Coumarin-derived Fe3+-selective fluorescent turn-off chemosensors: Synthesis, properties, and applications in living cells

Article abstract of DOI:10.1039/c3ra44843f

A novel coumarin-based fluorogenic probe bearing the tris unit (DAT-1) with high selectivity and suitable affinity toward Fe³⁺ over other cations tested was developed in biological systems. The sensing mechanism was studied by quantum calculations. The probe can be applied to the monitoring of Fe³⁺ in aqueous solution with a pH span of 3-8. In addition, biological imaging, membrane permeability and nontoxicity demonstrate that DAT-1 could act as a turn-off fluorescent chemosensor for Fe³⁺ in living cells.

Full text of DOI:10.1039/c3ra44843f

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PAPER
Coumarin-derived Fe3+-selective fluorescent turn-
off chemosensors: synthesis, properties, and
applications in living cells†
Cite this: RSC Adv., 2014, 4, 248
a
ab
Bo-Ting Chen,c Biao Dongc and Meng-Jiao Penga
*
Da En, Yuan Guo,
A novel coumarin-based fluorogenic probe bearing the tris unit (DAT-1) with high selectivity and suitable
affinity toward Fe3+ over other cations tested was developed in biological systems. The sensing
mechanism was studied by quantum calculations. The probe can be applied to the monitoring of Fe3+ in
aqueous solution with a pH span of 3–8. In addition, biological imaging, membrane permeability and
nontoxicity demonstrate that DAT-1 could act as a turn-off fluorescent chemosensor for Fe3+ in living cells.
Received 2nd September 2013
Accepted 6th November 2013
DOI: 10.1039/c3ra44843f
www.rsc.org/advances
known that anemia is due to iron deciency. Therefore, a
convenient and rapid method for detecting the concentration of
Fe3+ in biological samples is of great interest in biological and
Introduction
The design and synthesis of high selectivity and sensitivity
chemosensors to detect metal ions in the ecological environ-
ment and biology have attracted a great deal of attention.1 A
chemosensor shows a signicant change in electrical, elec-
tronic, magnetic, or optical signal when it binds to a specic
guest counterpart.2 Current chemosensors have many advan-
tages, including high sensitivity, low cost, easy detection, and
suitability as a diagnostic tool for biological purposes.3 As an
essential trace element in biological systems, Fe3+ plays an
important role in living organisms and metabolism. Many
enzymatic reactions involving iron are related to oxygen
metabolism, electron transfer and the mitochondrial respira-
tory chain.4 Although iron is indispensable for the proper
functioning of all living cells, it is disadvantageous when
present in excess. Excessive Fe3+ in the human body has been
found to be related to an increased incidence of certain cancers
and the dysfunction of certain organs.5 Moreover, hereditary
hemochromatosis is characterized by excess iron that causes
tissue damage and brosis with irreversible damage to various
organs.4a,6 Furthermore, iron homeostasis is an important
factor involved in neuroinammation and the progression of
Alzheimer’s disease.4a,7 At the same time, iron deciency can be
as harmful as iron overload, or even more harmful. It is well
environmental concerns. Recently, many uorescent chemo-
sensors for Fe(III)-selective detection were reported and have
been used with some success in biological applications.8
Nevertheless, a few of them have defects in actual practice such
as cross-sensitivity toward other metal cations, long response
times, poor water solubility, a narrow pH detection span, a low
uorescence quantum yield in aqueous media, and ligand
cytotoxicity.9
Of late, coumarin-based chemosensors have been extensively
used as uorescent labeling reagents for their large molar
extinction coefficient, relatively long excitation and emission
wavelengths and high uorescence quantum yield.10 Here, we
synthesized a novel coumarin-derived Fe(III)-selective chemo-
sensor
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-7-hydrox-
ycoumarin-3-carboxamide (DAT-1) which is highly selective and
sensitive toward Fe3+ over other common metal ions such as
Na+, K+, Ca2+, Fe2+, Mg2+, Al3+, Co2+, Ni2+, Ag+, Cu2+, Zn2+, Cr3+,
Cd2+, Mn2+, Hg2+ and Pb2+ in buffered aqueous solutions, and
the sensing mechanism was studied by quantum calculations.
Experiment
Ethyl-7-hydroxy-3-carboxylate
2 (ref. 10) was synthesized
according to the procedures reported in the literature. 2-Amino-
2-(hydroxymethyl)-3-propanediol (tris) was purchased. DAT-1
was prepared by the standard method with a minor modica-
tion.11 Compound 2 (234 mg, 1 mmol) was treated with tris
(121 mg, 1 mmol) in anhydrous ethanol (5 mL). The reaction
was then reuxed for 8 h. The mixture was concentrated under
vacuum and the crude product was puried by column chro-
matography (CH2Cl2 : CH3OH ¼ 10 : 1) to give DAT-1 as a yellow
solid (185 mg, yield: 60 %), mp. 239–241 ꢀC. 1HNMR (400 MHz,
DMSO-d6): d ¼ 11.09 (s, 1H), 8.96 (s, 1H), 8.81 (s, 1H), 7.81 (d,
aKey Laboratory of Synthetic and Natural Functional Molecule Chemistry of the
Ministry of Education, College of Chemistry and Materials Science, Northwest
University, 229 Taibai Road (North), Xi’an 710069, P. R. China. E-mail: guoyuan@
nwu.edu.cn; Fax: +86 29 8830 2122; Tel: +86 29 8830 2122
b
´
´
´
Institut de Chimie Organique et Analytique, Universite d’Orleans, 45067 Orleans
cedex 2, France
cState Key Laboratory on Integrated Optoelectronics, College of Electronic Science and
Engineering, Jilin University, Changchun 130012, P. R. China
† Electronic supplementary information (ESI) available. CCDC 939460. For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/c3ra44843f
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Scheme 1 Synthetic procedure of DAT-1.
J ¼ 8.5 Hz, 1H), 6.89 (dd, J ¼ 8.5, 1.7 Hz, 1H), 6.82 (d, J ¼ 1.6 Hz,
1H), 4.86 (s, 3H), 3.65 (s, 6H). 13CNMR (400 MHz, DMSO-d6): d
169.08, 166.84, 166.39, 161.52, 153.23, 137.19, 119.62, 118.82,
116.19, 106.96, 67.44, 65.30, 45.27, 45.06, 44.86, 44.65, 44.44,
44.23, 44.02. HRMS (ESI): m/z, calcd for (M ꢁ H)ꢁ 308.0770;
found 308.0798 (Scheme 1).
Results and discussion
The DAT-1 chemosensor contain a coumarin group used as the
uorescent signal unit and a 2-amino-2-(hydroxymethyl)-1,3-
propanediol (tris) acting as a possible recognition unit. Since
coumarin derivatives exhibit several advantages such as large
Stokes shis, good photostability and high uorescence
quantum yields (the quantum yield of the system was calculated
to be 0.75), they could be used as efficient uorophores. And the
tris which includes three hydroxyl groups and an amide group is
expected to serve as a potential binding group.11 Furthermore,
the tris group can improve the water solubility of chemosensors
due to its hydrophilic properties.
The structure of DAT-1 was conrmed by 1H NMR, 13C NMR,
FT-IR, ESI-MS and X-ray analysis (Fig. S7–S10†). The single
crystal of DAT-1 was obtained under vapor diffusion of CH3OH
into a solution of water and characterized using X-ray crystal-
lography (Fig. 1).
Fig. 2a shows the change in the uorescence spectra of
DAT-1 upon the addition of Fe3+ in the Na2HPO4–citric acid
buffer solution at pH 4.8. Upon the addition of an increasing
amount of Fe3+, the emission intensity of DAT-1 at 448 nm
gradually decreased. The uorescence emission intensity were
sensitively and proportionately decreased with an increasing
concentration of Fe3+ as noted from the relationship between
the uorescence emission intensity at 350 nm and Fe3+
concentration (Fig. 2b). Therefore, the DAT-1 could be used for
Fig. 2 (a) Fluorescence titration spectra (lex ¼ 350 nm) of DAT-1
(20 mM) with the addition of Fe3+ (0–200 equiv.) in the 0.2 M
Na2HPO4–citric acid buffer. (b) Titration data points and the nonlinear
least-squares fitting curve. Insert: the Stern–Volmer plot of fluores-
cence quenching of the DAT-1 by Fe3+. R2 ¼ 0.996.
the development of an efficient method for Fe3+ ion detection.
The linear relation of the obtained experimental data for Fe3+
detection could be best described as the Stern–Volmer equa-
tion. The Stern–Volmer relationship with an R2 of 0.996 is given
in the following equation (Fig. 2b, inset):
I0/I ¼ 1 + KSV[Q]
where I and I0 are the uorescence emission intensities of the
DAT-1 in the presence and absence of Fe3+ respectively. Q is the
Fe3+ concentration. KSV is the Stern–Volmer quenching constant
and is found to be 3.57 ꢂ 103 Mꢁ1. The linear range of Fe3+
concentration was from 1 ꢂ 10ꢁ5 M to 1 ꢂ 10ꢁ3 M and the
detection limit of Fe3+ was 3 ꢂ 10ꢁ7 M. It can be found that the
presented method has wider detection range and higher
sensitivity than many of uorescent chemosensors for Fe3+
.
There was a remarkable selectivity of DAT-1 for Fe3+ over
various other metal ions in the competition experiments, which
turned out to be applicable in environmental technology
(Fig. 3). The uorescence intensity of the chemosensor DAT-1
Fig. 1 View of the structure of DAT-1 with displacement atomic
ellipsoids drawn at the 30% probability level. All hydrogen atoms are
omitted for clarity.
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Fig. 3 Metal ions selectivity of DAT-1 (20 mM) in the Na2HPO4–citric
acid buffer solution. The black bars represent the fluorescence
emission intensity of DAT-1 and 100 equiv. of metal ions. The red bars
represent the fluorescence response of DAT-1 to 100 equiv. of Fe3+
containing 100 equiv. of other metal ions. lex ¼ 350 nm, lem ¼ 448 nm.
Fig. 6 Time-resolved fluorescence of DAT-1 with an excitation at 405
nm in the absence (black) and in the presence of 1 equiv. (green), 5
equiv. (blue), 25 equiv. (magenta) and 50 equiv. (red) of Fe3+ ions.
applied to the detection of iron cations by the naked eye (Fig. 4).
When the chemosensor (20 mM) was excited at 350 nm in the
presence of various cations (100 equiv.) in Na2HPO4–citric acid
buffer (pH 4.8), hardly any uorescence response of Fe3+ was
selectively observed (Fig. 4).
was substantially diminished by the addition of Fe3+, whereas
other cations such as K+, Ca2+, Fe2+, Mg2+, Al3+, Co2+, Ni2+, Ag+,
Cu2+, Zn2+, Cr3+, Cd2+, Mn2+, Hg2+ and Pb2+ did not cause any
signicant changes in the uorescence emission intensity, even
at a concentration of 100 equiv. of guest counterparts. Even with
highly concentrated Na+, K+, Ca+, and Mg2+ (ꢃ5.0 mM) under
physiological conditions,12 there were no uorescence changes
in DAT-1. The large decrease in uorescence intensity could be
It is well-known that heavy metal ions such as Fe3+, Cu2+,
Cd2+, Hg2+ and Pb2+ tend to quench the luminescence through
electron- and/or energy-transfer processes.13 Because of the
Table 1 Fluorescence decay time constants of 1 in the presence of
Fe3+ ions
Fe3+ (equiv.)
s (ns)
Fe3+ (equiv.)
s (ns)
0
1
5
3.68
3.60
3.42
25
50
2.76
2.30
Fig. 4 Fluorescent (bottom) and color (top) responses of DAT-1 (20
mM) in Na2HPO4–citric acid buffer solutions (0.2 M) upon the addition
of (100 equiv.) metal ions in water.
Fig. 7 Variation of fluorescence spectra of DAT-1 (20 mM) in aqueous
Fig. 5 The calculated frontier molecular orbitals of complex-23 (a–c) solution (Na2HPO4–citric acid buffer) (0.2 M) with and without Fe3+
and the best structure of complex-23 (d).
(100 equiv.) ion as a function of pH. Fluorescence intensity at 448 nm.
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specic structure, the recognition unit of our probe could many coordination sites, but it is difficult to determine the
complex with iron ions selectively. Thus the photoinduced structure of coordination between DAT-1 and Fe3+. We therefore
electron transfer (PET) effect on N atom causes uorescence designed 22 coordinations, considering all possible options
quenching of coumarin. The fact that our DAT-1 chemosensor except water as ligand. We found, however, that complex-23,
can not complex with other ions besides iron ions is attributed with water as ligand, was the optimum structure compared with
to the specic recognition of the recognition unit to iron ions.13e other complexes. This result agrees with the experimental data
Therefore, our probe cannot be quenched by other ions without reported by other authors.11 Thus we can assume that complex-
PET effect. To prove the mechanism of uorescence quenching, 23 was the best structure in this system. The electron distribu-
we carried out theoretical calculations (Fig. 5; details in tions of HOMO, LUMO, and LUMO+4 of complex-23 were
Fig. S3†). From the single crystal structure of DAT-1, there are calculated by B3LYP/6-31G(d, p) and are shown in Fig. 5. The
electronic distribution on LUMO+4 for complex-23 was
extended to Fe, indicating the electron transfer from HOMO to
LUMO+4. Thus the electrons transfer from DAT-1 to Fe3+,
making the uorescence quenching behavior possible. Indeed,
it has been demonstrated in the literature that uorescence
quenching of the ligand may occur by the excitation energy
transfer from the ligand to the metal d orbital and/or ligand to
metal charge transfer (LMCT).9,14
To elaborate upon the mechanism of uorescence quench-
ing which we speculate takes place by the energy and/or charge
transfer model, uorescence lifetime testing was conducted.
The uorescence decay behavior in the presence of Fe3+ is
shown in Fig. 6, and the exponential t results are summarized
in Table 1. In the absence of Fe3+, the uorescence decays single
exponentially by a 3.68 ns time constant, which is the lifetime of
the S1 state of free DAT-1. The time constant (s) of the lifetime
decay component drops off when Fe3+ is added.
For biological applications of the chemosensor, the sensing
should operate in a wide range of pH. Fig. 7 shows that in
aqueous solution the suitable pH range for Fe3+ determination
Fig. 8 Percentage of MCF-7 cell viability remaining after cell treat-
ment with DAT-1 (untreated cells were considered to have 100%
is pH 3–8 where the “on–off” uorescence can be operated by
the iron ion binding. Consequently, our present Fe3+-selective
receptor would be an ideal chemosensor for monitoring Fe3+.
survival). Cell viability was assayed by the MTT method (values: mean ꢄ
standard deviation).15
Fig. 9 Confocal fluorescence images of Fe3+ in MCF-7 cells (Olympus FV1000, 40ꢂ objective lens). (a) Bright-field transmission image of MCF-7
cells. (b) Fluorescence image of MCF-7 cells incubated with DAT-1 (20 mM). Further incubated with addition of various concentrations of FeCl3
[(c) 25, (d) 50, and (e) 100 equiv., respectively]. (f) Return of intracellular Fe3+ to the resting level was achieved by addition of EDTA (2 mM).
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The ability of biosensing molecules to selectively monitor
guest species in living cells is of great importance for biological
applications. First, we selected breast carcinoma cell lines
(MCF-7) to investigate the toxicity of DAT-1. The toxicity data
were obtained by performing 3-(4,5-dimethylthiazol-2-yl)-2,5
diphenyltetrazolium bromide (MTT) assays as shown in Fig. 8.
The cells were cultured in RPMI 1640 (Gibco) and supple-
mented with 10% fetal bovine serum (FBS; Gibco) at 37 ꢀC in a
humidied atmosphere of 5% CO2. For all experiments, cells
were harvested from subconuent cultures by the use of
trypsin and were resuspended in fresh complete medium
before plating. In vitro cytotoxicity was assessed by using MTT
reduction assays. In a typical procedure, about 2000 cells were
plated in 96-well plates for 24 h to allow the cells to attach, and
Acknowledgements
We are very grateful for the support of this work from the
National Natural Science Foundation of China (no. 21072158
and 20802056), the Special Foundation of the Education
Committee of Shaanxi Province (no. 12JK0580), the French
Chinese Foundation for Science and Applications (FFCSA) and
the China Scholars Council (CSC).
Notes and references
1 (a) D. H. Vance and A. W. Czarnik, J. Am. Chem. Soc., 1994,
116, 9397–9398; (b) Z. C. Xu, J. Yoon and D. R. Spring,
Chem. Soc. Rev., 2010, 39, 1996–2006; (c) J. S. Wu,
W. M. Liu, J. C. Ge, H. Y. Zhang and P. F. Wang, Chem.
Soc. Rev., 2011, 40, 3483–3495; (d) M. Baglan and
S. Atilgan, Chem. Commun., 2013, 49, 5325–5327; (e)
M. J. Peng, Y. Guo, X. F. Yang, L. Y. Wang and J. An, Dyes
Pigm., 2013, 98, 327–332.
2 (a) T. W. Bell and N. M. Hext, Chem. Soc. Rev., 2004, 33, 589–
598; (b) W. Wang, X. L. Wang, Q. B. Yang, X. L. Fei, M. D. Sun
and Y. Song, Chem. Commun., 2013, 49, 4833–4835; (c)
X. G. Li, Y. Z. Liao, M. R. Huang, V. Strong and
R. B. Kaner, Chem. Sci., 2013, 4, 1970–1978; (d) Z. Yang,
M. Y. She, B. Yin, J. H. Cui, Y. Z. Zhang, W. Sun, J. L. Li
and Z. Shi, J. Org. Chem., 2012, 77, 1143–1147.
3 (a) C. Y. Lai, B. G. Trewyn, D. M. Jeinija, K. Jeinija, S. Xu,
S. Jeinija and V. S. Y. Lin, J. Am. Chem. Soc., 2003, 125, 4451–
4459; (b) P. G. Sutariya, A. Pandya, A. Lodha and S. K. Menon,
Analyst, 2013, 138, 2531–2535; (c) G. Sivaraman, T. Anand
and D. Chellappa, Analyst, 2012, 137, 5881–5884; (d)
X. J. Wu, Z. Li, L. Yang, J. H. Han and S. F. Han, Chem.
Sci., 2013, 4, 460–467.
4 (a) S. K. Sahoo, D. Sharma, R. K. Bera, G. Crisponi and
J. F. Callan, Chem. Soc. Rev., 2012, 41, 7195–7227; (b)
S. P. McCormick, M. Chakrabarti, A. L. Cockrell, J. Park,
L. S. Lindahl and P. A. Lindahl, Metallomics, 2013, 5, 232–
241.
ꢀ
then incubated with DAT-1 (20 mM) in 5% CO2 at 37 C. At the
end of the incubation time, MTT solution (10 mL, diluted in
culture medium to a nal concentration of 1 mg mLꢁ1) was
added, and then the mixture was incubated for another 4 h.
Finally, the incubation solution was removed and dimethyl
sulfoxide (DMSO, 150 mL) was added to each well. The absor-
bance of MTT was determined (l ¼ 490, 630 nm) by using a
microplate reader (BioTek, ELX808). The cell viability obtained
was expressed as a percentage relative to the control. The
viability of untreated cells was assumed to be 100%. When the
concentration of chemosensor DAT-1 was 5 mM, more than
99% of the MCF-7 cells were alive. Even when the concentra-
tion of DAT-1 increased to 100 mM, there were still about 87%
of the MCF-7 cells alive. From the results of cytotoxicity
experiments, the chemosensor DAT-1 shows low toxicity even
at a high concentration of 100 mM particles and an incubation
time of 24 h, which means that DAT-1 possesses high
biocompatibility.
To determine the cell permeability of DAT-1, MCF-7 cells
were incubated with DAT-1 (20 mM) for 25–30 min at 37 ꢀC, and
washed with PBS to remove the remaining compound DAT-1.
The results are shown in Fig. 9b. One can clearly observe
signicant confocal imaging changes of the medium upon
addition of FeCl3 for 20 min (25, 50, and 100 equiv., respec-
tively) at 37 ꢀC. MCF-7 cells incubated with DAT-1 initially
display a strong uorescence image, but the uorescence
image immediately becomes faint in the presence of Fe3+
(Fig. 9c–e). Thus, the chemosensor DAT-1 can be a suitable
uorescence chemosensing probe for Fe3+ detection in bio-
logical systems.
5 (a) B. J. Halliwell, J. Neurochem., 1992, 59, 1609–1623; (b)
E. D. Weinberg, Eur. J. Cancer Prev., 1996, 5, 19–36; (c)
D. Galaris, V. Skiada and A. Barbouti, Cancer Lett., 2008,
266, 21–29.
6 E. Beutler, Blood Cells, Mol., Dis., 2007, 39, 140–147.
7 W. Y. Ong and A. A. Farooqui, J. Alzheimer’s Dis., 2005, 8, 183–
200.
8 (a) L. Huang, F. P. Hou, J. Cheng, P. X. Xi, F. J. Chen, D. Bai
and Z. Z. Zeng, Org. Biomol. Chem., 2012, 10, 9634–9638; (b)
S. K. Sahoo, D. Sharma, R. K. Bera, G. Crisponi and
J. F. Callan, Chem. Soc. Rev., 2012, 41, 7195–7227; (c)
B. K. Kanungo, M. Baral, R. K. Bera and S. K. Sahoo,
Monatsh. Chem., 2010, 141, 157–168.
9 H. S. Jung, P. S. Kwon, J. W. Lee, J. I. Kim, C. S. Hong,
J. W. Kim, S. H. Yan, J. Y. Lee, J. H. Lee, T. H. Joo and
J. S. Kim, J. Am. Chem. Soc., 2009, 131, 2008–2012.
Conclusion
In summary, this study has reported the synthesis, properties,
and cellular applications of a novel chemosensor DAT-1 for Fe3+
sensing. This chemosensor exhibits high sensitivity and selec-
tivity toward Fe3+ in aqueous solution. We simulated the
mechanism of uorescence quenching by quantum calcula-
tions. In terms of good optical properties, ne water solubility,
excellent membrane permeability and non-toxicity, DAT-1 could
be one of the most important chemosensors for the detection of 10 K. Y. Chen, Y. Guo, Z. H. Lu, B. Q. Yang and Z. Shi, Chin.
Fe(III) in living cells.
J. Chem., 2010, 28, 55–60.
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11 J. N. Yao, W. Dou, W. W. Qin and W. S. Liu, Inorg. Chem.
Commun., 2009, 12, 116–118.
12 T. D. Rae, P. J. Schmidt, R. A. Pufahl, V. C. Culotta and
T. V. O’Halloran, Science, 1999, 284, 805–808.
H. S. Jung, J. H. Yoon, S. Y. Lee and J. S. Kim, Bull. Korean
Chem. Soc., 2007, 28, 682; (e) T. E. Glass, J. Am. Chem. Soc.,
2000, 122, 4522–4523.
14 L. S. Villata, E. Wolcan, M. R. Feliz and A. L. Capparelli, J.
Phys. Chem. A, 1999, 103, 5661–5666.
13 (a) J. L. Bricks, A. kovalchuk, C. Trieinger, M. Nofz,
M. Buschel, A. I. Tolmachev, J. Daub and K. Rurack, J. Am. 15 (a) Y. Cetin and L. B. Bullerman, J. Agric. Food Chem., 2005,
Chem. Soc., 2005, 127, 13522–13529; (b) K. Kavallieratos,
J. M. Rosenberg, W. Z. Chen and T. Ren, J. Am. Chem. Soc.,
2005, 127, 6514–6515; (c) Y. Zheng, J. Orbulescu, X. Ji,
F. M. Andreopoulos, S. M. Pham and R. M. Leblanc, J. Am.
Chem. Soc., 2003, 125, 2680–2688; (d) D. T. Quang,
53, 6558–6563; (b) I. Roy, T. Y. Ohulchanskyy,
H. E. Pudavar, E. J. Bergey, A. R. Oseroff, J. Morgan,
T. J. Dougherty and P. N. Prasad, J. Am. Chem. Soc., 2003,
125, 7860–7865; (c) S. Andreescu, O. A. Sadik and
D. W. McGee, Chem. Res. Toxicol., 2005, 18, 466–474.
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