A novel calixsalen macrocycle: Metal sensing behavior for Zn2+ and intracellular imaging application

Article abstract of DOI:10.1016/j.tetlet.2011.12.005

The chiral [3+3] macrocycle 1 which displays a calixarene-like crystal structure has been synthesized. The UV/Vis and fluorescence spectral studies show that 1 and Zn²⁺ have a 1:3 complex stoichiometry. It exhibits high selectivity toward Zn

Full text of DOI:10.1016/j.tetlet.2011.12.005

Tetrahedron Letters 53 (2012) 804–807
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A novel calixsalen macrocycle: metal sensing behavior for Zn2+
and intracellular imaging application
Yong Fu a, Zhitao Xing a, Chengcheng Zhu a, Hongwei Yang a, Weijiang He a, Chengjian Zhu a,b,
,
⇑
Yixiang Cheng a,c,
⇑
a State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China
b State Key Laboratory of Organometallic Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China
c Key Laboratory of Mesoscopic Chemistry of MOE, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The chiral [3+3] macrocycle 1 which displays a calixarene-like crystal structure has been synthesized. The
UV/Vis and fluorescence spectral studies show that 1 and Zn2+ have a 1:3 complex stoichiometry. It
exhibits high selectivity toward Zn2+, but no significant responses toward other competitive cations.
The intracellular imaging ability has been tested in HeLa cells using a confocal microscope.
Ó 2011 Published by Elsevier Ltd.
Received 12 October 2011
Revised 25 November 2011
Accepted 2 December 2011
Available online 8 December 2011
Keywords:
Calixsalen
Fluorescence
Recognition
Living cells
Introduction
compounds have been studied extensively for their applications in
molecular recognition, host–guest chemistry, supermolecular
The design and synthesis of artificial receptors for metal ions
are of considerable importance in supermolecular chemistry due
to their fundamental role in medical, environmental, and biological
applications.1 Especially, as the second most abundant transition
metal ions in the human body, zinc(II) ion plays an essential role
in cellular metabolism, gene expression, apoptosis, and neuro-
transmission.2 Deregulation of zinc(II) homeostasis would be asso-
ciated with a number of diseases, ranging from Alzheimer’s
disease3 to prostate cancer4 and diabetes.5 Due to the importance
of Zn2+ in numerous biological systems, there is a great emphasis
structures, material chemistry, and catalysis. Among these macro-
cycles, especially, salen-based macrocycle molecules derived from
diamines and hydroxydialdehydes possess the advantage that they
can be obtained in different ring sizes and can be further function-
alized at well-defined molecular level.11 Although these salen-
based macrocycles named ‘calixsalens’ have been widely used to
synthesize mono-, di-, tri-, and tetranuclear macrocycle complexes,
the calixsalen macrocycles have not been utilized for the fluores-
cence sensors.
In this Letter, we report a novel chiral [3+3] macrocycle 1 used
as fluorescence probes specific for high selectivity to Zn2+ and the
intracellular imaging ability in HeLa cells. To the best of our knowl-
edge, this is the first calixsalen-type macrocycle sensor with turn-
on fluorescence, high selectivity, and rapid response to zinc ion.
placed on the development of chemosensors to determine Zn2+
.
Given that the closed-shell 3d10 electronic configuration of Zn(II)
renders it unable to give any spectroscopic or magnetic signals,
fluorescence or UV/Vis spectroscopy is best suitable for Zn2+ detec-
tion in biological contexts or living systems. In recent years, much
attention has been paid to the design of fluorescent probes for the
detection of zinc ion so as to understand their biological roles.6
A number of fluorescence sensors containing quinoline,7 fluores-
cein,8 and peptides9 for Zn2+ detection have been reported in previ-
ous literatures, but most of them are based on small molecules.
Nowadays, numerous heteroanalogues as well as homologues of
calixarenes exhibiting vase-like structures are known.10 These
Results and discussion
Synthesis and feature of the chiral [3+3] macrocycle 1
The synthesis procedure of sensor 1 is shown in Scheme 1.
Firstly, compound 2 is synthesized according to the literature.12
Then, macrocycle 1 was prepared from trans-(1R,2R)-1,2-cyclohex-
anediamine and compound 2 in 99% yield by Schiff-base formation
via nucleophilic addition–elimination reaction.13 In this Letter we
easily obtained the pure ligand [3+3] hexanuclear macrocycle 1
⇑
Corresponding authors. Tel.: +86 25 83594886; fax: +86 25 83317761 (C.Z.).
E-mail addresses: cjzhu@nju.edu.cna (C. Zhu), yxcheng@nju.edu.cn (Y. Cheng).
0040-4039/$ - see front matter Ó 2011 Published by Elsevier Ltd.
doi:10.1016/j.tetlet.2011.12.005

Y. Fu et al. / Tetrahedron Letters 53 (2012) 804–807
805
Figure 2. UV/Vis spectra of 1 (1 ꢁ 10ꢀ5 M) in THF with increasing amounts of
Zn(NO3)2 (0–3.0 equiv).
Zn2+ (0–3.0 ꢁ 10ꢀ5 M), as shown in Figure 2. Upon the addition
of increasing concentration of Zn2+ ion, the absorbance of 1 at
238 and 343 nm gradually decreases. Moreover, a new absorption
band appears at 406 nm, and its absorbance gradually increases
with the addition of Zn2+. This absorption peak is likely due to
the coordination of 1 and Zn2+. The changes that occur in the UV/
Vis spectra arise from the coordination of Zn2+ to the C@N sites,
which increase its coplanarity of the conjugated system.
Scheme 1. Synthesis procedures of sensor 1.
under ꢀ10 °C in high yield without further purification. The single
crystal X-ray structure of 1 shows the exact structure for calixare-
nes and the 27-membered macrocycle (Fig. 1). The C3-symmetric
neutral macrocycle contains three chiral-diamino backbones ar-
ranged in the form of an equilateral triangle, and the N2O cavities
are proximal to each other. The large size of these sub-cavities ap-
pears suitable for metal complexation as the two N and two O
atoms provide appropriate coordination sites.
CD spectra
Figure 3 shows the CD spectra of 1 in the absence and presence
of 3.0 equiv Zn2+ in THF. According to Figure 3, CD spectra of
1ꢀZn2+ appear great changes compared to free sensor 1. The CD
signal intensity gradually reduces by 30% and 42%, and the long
wavelength CD effect of metal-chelated 1 appears the gradual
red shift from 231 to 244 nm, 275 to 282 nm, respectively. The
intensity at 357 nm appears slight changes, only red shift from
357 to 393 nm. The result demonstrates that the CD spectra of 1
show high sensitivity to Zn2+. The obvious CD effect changes also
indicate that Zn2+ can coordinate with 1 and produce an induced
circular dichroism (ICD) in the absorption regions of 1.
Absorption study
Figure 2 shows the spectral variation of 1 upon the gradual
addition of Zn(NO3)2. The absorption spectrum of free sensor 1
exhibits two bands centered at 238 and 343 nm at room tempera-
ture in THF, which are associated with n–p⁄ transition of C@N and
p–
p⁄ transitions of benzene rings, respectively. To investigate the
binding property of 1 toward Zn2+, we measured the UV/Vis spec-
tra of 1 (1 ꢁ 10ꢀ5 M) in the presence of various concentrations of
Fluorescent spectra and titration
The fluorescence titration of 1 with Zn2+ is performed. As shown
in Figure 4, 1 displays a weak and broad emission band situated at
554 nm due to the extended
p-electronic structure. The fluores-
cence intensities of 1 show gradual enhancement as high as 68-fold
upon the concentration molar ratio addition of Zn2+ from 0 to
3.0 equiv. The nonlinear fitting of the titration curve also assumes
a 1:3 stoichiometry for the 1ꢀZn2+ complex (Fig. S2, electronic
Figure 1. Single-crystal structure of sensor 1. Hydrogen atoms and lattice solvent
molecules have been omitted for clarity.
Figure 3. CD spectra of 1 (1 ꢁ 10ꢀ5 M) in the absence and presence of 3.0 equiv
Zn2+ in THF solution.

806
Y. Fu et al. / Tetrahedron Letters 53 (2012) 804–807
Figure 4. Fluorescence spectra of 1 (1 ꢁ 10ꢀ5 M) in THF with increasing amounts of
Zn2+ (0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.3, 1.6, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 4.0, 5.0 equiv)
(kex = 343 nm). Inset: the fluorescence intensity at 497 nm of 1 as a function of Zn2+
concentration.
Figure 6. Confocal fluorescence imaging of HeLa cells: (a) brightfield transmission
image of cells labeled with 1 (10 M, PBS solution containing 10% DMSO) at 25 °C
for 20 min; (b) fluorescence image of (a); (c) fluorescence image after incubation
with 5 M ZnSO4/pyrithione (1:1) solution followed by rinse with 10 M sensor 1
solution; (d) fluorescence image of HeLa cells in (c) followed by further incubation
with 50 M TPEN solution for 20 min. kex = 488 nm. The transformation from light
blue to brown denotes the emission enhancement, Bar = 20 m.
l
l
l
l
l
and Mg2+, suggest that 1 may be a candidate for intracellular Zn2+
imaging. The intracellular Zn2+ imaging behavior of macrocycle 1
on HeLa cells is studied with a laser scanning confocal microscope.
Figure 5. Emission change at 497 nm of 1 (1 ꢁ 10ꢀ5 M) in THF induced by different
After incubation with 1 solution (10 lM in PBS, DMSO/water 1:9,
metal cations, kex = 343 nm. The concentration of Zn2+, Cd2+, Hg2+, Pb2+, Mn2+, Ni2+
,
v/v) at 25 °C for 20 min, the HeLa cells display very faint intracellular
fluorescence (Fig. 6). However, HeLa cells exhibit intensive fluores-
cence when exogenous Zn2+ is introduced into the cells via incuba-
tion with ZnSO4/pyrithione solution. Moreover, the intensive
fluorescence is deeply depressed by scavenging Zn2+ from the cells
with the cell permeable metal chelator, N,N,N0,N0-tetrakis(2-pyridyl-
methyl)ethylenediamine (TPEN). These results indicate that 1 is an
effective intracellular Zn2+ imaging agent with cell permeability. It
also implies that macrocycle 1 could be an effective model fluoro-
Fe3+, Fe2+, Cu2+, Co2+, and Ag+ is 3 ꢁ 10ꢀ5 M, while that of Na+, K+, Ca2+, and Mg2+ is
1 ꢁ 10ꢀ3 M, respectively.
Supplementary data). The quantum yield of Zn2+/1 complex is
ꢂ0.134.14 A competitive binding experiment gave an estimated Kd
of 1.44 ꢁ 10ꢀ14 M for Zn2+/1 complex.15 Moreover, the maximum
emission wavelength of 1 is remarkably blue shifted from 554 to
497 nm along with a dramatic enhancement of fluorescence inten-
sity when it is excited at 343 nm. The obvious fluorescent enhance-
ment can be attributed to the ICT (intramolecular charge transfer)
process of the Zn2+ coordination according to the correlative re-
view.16 Zn2+-coordination to 1 should also enhance the photo-in-
duced charge transfer (PCT) effect, trigger the emission change,
and increase the emission intensity.
phore to construct ratiometric sensors for Zn2+
.
Conclusions
In summary, a novel calixarene-like chiral [3+3] macrocycle 1 is
synthesized, which is an excellent fluorescent sensor for the detec-
tion of Zn2+. The UV/Vis and fluorescence spectral studies show that
1 and Zn2+ have a 1:3 complex stoichiometry. It produces a remark-
ably high selectivity toward Zn2+ ions over other competitive cat-
Fluorescence titration of 1 with different metal cations is also
investigated (Fig. 5). Other different kinds of metal ions (Na+, K+,
Mg2+, Ca2+, Ag+, Cd2+, Mn2+, Fe3+, Fe2+, Co2+, Ni2+, Hg2+, and Pb2+
)
which probably interfere with the detecting result is performed
under the similar conditions. As depicted in Figure S1 (ESI⁄), the
addition of 3.0 equiv of Fe3+, Hg2+, and Pb2+ shows slight changes
to the emission band of 1 at 497 nm. But Cu2+, Co2+, and Ni2+ cause
a little bit quenching of the fluorescence intensity. Ag+ shows grad-
ual enhancement as high as 3.6-fold at 539 nm. On the other hand,
the presence of 100 equiv of Na+, K+, Ca2+, and Mg2+ do not interfere
ions, such as Na+, K+, Mg2+, Ca2+, Mn2+, Fe3, Fe2+, Co2+, Ni2+, Cu2+
,
Ag+, Cd2+, Hg2+, and Pb2+. Most importantly, the large Stokes shift
and cell imaging ability of macrocycle 1 indicate that the novel cal-
ixarene-like structure could be a suitable platform to construct a no-
vel sensor for specific cation imaging in biological systems.
with its fluorescent response to Zn2+
.
Acknowledgments
Cellular experiment
We are very grateful for the support of this work from the Na-
tional Natural Science Foundation of China (Nos. 20832001,
20972065, 21074054) and the National Basic Research Program
of China (Nos. 2007CB925103, 2010CB923303) for their financial
The independent emission and the specific Zn2+-amplified emis-
sion which is not interfered by the cell abundance of Na+, K+, Ca2+
,

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807
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Supplementary data
Supplementary data associated with this article can be found, in
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