A coumarin-quinolinium-based fluorescent probe for ratiometric sensing of sulfite in living cells

Article abstract of DOI:10.1039/c4ob00132j

Based on a novel coumarin-quinolinium platform, probe 2 was rationally designed and synthesized as a novel ratiometric fluorescent sensor for sulfite anions. The probe exhibited a wide dynamic concentration range for sulfite anions in a PBS buffer (containing 1 mg mL^-1 BSA). More importantly, the probe was suitable for ratiometric fluorescence imaging in living cells with high sensitivity, favorable selectivity, and minimal cytotoxicity. This journal is

Full text of DOI:10.1039/c4ob00132j

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Biomolecular Chemistry
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A coumarin-quinolinium-based fluorescent probe
for ratiometric sensing of sulfite in living cells†
Cite this: Org. Biomol. Chem., 2014,
12, 4637
Li Tan, Weiying Lin,* Sasa Zhu, Lin Yuan and Kaibo Zheng
Based on a novel coumarin-quinolinium platform, probe 2 was rationally designed and synthesized as a
novel ratiometric fluorescent sensor for sulfite anions. The probe exhibited a wide dynamic concentration
range for sulfite anions in a PBS buffer (containing 1 mg mL−1 BSA). More importantly, the probe was suit-
able for ratiometric fluorescence imaging in living cells with high sensitivity, favorable selectivity, and
minimal cytotoxicity.
Received 18th January 2014,
Accepted 2nd May 2014
DOI: 10.1039/c4ob00132j
www.rsc.org/obc
Additives has regulated an acceptable daily intake of sulfite to
be lower than 0.7 mg kg−1 of body weight.10 So far, several ana-
Introduction
With the development of industries, sulfur dioxide (SO2) is an lytical techniques including the official Monier-Williams
inevitable product of the combustion of coal and fossil fuels methods,11 electrochemistry,12 spectrophotometry,13 chromato-
and has had a detrimental effect on the environment. Numer- graphy,14 capillary electrophoresis,15 and titration16 have
ous epidemiological studies have implied that frequent been developed to detect sulfites in food and beverages.
exposure to SO2 not only induces many respiratory responses,1 However, these methods usually require tedious sample and
but is also related to lung cancer, cardiovascular diseases, and reagent preparation or complicated instruments, and therefore
neurological disorders, such as migraine headaches, strokes, are not suitable for routine analysis. As an alternative, fluo-
and brain cancer.2 Furthermore, when SO2 is dissolved in solu- rescence sensing is appealing because of its high sensitivity,
tion or after inhalation, it is hydrated and an equilibrium is excellent selectivity, and simplicity. Furthermore, fluorescence
formed between two anions: sulfite and bisulfite. Sulphite, sensing is potentially applicable for bioimaging in living cells
because of its antioxidant and preservative properties, is com- and offers temporal and spatial resolution. Basically, mole-
monly used to prevent food from browning, discourage bac- cular fluorescence probes detecting biological species are
terial growth in wines, and maintain the stability and potency based on the unique increase or decrease of the emission
of some medications.3 However, exposure to high doses of intensity signal. A major limitation of intensity-based probes
sulfite may cause adverse reactions and acute symptoms, is that variations in the sample environment and probe distri-
including dermatitis, urticaria, flushing, hypotension, abdomi- bution may be problematic for utilization in quantitative
nal pain, and diarrhoea.4 In fact, there is evidence that some measurements.17 By contrast, ratiometric fluorescent probes
people may be extremely sensitive to sulfite even at very low allow the measurement of emission intensities at two different
levels,5 and that bronchoconstriction can occur in many asth- wavelengths, which should provide a built-in correction for
matic patients.6 Besides, toxicological studies also suggest that environmental effects and can also increase the dynamic
SO2 and its derivatives could change the characteristics of range of fluorescence measurements.18
voltage-gated sodium channels and potassium channels in rat
Up to now, several fluorescent probes for sulfites have been
hippocampal neurons7 and affect thiol levels, which can break designed on the basis of the selective deprotection of a levuli-
the redox balance in cells8 and produce a neuronal insult.9 nate group,19 complexation with amines,20 selective reaction
In view of the seriousness of the consequences of sulphite with aldehyde21 and Michael-type additions.22 However, most
exposure, the Joint FAO/WHO Expert Committee on Food of these fluorescent probes respond to sulfites with changes
only in fluorescent intensity; thus, it is still a challenge to
develop ratiometric fluorescent probes for sulfite. On the other
hand, coumarins, to the best of our knowledge, are a classic
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry
type of a push–pull dye, in which the intramolecular charge
transfer (ICT) process from the electron donor to the acceptor
and Chemical Engineering, Hunan University, Changsha, Hunan 410082,
P. R. China. E-mail: weiyinglin2013@163.com; Fax: (+)86-0731-88821464
proceeds upon excitation. Typically, for efficient ICT, the donor
†Electronic supplementary information (ESI) available: Experimental proce-
and acceptor are located in the 7- and 3-position, respect-
dures,
characterization
data, and additional spectra. See DOI:
10.1039/c4ob00132j
ively.23 We envisioned that diethylamino moieties at the
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Scheme 1 Design and synthesis of probe 2 as a new ratiometric fluore-
scent sulphite probe.
7-position and quinolyl moieties at the 3-position would
Fig. 1 Fluorescence spectra of probe 2 (5 μM) in PBS buffer (pH 7.4,
containing 1 mg mL−1 BSA) in the presence of SO32− (0–200 equiv.) with
excitation at 450 nm. Inset: fluorescence intensity ratio (I508/I610) of
probe 2 changes (5.0 μM) with the amount of Na2SO3.
be
a desirable coumarin push–pull system for an ICT
process. Therefore, we designed and synthesized probe 2
(Scheme 1) as a novel ratiometric fluorescent sulfite probe
based on a coumarin-quinolinium platform.
Results and discussion
Probe 2 was readily synthesized in two steps (Scheme 1). A
mixture of 2-methylquinoline with ethyl iodide yielded N-ethyl-
2-methylquinolinium iodide 1 in 87% yield,24 which was then
condensed with 7-(diethylamino)-2-oxo-2H-chromene-3-carb-
aldehyde in anhydrous ethanol to give target probe 2. The
structures of the synthesized compounds were fully character-
ized by standard NMR and mass spectrometry.
With probe 2, we evaluated its spectral properties in the
absence or presence of sulfite in an aqueous buffer (pH 7.4,
25 mM PBS buffer with 30% ethanol) (Fig. S1†). The response
of probe 2 towards sulfite was very good. Considering its appli-
cation in living systems, we checked the spectral response of
probe 2 (5 μM) to sulphite in pure PBS buffer at ambient temp-
erature (Fig. S2†). The results showed that probe 2 had weak
fluorescent intensity, which may be due to its limited solubility.
Fig. 2 Plot of the fluorescent ratio (I508/I610) of probe 2 in PBS buffer
(pH 7.4, containing 1 mg mL−1 BSA) as a function of the sulfite concen-
tration (0–200 equiv.).
Therefore, we added bovine serum albumin (BSA at 1 mg mL−1
)
in a PBS buffer solution.25 As displayed in Fig. S3,† the useful for the quantitative determination of sulfite concen-
free ratiometric probe 2 exhibited a main absorption band at trations in a large dynamic range and has high sensitivity in
around 515 nm, and almost no absorption appeared at living systems.
410 nm, consistent with the ICT signalling mechanism.
To shed light on the SO32−-induced fluorescence ratio-
2−
However, the addition of SO3 induced a large blue shift in metric response, we decided to characterize the addition
the absorption peak, indicating the formation of a new species product and carried out theoretical calculations. First, the
upon the treatment of probe 2 with SO32−. In good agreement product of probe 2 + Na2SO3 was isolated and then subjected
with the absorption findings, the probe exhibited a ratiometric to NMR analysis. The partial 1HNMR spectra of probe 2 and
fluorescent response to the SO32− anion. We chose the isosbes- the isolated compound 3 are shown in Fig. 3. The resonance
tic point (450 nm) as the optimal excitation wavelength. Upon signal corresponding to the alkene proton H2b at 8.52 ppm
excitation at 450 nm, the free sensor displayed an intense (doublet) disappeared, and a new peak at 4.43 ppm (triplet)
emission band at 610 nm. However, as shown in Fig. 1, fluo- assigned to the proton H3b emerged. The addition of sulfite to
rescent ratios of probe 2 at 508 and 610 nm (I508/I610) showed a CvC resulted in the formation of a chiral center of C3b, with
2−
large change from 0.34 to 4.81 upon treatment with the SO3
the nonequivalent protons of the methylene group at C3a.
anion. The emission ratios (I481/I659) were linearly proportional Therefore, the signal for H2a at 8.17 ppm (doublet) shifted to a
to the amount of sulfite (0.15–75 μM) (Fig. 2) with a detection higher field and appeared as two peaks at 3.77 ppm (dd) and
limit (S/N = 3) of 8.9 × 10−8 M in PBS buffer (pH 7.4, contain- 4.08 ppm (dd), respectively. At the same time, the resonance
ing 1 mg mL−1 BSA), suggesting that the probe is potentially signal of H2c at 8.47 ppm shifted to 8.275 ppm, due to the
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Fig. 3 Partial 1H NMR spectra of probe 2 and isolated compound 3 in DMSO-d6.
Fig. 4 DFT optimized structures of 2 and 3. In the ball-and-stick
representation, carbon, nitrogen, oxygen, and sulphur atoms are colored
in gray, blue, red and yellow, respectively.
shielding effect of the adjacent alkyl group. The results are in
good agreement with that of the reported paper.22 To better
understand the mechanism of probe 2’s response to sulfite,
density functional theory (DFT) and time-dependent DFT
(TD-DFT) by the Gaussian 09 program were employed to
explain the distinctions in the spectral profiles of probe 2 and
compound 3 (in water solvent at the B3LYP/6-31+G(d) (PCM)
level). The optimized structures in the ground states of 2 and 3
are shown in Fig. 4. The quinolyl and coumarinyl moieties of 2
were well coplanar via a conjugated bridge (–CvC–), and the di-
hedral angle between the quinolyl and coumarinyl moieties
was less than 1° (Fig. 4). The addition of sulfite to the double
bond of the conjugated bridge inhibited the addition of quino-
lyl and coumarinyl moieties, and the dihedral angle for reac-
tion product 3 was 84° (Fig. 4). This structural difference
between 2 and 3 showed the significant difference in π-con-
junction between 2 and 3. We then further compared the
highest occupied molecular orbital (HOMO) and the lowest
unoccupied molecular orbital (LUMO) of probe 2 and com-
pound 3 in the ground and excited states. In the case of free
probe 2, the electronic transition was mainly contributed by a
Fig. 5 Frontier molecular orbital plots of 2 (a) and 3 (b) in water (CPCM
model) involved in the vertical excitation (i.e., UV/Vis absorption, left
column) and emission (right column). The vertical-excitation-related
calculations are based on the optimized geometry of the ground state
(S0), and the emission-related calculations are based on the optimized
geometry of the excited state (S1). Excitation and radiative processes are
marked as solid lines, and the non-radiative processes are marked by
dotted lines.
HOMO–LUMO transition. The π electrons on the LUMO of ground state were mainly located on the π-conjugated cou-
probe 2 were mainly located on the quinolyl moiety, but the marin framework (Fig. 5b). Thus, the ICT process of com-
HOMO was mostly positioned at the coumarin unit (Fig. 5a). pound 3 was weaker than that of probe 2, which could lead to
Thus, an ICT took place through the conjugated bridge a blue shift in the absorption and fluorescence spectra.
between the coumarin and quinolinium groups. In addition, TDDFT calculations indicated that compound 3 showed the
TDDFT calculations indicated that probe 2 showed the absorp- absorption peak at 377 nm ( f = 0.6475) and a weak fluo-
tion peak at 534 nm ( f = 1.3175) and a high fluorescence band rescence band at 436 nm ( f = 0.0029), which was in good
at 588 nm ( f = 0.9409). However, the electronic transition of 3 agreement with the observation that the measured absorption
was mainly contributed by the HOMO–LUMO+1 transition. and emission wavelengths of compound 3 were shorter
The π electrons on both the HOMO and LUMO+1 in the than those of probe 2. Furthermore, the π electrons on the
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Fig. 6 Reaction time profiles of probe 2 (5.0 μM) in the absence [ ] or
presence of 20 equiv. of Na2SO3 [ ]). The fluorescent intensities ratio
I508/I610 were continuously monitored at time intervals in PBS buffer (pH
7.4, containing 1 mg mL−1 BSA). Time points represent 0, 0.5, 1, 1.5, 2, 3,
4, 6, 8, 10, 12 and 15 min.
HOMO/LUMO+1 of compound 3 were mainly distributed on
coumarin moieties, while those on the LUMO were located on
the quinoline moiety (Fig. S4†). Thus, upon the excitation of
compound 3, a process of d-PET involving the transfer of one
electron from the excited coumarin moieties to the quinoline
moieties leads to the fluorescence of compound 3 being
partially quenched, which explained the observation that the
measured emission intensity of compound 3 was weaker than
that of probe 2.
Fig. 7 Fluorescent spectra (a) and ratio I508/I610 (b) of probe 2 (5.0 μM)
to various biologically relevant species in PBS buffer (pH 7.4, containing
1 mg mL−1 BSA). Red bars represent the addition of the excess of rep-
resentative species. 1. Blank, 2. CH3COO−, 3. I−, 4. Br−, 5. Cl−, 6. cys,
7. N3−, 8. NO2−, 9. H2PO4−, 10. F−, 11. NO3−, 12. SO42−, 13. SCN−, 14.
S2O32−, 15. SO32−, 16. S2−, 17. Vc, 18. CO32−, 19. GSH.
The time profile of the fluorescence response of probe 2
2−
(5.0 μM) in the presence of SO3 (20 equiv.) in pH 7.4 PBS
buffer (containing 1 mg mL−1 BSA) are displayed in Fig. S5,† SO32− probes. In addition, we further examined the absorption
showing that the response of probe 2 to sulfite was very quick. and fluorescence response of the probe toward Na2SO3 in the
Fig. 6 shows the fluorescence intensity ratio I508/I610 of probe 2 presence of other potentially competing species. The other species
2−
towards SO3 (20 equiv.) at time intervals from 0 to 15 min, only displayed minimum interference (Fig. S7†). This suggests
with the pseudo-first-order kinetics constant calculated as that probe 2 is potentially useful for sensing Na2SO3 in the
kobs = 0.128 min−1 (Fig. S6†).
presence of other related species in pH 7.4 PBS buffer
To investigate the selectivity, probe 2 (5.0 μM) was treated (containing 1 mg mL−1 BSA).
with various biologically relevant species, including CH3COO−,
We envision that the desirable water solubility, high sensi-
I−, Br−, Cl−, F−,CO32−, N3−, NO2−, H2PO4−, NO3−, SO42−, SCN−, tivity, and favorable selectivity of probe 2 should benefit the
2−
S2O32−, S2−, Vc, Cys, GSH and SO32− in pH 7.4 PBS buffer (con- ratiometric imaging of SO3 anion in biological samples with
taining 1 mg mL−1 BSA). As exhibited in Fig. 7, the representa- high resolution. First, the MTT assays were conducted for
tive species CH3COO−, I−, Br−, Cl−, F−, CO32−, N3−, NO2
,
probe 2 at different concentrations, and the results showed
−
H2PO4−, NO3−, SO42−, SCN−, S2O32−, Vc, Cys, and GSH were that probe 2 had low cytotoxicity after a long period (24 h)
prepared at 1 mM, and S2− was prepared at 150 μM. Notably, (Fig. S8†), suggesting that the probe was desirable for imaging
all these relevant species displayed no response to probe 2 applications in living cells. Therefore, we performed a ratio-
2−
(Fig. 7). By contrast, upon treatment of Na2SO3 (100 μM) with metric fluorescence imaging experiment for SO3 anion in
the probe,
a large fluorescence ratiometric signal was living cells. The fluorescence ratio image of the intracellular
observed. Thus, these data demonstrate that probe 2 is highly SO32− anion with probe 2 is shown in Fig. 8. RAW 264.7 macro-
2−
selective for SO3 over the other biological species tested, phage cells incubated with probe 2 for 30 min showed a weak
including over glutathione (GSH) and cysteine at the biologi- emission ratio (Fblue/Fred) (Fig. 8b), which indicated that probe
cally relevant concentrations, validating the hypothesis that 2 could penetrate the cell. When cells pre-treated with probe 2
the SO32−-triggered addition reaction is chemoselective for were further incubated with exogenous Na2SO3 in PBS for
2−
SO3 over other biological species. The selectivity of probe 2 30 min and washed, a notable emission ratio (Fblue/Fred
)
was superior or comparable to already known fluorescent enhancement was observed (Fig. 8d). These preliminary experi-
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The initial geometries of the compounds were generated by
Gaussview software. Excited state calculations (UV-vis absorption)
were carried out with time dependent DFT (TDDFT) with the
optimized structure of the ground (DFT/6-31G(d)). The emis-
sion of the fluorophores was calculated on the basis of the
optimized S1 excited state geometry. All of these calculations
were performed with Gaussian 09 (Revision A.02).26
Fig. 8 Fluorescence images of RAW 264.7 macrophage cells. (a and b)
Cells incubated with probe 2 (2 µM) for 30 min: (a) bright-field trans-
mission image and (b) fluorescent ratio (Fblue/Fred) image generated from
the blue and red channels. (c and d) Cells incubated with probe 2 (2 µM)
for 30 min after preincubation with 100 μM Na2SO3 for 30 min:
Preparation of the test solution
(c) bright-field transmission image, and (d) fluorescence ratio (Fblue/Fred
image generated from the blue and red channels.
)
The stock solution of probe 2 was prepared at 0.5 mM in
DMSO. The solutions of various test species were prepared
from CH3COONa, KI, KBr, NaCl, NaF, Na2CO3, NaN3, NaNO2,
KH2PO4, KNO3, NaSO4, KSCN, Na2S2O3, Na2S, C6H5Na3O7,
GSH, cysteine and Na2SO3 in the twice-distilled water. The test
solution of probe 2 (5.0 μM) in 3 mL 25 mM PBS buffer pH 7.4
PBS buffer (containing 1 mg mL−1 BSA) was prepared by
placing 0.03 mL of the probe 2 stock solution and 3.0 mL of
the aqueous buffer with dissolved BSA (1 mg mL−1). The
resulting solution was shaken well and incubated with appro-
priate testing species for 30 min at 25 °C before recording the
spectra. Unless otherwise noted, for all measurements, the
excitation wavelength was 450 nm, the excitation slit widths
were 10 nm, and emission slit widths were 10 nm.
mental results demonstrated that probe 2 could be applied for
2−
the ratiometric imaging of SO3 in biological samples with
high resolution.
Conclusions
In summary, we presented a novel probe based on a coumarin-
quinolinium platform. This novel probe could be used as a
2−
ratiometric fluorescence sensor for SO3 anions with high
sensitivity, favorable selectivity, as well as a wide dynamic con-
centration range in a PBS buffer solution (containing 1 mg
mL−1 BSA). More importantly, the probe is permeable to the
cell membrane and can image living cells with low cytotoxicity.
We expect that this design concept will be further developed
for the detection of SO2 derivatives for food safety, clinical and
environmental applications.
RAW 264.7 macrophage cells culture and imaging using
probe 2
Raw 264.7 murine macrophages were obtained from the Third
Xiangya Hospital and cultured in DMEM (Dulbecco’s Modified
Eagle Medium) supplemented with 10% FBS (fetal bovine
serum) in an atmosphere of 5% CO2 and 95% air at 37 °C.
Immediately before the experiments, the cells were washed
with PBS, followed by incubating with probe 2 (2 µM) for
30 min at 37 °C (in PBS containing 0.5% DMSO) and then by
washing with PBS three times and imaged.
Experiment section
Materials and instruments
Unless otherwise stated, all reagents were purchased from
commercial suppliers and used without further purification.
The solvents used were purified by standard methods prior to
use. Twice-distilled water was used in all experiments. Mass
spectra were performed using an LCQ Advantage ion trap mass
spectrometer from Thermo Finnigan or an Agilent 1100 HPLC/
MSD spectrometer. NMR spectra were recorded on an
INOVA-400 spectrometer using TMS as an internal standard.
Electronic absorption spectra were obtained on a Labtech UV
Power PC spectrometer. Photoluminescence spectra were
recorded at room temperature with a HITACHI F4600 fluo-
rescence spectrophotometer with a 1 cm standard quartz cell.
pH measurements were carried out on a Mettler-Toledo Delta
320 pH meter. Cell imaging was performed with an inverted
microscope. TLC analysis was performed on silica gel plates,
and column chromatography was conducted over silica gel
(mesh 200–300), both of which were obtained from Qingdao
Ocean Chemicals.
Cytotoxicity assays
RAW 264.7 macrophage cells were cultured in DMEM sup-
plemented with 10% FBS (fetal bovine serum) in an atmos-
phere of 5% CO2 and 95% air at 37 °C. Immediately before the
experiments, the cells were placed in a 96-well plate, followed
by the addition of increasing concentrations of probe 2 (99.9%
DMEM and 0.1% DMSO). The final concentrations of the
probe were kept from 0.5 to 10 μM (n = 3). The cells were then
incubated at 37 °C in an atmosphere of 5% CO2 and 95% air
at 37 °C for 24 h, followed by MTT assays. An untreated assay
with DMEM (n = 3) was also conducted under the same
conditions.
Synthesis of N-ethyl-2-methylquinolinium iodide 1
Compound 1 was prepared according to the reported refer-
ence. A mixture of 2-methylquinoline (1.2 mmol) and ethyl
iodide (1.5 mmol) in 30 ml of toluene was heated at reflux for
20 h. The crude product was filtered and recrystallized from
absolute ethanol, washed with ether and dried under vacuum
DFT calculations
The ground state structures of 2 and 3 were optimized to give compound 1 (yield 87.1%) as a yellow powder, which
using DFT with a B3LYP functional and 6-31G(d) basis set. was used for the next step reaction without further purifi-
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cation. 1H NMR (400 MHz, d6-DMSO) δ 1.55 (t, J = 7.2 Hz, 3H),
3.14 (s, 3H), 5.02 (q, J = 7.2 Hz, 2H), 8.01 (t, J = 7.4 Hz, 1H),
8.16 (d, J = 8.8 Hz, 1H), 8.25 (t, J = 8.0 Hz, 1H), 8.44 (d, J = 8.0
Hz, 1H), 8.64 (d, J = 8.0 Hz, 1H), 9.13 (d, J = 8.4 Hz, 1H);
13C NMR (100 MHz, d6-DMSO) δ 13.94, 22.92, 47.71, 119.35,
126.05, 128.72, 129.50, 131.10, 135.76, 138.53, 146.07, 161.00;
MS (ESI): [M+] 172.1; HRMS m/z calcd for C26H27N2O2 [M+]:
172.1121. Found 172.1129.
Notes and references
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Synthesis of probe 2
1-Ethyl-2-methylquinolinium iodide 1 (59.8 mg, 0.2 mmol) was
treated with coumarin 7-(diethylamino)-2-oxo-2H-chromene-3-
carbaldehyde (49.1 mg, 0.2 mmol) in anhydrous ethanol
(10 mL). The reaction mixture was then refluxed for 10 h, and
the solvent was removed under reduced pressure. The resulting
residue was purified by column chromatography on silica gel
(CH2Cl2 to CH2Cl2–acetone = 50 : 1) to yield the product as a
purple powder (78.7 mg, yield: 74.8%). Mp 139–140 °C.
1H NMR (400 MHz, d6-DMSO) δ 1.15–1.18 (t, J = 7.2 Hz, 6H),
1.63 (t, J = 7.6 Hz, 3H), 3.49–3.54 (q, 4H), 4.96–5.01 (q, 2H),
6. 64 (d, J = 2.0 Hz, 1H), 6.83–6.86 (dd, J = 8.8 Hz, 2.0 Hz, 1H),
7.59–7.62 (d, J = 8.8 Hz, 1H), 7.89–7.94 (t, J = 8.4 Hz, 1H),
8.13–8.18 (m, 3H), 8.31–8.33 (d, J = 8.0 Hz, 1H), 8.46(s, 1H),
8.50–8.53 (d, J = 9.2 Hz, 2H), 8.96–8.98 (d, J = 8.8 Hz, 1H);
13C NMR (100 MHz, d6-DMSO) δ 12.70, 13.91, 44.80, 46.93,
96.57, 108.87, 110.79, 113.33, 116.84, 118.94, 120.82, 128.07,
128.95, 130.59, 131.69, 135.27, 138.36, 143.84, 143.88, 147.92,
153.01, 155.61, 157.09, 159.99. MS (ESI): [M+] 399.1; HRMS m/z
calculated for C26H27N2O2 [M+]: 399.2067. Found 399.2056.
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Synthesis of compound 3
Probe 2 (20.0 mg, 0.05 mmol) dissolved in 3 mL ethanol was
treated with a Na2SO3 (63.0 mg, 0.5 mmol) solution and stirred
at room temperature for 30 min. The solution was evaporated
under vacuum, and the resulting residue was then subjected to
preparative thin layer chromatography (CH2Cl2–CH3CH2OH =
10 : 1) to give compound 3 (20.1 mg, 84.1% yield) as a yellow
1
solid. H NMR (400 MHz, d6-DMSO) δ 1.12 (t, J = 6.0 Hz, 6H),
1.62 (t, J = 6.0 Hz, 3H), 3.41–3.46 (m, 4H), 3.76–3.80 (m, 1H),
4.41–4.45 (q, 1H), 4.43 (t, J = 8.0 Hz, 1H), 5.19–5.36 (m, 2H),
6.52 (d, 1H), 6.72–6.75 (m, 1H), 7.51 (d, J = 8.0 Hz, 1H),
7.98–8.02 (m, 2H), 8.22–8.28 (m, 2H), 8.40 (d, J = 8.0 Hz, 1H),
8.66 (d, J = 8.0 Hz, 1H), 9.07 (d, J = 8.0 Hz, 1H); 13C NMR
(100 MHz, d6-DMSO) δ 12.53, 14.51, 37.58, 44.08, 57.10, 96.41,
108.09, 109.09, 117.06, 119.64, 125.15, 128.48, 129.49, 129.58,
130.85, 135.26, 137.92, 141.17, 145.37, 150.44, 155.48, 161.90,
162.04. MS (ESI): [M+] 503.0.
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Acknowledgements
This work was financially supported by NSFC (20972044,
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