Fluorescent probe for biological gas SO2 derivatives bisulfite and sulfite

Article abstract of DOI:10.1039/c3cc39161b

A coumarin-hemicyanine dye 1 was reported for ratiometric fluorescent detection of SO₂ derivatives HSO₃^- and SO ₃^2- based on a novel addition-rearrangement cascade reaction. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3cc39161b

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Fluorescent probe for biological gas SO2 derivatives
bisulfite and sulfite†
Cite this: Chem. Commun., 2013,
49, 2637
a
a
Yuan-Qiang Sun,z Jing Liu,z Jingyu Zhang,a Ting Yangb and Wei Guo*a
Received 22nd December 2012,
Accepted 8th February 2013
DOI: 10.1039/c3cc39161b
www.rsc.org/chemcomm
A coumarinꢀhemicyanine dye 1 was reported for ratiometric
After several decades of development, fluorescent probes have
fluorescent detection of SO2 derivatives HSO3 and SO3 based been recognized as efficient molecular tools that can help monitor
ꢀ
2ꢀ
on a novel addition-rearrangement cascade reaction.
and visualize molecules with high sensitivity and spatial resolution.
In recent years, some excellent fluorescent probes for gasotrans-
Sulfur dioxide (SO2) is a common air pollutant, and human mitters NO,11 H2S12 and CO13 have been exploited for applications
exposure to SO2 has become increasingly widespread due to the in biological systems. As for SO2 derivatives HSO3ꢀ and SO32ꢀ, the
combustion of fossil fuels. Inhaled SO2 is easily hydrated to produce previously reported fluorescent probes are mainly focused on the
sulfurous acid in the respiratory tract and subsequently forms its in vitro assay, and function by their specific reactions with an
derivatives sulfite (SO32ꢀ) and bisulfite (HSO3ꢀ) (3:1 M/M, in aldehyde14 or levulinate group15 (details in the ESI†). However, the
neutral fluid),1 and the toxicity of SO2 is mainly affected by the aldehyde-based probes can only be operated in acidic conditions,
two derivatives. Epidemiological studies implied that SO2 exposure and may suffer from the interference from biothiols (Cys or Hcy);16
not only induces many respiratory responses,2 but is also linked the labile ester linkage in levulinate-type probes may induce a high
to lung cancer, cardiovascular diseases, and many neurological background signal in biological imaging, as it can potentially be
disorders, such as migraine headaches, stroke, and brain cancer.3 cleaved by proteases and esterases to produce active fluorophores.
Toxicological studies further suggested that SO2 and/or its deriva- Therefore, new methods are highly expected to overcome these
tives could change the characteristics of voltage-gated sodium shortcomings. However, the major challenge is to find an
channels and potassium channels in rat hippocampal neurons,4 appropriate receptor so that it can specifically bind SO2 derivatives
affect thiol levels and hence redox balance in cells,5 and produce a over other biologically related species, in particular, millimolar
neuronal insult.6 However, distinctive from its toxicological effects, concentrations of biothiols found inside most cells, under
it was also revealed that the blood pressure of male Wistar rats can physiological conditions.
be decreased by SO2 inhalation or by intraperitoneal injection of SO2
In 1952, it was reported that HSO3ꢀ or SO32ꢀ could add very
derivatives (Na2SO3 and NaHSO3) in a concentration-dependent rapidly and quantitatively to a,b-unsaturated compounds, such
manner.7 Furthermore, SO2 was shown to be a vasodilator, and as acrylonitrile and methyl acrylate, in aqueous solution.17 We
might regulate vascular smooth muscle tone in synergy with NO.8,9 envisioned that coupling the addition reaction with an appropriate
Although SO2 can be produced endogenously from in vivo fluorescence sensing mechanism would serve as the foundation for
ꢀ
sulfur-containing amino acids,10 a large number of its underlying a novel fluorescent probe for HSO3 or SO32ꢀ. Such consideration
molecular events remain unknown, and the status of SO2 remains reminded us of coumarinꢀhemicyanine dyes,18 not only because
to be confirmed. Since SO2 exists in aqueous solution at neutral pH they possess a similar a,b-unsaturated structure to acrylonitrile as
as an equilibrium between its two derivatives, i.e., bisulfite and well as their favorable photophysical properties, but also because,
sulfite, the sensitive and selective detection techniques that enable unlike the common Michael receptors,19 this type of dye has been
the distribution and function of bisulfite and sulfite in biological indicated to be inert to biothiols, such as Cys and GSH.20 It was
ꢀ
2ꢀ
systems to be probed are highly valuable.
envisioned that the nucleophilic attack of HSO3 or SO3 toward
this type of dye will interrupt the p-conjugation and block the ICT
process, and, as a result, twꢀo well-separated emission peaks
a School of Chemistry and Chemical Engineering, Shanxi University,
Taiyuan 030006, China. E-mail: guow@sxu.edu.cn
b Institute of Biotechnology, Shanxi University, China
2ꢀ
before and after adding HSO3 or SO3 could be obtained due
to the distinct emission between the hemicyanine dye and the
produced coumarin dye (Fig. 1). If so, this will enable the sensing
† Electronic supplementary information (ESI) available: Experimental proce-
dures, supplemental spectra, and the 1H-, 13C-NMR, and MS spectra. See DOI:
10.1039/c3cc39161b
ꢀ
2ꢀ
of HSO3 or SO3 using an attractive ratiometric fluorescence
ꢀ
2ꢀ
‡ These authors contributed equally to this work.
strategy.21 In addition, the high selectivity for HSO3 or SO3
c
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1 was dramatically shifted upfield to 4.9 ppmꢀ, corresponding to the
saturated –CH– proton adjacent to the SO3 group in the inter-
ꢀ
mediate, indicating that the initial nucleophilic attack of HSO3
towards 1 occurs at the 4-position of the coumarin moiety. However,
the intermediate is unstable because it gradualꢀly disappears along
with the simultaneous emergence of the 1–SO3 adduct. Thus, we
reasoned that the intermediate probably undergoes an intra-
molecular 1,3-rearrangement reaction, which restores conjugation
ꢀ
of the coumarin moiety, and leads to the 1–SO3 adduct (Fig. 1).
ꢀ
2ꢀ
Fig. 1 The proposed mechanism of 1 for SO2 derivatives HSO3 and SO3
.
Overall, the reaction of 1 with HSO3ꢀ could be completed within
5 min under the same conditions. With a time point of 5 min upon
addition of HSO3ꢀ, we performed the UV-vis titration studies of 1
towards HSO3ꢀ (Fig. 2B). The obtained results displayed that 6 equiv.
over the main competitive species, i.e., biothiols, was also highly
expected.
ꢀ
To test the above-mentioned possibility, we synthesized a
diethylaminocoumarinꢀhemicyanine dye 1 (Fig. 1), and examined
its reactivity towards HSO3ꢀ through time-dependent UV–vis of 1 in
PBS buffer (pH 7.4, 10 mM, containing 30% DMF). To our surprise,
the reaction is not a simple one-step process, but involves an
intermediate before completion. As shown in Fig. 2A (details in
Fig. S1, ESI†), upon addition of HSO3ꢀ, the absorption of 1 at
545 nm decreased promptly within 10 s, along with the simulta-
neous emergence of a new absorption at 463 nm; after that, the
absorption at 463 nm gradually decreased from 10 s to 240 s,
and, concomitantly, the absorption at 410 nm emerged. Also,
similar changes could be observed in time-dependent fluores-
cence spectra of 1 in the presence of HSO3ꢀ (Fig. S2, ESI†), where
three corresponding emissions at 633 nm, 491 nm and 478 nm
could be clearly observed one after another.
of HSO3 is enough to complete the reaction. Moreover, a well-
defined isosbestic point was noted at 445 nm, suggesting the clean
chemical transformation. In addition, the probe is stableꢀin a pH
region of 1–8, and displays the best response for HSO3 in the
physiological pH region (Fig. S4, ESI†). Noteworthy is that when
2ꢀ
SO3 was used, 1 displayed almost the same spectral responses;
also, with a synthetic SO2 donor,23 we could detect real-time SO2
release (Fig. S5 and S6, ESI†) by using 1.
Turning ouꢀr attention now to the fluorescent properties of 1
towards HSO3 under the same conditions (Fig. 3A), the free
probe displayed a red emission with the maximum at 633 nm;
upon addition of HSO3ꢀ, the emission intensity at 633 nm
gradually decreased with the simultaneous appearance of a new
blue emission peak at 478 nm, indicating that the chemical
reaction interrupted the p-conjugation of 1, after which the
fluorescence of the 7-diethlyaminocoumarin moiety recovered, in
good agreement with the aforementioned ratiometric fluorescent
The absorption at 410 nm (emission at 478 nm) is due to the
7-diethylaminocoumarin moiety, and could be assigned to the
1–SO3 adduct. Moreover, the adduct could be separated and
ꢀ
strategy. Essentially, the ratio of the emission intensities (I478/I633
)
characterized by 1H NMR and HRMS (ESI†). However, we speculated
that the absorption at 463 nm (emission at 491 nm) probably
resulted from the intermediate I (Fig. 1) due to the nucleophilic
attack of HSO3ꢀ to the 4-position of the coumarin moiety of 1. This
can be rationalized by the unique structural motif of 1, that is, the
4-position of the coumarin moiety in 1 is doubly activated, and, thus,
is more reactive.22 In fact, the intermediate was also observable by
1H NMR spectroscopy (Fig. S3, ESI†). Upon addition of NaHSO3,
the signals of 1 disappeared immediately with the concomitant
appearance of two sets of new signals. One could be assigned to
ꢀ
became constant when the amount of HSO3 added reached
6 equiv. Noteworthy is that the difference between the two emission
wavelengths is very large (DEm: 155 nm), which not only contributes
to the accurate measurement of the intensities of the two emission
peaks, but also results in a huge ratiometric value. In the
presence of 10 equiv. of HSO3ꢀ, a ca. 1110-fold enhancement
in the ratiometric value of I475/I635 (from 0.0999 to 110.9548)
is achieved with respect to the HSO3ꢀ-free solution. In
addition, the detection limit is determined to be 0.38 mM based
ꢀ
on S/N = 3 (Fig. S7, ESI†), which is sufficient to probe the HSO3
ꢀ
concentration in cells (ca. 16 mM).24
the 1–SO3 adduct, and the other, which gradually disappeared
after 10 min, was due to the intermediate. Moreover, the proton
(H1, at ca. d 8.3 ppm) at the 4-position of the coumarin moiety of
Fig. 3 (A) Fluorescence spectra of 1 (10 mM) upon addition of NaHSO3 (0–10 equiv.)
in PBS buffer (pH 7.4, 10 mM, containing 30% DMF). Each spectrum was recorded
after 5 min. lex = 445 nm. Slits: 5/10 nm. (B) Fluorescence spectra of 1 upon addition
of various species, including AcOꢀ, Brꢀ, Clꢀ, CNꢀ, CO32ꢀ, Fꢀ, S2ꢀ, Iꢀ, N3ꢀ, PO43ꢀ, SCNꢀ,
ꢀ
Fig. 2 (A) Time-dependent UV-vis of 1 (10 mM) in the presence of HSO3
(10 equiv.) in PBS buffer (pH 7.4, 10 mM, containing 30% DMF). (B) UV-Vis
SO42ꢀ, NO, O2 , H2O2, OHꢁ, ClOꢀ, NO2ꢀ (10 equiv. for each); Cys (1 mM), GSH (5 mM)
ꢀ
spectra of
1
(10 mM) upon addition of increasing concentrations of NaHSO3
ꢀ
(0–10 equiv.) under the same conditions. Each spectrum was recorded after 5 min.
and HSO3 (6 equiv.).
c
2638 Chem. Commun., 2013, 49, 2637--2639
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cascade reaction. The probe displays advantages such as being
easy-to-make, excellent ratiometric fluorescent response, and
high selectivity. Preliminary biological experiments indicate its
potential to probe SO2 chemistry in biological systems.
Notes and references
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Fig. 4 (A) Fluorescence imaging of HeLa cells incubated with probe 1 (10 mM)
from the green channel; (B) fluorescence imaging of (A) from the red channel;
(C) overlay of (A) and (B); (D) fluorescence imaging of HeLa cells incubated with 1
for 30 min, and further incubated with NaHSO3 (200 mM) for 30 min from the
green channel; (E) fluorescence imaging of (D) from the red channel; (F) overlap
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Next, we examined the fluorescence spectral changes of 1
(10 mM) incubated with various competitive species under the
same conditions. As shown in Fig. 3B (Fig. S8, ESI†), addition of
the representative anions, reactive oxygen species (ROS) and
biothiols, such as GSH and Cys, did not lead to any significant
fluorescence changes of 1.25 In contrast, upon treatment of 1
with HSO3ꢀ, a dramatic ratiometric fluorescence response was
ꢀ
observed. The high selectivity of 1 towards HSO3 is also
observable by the naked eye. When probe 1 was excited at
365 nm using a UV lamp in the presence of various species, only
HSO3ꢀ caused an obvious fluorescence change from red to blue
(Fig. S9, ESI†).
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ꢀ
tested the capability of 1 to image SO2 derivatives in living
cells. First, the MTT assay for 1 showed that the probe with a
concentration range of 2–20 mM has only minimal cytotoxicity
after 24 h (Fig. S12, ESI†), and thus the probe at 10 mM was
selected for imaging experiments. HeLa cells, incubated with 1
(10 mM) in culture medium for 30 min at 37 1C, showed strong
fluorescence in the red channel (Fig. 4B) and weak fluorescence in
the green channel (Fig. 4A), confirming that 1 is cell-permeable.
Since a certain concentration of SO2 derivatives has been indicated
in cells,24 the observed weak fluorescence in the green channel is
probably a result of the fact that 1 binds the two derivatives in cells.
In addition, incubation of excess GSH did not elicit obvious
fluorescence changes of 1 in HeLa cells (Fig. S13, ESI†), excluding
the possibility that the weak fluorescence in the green channel is
due to the cellular biothiols. In a control experiment, the cells were
pretreated with 1 for 30 min, and further incubated with NaHSO3
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the red channel (Fig. 4E) and a fluorescence increase in the green
channel (Fig. 4D), in agreement with the HSO3ꢀ-induced ratio-
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25 We proposed a C–HꢂꢂꢂO hydrogen-binding interactionꢀin intermediate
I for explaining the high selectivity of 1 towards HSO3 over thiols on
an anonymous reviewer’s advice.
In summary, we have reported a coumarin–hemicyanine dye
(1) as a ratiometric fluorescence probe for SO2 derivatives
ꢀ
2ꢀ
HSO3 and SO3 based on a novel addition-rearrangement
c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 2637--2639 2639