MHz) δ (ppm): 48.24, 31.79, 25.80, 14.84. ESI–MS: (m/z): 194.2
[M+H]+, calcd. for C8H19NS2=193.1.
Synthesis of 1
For the preparation of 1, compound 2 (220 mg, 1mmol), N, N- diisopropylethylamine (1.29 g, 10 mmol) and
potassium iodide (50 mg) were added to acetonitrile (20 ml). The reaction mixture was kept at 40 ℃ for 0.5 h
under nitrogen atmosphere. Compound 3 (232 mg, 1.2 mmol) dispensed in acetonitrile was added dropwise to
the mixture, and refluxed with stirring for another 10 h. The mixture was evaporated to dryness, and the desired
product 1 was obtained as yellow oil in 79% yield by column chromatography with silica gel (eluting with
petroleum ether: ethyl acetate = 3:1). 1H NMR (400 MHz, CDCl3) δ (ppm): 11.37 (s, 1H), 8.84 (t, J = 4.0 Hz,
1H), 8.78 (dd, J = 6.7, 2.1 Hz, 1H), 8.14 (dd, J = 8.2, 1.3 Hz, 1H), 7.59 – 7.49 (m, 2H), 7.44 (dd, J = 8.3, 4.2 Hz,
1H), 3.42 (s, 2H), 2.92 (dd, J = 9.9, 5.3 Hz, 4H), 2.81 (dd, J = 10.0, 5.3 Hz, 4H), 2.51 (q, J = 7.4 Hz, 4H), 1.18
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(t, J = 7.4 Hz, 6H). 13C-NMR (400 MHz, CDCl3) δ (ppm): 169.80, 148.35, 138.93, 136.02, 134.18, 127.96,
127.14, 121.75, 121.47, 116.53, 59.82, 55.45, 29.38, 26.04, 14.72. ESI–MS: 378.3 [M+H]+; HRMS (ESI) m/z
calcd for C19H27N3OS2, calcd. 378.1596, found 378.1664.
X-ray Crystallography
Synthesis of 1-Zn2+ complex
Equal molar ratio of the probe 1 and Zn(ClO4)2•6H2O were mixed in methanol with stirring. Then the
precipitate was collected by filtration. The crystals were obtained suitable for X-ray structure determination by
slow diffusion of diethyl ether into the acetonitrile solution of the product in several days.
Synthesis of 1-Cu2+ complex
The crystal of C19H26ClCuN3O5S2 was obtained by the same method as that described for 1-Zn2+ complex.
Crystal data. C57H78Cl3N9O15S6Zn3, M = 1624.10, monoclinic, a = 24.92 (2) Å, b = 17.435 (14) Å, c = 15.816
(13) Å, β = 97.185(9)°, V = 6818 (10) Å3, Dc =1.582 Mg m-3, ꢀ = 1.42 mm-1, F(000) = 3360, T = 293 K, space
group P21/c, Z = 4, 2.00 ≤θ≤ 25.50°, 12687 reflections measured, 7434 independent reflections (Rint = 0.072).
The final GooF = 1.007, R1 = 0.0533, wR2 = 0.1223, R indices based on 12687 reflections and 844 refined
parameters, with I > 2σ ( I ).CCDC.870318.
Crystal data. C19H26ClCuN3O5S2, M = 539.54, monoclinic, a = 24.913 (13) Å, b = 17.481 (9) Å, c = 15.586 (8)
Å, β = 96.076(6)°, V = 6749 (6) Å3, Dc = 1.593 Mg m-3, ꢀ = 1.31 mm-1, F(000) = 3348, T = 296 K, space group
P21/c, Z = 12, 2.31 ≤θ≤ 25.50°, 12551 reflections measured, 7330 independent reflections (Rint = 0.077). The
final GooF = 1.051, R1 = 0.0500, wR2 = 0.0849, R indices based on 12551 reflections and 900 refined
parameters, with I > 2σ ( I ).CCDC.870319.
Acknowledgements
The research supported by the National Natural Science Found of China (21002047), the Ministry of
Education of China (20100211110027), Lanzhou University (the Fundamental Research Funds for the Central
Universities, lzujbky-2012-59), and the 111 project.
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Notes and references
†Copes of 1H NMR, 13C NMR spectra, ESI–MS, and HRMS of compounds 1, 2, 3 and other additional
spectroscopic data, X-ray crystallographic data and cell images are available as Electronic Supplementary
Information (ESI) at http://xxxxxx.
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