Highly sensitive chemosensor for detection of PPi with improved detection limit

Article abstract of DOI:10.1016/j.tetlet.2011.07.067

We have developed a new chemosensor 1·2Zn for the detection of PPi in an aqueous solution by photo-induced electron transfer. Two coumarin units were introduced to enhance the fluorescent signals. Consequently, this chemosensor showed an improved detectio

Full text of DOI:10.1016/j.tetlet.2011.07.067

Tetrahedron Letters 52 (2011) 4944–4946
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Highly sensitive chemosensor for detection of PPi with improved
detection limit
⇑
Hoon Jun Kim, Jae Han Lee, 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 a new chemosensor 1Á2Zn for the detection of PPi in an aqueous solution by photo-
induced electron transfer. Two coumarin units were introduced to enhance the fluorescent signals. Con-
sequently, this chemosensor showed an improved detection limit as compared to the previously reported
chemosensor 2Á2Zn.
Received 30 May 2011
Revised 11 July 2011
Accepted 15 July 2011
Available online 22 July 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Chemosensor
Photo-induced electron transfer
Pyrophosphate
The development of chemosensors for the detection of biologi-
cally significant anions is a growing research topic.1 Chemosensor-
based detection of pyrophosphate (P2O74À, PPi) is particularly
important as PPi is involved in a range of bioenergetic and metabolic
processes, such as gene duplication, gene transcription, and signal
transduction.2 Additionally, PPi is closely related to diseases: Accu-
mulations of calcium pyrophosphate dihydrate crystals are fre-
quently found in patients with osteoarthropathy or psuedogout.3
of this sensor to PPi by more than hundred times that of 2Á2Zn
to PPi by introducing additional hydrogen bonds to this sensor.7d
Although the introduction of additional hydrogen bonds results
in a notable increase in the binding affinity, introducing a high
quantum yield signaling motif is expected to increase the sensitiv-
ity of PPi chemosensors, leading to an improved detection limit.
Therefore, two coumarin units were introduced in place of the
naphthyl group used in 2Á2Zn to amplify the fluorescent response
caused by the addition of PPi (Scheme 1).7b
Approximately, 3 and 6 lM of PPi is normally present in human
plasma and serum, respectively.4 Thus, the PPi in biological sam-
ples can be detected using the known PPi chemosensors because
these sensors respond to micromolar concentrations of PPi.5–7
However, there is a need to develop highly sensitive PPi sensors
with improved detection limits because enzymatic reactions that
involve the release of this anion often result in subtle changes in
the PPi concentration. Further, the improvement of the detection
limit results in efficient pyrosequencing, which is one of the most
promising methods of DNA sequencing.8 In this communication,
we propose a fluorescent chemosensor (1Á2Zn) for the detection
of PPi; the chemosensor has an improved detection limit as com-
pared to that of the existing PPi sensors and also shows selectivity
for PPi over other anions including phosphate anions in aqueous
media.
The synthesis of compound 1 is outlined in Scheme 2. The conver-
sion of 1,3-phenylenediacetic acid into its acyl chloride facilitated
a-bromination. The resulting halogenated acyl chloride was then
hydrolyzed to the corresponding carboxylic acid, which was con-
verted into its methyl ester product (compound 4). Compound 3
was obtained through the reaction between compound 4 and
di-(2-picolyl)amine in the presence of potassium carbonate and
potassium iodide. Hydrolysis of compound 3 followed by amide
bond formation between the resulting diacid and 2 equiv of 3-ami-
no-7-diethylaminocoumarin furnished compound 1.
The effect of metal coordination on the emission spectra was
measured through fluorescence spectroscopy (Supplementary
data): By blocking the photo-induced electron transfer (PET), com-
plexes with zinc and cadmium cations showed strong fluorescence
emissions compared to the fluorescence emissions of other metal
complexes.9 Blocking of the PET process through coordination of
a d10-metal with an amine (reductive quencher) is commonly
observed in fluorescence chemosensor studies (Fig. 1).10 We
expected that the addition of PPi would weaken the metal coordi-
nation with the reductive quencher and lead to a significant change
in the fluorescence intensity of 1Á2Zn and 1Á2Cd by restoring the
PET pathway (Fig. 1).
Generally, anion sensors consist of two basic elements.1 The
first one is the binding moiety, which overcomes strong hydration
in an aqueous medium. The other element converts these binding-
induced changes into optical signals via chromophores or fluoro-
phores. Previously, we were able to improve the binding affinity
⇑
Corresponding author. Tel.: +82 2 880 6682; fax: +82 2 889 1568.
E-mail address: jihong@snu.ac.kr (J.-I. Hong).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.07.067

H. J. Kim et al. / Tetrahedron Letters 52 (2011) 4944–4946
4945
Et2
N
O
O
O
O
NEt2
NH
HN
O
N
O
N
N
N
N
N
OH
N
N
N
N
N
N
1
2
Scheme 1. Introduction of two coumarin units to improve the detection limit of
PPi.
(a)
(b)
O
O
OMe
OMe
O
O
O
HO
OH
MeO
OMe
O
N
Figure 2. Fluorescence emission spectra of 1Á2Zn (10
anions (20
l
M) on addition of various
N
N
N
N
Br
Br
l
M, sodium salts) in an aqueous HEPES buffer solution (10 mM, pH 7.4).
N
4
3
Et2N
O
O
O
O
NEt2
NH
HN
(c)
O
N
O
N
N
N
N
N
1
Scheme 2. Reagents and conditions: (a) (i) (COCl)2, DMF, CH2Cl2, (ii) Br2, benzene,
(iii) H2O, (iv) cat. H2SO4, MeOH; (b) di-(2-picolyl)amine, K2CO3, KI, MeCN; (c) (i)
NaOH, MeOH/H2O, (ii) 3-amino-7-diethylaminocoumarin, EDC–HCl, pyridine/
CH2Cl2.
As expected, we found that the fluorescence of the complex
(1Á2Zn) between 1 and zinc cations was significantly reduced on
the addition of PPi (Fig. 2). The effect of other anions on the emis-
sion spectra of 1Á2Zn was examined by adding various anions to
1Á2Zn in an aqueous 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethane-
sulfonic acid (HEPES) buffer solution (0.01 M, pH 7.4) at 25 °C.11
However, other anions, including those of phosphate, showed no
detectable changes in the fluorescence of 1Á2Zn (Fig. 2). These re-
sults suggest that 1Á2Zn has a high selectivity for PPi over other
anions.
To measure the change in the fluorescence of 1Á2Zn upon the
addition of PPi, fluorescence titration was carried out in an aqueous
HEPES buffer solution (10 mM, pH 7.4, 25 °C). On the addition of
PPi, fluorescence decreased by a factor of 9.8 and was saturated
at 3.3 equiv (Fig. 3). 1:1 Complexation between 1Á2Zn and PPi
was confirmed by Job’s plot (Fig. 3 inset, refer to Supplementary
data for the Job’s plot for adenosine triphosphate)
Figure 3. Change in the fluorescence of 1Á2Zn (10
lM) upon addition of PPi (0–
33 M) in aqueous HEPES buffer solutions (10 mM, pH 7.4).
l
of 2Á2Zn upon the addition of PPi under the same conditions. The
detection limit was calculated to be three times that of the stan-
dard deviation of the background noise. As expected, the detection
limit of 1Á2Zn for PPi (49 nM; refer to Supplementary data) was
better than that of 2Á2Zn (83 nM).12 Considering the reduced bind-
ing affinity of 1Á2Zn compared to that of 2Á2Zn, the improved
detection limit of 1Á2Zn indicates that high quantum yield fluoro-
phores may have a dominant effect on the determination of the
detection limit.
In summary, we developed a new fluorescent chemosensor
(1Á2Zn) for the detection of PPi in an aqueous solution. The sensor
system shows an improved detection limit for PPi compared to that
of the naphthyl-based PPi sensor by the introduction of coumarin
dye. In addition, it shows good selectivity for PPi over other anions.
The emission spectra of 1Á2Zn containing coumarin dye were
expected to vary significantly upon the addition of PPi, resulting
in an improved detection limit. As depicted in Figure 4, the change
in fluorescence intensity of 1Á2Zn was 4.7 times greater than that
Figure 1. Proposed mechanism for controlling the fluorescence by blocking and restoring PET.

4946
H. J. Kim et al. / Tetrahedron Letters 52 (2011) 4944–4946
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Figure 4. Fluorescence spectra of sensor 1Á2Zn (10
l
M, solid lines) and 2Á2Zn
(10 lM, dotted lines) before and after the addition of PPis (20 lM) in an aqueous
buffer solution (10 mM HEPES, pH 7.4).
Acknowledgments
7. (a) Lee, D. H.; Im, J. H.; Son, S. U.; Chung, Y. K.; Hong, J.-I. J. Am. Chem. Soc. 2003,
125, 7752–7753; (b) Lee, D. H.; Kim, S. Y.; Hong, J.-I. Angew. Chem., Int. Ed. 2004,
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Kim, S. K.; Lee, D. H.; Hong, J.-I.; Yoon, J. Y. Acc. Chem. Res. 2009, 42, 23–31; (h)
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This work was supported by the Basic Science Research Pro-
gram through the National Research Foundation of Korea (NRF)
grant funded from the Ministry of Education, Science, and Technol-
ogy (MEST) of Korea (to the Center for Next Generation Dye-sensi-
tized Solar Cells, No. 2011-0001055, and No. 2009-0080734). We
also wish to acknowledge BK21 fellowship grants provided to H.J.K.
8. Zhou, G.; Kajiyama, T.; Gotou, M.; Kishimoto, A.; Suzuki, S.; Kambara, H. Anal.
Chem. 2006, 78, 4482–4489.
Supplementary data
9. There might be two types of metal complexes as proposed by Yoon and co-
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Supplementary data (spectral data for the new compounds, ef-
fect of metal cations and anions on the emission spectra, determi-
nation of the detection limit, and Job’s plot for ATP) associated with
this article can be found, in the online version, at doi:10.1016/
j.tetlet.2011.07.067.
References and notes
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