Rhodamine-based 'turn-on' fluorescent probe for Cu(II) and its fluorescence imaging in living cells

Article abstract of DOI:10.1016/j.bmcl.2013.03.052

A novel rhodamine spirolactam derivative 3′,6′- Bis(diethylamino)-2-(2-hydroxyethylamino) spiro[isoindoline-1,9′-xanthen]- 3-one (RO1) was synthesized, and characterized by high-resolution mass spectrometry (HRMS), X-ray crystallography, Infrared spectroscopy (IR), and ¹H NMR and ¹³C NMR spectroscopy. RO1 exhibited highly sensitive and exclusively selective fluorescence response toward Cu²⁺ over other metal ions with a detection limit of 0.56 ppb in mixed aqueous solution. The fluorescence was pH-independent in the wide range pH 3.1-11.6. The turn-on fluorescence enhancement of the probe is based on Cu²⁺ induced ring-opening mechanism of the rhodamine spirolactam. Moreover, by means of fluorescence microscopy experiments, it was demonstrated that RO1 could monitor trace Cu²⁺ changes by live cell imaging.

Full text of DOI:10.1016/j.bmcl.2013.03.052

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2916–2919
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Rhodamine-based ‘turn-on’ fluorescent probe for Cu(II)
and its fluorescence imaging in living cells
a
a
a
Mao-Zhong Tian a,b, Ming-Ming Hu a, Jiang-Li Fan a, , Xiao-Jun Peng , Jing-Yun Wang , Shi-Guo Sun ,
⇑
Rong Zhang a
a State Key Laboratory of Fine Chemicals, Dalian University of Technology, 2 Linggong Rd., Hi-tech Zone, Dalian 116024, People’s Republic of China
b College of Chemistry and Chemistry Engineering, Shanxi Datong University, Datong 037009, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
A novel rhodamine spirolactam derivative 30,60-Bis(diethylamino)-2-(2-hydroxyethylamino) spiro[iso-
indoline-1,90-xanthen]-3-one (RO1) was synthesized, and characterized by high-resolution mass spec-
trometry (HRMS), X-ray crystallography, Infrared spectroscopy (IR), and 1H NMR and 13C NMR
spectroscopy. RO1 exhibited highly sensitive and exclusively selective fluorescence response toward
Cu2+ over other metal ions with a detection limit of 0.56 ppb in mixed aqueous solution. The fluorescence
was pH-independent in the wide range pH 3.1–11.6. The turn-on fluorescence enhancement of the probe
is based on Cu2+ induced ring-opening mechanism of the rhodamine spirolactam. Moreover, by means of
fluorescence microscopy experiments, it was demonstrated that RO1 could monitor trace Cu2+ changes
by live cell imaging.
Article history:
Received 11 January 2013
Revised 10 March 2013
Accepted 15 March 2013
Available online 25 March 2013
Keywords:
Fluorescence
Probe
Cu2+
Ó 2013 Elsevier Ltd. All rights reserved.
Rhodamine
Cell imaging
The design and development of a fluorescent probe with high
selectivity and sensitivity targeting heavy and transition metal cat-
ions are of intense research activity, since they allow nondestruc-
ity, cross-sensitivity toward other metal cations, a narrow pH span,
a low fluorescence quantum yield, and cytotoxicities of ligand. In
addition, fluorescence detecting of Cu2+ is particularly challenging
since Cu2+ generally acts as quencher via the electron transfer and
facilitated intersystem crossing (isc) processes.1
tive and prompt detection of metal cations by
a simple
fluorescence enhancement (turn-on) or quenching (turn-off) re-
sponse.1–4 Among transition metal ions, copper is the third most
abundant trace element in the human body and many living organ-
isms, and is involved in redox processes of a number of biomole-
cules.4 Besides the beneficial effects, excess copper in the human
body has been reportedly linked to serious health threats such as
cellular or organ damage and neurodegenerative disease, like Wil-
son disease, and Alzheimer’s disease.5 Accordingly, the U.S. Envi-
ronmental Protection Agency (EPA) has set the maximum
allowable level of copper in drinking water at 1.3 ppm
As a fluorogenic unit (fluorophore), rhodamine derivatives are
one of the most promising dyes due to their pretty good photo-
physical properties, such as long absorption and emission wave-
length, large extinction coefficient, high fluorescence quantum
yield, and high light stability. Recently, many fluorescent probes
based on rhodamine B, which were driven by visible light excita-
tion have been studied. They can show turn-on response to the tar-
geted HTM cation,13 such as Cu2+ 14–19
,
Hg2+ 20,21 Fe3+ 22 Zn2+ 23
, , ,
Cr3+ 24 Pd2+ 25
,
.
The cation-sensing mechanism of these probes was
(ꢀ20
l
M).6 Nevertheless, copper is present in waste effluents gen-
based on the change in structure between spirocyclic and opency-
clic forms. Without cations, these probes existed in a spirocyclic
form, which was colorless and nonfluorescent. Addition of metal
cation lead to the ring-opening of the corresponding spirocycle,
and gave rise to a strong fluorescence enhancement and a color
change.
Fluorescence imaging technology was regarded as a significant
approach for visualizing subcellular distribution of Cu2+ in physio-
logical process by virtue of its outstanding properties.26 To image
intracellular copper ions, highly sensitive, selective, and cell mem-
brane-penetrable probes that exhibit an enhanced visible fluores-
cent emission in aqueous media need to be developed. In recent
years, a lot of fluorescence ‘off–on’ probes for imaging various
erated by various industries (i.e., electroplating, wood, painting,
textile, and paper industries), and can be accumulated in the envi-
ronment and food chain, especially in fish. It is, therefore, of con-
siderable significance to develop highly sensitive and selective
probes for Cu2+ determination. In recent years, many fluorescent
probes for copper ion selective detection have been explored.7–12
However, some of them suffered from drawbacks of practical
application in terms of complicated organic synthesis, low sensitiv-
⇑
Corresponding author. Tel.: +86 411 84986327; fax: +86 411 84983606.
E-mail address: fanjl@dlut.edu.cn (J.-L. Fan).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.052

M.-Z. Tian et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2916–2919
2917
Scheme 1. Synthesis route of probe RO1.
intracellular metal ions have been developed. However, just a few
probes reported for imaging Cu2+ in living cells.27–36 Thus, it is still
very necessary to design small-molecule fluorescent probes for
Cu2+ with favorable photophysical properties suitable for imaging
of copper ions in living cells.
Our research work involves the design, synthesis, spectroscopy,
and biological applications of fluorescent probes for selective sens-
ing of ions as well as evaluation of their switching behavior.3 In this
article, we have designed and synthesized a new rhodamine deriv-
ative RO1. The hydroxyethylhydrazine moiety is introduced into
RO1 due to its good binding affinity for transition metal ions.18
The catalytic hydrolysis of hydroxyethylhydrazine moiety in RO1
was selectively induced by Cu2+ ions to produce irreversible fluo-
rescence turn-on changes ascribed to a ring opened form of the
rhodamine. Especially, RO1 showed excellent sensitivity and selec-
tivity with a detection limit of 0.56 ppb in mixed aqueous media.
RO1 was pH-independent, but Cu2+ induced large increases in fluo-
rescence intensity of RO1 between pH 3.10 and 11.6. Herewith, we
also present the bioapplication of RO1 as a fluorescent probe to de-
tect Cu2+ in cultured cells.
As depicted in Scheme 1, Compound 1 was synthesized accord-
ing to the literature.37 Compound 1 was dissolved in absolute eth-
anol. Sodium borohydride was then added slowly. The mixture was
stirred under N2 atmosphere at room temperature for 2 h. The
crude product was purified to give RO1 as a white solid in 89.9%
yield.
The single crystal suitable for X-ray analysis was obtained by
slow evaporation of the CH2Cl2/n-hexane solution. Table S1 sum-
marizes the crystal data, data collection and refinement parame-
ters for RO1. The crystal structure of RO1 clearly reveals the
unique spirolactam ring formation,38 with the lactam and xan-
thene moiety form vertical planes (Fig. S1), which breaks the con-
jugation of the whole system, thus leading to the non-fluorescence
of the molecule.
Spectral changes of RO1 in CH3CN/H2O (1/1, v/v, 10 mM Tris–
HCl buffer, pH = 7.15) solution were tested (Fig. S2) upon addition
Figure 1. (A) Absorption spectra of RO1 (10
l
M) in CH3CN/Tris–HCl buffer (5:5, v/v,
pH 7.15) upon addition of different amounts of Cu2+. (B) Fluorescence spectra of
of various competitive metal ions, such as Na+, K+, Mg2+, Ca2+, Ba2+
,
.
RO1 (10
amounts of Cu2+ (kex = 510 nm). Inset: the changes of fluorescence intensity of RO1
(10
M) upon addition of Cu2+ (1–5 ppb).
lM) in CH3CN/Tris–HCl buffer (5:5, v/v, pH 7.15) upon addition of different
Mn2+, Cd2+, Cr3+, Co3+, Ni2+, Ag+, Pb2+, Zn2+, Hg2+, Fe3+, Cu+, Cu2+
l
From UV/vis spectra of RO1 (10 lM) (Fig. S2A), we can clearly ob-
serve a new absorption band centered at 562 nm in the presence of
5 equiv of Cu2+. In contrast, other metal ions did not lead to any
distinct spectral changes. On the other hand, fluorescence spectra
(Fig. S2B) also showed a similar result, which was well consistent
with that of UV/vis spectra. Addition of only 5 equiv Cu2+ resulted
in a dramatic change of fluorescence intensity peaked at 589 nm
these results indicated that the selectivity of RO1 for the Cu2+ over
other competitive cations in the water medium was remarkably
high, which was well consistent with that of UV/vis spectra.
Figure 1 shows a spectral variation of RO1 upon the gradual addi-
tion of Cu2+. The UV–vis titration spectra of Cu2+ was conducted
(off–on), while other metal ions including Na+, K+, Mg2+, Ca2+
,
using 10
Upon the addition of increasing concentrations of the Cu2+ (0–
70 M), a new absorption band centered at 562 nm appeared with
lM of RO1 in acetonitrile aqueous solution at pH = 7.15.
Ba2+, Mn2+, Cd2+, Cr3+, Co3+, Ni2+, Ag+, Pb2+, Zn2+, Hg2+, Fe3+, Cu+
did not give rise to any fluorescence increases. Moreover, in the
presence of miscellaneous competitive cations, the Cu2+ still re-
sulted in the similar fluorescence changes (Fig. S3). In addition,
the increases of fluorescence intensity resulting from the addition
of the Cu2+ were not influenced by the subsequent addition of mis-
cellaneous cations. More importantly, RO1 is selective for Cu2+ over
Cu+, showing that this probe has metal and redox specificity. All of
l
increasing intensity, which induced a clear color change from color-
less to pink (Fig. 1A). On the other hand, for the fluorescence titration
spectra of RO1, in the presence of Cu2+ there was also a new emissive
peak at 589 nm (Fig. 1B), which was in good consistency with the re-
sults of UV–vis spectra. Both UV–vis and fluorescence data lead to a
significant off–on signal. From the molecular structure and spectral

2918
M.-Z. Tian et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2916–2919
Figure 2. Fluorescence intensity (589 nm) of free RO1 (10
lM) (.) and after
addition of 50
l
M of Cu2+ (d) in the CH3CN/H2O (1:1, v/v, 10 mM Tris–HCl buffer,
pH 7.15) solution as a function of different pH values. Excitation at 510 nm.
results of RO1, it is probably concluded that the addition of Cu2+ in-
duced a ring-opening of the spirolactam in rhodamine framework,
observed by the distinct color change and fluorescence off–on. The
F/F0 was proportional to the amount of Cu2+ added in ppb level with
a detection limit of 0.56 ppb (Fig. 1B inset). The stoichiometry of a
binding event between RO1 and Cu2+ was also determined. The re-
sults obtained from Job’s plot show the 1:1 stoichiometry between
RO1 and Cu2+ (Fig. S4).
Figure 3. Fluorescence images of Cu2+ ion level using RO1 in living Hela cells; (a)
brightfield image of living Hela cells treated with RO1 (10 M); (b) fluorescence
image of cells in panel (a); (c) brightfield image of RO1 (10 M) loaded cell exposed
to 50 (d) fluorescence image of cells in panel (c). The excitation
Cu2+
l
l
l
M
;
wavelength was 515 nm and the emission was collected at 575–620 nm.
The binding interaction of RO1 with Cu2+ was investigated. RO1
of Cu2+, free RO1 showed no detectable fluorescence signal in living
cells. After incubation with Cu2+, a bright fluorescence was ob-
served in living cells. The results suggested that probe RO1 can
penetrate the cell membrane and be applied for in vitro imaging
of Cu2+ in living cells.
is a colorless, nonfluorescent compound. Upon the addition of Cu2+
,
a pink color was observed, and a bright fluorescence developed
simutaneously (Fig. S5). The above resulting colored solution was
subsequently treated with excess EDTA, and the strong fluores-
cence of the RO1–Cu2+ was almost not affected by the addition
of EDTA solution, indicating that the interaction of RO1 with Cu2+
is chemically irreversible. The formation of rhodamine B as product
was confirmed by comparison of the product’s TLC data with that
of authentic rhodamine B. The formation of rhodamine B as prod-
uct was also confirmed by HRMS: m/z [M+H]+ = 443.2337 (authen-
tic rhodamine B: m/z [M+H]+ = 443.2343) (Fig. S6). These results
clearly showed that rhodamine B was formed in the reaction. The
turn-on response of fluorescence in this system may be proposed
to proceed through the following route (Scheme S1): RO1 binds
Cu2+, and the subsequent complexation of Cu2+ promotes hydro-
lytic cleavage of the amide bond, leading to the release of rhoda-
mine B and thus the retrievement of fluorescence.
It is well known that pH-insensitivity of probes is extremely
valuable for practical applications.37 Therefore, the proper pH envi-
ronment of the probe RO1 was also evaluated (Fig. 2). The acid titra-
tion control experiments revealed that no obvious fluorescence
change of RO1 could be observed between pH 3.10 and 11.6, sug-
gesting that RO1 is insensitive to pH and that the spirolactam form
is still preferred in this range. Furthermore, the effect of pH on the
In summary, a new rhodamine spirolactam based fluorescence
probe RO1 for Cu2+ has been synthesized and structurally charac-
terized. Fluorescence changes of RO1 are remarkably specific for
Cu2+ in the presence of other commonly coexistent metal ions. Fur-
thermore, the parts per billion level fluorescent detection limit
suggests the possibility of practical applications in biology and
environmental sciences. In addition, RO1 shows pH-independent
in the wide range 3.10–11.6. Especially, the probe RO1 can be em-
ployed as a Cu2+-selective probe in the fluorescence imaging of liv-
ing cells. Such a sensor would become valuable in revealing the
roles of Cu2+ in biological systems under either in vitro or in vivo
conditions. The development of new rhodamine-based probes by
incorporating various ligand groups is in progress in our
laboratory.
Acknowledgments
This work was financially supported by NSF of China
(21136002, 20923006, and 21076032), National Basic Research
Program of China (2009CB724706 and 2013CB733702) and the
Shanxi Scholarship Council of China (No. 20090980).
fluorescence intensity of RO1 (10
lM) in the CH3CN/H2O solution in
the presence of Cu2+ (50 M) was performed. 5 equiv of Cu2+ in-
l
duced large increases in fluorescence intensity of RO1 between
pH 3.10 and 11.6. These results indicated that RO1 would provide
the potential applications in some environmental and physiological
regions for the detection of Cu2+ in the wide pH range.
To demonstrate the practical applicability of the probe in bio-
logical samples, fluorescence imaging experiments were carried
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
03.052.
out in living cells. Hela cells were incubated with RO1 (10
l
l
M)
M)
for 0.5 h at 37 °C, then followed by the addition of Cu2+ (50
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