An indicator displacement system for fluorescent detection of phosphate oxyanions under physiological conditions

Article abstract of DOI:10.1016/j.tetlet.2004.09.133

A fluorogenic chemosensing system is described and shown to selectively detect pyrophosphate under physiological conditions. In the best case, pyrophosphate and hydrogen phosphate are capable of displacing a fluorescent coumarin-derived indicator from a bis Zn²⁺-dipicolylamine coordination compound with association constants of 10⁷ and 10 ⁵ M^-1, respectively.

Full text of DOI:10.1016/j.tetlet.2004.09.133

Tetrahedron
Letters
Tetrahedron Letters 45(2004) 8721–8724
An indicator displacement system for fluorescent detection
of phosphate oxyanions under physiological conditions
Roger G. Hanshaw, Sarah M. Hilkert, Hua Jiang and Bradley D. Smith*
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA
Received 25August 2004; revised 20 September 2004; accepted 20 September 2004
Available online 7 October 2004
Abstract—A fluorogenic chemosensing system is described and shown to selectively detect pyrophosphate under physiological con-
ditions. In the best case, pyrophosphate and hydrogen phosphate are capable of displacing a fluorescent coumarin-derived indicator
2
+
7
5
À1
from a bis Zn -dipicolylamine coordination compound with association constants of 10 and 10 M , respectively.
2004 Elsevier Ltd. All rights reserved.
ꢀ
2
Zn -coordination compounds 1–4 (Fig. 1) were pre-
pared by literature methods and evaluated as receptors
for pyrophosphate in a fluorescence displacement assay
+
The selective recognition of anions with biological
importance is a topic of current interest to both chemists
and biologists. An increasingly popular strategy for
anion recognition is the displacement of a fluorogenic or
chromogenic indicator from an indicator–receptor com-
1
1
1
2
–8
plex by the analyte anion.
This displacement
approach has been used recently in the detection of
N
N
Zn2+
2
3
Zn2+
N
N
carbonate, hydrogen sulfate, and hydrogen phosphate,
4
N
4 NO3-
NO3-
N
Zn2+
N
2
5
6
nitrate, phosphate, pyrophosphate, and various ha-
7
8
lides, as well as the organic anions tartrate and malate.
Sensors for pyrophosphate in aqueous solution have
N
N
6
,9
been previously reported, but most available strategies
employ redox-active metal cations as fluorescence
quenchers, or use UV chromophores as the indicator.
As a part of our ongoing effort to develop sensing sys-
tems for cell apoptosis, we needed to develop a fluores-
cent displacement assay for phosphate oxyanions. The
specific goal of this study was to find a suitable fluores-
cent indicator that could be used in combination with
2
1
1
0
N
N
Zn
N
N
O
N
Zn
Zn2+
2+
Zn2+
2+
N
N
N
N
N
N
N
2
+
zinc coordination compounds 1–4. The Zn -coordina-
tion compound 4 has been used as a colorimetric sensor
for pyrophosphate in aqueous solution,
4 NO3-
3 NO3-
5
,9a
and we
4
report here that 4 can be used in a fluorescence dis-
placement assay to detect low concentrations of
pyrophosphate under physiological conditions (pH7.4,
3
SO3
O
5mM TES, 145mM NaCl). We have further identified
the related Zn -coordination compound, 3, as a recep-
tor with a stronger affinity for pyrophosphate.
HO
HO
HO
HO
2
+
O
O
O
5
6
2
+
Keywords: Fluorescence; Displacement assay; Pyrophosphate; Sensing.
*
Figure 1. Structure of the Zn -DPA coordination compounds 1
through 4 used as receptors, and the fluorescent molecules 5 and 6 used
as indicators in the displacement assays.
Corresponding author. Tel.: +1 574 631 8632; fax: +1 574 631
652; e-mail: smith.115@nd.edu
6
0
040-4039/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.09.133

8722
R.G.Hanshaw et al./ Tetrahedron Letters 45 (2004) 8721–8724
using the coumarin-based compounds 5 and 6 as indica-
1
tors. We first explored the changes in spectral properties
acquire a suitable absorption spectrum. Fluorescence
spectroscopy was used in order to evaluate the capacity
of small anions to displace the indicator from the recep-
tor at the low concentrations needed for physiological
utility. We found that receptors 1 through 4 were each
capable of quenching the fluorescence emission of 6 in
a concentration dependent fashion (Fig. 3). Jobs plots
were prepared for the complex of each receptor and 6,
and a 1:1 binding stoichiometry was found in each case.
We determined the association constant for each recep-
tor with 6 by fluorescence titration and subsequent non-
2
2
+
of each indicator upon addition of the Zn coordination
compounds. The requirement to operate under physio-
logical conditions lead us to eliminate the commercially
available 5 due to poor solubility in aqueous solution
and an unstable fluorescence emission. Sulfonate 6,
which is highly water soluble and exhibits very stable
fluorescence emission, was subsequently chosen as the
more suitable indicator. Compound 6 was easily pre-
pared in 65% yield by treatment of 6,7-dihydroxy-4-
1
3
15
chloromethyl coumarin with sodium sulfite. Initially
UV–vis titrations were performed at 25ꢁC in an aqueous
solution (buffered at pH7.4 using 5mM TES, 145mM
NaCl) of 6 (100lM) using 1 through 4 as the titrant.
Increasing receptor concentration lead to a decrease in
intensity of the indicator absorption band at 375nm
and a simultaneous increase in intensity of the band at
linear curve fitting to a standard 1:1 binding model
(Fig. 4). Association constants are given in Table 1.
8
b
Anslynandco-workershaverecentlyshown thatselectiv-
ity of a displacement assay for a given analyte can be
regulated by variation in the relative concentration of
receptor and indicator. In our assay for pyrophosphate,
we prepared a 1:1 receptor–indicator complex in aque-
ous solution buffered at pH7.4 (5mM TES, 145mM
NaCl) by mixing equimolar amounts of receptor and
indicator (10lM each). We then titrated this solution
with aliquots of the sodium salts of nitrate, phosphate,
hydrogen phosphate, pyrophosphate, sulfate, carbonate,
and acetate. Only pyrophosphate, and to a lesser extent
hydrogen phosphate, were able to displace the indicator
from the receptor–indicator complex, as reported by a
restoration of fluorescence intensity (Fig. 5). The recep-
tor–indicator complexes of 1 and 2 failed to respond to
any of the anions used in this investigation. Pyrophos-
phate was easily able to displace 6 from the receptor–
indicator complexes of 3 and 4, and addition of hydro-
gen phosphate resulted in a substantial, though signifi-
cantly diminished indicator displacement from these
same complexes. Fluorescence emission intensities from
these titrations (kex. = 347nm, kem. = 480nm) were then
analyzed by the standard method for competition as-
1
4
4
30nm (Fig. 2).
While this change in the absorption spectrum could be
used in a ratiometric assay, a drawback was the rela-
tively high indicator concentration (100lM) needed to
1
6
says to determine the association constants presented
in Table 2.
Figure 2. UV–vis spectra of 6 following the addition of increasing
amounts of 3. Compound 3 was titrated into a 100lM solution of 6
over the concentration range 0–250lM. All titrations were performed
at 25ꢁC in aqueous solution, pH7.4, 5mM TES, 145mM NaCl.
Receptors 3 and 4 bound pyrophosphate with nearly
two orders of magnitude greater affinity than hydrogen
Figure 3. Fluorescence emission spectra of 6 following the addition of increasing amounts of 3. Compound 3 was titrated into a 10lM solution of 6
over the concentration range 0–100lM. Excitation wavelength was 347nm. All titrations were performed at 25ꢁC in aqueous solution, pH7.4, 5mM
TES, 145mM NaCl.

R.G.Hanshaw et al./ Tetrahedron Letters 45 (2004) 8721–8724
Table 2. Kassociation for receptors and anionic species (·10 M
8723
5
À1 a
)
Receptor
Pyrophosphate
Hydrogen phosphate
1
2
3
4
—
—
—
—
152 ± 26
67.2 ± 18.2
7.3 ± 3.7
1.1 ± 0.3
a
All titrations were performed at 25ꢁC in aqueous solutions buffered
at pH7.4 with 5mM TES, 145mM NaCl. Each value represents the
average of at least three separate experiments.
the anions by enhancing the electrostatic interaction be-
tween the receptor and the analyte. The association con-
stant determined for 4 with hydrogen phosphate,
4
À1
5
(
11.4 ± 3.3) · 10 M , agrees remarkably well with the
4
À1
.
previously reported value of (11.2 ± 0.8) · 10 M
The association constant for 4 with pyrophosphate,
6.7 ± 1.8) · 10 M , also agrees well with previous
reports after consideration of the greater ionic strength
used in the present studies.
5
À1
(
9a
Figure 4. Fluorescence intensity of 6 at 480nm in the presence of
increasing amounts of 3. Excitation wavelength was 347nm. Inset:
Jobs plot for the receptor–indicator binding of 6 and 3, clearly
indicating a 1:1 stoichiometry. All experiments were performed at
In summary, a fluorescence-based displacement assay
for pyrophosphate has been developed that is fully func-
tional under physiological conditions. The chemosens-
ing ensemble formed from 3 and 6 detects pyro-
phosphate over hydrogen phosphate with a selectivity
difference of nearly two orders of magnitude. Practical
applications of this sensing system will be reported in
due course.
25ꢁC in aqueous solution, pH7.4, 5mM TES, 145mM NaCl.
5
À1 a
Table 1. Kassociation for receptors and indicator (·10 M
)
Receptor
Indicator 6
1
2
1.0 ± 0.1
6.0 ± 1.5
3
23.1 ± 16.0
1.8 ± 0.3
4
a
Acknowledgements
All titrations were performed at 25ꢁC in aqueous solutions buffered
at pH7.4 with 5mM TES, 145mM NaCl. Each value represents the
average of at least three separate experiments.
The National Science Foundation and the University of
Notre Dame are acknowledged for financial support.
phosphate. Furthermore, receptor 3 bound both pyro-
phosphate and hydrogen phosphate more tightly than
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8724
R.G.Hanshaw et al./ Tetrahedron Letters 45 (2004) 8721–8724
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