Development of highly sensitive fluorescent probes for detection of intracellular copper(I) in living systems

Article abstract of DOI:10.1021/ja100714p

We describe here the new copper-selective fluorescent probes FluTPA1 and FluTPA2, in which the tetradentate ligand tris[(2-pyridyl)methyl]amine (TPA) is connected to a reduced form of a fluorescein platform through a benzyl ether linkage. These probes selectively react with copper ions in the presence of submillimolar glutathione and emit intense green fluorescence from the reaction product, O-methylfluorescein. Confocal images of live cells further show that FluTPA2 is membrane-permeable and allows visualization of Cu^I present in living cells.

Full text of DOI:10.1021/ja100714p

Published on Web 04/08/2010
Development of Highly Sensitive Fluorescent Probes for Detection of
Intracellular Copper(I) in Living Systems
Masayasu Taki,*,†,‡ Shohei Iyoshi,† Akio Ojida,§ Itaru Hamachi,§ and Yukio Yamamoto†
Graduate School of Human and EnVironmental Studies and Graduate School of Global EnVironmental Studies,
Kyoto UniVersity, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan, and Graduate School of Engineering, Kyoto
UniVersity, Katsura Campus, Nishikyo-ku, Kyoto 615-8510, Japan
Received January 26, 2010; E-mail: taki@chem.mbox.media.kyoto-u.ac.jp
Scheme 1
Copper is the third most abundant transition metal in the human
body; because of its redox-active nature (CuI/CuII), it serves as an
essential cofactor for a variety of enzymes, including tyrosinase,
cytochrome c oxidase (CcO), and Cu/Zn superoxide dismutase
(SOD1), in all living organisms.1 However, when copper is present
in excess, its redox properties turn it into a biological hazard because
of its ability to generate reactive oxygen species (ROS), which
interfere with cellular metabolism.1 Sophisticated regulatory mech-
anisms are available for maintaining a critical balance between the
necessity of copper and its toxicity at both the organ and cellular
levels;2 therefore, disruption of copper homeostasis in cells results
in severe disorders such as Menkes syndrome,3 Wilson’s disease,4
amyotrophic lateral sclerosis,5 and Alzheimer’s disease.6 However,
many questions about the trafficking mechanisms, subcellular
localization, and quantification of copper in living cells remain
unanswered.
in the visible region and has negligible fluorescence; these behaviors
remained unchanged for several days under aerobic conditions,
indicating that the reduced structure of FluTPA1 can exist stably
under physiological conditions. When 20 µM CuI was added to
the solution of FluTPA1 at room temperature, a significant increase
(more than 100-fold) in the emission intensity was observed (Figure
1a). HPLC and electrospray ionization mass spectrometry (ESI-
MS) of the reaction mixture revealed that 3′-O-methylfluorescein
(OMF, Φ ) 0.37)14 was the only fluorescent product, and its yield
was determined to be ∼70% after reaction for 2 h. This indicates
that the benzyl ether linkage was cleaved and the resulting reduced
form of “deprotected” fluorescein was easily air-oxidized to afford
OMF, even in the presence of submillimolar GSH. Interestingly,
the ESI-MS spectrum of the reaction mixture revealed a set of peaks
at m/z 395.8 (Figure S4 in the Supporting Information) with an
isotope distribution pattern calculated for the copper(II) complex
having an oxidized ligand of TPA, [CuII(TPA-COO-)]+. Thus, it
can be suggested that the benzyl radical of the TPA ligand is
generated in this C-O bond cleavage reaction.15 We are currently
in the process of elucidating the reaction mechanism involving
copper ions. One of the possibilities we are considering is that the
copper-active oxygen species is generated as the reactive intermedi-
ate.16 Indeed, when the monodentate probe FluPy, in which the
TPA ligand of FluTPA1 was replaced with a 2-pyridylmethyl group,
was employed as the control compound, no emission increase for
CuI was observed. In addition, external oxidants such as H2O2,
hypochlorite, and hydroxyl radical did not cause fluorescence from
this probe (Figure S5). These results support the idea that C-O
bond cleavage of the probe proceeded in an intramolecular manner
through the formation of a CuI-FluTPA1 complex with the
tetradentate TPA moiety.
Optical imaging with fluorescent probes has been an efficient
approach for answering questions about how copper ions function
in biological systems.7 Although a number of satisfactory fluores-
cent probes for transition metals such as zinc,8 cadmium,9 and
mercury10 have been developed over the last few decades, relatively
few copper-selective probes have been reported.11,12 Among these
probes, membrane-permeable chemosensors that allow monitoring
of intracellular copper with a microscope are rare. Because CuI
rather than CuII is the dominant oxidation state in a cytosolic
reducing environment,11a CuI-selective probes, ideally with a turn-
on response in fluorescence intensity, are superior for cell
imaging.11a,b However, most of the reported chemosensors for CuI
have limitations such as a turn-off response, high background
fluorescence, and the requirement that two molecules have ap-
propriate concentrations for fluorescence enhancement. Herein, we
report the new copper-selective fluorescent probes FluTPA1 and
FluTPA2 (Scheme 1) in which the tetradentate ligand tris[(2-pyridyl)-
methyl]amine (TPA) is connected to a reduced form of a fluorescein
platform through a benzyl ether linkage. We have found that the C-O
bond of the benzyl ether of FluTPA1 is selectively cleaved by the
reaction with CuI under physiological reducing conditions and also
observed that the fluorescence emission is enhanced significantly. We
have further demonstrated that the membrane-permeable probe
FluTPA2 reacts with intracellular CuI and exhibits bright fluorescence
in living systems.
All of the spectroscopic measurements on FluTPA1 were
performed in an aqueous buffer solution (50 mM HEPES, pH 7.20)
containing 2 mM glutathione (GSH), which is the most abundant
cellular thiol compound (0.5-10 mM in cells),13 to simulate
intracellular environments. FluTPA1 does not exhibit absorption
The significant fluorescence enhancement of FluTPA1 is specific
for copper ions (Figure 1b). Because CuII is rapidly reduced by
GSH to provide the CuI oxidation state, the fluorescence intensity
for CuII is as high as that observed for CuI. In fact, in the absence
† Graduate School of Human and Environmental Studies.
‡ Graduate School of Global Environmental Studies.
§ Graduate School of Engineering.
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5938 J. AM. CHEM. SOC. 2010, 132, 5938–5939
10.1021/ja100714p  2010 American Chemical Society

C O M M U N I C A T I O N S
induces the debenzylation reaction of FluTPA probes. Further
studies of the reaction mechanism of the fluorogenic process are
in progress.
Acknowledgment. We thank Professor S. Itoh at Osaka
University for helpful advice. This work was financially supported
by a Grant-in-Aid for Young Scientists (B) (21750168 to M.T.)
and a Grant-in-Aid for JSPS Fellows (S.I.).
Note Added after ASAP Publication. Reference 11b was replaced
in the version published April 14, 2010.
Figure 1. (a) Fluorescence response of 1 µM FluTPA1 before (dotted line)
and after (solid line) reaction with 20 µM [CuI(CH3CN)4]PF6. The solid
spectrum was recorded after 2 h of reaction of FluTPA1 with CuI in 50 mM
HEPES (pH 7.20) containing 2 mM glutathione (GSH). The excitation
wavelength was 470 nm. (b) Metal ion selectivity of FluTPA1 in 50 mM
HEPES buffer (pH 7.20). The bars represent the fluorescence intensity at 513
nm after 2 h of reaction of 1 µM FluTPA1 with each type of metal ion (20
µM) in the absence (gray bars) or presence (black bars) of 2 mM GSH.
Supporting Information Available: Synthesis and characterization
of organic compounds, ESI-MS data, confocal images, and experimental
details. This material is available free of charge via the Internet at http://
pubs.acs.org.
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Figure 2. (a) Confocal fluorescence image of HeLa cells supplemented
with 100 µM CuCl2 for 8 h and then further incubated with 5 µM FluTPA2
for 3 h at 37 °C. (b) Bright-field transmission image of the cells shown in
(a). (c) Confocal fluorescence image of FluTPA2-loaded cells grown in a
basal medium.
of GSH, CuII did not show the fluorescence response. Other heavy
metal ions apart from CoII caused no discernible change in the
emission intensity. Only CoII caused small enhancements in
fluorescence intensity both in the presence and in the absence of
GSH; however, these enhanced intensities were still much lower
than those for copper ions.
The spectroscopic features as well as high selectivity of FluTPA1
for CuI are satisfactory for practical in vivo applications. However,
this probe was membrane-impermeable because of the hydrophi-
licity of the carboxyl group.17 Therefore, for application to living
cells, we synthesized FluTPA2, which has a hydrophobic character;
this probe is based on the TokyoGreen scaffold developed by Urano,
Nagano, and co-workers.18 To examine the performance of
FluTPA2, experiments were performed as follows. HeLa cells were
incubated with a various concentrations of CuCl2 (20-200 µM) in
the growth medium for 8 h at 37 °C, washed with phosphate-
buffered saline containing 200 mM EDTA to remove extracellular
CuII, and further incubated with 5 µM FluTPA2 for 3 h. A strong
fluorescence signal with a significantly high signal-to-noise ratio
was observed in the CuII-supplemented cells (Figure 2a and Figure
S8), whereas no detectable increase in fluorescence was observed
in cells grown in a basal medium (Figure 2c). These experiments
clearly demonstrate that FluTPA2 is membrane-permeable and can
react with intracellular CuI to afford the highly fluorescent xanthene
dye, which means that the C-O bond cleavage reaction with CuI
and the subsequent air-oxidation process as described for FluTPA1
also occur for FluTPA2 inside living cells.
(17) Fujikawa, Y.; Urano, Y.; Komatsu, T.; Hanaoka, K.; Kojima, H.; Terai,
T.; Inoue, H.; Nagano, T. J. Am. Chem. Soc. 2008, 130, 14533.
(18) Urano, Y.; Kamiya, M.; Kanda, K.; Ueno, T.; Hirose, K.; Nagano, T. J. Am.
Chem. Soc. 2005, 127, 4888.
In conclusion, we have developed the new copper-selective
fluorescent probes FluTPA1 and FluTPA2 and applied the latter
one for visualizing CuI present in living cells. From the results of
this study, it is obvious that only CuI is the key metal ion that
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