Design and synthesis of zinc-selective chelators for extracellular applications

Article abstract of DOI:10.1021/ja044697q

Zinc (Zn²⁺) is found in every cell in human bodies. A few millimolar of free Zn²⁺ exists in the vesicles of presynaptic neurons in the mammalian brain and is released by synaptic activity or depolarization, modulating the function of certain ion channels and receptors. Although various chemical tools for measuring Zn²⁺ in biological samples, such as fluorescent probes for Zn²⁺, have been developed, Zn²⁺-selective chelators have room to be improved. Research on Zn²⁺ signals in the brain has traditionally employed several chelators, which have several shortcomings for biological applications. Here we report the design, synthesis, and properties of new membrane-impermeable chelators selective for Zn²⁺ and describe biological applications in hippocampal slices. As a result, our newly designed chelator revealed the first biological implication that presynaptic Zn²⁺ can be released in the CA1 region. This confirms the utility of these new chelatotrs as extracellular Zn²⁺ chelators for biological applications. Copyright

Full text of DOI:10.1021/ja044697q

Published on Web 12/24/2004
Design and Synthesis of Zinc-Selective Chelators for Extracellular
Applications
Eri Kawabata,† Kazuya Kikuchi,†,‡ Yasuteru Urano,† Hirotatsu Kojima,† Akira Odani,§ and
Tetsuo Nagano*,†
Graduate School of Pharmaceutical Sciences, The UniVersity of Tokyo, 7-3-1 Hongo,
Bunkyo-ku,Tokyo 113-0033, Japan, Presto, JST Corporation, Kawaguchi, Saitama, Japan, and
Department of Chemistry, Graduate School of Science, Nagoya UniVersity, Nagoya, Aichi, Japan
Received September 2, 2004; E-mail: tlong@mol.f.u-tokyo.ac.jp
Zinc (Zn2+) is found in every cell in the human body and is
mostly tightly bound to proteins as a key component of numerous
enzymes and transcription factors.1,2 Chelatable Zn2+ co-localizes
with glutamate in the synaptic vesicles of certain glutamatergic
vesicles in the mammalian brain, including the hippocampus,
amygdala, and neocortex.3 Free Zn2+ exists at a concentration of a
Figure 1. Structures of new zinc-selective chelators.
few millimolar in the vesicles of presynaptic neurons and is released
during synaptic activity or depolarization, modulating the function
Table 1. Stability Constants of Chelators
of certain ion channels and receptors.4-7 Many reports describe the
log K
significance of Zn2+ in biological systems,8-13 but its mechanisms
of action are poorly understood.
+
Mn
DPESAa
TPESAa
TPENb
EDTAb
Although various chemical tools for measuring Zn2+ in biological
samples, such as fluorescence probes for Zn2+, have been
developed,14-23 better Zn2+-selective chelators are still needed.
Research on Zn2+ signals in the brain has traditionally employed
several chelators, though they have various shortcomings for
biological applications. Use of N,N,N′,N′-tetrakis(2-pyridylmethyl)-
ethylenediamine (TPEN), a classical membrane-permeable Zn2+
chelator, does not allow selective manipulation of extracellular or
intracellular Zn2+. On the other hand, calcium ethylenediamine-
tetraacetic acid (CaEDTA), an extracellular (membrane-imperme-
able) Zn2+ chelator, has the disadvantage of slow kinetics of Zn2+
binding. Concentrations of CaEDTA that are sufficient to chelate
synaptic Zn2+ at equilibrium do not effectively chelate Zn2+ within
the period of tens to hundreds of microseconds that it takes Zn2+
to cross the synapse and interact with various postsynaptic sites.24
In contrast, a higher concentration of CaEDTA markedly reduces
the Zn2+ and Ca2+ signals.25 This phenomenon makes it difficult
to interpret the action mechanism of Zn2+ under physiological
conditions, where change of Ca2+ concentration can also induce
various responses. Especially in electrophysiological studies of ion
channels and neuronal activities, reduction of extracellular Ca2+
can result in neurological side effects. The development of new,
more rapid Zn2+ chelators which have low affinity for Ca2+ is
required for the clarification of the mechanism of synaptic release
of Zn2+. We report here the design, synthesis, and properties of
new Zn2+ chelators, and we describe biological application in
hippocampal slices.
Ca2+
Mg2+
Zn2+
4.20 ( 0.11
3.97 ( 0.10
11.8 ( 0.01
2.47 ( 0.10
2.66 ( 0.10
12.3 ( 0.03
4.40
1.70
15.4
10.7
8.64
16.3
a Stability constants of DPESA and TPESA were determined at 25 °C,
I ) 0.1. The determined value ( SD is given. b From the SCDatabase
(IUPAC and Academic Software): 25 °C, I ) 0.1.
methods (Table 1 and Table S1, Supporting Information). The log
K values of DPESA and TPESA for Zn2+ are 11.8 and 12.3,
respectively, showing that these two compounds have high affinity
for Zn2+. The log K values for Ca2+ are 4.20 and 2.47, and those
for Mg2+ are 3.97 and 2.66, respectively, indicating low affinity
for these metal ions.
To compare the relative association rate constants of the new
chelators for Zn2+ with those of traditional chelators, competition
analysis was performed between Zn2+-fluorescence probe complex
and Zn2+-selective chelators (Figure S3, Supporting Information).
ZnAF-214a was used as a fluorescence probe for Zn2+; its log Kobs
value (Kobs is the apparent association constant at pH 7.4, I ) 0.10
M NaNO3) has been reported as 8.57. The fluorescence intensity
of ZnAF-2 (1.0 µM) linearly increased up to a 1:1 [ZnAF-2]/[ Zn2+
]
ratio, and the maximum fluorescence was obtained with 1.0 µM
Zn2+ addition. Then 10 µM chelator was added and the time course
of fluorescence decrease was compared among DPESA, TPESA,
TPEN, and CaEDTA. The addition of 10 µM TPESA rapidly
decreased the fluorescence with a half-life of 18.6 s, which is
comparable to that of TPEN (12.2 s). This result suggested that
TPESA can reduce the concentration of synaptically released Zn2+
rapidly, without changing the extracellular Ca2+ concentration. On
the other hand, DPESA reduced the fluorescence with a half-life
of 67.0 s, which is similar to the value of 65.4 s obtained for
CaEDTA. Considering that DPESA may have the same affinity
for Zn2+ as ZnAF-2, whereas the other chelators have higher
affinities (Table 1), the above result suggests rapid Zn2+ chelation.
To examine the membrane permeability of the new chelators,
we applied them to hippocampal slices (Figure S4, Supporting
Information). Acute rat hippocampal slices were incubated with
10 µM ZnAF-2 DA14a for 1.5 h at room temperature for dye loading.
We designed (4-{[2-(bis-pyridin-2-ylmethylamino)ethylamino]-
methyl}phenyl)methanesulfonic acid, sodium salt (DPESA), and
[4-({[2-(bis-pyridin-2-ylmethylamino)ethyl]pyridin-2-ylmethylamino}-
methyl)phenyl]methanesulfonic acid, sodium salt (TPESA) utilizing
TPEN structure for high Zn2+ selectivity and a sulfonic acid moiety
for hydrophilicity (Figure 1). The acid dissociation constants and
metal chelate stability constants were determined by potentiometric
† The University of Tokyo.
‡ Presto, JST Corporation.
§ Nagoya University.
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10.1021/ja044697q CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
presynaptic Zn2+ can be released in the CA1 region. This confirms
the utility of these new chelators as extracellular Zn2+ chelators
for biological applications.
In conclusion, we have developed new membrane-impermeable
Zn2+-selective chelators, DPESA and TPESA, with the advanta-
geous characteristics of high Zn2+ selectivity and rapid chelation,
that can be applied to cell-biological studies to shed additional light
on the function of synaptically released Zn2+
.
Acknowledgment. This work was supported in part by the
Ministry of Education, Culture, Sports, Science and Technology
of Japan (Grant Nos. 16370071 to T.N., 15681012 and 16048206
to K.K.). K.K. was also supported by the Sankyo Foundation, the
Kanagawa Academy of Science, and the Suzuken Memorial
Foundation.
Supporting Information Available: Synthesis, experimental de-
tails, and characterization of DPESA and TPESA. This material is
available free of charge via the Internet at http://pubs.acs.org.
Figure 2. Application of the chelators under ischemic conditions. Dye-
loaded slices were exposed to anoxic-aglycemiic ACSF for 17 min (from
2 to 19 min). (a) Fluorescence images at 0, 10, and 22 min after the
measurement. The fluorescence ratio in the plot (b) is the ratio of the
fluorescence intensity to the initial intensity of the corresponding area in
the image at 0 min. (c) Histogram showing the effects of the chelators on
the fluorescence change of ZnAF-2 DA-loaded slices under anoxia-
aglycemia. Fluorescence ratio in the histogram is the ratio of the fluorescence
intensity at 10 min after the start of the measurement to the initial intensity
of the corresponding area in the image at 0 min. Each column, except for
TPEN, shows the mean ( SE.
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