Molecules 2019, 24, 3465
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3.2.3. Synthesis of (E)-10-ethyl-7-(2-(pyridin-4-yl)vinyl)-10H-phenothiazine-3-carbaldehyde (EP)
Compound 2 (3.0 g, 9.0 mmol) and 4-vinylpyridine (1.4 g, 13.5 mmol) dissolved in toluene was
refluxed with palladium(II) acetate (202.0 mg, 0.9 mmol), tri(o-tolyl)phosphine (410.0 mg, 1.4 mmol),
and trimethylamine (5.0 mL, 35.8 mmol) for 72 h in N2 atmosphere. The crude mixture was concentrated
under reduced pressure. Silica gel column chromatography was run to purify the product by using
1
n-hexane and EA (v/v = 1:3). H NMR (400 MHz, acetone-d6)
δ
9.82 (s, 1H), 8.52 (d, J = 5.6 Hz, 2H),
7.72 (dd, J = 8.5, 1.9 Hz, 1H), 7.58 (d, J = 1.9 Hz, 1H), 7.51–7.38 (m, 7H), 7.20–7.06 (m, 3H), 4.09 (q,
J = 7.0 Hz, 2H), 1.43 (t, J = 7.0 Hz, 3H); 13C NMR (101 MHz, Acetone)
δ
190.97, 151.59, 150.75, 145.99,
144.67, 133.61, 133.06, 132.75, 131.60, 128.86, 128.52, 126.58, 126.44, 124.75, 124.56, 122.04, 117.44, 116.47,
43.68, 13.50.
3.2.4. Synthesis of 4-((1E,3E)-4-(benzo[d]thiazol-2-yl)buta-1,3-dien-1-yl)-N,N-dimethylaniline
(Compound 3)
2-Methylbenzothiazole (2.0 g, 13.7 mmol), 4-(Dimethylamino)cinnamaldehyde (2.0 g, 11.4 mmol)
and potassium tert-butoxide (1.5 g, 13.7 mmol) were mixed in 30 mL DMF at 80 ◦C overnight. Fifty
milliliters (50 mL) of deionized water was added to the crude mixture to obtain orange precipitate.
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The precipitate was collected by vacuum filtering and purified by recrystallization in ethanol. H NMR
(400 MHz, chloroform-d)
δ 7.98–7.95 (m, 1H), 7.87–7.82 (m, 1H), 7.46 (ddd, J = 8.3, 7.2, 1.3 Hz, 1H), 7.42
(d, J = 8.9 Hz, 2H), 7.38–7.34 (m, 2H), 6.89 (s, 1H), 6.86–6.83 (m, 2H), 6.71 (d, J = 8.9 Hz, 2H), 3.04 (s, 6H).
3.2.5. Synthesis of 3-(4-boronobenzyl)-2-((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,3-dien-1-
yl)benzo[d]thiazol-3-ium bromide (BT) was Synthesized by the Method Reported [12]
Compound 3 (1.0 g, 3.3 mmol) and 4-(bromomethyl) phenylboronic acid (0.6 g, 3.0 mmol) were
mixed in MeCN/THF (v/v = 1:1) under nitrogen atmosphere. The reaction was heated to 80 ◦C for two
days. The crude mixture was concentrated under reduced pressure and purified by recrystallization
in DCM to obtain dark violet product. 1H NMR (400 MHz, DMSO-d6)
δ 8.37 (dd, J = 7.9, 1.3 Hz,
1H), 8.12 – 8.04 (m, 4H), 7.79 (d, J = 8.0 Hz, 2H), 7.75–7.67 (m, 2H), 7.55 (d, J = 8.9 Hz, 2H), 7.50 (d,
J = 14.8 Hz, 1H), 7.38 (d, J = 14.4 Hz, 1H), 7.25 (d, J = 8.1 Hz, 2H), 7.17 (dd, J = 14.9, 11.1 Hz, 1H), 6.79
(d, J = 9.0 Hz, 2H), 6.01 (s, 2H), 3.05 (s, 6H). 13C NMR (101 MHz, DMSO)
δ 171.75, 152.88, 149.77, 141.74,
135.92, 135.26, 131.36, 129.75, 128.23, 127.88, 126.15, 124.74, 123.32, 116.58, 112.67, 111.82, 51.25. Q-TOF
m/z: calcd, 397.1733 [M − Br − BO2H2]+; found, 397.1718.
3.3. Preparation of ONOO− and Other Reactive Oxygen Species
ClO−, H2O2, NO , NO2−, NO3−, and cysteine obtained from commercial sources were diluted
·
or dissolved in water. ONOO- was prepared by mixing pre-cooled 0.6 M NaNO2, 0.6 M HCl, and
0.7 M H2O2 into 3 M NaOH at 0 ◦C. Manganese dioxide was then added to the ONOO− solution to
eliminate the residual H2O2 and was removed by simple filtration. The ONOO− solution was stored at
−
80 ◦C. The concentration of peroxynitrite was estimated by its extinction coefficient of 1670 M−1 cm−1
at 302 nm before use [19,20].
3.4. Optical Response of PB-PVA to ONOO−
The absorption and emission spectra of the probe at different concentrations of ONOO− were
measured at room temperature. The probe was measured 3 min after the addition of ONOO−.
The absorption spectra were scanned from 300 to 800 nm while the emission spectra were scanned from
510 to 800 nm. The ratio between the emission intensities of two peaks from the probe when exposed to
different concentrations of ONOO− was calculated in order to investigate the relationship between the
ratio and the concentration of ONOO− exposed. The probe was also tested for its selectivity towards
ONOO− by investigating the fluorescence intensity ratios when exposed to various ROS. The time