Communications
Synthesis of TDPP-mPEG
Experimental Section
mPEG-COOH (MW=2000 kDa; 0.8 g, 0.4 mmol) and tetrabutylam-
monium hydroxide (10% in water, 0.11 g, 0.4 mmol) were added to
doubly distilled water (30 mL) and stirred for 24 h. The resulting
mPEG+COOÀ was obtained by freeze-drying. Next, DPP-C6Br
(0.03 g, 0.1 mmol) and mPEG+COOÀ (0.4 g, 0.2 mmol) was dis-
solved in THF/acetone (3:1 v/v) and stirred for 24 h. After evapora-
tion of the solvent under reduced pressure, doubly distilled water
(50 mL) was added to the crude product under ultrasound agita-
tion and filtered to obtain the filtrate. The filtrate was purified by
dialysis (membrane MW cut-off: 3.5 kDa) for 48 h. The sample was
freeze-dried to obtain TDPP-mPEG.
All chemicals and reagents were used without further purification.
NMR spectra were measured on a Bruker Ultra Shield Plus 400 MHz
spectrometer using CDCl3 and [D6]DMSO as the solvent. GC–MS
was performed on a Shimadzu GCMS-QP2010 Plus mass spectrom-
eter. MALDI-TOF MS was performed on a Bruker Autoflex mass
spectrometer. UV/Vis absorption and photoluminescence spectra
were recorded on a Shimadzu UV-3600 UV/Vis/NIR spectrophotom-
eter and a RF-5301PC spectrofluorophotometer, respectively.
Synthesis of PDPP
In a typical procedure, potassium tert-butoxide (4.0 g, 35.71 mmol)
was added into the reaction vessel under a N2 atmosphere. Then,
1,1-dimethyl-1-propanol (25 mL) was added and the mixture
heated to 1088C to dissolve the potassium tert-butoxide. Benzoni-
trile (3.06 g, 29.71 mmol) and methyl succinate (1.46 g,
11.16 mmol) were added to the reaction mixture, which was then
stirred for 1 h. The reaction was cooled to 658C, then methanol
(50 mL) and acetic acid (2–3 mL) were added the reaction was
heated at reflux for 10 min. The reaction mixture was cooled to
room temperature, filtered and washed to obtain PDPP as
Cell lines and culture conditions
HeLa cells, provided by the Institute of Biochemistry and Cell Biol-
ogy, SIBS, CAS (China), were cultured in a regular growth medium
consisting of DMEM (Gibco) supplemented with 10% FBS at 378C
under a 5% CO2 atmosphere. The cells were routinely harvested by
treatment with a trypsin/ethylenediamine tetraacetic acid (EDTA)
solution (0.25%).
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a powder. H NMR (400 Hz, [D6]DMSO): d=8.49–8.44 (m, 4H), 7.58–
Fluorescence microscopy study
7.56 (m, 2H), 7.55–7.53 ppm (m, 4H); 13C NMR (100 Hz, [D6]DMSO):
d=162.9, 144.3, 132.5, 129.4, 128.3, 79.8, 79.4, 79.1 ppm; MS (EI):
m/z: calcd for C18H13N2O2: 289.09 [M+H]+; found: 289.46.
About 2105 HeLa cells in the culture medium (2 mL) were seeded
on a glass bottom Petri dish and incubated overnight at 378C.
Then the medium was removed. The cells were washed with PBS
and were incubated with a solution of TDPP-mPEG in the culture
medium (1 mgmLÀ1, 2 mL) for different durations (3, 9 and 24 h).
The cells were washed with PBS and imaged with an Olympus IX70
inverted microscope. The excitation light (559 nm) was provided
by a multi-wavelength illuminator (Polychrome IV, TILL Photonics;
Beijing, China). The emitted fluorescence at >600 nm was collect-
ed, digitized, and analyzed using MetaFluor 6.3 (Universal Imaging,
USA).
Synthesis of TDPP
TDPP was prepared by a similar method to that above. Typically,
potassium tert-butoxide (4.0 g, 35.71 mmol) was added into the re-
action vessel under a N2 atmosphere. Then, 1,1-dimethyl-1-propa-
nol (25 mL) was added and the mixture heated to 1088C to dis-
solve the potassium tert-butoxide. 2-Thiophenecarbonitrile (3.24 g,
29.71 mmol) and methyl succinate (1.46 g, 11.16 mmol) were
added to the reaction mixture, which was then stirred for 1 h. The
reaction was cooled to 658C, then methanol (50 mL) and acetic
acid (2–3 mL) were added the reaction was heated at reflux for
10 min. The reaction mixture was cooled to room temperature, fil-
Fluorescence studies with the ROS probe
Approximately 2105 HeLa cells in culture medium (2 mL) were
seeded onto a glass-bottom Petri dish and incubated overnight at
378C under 5% CO2. Then, the medium was removed, the cells
were washed with PBS and incubated with a solution of TDPP-
mPEG (1 mgmLÀ1) for 24 h. The cells were then washed with PBS
and further incubated with the ROS probe DCFH-DA (10 mM) for
1 h. Then, the cells were washed with PBS three times and refresh-
ed with culture medium (1 mL) before being illuminated at ambi-
ent temperature using a Xenon lamp light source. The cells were
imaged with an Olympus IX70 inverted microscope. The excitation
light source at 488 nm was provided by a multi-wavelength illumi-
nator. The emitted fluorescence from 500 to 600 nm was collected,
digitized, and analyzed using MetaFluor 6.3.
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tered and washed to yield TDPP. H NMR (400 Hz, [D6]DMSO): d=
11.22 (d, 2H), 8.20 (d, 2H), 7.95 (d, 2H), 7.29 ppm (m, 2H); 13C NMR
(100 Hz, [D6]DMSO): d=162.2, 133.4, 131.7, 131.3, 129.1, 109.1; MS
(EI): m/z: calcd for C14H9N2O2S2: 301.01 [M+H]+; found: 300.73.
Synthesis of TDPP-C6Br
TDPP (0.6 g, 2.0 mmol) and potassium hydroxide (0.11 g, 2.0 mmol)
were dissolved in DMF, and heated at 1208C for 30 min. Then, 1,6-
dibromohexane (0.98 g, 4.0 mmol) was added to the above solu-
tion, and the mixture was stirred until TDPP was completely con-
sumed (monitored by TLC). Then, the solvent was evaporated
under reduced pressure and the residue was purified by column
chromatography (silica gel, petroleum ether) to give TDPP-C6Br as
Cell nucleus damage study
Approximately 2105 HeLa cells in culture medium (2 mL) were
seeded onto a glass-bottom Petri dish and incubated overnight at
378C. Then, the medium was removed, the cells were washed with
PBS and incubated with a solution of TDPP-mPEG in culture
medium at different concentrations (0.2, 0.5 and 1.0 mgmLÀ1) for
24 h. The cells were washed with PBS and incubated with the cell
nucleus probe 4’,6-diamidino-2-phenylindole (DAPI) for 1 h. Then,
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a black solid. HNMR (400 Hz, CDCl3): d=8.94–8.91 (m, 2H), 7.67–
7.64 (m, 2H), 7.31–7.28 (m, 2H), 4.13–4.04 (m, 4H), 3.43–3.36 (m,
4H), 1.92–1.82 (m, 4H), 1.81–1.72 (m, 4H), 1.53–1.41 ppm (m, 8H);
13C NMR (100 Hz, CDCl3): d=161.1, 139.9, 135.3, 130.8, 129.7, 128.8,
42.0, 33.6, 32.5, 29.7, 27.6, 25.8 ppm; MS (EI): m/z: calcd for:
C26H30N2O2S2Br2: 626.01 [M]+; found: 626.12.
ChemPlusChem 2016, 81, 515 – 520
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