Macromolecules
Article
tetramethylsilane. Mass spectra were recorded using an Agilent 1100
LC/MSD Trap. Purification of intermediates and final products was
accomplished mainly by gravity column chromatography, using silica
gel (200−300 mesh). The UV−vis spectra were recorded using a
Shimadzu UV−vis−NIR spectrophotometer (UV-3600). The PL
spectra were recorded using Edinburgh instruments (FLSP920
spectrometers). The 302 nm UV irradiation for the “ring-closing”
reaction was performed with an trans-illuminator (UVP, LLC, US,
with four ultraviolet tubes, each 8 W 220 V; the measured power is
g, 7.60 mmol), water (5 mL), and dimethoxyethane (20 mL) under N2
atmosphere. Then the catalyst Pd(PPh ) (0.22 g, 0.19 mmol) was
3
4
added under N stream, and the reaction mixture was refluxed at 90 °C
2
for 24 h. Water (200 mL) and ether (200 mL) were added, and the
organic layer was washed with water (200 mL × 3), dried over
Na SO , and concentrated under reduced pressure. The crude product
2
4
was purified by gravity silica-gel column chromatography and eluted
1
with hexane to yield a white solid (0.77 g, 36.8%). H NMR (600
MHz, CDCl , ppm): δ = 7.52 (d, 1H, J = 8.3 Hz), 7.45 (d,, 1H, J = 8.3
3
2
about 0.85 mW/cm ) and the visible light for the “ring-opening”
Hz), 7.26 (s, 1H), 7.08 (s, 1H), 6.80−6.70 (dd, 1H, J = 17.6 Hz, J =
1
2
reaction is obtained from a common household table lamp (25 W) and
a long-wavelength pass filter (490 nm, accessory of the FLSP920
spectrometer). The 617 nm visible light used for chemical reaction
quantum yield measurements is from a Red-Orange (617 nm) LED
11.0 Hz), 5.81 (d, 1H, J = 17.6 Hz), 5.31 (d, 1H, J = 11.0 Hz), 1.99 (s,
1H), 1.92 (s, 2H). 13C NMR (150 MHz, CDCl , ppm): δ = 143.32,
3
142.18, 141.29, 137.31, 136.09, 132.56, 129.32, 126.83, 125.68, 125.60,
125.51, 122.14, 114.37, 109.84, 14.57, 14.41. IR (KBr pellet): 3121−
2854, 1904.4, 1809.9, 1626.1, 1548.7, 1510.7, 1470.1, 1455.2, 1439.7,
1334.2, 1269.4, 1186.3, 1113.0, 1054.9, 987.8, 902.4, 825.4, 742.8
−2
(
LUXEON Rebel 2.28 mW cm , Lumileds Lighting, USA). The
intermediates and monomers were purified using a Waters Breeze 2
HPLC equipped with a UV detector (254 nm) and a 5-μm CN
column, and HPLC-grade-eluting solvents such as DCM, EA and
hexane as either pure solvents or binary mixed solvents. The molecular
weight of the poly[NIPAM-co-(DTE-NI)] was determined by GPC
using an IR detector in DMF/LiBr at 353 K. IR spectra were recorded
using a Bruker VERTEX 70 spectrophotometer. The hydrodynamic
diameter was measured using a dynamic light scattering instrument
−
1
+
cm . MS (APCI, m/z): 549.3 ([M + 2] ). HPLC purity: 99.5%,
eluting with hexane.
Compound 8: 1-[(5-(4-Carboxylphenyl)-2-methyl-3-thienyl)]-2-
[2-methyl-5-(4-vinylphenyl)-3-thienyl]perfluorocyclopentene. To a
100-mL two-neck round-bottom flask were added compound 5 (0.70
g, 1.30 mmol), 4-carboxyphenylboronic acid (0.23 g, 1.40 mmol),
Na
mL) under N
mmol) was added under N
CO
(0.27 g, 2.50 mmol), water (5 mL), and dimethoxyethane (10
atmosphere. Then the catalyst Pd(PPh (0.07 g, 0.064
stream, and the reaction mixture was
2
3
(
ZEN3690, Malvern). The HOMO and LUMO energy levels were
2
)
3 4
obtained using the B3LYP/6-31G(d) basis set.
2
Confocal Laser Scanning Microscopy and Super-Resolution
Imaging. HeLa cells were cultured in Dublecco’s modified Eagle’s
medium (DMEM, Invitrogen), which contained 10% fetal bovine
serum (FBS) in a humidified incubator at 37 °C under 5% CO2
atmosphere. HeLa cells were transferred into glass bottom Petri dishes
grown to 80% confluence. After attachment, the cells were exposed to
DMEM containing open-form poly[NIPAM-co-(DTE-NI)] (1O, 50
μg/mL), and after a 24 h exposure, the monolayer of cells was washed
with PBS to remove external fluorescent dyes. Before imaging, we
incubated the cells with LysoTracker Red at 75 nM for 30 min to label
lysosomes. The polymer 1O in the cells were visualized by excitation
at 405 nm and fluorescence emission was collected between 510 and
refluxed at 90 °C for 24 h. After the reation was allowed to cool down
to room temperature, 0.1 M aqueous HCl (150 mL) was added. The
mixture was extracted with ether (150 mL), and the organic layer was
washed with water (150 mL × 3), dried over Na SO , and
2 4
concentrated under reduced pressure. The crude product was purified
by gravity silica-gel column chromatography and eluted with DCM:
1
methanol (20:1) to yield a white solid (0.60 g, 50%). H NMR (600
MHz, DMSO, ppm): δ = 13.02 (s, 1H), 7.97 (d, 2H, J = 8.4 Hz), 7.78
(d, 2H, J = 8.3 Hz), 7.68 (s, 1H), 7.62 (d, 2H, J = 8.3 Hz), 7.58−7.45
(m, 3H), 6.75 (dd, 1H, J = 17.6, 11.0 Hz), 5.88 (d, 1H, J = 17.7 Hz),
5.30 (d, 1H, J = 11.0 Hz), 2.01 (d, 6H, J = 10.8 Hz). 1 C NMR (151
MHz,DMSO, ppm): δ 167.27, 143.14, 141.91, 141.77, 140.93, 137.36,
136.92, 136.40, 132.38, 130.74, 130.41, 127.42, 125.93, 125.78, 125.71,
125.47, 124.69, 123.02, 115.31, 14.62, 14.57. IR (KBr pellet): 3429.7,
3081.5, 3014.6, 2919.6, 2671.9, 2546.5, 1689.0, 1607.1, 1421.1, 1318.6,
1273.8, 1193.0, 1115.0, 1055.2, 989.0, 897.2, 836.9, 774.3, 744.2, 699.2
3
540 nm using a confocal laser-scanning microscope (Olympus
FluoView FV1000, Japan). Lysotracker in the cells was visualized by
excitation at 561 nm, and fluorescence emission was collected between
5
80 and 640 nm using the same confocal laser-scanning microscope.
The optical setup for super-resolution microscopy imaging was
−
1
+
cm . MS (APCI, m/z): 591.2 ([M + 1] ). HPLC purity: 99.0%,
eluting with EA.
based on a home-built system consisting of an Olympus IX 71 inverted
optical microscope, a 100x/NA1.49 oil immersion TIRF objective
DTE-NI. To a mixture of compound 8 (0.20 g, 0.22 mmol),
compound 9 (0.13 g, 0.22 mmol) and triphenylphosphine (0.33g, 1.26
mmol) in anhydrous THF (10 mL) at 0 °C was added dropwise a
solution of diisopropyl azodicarboxylate (DIAD) (0.30 mL, 1.48
mmol) in THF (10 mL). The reaction was warmed to room
temperature and stirred for 24 h. Water was added, and the product
was extracted twice with dichloromethane. The combined organic
layers were washed with water three times, and dried over sodium
sulfate, filtered, and evaporated. The residue was chromatographed on
(
(
(
UAPON 100XOTIRF, Olympus), a 405 nm solid-state laser
CNILaser, China), a 302 nm UV lamp, and an EMCCD camera
Andor iXon 897). During super-resolution imaging, electronic
shutters (UNIBLITZ VS14, Vincent Associates) were used to control
the duration of laser irradiance and a dichroic mirror (FF509-FDi01,
Semrock) and a long pass filter (BLP01−488R-25, Semrock) were
used to separate the collected fluorescence from scattering laser and
impurity fluorescence. We used an ImageJ plugin written in Java
15
developed in our laboratory to analyze the image. The movies were
collected at a frame rate of ∼30 Hz and all durations were 2000
frames. When the 405 nm laser photoswitched on the molecular dyads
and imparted fluorescence but also photobleached molecular dyads
during the imaging, the weak 302 nm UV irradiation was used to
regulate the emitter density by flashing on and off. We then repeated
this cycle many times to collect enough data for the reconstruction of
the super-resolution image.
a silica-gel column using DCM: methanol (50:1) as an eluent to yield a
1
light yellow solid (0.20 g, 71%). H NMR (600 MHz, CDCl
, ppm): δ
3
= 8.60 (d, 1H, J = 6.9 Hz), 8.54 (d, 1H, J = 8.0 Hz), 8.38 (d, 1H, J =
8.3 Hz), 7.98 (d, 2H, J = 8.4 Hz), 7.71 (t, 1H, J = 7.8 Hz), 7.54 (d, 2H,
J = 8.4 Hz), 7.49 (d, 2H, J = 8.2 Hz), 7.42 (t, 2H, J = 6.5 Hz), 7.36 (s,
1H), 7.28 (s, 2H), 6.71 (dd, 1H J = 17.6, J = 10.9 Hz), 5.78 (d, 1H, J
1 2
= 17.6 Hz), 5.28 (d, 1H, J = 10.9 Hz), 4.65 (s, 4H), 3.45 (s, 4H), 2.99
(s, 4H), 2.61 (s, 3H), 1.97 (d, 6H, J = 8.8 Hz). 13C NMR (151 MHz,
General Synthesis. Compounds 6 (4-methylphenylboronic
CDCl , ppm) δ = 165.97, 164.40, 163.86, 142.68, 142.08, 141.27,
3
11
acid), 3 (1,2-bis[2-bromo-5-methyl-3-thienyl]perfluorocyclopentene
141.01, 137.35, 137.26, 136.08, 132.66, 132.59, 131.39, 130.50, 129.96,
129.18, 126.81, 126.23, 126.17, 126.04, 125.78, 125.67, 125.18, 123.77,
123.23, 122.24, 114.33, 62.63, 54.67, 38.90, 29.71, 14.66, 14.61. IR
(neat): 3419.4, 3075.3, 2960.3, 2925.4, 2852.1, 2744.3, 1719.5, 1698.6,
1658.6, 1589.4, 1514.0, 1456.7, 1338.5, 1270.3, 1190.5, 1111.3, 1055.0,
10
12
or Di-Br-DTE), 7 (4-carboxyphenylboronic acid), and 9 (2-(2-
hydroxyethyl)-6-(4-methylpiperazin-1-yl)benzo[de]isoquinoline-1,3-
13
dione) were synthesized according to the corresponding literatures
while the synthetic details of other compounds are given below.
Compound 5: 1-(5-Bromo-2-methyl-3-thienyl)-2-[2-methyl-5-(4-
vinylphenyl)-5-thienyl]perfluorocyclopentene. To a 100 mL two-
neck round-bottom flask were added compound 3 (2.00 g, 3.80
mmol), 4-vinylphenylboronic acid (0.56 g, 3.80 mmol), Na CO (0.81
−1
1019.2, 989.7, 902.3, 792.8, 760.7, 737.2 cm . MS (APCI, m/z):
+
912.6 ([M + 1] ). HPLC purity: 99.7%, eluting with 20% EA in DCM.
Poly[NIPAM-co-(DTE-NI)] (1O). DTE-NI (10 mg, 0.011 mmol),
N-isopropylacrylamide (0.50 g, 4.42 mmol), 4,4′- azobis(4-cyano
2
3
B
dx.doi.org/10.1021/ma501505w | Macromolecules XXXX, XXX, XXX−XXX