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A.K. Mahapatra et al. / Journal of Photochemistry and Photobiology A: Chemistry 334 (2017) 1–12
anhydride (0.019 g, 0.18 mmol) was added to this reaction mixture.
The reaction mixture was stirred at rt for 2 h. TLC showed SM was
consumed. The reaction mixture was diluted with DCM and
washed with water. The combined organic solution was washed
with brine, dried over sodium sulfate and concentrated. The crude
mass was purified by column chromatography on silica, product
eluted with 10% ethyl acetate in hexane to afford 2-(4-Acetoxy-3-
benzothiazol-2-yl-phenyl)-4-methyl-thiazole-5-carboxylic acid
ethyl ester (CD1) (0.04 g, 72%) as white solid.
127.31, 126.46, 126.41, 125.57, 125.42, 123.55, 123.33, 121.67, 121.38,
21.32, 16.57. MS (ESI): m/z calc. for C23H16N2O2S2+: 416.5; found:
417.1 [M+H]+.
2.5. Preparation of test solution for UV–vis and fluorescence study
For UV–vis and fluorescence titrations, stock solution of Probe
CD1/CD2was prepared (c = 10.0 mM) in H2O–DMSO (1:2, v/v)
solution (10 mM HEPES buffer, pH = 7.4). The solution of the guest
anions, cations and amines in the order of 1.0 ꢃ10ꢀ4 MLꢀ1 was also
prepared in H2O–DMSO (1:2, v/v) solution (10 mM HEPES buffer,
pH 7.4). The test solution of sensor Probe CD1/CD2 was prepared by
proper dilution method. The spectra of these solutions were
recorded by means of UV–vis and the fluorescence methods. All the
solvents were purchased from local suppliers and were distilled by
standard procedure before use.
1H NMR (400 MHz, CDCl3)
d (ppm): 8.91 (s, 1H), 8.14–8.10 (m,
2H), 7.94 (d, J = 8.08 Hz, 1H), 7.55–7.51 (m, 1H), 7.45–7.41 (m, 1H),
7.35 (d, J = 8.52 Hz,1H), 4.36 (q, J = 7.12 Hz, 2H), 2.80 (s, 3H), 2.5 (s,
3H), 1.39 (t, J = 7.04 Hz, 3H). 13C NMR (100 MHz, CDCl3):168.65,
167.80, 162.12, 161.18, 152.79, 149.84, 135.43, 131.32, 129.21, 128.61,
126.80, 126.46, 125.61, 124.48, 123.58, 121.35, 61.31, 21.66, 17.50,
14.31. MS (ESI): m/z calc. for C22H18N2O4S2+: 438.0; found: 439.3
[M+H]+.
2.6. Computational studies
2.4. Synthesis of receptor CD2
All geometries for CD1 and CD2 and their corresponding anion
products (A1 and A2) after reaction with hydrazine were optimized
by density functional theory (DFT) calculations using Gaussian 09
(B3LYP/6-31G(d,p)) software package.
2.4.1. Preparation of compound 4-Hydroxy-5-methyl-benzene-1,3-
dicarbaldehyde (7)
To a stirred solution of 2-methyl-phenol (6) (5 g, 46.29 mmol) in
TFA (52 mL) was added Hexamethylenetetramine (12.96 g,
55.55 mmol). The reaction mixture was heated at 130 ꢁC for 4 h.
The reaction mixture was cooled down to rt and then added 10%
sulfuric acid (ꢂ100 mL). Then the reaction mixture was heated at
95 ꢁC for 2 h and the reaction mixture was left at rt for overnight.
The resultant white solid was filtered and washed with cold
ethanol, dried under vaccum pump to afford 4-Hydroxy-5-methyl-
benzene-1,3-dicarbaldehyde (7) (2.5 g, 33%) as white solid.
2.7. Cell culture
Vero cell (very thin endothelial cell) (Vero 76, ATCC No CRL-
1587) lines were prepared from continuous culture in Dulbecco’s
modified Eagle’s medium (DMEM, Sigma Chemical Co., St. Louis,
MO) supplemented with 10% fetal bovine serum (Invitrogen),
penicillin (100 mg/mL), and streptomycin (100 mg/mL). The Vero 76
1H NMR (400 MHz, CDCl3)
d
(ppm): 11.82(s, 1H), 9.97 (s, 1H),
were obtained from the American Type Culture Collection (Rock-
ville, MD) and maintained in DMEM containing 10% (v/v) fetal
bovine serum and antibiotics in a CO2 incubator. Cells were initially
propagated in 75 cm2 polystyrene, filter-capped tissue culture flask
in an atmosphere of 5% CO2 and 95% air at 37 ꢁC in CO2 incubator.
When the cells reached the logarithmic phase, the cell density was
adjusted to 1.0 ꢃ105 per/well in culture media. The cells were then
used to inoculate in a glass bottom dish, with 1.0 mL (1.0 ꢃ104
cells) of cell suspension in each dish. After cell adhesion, culture
medium was removed. The cell layer was rinsed twice with
phosphate buffered saline (PBS), and then treated according to the
experimental need.
9.90 (s, 1H), 7.96 (s, 1H), 7.93 (s, 1H), 2.33 (s, 3H).
2.4.2. Preparation of compound 2, 4-Bis-benzothiazol-2-yl-6-methyl-
phenol (8)
To a mixture of 4-Hydroxy-5-methyl-benzene-1,3-dicarbalde-
hyde (7) (300 mg, 1.82 mmol) and 2-aminothiophenol (572 mg,
4.57 mmol) in ethanol (10 mL) was added cat. Amount KHSO4. The
reaction mixture was heated to reflux for 12 h. The resultant solid
was filtered and washed with cold ethanol, dried under rotavapour
to afford 2, 4-Bis-benzothiazol-2-yl-6-methyl-phenol (8) (0.25 g,
36%) as white solid.
1H NMR (400 MHz, CDCl3)
d (ppm): 13.27 (bs, 1H), 8.31 (s, 1H),
8.06 (d, J = 8.0 Hz,1H), 8.01 (d, J = 8 Hz,1H), 7.97–7.93 (m, 2H), 7.9 (d,
J = 8.8 Hz,1H), 7.55–7.49 (m, 2H), 7.47–7.36(m, 2H), 2.45(s, 3H). MS
(ESI): m/z calc. for C21H14N2OS2+: 374.4; found: 375.3 [M+H]+.
2.8. Cell imaging study
For fluorescence imaging studies Vero cell (Vero 76, ATCC No.
CRL-1587) lines, were seeded on sterile 35 mm m-Dish, glass
2.4.3. Preparation of compound acetic acid 2, 4-bis-benzothiazol-2-yl-
6-methyl-phenyl ester (CD2)
bottom culture dish (ibidi GmbH, Germany), and incubated at 37 ꢁC
in a CO2 incubator for 24–30 h. The next day, cells were washed
three times with phosphate buffered saline (PBS) and fixed using
4% paraformaldehyde in PBS (pH 7.4) for 10 min at room
temperature. Thereafter the cells were washed with PBS followed
by permeabilization using 0.1% saponin for 10 min followed by
A solution of 2, 4-Bis-benzothiazol-2-yl-6-methyl-phenol (8)
(0.1 g, 0.267 mmol) in DCM (0.5 mL) was added dry pyridine
(0.053 g, 0.668 mmol) at 0 ꢁC condition. Under same condition, dry
acetic anhydride (0.038 g, 0.373 mmol) was added to this reaction
mixture. The reaction mixture was stirred at rt for 2 h. TLC showed
SM was consumed. The reaction mixture was diluted with DCM
and washed with water. The combined organic solution was
washed with brine, dried over sodium sulfate and concentrated.
The crude mass was purified by column chromatography on silica,
product eluted with 10% ethyl acetate in hexane to afford 2-(4-
Acetoxy-3-benzothiazol-2-yl-phenyl)-4-methyl-thiazole-5-car-
boxylic acid ethyl ester (CD2) (0.07 g, 63%) as white solid.
incubation with 1.0 ꢃ10ꢀ6 M of probe 1 dissolved in 100
mL DMEM
at 37 ꢁC for 1 h in a CO2 incubator. Before microscopic imaging, all
the solutions were aspirated and mounted on slides in a mounting
medium containing DAPI (1 mg/mL) and stored in dark before
microscopic images were acquired. The cells were observed under
Andor spinning disk confocal microscope (SD-CM) with excitation
at 400 nm monochromatic laser beam and the collected range of
emission wavelength was between 420 and 480 nm (blue channel).
Cells were imaged live by SD-CM 63 ꢃ oil-immersion objective.
1H NMR (400 MHz, CDCl3)
d (ppm): 8.73 (s, 1H), 8.15–8.09 (m,
3H), 7.95–7.92 (m, 3H), 7.55–7.49 (m, 2H), 7.45-7.39 (m, 2H), 2.49(s,
3H), 2.37(s, 3H).. 13C NMR (100 MHz, CDCl3): 168.50,166.36,162.14,
153.97, 153.15, 148.92, 135.35, 135.19, 133.29, 131.77, 131.56, 127.49,
Images were acquired in z-stacks of 28 planes at 0.3-mm intervals
with 400-ms exposure times every 20 s over a period of 30 min. In
another dish new cells were again cultured in the same manner