Highly zinc-selective fluorescent sensor molecules suitable for biological applications

Full text of DOI:10.1021/ja002467f

J. Am. Chem. Soc. 2000, 122, 12399-12400
12399
Highly Zinc-Selective Fluorescent Sensor Molecules
Suitable for Biological Applications
Tomoya Hirano, Kazuya Kikuchi, Yasuteru Urano,
Tsunehiko Higuchi, and Tetsuo Nagano*
Graduate School of Pharmaceutical Sciences
The UniVersity of Tokyo, 7-3-1 Hongo, Bunkyo-ku
Tokyo 113-0033, Japan
ReceiVed July 11, 2000
Figure 1. Structure of newly synthesized fluorescent Zn2+ sensor
molecules.
Zinc (Zn2+) is the second most abundant heavy metal ion after
iron, and it is an essential component of many protein scaffolds
(e.g., carbonic anhydrase and zinc finger proteins).1 Chelatable
Zn2+ is released from nerve terminals by excitatory signals,2 and
binds to the N-methyl-D-aspartate (NMDA) receptor, changing
its function.3 Zn2+ is co-stored with insulin in secretory vesicles
of pancreatic â-cells, and is released when insulin is secreted.4
Zn2+ also suppresses apoptosis,5 and induces the formation of
â-amyloid,6 which is thought to be related to the etiology of
Alzheimer’s disease.
Although Zn2+ has many important cellular roles, little is known
about the cellular regulation of Zn2+ in comparison with other
cations such as Ca2+, Na+, K+, etc. Therefore, several chemical
tools for measuring Zn2+ in living cells have recently been
developed to clarify its physiological significance.7-14 There are
two types of fluorescent sensor molecules for Zn2+, one based
on a quinoline structure excitable with UV light (TSQ,7 Zinquin,8
and TFLZn9) and the other based on fluorescein excitable with
visible light.10-12 A cell-permeable Zn2+ sensor molecule based
on fluorescein (Zinpyr-1) was reported recently.10 Zinpyr-1
fluoresces strongly upon addition of Zn2+ to cells. However, it
has the disadvantages that the basal fluorescence is high (quantum
yield, 0.39) and is pH-sensitive with a pKa of 8.3. Thus, the
fluorescence can be changed by intracellular pH changes under
physiological conditions, and such pH changes are observed in
many cells exposed to certain biological stimuli.15
Fluorescein is a convenient fluorophore for biological experi-
ments, since it has a high fluorescence quantum yield in aqueous
solution, and its excitation wavelength is in the visible range,
which does not cause severe cell damage. This wavelength is free
from interference by autofluorescence from biological molecules
and is also suitable for fluorescence confocal microscopy with
an Ar laser. We have explained the fluorescence quenching of
aminofluorescein in terms of photoinduced electron transfer
(PET).11 Aminofluorescein itself does not fluoresce due to the
high HOMO level of its electron-donating group. If this electron
donation is hindered by complex formation with a cation using
the lone pair of nitrogen or by chemical conversion to a less
electron-donating group, the HOMO level is lowered, resulting
in fluorescence with a high quantum yield. On the basis of this
approach, we have developed several fluorescent sensor molecules
for nitric oxide,16 singlet oxygen,17 and Zn2+ 11
This method is
.
advantageous for providing a wide range of sensor molecules,
because if we can design and synthesize a suitably specific
reactive moiety which is linked to aminofluorescein, we can obtain
sensor molecules suitable for different types of analytes. It is also
advantageous that the fluorescence quantum yield of aminofluo-
rescein is very low, so if the sensor molecule is converted to
fluorescent form, the measured fluorescence intensity is essentially
entirely due to the analyte.
We have already reported fluorescent Zn2+ sensor molecules,
ACF-1 and ACF-2, which are excitable with visible light, for
biological applications.11 However, improvements are desirable
in two respects, i.e., the slow complex formation rate and the
small quantum yield. ACFs require about 100 min for completion
of the complex formation due to the properties of the macrocyclic
polyamine ring, which is the acceptor of Zn2+. We therefore
designed ZnAF-118 and ZnAF-2, utilizing N,N,N′,N′-tetrakis(2-
pyridylmethyl)ethylenediamine (TPEN) as the acceptor of Zn2+
(Figure 1). Fluorescein was employed as a fluorophore instead
of 6-hydroxy-9-phenylfluorone, the fluorophore of ACFs, because
of its larger quantum yield.
ZnAF-1 and ZnAF-2 were synthesized from the corresponding
aminofluoresceins by using 4-nitrobenzenesulfonyl chloride for
alkylation of the amine.19 At pH 7.5 (100 mM HEPES buffer, I
) 0.1 (NaNO3)), both compounds showed almost no fluorescence.
However, upon addition of Zn2+, the fluorescence intensity was
increased by 17-fold for ZnAF-1 and 51-fold for ZnAF-2 (Table
2). The fluorescence intensity of the free bases was little
influenced by pH. Thus, the basal fluorescence intensity should
be little affected by physiological pH changes. The excitation and
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(18) AF stands for Aminofluorescein.
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10.1021/ja002467f CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/28/2000

12400 J. Am. Chem. Soc., Vol. 122, No. 49, 2000
Communications to the Editor
may be as follows. When Zn2+ is added to ZnAF-1, the four
nitrogen atoms of the acceptor, N,N-bis(2-pyridylmethyl)ethyl-
enediamine, form a four-coordinate complex, resulting in strong
fluorescence augmentation. However, the ionic radius of Cd2+ is
larger than that of Zn2+, so carboxylate coordinates instead of
nitrogen adjacent to the benzene ring. This blocks the on-off
mechanism of fluorescence, resulting in no fluorescence aug-
mentation, because there is no large HOMO change. In the case
of ZnAF-2, the carboxylate cannot participate in complex
formation because of its geometry. To test this hypothesis, the
carboxylate of ZnAF-1 was derivatized to its methyl ester using
methanol and concentrated sulfuric acid23 to hinder the participa-
tion of the carboxylate in complex formation. The selectivity of
ZnAF-1 methyl ester (Me-ZnAF-1) against Cd2+ was almost the
same as that of ZnAF-2, supporting the idea that the carboxyl
group is important for the selective augmentation of fluorescence
intensity by Zn2+. Neither Fe2+ nor Fe3+ enhanced the fluores-
cence intensity. Cu2+ formed complexes with ZnAF-1 and ZnAF-
2, resulting in quenching of fluorescence. Other cations, which
Figure 2. (a) Excitation spectra (emission at 514 nm) and (b) emission
spectra (excitation at 492 nm) of ZnAF-2 (5 µM) in the presence of
various concentrations of Zn2+ ranging from 0 to 5.0 µM. These spectra
were measured at pH 7.5 (100 mM HEPES buffer, I ) 0.1 (NaNO3)).
a
Table 1. Chemical Properties of ZnAFs with and without Zn2+
free
Zn2+
ꢀb
compd
ꢀb
Φc
Φc
Kd (M)
ZnAF-1 7.4 × 104 (489) 0.022 6.3 × 104 (492) 0.23 7.8 × 10-10
exist at high concentration under physiological conditions, Ca2+
,
ZnAF-2 7.8 × 104 (490) 0.023 7.6 × 104 (492) 0.36 2.7 × 10-9
Mg2+, Na+, and K+, did not enhance the fluorescence intensity
even at very high concentration (5 mM) (Table 2). It was also
confirmed that these ions did not interfere with Zn2+-enhanced
fluorescence (data not shown).
a All data were obtained at pH 7.5 (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 (0.95) in 0.1 N NaOH
as a standard.20
The complexes of ZnAFs and Zn2+ were formed immediately
upon Zn2+ addition, as determined from the time course of
fluorescence intensity.24 This characteristic is convenient for
biological applications. The fluorescence intensity of the Zn2+
complex was dependent on pH, with a pKa value of 6.2 for both
ZnAF-1 and ZnAF-2,24 but was almost stable over the physi-
ological pH range.
Table 2. Selectivity of ZnAF-1, ZnAF-2, and Me-ZnAF-1 toward
Other Cationsa
b
b
b
b
c
c
compd
free Zn2+ Cd2+ Co2+ Ni2+ Ca2+ Mg2+
ZnAF-1
ZnAF-2
110 1881
80 4105 1462
181
118
173
121
218
741
225
104
89
90
115
116
82
To determine the cell permeability of ZnAFs, cultured mac-
rophage (RAW 264.7) were incubated with PBS (Phosphate
Buffered Saline) containing ZnAF-2. As a result, the cells did
not stain, that means ZnAF-2 could not permeate through the cell
membrane. So we prepared a diacetyl derivative of ZnAF-2
(ZnAF-2 DA), which was synthesized from ZnAF-2, acetic
anhydride, and cesium carbonate.16 ZnAF-2 DA was more
lipophilic, and once ZnAF-2 DA permeates into the cell, it will
be transformed into ZnAF-2 by esterase in the cytosol. RAW
264.7 were incubated with PBS containing ZnAF-2 DA, and the
cells stained by ZnAF-2 (data not shown). These data indicate
Me-ZnAF-1 61 4039 2398
a All data were obtained at pH 7.5 (100 mM HEPES buffer, I ) 0.1
(NaNO3)) and expressed as arbitrary units. b Those ions (5 µM) were
added to 5 µM sensor molecule. c Those ions (5 mM) were added to 5
µM sensor molecule.
emission maxima did not change upon addition of Zn2+: excitation
at 492 nm and emission at 514 nm for both ZnAF-1 and ZnAF-2
(Table 1 and Figure 2). The apparent dissociation constant, Kd,
was determined as 0.78 nM (ZnAF-1) or 2.7 nM (ZnAF-2) using
Zn2+ and pH-buffered solutions (Table 1).21 These values indicate
that these molecules can be used in the sub-nM range, which is
the same as that for Zinquin in undifferentiated eukaryotic cells.22
The detection limit of ZnAFs is also in the sub-nM range, which
affords sufficient sensitivity for application in mammalian cells.
Selectivity toward other cations was next examined (Table 2).
Although the Zn2+-induced fluorescence augmentation was
smaller with ZnAF-1 than with ZnAF-2, the selectivity of ZnAF-1
against Cd2+ was much higher than that of ZnAF-2 (Table 2).
Upon addition of Cd2+, the fluorescence intensity of ZnAF-1
almost did not increase. However, other fluorescent sensor
molecules for Zn2+ fluoresced upon addition of Cd2+, for example,
10-fold for Zinquin8 and 6-fold for Newport Green.12 ZnAF-1 is
the first Zn2+ sensor molecule that can distinguish Cd2+ from
Zn2+. This selectivity may originate from the carboxylic acid
moiety on the benzene ring. We speculate that the mechanism
that ZnAF-2 DA can be used as a cellular probe for Zn2+
.
In conclusion, we have developed new fluorescent Zn2+ sensor
molecules, ZnAFs, which possess the characteristics of improved
selectivity and faster complex formation. Since these sensor
molecules only fluoresce after the coordination with Zn2+, they
should be useful for studies on the biological functions of Zn2+
.
Acknowledgment. We thank Dr. T. Kan for advice on the synthesis.
This work was supported in part by the Ministry of Education, Science,
Sports and Culture of Japan (grant numbers 11794026, 12470475,
12557217 to T.N. and 11771467 and 12045218 to K.K.), as well as the
Mitsubishi Foundation and the Research Foundation for Opt-Science and
Technology. K.K. gratefully acknowledges financial support from Nissan
Science Foundation, Shorai Foundation of Science and Technology, and
Uehara Memorial Foundation.
Supporting Information Available: Synthesis, experimental details,
and characterization of ZnAF-1 and ZnAF-2 (PDF). This material is
available free of charge via the Internet at http://pubs.acs.org.
(20) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.;
Kluwer Academic/Plenum: New York, 1999.
JA002467F
(21) Fahrni, C. J.; O’Halloran, T. V. J. Am. Chem. Soc. 1999, 121, 11448-
11458.
(23) Adamczyk, M.; Grote, J.; Moore, J. A. Bioconj. Chem. 1999, 10, 544-
(22) Nasir, M. S.; Fahrni, C. J.; Suhy, D. A.; Kolodsick, K. J.; Singer, C.
P.; O’Halloran, T. V. J. Biol. Inorg. Chem. 1999, 4, 775-783.
547.
(24) For experimental details, see Supporting Information.