Dual signal (color change and fluorescence ON-OFF) ensemble system based on bis(Dpa-CuII) complex for detection of PPi in water

Article abstract of DOI:10.1016/j.tetlet.2009.02.036

We have developed simple dual signal (color change and fluorescence ON-OFF) ensemble systems based on a bis(Dpa-Cu^II) complex 1 for the detection of PPi in water. Dual signal takes place because of weak binding and fluorescence quenching effect

Full text of DOI:10.1016/j.tetlet.2009.02.036

Tetrahedron Letters 50 (2009) 1951–1953
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Dual signal (color change and fluorescence ON–OFF) ensemble system based
on bis(Dpa-CuII) complex for detection of PPi in water
*
Soon Young Kim, Jong-In Hong
Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-747, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
We have developed simple dual signal (color change and fluorescence ON–OFF) ensemble systems based
on a bis(Dpa-CuII) complex 1 for the detection of PPi in water. Dual signal takes place because of weak
binding and fluorescence quenching effect of coordinatively unsaturated CuII complex for indicators
and replacement of the indicators by more strongly binding PPi. As a consequence, these ensembles show
a high selectivity and sensitivity for PPi over various anions including phosphate and its derivatives (AMP,
ADP, and ATP) in water.
Received 11 December 2008
Revised 4 February 2009
Accepted 6 February 2009
Available online 10 February 2009
Ó 2009 Elsevier Ltd. All rights reserved.
In these days, a growing number of research groups are becom-
ing interested in the development of sensors for biologically
important anions using color and fluorescence responses.1 In par-
ticular, pyrophosphate (P2O74ꢀ, PPi) is known to be involved in sev-
eral biochemical reactions, such as the hydrolysis of adenosine
triphosphate (ATP), DNA polymerization, and other metabolic pro-
cesses.2,3 Hence, the selective detection of pyrophosphate has been
a matter of investigation by several groups.4,5a
Among the known design principles for anion sensing, the dis-
placement (ensemble) approach is simple and convenient because
a signaling unit (indicator) is bound to a binding site (receptor) by
noncovalent interactions.1a,c There are several advantages of this
approach because of the noncovalent binding between receptor
and indicator: (1) No synthetic effort is required to chemically link
an indicator and a receptor; (2) there is considerable freedom in
the choice of an appropriate indicator; and (3) signal transduction
is induced by the simple addition of an analyte to the receptor-
indicator mixture.5
Recently, a few fluorescent ensembles that can recognize PPi
were reported.5d–f These ensembles, which used a bis(Dpa-ZnII)
(Dpa = dipicolylamine) complex, displayed quenching phenomena
due to either binding effect with receptor and indicator or energy
transfer between quencher and fluorophore. However, there were
a few disadvantages in these systems. First, several synthetic steps
were required to link the quencher with receptor and to synthesize
an indicator unit, which was time consuming and tedious.5d Sec-
ond, because quenching should be accompanied by binding, the
screening process required to choose the right indicator was re-
stricted.5e,f Also, the above ensembles showed only fluorescence
ON–OFF.5d–f
Fabbrizzi et al. reported a fluorescent PPi ensemble based on a
Cu(II) complex,5a where the Cu(II) ion was used to completely
quench the fluorescence emission of a bound indicator and a coor-
dinately unsaturated Cu(II) complex showed the capability to bind
anionic substrates. However, Fabbrizzi’s system showed only fluo-
rescence ON–OFF, in comparison with our dual signaling effect,
upon PPi binding. Further, no selectivity was reported for PPi over
strong biological competitors such as AMP, ADP, and ATP.
Using these characteristics of the Cu(II) ion, we developed a dual
signal (color change and fluorescence ON–OFF) ensemble system
based on a bis(Dpa-CuII) complex (1) for detection of PPi. In order
to realize a dual signal, the well-known indicators, pyrocatechol vio-
let (PV) as a chromophore capable of instantly detecting by naked
eyes and fluorescein disodium salts as a fluorophore, were used.
These ensembles showed a high selectivity and sensitivity for PPi
over various anions including phosphate and its derivatives (AMP,
ADP, and ATP) in water.
The synthesis and characterization of complex 1 had been re-
ported previously.6 a0-Dibromo-m-xylene was reacted with
a,
Dpa, KI and Cs2CO3 in acetonitrile to give rise to 1,3-bis[(bis(2-pyr-
idylmethyl)amino)methyl]benzene (2). Finally, complex 1 was pre-
pared by complexing 2 with Cu(ClO4)2 in DMSO.7
First, the UV–vis absorption changes of PV (1.98 ꢁ 10ꢀ5 M) for
complex 1 (4.41 ꢁ 10ꢀ4 M) were observed in a 10 mM HEPES buffer
(pH 7.4) at 25 °C (Fig. 1). The UV–vis absorption curves of PV gradu-
ally decreased at 442 nm and increased at 664 nm upon the addition
of complex 1, showing a bathochromic shift. When 1.2 equiv of com-
plex 1 was added into PV solution, the UV–vis absorption changes
were completely saturated. Also, the color of PV solution turned
from pale yellow to sky blue upon the addition of complex 1. The
association constant (Ka) between PV (abs = 664 nm) and complex
1 was estimated to be 1.01 ꢁ 106 Mꢀ1 8
.
The UV–vis absorption spectra of ensemble 1 ([1] = 2.1 ꢁ 10ꢀ5 M,
ensemble 1 = complex 1 + PV) for various anions (2 equiv, sodium
* Corresponding author. Tel.: +82 28806682; fax: +82 28891568.
E-mail address: jihong@snu.ac.kr (J.-I. Hong).
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.02.036

1952
S. Y. Kim, J.-I. Hong / Tetrahedron Letters 50 (2009) 1951–1953
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
N
N
N
N
Cu
N
Cu
N
Complex 1
Figure 3. Color changes of ensemble 1 ([1] = 2.1 ꢁ 10ꢀ5 M) in a 10 mM HEPES
buffer (pH 7.4) at 25 °C after the addition of various anions (2 equiv, ꢀsodium salts).
From left to right: Ensemble 1, PPi, ATP, ADP, AMP, Pi, AcOꢀ, NO3ꢀ, I , Brꢀ, Clꢀ, Fꢀ.
600
600
500
350
400
450
500
550
600
650
700
750
800
500
400
300
Wavelength (nm)
Figure 1. Changes of UV–vis absorption of pyrocatechol violet (PV, 1.98 ꢁ 10ꢀ5 M)
upon the addition of complex 1 (4.41 ꢁ 10ꢀ4 M) in a 10 mM HEPES buffer (pH 7.4)
400
200
100
0
at 25 °C: Complex 1 (lL) = 0, 10, 20, 40, 60, 80, 100, 130, 160, 200.
300
0
50
100
150
200
250
300
Complex 1 (equiv)
200
100
0
salts) were investigated in a 10 mM HEPES buffer (pH 7.4) at 25 °C
(Fig. 2). When PPi was gradually added to the solution of ensemble
1, the UV–vis absorption curves increased at 441 nm and decreased
at 663 nm (Hypsochromic shift). The UV–vis absorption spectrum
(at 663 nm) was completely saturated at 2 equiv of PPi (a 20-fold de-
crease). According to displacement titration, the association con-
stant (Ka) between complex 1 and PPi was estimated to be
500
520
540
560
580
600
Wavelength (nm)
3.93 ꢁ 107 Mꢀ1 9
. Also, the color of ensemble 1 returned from sky
Figure 4. The fluorescence emission changes (kex = 495 nm) of fluorescein
(1.25 ꢁ 10ꢀ6 M, disodium salts) upon the addition of complex 1 (4.41 ꢁ 10ꢀ4 M)
blue to pale yellow upon the addition of PPi (Fig. 3). However, each
addition of 2 equiv of ATP or ADP showed only a 1.9-fold or 1.3-fold
decrease in UV–vis absorbance at 663 nm, respectively. Upon the
addition of 2 equiv of ATP, ensemble 1 turned from sky blue to pale
green, but the solution of ensemble 1 after the addition of ADP
(2 equiv) showed no color changes (Fig. 3). Other anions, such as
Fꢀ, Clꢀ, Brꢀ, Iꢀ, NO3ꢀ, AcOꢀ, HPO42ꢀ, and AMP, did not give rise to
any UV–vis spectral changes, nor, accordingly, induced any color
changes (Fig. 3).
In the second place, the fluorescence emission changes
(kex = 495 nm) of fluorescein (1.25 ꢁ 10ꢀ6 M, disodium salts) for
complex 1 (4.41 ꢁ 10ꢀ4 M) were examined in a 10 mM HEPES buf-
fer (pH 7.4) at 25 °C (Fig. 4). According to the quenching effect of a
Cu(II) ion, the fluorescence emission intensity of fluorescein de-
creased at 514 nm upon the addition of complex 1. When
52.9 equiv of complex 1 was added to fluorescein solution, about
80% of the fluorescence emission intensity was quenched. The
in a 10 mM HEPES buffer (pH 7.4) at 25 °C: Complex 1 (lL) = 0, 10, 30, 50, 70, 100,
150, 200, 300, 450, 650, 1000, 1500.
association constant (Ka) between 1 and fluorescein was estimated
to be 5.83 ꢁ 104 Mꢀ1 8
.
The fluorescence emission spectra of ensemble 2 ([1] = 7.35 ꢁ
10ꢀ5 M, ensemble 2 = 1 + fluorescein disodium salts) for various
anions (1.2 equiv, sodium salts) were examined in a 10 mM HEPES
buffer (pH 7.4) at 25 °C (Fig. 5). The fluorescence emission at
514 nm was completely saturated at 1.2 equiv of PPi, which corre-
sponded to a 5.7-fold increase in the emission intensity (Fig. 5).
Each addition of 1.2 equiv of ATP or ADP caused a 3.6-fold or 3.2-
fold increase in fluorescence emission intensities at 514 nm,
500
PPi
0.8
0.7
400
ATP
Ensemble, F--, Cl--, Br--,
ADP
I--, NO3--, AcO--, Pi, AMP
0.6
ADP
300
0.5
Pi, AMP
ATP
AcO--, NO3--, F--, Cl--,
0.4
0.3
200
Br--, I--, Ensemble
0.2
100
PPi
0.1
0
0
500
520
540
560
580
600
350 400 450 500 550 600 650 700 750 800
Wavelength (nm)
Wavelength (nm)
Figure 2. Changes of UV–vis absorption for ensemble 1 ([1] = 2.1 ꢁ 10ꢀ5 M) upon
the addition of various anions (2 equiv, sodium salts) in a 10 mM HEPES buffer (pH
7.4) at 25 °C (Ensemble 1 = complex 1 + PV).
Figure 5. Changes of fluorescence emission for ensemble 2 ([1] = 7.35 ꢁ 10ꢀ5 M)
upon the addition of various anions (1.2 equiv, sodium salts) in a 10 mM HEPES
buffer (pH 7.4) at 25 °C (Ensemble 2 = complex 1 + Fluorescein disodium salts).

S. Y. Kim, J.-I. Hong / Tetrahedron Letters 50 (2009) 1951–1953
1953
Acknowledgments
N
N
N
N
+2
2+
This work was supported by the KRF (2006-312-C00592) and
Seoul R&BD. S.Y.K. is grateful to the Ministry of Education for the
award of the BK 21 fellowship.
Cu
Cu
N
N
O
OH
OH
Complex 1
Ensemble 1
Sky blue
Supplementary data
PPi
-
SO
OH
3
Synthesis and HRMS data of complex 1, color change of PV for
addition of complex 1, fluorescence emission change of ensemble
2 after addition of PPi, and the determination of association con-
stant by displacement titration are available. Supplementary data
associated with this article can be found, in the online version, at
doi:10.1016/j.tetlet.2009.02.036.
Pale yellow
+N2
N
N
N
N
2+
Cu
Cu
N
References and notes
O
O
ONa
Complex 1
Ensemble 2
Quenching
1. (a) Martínez-Máñez, R.; Sancenón, F. Chem. Rev. 2003, 103, 4419–4476; (b) Gale,
P. A. Coord. Chem. Rev. 2003, 240, 1–226; (c) Wiskur, S. L.; A-Haddou, H.; Lavigne,
J. J.; Anslyn, E. V. Acc. Chem. Res. 2001, 34, 963–972; (d) Yoon, J.; Kim, S. K.; Singh,
N. J.; Kim, K. S. Chem. Soc. Rev. 2006, 35, 355–360.
2. (a) Limpcombe, W. N.; Sträter, N. Chem. Rev. 1996, 96, 2375–2434; (b) Nyrén, P.
Anal. Biochem. 1987, 167, 235–238; (c) Tabary, T.; Ju, L. J. Immunol. Methods 1992,
156, 55–60.
CO Na
2
PPi
Fluorescence
3. (a) McCarty, D. J. Arthritis Rheum. 1976, 19, 275–285; (b) Doherty, M. Ann.
Rheum. Dis. 1983, 42, 38–44.
Scheme 1. A schematic representation of dual signal PPi ensemble systems.
4. (a) Nishizawa, S.; Kato, Y.; Teramae, N. J. Am. Chem. Soc. 1999, 121, 9463–9464;
(b) Lee, D. H.; Im, J. H.; Son, S. U.; Chung, Y. K.; Hong, J.-I. J. Am. Chem. Soc. 2003,
125, 7752–7753; (c) Lee, D. H.; Kim, S. Y.; Hong, J.-I. Angew. Chem., Int. Ed. 2004,
43, 4777–4780; (d) Lee, H. N.; Xu, Z.; Kim, S. K.; Swamy, K. M. K.; Kim, Y.; Kim, S.-
J.; Yoon, J. J. Am. Chem. Soc. 2007, 129, 3828–3829.
5. (a) Fabbrizzi, L.; Marcotte, N.; Stomeo, F.; Taglietti, A. Angew. Chem., Int. Ed. 2002,
41, 3811–3814; (b) Han, M. S.; Kim, D. H. Angew. Chem., Int. Ed. 2002, 41, 3809–
3811; (c) Folmer-Andersen, J. F.; Lynch, V. M.; Anslyn, E. V. J. Am. Chem. Soc.
2005, 127, 7986–7987; (d) Lee, D. H.; Kim, S. Y.; Hong, J.-I. Tetrahedron Lett. 2007,
48, 4477–4480; (e) McDonough, M. J.; Reynolds, A. J.; Lee, W. Y. G.; Jolliffe, K. A.
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respectively. When 1.2 equiv of AMP or Pi was added into solution
of ensemble 2, the fluorescence emission intensities increased two-
fold or 1.9-fold,ꢀrespectively. Other anions such as Fꢀ, Clꢀ, Brꢀ, Iꢀ,
NO3ꢀ, and AcO barely showed any changes in the fluorescence
emission intensities.
The schematic representation for the dual signal ensemble sys-
tem is illustrated in Scheme 1. Ensemble 1, composed of complex 1
and PV, turned from yellow to blue upon the addition of PPi to the
ensemble, which was caused by the displacement of weakly bound
PV by PPi. Similarly, ensemble 2, consisting of complex 1 and fluo-
rescein, revived the original fluorescence of fluorescein upon the
addition of the more strongly binding PPi to ensemble 2.
In conclusion, using quenching effect of a Cu(II) ion, we devel-
oped simple dual signal (color change and fluorescence ON–OFF)
ensemble system for detection of PPi, based on a bis(Dpa-CuII)
complex 1. There are several advantages to this system: (1) an easy
synthesis of the receptor molecules; (2) the use of commercially
available indicators; and (3) dual signaling effect (color and fluo-
rescence changes).
6. Yoshiro, M. Bull. Chem. Soc. Jpn. 2002, 75, 1383–1384.
7. Synthesis of complex 1. To
a solution of a,
a0-dibromo-m-xylene (200 mg,
0.76 mmol) in MeCN were added 2.1 equiv of Cs2CO3 (520 mg), di-2-(picolyl)
amine (318 mg) and 1.5 equiv of KI (189 mg). The reaction mixture was stirred
at 60 °C overnight, and then all the volatile components were evaporated. The
residue was partitioned between CH2Cl2 and brine (ꢁ2). The combined organic
phase was washed with water, and then dried in anhydrous Na2SO4. Flash
chromatographic purification (only CH2Cl2ꢀCH2Cl2:MeOH = 20:1) afforded
2
(52% yield). Finally, complex 1 was prepared by adding aqueous solution of
Cu(ClO4)2 (2 equiv) to 2 (1 mg) in DMSO (1 ml). Characterization for complex 1
was reported previously.6 HRMS (FAB): m/e calcd for C32H32Cl3N6O12 [M]+
922.9736, found 922.9734.
8. Conners, K. A.. Binding Constants. In The Measurement of Molecular Complex
Stability;; John Wiley and Sons: New York, 1987.
9. Zhong, Z.; Anslyn, E. V. J. Am. Chem. Soc. 2002, 124, 9014–9015.