C. Zhang et al. / Tetrahedron xxx (2017) 1e6
5
from commercial suppliers and applied directly in the experiments
without further purification. Merck silica gel 60 (100e200 mesh)
was used for general column chromatography purification. 1H NMR
and 13C NMR spectra were recorded on a Bruker 400 spectrometer.
Chemical shifts are reported in parts per million relative to internal
standard tetramethylsilane (Si(CH3)4 ¼ 0.00 ppm) or residual sol-
vent peaks (CDCl3 ¼ 7.26 ppm; DMSO-d6 ¼ 2.50 ppm). 1H NMR
coupling constants (J) are reported in Hertz (Hz), and multiplicity is
indicated as the following: s (singlet), d (doublet), t (triplet), dd
(doublet of doublets), m (multiple). High-resolution mass spectra
(HRMS) were obtained on an Agilent 6540 UHD Accurate-Mass Q-
TOFLC/MS. The UVevisible spectra were recorded on a UV-3600
UV-VIS-NIR spectrophotometer (SHIMADZU, Japan). Fluorescence
studies were carried out using F-280 spectrophotometer (Tianjin
Gangdong Sci & Tech., Development. Co., Ltd).
8.21 (d, J ¼ 7.1 Hz, 1H), 7.55 (t, J ¼ 7.7 Hz, 3H), 7.19 (d, J ¼ 7.5 Hz, 1H),
4.14 (s, 2H), 3.65 (dd, J ¼ 11.0, 5.6 Hz, 4H), 3.32 (s, 2H), 3.19 (t,
J ¼ 4.8 Hz, 2H), 2.89 (s, 6H); 13C NMR (151 MHz, CDCl3)
d 164.8,
152.1, 149.3, 142.6, 138.3, 133.7, 132.2, 131.4, 131.1, 130.7, 130.4, 130.2,
128.5, 123.6, 123.3, 119.3, 115.6, 46.3, 45.6, 45.6, 45.4, 42.1, 34.4.
HRMS calculated for (M þ H)þ C24H25N6O6S2þ: 557.1272, found
557.1223.
4.2. General procedure for spectroscopic studies
Measurements were performed in degassed phosphate-
buffered saline buffer (PBS, 50 mM, pH 7.4, containing 10%
DMSO). Probes 1 and 2 were dissolved in DMSO to prepare 5 mM
stock solutions. Probes were diluted in PBS buffer to afford the final
concentrations of 1e25
mM. The quantum yield tests were carried
out in PBS buffer using fluorescein (
f
, 0.95) as reference. For the
4.1. Synthesis
selectivity experiment, different bio-relevant molecules (100 mM
or 25 mM) were prepared as stock solutions in PBS buffer. Appro-
priate amount of bio-relevant species were added to separate
portions of the probe solution and mixed thoroughly. The reaction
mixture was shaken uniformly before emission spectra were
measured. All measurements were performed in a 3 ml corvette
with 2 ml solution.
4.1.1. Synthesis of 3
To a CH2Cl2 solution (25 ml) of dansyl chloride (405 mg,
1.5 mmol) and tert-butyl piperazine-1-carboxylate (419 mg,
2.25 mmol), DIPEA (1.5 mmol) was added drop-by-drop at 0 ꢀC. The
mixture was stirred at room temperature for 2 h. After that, the
reaction solution was washed with water and brine. The organic
phase was dried by Na2SO4. Then, organic solvent was evaporated
under reduced pressure. The resulted residue was purified by silica
gel column chromatography with CH2Cl2/MeOH ¼ 100/1 to get a
4.3. Cell cultures and fluorescence imaging
HeLa cells were cultured at 37 ꢀC, 5% CO2 in DMEM/HIGH
GLUCOSE (GIBCO) supplemented with 10% fetal bovine serum
(FBS), 100 U/ml penicillin, 100 mg/ml streptomycin, and 4 mM L-
glutamine. The cells were maintained in exponential growth, and
then seeded in glass-bottom 35 mm plate at the density about
2 ꢁ 104/well. Cells were passaged every 2e3 days and used be-
tween passages 3 and 10. Cells were imaged on a confocal micro-
scope (Olympus FV1000 UPLSAPO40X) with a 40 ꢁ objective lens.
Emission was collected at green channel (500e600 nm) with
488 nm excitation. All images were analyzed with Olympus
FV1000-ASW.
yellow power 3 (284 mg, 45%). 1H NMR (400 MHz, CDCl3)
d 8.59 (d,
J ¼ 8.5 Hz, 1H), 8.40 (d, J ¼ 8.7 Hz, 1H), 8.20 (dd, J ¼ 7.3, 1.1 Hz, 1H),
7.55 (dd, J ¼ 12.0, 4.4 Hz, 2H), 7.19 (d, J ¼ 7.5 Hz, 1H), 3.48e3.41 (m,
4H), 3.19e3.11 (m, 4H), 2.89 (s, 6H), 1.39 (s, 9H); 13C NMR (101 MHz,
CDCl3)
d 154.3, 132.7, 131.1, 130.9, 130.5, 130.2, 128.3, 123.4, 119.8,
115.5, 80.5, 77.5, 77.2, 76.8, 45.6, 45.6, 28.4.
4.1.2. Synthesis of 6
Compound 3 (120 mg, 0.29 mmol) was dissolved by a solution
consisted of CH2Cl2 (2 ml), TFA (2 ml) and H2O (20 ml), and the
resulted mixture was stirred at room temperature for 40 min. After
that, the reaction solution was removed under reduced pressure to
get product 4, which was used in following synthesis directly.
Compound 5 (52 mg, 0.16 mmol) was dissolved by 4 ml DMF and
then HATU (77 mg, 0.20 mmol) and DMAP (50 mg, 0.40 mmol)
were added to the solution. After stirring for 5min, compound 4
(45 mg, 0.14 mmol) was added. The mixture was stirred at room
temperature overnight, and then poured into 80 ml H2O to give
yellow-green precipitate by centrifugation. The precipitate was
dried and then purified by silica gel column chromatography with
CH2Cl2/MeOH ¼ 1000/5 to get a yellow-green solid 6 (50 mg, 58%).
Acknowledgments
This work was supported by the MOST (2010CB126102), NSFC
(21332004, 21402007), 111 project (B14004).
Appendix A. Supplementary data
Supplementary data related to this article can be found at
1H NMR (400 MHz, CDCl3)
d
8.61 (d, J ¼ 8.5 Hz, 1H), 8.35 (d,
References
J ¼ 8.7 Hz,1H), 8.20 (d, J ¼ 7.2 Hz,1H), 7.56 (t, J ¼ 8.0 Hz, 2H), 7.43 (d,
J ¼ 7.7 Hz, 6H), 7.29 (t, J ¼ 7.5 Hz, 6H), 7.22 (t, J ¼ 7.1 Hz, 4H),
3.59e3.53 (m, 2H), 3.17e3.10 (m, 2H), 3.06 (s, 4H), 2.92 (s, 6H), 2.88
(s, 2H).
4.1.3. Synthesis of probe 2
To a solution of 6 (20 mg, 0.03 mmol) in 0.8 ml CH2Cl2 were
added Et3SiH (100 ml) and then TFA (200 ml) under nitrogen gas, and
the mixture was stirred at room temperature for 1 h. After evapo-
ration of the organic solvent, the residue was dissolved in CH2Cl2
(5 ml), NBD-Cl (30 mg, 0.15 mmol) and Et3N (200 ml) were added
under nitrogen gas at 0 ꢀC. After stirring overnight, the mixture was
evaporated under reduced pressure. The resulted residue was pu-
rified by silica gel column chromatography with CH2Cl2/
MeOH ¼ 1000/5 to get a yellow solid 2 (8 mg, 42%). 1H NMR
(400 MHz, CDCl3)
d
8.60 (d, J ¼ 8.4 Hz, 1H), 8.34 (d, J ¼ 7.7 Hz, 2H),
Please cite this article in press as: Zhang C, et al., Design and synthesis of NBD-S-dye dyads for fluorescently discriminative detection of biothiols