Development of an ICT-based ratiometric fluorescent hypochlorite probe suitable for living cell imaging

Article abstract of DOI:10.1039/c1cc15762k

We have judiciously constructed a novel ICT-based ratiometric OCl ^- probe capable of ratiometric imaging in the live cells based on the new OCl^--promoted de-diaminomaleonitrile reaction. The Royal Society of Chemistry 2011.

Full text of DOI:10.1039/c1cc15762k

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Development of an ICT-based ratiometric fluorescent hypochlorite probe
suitable for living cell imagingw
Lin Yuan, Weiying Lin,* Jizeng Song and Yueting Yang
Received 18th September 2011, Accepted 17th October 2011
DOI: 10.1039/c1cc15762k
We have judiciously constructed a novel ICT-based ratiometric
OClÀ probe capable of ratiometric imaging in the live cells based
on the new OClÀ-promoted de-diaminomaleonitrile reaction.
Scheme 1 Transformation of 2,4-dinitrophenylhydrazones to aldehydes
Hypochlorite anion (OClÀ)/hypochlorous acid (HOCl), a
by an oxidant.
fluorescent OClÀ probes. With this in mind, we initially
biologically significant reactive oxygen species (ROS), is produced
by peroxidation of chloride ions catalyzed by the enzyme
myeloperoxidase (MPO).1 Although OClÀ/HOCl plays a critical
constructed compound 1a (Scheme 2) as a candidate for
fluorescent OClÀ probes. The selection of aminocoumarin
role in many biological processes,2 it is implicated in a variety
dye is due to the consideration that this dye possesses several
of pathological diseases including atherosclerosis, neuron
favorable fluorescence properties such as excitation and emission
degeneration, cystic fibrosis, arthritis, and cancers.3,4 In addition,
wavelengths in the visible region as well as a high fluorescence
OClÀ/HOCl is often used as a disinfectant, antimicrobial or
quantum yield. These advantageous fluorescence properties
bleaching agent.1a,4,5 Therefore, it is of high importance to
are desirable for the intended bioapplications of the probe. In
detect OClÀ/HOCl in living systems.
addition, we envisioned that OClÀ may promote the transformation
Toward this end, a number of small-molecule fluorescent
probes for OClÀ/HOCl have been devised.6 However, these
(Scheme 2) based on the reaction illustrated in Scheme 1. Since
of coumarin-2,4-dinitrophenylhydrazone 1a to coumarin aldehyde 7
probes respond to OClÀ/HOCl with changes only in fluores-
the aldehyde and the dinitrophenylhydrazone groups have
cence intensity. Thus, these fluorescent ‘‘turn-on’’ probes, in
different electron withdrawing abilities, we reasoned that com-
spite of being appealing in terms of design, have fluorescence
pounds 1a and 7 may have distinct fluorescence properties.
Thus, compound 1a may act as a ratiometric probe for OClÀ.
signals that may be significantly influenced by some factors
including probe concentration, sample environment, and light
We then proceeded to examine the fluorescence response
of compound 1a to OClÀ. However, compound 1a is essen-
scattering. By contrast, these factors may be potentially
avoided by employing ratiometric fluorescent probes, as they
tially non-fluorescent (Fig. S1, ESIw). Thus, only a fluorescence
allow the measurement of fluorescence intensities at two
wavelengths.7 Previously, we have reported a ratiometric
fluorescent OClÀ probe.8 However, the probe only functioned
appropriately at a high pH value (pH 9.0), which renders it
unsuitable for applications in living cells. Thereby, it is
necessary to develop new ratiometric fluorescent probes capable
of ratiometric imaging of OClÀ in living cells.
To rationally design a new ratiometric fluorescent probe for
OClÀ, a selective chemical reaction mediated by OClÀ is highly
sought. It is known that transformation of 2,4-dinitrophenyl-
hydrazones to aldehydes can be proceeded by an oxidant
(Scheme 1).9 We reasoned that this reaction may be exploited
as a starting point to judiciously develop new ratiometric
Scheme 2 Design and synthesis of fluorescent OClÀ probes 1a and 1b.
State Key Laboratory of Chemo/Biosensing and Chemometrics,
College of Chemistry and Chemical Engineering, Hunan University,
Changsha, Hunan 410082, China. E-mail: weiyinglin@hnu.edu.cn;
(b) HCl/HI, n, 18 h; (c) POCl3, DMF, 60 1C, 6 h; (d) diethylmalonate,
Fax: +86 88821464; Tel: +86 88821464
w Electronic supplementary information (ESI) available: Experimental
procedures, characterization data, and additional spectra. See DOI:
10.1039/c1cc15762k
Reaction conditions: (a) 1-bromo-3-chloropropane, Na2CO3, n, 12 h;
piperidine; (e) concentrated HCl, HAc, n, 6 h; (f) POCl3, DMF, 60 1C,
6 h; (g) diaminomaleonitrile or (2,4-dinitrophenyl)hydrazine; (h) PBS/
DMF, NaOCl.
c
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Chem. Commun., 2011, 47, 12691–12693 12691

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turn-on instead of ratiometric response was observed (Fig. S1,
ESIw). We reasoned that the fluorescence of compound 1a is
quenched by the nitro group, a notorious fluorescence quencher.10
Obviously, to successfully design a ratiometric OClÀ probe,
the dinitrophenylhydrazone group has to be replaced by
another strong electron withdrawing group, which should not
quench the coumarin dye. It seems that the diaminomaleonitrile
group is a good candidate for this purpose. Thus, compound
1b was then designed (Scheme 2). Compound 1b may function
as a ratiometric fluorescent probe for OClÀ provided that the
de-diaminomaleonitrile reaction can be selectively mediated
by OClÀ. Notably, the OClÀ-promoted de-diaminomaleo-
nitrile reaction is unprecedented.
Fig. 1 The emission spectra of probe 1b (3 mM) upon addition of
increasing concentrations of NaOCl (0 to 40 equiv.) in PBS/DMF
(pH 7.4, 8 : 2) excited at 540 nm (a) or 464 nm (b), respectively. The
arrows indicate the change of the emission intensities with the increase
of NaOCl from 0 to 40 equiv.
The synthesis of compounds 1a and 1b is outlined in
Scheme 2. Briefly, compound 2 was converted to compound
5 in three steps.11,12 However, we employed an improved
work-up/purification procedure to afford pure compounds
(see the ESIw for details). Hydrolysis and the subsequent
decarboxylation of compound 5 under concentrated HCl gave
compound 6, which was converted into aldehyde 7 via a
Vilsmeier-type reaction. Finally, condensation of coumarin
aldehyde 7 with (2,4-dinitrophenyl)hydrazine or diaminomaleo-
nitrile afforded compound 1a or 1b, respectively.
graph of the response to the OClÀ concentrations from 0.9 to
90 mM was obtained (Fig. S6b, ESIw), indicating that probe 1b
can be potentially employed to quantitatively detect OClÀ
concentrations. The probe is highly sensitive to OClÀ with a
detection limit of 0.2 mM (S/N = 3, see ESIw). The ratiometric
fluorescence response of 1b to OClÀ was also corroborated by
the emission colour change of the solution from orange to
green upon addition of NaOCl (Fig. S7, ESIw). To confirm
that the ratiometric response of probe 1b to OClÀ is indeed
due to the transformation of compound 1b to compound 7 as
designed, compound 1b was treated with NaOCl, and the
With compounds 1a, 1b, and 7 in hand, we set out to
investigate their optical properties in PBS/DMF (pH 7.4, v/v
8 : 2) at room temperature. The photophysical data for
compounds 1a–b and 7 are compiled in Table S1 (ESIw). The
absorption spectra of compounds 1a–b exhibited a marked red-
shift relative to that of the reference 7 (Fig. S2a, ESIw). This
bathochromic shift is apparently attributed to intramolecular
charge transfer (ICT).13 The ICT feature of compound 1b was
1
reaction product was isolated and subjected to the H NMR
and MS analysis. The 1H NMR spectrum of the isolated
product is identical to that of the standard compound 7
(Fig. S8, ESIw). In addition, the mass spectrometry analysis
further substantiates the formation of compound 7 upon
treatment of probe 1b with OClÀ (Fig. S9, ESIw). These results
demonstrate that the ratiometric response is due to OClÀ-
promoted de-diaminomaleonitrile reaction. Significantly, the
de-diaminomaleonitrile reaction has not been previously
exploited in fluorescent OClÀ probe design.
14
further confirmed by the studies of the solvent effect and the
charge distribution based on the density functional theory (DFT)
calculations. As shown in Fig. S3 (ESIw), a red-shift in both the
absorption and emission was observed with increasing the
polarity of the solvents. In addition, DFT calculations indicate
that the donor unit bears partial positive charges and the
acceptor unit contains partial negative charges (Fig. S4, ESIw).
This is in good agreement with the intramolecular charge transfer
according to the Mulliken theory.15
To test the selectivity, probe 1b (3 mM) was incubated with the
representative species including Mg2+, Cu2+, Fe3+, Zn2+, Co2+
,
Ca2+, FÀ, SO42À, HCO3À, NO2À, NO3À, SO32À, ClÀ, PO43À, IÀ,
OClÀ, hydrogen peroxide, hydroxyl radicals, superoxide, and
CH3COOOH in PBS buffer/DMF (pH 7.4, 8 : 2). As shown in
Fig. 2, OClÀ elicited a large ratiometric signal with I505/I585
=
Compounds 1b and 7 have maximal emission at 505 and 585 nm
(Fig. S2b, ESIw), respectively. Thus, consistent with the apparent
bathochromic shift in the absorption spectra, the fluorescence
emission spectrum of compound 1b displayed an 80 nm redshift
when compared with that of compound 7. DFT calculations at the
B3LYP/6-31G(d) level further support the bathochromic shift
spectra of compound 1b. The HOMO–LUMO energy gap of
compound 1b is smaller than that of compound 7 (Fig. S5, ESIw).
The optical data and DFT calculations indicate that compound 1b
is promising as a novel ratiometric fluorescent probe.
18.1. By contrast, other ROS species (hydrogen peroxide, hydroxyl
radicals, superoxide, and CH3COOOH) induced a very modest
ratiometric response with I505/I585 o 1.6, and the remaining
species caused only negligible changes in the emission ratio (I505
/
I585 o 0.5). Thus, these data indicate that the probe has high
selectivity for OClÀ.
The ratiometric responses of probe 1b to OClÀ at different
pH values were also investigated. In the absence of OClÀ, the
free probe is stable over a wide range of pH values from 5.5 to
9.8 (Fig. S10, ESIw). However, the ratiometric response of the
probe toward OClÀ was pH-dependent. At pH 7.4, probe 1b
exhibited a drastic change of the emission ratio (I505/I585) from
0.12 in the absence of OClÀ to 28.2 in the presence of OClÀ
(40 equiv.), a very large (235-fold) enhancement. This indicates
that the probe may be suitable for bio-applications at the
physiological pH. Furthermore, the observation that the probe
has a larger ratiometric signal in aqueous solutions under basic
conditions suggests that the probe detects OClÀ rather than
HOCl, as the pKa of HOCl is around 7.6.6b,c,16 We further
To examine the ratiometric fluorescence response of probe 1b
to OClÀ, the probe was titrated with different concentrations of
NaOCl in PBS/DMF (pH 7.4, v/v 8 :2). As shown in Fig. 1, the
free probe displayed an emission peak with maximum at around
585 nm. By contrast, upon addition of OClÀ, the intensity of the
emission peak at 585 nm gradually decreased with the simulta-
neous appearance of a new blue-shifted emission band centered
at 505 nm (Fig. 1). Consistently, the blue-shift was also observed
in the absorption spectra (Fig. S6a, ESIw). A linear calibration
c
12692 Chem. Commun., 2011, 47, 12691–12693
This journal is The Royal Society of Chemistry 2011

View Article Online
This research was partially supported by NSFC (20872032,
20972044, 21172063), NCET (08-0175), the Doctoral Fund of
Chinese Ministry of Education (20100161110008), and the
Fundamental Research Funds for the Central Universities,
Hunan University.
Notes and references
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Fig. 2 Emission intensity ratio (I505/I585) of probe 1b (3 mM) in PBS/
DMF (pH 7.4, 8 : 2) in the presence of various species. 1, blank; 2, Mg2+
(0.1 mM); 3, Cu2+ (60 mM); 4, Fe3+ (60 mM); 5, Zn2+ (60 mM); 6, Co2+
(60 mM); 7, Ca2+ (0.1 mM); 8, FÀ (0.1 mM); 9, SO4 (0.1 mM); 10,
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examined the time course of the ratiometric response of probe
1b to OClÀ (Fig. S11, ESIw). The free probe is highly stable
under the assay conditions. However, upon addition of OClÀ,
a dramatic increase in the ratio was observed within 1 min, and
the ratio signal essentially reached maximum within 10 min.
This finding suggests that probe 1b is a ‘‘fast-response’’ probe
for OClÀ and may be suitable for real-time sensing of OClÀ.
The favorable fluorescence properties and appropriate
amphipathicity of probe 1b prompted us to test its potential
use for OClÀ ratiometric imaging in living cells. MCF-7 cells
incubated with only probe 1b exhibited a very weak ratio-
metric response (Fig. 3b), whereas MCF-7 cells treated with
probe 1b and then further incubated with OClÀ afforded an
intense ratiometric response (Fig. 3d). These data indicate that
probe 1b is cell membrane permeable and capable of ratio-
metric imaging of OClÀ in the living cells.
In summary, we have developed compound 1b as the novel
ratiometric fluorescent OClÀ probe based on the OClÀ-promoted
de-diaminomaleonitrile reaction. Probe 1b exhibited high sensi-
tivity and selectivity to OClÀ. Importantly, we have demonstrated
that, unlike the previous ICT-based ratiometric OClÀ probes,
probe 1b could be employed for ratiometric imaging of OClÀ in
living cells. Thus, probe 1b represents the first ICT-based ratio-
metric OClÀ probe suitable for ratiometric imaging in live cells.
We expect that the new OClÀ-promoted de-diaminomaleonitrile
reaction introduced herein will be widely used for design of OClÀ
fluorescent probes.
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Fig. 3 (a) Bright-field image of live MCF-7 cells incubated with only
probe 1b (5 mM) for 30 min; (b) fluorescence ratio image (I505/I585) of
(a); (c) bright-field image of live MCF-7 cells incubated with probe 1b
(5 mM) for 30 min, then with NaOCl (6 equiv.) for 30 min;
(d) fluorescence ratio image (I505/I585) of (c).
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c
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Chem. Commun., 2011, 47, 12691–12693 12693