Two new colorimetric and ratiometric fluorescent probes based on diketopyrrolopyrrole (DPP) for detecting and imaging of mitochondrial SO2 derivatives in cancer cells

Article abstract of DOI:10.1016/j.jphotochem.2017.06.007

Two new fluorescent probes (DPP-FI/DPP-BI) based on diketopyrrolopyrrole (DPP) were designed and synthesized for colorimetric and ratiometric detection of SO₂ derivatives. Both of them displayed obvious ratiometric changes with about 21 folds o

Full text of DOI:10.1016/j.jphotochem.2017.06.007

Journal of Photochemistry and Photobiology A: Chemistry 346 (2017) 265–272
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Two new colorimetric and ratiometric fluorescent probes based on
diketopyrrolopyrrole (DPP) for detecting and imaging of mitochondrial
SO2 derivatives in cancer cells
Jian Wanga, Yandi Hanga, Haoqi Tanb, Tao Jianga, Xue Qub, Jianli Huaa,
*
a
Key Laboratory for Advanced Materials & Institute of Fine Chemicals, College of Chemistry and Molecular Engineering, East China University of Science and
Technology, 130 Meilong Rd., Shanghai 200237, China
b
The State Key Laboratory of Bioreactor Engineering, Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and
Technology, Shanghai, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Two new fluorescent probes (DPP-FI/DPP-BI) based on diketopyrrolopyrrole (DPP) were designed and
synthesized for colorimetric and ratiometric detection of SO2 derivatives. Both of them displayed obvious
ratiometric changes with about 21 folds of fluorescent ratio transformation from red to yellow upon
Received 24 April 2017
Received in revised form 5 June 2017
Accepted 5 June 2017
Available online 10 June 2017
treating with bisulfite to interrupt the p-conjugation of probes. In addition, the calculated detection limit
of DPP-FI and DPP-BI was down to 2.34 Â10À6 M and 1.73 Â10À6 M towards HSO3À, respectively. Besides,
2À
DPP-FI/DPP-BI showed outstanding selectivity toward HSO3À/SO3 over other nuÀcleophilic anions.
Keywords:
Diketopyrrolopyrrole
Fluorescent probes
Colorimetric
Ratiometric
SO2 derivatives
Mitochondrial
More importantly, these two probes could successfully detect mitochondrial HSO3 in HepG2 cells,
which is to our knowledge the first example using DPP as colorimetric and ratiometric fluorescent probes
to detect small molecules in organelles.
© 2017 Elsevier B.V. All rights reserved.
1. Introduction
To date, a variety of disparate approaches have been applied to
SO2 derivatives determination, including spectrophotometry
Sulfur dioxide (SO2), a kind of industrial waste, has been
considered as toxic environmental pollutant. Recent studies have
demonstrated that SO2 even at low concentrations in the
environment will cause not only some respiratory diseases, but
also induced lung cancer [1], cardiovascular disease [2,3], and
nervous system diseases (such as migraine, stroke, and brain
tumors) [4–6]. Endogenous biological SO2 mainly generated
enzymatically in the mitochondria of cells, which was as an
oxidation product from hydrogen sulfide [7,8] and some other
sulphur-containing amino acids [9], can regulate the structure and
function of cardiovascular, such as lowering blood pressure and
vasodilation [10]. However, it also destroys the enzyme activity
[14,15], electro-chemistry [16,17], chromatography [18,19], chemi-
luminescence [20,21], capillary electrophoresis [22] and enzymat-
ic techniques [23]. However, these methods are usually unsuitable
for routine analysis due to the requirement of tedious sample and
reagent preparation or complicated instruments. Therefore, new
techniques for accurate detection of HSO3À/SO32À levels with good
selectivity and high sensitivity are in high demand to provide
2À
valuable information on the functions of HSO3À/SO3 in biology.
Fluorescent probes provided an indispensable real tool for real-
time sensing and visualization of some biological species because
of its high sensitivity, non-invasiveness, high temporal and spatial
2À
resolution. Up to now, several fluorescent probes for HSO3À/SO3
À
once exceed normal concentrations [11] (16.77 Æ 8.24
m
mol/L in
have been reported based on HSO3 nucleophilic reaction with
plasma), thus affecting human metabolism [12]. Therefore, it is
important to develop a reliable assay method for detecting SO2 and
its derivatives in living systems. As the mainly exists form of SO2 in
aldehydes [24–26], coordinative interactions [27,28] and the
selective deprotection of levulinate [29]. But they have some
disadvantages such as too long response time (5min–10 h),
unsatisfactory detection limit. And also suffered interference from
hydrogen sulfide (H2S), cysteine (Cys), homocysteine (Hcy),
glutathione (GSH), protease or esterase. Besides, these probes
display changes only in the fluorescence emission intensity,
thereby being subjected to environmental conditions and instru-
mental efficiency.
2À
living cells, HSO3À/SO3 has been widely concerned [13].
* Corresponding author.
E-mail address: jlhua@ecust.edu.cn (J. Hua).
http://dx.doi.org/10.1016/j.jphotochem.2017.06.007
1010-6030/© 2017 Elsevier B.V. All rights reserved.

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J. Wang et al. / Journal of Photochemistry and Photobiology A: Chemistry 346 (2017) 265–272
Diketopyrrolopyrrole (DPP) was a robust chromophore and had
silica gel (200–300 mesh). Dry N,N-Dimethylformamide (DMF)
were also commercially obtained and without further purification.
1H and 13C NMR spectra were recorded on a Bruker AM 400 MHz
spectrometer using CDCl3 or DMSO-d6 as solvent and tetrame-
thylsilane (TMS) as the internal reference. Absorption spectra were
measured with a Varian Cary 500 UV–vis spectrophotometer.
been extensively used as organic photovoltaics [30–32], semi-
conductors [33–35] and two-photon absorption materials [36,37].
Furthermore, on the one hand, unmodified DPP emits green
fluorescence but it can reach the near-infrared region after
modification. On the other hand, compared with other commercial
dyes, the structure of DPP has a certain rigidity. As a result, it
improves the light stability under high power excited source and
ensuring quantum efficiency at the same time, which makes it the
potential material of chemical and biological probes. Therefore,
since the first example using DPP as fluorescent probe reported by
our group [38], it has been widely developed on reaction-based
fluorescent probes by our group [39,40] and other groups [41–45].
However, DPP was rarely reported in the field of organelles-
targeted [46], especially none of it has been applied to detect small
molecules in organelles. Recently, Zhang’s group has reported a
new fluorescent probe for sulphite detection based on DPP
derivative [47], but it takes too long time to response, easily being
affected by the auto-fluorescence after nucleophilic addition of
sulfite, and had not been applied in living cells imaging. Therefore,
Fluorescence measurements were collected using
a Horiba
Fluoromax À 4 fluorescence spectrometer.
2.2. Calculation of LOD
Plotting the ratio of I1/I2(I1 and I2 refers to value of the
corresponding emission maxima before and after reaction with
HSO3À) as a function of HSO3À concentration for determination of
the limit of detection (LOD). LOD were calculated by the equation
3d/slope (d is a standard deviation of the measured value of the
emission maxima of blank solution for seven times).
2.3. Determination of fluorescence quantum yields
2À
two new probes for HSO3À/SO3 were designed and synthesized
The relative fluorescence quantum yields of DPP-FI/DPP-BI
were respectively determined in ethanol before and after the
reaction with NaHSO3. The fluorescence of Rhodamine B (Ff = 0.97)
in ethanol was chosen as a standard [50].
in our present work, in which DPP was used as the fluorophore, and
the
a
,
b
-unsaturated structure between DPP and indolium moiety
was designed to detect HSO3À/SO3 (Scheme 1). Meanwhile, the
introduced indolium improved the water solubility, and made it a
potential mitochondria-targeted probe due to its positively
charged [48,49]. Results indicated that these two probes had
good biocompatibility, selectivity and good competition for sulfite
over other some anions and biothiols just within 4 min. And their
ratiometric properties gave them much lower detection limit, less
auto-fluorescence interference and higher accuracy. In addition,
these two probes have been demonstrated to realize mitochon-
2À
2.4. Synthesis
2.4.1. Synthesis of compound 1
A mixture of ethyl benzoylacetate (4.81 g, 25.0 mmol), K2CO3
(3.59 g, 26.0 mmol), KI (0.53 g, 3.2 mmol) and ethyl bromide
(4.34 g, 26 mmol) in acetone (20 mL) and 1,2-dimethoxyethane
(10 mL) was stirred at 75 ꢀC for 24 h, then cooled to room
temperature and filtered. The filter was collected and acetone
was removed via rotatory evaporation. The residue was diluted
with CH2Cl2 and washed with water. The organic layer was dried
with anhydrous MgSO4 and the solvent was removed via rotatory
evaporation to give yellow liquid crude product. The crude product
and ammonium acetate (18.69 g, 242.5 mmol) were dissolved in
50 mL of acetic acid, and then the solution was stirred under argon
protection at 120 ꢀC for 8 h. After cooling to room temperature, the
mixture was poured into ice water. The precipitate was collected
and dried under vacuum to give 1 as gray solid (3.46 g, 60.49%).
dria-localized and be used for direct visualization of HSO3À/SO3
2À
in living cells.
2. Experimental section
2.1. Reagents and measurements
Unless otherwise stated, reagents were commercially obtained
and used without further purification. Reactions were monitored
by TLC. Flash chromatography separations were carried out using
2À
Scheme 1. Chemical structure of probe DPP-FI/DPP-BI and reaction mechanism of probe DPP-FI/DPP-BI with HSO3À/SO3
.

J. Wang et al. / Journal of Photochemistry and Photobiology A: Chemistry 346 (2017) 265–272
267
2.4.2. Synthesis of compound 2
CDCl3) d (ppm): 10.09 (s, 1H), 8.04 (d, J = 8.3 Hz, 2H), 7.98 (d,
Sodium (180.0 mg, 7.5 mmol) was added in t-amyl alcohol
(20 mL) at 50 ꢀC, the mixture was heated to 100 ꢀC until the sodium
disappeared completely. Then the solution was cooled to 50 ꢀC,
compound 1 (462 mg, 2.0 mmol) and 4-(1,3-dioxolan-2-yl) benzo-
nitrile (525 mg, 3.0 mmol) was added. Subsequently, the resulting
suspension was stirred at 100 ꢀC overnight under the protection of
an argon atmosphere, and cooled to room temperature. The
mixture was poured into a mixture of hydrochloric acid and
methanol. The precipitate was filtered and washed with water and
methanol, dark-red solid was obtained after dried under vacuum
(300 mg, 41.63%). The product was used without further purifica-
tion.
J = 8.3 Hz, 2H), 7.82 (dd, J = 6.5, 3.1 Hz, 2H), 7.56 À 7.52 (m, 3H), 3.77
(t, J = 8.3 Hz, 4H), 1.60 À 1.53 (m, 4H), 1.30 À 1.25 (m, 4H), 0.84 (td,
J = 4.1 Hz, 4.1 Hz, 6H). 13C NMR (100 MHz, CDCl3),
d (ppm): 191.31,
162.66, 162.37, 150.09, 146.08, 137.43, 133.70, 131.49, 130.01, 129.21,
128.96, 128.75, 127.93, 111.21, 109.70, 41.74, 31.58, 31.49, 19.96,
13.57. HRMS (ESI, m/z): [M+H]+ calcd. for C27H29N2O3 429.2178,
+
found 429.2193.
2.4.8. Synthesis and characterization of compound DPP-FI
Compound 6 (218 mg, 0.5 mmol), 1-butyl-2,3,3-trimethyl-3H-
indolium (108 mg, 0.5 mmol) and three drops of piperidine were
dissolved in acetonitrile. The reaction mixture was stirred at 80 ꢀC
for 7 h under the protection of an argon atmosphere. Then the
mixture was cooled to room temperature, and the reaction solvent
was evaporated under vacuum, then the residue was purified by
flash column chromatography on silica gel (DCM/ethanol, 30/1, v/
v) to provide the corresponding pure product DPP-FI (110 mg,
2.4.3. Synthesis of compound 3
Sodium (4.00 g, 174 mmol) was added in t-amyl alcohol
(100 mL) at 50 ꢀC, the mixture was heated to 100 ꢀC until the
sodium disappeared completely. Then the solution was cooled to
50 ꢀC, 4-(1,3-dioxolan-2-yl) benzonitrile (13.8 g, 78.86 mmol) was
added, diisopropyl succinate (8.07 mL, 39.43 mmol) in t-amyl
alcohol (30 mL) was added dropwise under the protection of an
argon atmosphere. Subsequently, the resulting suspension was
stirred at 100 ꢀC overnight, and cooled to room temperature. The
mixture was poured into a mixture of hydrochloric acid and
methanol. The precipitate was filtered and washed with water and
methanol, dark-red solid was obtained after dried under vacuum
(5.96 g, 35%). The product was used without further purification.
33.64%) as a dark-red solid. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
10.13 (s, 1H), 8.55 (d, J = 16.4 Hz, 1H), 8.45 (d, J = 8.5 Hz, 2H), 8.15-
8.05 (m, 6H), 8.03 À 7.99 (m, 1H), 7.96 À 7.93 (m, 1H), 7.87 (d,
J = 16.4 Hz, 1H), 7.69 À 7.65 (m, 2H), 4.78 (t, J = 7.2 Hz, 2H), 3.79 (dt,
J = 15.9, 7.5 Hz, 4H), 1.85 (s, 6H), 1.49 À 1.37 (m, 8H), 1.19 À 1.13 (m,
4H), 0.95 (t, J = 7.3 Hz, 3H), 0.76 (dd, J = 15.9, 7.4 Hz, 6H). 13C NMR
(100 MHz, DMSO-d6),
d (ppm): 192.65, 181.81, 161.31, 147.39,
144.20,140.67,137.45,132.69,130.81,129.74,129.30,129.22,129.16,
123.18, 115.72, 114.43, 110.08, 110.04, 52.55, 30.72, 30.67, 25.50,
19.28, 19.21, 13.70, 13.37, 13.29. HRMS (ESI, m/z): [M+H]+ calcd. for
+
2.4.4. Synthesis of compound 4
C
43H48N3O3 654.3696, found 654.3687.
Compound 3 (864 mg, 2.0 mmol), NaH (200 mg, 8.3 mmol) were
dissolved in dry DMF (20 mL), the mixture was then stirred at 0 ꢀC
for 1 h under argon protection. Then the reaction mixture was
warmed to room temperature and n-iodobutane (0.91 mL,
8.0 mmol) was added dropwise. The mixture was kept at 100 ꢀC
for 8 h before being allowed to cool room temperature. The mixture
was poured into ice water, the precipitate was collected and dried
under vacuum to give 4 as an orange solid (505 g, 46.28%) which
was used directly without further purification.
2.4.9. Synthesis and characterization of compound DPP-BI
From 7 (100 mg, 0.24 mmol) and 1-butyl-2,3,3-trimethyl-3H-
indolium (51.84 mg, 0.24 mmol), column chromatography eluent:
dichloromethane/ethanol (40:1, v/v) gave DPP-BI (94.24 mg, 64.8%
yield) as a dark-red solid. 1H NMR (400 MHz, DMSO-d6) (ppm): 1H
d
NMR (400 MHz, DMSO-d6) d (ppm): 8.55 (d, J = 15.5 Hz,1H), 8.45 (d,
J = 7.2 Hz, 2H), 8.07 (d, J = 7.6 Hz, 2H), 8.04 À 7.93 (m, 2H), 7.87 (dd,
J = 7.7 Hz, 3.9 Hz, 3H), 7.65 (d, J = 17.7 Hz, 5H), 4.78 (t, J = 8.4 Hz, 2H),
3.84 À 3.72 (m, 4H), 1.85 (s, 6H), 1.48 À 1.39 (m, 8H), 1.18 À 1.12 (m,
4H), 0.95 (t, J = 4.2 Hz, 3H), 0.77 (t, J = 8.4 Hz, 6H). 13C NMR
2.4.5. Synthesis of compound 5
From 2 (720.72 mg, 2.0 mmol) and n-iodobutane (0.91 mL,
8.0 mmol), the mixture was poured into ice water, the precipitate
was collected and dried under vacuum to give 5 as an orange solid
(380 mg, 40.25%) which was used directly without further
purification.
(100 MHz, DMSO-d6), d (ppm): 181.79, 161.53, 161.36, 148.96,
146.14, 144.17, 140.67, 136.59, 130.80,129.08, 128.95, 128.60, 127.55,
123.17, 115.69, 114.28, 110.02, 108.84, 52.52, 30.75, 30.64, 25.50,
19.27, 19.22,13.69, 13.37, 13.29. HRMS (ESI, m/z): [M+H]+ calcd. for
+
C
42H48N3O2 626.3747, found 626.3749.
2.4.6. Synthesis and characterization of compound 6
3. Results and discussion
Compound 4 (400 mg, 0.73 mmol), THF (20 mL), and HCl (2 M,
10 mL) were added into a two-necked flask, the reaction mixture
was stirred for 2 h at 60 ꢀC, then cooled to room temperature and
saturated brines (30 mL) was added, the organic layer was
collected and dried over Na2SO4. Removal of the solvent under
vacuum and the crude product was purified by column chroma-
tography (silica gel; DCM/ethyl acetate, 30/1, v/v) to provide the
corresponding pure product 6 (274 mg, 85.62%) as an orange solid.
3.1. Molecular design and synthesis
It is well known that
a,b-unsaturated indolium dyes possess
similar structure to acrylonitrile which could rapidly and
quantitatively response to HSO3À/SO3 [51,52], and this type of
2À
dye has been demonstrated to be inert to biothiols, such as Cys and
À
GSH [53–55]. Meanwhile, after nucleophilic addition of HSO3
/
1H NMR (400 MHz, CDCl3)
d
(ppm): 10.03 (s, 2H), 7.98 (d, J = 8.4 Hz,
SO3
toward the unsaturated bonds, p-conjugation and ICT
2À
4H), 7.92 (d, J = 8.4 Hz, 4H), 3.71 (t, J = 8.4 Hz, 4H), 1.52-1.46 (m, 4H),
process was broken and blocked, resulting in two well-separated
emission peaks which would enable the ratiometric sensing of
HSO3À/SO32À. In order to obtain the sensitive and selective
fluorescent probes with ratiometric and colorimetric for the
detection of SO2 derivatives, DPP-FI was designed and synthesized
(Scheme 2) in which the aldehyde group could affect the
1.24-1.15 (m, 4H), 0.77 (t, J = 7.3 Hz, 6H). 13C NMR (100 MHz, CDCl3),
d
(ppm): 190.21, 161.23, 146.60, 136.62, 132.25, 128.96, 128.26,
110.01, 40.73, 30.47,18.90,12.53. HRMS (ESI, m/z): [M+H]+ calcd. for
+
C
28H29N2O4 457.2127, found 457.2130.
À
2.4.7. Synthesis and characterization of compound 7
nucleophilic addition of HSO3 and the emission wavelength. As
From 5 (120 mg, 0.29 mmol) by treated with HCl, column
chromatography (eluent: DCM/ethyl acetate, 40/1, v/v) gave 9
(149.22 mg, 84.89% yield) as an orange solid. 1H NMR (400 MHz,
a contrast, DPP-BI without aldehyde group was also synthesized
(Scheme 2). For these two probes, the introduction of indolium
group would be beneficial to improve the water solubility of the

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J. Wang et al. / Journal of Photochemistry and Photobiology A: Chemistry 346 (2017) 265–272
Scheme 2. Synthesis of the target DPP-FI and DPP-BI.
molecule in some degree, additionally, the indolium moiety as a
mitochondria-targeted functional group could realize the cellular
organelle localization.
The synthesis of targets DPP-FI and DPP-BI are shown in
Scheme 2. In the first step, the two different DPP cores were
synthesized by one step reaction of cyclization to form (com-
pounds 2 and 3) from compound 1 and 4-(1,3-dioxolan-2-yl)
benzonitrile, respectively, then 1-iodobutane was attached to the
core of DPP with the catalysis of NaH to produce the alkyl DPP
of DPP-BI decreased and a new emission band at 535 nm
Ff = 0.27) developed, it is obvious that DPP-BI showed slight
(
blue shift of fluorescent emission both before and after react with
HSO3À, which may ascribe to the weaker electron-withdrawing
ability of hydrogen atom towards aldehyde group. The distinct
emission spectral shift (about 145 nm) of DPP-FI and DPP-BI
showed that they were favorable for ratiometric fluorescence
detection. In addition, the time-dependent fluorescence responses
were also detected with 10 equiv. of HSO3À from 0 to 8 min, and the
results showed that the reaction was almost completed within
4 min (Fig. S1).
intermediates
4 and 5. Then deprotection reactions were
conducted by treated with HCl to provide corresponding com-
pounds 6 and 7, which were finally react with indolium dyes to
produce the desirable compounds DPP-FI and DPP-BI according to
Knoevenagel reaction. All of the structures of new compounds
were well-characterized by 1H NMR, 13C NMR, and HRMS (ESI).
3.3. Selectivity towards different species
To investigate the selectivity of DPP-FI and DPP-BI towards
HSO3À/SO32À, the emission intensity ratio of DPP-FI and DPP-BI
À
À
3.2. Characterization of DPP-FI and DPP-BI for SO32À/HSO3
were measured toward various species. As shown in Fig. 2, HSO3
/
recognition
SO32À exclusively produced a dramatic increment in the emission
À
intensity ratio. The responsiveness of DPP-FI and DPP-BI to HSO3
/
UV-vis absorption spectra properties were studied firstly in
SO32À were approximately 21-fold versus other species. The other
nucleophilic anions (FÀ, ClÀ, BrÀ, IÀ, HCO3À, AcOÀ, NO3À, S2O3
,
2À
aqueous solution (PBS: DMSO = 7:3, pH = 7.4). The changes of the
À
absorption spectra in the absence and presence of HSO3 (0–
SO42À, HSO4À, PO43À, HPO42À, H2PO4À, Citrate, HSÀ), oxidizing
reagents (H2O2, ClOÀ) and three sulphur-containing amino acids
(Hcy, Cys, GSH) did not cause the ratiometric change. The emission
intensity ratio of these species were almost an order of magnitude
lower than that generated by HSO3À/SO32À, despite their 5-fold
concentrations versus HSO3À/SO32À. All above considered, DPP-FI/
DPP-BI showed outstanding selectivity towards HSO3À/SO32À over
other common biological species.
20equiv.) were displayed in Fig. 1. The probe DPP-FI showed an
absorption band at 520 nm (
e
= 1.43 Â104 MÀ1cmÀ1), but decreased
when HSO3À was added to the solution, while an absorption band
at 480 nm (
e
= 1.25 Â104 MÀ1cmÀ1) developed, which was visible
to the naked eye with a clear color change from pink to yellow
(Fig. 1a). Similar to DPP-FI, DPP-BI demonstrated same changes
that absorbance shifted from 505 nm (
e
= 2.10 Â104 MÀ1cmÀ1) tÀo
475 nm
(e
= 1.83 Â104 MÀ1cmÀ1
) after addition with HSO3
(Fig. 1d).
3.4. Effect of pH on the fluorescence of DPP-FI and DPP-BI
The emission spectra and fluorescence titration experiments of
À
probe DPP-FI and DPP-BI with HSO3 were then recorded in
In mitochondria, the internal environment pH was about
8.05Æ 0.11, so the probe should remain stable in slightly alkaline
environment with no fluorescence response. Firstly, we investigated
theinfluenceofpHonthefluorescentpropertiesofDPP-FI/DPP-BIin
a wide range of pH values in aqueous solution (PBS: DMSO = 7:3)
(Fig. 3). What we can see from the fluorescent ratio of DPP-FI/DPP-
BI is that these two probes exhibited a weak emission band with a
maximum at 545 nm and 535 nm respectively, thus increased
sharply after addition of HSO3À. Hence, the stable fluorescence of
aqueous solution (PBS: DMSO = 7:3, pH = 7.4) (Fig.1). The free probÀe
DPP-FI displayed weak red fluorescence. With titration of HSO3
from 0 to 20 equiv. to the solution, the probe was converted
efficiently to an additive product. And an obvious decrease in
fluorescence intensity at 720 nm
(Ff = 0.12) was observed;
Meanwhile, the fluorescence intensity of the emission band at
545 nm (Ff = 0.18) increased significantly. Compared with DPP-FI,
in the presence of HSO3À, the emission band at 670 nm (Ff = 0.43)

J. Wang et al. / Journal of Photochemistry and Photobiology A: Chemistry 346 (2017) 265–272
269
Fig. 1. UV-vis absorption spectra of (a) DPP-FI (10
m
M) and (d) DPP-BI (10
m
M) with different equiv. (from 0 to 20 eq.) HSO3À in DMSO-buffer solution (PBS: DMSO = 7:3, v/v,
À
pH = 7.4) at 37 ꢀC for 4 min. Insert: photograph showing the visual color of DPP-FI and DPP-BI when treated without (left) or with (right) HSO3 under visible light.
Fluorescence spectra of (b) DPP-FI (10 mM) and (e) DPP-BI (10 m
M) with different equiv. (from 0 to 10 eq.) HSO3À in DMSO-buffer solution (PBS: DMSO = 7:3, v/v, pH = 7.4) at
37 ꢀC for 4 min. lex = 480 nm and 475 nm respectively. Insert: photograph showing the visual fluorescence color of DPP-FI and DPP-BI when treated without (left) or with
(right) HSO3À under a 365 nm UV lamp. (c) Linear relationship between the fluorescence ratio (I545/I720) changes and the determination of the limit of detection (LOD) of DPP-
FI. (f) Linear relationship between the fluorescence ratio (I535/I670) changes and the determination of the limit of detection (LOD) of DPP-BI.
Fig. 2. (a) Fluorescence ratio (I545/I720) of DPP-FI (10 m
M) and (b) ratio variation of I535/I670 of DPP-BI in 10 equiv. HSO3À/SO32À and 50 equiv. various other additives in DMSO-
buffer solution (PBS: DMSO = 7:3, v/v, pH = 7.4). lex = 480 nm and 475 nm respectively.
DPP-FI and DPP-BI in the pH range 3.0–9.0 can provide its
application in monitoring intracellular SO2 derivatives in living
cells.
proton signals at 10.13 ppm was assigned to Ha in DPP-FI was
retained (Fig. 4 middle row). After addition of 5 equiv. HSO3À, the
proton signal of
d 10.13 ppm was shifted to 5.10 ppm attributed to
the nucleophilic addition to the formyl group, and the chemical
shift of Hb and Hc shifted to 5.00 and 4.92 ppm completely,
meanwhile, a new peak at 3.54 ppm (triplet, Hd’) appeared due to
the disappeared positively-charged of indolium group (Fig. 4 under
row). Furthermore, an MS titration was also performed to confirm
the reaction mechanism (Fig. S6 and S7). Similar results were also
obtained for DPP-BI according to its 1H NMR and MS titrations
(Fig. S2 and S8). All these results indicate that bisulfite ion attack
À
3.5. Detection mechanism in sensing HSO3
To further explain the reaction pathway, 1H NMR and MS
titrations were carried out in DMSO-d6. The partial 1H NMR spectrÀa
of DPP-FI before and after treatment with different equiv. of HSO3
is shown in Fig. 4. When 0.5 equiv. HSO3À (dissolved in D2O) were
added, the resonance signal corresponding to the vinylic proton Hb
at 8.55 ppm (doublet) and Hc at 7.87 ppm (doublet) significantly
decreased because of the nucleophilic attack of HSO3À, while the
the
a, b-unsaturated bond between DPP and indolium moiety first,
then nucleophilic addition to the extra formyl group if exist.

270
J. Wang et al. / Journal of Photochemistry and Photobiology A: Chemistry 346 (2017) 265–272
À
Fig. 3. (a) Fluorescence ratio (I545/I720) of DPP-FI (10 mM) and (b) ratio (I535/I670) of DPP-BI (10 mM) before (black squares) and after (red dots) the addition of 100 mM HSO3
for 4 min at different pH values. lex = 480 nm and 475 nm respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
with 50 m
M HSO3À. As shown in Fig. 5b, a clear mitochondria
profile with strong red fluorescence was observed from the red
channel, which was ascribed to Mito Tracker Red CMXRos. A
similar mitochondria profile with green fluorescence was obtained
after incubation with DPP-FI and HSO3À (Fig. 5a). From Fig. 5e, we
can see that the changes in the intensity profiles of linear regions of
interest (ROIs) (DPP-FI and Mito Tracker Red CMXRos co-staining)
were synchronous, and the fluorescence signals of the probe
overlaid well with those of MitoTracker Red CMXRos. Furthermore,
co-localization was quantified using the Pearson's co-localization
coefficient. The intensity of the correlation plot (Fig. 5f), which
described the distribution between the green channel (Fig. 5a) and
red channel (Fig. 5b), revealed a high co-localization coefficient
(0.84), Similar experiments were also conducted for DPP-BI, and it
showed same satisfactory results (Fig. S4), all these proved that
DPP-FI/DPP-BI was site specifically internalized in mitochondria
and it could be used to detect mitochondrial SO2 derivatives in
cancer cells.
Next, we attempted to apply DPP-FI/DPP-BI to ratiometric
fluorescence imaging of SO2 derivatives in vivo systems. As shown
in Fig. 6, we incubated 10 mM DPP-FI with HepG2 cells for 30 min
at 37 ꢀC in PBS, and confocal laser scanning microscopy was carried
out in living cells. On excitation at 480 nm, DPP-FI exhibited red
fluorescence in the red channel from 690 to 750 nm, but negligible
through green channel from 525 to 575 nm on excitation at
480 nm. After addition of the HSO3À to DPP-FI loaded HepG2 cells
for 30 min incubation, DPP-FI stained cells exhibited distinct
changes of ratiometric fluorescence responses generated from
green channel and red channel in living cells, thus leading to a
significant increase in fluorescence ratio (from 0.21 to 4.51, about
21-fold). For DPP-BI, similar experiments were also conducted,
and we obtained similar fluorescence changes in living cells
(Fig. S5). All these results confirmed that both DPP-FI and DPP-BI
could be used for ratiometric fluorescence imaging of mitochon-
drial SO2 derivatives in living cells.
Fig. 4. 1H NMR spectra titration of DPP-FI with different equivalent HSO3 in
DMSO-d6.
À
3.6. Intracellular detection
The cytotoxicity of two probes was evaluated by MTT with the
probe concentration at 10 m mM,
M and HSO3À concentration at 50
and the results were shown in Fig. S3. The results demonstrated
that DPP-FI and DPP-BI showed little cytotoxicity for long period
incubation and should be safe when used for imaging.
4. Conclusions
In summary, two new DPP-based derivatives (DPP-FI/DPP-BI)
for the imaging of mitochondrial SO2 derivatives in living cells have
been developed. The ratiometric and colormetric sensing is
To prove the cellular organelle localization of DPP-FI/DPP-BI
with a positively-charged of indolium moiety as a mitochondria-
targeted group, a commercially available mitochondrial dye (Mito
Tracker Red CMXRos), was employed for a co-localization study.
realized by broken the p-conjugation structure due to nucleophilic
attack of HSO3À, which were determined by 1H NMR and MS
titration analysis. Both probes could detect SO2 derivatives with
HepG2 cells were co-incubated with DPP-FI (10
mM) and Mito
Tracker Red CMXRos (500 nM) at 37 ꢀC for 30 min, then treated

J. Wang et al. / Journal of Photochemistry and Photobiology A: Chemistry 346 (2017) 265–272
271
Fig. 5. Confocal fluorescence images of HepG2 cells stained with (a) 10 m mM
M DPP-FI (green channel: lex = 480 nm, lem = 525–575 nm) at 37 ꢀC for 30 min then added 50
NaHSO3 solution for 30 min and (b) 500 nM MitoTracker Red CMXRos (red channel: lex = 579 nm, lem = 570–630 nm) at 37 ꢀC for 30 min; (c) Merged image of (a) and (b); (d)
Bright field image; (e) Intensity profiles of regions of interest (ROI) across HepG2 cells. Green lines represent the intensity of the probe DPP-FI treated with HSO3À and red
lines represent the intensity of MitoTracker Red CMXRos; (f) Correlationplot of MitoTracker Red CMXRos and DPP-FI intensities. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)
Fig. 6. (a) Fluorescence imaging of HepG2 cells incubated with DPP-FI (10
of (a). (d) Fluorescence intensity ratio signals of (a) and (b); (e) Fluorescence imaging of HepG2 cells incubated with DPP-FI (10
NaHSO3 (50 M) for 30 min from the green channel; (f) Fluorescence imaging of (e) from the red channel; (g) Bright field of (e). (h) Fluorescence intensity ratio signals of (e)
m
M) from the green channel; (b) Fluorescence imaging of (a) from the red channel; (c) Bright field
m
M) for 30 min and further incubated with
m
and (f). Green channel: lex = 480 nm, lem = 525–575 nm, Red channel: lex = 480 nm, lem = 690–750 nm (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
excellent sensitivity and selectivity. Probes DPP-FI/DPP-BI dis-
played a significant blue shift (about 145 nm) in its fluorescence
spectra in the presence of SO2 derivatives within few minutes
(4 min). More importantly, Co-staining experiments of DPP-FI/
DPP-BI and Mito Tracker Red CMXRos (co-localization coefficient:
0.84 and 0.81) revealed that DPP-FI/DPP-BI were predominantly
present in mitochondria, which is to our knowledge the first
example employing DPP probes DPP-FI/DPP-BI for imaging of
mitochondrial SO2 derivatives in HepG2 cells. Hence, we expect
this research paves a significant way for designing more DPP-based
colorimetric and ratiometric fluorescent probes with effective
sensing capability and specific organelle localization for biological
applications.
Acknowledgements
For financial support of this research, we thank NSFC/China
(21372082, 21421004 and 21572062), the National Basic Research
973 Program (2013CB733700), the Fundamental Research Funds
for the Central Universities (222201717003) and the Programme of
Introducing Talents of Discipline to Universities (B16017).

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J. Wang et al. / Journal of Photochemistry and Photobiology A: Chemistry 346 (2017) 265–272
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