Improvement and biological applications of fluorescent probes for zinc, ZnAFs

Article abstract of DOI:10.1021/ja025567p

The development and cellular applications of novel fluorescent probes for Zn²⁺, ZnAF-1F, and ZnAF-2F are described. Fluorescein is used as a fluorophore of ZnAFs, because its excitation and emission wavelengths are in the visible range, which m

Full text of DOI:10.1021/ja025567p

Published on Web 05/17/2002
Improvement and Biological Applications of Fluorescent
Probes for Zinc, ZnAFs
Tomoya Hirano,† Kazuya Kikuchi,†,‡ Yasuteru Urano,† and Tetsuo Nagano*,†
Contribution from the Graduate School of Pharmaceutical Sciences, The UniVersity of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, and Presto, JST Corporation,
Kawaguchi, Saitama, Japan.
Received January 11, 2002
Abstract: The development and cellular applications of novel fluorescent probes for Zn2+, ZnAF-1F, and
ZnAF-2F are described. Fluorescein is used as a fluorophore of ZnAFs, because its excitation and emission
wavelengths are in the visible range, which minimizes cell damage and autofluorescence by excitation
light. N,N-Bis(2-pyridylmethyl)ethylenediamine, used as an acceptor for Zn2+, is attached directly to the
benzoic acid moiety of fluorescein, resulting in very low quantum yields of 0.004 for ZnAF-1F and 0.006 for
ZnAF-2F under physiological conditions (pH 7.4) due to the photoinduced electron-transfer mechanism.
Upon the addition of Zn2+, the fluorescence intensity is quickly increased up to 69-fold for ZnAF-1F and
60-fold for ZnAF-2F. Apparent dissociation constants (Kd) are in the nanomolar range, which affords sufficient
sensitivity for biological applications. ZnAFs do not fluoresce in the presence of other biologically important
cations such as Ca2+ and Mg2+, and are insensitive to change of pH. The complexes with Zn2+ of previously
developed ZnAFs, ZnAF-1, and ZnAF-2 decrease in fluorescence intensity below pH 7.0 owing to protonation
of the phenolic hydroxyl group of fluorescein, whose pKa value is 6.2. On the other hand, the Zn2+ complexes
of ZnAF-1F and ZnAF-2F emit stable fluorescence around neutral and slightly acidic conditions because
the pKa values are shifted to 4.9 by substitution of electron-withdrawing fluorine at the ortho position of the
phenolic hydroxyl group. For application to living cells, the diacetyl derivative of ZnAF-2F, ZnAF-2F DA,
was synthesized. ZnAF-2F DA can permeate through the cell membrane, and is hydrolyzed by esterase
in the cytosol to yield ZnAF-2F, which is retained in the cells. Using ZnAF-2F DA, we could measure the
changes of intracellular Zn2+ in cultured cells and hippocampal slices.
associated with certain acute conditions, including epilepsy,5
transient global ischemia,6 and brain injury.7 Although many
Introduction
Zinc ion (Zn2+) is an essential component of many enzymes
and transcription factors (e.g., carbonic anhydrase, zinc finger
proteins, etc.), in which it plays structural or catalytic roles.1 In
addition to such protein-bound Zn2+, free or loosely bound
(labile, chelatable) Zn2+ exists at high concentration especially
in brain,2 pancreas,3 and spermatozoa.4 In brain, a few millimolar
of free Zn2+ exists in the vesicles of presynaptic neurons, and
is released by synaptic activity or depolarization, modulating
the function of certain ion channels and receptors. Zn2+ has
been reported to induce selective neuronal cell death that is
reports describe the significance of Zn2+ in biological systems,
its mechanisms of action are poorly understood.
Fluorescent probes, which allow visualization of cations, small
molecules, or enzyme activity in living cells by fluorescence
microscopy, are useful tools for clarifying the function in
biological systems.8 Therefore, various fluorescent probes for
Zn2+ have been developed.9-11,13-16 The most widely used
fluorescent probes for Zn2+ are TSQ9 (6-methoxy-8-(p-tolu-
(5) Frederickson, C. J.; Hernandez, M. D.; McGinty, J. F. Brain Res. 1989,
480, 317-321.
(6) Koh, J.-Y.; Suh, S. W.; Gwag, B. J.; He, Y. Y.; Hsu, C. Y.; Choi, D. W.
Science 1996, 272, 1013-1016.
* Corresponding author. E-mail: tlong@mol.f.u-tokyo.ac.jp. Phone:
+81-3-5841-4850. Fax: +81-3-5841-4855.
(7) (a) Yeiser, E. C.; Lerant, A. A.; Casto, R. M.; Levenson, C. W. Neurosci.
Lett. 1999, 277, 75-78. (b) Suh, S. W.; Chen, J. W.; Motamedi, M.; Bell,
B.; Listiak, K.; Pons, N. F.; Danscher, G.; Frederickson, C. J. Brain Res.
2000, 852, 268-273.
† The University of Tokyo.
‡ Presto, JST Corporation.
(1) (a) Vallee, B. L.; Falchuk, K. H. Physiol. ReV. 1993, 73, 79-118. (b) da
Silva, J. J. R. F.; Williams, R. J. P. The Biological Chemistry of Elements:
The Inorganic Chemistry of Life, 2nd ed.; Oxford University Press: Oxford,
2001; pp 315-339.
(8) Mason, W. T. Fluorescent and Luminescent Probes for Biological ActiVity,
2nd ed.; Academic Press: New York, 1999.
(9) (a) Frederickson, C. J.; Kasarskis, E. J.; Ringo, D.; Frederickson, R. E. J.
Neurosci. Methods 1987, 20, 91-103. (b) Savage, D. D.; Montano, C. Y.;
Kasarskis, E. J. Brain Res. 1989, 496, 257-267.
(2) (a) Frederickson, C. J. Int. ReV. Neurobiol. 1989, 31, 145-138. (b)
Cuajungco, M. P.; Lees, G. J. Neurobiol. Dis. 1997, 4, 137-169. (c)
Frederickson, C. J.; Suh, S. W.; Silva, D.; Frederickson, C. J.; Thompson,
R. B. J. Nutr. 2000, 130, 1471S-1483S.
(10) (a) Zalewski, P. D.; Forbes, I. J.; Betts, W. H. Biochem. J. 1993, 296,
403-408. (b) Zalewski, P. D.; Forbes, I. J.; Borlinghaus, R.; Betts, W. H.;
Lincoln, S. F.; Ward, A. D. Chem. Biol. 1994, 1, 153-161.
(11) (a) Budde, T.; Minta, A.; White, J. A.; Kay, A. R. Neuroscience 1997, 79,
347-358. (b) Kimber, M. C.; Mahadevan, I. B.; Lincoln, S. F.; Ward, A.
D.; Tiekink, E. R. T. J. Org. Chem. 2000, 65, 8204-8209. (c) Pearce, D.
A.; Jotterand, N.; Carrico, I. S.; Imperiali, B. J. Am. Chem. Soc. 2001,
123, 5160-5161.
(3) Zalewski, P. D.; Millard, S. H.; Forbes, I. J.; Kapaniris, O.; Slavotinek,
A.; Betts, W. H.; Ward, A. D.; Lincoln, S. F.; Mahadevan, I. J. Histochem.
Cytochem. 1994, 42, 877-874.
(4) Zalewski, P. D.; Jian, X.; Soon, L. L. L.; Breed, W. G.; Seamark, R. F.;
Lincoln, S. F.; Ward, A. D.; Sun, F.-Z. Reprod. Fertil. DeV. 1996, 8, 1097-
1105.
9
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J. AM. CHEM. SOC. 2002, 124, 6555-6562
6555

A R T I C L E S
Hirano et al.
ZnAF-1 or ZnAF-2 is decreased below pH 7.0. So, although
ZnAF-1 or ZnAF-2 is useful above pH 7.0, the signal is affected,
for example, under conditions of acidosis.
Here we report the design and synthesis of fluorine-substituted
derivatives of ZnAFs, ZnAF-1F, and ZnAF-2F, whose com-
plexes with Zn2+ exhibit stable fluorescence above pH 6.0,
allowing detection of Zn2+ regardless of physiological pH
changes in living cells. We also report the fluorescence and
kinetic properties of these ZnAFs and describe biological
applications in cultured cells and hippocampal slices.
Figure 1. Structures of the fluorescent probes for Zn2+: ZnAF-1 and
ZnAF-2.
Results and Discussion
enesulfonamide)quinoline) and its derivatives.3,10,11 One of these
derivatives, Zinquin, can detect intracellular Zn2+ in living
cells.3,10 Although quinoline-based probes are useful, they are
not ideal because the excitation wavelength is in the ultraviolet
range, which may cause cell damage and is subject to interfer-
ence by autofluorescence from biological molecules such as
pyridine nucleotides. Fluorescein is the most widely used
fluorophore for labeling and sensing biomolecules,12 because
it has a high extinction coefficient and a high fluorescence
quantum yield in aqueous solution, and its excitation wavelength
is in the visible range, which is preferable to UV excitation.
Recently, fluorescein-based probes, Zinpyr-1 and Zinpyr-2,13
which fluoresce in the presence of Zn2+, have been reported.
However, the basal fluorescence of Zinpyrs is pH-sensitive
around neutral conditions with pKa values of 8.4 for Zinpyr-1
and 9.4 for Zinpyr-2, and its intensity is strong (quantum yield
is 0.39 for Zinpyr-1 and 0.25 for Zinpyr-2) at pH 7.0, so cellular
pH change induced by biological stimuli17 can cause difficulty
in interpreting fluorescence intensity change. Therefore, Zin-
pyr-1 and Zinpyr-2 cannot reliably monitor Zn2+ in living cells.
We previously reported fluorescein-based probes, ZnAF-1 and
ZnAF-214 (Figure 1). Upon addition of Zn2+, the fluorescence
intensity was increased by 17-fold for ZnAF-1 and 51-fold for
ZnAF-2 at pH 7.5. At this pH, the fluorescence intensity of
ZnAF-1 or ZnAF-2 itself is very small; the quantum yield is
only 0.02 for both ZnAFs, and is not increased by pH change.
However, the fluorescence intensity of the Zn2+ complex with
Design and Synthesis of ZnAF-1F and -2F. The fluores-
cence intensity of fluorescein, used as a fluorophore of ZnAF-1
and ZnAF-2, decreases under acidic conditions. This property
arises from protonation of the phenolic hydroxyl group of
fluorescein, whose pKa value is 6.43.18 The Zn2+ complex of
ZnAF-1 or ZnAF-2 exhibits similar properties, and the pKa
values of these ZnAFs are almost the same, 6.2. So, we designed
ZnAF-1F and ZnAF-2F with electron-withdrawing fluorine at
the ortho position of the phenolic hydroxyl group to lower the
pKa value, and thereby obtain stable fluorescence under near-
neutral conditions.
The synthetic scheme for ZnAF-1F and ZnAF-2F is shown
in Scheme 1. 2′,7′-Difluoro-5- and 6-nitrofluorescein were
formed from 4-nitrophthalic acid anhydride and 4-fluororesor-
cinol by heating in methanesulfonic acid. Then the nitro group
was reduced to an amino group with Na2S and NaSH in water,
as in the synthesis of aminofluorescein.19 The phenolic hydroxyl
group of fluorescein was protected as the pivaloate ester. Direct
alkylation of the nitrogen atom of aminofluorescein dipivaloate
ester (1) was difficult because of its low electron density, so
the amino group was converted to 4-nitrobenzenesulfonamide.
At this stage, the 5-isomer (2) and 6-isomer (2′) could be easily
separated by silica gel chromatography. Then, reaction with 1,2-
dibromoethane and 2,2′-dipicolylamine afforded 4. 4-Nitroben-
zenesulfonamide was converted to the secondary amine and the
pivaloate ester was cleaved to yield ZnAF-1F. The 6-substituted
isomer ZnAF-2F was also similarly obtained from 2′.
(12) (a) Kojima, H.; Nakatsubo, N.; Kikuchi, K.; Kawahara, S.; Kirino, Y.;
Nagoshi, H.; Hirata, Y.; Nagano, T. Anal. Chem. 1998, 70, 2446-2453.
(b) Kojima, H.; Urano, Y.; Kikuchi, K.; Higuchi, T.; Nagano, T. Angew.
Chem., Int. Ed. 1999, 38, 3209-3212. (c) Umezawa, N.; Tanaka, K.; Urano,
Y.; Kikuchi, K.; Higuchi, T.; Nagano, T. Angew. Chem., Int. Ed. Engl.
1999, 38, 2899-2901. (d) Tanaka, K.; Miura, T.; Umezawa, N.; Urano,
Y.; Kikuchi, K.; Higuchi, T.; Nagano, T. J. Am. Chem. Soc. 2001, 123,
2530-2536.
(13) (a) Walkup, G. K.; Burdette, S. C.; Lippard, S. J.; Tsien, R. Y. J. Am.
Chem. Soc. 2000, 122, 5644-5645. (b) Burdette, S. C.; Walkup, G. K.;
Spingler, B.; Tsien, R. Y.; Lippard, S. J. J. Am. Chem. Soc. 2001, 123,
7831-7841. (c) Burdette, S. C.; Lippard, S. J. Coord. Chem. ReV. 2001,
216-217, 333-361.
(14) Hirano, T.; Kikuchi, K.; Urano, Y.; Higuchi, T.; Nagano, T. J. Am. Chem.
Soc. 2000, 122, 12399-12400.
(15) Hirano, T.; Kikuchi, K.; Urano, Y.; Higuchi, T.; Nagano, T. Angew. Chem.,
Int. Ed. 2000, 39, 1052-1054.
(16) (a) Walkup, G. K.; Imperiali, B. J. Am. Chem. Soc. 1996, 118, 3053-
3054. (b) Godwin, H. A.; Berg, J. M. J. Am. Chem. Soc. 1996, 118, 6514-
6515. (c) Koike, T.; Watanabe, T.; Aoki, S.; Kimura, E.; Shiro, M. J. Am.
Chem. Soc. 1996, 118, 12696-12703. (d) Walkup, G. K.; Imperiali, B. J.
Am. Chem. Soc. 1997, 119, 3443-3450. (e) Tompson, R. B.; Whetsell,
W. O., Jr.; Maliwal, B. P.; Fierke, C. A.; Frederickson, C. J. J. Neurosci.
Methods 2000, 96, 35-45. (f) Haugland, R. P. Handbook of Fluorescent
Probes and Research Products, 8th ed.; Molecular Probes, Inc.: Eugene,
OR, 2001; Section 20.7.
(17) (a) Li, J.; Eastman, A. J. Biol. Chem. 1995, 270, 3203-3211. (b) Shepherd,
R. M.; Henquin, J.-C. J. Biol. Chem. 1995, 270, 7915-7921. (c) Zhao, H.;
Xu, X.; Diaz, J.; Muallem, S. J. Biol. Chem. 1995, 270, 19599-19605. (d)
Llopis, J.; McCaffery, J. M.; Miyawaki, A.; Farquhar, M. G.; Tsien, R. Y.
Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 6803-6808.
Derivatives of fluorescein that are amino-substituted at the
benzoic acid moiety exhibit almost no fluorescence, but when
the amino group is converted to a less electron-donating group,
such as an amide group, or complexed with a cation via the
lone pair of nitrogen, fluorescence with a high quantum yield
is obtained. If we can design and synthesize a suitably specific
reactive moiety, which is linked to aminofluorescein, we should
be able to obtain sensor molecules suitable for different types
of analytes. It is also advantageous that the fluorescence
quantum yield of aminofluorescein is very low, so if the sensor
molecule is converted to fluorescent form, the measured
fluorescence intensity will be mainly due to the analyte. For
such a strategy, 2 and 2′ could be good intermediates, because
4-nitrobenzenesulfonamide can be alkylated efficiently with
alcohol or alkyl halide, and deprotected under mild conditions.20
(18) Sun, W.-C.; Gee, K. R.; Klaubert, D. H.; Haugland, R. P. J. Org. Chem.
1997, 62, 6469-6475.
(19) Mckinney, R. M.; Spillane, J. T.; Pearce, G. W. J. Org. Chem. 1962, 27,
3986-3988.
(20) (a) Fukuyama T.; Jow, C.-K.; Cheung, M. Tetrahedron Lett. 1995, 36,
6373-6374. (b) Fukuyama T.; Cheung, M.; Jow, C.-K.; Hidai, Y.; Kan,
T. Tetrahedron Lett. 1997, 38, 5831-5834.
9
6556 J. AM. CHEM. SOC. VOL. 124, NO. 23, 2002

Biological Applications of Fluorescent Probes for Zinc, ZnAFs
A R T I C L E S
Scheme 1. Synthetic Scheme for ZnAF-1F and ZnAF-2Fa
a Ns ) 4-nitrobenzenesulfonyl, Pv ) Pivaloyl.
Table 1. Spectroscopic Properties of ZnAFs with and without
a
Zn2+
free
+Zn2+
compd
ꢀb
Φc
ꢀb
Φc
ZnAF-1
ZnAF-2
ZnAF-1F
ZnAF-2F
7.4 × 104 (489)
7.8 × 104 (490)
7.7 × 104 (489)
7.4 × 104 (490)
0.022
0.023
0.004
0.006
6.3 × 104 (492)
7.6 × 104 (492)
7.0 × 104 (492)
7.3 × 104 (492)
0.21
0.32
0.17
0.24
a All data were obtained at pH 7.4 (100 mM HEPES buffer, I ) 0.1
(NaNO3)). b ꢀ stands for extinction coefficient (M-1 cm-1). Measured at
each λmax, which is shown in parentheses (nm). c Φ stands for quantum
yield, determined using Φ of fluorescein (0.85) in 0.1 N NaOH as a
standard.21
Fluorescence Properties of ZnAF-1F and -2F. ZnAF-1F
and ZnAF-2F themselves showed almost no fluorescence under
physiological conditions (pH 7.4, I ) 0.1 (NaNO3)), exhibiting
fluorescence quantum yields of only 0.004 for ZnAF-1F and
0.006 for ZnAF-2F (Table 1). These values are much lower
than those of existing Zn2+ fluorescent probes, Zinpyr-1 (0.38),
Zinpyr-2 (0.25), ZnAF-1 (0.02), and ZnAF-2 (0.02), suggest-
ing that ZnAF-1F and ZnAF-2F will exhibit very low back-
ground fluorescence. Upon addition of Zn2+, the fluorescence
intensity was increased by 69-fold for ZnAF-1F and 60-fold
for ZnAF-2F, and the quantum yield of the Zn2+ complex was
Figure 2. Emission spectra (excitation at 492 nm) of 1 µM ZnAF-2F in
the presence of various concentrations of Zn2+ ranging from 0 to 1 µM.
These spectra were measured at pH 7.4 (100 mM HEPES buffer, I ) 0.1
(NaNO3)).
0.17 for ZnAF-1F and 0.24 for ZnAF-2F. The absorption maxi-
mum wavelength was very slightly bathochromically shifted
(2-3 nm). The emission maximum did not change upon the
addition of Zn2+ for either ZnAF-1F or ZnAF-2F (Figure 2 and
Table 1).
9
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A R T I C L E S
Hirano et al.
Figure 4. Effect of pH on the fluorescence intensity of ZnAF-2 and ZnAF-
2F: open circle, 1 µM ZnAF-2; closed circle, 1 µM ZnAF-2 + 1 µM Zn2+
;
open triangle, 1 µM ZnAF-2F; closed triangle, 1 µM ZnAF-2F + 1 µM
Zn2+. These data were fitted to the following equations: ZnAF-2F + Zn2+
(shown as a solid line), F ) 973/(1 + 104.9-pH), R ) 1.00; ZnAF-2 + Zn2+
(shown as a broken line), F ) 1456/(1 + 106.2-pH), R ) 1.00. F )
fluorescence intensity.
Figure 3. The relative fluorescence intensity of 1 µM ZnAF-2F in the
presence of various cations. These data were measured at pH 7.4 (100 mM
HEPES buffer, I ) 0.1 (NaNO3)).
the benzoic acid moiety by proton or metal cations, resulting
in a change of HOMO levels. The pKa value of this pivotal
nitrogen for fluorescence seems to be as low as 5.0, since ZnAFs
do not fluoresce upon protonation above this pH. Under strongly
acidic conditions this nitrogen would be protonated, but the
fluorescence would be quenched because the phenolic hydroxyl
group of fluorescein would also be protonated. This insensitivity
to pH of the fluorescence is extremely useful for applications
to living cells, where pH changes are caused by certain
biological stimuli.17
Other cations which exist at high concentration in living cells,
Ca2+, Mg2+, Na+, and K+, did not enhance the fluorescence
intensity even at high concentration (5 mM), as shown in Figure
3. These results are presumably due to the poor complexation
of alkaline metals or alkaline earth metals with the chelator of
ZnAFs, based on the absence of any obvious change of UV-
visible spectrum upon addition of these cations, which also did
not interfere with the Zn2+-induced fluorescence enhancement.
Among first-row transition metal cations, Fe2+, Co2+, and Ni2+
induced a slight enhancement of the fluorescence intensity, and
Cu2+ quenched the fluorescence. Like TPEN (N,N,N′,N′-tetrakis-
(2-pyridylmethyl)ethylenediamine), ZnAFs probably form com-
plexes with these transition metal cations, but the fluorescence
is weakened because of an electron or energy transfer between
metal cation and fluorophore, which is known as the fluores-
cence quenching mechanism.22
The Effect of pH on the Fluorescence Intensity. The
fluorescence intensity of the Zn2+ complex with ZnAF-2
decreased below pH 7.0, whereas that of ZnAF-2F hardly
changed above pH 6.0 (Figure 4). The pKa values of the phenolic
hydroxyl group of fluorescein were calculated to be 6.2 for both
ZnAF-1 and ZnAF-2, and 4.9 for both ZnAF-1F and ZnAF-
2F. These shifts of pKa presumably arise from the fluorine
substitution. So, the fluorescence intensity of the Zn2+ complex
with ZnAF-1F or ZnAF-2F is not subject to interference by pH
changes under near-neutral or slightly acidic conditions.
The fluorescence intensity of ZnAF-1F or ZnAF-2F in the
absence of Zn2+ did not change above pH 5.0. Fluorescence
properties of fluorescein derivatives are believed to be controlled
by a photoinduced electron transfer (PET) process from the
benzoic acid moiety to the xanthene ring, and fluorescence on/
off switching should depend on the highest occupied molecular
orbital (HOMO) levels of the benzoic acid moiety.12d The
enhancement of fluorescence intensity of ZnAFs might be
controlled by the coordination of nitrogen directly attached to
Kinetic Analysis of the Complex Formation with Zn2+
.
Upon addition of various concentrations of Zn2+, the fluores-
cence intensity of ZnAF (1 µM) linearly increased up to a 1:1
[ZnAF]/[Zn2+] ratio, and the fluorescence and absorption spectra
did not change between 1 and 100 µM Zn2+ addition. Further-
more, a Job’s plot analysis revealed maximum fluorescence
obtained at a 1:1 ratio. These data suggested that ZnAF should
form a 1:1 complex with Zn2+. So, the apparent dissociation
constants, Kd, were determined from the fluorescence intensity
in 100 mM HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]e-
thanesulfonic acid) buffer (pH 7.4, I ) 0.1 (NaNO3)) with 10
mM NTA (nitrilotriacetic acid) and 0-9 mM ZnSO4 at 25 °C.
The fluorescence intensity data (Figure 5) were fitted to eq 1,
and Kd was calculated,
[Zn2+]f
Kd + [Zn2+]f
F ) F0 + (Fmax - F0)
(1)
where F is the fluorescence intensity, Fmax is the maximum
fluorescence intensity, F0 is the fluorescence intensity in the
absence of Zn2+, and [Zn2+]f is the free Zn2+ concentration
calculated according to the reported method.14,22 The calculated
constants shown in Table 2 suggested that ZnAFs can quanti-
tatively measure the concentration of Zn2+ around the nanomolar
range (0.1-10 nM order) as shown in Figure 5, which is
sufficient sensitivity for application in mammalian cells. Above
this range, they maximally fluoresced, and can detect the change
of Zn2+ qualitatively, although the precise concentration cannot
be determined. And the values of Kd almost did not change with
the addition of Ca2+ or Mg2+ (total concentration: 1 mM). The
(21) Paeker, A.; Rees, W. T. Analyst 1960, 85, 587-600.
(22) (a) Fabbrizzi, L.; Licchelli, M.; Pallavicini, P.; Sacchi, D.; Taglietti, A.
Analyst 1996, 121, 1763-1768. (b) de Silva, A. P.; Gunaratne, H. Q. N.;
Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C. P.; Rademacher, J. T.;
Rice, T. E. Chem. ReV. 1997, 97, 1515-1566.
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Biological Applications of Fluorescent Probes for Zinc, ZnAFs
A R T I C L E S
Figure 5. Fluorescence intensity of 1 µM ZnAF-1F (open square) or ZnAF-
2F (open triangle) as a function of the concentration of free Zn2+ in 100
mM HEPES buffer (pH 7.4, I ) 0.1 (NaNO3)) with 10 mM NTA
(Nitrilotriacetic acid) and 0-9 mM Zn2+ at 25 °C. These data were fitted
to eq 1 (shown as a solid line) and Kd was calculated.
Figure 6. Time course measurement of the fluorescence intensity. ZnAF
mixed with Zn2+ (final concentration: 1 µM ZnAF; 50 µM Zn2+) at pH
7.4 (100 mM HEPES buffer, I ) 0.1 (NaNO3)) and 25 °C. These data
were fitted to eq 5 (shown as a solid line) and kobs was calculated.
calculated by using eq 6, and koff was calculated by using eq 7.
Table 2. Apparent Dissociation Constants (Kd) and Association
and Dissociation Rate Constants (kon and koff) of ZnAFs in 100
mM HEPES Buffer (pH 7.4, I ) 0.1 (NaNO3)) at 25 °C
kobs
[Zn2+] + Kd
kon
)
(6)
(7)
compd
K (nM)
d
kona (M-1 s-1
)
koff (s-1
)
ZnAF-1
ZnAF-2
ZnAF-1F
ZnAF-2F
0.78
2.7
2.2
5.5
4.3 × 106
3.1 × 106
3.5 × 106
3.2 × 106
3.4 × 10-3
8.4 × 10-3
7.7 × 10-3
1.8 × 10-2
koff ) Kdkon
The calculated values of these constants are shown in Table
2. The kon values of ZnAFs were almost the same, (3∼4) ×
106 M-1 s-1, implying that the complexation is sufficiently fast
to detect an increase of Zn2+ concentration within a few hundred
milliseconds. These values are 2 orders of magnitude smaller
than those of complexation of Ca2+ with fluorescent probes for
Ca2+ such as fura-2, indo-2 and quin-2,24 possibly because of
the nature of the metal cations, for example, the water exchange
rate constant, which correlates with association rates, of Zn2+
(3 × 107 s-1) is smaller than that of Ca2+ (108 s-1). Although
the values of koff are relatively small, this is inevitable for high-
affinity probes such as ZnAFs, whose Kd values are in the
nanomolar range, and the dissociation rates are considered to
be not too slow for the reversible assay of Zn2+ concentration.
If other probes, with different properties, were needed, they
could be easily synthesized from compound 2 or 2′ and a
suitable chelating group. For example, the probe, whose acceptor
for Zn2+ is N-(2-pyridylmethyl)ethylenediamine instead of N,N-
bis(2-pyridylmethyl)ethylenediamine, was synthesized via a
similar synthetic route. It had almost the same kon value as
ZnAFs and a 5 orders of magnitude larger Kd, so that its
dissociation rate was sufficiently fast (data not shown).
Biological Applications of ZnAFs. We examined the ap-
plication of ZnAF-2F to cultured cells or hippocampal slices.
Cultured macrophages (RAW 264.7) were incubated with ZnAF-
2F for dye loading, but the cells did not stain, indicating that
ZnAF-2F could not permeate through the cell membrane (data
not shown). So, we newly synthesized a diacetyl derivative,
ZnAF-2F DA. ZnAF-2F DA is more lipophilic, and so was
expected to permeate well into cells and then be transformed
to ZnAF-2F by esterase in the cytosol, where the dye would be
a These data were measured under pseudo-first-order conditions, final
concentrations: 1 µM ZnAFs; 50 µM ZnSO4.
Kd of ZnAF-1F and ZnAF-2F were slightly lower than that of
ZnAF-1 or ZnAF-2, probably because of the electron-withdraw-
ing property of fluorine.
Next, we measured the association (kon) and dissociation (koff)
rate constants of ZnAF-1, ZnAF-2, ZnAF-1F, and ZnAF-2F at
pH 7.4 (100 mM HEPES buffer, I ) 0.1 (NaNO3)), 25 °C using
a stopped-flow spectrofluorimeter. The rate constants of the
ZnAF-Zn2+ complex formation are given by:
d[ZnAF - Zn2+]
) kon[ZnAF][Zn2+] - koff[ZnAF - Zn2+]
dt
(2)
In this experiment, the final concentration of Zn2+ (50 µM) is
much higher than that of ZnAF (1 µM), so eq 2 can be
approximated to
d[ZnAF - Zn2+]
) kon′[ZnAF] - koff[ZnAF - Zn2+] (3)
dt
where kon′ ) kon[Zn2+]. Thus,
Fmax - F
Fmax - F0
ln
) - kobs
t
(4)
(5)
(
)
F ) Fmax - (Fmax - F0) exp(- kobst)
where kobs is kon′ + koff, F is the fluorescence intensity, Fmax is
the maximum fluorescence intensity, and F0 is the fluorescence
(23) Fahrni, C. J.; O’Halloran, T. V. J. Am. Chem. Soc. 1999, 121, 11448-
11458.
(24) (a) Quast, U.; Labhardt, A. M.; Doyle, V. M. Biochem. Biophys. Res. Comm.
1984, 123, 604-611. (b) Jackson, A. P.; Timmerman, M. P.; Bagshaw, C.
R.; Ashley, C. C. FEBS Lett. 1987, 216, 35-39. (c) Kao, J. P. Y.; Tsien,
R. Y. Biophys. J. 1988, 53, 635-639.
intensity in the absence of Zn2+
.
The time-dependent fluorescence intensity data (Figure 6)
were fitted to eq 5, and kobs was obtained. Then, kon was
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A R T I C L E S
Hirano et al.
Figure 7. (a) Bright-field and (b, c) fluorescence images of RAW 264.7 loaded with ZnAF-2F DA. The cells were incubated with 10 µM ZnAF-2F DA for
0.5 h at 37 °C. Then the cells were washed with PBS and fluorescence excited at 470-490 nm was measured at 20 s intervals. At 2 min, 5 µM pyrithione
and 50 µM ZnSO4 were added to the medium, and 100 µM TPEN was added at 10 min. Fluorescence images are shown in pseudo-color and correspond to
the fluorescence intensity data in (d), which showed the average intensities of the corresponding areas (1-3, intracellular region; 4, extracellular region).
Conclusion
retained for a long time. The cells were incubated with 10 µM
ZnAF-2F DA for 30 min, which was a sufficient time for
intracellular accumulation of ZnAF-2F judging from the weak
fluorescence seen in the intracellular regions (Figure 7b). After
washing the stained cells, we added Zn2+ (50 µM) and the Zn2+
ionophore, pyrithione (2-mercaptopyridine N-oxide, 5 µM), to
the medium at 2 min, which induces an increase of intracellular
Zn2+. As a result, the fluorescence in intracellular regions
(Figure 7d, 1-3) was increased, although that in extracellular
regions (Figure 7d, 4) almost did not change. This fluorescence
was decreased by extracellular addition of the cell-permeable
transition metal chelator TPEN (100 µM) at 10 min. These
results suggested that ZnAF-2F DA can be used to monitor
changes of intracellular Zn2+, and should therefore be useful
for clarifying the role of Zn2+ in biological processes in which
the intracellular concentration of Zn2+ is important, for example,
cell death induced by ischemia.6
In addition to the cultured cell systems, we applied ZnAF-
2F DA to hippocampal slices. Acute rat hippocampal slices were
incubated with 10 µM ZnAF-2F DA for 1.5 h at room
temperature for dye loading. The fluorescent image of a dye-
loaded slice is shown in Figure 8a. The fluorescence was dense
in the hilus and the stratum lucidum of CA3. This distribution
is also detected by other staining methods, such as TSQ,9b where
Zn2+ is concentrated in the vesicles, which is numerous in mossy
fiber synapses at the CA3 region. The fluorescence was
quenched by incubation of the slices with 150 µM TPEN for
30 min, as shown in Figure 8b. Thus, ZnAF-2F DA can be
used to detect intracellular free Zn2+ in hippocampal slices.
We have developed fluorescent probes for Zn2+, ZnAF-1,
ZnAF-2, ZnAF-1F, and ZnAF-2F by utilizing N,N-bis(2-
pyridylmethyl)ethylenediamine as an acceptor for Zn2+ and
fluorescein as a fluorophore. The selectivity for Zn2+ is very
high, and fluorescence is not induced by other biologically
important cations such as Ca2+ or Mg2+. The fluorescence
intensity of ZnAFs in the absence of Zn2+ was small and was
affected by pH change. The complex with Zn2+ of ZnAF-1F or
ZnAF-2F emitted stable fluorescence under near-neutral and
slightly acidic conditions, which is favorable for detecting Zn2+
quantitatively in living cells. By the incubation of cultured cells
or hippocampal slices with a cell-permeable derivative, ZnAF-
2F DA, which is hydrolyzed to ZnAF-2F in the cytosol,
intracellular Zn2+ concentration, could be monitored. In addition
to the intracellular studies, extracellular Zn2+ concentration could
be detected by the addition of ZnAF-2F to the medium, because
ZnAF-2F does not permeate through the cell membrane. This
property should be useful especially for neuroscience experi-
ments, because extracellular Zn2+ modulates the function of ion
channels or receptors, and its effect is dependent on the
concentration.2 ZnAFs have appropriate sensitivity and associa-
tion rate constants for studies on the biological functions of
Zn2+
.
Experimental Section
General Information. All reagents and solvents were of the highest
commercial quality and were used without purification. ZnAF-1 and
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Biological Applications of Fluorescent Probes for Zinc, ZnAFs
A R T I C L E S
acid) buffer (pH 8.5).25 The fluorescence intensity (excitation 492 nm;
emission 514 nm) of samples diluted with these buffers was measured.
Stopped-Flow Measurements. Stopped-flow experiments were
performed with a SF-61 DX2 double-mixing stopped-flow spectrof-
luorimeter (Hi-Tech, Salisbury, UK) equipped with a monochromator
between the 75 W xenon light source and the reaction cuvette excitation
window. The quartz sample cuvette and syringes containing the
reactants were maintained at 25 ( 0.1 °C by a circulating water bath.
The pneumatic cylinder was driven by a nitrogen pressure of 6 bar,
which resulted in an instrument dead-time of 1 ms. A solution of 2
µM ZnAF in 100 mM HEPES buffer (pH 7.4, I ) 0.1 (NaNO3)) was
combined with an equal volume of 100 µM ZnSO4. Fluorescence was
measured with excitation at 492 nm and with emission light (>530
nm), using a 530 cutoff filter.
Imaging System. The imaging system was comprised of an inverted
fluorescence microscope (IX-70, Olympus, Tokyo, Japan), cooled
charge-coupled device (cooled CCD), camera (Hamamatsu Photonics,
Hamamatsu, Japan), and an image processor (Argus 50, Hamamatsu
Photonics). The microscope was equipped with a xenon lamp, an
objective lens (×40 (RAW 264.7) or ×4 (hippocampus slice)), an
excitation filter (470-490 nm), a dichroic mirror (505 nm), and an
emission filter (515-550 nm).
Preparation of Cells. RAW 264.7 mouse macrophage cells were
kindly provided by Prof. M. Iino (Graduate School of Medicine, The
University of Tokyo). Cells were passed in Dulbecco’s modified Eagle’s
medium (DMEM, Gibco BRL) containing 10% fetal bovine serum (JRH
Bioscience), 1% penicillin (Gibco BRL), and 1% streptomycin (Gibco
BRL) at 37 °C in a 5% CO2/95% air incubator. Cells were passed 12
h before dye loading onto a glass-bottomed dish at the density of 3 ×
105 cells/mL. Then the cells were rinsed with PBS (phosphate-buffered
saline, Sigma Diagnostics) and incubated with PBS containing 10 µM
ZnAF-2F DA for 30 min at 37 °C. The cells were then washed with
PBS twice, and mounted on a microscope stage.
Preparation of Rat Hippocampal Slices. The whole brains of adult
Wistar rats (male, 200-250 g) were removed quickly under ether
anesthesia and placed in ice-cold ACSF (artificial cerebrospinal fluid),
which was aerated with 95% O2/5% CO2. The composition of ACSF
was 124 mM NaCl, 2.5 mM KCl, 26 mM NaHCO3, 1.25 mM NaH2-
PO4, 2.0 mM CaCl2, 1.0 mM MgCl2, and 10 mM glucose. The
hippocampus was isolated, placed on an agar plate, and sliced into 300
µm thick slices with a rotary slicer (Model DTY 7700, Dosaka Co.,
Ltd., Osaka, Japan). The fresh hippocampal slices were incubated in
ACSF equilibrated with 95% O2/5% CO2 for more than 30 min at room
temperature. Then they were loaded with 10 µM ZnAF-2F DA for 1.5
h at room temperature. Postincubation with ACSF for 30 min was
carried out to wash out the extracellular dye. A small flow-through
chamber, whose base consisted of a thin glass coverslip, was placed
on a microscope stage. Then, a dye-loaded slice was placed in the
chamber and continuously perfused (2.5 mL/min) with equilibrated
ACSF at 33-34 °C. The slice was held in place by a metal wire ring
with a stretched nylon net.
Figure 8. Fluorescence images of ZnAF-2F DA loaded rat hippocampus
slices. The slices were incubated with 10 µM ZnAF-2F DA for 1.5 h at
room temperature. Then the slices were washed with ACSF for 0.5 h and
fluorescence images excited at 470-490 nm were measured (a) before and
(b) after incubation with 150 µM TPEN for 0.5 h at room temperature.
ZnAF-2 were prepared as described previously.14 ZnAF-1F, ZnAF-
2F, and ZnAF-2F DA were prepared as described in the Supporting
Information.
Fluorometric Analysis. Fluorescence spectroscopic studies were
performed with an Hitachi F4500 (Tokyo, Japan). The slit width was
2.5 nm for both excitation and emission. The photomultiplier voltage
was 950 V. ZnAFs were dissolved in DMSO (for fluorometric analysis;
Dojindo, Kumamoto, Japan) to obtain 10 mM stock solutions. Relative
quantum yields of fluorescence were obtained by comparing the area
under the corrected emission spectrum of the test sample at 492 nm
excitation with that of a solution of fluorescein in 0.1 M NaOH
(quantum yield: 0.85).24 UV-visible spectra were measured with a
Shimadzu UV-1600 (Tokyo, Japan).
Effect of pH on the Fluorescence Intensity. The following buffers
were prepared: 100 mM Cl2CHCOOH-Cl2CHCOONa buffer (pH 2.0),
100 mM ClCH2COOH-ClCH2COONa buffer (pH 3.0), 100 mM
AcOH-AcONa buffer (pH 4.0-5.0), 100 mM MES (2-morpholino-
ethanesulfonic acid) buffer (pH 5.5-6.5), 100 mM HEPES buffer (pH
7.0-8.0), and 100 mM CHES (N-cyclohexyl-2-aminoethanesulfonic
Acknowledgment. We thank Prof. K. Yamaguchi and Mr.
S. Sakamoto of Chiba University for HRMS measurement. We
are also grateful to Prof. Y. Kudo, Mr. Y. Kawamura, and Ms.
N. Matsumoto of the Tokyo University of Pharmacy and Life
Science for advice on the preparation of rat hippocampus slices,
and to Prof. M. Iino of The University of Tokyo for donating
RAW 264.7. This work was supported in part by the Ministry
of Education, Science, Sports and Culture of Japan (Grant Nos.
11794026, 12470475, 12557217, 11771467, and 12045218), by
(25) (a) Good, N. E. Arch. Biochem. Biophys. 1962, 96, 653-661. (b) Good,
N. E.; Winget, G. D.; Winter, W.; Connolly, T. N.; Izawa, S.; Singh, R.
M. M. Biochemistry 1966, 5, 2, 467-477.
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A R T I C L E S
Hirano et al.
the Mitsubishi Foundation, and by the Research Foundation for
Opt-Science and Technology. T.H. thanks the Japan Society
for the Promotion of Science for a JSPS Research Fellowship
for Young Scientists. K.K. is grateful for financial support from
Nissan Science Foundation, Shorai Foundation of Science and
Technology, and Uehara Memorial Foundation.
Supporting Information Available: Synthetic details and
characterization of ZnAF-1F, ZnAF-2F, and ZnAF-2F DA
(PDF). This material is available free of charge via the Internet
at http://pubs.acs.org.
JA025567P
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6562 J. AM. CHEM. SOC. VOL. 124, NO. 23, 2002