Fluorescent sensing of IP3 with a trifurcate Zn(II)-containing chemosensing ensemble system

Article abstract of DOI:10.1021/ol801290g

A trifurcate receptor containing Zn(ll)-dipicolylamine ligands is developed for the fluorescent sensing of IP₃, myo-inositol 1,4,5-tris(phosphate), through an indicator displacement approach. The chemosensing ensemble containing the Zn(II) complex and eosin Y as indicator shows the maximum fluorescence restoration for IP₃ among various other anions including phosphate derivatives in water buffered at pH - 7.

Full text of DOI:10.1021/ol801290g

ORGANIC
LETTERS
2008
Vol. 10, No. 16
3539-3542
Fluorescent Sensing of IP3 with a
Trifurcate Zn(II)-Containing
Chemosensing Ensemble System
Dong Ju Oh and Kyo Han Ahn*
Department of Chemistry and Center for Integrated Molecular Systems, Pohang
UniVersity of Science and Technology (POSTECH), San 31 Hyoja-dong,
Pohang 790-784, Republic of Korea
ahn@postech.ac.kr
Received June 9, 2008
ABSTRACT
A trifurcate receptor containing Zn(II)-dipicolylamine ligands is developed for the fluorescent sensing of IP3, myo-inositol 1,4,5-tris(phosphate),
through an indicator displacement approach. The chemosensing ensemble containing the Zn(II) complex and eosin Y as indicator shows the
maximum fluorescence restoration for IP3 among various other anions including phosphate derivatives in water buffered at pH ) 7.
myo-Inositol 1,4,5-tris(phosphate) (IP3) is an important
signaling molecule involved in intracellular signal transduc-
tion.1 The biological importance of IP3 and its analogues
makes them attractive targets for the development of
chemosensors. Significant progress has been made in the
development of chemosensors for simple mono- and diphos-
phate ions and their nucleic acid derivatives.2 However,
chemosensors for tris(phosphate) such as IP3 or higher
homologues have been less explored, and only a few
chemical sensing systems for IP3 have been reported so far.3,4
To develop an abiological sensing system for tris(phosphate)
analytes such as IP3, we need a recognition motif that
recognizes the three asymmetrically substituted phosphate
groups on the myo-inositol backbone, cis-1,2,3,5-trans-4,6-
cyclohexanehexol. Anslyn and co-workers devised a cleft-
like receptor that provides six peripheral guanidinium groups
for chemical sensing of IP3 through a competition assay.3a
(1) (a) Berridge, M. J. Nature 1993, 361, 315. (b) Clapham, D. E. Cell
1995, 80, 259. (c) Hinterding, K.; Alonso-Diaz, D.; Waldmann, H. Angew.
Chem., Int. Ed. 1998, 37, 688. (d) Irvine, R. Curr. Biol. 2001, 11, R172.
(e) Taylor, C. W.; Thorn, P. Curr. Biol. 2001, 11, R352.
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J. Am. Chem. Soc. 1999, 121, 9463. (c) Aldakov, D.; Anzenbacher, P, Jr.
J. Am. Chem. Soc. 2004, 126, 4752. (d) Zyryanov, G. V.; Palacios, M. A.;
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Lee, K. H.; Hong, J.-I. Org. Lett. 2001, 3, 5. (f) Mizukami, S.; Nagano, T.;
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Chem. Soc. 2005, 127, 11835. (h) Han, M. S.; Kim, D. H. Angew. Chem.,
Int. Ed. 2002, 41, 3809. (i) Lee, D. H.; Im, J. H.; Son, S. U.; Chung, Y. K.;
Hong, J.-I. J. Am. Chem. Soc. 2003, 125, 7752. (j) Lee, H. N.; Swamy,
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10.1021/ol801290g CCC: $40.75
Published on Web 07/11/2008
2008 American Chemical Society

a new platform 2 on the base of a molecular modeling study.
Figure 1. Modeled structures of 3 (ZnCl2-complex) (left) and IP3
stacked on 3 (right).
Kimura and co-workers devised a luminescent ruthenium(II)-
templated assembly that provides two tripodal Zn(II)-cyclen
units for fluorescent sensing of IP3 and its analogues.3b This
ruthenium complex recognizes two molecules of a C3-
symmetric cis,cis-1,3,5-cyclohexanetriol triphosphate (CTP3)
in a stepwise manner. We were interested in a simpler
tripodal recognition system that has three phosphate-binding
units in a matched geometry with the three phosphate groups
of IP3 or its sereoisomers. Here, we report the new tripodal
platform for chemical sensing of IP3 through the competition
assay.
Initially, we focused on a benzene-based tripodal system
such as 1,5 which readily provides three binding units (L)
in the same hemisphere through conformational preorgani-
zation owing to the steric hindrance between the adjacent
substituents. As the phosphate binding unit, we chose metal
complexes of dipicolylamine (DPA), which have been
successfully used for the recognition of phosphate
derivatives.2g–j Thus, we synthesized a tripodal Cu(II)-
complex of 1 (L ) DPA) and its application to a competition
assay for phytate, a hexakis(phosphate) anion.6 However,
the complex could not provide geometry wide enough to
accommodate phytate or its analogues such as IP3 in a 1:1
chelation mode. We need an extended tripodal platform to
accommodate IP3 or its stereoisomers. Thus, we have devised
Although the tripodal system 2 (L ) DPA) has the
extended structure, its metal complex shows a well-preor-
ganized conformation, particularly suitable for the recognition
of IP3 or CTP3. A modeled structure for a Zn(II) complex
of 2 (L ) DPA) shows that the three Zn(II)-DPA groups
can have the “all-syn” conformation with little distortion.
Furthermore, this extended structure seems to adequately
accommodate IP3, as shown in the stacked structure (Figure
1).
The ligand 2 (L ) DPA) can be synthesized readily by
reductive amination of tris(formylmethyl)benzene 5 with dipi-
colylamine using sodium acetoxyborohydride in 70% yield.
Aldehyde 5 was prepared from the tris(cyanomethyl)benzene
45g by treatment with DIBAL in 68% yield. Treatment of
tris(dipicolylamine) 2 with zinc perchlorate hexahydrate in
methanol afforded the corresponding Zn(II) complex 3 in
80% isolated yield (Scheme 1).
(4) For biosensors of IP3, see: (a) Hirose, K.; Kadowaki, S.; Tanabe,
M.; Takeshima, H.; Iino, M. Science 1999, 284, 1527. (b) Luzzi, V.;
Murtazina, D.; Allbritton, N. L. Anal. Biochem. 2000, 277, 221. (c) Cheley,
S.; Gu, L.-Q.; Bayley, H. Chem. Biol. 2002, 9, 829. (d) Yamakoshi, M.;
Takahashi, M.; Kouzuma, T.; Imanmura, S.; Tsuboi, I.; Kawazu, S.;
Yamagata, F.; Tominaga, M.; Noritake, M. Clin. Chim. Acta 2003, 328,
163. (e) Morii, T.; Sugimoto, K.; Makino, K.; Otsuka, M.; Imoto, K.; Mori,
Y. J. Am. Chem. Soc. 2002, 124, 1138. (f) Sugimoto, K.; Nishida, M.;
Otsuka, M.; Makino, K.; Ohkubo, K.; Mori, Y.; Morii, T. Chem. Biol. 2004,
11, 475.
Scheme 1. Synthesis of Zn(II) Complex 3
(5) For selected examples of benzene-based tripodal receptors used in
molecular recognition, see: (a) Metzger, A.; Lynch, V. M.; Anslyn, E. V.
Angew. Chem., Int. Ed. 1997, 36, 862. (b) Kimura, E.; Aoki, S.; Koike, T.;
Shiro, M. J. Am. Chem. Soc. 1997, 119, 3068. (c) Chin, J.; Walsdorff, C.;
Stranix, B.; Oh, J.; Chung, H. J.; Park, S.-M.; Kim, K. Angew. Chem., Int.
Ed. 1999, 38, 2756. (d) Lavigne, J. L.; Anslyn, E. V. Angew. Chem., Int.
Ed. 1999, 38, 3666. (e) Abouderbala, L. O.; Belcher, W. J.; Boutelle, M. G.;
Cragg, P. J.; Steed, J. W.; Turner, D. R.; Wallace, K. J. Proc. Natl. Acad.
Sci. U.S.A. 2002, 99, 5001. (f) Tobe, S. L.; Anslyn, E. V. Org. Lett. 2003,
5, 2029. For some of our own contributions, see: (g) Kim, S.-G.; Ahn,
K. H. Chem.-Eur. J. 2000, 6, 3399. (h) Kim, S.-G.; Kim, K.-H.; Jung, J.;
Shin, S. K.; Ahn, K. H. J. Am. Chem. Soc. 2002, 124, 591. (i) Kim, S. J.;
Kim, K. H.; Kim, Y. K.; Shin, S. K.; Ahn, K. H. J. Am. Chem. Soc. 2003,
125, 13819. (j) Kim, J.; Kim, S.-G.; Seong, H. R.; Ahn, K. H. J. Org.
Chem. 2005, 70, 7227. (k) Kim, K.; Raman, B.; Ahn, K. H. J. Org. Chem.
2006, 71, 38.
The NMR spectrum of ligand 2 (L ) DPA) taken at room
temperature showed a C3 symmetric structure in solution,
(6) Oh, D. J.; Han, M. S.; Ahn, K. H. Supramol. Chem. 2007, 19, 315–
320.
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Org. Lett., Vol. 10, No. 16, 2008

whereas that of Zn(ClO4)2 complex 3 showed broad peaks
in the temperature range of 25-73 °C, indicating that the
metal complex is sterically bulky and thus conformational
change is slow.
Because Zn(II) complex 3 does not contain a good
chromo- or fluoro-genic moiety, a direct sensing of IP3 is
not feasible. Therefore, we have investigated the competition
assay using a chemosensing ensemble system that consists
of a receptor for an analyte and a dye competitive with the
analyte. Such a chemical ensemble system has been dem-
onstrated to be useful in sensing various analytes for its
operational simplicity and versatility in the receptor design.7
The chemosensing ensembles based on metal coordination
complexes seem to be promising for the development of
assay systems for phosphate groups, because such coordina-
tion complexes can provide strong binding affinity toward
the anions even in aqueous media.8 Thus far, only a few
trifurcated sensing systems containing metal complexes as the
binding units have been reported.6,9
Figure 2. Fluorescence titration of the chemical ensemble (a
solution containing 3.0 µM eosin Y and 2.0 µM Zn(II) complex 3)
with IP3 (0-6.0 µM) in water buffered at pH 7.0 (HEPES 10 mM),
with the excitation wavelength at 517 nm. (Inset) Plot of the
fluorescence intensity (peak height at 536 nm) against concentration
of IP3.
First, we evaluated several dyes such as eosin Y, 5-car-
boxy-fluorescein, and coumarine 343 to find an optimal
chemosensing ensemble for Zn(II)-DPA 3. Fluorescence
titrations of eosin Y (3.0 µM) with 3 (0-6.0 µM) in water
buffered at pH 7.0 (HEPES, 10 mM) resulted in continuous
quenching up to two equivalent points, which suggested a
1:2 binding mode (Figure S1). The other dyes showed small
or little quenching. A Job plot for the complex formation
between eosin Y and Zn(II) complex 3 supported the 1:2
(3/eosin Y) binding stoichiometry (Figure S2). By carrying
out UV/vis titrations between eosin Y and Zn(II) complex 3
(Figure S3), we could determine association constants for
the dye binding processes: K1 ) 1.2 × 106 M-1, K2 ) 8.2 ×
104 M-1.
On the basis of the titration experiments, an ensemble
system containing Zn(II) complex 3 and eosin Y was chosen
for the chemical sensing study. Thus, a chemical ensemble
system composed of 3.0 µM of eosin Y and 2.0 µM of 3 in
water at pH 7.0 (HEPES, 10 mM) was titrated with
increasing amount of IP3 (0-6.0 µM) and the resulting
changes in the fluorescence spectra were recorded with an
excitation wavelength at 517 nm. As shown in Figure 2, the
fluorescence of eosin Y restores as it is replaced by IP3 added,
showing a saturation behavior after about 2.2 equivalents of
IP3 were added.
cence intensity before and after addition of IP3 becomes
smaller both at the lower and higher pH ranges.
An attempt to determine the association constant between
Zn(II)-DPA 3 and IP3 (K5) directly by isothermal titration
calorimetry (ITC) was not successful. To carry out a
competition calorimetry we needed a competitive analyte that
binds Zn(II) complex 3 in a 1:1 stoichiometry and with lower
binding affinity. We have found that benzene-1,3,5-tricar-
boxylate (BTC), trimesic acid sodium salt, can be used as
the competitive analyte. The Zn(II) complex 3 was found to
bind BTC with an association constant of 2.0 × 104 M-1
and with near 1:1 binding stoichiometry (n ) 0.90), as
determined by ITC titrations carried out in 20% DMSO-
water buffered at pH 7.0 (HEPES, 10 mM) at 303 K (Figure
S4). Although the n value deviated a little from unity,
indicating that other minor binding modes are involved, we
used BTC as the competition ligand to determine the binding
The fluorescence titration was carried out at different pH,
from 6.0 to 8.5 (Figure 3). The data indicate that the present
chemosensing ensemble system works better for the physi-
ological pH range (pH 7.3-7.4). The difference in fluores-
(7) (a) Metzger, A.; Anslyn, E. V. Angew. Chem., Int. Ed. 1998, 37,
649. (b) Niikura, K.; Metzger, A.; Ansyln, E. V. J. Am. Chem. Soc. 1998,
120, 8533. (c) Lavigne, J. J.; Anslyn, E. V. Angew. Chem., Int. Ed. 2001,
40, 3119. (d) Wiskur, S. L.; Ait-Haddou, H.; Lavigne, J. J.; Anslyn, E. V.
Acc. Chem. Res. 2001, 34, 963.
(8) (a) Han, M. S.; Kim, D. H. Angew. Chem., Int. Ed. 2002, 41, 3809.
(b) Han, M. S.; Kim, D. H. Bioorg. Med. Chem. Lett. 2003, 13, 1079. (c)
Hanshaw, R. G.; O’Neil, E. J.; Foley, M.; Carpenter, R. T.; Smith, B. D. J.
Mater. Chem. 2005, 15, 2707.
Figure 3. Fluorescence changes depending on pH; b, the ensemble
only (2.0 µM eosin Y and 2.0 µM3); 9, the ensemble with IP3
(2.0 µM) in water buffered at various pH’s: pH 6.0 (MES 10 mM),
pH 6.5 (MES 10 mM), pH 7.0 (HEPES 10 mM), pH 7.5 (HEPES
10 mM), pH 8.0 (HEPES 10 mM), and pH 8.5 (CHES 10 mM).
(9) (a) Kimura, E.; Aoki, S.; Koike, T.; Shiro, M. J. Am. Chem. Soc.
`
1997, 119, 3068–3076. (b) Morey, J.; Orell, M.; Barcelo´, M. A; Deya`, P. M.;
Costa, A.; Ballester, P. Tetrahedron Lett. 2004, 45, 1261. (c) Fabbrizzi, L.;
Foti, F.; Taglietti, A. Org. Lett. 2005, 7, 2603
Org. Lett., Vol. 10, No. 16, 2008
.
3541

affinity of Zn(II) complex 3 toward IP3. Thus, from competi-
tive ITC titrations for a 1:1 mixture of 3 and BTC with IP3,
we were able to obtain an approximate association constant
between 3 and IP3 in an aqueous buffer solution (pH 7.0,
HEPES 10 mM) containing 20% DMSO at 303 K: K5 ) 4.6
((1.4) × 108 M-1. The complexation involved a large
enthalpy change, ∆H° ) 8.8 ((0.3) kcal·mol-1 (Figure S5).
With the K1, K2, K5 values, we could also determine
equilibrium constants for K3 and K4 according to the literature
precedent:10
I + H h HI (K1 ) 1.2 106 M-1)
HI + I h HI2 (K2 ) 8.2 × 104 M-1)
HI2 + A h HI + HA (K3 ) 5.6 × 103 M-1)
HI + A h I + HA (K4 ) 3.9 × 102 M-1)
H + A h HA (K5 ) 4.6 × 108 M-1)
Figure 4. Comparison of the fluorescence intensity (peak height at
536 nm) of the ensemble system (2.0 µM eosin Y and 2.0 µM 3)
toward various anions (2.0 µM). F0 is the intensity of the ensemble
itself.
where H, I, and A represent Zn(II) complex 3, eosin Y, and
IP3, respectively. In this calculation, we have assumed that
Zn(II) complex 3 binds IP3 in a 1:1 stoichiometry. An
evidence for the 1:1 complexation between 3 and IP3 was
obtained by electrospray ionization mass spectrometry, which
gave peaks responsible for 1:1 complexes (Figure S6). The
competitive ITC titration data shows an inflection point at
[IP3]/[3] ) 1.24, a small deviation from the equimolar ratio.
This deviation indicates that other higher-order complexes
exist even though they are minor components. However, to
a good approximation, we assumed that Zn(II) complex 3
interacts with IP3 in a 1:1 binding mode.
for the ensemble system. A higher selectivity of PPi over
ATP or ADP suggests that steric bulkiness of the latter
phosphates may influence the binding negatively. Regarding
to the binding modes of such linear di- and triphosphate
anions, not a 1:1 but a complex binding mode is expected.
Indeed, ITC titrations of Zn(II) complex 3 with PPi gave a
complex binding isotherm (Figure S7).
In summary, we have devised a novel chemosensing
ensemble system for IP3 based on a new tripodal receptor
containing three Zn(II)-dipicolylamine ligands. The addition
of IP3 to the chemosensing ensemble system, a mixture of
the Zn(II) complex and eosin Y, in an aqueous medium at
pH 7 resulted in the restoration of fluorescence of eosin Y
as it is expelled by the anion added. The ensemble system
shows the maximum fluorescence enhancement in the case
of IP3, while it shows reduced changes in the cases of PPi,
ATP, ADP, and monovalent anions (HPO42-, CH3CO2-,
CO32-, Cl-, Br-, ClO4-, N3-, and NO3-). The present
chemical ensemble system is structurally simple, yet it can
be used for fluorescence sensing of IP3 in aqueous medium
of physiological pH. A further exploration of the new tripodal
platform in chemical sensing of other analytes is under
investigation.
On the basis of the fluorescence titration results, we
evaluated chemical sensing ability of the ensemble system
toward various other anions such as pyrophosphate (PPi),
ATP, ADP, AMP, chloride, bromide, nitrate, sulfate, azide,
acetate, perchlorate, hydrogen phosphate, and carbonate ions
as their sodium salts. The fluorescence titration toward each
of the anions was carried out using an ensemble system
containing 2.0 µM each of Zn(II) complex 3 and eosin Y in
an aqueous solution buffered at pH 7.0 (HEPES, 10 mM) at
room temperature. The results are summarized in Figure 4.
The ensemble system shows the highest selectivity for IP3
among the anions including mono- and diphosphate anions.
Clearly, the tripodal nature of ensemble system discriminates
2-
monophosphate compounds such as AMP and HPO4 as
well as all the nonphosphate anions, exhibiting slight
fluorescence response. Also, the ensemble system has
decreased sensitivity toward ATP and ADP, showing relative
fluorescence intensity of 58% and 48% with respect to that
of IP3, respectively. This lower selectivity seems to be
originated from the tripodal nature of the ensemble system,
of which geometry does not match with the linear tri- or
diphosphates. Interestingly, PPi shows the next best analyte
Acknowledgment. This work was supported by grants
from Korea Health Industry Development Institute (A05-
0426-B20616-05N1-00010A) and Korea Research Founda-
tion (KRF-2005-070-C00078).
Supporting Information Available: Details for the
synthesis of Zn(II) complex 3 and Figures S1-S7. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(10) McCleskey, S. C.; Metzger, A.; Simmons, C. S.; Anslyn, E. V.
Tetrahedron 2002, 58, 621.
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