A highly selective pyrophosphate sensor based on ESIPT turn-on in water

Article abstract of DOI:10.1021/ol200054w

Pyrophosphate (PPi) is a biologically important target. A binuclear system 3?2Zn is found to selectively recognize PPi, leading to a ratiometric fluorescent sensor at pH 7.4 in water. The binding event triggered a large fluorescence response (～100 nm bath

Full text of DOI:10.1021/ol200054w

ORGANIC
LETTERS
2011
Vol. 13, No. 6
1362–1365
A Highly Selective Pyrophosphate Sensor
Based on ESIPT Turn-On in Water
Wei-Hua Chen,† Yu Xing,‡ and Yi Pang*,†
Department of Chemistry and Maurice Morton Institute of Polymer Science,
The University of Akron, Akron, Ohio 44325, United States, and Institute for Integrative
Genome Biology and Department of Botany and Plant Sciences, University of California
at Riverside, Riverside, California 92521, United States
yp5@uakron.edu
Received January 7, 2011
ABSTRACT
Pyrophosphate (PPi) is a biologically important target. A binuclear system 3•2Zn is found to selectively recognize PPi, leading to a ratiometric
fluorescent sensor at pH 7.4 in water. The binding event triggered a large fluorescence response (∼100 nm bathochromic shift) by turning on the
excited state intramolecular proton transfer (ESIPT). Detection of PPi released from a PCR experiment indicated that this new probe could be a
useful tool in bioanalytical applications.
Pyrophosphate (P2O74-, PPi) isa biologically significant
anion which isinvolved inmany cellular processes,1 suchas
cellular ATP hydrolysis, DNA and RNA polymerizations,
and enzymatic reactions. It has been reported that abnor-
mal PPi levels can lead to vascular calcification resulting in
severe medical conditions.2 Since Czarnik’s pioneering
work in fluorescent sensing of PPi in 1994 by using
a polyamine-attached anthracene derivative in 100%
aqueous solution,3 considerable effort has been made to
develop chemosensors for the optical detection of PPi.4
In order to achieve selective binding for PPi, the cur-
rent strategy utilizes a binuclear metal5a-j complex in
conjunction with bis(2-pyridylmethyl)amine (DPA) ligands
as shown in 1.
An example of 1 (R = ;NdN;Ar)5a illustrates the
selective binding of PPi to give a non-fluorescent 2 and
shows a color response (λmax red shift by 48 nm). Despite
strong interest in developing fluorescent PPi sensors,5d,h
the PPi binding has induced a relatively small red shift in
fluorescence signal (∼11 nm from a fluorescein-deriva-
tive5d or ∼20 nm from a coumarin derivative).5h Lack of
significant fluorescence signal shift lowers the sensitivity
and hampers the reliable fluorescent detection for PPi. In
(5) (a) Lee, D. H.; Im, J. H.; Son, S. U.; Chung, Y. K.; Hong, J.-I.
J. Am. Chem. Soc. 2003, 125, 7752. (b) Lee, D. H.; Kim, S. Y.; Hong,
J.-I. Angew. Chem., Int. Ed. 2004, 43, 4777. (c) Lee, J. H.; Park, J.; Lah,
M. S.; Chin, J.; Hong, J.-I. Org. Lett. 2007, 9, 3729. (d) Jang, Y. J.; Jun,
E. J.; Lee, Y. J.; Kim, Y. S.; Kim, J. S.; Yoon, J. J. Org. Chem. 2005, 70,
9603. (e) Lee, H. N.; Swamy, K. M. K.; Kim, S. K.; Kwon, J.-Y.; Kim,
Y.; Kim, S.-J.; Yoon, Y. J.; Yoon, J. Org. Lett. 2007, 9, 243. (f) Carolan,
J.; Butler, S.; Jolliffe, K. J. Org. Chem. 2009, 74, 2992. (g) Zeng, Z.;
Torriero, A.; Bond, A.; Spiccia, L. Chem.-Eur. J. 2010, 16, 9154. (h)
Kim, M. J.; Swamy, K. M. K.; Lee, K. M.; Jagdale, A. R.; Kim, Y.; Kim,
S.-J.; Yoo, K. H.; Yoon, J. Chem. Commun. 2009, 45, 7215. (i) Fabbrizzi,
L.; Marcotte, N.; Stomeo, F.; Taglietti, A. Angew. Chem., Int. Ed. 2002,
41, 3811. (j) Jose, D. A.; Stadlbauer, S.; Konig, B. Chem.;Eur. J. 2009,
15, 7404.
† The University of Akron.
‡ University of California at Riverside.
(1) Heinonen, J. K. Biological Role of Inorganic Pyrophosphate;
Kluwer Academic Publishers: Norwell, 2001.
(2) (a) Hessle, L.; Johnson, K. A.; Anderson, H. C.; Narisawa, S.;
Sali, A.; Goding, J. W.; Terkeltaub, R.; Millan, J. L. Proc. Natl. Acad.
Sci. 2002, 99, 9445. (b) Kim, I.-B.; Han, M. H.; Phillips, R. L.; Samanta,
B.; Rotello, V. M.; Zhang, J. Z.; Bunz, U. H. F. Chem.-Eur. J. 2009, 15,
449.
(3) Vance, D. H.; Czarnik, A. W. J. Am. Chem. Soc. 1994, 116, 9397.
(4) Kim, S. K.; Lee, D. H.; Hong, J.-I.; Yoon, J. Acc. Chem. Res.
2009, 42, 23.
r
10.1021/ol200054w
Published on Web 02/21/2011
2011 American Chemical Society

Scheme 1. Molecular Design of PPi Sensor with ESIPT
the current PPi sensing systems (e.g., from 1 to 2), the PPi
only induces a minor structural change on the chromo-
phore, thereby resulting in the limited spectral shift.
Intramolecular hydrogen bonding in 2-(2-hydroxy-
phenyl)-1,3-benzoxazole (HBO) derivatives is known to
exhibit excited state intramolecular proton transfer
(ESIPT),6 which gives a large Stokes shift (100-200 nm).
Extending our general interest in exploring the analyte
binding-enabled ESIPT,7 we now report the PPi sensor
3 2Zn, in which the ESIPT is used as a switching mechan-
ism in a buffered aqueous solution (Scheme 1). In the PPi
sensor 3 2Zn, the phenoxide “oxygen” is used to chelate
3
2þ3
Figure 1. (a) FL spectra of sensor 3 2Zn (12 μM) in aqueous
3
the Zn center to disable ESIPT. The analyte recognition
requires PPi to simultaneously connect to the two Zn2þ
sites in 4, which moves the Zn2þ away to free the phenol
moiety for ESIPT. The well-defined chemical event in the
molecular design leads to a highly selective probe for PPi,
with fluorescence changing from blue to green (corres-
ponding to the bathochromic shift of ∼100 nm, Figure 1).
The sensor 3•2Zn was synthesized from 2-(20-hydro-
xyphenyl)benzoxazole 5 (Scheme 2). The dialdehyde inter-
mediate 6 was obtainedby a dual-Duff reaction, which was
further transformed into 3 by a reductive amination to
install the two bis(2-pyridylmethyl)amino groups. The
solution of 10 mM HEPES buffer (pH 7.4) at 25 °C in the
presence of various anions (600 μM) and their fluorescent
images (irradiated at 365 nm). (b) Change in fluorescence
emission of 3 2Zn (12 μM) upon addition of PPi (sodium salt) in
3
an aqueous solution of HEPES buffer (10 mM, pH 7.4) at 25 °C.
Scheme 2. Synthetic Strategy for Sensor 3•2Zn and 8•Zn
dinuclear Zn2þ complex 3 2Zn was formedby the addition
3
of a methanolic solution of 3 to an aqueous solution of 2.0
equiv of Zn(NO3)2. The overall yield is 55% from 5. For
comparison, a mononuclear Zn2þ complex 8•Zn was
synthesized in a similar route in an overall yield of 49%
from 2-hydroxy-5-methylbenzaldehyde 7. The experimen-
tal details and characterization data are given in the
Supporting Information (SI).
solution (pH = 7.4). In the absence of an anion guest,
senor 3•2Zn gives emission at 420 nm. This fluorescent
emission was not changed upon addition of excessive
HPO42-, CH3CO2-, citrate, etc. In contrast, the PPi anion
caused the emission band to be shifted to a longer wave-
length (at 518 nm), attributed to the keto emission arising
from ESIPT (SI, Scheme S1). Although the related phos-
phate analogues ATP slightly increased the fluorescence
Figure 1 shows the effect of anions (sodium salts) on the
fluorescence spectrum of 3•2Zn in a buffered aqueous
(6) (a) Pang, Y.; Chen, W.-H. In Hydrogen Bonding and Transfer in
the Excited State; Han, K.-L., Zhao, G.-J., Eds.; Wiley: West Sussex, 2011;
Vol. 2 pp 747-760. (b) Seo, J.; Kim, S.; Park, S. Bull. Korean Chem. Soc.
2005, 26, 1706. (c) Seo, J.; Kim, S.; Park, S. J. Am. Chem. Soc. 2004, 126,
11154.
(7) Xu, Y.; Pang, Y. Chem. Commun. 2010, 46, 4070.
Org. Lett., Vol. 13, No. 6, 2011
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Figure 2. 1H NMR spectra of 3•2Zn (1.5 mM) with various
molar ratios of hydrogen pyrophosphate (Na3HP2O7, HPPi)8 in
DMSO-d6.
Scheme 3. Proposed ESIPT Turn on Mechanisma
Figure 3. (A) Mechanism of sensing PPi released from PCR by
3•2Zn. (B) Gel electrophoresis of finished PCR mixtures. M:
shown as DNA marker. (C) Fluorescence intensity of sensor
3 2Zn at 518 nm upon addition of the finished PCR product
3
mixture. W: 10 μL of finished PCR product mixture performed
without template DNA; 10 μL of finished PCR product mixture
performed with template DNA after B: 21 cycles; C: 22 cycles;
D: 23 cycles; E: 24 cycles; F: 25 cycles.
a For clarity, the NO3 ligands to zinc were omitted. Molecular
modeling of 9 (optimized by using AM1 setting on HyperChem) shows
that two vertical pyridyl rings on the top of plane form a narrow
entrance gate.
-
(= [3•2Zn þ PPi þ Hþ]þ) and a peak at m/z = 934
corresponding to the deprotonated phenoxide, [C39H35-
N7O9P2Zn2]- (= [3•2Zn þ PPi - Hþ]-).
Upon titration of 3•2Zn withhydrogen pyrophosphate,8
the 1H NMR spectra (Figure 2) revealed a new peak at
11.6 ppm corresponding to the intramolecular H-bond-
ing between the phenolic proton and benzoxazole
nitrogen.9 The intensity of this peak increased with the
amount of HPPi. The result indicated that the phen-
oxide oxygen was released from the metal-binding by
the cooperative coordination of PPi with the two Zn2þ
ions as shown in Scheme 1.
intensity, its binding with 3•2Zn was not sufficiently strong
to enable the ESIPT. Job’s plot (SI, Figure S3) revealed
that the complex had a 1:1 stoichiometry for 3•2Zn and
PPi. The formation of a 1:1 complex was further sup-
ported by electrospray mass spectrometry (ES-MS) data
(SI, Figures S5 and S6), which detected the complex
at m/z = 936 corresponding to [C39H36N7O9P2Zn2]þ
(8) The four pKa’s of pyrophosphoric acid (H4P2O7) were reported to
be 0.9, 2.0, 6.6, 9.4 (McElroy, W.D.; Glass, B. Phosphorus Metabolism;
Johns Hopkins University Press: Baltimore, 1951; Vol. I). At pH 7.4, an
(9) The chemical shift of the intramolecular H-bonding in the HBO
system is typically around 11 ppm. Chen, W.-H.; Pang, Y. Tetrahedron
Lett. 2009, 50, 6680.
aqueous solution of pyrophosphoric acid consists of 85.6% HA3-
,
13.6% H2A2-, 0.7% A4-
.
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Org. Lett., Vol. 13, No. 6, 2011

To further understand the role of the binuclear Zn(II)...
Zn(II) structure on the observed PPi selectivity, mononu-
lear 8•Zn was prepared and titrated with PPi under the
same conditions. Addition of PPi increased the fluores-
cence intensity at 427 nm without inducing a notable
bathochromic shift (SI, Figure S1). The result indicated
that no ESIPT was induced from the 8•Zn complex. In
other words, PPi was bound to the Zn2þ in the mono-
nuclear complex without breaking the existing Zn-O
bond. From the fluorescence titration experiments, the
association constants of 3•2Zn with PPi and 8•Zn with PPi
were calculated to be 9.2 ꢀ 107 and 1.8 ꢀ 104 M-1
respectively.10 It therefore can be concluded that the
principle, is coming into fashion.12 The pyrosequencing
technique relies on the enzymatic detection of the pyr-
ophosphate released during the DNA polymerase chain
reaction (PCR). The application requires the probe to
specifically recognize the released PPi, in the presence of
structurally similar anionic nucleotides 11 (Figure 3). To
demonstrate the applicability, sensor 3•2Zn was used and
found to be a simple and rapid method to fluorescently
detect the PPi released from dNTPs in a PCR. Gel
electrophoresis revealed the production of DNA of the
same molecular weight (Figure 3b), where the band
intensity was proportional to the amount of PPi released
from PCR and subsequently detected by the fluorescence
intensity at 518 nm (Figure 3c). This result showed that
the sensor 3•2Zn had potential application in the new
generation of DNA sequencings by virtue of its selectiv-
ity and sensivitity.
In summary, we have developed a new ratiometric sen-
sor based on a unique ESIPT turn-on mechanism. The
two DPA-Zn2þ groups in the sensor 3•2Zn, which were
located at a suitable distance, create a strong binding
environment to selectively recognize PPi over structu-
rally similar phosphate ATP and other anions. Utiliza-
tion of this molecular recognition event to trigger the
ESIPT results in a large fluorescent bathochromic shift
(∼100 nm). A large fluorescence response, in addition to
its excellent selectivity toward PPi, points to the probe
molecule’s potential use for biochemical and analytical
applications.
ESIPT turn-on in 3 2Zn required the analyte to simulta-
3
neously bind to two Zn2þ sites. It was assumed that
interaction of 3•2Zn with the PPi anion initially led to 9
(Scheme 3). The negative charged oxygen at the other end
of PPi then reached out to the second Zn2þ site to form 10.
Molecular modeling of 9 showed that the Zn1 and Zn2
˚
centers were separated by 7.35 A, which was beyond the
2-
-
distance a monoanion such as HCO3 and H2PO4 can
bridge. As seen from the model of 9, the pyridyl ring at the
second zinc center (labeled Zn2) acted as a gate to allow the
less hindered phosphate anion to pass. In contrast, the
bulky tail of ATP would have increased steric interaction
with the pyridyl ring at the gate, which prevents the
associated phosphate anion from entering into the cavity
to bind to the second Zn2þ site. In other words, the unique
steric interaction posed by the pyridylmethyl group plays
an important role in the sensor’s ability to differentiate PPi
from the structural analogue ATP.
Acknowledgment. Financial support has been provided
by The University of Akron and the Coleman endowment.
We thank Dr. Xiaopeng Li for assistance in acquiring
ES-MS data.
Recently, pyrosequencing,11 a revolutionary DNA seq-
uencing technique based on the “sequencing by synthesis”
(10) (a) Conners, K. A. Binding constants; Wiley: New York, 1987. (b)
Association constants were obtained by a global nonlinear least-squares fit of
the data with the program Specfit/32, available from Spectrum Software
Associates, Marlborough, MA, U.S.A.
(11) Ronaghi, M.; Uhlen, M.; Nyren, P. Science 1998, 281, 363.
(12) Guo, J.; Yu, L.; Turro, N.; Ju, J. Acc. Chem. Res. 2010, 43,
551.
Supporting Information Available. Experimental pro-
cedure and spectral data for all new compounds, optical
spectra of 3•2Zn and 8•Zn. This material is available free
of charge via the Internet at http://pubs.acs.org.
Org. Lett., Vol. 13, No. 6, 2011
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