Highly selective fluorescent recognition of pyrophosphate in water by a new chemosensing ensemble

Article abstract of DOI:10.1007/s10895-010-0758-2

A new chemosensing ensemble that displays sensitive and selective fluorescent recognition of pyrophosphate in water at pH 7.4 has been developed. The ensemble is constructed by a copper complex (receptor) and eosin Y (indicator), the constructed ensemble is capable of highly selectively discriminate pyrophosphate from other common existing anions such as CH ₃COO^-, HSO₄^-, NO₃ ^-, H₂PO₄^-, HPO₄ ^2-, PO₄^3-, NCS^-, I^-, Cl^-, Br^-, F-as well as some structurally similar carboxylates such as citrate, tartrate, oxalate, malonate, succinate and glutarate.

Full text of DOI:10.1007/s10895-010-0758-2

J Fluoresc (2011) 21:701–705
DOI 10.1007/s10895-010-0758-2
ORIGINAL PAPER
Highly Selective Fluorescent Recognition of Pyrophosphate
in Water by a New Chemosensing Ensemble
Lijun Tang & Minghui Liu & Fangfang Li &
Raju Nandhakumar
Received: 5 August 2010 /Accepted: 18 October 2010 /Published online: 30 October 2010
#
Springer Science+Business Media, LLC 2010
Abstract A new chemosensing ensemble that displays
sensitive and selective fluorescent recognition of pyro-
phosphate in water at pH 7.4 has been developed. The
ensemble is constructed by a copper complex (receptor)
and eosin Y (indicator), the constructed ensemble is
capable of highly selectively discriminate pyrophosphate
from other common existing anions such as CH3COO−,
(PPi) is a major research focus, as it participates in several
bioenergetic and metabolic processes [4]. It is also well
known that patients with calcium pyrophosphate dihydrate
crystals and chondrocalcinosis have been shown to have
high synovial fluid PPi level [5, 6]. Moreover, the ability to
detect PPi has become important in cancer research [7]. As
a consequence, many researchers have made an effort to
develop selective chemosensors for biologically potent PPi
[8–24].
On the other hand, fluorescent sensing of PPi has
become particularly attractive because of its simplicity
and low detection limit [1–3, 25–30]. A convenient and
increasingly popular method for sensing of anions is the
displacement (ensemble) approach, which is based on
competitive binding of an indicator and the analyte to a
receptor [28, 31–33]. In this protocol, a signaling unit
(indicator) is bound to a binding site (receptor) by non-
covalent interactions to form a chemosensing ensemble.
Treatment of the ensemble with the analyte results in
displacement of the indicator from the receptor and
restoration of the indicator’s original spectral properties.
This strategy avoids the complicated synthetic process for
indicator-receptor chemically linked type sensors. Thus,
one can focus on design of receptors having complementary
shape with the target anion.
−
−
−
HSO4 , NO3 , H2PO4 , HPO42−, PO43−, NCS−, I−, Cl−,
Br−, F−as well as some structurally similar carboxylates
such as citrate, tartrate, oxalate, malonate, succinate and
glutarate.
.
.
Keywords Pyrophosphate Fluorescent recognition
.
Chemosensing ensemble Dicarboxylate
Introduction
During the past two decades, considerable attention has
been devoted to the development of chemosensors for
anionic species, because they are ubiquitous in biological
systems and play significant roles in a wide area of
pharmacy, biology and environmental sciences [1–3]. In
particular, selective detection of the anion pyrophosphate
:
:
Anion sensing in water is generally regarded as a
challenging aspect because of the strong hydration effects
of anions as well as the ability to convert the anion
recognition into a fluorescent or colorimetric signal [34].
Nevertheless, an effective strategy to solve this problem is
by utilizing some of the carefully designed metallic
receptors which can bind a target anion selectively and
tightly in water [35–38]. Even though various types of
chemosensors for selective recognition of PPi have been
L. Tang (*) M. Liu F. Li
College of Chemistry and Chemical Engineering, Liaoning Key
Laboratory for the Synthesis and Application of Functional
Compounds, Bohai University,
Jinzhou 121013, China
e-mail: lijuntang@tom.com
R. Nandhakumar (*)
Division of Nano Sciences, Ewha Womans University,
Seoul 120-750, South Korea
e-mail: rajunandhakumar@yahoo.com

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J Fluoresc (2011) 21:701–705
Scheme 1 Synthesis of
complex 1
O
O
H
N
N
H
NH2
NH2
N
H
NH
NH
O
1) MeOH
2) NaBH4
OMe
OMe
OMe CuCl2
Cu2+
OHC
N
H
EtOH
OMe
H
H
N
OMe
N
N
H
O
O
2
3
1
4
COO
Br
Br
O
Br
O
O
Br
5
Structure of 5
reported [8–24, 39], only a very few of them have taken
structurally similar carboxylate anions as competing analy-
tes [40, 41]. It is noteworthy that the sensors reported in
literature 40 and 41 were all designed by the PET
mechanism, they also have some disadvantages such as
only could be used in pure organic solvent, and could not
effectively distinguish PPi from dicarboxylates, for exam-
ple, malonate and glutarate. As far as we are aware, there
are no chemosensors that can distinguish PPi from
competitive carboxylate anions in 100% aqueous solution
at physiological pH has been documented.
Herein, we report a new chemosensing ensemble for
fluorescent recognition of PPi in water at physiological pH.
The ensemble is constructed by a copper complex 1 and
eosin Y (5), which exhibits a high selectivity to PPi over
other common existing anions and some structurally similar
carboxylate anions.
Syntheses
Synthesis of Ligand 4
Compounds 2 (150 mg, 1.31 mmol) and 3 (500 mg,
3.27 mmol) were dissolved in anhydrous methanol (20 mL)
in a 50 mL round bottomed flask and stirred under nitrogen
atmosphere for 6 h at room temperature. Then the reaction
mixture was cooled to 0 °C and NaBH4 (160 mg, 4.10 mmol)
was added portion-wise during 1 h, and the reaction mixture
was allowed to stir for further 1 h. After the completion of
the reaction, the solvent was removed under reduced
pressure to give a white residue, which on recrystallization
from ethyl acetate-hexane (1:19, v/v) gave 4 as white solid.
Yield: 77%, mp: 107–108 °C. 1H NMR (400 MHz, CDCl3):
δ 6.83 (d, J=3.6 Hz, 2 H), 5.97 (d, J=3.6 Hz, 2 H), 4.26 (d,
J=16 Hz, 2 H), 4.01 (d, J=16 Hz, 2 H), 3.78 (s, 6 H), 2.24
800
Experimental
800
600
Apparatus and Chemicals
400
600
1H NMR spectra and 13C NMR spectra were obtained on a
Varian INOVA-400 MHz and Bruker AV-300 MHz Spec-
trometer, respectively. Chemical shifts were expressed in ppm
and tetramethylsilane (TMS) was used as internal standard.
High-resolution mass spectrometry (HRMS) was carried out
on a UPLC/Q Tof mass spectrometer. Fluorescence spectra
were obtained with a Hitachi F-4500 FL spectrophotometer at
room temperature for aerated solutions.
200
0
400
0.0000 0.0003 0.0006 0.0009
[1] / M
200
0
550
600
650
Compound 3 was prepared according to a literature
procedure [42]. Other reagents for synthesis obtained
commercially were used without further purification. In
the titration experiments, all the anions were added in the
form of sodium salts.
Wavelength (nm)
Fig. 1 Fluorescence changes of 5 (1.0×10−5 M, λex=523 nm, λem
543 nm) upon the addition of complex 1 in aqueous buffer solution
(HEPES 10 mM, pH=7.4). (Inset: plot of F543 vs concentration of 1.)
=

J Fluoresc (2011) 21:701–705
703
600
600
400
200
0
PPi
500
400
300
PPi
Citrate
Oxalate
Malonate
ensemble + other anions
Citrate
Oxalate
200
100
0
Malonate
0.0000
0.0001
0.0002
0.0003
0.0004
0.0005
550
600
650
[Anion]/M
Wavelength (nm)
Fig. 4 Fluorescence changes of the ensemble recorded at 543 nm upon
titration by some representative anions in buffered water solution (HEPES
10 mM, pH=7.4). PPi (black circle), Citrate (black up-pointing triangle),
Oxalate (black square), Malonate (black star)
Fig. 2 Fluorescence changes of the ensemble upon the addition of
various anions (4.0×10−4 M for each anion)
(d, J=9.2 Hz, 2 H), 2.18 (d, J=13.2 Hz, 2 H), 1.81 (b,
4 H), 1.72 (d, J=8.4 Hz, 2 H), 1.17 (t, J=10 Hz, 2 H), 1.01
(d, J=8.4 Hz, 2 H). 13C NMR (75 MHz, CDCl3): δ 163.4,
137.5, 121.4, 116.2, 106.8, 61.4, 51.1, 43.6, 32.9, 25.2.
HRMS (ESI+) m/z calcd. for C20H29N4O4 [4+H]+:
389.2189 and found: 389.2193.
Results and Discussion
Determination of Chemosensing Ensemble
After the screening of several of fluorescent indicators for
this chemosensing ensemble, eosin Y (5) was finally
selected for this study. The fluorescence emission of 5
was quenched when it was mixed with copper complex 1,
which is attributed to the energy or electron transfer effect
between 1 and 5 [43]. Fluorescence changes of 5 (1.0×
10−5 M, HEPES 10 mM, pH=7.4) upon the addition of an
aqueous buffer solution (HEPES 10 mM, pH=7.4) of
complex 1 was firstly investigated (Fig. 1). Upon incre-
mental addition of copper complex 1 to solution 5, the
fluorescence intensity of 5 gradually decreased at 543 nm
(excited at 523 nm) and completely quenched when 60
equiv of complex 1 was used, accompanied with an
obvious color change from orange to pink. Nonlinear
least-squares fitting of the titration profiles (inset of
Synthesis of Copper Complex 1
To an ethanol solution of 4 (100 mg, 0.26 mmol), an
ethanol solution of CuCl2⋅2H2O (56 mg, 0.33 mmol) was
added and stirred for 1 h at room temperature. The blue
precipitate formed was filtered and washed thoroughly
with cold diethyl ether to give complex 1 in 70% yield
(102 mg). UPLC/Q-TOF MS (ESI+) m/z calcd. for
C20H27CuN4O4 [1-H]+: 450.1317 and found: 450.2875.
Scheme 1.
600
40 equiv
500
400
0 equiv
300
200
100
0
550
600
650
Fig. 5 Fluorescence response of the ensemble to selected anions in
buffered water solution (HEPES 10 mM, pH=7.4). From left to right:
I−, Cl−, Br−, F−, CH3COO−, HSO4−, HPO42−, PO43−, malonate, SCN−,
glutarate, NO3−, H2PO4−, oxalate, succinate, tartrate, citrate and PPi
Wavelength (nm)
Fig. 3 Titration of an aqueous solution of the ensemble (HEPES
10 mM, pH=7.4) with standard PPi solution

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J Fluoresc (2011) 21:701–705
Fig. 1) employing the 1:1 binding mode equation strongly
support the formation of a 1:1 complex of 1 and 5 and the
association constant Ka was calculated to be 5.15×104 M−1
[44, 45]. Thus, a chemosensing ensemble solution (HEPES
10 mM, pH=7.4) composed of 5 (1.0×10−5 M) and
complex 1 (6.0×10−4 M) can be readily prepared.
competitive anions were carried out. As shown in Fig. 5,
except PPi, other anions (40 equiv to 5) did not produce
dramatic fluorescence emission changes. However, upon
addition of PPi (40 equiv to 5) to the solution containing
both ensemble and other anion, a significant fluorescence
emission enhancement is observed. These results indicate
that the recognition of PPi by the ensemble is not
significantly influenced by other coexisting anions, which
strongly proves that the ensemble exhibits a high selectivity
toward PPi.
Selective Fluorescent Recognition of PPi
Fluorescence intensity changes of the prepared chemosensing
ensemble upon addition of various anions (4.0×10−4 M for
each anion) are illustrated in Fig. 2. A significant fluores-
cence revival of 5 was observed upon addition of PPi. This
result indicates the successful competitive binding of the PPi
anion and displacement of 5 from the receptor. Some
carboxylate anions such as citrate, oxalate and malonate
can also induce, to some extent, fluorescence revival of 5,
but the revival levels are very small compared with that of
PPi. Whereas, no noticeable fluorescence changes were
observed upon addition of other anions such as CH3COO−,
Conclusions
In summary, we have developed a new chemosensing
ensemble which exhibits high selectivity toward PPi in
100% aqueous solution at pH 7.4. The readily prepared
chemosensing ensemble can effectively differentiate PPi from
−
other common existing anions such as CH3COO−, HSO4 ,
NO3 , H2PO4 , HPO42−, PO43−, NCS−, I−, Cl−, Br−, F− and
some potentially competitive carboxylates such as citrate,
tartrate, oxalate, malonate, succinate and glutarate. More-
over, the PPi recognition process by the ensemble is proved
to be hardly influenced by other coexisting anions.
−
−
HSO4 , NO3 , H2PO4 , HPO42−, PO43−, NCS−, I−, Cl−, Br−,
F− as well as other dicarboxylates such as succinate, glutarate
and tartrate. The PPi induced displacement of indicator 5
from the receptor also can be easily conformed by visual
detection, upon the addition of PPi, the pink color of the
ensemble changed to orange. These results revealed that the
chemosensing ensemble has a high selectivity toward PPi.
−
−
−
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