Development of azo-based fluorescent probes to detect different levels of hypoxia

Article abstract of DOI:10.1002/anie.201305784

Let it shine: New hypoxia-sensitive fluorescent probes were developed; they consist of a rhodamine moiety with an azo group directly conjugated to the fluorophore. Because of an ultrafast conformational change around the N=N bond, the compounds are nonflu

Full text of DOI:10.1002/anie.201305784

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Angewandte
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DOI: 10.1002/anie.201305784
Fluorescent Probes
Development of Azo-Based Fluorescent Probes to Detect Different
Levels of Hypoxia**
Wen Piao, Satoru Tsuda, Yuji Tanaka, Satoshi Maeda, Fengyi Liu, Shodai Takahashi,
Yu Kushida, Toru Komatsu, Tasuku Ueno, Takuya Terai, Toru Nakazawa, Masanobu Uchiyama,
Keiji Morokuma, Tetsuo Nagano, and Kenjiro Hanaoka*
Hypoxia is due to an inadequate supply of oxygen in the body,
this fluorescent probe still entailed some drawbacks. First,
QCy5 responds only to very low oxygen concentrations (less
[
1]
and is associated with various diseases. In some solid tumors,
for example, the median oxygen concentration has been
reported to be around 4%, and locally, it may even decrease
than 1% O ; Supporting Information, Figure S1). However,
2
hypoxic responses do not always occur only under such severe
conditions, so higher sensitivity is desirable. Second, QCy5
utilizes the Fçrster resonance energy transfer (FRET)
mechanism for the off/on switching of fluorescence, and the
fluorescence quencher, Black Hole Quencher 3 (BHQ-3),
which contains an azo group, is used as the hypoxia-sensitive
moiety. This quencher, however, is not susceptible to further
chemical modification. Indeed, to the best of our knowledge,
there has been only one report of a chemical modification of
BHQ-3, and even in that case, the basic scaffold was not
[
2]
to 0%. This induces various biological phenomena, such as
the stabilization of the hypoxia-inducible factor 1a (HIF-1a)
[
3,4]
and the modulation of HIF-mediated gene expression.
Therefore, methods to detect and visualize hypoxia are
important for investigating its biological effects. Several
organic molecular probes have been used as indicators of
hypoxia. For example, pimonidazole contains a nitroaryl
group that is selectively reduced by reductases under
[
5]
hypoxia. However, detection requires immunostaining of
the reduction product, and this method is thus not applicable
to living cells.
[
12]
changed. As we wished to develop a series of probes with
different colors that would be suitable for the detection of
different levels of hypoxia, a different probe design was
needed that would not require the use of BHQ-3. For this
purpose, we focused on the photophysical properties of azo
compounds. In general, azo dyes, such as azobenzene
derivatives, are nonfluorescent owing to ultrafast conforma-
tional change around the N=N bond after photoexcitation.[
On the other hand, reaction-based fluorescent probes can
[6]
be powerful tools to elucidate processes in living systems,
and several hypoxia-sensing fluorescent probes have been
[
7]
developed to detect hypoxia in living cells. Most of them
contain a nitroaryl or a quinone group as the hypoxia-
sensitive moiety. The photoswitching mechanism of these
probes is mostly based on a photoinduced electron transfer
13]
Although this photophysical property of azo compounds is
well-known, to the best of our knowledge, there has been no
attempt to conjugate an azo moiety directly to the conjugated
system of a fluorophore to quench the fluorescence. We
thought that this simple idea would be useful for the
development of a variety of hypoxia-sensitive probes, because
reductive cleavage of the azo bond would restore fluores-
cence by regenerating the original fluorophore (Figure 1).
Such a strategy should significantly extend the scope of
hypoxia sensors with controllable off/on switching of the
(
PeT) mechanism, but it is susceptible to environmental
[
8,9]
conditions, such as pH or polarity.
Furthermore, some of
these probes employ fluorophores that are known to be pH-
sensitive. Therefore, a new design strategy is required to
develop more reliable and versatile tools.
Recently, we reported that an azo group has excellent
properties as a hypoxia-sensitive moiety, and we developed
[
10]
a hypoxia-sensitive fluorescent probe QCy5 that makes use
[
11]
of the reduction of an azo group under hypoxia. However,
[
*] W. Piao, S. Takahashi, Y. Kushida, Dr. T. Komatsu, Dr. T. Ueno,
Dr. T. Terai, Prof. M. Uchiyama, Prof. T. Nagano, Dr. K. Hanaoka
Graduate School of Pharmaceutical Sciences
Prof. M. Uchiyama
Advanced Elements Chemistry Laboratory
RIKEN 2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan)
The University of Tokyo
[
**] This work was supported in part by MEXT (Specially Promoted
Research Grant 22000006 to T.N., 24689003 and 24659042 to K.H.,
and 24655147 to T.K.) and SENTAN and JST( K.H.). K.H. was also
supported by The Asahi Glass Foundation, The Uehara Memorial
Foundation, the Tokyo Biochemical Research Foundation, the Inoue
Foundation for Science, the Takeda Science Foundation, and The
Cosmetology Research Foundation. The authors thank Mr. Yu
Harabuchi of Hokkaido University for helpful discussions on
photoreactions of azobenzene. Some of the theoretical calculations
were carried out using the computational resources of the Institute
for Molecular Science, Okazaki (Japan). W.P. was supported by
a Grant-in-Aid for JSPS Fellows.
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)
E-mail: khanaoka@mol.f.u-tokyo.ac.jp
S. Tsuda, Dr. Y. Tanaka, Prof. T. Nakazawa
Department of Ophthalmology, Tohoku University
Graduate School of Medicine
1-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8574 (Japan)
Dr. S. Maeda
Department of Chemistry, Faculty of Science, Hokkaido University
Kita 10 Nishi 8, Kita-ku, Sapporo, Hokkaido 060-0808 (Japan)
Dr. F. Liu, Prof. K. Morokuma
Fukui Institute for Fundamental Chemistry, Kyoto University
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201305784.
34-4 Takano Nishihiraki-cho, Sakyo, Kyoto 606-8103 (Japan)
1
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[
13d]
ene.
To simplify the calculations, we employed a model
molecule of MAR, namely a MAR derivative without the
methylbenzene moiety. We first confirmed that the molecular
orbitals and vertical excitation energies for the model
molecule were very similar to those obtained for MAR
(Figure S4a). Moreover, for the model molecule, the oscil-
lator strengths to the S and S states are 0.0030 and 1.7647,
1
2
respectively; these values are in good agreement with those
for MAR (0.0085 and 1.7015, respectively; Table S1). In both
systems, excitations to S and S correspond mainly to n!p*
1
2
(
(
HOMOꢀ2!LUMO and LUMO + 1) and p!p*
HOMO!LUMO) transitions, respectively (Table S1). The
results of the calculations also indicate that excitation almost
Figure 1. Design strategy, chemical structure, and proposed detection
mechanism for azo rhodamines (MAR and MASR).
exclusively occurs to the S state, judging from the oscillator
2
strength (Table S1). Starting from the S state, the system
2
moves to the S state through the S /S conical intersection
1
2
1
fluorescence to afford probes that respond to different levels
of hypoxia. Herein, we report the development of novel
fluorescent probes that employ this new, non-FRET-based
photo switching concept, with a scaffold amenable to
chemical modification.
(CI), then it undergoes CꢀN=NꢀC bond rotation at the S
1
state, and the system finally reaches the S state through a
0
S /S CI (Figure S4b,c). In the case of azobenzene, a compli-
1
0
cated S !S pathway that involves higher excited states was
2
1
proposed, as S (p!p*) and S (n!p*) do not cross near the
2
1
[
16]
Rhodamine derivatives were employed as scaffold fluo-
rophores, because there are numerous reports of fluorescent
probes that are based on this fluorophore, and chemical
initial (S -MIN) geometry. However, in the present case,
0
the S /S CI is very close to the initial geometry, and the decay
2
1
simply occurs through it. There is a very small barrier on S
1
[
14]
ꢀ1
modification is straightforward.
2Me RG) and 2Me Si-rhodamine600 (2Me SiR600) were
employed as scaffold fluorophores for fluorescent probes
Figures 1, S2 and Table 1). 2Me RG is a derivative of
2Me rhodamine green
(1.9 kJmol from S -MIN to S -TS), which is negligible
1
1
[
15]
(
compared to the excitation energy to S . Overall, the
2
potential-energy profile is generally downhill along the
present pathway (for potential energy curves along the path,
(
rhodamine green that is obtained by converting the carboxylic
acid substituent into a methyl group to increase its hydro-
see Figures S5 and S6). Thus, once excited to the S state, the
2
system is expected to decay very quickly to the S0 state
without emission. Based on the high similarity between MAR
and the model molecule, we consider it very likely that
a similar ultrafast radiationless decay also occurs in MAR.
To synthesize the fluorescent probes, we converted one
amino group of the xanthene moiety of the fluorophores into
Table 1: Photophysical Properties in PBS at pH 7.4
[
a]
Dye
labs
nm]
lfl
e
Ffl
ꢀ
1
ꢀ1
[
[nm]
[m cm ]
[17]
an azo group by means of an azo coupling reaction. This
reaction provided the desired fluorescent probes, MAR and
MASR. As expected, these two compounds showed no
fluorescence (Table 1; see also Table S2 and Figure S7),
were unaffected by a change in pH between pH 3.0 and 10.0
4
2
2
Me RG
Me SiR600
498
593
601
427
520
613
n.d.
n.d.
8.2ꢀ10
9.1ꢀ10
3.8ꢀ10
2.0ꢀ10
0.84
0.38
<0.001
<0.001
4
4
4
MAR
MASR
[
a] For the determination of the fluorescence quantum efficiency (F ),
fluorescein in NaOH solution (0.1n; F =0.85) was used for 2Me RG
and MAR, and rhodamine B in EtOH (F =0.65) was used for
Me SiR600 and MASR as fluorescence standards. n.d.=not detectable,
fl
(Figure S8), and possess high photostability (Figure S9). The
fl
absorption spectrum of MASR in a phosphate buffer at
pH 7.4 indicated a less intense absorbance at approximately
620 nm, when compared to the spectrum recorded in CHCl3
fl
2
PBS=phosphate-buffered saline.
(
Figure S10). This might be the result of nucleophilic attack of
[15]
the hydroxide ion at the 9 position of the xanthene moiety.
phobicity, thus affording higher cell-membrane permeability.
These results indicate that our design strategy is a promising
method to control the off/on switching of fluorescence for
hypoxia sensors.
2
Me SiR600 is a red fluorescent dye that we recently
developed, in which the oxygen atom in the xanthene
moiety of 2Me RG is replaced by a dimethylsilyl group.
Both of these dyes are insensitive to a change in pH in the
range of pH 3.0–10.0, and exhibit high fluorescence emission
To examine whether these probes can detect hypoxia in
biological systems, we first conducted an in vitro assay using
rat liver microsomes, which contain various reductases. After
adding NADPH (50 mm) as a cofactor for the reductases to
aqueous solutions of MAR or MASR (1 mm) in the presence
of rat liver microsomes (226 mg/3 mL), a rapid increase in the
fluorescence intensity of MAR or MASR was observed only
(
Figure S3). Based on these fluorophores, we designed mono-
azo rhodamine (MAR) and mono-azo Si-rhodamine (MASR;
Figure 1).
We conducted quantum-chemical calculations to examine
whether quenching would occur through an ultrafast con-
formational change of the N=N bond of these fluorescent
ꢀ
3
ꢀ1
under hypoxia with an initial reaction rate of 1.1 ꢀ 10 mm s
ꢀ
3
ꢀ1
or 2.1 ꢀ 10 mm s ; the fluorescence intensity increased by
probes (Figure S4), as seen for azo systems such as azobenz-
630-fold or 20-fold, respectively (Figure 2). The absorption
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tion: As evaluated by fluorescence microscopy imaging, the
fluorescence intensity of MASR increased at oxygen concen-
trations of 1% or less, whereas that of MAR increased at
oxygen concentrations of 5% or less, which corresponds to
relatively mild hypoxia (Figure 3). Pimonidazole is reported
to detect hypoxia at oxygen concentrations of 10 mmHg
[23]
(
1.3%) or less; thus, whereas MASR, like QCy5, is suitable
Figure 2. Time-dependent changes in the fluorescence intensity of
MAR or MASR (1 mm) in the presence of rat liver microsomes (226 mg/
3
mL) under hypoxia (c) or normoxia (c). Measurements were
performed in a potassium phosphate buffer (0.1m; pH 7.4) containing
DMSO (0.1%) as a cosolvent. As a cofactor for reductases, NADPH
(
50 mm) was added after 5 min. The hypoxic conditions were prepared
by bubbling argon gas into the reaction solution for 30 min. The
excitation and emission wavelengths were 498 and 520 nm for MAR,
and 593 and 612 nm for MASR, respectively.
spectra also indicated that the reduction proceeded only
under hypoxia (Figure S11). We further confirmed that the
fluorophores, 2Me RG or 2Me SiR600, were generated from
MAR or MASR under hypoxia by HPLC analysis of the
reaction mixtures (Figure 1; see also Figure S12). Further-
more, these probes showed a negligible fluorescence increase
in the presence of an excess of GSH, hydrogen sulfide, or
reactive oxygen species (ROS), which are generated during
Figure 3. Fluorescence confocal microscopy images of MAR or MASR
in live A549 cells. A549 cells were incubated with MAR or MASR
(1 mm) containing DMSO (0.1%) as a cosolvent at various oxygen
concentrations for six hours. Scale bar: 30 mm. The excitation and
emission wavelengths were 488 and 515–553 nm for MAR, and 600
and 620–700 nm for MASR, respectively.
[
18]
reperfusion (Figure S13). In the case of MASR, a fluores-
cence intensity decrease occurred after approximately
for the detection of severe hypoxia, MAR features a higher
sensitivity and can detect mild hypoxia, as compared to
pimonidazole (Figure S19). This oxygen concentration
dependency of the fluorescence intensity increase of MAR
and MASR was also confirmed by flow-cytometric analysis
(Figure S20). The same oxygen concentration dependency of
MAR and MASR was observed in both HeLa cells and
Colon26 cells (Figure S21), as well as in A549 cells. Although
the chemical structures of MAR and MASR are similar, the
localizations of the scaffold fluorophores, 2Me RG and
2Me SiR600, inside cells were different: 2Me RG was local-
ized in the mitochondria, whereas 2Me SiR600 was mainly
localized inside the lysosomes (Figure S22). Thus, we spec-
ulate that the localizations of MAR and MASR inside cells
are also different, and that this may at least partially influence
their sensitivity to the oxygen concentration.
1
000 seconds (Figure 2), and we considered this observation
to be due to the reduction of the fluorophore itself
2Me SiR600), as a similar fluorescence intensity decrease
(
was observed when 2Me SiR600 was incubated with micro-
somes under hypoxia (Figure S14). This can explain the weak
absorbance increment at approximately 610 nm that is seen
after reduction under hypoxic conditions (Figure S11b). As
replacing the oxygen atom with a dimethylsilyl moiety lowers
[
19,20]
the LUMO energy level of the xanthene moiety,
Me SiR600 is more susceptible to reduction than 2Me RG.
2
Nevertheless, no significant decrease in fluorescence intensity
was observed when cells were incubated with 2Me SiR600
under hypoxia (Figure S15). This result indicates that MASR,
as well as 2Me SiR600, is practical and useful for live-cell
fluorescence imaging.
Based on the above findings, we next tried to visualize the
oxygen gradient in cultured cells under a cover glass using
both MAR and MASR, as it has been reported that mounting
a thin quartz cover glass on top of cultured cells can create an
We next applied these probes to living cells to monitor
cellular hypoxia. Under hypoxia (0.1% O ), a time-depen-
2
dent increase in fluorescence intensity was observed in A549
cells, whereas almost no fluorescence enhancement inside
cells was seen under normoxia (Figure S16). For both probes,
their low toxicity was confirmed by a live/dead staining assay
[24]
oxygen gradient. The cover glass prohibits oxygen supply
from above, so that the oxygen concentration gradually
decreases with the distance from the edge of the cover glass
(Figure 4a). A549 cells were simultaneously loaded with both
MAR and MASR, then a cover glass was placed on top. We
observed that the fluorescence intensities of MAR (green
channel) and MASR (red channel) gradually increased with
increasing distance from the edge of the cover glass (Fig-
ure 4b), whereas almost no fluorescence enhancement was
observed in the absence of the cover glass (Figure S23).
Strong fluorescence of both MAR and MASR was observed
from cells at the center of the cover glass, while only
(
Figure S17). The flavoprotein inhibitor diphenyliodonium
[
21]
chloride (DPI) dramatically suppressed the fluorescence
increase both in the cuvette and in cells (Figure S18). Despite
the low specificity of the inhibitor, this result suggests that
these probes are reduced by flavoproteins, such as NADPH-
cytochrome P450 reductase, which are thought to be respon-
sible for the metabolic activation of bioreductive compounds
[22]
under hypoxia.
Intriguingly, we found that MAR and
MASR showed different sensitivities to oxygen concentra-
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Figure 5. Fluorescence imaging of branch retinal artery occlusion
BRAO) in a rat model with MAR. a) Hypoxia imaging method after
(
BRAO. MAR (2 mm, 2 mL) was injected into the vitreous humor of
a BRAO model rat, and the retinal whole-mount was observed under
a fluorescence microscope. b,c) Fluorescence imaging of the isolated
retina two hours after MAR treatment with a low (b)- or high (c)-
magnification lens. Scale bars: 1 mm (b) and 100 mm (c). The arrow in
Figure 4. Detection of a gradient of cellular hypoxia generated by
placing a cover glass on top of the cells. a) Cellular hypoxia being
caused by the cover glass. b) Bright-field and fluorescence confocal
microscopy images of A549 cells onto which the cover glass was
placed. Cells were incubated under normal culture conditions with the
cover glass for three hours in the presence of MAR (0.5 mm) and
MASR (1 mm) containing DMSO (0.15%). Twenty images were com-
bined into one image with the Leica SP5 software. Scale bar: 2.5 mm.
c) Magnified images of A549 cells near the boundary of the cover
glass (periphery) and in the central part of the cover glass (center).
Scale bar: 100 mm. The excitation and emission wavelengths were 488
and 515–553 nm for MAR, and 600 and 620–700 nm for MASR,
respectively.
(b) indicates the point of laser irradiation. d) Comparison of the
fluorescence intensities observed in hypoxic and normoxic areas
(n=6, * Mann–Whitney U test, p=0.007).
In conclusion, by taking advantage of the photochemical
properties of an azo bond, which include sensitivity to
hypoxia, we have developed two novel fluorescent probes,
MAR and MASR, which are based on a new photo switch
concept to detect hypoxia. Owing to quenching by ultrafast
azo-bond rotation after photoexcitation, MAR and MASR
showed no fluorescence, which was supported by quantum-
chemical calculations. Reduction of MAR or MASR by
reductases under hypoxia resulted in the generation of highly
fluorescent rhodamine derivatives, 2Me RG or 2Me SiR600,
respectively, accompanied by a large enhancement in the
fluorescence intensity. Thus, direct conjugation of an azo
group to the conjugated system of the fluorophore is a useful
design strategy for hypoxia-sensitive fluorescent probes.
Furthermore, MAR and MASR showed different hypoxia
detection thresholds: Their fluorescence intensity increased
as the oxygen concentration fell below 5% and 1%,
respectively. Multicolor imaging with both probes allowed
us to visualize the oxygen concentration gradient in living
cells under a cover glass. Moreover, we showed that MAR
could visualize retinal hypoxia in a rat model of retinal artery
occlusion. Thus, we believe MAR and MASR will be
practically useful tools for investigating a variety of hypo-
xia-related biological phenomena. Because of their simple
chemical structures, further chemical modification would be
straightforward. Our strategy should also be applicable to the
development of a range of hypoxia-sensitive probes with
different threshold levels.
fluorescence of MAR was detected for cells closer to the edge
of the cover glass (Figure 4c). Although it is still difficult to
quantify the oxygen concentration, the combined use of the
two probes allows the visualization of the oxygen-concen-
tration gradient by means of multicolor imaging.
Finally, we attempted to visualize retinal hypoxia in a rat
model of retinal artery occlusion, where rapid development of
[25]
retinal ischemia quickly damages the inner layer neurons.
We used MAR in this experiment, because it is more sensitive
to hypoxia than MASR. The rat model of branch retinal
artery occlusion (BRAO) was prepared by intravenous
administration of the photosensitizing dye Rose Bengal,
followed by green-laser irradiation of a retinal artery (Fig-
[
25b,26]
ure 5a).
BRAO was confirmed by observing the blood
flow in the retinal vessels before and after BRAO with laser
speckle flowgraphy (LSFG), which can noninvasively mea-
sure the blood-flow velocity in the choroid, retina, and optic
[27]
nerve head (Figure S24). After BRAO, MAR was injected
into the vitreous humor, and two hours later, the rat was
sacrificed for histological observation. The fluorescence
intensity in the area of retinal hypoxia was higher than that
in a normoxic area (Figure 5b). The cells of the inner retinal
layer in the area were stained (Figure 5c). In this fluorescence
image, the mean ratio of the fluorescence brightness in the
area of retinal hypoxia was 389.8 ꢁ 189.8% (mean ꢁ SD),
which was significantly higher than that in the area of retinal
normoxia (100.0 ꢁ 17.2%; Figure 5d). These results indicate
that MAR can be a useful tool to detect hypoxia not only
in vitro, but also in vivo.
Received: July 4, 2013
Published online: October 14, 2013
Keywords: azo compounds · bioorganic chemistry ·
.
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
fluorescent probes · imaging agents · oxygen concentration
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