A series of water-soluble A-π-A′ typological indolium derivatives with two-photon properties for rapidly detecting HSO3-/SO32- in living cells

Article abstract of DOI:10.1039/c7tb00726d

It is believed that HSO₃^- and SO₃^2- play important roles in several physiological processes. However, probes with two-photon absorption to detect HSO₃^- or SO₃^2- in living cells are still limited. Herein, a series of novel indolium derivatives (L1-L4) with an A-π-A′ structure was designed and synthesized as ratiometric probes to detect HSO₃^-/SO₃^2-in vitro. L3 and L4 display a colorimetric and ratiometric fluorescence dual response to HSO₃^-/SO₃^2- with a very fast (～15 s) and high specificity, as well as low detection limits (～22 nM). Furthermore, their detection is also carried out by using a two-photon excited fluorescence method. A nucleophilic addition reaction is proposed for the sensing mechanism, which is supported by MS, ¹H NMR, and density functional theory (DFT) investigations. Importantly, L3 was successfully used for detecting intrinsically generated intracellular HSO₃^-/SO₃^2- in cancerous cells under one- and two-photon excited fluorescence imaging.

Full text of DOI:10.1039/c7tb00726d

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DOI: 10.1039/C7TB00726D
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ARTICLE
A series of waterꢀsoluble AꢀπꢀA′ typological indolium derivatives
ꢀ
2ꢀ
with twoꢀphoton properties for rapidly detecting HSO3 /SO3 in
living cells
Received 00th January 20xx,
Accepted 00th January 20xx
‡
‡
Yingzhong Zhua , Wei Dua , Mingzhu Zhanga, Ying Xua, Leilei Songa, Qiong Zhanga*, Xiaohe
DOI: 10.1039/x0xx00000x
Tianb, Hongping Zhoua, Jieying Wua, Yupeng Tiana, c*
www.rsc.org/
It is believed that HSO3ꢀ and SO32ꢀ play important roles in several physiological processes. However, the probes with twoꢀ
photon absorption to detect HSO3ꢀ or SO32ꢀ in living cells are still limited. Herein, a series of novel indolium derivatives (L1ꢀ
ꢀ
2ꢀ
L4) with AꢀπꢀA′ model were designed and synthesized as ratiometric probes to detect HSO3 /SO3 in vitro. L3 and L4
ꢀ
display colorimetric and ratiometric fluorescence dual response to HSO3 /SO32ꢀ with very fast (~15 s) and high specificity, as
well as low detection limits (~22 nM). Furthermore, their detection is also carried out by using twoꢀphoton excited
fluorescence method. A nucleophilic addition reaction is proposed for the sensing mechanism, which is supported by MS,
1H NMR, and density function theory (DFT) investigation. Importantly, L3 is successfully used for detecting intrinsically
ꢀ
generated intracellular HSO3 /SO32ꢀ in cancerous cells under oneꢀ and twoꢀphoton excited fluorescence imaging.
Fluorescent technique offers a powerful tool for realꢀtime
sensing and visualizing biological species due to its nonꢀ
invasiveness, high sensitivity, temporal, and spatial resolution.17ꢀ23
Introduction
Sulfur oxide (SO2) is usually regarded as an air pollutant with
the potential to form acid rain.1,2 Actually, with the combustion of
fossil fuels, SO2 has become increasingly widespread and been
involved in the vital process of human. Inhaled SO2 will be
Recently, numerous of fluorescent probes for the detection and
ꢀ
2ꢀ
visualization of HSO3 /SO3 in living cells have been reported,
which based on nucleophilic reaction with aldehyde, the selective
deprotection of levulinate, Michaelꢀtype additions, and coordinative
interactions.24ꢀ26 However, drawbacks still remained including poor
waterꢀsolubility, unsatisfactory detection limit, long response time
(30 min to 10 h), and interference of biothiols.27 Besides, their
fluorescent spectra were easily influenced by environmental
conditions, probe distribution, and instrumental efficiency.14,28 All
2ꢀ
dissolved in the body liquid and produce its derivatives sulfite (SO3
ꢀ
) and bisulfite (HSO3 ) (3:1 M/M, in neutral fluid), which is mainly
responsible for its toxicity.3ꢀ5 Clinical investigation confirmed that
ꢀ
HSO3 /SO32ꢀ can not only cause respiratory disease but also result in
cardiovascular diseases, as well as many neurological disorders, such
as migraine headaches, stroke, and brain cancer.6ꢀ8 Research have
indicated that the endogenous HSO3 /SO3 could also be generated
ꢀ
these factors limit their further application in monitoring HSO3
/SO32ꢀ in vivo and in vitro.
ꢀ
2ꢀ
in biological metabolism.9 For instance, the HSO3 /SO3 could be
endogenously produced enzymatically in cytosols and mitochondria
of cells by oxidation of hydrogen sulfite, sulphurꢀcontaining amino
acids, and decomposition of sulfinylpyruvate to pyruvate in
ꢀ
2ꢀ
According to the literature reported before (Table S1),7,12,14,27,29ꢀ
the benzoindolium derivatives used for detecting HSO3 /SO3
35
ꢀ
2ꢀ
share similar features as below. i) The molecules were generally
designed with DꢀπꢀA model. ii) The investigation was only focus on
detection. iii) Most of the detection time would complete way over
~40 s and the mechanisms were studied by experimental methods. In
regard to these features, we designed and synthesized a series of new
mammals.10,11 Of note, it is well established that endogenous HSO3
/SO32ꢀ, like other gaseous signal molecules (NO, CO, and H2S), do
ꢀ
play
a
positive role in physiological process, including
vasorelaxation, antihypertensive effect, inhibition of vascular smooth
muscle, and regulation of cardiac channel function.12ꢀ16 Therefore,
quantitative detection, especially in situ detection of HSO3 /SO3 in
cells is of significant interest.
ꢀ
indolium derivatives to detect HSO3 /SO32ꢀ. The molecules adopted
ꢀ
2ꢀ
AꢀπꢀA′ model which might make the nucleophilic addition easier to
occur. The systemic terminal group effects, photophysical property
and the relationships between structure and properties were
discussed in detail. These compounds presented selective, sensitive,
ꢀ
2ꢀ
fast and eyeꢀnaked response to HSO3 /SO3
over other
physiologically anions and biothiols. More importantly, the detection
could be realized by ratiometric as well as twoꢀphoton in dualꢀ
methods, and the detection mechanisms were studied by
experimentally and theoretically. To our best knowledge, the
molecules adopted AꢀπꢀA′ model for ratiometric and twoꢀphoton
ꢀ
dualꢀresponse HSO3 /SO32ꢀ probes were still rarely existed.
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emission energies were investigated by timeꢀdependent density
functional theory (TDꢀDFT). All calculations were performed by the
use of the Gauss 09 suite of programs.
Experimental section
Measurements and Methods
Cell culture
All chemicals and solvents were dried and purified by standard
methods. IR spectra (4000ꢀ400 cmꢀ1), as KBr pellets, were recorded
on a Nicolet FTꢀIR 870 SX spectrophotometer. The 1HꢀNMR and
13CꢀNMR spectra recorded on at 25 °C using Bruker 400 Ultrashield
spectrometer were reported as parts per million (ppm) from TMS
(d). ESIꢀMS mass spectra were recorded using Bruker Autoflex III
Smartbeam. Elemental analyses were carried out on a PerkineElmer
240 analyzer. Melting point is measured on FP62 instruments. The
UVꢀvisible absorption spectra of dilute solutions (10 ꢀM) were
HepG2 and Hela cells were chosen to culture in Dulbecco’s
modified Eagle’s medium supplemented with penicillin/
streptomycin and 10 % bovine calfꢀserum in a 5 % CO2 incubator at
37 °C. L3, L4, and commercial dyes were prepared in phosphate
buffered saline (PBS) buffer with concentration of 10 ꢀM.
Synthesis
The synthetic routes for the four indolium derivatives were
recorded using a SHIMADZU UVꢀ3600 spectrophotometer using a shown in Scheme S1, and the molecular structures were presented in
Scheme 1.
quartz cuvette with a 1 cm path length. Oneꢀphoton fluorescence
spectra were obtained using a HITACHI Fꢀ7000 spectrofluorimeter
Synthesis of intermediate
equipped with
a 450 W Xe lamp. When the fluorescence
The intermediate was synthesized according to the literature
reported before.9 2,3,3ꢀtrimethylꢀ3Hꢀindole 1.59 g (10 mmol) mixed
with CH3I 2.82 g (20 mmol) and stirred at roomꢀtemperature for 24
h. After the reaction, white solid would achieved by filtering. O1,
2.75 g, Yield: 91 %.
measurements were taken, the concentration of samples was 10 ꢀM
with quartz cuvette (path length = 1 cm). In the measurements of
emission spectra, the slit width was 5 nm. The exciting voltage of
emission spectrum was 400 V. Timeꢀresolved fluorescence
measurements were recorded on HORIBA HuoroMaxꢀ4P. Twoꢀ
photon excited fluorescence was measured using a SpectroPro300i
and the pump laser beam came from a modelocked Ti:sapphire laser
system at the pulse duration of 140 fs and a repetition rate of 80
MHz (Coherent Mira900ꢀD). Cell survival was assayed using the
cell proliferation Kit I with the absorbance at 570 nm detected using
a PerkinꢀElmer Victor plate reader. Confocal fluorescence imaging
was obtained with an LSM 710 (Zeiss) confocal laser scanning
microscope.
The synthetic rout for 1, 1, 2ꢀtrimethylꢀ1Hꢀbenz[e]indole was
similar to that of O1. Hoar solid, O2, 3.30 g, Yield: 94 %.
Synthesis of L1
O1 3.01g (10 mmol) and 4ꢀPyridinecarboxaldehyde 1.07g (10
mmol) were dissolved in 30 mL ethanol. Then, two drops of
hexahydropyridine were added to the solution. After that, the
mixture was heated to 60 °C and stirred for 6 h. Red solid was
obtained after the reaction solution cold to roomꢀtemperature. L1,
3.20 g. Yield: 82%. Melting point: 218 – 221 °C. 1H NMR (400
MHz, d6ꢀDMSO) δ 8.79 (d, J = 5.9 Hz, 2H), 8.37 (d, J = 16.6 Hz,
1H), 8.07 (d, J = 6.0 Hz, 2H), 7.97 – 7.91 (m, 2H), 7.88 – 7.82 (m,
2H), 7.76 – 7.61 (m, 2H), 4.27 (s, 3H), 1.84 (s, 92H), 1.84 (s, 6H).
13C NMR (100 MHz, d6ꢀDMSO) δ 173.1, 149.7, 148.0, 144.5, 141.7,
137.9, 128.5, 126.2, 125.7, 120.7, 119, 54.6, 32.6, 27.3. ESIꢀMS:
263.1542. ESIꢀMS (m/z) Calculated: 263.1543; Found: 263.1542. IR
(KBr, cmꢀ1): 3400, 3033, 2974, 1618, 1595, 1556, 1476, 1459, 1415,
1371, 1240, 807, 761, 708, 514. Anal. Calcd. for C18H19N2I: C,
55.40; H, 4.91; N,7.18. Found: C, 55.46; H, 4.94; N, 7.15.
Crystal analysis and TDꢀDFT calculation
The Xꢀray diffraction measurements were performed on a
Bruker SMART CCD area detector using graphite monochromated
MoꢀKα radiation (λ = 0.71069 Å) at 298(2) K. Intensity data were
collected in the variable ωꢀscan mode. The structures were solved by
direct methods and difference Fourier transformations. The nonꢀ
hydrogen atoms were refined anisotropically and hydrogen atoms
were introduced geometrically. Calculations were performed with
SHELXTLꢀ97 program package.
The ground states for each molecule were calculated using the
density functional theory level with the B3LYP functional
employing a 6ꢀ31G* basis set. No symmetry or internal coordinate
constraints were applied during optimization. The absorption and
Synthesis of L2
The synthetic rout of L2 was similar to that of L1. Red solid, L2,
3.90 g. Yield: 89 %. Melting point: 230 ꢀ 233 °C. 1H NMR (400
MHz, d6ꢀDMSO) δ 9.15 (d, , J = 4.4 Hz, 1H), 8.98 (d, J = 16.3 Hz,
1H), 8.44 (d, J = 8.3 Hz, 1H), 8.24 (d, J = 4.4, 1H), 8.18 (d, J = 8.4,
1H), 8.04 – 7.90 (m, 4H), 7.83 (t, J = 7.5 Hz, 1H), 7.75 – 7.70 (m,
2H), 4.28 (s, 7H), 4.28 (s, 3H), 1.88 (s, 6H). 13C NMR (100 MHz,
d6ꢀDMSO) δ 173.1, 150.0, 149.5, 148.0, 146.7, 141.7, 137.9, 129.6,
129.5, 128.5, 127.0, 126.2, 125.7, 124.0, 119.2, 113.0, 52.9, 32.6,
27.3. ESIꢀMS (m/z) Calculated: 313.1699; Found: 313.1699. IR
(KBr, cmꢀ1): 3414, 3047, 2973, 2925, 1612, 1563, 1505, 1460, 1420,
1350, 1303, 1247, 954, 844, 751, 532. Anal. Calcd. for C22H21N2I:
C, 60.01; H, 4.81; N, 6.36. Found: C, 60.10; H, 4.78; N, 6.39.
Synthesis of L3
Scheme 1. The molecular structures of L1
2 | J. Name., 2012, 00, 1-3
ꢀL4.
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The synthetic rout of L3 was similar to that of L1. L3 was red density of the reactive site of the alkenylꢀbridge, leading to easier for
1
the nucleophilic addition. The designed molecular structures of L1ꢀ
L4 were shown in Scheme 1, and the corresponding synthetic routes
were presented in Scheme S1. As best known, the trend of electronꢀ
withdrawing ability was indolium > benzoindolium > pyridine>
solid, 3.80 g. Yield: 86 %. Melting point: 215 – 218 °C. H NMR
(400 MHz, d6ꢀDMSO) δ 8.84 (d, J = 5.9 Hz, 2H), 8.46 (dd, J = 16.8,
12.6 Hz, 2H), 8.35 (d, J = 9.0 Hz, 1H), 8.26 (d, J = 8.1 Hz, 1H), 8.18
(d, J = 9.0 Hz, 1H), 8.13 (d, J = 6.0 Hz, 1H), 7.96 (d, J = 16.7 Hz,
1H), 7.85 (dd, J = 11.3, 4.1 Hz, 1H), 7.78 (t, J = 7.5 Hz, 1H), 4.40 – chinoline moiety. Hence, the indolium and benzoindolium were of
4.37 (s, 3H), 2.03 (s, 6H). 13C NMR (100 MHz, d6ꢀDMSO) δ 182.9, the acceptor “A”, while the pyridine and chinoline groups were of
151.2, 148.7, 141.8, 139.9, 139.4, 134.0, 131.5, 130.5, 129.0, 128.1,
127.0, 123.9, 123.3, 117.4, 114.1, 54.8, 36.2, 24.9. ESIꢀMS (m/z)
Calculated: 313.1699; Found: 313.1698. IR (KBr, cmꢀ1): 3414, 2996,
1616, 1595, 1550, 1465, 1417, 1386, 1350, 11319, 1236, 1213, 975,
806, 763, 669, 524. Anal. Calcd. for C22H21N2I: C, 60.01; H, 4.81;
N, 6.36. Found: C, 59.96; H, 4.77; N, 6.39.
the acceptor “A′”.
Crystal structure of L1 and L2
The single crystals of L1 and L2 suitable for Xꢀray diffraction
analysis were obtained by slow inꢀdiffusion from isopropanol to
ethanol at roomꢀtemperature for five days. Crystal data collection
and refinement parameters were summarized in Table S2 and S3.
The crystal structures of L1ꢀL2 were shown in Figure 1.
Synthesis of L4
The synthetic rout of L4 was similar to that of L1. Red solid, L4,
L1 belonged to orthorhombic system with Cmc21 space group,
while L2 crystalized in monoclinic system with P21/c space group.
With regard to L1, the pyridine and indoium moieties were almost in
a coplanar (Figure 1C directly displayed the planarity of L1, the
dihedral angle between P1 and P2 was < 2.0°). In contrary, the
introduction of chinoline ring would alter the planarity of the
molecule (L2 in Figure 1D), and the dihedral angle between P3
(chinoline moiety) and P4 (indolium moiety) was 7.12°, which might
lead to changes in optical properties. In addition, the main CꢀC/CꢀN
bond distance was located between normal CꢀC/CꢀN single bond and
C=C/C=N double bond length (especially C8ꢀN2, N2ꢀC11 in L1;
and C12ꢀN2, N2ꢀC15 in L2), indicating high πꢀelectron
delocalization within the molecules, which would be in favour of
their good nonlinear optical properties.
1
4.10 g. Yield: 84 %. Melting point: 222 – 225 °C. H NMR (400
MHz, d6ꢀDMSO) δ 9.17 (d, J = 4.5 Hz, 1H), 9.04 (d, J = 16.4 Hz,
1H), 8.49 (dd, J = 8.2, 3.5 Hz, 2H), 8.38 (d, J = 9.0 Hz, 1H), 8.33 –
8.15 (m, 4H), 8.08 – 7.74 (m, 5H), 4.43 (s, 3H), 2.10 (s, 6H). 13C
NMR (100 MHz, d6ꢀDMSO) δ 173.1, 150.0, 149.5, 146.7, 137.9,
134, 133.5, 132.6, 129.6, 129.5, 128.6, 126.5, 125.8, 125.6, 124.2,
118.7, 113.0, 52.9, 32.6, 27.7. ESIꢀMS (m/z) Calculated: 363.1856;
Found: 363.1852. IR (KBr, cmꢀ1): 3424, 3062, 2988, 1613, 1560,
1504, 1462, 1396, 1354, 1248, 1211, 955, 876, 815, 792, 756, 526.
Anal. Calcd. for C26H23N2I: C, 63.68; H, 4.73; N,5.71. Found: C,
63.72; H, 4.76; N, 5.67.
Results and Discussion
Design and synthesis
UVꢀvis absorption spectra
Indolium derivative, with AꢀπꢀD model, were considered as
The absorption spectra of indolium derivatives (L1ꢀL4) in
different solvents were shown in Figure S1, and the corresponding
spectroscopic data were listed in Table 1 and S4.
ꢀ
2ꢀ 1,4,9,14
good ratiometric fluorescence probes to detect HSO3 /SO3
.
ꢀ
2ꢀ
During this detecting process, HSO3 /SO3 were utilized as
nucleophilic reagents to attack the alkenylꢀbridge of the molecules.
Hence, the key point was the electron density of alkenylꢀbridge. The
“donor group” was replaced by pyridine and chinoline moieties as
acceptor groups, which could significantly decrease the electron
As shown in Figure 1, the UVꢀvis absorption spectra of L1ꢀL4
displayed two kinds of bands between 275 nm and 500 nm. The low
energy bands could be ascribed as the intramolecular charge transfer
(ICT) transition, while the high energy bands ranging from 290ꢀ300
nm could be assigned as πꢀπ* transition of the indolium moiety.36
Interestingly, all the low energy bands located at about 360 nm with
slight changes from L1 to L4, revealing that the terminal moiety had
little effect on absorption bands of these AꢀπꢀA′ typological
indolium derivatives. Meanwhile, the experimental data also
confirmed the affection of solvents. As shown in Figure S1, the
Figure 1. Crystal structures of compounds L1 (A) and L2 (B) with
thermal ellipsoids at 50% probability (H atoms and anions have been
omitted for clarity). The illustrative diagrams of planarity for L1 (C)
and L2 (D).
Figure 2. The UVꢀvis absorption spectra (A) and fluorescence
emission spectra (B) of L1ꢀL4 in tetrahydrofuran with concentration
10 ꢀM.
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