A Colorimetric Fluorescent Probe for SO2 Derivatives-Bisulfite and Sulfite at Nanomolar Level

Article abstract of DOI:10.1007/s10895-017-2115-1

A colorimetric fluorescent probe with fluorescence emission feature sensitive to SO₂ derivatives, i.e. bisulfite (HSO₃ ^?) and sulfite (SO₃ ^2?), was developed based on the HSO₃ ^?/SO₃ ^2?-mediated nucleophilic addition reaction of the probe that. This probe exhibited SO₃ ^2? sensing ability with detection limit down to 46?nM and desired selectivity over other reference anions and redox species. The preliminary fluorescence bioimaging experiments have validated the practicability of the as-prepared probe for SO₂ derivatives sensing in living cells.

Full text of DOI:10.1007/s10895-017-2115-1

J Fluoresc
DOI 10.1007/s10895-017-2115-1
ORIGINAL ARTICLE
A Colorimetric Fluorescent Probe for SO Derivatives-Bisulfite
2
and Sulfite at Nanomolar Level
1
2
1
1
1
Jian Zhang & Aidong Peng & Yanlin Lv & Yuanlin Zhang & Xuefei Wang
&
3
1
Guangjin Zhang & Zhiyuan Tian
Received: 13 February 2017 /Accepted: 8 May 2017
Springer Science+Business Media New York 2017
#
Abstract A colorimetric fluorescent probe with fluorescence
Introduction
emission feature sensitive to SO derivatives, i.e. bisulfite
2
−
2−
(
HSO ) and sulfite (SO ), was developed based on the
HSO /SO -mediated nucleophilic addition reaction of the
probe that. This probe exhibited SO3 sensing ability with
detection limit down to 46 nM and desired selectivity over
other reference anions and redox species. The preliminary
fluorescence bioimaging experiments have validated the prac-
Bisulfite and sulfite are substances widely involved in daily
life and the general population is generally exposed to them
owing to their widespread use as additives in food and bever-
age, cosmetic, drugs, and some fields such as leather process-
ing, textile, and mining [1]. However, it has demonstrated that
exposure to high dose of sulfite may cause adverse effects
such as dermatitis, urticarial, hypotension, stomachache,
diarrhea and so on [2, 3]. Specifically, sulfite may cause
breathing difficulty within minutes after intake of sulfite-
containing food and even low dose of sulfite would trigger
bronchoconstriction of some asthma patients extremely sensi-
tive to sulfite [4–7]. Taking these adverse effects on human
health, the use of bisulfite and sulfite as additives is strictly
regulated. In clinical diagnostics, high sulfite content in the
blood and urine of babies has been demonstrated to be asso-
ciated with molybdenum cofactor deficiency disease, which
leads to neurological damage and early death unless treated [8,
3
3
−
2−
3
3
2−
ticability of the as-prepared probe for SO derivatives sensing
in living cells.
2
.
.
Keywords Sulfur dioxide derivatives Fluorescent probe
Bioimaging
Electronic supplementary material The online version of this article
doi:10.1007/s10895-017-2115-1) contains supplementary material,
which is available to authorized users.
(
9
]. On the other hand sulfur dioxide (SO ) is a kind of primary
*
*
*
Xuefei Wang
wangxf@ucas.ac.cn
2
air pollutant generated from the widespread combustion of
fossil fuel. The inspiratory SO will hydrate and generate sul-
furous acid, and then hydrolyze immediately to the corre-
sponding bisulfite and sulfite derivatives, which mainly deter-
Guangjin Zhang
zhanggj@home.ipe.ac.cn
2
Zhiyuan Tian
zytian@ucas.ac.cn
mine the toxicity of SO [10]. Epidemiology studies indicated
2
that the contact of SO typically gives rise to a variety of
respiratory tract reaction [11] and is related to lung cancer,
angiocardiopathy and many kinds of neurological disorders
2
1
2
3
School of Chemistry & Chemical Engineering, University of Chinese
Academy of Sciences (UCAS), Beijing 100049, People’s Republic of
China
(migraine headaches, stroke, brain tumor) [12]. Toxicology
College of Materials Science and Opto-Electronic Technology,
University of Chinese Academy of Sciences (UCAS),
Beijing 100049, People’s Republic of China
studies demonstrated that SO and its derivatives are capable
2
of changing the voltage-gated features of the sodium and po-
tassium channels in rat hippocampus neurons [13], affecting
the intracellular mercaptan content [14] and causing the neu-
ron damage [15]. Thus, it is unequivocally crucial to develop
strategies for the facile detection of bisulfite and sulfite with
Key Laboratory of Green Process and Engineering, Beijing Key
Laboratory of Ionic Liquids Clean Process, Institute of Process
Engineering, Chinese Academy of Sciences, Beijing 100190,
People’s Republic of China

J Fluoresc
high sensitivity and selectivity in biological systems. Various
and iodomethane, the target HBT-HcD probe was eventually
obtained. Detailed synthetic procedures and characterization
data were given in the Experimental Section.
types of fluorescent probes selectively sensitive to SO and its
2
derivatives have been exploited [16–24]. Amoros et al. report-
2
−
ed detection of SO3 in aqueous milieu using organic-
inorganic silica nanoparticles with limit of detection (LOD)
of 0.32 ppm [16]. Based on the mechanism of Förster reso-
nance energy transfer (FRET) between the carbon quantum
dots (CDs) and merocyanine dye, Wang et al. developed fluo-
Time-Dependent Tracking and Titration
To evaluate the response of the as-prepared HBT-HcD probe
2
−
to SO3 , fluorescence evolution features of the probe in
DMSO/PBS buffer (3:7, v/v, 10 mM, pH 7.4) were acquired
firstly. As shown in Fig. 1, the aqueous sample of HBT-HcD
−
rescent probes for HSO3 sensing with LOD of 1.8 μM [17].
By taking advantage of the iridium metal complexes, Chao
2−
2−
et al. achieved the detection of SO3 with LOD of 0.24 μM in
phosphorescence mode [19].
probe (10 μM) in the absence of SO3 exhibited an emission
band in the range of 550–650 nm with a peak around 590 nm.
2−
In this work, a new type of colorimetric fluorescent probe
Upon addition of SO3 (15 equiv), obvious changes in the
fluorescence emission features of the probe was clearly ob-
served. Specifically, the fluorescence emission intensity of the
solution sample at 465 nm increased consecutively at the ex-
pense of the fluorescence emission intensity at 590 nm and
for SO derivatives sensing was developed based on 2-(2-
2
Hydroxyphenyl) benzothiazole (HBT) conjugated with
hemicyanine dye (HcD). Such type of probe, hereafter abbre-
2−
viated as HBT-HcD, displayed SO3 sensing sensitivity with
detection limit down to 46 nM, obviously outperforming the
counterpart probes described to date, and desired selectivity
over other reference anions and redox species. It was demon-
strated that the electron-deficient double bond of HBT-HcD
2−
reached a plateau ∼30 min after the addition of SO3 . Taking
such fluorescence evolution feature, fluorescence measure-
ment of the probe sample was performed 30 min after the
2−
addition of SO3 in the following experiments for the fluo-
rescence titration.
2
−
underwent SO3 -mediated nucleophilic addition reaction,
which typically altered the π-conjugation feature of HBT-
HcD and its fluorescence emission. As a typical class of
fluorophore with salient excited-state intramolecular proton
transfer (ESIPT) feature, HBT and its derivatives have been
widely applied in the design of various fluorescent probes.
Owing to the high fluorescence quantum yield of HBT [25],
Bturn-on^ type fluorescent probe using HBT as signaling unit
is anticipated to enable large contrast in analyte-triggered fluo-
rescence response and therefore high sensitivity. On the other
hand, the electron-withdrawing nature of HBT fragment
2−
To evaluate the ability of HBT-HcD probe for SO3 sens-
ing, fluorescence titration was carried out with the result
displayed in Fig. 2. It can be seen from Fig. 2a that the probe
sample (10 μM) in DMSO/PBS buffer (3:7, v/v, 10 mM,
pH 7.4) displayed strong absorption band centered at
2−
517 nm. Upon increasing the concentration of SO3 in the
sample, this characteristic absorption peak gradually attenuat-
ed accompanying with a slight increment in absorption band
centered at 335 nm. As a result of such change in the absorp-
tion feature, the aqueous sample of the probe clearly changed
from pale pink to colorless, as illustrated in the inset in Fig. 2a,
2−
[
26–28] is anticipated to facilitate the SO3 -mediated nucle-
2
−
ophilic addition reaction that double bond undergoes by de-
creasing the electron density of the attacked atom, which was
demonstrated to be efficient in improving the sensing sensi-
tivity of HBT-HcD as compared to the previous counterpart
probes as discussed in next section.
suggesting the SO3 -mediated formation of adduct. Upon
400-nm excitation, the HBT-HcD probe in DMSO/PBS buffer
displayed weak orange fluorescence emission features cen-
tered at ~590 nm, as shown in Fig. 2b. Upon incremental
2
−
addition of SO3 , a new emission band centered at
465 nm emerged and became gradual predominance at the
~
expense of the orange emission band. Figure 2c displayed the
evolution of fluorescence intensities of the sample at 465 nm
and 590 nm, respectively, as a function of the apparent con-
Results and Discussion
2−
Synthesis of HBT-HcD Probe
centration of SO3 in the aqueous sample. It can be seen that
2−
upon addition of 15 equiv. of SO3 , the blue fluorescence
intensity reached a plateau accompanying with the nearly
complete disappearance of the orange emission band, suggest-
HBT-HcD was synthesized following strategy as depicted in
Scheme 1. In brief, the condensation reaction of 2-
aminobenzenethiol with 5-methylsalicylaldehyde yielded the
intermediate HBT-Me. After the protection of the hydroxyl of
HBT-Me by MOMCl, the oxidation of the methyl was
achieved via Sommelet reaction and HBT-CHO was therefore
obtained. Following the condensation between HBT-CHO
and ionized 2, 3-dimethylbenzo[d]thiazol-3-ium iodide which
was derived from the reaction between 2-methylbenzothiazole
2−
ing the SO3 -mediated complete conversion from the probe
to the adduct. Specifically, the fluorescence intensity at
465 nm of the HBT-HcD probe dramatically increased with
a 150-fold enhancement factor upon the addition of 15 equiv.
2−
SO3 . Fig. 2d displays a plot of the fluorescence intensity at
2−
465 nm of the sample against the concentration of SO and
3
2
the corresponding linear fit (R = 0.986) to the experimental

J Fluoresc
Scheme 1 Synthetic routes for
the target HBT-HcD probe. a,
H O , HCl (35%), EtOH, rt.; b,
2 2
MOMCl, DIPEA, DCM, 0 °C,
then reflux; c, (i) NBS, AIBN,
CCl
reflux, (iii) AcOH, reflux; d,
CH I, CH CN; e, EtOH, reflux
4 3
, reflux, (ii) HMTA, CHCl ,
3
3
data. From such titration result, a detection limit of ~46 nM of
HcD probe sample, as shown in Fig. 3. In sharp contrast, the
addition of SO3 with identical concentrations dramatically
2−
2−
HBT-HcD for SO3 sensing was determined based on the 3-
sigma method, namely, the equation of detection limit = 3σ/k,
where σ is the standard deviation of blank measurement and k
is the slope acquired from the linear fit illustrated in Fig. 2d.
induced the increase of fluorescence intensity quotient I465
/
I590 up to ~118, presenting a pure blue color. Thus, the fluo-
rescence intensity quotient altered by 656 times upon addition
2−
2−
of SO3 to the probe sample. Similarly, the addition of SO3
The Selectivity
with identical concentrations led to remarkable fluorescence
change from orange to blue with significant fluorescence in-
tensity quotient alteration. These results suggest a high selec-
To evaluate the sensing selectivity of HBT-HcD probe for SO2
derivatives, the probe was tested against various typical refer-
tivity of the as-prepared probes for SO
derivatives over other
2
3
−
−
ence anions and redox species such as PO , H PO ,
anions and indicate the diagnostic potential of the probe for
4
2
4
−
−
−
−
2−
2−
−
−
−
−
2−
HPO , HSO , NO , NO , S O , SO4 , Cl , Br , I ,
HSO
3
/SO
3
sensing in biological samples. Inset in Fig. 3b
4
4
3
2
2 3
−
2−
−
−
−
−
F , CO , OAc , HCO , PPi, ClO , H O , SH , N H ,
O , Cys, and GSH. Taking the aforementioned evolution
illustrates the photographs of HBT-HcD probe (10 μM) with
various anions and redox species under the illumination of a
hand-held UV lamp with λex = 365 nm. Obviously, only the
3
3
2
2
2 4
−
2
feature of two characteristic emission peak as illustrated in
Fig. 2b, we acquired the fluorescence intensity ratio as the
measure of the fluorescence color purity of the sample in the
sensing selectivity evaluation. For the fluorescence feature of
free HBT-HcD probe, the fluorescence color is typically or-
ange because the blue/orange fluorescence intensity quotient
I465/I590 = ~0.18. Upon addition of these reference anions and
redox species with identical concentrations, the counterpart
fluorescence intensity quotient, I465/I590, nearly kept un-
changed or minimally affected as compared to the free HBT-
−
2−
probe sample in the presence of HSO
/SO
displayed vivid
3
3
cyan fluorescence while the sample in the presence of refer-
ence anions and redox species exhibit orange-red as the same
color to the free aqueous HBT-HcD probe sample, indicating
−
2−
the reliable selectivity of the probe for HSO
To evaluate the recognition specificity of HBT-HcD probe
to SO derivatives, competitive reaction experiments of the
/SO
sensing.
3
3
2
probe with sulfite in the presence of interference anions and
redox species were performed. Typically, fluorescence
Fig. 1 Time-elapsed fluorescence emission spectra of the aqueous HBT-HcD probe (10 μM) in DMSO/PBS buffer (3:7, v/v, 10 mM, pH 7.4, 37 °C)
2−
sample upon addition of SO
3
(15 equiv)

J Fluoresc
Fig. 2 (a) Absorption and (b),
c) fluorescence titration profile of
(
HBT-HcD probe (10 μM) in
DMSO/PBS buffer (3:7, v/v,
2
1
0 mM, pH 7.4, 37 °C) with SO
3
−
with different concentrations
and (d) the corresponding plot of
fluorescence intensity (at 465 nm)
of the aqueous HBT-HcD probe
2−
as a function of SO
tion. λex = 400 nm
3
concentra-
intensity quotient (I465/I590) of the probe in DMSO/PBS buffer
The Effect of pH on HBT-HcD
2−
in the presence of 10 equiv. of SO3 and various interference
anions, respectively, were acquired firstly and then the coun-
terpart fluorescence intensity ratio of the abovementioned
The effect of pH on the fluorescence emission features of the
HBT-HcD probe was also investigated. Figure 4 displayed the
fluorescence intensity quotient of HBT-HcD probe in DMSO/
PBS buffer (3:7, v/v, 10 mM) with various pH values in the
2−
samples with subsequent addition of 10 equiv. of SO3 were
acquired under the identical condition. By comparing the
change in the fluorescence intensity ratio of each pair of probe
samples, namely, samples before and after the addition of
2−
absence and presence of SO3 . It can be clearly seen that the
fluorescence intensity quotient (I4 /I ) of the free probe sample
65 590
2
−
2−
SO3 , the response performances of the probe to SO3 at
the presence of various interference anions were obtained.
As illustrated in Fig. S1(ESI), these reference anions could
did not displayed discernable change upon changing the pH
value in the range of pH 4.0–10.0, suggesting the good stability
of the probe even in such broad range of acidic/alkaline milieu.
−
2−
2
3
hardly interfere with the HSO /SO -induced fluorescence
Furthermore the HBT-HcD probe sample in the presence of SO
3
3
−
evolution of HBT-HcD probe, definitely indicating the recog-
exhibited excellent response feature in the range of pH 6.0–8.0,
nition specificity of the probe to SO derivatives.
indicating its potential in biological applications.
2
Fig. 3 (a) Fluorescence emission spectra of HBT-HcD probe (10 μM) in
DMSO/PBS buffer (3:7, v/v, 10 mM, pH 7.4, 37 °C) in the absence and
and anions. Inset: photographs of HBT-HcD probe (10 μM) in the ab-
−
2−
3 3
sence and presence of HSO /SO , reference anions under the illumi-
−
2−
presence of HSO
3 3
/SO , reference anions (10 equiv). (b) Fluorescence
nation of a hand-held UV lamp with λex = 365 nm
−
2−
intensity ratio (I465/I590) response of the probe to HSO
3
3
/SO , reference

J Fluoresc
Fig. 4 The pH-dependent fluorescence intensity quotient (I465/I590) of
the probe in DMSO/PBS buffer (3:7, v/v, 10 mM) in the absence and
2−
presence of SO
3
2
−
Fig. 5 The proposed response mechanism of HBT-HcD to SO
3
and
1
partial H NMR spectra of HBT-HcD in deuterated DMSO/D
2
O upon
2−
The Sensing Mechanism
addition of SO
3
(10 equiv)
1
For molecular structure of HBT-HcD probe, HBT moiety is
integrated into the molecular skeleton of HcD and is a part of
the delocalized electron system. Electron communication be-
tween these two moieties enables extended π-conjugation that
typically generates low energy fluorescence emission. Upon
Definitely, the H NMR and ESI-Mass characterization results
validated the abovementioned proposed sensing mechanism
of the as-prepared HBT-HcD probe. Based on the similar
2−
mechanism, namely SO3 -mediated addition reaction, Yang
et al. developed another type of fluorescent probe for sulfite
sensing characterized with sulfite-induced fluorescence de-
crease and detection limit of 0.1 μM [29].
2
−
addition reaction with nucleophilic agent SO3 , the π-
conjugate bridge (double bond) is broken and the delocaliza-
tion of π-electron is therefore interrupted. For the adduct prod-
uct, HBT moiety is not part of the delocalized electron system
and the excitation is localized on the HBT moiety. As a result,
fluorescence emission originating from the locally excited
state of HBT moiety is restored, shifting the fluorescence
emission from orange (centered at ~590 nm) to cyan (centered
at ~465 nm). To verify such proposed sensing mechanism of
Taking the nature of analyte-mediated nucleophilic addi-
tion reaction that HBT-HcD and other fluorescent probes with
similar structural features undergo, decreasing the electron
density of the active sites in the molecular skeleton of the
probes is anticipated to facilitate the nucleophilic attack and
therefore improve the sensing sensitivity. To verify such spec-
ulation, the natural bonding orbital (NBO) charge distribution
of HBT-HcD was acquired via geometry optimization at the
level of B3LYP/6-31G (d) and the counterpart feature of an-
other model molecule with molecular structure closely related
to that of HBT-HcD, abbreviated as coumarin-HcD, was in-
vestigated for comparison. It is noted that the replacement of
HBT moiety by coumarin constitutes the whole difference
between the molecular structure of HBT-HcD and that of
coumarin-HcD, as illustrated in Fig. 6. It also deserves
mentioning that the coumarin groups without specific
functionalization typically do not display appreciable
electron-withdrawing characteristics [30] and they may
function as electron-donating moieties in some cases [31]. It
can be clearly seen from the optimization results that the net
atomic charge of the target double bond carbon atom residing
in HBT-HcD skeleton is approximately 0.1 less than the coun-
terpart charge in coumarin-HcD (−0.075 vs. -0.173). Such
significant difference in net atomic charge of the active carbon
atoms is expected to lead to great discrepancy in reactivity of
the probes with nucleophilic agents. Specifically, the much
2− 1
HBT-HcD probe for SO3 , H NMR titration was carried out
in mixed deuterated DMSO and D O with the results shown in
2
2−
−
Fig. 5. It is conjectured that the hydrolysis of SO3 to HSO3
occurred before the nucleophilic attack to HBT-HcD.
Specifically, the signals at 7.26–7.27 and 7.48–7.50 ppm (de-
1
noted a and b) in the H NMR spectrum attributable to the
double bond in HBT-HcD vanished and simultaneously the
signals at 4.07/4.33 ppm that can be assigned to the methyne
(
denoted a’ and b’) of the addition product respectively, clear-
ly appeared. Additionally, other signals belong to the aromatic
ring were found up-shifted to the high field. In addition to the
1
information from the H NMR titration, the proposed mecha-
2−
nism of HBT-HcD probe for SO3 sensing also found support
from the ESI-MS characterization results of the HBT-HcD
2
−
probe sample in the absence and presence of SO3
.
Specifically, the adduct product of the probe upon addition
2
−
of SO3 displayed characteristic mass-to-charge ratios at
+
m/z = 505.2 [M + Na ], which is in well agreement with the
−
calculated m/z values of the structures of HBT-HcD + HSO .
3

J Fluoresc
Bioimaging
The applicability of the HBT-HcD probe for intracellular
2
−
SO3 sensing was also confirmed. Prior to the fluores-
cence cell imaging experiment, the cytotoxicity of the
probe in the milieu of DMSO/PBS buffer we evaluated.
Specifically, MTT assay was used to evaluate the via-
bility of MCF-7 cells treated with various concentrations
of HBT-HcD ranging from 0 to 10 μΜ. As illustrated in
Fig. S2 (ESI), although the cell viability displayed grad-
ual decrease upon increasing the feeding amount of
HBT-HcD probe stock solution in DMSO/PBS buffer,
nearly 84% cells survived in the case of cells incubation
with the HBT-HcD probe with the concentration up
to10 μM for 24 h. Thus, the HBT-HcD probe in the
milieu of DMSO/PBS buffer possesses good biocompat-
2
−
ibility for cell imaging. In a typical intracellular SO3
sensing experiment, MCF-7 cells were incubated with
0 μM HBT-HcD probe in Dulbecco’s Modified Eagle’s
1
Medium (DMEM medium) at 37 °C for 30 min before the
acquisition of the differential interference contrast (DIC)
Fig. 6 Net Atomic Charges of HBT-HcD (a) and coumarin-HcD (b). The
arrows indicate the attacked carbon atoms. Optimization of the structures
were performed using B3LYP/6-31G* with Gaussian 03
(
Fig. 7a) and fluorescence images of the cells via con-
focal laser scanning microscopy (CLSM). It can be seen
that the cells with internalized HBT-HcD probes exhibit
discernible fluorescence in red channel (Fig. 7c), but not
in blue channel (Fig. 7b). Subsequently, the cells were
lower electron density of the active carbon atom in HBT-HcD
owing to the more prominent electron-withdrawing character-
istics of HBT fragment is anticipated to impart higher reaction
activity of HBT-HcD in nucleophile-mediated addition reac-
tion [28]. Such improved reaction activity, together with the
high fluorescence quantum yield of HBT, contributes to the
2
−
treated with SO3 for another 30 min at 37 °C and
then the fluorescence images of the cells in both chan-
nels were acquired. In sharp contrast to the favorable
fluorescence of the cells in red channel before the ad-
2−
−
2−
excellent sensitivity of HBT-HcD to SO3 /HSO .
dition of SO3 , the fluorescence the cells acquired after
3
Fig. 7 Differential interference
contrast (DIC) and confocal laser
scanning microscopy (CLSM)
fluorescence images of HBT-HcD
2−
probe in intracellular SO
3
sens-
ing. (a) The DIC image of MCF-7
cells after incubation with HBT-
HcD probe; (b) and (c) the CLSM
fluorescence images of MCF-7
cells after incubation with HBT-
HcD probe acquired via blue- and
orange-channel, respectively. (d)
The DIC image of cells shown in
(
a, b and c) upon further incuba-
2−
tion with SO
3
; (e and f) the
CLSM fluorescence images of
cells shown in (d) upon further
2−
incubation with SO
3
acquired
via blue- and orange-channel,
respectively

J Fluoresc
2
−
the addition of SO3 in red channel was vanished (Fig. 7f),
meanwhile exhibited cellular contours with much stronger
brightness in blue channel (Fig. 7e). Unequivocally, such a
dramatic contrast in two fluorescence channels clearly dem-
onstrated the usefulness of the as-prepared HBT-HcD probe
Synthesis of Probe HBT-HcD
2-(benzo[d]thiazol-2-yl)-4-methylphenol (HBT-Me)
To the solution of 2-aminobenzenethiol (832 mg, 6.6 mmol)
and 5-methylsalicylaldehyde (900 mg, 6.6 mmol) in absolute
ethanol (30 mL) was added H O (0.915 mL, 30 mmol) and
2
−
for intracellular SO3 sensing.
2
2
concentrated hydrochloric acid (0.465 mL,15 mmol) at room
temperature. After 3 h the mixture was filtered off and washed
Conclusion
with ethanol for three times and then dried in vacuum to afford
1
In conclusion, a new type of colorimetric fluorescent
HBT-Me for 1.2 g (yield, 75%). H NMR (600 MHz, CDCl ):
3
2
−
−
probe HBT-HcD for SO2 derivatives (SO3 /HSO )
δ 12.30(1H, s), 7.97–7.98 (1H, d, J = 8.4 Hz), 7.89–7.90 (1H,
d, J = 7.8 Hz), 7.47–7.50 (2H, m), 7.40–7.41 (1H, m), 7.18–
7.19 (1H, m), 7.00–7.02 (1H, d, J = 8.4 Hz), 2.36 (3H, s);
3
sensing with high selectivity over other reference anions
and redox species was developed in the present work.
1
3
C
2
−
−
Upon the SO3 /HSO -mediated nucleophilic addition
NMR (150 MHz, CDCl ): δ 163.38, 154.78, 150.90, 132.70,
3
3
reaction of the double bond in HBT-HcD molecular
skeleton, the aqueous sample of the probe clearly
changed from pale pink to colorless; on the other hand
the fluorescence emission peak at 465 nm attributable to
the locally excited state of HBT moiety lighted up at
the expense of the emission band centered at 590 nm
originating from HBT-HcD with extended π-conjugation
system. The geometry optimization result indicated that
the HBT fragment characterized with electron-
withdrawing feature contributed to the decreased elec-
tron density of the active carbon atoms in the HBT-
HcD molecular skeleton, which was anticipated to facil-
131.58, 127.64, 127.29, 125.59, 124.39, 121.09, 120.45,
116.63, 115.31, 19.44. MS (ESI) m/z 240.1(M-1).
2-(2-(methoxymethoxy)-5-methylphenyl)benzo[d]thiazole
(HBT-MOM)
To the solution of HBT-Me (747 mg, 3.1 mmol) and DIPEA
(4.1 mL, 23.25 mmol) in dried dichloromethane was added
MOMCl (1.8 mL, 23.25 mmol) dropwise at 0 °C. Then the
mixture was refluxed for 10 h, after the solution was extracted
with saturated sodium bicarbonate. The organicphase was
concentrated in vacuum and then the residue was purified by
column chromatography on silica gel (dichloromethane: pe-
2
−
−
itate the nucleophilic attack in the SO3 /HSO -mediat-
3
ed nucleophilic addition reaction. Owing to the salient
electron characteristics and high fluorescence quantum
yield of HBT moiety, improved sensing sensitivity of
troleum ether = 2:1) to afforded HBT-MOM for 857 mg
1
(yield, 97%). H NMR (600 MHz, CDCl ): δ 8.34–8.35(1H,
3
m), 8.10–8.11(1H, d, J = 8.1 Hz), 7.92–7.93 (1H, d,
J = 7.6 Hz), 7.48–7.49 (1H, m),7.38–7.39 (1H, m),7.22–
7.26 (1H, m), 7.17–7.18 (1H, d, J = 8.4 Hz), 5.39 (2H, s),
2
−
the probe to SO3 with limit of detection down to
6 nM was achieved. The preliminary dual-channel
4
1
3
fluorescence bioimaging experiments have validated the
3.56 (3H, s), 2.41 (3H, s); C NMR (150 MHz, CDCl ): δ
3
practicability of the as-prepared HBT-HcD for SO de-
163.22, 152.79, 152.12, 136.08, 132.36, 131.66, 129.57,
125.91, 124.61, 122.77, 122.51, 121.14, 114.75, 94.53,
2
rivatives sensing in living cells.
Experimental Section
General
56.56, 20.50. MS (ESI) m/z 286.1 (M + 1).
3-(benzo[d]thiazol-2-yl)-4-hydroxybenzaldehyde
(HBT-CHO)
All chemicals were purchased from J&K Chemicals or
Energy and used as received. PL spectra were recorded
on a spectrofluorophotometer (F-7000, Japan). UV ab-
sorption spectra were taken on U-4100 UV-Vis spectro-
photometers. Both UV-vis spectra and fluorescent spec-
tra were acquired with 10 mm-light path standard
quartz cuvettes. H and C NMR spectra were mea-
sured on a Bruker Avance 600 MHz NMR spectrome-
ter in deuterated CDCl3 and deuterated DMSO with
tetramethylsilane (TMS; δ = 0 ppm) as internal stan-
dard. Electrospray mass spectra (ESI-MS) were record-
ed on Agilent 1100 Series.
To a solution of HBT-MOM (612 mg, 2.15 mmol) in tetra-
chloromethane (50 mL) was added NBS (463 mg, 2.6 mmol)
and AIBN (107 mg, 0.65 mmol). The resulting solution was
refluxed for 24 h, after which the tetrachloromethane was
distilled to afford yellow residue, which was extracted with
dichloromethane of 100 mL and saturated sodium thiosulfate.
The organic phase was concentrated in vacuum and then the
residue was added to the solution of hexamethylenetetramine
(HMTA) (900 mg, 6.5 mmol) in chloroform (30 mL). The
resulting solution was refluxed for 18 h, after which the chlo-
roform was distilled to afford white residue, to which 20 mL
acetic acid was added. The mixture was refluxed for 2 h and
1
13

J Fluoresc
then cooled to room temperature and 100 mL ethyl acetate
was added. The solution was neutralized with saturated
sodium carbonate and then washed with saturated brine.
The organic phase was concentrated in vacuum and the
residue was purified by column chromatography on sil-
ica gel (dichloromethane: petroleum ether = 1:1) to
General Procedure for Cell Imaging
MCF-7 cells were cultured in media (GIBCO RPMI 1640
supplemented with 10% FBS, 100 units per mL of penicillin
and 100 units per mL of streptomycin) at 37 °C in a humidi-
fied incubator, and culture media were replaced with fresh
media every day. The cells were further incubated with
10 μM of probe in culture media for 15 min at 37 °C and then
washed 3 times with warm PBS buffer before cell fluores-
cence imaging experiments with confocal laser scanning
microscopy.
1
afforded HBT-CHO for 97 mg (yield, 18%). H NMR
(
600 MHz, CDCl ): δ 13.36 (1H, s), 9.95 (1H, s),
3
8
.25–8.26(1H, d, J = 1.8 Hz), 8.03–8.04 (1H, d,
J = 7.8 Hz),7.96–7.97 (1H, d, J = 7.8 Hz),7.91–7.93
(
7
1H, dd, J = 1.8 Hz, J = 8.4 Hz), 7.54–7.57 (1H, m),
1
3
.46–7.49 (1H, m), 7.22–7.23 (1H, d, J = 8.4 Hz);
C
Acknowledgements Financial support from the National Natural
Science Foundation of China (grant no. 21373218 and 21573234) is
acknowledged.
NMR (150 MHz, CDCl ): δ 189.89, 168.24, 163.09,
3
1
1
2
51.34, 133.91, 132.58, 130.68, 128.84, 127.05,
26.15, 122.35, 121.72, 118.71, 117.10. MS (ESI) m/z
54.1 (M-1).
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