A "switching on" fluorescent chemodosimeter of selectivity to Zn2+ and its application to MCF-7 cells

Article abstract of DOI:10.1039/c0cc01687j

A highly sensitive and selective 1,8-naphthyridine-based fluorescent chemodosimeter for Zn²⁺ has been designed, synthesized, and applied to the detection of intracellular Zn²⁺.

Full text of DOI:10.1039/c0cc01687j

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A ‘‘switching on’’ fluorescent chemodosimeter of selectivity to
Zn2+ and its application to MCF-7 cellsw
Zhanxian Li,a Mingming Yu,*a Lifeng Zhang,a Ming Yu,a Jinxia Liu,a Liuhe Wei*a and
Hongyan Zhang*b
Received 1st June 2010, Accepted 13th August 2010
DOI: 10.1039/c0cc01687j
A highly sensitive and selective 1,8-naphthyridine-based fluorescent
chemodosimeter for Zn2+ has been designed, synthesized, and
Zn2+ to make the acyclic CQN bond become to cyclic
CQN bond, resulting in a fluorescent product.
applied to the detection of intracellular Zn2+
.
Herein, we report the Zn2+-selective recognizing properties
of compound 1. Compound
1 possesses an efficient
Zn2+-selective OFF–ON fluorescent behavior both in solution
The design and synthesis of fluorescent probes with high
selectivity and sensitivity is a vibrant field of supramolecular
chemistry for their fundamental role in medical, environ-
mental and biological applications.1 The zinc ion (Zn2+), the
second most abundant transition metal in the human body,
plays vital roles in numerous biological processes, such as gene
transcription, regulation of metalloenzymes, and neural signal
transmission.2 Hence, the development of Zn2+-probes is a
promising field. Rapid progress has been made in the
development of fluorescent Zn2+-probes in solution,4,5 due
to their simplicity, high sensitivity and instantaneous
response,3 and based on ion-induced fluorescence changes.
However, reports of their intracellular application are rare.5 It
is desired to develop new fluorescent indicators with improved
properties, especially with high efficiency in the spectral visible
region and study of Zn2+-probes in cell.
and cell.
Compound 1 was synthesized according to the reported
method.9 The synthetic route (Scheme S1), detailed character-
ization and the crystal structure of 110 (Fig. S1) are shown in
the ESI.w
The absorption spectra change of 1 in ethanol upon
addition of Zn2+ is shown in Fig. S2.w Upon gradual addition
of Zn2+ into the ethanol solution of 1, a new absorption peak
at about 428 nm appeared and its intensity increased while the
original absorption peak at 378 nm weakened (e = 1.99 Â
104 MÀ1 cmÀ1). The red-shift of the absorption peak can be
ascribed to reformed electronic orbits and new energy gaps in
the product of compound 1 and Zn2+
.
Compound 1 hardly exhibits fluorescence emission in
ethanol upon excitation at 370 nm (Fig. 1). Upon titration
of Zn2+, a new fluorescence emission peak at about 505 nm
appeared and the intensity was dramatically enhanced,
indicating a Zn2+-selective fluorescent signaling behavior.
Because of the great enhancement of emission at 505 nm
(F/F0 = 15) upon addition of zinc ions, compound 1 indicates
an efficient Zn2+-selective OFF–ON fluorescent behavior. The
increased emission intensity is probably due to the complex
formed between ZnCl2 and 1, in which the acetamide group
was hydrolyzed to amino group and the rotation of acyclic
CQN is inhibited.4c,7 The possible reaction mechanism of
Zn2+ and 1 is depicted in Scheme 1, which was further
confirmed by ESI data measured after addition of Zn2+ into
the ethanol solution of 1.11 The insets in Fig. 1 demonstrate
the relationship between the concentration of Zn2+ and the
emission intensity at a special wavelength.
The aim of this communication is to design and construct a
fluorometric assay to highly effectively detect Zn2+ both in
solution and cell. Because of their various photoluminescent
properties and short metalÁ Á Ámetal distance formed in their
metal building blocks, 1,8-naphthyridine and its derivatives
have been widely used as guanine recognition regents and
bidentate ligands.6 However, there are very few reports about
the derivatives of 1,8-naphthyridine as fluorescent probes for
transition metals. In our previous work, a 1,8-naphthyridine-
based Zn2+-chemodosimeter was designed and studied.7
Considering the above, 1,8-naphthyridine group was used
herein as the reaction unit with Zn2+. On the other hand, in
general, compounds containing acyclic CQN bonds are non-
fluorescent, and cyclic CQN bonds significantly fluorescent.8
(E)-2-(4-Hydroxyphenylimino)-4-methyl-7-acetamidyl-1,8-
naphthyridine (1; Chart 1) was designed, in which the
naphthyridine group and the CQN bond can react with
To validate the proposed reaction mechanism of 1 and
1
Zn2+, H NMR spectra of 1 in DMSO-d6, 1 in the mixture
of CD3OD and DMSO-d6, and 1 with Zn2+ in the mixture of
CD3OD and DMSO-d6 were measured, as shown in Fig. 2.
The difference between Fig. 2a and b is the decrease of signal
a Department of Chemistry, Zhengzhou University, Zhengzhou, China.
E-mail: yumm@zzu.edu.cn, weiliuhe@zzu.edu.cn;
Fax: (+)86-371-67781205; Tel: (+)86-371-67781205
b Key Laboratory of Photochemical Conversion and Optoelectronic
Materials Technical Institute of Physics and Chemistry,
Chinese Academy of Sciences, Beijing, China.
E-mail: zhanghongyan@mail.ipc.ac.cn
w Electronic supplementary information (ESI) available: Experimental
details and additional figures. CCDC 783331. For ESI and crystallo-
graphic data in CIF or other electronic format see DOI: 10.1039/
c0cc01687j
Chart 1
c
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Fig. 2 1H NMR spectra (400 MHz, DMSO-d6, 298 K) of (a) 1;
Fig. 1 Changes in fluorescence emission spectra of 1 (5.0 Â 10À5M) in
ethanol upon addition of Zn2+. The final ratio of zinc ion to 1 is
12 equiv. Inset: Emission intensities of 1 at 505 nm (black dot) as a
function of addition of Zn2+. The concentration of Zn2+ changed
from 0 to 6.0 Â 10À4 M and excitation was at 370 nm.
(b) 1+CD3OD-d4; (c) 1+CD3OD-d4+2 eq Zn2+
.
Zn2+ (Fig. S4–S6w). As for the sulfate anion, the addition of
ZnSO4 enhanced the fluorescence of 1 very little, which may be
ascribed to the uncoordinated ability of sulfate anion.
The selectivities of 1 to the specific metal ions were
examined in ethanol. As illustrated in Fig. S3w and in the left
part of Fig. 3, upon addition of the same amount of the
various metal ions, only Zn2+ enhanced the emission of 1.
Compound 1 did not respond distinctly to alkali and alkaline
earth metal ions such as Ca2+, Mg2+, Na+, and K+. As for
the first-row transition-metal ions, Fe2+, Cu2+, Co2+ and
Ni2+ decreased the emission intensity while Mn2+and Cr2+
had little influence on the fluorescence intensity of 1. The
selectivity of 1 toward Zn2+ suggests that 1 has potential
application in sensing Zn2+
.
Because Ca2+, Mg2+, Na+ and K+ exist at high concen-
tration under physiological conditions, it is necessary to
Scheme 1 Proposed reaction mechanism of Zn2+ with 1.
intensity at around 9.73, which was assigned to the proton
exchange between Hb and CD3OD. After addition of Zn2+
into DMSO-d6 and CD3OD solution of 1, the decrease of the
signal intensity at around 9.73 and the shift to downfield of the
signal at around 2.17 (Fig. 2b and c) are the most significant.
Such changes are ascribed to the reaction between acetylamide
group and Zn2+. Combined with the ESI data, the reaction
mechanism of Zn2+ and 1 was further confirmed. Moreover,
Fig. 3 Fluorescence responses of 1 to various metal ions (left part
from 1 to 13 represents compound 1 only, Zn2+, Cu2+, Co2+, Cr3+
,
Fe2+, Hg2+, Mn2+, Ni2+, Na+, Mg2+, K+ and Ca2+, respectively)
and fluorescence change of the mixture of 1 and Zn2+ after addition of
an excess of the appropriate metal ions (right part from 14 to 23
represents Na+, Mg2+, K+, Ca2+, Cu2+, Fe2+, Mn2+, Co2+, Cr3+
and Ni2+, respectively. The equiv. ratio of Zn2+ to each of Ca2+
Mg2+, Na+, K+ is 1 : 10 and the equiv. ratio of Zn2+ to each of the
other competing metal ions is 5) (right part from Zn2+ represented in
Fig. 3). Bars represent the ratio of the difference between F to F0.
F and F0 represent the emission intensity at 505 nm. The overall
emission spectra were measured at excitation of 370 nm.
1
the H NMR spectra of 1 and Zn2+ (Fig. 2) and Job’s plot
(Fig. S7, Table S1w) reveal a 1 : 2 binding model between 1 and
Zn2+. The binding constants of 1 with Zn2+, Cu2+, Co2+ and
Ni2+ were calculated (Fig. S7–S10, Table S1w).
The experiments of the counterion effect on the selective
properties of Zn2+ demonstrate that nitrate and acetate
anions have little influence on the selective effect of 1 on
c
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Fig. 4 The intracellular Zn2+ was imaged in living cells at 37 1C with
use of confocal microscopy. (a) MCF-7 cells incubated with 10 mM of
compound 1 solution (the volume ratio of ethanol and water is 4 to 6)
for 30 min. (b) MCF-7 cells in part a 10 min after being treated with
2 Â 10À2 mmol of Zn2+ aqueous solution. (c) Bright field image of
living MCF-7 cells in parts a and b.
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explore the disturbance of such metal ions on the detection of
Zn2+. The titration of Zn2+ and 1 in the presence of various
metal ions was conducted, and the experimental results (the right
part in Fig. 3) indicate that the fluorescence of the complex
resulting from the reaction of Zn2+ and 1 was not influenced by
the alkali and alkaline earth metal ions, even at a high concen-
tration of 6 mM. Furthermore, the other physiological necessary
metal ions such as Fe2+, Cu2+, Mn2+, Cr2+, Co2+, Ni2+ were
also applied to explore their disturbance on the detection of
Zn2+. When the ratio of Zn2+ to such metal ions is no less
than 5, no influence was observed on the detection of Zn2+
.
The sensitivity of 1 to Zn2+ was examined in living cells by
using confocal microscopy. Fluorescence images were
recorded with excitation at 408 nm by an diode laser, Spinhole
aperture, 100% gain of detector, and an oil objective with 60Â
magnification and 1.40 NA. The qualitatively in vitro results
are exhibited in Fig. 4. After MCF-7 cells were incubated with
10À3 mmol 1 and 1 mL PBS for 30 min at 37 1C, no obvious
fluorescence can be imaged (Fig. 4a). At the same experimental
conditions, 10 min after 2 Â 10À2 mmol of Zn2+ was intro-
duced into the same MCF-7 cells, the strong fluorescence was
imaged (Fig. 4b), which resulted from the reaction of 1 and
Zn2+. The bright field transmission images of these MCF-7
cells in Fig. 4c is exactly the same as the fluorescence image in
Fig. 4b, confirming an intracellular fluorescence imaged.
In summary, a novel 1,8-naphthyridine-based fluorescent
Zn2+-chemodosimeter 1 has been designed and synthesized,
and it displays high selectivity and sensitivity for Zn2+ in the
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,
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significant fluorescence enhancement is achieved. The reason is
probably that the acetamide group of 1 was hydrolyzed to
amino group and the rotation of acyclic CQN is frozen. The
sensitivity of 1 to Zn2+ was demonstrated in living cells,
indicating its potential application for the intracellular
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imaging of Zn2+
.
10 Crystal data for 1: C36H32N8O5, M = 656.70, orthorhombic, space
ꢀ
group P1, a = 7.5496(15) A, b = 13.657(3) A, c = 30.427(6) A,
We thank the NSFC (50903075, 60978034 and 50873093)
for financial support and Professor Chenjie Fang at Capital
Medical University for her kind help.
a = 901, b = 901, g = 901, V = 3137.1(11) A3, Z = 4, Dc
=
1.390 Mg mÀ3, F000 = 1376, MoKa radiation, l = 0.71073 A,
T = 273(2) K, 2ymax = 55.64. 32 427 reflections measured, 13 839
unique (Rint = 0.0456). The structures were solved by direct
methods and refined by a full-matrix least-squares technique on
Notes and references
F2 using the SHELXH97 program. Final GooF = 1.195, R1
0.0937, wR2 = 0.1995, R indices based on 8981 reflections and 912
=
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refined parameters, with I 4 2s(I). CCDC 783331.
11 ESI data of the final compound from reaction of 1 and ZnCl2: m/z)
514.5 [M], 279 [M+ + 1 À ZnCl2 À ZnCl À CH2CO].
c
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 7169–7171 7171