Ratiometric fluorescent detection of intracellular hydroxyl radicals based on a hybrid coumarin-cyanine platform

Article abstract of DOI:10.1039/c0cc02390f

The first ratiometric fluorescent probe for hydroxyl radical ratiometric imaging in living cells was rationally designed based on a hybrid coumarin-cyanine platform.

Full text of DOI:10.1039/c0cc02390f

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www.rsc.org/chemcomm | ChemComm
Ratiometric fluorescent detection of intracellular hydroxyl radicals based
on a hybrid coumarin–cyanine platformw
Lin Yuan, Weiying Lin* and Jizeng Song
Received 6th July 2010, Accepted 2nd September 2010
DOI: 10.1039/c0cc02390f
The first ratiometric fluorescent probe for hydroxyl radical
ratiometric imaging in living cells was rationally designed based
on a hybrid coumarin–cyanine platform.
excitation backscattering effects on fluorescence detection. We
reasoned that compound 1 may show the typical emission
(usually o500 nm) of coumarin dyes as the ‘‘half’’ cyanine is
not conjugated with the coumarin chromophore. By contrast,
compound 2 may have the characteristic emission of hybrid
coumarin–cyanine at NIR as the ‘‘half’’ cyanine is conjugated
with the coumarin chromophore. Thus, we should observe a
significant red-shift in emission provided that the conversion
of compound 1 to compound 2 can be mediated by hydroxyl
radicals. It is worthy to note that, although hybrid coumarin–
cyanine dyes have been broadly used in fluorescent labeling,
Reactive oxygen species (ROS) have very critical biological
functions and are involved in a variety of pathological
1
diseases. Hydroxyl radicals, one type of biologically important
ROS, are produced by successive monovalent reduction
2
of dioxygen (O ) under various stress conditions in cell
2
metabolism. The damage of DNA, proteins, or membrane
lipids by hydroxyl radicals is associated with many diseases
such as inflammations, embryo teratogenesis, herbicide effects,
cell death, and killing of micro-organisms in pathogen-defence
7
,9
fluorescent imaging and PCR,
to our best knowledge,
hybrid coumarin–cyanine dyes have not been previously
employed to design ratiometric fluorescent probes.
2
,3
reactions. Thus, it is very important to develop new methods
for detecting hydroxyl radicals in living cells.
The synthesis of probe 1 started with the preparation of
indole 5, which was obtained using a standard Fischer indole
10
Recently, a few fluorescent probes for detecting hydroxyl
4
radicals in living cells were reported. However, some of them
synthesis procedure. Treatment of indole 5 with ethyl iodide
have poor selectivity for hydroxyl radicals over other ROS.
Furthermore, most of the reported fluorescent probes sense
hydroxyl radicals with optical signal changes only in fluorescent
intensity. In contrast to intensity-based fluorescent probes,
ratiometric fluorescent probes can eliminate most or all
environmental effects (i.e. canceling artifacts due to variations
in probe concentration, probe environment, and excitation
intensity.) by self-calibration of two emission bands and can
afforded the key intermediate, indolium 4. With the key
intermediate 4 in hand, the stage was set to prepare hybrid
coumarin–cyanine dye 2. Condensation of indolium 4 with
coumarin aldehyde 3 gave compound 2. Then the probe 1 was
synthesized in one step by reduction of compound 2. The
structures of hybrid coumarin–cyanine dye 2 and probe 1 were
1
13
characterized by H NMR, C NMR and ESI-MS, and were
further unambiguously confirmed by HRMS analysis.
1
Interestingly, in the H NMR spectrum of compound 2 in
5
also increase the dynamic range of fluorescence measurement.
A FRET-based ratiometric fluorescent probe for hydroxyl
6
radicals was reported. However, to our best knowledge,
CDCl
3
(Fig. S1A, ESIw), the singlet peak at 10.16 ppm
) of compound 2 (Scheme 1) is
assigned to the proton (H
4
probes which can be used for ratiometric detection of intra-
cellular hydroxyl radicals have not been previously developed.
Thus, it is of intense interest to construct new ratiometric
probes for detection of hydroxyl radicals in living systems.
In this communication, we present compound 1 as the first
ratiometric probe capable of ratiometric detection of hydroxyl
radicals in living cells based on a hybrid coumarin–cyanine
platform. The selection of the hybrid coumarin–cyanine platform
is due to its favorable photophysical properties including
markedly down-shifted when compared to the singlet peak at
0
7.66 ppm corresponding to the proton (H
(Fig. S3, ESIw). However, in d
the proton (H ) of compound 2 is around 8.82 ppm (Fig. S1B,
) of compound 1
4
6
-DMSO, the chemical shift of
4
ESIw), consistent with those of other hybrid coumarin–cyanine
emission maximum beyond 650 nm in the near-infrared
7
(
NIR, 650–900 nm) region and a relatively large Stokes shift.
Notably, the ratiometric fluorescent probes with emission in
NIR are very attractive, as biological samples have relatively
8
low background fluorescence in the NIR region. Furthermore,
large Stokes shifts may effectively prevent any influence of
State Key Laboratory of Chemo/Biosensing and Chemometrics,
College of Chemistry and Chemical Engineering, Hunan University,
Changsha, Hunan 410082. E-mail: weiyinglin@hnu.cn;
Fax: (+)86 88821464; Tel: (+)86 88821464
w Electronic supplementary information (ESI) available: Experimental
procedures, characterization data, and some spectra. See DOI:
1
0.1039/c0cc02390f
Scheme 1 Synthesis of ratiometric fluorescent probe 1.
This journal is c The Royal Society of Chemistry 2010
7
930 Chem. Commun., 2010, 46, 7930–7932

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7
a
derivatives reported. This phenomenon may be ascribed to
the deshielding effect in light of the resonance delocalization of
the positive charge on the nitrogen atom of compound 2
variation in the emission ratios. It is worthy to note that such a
huge change of signal ratios at two wavelengths is highly
desirable for ratiometric fluorescent probes, as the sensitivity
and the dynamic range of ratiometric probes are controlled by
the ratios. The observed rate constant for hydroxyl radical-
mediated oxidation of 1 under pseudo-first-order conditions is
(
Scheme S1, ESIw).
With the hybrid coumarin–cyanine dye 2 and its reductive
form 1 in hand, we then examined their spectral properties
Fig. S5, ESIw). As shown in Fig. S5b, compounds 1 and 2
have maximal emission at around 485 and 651 nm in
PBS–CH CN (1 : 1 v/v, pH 7.4), respectively. Thus, as designed,
0
ꢁ4 ꢁ1
(
k = 5.14 ꢀ 10
s
1
(Fig. S7, ESIw) according to pseudo-
1
first-order eqn (1):
3
compound 1 has a large blue-shift in emission relative to
compound 2 indicating that compound 1 is promising as
a ratiometric fluorescent probe for hydroxyl radicals with
outstanding spectral properties.
ln[(Fmax ꢁ F
t
)/Fmax) = ꢁk0t
(1)
where F and F
t
are the fluorescence intensities at 651 nm at
max
times t and the maximum value obtained after the reaction was
0
11b
Free probe 1 displayed an emission band centered at 495 nm
complete; k is the observed rate constant. To confirm that
the ratiometric response of the probe to hydroxyl radicals is
indeed due to the transformation of compound 1 to compound
2, compound 1 was treated with hydroxyl radicals in the
assay conditions. The mass spectroscopy studies indicate
the formation of compound 2 (Fig. S8, ESIw). In addtion, the
(
Fig. 1a). However, introduction of hydroxyl radicals elicited
a marked decrease of emission intensities at 495 nm and
concurrently a significant enhancement of emission intensities
at 651 nm. Thus, probe 1 exhibited a remarkable emission shift
(
156 nm), which is much larger than that of the FRET-based
6
ratiometric hydroxyl radical probe reported. Furthermore,
1
H NMR of the isolated product is identical with that of
the fluorescence intensity ratios of probe 1 at 495 and 651 nm
the standard compound 2 (Fig. S9, ESIw), supporting that the
spectra shift in the presence of hydroxyl radicals is indeed due
to the transformation of compound 1 to compound 2.
(I651/ I495) showed a drastic change from 0.018 to 3.8 upon
treatment with hydroxyl radicals (Fig. S6, ESIw), a 210-fold
Fig. 1b shows the fluorescent ratios of probe 1 in the
presence of a wide variety of species represented by hydroxyl
radicals, superoxide, singlet oxygen, hydrogen peroxide,
ꢁ
2+
2+
2+
2+
ꢁ
CH
3
3ꢁ
COOOH, ClO , Fe , Ca , Co , Zn , NO ,
2
NO and GSH in H
hydroxyl radicals provides a large ratiometric ratio with
651/I495 = 3.8. By contrast, superoxide induced a very modest
2 3
O–CH OH (1 : 1, v/v). Introduction of
I
ratiometric response with I651/I495 = 0.11, and negligible
changes in the emission ratio (I651/I495 o 0.04) were noted
upon addition of singlet oxygen, hydrogen peroxide,
ꢁ
2+
2+
2+
2+
ꢁ
CH
3
ꢁ
COOOH, ClO , Fe , Ca , Co , Zn , NO
2
,
NO3 and GSH, indicating that the probe has a high selectivity
for hydroxyl radicals over other biologically relevant species.
Thus, probe 1 is superior to other fluorescent hydroxyl radical
probes in terms of selectivity.
To be useful, fluorescent ROS probes should have minimal
auto-oxidation in the air. We investigated the stability of the
free probe 1 in the assay solution. There were no marked
variations in the emission intensities at 495 and 651 nm for free
probe 1 in the assay solution for three days at room temperature
(Fig. S10, ESIw), indicating that probe 1 has very high stability
against auto-oxidation. Thus, probe 1 exhibited better stability
than some existing ROS fluorescent probes.
In light of the afore-mentioned favorable properties of
probe 1, we proceeded to examine its potential utility for
fluorescent imaging of hydroxyl radicals in living cells. Hela
Fig. 1 (a) Ratiometric fluorescence response of probe 1 (20 mM)
to hydroxyl radicals (25 equiv.). The spectra were recorded after
incubation of probe 1 with hydroxyl radicals for 0 to 120 min at
cells incubated with phorbol myristate acetate (PMA, a
4a,12
stimulator for production of ROS)
and probe 1 exhibited
2 3
25 1C in H O–CH OH (1 : 1, v/v), with excitation l = 460 nm. The
very faint fluorescence in the green channel (Fig. 2e) and
intense fluorescence in the red channel (Fig. 2f), whereas Hela
cells treated with only probe 1 showed modest fluorescence in
both the green channel (Fig. 2b) and the red channel (Fig. 2c).
Notably, cells incubated sequentially with PMA, superoxide
arrows indicate the change of the emission intensities with incubation
time variation from 0 to 120 min. (b) The emission intensity ratios
(
(
(
I651/I495) of probe 1 (20 mM) in the presence of various analytes
25 equiv.). Bars represent emission intensity ratios (I651/I495) at 60 min
black) and 120 min (red) after addition of the various analytes to
4
a
dismutase mimetic TEMPOL, and probe 1, gave intense
green fluorescence (Fig. 2h) but very faint red fluorescence
(Fig. 2i), indicating that the cellular red fluorescence was due
2 3
probe 1. Data were acquired at 25 1C in H O–CH OH (1 : 1, v/v).
Excitation was provided at 460 nm, and the ratio of emission
intensities at 651 nm and 495 nm was measured (see ESIw).
This journal is c The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 7930–7932 7931

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platforms for construction of ratiometric probes. We expect
that this design concept will be broadly employed to develop a
variety of ratiometric probes.
This research was supported by NSFC (20872032,
2
0972044), NCET (08–0175), and the Key Project of Chinese
Ministry of Education (108167).
Notes and references
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Fig. 2 (a) Bright-field image of live Hela cells incubated with only
probe 1 (20 mM) for 30 min; (b) fluorescence image of (a) with blue
light excitation; (c) fluorescence image of (a) with green light excitation;
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(
d) bright-field image of live Hela cells pre-treated with PMA
ꢁ1
(
2 ng mL ) for 2 h and then incubated with probe 1 (20 mM) for 30 min;
e) fluorescence image of (d) with blue light excitation; (f) fluorescence
image of (d) with green light excitation; (g) bright-field image of live
(
ꢁ1
Hela cells incubated sequentially with PMA (2 ng mL ) for 2 h, then
with TEMPOL (5 mM) for 1 h, and finally with probe 1 (20 mM) for
3
0 min; (h) fluorescence image of (g) with blue light excitation;
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4
a
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radicals in living cells. Significantly, this is the first report of
ratiometric fluorescent detection of hydroxyl radicals in
living cells.
(
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8
9
5
In summary, based on a hybrid coumarin–cyanine platform,
we have developed ratiometric probe 1 as the first probe
capable of ratiometric fluorescent imaging of intracellular
hydroxyl radicals. The new probe features high selectivity
for hydroxyl radicals, a very large emission shift (156 nm), a
large ratiometric signal (a 210-fold variation), emission in
the NIR, high stability in the solution, excitation in the
visible light region, cell membrane permeability, and, more
importantly, ratiometric fluorescent detection of hydroxyl
radicals in living cells. These prominent features should render
the probe as a useful biological tool. Furthermore, this work
also represents the first utilization of hybrid coumarin–cyanine
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932 Chem. Commun., 2010, 46, 7930–7932
This journal is c The Royal Society of Chemistry 2010