A fluorescent pyrophosphate sensor with high selectivity over ATP in water

Article abstract of DOI:10.1002/anie.200453914

High affinity and selective detection for PPi is exhibited by a new fluorescent sensor based on a naphthalene-dpa system (see picture). The binuclear zinc complex is remarkably selective toward PPi over other anions. For example, PPi can be detected at mi

Full text of DOI:10.1002/anie.200453914

Angewandte
Chemie
discrimination of these anions has been the main focus of the
efforts of several research groups. Although traditional
methods of anion sensing such as the use of ion-selective
electrodes continue to hold their ground, there is an increas-
ing incentive to find alternative means of analysis, including
those based on the use of selective chemosensors.[1b,4] Among
the various chemosensors, fluorescent chemosensors present
many advantages, as they benefit from high sensitivity, low
cost, easy detection, and versatility.[1,5] Unfortunately, few, if
any, fluorescent anion sensors exist that display both high PPi/
ATP selectivities and function in aqueous solution over a wide
range of pH values.[6,7] Herein, we present a new fluorescent
PPi sensor based on a naphthalene–dpa system (dpa = bis(2-
pyridylmethyl)amine), which exhibits high sensitivity and
selectivity over a wide range of pH values for PPi ions relative
to other anions, including ATP and ADP (which are
structurally similar to PPi in aqueous solution).
A fluorescent reporting naphthalene group was easily
introduced into the sensor by a Suzuki coupling reaction.
Compound 1 was obtained in 35% overall yield from
acetonide.[8] ZnII complexation with the dpa moieties gen-
erates an anion-binding site through the formation of a well-
known phenoxo-bridged binuclear metal complex
(Scheme 1):[9] Sensor 1·2Zn, a binuclear ZnII complex of
compound 1, was easily formed by the addition of a
Fluorescent Sensors
A Fluorescent Pyrophosphate Sensor with High
Selectivity over ATP in Water**
methanolic solution of
Zn(NO3)2 (2 equiv).
First, the effect of anions (sodium salts) on the absorption
spectrum of sensor 1·2Zn (30 mm) was examined in an
aqueous solution of HEPES buffer (0.01m, pH 7.4)
(HEPES = 2-[4-(2-hydroxyethyl)-1-piperazinyl]-
1 to an aqueous solution of
Dong Hoon Lee, Soon Young Kim, and Jong-In Hong*
Recently, considerable attention has been focused on the
design of receptors and sensors that have the ability to bind
and sense biologically important anions selectively through
electrochemical and optical responses.[1] Anions such as
pyrophosphate (P2O74À, PPi) and adenosine triphosphate
(ATP) play an important role in energy transduction in
organisms and control metabolic processes by participation in
enzymatic reactions.[2,3] ATP hydrolysis with the concomitant
release of PPi is central to many biochemical reactions, such
as DNA polymerization and the synthesis of cyclic adenosine
monophosphate (AMP) catalyzed by DNA polymerase and
adenylate cyclase, respectively.[2] Therefore, the detection and
ethanesulfonic acid) at 258C (Figure 1). In the absence of an
anion guest, the absorption spectrum of sensor 1·2Zn is
characterized by an intense band centered at 305 nm. Upon
the addition of PPi in increasing amounts, the peak at 305 nm
decreases and a new peak appears at 316 nm. The degree of
absorption change is not affected even by the addition of
more than 1 equiv of PPi (Figure 1a). Unlike the large 11-nm
red shift observed with PPi, relatively small red shifts are seen
with ATP (5 nm) and ADP (2 nm) upon complexation.
Furthermore, sensor 1·2Zn does not exhibit any clear spectral
change upon addition of AMP, HPO42À; other monovalent
anions such as CH3CO2À, FÀ, HCO3À, and ClÀ do not affect
the spectrum, even when present in excess (up to 100 equiv).
The degree of red shift was determined to be PPi @ ATP >
ADP > AMP ꢀ HPO42À. These results suggest that sensor
1·2Zn has a higher selectivity for PPi over other anions.
The optical sensing of anions was more drastically
demonstrated by virtue of the fluorescent response of 1·2Zn
to complex formation. The effect of anions (sodium salts) on
the fluorescent emission spectrum of sensor 1·2Zn (6 mm) was
investigated in an aqueous solution of HEPES buffer (0.01m,
pH 7.4) at 258C (Figure 2). When PPi was added to an
aqueous solution of 1·2Zn, the fluorescent emission spectrum
shifted in a dose-dependent manner toward longer wave-
lengths. As shown in Figure 2a, the lmax shifted from 436 nm
to 456 nm. An increase in the PPi concentration up to 1 equiv
resulted in a 9.5-fold fluorescence enhancement. However,
the addition of more than 4 equiv of ATP shows only twofold
[*] D. H. Lee, S. Y. Kim, Prof. Dr. J.-I. Hong
Department of Chemistry, College of Natural Sciences
Seoul National University
Seoul 151-747 (Korea)
Fax: (+82)2-889-1568
E-mail: jihong@plaza.snu.ac.kr
Prof. Dr. J.-I. Hong
Center for Molecular Design and Synthesis
KAIST, Daejon 305-701 (Korea)
[**] Support for this work in the form of a grant from the Basic Research
Program of the KOSEF (Grant No. R02-2003-000-10055-0) is
gratefully acknowledged. D.H.L. and S.Y.K. thank the Ministry of
Education for the award of a BK 21 fellowship. ATP=adenosine
triphosphate.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Angew. Chem. Int. Ed. 2004, 43, 4777 –4780
DOI: 10.1002/anie.200453914
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Scheme 1. Synthetic strategy for sensors 1·2Zn and 2·Zn.
Figure 1. a) Change in absorbance for sensor 1·2Zn (30 mm) upon
addition of PPi (sodium salt): [PPi]=0, 3, 6, 9, 12, 15, 18, 21, 24, 27,
30, 32, 34, 36, 38 mm. The spectra were measured in an aqueous sol-
vent of HEPES buffer (10 mm, pH 7.4) at 258C. b) UV/Vis spectra of
sensor 1·2Zn (30 mm) in HEPES buffer (0.01m, pH 7.4) at 258C in the
presence of various anions (30 mm).
Figure 2. a) Change in fluorescence emission for sensor 1·2Zn (6 mm)
upon addition of PPi (sodium salt): [PPi]=0, 0.6, 1.2, 1.8, 2.4, 3.0, 3.6,
4.2, 4.8, 5.4, 6.0, 6.6, 7.2 mm. The spectra were measured in an aque-
ous solvent of HEPES buffer (0.01m, pH 7.4) at 258C. (Inset: Job plot
~
^
of 1·2Zn and anions: PPi, ATP.) b) Fluorescence emission spectra
of sensor 1·2Zn (6 mm) in HEPES buffer (10 mm, pH 7.4) at 258C in
the presence of various anions (8 mm). I=Intensity (arbitrary units).
enhancement accompanied by a 12-nm red shift. In the case of
ADP, sensor 1·2Zn shows only a subtle emission change (1.5-
fold increase) upon addition of a 50-fold excess of ADP. The
(2.9 Æ 0.7) ” 108 mÀ1 for PPi–1·2Zn by a standard algorithm for
2À
2À
addition of AMP and HPO4 does not lead to an emission
competitive binding in the presence of excess HPO4 in an
enhancement, even after the addition of 100 equivalents of
each anion (Figure 2b).
The Job plot for the binding between 1·2Zn and anions
(PPi and ATP) shows a 1:1 stoichiometry (inset of Figure 2a).
The apparent association constant (Ka) was determined to be
aqueous solvent of HEPES buffer (0.01m, pH 7.4) at
258C.[8,10] By the same method, Ka for ATP–1·2Zn was
found to be (7.2 Æ 1.0) ” 106 mÀ1, which is 40-fold lower than
for PPi–1·2Zn. This observation means that 1·2Zn can even
detect PPi at nanomolar concentrations in water.
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The same selectivity trend found in the absorbance
spectra (Figure 1b) is observed in the fluorescence spectra
(Figure 2b). However the signal difference is greatly
enhanced because of the high sensitivity of fluorescence
spectroscopy in favor of PPi. This selectivity enables PPi
detection by fluorescence, even in the presence of a large
excess of ATP (Figure 3). Figure 3 shows that 1·2Zn can
complex by bridging the two metal ions to give rise to two
hexacoordinated ZnII ions in 1·2Zn.[11] PPi induces a pro-
nounced red shift of lmax of 1·2Zn because the weakening of
the bond between the phenolate oxygen atom and ZnII
induces a more-negative charge characteristic on the pheno-
late oxygen atom and thus the bathochromic shift of lmax of
1·2Zn occurs. Simultaneously, an increased charge character-
istic on the phenolate oxygen atom induces a fluorescence
enhancement.
The selectivity for PPi over ATP can be understood on the
basis of the structure of the guest and the charge density of
four O-P oxygen atoms of the guest involved in the complex-
ation. The total anionic charge density of the four O-P oxygen
atoms involved in the complexation of ATP with 1·2Zn is
relatively smaller than that of the four O-P oxygen atoms of
PPi (Scheme 2). Therefore, the binding affinity of ATP is
drastically reduced, and the degree of fluorescence change
becomes smaller relative to PPi binding.
A control sensor, mononuclear 2·Zn (Scheme 1), does not
show emission lmax shift and fluorescence enhancement upon
addition of PPi. This result means that the cooperative action
of two ZnII–dpa units is required for the selective sensing of
PPi. Finally, to check for the working pH range for sensing,
the effect of the pH value of the medium on the PPi sensing
was investigated. Fortunately, fluorescence emission changes
shown in Figure 2b were also observed over a wide range of
pH values (6.5–10.1) with a similar tendency. This result
shows that even if the external pH value is disturbed, sensor
1·2Zn can still detect PPi.[8]
Figure 3. a) Change in fluorescence emission for sensor 1·2Zn (6 mm)
in the presence of ATP (300 mm) upon addition of PPi (sodium salt):
[PPi]=0, 1.2, 2.4, 3.6, 4.8, 6.0, 8.0, 11, 17, 24, 34, 45, 65, 85 mm. The
spectra were measured in a pure aqueous solution of HEPES buffer
(0.01m, pH 7.4) at 258C. I=Intensity (arbitrary units).
detect less than 1 equivalent of PPi even in the presence of a
50- to 250-fold excess of ATP (based on the amount of PPi
detected). In other words, 1·2Zn can selectively detect PPi in
an aqueous solution with remarkable selectivity over ATP
with a detection limit at micromolar concentrations. This is
significant in view of the fact that there are many biochemical
reactions in which PPi is released in the presence of ATP. An
efficient PPi sensor for bioanalytical applications requires
that PPi be detected in small amounts in the presence of a
large excess of ATP,[8] thus making our sensor suitable for
bioanalytical applications.
The binding mode for PPi–1·2Zn is illustrated in
Scheme 2, which is based on our previous work involving a
structurally similar sensor and its X-ray crystal structure.[11]
The proposed complex shows that the two sets of oxygen
anions on each P atom of PPi bind to the binuclear zinc
In summary, we have developed a naphthalene-based
fluorescent sensor that selectively detects PPi with high
affinity in aqueous solution over a wide pH range. This system
shows remarkable selectivity for PPi over other anions,
including strong competitors such as HPO4 and ATP. This
system can be applied for biochemical and analytical enzyme
assays involving ATP and PPi.
2À
Received: February 3, 2004
Revised: June 25, 2004 [Z53914]
Keywords: binuclear complexes · fluorescent probes ·
.
molecular recognition · pyrophosphates · zinc
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