Colorimetric and orange light-emitting fluorescent probe for pyrophosphate in water

Article abstract of DOI:10.1016/j.tetlet.2012.08.018

A dual-mode probe based on a benzothiazolium hemicyanine chromophore was designed and synthesized for the detection of pyrophosphate (PPi) in water. The use of a fluorescent probe for colorimetric and long-wavelength fluorescence detection of PPi could be

Full text of DOI:10.1016/j.tetlet.2012.08.018

Tetrahedron Letters 53 (2012) 5528–5530
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Colorimetric and orange light-emitting fluorescent probe for pyrophosphate
in water
Dong-Nam Lee a, Ala Jo a, Seung Bum Park a,b, , Jong-In Hong a,
⇑
⇑
a Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-747, Republic of Korea
b Department of Biophysics and Chemical Biology/BioMAX Institute, 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:
A dual-mode probe based on a benzothiazolium hemicyanine chromophore was designed and synthe-
sized for the detection of pyrophosphate (PPi) in water. The use of a fluorescent probe for colorimetric
and long-wavelength fluorescence detection of PPi could be suitable for both rapid in-field and bioimag-
ing experiments.
Received 18 June 2012
Revised 28 July 2012
Accepted 3 August 2012
Available online 10 August 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Colorimetric detection
Orange fluorescence
Pyrophosphate
Cellular imaging
Pyrophosphate (PPi) is involved in a number of important cellu-
lar metabolic processes such as DNA and RNA polymerization reac-
tions and ATP hydrolysis.1 In addition, researchers have recently
reported a relationship between PPi concentration and cancer.2
In this regard, there is a growing interest in using different detec-
tion methods to monitor PPi in biological processes, with fluores-
cence techniques being the most popular.3–5 Although many
fluorescent probes for PPi have already been reported, both by
our group and others,6–11 there is a continuous need for the devel-
opment of novel probes for this species that can facilitate the study
of biological processes involving its release.
It is well known that a fluorophore with near infrared (NIR) or
long-wavelength emission is desirable for the detection of cellular
PPi because of its favorable cell penetrating ability and the minimal
overlap with the wavelengths at which cells exhibit autofluores-
cence.12,13 In addition, a probe that can detect PPi using a colori-
metric method could facilitate rapid in-field analysis without
sophisticated instrumentation.14 Currently, there is considerable
interest in developing chemosensors that emit at longer wave-
lengths and can be visualized with the naked eye.15–17 However,
there are only a few successful PPi sensors reported to date.18,19
Herein, we report a colorimetric and orange light-emitting fluores-
cent probe for the detection of PPi in cells.
Figure 1. Synthesis of 1ꢁ2Zn(II): (a) 2,20ꢂDipicolylamine, aq formaldehyde, 1,4-
dioxane, 110 °C, 2 days, 48% yield (b) 3-Ethyl-2-methylbenzothiazolium iodide,
pyridine, EtOH, 80 °C, 1 day, 12% yield (c) Zn(NO3)2, DMSO, RT, 30 min, quantitative
yield.
emission (kem ꢀ 560 nm).20 Thus, by combining this with a Zn(II)
ꢁ2,20-dipycolylamine (DPA) complex as a binding site, an effective
fluorescent probe for PPi could be achieved (Fig. 1).4,7 Treatment
of 4-hydroxybenzaldehyde with DPA in the presence of formalde-
hyde gave compound 2. The benzothiazolium hemicyanine fluoro-
phore was then introduced by Knoevenagel condensation with
3-ethyl-2-methylbenzothiazolium iodide. Sensor 1ꢁ2Zn(II) was
obtained by the addition of Zn(NO3)2 to a solution of 1 in DMSO
(See Supplementary Material).
It was expected that benzothiazolium hemicyanine dye would
be a suitable signaling unit for use in biological applications be-
cause of its cell permeability, non-toxicity, and long-wavelength
Photophysical properties of 1ꢁ2Zn(II) in the presence of PPi were
⇑
Corresponding authors. Tel.: +82 2 880 6682; fax: +82 2 889 1568.
E-mail addresses: sbpark@snu.ac.kr (S.B. Park), jihong@snu.ac.kr (J.-I. Hong).
monitored by UVꢂvis and fluorescence spectrometry. UVꢂvis
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.08.018

D.-N. Lee et al. / Tetrahedron Letters 53 (2012) 5528–5530
5529
titration of 1ꢁ2Zn(II) revealed that the maximum intensity wave-
length (k = 454 nm) underwent a 27 nm bathochromic shift with
an increased intensity upon the addition of PPi in 10 mM HEPES
buffer (pH 7.4). The intensity of the fluorescence emission of
1ꢁ2Zn(II) at 548 nm was relatively weak (Uf = 0.08), whereas the
addition of 2 equiv. of PPi caused a prominent increase (ca. 5 times)
in fluorescence centered at 558 nm (Uf = 0.10)21 (Fig. 2A). The fluo-
rescence intensity exhibited a sharp increase upon the addition of
an increasing amount of PPi to the 1ꢁ2Zn(II) solution, with the
maximum intensity being exhibited when 1 equiv of PPi was added
(Fig. 2B). The binding constant was estimated to be 4.4 ꢃ 107 Mꢂ1
(See Supplementary Material).
To evaluate the selectivity of 1ꢁ2Zn(II) toward PPi, various bio-
logical competitive analytes were prepared. As expected, there
were no significant changes in the fluorescence intensity upon
the addition of an excess amount of phosphate (Pi), CNꢂ, citrate,
Clꢂ, Fꢂ, HCO3ꢂ, OAcꢂ, N3ꢂ, and AMP, whereas a detectable response
was observed due to ATP. This competitive experiment clearly
demonstrated that 1ꢁ2Zn(II) has a high selectivity for PPi over
many other biological competitive analytes, apart from nucleoside
triphosphates (NTPs) that are present in cells (Fig. 3).
Furthermore, the selective recognition of PPi by 1ꢁ2Zn(II) can be
visualized by the naked eye owing to the colorimetric change.
Solutions of 1ꢁ2Zn(II) alone and those of it mixed with other ana-
lytes, except PPi and ATP, in 10 mM HEPES buffer, appeared yellow,
Figure 4. Colorimetric detection of 1ꢁ2Zn(II) (5
lM, pH 7.4, 10 mM HEPES buffer) in
the presence of (a) probe only, (b) PPi, (c) PO43ꢂ, (d) CNꢂ, (e) citrate, (f) Clꢂ, (g) Fꢂ,
(h) HCO3ꢂ, (i) OAcꢂ, (j) N3ꢂ, (k) AMP, (l) ATP. 20 equiv. of analyte were added except
for PPi (2 equiv.).
Figure 2. (A) UVꢂvis absorption (blue dashed line) and fluorescence emission (red
dashed line) spectra of 1ꢁ2Zn(II) (10
lM) in the presence of PPi (2 equiv., solid line)
in 10 mM HEPES buffer (pH 7.4). (B) Fluorescence emission intensity of 1ꢁ2Zn(II)
(10 M, pH 7.4, 10 mM HEPES buffer).
l
M) in various concentrations of PPi (1 ꢀ 100 l
Inset shows increasing fluorescence intensities of 1ꢁ2Zn(II), which are measured at
560 nm with excitation at 500 nm.
Figure 5. Fluorescence live-cell pseudo-color images of C2C12 myoblast cells. (A
and C) Cells stained by Hoechst nuclear dye. (B) Cells incubated with Hoechst
Figure 3. Fluorescence emission intensity of 1ꢁ2Zn(II) (5
l
M, pH 7.4, 10 mM HEPES
nuclear dye and then with 1ꢁ2Zn(II) (80
Hoechst nuclear dye followed by 1ꢁ2Zn(II) (80
with PPi (Na4P2O7, 200 M) for 30 min. Emission was collected at (A, C) blue
channel and (B, D) Cy3 channel upon excitation at (A, C) 350 25 nm (200 W metal
halide arc lamp) and (B, D) 543 11 nm (200 W metal halide arc lamp). All images
were acquired using a 20ꢃ objective. (E) Quantification data from (B) and (D).
l
M) for 30 min. (D) Cells incubated with
buffer) in the presence of (a) probe only, (b) Pi, (c) CNꢂ, (d) citrate, (e) Clꢂ, (f) Fꢂ, (g)
HCO3ꢂ, (h) OAcꢂ, (i) N3ꢂ, (j) AMP, (k) ATP. Blue bars represent the addition of
analytes (20 equiv.). Red bars represent the subsequent addition of PPi (2 equiv.) to
the mixture. All data were measured using a fluorescence spectrometer at 560 nm
with excitation at 500 nm.
l
M) for 30 min, and subsequently,
l

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D.-N. Lee et al. / Tetrahedron Letters 53 (2012) 5528–5530
whereas mixtures of 1ꢁ2Zn(II) and either PPi or ATP were pale
brown. Therefore, it is clear that 1ꢁ2Zn(II) can distinguish PPi or
ATP from other anions (Fig. 4). The color change may result from
the observable bathochromic shift22,23 of 1ꢁ2Zn(II) triggered by
the addition of either PPi or ATP (Fig. 2A).
References and notes
1. Heinonen, J. K. Biological Role of Inorganic Pyrophosphate; Kluwer Academic
Publishers: Norwell, 2001.
2. Xu, S.; He, M.; Yu, H.; Cai, H.; Tan, X.; Lu, B.; Shu, B. Anal. Biochem. 2001, 299,
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3. Vance, D. H.; Czarnik, A. W. J. Am. Chem. Soc. 1994, 116, 9397.
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125, 7752.
5. Nyrén, P.; Pettersson, B.; Uhlén, M. Anal Biochem. 1993, 208, 171.
6. Kim, S. K.; Lee, D. H.; Hong, J.-I.; Yoon, J. Acc. Chem. Res. 2009, 42, 23.
7. Lee, D. H.; Kim, S. Y.; Hong, J.-I. Angew. Chem., Int. Ed. 2004, 43, 4777.
8. Lee, J. H.; Park, J.; Lah, M. S.; Chin, J.; Hong, J.-I. Org. Lett. 2007, 9, 3729.
9. Ojida, A.; Nonaka, H.; Miyahara, Y.; Tamaru, S.; Sada, K.; Hamachi, I. Angew.
Chem., Int. Ed. 2006, 45, 5518.
10. Cho, H. K.; Lee, D. H.; Hong, J.-I. Chem. Commun. 2005, 1690.
11. 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.
12. Karton-Lifshin, N.; Segal, E.; Omer, L.; Portnoy, M.; Satchi-Fainaro, R.; Shabat, D.
J. Am. Chem. Soc. 2011, 133, 10960.
13. Fu, M.; Xiao, Y.; Qian, X.; Zhao, D.; Xu, Y. Chem. Commun. 2008, 1780.
14. Li, Y.; Ashizawa, M.; Uchida, S.; Michinobu, T. Macromol. Rapid Commun. 1804,
2011, 32.
15. Yuan, M.; Li, Y.; Li, J.; Li, C.; Liu, X.; Lv, J.; Xu, J.; Liu, H.; Wang, S.; Zhu, D. Org.
Lett. 2007, 9, 2313.
16. Wang, Y.; Wu, H. Q.; Sun, J. H.; Liu, X. Y.; Luo, J.; Chen, M. Q. J. Fluoresc. 2012, 22,
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18. Zhu, W.; Huang, X.; Guo, Z.; Wu, X.; Yu, H.; Tian, H. Chem. Commun. 2012, 48,
1784.
19. Feng, X.; An, Y.; Yao, Z.; Li, C.; Shi, G. ACS Appl. Mater. Interfaces 2012, 4, 614.
20. Zhu, B.; Yuan, F.; Li, R.; Li, Y.; Wei, Q.; Ma, Z.; Du, B.; Zhang, X. Chem. Commun.
2011, 47, 7098.
21. Fluorescence quantum yields were determined using rhodamine B (Uf = 0.49 in
ethanol) as a standard: Casey, K. G.; Quitevis, E. L. J. Phys. Chem. 1988, 92, 6590.
22. Leontiev, A. V.; Rudkevich, D. M. J. Am. Chem. Soc. 2005, 127, 14126.
23. Sola, A.; Tárraga, A.; Molina, P. Dalton Trans. 2012, 41, 8401.
24. We believe that the observed weak fluorescence prior to PPi addition may be
due to the presence of cellular PPi and NTPs.
To determine whether 1ꢁ2Zn(II) was suitable for use in biologi-
cal applications, in vitro testing using the C2C12 myoblast cell line
was carried out (Fig. 5). The cells were incubated with 1ꢁ2Zn(II)
(80 lM) for 30 min, and subsequently with PPi (Na4P2O7,
200 lM) for 30 min. Prior to the addition of PPi, the cells showed
only a weak level of fluorescence (Fig. 5B),24 however, a clear intra-
cellular fluorescence increase was observed after the addition
(Fig. 5D). The viability of the cells after treatment with the probe
was verified using the Hoechst nuclear stain (Fig. 5A, C).25 These
results show the potential for using 1ꢁ2Zn(II) for the detection of
PPi within cells.
In conclusion, we have developed a highly selective colorimetric
and fluorescent probe for PPi. This probe can be applied in biological
fluorescence imaging, and its orange emitting light and good cell
permeability make it highly desirable for other bio-studies. This
would be attractive to biological and medical researchers who are
studying the biological roles and diagnostic relevance of PPi.
Acknowledgments
This work was supported by an NRF grant funded by the MEST
(Grant No. 2012-0000159). D.-N. Lee was supported by Hi Seoul
Science/Humanities Fellowship from the Seoul Scholarship
Foundation.
25. The cell imaging experiments with lower concentrations of 1ꢁ2Zn(II) (640
lM)
Supplementary data
were not successful as the fluorescence was not high enough for monitoring
the fluorescence differences. After treatment with higher concentrations of
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2012.08.
018.
1ꢁ2Zn(II) (P160
lM) and PPi (P400 lM), denaturation of cells was
occasionally observed.