A reversible near-infrared fluorescence probe for reactive oxygen species based on Te-rhodamine

Article abstract of DOI:10.1039/c2cc18011a

We have designed and synthesized a reversible near-infrared (NIR) fluorescence probe, 2-Me TeR, for reactive oxygen species (ROS), utilizing the redox properties of the tellurium (Te) atom. 2-Me TeR is oxidized to fluorescent 2-Me TeOR by various ROS, whi

Full text of DOI:10.1039/c2cc18011a

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A reversible near-infrared fluorescence probe for reactive oxygen species
based on Te–rhodaminew
a
a
Yuichiro Koide,z Mitsuyasu Kawaguchi,z Yasuteru Urano,b Kenjiro Hanaoka,a
Toru Komatsu,a Masahiro Abo,a Takuya Teraia and Tetsuo Nagano*a
Received 22nd December 2011, Accepted 6th February 2012
DOI: 10.1039/c2cc18011a
We have designed and synthesized a reversible near-infrared
(NIR) fluorescence probe, 2-Me TeR, for reactive oxygen
species (ROS), utilizing the redox properties of the tellurium
(Te) atom. 2-Me TeR is oxidized to fluorescent 2-Me TeOR by
various ROS, while the generated 2-Me TeOR is quickly
reduced in the presence of glutathione to regenerate 2-Me
TeR. This redox-induced reversible NIR-fluorescence response
of 2-Me TeR allowed us to detect the endogenous production of
ROS and subsequent homeostatic recovery of the intracellular
reductive environment in hydrogen peroxide-stimulated HL-60
cells. This probe is expected to be useful for monitoring the
dynamics of ROS production continuously in vivo.
(reductive environment). Development of such reversible
fluorescence probes for ROS is currently one of the most
challenging topics in the field.
On the other hand, Cy-PSe and BzSe-Cy, both of which
contain selenium atoms as a redox-sensitive, fluorescence
off/on switch moiety, have been reported as cyanine-based
reversible fluorescence probes for peroxynitrite.7 We considered
that compounds with various oxidation states, such as those
containing chalcogen atoms, sulfur (S) and selenium (Se), might
also be applicable to the design of novel reversible ROS probes.8
In particular, we focused on organic tellurium (Te)-containing
compounds; we expected that these would be more sensitive to
redox reactions than the Se-containing ones, because they
exhibit both a high HOMO energy level and a low LUMO
energy level.9 For example, Oba et al. reported that diaryl
telluride was converted to diaryl telluroxide by singlet oxygen
(1O2).9,10 Therefore, we considered that the Te atom would be
suitable as the functional core of reversible ROS probes.
We synthesized rhodamine-based 2-Me TeR as a candidate
reversible ROS probe. It is known that the O atom at the
10 position of the xanthene moiety of typical rhodamines can
be replaced by a Te atom to afford tellurium rhodamines
(TeRs).11 2-Me TeOR was also synthesized by the oxidation of
2-Me TeR with H2O2 (Scheme S1 in the ESIw). 2-Me TeOR
was relatively stable, so we could characterize its structure by
means of NMR and MS. Our strategy to reversibly detect
ROS with 2-Me TeR is illustrated in Scheme 1. As shown in
Fig. 1, 2-Me TeR exhibited a relatively long labs (600 nm) and
showed no fluorescence (Ffl o 0.001), due to the heavy-atom
effect.12 In contrast, 2-Me TeOR, the oxidized form of 2-Me
TeR, exhibited a large red shift (labs = 669 nm) compared to
2-Me TeR and showed strong fluorescence (Ffl = 0.18). This
result indicates that the heavy-atom effect in 2-Me TeOR owing
to the Te atom was weakened by the binding of an oxygen atom.
This in turn means that we can detect the oxidative environment
Living cells are exposed to various stresses, such as reactive
oxygen species (ROS), UV light, and metal ions.1 Among
them, ROS especially play important roles in a variety of
biological and pathological systems.2 Overproduction of ROS
is associated with many diseases, including acute and chronic
inflammation, cancer, cardiovascular disease, and neuro-
degenerative disorders.3 On the other hand, ROS have key
roles as signal transducing molecules.4 Therefore, visualizing
ROS or oxidative environments would be a powerful approach
for elucidating the biological functions of ROS and for diagnosis
of related diseases. Generally, living cells are protected from
oxidative stresses by intracellular redox systems, such as
glutathione (GSH) and thioredoxin.5 An understanding of
the mechanisms that maintain intracellular redox homeostasis
would be helpful in developing novel therapeutics for various
ROS-related diseases. Although reported fluorescence ROS
probes are very useful for sensing ROS, the generated fluorescent
products are generally irreversibly fluorescent,6 i.e., the probes
are unsuitable for continuous monitoring of real-time ROS
dynamics. For this purpose, we require reversible ROS probes,
which detect ROS (oxidative environment) and also GSH
a Graduate School of Pharmaceutical Sciences, The University of
Tokyo, 7-3-1 Hongo, Bunkyo-ku Tokyo 113-0033, Japan.
E-mail: tlong@mol.f.u-tokyo.ac.jp; Fax: +81 3 5841 4855;
Tel: +81 3 5841 4850
b Graduate School of Medicine, The University of Tokyo,
7-3-1 Hongo, Bunkyo-ku Tokyo 113-0033, Japan
w Electronic supplementary information (ESI) available: Detailed
descriptions of materials and general methods, and synthetic procedures.
See DOI: 10.1039/c2cc18011a
z These authors contributed equally to this work.
Scheme 1 Principle of our reversible fluorescence probe for ROS.
Chem. Commun., 2012, 48, 3091–3093 3091
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Fig. 3 Reversibility of the reactions of the probe with ꢁOCl and GSH.
Reactions were performed with 5 mM 2-Me TeOR in PBS (pH 7.4) at
37 1C. GSH (final 3 mM each) and ꢁOCl (final 10 mM each) were
successively added to the reaction mixture three times each. The shown
fluorescence intensities are averages after each addition. The fluorescence
intensity was measured at 690 nm with excitation at 660 nm.
Fig. 1 Photophysical properties of 2-Me TeR and 2-Me TeOR.
(a) Absorption and (b) emission spectra of 1 mM 2-Me TeR containing
3 mM GSH (reduced form) and 1 mM 2-Me TeOR in PBS at pH 7.4.
Excitation wavelength for fluorescence spectra was 660 nm. (c) Photo-
physical properties of 2-Me TeR and 2-Me TeOR, measured in PBS at
pH 7.4. For the determination of fluorescence quantum yields, Cy5.5 in PBS
(Ffl = 0.23) was used as a fluorescence standard. N.D. = not determinable.
2-Me TeR by GSH (see Fig. S9 and S10 in the ESIw). These
results suggest that 2-Me TeR can work as a reversible ROS
probe with high sensitivity (FIhROS/FIbackground = B200-fold).
We further examined the reversibility of the reaction of
2-Me TeOR with GSH. As shown in Fig. 3, 2-Me TeOR was
reduced upon addition of GSH and exhibited almost no
fluorescence. The generated 2-Me TeR showed reactivity with
subsequently added ꢁOCl and exhibited a large fluorescence
increment, with recovery of the fluorescence almost to the
original level. Furthermore, the generated 2-Me TeOR in turn
showed the same reactivity with GSH as before. This cycle
could be repeated at least three times with only a modest
fluorescence decrement (20% of 2-Me TeOR was bleached
during three cycles). These results suggest that 2-Me TeR
could be used to detect ROS continuously in vitro.
as a large fluorescence increment (B200-fold), together with a
large red shift of the absorption spectrum of the probe.13 We also
evaluated the effect of pH on the fluorescence. As shown in
Fig. S1 and S2 in the ESIw, the pH hardly influenced the
fluorescence properties of 2-Me TeR and 2-Me TeOR under
physiological conditions (pH 5.0 to 8.0).
Next, we examined the reactivity of 2-Me TeR with various
ROS, as shown in Fig. 2. Upon addition of hydroxyl radical
(ꢀOH), peroxynitrite (ONOOꢁ), or hypochlorite (ꢁOCl) in
phosphate-buffered saline (PBS, pH 7.4), 2-Me TeR showed a
large fluorescence increment and we confirmed that the resulting
product was 2-Me TeOR (see Fig. S7 in the ESIw). On the other
Finally, we applied 2-Me TeR to live HL-60 cells, which are
known to produce large amounts of ROS, such as ꢁOCl, upon
H2O2 stimulation (Fig. 4).6e,14 Although 2-Me TeR existed as
the reduced form in HL-60 cell suspension, a fluorescence
increment was observed following stimulation of the cells with
100 mM H2O2. This fluorescence increment was short-lived,
because the added H2O2 was consumed by myeloperoxidase
(MPO) and/or quenched by intracellular reductants. This
result indicates that 2-Me TeR was oxidized in the oxidative
environment after addition of H2O2, then reduced as the
ꢁ
hand, in the presence of superoxide (O2 ꢀ), hydrogen peroxide
(H2O2), or nitric oxide (ꢀNO), 2-Me TeR showed a weak
fluorescence increment. We also examined whether or not
2-Me TeOR could be reduced to 2-Me TeR by an excess amount
of GSH and found that 2-Me TeOR was quickly converted to
Fig. 2 Fluorescence intensity of 5 mM 2-Me TeR after reaction with
various ROS in PBS (pH 7.4), measured at 690 nm with excitation at
660 nm. ꢀOH: ferrous perchlorate (10, 50, 100, and 500 mM) and H2O2
(500 mM) were added at rt. ONOOꢁ: ONOOꢁ (final 0.3, 1, 3, and 5 mM)
was added and the mixture was stirred at 37 1C. ꢁOCl: NaOCl (finꢁal 0.3, 1,
3, and 5 mM) was added and the mixture was stirred at 37 1C. O2 ꢀ: KO2
(10, 30, and 100 mM) was added and the mixture was stirred at 37 1C for
30 min. H2O2: H2O2 (10, 30, and 100 mM) was added and the mixture was
stirred at 37 1C for 30 min. ꢀNO: NOC7 (10, 30, and 100 mM) was added
and the mixture was stirred at 37 1C for 30 min. The results are mean ꢂ SD
(n = 3). BG = background. For details, see Fig. S3–S8 in the ESI.w
Fig. 4 Time courses of the change in fluorescence intensity
observed with 5 mM 2-Me TeR for H2O2-stimulated or unstimulated
HL-60 cells (2.5 ꢃ 105 cells mLꢁ1), or H2O2-stimulated HeLa cells
(2.5 ꢃ 105 cells mLꢁ1) in HBSS at 37 1C. H2O2 (100 mM and 200 mM
final concentration) was added at 100 s and 2400 s, respectively.
MPOI is aminobenzoic acid hydrazide (ABAH; 2 mM), added as a
myeloperoxidase inhibitor. The fluorescence intensity was measured at
690 nm with excitation at 660 nm.
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intracellular reductive environment was homeostatically restored.
After the fluorescence intensity had returned to the original level,
a second cycle of fluorescence increment and decrement was
observed upon further addition of 200 mM H2O2. This result
demonstrates that 2-Me TeR was not bleached by ROS during
the first redox cycle. Both fluorescence increments were sup-
pressed in the presence of a myeloperoxidase inhibitor, amino-
benzoic acid hydrazide (ABAH).15 Also, no fluorescence
increment was observed in the case of H2O2-stimulated HeLa
cells; this is as expected, because HeLa cells do not produce ROS
upon H2O2 stimulation.6e In contrast, APF, which is a previously
reported fluorescence probe for highly reactive oxygen species
(hROS), produced only a monotonic fluorescence increment in
the H2O2-stimulated HL-60 cell assay (see Fig. S11 in the
ESIw).6d Taken together, these results indicate that 2-Me TeR
allows the reversible detection of endogenously produced ROS in
HL-60 cells with high sensitivity and reversibility.
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In conclusion, we have developed 2-Me TeR as a reversible
NIR fluorescence probe for ROS, based on the redox properties
of the Te atom. 2-Me TeR is oxidized by various ROS and
quickly converted to fluorescent 2-Me TeOR. In turn, 2-Me
TeOR is reduced by GSH, quickly regenerating 2-Me TeR. This
redox cycle between 2-Me TeR and 2-Me TeOR can be run
repeatedly. This system was confirmed to work in living cells.
Interestingly, the reactivity of this Te-rhodamine-based rever-
sible ROS probe toward hROS is different from that of
Se–cyanines, which exhibit selective reactivity toward ONOOꢁ.
Furthermore, the photochemical properties of 2-Me TeOR,
whose absorption and emission maxima are in the NIR region
of 650–900 nm, are favorable, because light in this region shows
high tissue penetration.16 We believe that the redox character
and reversible NIR-fluorescence response of 2-Me TeR mean
that this probe will be useful for monitoring the dynamics of
ROS production continuously in vivo.
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This research was supported in part by the Ministry of
Education, Culture, Sports, Science and Technology of Japan
(Grant No. Specially Promoted Research 22000006 to T.N.), and
by a grant from the Industrial Technology Development Organi-
zation (NEDO) of Japan (to T.T.). K.H. was supported by Inoue
Foundation for Science, Konica Minolta Science and Technology
Foundation and The Asahi Glass Foundation. Y.K. and M.K.
were supported by a Grant-in-Aid for JSPS Fellows.
13 T. Egawa, Y. Koide, K. Hanaoka, T. Komatsu, T. Terai and
T. Nagano, Chem. Commun., 2011, 47, 4162.
14 S. Izumi, Y. Urano, K. Hanaoka, T. Terai and T. Nagano, J. Am.
Chem. Soc., 2009, 131, 10189.
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c
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Chem. Commun., 2012, 48, 3091–3093 3093