Dicyanostilbene-derived two-photon fluorescence probe for free zinc ions in live cells and tissues with a large two-photon action cross section

Article abstract of DOI:10.1021/ol200146j

A novel two-photon fluorescence probe for Zn²⁺ derived from dicyanostilbene as a two-photon fluorophore and 4-(pyridine-2-ylmethyl) piperazine as a novel Zn²⁺ ligand was developed. The probe shows a 72.5-fold fluorescence enhancement in response to Zn²⁺, a large two-photon action cross-section (580 GM), a noncytotoxic effect, and pH insensitivity in the biologically relevant range, and its dissociation constant (K_d^TP) is 0.52 ± 0.01 μM. The probe can selectively detect free Zn²⁺ ions in live cells for 1500 s or so and in living tissues at a depth of 80-150 μm without interference from other metal ions and the membrane-bound probes.

Full text of DOI:10.1021/ol200146j

ORGANIC
LETTERS
2011
Vol. 13, No. 6
1462–1465
Dicyanostilbene-Derived Two-Photon
Fluorescence Probe for Free Zinc Ions
in Live Cells and Tissues with a Large
Two-Photon Action Cross Section
Chibao Huang,* Junle Qu, Jing Qi, Meng Yan, and Gaixia Xu
Key Laboratory of Environmentally Friendly Chemistry and Applications of Ministry of
Education, College of Chemistry, Xiangtan University, Xiangtan 411105, China,
Department of Agricultural Engineering, Yingdong Bioengineering College,
Shaoguang University, Shaoguan 512005, China, and
Department of Instrument Science and Technology, Optoelectronic Technology College,
Shenzhen University, Shenzhen 518060, China
huangchibao@163.com
Received January 18, 2011
ABSTRACT
A novel two-photon fluorescence probe for Zn2þ derived from dicyanostilbene as a two-photon fluorophore and 4-(pyridine-2-ylmethyl)piperazine
as a novel Zn2þ ligand was developed. The probe shows a 72.5-fold fluorescence enhancement in response to Zn2þ, a large two-photon action
cross-section (580 GM), a noncytotoxic effect, and pH insensitivity in the biologically relevant range, and its dissociation constant (KdTP) is 0.52 (
0.01 μM. The probe can selectively detect free Zn2þ ions in live cells for 1500 s or so and in living tissues at a depth of 80-150 μm without
interference from other metal ions and the membrane-bound probes.
Two-photon excitation fluorescence microscopy (TPM),
which uses two photons of lower energy as the excitation
source, has rapidly evolved into a widely used tool in biological
and biomedical research and is increasingly popular. Com-
pared to traditional fluorescence microscopy, TPM offers
intrinsic three-dimensional (3D) resolution combined with
reduced phototoxicity and photobleaching, increased speci-
men penetration, and negligible background fluorescence.1
However, most fluorophores presently used as labels or
sensor platforms in TPM belong to one-photon (OP) ones
which have small two-photon absorption cross sections (δ)
that limit their usage.2 Therefore, to make TPM a more
versatile tool in biology, researchers need a wider variety
of two-photon (TP) probes with large δ for specific
applications.
Molecular design strategies of organic molecules with
large δ are well-established.3 In general, the magnitude of δ
increases with an increasing degree of intramolecular
charge transfer (ICT) upon excitation.
We have reported a TP fluorophore, 4-methyl-2,5-
dicyano-40-amino stilbene (DCS) with remarkably large
δ, which has been successfully employed in the design of
(1) So, P. T. C.; Dong, C. Y.; Masters, B. R.; Berland, K. M. Annu.
Rev. Biomed. Eng. 2000, 2, 399.
(2) (a) Zhang, M.; Gao, Y. H.; Li, M. Y.; Yu, M. X.; Li, F. Y.; Li, L.;
Zhu, M. W.; Zhang, J. P.; Yi, T.; Huang, C. H. Tetrahedron Lett. 2007,
48, 3709. (b) Miller, E. W.; Albers, A. E.; Pralle, A.; Isacoff, E. Y.;
Chang, C. J. J. Am. Chem. Soc. 2005, 127, 16652.
(3) Rumi, M.; Ehrlich, J. E.; Heikal, A. A.; Perry, J. W.; Barlow, S.; Hu,
Z. Y.; McCord-Maughon, D.; Parker, T. C.; Rockel, H.; Thayumanavan,
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r
10.1021/ol200146j
Published on Web 02/18/2011
2011 American Chemical Society

As an efficient bioimaging TPF probe, it should have
long-wavelength emission (near the ideal NIR imaging
window 650-900 nm), large δ, excellent photostability,
proper water solubility, and good cell permeability.
Scheme 1. Structure of DZn and Its Two-Photon PET
Mechanism
Herein, we extend our earlier work4 and report a new
TPF probe for Zn2þ derived from DCS as a TP fluoro-
phore and 4-(pyridine-2-ylmethyl)piperazine (PMP) as a
novel Zn2þ ligand. PMP is a original receptor and exhibits
a good affinity to Zn2þ. To the best of our knowledge, no
probes for Zn2þ using PMP as a receptor have been
reported. We report that DZn (Scheme 1) is capable of
imaging the intracellular free Zn2þ ions in live cells for a
long period of time and in living tissue ata depth of >80 μm
without mistargeting and photobleaching problems.
Scheme 2. Synthetic Procedure of DZn
TP fluorescence (TPF) probes for Hg2þ and Agþ ions.4
DCS is a push-pull chromophore with the strong electron
donor of a N,N0-disubstitutedamino group at its extremity
and the strong electron acceptor of two cyano groups on its
single aromatic ring. The interaction between the amino
group and cyano groups can significantly improve ICT
and increase the excited state dipole moment, which
drastically boosts the δ value of the fluorophore.
Zinc is a vital component of enzymes and proteins.5 In
the brain, a few millimoles of intracellular free Zn2þ ions
are stored in the presynaptic vesicles, which are released
with synaptic activation, and seem to modulate excitatory
neurotransmission.5a To understand the biological roles
ofZn2þ, a variety ofOPfluorescence(OPF) probesderived
from quinoline (TSQ, Zinquin and TFLZn) and fluo-
rescein (FluoZin-3, Zinpyr, ZnAF, etc.) have been
developed.6,7 However, most of them require a rather short
excitation wavelength or suffer from pH-sensitivity.
To overcome these problems, people began to develop
TPF probes for Zn2þ, and four such probes have been
reported.8 Although two among them have good achieve-
ment in application and one can be used for bioimaging,
the values of their δ are comparatively small, and the
largest δ is just 193 GM.
(4) (a) Huang, C.; Ren, A.; Feng, C.; Yang, N. Sens. Actuators, B
2010, 151, 236. (b) Huang, C.; Fan, J.; Peng, X.; Lin, Z.; Guo, B.; Ren,
A.; Cui, J.; Sun, S. J. Photochem. Photobiol. A: Chemistry 2008, 199, 144.
(5) (a) Frederickson, C. J.; Koh, J.-H.; Bush, A. I. Nat. Neurosci.
2005, 6, 449. (b) Vallee, B. L.; Falchuk, K. H. Physiol. Rev. 1993, 73, 79.
(6) (a) Hendrickson, K. M.; Geue, J. P.; Wyness, O.; Lincoln, S. F.;
Ward, A. D. J. Am. Chem. Soc. 2003, 125, 3889. (b) Fahrni, C. J.;
O’Halloran, T. V. J. Am. Chem. Soc. 1999, 121, 11448. (c) Budde, T.;
Minta, A.; White, J. A.; Kay, A. R. Neuroscience 1997, 79, 347–358.
(7) (a) omatsu, K.; Kikuchi, K.; Kojima, H.; Urano, Y.; Nagano, T.
J. Am. Chem. Soc. 2005, 127, 10197. (b) Burdette, S. C.; Frederickson,
C. J.; Bu, W.; Lippard, S. J. J. Am. Chem. Soc. 2003, 125, 1778. (c) Gee,
K. R.; Zhou, Z.-L.; Qian, W.-J.; Kennedy, R. J. Am. Chem. Soc. 2002,
124, 776.
The preparation of DZn is given in the Supporting
Information (SI) and Scheme 2. The solubility of DZn in
water was 20 μM, which is sufficient to stain the cells (Figure
S1, SI). The absorption intensities of DZn are almost
independent of Zn2þ (Figure S2, SI), indicating DZn is a
PET (photoinduced electron transfer)-based probe. How-
ever, DZn exhibits an unexpected strong sensitivity to Zn2þ
in the OP and TP processes (Figures 1a, S3, SI). As expected,
the TP action cross section or fluorescence brightness (Φδ)
(8) (a) Belfield, K. D.; Bondar, M. V.; Frazer, A.; Morales, A. R.;
Kachkovsky, O. D.; Mikhailov, I. A.; Masunov, A. E.; Przhonska, O. V.
J. Phys. Chem. B 2010, 114, 9313. (b) Kim, H. M.; Seo, M. S.; An, M. J.;
Hong, J. H.; Tian, Y. S.; Choi, J. H.; Kwon, O.; Lee, K. J.; Cho, B. R.
Angew. Chem., Int. Ed. 2008, 47, 5167. (c) Sumalekshmy, S.; Henary,
ꢀ
M. M.; Siegel, N.; Lawson, P. V.; Wu, Y.; Schmidt, K.; Bredas, J.-L.;
Perry, J. W.; Fahrni, C. J. J. Am. Chem. Soc. 2007, 129, 11888. (d)
Bhaskar, A.; Ramakrishna, G.; Twieg, R. J.; Goodson, T. J. Phys.
Chem. C 2007, 111, 14607.
Org. Lett., Vol. 13, No. 6, 2011
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Figure 1. (a) TP emission spectra of 1 μM DZn (MOPS buffer) in
the presence of free Zn2þ (0-6 μM). (b) TP action spectra of
1 μM DZn in the presence of 6 μM free Zn2þ
.
and δ of DZn increased 72.5-fold from 8 to 580 GM and 2.3-
fold from 400 to 935 GM (Table S1, SI) upon saturation
with Zn2þ in 30 mM MOPS buffer (100 mM KCl, 10 mM
EGTA, pH 7.2; EGTA = ethylene glycol bis(2-aminoethyl
ether) N,N,N0,N0-tetraacetic acid, and MOPS = 3-(mor-
pholino)-propanesulfonic acid), respectively. This exceeds
the brightness of many fluorophores widely used in biology,
including fluorescein, BODIPY, or Acedan (2-acetyl-6-
(dimethylamino)naphthalene), and the TP action cross
section of DZn-Zn2þ is more than six times that of AZn2-
Zn2þ (Table S1, SI). AZn2 is a TPF probe for Zn2þ derived
from acedan and N,N-di-(2-picolyl) ethylenediamine
(DPEN).8b TP emission spectra of DZn are similar to those
of OP (Figures 1a, S3, SI), and its TPF intensities enhanced
with increasing Zn2þ concentrations. Likewise, its two-
photon excitation spectrum in the presence of 6 μM Zn2þ
is analogous to the OP absorption spectrum, and it had a TP
excitation maximum at 810 nm (Figure 1b) (OP absorption
and emission maximum at 403 and 610 nm, respectively).
DZn is pH-insensitive in the biologically relevant pH range
(Figure S5, SI) and highly selective toward zinc ions and
does not yield a positive fluorescence response to Cd2þ
(Figure S4, SI), which has been a challenge in the selective
detection of zinc ions.
Figure 2. (a) Bright-field image. (b-d) TPM images of 1 μM
DZn-labeled mouse fibroblast collected at 550-650 nm, before
(b) and after (c) addition of 10 mM SNOC to the imaging
solution; (d) After addition of 0.1 mM TPEN to (c). The TP
excitation fluorescence (TPEF) images were collected upon
excitation at 810 nm with a femtosecond pulse. Cells shown are
representative images from replicate experiments (n = 5).
The TPM images of mouse fibroblast labeled with DZn
showed considerably poor TPF at 550-650 nm (Figure 2b),
which was due to fluorescence quenching based on the
PET mechanism after the addition of 10 mM S-nitroso-
cysteine (SNOC), an endogenous NO donor that triggers
the release of Zn2þ, to the above cell culture fluids; the TPF
intensity increased gradually with time (Figure 2c) and
then decreased abruptly upon the addition of 0.1 mM
TPEN (N,N,N0,N0-tetrakis(2-pyridyl) ethylenediamine)
(Figure 2d), a membrane-permeable Zn2þ chelator that
can effectively remove Zn2þ 8b
Without interference from
.
OP
The dissociation constants (Kd and KdTP) for DZn
the membrane-bound probes (no fluorescence at
360-460 nm) in this visual window,8b therefore, DZn
can detect Zn2þ concentrations in live cells and has no
cytotoxic effect.
calculated from the OP and TP fluorescence titration
curves (Figures 1a, S3, S6, S8, SI) are 0.51 ( 0.02 μM
and 0.52 ( 0.01 μM (Figures S7, S9, SI), respectively; the
detection limit of the probe is in the submicromolar range.
A Job plot (Figure S10, SI) indicated that DZn coordi-
nated to Zn2þ with 1:1 stoichiometry in water solution.
H NMR titration data clearly indicated that, except for
Hb, Hc, and Hm, all the peaks of the other 10 types of Hs
(Ha, Hd-Hl) experienced distinct downfield shifts and
broadened with increasing Zn2þ concentration, and the
chemical shifts for two sorts of hydrogen atoms (Hk and
Hl) of the piperazidine group evinced a great increase.
More surprisingly, upon increasing Zn2þ concentration
from 1 to 10 equiv, the chemical shifts of Hd and Hg caught
up and overtook those of Hc, and He and Hf, respectively
(Scheme 2, Figure S11 and Table S2, SI). This could be
attributed to a deshielding effect, arising from the decrease
of the electron density in the DCS fluorophore caused by
To further investigate the utility of this probe in deep
tissue imaging, TPM images were obtained from a part of a
mouse brain tissue slice incubated with 10 μM DZn for
30 min at 37 °C. Three TPM images were obtained
in the same plane at a depth of about 120 μm. Without
the addition of SNOC, the TPM image was dimmish
(Figure 3a), while, after the addition of 30 mM SNOC, it
became extremely bright (Figure 3b). Subsequently,
upon the addition of 0.3 mM TPEN, the TPM image
became rapidly faint (Figure 3c). The fluorescence of the
DZn-Zn2þ complex lasted 1500 s or so (Figure 3d).
Similarly, the fluorescence lasted for a long time in the
cell-imaging process. Moreover, the TPM images ob-
tained at a depth of 80-150 μm revealed the [Zn2þ
]
distribution in the brain tissues in the given plane along
the strong complexation of N-Zn2þ
.
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Org. Lett., Vol. 13, No. 6, 2011

Figure 3. TPM images of a mouse brain tissue slice stained with
10 μM DZn. (a) At a depth of ca. 120 μm with magnification
100ꢀ. Before (a) and after (b) addition of 30 mM SNOC to the
imaging solution; (c) After addition of 0.3 mM TPEN to (b).
(d) Relative TPEF intensity of a mouse brain tissue slice stained
with 10 μM DZn collected at 550-650 nm as a function of time.
The TPEF images were collected at 550-650 nm upon
excitation at 810 nm with a femtosecond pulse.
Figure 4. TPM images of a mouse brain tissue slice stained with
10 μM DZn by magnification 100ꢀ at different penetration
depths of (a) 80, (b) 100, (c) 120, and (d) 150 μm. The TPEF
images were collected at 550-650 nm upon excitation at 810 nm
with fs pulses.
its dissociation constant (KdTP) is 0.52( 0.01μM. DZn can
selectively detect intracellular free Zn2þ ions in live cells for
1500 s or so and in living tissues at a depth of 80-150 μm
without interference from other metal ions and the mem-
brane-bound probes.
the z direction (Figure 4). These findings demonstrate
that DZn is capable of detecting intracellular free
Zn2þ ions at a depth of 80-150 μm in living tissues by
using TPM.
Inconclusion, wehavedeveloped aTPF probe DZn with
small molecule size, large Φδ (580 GM), noncytotoxic
effect, long-wavelength emission at 610 nm (adjacent to
the ideal imaging visual window 650-900 nm), large
Stokes shift (207 nm), excellent photostability, moderate
water solubility, and good cell permeability. DZn shows a
72.5-fold fluorescence enhancement in response to Zn2þ
and pH insensitivity in the biologically relevant range, and
Acknowledgment. This work was supported by a China
Postdoctoral Science Foundation funded project (No.
20100471224).
Supporting Information Available. Synthesis, photo-
physical study, cell culture, and two-photon imaging.
This material is available free of charge via the Internet
at http://pubs.acs.org.
Org. Lett., Vol. 13, No. 6, 2011
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