A fluorescent ratiometric chemodosimeter for Cu2+ based on TBET and its application in living cells

Article abstract of DOI:10.1021/ol3032889

Based on a through bond energy transfer (TBET) between Rhodamine and a naphthalimide fluorophore, a fluorescent ratiometric chemodosimeter RN1 was designed and prepared for single selective detection of Cu²⁺ in aqueous solution and in living ce

Full text of DOI:10.1021/ol3032889

ORGANIC
LETTERS
2013
Vol. 15, No. 3
492–495
A Fluorescent Ratiometric
Chemodosimeter for Cu2þ Based on TBET
and Its Application in Living Cells
Jiangli Fan,* Peng Zhan, Mingming Hu, Wen Sun, Jizhou Tang, Jingyun Wang,
Shiguo Sun, Fengling Song, and Xiaojun Peng*
State Key Laboratory of Fine Chemicals, Dalian University of Technology, No. 2
Linggong Road, High-tech District, Dalian 116024, China
fanjl@dlut.edu.cn; pengxj@dlut.edu.cn
Received November 30, 2012
ABSTRACT
Based on a through bond energy transfer (TBET) between Rhodamine and a naphthalimide fluorophore, a fluorescent ratiometric chemodosimeter
RN1 was designed and prepared for single selective detection of Cu2þ in aqueous solution and in living cells, as Cu2þ acts as not only a selective
recognizing guest but also a hydrolytic promoter.
Copper, the third most abundant transition metal in the
human body, isvital for bothenvironmental and biological
systems. Nevertheless, at high concentrations, copper be-
comes a toxic and hazardous element to organisms such as
many bacteria.1 Owing to its toxicity to bacteria, elevated
concentrations of copper hamper the self-purification
capability of the sea or rivers and destroys the biological
reprocessing systems in water. Furthermore, alterations in
the cellular homeostasis of copper ions can cause neuro-
degenerative diseases, such as Menkes and Wilson’s
diseases,2 familial amyotrophic lateral sclerosis, prion dis-
ease, and Alzheimer’s disease,3 probably by its involve-
ment in the production of reactive oxygen species.
Therefore, considerable efforts have been devoted to de-
velop Cu2þ-selective fluorescent probes due to the
nondestructive, quick, and sensitive advantages of emis-
sion signals.4
Most reported Cu2þ fluorescent probes were based on
fluorescence intensity.5 Although turn-on probes are more
sensitive due to the lack of background signal, a major
limitation is that variations in the sample environment
(pH, polarity, temperature, and so forth) might influence
the fluorescence intensity measurements. Besides an inter-
nal charge transfer (ICT) mechanism using a single fluoro-
phore to obtain ratiometric changes, the exploration of
€
(4) Kramer, R. Angew. Chem., Int. Ed. 1998, 37, 772.
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9220. (c) Swamy, K. M. K.; Ko, S.-K.; Kwon, S. K.; Lee, H. N.; Mao, C.;
Kim, J.-M.; Lee, K.-H.; Kim, J.; Shin, I.; Yoon, J. Chem. Commun. 2008,
45, 5915. (d) Wang, D.; Shiraishi, Y.; Hirai, T. Chem. Commun. 2011, 47,
2673. (e) Yin, S.; Leen, V.; Snick, S. V.; Boens, N.; Dehaen, W. Chem.
Commun. 2010, 46, 6329. (f) Yu, M.; Shi, M.; Chen, Z.; Li, F.; Li, X.;
Gao, Y.; Xu, J.; Yang, H.; Zhou, Z.; Yi, T.; Huang, C. Chem.;Eur. J.
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Wu, Y.; Bai, D.; Zeng, Z. Dalton Trans. 2010, 39, 7894. (i) Dujols, V.;
Ford, F.; Czarnik, A. W. J. Am. Chem. Soc. 1997, 119, 7386.
(1) Barranguet, C.; van den Ende, F. P.; Rutgers, M.; Breure, A. M.;
Greijdanus, M.; Sinke, J. J.; Admiraal, W. Environ. Toxicol. Chem. 2003,
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r
10.1021/ol3032889
Published on Web 01/22/2013
2013 American Chemical Society

multifluorophores with energy donorꢀacceptor architec-
tures can achieve large pseudo-Stokes shifts, meanwhile
affording simultaneous recorded ratio signals of two emis-
sion intensities at different wavelengths, which could pro-
vide a built-in correction for the environmental effects.
process could change the emission maximum of the
system from 535 nm (the characteristic peak of naphth-
alimide) to 577 nm (the characteristic peak of rhodamine).
This wavelength shift allows highly selective ratiometric
detection of Cu2þ both in acetonitrile/water solution and
in living cells.
To explore the mechanism, an RN1-Cu2þ complex was
detected by TOF mass spectrum analysis (Figure S14). A
peak at m/z 725.2745 corresponding to intermediate 6 was
observed after the addition of Cu2þ to RN1 aqueous
solution, which suggested that Cu2þ induces the hydrolysis
and opening of the spirolactam ring of the rhodamine
moiety.
RN1 was synthesized on the basis of the route shown in
Scheme 1. Compounds 4 and 3 were successfully linked
together by a Sonogashira reaction between terminal
alkynyl and bromine in the presence of PdCl2(PPh3)2,
PPh3, and CuI as the catalyst to afford 6 in 75% yield.
Finally, RN1 was obtained by refluxing 6 and hydrazine
hydrate for 6 h in ethanol with a yield of 74%. All the
new intermediates and RN1 were well characterized by
1H NMR, 13C NMR, and TOF-MS (Figures S9ꢀS24).
€
Forster Resonance Energy Transfer (FRET) is generally
the most adopted methodology for addressing this issue.
€
Forster Resonance Energy Transfer (FRET) is generally
the most adopted methodology for addressing this issue.
Normally, dyes based on FRET processes are usually
linked by a nonconjugated spacer, and the energy transfer
occurs through space. For FRET to be effective, a sub-
stantial spectral overlap for the donor emission and ac-
ceptor absorption bands is required, which sometimes
restrict the choice and the design of these kinds of probe
molecules. The dyesbasedon TBET(through-bondenergy
transfer) are the ones which have a donor connected to an
acceptor via electronically conjugated linkers which pre-
vent the donor and acceptor fragments from becoming
planar, and the energy transfer occurs through a bond. For
a TBET system there is no such prerequisite, thus it exhi-
bits fast energy transfer rates, large pseudo-Stokes shifts,
and flexibility in fluorophores.6 Actually, to the best of our
knowledge, there are a few fluorescent probes for Hg2þ
reported on TBET (naphthalimide appended rhodamine)7
and just only one for Cu2þ with two different approaches
which has not been applied in living cells.8 Thus, it is
important to develop ratiometric fluorescent probes for
Cu2þ with favorablechemicaland spectroscopicproperties
suitable for the imaging of Cu2þ in living cells.
As the fluorescence of the naphthalimide moiety was
often quenched probably due to the efficient photoinduced
electron transfer (PET) from the amide of rhodamine to
the naphthalimide fluorophore, the probes could not
exhibit any ratiometric fluorescence for metal ion detec-
tion. To solve this problem, herein, we report a ratiometric
fluorescent chemodosimeter RN1 for Cu2þ based on
TBET, in which (4-N,N-dimethylamino)-1,8-naphthalide
(energy donor) and rhodamine (energy acceptor) were
linked by a rigid and conjugated spacer 4-ethynylaniline
at the naphthalic anhydride position. A hydrazide func-
tional group was selected as the potential reaction site for
Figure 1. Fluorescence ratio of RN1 (5 μM) in response to the
presence of Cu2þ (0ꢀ20 equiv) in CH3CN/H2O (20:1, v/v)
buffered with Tris-HCl (pH 7.4, 10 mM). λex = 420 nm. Inset
showing the fluorescence before and after the addition of Cu2þ
.
Cu2þ 9
.
The absorption and emission properties (Figure S1) of
RN1 (5 μM) were investigated in CH3CN/Tris-HCl buffer
(v/v = 20:1, pH 7.4). In the absence of Cu2þ, RN1 showed
one absorption band at 431 nm due to the naphthalimide
moiety. On addition of Cu2þ (0ꢀ20 equiv), a new absorp-
tion band appeared at 546 nm corresponding to the
rhodamine acceptor. Such a large red shift (115 nm) in
absorption behavior changed the color of the solution
from yellow-green to pink, allowing colorimetric detection
of Cu2þ by the naked eye. Accordingly, as shown in Figure 1,
upon excitation at 420 nm, the free RN1 displays a single
emission band centered at 535 nm, attributed to the emis-
sion of the naphthalimide moiety. There is no TBET in the
free RN1, as the rhodamine acceptor is in a ring-closed
form. Addition of Cu2þ significantly decreases the fluo-
rescence intensity at around 535 nm, and simultaneously a
Fortunately, this connection efficiently prevented the
fluorescence quenching of naphthalimide. In the absence
of Cu2þ, the excited energy of the naphthalimide donor could
not be transferred to the rhodamine acceptor, as the rhoda-
mine acceptor is in the closed form. Thus, only the emission of
the dye naphthalimide is observed. A Cu2þ-induced
(6) Fan, J.; Hu, M.; Zhan, P.; Peng, X. Chem. Soc. Rev. 2013, 42, 29.
(7) (a) Kumar, M.; Kumar, N.; Bhalla, V.; Singh, H.; Sharma, P. R.;
Kaur, T. Org. Lett. 2011, 13, 1422. (b) Mahato, P.; Saha, S.; Suresh, E.;
Di Liddo, R.; Parnigotto, P. P.; Conconi, M. T.; Kesharwani, M. K.;
Ganguly, B.; Das, A. Inorg. Chem. 2012, 51, 1769. (c) Luxami, V.;
Verma, M.; Rani, R.; Paul, K.; Kumar, S. Org. Biomol. Chem. 2012, 10,
8076.
(8) Zhang, J. F.; Zhou, Y.; Yoon, J.; Kim, Y.; Kim, S. J.; Kim, J. S.
Org. Lett. 2010, 12, 3852.
(9) (a) Dujols, V.; Ford, F.; Czarnik, A. W. J. Am. Chem. Soc. 1997,
119, 7386. (b) Yuan, L.; Lin, W.; Chen, B.; Xie, Y. Org. Lett. 2012, 14,
432.
Org. Lett., Vol. 15, No. 3, 2013
493

change in emission behavior. They also did not interfere
with the fluorescence ratiometric detection of Cu2þ by the
fluorescence spectrum of RN1 in CH3CN/H2O (20:1, v/v)
buffered with Tris-HCl (pH 7.4, 10 mM).
Scheme 1. Design and Synthesis of TBET-Based Ratiometric
Fluorescent Cu2þ Chemodosimeter RN1
Figure 2. Fluorescence ratio (F577/F535) of RN1 (5 μM) in the
presence of various analytes (50 μM) (Cu2þ, Naþ, Cd2þ, Ni2þ
,
Pd2þ, Zn2þ, Kþ, Hg2þ, Co2þ, Fe3þ, Mn2þ, Cr3þ, Mg2þ, Agþ)
in CH3CN/H2O (20:1, v/v) buffered with Tris-HCl (pH 7.4,
10 mM); λex = 420 nm. Red bars represent the addition of 10 equiv
of the appropriate metal ion to a 5 μM solution of RN1. Black
bars represent the subsequent addition of 50 μM Cu2þ to the
solution.
new red-shifted emission band at around 577 nm gradually
increases. These changes in the fluorescence spectrum
stopped when the amount of added Cu2þ reached 20 equiv
of the probe. At this amount the ratio of the emission
intensities at 577 and 535 nm (F577/F535) became as high
as 10 times that in the absence of Cu2þ. The optimized
spatial structure of compound6 (byGaussView 5.0, Figure
S7) shows that there is a 5° torsion angle between the
naphthalimide donor and rhodamine acceptor, which
further ensures the TBET process between the two fluoro-
phores. Therefore, although the donor emission has
overlap with the acceptor absorption in the system, the
energy transfer should be mainly based on TBET. Actu-
ally, TBET is sometimes accompanied by FRET.10 The
energy transfer efficiency was calculated as 81.3%.11 The
detection limit (3σ/slope)12 was as low as 3.88 ꢁ 10ꢀ7 M
(Figure S4) for Cu2þ which is sufficiently low for the
detection of the submillimolar concentration range of
Cu2þ found in many chemical systems.
To apply RN1 in more complicated systems, such as in
organisms and the environment, the fluorescence re-
sponses of RN1 in the absence and presence of Cu2þ in
different pH values were evaluated. The pKa of RN1 alone
is 1.2 from the sigmoidal curve. The fluorescence responses
(F577/F535) of RN1-Cu2þ were not changed for pH values
above 5.5, which means that RN1 can work in near-neutral
and weak acidic media. The time course of the respective
fluorescence intensity at 535 and 577 nm (Figure 4) in the
presence of Cu2þ indicates that the reaction essentially
Other metal ions such as Naþ, Cd2þ, Ni2þ, Pd2þ, Zn2þ
Kþ, Hg2þ, Co2þ, Fe3þ, Mn2þ, Cr3þ, Mg2þ, and Agþ
,
h
h
(Figure 2) as well as anions such as Sh2, ClO, hI, Cl, HPOh4,
Ac, Bhr, ClOh4, CO3 , NOh3, and H2POh4 (Figure 3) found in
environmental and biological settings exhibited almost no
h
h2
(10) (a) Wan, C. W.; Burghart, A.; Chen, J.; Bergstrom, F.; Johans-
son, L. B. A.; Wolford, M. F.; Kim, T. G.; Topp, M. R.; Hochstrasser,
R. M.; Burgess, K. Chem.;Eur. J. 2003, 9, 4430. (b) Speiser, S. Chem.
Rev. 1996, 96, 1953.
(11) Bhalla, V.; Roopa; Kumar, M.; Sharma, P. R.; Kaur, T. Inorg.
Chem. 2012, 51, 2150.
Figure 3. Fluorescence ratio (F577/F535) of RN1 (5 μM) with
addition of 5 μM Cu2þ subsequent addition of various appro-
priate anions in CH3CN/H2O (20:1, v/v) buffered with Tris-HCl
(pH 7.4, 10 mM). λex = 420 nm.
(12) Zhu, B.; Gao, C.; Zhao, Y.; Liu, C.; Li, Y.; Wei, Q.; Ma, Z.; Du,
B.; Zhang, X. Chem. Commun. 2011, 47, 8656.
494
Org. Lett., Vol. 15, No. 3, 2013

reached completion within minutes so that it could be used
as a fluorescent probe for the fast detection of Cu2þ
.
Figure 5. Images of MCF-7 cells treated with the ratiometric
RN1: (a) bright field image of MCF-7 cells incubated with RN1
(5 μM); (b) fluorescence image (a) from green channel; (c)
fluorescence image (a) from red channel; (d) overlay image of
(b) and (c); (e) bright field image of MCF-7 cells incubated with
RN1 (5 μM) for 30 min, and then further incubation with Cu2þ
(50 μM) for 30 min at 37 °C; (f) fluorescence image (e) from
green channel; (g) fluorescence image (e) from red channel; (h)
overlay image of (f) and (g). λex = 405 nm.
Figure 4. Time-dependent fluorescence ratio (F577/F535) of RN1
(5 μM) with Cu2þ (10 equiv) in CH3CN/H2O (20/1, v/v) buffered
with Tris-HCl (pH 7.4, 10 mM). λex = 420 nm.
Finally, due to the good chemical and spectroscopic
properties of the probe, the ratiometric RN1 was applied
for ratiometric fluorescence imaging in living cells. When
MCF-7 cells were incubated with only RN1 (5 μM) for 30
min at 37 °C, the cells showed intense fluorescence in the
green channel (Figure 5b) and weak fluorescence in the red
channel (Figure 5c). However, treatment of Cu2þ (50 μM)
with RN1-loaded cells elicited a partial fluorescence de-
crease in the green channel (Figure 5f) and strong fluores-
cence in the red channel (Figure 5g). Cytotoxicity testing in
MCF-7 cells (Figure S8) shows that RN1 is almost non-
toxic to living cells in our cell imaging conditions. Thus, all
the results demomstrated that RN1 was cell membrane
permeable and could be used to image Cu2þ in living cells.
In conclusion, by changing the connection between the
naphthalimide (energy donor) and rhodamine (energy
acceptor), a novel fluorescent ratiometric chemodosimeter
for Cu2þ based on TBET was synthesized and could be
used to image Cu2þ in living cells which will help in the
understanding of biological processes of Cu2þ at the
molecular level. Unlike standard FRET-based probes,
TBET is apparently not constrained by the overlap re-
quirement and enables greater freedom in the selection of
fluorophores, thus enabling the development of more
wonderful probes based on TBET to satisfy different
applications.
Acknowledgment. This work was financially supported
by NSF of China (21136002, 20923006, 21222605,
21272030, and 21076032), National Basic Research Pro-
gram of China (2009CB724706 and 2013CB733702), and
National High Technology Research and Development
Program of China (863 Program, 2011AA02A105).
Supporting Information Available. Experimental pro-
cedures, characterization data of compounds, and addi-
tional spectroscopic data. This material is available free
of charge via the Internet at http://pubs.acs.org.
The authors declare no competing financial interest.
Org. Lett., Vol. 15, No. 3, 2013
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