B. Zhu, X. Wu, J. Rodrigues et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 246 (2021) 118953
response of PTMQ to ClO− and N2H4, respectively. Moreover, PTMQ was
used to in-situ image ClO− and N2H4 in living human cervical cancer
HeLa cells.
temperature. The precipitate was collected by filtration, followed by re-
crystallization in ethanol to give PTMQ as purple solid (332 mg, yield:
68%). 1H NMR (400 MHz, DMSO‑d6) δ 9.26 (d, J = 6.6 Hz, 1H), 9.03 (d,
J = 8.6 Hz, 1H), 8.52 (d, J = 8.9 Hz, 1H), 8.41 (d, J = 6.6 Hz, 1H), 8.25
(d, J = 7.2 Hz, 1H), 8.20 (d, J = 5.8 Hz, 2H), 8.03 (s, 2H), 7.30 (t, J =
7.7 Hz, 1H), 7.25 (d, J = 7.8 Hz, 2H), 7.10–7.04 (m, 1H), 6.94 (s, 1H),
4.99 (d, J = 7.2 Hz, 2H), 4.83 (s, 2H), 4.01 (s, 3H), 3.59 (s, 1H), 1.57 (t,
J = 7.1 Hz, 3H). 13C NMR (100 MHz, DMSO‑d6) δ 159.0, 153.4, 147.9,
147.3, 142.8, 138.1, 136.5, 135.5, 129.4, 128.2, 127.42, 127.2, 127.1,
126.6, 124.2, 122.4, 119.4, 119.2, 118.1, 116.3, 116.1, 113.8, 100.3, 79.5,
77.8, 56.6, 52.4, 38.0, 15.6. HR-MS (ESI, m/z): calcd. for
[C29H25N2OS]+: 449.1682; found: 449.1699.
2. Experimental
2.1. Chemicals and instrumentations
All chemicals were commercially purchased from Sigma-Aldrich and
used directly without further purification. Compound 1 (N-ethyl-4-
methylquinolinium iodide, MQ) was prepared according to the litera-
ture [29]. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were
measured on a Bruker AV spectrometer. High resolution mass spec-
trometry (HRMS) was obtained on HP-1100 LC-MS spectrometer. UV–
Vis absorption spectra were performed on a Hitachi UV-3310 spectrom-
eter. Fluorescence spectra were obtained with a FL-4500 fluorescence
spectrometer. The cells imaging experiments were taken under a
Nikon A1 confocal laser-scanning microscope with a 100 × objective
lens.
2.5. General procedures for spectral measurements
10 μM of PTMQ solution in DMSO/PBS (v/v = 2/8, pH = 7.4) was
used for spectral measurements. Optical tests of PTMQ for ClO− and
N2H4 were performed as follows: a) PTMQ (10 μM) was pre-
incubated with ClO− for 5 min at room temperature. The UV–Vis ab-
sorption and the fluorescence spectra were recorded directly. Excitation
wavelength was 460 nm with slit widths 5/5 nm; b) PTMQ (10 μM) was
pre-incubated with N2H4 for 30 min at room temperature. Then the UV–
Vis absorption and the fluorescence spectra were measured directly. Ex-
citation was at 360 nm with slit widths 2.5/5 nm.
2.2. Synthesis of compound 2 (N-propagyl-8-methoxy-phenothiazine)
In a 250 mL flask, 2-methoxyphenothiazine (500 mg, 2.18 mmol), 3-
bromopropyne (1.30 g, 10.90 mmol) and KI (166 mg, 1.0 mmol) were
dissolved in 15 mL anhydrous DMF. The mixture was stirred at 100 °C
for 8 h under N2 atmosphere. After cooling down to room temperature,
the resulted mixture was poured into 100 mL H2O and extracted three
times with CH2Cl2. Then, solvent of the collected organic layer was re-
moved under reduced pressure. The obtained crude product was further
purified by silica gel flash chromatography (petroleum ether/ethyl
acetate = 15:1, v/v) to give compound 2 (N-propagyl-8-methoxy-
phenothiazine) as colorless solid (495 mg, yield: 85%). 1H NMR (400
MHz, DMSO‑d6) δ 7.24 (t, J = 7.7 Hz, 1H), 7.21–7.12 (m, 2H), 7.07 (d, J
= 8.4 Hz, 1H), 6.99 (t, J = 7.4 Hz, 1H), 6.79 (s, 1H), 6.62 (d, J = 8.4
Hz, 1H), 4.64 (s, 2H), 3.76 (s, 3H), 3.50 (s, 1H). 13C NMR (100 MHz,
DMSO‑d6) δ 159.9, 145.4, 143.8, 127.9, 127.7, 127.2, 123.4, 123.1,
115.6, 113.2, 107.8, 103.4, 79.9, 77.4, 55.8, 38.0.
2.6. Fluorescence imaging of ClO− and N2H4 by PTMQ in HeLa cells
HeLa cells were seeded in glass-bottomed dishes and cultured in
DMEM culture medium with 10% FBS at 37 °C for 24 h. For imaging
ClO− and N2H4, the HeLa cells were washed with PBS three times to re-
move culture medium and then pre-treated with PTMQ (10 μM) for 30
min. Afterwards, the cells-containing dishes were washed there times
with PBS, and mounted on the stage of confocal laser-scanning micro-
scope. Before fluorescence imaging, the HeLa cells were further co-
incubated with different concentrations of ClO− and N2H4 for another
30 min, respectively.
3. Results and discussion
2.3. Synthesis of compound 3 (N-propagyl-8-methoxy-phenothiazine-7-al-
dehyde, PT)
3.1. Design and synthesis of PTMQ
Phosphorous oxychloride (0.52 mL, 5.61 mmol) was dropwise
added to anhydrous dimethylformamide (0.43 mL, 5.61 mmol) at 0 °C
under a N2 atmosphere. After the mixture was stirred at 0 °C for 30
Herein, a simple synthetic route was established to prepare the de-
sired dual-analytes responsive fluorescent probe PTMQ, as depicted in
Scheme 1. 4-Methyl-quinoline was quaternized with ethyl iodide to
prepare N-ethyl-4-methyl-quinolinium iodide (compound 1, MQ) via
N-alkylation reaction. The fluorescent precursor 8-methoxy-phenothia-
zine was coupled with propagyl bromide to prepare compound 2, which
underwent a Vilsmeier reaction (POCl3/DMF) to prepare N-propagyl-8-
methoxy- phenothiazine-7-aldehyde (compound 3, PT). Furthermore,
MQ reacted with PT to prepare the dual-analytes responsive fluorescent
probe PTMQ via one-step condensation approach. The structure of
PTMQ was analyzed by 1H NMR, 13C NMR and HR-MS (shown in
Figs. S1–S7, Supporting information).
min, compound
2 (300 mg, 1.12 mmol) in 5 mL anhydrous
dimethylformamide was dropwise added to the above solution. The
reacting mixture was heated to 60 °C and stirred for 5 h. Then the
resulting mixture was poured into ice water, and the reaction solution
was neutralized to pH 7 by NaOH solution (20%) until a large amount
of solid precipitated. After filtration, the solid was washed with distilled
water (3 × 10 mL), and dried in vacuum and then recrystallized from
ethanol to offer compound 3 (N-propagyl-8-methoxy-phenothiazine-
7-aldehyde, PT) as yellow solid (252 mg, yield: 78%). 1H NMR (400
MHz, DMSO‑d6) δ 10.14 (s, 1H), 7.40 (s, 1H), 7.32–7.26 (m, 1H), 7.24
(d, J = 8.3 Hz, 1H), 7.21 (d, J = 6.2 Hz, 1H), 7.06 (t, J = 8.1 Hz, 1H),
6.91 (s, 1H), 4.83 (s, 2H), 3.97 (s, 3H), 3.58 (s, 1H)·13C NMR (100
MHz, DMSO‑d6) δ 186.8, 162.6, 151.0, 142.2, 128.3, 127.4, 125.9, 124.5,
122.3, 119.7, 116.3, 113.6, 100.3, 79.3, 77.9, 56.5, 38.6.
3.2. Sensing performance of PTMQ for ClO− and N2H4
The UV–Vis and fluorescent spectra titration of PTMQ with HClO and
N2H4 were measured, respectively (Figs. 1 and 2). As shown in Fig. 1,
PTMQ itself is non-fluorescence in DMSO/PBS solution due to the strong
intramolecular charge transfer (ICT) effect from sulfur (\\S\\) and ni-
trogen (\\N\\) atoms in phenothiazine to quinolinium [30]. Upon the
addition of an increasing amount of ClO−, the absorption band of
PTMQ at 518 nm gradually decreased with the increase of a new band
at 465 nm, and simultaneously the color of the PTMQ solution turned
from deep purple to light pink. The isosbestic point in the absorption ti-
tration spectra implied that PTMQ has transformed to a new compound
2.4. Synthesis of probe PTMQ
Compound 1 (MQ, 380 mg, 1.27 mmol) and compound 3 (PT, 250
mg, 0.85 mmol) were dissolved in 15 mL anhydrous EtOH. Afterward,
piperidine (0.1 mL) and AcOH (0.1 mL) were added to the solution as
catalysts. The mixture was refluxed at 80 °C for 6 h under nitrogen at-
mosphere. Precipitate was formed after cooling down to room
2