Development of ratiometric fluorescent probe for zinc ion based on indole fluorophore

Article abstract of DOI:10.1016/j.tetlet.2009.01.026

A water-soluble ratiometric fluorescent probe ZID-1 has been developed on the basis of an internal charge transfer (ICT) mechanism. Upon complexation with Zn²⁺ under physiological conditions, ZID-1 exhibits a significant blue shift of 77 nm in

Full text of DOI:10.1016/j.tetlet.2009.01.026

Tetrahedron Letters 50 (2009) 1345–1347
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Development of ratiometric fluorescent probe for zinc ion based
on indole fluorophore
a
a
Masayasu Taki a,b, , Yasumasa Watanabe , Yukio Yamamoto
*
a Graduate School of Human and Environmental Studies, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan
b Graduate School of Global Environmental Studies, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A water-soluble ratiometric fluorescent probe ZID-1 has been developed on the basis of an internal
charge transfer (ICT) mechanism. Upon complexation with Zn2+ under physiological conditions, ZID-1
exhibits a significant blue shift of 77 nm in the emission spectrum. The fluorescent behavior of ZID-1 sug-
gests that the pyridyl group incorporated into the fluorophore coordinates the metal ion as the fourth
Received 2 December 2008
Revised 7 January 2009
Accepted 9 January 2009
Available online 14 January 2009
ligand and affords an appropriate binding affinity (Kd = 17.1 nM) for the intracellular imaging of Zn2+
.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Ratiometric probe
Zinc ion
Indole
High affinity
Fluorescent chemosensors are used for the detection of a spe-
cific metal ion in living cells and are excellent tools for elucidating
the functions of metal ions in biological systems.1 These chemo-
sensors can be used to visualize pools of labile metal ions with high
optical sensitivity by means of fluorescence microscopy.2 In partic-
ular, ratiometric fluorescent probes that exhibit a shift in either
excitation or emission maxima upon the formation of a complex
can be used to perform quantitative measurement of a change in
metal ion concentration.3 Determination of the ratio of fluores-
cence intensities measured at two suitably selected wavelengths
results in the cancellation of artifactual factors, such as illumina-
tion intensity, photobleaching of the probe, cell thickness, and
dye concentration within cells, in the fluorescent signals. An inter-
nal charge transfer (ICT) mechanism has been widely used as the
basis for the design of ratiometric fluorescent sensors.1 Fura-2
and Indo-1 are typical examples as the ICT probe in which Ca2+
binding alters the electron-donating properties of the electron-rich
chelating group in the excitation state and causes a significant blue
shift in its fluorescence spectrum.3
that are based on quinoline,8 fluorescein,9 benzazole,10 coumarin,11
other fluorophores,12 or proteins13 have been developed. However,
the functions of Zn2+ in these tissues or even within single-celled
organisms are not fully investigated, because of the lack of suitable
ratiometric probes comparable with Fura-2 and Indo-1 for Ca2+ 3
,
using which valuable information about its intracellular behavior
has been obtained. Therefore, the development of efficient ratio-
metric probes for Zn2+, matching for Fura-2 and Indo-1, is indis-
pensable for the investigation of the biological roles of Zn2+
.
Although several kinds of ratiometric fluorescent probes have re-
cently been developed for the detection of Zn2+ 14,15
,
most of these
suffer from some weak points such as small shift in the fluores-
cence wavelength, large fluorescence increase or decrease in the
Zn2+-bound form, low water solubility, or low binding affinity to
Zn2+. Furthermore, an emission ratiometric probe is required for
imaging using two-photon excitation (TPE) fluorescence micros-
copy, which provides significant advantages over standard laser
confocal approaches.16 Therefore, there is still a requirement for
ratiometric probes that can provide a large peak shift in emission
Among a series of biologically important metal ions, zinc (Zn2+
)
spectra upon their binding with Zn2+
.
has attracted considerable attention because of its structural sig-
nificance and catalytic functions in metalloproteins.4 Although
these Zn2+ ions are often tightly bound, chelatable (weakly bound)
Zn2+ ions also exist in several tissues, including brain tissue,5 pan-
creatic tissue,6 and seminal plasma.7 To understand the roles of
Zn2+ in these tissues, a variety of Zn2+-selective fluorescent probes
Herein, we report the development of a new Zn2+-selective fluo-
rescent probe ZID-1, which utilizes a 2-(4-aminophenyl)indole
derivative as the fluorophore. Such a derivative is also utilized in
Indo-1,3 Mag-indo,17 and IndoZin,15a which are sensor molecules
for Ca2+, Mg2+, and Zn2+, respectively. In order to achieve high selec-
tivity and affinity for Zn2+ detection, we introduced a pyridylmethyl
arm into the fluorophore of ZID-1 through an oxygen atom at the o-
position of the amino group. The fluorescence properties, metal ion
selectivity, and dissociation constant for Zn2+, determined under
* Corresponding author. Tel.: +81 75 753 7871; fax: +81 75 753-6833.
E-mail address: taki@chem.mbox.media.kyoto-u.ac.jp (M. Taki).
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.01.026

1346
M. Taki et al. / Tetrahedron Letters 50 (2009) 1345–1347
physiological conditions, strongly suggest that ZID-1 can be used as
an effective ratiometric fluorescent probe for intracellular imaging
1.5
1.0
0.5
0
351
of Zn2+
.
329 nm
351 nm
1.0
0.5
329
335
The synthesis of ZID-1 started from 2-aminophenol and per-
formed in 6 steps, as outlined in Scheme 1. The amino group was
alkylated by the reaction with 2 equiv of tert-butyl bromoacetate
according to the reported procedure.18 The resulting phenol 1
was then treated with NaH and 2-picolyl chloride to yield com-
pound 2, which composes the Zn2+ binding moiety of the probe.
Successive formylation under the Vilsmeier conditions, the Wittig
reaction, and reductive cyclization using triethyl phosphite gives
indole-triester 5 as a white powder. Finally, the ester was succes-
sively hydrolyzed by boron trifluoride diethyl ether complex
(BF3ÁEt2O) and KOH to yield ZID-1, which was purified by size-
exclusion chromatography and reverse-phase chromatography.
Under physiological conditions (50 mM HEPES, pH 7.2, 0.1 M
KNO3), ZID-1 exhibited an absorption maximum at 351 nm
0
0.5
1.0
1.5
2.0
[Zn2+]/[ZID-1]
250
300
350
400
450
500
= 3.84 Â 104 MÀ1 cmÀ1), which is in good agreement with those
Wavelength, nm
(
e
reported for the apo forms of Indo-13 and its derivatives.15a,17 Upon
Figure 1. UV–vis spectral change by addition of Zn2+ to 30
HEPES buffer (pH 7.20, 0.1 M KNO3). Inset: mol ratio plots of absorbance at 329 nm
l
M of ZID-1 in 50 mM
addition of Zn2+, a decrease in the absorbance of this band and a
concomitant increase in that of
a new band at 329 nm
and 351 nm.
(e
= 3.26 Â 104 MÀ1 cmÀ1) were observed with a distinct isosbestic
that of IndoZin (3 l
M),19 in which a methyl group replaces pyridyl-
point at 335 nm ( Fig. 1). The absorption bands at 351 nm and
329 nm linearly decreased and increased, respectively, up to a
1:1 [Zn2+]/[ZID-1] ratio (Fig. 1 inset), indicating the formation of
a 1:1 complex with a strong binding affinity.
methyl group. This result reveals that the incorporated pyridine
moiety coordinates the metal ion as the fourth ligand to give a sta-
ble chelate. Furthermore, this nonlinear fitting analysis reveals that
ZID-1 is suitable for detecting [Zn2+
]
free
between 4.1 and 68 nM.20
Figure 2a shows emission spectra of ZID-1 excited at 335 nm,
which is the isosbestic point of the UV–vis traces, at various free
Zn2+ concentrations. The apo form exhibits a characteristic band
We then examined the fluorescence responses of 1 lM ZID-1 to
various biologically relevant metal ions, shown in Figure 3. Na+, K+,
Mg2+, and Ca2+, which exist in high concentrations in cells, had a very
small effect on the fluorescence spectrum, even at metal concentra-
tions as high as 5 mM; furthermore, they did not interfere with Zn2+
binding, indicating that this probe can be employed for a wide range
of biological applications using microscopic techniques. Other tran-
sition metal ions such as Fe3+, Co2+, Ni2+, and Cu2+ formed complexes
and quenched the fluorescence of ZID-1. Cd2+ induced the emission
shiftof ZID-1asobservedforZn2+. However, thesefree cationswould
have little influence on intracellular Zn2+ imaging, because of their
presence in very low concentrations.21
at 470 nm (
U = 0.097) that shifts to 393 nm (U = 0.11) with an
isoemissive point upon binding with Zn2+. The fluorescence re-
sponse of ZID-1 fits a Hill coefficient of ꢀ1 (Fig. 2a inset); it is again
confirmed that ZID-1 forms a 1:1 complex with Zn2+ even at the di-
luted concentration of ZID-1 such as 1 lM. A significant hypso-
chromic shift (77 nm) of the emission wavelength indicates that
the ICT excited state of ZID-1 (apo form) is strongly affected by
coordination with Zn2+; this in turn indicates that ZID-1 would
be more useful than reported emission ratiometric fluorescent
Zn2+ probes, the fluorescence shifts of which are much smaller than
the present value. From the plot of the fluorescence intensities at
In summary, we have developed a new ratiometric fluorescent
probe for Zn2+, ZID-1, on the basis of an ICT mechanism. Upon com-
plexation with Zn2+, this probe exhibits a significantly large blue
shift (77 nm) in the emission spectrum. It also has a binding affin-
ity such that it is suitable for biological applications; such an affin-
395 nm or 470 nm against log[Zn2+
]
(Fig. 2b), the apparent dis-
free
sociation constant Kd for Zn2+ was determined to be 17.1 0.2 nM
at pH 7.20. The Kd value of ZID-1 is comparable to those of reported
tetradentate ligands (0.1–20 nM) but considerably smaller than
t-BuOOC
COOt-Bu
t-BuOOC
COOt-Bu
NH2
N
N
N
O
a
b
c
OH
OH
1
2
t-BuOOC
COOt-Bu
R1OOC
N
COOR1
O
t-BuOOC
COOt-Bu
N
N
O
N
N
N
O
d
e
5: R1 = t-Bu
NH
f
3
5: R2 = Et
CHO
NO2
ZID-1:
R
1 = R2 = H
4
COOR2
COOEt
Scheme 1. Synthetic procedure of ZID-1. Reagents and conditions: (a) t-butyl bromoacetate, proton sponge, KI, CH3CN, reflux, overnight, 80%; (b) NaH, KI, 2-picolyl
chlorideÁHCl, THF, room temperature, overnight, 22%; (c) POCl3, DMF, room temperature, overnight, 77%; (d) Wittig reagent, K2CO3, DMF, 90 °C, overnight, 78%; (e) P(OEt)3,
125 °C, 4 h, 46%; (f) BF3ÁEt2O, KOH, then 0.1 M HCl; 100%.

M. Taki et al. / Tetrahedron Letters 50 (2009) 1345–1347
1347
2.0
1.5
1.0
ity is successfully achieved by incorporating a pyridylmethyl group
(as the binding ligand) into the fluorophore. Intracellular imaging
of zinc ions using this probe is currently in progress for evaluating
the suitability of ZID-1 for biological applications.
a
1
0
n = 0.97
395
Zn2+-bound
Acknowledgment
—1
This work was financially supported by Grant-in-Aid for Young
Scientists (B) (No. 17750155 to M.T.) from JSPS.
470
—9
—8
—7
]
free
log[Zn2+
Supplementary data
Zn2+ free
0.5
0
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2009.01.026.
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